EPA-600/1-76-004
January 1976
Environmental Health Effects Research Series
CHEMISTRY AND MODE OF
ACTION OF INSECTICIDES
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina 27711
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development,
U.S. Environmental Protection Agency, have been grouped into
five series. These five broad categories were established to
facilitate further development and application of environmental
technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in
related fields. The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS
RESEARCH series. This series describes projects and studies relating
to the tolerances of man for unhealthful substances or conditions.
This work is generally assessed from a medical viewpoint, including
physiological or psychological studies. In addition to toxicology
and other medical specialities, study areas include biomedical
instrumentation and health research techniques utilizing animals -
but always with intended application to human health measures.
This document is available to the public through the National
Technical Information Service, Springfield, Virginia 22161.
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EPA-600/1-76-004
January 1976
CHEMISTRY AND MODE OF ACTION OF INSECTICIDES
by
T. R. Fukuto
Department of Entomology
University of California
Riverside, California 92502
R-801837
Project Officer
John A. Santolucito
Environmental Toxicology Division
Health Effects Research Laboratory
Research Triangle.Park, North Carolina 27711
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
HEALTH EFFECTS RESEARCH LABORATORY
Research Triangle Park, North Carolina 27711
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DISCLAIMER
This report has been reviewed by the Health Effects
Research Laboratory, U.S. Environmental Protection Agency,
and approved for publication. Approval does not signify
that the contents necessarily reflect the views and policies
of the U.S. Environmental Protection Agency, nor does mention
of trade names or commercial products constitute endorsement
or recommendation for use.
n
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CONTENTS
Page
List of Figures iy
List of Tables VI
Chapters
A. Summary 1
B. Studies on Selective Toxicity 2
C. Studies on the Metabolism of Insecticides 61
D. Basic Studies on the Inhibition of the Cholinesterase Enzymes 103
E. Structure-Activity Relationships in Insecticides 145
F. Studies on the Oxidative Conversion of PS to PO Esters 186
G. Studies on Insecticide Synergism and Insect Growth Regulators 197
H. Studies on Chemical Reactions Involving Carbamate and
Organophosphate Esters 214
I. Insecticide Cyclic Nucleotide Interactions 235
J. Insecticide Penetration and its Specificity and Resistance
Mechanism 250
K. Gas Chromatography of Insecticides on Modified Supports 262
L. Neurophysiological Studies on the Mode of Action of
Insecticides 267
M. Insect Resistance to Chemicals: Resistance to Insect
Growth Regulators 280
N. Insect Resistance to Chemicals: Insecticide Resistance in
Mosquitoes 297
0. Joint Action of Herbicides and Insecticides 310
P. List of Inventions or Publications 317
ill
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FIGURES
No.
C 1 Rate of elimination of radioactivity...after leptophos 72
C 2 Rate of penetration of leptophos...house flies 81
C 3 Proposed alteration pathways for phenthoate... 98
D 1 Effect of pH on the decarbamylation rate constant...! 106
D 2 Effect of pH on the decarbamylation rate constant...II 107
D 3 Effect of pH on the decarbamylation rate constant...! 108
D 4 Effect of pH on the decarbamylation rate constant...II 109
D 5 Plots showing residual AChE activity V 117
D 6 Long-term time effects on the inhibition of AChE... 113
D 7 Double-reciprocal plot for the inhibition of AChE by VII 119
D 8 Absolute configuration of...XVIId 142
E 1 Hypothetical models showing...DDT receptor site 146
E 2 Correlation between observed. . .DDT analogs 15.1
E 3 Correlation between observed...DDT analogs 153
E 4 Relationship between observed... 154
E 5 Absorption spectra...in absolute ethanol at 70°. 165
E 6 Relationship between ...for phosphoramidothioate esters 179
E 7 Relationship between.. .for C^,j>-dimethyl N-alkylphosphor-
amidothioates 180
F 1 Mass spectrum of compound II. 188
F 2 Stereoscopic view...of ()-ethyl ethylphosphonothioic acid 194
F 3 Stereoscopic view...in the unit cell 195
H 1 Relationship between Grunwald-Winstein Y values... 224
H 2 Relationship between k^s and k1n and Grunwald-Winstein... 225
IV
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Figures continued
No.
I 1 Chemical structures of quinoxalinedithiol derivatives 239
I 3 Inhibition of cockroach brain phosphodiesterase by SAS
2079 240
I 3 Inhibition of rat brain phosphodiesterase by SAS 2079,
SQ 65,442, and aminophylline. 242
J 1 Absorption of ronnel by various house fly strains 251
J 2 Absorption of dimethoate by various house fly strains 252
LI 501 c 67 268
L 2 Picrotoxinin, picrotin, tutin 270
L 3 Bicuculline, bicucine, bicucine methyl ester, bicuculline
methiodide 271-2
L 4 Control of the activity of the flight muscles of Musca 275
L 5 Flight muscle potentials 2% hours... 276
L 6 Flight muscle activity 10 rain... 276
L 7 Flight muscle activity 6 hours... 276
L 8 Amounts of radiolabel incorporated... 277
M 1 Dose-mortality relationships of R, S,... 289
M 2 Development of resistance...selections, I and II 291
M 3 Development of resistance...selections, I and II 292
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TABLES
No. Page
B 1 Summary of autopsy data...114 mg/kg of phoxim 3
B 2 Metabolites found in urine...at 955 mg/kg 4
B 3 Metabolites in extract fraction...application of phoxim 6
B 4 Summary of toxicity data...and white mice 7
B 5 Cholinesterase inhibition data...at 37.5° 9
B 6 The chemical and toxicological properties of Abate and
its analogs 11
B 7 Bimolecular rate constants...by PO analogs of Abate 13
B 8 Amounts of Abate...after treatment with Abate 14-5
B 9 Amounts of Abate...after exposure to 0.687 p£/g Abate 17
BIO Metabolites recovered.. .dimethoxyphosphinothioyl carbofuran 21
14
Bll Metabolites present.. .application (8 |j,g/g) of CH_-N-PSC 23
B12 Summary of metabolites...with C H -N-PSC 24
B13 Metabolic products...carbofuran (XXXIV) 28
B14 Metabolic products...carbofuran (XXXIX) 30
B15 Internally recovered metabolites from house flies 31
B16 Toxicological properties...metabolites and derivatives 35
B17 Degree of N-S bond cleavage...after 10 min at 30°C 37
B18 Relation between estimated...after 10 min at 30°C 39
B19 Second-order rate constants...at pH 7.0 and 30°C 40
B20 Comparison of rates...carbofuran derivatives 43
B21 Toxicological properties...bis-carbamoyl sulfides 47
B22 Relative values — bis-carbamoyl sulfides 48
B23 Cumulative distribution..a single indicated dose 50
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Tables continued
No.
B24 Metabolites isolated... and 161 mg/kg 52
B25 Metabolites isolated...and 161 mg/kg 53
32
B26 Distribution of radioactivity... P-labeled phenthoate 55
14
B27 Metabolic products recovered... C phenthoate 56-7
C 1 Amounts of R-16,661...varying time intervals 63
C 2 Distribution of I...after treatment 65
C 3 Metabolism of I...enzyme preparation 66
C 4 Toxicological properties of I and its 4-keto (II)
metabolite 68
C 5 Distribution of leptophos...at 25 mg/kg 70
C 6 Metabolites found at various...at 25 mg/kg 73
C 7 Metabolites found at various...at 25 mg/kg 74
C 8 Distribution of leptophos...to cotton plants 75
C 9 Metabolites found at various times..to cotton plants 76
CIO Metabolites found at various times...to cotton plants 77
Cll Metabolic products recovered.. C-phenoxy leptophos 80
C12 Summation of metabolic products..radiolabeled leptophos 84
1 / Q O
C13 The penetration of C and P phenthoate...solid residue 88
C14 Amount of C metabolites...indicated time intervals 90
32
C15 Amount of P metabolites...indicated time 91
C16 Amount of C metabolites...indicated time 92
32
C17 Amount of P metabolites...indicated time 93
C18 The breakdown products...indicated time 94-5
Vll
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Tables continued
No. Page
1 / O O
C19 The metabolites of C and P phenthoate, , .sunlight 97
D 1 Rate constants. . .3-nitrophenyl dimethylcarbamate at pH 7.9
D 2 Effect of deuterium oxide. . .at pD of 8.42 112
D 3 Dissociation constant. . .irreversible carbamates 121
D 4 Optical rotation... XVIII 123
D 5 Anticholinesterase properties. .. (HSChE) 125
D 6 Toxicity of XVIII... and white mouse (oral) 127
D 7 Plant systemic activity — test arthropods 129
D 8 Optical rotation. . .resolved (XXIa-d) compounds 131
D 9 Affinity, equilibrium. ..at 30°C, pH 7.55 136-7
D10 Final positional parameters... 141
E 1 Effect of variations. . .activity of DDT analogs 148-9
E 2 Effect of variation. . .activity of DDT analogs 150
E 3 Physical and biological properties. . .and related ketones 158-9
E 4 Toxicological properties of (),()-diethyl Ifl-acetyl-lfl-
phenylphosphoramidates 167
E 5 Toxicological and hydrolytic properties — A_ 173
E 6 Physical and chemical properties. . .insecticides 175-6
E 7 Translocation data... other organophosphorus insecticides 178
F 1 Yields of products from the oxidation of III 191
G 1 Toxicological and oxidase. . ,1,3-benzodioxoles 199
G 2 Toxicological and oxidase. . .1,3-dioxoles 200-1
G 3 Oxidase inhibition data. . .corresponding catechols 202-3
G 4 Toxicological data ...Culex pipiens larvae 206
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Tables continued
No. Page
G 5 Toxicological data...Culex pipiens larvae 207
G 6 Toxicological data...Culex pipiens larvae 208
G 7 Toxicological data...Culex pipiens larvae 209-10
H 1 Salt distribution...solvent mixtures at 23° 216
H 2 Product distribution...in a sealed ampoule 217
H 3 Second-order rate constants...potassium hydroxide at 27° 219
H 4 Pseudo first-order rate constants... 219
H 5 Products obtained...triethyl phosphite at 25° 230
H 6 Effect of HBr...triethyl phosphite in benzene 232
I 1 Direct effects of insecticide-acaricides on cockroach
brain adenyl cyclase 237
I 2 Direct effects of insecticides-acaricides on cockroach
brain phosphodiesterase 238
I 3 Inhibition of cockroach brain phosphodiesterase by
quinoxalinedithiol derivatives 240
I 4 The effects of methylxanthines... from various sources 241
I 5 Normal (control) levels ... the Madagascar cockroach 243
I 6 Levels of cyclic nucleotides...aminophylline/g body weight 244
I 7 Levels of cyclic nucleotides...parathion per cockroach 246
J 1 Percent insecticide absorbed...1.0 ^/female fly 253-5
J 2 Rank order of house fly strains...(from Table 1) 256
J 3 Water solubility, partitioning coefficients...house fly 257
J 4 Toxicity and resistance ratios...10 yg/female fly 258
K 1 Retention time in minutes...surface-modified GLC column 263
K 2 Retention time in minutes...surface-modified GLC column 265
L 1 Effects of respiratory inhibitors on spontaneous discharge 27
IX
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Tables
No.
M 1
M 2
M 3
M 4
M 5
M 6
M 7
M 8
N I
N 2
N 3
N 4
N 5
N 6
0 1
0 2
0 3
continued
Comparative activity of various. . .house fly
Response of various strains.,. at 0.025 |j,g/prepupa
Response of four strains of the house fly toward
methoprene
Response of various strains of the house fly to treatment
of prepupae with JHAs
Susceptibility of various strains... 10 ^g per prepupa
Genetic analysis. . .house fly
Effect of methoprene selection. . .house fly
Development of resistance. . .house fly
Comparative levels of resistance. . .propoxur (Carb.-R)
K^ values for larvae of four strains of A. albiraanus
l
Michaelis constants. . .at 30 ± 1°C, pH 7.5
Resistance spectra. . .Hanford, California, 1974
Synergism of organophosphates. . .Culex p. quinquefasciatus
larvae
Relative toxicity. . .Culex p. quinquefasciatus
Toxicity of herbicides. . .and Anopheles albimanus
Joint action of propoxur .. .Culex p. fatigans
Joint action of propoxur. . .Anopheles albimanus
Page
282
283
283
285
287
288
293
293
298
299
300
302
304
305
312
313
314
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A. SUMMARY
This report summarizes research accomplishments for the period
January 1, 1971, to September 1, 1975. In gross aspects, our studies
on the chemistry and mode of action of insecticides have been
concerned with the intoxication and detoxication processes which
take place when an animal or plant is exposed to different organic
insecticides. Progress in the following general areas is reported.
1. Insecticide selectivity
2. Insecticide metabolism
3. Inhibition of the cholinesterase enzymes
4. Structure-activity relationships in insecticides
5. Oxidative conversion of PS to PO esters
6. Insecticide synergism and insect growth regulators
7. Chemical reaction involving carbamate and organophosphorus
esters
8. Insecticide cyclic nucleotide interactions
9. Insecticide penetration and its relation to resistance
10. Gas chromatography of insecticides
11. Neurophysiological studies on insecticide mode of action
12. Insecticide resistance
13. Joint action of herbicides and insecticides
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B. STUDIES ON SELECTIVE TOXICITY
1. Selective Toxicity of Phenylglyoxylonitrile Oxime 0,0-Diethyl
Phosphorothioate (Phoxim)
Introduction - Phoxim is a promising new organophosphorus insecticide
which has shown excellent activity for the control of a variety of
insects of hygienic and agricultural importance and is extremely safe
to warm-blooded animals (rat oral LD5Q 8500 mg/kg). In contrast to
most "safe" organophosphorus insecticides, Phoxim is unusual in that
it is a diethyl ester rather than a dimethyl ester. Because of its
outstanding properties of selectivity, an investigation on the mode
of action of Phoxim in the white mouse and house fly was conducted to
determine the basis for selectivity. In addition, a number of other
dialkyl esters analogous to Phoxim were synthesized and examined for
toxicity to mice and insects.
32
Metabolism in the White Mouse - P-Labeled Phoxim was given orally
to white mice at 3 dosages; 10.5, 114 and 955 mg/kg. At these dosage
levels, the ultimate recovery of administered radioactivity in the
urine and feces ranged from 73 to 827o over a period of 140 hours. In
general, excretion of radioactivity was substantially slower compared
to other organophosphorus insecticides previously studie. For example,
24 hours after oral treatment of mice with 10.5 and 114 mg/kg of 32p_
Phoxim only 43% and 22%, respectively of the administered radioactivity
was excreted in the urine. At the high dosage of 955 mg/kg only 17%
of the administered dosage was excreted after 30 hours.
Because of the problem presented by the abnormally slow rate of
Phoxim excretion, an autopsy was performed on a mouse treated at 114
mg/kg after 48 hours to determine the fate of the administered
material. At this time 437o of the dose had been excreted. The
results of the autopsy are presented in Table 1 and indicate that
most of the radioactivity was localized in the gut or urinary bladder.
Further, virtually all of the radioactivity was in the form of water
soluble material, indicating that most of the administered dose was
converted to detoxified materials. The slower than expected excretion
rates observed after treatment with Phoxim is attributed to storage of
metabolic products in the urinary bladder which was abnormally large
owing to the accumulation of urine. An explanation for this observation
is not available.
Table 2 gives data on the identity and relative amounts of the various
metabolites obtained at 114 and 955 mg/kg. Radioactive urine from
white mice was analyzed by ion-exchange and thin-layer chromatography
(tic). As indicated in the table, at least 5 metabolites were identi-
fied as (1) diethyl phosphoric acid, (2) Phoxim, (3) Phoxim carboxylic
acid, (4) 0,0-diethyl phosphorothioic acid, and (5) either desethyl
Phoxim or desethyl PO Phoxim. Of the products isolated, diethyl
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Table 1. SUMMARY OF AUTOPSY DATA ON A WHITE MOUSE 48 HR
AFTER ORAL TREATMENT WITH 114 MG/KG OF PHOXIM
Organ
Brain
Thymus gland
Hind leg muscle
Heart
Kidney
Liver
Gut
Urinary bladder
Total
Organic-
soluble
0.07
0.01
0.00
0.01
0.03
0.05
0.17
2.10
2.44
7» Recovered internal
radioactivity
Water-
soluble
0.14
0.05
0.54
0.23
0.13
1.60
8.60
86.30
97.59
Sum of
organic-
+ water-
soluble
0.21
0.06
0.54
0.24
0.16
1.65
8.77
88.40
100.00
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Table 2. METABOLITES FOUND IN URINE FROM WHITE MICE 24 HR AFTER TREATMENT WITH PHOXIM AT
114 MG/KG AND AFTER 30 HR AT 955 MG/KG
1.
2.
3.
4.
5.
Anion exchange
tube numbers
0
(C2H50)2P-OH 21-35
Phoxim 47-57
S COOH
(C2H50)2PON=C-C6H5 47-57
S
(C2H50)2POH 59-64
S(0)
(C2H50) (HO)PON=C-C6H5 84-93
CN
Radioactivity not in peaks
Unrecoverable radioactivity
114 mg/kg
%a
58.9
1.1
2.8
20.0
6.2
4.3
6.7
dose
mgb
0.62
0.01
0.03
0.21
0.07
0.05
0.07
955
%
43.1
2.1
23 . 6
17.7
5.0
5.4
3.1
mg/kg dose
mg
2.36
0.12
1.29
0.97
0,27
0.30
0.17
a % of radioactivity in urine applied to column.
° Values expressed in terms of Phoxim equivalents based on average dose given to each mouse.
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phosphoric acid and 0,0-diethyl phosphorothioic acid were by far the
most abundant metabolites.
Because of the large amounts of diethyl phosphoric acid present in the
urine, J P-0,0-diethyl phosphorothioic acid was given orally to the
mouse to determine whether this material served as a precursor to diethyl
phosphoric acid. However, this study showed that more than 95% of
administered phosphorothioic acid was recovered in the urine as un-
changed material suggesting that diethyl phosphoric acid was produced
from PO Phoxim.
The most striking change in the relative amounts of any metabolite
upon increasing the dose from 114 to 955 mg/kg occurred with Phoxim
carboxylic acid. In terms of actual amounts of material present in the
urine, Phoxim carboxylic acid was 43 times more abundant at the higher
than at the lower dosage. The results point out the possible importance
of this metabolic reaction in making Phoxim safe to mammals.
Metabolism in House Flies - The metabolism of Phoxim also was examined
in susceptible (SNAIDM) and resistant (Kgc) strains of house flies.
RSC is the Stauffer chlorthion resistant strain to which Phoxim was
about 100-fold less effective (LD5Q 215 |_ig/g) compared to the SWAIDM
strain (LD^Q 2.1 (ig/g) . House flies were treated topically, Sj^j™ at
1.75 p.g/g and Rg(-, at 1.75 and 150 fig/g. Penetration as measured by the
rate of fly surface loss was rapid in both susceptible and resistant
house flies, 80-90% being penetrated after 30 minutes. Penetration
probably is not a factor contributing to the difference in Phoxim
toxicity between the two strains.
The results showed conclusively that differences in the rates of detoxi-
cation of Phoxim exist between susceptible flies and the lower dosage
and there are considerably larger amounts of hydrolytic products in the
resistant strain at all times. Table 3 gives data on the nature and
amounts of the metabolites found internally in flies 2 hours after
treatment. The data show that five metabolites were found internally
in the susceptible strain and six in the resistant strain. Phoxim,
PO Phoxim, 0,0-diethyl phosphorothioic acid, diethyl phosphoric acid,
and phosphoric acid were found in both strains. A metabolite tenta-
tively identified as ethyl phosphoric acid was found only in resistant
flies. At the same dosage of 1.75 |ag/g much smaller amounts of intact
Phoxim and its PO analog were found in the internal extract of resistant
compared to susceptible flies. The greatly reduced amounts of Phoxim
and PO Phoxim in the resistant strain must be the result of more rapid
metabolic 'degradation, resulting in significantly greater amounts of
hydrolysis products in the resistant compared to the susceptible strain.
Toxicity of Phoxim Analogs - Toxicity data for Phoxim and some of its
analogs against SNATnM and R flies, German cockroach and the white
mouse are summarized in Table 4. With the exception of the diethyl-
phosphinothioate (IX) and phosphinate (X) analogs, all of the compounds
were quite toxic to susceptible flies with LD^g values between 2.1-16
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Table 3. METABOLITES IN EXTRACT FRACTION FROM SUSCEPTIBLE
AND RESISTANT HOUSE FLIES 2 HR AFTER TOPICAL APPLICATION
OF PHOXIM
Metabolite
Mg/g
HO /O 7»
?' (?) Mg/g
bNAIDM
1.75 |_ig/g
Dose
3.1
0.04
-
RSC
1.75 pg/g
Dose
3.2
0.04
9.2
0.11
150 Mg/g
Dose
0.9
0.38
-
GEN =
NON=C
70 26.9 43.3 11.9
Mg/g 0.38 0.53 5.1
7o 7.4 2.4 2.9
Mg/g 0.11 0.03 1.2
30.3 5.9 47.9
0.39 0.38 14.7
C,H,-0 /$ I 27.6 30.6 34.4
P' Ug/g 0.39 0.38 14.7
CJ3.rO OH
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Table 4. SUMMARY OF TOXICITY DATA FOR PHOXIM AND ITS ANALOGS TO HOUSE FLIES (SflAIDM
GERMAN COCKROACHES, AND WHITE MICE
R ^X
R1 ON=C — (' M
Analog R R1
IP 11 n P T4 n
Vjotlcv/ L/oHrVJ
T T /"* TJ /"^ P U /"^
x. -L ^» n f \J ^* n \j
III CH 0 CH 0
IV CH30 CH 0
V-I f~> TJ f\ ~ (** TT /^
X ~ V* ^. -Tl-^ \J JL \J ,* Li^\J
VI i-C3H7° i"C3H7°
VII C2H50 C2H5
TTTTT P TJ n P 14
V 111 L.-n[-U ? S
TV P TJ P 1J
1A L..."^ L^rtrlc-
X C H C H
LD50 Values
X
SNAIDM
S 2
0 3
S 3
0 5
S 16
0 5
S 5
0 5
S 125
0 410
Insects
flies Rg£
.1
.4 >2,
.3 >2,
.0 >2,
.0
.0
.8
.9
>2,
>2,
(ug/g)
flies
215
500
500
500
220
53.5
21.5
150
500
500
German
cockroach
6.1
5.5
7.8
7.6
209
12.5
9.7
5.5
-
-
White mice
(mg/kg)
>2,000
1,000
>2,000
>1,000
>1,500
1,250
>500
70-73
>500
>500
-------
Hg/g. Toxicities against resistant flies were variable, ranging from
21.5 to >2,500 (j.g/g, and with the exception of V, the analogs showed
high toxicity to the German cockroach. However, with the exception of
the ethylphosphonate analog, all compounds were of low toxicity to the
white mouse.
The data in Table 5 show that there are substantial differences in the
sensitivity of insect and mammalian ChE to inhibition by the various
PO esters. The magnitude of these differences is represented in Table
5 by the ratios of inhibition of the two cholinesterases, i.e., ke (fly-
head ChE)/k (bovine erythrocyte ChE) defined as the selective inhibi-
tion ratio. The largest ratio was found with X, the diethylphosphinate
analog of Phoxim. However, in spite of the large difference in inhibi-
tion rates, X was nontoxic to both the house fly and the mouse probably
because of its high susceptibility to hydrolysis. Compounds II, IV, and
VI all showed large selective inhibition ratios and all these compounds
were substantially more toxic to house flies than to mice. The phosphon-
ate analog VIII, the compound most toxic to the mouse, showed the lowest
inhibition ratio of 49.
Conclusion - Our results indicate the metabolic factors play an im-
portant role in the biocidal activity of Phoxim to insects and the
mouse. The equations below show the proposed scheme for the metabo-
lism of Phoxim in the white mouse and in house flies.
CN
mice
flies
mice _
mice
•-
2 5 Hp*
CH d N
2 5
C2H5V
C2H5d ^
C2H5Ov ,(
3
OH
s
°~Nz(r~C=
COOH
D
mice flies
, C2H5°
.8(0)
mice PO analog of
* corboxylic acid
i
'mice
V
mice
HO
CN
H3P04
flies
flies
CN
(either PS or .PO esfer)
flies
HO OH
(tentative)
•Hypothetical pathway
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Table 5. CHOLINESTERASE INHIBITION DATA FOR PHOXIM AND ITS ANALOGS AT 37.5°
R ^-JO
R,/-ON=C_^J>
Analog R R'
I C2H50 C2H50
IV CH 0 CH 0
VI i-C3H70 i-C3H?C
VIII C2H5° C2H5
X C2H5 C2H6
SNAIDM Fly-head ChE
i50 OS)
1.9 X 10"9
1.1 X 10"9
) 7.8 X 10"10
2.5 X 10"9
1.2 X 10"8
min"1)
8.4 X 107
1.3 X 108
8.1 X 107
5.9 X 107
1.9 X 106
Bovine-erythrocyte ChE
I50
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The basis for the extreme safety of Phoxim to mice is readily apparent
from the metabolism data. Only very small amounts of Phoxim were found
in mouse urine 24 hr after treatment and in no case was any PO Phoxim
detected. Further, only insignificant amounts of intact ester were
noted in the internal organs of the mouse autopsied 48 hr after treat-
ment with 114 mg/kg Phoxim. The evidence clearly indicates that Phoxim
is peculiarly suited for metabolic detoxication by the white mouse.
Owing to the large amounts of diethyl phosphoric acid found in mouse
urine, it is evident that Phoxim is desulfurated to PO Phoxim which in
turn must be hydrolyzed with remarkable rapidity to diethyl phosphoric
acid and at no time does PO Phoxim reach a sufficiently high concentra-
tion to intoxicate the mouse. Support for rapid degradation of PO
Phoxim is found in the fact that PO Phoxim itself is quite nontoxic
with an I^Q value of 1000 mg/kg. In addition to the above pathway
for detoxication, the mouse also is capable of degrading Phoxim directly
to C),0-diethyl phosphorothioic acid and to Phoxim carboxylic acid. The
hydrolysis of Phoxim to 0,0-diethyl phosphorothioic acid appears to be
relatively constant with respect to the administered dose. Hydrolysis
to Phoxim carboxylic acid, on the other hand, appears to increase with
increase in administered dosage, and evidently becomes an important
detoxication pathway at high dosages. The net result is that PO Phoxim
in spite of its strong anticholinesterase properties, is not allowed
to reach a level critical in the mouse.
2. Metabolism of Abate in Mosquito Larvae and House Flies.
Introduction - Abate (0,0,0',0'-tetramethyl 0,0' -thiodi-p_-phenylene
phosphorothioate) is currently being used for the control of aquative
larvae. Based on extensive toxicological evaluations, Abate has proved
to be highly effective against a variety of mosquito larvae but is
relatively nontoxic to other insects, fish, and mammals. The reported
LD,-0 of Abate to rats and mice is approximately 4000 mg/kg. Abate,
because of its peculiar specificity toward aquatic larvae, provides
an interesting example of selectivity between insects. The following
discussion is concerned with the absorption and metabolism of Abate in
the larvae of the mosquito (Aedes aegypti) and common house fly.
Toxicity of Abate and Some of its Oxidation Products - The principal
oxidation products of Abate were synthesized and examined for toxicity
to mosquito larvae and house flies. The data in Table 6 show that these
compounds are relatively nontoxic to the house fly. Against the 4th
instar mosquito larvae, Aedes aegypti, Abate also was more effective
than its oxidation products and the order of effectiveness may be
summarized as follows:
PS(S) > PS(SO) > PS(SOa) > PO(S) > PO(SO) > PO(S02).
Evidently, the order of toxicity to mosquito larvae is directly related
to the oxidation state of the molecule. Although this order may be
purely fortuitous, i.e., decreasing toxicity with increasing oxidation
10
-------
Table 6. THE CHEMICAL AND TOXICOLOGICAL PROPERTIES OF ABATE AND ITS ANALOGS
Compound structure
XI [(CH30)jO..
0
XII [ (CH 0) PO-^Jy-
S
XIII [(CH30)2PO-^^-
0
xiv [ (CH o) po-//_y-
XV [ (CH 0) 2PO~vJ/'
0
XVI [(CH 0) PO-^jV
Designated
symbol
-+28 PS(S)
-J-jS PO(S)
-J-^SO PS (SO)
-^jSO PO(SO)
•*2S02 PS(S02)
-^so2 po(so2)
fOr\ Elemental
Calcd
36
1.5522a C 44.22
H 4.60
41-42 C 39.65
H 4.14
32-33 C 42.60
H 4.44
61-62 C 38.52
H 4.03
85-87 C 41.15
analysis
Found
-
44.50
5.56
39.53
4.18
42.46
4.24
38.38
4.07
41.36
Insect
House fly
LD5Q
(ug/g)
205
>500
>500
>500
>500
>500
toxicity
Mosquito
larvae
LC5Q (ppm)
6.3 X 10"3
6.3 X 10"2
3.3 X 10"2
2.4 X 10"1
3.9 X 10"2
2.9 X 10"1
a Liquid, refractive index.
-------
state (and also polarity), it nevertheless suggests that absorption of
the toxicant into the larvae from the aqueous phase may. strongly in-
fluence the effectiveness of the compound. PS esters, being more
lipophilic than PO esters, should be able to penetrate the hydrophobic
epicuticular wax layer of the larvae at a faster rate compared to PO
esters. The same reasoning also applies to the relative toxicities of
the sulfide, sulfoxide, and sulfone.
No obvious relationship was evident between toxicity and anticholin-
esterase activity of the PO esters (XIV-XVI) and it appears that
sensitivity of fly-head and mosquito larvae cholinesterase toward these
has little to do with the selective properties of Abate. The pattern
of anticholinesterase activity against mosquito larvae cholinesterase
was in the order expected, i.e., PO(SOg) > PO(SO) > PO(S). Overall,
however, the series of PO esters were equally effective in inhibiting
fly-head and mosquito larvae cholinesterase, suggesting that cholinester-
ase inhibition has little to do with selectivity.
3
Metabolism in the House Fly - House flies were treated topically with H-
Abate at the dosages of 4.23 and 53.8 |_ig/g. Recovery of total radio-
activity was quite good at the higher dosage (84-100%) but was less
satisfactory at the lower dosage which and an average recovery of only
60%. The fraction of applied Abate penetrated in the house fly was much
greater at the lower dosage and was approximately equal to that of other
organophosphorus insecticides previously studied, e.g., Phoxim. At the
higher dosage, the amount of penetrated Abate relative to the applied
dose was low and the rate of penetration was approximately zero-order,
indicating that the penetration process was near saturation at this
dosage.
Virtually all of the expected metabolic products were found internally
in the fly, either as the intact ester, or as hydrolyzed material.
Table 8 gives the various amounts, in terms of microgram equivalents of
Abate per gram of fly for each metabolite found in the internal fraction
after treatment with Abate. The data show that there is a significant
lag in the accumulation of total intact esters (XI-XVI) at the higher
dosage relative to the amount of applied Abate. For example, at the
point where approximately maximum accumulation occurs (5 hr) and where
comparison can be made, the total amount of intact esters at the high
dosage was 5.88 n§/g compared to 0.85 |ag/g at the low dosage. Thus, the
ratio of intact ester present internally at the two dosages is 6.8, a
significantly smaller number than the 12.3-fold difference in the applied
dosages. At the earlier time periods, the ratio was even smaller. These
results serve to point out the limiting effect that penetration has on
house fly intoxication by Abate.
The largest amount of radioactivity present in the fly was in the form
of unchanged Abate at all time intervals after treatment, ranging from
75-90% of the total intact esters, indicating that the metabolism of
12
-------
Table 7. BIMOLECULAR RATE CONSTANTS (k^ FOR THE INHIBITION OF
HOUSE FLY-HEAD AND MOSQUITO LARVAE CHOLINESTERASE
BY PO ANALOGS OF ABATE
Compound
k (M"1 min"1) X 10"6
House fly
Mosquito
larvae
XIV
XV
XVI
PO(S)
PO(SO)
PO(S02)
6.99
0.59
1.20
0.37
1.10
1.56
13
-------
Table 8. AMOUNTS OF ABATE AND ITS METABOLITES FOUND INTERNALLY IN THE FLY AFTER TREATMENT WITH ABATE
Compound
XI PS(S) (Abate)
XII PS (SO)
XIII PS(S02)
XIV PO(S)
XV PO(SO)
XVI PO(S07)
XVII HO(S)
XVIII HO(SO)
XIX HO(S02)
Conjugates (RfO)
Abate
dosage
53.8
4.23
53.8
4.23
53.8
4.23
53.8
4.23
53.8
4.23
53.8
4.23
53.8
4.23
53.8
4.23
53.8
4.23
53.8
4.23
Amount (ng Abate equiv/g) of recovered radioactivity at
indicated time intervals (hr)
0.25
0.62
0.22
0.04
0.01
0.02
ND
ND
Tr
ND
Tr
ND
Tr
ND
Tr
ND
0.01
ND
Tr
0.09
0.02
0.5 1.0
0.98 1.37
0.04 0.07
Tra NDb
Tr
Tr 0.01
ND 0.01
ND Tr
ND ND
0.01 0.04
ND ND
0.11 0.33
2.0
1.90
0.50
0.13
0.04
0.02
0.01
0.04
0.01
0.04
0.01
0.02
Tr
ND
Tr
0.19
0.02
ND
Tr
0.51
0.19
4.0 5.0
4.25 4.67
0.68
0.46 0.61
0.08
0.10 0 . 20
0.04
0.11 0.14
0.02
0.12 0.14
0.02
0.09 0.12
0.01
ND Tr
Tr
0.18 Oo24
0.03
ND Tr
0.01
1.10 1.14
0.11
6.0 7.0 10.0
5.16 4.55
0.46
0.70 0.85
0.04
0.19 0.21
0.02
0.13 0.12
0.01
0.22 0.18
0.02
0.10 0.11
0,01
Tr Tr
Tr
0.32 0.14
0.02
0.02 0.02
0.01
1.83 1.34
0.01
-------
Table 8 (continued).
Compound
Total PS esters
Total PO esters
Abate
dosage
(us/g)
53.8
4.23
53.8
4.23
Amount (jjg Abate equiv/g) of recovered radioactivity at
indicated time intervals (hr
0.25 0.5
0.68 1.02
0.23
ND Tr
Tr
1.0 2.0
1.44 2.05
0.55
0.02 0.10
0.02
4.0
4.81
-
0.32
—
5.0
5.48
0.80
0.40
0.05
6.0
6.05
-
0.45
—
7.0
5.62
-
0.41
—
10.0
_
0.52
_
0.04
a Trace.
b Not detectable,
-------
Abate to oxidation products is quite slow in the house fly. Of the
various oxidative metabolites, Abate sulfoxide (XII) was present in
substantially larger amounts than any of the other intact esters.
Significant amounts of the PO esters (XIV-XVI) were not found until 1
hr after treatment and the amount reached a maximum at around 5-6 hr.
The total amount of PO esters accumulating in the house fly was much
lower than that found for PO esters generated from other phosphoro-
thionates previously studied in this laboratory. Since intoxication
of house flies probably occurs through the PO esters, i.e., by inhibi-
tion of cholinesterase, the low level of PO esters present after Abate
treatment is consistent with the high tolerance of house flies toward
Abate.
Metabolism in Mosquito Larvae - A single dose of 0.0565 ppiti Abate in
3.0 ml water containing 0.25 g larvae was used for the metabolism study
in mosquito larvae. Because of its aqueous habitat, it was necessary to
examine the water medium for metabolites as well as the larvae. It
became evident early in our studies that the larvae absorbed Abate at
a rapid rate and that the metabolites appear both internally in the
larvae and externally in the water medium after short intervals follow-
ing exposure. This absorption and desorption phenomenon is termed
"recycling." The absorption of Abate into mosquito larvae was extremely
rapid and over 99% was absorbed within 1 hour after exposure. In deter-
mining the rate of absorption, the amount of Abate remaining in the water
was assumed to be unabsorbed material.
In spite of the recycling phenomenon, substantial amounts of radio-
activity remained inside the larvae. Data for the distribution of Abate
and its metabolites in larvae and the water medium are summarized in
Table 9. The amount present in water was divided into an ether-
extractable and an ether-unextractable fraction to differentiate between
metabolites and conjugates since compounds XI-XIX all partitioned in
favor of ether. The total recovery of radioactivity, including that
found in the larvae, ether-extractables and ether-unextractables in the
water medium, ranged from 80-91%. Examination of the data will show
that substantial amounts of radioactivity accumulate in the larvae,
even as early as 1/4 hr after exposure, rising to a maximum after 1 hr
(55% of recovered dosage). Evidently, Abate or its metabolites accumu-
late in mosquito larvae at a rate some 2-3 times faster than in house
flies.
The data in Table 9 show that the largest amount of radioactivity found
internally in mosquito larvae was in the form of Abate. However, in
contrast to house flies, Abate appeared to be more rapidly metabolized
in mosquito larvae. This is apparent from the amounts of phenols (XVII-
XIX) and conjugates listed in the table. The amount of PO esters found
at any time after treatment is the result of the difference in formation
and degradation of these esters. Since mosquito larvae are able to
degrade Abate and its intact metabolites to hydrolytic products faster
than house flies, it appears that the conversion of PS to PO esters takes
16
-------
Table 9. AMOUNTS OF ABATE AND ITS METABOLITES IN MOSQUITO LARVAE AND WATER MEDIUM (ETHER EXTRACT)
AFTER EXPOSURE TO 0.687 |aG/G ABATE
Compound
XI Abate
XII PS(SO)
XIII PS(S02)
XIV PO(S)
XV PO(SO)
XVI PO(S02)
XVII HO(S)
XVIII HO (SO)
XIX HO(S02)
Conjugates (R^O)
Total PS esters
Total PO esters
Amount (M-g Abate equiv/g) of recovered radioactivity at indicated time
intervals (hr)
0.25
Larvae
0.183
0.004
0.002
0.003
0.005
0.001
Tr
0.023
0.003
0.061
0.189
0.009
0
Larvae
0.211
0.002
0.001
0.001
0.002
0.001
0.001
0.019
0.003
0.059
0.214
0.004
.5
Water
0.065
0.111
0.002
0.002
0.001
0.001
Tr
0.001
0.002
0.003
0.178
0.004
1
Larvae
0.120
0.003
0.009
0.001
0.026
0.005
ND
0.022
Tr
0.184
0.132
0.032
.0
Water
0,009
0.059
0.010
0.010
0.004
0.002
ND
0.005
0.002
0.010
0.078
0.016
2
Larvae
0.071
0.004
0.008
0.001
0.005
0.002
0.001
0.048
ND
0.205
0.083
0.008
.0
Water
0.011
0.026
0,002
0.003
0.003
0 = 001
ND
0.009
0.005
0.013
0.039
0.007
4
Larvae
0.049
0.004
0.001
0.001
0.001
0.001
ND
0.021
0.003
0.170
0.055
0.003
.0
Water
0.004
Oo016
0.004
0.007
0.005
0.005
ND
0.012
0.017
0.016
0.024
0.017
5
Larvae
0.053
0.003
Tra
Tr
0.001
0.001
Tr
0.033
0.007
0.144
0.056
0.002
.0
Water
0.002
0.011
0.014
0.003
0.001
0.003
Tr
0.002
0.017
0.016
0.027
0.007
a Trace.
b Not detectable.
-------
place more rapidly in mosquito larvae than in house flies. For example,
at 1 hr after treatment, approximately 7.1% (0.048 |ag/g) of the applied
dose of 0.678 |ig/g was present as PO esters (XIV-XVI) in the total system
(larvae and water medium). In comparison, house flies treated at 4.23
(jg/g, a dosage some 16-fold greater than for mosquito larvae on a weight
basis, contained 0.02 (ig/g (0.59% of applied dosage) at 2 hr following
treatment.
The data in Table 9 show that significant amounts of intact esters are
present in the water at all time periods, the largest fraction consist-
ing of the PS(SO) derivative (XII). These results indicate that Abate,
after absorption in the larvae, is rapidly converted to metabolic
products and a substantial portion of the metabolites is eliminated into
the water medium, including intact esters. These materials, then, are
available to the mosquito larvae for reabsorption and further metabolism.
Conclusion - The pathways for the metabolism of Abate in the house fly
and mosquito larvae are qualitatively similar as indicated in the scheme
below. No gross difference in metabolic pathways or products was noted
which would readily explain the selective action of Abate.
P=S(S) >P=S(SOl >P=S(S02)
! p=0(S) >P=0(SO) >P=0(S02)
V -L '
HO(S) >HO(SO) >HO(SO,)
•"Conjugates4
In spite of the overall similarity in the behavior of Abate in house
flies and mosquito larvae, there are distinct quantitative differences
which may account, at least in part, for the toxicity of Abate to these
insects. A notable difference is the apparently greater rate of Abate
absorption into mosquito larvae compared to flies. At the dosage of
0.678 |jg/g (0.3 ml of 0.0565 ppm Abate containing 0.25 g larvae), the
penetration of Abate into mosquito larvae was extremely rapid and vir-
tually all of the Abate in the water medium was absorbed into the larvae
in less than 1 hr after exposure. The dosage of 0.678 |og/g is approx
27-fold less than the calculated LC^Q value of 18 (ig/g Abate to larvae
(from an LC.-Q value of 0.0063 ppm Abate in 100 ml water containing 20
larvae.
18
-------
In terms of actual amounts penetrated, house flies absorbed only 2.6
times as much Abate in 1 hr compared to mosquito larvae, although the
dosage applied to flies (4.23 ng/g) was more than 6-fold greater than
that applied to mosquito larvae. The relative rate of penetration in
house flies after treatment at 53.8 |_ig/g, a dosage approx 4-fold less
than the LDcQ value, was much slower and may be the limiting step in
the intoxication process.
The accumulation of intact PO esters produced by desulfuration of PS
esters also was greater in mosquito larvae compared to house flies,
particularly in the early stages after treatment where as much as 8-fold
more PO ester was present in the larvae. The rapid buildup of PO esters
in the mosquito larvae may be attributed in part to rapid initial absor-
ption and the recycling process which allows reabsorption of previously
eliminated intact esters from the water medium. The recycling phenom-
enon particularly applies to the PS(SO) ester which was present in
relatively large amounts in the water medium shortly after larvae
exposure. Rapid absorption of Abate and conversion to intact PO esters
in the larvae undoubtedly are important factors which contribute to the
peculiar specificity of Abate to mosquito larvae.
3. Metabolism of 2,2-Dimethy1-2,3-Dihydrobenzofuranyl-7 N-Dimethoxy-
phosphinothioyl-N-Methylcarbamate (PSC) in the House Fly, Rat and Mouse
Introduction - A previous investigation in this laboratory demonstrated
that toxic methylcarbamate insecticides may be converted into derivatives
possessing favorable properties of selectivity by appropriate substitu-
tion of the proton on the carbamyl nitrogen atom-'-. Compared to the
original methylcarbamate, the N-dialkoxyphosphinothioyl moieties are
substantially less toxic to the white mouse but on the whole are quite
effective insecticides. In seeking additional information concerning
the underlying basis for the selective toxicity of derivatized methyl-
carbamate esters, a study of the comparative metabolism of the dimethoxy-
phosphinothioyl derivative of carbofuran (XXX, 2,2-dimethyl-2,3-dihydro-
benzofuranyl-7 N-dimethoxyphosphinothioyl-N-methylcarbamate, or PSC)
was conducted in the house fly, mouse, and rat.
Metabolism in the White Mouse and Rat - White mice and rats were treated
orally at a dose of 100 mg/kg of C ring-labeled PSC. At this dosage,
PSC was metabolized rapidly and excreted principally via the urine. For
example, in the mouse 64% of the administered dose was recovered after 21
hours, 78% after 24 hours, and 88% after 96 hours. A maximum of 5% of
the dose was recovered in the feces during a 96-hour collection period.
The total recovery of 14C ranged from 93-95%.
Of the total radioactivity present in the 24-hr urine sample, less than
1% was extractable into chloroform and the bulk of the radioactivity re-
mained in the aqueous phase, presumably in the form of conjugates. Mild
acid hydrolysis of the aqueous phase, however, afforded products in vir-
tually quantitative conversion which were extractable into chloroform.
19
-------
Analysis of the chloroform extract by tic revealed the presence of the
seven metabolites listed in Table 10, of which two remain as minor
unknown components. 3-Ketocarbofuran and 3-hydroxycarbofuran were
identified by cochromatography with authentic samples and cochroma-
tography, infrared and mass spectral analyses were used to verify the
structures of carbofuran phenol, 3-hydroxycarbofuran phenol and 3-keto-
carbofuran phenol.
The data in Table 10 reveal that a major fraction of the administered
4C-ring PSC is eliminated in the urine as conjugated hydrolytic products.
For example, approximately 72% of the radioactivity recovered from the
mouse was isolated as the phenols of carbofuran, 3-hydroxycarbofuran,
and 3-ketocarbofuran, and these materials represented 76% of the radio-
activity recovered from the rat. Although significant amounts of 3-
hydroxycarbofuran and 3-ketocarbofuran also were detected, these metabo-
lites appeared after mild acid hydrolysis and evidently were present in
the urine as conjugates. 3-Hydroxycarbofuran is somewhat toxic to the
white mouse (LDr0 7 mg/kg) and conjugation evidently is necessary to avoid
or reduce intoxication by this metabolite.
32P-PSC also was administered to mice at 100 mg/kg in order to establish
the nature of the fragmentation products associated with the dimethoxy-
phosphinothioyl portion of the molecule. After treatment, radioactivity
was excreted in the urine with up to 70% of the dose recovered in 24 hr.
This was followed by a slower excretion rate, resulting in 93% total
recovery in 72 hr. In contrast to the 14C ring-labeled metabolites,
55-757,, of the radioactivity in the 24-hr urine sample was chloroform
extractable, the rest remained in the aqueous phase even after adjustment
of acidity to pH !„ Throughout the remainder of the observation period,
the fraction of chloroform extractable radioactivity decreased to 20%
for the 48-hr sample (24-48 hr) and 12% for the 72-hr sample (48-72 hr).
A similar metabolic behavior was observed in the rat and pooled chloro-
form extracts were used for isolation of the major metabolite.
A maximum of 40% of the dose was present in the feces, most of which was
unchanged PSC along with minor amounts of unidentified polar metabolites.
The pooled chloroform-soluble extract was analyzed by silica gel tic
using ethanol-acetone (9:1) for development. After repeated tic and
extraction of the R 0.71 zone with chloroform, a single metabolite was
obtained which was further purified by preparative glc. Subsequent
analysis by glc-ms revealed a parent ion of 139 which, along with its
fragmentation products, verified the structure of the metabolite as
dimethyl ^J-methylphosphoramide (XXXVIII). A similar correspondence was
obtained between the metabolite and an authentic sample of dimethyl N-
methylphosphoramide by infrared and tic analysis.
additional P metabolites were present in the aqueous phase, each in
Preliminary ion exchange chromatography revealed that at least four
additional 2P metabolites were present :
minor amounts, but none were identified.
20
-------
Table 10. METABOLITES RECOVERED FROM WHITE MOUSE AND RAT
EXCRETA 24 HOURS AFTER ORAL ADMINISTRATION OF 100
MG/KG 14C-RING LABELED DIMETHOXYPHOSPHINOTHIOYL
CARBOFURAN
Total
XXXII
XXXIII
XXXIV
XXXV
XXXVI
XXX
XXXII
XXXIV
XXXV
XXXVI
recovery (96 hr)
Urinary metabolites
3-Hydroxycarbofuran
3-Ketocarbofuran
Carbofuran phenol
3-Hydroxycarbofuran phenol
3-Ketocarbofuran phenol
Unknown 1
Unknown 2
Fecal metabolites
PSC
3-Hydroxycarbofuran
Carbofuran phenol
3-Hydroxycarbofuran phenol
3-Ketocarbofuran phenol
Unknown 1
Unknown 2
Total recovered radioactivity
Per
Mouse
93.4
9.0
11.0
42.6
2.0
27.5
2.0
1.0
1.7
0.4
0.6
0.4
1.0
0.3
0.4
100.0
cent
Rat
95.2
6.4
9,2
39.2
20.1
16.4
3.0
1.0
2.6
-
1.5
-
-
-
0.6
100.0
Mean from two separate experiments.
21
-------
Metabolism in House Flies - Results of the metabolism of 14CHg-N-PSC
in female susceptible house flies (S^AIDM strain) treated topically
at a dosage of 10 ug/g are presented in Tables 11 and 12. Presented
are data for the distribution of radioactivity and the nature of the
various metabolites recovered 4 hr after treatment. Recovery of the
applied radioactivity in several experiments ranged from 88 to 95%
The data in Table 11 reveal that PSC is rapidly absorbed by the flies.
At zero time, i.e., immediately after treatment, an external methanol-
acetone rinse returned 84% of the applied radioactivity. This decreased
to 46, 35 and 21% after 1, 2, and 4 hr, respectively.
Detailed analysis for metabolic products was made after 4 hr, at which
time most of the flies were in a knockdown state and exhibited typical
cholinergic symptoms of intoxication. The flask rinse contained un-
changed PSC (5.7%) which was likely physically rubbed from the house
fly pronota. An additional amount of water soluble material was observed
(2.3%) which was not characterized. The external fly rinse fraction,
representing approximately 20% of the total applied radioactivity,
contained a lesser amount of PSC (1.9%) and three metabolites, i.e.,
3-hydroxycarbofuran (5.3%), N-hydroxymethylcarbof uran (6.1%), and a
polar unknown (4.6%) which did not migrate in the tic systems employed.
The remaining 4.6% in the fly rinse fraction was water soluble even
after acid treatment.
Approximately 70% of the total recovered radioactivity was present in
the fly extract after homogenization of rinsed flies with methanol-
acetone solvent, of which approximately one-third partitioned in favor
of chloroform over water. The chloroform fraction of the fly homogenate
contained PSC (9.9%) and carbofuran (13.5%), the latter undoubtedly being
the agent responsible for intoxication owing to its strong anticholin-
esterase activity. The principal metabolites which were extractable
into chloroform after mild acid treatment of the aqueous phase were 3-
hydroxycarbofuran (22.7%), 3-ketocarbofuran (7.1%), N-hydroxymethyl-
carbofuran (6.5%) and a polar unknown (3.5%) which stayed at the origin
of the tic plate. An additional 7.8% of the radioactivity remained in
the aqueous phase even after acid treatment.
A summation of the amounts of individual metabolites recovered from the
house fly, i.e., sum of internal extract, flask and fly rinse, is pre-
sented in Table 12. Examination of the data shows that at least 78% of
the recovered radioactivity (91%) was in a form of a metabolite in which
the carbamate ester linkage was intact. This is in striking contrast
to the spectrum of metabolites which were isolated from rodents (cf
Table 10) where 72-76% of the radioactivity was in the form of hydrolytic
products, e.g., carbofuran phenol, 3-ketocarbofuran phenol, and 3-hydroxy-
carbofuran phenol. Thus, the results point to a marked difference in
the pathways for the metabolism of PSC in the house fly and mouse or
rat. This difference in metabolism is in agreement with the selectivity
of PSC toward insects and its relative safety to mammals „
22
-------
Table 11. METABOLITES PRESENT IN EACH EXTRACT FRACTION FROM
FEMALE HOUSE FLIES 4 HOURS AFTER TOPICAL APPLICATION
14,
(8 |_iG/G) OF 14fCH3-N-PSC
Per cent
ft
Total recovery
Flask rinse
XXX PSC
Water soluble (unhydrolyzed)
Fly rinse
XXX PSC
XXXII 3-Hydroxycarbofuran
XXXVII N-Hydroxymethylcarbofuranb
Unknown
Water soluble
Fly extract
XXX PSC
XXXI Carbofuran
XXXII 3-Hydroxycarbofuran
XXXIII 3-Ketocarbofuran
XXXVII N-Hydroxymethylcarbofuran
Unknown
Water soluble
Total recovered radioactivity
91.0
5.7
2.3
Total 8.0
1.9
5.3
6.1
3.1
4.6
Total 21.0
9.9
13.5
22.7
7.1
6.5
3.5
7.8
Total 71.0
100.0
a Based upon amount administered.
b Rf ether-hexane, 0.14; Rf CH2C12-CH3CN, 0.10.
23
-------
Table 12. SUMMARY OF METABOLITES RECOVERED FROM HOUSE FLIES
TREATED WITH C14H3-N-PSC
^. Per cent
XXX PSC 17.5
XXXI Carbofuran 13.5
XXXII 3-Hydroxycarbofuran 28.0
XXXIII 3-Ketocarbofuran 7.1
XXXVII N-Hydroxymethylcarbofuran 12.6
Unknowns 6.6
Unextractable metabolites 14.7
-------
Conclusion - The selective toxicity of PSC is clearly associated with
fundamentally different pathways of metabolism in house flies and in
rats and mice. The basis for selectivity is shown in the metabolic
schemes given below
Housefly
0
0
OCNHCH OH
(XXXVII)
conjugate
0
II
OCNHC1L
S
II
(XXXI)
(CH30)2PNHCH
(XXXIII)
Rodent
conjugate
(XXXIV)
r ? l
(CH30)2PNHCH3I
J
0
il
(XXXVIII)
HO
1°.'
OH
(XXXV)
(XXXVI)
conjugate
conjugate
25
-------
In house flies, cleavage of the P-N in the phosphoramide moiety occurs
resulting in the in vivo liberation of the toxic carbamate carbofuran
and other intact carbamate metabolites. Thus, the metabolic activa-
tion of PSC which occurs in house flies is analogous to the activation
of N-acetyl Zectran in the spruce budworm and N-(2-toluenesulfenyl)
carbofuran in house flies .
In rodents, PSC is largely degraded to nontoxic phenolic products by
hydrolytic and oxidative reactions. It is not certain whether de-
sulfuration precedes or follows hydrolysis of the carbamate ester moiety.
Conceivably, PSC may undergo desulfuration first to give the dimethoxy-
phosphinyl derivative (in brackets) which subsequently is hydrolyzed to
carbofuran phenol and dimethyl N-methylphosphoramidate. Alternatively,
hydrolysis may occur first to yield ^),()-dimethyl fl-methylphosphoramido-
thioate which is then converted to the phosphoramidate. 0,p-Dimethyl
^-methylphosphoramidothioate partitions into chloroform from water and,
therefore, it, as well as PSC, probably is sufficiently lipophilic to
act as a substrate for mixed function oxidase systems which undoubtedly
carry out the desulfuration reaction. It should be pointed out that
both of the intermediates in brackets were synthesized by independent
methods and neither of these compounds corresponded by tic to any of
the unknown metabolites.
Phenolic hydrolysis products of PSC constituted approximately 757o of
the radioactivity recovered from both the mouse and rat after adminis-
tration of 100 mg/kg 14:C-ring PSC. The relative amounts of hydrolysis
products are greater than that recovered from mice (24% phenolics)
treated with carbofuran at the LD^ dosage of 2 mg/kg, but are similar
to that recovered from rats (73% phenolics) also treated with 2 mg/kg
carbofuran (one-half LDrQ level). Since the amount of PSC used in this
study, even on a molar oasis, is much larger than carbofuran used in
earlier studies, the large quantities of phenolic products from PSC
suggest that the dimethoxyphosphinothioyl moiety in PSC provides an
opportunity for the rat and mouse to carry out metabolic reactions lead-
ing to detoxified products.
4. Metabolism of 2,2-Dimethyl-2,3-Dihydrobenzofuranyl-7 N-Methyl-N-
(2-Toluenesulfenyl)carbamate in the House Fly and White Mouse.
Introduction - The preceding section summarized results on the compara-
tive metabolism of PSC in the house fly, rat, and mouse. As in the
case of dialkoxyphosphinothioyl derivatives of toxic carbamate insecti-
cides, derivatives produced by aryl- or alkylsulfenylation also are
relatively safe to mammals and, further, often exhibit improved insecti-
cidal activity compared to the parent carbamate. In continuation of our
efforts in seeking basic information for the design of selectively toxic
compounds, a study of the comparative metabolism of 2,2-dimethyl-2,3-
dihydrobenzofuranyl-7 N-methyl-N-(2-toluenesulfenyl)carbamate fXXXIX, or
IJ-(2-toluenesulfenyl)carbofuran] in the house fly and white mouse was
conducted. Compared to carbofuran (XXXI), XXXIX, with an oral
26
-------
100 mg/kg, is 50-fold less toxic to the white mouse and yet is almost
twice as toxic to the common house fly.
Metabolism in the White Mouse - Studies were conducted with C carbonyl
and ring-labeled N-(2-toluenesulfenyl)carbofuran (XXXIX) administered
orally at a dosage of 15 mg/kg. XXXIX was metabolized rapidly in the
white mouse with by far the major portion of the administered radio-
activity appearing in the urine within 24 hours. Little, if any, radio-
activity appeared in the urine after this time. For each label the
ultimate recovery of radioactivity was at least 907» of the dose admin-
istered.
Table 13 summarizes data for the distribution, identity, and relative
amounts of the various metabolites found after treatment of the mouse
with a single dose of XXXIX at 15 mg/kg. By far, the greatest amount
of radioactivity was present in the urine as water-soluble conjugates.
The feces contained only 3.2 and 5.670 of the recovered radioactivity
for ring-l^C and carbonyl-^C labels, respectively. The identities of
the various metabolites were confirmed by cochromatography in at least
four different solvent systems and in one case by mass spectroscopy.
The low recovery of XXXIX in these experiments indicates that the mouse
is able to metabolize this compound effectively. Further, the relatively
low amount of N-sulfenyl metabolites dectected suggests that N-S bond
cleavage occurs to a significant extent. By far, the greatest portion
of the recovered radioactivity was in the form of conjugated metabolites
of carbonfuran as found in previous studies >>.
Metabolites detected in this investigation corresponded closely to those
found in earlier studies of carbofuran metabolism in the mouse and
rat^>5 although significant quantitative differences were found. For
example, the 3-ketocarbofuran from the rat^, was much less evident in
the present study. Although 3-hydroxycarbofuran (XXXII) is a major
metabolite of XXXIX from the mouse and of carbofuran from both the mouse
and rat^>5, the dihydroxy metabolite (XLII) in rat urine comprised only
5.3% of the applied dose of carbofuran but was present to a much larger
degree (26.8-31.0%) in the present study. These quantitative differences
in the metabolism of XXXIX and carbofuran probably are responsible, at
least in part, for the reduced toxicity of XXXIX to the mouse.
The three phenolic metabolites XXXIV, XXXV and XXXVI were each present
in significant amounts as conjugates in mouse urine. Together, these
metabolites account for almost one-third of administered ring labeled
XXXIX. The reasonable agreement between the total amount of expired
l^-C-COo trapped suggests that the latter can serve as a good indicator
for metabolism of XXXIX by the mouse.
In order to compare further the metabolism of XXXIX with that of carbo-
furan under similar conditions, additional data for the expiration of
14-C02 was obtained. Mice were treated individually with 0.20 mg/kg
each of carbonyl labeled XXXIX and carbofuran and the expiration of
27
-------
Table 13. METABOLIC PRODUCTS RECOVERED FROM THE WHITE MOUSE AFTER ORAL
ADMINISTRATION OF 15 MG/KG N-(2-TOLUENESULFENYL) CARBOFURAN (XXXIV)
Ring label (%) CO label (%)
Total recovery 98.7 90.0
Urinary metabolites
a
a. Organo-soluble
XLI N-(2-Toluenesulfenyl) 3-keto- - 0.2 .
carbofuran
J>
XXXII 3-Hydroxycarbofuran 1.0 3.3
XXXIII 3-Ketocarbofuran 0.3 0.3
XLII 3-Hydroxy-N-CH2OH-carbofuran 5.9 10.6
b. Water-soluble conjugates
XXXIV Carbofuran phenol 12.9
XXXV 3-Hydroxycarbofuran phenol 11.7
XXXVI 3-Ketocarbofuran phenol 4.5
XXXII 3-Hydroxycarbofuran 29.2 13.3
XLII 3-Hydroxy-N-CH2OH-carbofuran 20.9 20.4
Unextractables 10.4 11.8
Fecal metabolites
XXXIX
XL
XLI
XXXI
N- (2-Toluenesulfenyl) carbofuran 0.5
N- (2-Toluenesulf inyl) carbofuran
N-(2-Toluenesulfenyl) 3-keto
carbofuran
Carbofuran 1.0
Unextractables 1.7
co2a
Total 100.0
0.3
1.4
2.7
-
1.2
34.5
100.0
Based on total recovered radioactivity.
28
-------
14CO^ was monitored for 24 hr. The results showed that at this lower
dose, XXXIX is metabolized more rapidly and to a greater extent than
is carbofuran. In the 4 hr following treatment, the mouse treated
with XXXIX produced 14COg at twice the rate of a mouse similarly
treated with carbofuran. After 6 hr, expiration of C02 occurred
slowly and finally yielded 41 and 367» of the administered radioactivity
for XXXIX and carbofuran, respectively, in 24 hr. The enhanced sus-
ceptibility of the N-arylsulfenyl carbamate to this manner of detoxi-
cation is doubtlessly a significant factor contributing to the reduced
toxicity of XXXIX to mice.
Metabolism in House Flies - Results of metabolism studies with sus-
ceptible Sj^-j-jj^ house flies are presented in Table 14. In each case
the flies were treated with radiolabeled XXXIX at 2.85 |j,g/g. The
distribution of radioactivity found in the various fractions 3 hours
after treatment is shown in the table. Recoveries of applied radio-
activity of greater than 90% were achieved.
All fractions were analyzed by tic. The conjugated metabolites re-
covered from the internal extracts were subjected to enzymatic hydrolysis
before analysis. The conjugated material recovered from the cage rinse
was not analyzed further.
Significant amounts of XXXIX, carbofuran (XXXI) and 3-hydroxycarbofuran
(XXXII) were recovered. 3-Ketocarbofuran (XXXIII) was detected only
in the hydrolysate of the internal fraction. Almost certainly, this
material results from oxidation of XXXII which was also present. Oxi-
dation of XXXII to XXXIII on tic plates was noted during the identi-
fication procedures. Only trace amounts of the dihydroxy metabolite
(XLII) were recovered.
Table 15 summarizes the total amounts of the various metabolites identi-
fied in all fractions obtained from the house flies. XXXIX was recovered
only from the two external fractions (cag;e and body rinse) and none
was detected internally in the fly. The relatively low recovery of this
material, after only 3 hr suggests that it is rapidly metabolized by the
fly. The detection of small amounts of carbofuran in both of the
external fractions further suggests rapid formation of this carbamate
from the applied compound. The appreciable amounts of carbofuran accu-
mulated internally in the house fly, corresponding to 0.32 ^g/g and
0.68 u.g/g for the ring and carbonyl lables, respectively, indicate its
role as the toxic agent. Such amounts are comparable and probably even
greater than the amounts recoverable when house flies are treated in a
similar manner with carbofuran itself. For example, 1 hr after treat-
ment at 2.5 p,g/g Borough^ found 0.31 u.g/8 of unchanged carbofuran
internally. Since carbofuran is most certainly responsible for intoxi-
cation, i.e., by inhibition of cholinesterase, the high amounts recovered
after treatment with XXXIX are consistent only with the rapid metabolism
of this compound to produce lethal amounts of carbofuran in vivo.
29
-------
Table 14. METABOLIC PRODUCTS RECOVERED FROM SUSCEPTIBLE
HOUSE FLIES AFTER TOPICAL APPLICATION OF 2.85
UG/G N-(2-TOLUENESULFENYL) CARBOFURAN (XXXIX)
Ring label (%) CO label (%)
Total recovery
Analysis?
Cage rinse
90.4
a Based on administered radioactivity.
b Based on recovered radioactivity.
30
93.7
XXXIX
XXXII
XXXIX
XXXI
XXXII
XXXI
XXXII
XLII
N- (2-Toluenesulfenyl) carbofuran
Carbofuran
Conjugates
Body rinse
N- (2-Toluenesulfenyl) carbofuran
Carbofuran
3-Hydroxycarbofuran
Internal extracts
Carbofuran
3-Hydroxycarbofuran
3 -Hydroxy-N-CH^OH- carbofuran
Conjugates:
XXXII 3-Hydroxycarbofuran
XXXIII 3-Ketocarbofuran
XLII 3-Hydroxy-N-CH2OH-carbofuran
Unextractables
co2
Total
1.6
0.6
2.5
26.1
1.2
5.9
17.2
8.1
1.6
28.2
3.4
1.2
2.4
100.0
2.3
0.5
1.5
22.8
2.9
7.2
37.3
9.4
trace
3o5
1.4
1.3
5.3
4.6
100.0
-------
Table 15. INTERNALLY RECOVERED METABOLITES FROM HOUSE FLIES
Ring label CO label
Carbofuran 17.2 37.3
3-Hydroxycarbofuran 36.3 12.9
3-Ketocarbofuran 3.4 1.4
3-Hydroxy-N-CH2OH-carbofuran 2.8 1.3
31
-------
Supporting evidence for the rapid action of XXXIX was obtained by
electrophysiological techniques?. The intoxication of house flies
treated topically with 0.5 |j,g of XXXIX was monitored and closely
compared to the effects of carbofuran measured under the same conditions
Application of XXXIX produced effects indistinguishable from the effects
of carbofuran poisoning except that XXXIX acted more rapidly. Intoxi-
cation by XXXIX was evident only 6 min after application, whereas
carbofuran required 12 min to produce the same physiological response.
If carbofuran is the common toxicant, this result leaves little doubt
of the rapid absorption and metabolism of XXXIX to carbofuran in house
flies.
Toxicology - Toxicological data for various metabolites and derivatives
of XXXIX and carbofuran are presented in Table 16. XXXIX and its
derivatives are generally more toxic to the house fly, yet are still
relatively safer to the mouse than the corresponding carbofuran deriva-
tives. The insecticidal activity of N-(2-toluenesulfenyl) 3-ketocarbo-
furan as compared with 3-ketocarbofuran is particularly noteworthy.
Oxidation of the sulfenamide XXXIX to the sulfinamide reduces insecti-
cidal activity to some degree but, with an oral LDcQ of 75 mg/kg, the
sulfinamide is still reasonably safe to the mouse. Further oxidation
to the sulfonamide destroys toxicity to both the insect and mammal.
The appreciable oral toxicities of the carbofuran metabolites are notable
and doubtlessly contribute to the high mammalian toxicity of carbofuran.
In accord with reduced mammalian toxicities, XXXIX is 19-fold less
effective as an inhibitor of bovine erythrocyte acetylcholinesterase
(BAChE) than is carbofuran. On the other hand, XXXIX is 76-fold less
inhibitory to house fly acetylcholinesterase (HAChE) than is carbofuran
and this decreased potency further supports the view that carbofuran,
and not XXXIX is the primary toxicant to house flies. Oxidation at the
sulfur atom to give the sulfinamide influences anticholinesterase activity
but does not greatly affect toxicities. Further oxidation to the sulfon-
amide destroys toxicity and severely reduces anticholinesterase activity.
N-(2-Toluenesulfenyl) 3-ketocarbofuran is surprisingly almost as effec-
tive as an inhibitor of BAChE as is 3-ketocarbofuran, although it is
somewhat less active toward HAChE. As expected, the carbofuran metabo-
lites are all effective inhibitors of both enzymes. In summary, the N-
substituted-^-methylcarbamates are generally poorer inhibitors of both
insect and mammalian acetylcholinesterases, a result which again suggests
that processes other than direct enzyme inhibition are responsible for
the selective toxicity of XXXIX.
Conclusion - The basis for the selective toxicity of N-(2-toluenesulfenyl)
carbofuran (XXXIX) is undoubtedly attributable to different paths of
metabolism in the mouse and house fly. Clearly, XXXIX is rapidly
absorbed and metabolized in house flies to produce carbofuran by cleav-
age to the N-S bond. This process probably occurs more slowly in the
32
-------
mouse and thereby provides an opportunity for detoxication processes
to occur.
Presented below is the scheme for the metabolism of XXXIX in the mouse.
MOUSE METABOLISM
OH
0 ™
" x- CH3
oJ^SX
^ ' — ^ CH;
1 XXXIV 1 XXXIX
conjugates r~ "•
rt /CH
Q 9™\sJ-\)
HO'^ ^<^ CH_
/' \
1 "-^ ocV
XL
0 TH
II / CH3
OCN^ > — .
^o.^^sV^
; <3 >^
0^ ^^ CH3
XL I
carbofuran
XXXI
If .CH OH
OGN^ ^
XXXII
conjugates
XXXIII
XXXVI
conjugates
33
-------
The primary route of metabolism is a series of detoxication steps and
probably proceeds via the key intermediate ^-(2-toluenesulfenyl) 3-
hydroxycarbofuran (in brackets). Although this intermediate was not
isolated, evidence for its formation rests with the recovery of signifi-
cant amounts of N-(2-toluenesulfenyl) 3-ketocarbofuran (XLI) and the
relatively large amounts of 3-hydroxycarbofuran (XXXII) and the
dihydroxycarbofuran (XLII). Although XXXII is a major metabolite of
carbofuran in both the rat and mouse ' , small amounts of XLII were
found only with the rat. The formation of XLII by hydroxylation of
N-(2-toluenesulfenyl) 3-hydroxycarbofuran and subsequent cleavage of
the N-S bond would be consistent with the observed metabolites.
Conjugation mechanisms almost certainly play an important role in the
further detoxication of hydroxylated carbofuran metabolites. XXXII
is quite toxic to the mouse and is recovered in significant amounts
from mouse urine. However, by far the greater proportion of hydroxy
metabolites recovered were in the conjugated form. The formation of
conjugates would seem, therefore, tc provide an important additional
pathway in detoxication of XXXIX.
The ability of the mouse to metabolize XXXIX at a rate faster than
carbofuran, as estimated by l4CC^ respiration studies, may well provide
the most important reason for its reduced toxicity to mammals.
The basis for the toxicity of XXXIX to house flies is apparent from
the metabolism data. Presented below is the scheme proposed for its
metabolism in house flies and it appears that XXXIX is peculiarly
suited for liberation of carbofuran in the fly. The recovery of large
amounts of carbofuran in the house fly agrees with its role as the toxic
agent. The complete absence of the applied compound, or any of the
other N-sulfenyl derivatives in the internal fraction, further implicates
carbofuran and not the sulfenamides as the toxicant. The liberation
of carbofuran by cleavage of the N-S bond of XXXIX occurs as an activa-
tion step in house flies.
conjugates
/ \
XCH OH
OCN
- conjugates
HO
-------
Table 16. TOXICOLOGICAL PROPERTIES OF N-(2-TOLUENESULFENYL) CARBOFURAN
(XXXIX) AND ITS CARBAMATE METABOLITES AND DERIVATIVES
Toxicity
Anticholinesterase activity Mouse House-
Compound
Jfl-(2-Toluenesulfenyl)
carbofuran
IJ- (2-Toluenesulf inyl)
carbofuran
N- (2-Toluenesulf onyl)
carbofuran
If- (2-Toluenesulf enyl)
3-keto-carbofuran
Carbofuran
3-Hydroxy-carbofuran
N-Hydroxymethyl-
carbofuran
3-Keto-carbofuran
5-Hydroxy-carbofuran
BAChE
k. (Mf1 min"1)
1.0 x
3.7 x
2.3 x
2.3 x
1.9 x
3.2 x
2.4 x
3.3 x
1.2 x
io5
io4
io3
io5
6
10
io5
5
10
io5
io5
HAChE
k-j_ (M"1 min"1
1.7 x
3.5 x
1.5 x
8.6 x
1.3 x
1.4 x
6.3 x
2.3 x
1.8 x
io5
io5
10*
io4
7
10
io6
5
10
io5
io6
oral fly
) mg/kga p£/g
100-25 3.2
75 9.0
>150 >500
150 22.0
2 6.7
7a >500
a
30-75 >500
10-303 >500
-
3.
Determined in propylene glycol solvent.
35
-------
5. Thiolysis as an Activation Process in N-Sulfenylated Derivatives
of Methylcarbamate Esters
Introduction - On the basis of its comparative metabolism in the
white mouse and house fly, the selective toxicity of N-(2-toluene-
sulfenyl) carbofuran (XXXIX) was attributed to differning pathways of
metabolism between the mammal and insect. In house flies, lethal
quantities of carbofuran were produced j.n vivo after topical applica-
tion of XXXIX, while the mouse degraded XXXIX largely to detoxified
conjugated products. Owing to the nature of the metabolic products
isolated from the mouse, it was postulated that the initial step in
the metabolic sequence was the formation of the intermediate N-(2-
toluenesulfenyl)-3-hydroxycarbofuran, even though this material was
not isolated. A study was conducted in an attempt to establish the
CH,,
XXXIX
intermediacy of N-(2-toluenesulfenyl)-3-hydroxycarbofuran in the
metabolic pathway by examining the fate of XXXIX in vitro in sub-
cellular fractions of mouse liver. This section describes the un-
expected results which were obtained and focuses on the nature of the
cleavage of the N-S bond in sulfenylated carbamate esters.
N-S Bond Cleavage by Biological Tissue - Contrary to expectation, the
various subcellular fractions of mouse liver indicated in Table 17
rapidly effected the cleavage of the N-S bond of N-(2-toluenesulfenyl)
carbofuran (XXXIX) to generate carbofuran as the principal product.
Evidently, XXXIX is stable in tris buffer (pH 7.4) since 100% un-
changed material was recovered after a fixed incubation period of 10
min. In the presence of different liver tissue, however, XXXIX was
quite unstable, the amount of XXXIX recovered ranging from 0 to 26%.
The addition of NADPH cofactor markedly reduced the amount of carbo-
furan produced owing to oxidation of carbofuran to other metabolic
products, e.g., to 3-hydroxycarbofuran. This was particularly appar-
ent with the homogenate and microsomal fractions, providing evidence
that each of these fractions consisted of active preparations of mixed
function oxidase. Based on the quantity of XXXIX recovered, the super-
natant appeared to be more effective in producing carbofuran than the
microsomal fraction, although the microsomal preparation after heat
denaturation showed the highest N-S bond cleaving activity after 1
hour incubation. The high activity of denatured microsomes suggests
that the N-S bond is cleaved by a chemical rather than an enzymatic
process.
36
-------
Table 17. DEGREE OF N-S BOND CLEAVAGE OF N-(2-TOLUENESULFENYL)
CARBOFURAN (XXXIX) BY SUBCELLULAR FRACTIONS OF MOUSE
LIVER AFTER 10 MIN AT 30°C
Subcellular fraction
Buffer alone
Homogenate
Homogenate -f- NADPH
Microsome
Microsome + NADPH
Supernatant
Supernatant + NADPH
f\
Boiled microsome + NADPH
Relative
XXXIX
100
11
8
26
22
8
9
0
amounts of products
Carbofuran
0
84
52
72
48
91
89
100
obtained (%)
Other
0
5
40
2
30
1
2
0
a Liver microsomal fraction was heated in boiling water for 10 min and
incubated with XXXIX and NADPH for 1 hr.
37
-------
The reported lability of the N-S bond in the fungicide Captan to
sulfur nucleophiles ' prompted the examination of the effect of
L-cysteine and reduced glutathione. Each of these reagents at 10
M in tris buffer, pH 7.4, quantitatively converted XXXIX into carbo-
furan under the usual conditions of 10 minute incubation at 30°C. This
drew attention to the examination of different biological preparations,
including bovine serum albumin (BSA), house fly homogenate, and mouse
blood for total thiol titre and its relationship to degree of N-S
bond cleavage. The results, summarized in Table 18, indicate a fairly
good linear relationship between thiol titre, as expressed by GSH
equivalency, and the level of carbofuran produced. Of the biological
preparations examined, mouse blood, with a thiol titre equivalent to
3.1 X 10"^ M GSH gave 100% carbofuran compared to 29% for BSA whose
GSH equivalent was 0.75 X 10"4 M.
In the presence of sulfhydryl inhibitors, e.g., N-ethylmaleimide (NEM)
and £-chloromercuribenzoate (PCMB), the production of carbofuran was
completely inhibited and XXXIX was quantitatively recovered. This
suggests that free thiol groups are required in biological tissues to
effect N-S bond cleavage.
Kinetic Analysis of N-S Bond Cleavage - The reaction between the fungi-
cide Captan (N-trichloromethylthio-4-cyclohexane-l,2-dicarboxyimide)
and Folpet (N-trichloromethylthiophthalimide), each containing an N-S
bond, and p_-nitrobenzenethiol (NBT) and other thiols has been previously
described". Because of the convenience with which NBT may be estimated
colorimetrically (410 nm), this reagent was selected for analysis of
N-S bond cleavage of sulfenylated methylcarbamates by thiolate nucleo-
philes. When XXXIX and other sulfenylated methylcarbamates were
allowed to stand with NBT, the absorption of the yellow anion decreased
rapidly with time, giving as products the methylcarbamate and a mixture
of disulfides (cf section on product analysis). For example, the
absorption of a buffered solution of 8 X 10"5M NBT and 1.34 X 10~4 M
XXXIX diminished virtually to zero within a period of 5 min. No
detectable decrease in absorption of NBT was noted when carbofuran was
substituted for the sulfenylated derivative.
In addition to XXXIX, the N-(2-toluenesulfinyl) (XLI), and N-(2-toluene-
sulfonyl) (XLII) derivatives of carbofuran also were examined for their
reaction with NBT. These derivatives also react with NBT but at re-
duced rates and in the decreasing order of sulfenyl > sulfinyl »
sulfonyl. The reaction between NBT and the sulfenylated carbamate
derivatives proceeded with second-order kinetics and values for the
rate constants for a number of different derivatives are summarized in
Table 19. As expected, arylsulfenyl derivatives substituted with
electron withdrawing substituents, e.g., the halogen and cyano deriva-
tives, were most susceptible to reaction with NBT and an orderly de-
crease in rate constants was observed as the substituents became less
electron withdrawing. The reaction between NBT and the pentachloro-
benzenesulfenyl derivative occurred in such a rapid manner that a rate
38
-------
Table 18. RELATION BETWEEN ESTIMATED CONCENTRATION OF FREE THIOL
GROUPS IN DIFFERENT BIOLOGICAL TISSUE AND DEGREE OF
N-S BOND CLEAVAGE OF N-(2-TOLUENESULFENYL)
CARBOFURAN (XXXIX) AFTER 10 MIN AT 30°C
Tissue or Reagent
& (by O.D.)
10"3 M L-Cysteine
10~3 M GSH
0.1 ml Mouse blood 4.1
0.5 ml 20% Liver homog. 3.3
0.5 ml 20% Fly homog. 1.4
50 mg BSA 1.0
10"2 M NEM in each
SH Equiv.
to GSH (M)
-
-
3.1 X 10"4
2.5 X 10"4
1.1 X 10"4
7.5 X 10"5
—
XXXIX
(%)
0
0
0
11
61
71
100
Carbofuran
cy\
\«/
100
100
100
86a
39
29
0
tissue prep.
10"3 M PCMB in each - 100 0
tissue prep.'3
a Approximately 3% of the product was 3-hydroxycarbofuran.
k Each tissue preparation was incubated with the indicated concentration
of NEM and PCMB for 10 min prior to reaction with XXXIX.
39
-------
Table 19. SECOND-ORDER RATE CONSTANTS (k) FOR THE CLEAVAGE OF
THE N-S BOND IN N-SULFENYLATED DERIVATIVES OF
CARBOFURAN BY £-NITROBENZENETHIOL AT pH
7.0 AND 30°C
R
k (M*1 rain"1)
SC,H,-2-CH0 (XXXIX)
OH j
SOC,H,-2-CH0 (XL)
o 4 j
SO-C,H,-2-CH, (XLIII)
L o 4 j
SC6H4-4-OCH3
SC,H,-4-t-butyl
64 —
SC,H.-4-Cl
6 4
SC,H.-4-Br
6 4
SC,H.-4-CN
6 4
S-2-naphthyl
sc6ci5
S-2-(1,3-benzothiazolyl)
6.6
8.7
7.6
2.4
0.083
7.1
7.6
24.0
27.0
35.0
11.3
too rapid to measure
too rapid to measure
40
-------
constant could not be determined. Relatively little difference was
noted between the unsubstituted benzenesulfenyl derivative and those
containing electron donating substituents.
A comparison of the rate constants for N-S bond cleavage and the
toxicological properties of a limited number of carbofuran derivatives
is made in Table 20. The data show that the derivatives were all
significantly less effective in inhibiting both bovine erythrocyte
(BAChE) and house fly-head (HAChE) acetylcholinesterase than carbo-
furan. While a direct relationship between the rates of N-S bond
cleavage and toxicity to the house fly and white mouse is not
apparent, evidently rapid N-S bond cleavage is required for insecti-
cidal activity. The poor toxicity of the N-(2-toluenesulfonyl)
derivative is consistent with its poor anticholinesterase activity
and resistance to N-S bond cleavage. This suggests that sulfonyl
derivatives containing a more labile N-S bond should be examined for
insecticidal activity and work along this line is in progress.
Product Analysis - The experimental condition employed for the analysis
of products obtained from the reaction between XXXIX and NET was similar
to that for the kinetic studies except proportionately large quantities
of reactants were used. For convenience of analysis, nonradioactive
XXXIX was charged with a small amount of 14C carbonyl label. The
reaction proceeded rapidly and carbofuran was recovered quantitatively
after 5 minute reaction, along with a mixture of disulfides. The
products were separated and identified by cochromatography (tic) as
(2-tolyl) disulfide, 2-tolyl-4'-nitrophenyl disulfide, bis-(4-nitro-
phenyl) disulfide, and carbofuran. Of the three disulfides recovered,
approximately 80% was 2-tolyl-4'-nitrophenyl disulfide, each of the
other two being obtained in about 10%. Based on the products observed,
the following equations reasonably depict the reactions which take place.
41
-------
XXXIX
\VNO
H-0
carbofuran
(1)
•P-
S3
s-s-
CH,
-S<' X)-NO,
-s
CH,
(2)
CH
-NO,
(3)
-------
OJ
Table 20. COMPARISON OF RATES OF N-S BOND CLEAVAGE (k) AND TOXICOLOGICAL PROPERTIES OF
N-SUBSTITUTED CARBOFURAN DERIVATIVES
Compound
Carbofuran
N-Benzenesulf enyl- carbofuran
N-(
£-t-Butylbenzenesulfenyl)
Anticholinesterase activity
k. (M"1 min"1)
i
BAChE HAChE
1
8
7
.9
.1
.8
X
X
X
IO6
io5
IO4
1.3
9.8
6.7
X
X
X
io7
io5
io5
Toxicity, LD5Q (mg/kg)
Mouse oral
2
25-50
75
House fly
6.7
72.5
2.7
N-S bond
k
(M"1 min"1)
-
8.7 X IO3
7.6 X IO3
carbofuran
c
N-(2-Toluenesulfenyl) carbofuran 1.0 X 10"
N-(2-Toluenesulfinyl) carbofuran 3.7 X 10
N-(2-Toluenesulfonyl) carbofuran 2.3 X 10"
1.7 X 10-
3.5 X 10'
1.5 X 10
100-125 3.2
75 9
>150 >500
7.6 X 10-
2.4 X 10-
8.3 X 10
-------
In addition to reactions 1-3, all three disulfides also may result
from atmospheric oxidation since the experiments were not conducted
under anaerobic conditions. Although the overall process is probably
quite complex, the reaction depicted by equation 1 is undoubtedly of
major significance owing to the preponderance of carbofuran and the
unsymmetrical disulfide as products and the second order nature of the
reaction.
Conclusion - The results described in section 4 revealed that the
toxicity of N-(2-toluenesulfenyl) carbofuran (XXXIX), was attributable
to i.n vivo conversion of XXXIX, a weak anticholinesterase, to the potent
anticholinesterase carbofuran. The present study indicates that this
activation process is nonenzymatic and probably involves thiolytic
cleavage of the N-S bond. All tissues, including subcellular fractions
of mouse liver, heparinized mouse blood, commercial protein, and house
fly homogenate, as well as thiol reagents, readily effected the cleavage
of the N-S bond. In limited studies, this was also found to be true
with homogenates of mouse stomach and intestine. The fact that no
less activity was observed with the heat denatured microsomal fraction
of mouse liver and high activity was observed with the supernatant
virtually eliminates involvement of oxidase enzymes in the activation
process. Further, the inhibitory action of such sulfhydryl inhibitors
as NEM and PCMB strongly suggests that thiol residues in biological
tissue are involved in the generation of the carbamate from the sulfenyl-
ated derivative.
Although the results of this study provide insight into the nature of
the alteration reactions which XXXIX and other sulfenylated carbamates
undergo in mammals, they are, however, contrary to the rationale
proposed for the selective toxicity of these materials. While toxicity
to insects may be explained on the basis of in vivo thiolysis of the N-S
bond to generate the toxic methylcarbamate, the same explanation argues
against reduced mammalian toxicity of sulfenylated carbamates owing to
the ubiquitous nature of sulfhydryl groups in mammalian tissue. Since
the results show that carbofuran is rapidly generated from XXXIX in vitro
by a variety of mammalian tissue, the much lower mammalian toxicity of
XXXIX compared to carbofuran is difficult to explain, particularly since
the cleavage of the N-S bond appears to be a nonenzymatic process. It
should be reiterated that in the in vivo study of XXXIX in the mouse,
virtually no carbofuran was recovered and, further, about 90% of the
metabolites were in the form of water soluble conjugates of hydroxylated
carbofuran or the phenol. However, the facility with which various
mammalian tissues are able to catalyze the cleavage of the N-S bond is
disturbing and further work is required to explain the discrepancy
between in vivo and in vitro results.
44
-------
6. Selective Toxicity of N- Substituted Biscarbamoyl Sulfides.
Introduction - In a separate study, it was demonstrated that when 2,2-
dimethyl-2,3-dihydrobenzofuranyl N-methyl-N- (dimethoxyphosphinothioyl)
carbamate (XXX or PSC) was treated with m-chloroperbenzoic acid, the
principal products observed are the expected desulfuration product
2, 2-dimethyl-2,3-benzofuranyl-7 N-methyl-N- (dimethoxyphosphinyl)
carbamate (XLIV) and an unexpected rearrangement product, the corres-
ponding N- (dime thoxyphosphinylthio) carbamate (XLV) (cf part F-l)
XLV was unstable on the plates and decomposed to carbofuran (XXXI) and
to the N-substituted biscarbofuran disulfide (XLV). In an attempt to
prepare XLV by an independent method, 2 moles of carbofuran was reacted
with 1 mole of sulfur monochloride in pyridine solvent. This reaction,
however, did not produce XLV but gave the biscarbamoyl sulfide (XLVI)
as the principal product according to the equation below.
0
l»
OCNHCI
3 pyridine
+ C1SSC1 >
XXXI XLVI
To our surprise, XLVI, although much less toxic to the white mouse, was
still highly insecticidal, comparable in activity to carbofuran. There-
fore, other N-substituted biscarbamoyl sulfides were prepared and
examined. The following section is concerned with the toxicological
properties of these compounds, with particular emphasis on their
selective toxicity.
Insecticidal Activity - Toxicological properties of the various N-
substituted biscarbamoyl sulfides are presented in Table 21, along with
those of the parent methylcarbamates. From examination of the data in
the table, it is apparent that the N-substituted biscarbamoyl sulfides
retained much of the insecticidal activity exhibited by the correspond-
ing methylcarbamate esters. Although all of the biscarbamoyl sulfides,
used alone or in combination with piperonyl butoxide(PB), were slightly
less toxic to house flies compared to the methylcarbamates, they were,
however, significantly more toxic to mosquito larvae. Except for bis-
aldicarb sulfide (LIV), a five- to eightfold increase in larvicidal
activity was observed with the biscarbamoyl sulfide.
Since aldicarb (LIII) is used primarily as a systemic insecticide, the
corresponding bisaldicarb sulfide (LIV) also was examined for systemic
activity in the cotton plant. Laboratory tests against the cotton aphid
(Aphis gossypii), perforator (Bucculatrix thurberiella), mite (Tetranychus
45
-------
cinnabarinus), and salt marsh caterpillar (Estigmene acrea) revealed
that LIV applied to soil containing cotton plants was significantly
more effective in controlling the first three pests than aldicarb.
For example, at the approximate dosage of 1.2 Ib of actual material
per acre, LIV gave virtually complete control of cotton aphids and
perforator for over 21 weeks, as compared to 15 weeks for aldicarb.
Aldicarb and LIV were about equal in their activity against the salt
marsh caterpillar.
Biscarbofuran sulfide (XLVI) also was examined as a cotton systemic
insecticide and although it was outstandingly effective in controlling
the cotton leaf perforator, giving 100% control for more than 20 weeks,
it was almost totally ineffective against the other pests.
Mouse Toxicity - The oral mouse toxicity data presented in Table 21
show that compared to the original methylcarbamates, the N-substituted
biscarbamoyl sulfides are in most cases substantially less toxic to
the white mouse. Depending on the compound, the biscarbamoyl sulfides
were from 5- to 50-fold less toxic to the mouse than the parent methyl-
carbamate,, The largest difference (50-fold) in mouse toxicity was
observed between carbofuran (XXXI) and biscarbofuran sulfide (XLVI).
Bisaldicarb sulfide (LIV) was approximately fivefold less toxic to
mice than aldicarb (LIII). In general, improvement in mouse toxicity
by conversion of the parent carbamates to the biscarbamoyl sulfides
paralleled results obtained in other related derivatization studies.
Anticholinesterase Activity - Bimolecular rate constants (k.) for the
inhibition of house fly-head (HAChE) and bovine erythrocyte (BAChE)
acetylcholinesterase by the methylcarbamate insecticides and N-
substituted biscarbamoyl sulfides are presented in Table 21. The data
clearly indicate that on an overall basis the biscarbamoyl sulfides are
less effective anticholinesterases than the parent methylcarbamates
against both bovine erythrocyte and house fly-head AChE. Differences
in anticholinesterase activity between respective biscarbamoyl sulfides
and methylcarbamates were quite variable, ranging from 4.7-fold for
aldicarb-bisaldicarb sulfide (LIIIrLIV) against BAChE to 141-fold for
carbofuran-biscarbofuran sulfide (XXXI:XLVI) against HAChE.
In the case of BAChE, i.e., an AChE from a mammalian source, the
reduction in anticholinesterase activity of the biscarbamoyl sulfide
compared to the methylcarbamate is more or less in agreement with the
reduction in mouse toxicity (cf Table 22). Although probably fortui-
tous, the correlation between mouse toxicity and BAChE inhibition by
aldicarb (LIII) and bisaldicarb sulfide (LIV) is quite good. Correla-
tion between the other pairs, particularly XLIX:L and LI:LII, however,
is much less satisfactory.
On the other hand, differences in inhibition between the methylcarbamate-
biscarbamoyl sulfide pairs against HAChE were far greater than their
respective relative toxicities to house flies. This suggests that
factors other than cholinesterase inhibition must be considered to
46
-------
Table 21. TOXICOLOGICAL PROPERTIES OF INSECTICIDAL METHYLCARBAMATE ESTERS AND THEIR
CORRESPONDING N-SUBSTITUTED BIS-CARBAMOYL SULFIDES
Compound
House
Alone
XXXI
XLVI
XLVII
XLVIII
XL IX
L
LI
LII
LIII
LIV
Carbofuran
Bis-carbofuran
sulf ide
6.7
19
Carbaryl >500
Bis-carbaryl >500
sulf ide
3-Isopropylphenyl
methylcarbamate (MIP)
Bis-MIP sulfide
Propoxur
Bis-propoxur sulfide
Aldicarb
Bis-aldicarb sulfide
41
85
22
35
5.5
8.5
LD
fly
+P.B.a
0.9
1.2
12.5
42.0
1.6
4.6
0.9
2.5
1.0
3.2
50 Ong /kg)
Mosquito
larvae
0
0
1
0
0
0
0
0
0
0
.052
.007
.0
.22
.038
.0056
.33
.041
.16
.17
Mouse
2
50-100
560b
c
16
200
24
700
0.3-0.5
1.6-2.5
Anticholinesterase activity
kt (M'1 min'1)
Bovine
erythrocyte Fly-head
1.
2.
4.
6.
7.
2.
4.
4.
3.
7.
9 X
5 X
0 X
1 X
5 X
7 X
3 X
6 X
5 X
4 X
106
IO4
104
103
105
io4
104
IO3
io4
IO3
1.
9.
4.
5.
7.
2.
1.
2.
2.
2.
3 X IO7
2 X IO4
9 X IO5
4 X IO4
7 X IO5
3 X IO4
2 X IO6
8 X IO4
0 X IO4
1 X IO3
a Piperonyl butoxide(P.B.) was applied at a constant dose of 40 jog/fly in combination with varying
doses of insecticide.
b Toxicity to the rat.
c Compound was too insoluble in olive oil or propylene glycol for accurate evaluation.
-------
Table 22. RELATIVE VALUES FOR THE INHIBITION OF BAChE AND HAChE AND TOXICITY TO THE MOUSE AND
HOUSE FLY FOR THE INDICATED PAIRS OF METHYLCARBAMATE AND BIS-CARBAMOYL SULFIDES
Compounds
XXXI/XLVI
XLIX/L
LI/LII
LIII/LIV
BAChE
k. ratio
76
28
9.3
5.2
Mouse
relative toxicity
25-50
13
29
4.7
HAChE
k^ ratio
141
33
42
9.5
House fly
relative toxicity
2.8
2.1
1.5
1.5
GO
-------
account for the comparatively high insecticidal activity of the
biscarbamoyl sulfides relative to their anticholinesterase activity.
This is particularly true in connection with mosquito larvae where
the biscarbamoyl sulfides are observed to be more toxic than the
methylcarbamates .
Conclusion - The results show that the conversion of insecticidal
methylcarbamate esters to the corresponding biscarbamoylsulfides by
treatment with sulfur monochloride in pyridine produced compounds
with significantly improved mammalian toxicity while still retaining
insecticidal activity,, By analogy with the derivatized methylcarbamates
reported earlier (Parts B-3 and B-4), the favorable toxicological
properties of the biscarbamoyl sulfides may be attributed to opportun-
ities which derivatization may provide in allowing additional metabolic
detoxication and intoxication processes to take place in mammals and
insects. Although reduced mammalian toxicity of the biscarbamoyl
sulfides may be accounted for in part by their lower anticholinesterase
activity, it is quite probable that they are detoxified before large
amounts of the parent methylcarbamate are produced in vivo owing to the
unlikelihood that a molecule such as XLVI will pass through the mouse
unchanged .
7. Metabolism of 0,0-Dimethyl S-[o'-(Carboethoxy)ben2yl'| Phosphoro-
dithioate (Phenthoate) in the White Mouse and House Flies
Introduction - Phenthoate or ^,^-dimethyl ^-[ck'-(carboethoxy)benzyl
phosphorodithoate (LV) is a relatively new organophosphorus insecticide
of low mammalian toxicity. Its rat oral LD..Q is reported to be 4728 rag/kg*"
a value which places it approximately 950-fold less toxic to the rat than
to the common house fly (LD,.- 5 mg/kg). Because of its overall high
insecticidal activity and low mammalian toxicity, a study of the compara-
tive metabolism of phenthoate in mammals and insects was carried out
to determine the underlying basis for the selective properties of this
compound .
Metabolism in the White Mouse - Two different radiolabels, C ring-
labeled phenthoate (LV) and 32p phenthoate were used. 32p phenthoate
was administered orally to mice at the dosages of 30 and 161 mg/kg,
and l^C phenthoate at 17 and 161 mg/kg „
Phenthoate is metabolized rapidly in the white mouse with the major
portion of the administered radioactivity appearing in the urine
within 24 hours after treatment. Table 23 summarizes data for the
distribution of excreted radioactivity 12 and 24 hours following
treatment with different single doses of ^ P or ^C phenthoate. For
the 24-hour period, the total recovery of radioactivity ranged from
85-100%, of which 85-94% was present in the urine in the form of water
soluble materials. The feces contained approximately 5-13% of the
administered radioactivity and the fraction present in the feces appeared
to be significantly higher at the high dosage (161 mg/kg) than at the
lower dosages .
49
-------
Table 23. CUMULATIVE DISTRIBUTION 14C- OR 32P-LABELED PHENTHOATE (LV) IN
THE WHITE MOUSE RECEIVING A SINGLE INDICATED DOSE
Ul
o
% of Administered dose
Dose Time
(mg/kg) (hours)
32
P Phenthoate
30 12
24
161 12
24
14
C Phenthoate
17 12
24
161 12
24
Urine
Aqueous
66.5
70.2
52.3
64.5
61.0
78.6
39.7
56.2
Organic Feces
16
16
7
12
14
15
14
20
.4 5.0
.5 5.3
.8 8.1
.3 8.2
.6 3.8
.6 7.6
.7 11.7
.6 13.0
Total
87
92
68
85
79
101
66
89
.9
.0
_ o
.0
. 4
.8
.1
.8
-------
Identification of the various metabolites was achieved by co-
chromatography with independently synthesized model metabolites in
at least four different solvent systems. Table 24 gives the identity
and relative amounts of the metabolites detected in the urine and feces
of the white mouse treated with 32p phenthoate at two dosages and
Table 25 gives analogous data for the ^C label. 32P-labeled metabo-
lites identified in varying amounts in either urine or feces were:
demethyl phenthoate (LVII), demethyl phenthoate oxon (LVIII), phenthoate
acid (LXI), t),0-dimethyl phosphorodithioic (LXII) and phosphorothioic
(LXIII) acids. Unchanged phenthoate was present only in the feces.
No phenthoate oxon (LVI) was detected in either urine or feces, despite
the presence of other metabolites containing the P=0 linkage such as
LVIII and LXI. Of the various unknowns, D and E were common to both
32p and 14-C labels. Based on its chromatographic behavior, which was
similar to that of phenthoate acid (LIX) in the different TLC systems
employed, the most likely candidate for the structure of E is 0,0-
dimethyl S-[ot-(carboxy)phenyl j phosphorothioate. However, this could
not be confirmed since synthesis of this compound was not achieved.
The other unknowns were highly polar substances which remained close
to the origin of tic plates developed with nonpolar solvents.
Metabolites LVII-LXI also were obtained from the white mouse treated
with phenyl ring ^C phenthoate (Table 25) along with metabolites
produced after removal of the dimethoxyphosphinothioyl moiety (LXIV-
LXVII). A trace amount of phenthoate oxon (LVI) was observed in
feces. Although noticeable differences in the relative amounts of
several of the metabolites were observed between the ^C and -^ P labels,
on an overall basis the pattern of metabolism for the two labels was
similar. It should be pointed out that direct comparisons between the
two labels should not be made since the level of treatment at the two
lower doses was different (17 and 30 mg/kg) and data presented for the
aqueous phase for the -^C label are from analyses conducted 12 hours after
treatment instead of the customary 24 hours. In this regard, numerous
attempts to analyze the aqueous phase of the 24-hour ^C urine sample
proved unsatisfactory owing to poor separation on tic plates. Attempts
to remove interfering materials by column chromatography, including
the use of Sephadex G-50, or preparative tic proved fruitless, and
even in these cases undefined smears were obtained on analytical tic
plates. Mild acid treatment of the aqueous phase also had little effect
in improving tic separation. For reasons which were not obvious, this
difficulty was not experienced with the urine samples containing ^2p
label nor with the 12-hour urine sample.
Examination of the data in Tables 24 and 25 reveals that the most prom-
'inent reactions leading to metabolic degradation of phenthoate in the
mouse involves hydrolysis of the carboethoxy moiety or cleavage of the
P-0 (or possibly 0-C), P-S, and C-S bonds. The metabolites produced
from these reactions probably represent detoxication products. Of key
significance is the virtual absence of phenthoate oxon (LVI), the
metabolite which presumably is responsible for intoxication. Owing to
51
-------
Table 24. METABOLITES ISOLATED FROM THE WHITE MOUSE 24 HOURS
AFTER TREATMENT WITH 32P PHENTHOATE AT 30 AND 161 MG/KG
7, of Total recovered radioactivity
Urine
Metabolite
LV
LVII
LVIII
LIX
LX
LXI
LXII
LXIII
Unknown A
Unknown B
Unknown C
Unknown D
Unknown E
Unknown F
Total
Dose
30
161
30
161
30
161
30
161
30
161
30
161
30
161
30
161
30
161
30
161
30
161
30
161
30
161
30
161
30
161
Aqueous
phase
-
4.1
7.1
4.9
8.3
0.3
24.2
9.4
16.9
4.6
15.3
7.9
7.7
24.4
2.9
1.3
0.2
1.8
6.8
4.1
0.3
76.3
76.2
Organo-
soluble
-
0.1
4.5
3.9
11.7
1.6
0.2
1.5
0.2
0.5
0.1
0.3
-
2.9
2.7
0.4
1.4
_
17.9
14.1
Feces
2.6
1.9
-
0.4
0.5
0.4
0.2
0.4
1.1
3.4
1.2
1.6
0.4
0.4
0.6
0.4
-
-
-
5.8
9.7
Total
2.6
1.9
4.1
7.2
9.4
8.7
4.7
12.1
25.8
9.8
18.4
5.2
16.9
11.4
9.2
26.0
3.3
1.7
2.9
0.8
2.7
2.2
7.2
5.5
0.3
100
100
52
-------
Table 25. METABOLITES ISOLATED FROM THE WHITE MOUSE 12 AND 24
HOURS AFTER TREATMENT WITH 14C PHENTHOATE AT 17 AND 161 MG/KG
°l, of Total Recovered Radioactivity
Urine
Metabolite Dose
LV
LVI
LVII
LVI II
LIX
LX
LXI
LXIV
LXV
LXVI
LXVII
Phenthoate
Phenthoate oxon
Demethyl phenthoate
Demethyl phenthoate
oxon
Phenthoate acid
Demethyl phenthoate
acid
Demethyl phenthoate
oxon acid
[ -SCH(C6H5)COOH]2
[ -SCH(C,H,)COOEt]
O J £-
Mandelic acid
CH,SCH(C,H,)COOH
3 03
Unknown D
Unknown E
Total
17
161
17
161
17
161
17
161
17
161
17
161
17
161
17
161
17
161
17
161
17-
161
17
161
17
161
17
161
Aqueous
phase3
-
8.5
28.3
9.6
4.9
5.8
3.7
3.9
9.3
2.3
4.7
1.6
-
-
2.6
4.8
7.8
4.4
-
_
-
59.9
44.3
Organo-
soluble
-
12.2
6.7
1.7
1.6
1.4
1.1
-
1.4
-
1.9
5.0
-
-
1.4
2.6
0.7
_
-
0.8
-
15.6
22.9
Feces
3.2
5.1
0.3
0.6
0.5
0.1
0.1
0.8
4.6
0.2
-
-
-
1.3
2.0
0.2
0.2
0.7
1.1
-
-
-
_
0.8
7.4
14.4
Total
3.2
5.1
0.3
0.6
21.2
35.1
11.4
7.3
11.8
7.0
3.9
10.7
2.3
7.9
8.6
0.2
0.2
4.7
8.5
0.7
7.8
4.4
-
0.8
0.8
82.9
81.6
a Data for aqueous phase is for 12-hr urine sample.
53
-------
the amounts of LVIII and LXI detected, desulfuration undoubtedly takes
place, but it is not clear whether the thiono sulfur is replaced by
oxygen before or after (D-demethylation or hydrolysis of the carboethoxy
moiety. If phenthoate oxon is produced by direct oxidative desulfuration
of phenthoate, evidently it is rapidly metabolized further to LVIII and
LXI and does not reach a critical level in the mouse.
Metabolism in House Flies - Data for the distribution and recovery of
32p or 1'4-C phenthoate 4 hours after topical application to susceptible
^NAIDM^ anc* dimethoate resistant (R ) strains of house flies are
presented in Table 26. Treatment was at 5.0 and 50 M-g/g, the respective
LD5Q values for the SNAIDM and Rd strains. At the time of treatment
the LDrQ of dimethoate to the Rd strain was approximately 150 M-g/g.
Based on recoveries in the internal fraction of flies treated at the
lower dose, it is apparent that phenthoate is absorbed by the two strains
at approximately equal rates. Thus, it appears that penetration is not
a significant factor contributing to the 10-fold greater tolerance
exhibited by the R^ strain to phenthoate.
Results of the metabolism of 1^C phenthoate in the two strains are
presented in Table 27« Because of the low specific activity of the P
label at the time of analysis, data from this label were less precise
than those of the -^C label and discussion will be restricted to data
from the latter.
Qualitatively, the metabolism of phenthoate in house flies was
similar to that in the white mouse. While slightly fewer metabolites
were isolated from house flies, with the exception of the unknowns,
those which were isolated from flies also were found in mice. The
same metabolic reactions leading to detoxication products such as
carboethoxy ester hydrolysis and cleavage of the P-0, P-S, and C-S
bonds evidently also take place in house flies . Quantitatively,
perhaps the most significant difference between mice and flies was the
substantial amounts of phenthoate oxon detected in the latter. This
metabolite, present at sufficiently high levels, readily accounted for
the toxicity of phenthoate to house flies. Phenthoate also appeared
to be relatively more stable in flies than in the mouse but direct
comparisons were not possible from the data available owing to differ-
ences in the time and type of analyses. Total recovery of phenthoate,
which includes internal extract, cage and external rinse, ranged from
35-67% of the applied dose 4 hours after treatment.
Owing to variability in the data, distinct differences in phenthoate
metabolism between S^AIDM ancl Rd h°use flies were not readily discernible.
From the l^C data (Table 27) penetration of phenthoate appeared to be
slightly slower in the Rd strain, but it is doubtful that this difference
is significant., Of possible significance was the recovery of 2-fold
more phenthoate oxon from SNAIDM (0.31 M-g/g) than from R, (0.16 M.g/g)
flies treated at the same dose of 5 |J.g/g. While this difference is not
large, it is in the proper direction. At the higher dose of 50
54
-------
Table 26. DISTRIBUTION OF RADIOACTIVITY 4 HOURS AFTER TOPICAL
TREATMENT OF SUSCEPTIBLE (SNAIDM) ^D DIMETHOATE-RESISTANT
(Rd) HOUSE FLIES WITH ^C- AND 32P-LABELED PHENTHOATE
7o of Applied radioactivity recovered
Label
32p
14c
32P
14c
32p
14c
Fly
strain
SNAIDM
SNAIDM
Rd
Rd
Rd
Rd
Dose
(ug/g)
5.0
5.0
5.0
5.0
50.0
50.0
Cage
rinse
24.1
11.2
21.9
12.3
16.8
17.8
External
rinse
29.1
26.7
29.4
40.1
45.9
55.2
Internal
extract
28.3
54.2
34.2
42.8
28.2
24.1
Total
81.5
92.1
85.5
95.2
90.9
97.1
55
-------
Table 27. METABOLIC PRODUCTS RECOVERED FROM HOUSE FLIES AFTER TOPICAL TREATMENT WITH UC PHENTHOATE
7o of Total recovered radioactivity3
SNAIDM ^5'° ^g/8^
Metabolite
LV
LVI
LVII
LVI 1 1
LIX
LX
LXI
LXIV
LXVI
Cage
rinse
4
(0
1
(0
1
(0
1
(0
2
(0
1
(0
.1
.19)
.8
.08
.2
.05)
_
.8
.08)
_
_
.0
.09)
.3
.06)
External
rinse
8.3
(0.38)
1.8
(0.08)
1.6
(0.07)
5.8
(0.26)
1.2
(0.05)
6.3
(0.29)
_
1.8
(0.08)
0.6
(0.03)
• Internal
rinse
23.2
(1.07)
3.3
(0.15)
9.2
(0.42)
5.9
(0.27)
3.3
(0.15)
3.9
(0.18)
_
2.9
(0.13)
4.9
(0.23)
Rd (5
.0 M8/8)
Cage External
rinse rinse
1.9 24
(0.09) (1
0
(0
5.5 1
(0.26) (0
5.5 5
(0.26) (0
2
(0
_
_
5
<°
2
(0
.3
.16)
.9
.04)
.8
.09)
.6
.27)
.2
.11)
_
_
.0
.24)
.2
.11)
Internal
rinse
26.
(1.
2.
(0.
2.
(0.
3.
(0.
2.
(0.
1.
(0.
0.
(0.
3.
(0.
3.
(0.
6
27)
5
12)
2
11)
3
16)
2
11)
2
06)
6
03)
5
17)
1
15)
Rd (50 |j.g/g)
Cage
rinse
10.5
(5.10)
0.3
(0.15)
1.8
(0.87)
4.3
(2.09)
_
_
0.8
(0.39)
0.3
(0.15)
0.3
(0.15)
External
rinse
50.9
(24.71)
_
1.5
(0.73)
1.5
(0.73)
_
0.2
(0.10)
0.5
(0.24)
0.3
(0.15)
0.3
(0.15)
Internal
rinse
5.9
(2.86)
1.3
(0.63)
6.7
(3.25)
4.1
(1.99)
1.4
(0.68)
-
1.0
(0.49)
0.9
(0.44)
0.5
(0.24)
-------
Table 27. continued
Cage External Internal Cage External Internal Cage External Internal
Metabolite rinse rinse rinse rinse rinse rinse rinse rinse rinse
Unknown G - 1.6 2.2 - - - - 1.7 3.0
(0.07) (0.10) (0.83) (1-46)
Total 12.2 29.0 58.8 12.9 42.0 45.2 18.3 56.9 24.8
o
Parenthetical values represent actual amounts (|ag) of metabolites in terms of phenthoate equivalents,
-------
(R strain LDsn), approximately 2.5-fold more ^C oxon was recovered
from the R, strain (0078 u,g/g) compared to the S^AIDM) strain treated
at 5.0 (J,g/g.
Conclusion - On the basis of the metabolic products identified, the
metabolism of phenthoate in the white mouse and house fly appears to
be similar to that which occurs in plants (see Part C-3). Further,
in gross aspects the various metabolic reactions which phenthoate
undergoes in these test animals correspond to those reported previously
for malathion , a compound of similar structure and properties of
selective toxicity0 Indication of the functional groups in the phenthoate
molecule which are metabolically labile is given in the structure below.
P-0 or C-0 px ^^.oxidative desulfuration
cleavage CH.j-0 \. ,$*
^,-P 0 /"^ester hydrolysis
CH,o' /I S-CHC-OC0HC
3 / \\ 25
C6H5
P-S cleavage C-S cleavage
Metabolites generated from each or different combinations of these
various reactions were identified. Secondary reactions involving the
metabolic fragments, e.g., oxidative coupling to produce b_is_-[a-(carbo-
ethoxy)benzyl] disulfide (LXV) and bis-[or-(carboxy)benzyl] disulfide
(LXIV), also occurred.
From the relative amounts of metabolites isolated from the mouse, no
single reaction appeared to predominate over any of the others in the
degradation of phenthoate. It is clear that the mouse is able to
degrade phenthoate with almost equal facility by hydrolysis of the
carboethoxy moiety, cleavage of the P-S or C-S bond, and removal of
the methoxy group by either direct demethylation or hydrolytic cleavage
of the P-0 bond. As indicated earlier, little, if any, phenthoate oxon
was detected, a finding which is in agreement with the low mammalian
toxicity of phenthoate.
Comparison of the data for mouse metabolism with those of house flies
reveals that hydrolysis of the carboethoxy moiety takes place to a
greater extent in the mouse than in flies (compare metabolites LIX, LX,
and LXI). This is in accord with earlier reports 12,13 where it was
pointed out that carboxyesterase activity is substantially lower in
insects than in mammals. On the other hand, degradation by loss of
one of the methoxy groups appeared to be of major significance in flies.
58
-------
Although a reasonable explanation for the selective toxicity of
phenthoate may be based on differences in the amounts of phenthoate
oxon recovered between the white mouse and house fly, the greater
tolerance of the R, strain compared to the SN.,-DM strain to phenthoate
intoxication is less readily explainable owing to the relatively large
amounts of oxon recovered from R^ flies at the higher dosage. This
phenomenon has been observed before in studies with other organophosphorus
insecticides-^> 15 an(j mav be attributed to compartmentalization of the
oxon at a locale distant from the site of action or possibly to decreased
sensitivity of fly cholinesterase to inhibition-*-".
Overall, the evidence indicates that phenthoate is metabolized through
similar pathways in mice and house flies but with significant quanti-
tative differences to account for its selective action.
8. References
1. Fahmy, M. A. H., T. R. Fukuto, R. 0. Myers, and R. B. March. The
Selective Toxicity of New N-Phosphorothioylcarbamate Esters. J. Agr.
Food Chem. 18:793-6 (1970).
2. Miskus, R. P., T. L. Andrews, and M. Look. Metabolic Pathways
Affecting Toxicity of N-Acetyl Zectran. J. Agr. Food Chem. 17:842-4
(1969).
3. Black, A. L., Y. C. Chiu, T. R. Fukuto, and T. A. Miller. Metabolism
of 2,2-Dimethyl-2,3-Dihydrobenzofuranyl-7 N-Methyl-N-(2-Toluenesulfenyl)-
carbamate in the Housefly and White Mouse. Pestic. Biochem. Physio1.
3:435-46 (1973).
4. Metcalf, R. L., T. R. Fukuto, C. Collins, K. Borck, S. A. El-Aziz,
R. Munoz, and C. C. Cassill. Metabolism of 2,2-Dimethyl-2,3-Dihydro-
benzofuranyl-7 N-Methylcarbamate (Furadan) in Plants, Insects, and
Mammals. J. Agr. Food Chem. 16:300-11 (1968).
5. Dorough, H. W. Metabolism of Furadan (NIA-10242) in Rats and
Houseflies. J. Agr. Food Chem. 16:319-25 (1968).
6. Ivie, G. W., and H. W. Dorough. Furadan-C Metabolism in a
Lactating Cow. J. Agr. Food Chem. 16:849-55 (1968).
7. Miller, T. and J. M. Kennedy. Flight Motor Activity of Houseflies
as Affected by Temperature and Insecticides. Pestic. Biochem. Physiol.
2:206-22 (1972).
8. Richmond, D. V., and E. Somers. Studies on the Fungitoxicity of
Captan. IV. Reactions of Captan with Cell Thiols. Ann. Appl. Biol.
57:231-48 (1966).
59
-------
9. Liu, M. K., and L. Fishbein. Reactions of Captan and Folpet with
Thiols. Experientia 23:81-2 (1967).
10. Pellegrini, G., and R. Santi. The Potentiation of Toxicity of
Organophosphorus Compounds Containing Carboxylic Ester Functions Toward
Warm-Blooded Animals by Some Organophosphorus Impurities. J. Agr. Food
Chem. 20:944-50 (1972).
11. Krueger, H. R., and R. D. O'Brien. Relationship Between Metabolism
and Differential Toxicity of Malathion in Insects and Mice. J. Econ.
Entomol. 52:1063-7 (1959). ~
12. Seume, F. W., and R. D. O'Brien. Metabolism of Malathion by Rat
Tissue Preparation and Its Modification by EPN. .J. Agr. Food Chem.
8:36-41 (1960).
13. Kojima, K. Studies on the Selective Toxicity and Detoxication
of Organophosphorus Compounds. Odawa, Japan. Special Report of the
TOA Noyaku Co. Ltd. (1961).
14. Hollingworth, R. M., T. R. Fukuto, and R. L. Metcalf. Selectivity
of Sumithion Compared with Methyl Parathion. Metabolism in Susceptible
and Resistant Houseflies. J. Agr. Food Chem. 15:250-5 (1967).
15. Vinopal, J. H. and T. R. Fukuto. Selective Toxicity of Phoxim
(Phenylglyoxylonitrile (3,0-Diethyl Phosphorothioate). Pestic.
Biochem. Physiol. 1:44-60 (1971).
16. Tripathi, R. K., and R. D. O'Brien. Insensitivity of Acetylcholin-
esterase as a Factor in Resistance of Houseflies to the Organophosphate
Insecticide Rabon. Pestic. Biochem. Physiol. 3:495-9 (1973).
60
-------
C. STUDIES ON THE METABOLISM OF INSECTICIDES
In Part B of this report a number of studies on the comparative metabo-
lism of selectively toxic insecticides in mammals and insects are
described. These studies were conducted for the elucidation of the
fundamental mechanisms responsible for insecticide selectivity. In
addition to metabolism studies of this nature, work also was carried
out for toxicological purposes and the assessment of hazards arising
from the use of insecticidal chemicals under current development. This
part of the report is concerned with these studies.
1. Metabolism of 2-[Methoxy(methylthio)phosphinylimino]-3-Ethyl-5-Methyl-
1,3-Oxazolidine in the Cotton Plant and House Flies
Introduction - 2-[Methoxy(methylthio)phosphinylimino]-3-ethyl-5-methyl-
1,3-oxazolidine (I) or Stauffer R-16,661, is a new experimental insecti-
cide which in our screening tests has demonstrated outstanding systemic
activity in cotton plants against a wide variety of insects. The
structure of I is given below.
?2«5
H.CO XN=^C CH.
3 II2
0 -- CHCH
In greenhouse screening tests this material was equal, or superior, to
aldicarb as a systemic insecticide, following granular treatment of the
soil or topical application on the stem, against such typical cotton
pests as the red spider mite (Tetranychus cinnabarinus), cotton aphid
(Aphis %ossypii), and cotton leaf perforator (Bucculatrix thurberiella).
I also was effective against the pink bollworm (Pectinophora gossypiella),
giving 1007o control for at least 40 days after treatment.
The principal drawback which prevents the use of this potent systemic
material in practical field application is its extremely high mammalian
toxicity. In our tests the LD,-Q of I to the white mouse was 0.1-0.2
mg/kg, a value which makes it approximately 10-fold more toxic to the
mouse than aldicarb. However, because of its outstanding systemic
properties, it was decided to study the metabolism of this material
in plants and insects in an effort to determine whether the compound
itself or whether metabolic products were responsible for insecticidal
activity. Also, it was important to assess the toxicological properties
of the metabolites, owing to the possibility that the metabolite might
be less toxic to mammals but still retain toxicity to insects.
61
-------
32
Metabolism in the Cotton Plant - P-labeled I, prepared through a
2-step synthesis beginning with ^2p (3,0-dimethyl phosphorochlorido-
thioate, was used for this study. For the study of the metabolism of
I in the cotton plant, two different methods of treatment of the plant
were used. In the first method, the base of a young cotton plant
containing developing bolls was treated with a total of 25 jj.1 (37.5 mg)
of 32p-I. xhe plant was maintained in the greenhouse and leaves
were taken from the upper and lower sections of the plant for periodic
assay. In the second method, ten freshly cut cotton'petioles were
placed in 10 ml water containing 10 mg P-I and allowed to stand
overnight. The aqueous solution containing I was completely absorbed
after 24 hr and the petioles were removed to fresh water and kept in
the greenhouse. At various time intervals,.petioles were removed and
examined for metabolism.
I is very stable in the cotton plant. For example, 14 days after stem
application 86-93% of the total radioactivity isolated from cotton
leaves was in the form of unchanged I. After 51 days the major
component was still I but quantitation was not possible owing to low
radioactivity from decay. Similar results were obtained when isolated
cotton petioles were treated, as shown in Table 1. The Rf values
reported in this table were determined on silica gel HF-254 (Merck,
Germany) using 95% ethanol as the developing solvent. The data in the
table show that I is the principal component in the leaf although
several metabolites also are formed but in relatively small amounts.
The metabolites at Rf 0.0 and 0.05 are probably hydrolysis products
since the compound 2-[hydroxy(methylthio)phosphinylimino]-3-ethyl-5-
methyl-1,3-oxazolidine, the ()~demethylated product isolated from the
alkaline hydrolysis of I showed an R£ of 0.1 on the same tic system.
However, it is possible also that these metabolites may be plant con-
jugates. The principal intact metabolite was found at Rf 0.58 and this
compound undoubtedly is the 4-keto derivative, II, formed by oxidation
of the 4-position in the oxazolidine ring. Support for this structure
will be presented later. Another metabolite which appeared in small
but varying amounts was found at R£ 0.33. The structure of this compound
is tentatively suggested as the 4-OH derivative, III, the precursor to
the 4-keto metabolite.
The major tic component found at Rf 0.48 was unequivocably established
as unchanged I. This material was isolated from cotton leaves by means
of column and repeated thin-layer chromatography. The PMR spectrum of
the isolated material was virtually identical to that of I with a H
triplet at 6 1.20 for the methyl protons on N-ethyl, a H doublet
centered at 6 1.53 for the 5-methyl, a H multiplet at 6 4.80 for the
proton on the 4-carbon atom, a multiplet near 6 3.2 for the pair of
methylene protons adjacent to the ring nitrogen, a ^H doublet for S-CH,
protons at 6 2.3 (J = 14 Hz), and a 3H doublet at 6 3.81 (J = 13 Hz)
for 0-CHo protons.
62
-------
Table 1. AMOUNTS OF R-16,661 (I) AND METABOLITES FOUND IN COTTON PETIOLES AFTER VARYING TIME INTERVALS
Compound
4-Keto R-16,661
R-16,661 (I)
4-OH R-16,661 (tentative)
(III)
Unknown
Unknown
Rf
0.58
0.48
0.33
0.05
0
7o of Recovered radioactivity after
days following treatment
7
5.2
89
trace
2.4
1.0
14 21
3.3 3.7
75 73
trace trace
8.0 7.5
7.7 5.2
indicated
28
6.2
80
trace
7.2
1.6
-------
Metabolism in the House Fly and by In Vitro Enzyme Systems - In general,
the metabolism of I in the house fly was similar to that occurring in
the cotton plant. I penetrated very slowly into the fly and 20 hr
after a 2 |_ig/$ topical application only 12% and 7% of the applied
dose was found internally in the susceptible S^AIDM anc^ parathion
resistant Rp strains, respectively (Table 2). As in the case with
the cotton plant, unchanged I was the major component found along with
a significant amount of the 4-keto derivative (II). The unknown at
Rf 0.0 again probably represents hydrolyzed material or possibly con-
jugates. The metabolite suggested as the 4-OH derivative (III),
however, was not detected in flies.
In vitro metabolism studies using house fly and mosquito larvae
homogenate resulted in tic patterns of metabolites similar to those
obtained with living houseflies. In all these cases, the metabolite
believed to be the 4-OH derivative was not detected. Mouse liver
microsome-plus-soluble enzyme fraction, on the other hand, produced
a metabolic pattern virtually identical to that found in the cotton
plant. The results are indicated in Table 3.
Oxidation by the Udenfriend Model System - Incubation of I in the
Udenfriend model oxidation system gave products with identical chromato-
graphic behavior to those obtained from the cotton plant and mouse liver
preparation. Comparison was made by use of two different tic systems.
Since earlier attempts to isolate the pure 4-keto derivative (II), (R^
0.48 using HF-254-9570 ethanol; Rf 0.8 using silica gel G-chloroform-
ethanol 4:1) from cotton leaves treated with I proved unsuccessful owing
to contamination by plant material, the Udenfriend system was turned to
for the synthesis of this metabolite. A variety of other synthetic
approaches for the preparation of II were tried earlier but failed.
After incubating 0.79 g of I in the Udenfriend system for a total of 72
hr, and after repeated thin-layer chromatography approximately 5 mg of
a yellow oil was isolated without impurity which from spectroscopic
evidence was proved to be the 4-keto derivative. This material was
chromatographically identified by two tic systems to the material
isolated from the cotton plant. The mass spectrum of this compound
was consistent with the 4-keto structure shown below. In addition
to the parent ion (M = 266), the cleavage of -CH (M-15), -OCH3
H3CS 0 f2H5
/®'\
H-CO N=C' C=0
3 I I
0 CHCH2
(M-31) and -SCH. (M-47) radicals from the parent ion confirmed the
presence of the 0,^-dimethyl phosphorothioyl moiety. The cyclic amide
64
-------
Table 2. DISTRIBUTION OF I IN SUSCEPTIBLE (SNAIDM) AND
PARATHION-RESISTANT (Rp) HOUSE FLIES 20 HR
AFTER TREATMENT
.Fraction
External
Internal
Compound
-
Unknown
I
4-Keto (II)
R a
Rf
-
0.0
0.5
0.6
% of Recovered
SNAIDM
89.1
0.5
8.9
1 .5. .
radioactivity
Rp
92.7
0.1
7.0
. .. 0.2
a tic; silica gel G, developed first with benzene, then with 95%
ethanol.
65
-------
Table 3. METABOLISM OF I BY MOUSE LIVER MICROSOME-
PLUS-SOLUBLE ENZYME PREPARATION
Compound R,
Unknown 0 . 0
4-OH (tentative) (III) 0.6
T 0.7
4-Keto (II) 0.8
% of Recovered radioactivity
4.3
1.5
91.0
3.1
a tic; silica gel G, developed first with 1:2 benzene-ethyl acetate,
then with 4:1 ethanol-chloroform.
66
-------
structure was confirmed by loss of ethylene (C H, = 28) and carbon
monoxide (CO = 28), and the presence of a base peak (m/e = 43) for
CH CO+. A PMR spectrum taken in d-chloroform (TMS) also confirmed
the 4-keto structure.
Attempt also was made to isolate the less important metabolite Rf 0.6 in
Table 3 (Rf 0.33 using HF-254-957» of ethanol system), but not enough
material could be obtained free from impurity for definitive spectro-
scopic analysis. The assignment of a hydroxylated structure, probably
4-OH, to the metabolite is based primarily on tic behavior relative to
I and to its likelihood as a logical precursor to the 4-keto metabolite.
In the various tic systems used in this study, the rate of movement of
each compound is inversely related to its polar property and, therefore,
the 4-OH compound would be expected to have a lower Rf value compared
to I.
Toxicological Properties - Toxicological properties for I and II are
presented in Table 4. The data show that compared to I, the 4-keto
metabolite is approximately 10-fold less effective in inhibiting
insect and mammalian cholinesterase and is 15-fold less toxic to the
house fly. On the other hand, the two compounds were almost equally
toxic to the white mouse. Thus, the 4-keto metabolite appears to be
selectively toxic in the wrong direction, i.e., it is nontoxic to the
house fly but highly toxic to the mouse.
Conclusion - R 16,661 (I), an outstanding systemic insecticide, evidently
is quite stable in the cotton plant and in the house fly. It is, however,
slowly metabolized in these biological systems to give as one of the
eventual principal metabolites the 4-keto derivative, probably according
to the metabolic scheme given below.
?2H5
H3CS\ ^° I
H3CO/ ^N=C CH >
0 CHCH
R 16,661 (I) 4-OH derivative
I
H3CS\ ^° ^5
HCOxP\N=c/Nxc=0
3 I i
rwTj
^noti«
4-keto derivative (II)
" H CSX /O
H3COX XN=CX
0-
I2"5 1
XCHOH
1
fUpTJ
67
-------
Table 4. TOXICOLOGICAL PROPERTIES OF I AND ITS
4-KETO (II) METABOLITE
II
Anti-ChE (T5f) M)a
Fly-head ChE 3.0 X 10"8 2.7 X 10"7
Bovine erythrocyte ChE 5.3 X 10~7 5.6 X 10~6
Toxicity, LD5Q (rag/kg)
House fly 16 240
White mouse 0.1-0.2 0.4-0.6
value after 10-min incubation period.
68
-------
Because of the stability of I in the cotton plant and the much lower
insecticidal activity of the major metabolite, the 4-keto derivative
(II), it appears that I itself is responsible for systemic activity.
Based on our toxicological data the metabolic coversion of I to the
4-keto derivative in the plant is an undesirable occurrence owing
to the relatively poor insecticidal activity and high mammalian
toxicity of the metabolite.
The structure of I is unusual among the contemporary organophosphorus
insecticides and compounds of this type deserve further study. For
example, based on its structure it is not obvious which moiety will
be displaced when this compound reacts to inhibit the cholinesterase
enzyme. The fact that CH^O-P bond cleavage occurs when I is treated
with sodium hydroxide suggests that methoxide ion is the leaving group,
even though thiomethylate ion is a more likely candidate based on
reactivity grounds.
In many respects I is similar in structure and properties to the 0-alkyl
Stalky 1 phosphoramidothioates, which have recently gained prominence as
effective insecticides^. The mode of action of the phosphoramidothioates,
at this stage, however, is not well understood.
2. Metabolism of 0-(4-Bromo-2,5-Dichlorophenyl) 0-Methyl Phenylphosphono-
thioate (Leptophos) in White Mice, on Cotton Plants, and in House Flies
Introduction - Leptophos [0-(4-bromo-2,5-dichlorophenyl) ()-methyl phenyl-
phosphonothioate] (IV) is a new phosphonate insecticide which has shown
promise for the control of a broad spectrum of insect pests and as a
fungicide against rice stem blast. However, a recent report-^ has
implicated it as a material possessing delayed neurotoxic activity.
Leptophos has been accused as the probable cause of paralysis and
death of about 1,300 water buffalo in the Nile delta during the summer,
1971^. In order to develop information on the mode of action and
metabolism of leptophos in animals and plants, this material was
examined in the white mouse, cotton plant, and house fly.
Metabolism in the White Mouse - White mice were treated orally with two
different labels of i4C leptophos. i.e., 0-(4-bromo-2,5-dichlorophenyl-
•^C) (phenoxy label), and phenyl-l^C-phosphonothioate (phenyl label).
The results of metabolism studies performed in the white mouse treated
with 25 mg/kg phenoxy and phenyl ^C-labeled leptophos are summarized
in Tables 5, 6, and 7.
In the case of phenoxy-labeled leptophos, radioactivity was rapidly
eliminated from the mouse and elimination was virtually complete 48
hours after treatment, the bulk of the excreted material being present
in the urine (Table 5). In comparison, elimination of phenyl-labeled
radioactivity was notably slower and radioactivity was detected in the
urine as long as 144 hours following treatment. It was estimated that
69
-------
Table 5. DISTRIBUTION OF LEPTOPHOS AND ITS METABOLITES AT
DIFFERENT TIME INTERVALS AFTER ORAL TREATMENT OF A
WHITE MOUSE AT 25 MG/KG
Position of label Time (hr)
phenoxy 7
" 24
" 48
phenyl 7
24
48
72
" 144
cy . oy •
A, in /o in
urine feces
58
29
18
43
23
6
2
1
.1 1.0
.9 2.2
.1 1.1
Total recovery
.8 1.1
.9 7.7
.9 trace
.7 traceb
.8 0
Total recovery
Totala
59.1
32.1
1.9.2
110.3
44.9
31.6
6.9
2.7
1.8
87.9
a The total recovery shown was based on the amount administered. The
error in administered dose is estimated to be 10%.
"trace" refers to less than 0.17= radioactivity administered.
70
-------
the error in the administered dose was approximately ± 10% and,
therefore, that the total amounts recovered were close to complete
recovery of radioactivity. The rate in which the two labels in
leptophos are eliminated, in urine and feces, is presented graphically
in Fig. 1. In spite of the possible error in administration of the
radioactive compound, there is little doubt that the carbon-14 in the
phenyl group is eliminated at a slower rate than that in the phenoxy
group. A possible explanation for the difference in elimination rates
is that the conjugate of the phenoxy-labeled phenol (IX) is eliminated
more rapidly than the phenyl-labeled phenylphosphonic acids (VI, VII,
and VIII).
The major portion of the radioactivity eliminated from the mouse was in
the form of degraded products present primarily in the urine (Table 6
and Table 7). Leptophos and the oxon (V) were present in small amounts
but essentially all in the feces. With phenoxy-labeled leptophos, more
than 90% of the urinary metabolite was in the form of conjugated 4-bromo-
2,5-dichlorophenol, although a small amount of free phenol also was
present. The exact nature of the conjugate was not established but
both enzymatic ([3-glucuronidase/aryl sulfatase) and acid hydrolysis
yielded only the phenol (IX).
The phenyl-labeled leptophos gave 0-methyl phenylphosphonothioic acid
(VI), 0-methyl phenylphosphonic acid (VII), and phenylphosphonic acid
(VIII) as the principal degradative products (Table 7) present entirely
in the urine. Compound VI was the major component present in the early
collection periods but its amount decreased rapidly with time, eventually
reaching a level about equal to that of VIII. As in the phenoxy label,
both leptophos and leptophos oxon were found in the feces, although
unexplainably larger amounts of unchanged leptophos were detected with
the phenyl label.
The unknown metabolite(s) listed in Table 6 and 7 cochromatographed
with an authentic sample of 0-(4-bromo-2,5-dichlorophenyl) phenylphos-
phonic acid on tic using Silica HF-254 with a developing solvent of
benzene-chloroform (1:1).
Cotton Plant Metabolism - Data for the metabolism of leptophos after
application on the surface of cotton leaves at a dosage equivalent to
one Ib/acre are presented in Tables 8, 9, and 10. From the data in
Table 8, it is evident that leptophos does not penetrate readily into
the leaf but remains on the surface or is volatilized. The primary
mechanism by which leptophos is lost appears to be by volatilization
since there was a steady decrease in total measurable radioactivity
with time. For example, 73-75% of the applied leptophos was recovered
one week after treatment compared to 19-21% after five weeks. Rela-
tively small amounts of radioactivity were found inside the leaf
either as benzene or methanol extracts or bound to the pulp. It should
be pointed out that control experiments were carried out to show that
71
-------
100
80
8
Q
Q
LU
X
uu
60
40
20
20
40
60 80
TIME (hours)
100
120
140
Fig. 1. Rate of elimination of radioactivity from the white mouse, in urine and feces, after
oral administration of phenoxy-labeled ( O ) and phenyl-labeled ( • ) leptophos
-------
Table 6. METABOLITES FOUND AT VARIOUS TIMES3 AFTER ORAL
TREATMENT OF A WHITE MOUSE USING ^C-PHENOXY-
LABELED LEPTOPHOS AT 25 MG/KC
Compound
IV Leptophos
V Leptophos oxon
Cl
7 hr
0.7
0.7
24 hr
0.8
0.2
48 hr
0.2
0
Total
1.7
0.9
IX HO
Br
Cl
Cl
conj-0-c
V— /
\
Cl
4.5
47.0
1.2
26.5
1.5
15.6
7.2
69.1
Unknown
0.6
0.5
trace
1.1
a Values are given as per cent of recovered ^C material.
k The unknown material was not conclusively identified but it c.o-
chromatographs with an authentic sample of 0-(4-bromo-2,5-
dichlorophenyl) phenylphosphonic acid.
c "trace" refers to less than 0.1% of recovered materials.
73
-------
Table 7. METABOLITES FOUND AT VARIOUS TIMESa AFTER ORAL
TREATMENT OF A WHITE MOUSE USING 14C-PHENYL-
LABELED LEPTOPHOS AT 25 MG/KG
Compound 7 hr 24 hr 48 hr 7.2 hr
IV Leptophos 1.2 8.2 traceb traceb
V Leptophos oxon 0 0.4 trace trace
S
ii
VI C H -P-OH 35.4 15.0 4.6 1.0
65,
OCH3
0
II
VII C,H,-P-01! 8.9 5.7 1.5 1.1
,65,
OCH3
0
VIII C,H,-P-OH 5.5 6.3 1.8 1.1
6 5 ,
OH
Unknown0 0 0.2 0 0
144 hr Total
traceb 9.4
traceb 0.4
0.6 56.6
0.7 17.9
0.8 15.5
0 0.2
Values are given as per cent of recovered L^C material.
"trace" refers to less than 0.170 recovered material.
The unknown material was not conclusively identified but it
cochromatographs with an authentic sample of 0_- (4-bronio-2,5~
dichlorophenyl) phenylphosphonic acid.
74
-------
Table 8. DISTRIBUTION3 OF LEPTOPIIOS AND ITS METABOLITES AT
DIFFERENT TIME INTERVALS AFTER TOPICAL APPLICATION
TO COTTON PLANTS
Phenoxy label
1 week 3 week 5 week
Wash 64.3 16.5 9.0
Benzene extract 5.6 6.2 4.5
Methanol extract 4.9 9.9 4.2
Pulp (bound) traceb 1.3 2.9
Total 74.8 34.4 20.6
Phenyl label
Wash 62.1 18.9 7.8
Benzene extract 7.0 8.1 4.8
Methanol extract 4.3 10.0 3.7
Pulp (bound) traceb 1.1 2.2
9 week
1.8
2.4
3.7
3.1
11.0
Total 73.4 38.1 18.5
a Values are given in per cent based on amount of radioactive compound
applied.
b "trace" refers to less than 0.1%.
75
-------
Table 9. METABOLITES FOUND AT VARIOUS TIMES AFTER TOPICAL
APPLICATION OF 14C PHENOXY-LABELED-LEPTOPHOS TO
COTTON PLANTS
Leptophos (IV)
Surface wash
Solvent extract
Pulp
1 week 3 week 5 week
80.7 37.8 35.1
10.7 23.6 22.8
00 0
9 week
29.3
13.6
0
Leptophos oxon (V)
Surface wash
Solvent extract
Pulp
0
0
0
trace
0
0
0
trace
0
0
0
0
Salt of phenol (IX)
Surface wash
Solvent extract
Pulp
5.3
3.3
0
9.9
23.2
0
8.7
19.4
0
7.2
21.8
0
Unknown
Surface wash
Solvent extract
Pulp
0
0
0
trace
0.3
5.2
0
0
14.0
0
0
28.1
a Values are given as per cent based on the total amount of radioactivity
recovered.
The values reported are a sum of the free phenol (IX) and a
salt of phenol (IX).
These values reflect the amount of unextractable radioactive material
bound in the pulp.
76
-------
Table 10. METABOLITES3 FOUND AT VARIOUS TIMES AFTER TOPICAL APPLICATION OF l^C-PHENYL LABELED
LEPTOPHOS TO COTTON PLANTS
Leptophos (IV)
Surface wash
Solvent extract
Pulp
Leptophos oxon (V)
Surface wash
Solvent extract
Pulp
Acid (VI)
Surface wash
Solvent extract
Pulp
1 week
82.1
14.3
0
0
0
0
1.6
0.7
0
3 week
48.2
30.1
0
0
0
0
1.3
0.8
0
5 week
37.5
31.3
0
0
0
0
4.3
2.7
0
Acid (VII)
Surface wash
Solvent extract
Pulp
Acid (VIII)
Surface wash
Solvent extract
Pulp
Unknown
Surface wash
Solvent extract
Pulp
1 week
0.8
0.5
0
0
trace
0
0
0
trace
3 week
0.3
0.3
0
0
16.2
0
0
0
2.8
5 week
0
1
0
0
10
0
0
0
11
.5
.6
.3
.8
Values are given as per cent based on the total amount of radioactive compound recovered.
These values reflect the amount of unextractable radioactive material bound in the pulp.
-------
radioactivity was not being lost in the work-up procedure, i.e.,
greater than 90% of the applied radioactivity was recovered.
Qualitatively, the degradation of leptophos in or on the cotton leaf
is similar to that occurring in the white mouse (cf Tables 9 and 10) .
The major component present in the recoverable radioactivity one week
after application was unchanged leptophos (91-96%) and this substance
gradually diminished with time to approximately 29% in 9 weeks. Most
of the leptophos remained on the leaf surface although significant
but small amounts evidently were absorbed into the leaf. Little, if
any, leptophos oxon (V) was present at any sampling period although
other alteration products, i.e., the phenol (IX), in the form of an
unknown salt, and the phenylphosphonic acid derivatives VI, VII, and
VIII were present in varying amounts. No free or conjugated phenol
was detected and essentially all of the phenol was isolated as a salt
(Table 9) . This substance was the principal degradation product
present in the leaf but substantial amounts also were found on the
leaf surface. The unknown material (s) from the phenoxy label treatment
was present almost entirely in the pulp and, because it was not extract
able with methanol, it was considered bound to the parent plant pulp.
Results obtained with the phenyl labeled leptophos show that all three
phenylphosphonic acid derivatives (VI, VII, VIII) were present both
in and on the cotton leaf. The largest component among the acids was
phenylphosphonic acid, particularly at the later sampling periods.
As in the case of the phenoxy label, a significant amount of the
measurable radioactivity was inseparable from the pulp by methanol
extraction and also was considered to be bound to the pulp. The
nature of this bound material, of course, is unknown but it undoubtedly
is not the same as the bound material obtained from plants treated with
the phenoxy- labeled leptophos.
Metabolism in House Flies - Two different strains of house flies were
used in this study, a susceptible (SNAIDM) and parathion resistant (Rp)
strain. The toxicity of leptophos to the SN^JDJ^ strain was 11 ng/g and
>500 p,g to the Rp strain.
The penetration of different dosages of leptophos into susceptible and
resistant flies was determined 1, 2, 4, and 6 hours after application
using l^C -phenoxy leptophos. Penetration rate, as estimated from the
amount of radioactivity present in the "external recovery" and "internal
recovery," was remarkably slow in both strains of flies. For example,
at a dosage of 10 |_ig/g the disappearance of radioactivity from the
external surface of the Rp strain was less than 5% after 1 hour. After
4 hours, 64.7% and 88.8% of the applied radioactivity were recovered
from the external surface of SfjAjn^ and Rp flies, respectively, and,
correspondingly, 30% and 107<> were recovered in the internal fraction.
78
-------
Proportionately lesser amounts of leptophos penetrated into
house flies treated at 500 |dg/g. This is shown graphically in
Fig. 2 where comparison is made between the rate of penetration
of leptophos into house flies treated at 10 and 500 |ig/g. The
figure clearly reveals a marked difference in the pentration rate
of leptophos into the two strains of house flies. Penetration into
the resistant Rparathion strain was abnormally slow and even after
6 hours approximately 85 and 95% of the applied dosages of 10 and
500 |_ig/g were recovered from the external surface of susceptible
and resistant flies, respectively. The unusually low rate of
penetration of leptophos into the Rp strain suggests that reduced
penetration is probably an important contributing factor to resistance
in this strain. Further, the generally poor penetrating ability of
leptophos into both strains is consistent with the observed delay
in onset of toxic symptoms and the relatively poor toxicity of
leptophos to house flies compared to parathion.
Results indicating the nature and quantity of the various metabolites
recovered from house flies 4 hours after treatment with C-phenoxy
leptophos are presented in Table 11. Data are given for susceptible
(SNAIDM^ flies treated at 10 jag/g and resistant (Rp) flies treated at
10 (R^) and 500 (R2> M,g/g. A brief explanation of the data under the
heading "Fly homogenate, water soluble" is appropriate since the
results under this column may be misunderstood. In the experimental
procedure for the extraction of metabolites present internally in the
fly, the methanol-acetone extract was concentrated to dryness, the
residual material was suspended in distilled water, and the mixture
was extracted twice with a benzene-hexane-chloroform mixture. This
extract was labeled the "organosoluble" fraction. The remaining
aqueous phase was acidified, placed in a boiling water bath for 15
minutes to hydrolyze conjugates, cooled and extracted with the same
organic solvent mixture. The material in this extract was labeled
"water soluble." Brief reiteration of the experimental procedure is
made here because of the substantial amounts of leptophos and leptophos-
oxon which were recovered after acid treatment. Since it is not
reasonable for these compounds to form conjugates, their retention
in the aqueous phase after extraction with organic solvent prior to
acid treatment is not readily accountable. Evidently, leptophos and
leptophos-oxon are tightly bound in some manner to house fly tissue
and treatment with dilute acid is required to release them for extrac-
tion by organic solvents.
Except for significant differences in the penetration rate, large
quantitative differences in the metabolism of leptophos between
susceptible and resistant house flies were not observed. At the lower
dosage of 10 |_ig/g almost 6-fold more radioactivity was detected
internally in susceptible than in resistant house flies. The data
in Table 11 show that leptophos is quite stable in both strains of
flies and a major portion of the radioactivity found internally in
the flies was the parent material. The data reveal that slightly more
leptophos-oxon was present in the internal fly homogenate of susceptible
79
-------
Table 11. Metabolic Products Recovered from S . and S ^>._ Houseflles 4 Ho-jra After Topical Application of C-Fher.oxy I-eot6?ho3
NAIL"
— paratnion -
at «* a** ,^ ^.^^
*I
K^IU* xuu a^ -
00 C6H5^ ^°\ /Br °'65 27'9 S
ciaae fix fJ-sss
g b ^c
.53 4.70
° \=^! (2.79) (0.55) (23.50)
C.H.^ ^O-// V-Br 0.25 • 1.33 0
65 \ /
^^ci • <°-13> <°
Cl
HO-^ ^-Br 0.39 - 2
^^l (0.
C,H ^ ^0-^ ^VBr 0.13 0.66 0
\=/ d (0.07) (0
U1 Total" 4.78 8
.56
.06)
.51
.25)
.23 0.42
.02) (2.10)
.77 5.12
5mi*
45.91
(4.59)
1.80
(0.18)
0.46
(0.05)
2.33
(0.23)
50.50
^
60.48
(6.05)
2.53
(0.25)
14.61
(1.46)
1.34
(0.13)
78.96
*z
75.48
(377.4)
1.35
(6.75)
1.72
(8.60)
2.15
(10.75)
80.70
toyms
10.55
(1.06)
0.42
(O.Ci)
0.30
(0.03)
0.41
(0.04)
11.68
In,tju>ial
) p o 1 u b 1 e
*ib J
4
(20.
0
(1
1
(5
0
(0
~1.0 5
£1* i-
^c
.00
,00)
.24
.20)
.03
•15)
.03
.15)
.3
LS.~t^
i!A.
21
(2
1
(0
4
(0
0
(°
27
ISFi&t.
Kntf
.80
.18)
.60
.16)
.08
.41)
.08
.01)
.55
'A
? j&ais!
hb
5.23
(0.52)
0.46
(0.05)
0.26
(0.03
0.05
(0.01)
6.0
US
^
4.35
(21.75)
1.53
(7.65)
1.00
(5.00)
1.C8
(5.40)
7.96
Parenthetical values represent amounts In Vg/g
7. Recovery.
94.57., Rt -.94.77., RZ - 99.17.
-------
oo
LJ
O
CC
UJ
60_
40_
20_
0
10 jjg/g
2 4
TIME (hr)
80_
60_
40_
20_
0
SOOyg/g
EXTERNAL
INTERNAL
Z 4
TIME (hr)
Figure 2. RATE OF PENETRATION OF LEPTOPHOS AT LOW AND HIGH DOSAGE
INTO S
NAIDM
(Q)
(©) HOUSE FLIES
-------
flies, but a larger amount of the oxon was present in the external
rinse of the resistant flies. The most striking difference between
the two strains, however, was the relatively large quantity of the
phenol in the external rinse of resistant flies. In general,
surprisingly large amounts of metabolites were found in the external
rinse and these materials probably were readsorbed on to the flies
through physical contact with fly excreta.
Rp flies treated with leptophos at 500 (j,g/g developed approximately
the same symptoms of intoxication as S^jj)^ flies treated at 10 |_ig/g.
At this higher dosage, the percentage of applied leptophos penetrating
into resistant flies was less than that for the susceptible strain
at the lower dosage but owing to the 50-fold more leptophos applied,
the actual quantity of penetrated material was substantially greater.
This is particularly significant with respect to leptophos-oxon since
this metabolite is undoubtedly the agent responsible for cholinesterase
inhibition and intoxication. For example, the actual amount of the
oxon detected in the homogenate of resistant flies treated with 500
ug/g was 0.177 [ig/fly while the amount detected in susceptible flies
treated with 10 jj.g/g was 0.004 (jg/fly, a difference of approximately
40 fold. Since the sensitivity of the cholinesterases from the two
strains of flies to inhibition by leptophos-oxon is virtually identi-
cal, the large difference in oxon levels in the two strains is in
strong contrast to the toxic symptoms observed.
Concurrent studies with C-phenyl labeled leptophos were conducted
with SNAIDM flies treated with 10 ug/g and Rp flies treated with 500
ug/g. The total amount of each metabolite recovered with this label
also are presented in Table 12. On the whole, the results are quite
similar to those obtained with the 1^C-p_henoxy_ label. With the label
in the phenyl ring the various expected derivatives of phenylphosphonic
acid were detected and the total percentage of these metabolites are
reasonably consistent with the percentage of phenol recovered when the
phenoxy label was used.
Conclusion - Overall, the results reveal that the pathways for the
metabolism of leptophos are similar in the white mouse, cotton plant,
and house flies. The various metabolic products which were obtained
are indicated in the scheme below. The pathways are similar to those
proposed for other related organophosphorus esters such as bromophos
and ronnel".
The formation of 0-methyl phenylphosphonothioic acid (VI) probably
occurs by oxidative dearylation or by esterase catalyzed hydrolysis
of leptophos, or a combination similar to that demonstrated with
other phosphorothionate insecticides ''. The desulfurated acid
VII may be formed by several possible mechanisms, (1) by direct
oxidative hydrolysis of (IV), (2) oxidative desulfuration of IV to
the oxon V followed by hydrolysis, or (3) by desulfuration of the
thionic acid VI. The oxidative hydrolysis of phosphorothionate esters
82
-------
,_/ S Cl
// \\
^s
i°xp^ °^IVr
Cl
leptophos (IV)
OH
(VI)
Cl
CH
_
°-
Cl
Icptophos-oxon (V)
CH
;0
OH
(VII)
Cl
+ HO
Cl
(IX)
conjugate
HO
•>o^V>
Cl
(VIII)
83
-------
Table 12. SUMMATION OF METABOLIC PRODUCTS RECOVERED FROM
SUSCEPTIBLE AND RESISTANT HOUSEFLIES AFTER
TOPICAL APPLICATION WITH RADIOLABELED LEPTOPHOS
14 u
C-Phenoxy D
leptophos f
IV 0.65
V 0.25
IX 0 . 39
X 0.13
Total
~ 'c-Phenyl leptophos
*,'
IV 0.89
V 0.78
VI 0.44
VII 0.27
VIII 0.10
X 0.62
Total
Per cent
SNAIDM
(10 ug/g)
81.05
(8.11)
5.15
(0.52
4.84
(0.48) '
3.48
(0.35)
94.52
91.63
(9.16)
5.62
(0.56)
2.49
(0.25)
2.34
(0.23)
2.32
(0.23)
1.08
(0.11
103.51
total applied
D
parathion
(10 ug/g)
74.24
(7.42)
3.08
(0.31)
14.50
(1.45)
2.80
(0.28)
94.62
,.
-
-
-
-
radioactivity3
parathion
(500 Mg/g)
88.53
(442.65)
3.12
(15.60)
3.75
(18.75)
3.68
(18.40)
99.08
84.24
(421.20)
4.13
(20.65)
0.81
(4.05)
7.06
(35.30)
0.48
(2.40)
0.36
(1.80)
97.08
a Parenthetical values represent amounts in .
" Benzene-chloroform (1:1) solvent. Acetdnitrile-water-ammonium
hydroxide (8:1.8:0.2)solvent.
84
-------
by microsomal oxidase enzymes to give directly the dialkyl phosphoric
acid has been demonstrated by Ify) studies with parathionlO,H.
Results with the phenoxy-labeled leptophos showed that 4-brorno-2,5-
dichlorophenol (or its conjugated form) is the sole degradation
product of this part of the leptophos molecule. No evidence was
obtained for any hydroxylated or dehalogenated products. Thus, the
overall metabolism of leptophos in mice is similar to that of parathion
and related insecticides, and no unexpected products were observed.
In general, leptophos is metabolized rapidly in the mouse, yielding
relatively nontoxic excretion products.
Leptophos evidently penetrates very slowly into the cotton leaf and
the term "alteration" is perhaps a better expression than "metabolism"
for its behavior on cotton plants. Separate experiments in this
laboratory have demonstrated that leptophos virtually has no systemic
movement when applied to the stem of cotton leaves, a finding which is
consistent with its poor penetration properties. It is difficult to
ascertain whether the alteration of leptophos occurs primarily on
the surface of the leaf by photochemical processes or within the leaf
by plant processes. The evidence indicates that both occur. Neverthe-
less, the principal component recoverable at all time intervals was
leptophos itself, the majority of which was present on the leaf
surface.
Penetration of leptophos into resistant house flies is markedly slower
than its penetration into susceptible flies. While the difference
can account for the tolerance of the resistant strain to low dosages
of leptophos, it cannot explain the ineffectiveness of leptophos at
high dosages, i.e., as the dosage approaches the LD^g level, since
substantially larger amounts of leptophos-oxon were recovered from
resistant flies treated at 500 fig/g. However, situations similar to
this have been observed before with other phosphorothionate insecti-
cides ' . For example, 10-fold more P(0) Phoxim was recovered from
resistant flies treated with 150 u£/g Phoxim than from susceptible
flies treated with 1.75 jag/g.
The question remains, by what mechanism can resistant house flies
tolerate such large amounts of leptophos-oxon? There are several
possible explanations. The desulfuration of leptophos to the oxon
may occur at a site which is quite remote from the critical lesion
and, therefore, the total amount of oxon found in the fly extract
does not represent the actual amount of oxon at the target site.
It is possible that leptophos or the oxon is partitioned into the fat
body of the fly which prevents it from being transported to the
target site. Support for this possibility is found in the apparent
difficulty encountered in the extraction by organic solvent of
leptophos and leptophos-oxon from the internal parts of the flies.
In this regard, more recent observations using neurophysiological
techniques have revealed that the time required to produce convulsions
85
-------
was slightly less in the susceptible than in the resistant house fly
topically treated with leptophos and leptophos-oxon.
3. Alteration of 0,0-Dimethyl S-[cy-(Carboethoxy)benzyl] Phosphoro-
dithioate (Phenthoate) in Citrus, Water, and Upon Exposure to Air
and Sunlight
Introduction - Phenthoate (XI) or 0,0-dimethyl S-[<-K- (carboethoxy)
benzyl] phosphorodithioate (Cidial®) is an effective broad spectrum
organopho sphorus insecticide of low mammalian toxicity. Its rat
oral LDcQ is reported to be 4728 mg/kgl\ a value which places it
approximately 950-fold less toxic to the rat than to the common house
fly, Musca domestica L., (LD5Q 5 mg/kg). Owing to the proven useful-
ness of phenthoate in controlling citrus scale insects, a study was
undertaken to determine the fate and environmental alteration of this
material in and on citrus trees. Also examined were the hydrolytic
breakdown of phenthoate in an aqueous environment and its photo-
chemical alteration upon exposure to air and sunlight. This section
summarizes the results obtained from this study.
CH3°\
/\ H
CH.,0 NSC-COOC0HC
j x 2 j
phenthoate
Synthesis of Radiolabels and Model Metabolites - Two different radio-
labels were used in this study,32p ancj ring-labeled C phenthoate.
32p phenthoate was prepared by reacting ^2p 0,0-dimethyl phosphorodi-
thioic acid (Amersham-Searle) with an equivalent amount of ethyl
crbromophenylacetate. Phenyl ring-labeled ^C phenthoate was obtained
from New England Nuclear Corp. (Boston). Each label was >9970 purity
as estimated by tic.
The following compounds were synthesized by unequivocal methods for
use as model compounds in the identification of the various metabolites
by cochromatography. The structure of all compounds was verified by
proton magnetic resonance spectroscopy (pmr), elemental analysis, and
in special cases by mass spectroscopy.
XII Phenthoate oxon, 0,0-dimethyl j5-[cr (carboethoxy)benzyl]
phosphorothioate.
XIII Demethyl phenthoate, potassium 0-methyl j3-[cr (carboethoxy)
benzyl phosphorodithioate.
86
-------
XIV Demethyl phenthoate oxon, sodium 0-methyl S-[cr (carbo-
ethoxy)benzyl] phosphorothioate.
XV Phenthoate acid, 0,0-dimethyl £}- [-[or
(carboxy)benzyl phosphorodithioate.
XVII Demethyl phenthoate oxon acid, sodium 0-methyl S-
[cr (carboxy)benzyl] phosphorothioate.
XVIII 0,0-Dimethyl phosphorodithioic acid.
XIX 0,0-Dimethyl phosphorothioic acid (sodium salt).
XX Ethyl mandelate.
XXII Bis-[pr (carboxy)benzyl] disulfide.
XXIII Bis-[cr (carboethoxy)benzyl] disulfide.
XXIV a~(Methylthio)phenylacetic acid.
Mandelic acid (XXI) was purchased from Aldrich Chemical Co.
Metabolism in Citrus - Selected leaves and fruit of a Valencia orange
tree growing in an experimental field plot on this campus were treated
by brush application with a solution of 30 mg phenthoate (^2p or ^C)
in 50 ml water containing 0.06% emulsifier. This concentration
approximated-a field application of \ Ib active ingredient per 100 gals
of spray solution. Samples were taken at 0, 3, 7, 10, and 14 days after
treatment and analyzed.
From the data for total recovery of radioactivity presented in Table
13, it appears that the major portion of phenthoate applied to orange
tree leaves is lost by volatilization. The data show a gradual decline
in total radioactivity for both labels and after 14 days a reduction
of 73% of applied ^C phenthoate and approximately 50% of applied 32P
phenthoate was observed. The rate of decline of total radioactivity
recovered from the leaves was greater for the ^C label than for the
32p label and the source for this difference is attributable to greater
loss of radioactivity from the surface of leaves treated with the ^C
label. It should be noted that the two labels were applied to the
orange trees at different times of the year, treatment of ^C phenthoate
was in March and 32p phenthoate was in September, and weather conditions
were quite different during these periods.
The amounts of radioactivity detected in the internal fractions, i.e.,
in the acetone extract and solid residue, were in closer agreement
87
-------
Table 13. THE PENETRATION OF C and P PHENTHOATE INTO ORANGE LEAVES
AND FRUIT, AND THE DISTRIBUTION OF RADIOACTIVITY IN THE
SURFACE WASH, ACETONE EXTRACT AND SOLID RESIDUE
.
Orange Leaves
14
C labeled phenthoate
External surface wash
Internal acetone extract
Solid residue
Total recovered
32
P labeled phenthoate
External surface wash
Internal acetone extract
Solid residue
Total recovered
Orange Fruit
14
C labeled phenthoate
External surface wash
Internal acetone extract
Solid residue1*
Total recovered
32
P labeled phenthoate
External surface wash
Internal acetone extract
Solid residue15
Total recovered
Percentage of the applied radioactivity
recovered at indicated time
0
96.7 (63.3)a
-
3.3 (2.2)
100. 0 (65.5)
94.4 (42.80)
. -
5.6 (2.54)
100.0 (45,34)
72.5 (2.14)
27.5 (0.81)
100.0 (2.95)
96.4 (1.62)
3.6 (6.06)
100.0 (1.68)
3 dy
41.8
2.4
16.9
61.1
79.5
9.5
4.5
93.5
66.1
5.4
21.7
93.2
63.4
5.9
19.4
88.7
7 dy
5.0
5.5
20.8
31.3
69.6
9.6
8.0
87.2
9.8
15.6
21.6
47.0
17.4
22.0
13.9
53.3
10 dy
2.1
2.6
15.5
20.2
49.0
6.3
2.5
57.8
8.1
12.8
34.7
55.6
13.6
24.2
16.3
54.1
14 dy
3.0
1.8
22.2
27.0
29.0
8.8
13.0
50.8
4.6
7.8
28.3
40.7
11.6
18.2
13.2
43.0
( ) is the amount of phenthoate in ppm wet weight of leaves or fruit.
Includes peel only. Radioactivity in pulp was less than 1% of the applied.
88
-------
although amounts found in the acetone extracts were consistently larger
for the P label while lesser amounts of this label were found in
the solid residue.
Analogous data for the recovery of phenthoate radioactivity from
orange fruit are presented in Table 14. Significant loss in total
recovery also was evident with the fruit but in this case the
rates of decline of radioactivity for the two labels were in
reasonably close agreement. Noteworthy is the observation that less
than l~7, of the applied radioactivity was detected in the orange pulp
for both *••+€ and 3-P labels. Control experiments were conducted to
show that very little radioactivity was lost during the work-up.
The various phenthoate metabolites observed in the surface wash and
acetone extracts of orange leaves are listed in Tables 15 (^C label)
and 16 (-*-p label). Qualitatively, the C metabolites isolated from
these two fractions are the same. The major compound isolated was
unchanged phenthoate (XI). Metabolites present in varying quantities
were phenthoate-oxon (XII), demethyl phenthoate (XIII), mandelic acid
I.XXI), and bis-fa~ (carboethoxy) benzyl] disulfide (XXIII). Recovered
from the 32p label were XI, XII, 0,0-dimethyl phosphorodithioic adid
(XYIII) and 0,0-dimethyl phosphorothioic acid (XIX). The detection of
XVIII and XIX from the 32P label and XXI and XXIII from the 14C label
is consistent with the products expected from the hydrolytic degra-
dation of phenthoate. One unknown of very minor significance was
isolated from the ^C label and two unknowns from the 32 label, both
of which were present in the surface wash.
Tables 16 and 17 provide similar data for the metabolism of C and ^2p
phenthoate in orange fruit. Again, as in the case of leaves, unchanged
XI was the major product recovered, followed by XII in order of
importance. The various other metabolites which were present in
orange leaves also were found in fruit.
Alteration in Water - The behavior of phenthoate in water was quite
complex and the identity and amounts of the various products isolated
after allowing *^C phenthoate to stand in 0.01 M phosphate buffer at
pH 6, 7. and 8 for intervals of up to 28 days are presented in
Table IS. Evidently, phenthoate is fairly stable in water at the
indicated pH values. Decomposition appeared to be fastest at pH 8
and the rate of decomposition was first-order with respect to phenthoate.
At this pH, the calculated half-life of phenthoate was approximately 12
days.
Of the various hydrolytic products, phenthoate acid (XV) was observed
in greatest abundance, irrespective of pH, indicating that the carbo-
ethoxy moiety was the most vulnerable part of the molecule to hydro-
lytic decomposition. The rate of formation of XV was significantly
slower at pH 6 and, in spite of the obvious experimental variation in
89
-------
Table 14. AMOUNT OF C METABOLITES FOUND IN THE SURFACE WASH ANT)
ACETONE EXTRACT OF ORANGE LEAVES AFTER INDICATED TIME INTERVALS
Compound found in
indicated material
Phenthoate (XI)
Surface wash
Acetone extract
Phenthoate oxor. (XII)
Surface wash
Acetone extract
CH 0 /S
/< H ,0 (XIII)
HO s£-C^
Surface wash
Acetone extract
HOC-C^0 (XXI)
Surface wash
Acetone extract
*V\\ OC^5 (xxin)
Surface wash
Acetone extract
Unknown I
Surface wash
Acetone extract
Totjl
Percent of recovered radioactivity
after indicated time
3 dy
84.4
4.6
0
0.2
0
0
0
0.6
10.2
0
0
0
100
7 dy
45.5
46.1
0
0
0.2
0
0.1
6.2
1.5
0
0.4
0
100
10 dy
35.4
37.7
2.8
7.2
0.9
4.0
1.3
6.4
4.3
0
0
0
100
14 dy
41.6
17.0
5.1
12.4
2.C
2.5
3.6
5.7
9.7
0
0.4
0
100
90
-------
Table 15. AMOUNT OF 32P METABOLITES FOUND IN THE SURFACE WASH AND
ACETONE EXTRACT OF ORANGE LEAVES AT THE INDICATED TIME
Compound found in
indicated material
Phenthoate (XI)
Surface wash
Acetone extract
Phenthoate oxon (XII)
Surface wash
Acetone extract
CH 0 /S
^\ H n (XIII>
HO NS£-C*
Surface wash
Acetone extract
CH3°xs
Surface wash
Acetone extract
CH-0V ,S
3 y* (XIX)
CH30 OH
Surface wash
Acetone extract
Unknown I
Surface wash
Acetone extract
Unknown II
Surface wash
Acetone extract
Total
Percent of recovered material
at the indicated time
3 dy
63.4
9.4
12.8
0.9
2.4
0
7.2
0.4
0
0
3.5
0
0
0
100
7 dy
39.1
10.3
34.4
1.0
4.1
0
4.1
0.8
0
0
1.1
0
i.l
0
100
10 dy
52.1
7.2
22.8
2.6
4.6
0
0
0
4.2
1.6
4.9
0
0
0
100
14 dy
43.5
14.9
17.6
4.5
4.2
0
4.5
3.4
3.4
0.5
3.5
0
0
0
100
91
-------
Table 16. AMOUNT OF C METABOLITES FOUND IN THE SURFACE WASH AND
ACETONE EXTRACT OF ORANGE PEELS AT THE INDICATED TIME
Compound found in
indicated material
Phenthoate (XI)
Surface wash
Acetone extract
Phenthoate oxon (XII)
Surface wash
Acetone extract
vrf ^er> r-1^ fYTT"O
liU olj"*U V"^^"*-/
Surface wash
Acetonr. extract
it
Y\\°*
Surface wash
Acetone extract
2[»^o
Surface wash
Acetone extract
Unknown I
Surface wash
Acetone extract
Total
Percent of recovered radioactivity
at indicated time
3 dy
90.3
7. A
0
0.2
0
0
0
0
2.1
0
0
0
100
7 dy
31.3
52.5
0
2.0
0.7
5.8
0.8
1.5
5.2
0
0.2
0
100
10 dy
37.9
5A.7
0
2.2
0.3
2.3
0
2.1
0.5
0
0
0
100
1A dy
35.6
58.2
0.5
2.2
0.2
0
0.2
2.3
0.7
0
0.1
0
100
92
-------
Table 17. AMOUNT OF 32P METABOLITES FOUND IN THE SURFACE WASH AND
ACETONE EXTRACT OF ORANGE PEELS AT THE INDICATED TIME.
Compound found in
indicated material
Phenthoate (XI)
Surface wash
Acetone extract
Phenthoate oxon (XII)
Surface wash
Acetone extract
Surface wash
Acetone extract
S\ (XVIII)
r*ti A ^ou
\sii-\j on
Surface wash
Acetone extract
CH3VS
x"v (XIX)
CH30 OH
Surface wash
Acetone extract
Unknown I
Surface wash
Acetone extract
Unknown II
Surface wash
Acetone extract
Total
Percent of recovered material
at the indicated time
3 dy
74.0
8.5
12.7
0
0
0
0
0
0
0
4.8
0
0
0
100
7 dy
33.3
39.0
6.1
9.5
0
7.4
1.7
0
2.3
0
0.7
0
0
0
100
10 dy
24.6
42.9
6.3
5.3
0
0
1.5
12.2
0
3.6
2.2
0
1.4
0
100
14 dy
23.7
22.1
7.8
4.0
0
0
2.9
13.2
3.0
6.5
1.6
15.2
0
0
100
93
-------
Table 18. THE BREAKDOWN PRODUCTS OF 14C LABELED PHENTHOATE IN 0.01 M SODIUM PHOSPHATE
BUFFER AT pH 6, 7, and 8 AFTER THE INDICATED TIME. (TEMPERATURE 24.5°C)
Compound
Phenthoate 'XI
•
CH30S ,S XIII
Sf\ H ,0
m s£-c^"
CH30N ^0 XIV
HO SC-C^°
6x"s
CH3Ox XS XV
3 rff°H
Combined compounds
XVI and XVII
TT -»
HOC-C^ XX
f^T\ OC?Hs
_xO VVT
HOC— C'** AAJL
^T^^OH -
kJ
PH
6
7
8
6
7
8
6
7
8
6
7
8
6
7
8
6
7
8
6
7
8
0.25
98.9
97.2
-
0
0
0
0
0
0.5
0
0
—
0
0
-
0
0
-
Percent of
dy 0.5 dy
96.9
95.1
92.0
0
0.3
0.6
0
0.6
2.2
0.8
0.5
3.5
0
0
0
0
0.3
0
0
0
0
recovered radioactivity after indicated tiir.e
1 dy 2 dy 3 dy 7 dy 14 dy 21 dy 28 dy
96.6 95.9 98.7 95.7 55.6 57.0 45.1
97.7 91.6 83.6 75.6 26.7 44.3 21.3
89.2 84.0 81.6 39.5 54.5 31.6 22.2
0 0.7 0.3 0.6 10.6 15.7 18.6
0.2 0.8 4.7 2.8 7.1 7.3 4.1
0.4 0.3 0 0 2.0 3.2 7.4
0 1.9 0.5 1.8 4.9 0.9 1.0
0 0 4.9 3.6 13.1 9.0 7.3
1.0 1.1 5.5 16.1 7.7 9.5 11.2
1.7 0.7 0.5 1.2 28.9 22.7 30.4
0.5 2.1 6.8 16.7 46.5 35.7 55.8
5.1 9.8 12.9 31.9 32.6 46.5 50.3
00000 2.3 3.3
0 0 0 0 2.7 1.3 4.2
00000 1.6 1.6
00000 0 0
0.2 0 0 0 0 0.7 0
00000 2.3 0.7
00000 0 0
0.3 1.7 0 0 0 0 0
0000 1.4 2.1 1.3
94
-------
Table 18. continued.
Compound
[SC-C^0 XXIII
pf0'2"5 '»
-§c -.
Unknown I
Unknown II
Unknown III
Unknown IV
PH
6
7
8
6
7
8
Percent of
0.25 dy 0.5 dy
1.1 2.3
2.3 3.3
1.6
0 0
0 0
0
recovered
1 dy
1.7
1.1
4.2
0
0
0
2
0
3
4
0
0
0
radioactivity
dy 3 dy
.8 0
.8 0
.2 0
0
0
0
7 dy
0
0
0
0.7
0
12.6
after
14 dy
0
0
0
0
1.9
1.8
indicated
21 dy
0
0
0
1.4
0.5
3.0
time
28 dy
0
0
0
1.6
0
1.1
0 0
0 0
0 0
0 0
0
0
0
0
0
0
0
0
0
0
0
0
1.4a
0
0
0
0.73
1.2a
0
0
1.33
0
0
f
0
2.13
0
2.7b
5'2b
1.4b
unknown found at pH 7.0.
Unknown found at pH 8.0.
95
-------
the data, hydrolysis of the carboethoxy moiety appeared to be fastest
at pH 8.0. On the other hand, the rate of formation of demethyl
phenthoate (XIII), the second most prominent hydrolysis product,
was fastest at pH 6.
Significant amounts of demethyl phenthoate oxon (XIV) were detected,
particularly at pH 7 and 8. Since at no time was any phenthoate oxon
(XII) noted, it is most likely that XIV was formed from XIII, possibly
by hydrolytic desulfuration. Other products observed in small amounts
were mandelic acid (XXI), bis-[or(carboethoxy)benzyl] disulfide (XXIII),
or(methylthio)phenylacetic acid (XXIV), demethyl phenthoate acid (XVI)
and demethyl phenthoate oxon acid (XVII). Several unknowns of minor
significance also were detected, particularly after standing for long
time intervals.
32
A single study of the hydrolysis of P phenthoate was conducted at pH
8. The products from the -^P label largely supported results from the
C^ label. XV was confirmed as the principal hydrolytic product,
followed by XIII and XIV. Other products included XVI, XVII, 0,0-
dimethyl phosphorodithioic acid (XVIII), and 0,0-dimethyl phosphoro-
thioic acid (XIX).
14 32
Photodegradation - The photoalteration of C and P phenthoate in
sunlight under atmospheric conditions was examined as thin films on
glass plates. Air temperatures during the exposure period ranged
from 23.3° to 7.2°C (typical day in March). Under these conditions
phenthoate was quite volatile and approximately 90% of the applied
material was lost after 40 hours of exposure. The high rate of
loss of phenthoate from a glass surface adds support to an earlier
conclusion that the principal route for the loss of phenthoate from
orange leaves and fruit was through volatilization.
The products recovered from the glass plates for both labels are given
in Table 19. The principal constituent present in the residue was un-
changed phenthoate which gradually decreased in proportion to the other
alteration products during the period of exposure. Phenthoate oxon
(XII) also was a significant product and the material increased in
amount as time progressed. Thus, after 35-hr exposure to sunlight,
19% and 28% of the residual material consisted of XII derived from
14-c and 32p phenthoate, respectively. Other products isolated were
demethyl phenthoate (XIII) with both labels; mandelic acid (XXI),
bis~rg~(carboxy)benzyl] disulfide (XXII), and bis-[cr(carboethoxy)benzyl]
disulfide (XXIII) from the 1^C label; and 0,0-dimethyl phosphorothioic
acid XIX from the 32P label.
Conclusion - Based on this study, the proposed pathways for the alter-
ation of phenthoate in orange leaves and fruit, in water, and upon
exposure to sunlight and the atmosphere are presented in Figure 3.
In gross aspects, the alteration scheme is similar to those reported
for phenthoate metabolism in cabbage, apples, and strawberries^.
96
-------
Table 19. THE METABOLITES OF l**C and 32P PHENTHOATE APPLIED
TO THE SURFACE OF GLASS PLATES AND EXPOSED TO SUNLIGHT
Compound found at
indicated time
14
C phenthoate
Phenthoate (XI)
Phenthoate oxon (XII)
CH Ox ^S
V fVTTT\
*\ u f\ \J\LLJ*)
HO SC-C^
H ,0
Ho-c-c'
X>\\)H (XXI)
H /\
^^^^ rw"1 U ^YYTTT\
(•V*^ T UC~n,. ^AAlllJ
II 25
IcsJ
Total
32
P phenthoate
Phenthoate (XI)
Phenthoate oxon (XII)
CH3%*SH (XIII)
HOX XSC-C^°
'CH o xs
, F (XIX)
CH 0^ OH
i -J
Unknown
Total
Percent of recovered radioactivity
after indicated time of exposure
7 hr
92.2
3.7
2.1
0
0
2.0
100
84.5
13.4
0
2.1
0
00
14 hr
84.4
8.8
2.7
0.6
0.8
2.7
100
76.8
15.2
3.0
'• .
5.0
0
100
21 hr
89.5
5.3
1.5
0.7
1.5
1.5
100
61.7
30.0
3.1
5.2
0
100
28 hr
76.9
11.7-
4.3
1.0
2.0
4.1
100
51.1
29.2
5.2
14.5
0
100
35 hr
71.7
19.8
1.9
2.2
2.5
1.9
100
51.1
28.0
10.6
10.3
0
100
42 hr
35.5
35.4
6.3
6.1
16.7
100
97
-------
CH-SC-C^
H x,
HO£-C^
"
(0
(XXII)
CH
/ NSH
(XVIII)
(XX)
CH_
(a) (c)
(c)
CH
CH,
(XXIII)
:H3os /s
m3ox XOH
(XIX)
ciLsc-c
(b)
(XII)
(XI)\(a)(b)(c)
CH,
CH30
jO
"OH
(b)
HO
(b)
(XIII)
CH30
'
HO
OH
(b)
(XVII)
(XIV)
x0
HSC-C-
-^ CH,
OH
(XV)
(b)
v(b)
(XVI)
Fig. 3. Proposed alteration pathways for phenthoate (a) in citrus, (b) in
s-
buffered water, and (c) after exposure to sunlight. * denotes hypothetical
intermediate that is not isolated.
98
-------
Also, the proposed scheme is similar in many respects to that reported
for the metabolism of dimethoate [0,0-dimethyl ^-(N-methylcarbamoyl-
methyl) phosphorodithioate], a compound of closely related structure,
in a variety of plants.
As indicated in the scheme, the initial step in the degradation of
phenthoate is hydrolytic in nature. The various places in the mole-
cule which are vulnerable to hydrolytic cleavage are indicated (below)
by the dotted lines. Evidently, reactions involving each of these
bonds take place, giving rise to the complex array of products.
.,.
2 5
Oxidative reactions also occurred, as evident by the formation of
phenthoate oxon (II) by desulfuration and the coupling reaction
leading to the disulfides XII and XIII.
According to the scheme the alteration products of phenthoate in
citrus and upon exposure to sunlight and air on glass plates are
essentially the same. Thus, it appears that the formation of the
various products recovered from the surface of orange leaves and
fruit is attributable to environmental effects. Products found
internally in citrus are probably the result of both biological and
nonbiological transformation since alteration products generated
on the surface undoubtedly penetrate into the leaves or the fruit.
It has been speculated that oxidants in air pollution may have a
significant effect on the alteration of pesticides in the environment.
Therefore, there is a possibility that some of the discrepancies
observed in this study, particularly in citrus and on glass plates,
may be accountable by effects from photochemical smog. For example,
the relative amounts of phenthoate oxon which were recovered from
citrus (surface wash) and glass plates were substantially greater
for the 32P than for the ^C label. The studies with the P label
were conducted in August-September, a period when air pollution levels
are high, while the C studies were conducted during months (March-
April) which are relatively free of air pollution. While definite
conclusions cannot be reached from this study owing to the absence of
proper controls, the results cited above suggest that oxidants present
in air pollution contribute to the oxidative desulfuration reaction.
99
-------
Under near neutral pH conditions the most susceptible linkage in the
phenthoate molecule to hydrolysis appears to reside in the carbo-
ethoxy moiety and not with linkage associated with the phosphorus
atom. Although comparable studies under similar conditions have not
been conducted with malathion (diethyl mercaptosuccinate S-ester with
0,0-dimethyl phosphorodithioate) and other organophosphorus insecti-
cides which contain carbonyl ester moieties, under strongly basic
or acid conditions malathion has been reported to hydrolyze to
0,0-dimethyl phosphorodithioic acid and diethyl fumarate, and to 0,0-
dimethyl phosphorothioic acid and diethyl 2-mercaptosuccinate,
respectively . Thus, malathion undergoes C-S bond cleavage in base,
probably through a p-elimination reaction, and P-S bond cleavage in
acid. While direct comparison with phenthoate cannot be made owing
to the different conditions employed, the two compounds appear to
differ in the relative susceptibility of their respective carboethoxy
moieties to hydrolytic attack.
In general, the various alteration products which were isolated and
identified in this study represent compounds which would normally be
anticipated from the structure of phenthoate. With the exception of
phenthoate oxon, all of the alteration products represent materials
which may be regarded as detoxication products. Because of its very
low acute mammalian toxicity, high susceptibility to environmental
degradation, and general effectiveness against a variety of insect
pests, phenthoate should prove to be a useful insecticide of minimum
threat to public health and the environment.
4. References
1. Udenfriend, S., C. I. Clark, J. Axelrod, and B. B. Brodie. Ascorbic
Acid in Aromatic Hydroxylation. I. A Model System for Aromatic
Hydroxylation. J. Biol. Chem. 208:731-9 (1954).
2. Quistad, G. B., T. R. Fukuto, and R. L. Metcalf. Insecticidal
Anticholinesterase, and Hydrolytic Properties of Phosphoramidothiolates.
J. Agr. Food Chem. 18:191-4 (1970).
3. Abou-Donia, M. B., M. A. Othman, G. Tantawy, A. Zaki Khalil, and
M. F. Shawer. Neurotoxic Effect of Leptophos. Experientia 30:63-5
(1974).
4. Shea, K. P. Nerve Damage. Environment 16:6-10 (1974).
5. Stiasni, M., D. Rehbinder, and W. Deckers. Absorption, Distribution,
and Metabolism of 0-(4-Bromo-2,5-Dichlorophenyl) 0,0-Dimethyl Phosphoro-
thioate in the Rat. J. Agr. Food Chem. 15:474-8 (1967).
100
-------
6. Plapp, F. W. and J. E. Casida. Bovine Metabolism of Organo-
phosphorus Insecticides: Metabolic Fate of C),0-Dimethyl C)-(2,4,5-
Trichlorophenyl) Phosphorothioate in Rats and a Cow. J_. Agr. Food
Chem. 6:662-7 (1958).
7. Yang, R. S. H., E. Hodgson, and W. C. Dauterman. Metabolism In
Vivo of Diazinon and Diazoxon in Rat Liver. .J. Agr. Food Chem. 19:
10-13 (1971).
8. Matsumura, F. and C. J. Hogendijk. The Enzymatic Degradation of
Parathion in Organophosphate-Susceptible and -Resistant Houseflies.
J. Agr. Food Chem. 12:447-53 (1964).
9. Nakatsugawa, T., N. M. Tolman, and P. A. Dahm. Degradation of
Parathion in the Rat. Biochem. Phartnacol. 18:1103-14 (1969).
10. Ptashne, K. A., R. M. Wolcott, and R. A. Neal. Oxygen-18 Studies
on the Chemical Mechanisms of the Mixed Function Oxidase Catalyzed
Desulfuration and Dearylation Reactions of Parathion, J. Pharmacol.
Exp. Ther. 179:380-5 (1971).
11. McBain, J. B., I. Yamamoto, and J. E. Casida. Mechanism of Acti-
vation and Reactivation of Dyfonate® (0-Ethyl S-Phenyl Ethylphosphono-
dithioate) by Rat Liver Microsomes. Life Sciences 10:947-54 (1971).
12. Hollingworth, R. M., T. R. Fukuto, and R. L. Metcalf. Selectivity
of Sumithion Compared with Methyl Parathion. Metabolism in Susceptible
and Resistant Houseflies. J. Agr. Food Chem. 15:250-5 (1967).
13. Vinopal, J. H., and T. R. Fukuto. Selective Toxicity of Phoxim
(Phenylglyoxylonitrile Oxime 0,0-Diethyl Phosphorothioate). Pestic.
Biochem. Physiol. 1:44-60 (1971).
14. Pellegrini, G., and R. Santi. The Potentiation of Toxicity of
Organophosphorus Compounds Containing Carboxylic Ester Functions
Toward Warm-Blooded Animals by Some Organophosphorus Impurities. J. Agr.
Food Chem. 20:944-50 (1972).
15. Hirose, M., I. Miyata, T. Saito, and M. Hayoshi. Residue, Degra-
dation and Metabolism of ^C Labeled Elsan® 0,0-Dimethyl £-[cv-(Carbo-
ethoxy)benzyl] Phosphorodithioate in Cabbage, Hime-apples and Straw-
berries. Botyu-Kagaku 36:43-51 (1971).
16. Dauterman, W. C., G. V. Viado, J. E. Casida, and R. D. O'Brien.
Persistence of Dimethoate and Metabolites Following Foliar Application
to Plants. J. Agr. Food Chem. 8:115-9 (1960).
17. Crosby, D. G. The Non-Metabolic Decomposition of Pesticides.
Ann. N.Y. Acad. Sci. 160:82-95 (1969).
101
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18. Bender, M. E. The Effect of Malathion on Fishes. Ph.D.
Dissertation. Dept. of Environ. Sci., Rutgers University, N.J. (1968)
102
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D. BASIC STUDIES ON THE INHIBITION OF THE CHOLINESTERASE ENZYMES
A number of investigations were completed on the basic aspects of the
inhibition of cholinesterase enzymes derived from mammalian and insect
sources. These studies were aimed at the elucidation of the fundamental
mechanisms involved in cholinesterase inhibition and reactivation
involving carbamate and organophosphorus esters.
1« The Reactivation of Carbamate Inhibited Cholinesterase
Introduction - The inactivation of acetylcholinesterase (AChE) by esters
of methyl- or dimethyIcarbamic acid has been demonstrated beyond reason-
able doubt to be the result of an actual chemical reaction between the
enzyme and ester as shown by the equation below.
ko
El > E + P
IX is the carbamate ester, E is free AChE, X is the leaving group, EIX
is the reversible complex, El is carbamylated enzyme, and P is alkyl-
carbamic acid.
During the assay of AChE activity in vitro the primary form of inhibited
enzyme is most probably the carbamylated enzyme which is responsible
for the eventual return of enzymatic activity after the effect of the
residual carbamate has been minimized or removed by dilution or gel
filtration.
Previous studies on the spontaneous reactivation of AChE inhibited by
carbamate esters were carried out by means of dilution techniques and
in the absence of the substrate acetylcholine (or acetylthiocholine).-'-
In the present study, the carbamylated AChE was separated from excess
inhibitor by Sephadex gel filtration and decarbamylation of the
inhibited enzyme was determined in the presence of the substrate
acetylthiocholine. The effect of different conditions of pH, salt
concentrations and temperature were determined in an attempt to
elucidate the mechanism of the decarbamylation process.
Carbamate Inhibitors - The carbamates used are indicated as follows:
3-isopropylphenyl methylcarbamate (I), 3-isopropyl-4-nitrophenyl
dimethylcarbamate (II), ring-labeled tritiated carbofuran (III) and
carbonyl-labeled ^C Banol (IV) (6-chloro-3,4-xylyl methylcarbamate).
103
-------
Kinetic Procedure - After the residual carbamate was removed from the
AChE by Sephadex gel filtration, an aliquot was added to the reaction
cuvette and hydrolysis of the substrate was monitored according to the
procedure of Ellman ej: al. at 412 m^. The k_ step indicates that
decarbamylation will result in a first-order increase in AChE (or a
decrease in carbamylated enzyme). Since the amount of AChE present
in successive time intervals is linearly related to the change in
absorbance at 412 mu., it follows that as AChE concentration increases
substrate hydrolysis should accelerate and the plot of absorbance versus
time should have a positive curvature. The plot would have the form
of an integrated first-order equation, i.e.,
y = At - A/k(l - e"kt), (2)
where y is absorbance at time t, A is the rate when all the enzyme is in
the uninhibited form, and k is equal to the constant of acceleration
or the regeneration rate constant. The value of A, the limiting
velocity for ATCh hydrolysis, was estimated from the absorbance versus
time plot after the curvilinear line became linear. In all experiments
the regeneration reaction was followed until this relationship was linear
with time, i.e., when no acceleration component in the curve was
detectable.
The data also may be fitted to the following multicompartmental mathe-
matical model and this model was employed in this study to obtain the
decarbamylation constant (k ), which is identical to k in Eq. (2).
k ,
(D) - 3 - > (E)
(A)
V (3)
In this model, compartment (A) is inhibited enzyme, (B) is free enzyme,
(C) and (E) are the coupled substrated reactions, (D) is free enzyme at
t = 0, and (F) is optical density. The appropriate rate constant for
regeneration of free enzyme is k , while the k and k , are indicative
of the reaction of this substrate (ATCh) and tne k and k , are charcter-
istic of the reaction of this substrate hydrolysisSproducc and the
reagent DTNB. The data fitting to the model was done utilizing the
systems analysis and modeling program of Berman and Weiss-^*^. In this
least- squares program, upper and lower limits for k were set by
employing values selected from previous studies, e.g., Reiner and
104
-------
Aldridge . The values so chosen were 0.001 min and 0.300 rain
for the lower and upper limits, respectively, for both carbamate
derivatives of AChE.
Sephadex Gel Filtration Using Radioisotopes - Since this was a study
of the regeneration reaction (k-j or kr), it was of fundamental import-
ance to be sure that the carbamylated enzyme was being isolated from
noncovalently bound carbamate. The crucial point, therefore, was to
determine the proper functioning of the Sephadex column. The first
control experiment was to ascertain the volume that would precede
the protein fraction when a 1.0-ml sample was applied. The position
of the protein was readily determined by the addition of the dye,
Dextran blue, which is excluded from all Sephadex gels and, therefore,
flows with the protein. The enzymatic activity of this fraction was
assayed by withdrawing an aliquot and adding it to a reaction mixture.
One-milliliter samples were collected starting at the first appearance
of blue color, the color continuing for four successive milliliters
of eluant. The majority of the activity occurred in the first few
milliliters after appearance of the color. This experiment indicates
two important points. First, the passage through the column apparently
does not alter the activity of the enzyme and second, the enzymatic
activity is associated with the Dextran blue fraction, as was expected.
The enzymatic scheme (Eq. 1) indicates that the leaving group (X) should
be lost from the enzyme during the inhibition process. This was checked
by using carbonyl-labeled ^C Banol (IV) and ring-labeled 4,5,6-H^-
carbofuran (III). When Banol was used as an inhibitor, radioactivity
was associated with the enzyme fraction; however, when carbofuran was
used, no radioactivity appeared in the enzyme fraction. This provided
evidence that the carbamylated enzyme was being isolated.
pH-Rate Dependence - The accepted role of carbamates as alternate sub-
strates for AChE predicts that the decarbamylation reaction might have
the same pH-rate profile as noninhibited substrate hydrolysis. The
substrate here, acetylthiocholine (ATCh), has been shown to have a
pH-rate profile which gives a plateau at high pH in contrast to the bell-
shape curve found in ACh hydrolysis. The plateau was not observed in this
study.
The pH-rate profiles for decarbamylation of a methyl- and dimethylcarbamyl
bovine erythrocyte AChE inhibited by 3-isopropylphenyl methylcarbamate
(I) and 3-isopropyl-4-nitrophenyl dimethylcarbamate (II) appear in Figs.
1 and 2 respectively. The maximum rate is found between pH 8.6 and 8.9
for both of the figures. The corresponding regeneration of methyl- and
dimethylcarbamyl derivatives of fly-head AChE appear in Figs. 3 and 4
respectively. In this case the methylcarbamyl derivative has a maxima
between pH 8.0 and 8.5 and the dimethylcarbamyl enzyme at pH 8.3-8.8.
It may be noted that the regeneration rate constant for the methyl-
carbamyl AChE has a more precipitous decline with increasing pH for
both the bovine and house fly enzyme than do the dimethylcarbamyl
AChE derivatives.
105
-------
0.03
J 0.02
i
"E
0.01
7.0
o o
3.0
pH
9.0
10.0
Figure 1, EFFECT OF pH ON THE DECARBAMYLATION HATE CONSTANT OF BOVINE ERYTHROCYTE AChE INHIBITED BY I,
-------
0.02
0.01
oo
7.0
8.0
9.0
PH
10.0
Figure 2.
EFFECT OF pH ON THE DECARBAMYLATION RATE. CONSTANT OF BOVINE
ERYTHROCYTE ACRE INHIBITED BY II
-------
Q
00
0.03
0.02
0.01
7.0
8.0
9.0
pH
10.0
Figure 3. EFFECT OF pH ON THE DECARBAMYLATION RATE CONSTANT FOR HOUSE FLY-HEAD AChE
INHIBITED BY I
-------
0.01
7.0
8.0
PH
9.0
10.0
Figure 4V EFFECT OF pHL ON THE DECAR£AMYLATI.ON RATE CONSTANT FOR HOUSE FLY^HEAD
AChE IHIBITED BY II
-------
Activation Parameters - Data showing the effect of temperature on
the rate constant for regeneration of house fly and bovine erythrocyte
AChE inhibited by 3-isopropylphenyl methylcarbamate (I) and 3-iso-
propyl-4-nitrophenyl dimethylcarbamate (II) are presented in Table 1.
Included in the table are the values for the Arrhenius activation
energy (E ) calculated by regression analysis of a plot of log kr vs
1/T (°K), and values for the entropy of activation (AS±). Satisfactory
Arrhenius plots were obtained with correlation coefficients ranging
from 0.94 to 0.99.
Values for the regeneration constants (kr) show that the rate of re-
generation of house fly AChE inhibited by I or II is about twice as
fast as the corresponding bovine erythrocyte enzyme (compare rate
constants at 26.0°C). The values of kr (min"1) for the spontaneous
recovery of the methylcarbamyl and dimethylcarbamyl bovine AChE at 26°C
are 0.020 and 0.012, respectively, in good agreement with the values
of 0.0234 and 0.0123 (25°C) reported by Reiner and Aldridge1 for the
same enzyme, although determined by a different procedure.
The magnitude of E_ and AS± for the regeneration of house fly and bovine
AChE reveal distinct differences between the two enzymes. Ea for the
regeneration of carbamylated house fly AChE is substantially greater
than that for bovine AChE, in spite of the faster rate of decarbamylation
by the former. Evidently, the larger activation energy for house fly
AChE is compensated by favorable entropy effects.
The activation energies for decarbamylation of house fly and bovine AChE
are larger than the 14 kcal/mole observed for the alkaline hydrolysis
of £-nitrophenyl methylcarbamate6, but they approach those reported for
the alkaline hydrolysis of ethyl methylcarbamate and dimethylcarbamate •
This is consistent with the general belief that carbamylation, acylation,
and phosphorylation takes place on an aliphatic hydroxyl moiety in the
active site, i.e., the serine hydroxyl.
Salt Effects - The primary and secondary salt effects on reaction rates
have been well investigated. More recently Bruice et al. , have in-
vestigated the structure-forming properties of different salts on
aqueous transition states. While the interpretation of primary or
secondary salt effects could lend information regarding the mechanism
of decarbamylation, pronounced salt effects were not found in this
study. There was probably a secondary nonspecific salt effect which
is consistent with acid-base catalysis, but this is to be expected.
Deuterium Oxide Effect - Deuterium oxide has been employed previously
in the differentiation of general base and nucleophilic catalysis of
hydrolysis. The values of kH/kD are generally less than 2.0 for
nucleophilic catalysis due to the secondary isotope effect. Deuterium
oxide was used as the sole solvent in the experiments cited in Table 2.
Inhibitors 3-isopropylphenyl methylcarbamate (I) and 3-isopropyl-4-
nitrophenyl dimethylcarbamate (II) were used to carbamylate bovine
' 110
-------
Table 1. RATE CONSTANTS (k ) AND ACTIVATION PARAMETERS FOR THE REGENERATION OF INHIBITED
CHOLINESTERASE IY 3-ISOPROPYLPHENYL METHYLCARBAMATE AND 3-NITROPHEHYL
DIMETHYLCARBAMATE AT pH 7.9
Enzyme
Fly
Fly
Bovine
Bovine
Carbamate
I 3-isopropylphenyl
me thy 1 carbamat e
II 3-isopropyl-4-
nitrophenyl
dime thy 1 car bamate
I 3-isopropylphenyl
methylcarbamate
II 3-isopropyl-4-
nitrophenyl
dimethylcarbamate
T(°C)
22.0
26.0
29.5
32.5
18.5
22.0
26.0
29.5
32.5
18.5
22.0
26.0
33.0
18.5
26.0
33.0
kr (min"1) Ea (Kcal/mole) AS± (e.u.)
0.0258 25.7 11.3
0.0465
0.104
0.105
0.00735 27.9 17.2
0.0135
0.0225
0.0400
0.0707
0.00539, 0.00583 19.8 -2.8
0.00675, 0.00863
0.0200
0.0242
0.00394, 0.00450 16.4 -3.5
0.0122
0.0142
-------
Table 2. EFFECT OF DEUTERIUM OXIDE ON THE REGENERATION RATE OF
CARBAMATE INHIBITED BOVINE ERYTHROCYTE
ACETYLCHOLINESTERASE AT pD of 8.42
Inhibitor Solvent k k, /k ,
r h d
3-Isopropylphenyl water 0.0508±0.0048
methylcarbamate
deuterium oxide 0.00674±0.00033 7.5
3-Isopropyl-4- water 0.0345±0.00070
nitrophenyl
dimethylcarbamate deuterium oxide 0.0060±0.00046 5.7
112
-------
erythrocyte ACHE. The isotope effect of 5.7 for dimethylcarbamoyl
enzyme regeneration and 7.5 for monomethylcarbamyl enzyme is con-
sistent with a single proton transfer mechanism being operative in
both cases and that general acid-base assisted nucleophilic attack
by water is probably involved". The magnitude and direction of the
isotope effect supports this preliminary conclusion.
Conclusion - The method used in this study of the regeneration of AChE
inhibited by methyl- and dimethylcarbamate esters differs from that
normally used^ in two basic respects. First, excess inhibitor (carbam-
ate) was separated from the enzyme-carbamyl enzyme mixture by Sephadex
chromatography. The general procedure used to minimize or terminate
continuous carbamylation has been the "dilution" technique. Second, the
rate of spontaneous regeneration of the carbamyl enzyme was monitored in
the presence of the substrate ATCh by following the increase in the vel-
ocity of substrate hydrolysis with time. As expected, a positive curva-
ture was obtained in the relationship between enzymatic activity and time.
In the method normally used, the diluted enzyme-inhibitor mixture is
allowed to stand and aliquot samples are taken for assay of enzymatic
activity after fixed time intervals by addition of substrate (ACh or
ATCh). This method requires that initial velocities for substrate hydrol-
ysis be used to calculate the first-order regeneration constant since the
velocity surely will increase with time as regeneration occurs.
The major difficulty in the present method resides in the proper
analysis of the data. For this purpose, a mathematical model was
established and regeneration constants were calculated by computer
analysis. Another problem lies in the possibility that the reagents
used to estimate substrate hydrolysis, i.e., ATCh, DTNB and its
products, may have some effect on the regeneration rate. This may
be especially true with respect to the substrate (ATCh) since it has
been shown^^ that decarbamylation or methylcarbamylated AChE is in-
hibited by acetylcholine. However, in spite of these possible effects,
the values obtained for k by this method are similar to those reported
by other investigators „
Models for the hydrolysis of ACh by AChE propose that both acetylation
and deacetylation of AChE are mediated by acid-base catalysis. The
mechanism of deacetylation is suggested as being the reverse of acetyla-
tion. The results from this study using l^C-labeled carbamates and of
the effect of pH, deuterium oxide, and salts on the regeneration of
carbamylated AChE suggest that a similar mechanism is in operation
in the carbamylation and decarbamylation of AChE by carbamate esters.
A mechanism for these processes consistent with that proposed for ACh
hydrolysis is presented by the scheme below. Although the actual
inhibition reaction was not examined in this study, the scheme in-
cludes the carbamylation process based on analogy with the model for
ACh hydrolysis. In particular, the effect of pH and deuterium oxide
113
-------
0
k
H H
A
i
s
i
E+I
0
II
>NCOH
D
RZ
HX-
0 A
J L
E+I
B..U .' Q-
".: H
9 ft
S
1
El
R2
R,-NX .0
D-
-
n
o-H-A
(ED
s
R2
v
C +R3OH
-------
indicates that decarbamylation is acid-base catalyzed as depicted in
the scheme.
The pH-rate profiles for decarbamylation for methyl- and dimethyl-
carbamyl AChE is compatible with a mechanism in which a water
molecule attacks the carbonyl carbon of the carbamate moiety with
assistance by the basic (imidazole) and acid (tyrosine OH) groups
in the active site. This sort of bifunctional mechanism would not
develop a formal charge and so a salt effect would be expected to be
slight, if existent. The relatively small salt effect is perhaps
consistent in this regard.
The deuterium oxide effect of 5.7-7.5 for kR/kD is of the magnitude
cited for a kinetic isotope effect which would be consistent with a
proton transfer being rate limiting. The values of 6-10 k^/k^ in
the reactions of normal proton transfer with nucleophiles has been
cited as indicative of nucleophilic attack involving proton transfer.
The deuterium oxide effect observed in this study is consistent with
water being directly involved in decarbamylation as indicated in the
scheme.
2. Aryl N-Hydroxv- and N-Methoxy-N-Methylcarbamates as Potent Reversible
Inhibitors of Acetylcholinesterase
Introduction - In the preceding section a mechanism for the inhibition
of AChE by carbamate esters and spontaneous regeneration of the in-
hibited enzyme was described. As indicated, the inhibition of AChE
undoubtedly occurs by a carbamylation process in which an actual
covalent bond is formed between the enzyme and inhibitor. In the
course of our investigations on the effect of structural modification
of the carbamoyl moiety of known carbamate insecticides on anticholin-
esterase and insecticidal activity, we have discovered a number of
new aryl N-hydroxy- and N-methoxy-N-methylcarbamates which behave
distinctly as reversible, competitive inhibitors of AChE. This section
provides evidence that these carbamate derivatives inhibit AChE exclu-
sively by a reversible competitive mechanism, i.e., by virtue of the
complex El as depicted in the first step of the scheme in part D-l.
Evidence for a Reversible, Competitive Mechanism for AChE Inhibition -
Evidence supporting a reversible, competitive mechanism for the
inhibition of bovine erythrocyte AChE by aryl N^-hydroxy- and N-methoxy-
N-methylcarbamates is given below for the m-isopropylphenyl methyl-
carbamate derivatives.
The degree of AChE inhibition by a fixed concentration of inhibitor was
the same whether the enzyme was incubated with inhibitor for different
time intervals before substrate was added or when the substrate and
inhibitor were added simultaneously to the enzyme. Plots showing the
115
-------
rate of ATCh hydrolysis after inhibition with a fixed amount of m-
isopropylphenyl N-hydroxy-N-methylcarbamate (V) are given in Fig. 5A.
The parallel lines show that the activity of the enzyme was constant
after different time intervals for the inhibition reaction and that
equilibrium between enzyme, inhibitor, and substrate was established
virtually instantaneously. As expected, residual enzymatic activity
clearly was a function of the concentration of V (Fig..5B). The
characteristic time-independent and concentration-dependent behavior
of AChE inhibition by V satisfied our first criterion that inhibition
occurred in a reversible manner. The same phenomenon was observed with
all other aryl N-hydroxy- or N-methoxy-N-methylcarbamates examined.
Incubation of enzyme and V for long periods of time (12 hr) had no
effect on the degree of AChE inhibition but marked differences were
noted when the enzyme was allowed to stand with m-isopropylphenyl
N-methylcarbamate (VI), a typical carbamylating inhibitor (cf. Fig. 6).
The inhibition profile obtained with VI shows an initial exponential
decrease in enzymatic activity (to about 1 hr), eventually followed by
a gradual increase in activity owing to regeneration of the enzyme by
decarbamylation (k^ step in scheme).
It is worth mentioning that N-hydroxypropoxur (XI in Table 3) has been
reported as a carbamylating agent with spider mite acetylcholinesterase •
In this study the inhibitor (XI) and substrate were added simultaneously
to the enzyme and the decrease in enzymatic activity was monitored with
time. The author reported that the reaction between XI and spider mite
AChE reached a steady-state situation unusually fast and decarbamylation
occurred very rapidly, therefore, accurate kinetic data for Ka and ^
could not be obtained. However, the author failed to investigate the
possibility that XI was behaving as a reversible inhibitor by pre-
incubating the inhibitor and enzyme or examine long-term incubation
effects on enzyme inhibition.
Gel filtration provided additional evidence that V and related esters
are reversible AChE inhibitors. For example, 15-min incubation of AChE
in a solution containing 5 X 10"^ M m-isopropylphenyl N-methoxy-N-
methylcarbamate (VII) and analysis for enzymatic activity by adding
ATCh (final concentration of VII was 3.3 X 10"6 M) showed that 56% of
the enzyme was inhibited. However, analysis of the eluant obtained
after passage of the original enzyme-inhibitor mixture through
Sephadex resulted in virtually 100% recovery of enzymatic activity.
On the other hand, AChE treated under the same conditions with 10~^ M
m-isopropylphenyl N-(methoxy)-methyl-N-methylcarbamate (VIII), a
carbamylating inhibitor, gave 7 and 15% enzyme activity before and
after column filtration, respectively.
Finally, several of the N-hydroxy- and N-methoxy-derivatives were
examined by the conventional kinetic methods to test for competitive
inhibition. Figure 7 provides the double reciprocal plot for the
inhibition of AChE by VII. The results clearly show that
116
-------
1.0
c -o
o
-Q
o-6
to
3.4
.2
100 200
Time (sec)
1,0
o>
u
c
o
.£>
v_
O
,8
.2
B
0 100 200
Time (sec)
Figure 5. PLOTS SHOWING RESIDUAL AChE ACTIVITY IN THE PRESENCE OF m-ISOPROPYLPHENYL N-METHYLCARBAMATE
(V). A. EFFECT OF TIME ON RESIDUAL ACTIVITY IN THE PRESENCE OF 1.33 X 10"6 M V; (1) CONTROL ENZYME,
(2) ENZYME ADDED TO MIXTURE OF SUBSTRATE AND INHIHITOR, (3) AND (A) PREINCUBATION WITH INHIBITOR FOR
10 AND 30 MIN, RESPECTIVELY. (PLOTS 1, 2 AND 3 WKR" ARBITRARILY TRANSLOCATED FROM THE ORIGIN FOR BETTER
ILLUSTRATION) B. EFFECT OF INHIBITOR CONCENTRATION ON RESIDUAL AChE ACTIVITY; (1) CONTROL ENZYME (2)-
(6) 1.33'X 10"7, 3.33 X 10'7, 6.67 X 10'7, 13.3 X 10"7 AND 16.7 X 10"7 M INHIBITOR, RESPECTIVELY
117
-------
100
6 8
Time(hr)
to 12
Figure 6. LONG-TERM TIME EFFECTS ON THE INHIBITION OF AChE BY m-ISOPROPYLPHENYL
N-HYDROXY-N-METHYLCARBAMATE (V) AND N-METIIYLCARBAMATE (VI), (I) AChE'ACTIVITY IN
THE PRESENCE OF 5
AND 1.5 X 10'
X Itr8 M V, (2) and (3) ACTIVITY IN THE PRESENCE OF '3 X 10
"° M vi TPREINCUBATION CONCENTRATION) , RESPECTIVELY
118
-------
V
-I
14
12
10
8
6
4
2
0
10
S"1 x 10
15
3M
20
Figure 7. DOUBLE-RECIPROCAL PLOT FOR THE INHIBITION OF AChE BY
m-ISOPROPYLPHENYL N-METHOXY-METHYLCARBAMATE (VII), O CONTROL
ENZYNE, 0 8.33 X 1(T7 M VII, & 1.67 X 10~? M VII
119
-------
inhibition is reversible and competitive.
Values for K , or the dissociation constant for enzyme-inhibitor
d.
complex (El) , are given in Table 3 for the various N-hydroxy-
and N-methoxy-N-methylcarbamates. Kfl in the case of reversible
inhibitors is identical to K.^, the conventional expression for
the reversible inhibition constant. As indicated in the table,
the N-hydroxy- and N-methoxy-derivatives were unusually strong
reversible inhibitors of AChE with Ka (K-[) values in the range of
2 X 10" -1 X 10"' M. Also, the N-hydroxy esters were generally
more effective than the corresponding N-methoxy analogs. The data
show that the affinity of these compounds for the enzyme is sub-
stantially greater than that previously reported for eserine and
neostigmine, both of which are strong irreversible inhibitors of
AChE12.
Evidence also was obtained which showed that these reversible inhibitors
are able to protect AChE from inhibition by irreversible inhibitors.
This was demonstrated by examining the effect of V on the rate of
inhibition of AChE by the potent anticholinesterase, Amiton (0,0-
diethyl S^-2-diethylaminoethylphosphorothioate oxalate salt), and by
VI (Fig. 5). In the presence of 1 X 10"7 M (V), the first-order
rate of inhibition of AChE by 3 X 10 M Amiton was reduced 3.3-fold,
i.e., from 0.39 to 0.12 min'l. Similarly, using 1 X 10"7 M (V), the
rate of inhibition of AChE by 1 X 10~ M (VI) was reduced approximately
5-fold, from 0.58 to 0.12 min~ . Although IV} at a concentration
approximately equal to its K^ value, markedly reduces the rate which
AChE is inactivated by (VI) or Amiton, it is not certain whether
these compounds are competing with (I) for the same active site of
the enzyme. The 3.3- and 5-fold reduction in rate constants for in-
hibition by Amiton and (VI) in the presence of 10 M (V) suggests
that (V) also may protect AChE from irreversible inhibition by more
than one mechanism.
Limited studies on the stability of N-methoxy-N-methylcarbamate esters
to alkali have revealed that they are substantially less susceptible
to hydrolysis than the corresponding methylcarbamate. For example,
the second-order constant for hydroxide ion catalyzed hydrolysis of
£-nitrophenyl N-methoxy-N-methylcarbamate is 4.5 M min compared
to 3.5 X 10-5 M"1 min~l for £-nitrophenyl methylcarbamate, a difference
of approximately 7.6 X 10 -fold. The N-methoxycarbamates evidently
are quite stable and the low reactivity of the N-methoxycarbamate
moiety to nucleophilic attack is consistent with the finding that
these compounds evidently do not carbamylate AChE.
120
-------
Table 3. DISSOCIATION CONSTANT (Ka) OF ARYL N-HYDROXY- AND
N-METHOXY-N-METHYLCARBAMATES AND OTHER
IRREVERSIBLE CARBAMATES
/*2
RiOCN
H ^CH,
0 3
V
VI
VII
VIII
IX
X
XI
XII
XIII
XIV
Rl
m-isopropylphenyl
m-isopropylphenyl
m-isopropylphenyl
m-isopropylphenyl
m-trimethylammonium-
phenyl
m-dimethylaminophenyl
o- isopropoxypheny 1
o- isopropoxyphenyl
(Eserine)
m-trimethylammonium-
Type of
0
L inhibition
OH Reversible
H Irreversible
OCH3 Reversible
CH2OCH3 Irreversible
OCH3 Reversible
OCH3 Reversible
\
OH Reversible
OCH3 Reversible
H Irreversible
CH-i Irreversible
K
a
1.1
1.8
6.7
1.6
1.9
1.6
9.5
3.3
1.2
(M)
X 10"7
X 10~4
X ID"7
-
x io"7
x io"5
x io"7
x io"6
X 10~
x io"5
121
-------
Conclusion - The results clearly show that the aryl N^-hydroxy- and
N-methoxy-N^-methylcarbamates examined in this study are potent,
reversible inhibitors of AChE with K^ values as low as 1 X 10"? M.
While none of these carbamate derivatives showed any insecticidaT
activity, they are of potential usefulness in basic studies on the
nature of the active site in the cholinesterase enzymes. Indeed,
they may be used in protecting the active site from phosphorylating
and carbamylating agents. Since these carbamate derivatives are
reversible inhibitors, the relative values of their affinity constant
(Ka) may provide a direct measure of substituent effects on the
binding of the carbamate to the enzyme. Aryl N-hydroxy- and N-methoxy-
N-methylcarbamates, therefore, may be used to assess the effect of the
nature and position of ring substituents on carbamate binding to AChE
without complication from the carbamylation reaction. Further work
along these lines is in progress.
3. Stereoselectivity in Cholinesterase Inhibition
Introduction - Stereoselectivity in the interaction of molecules con-
taining asymmetric centers with biological systems is a well known
phenomenon, and the interaction of asymmetric substrates and inhibitors
with cholinesterase is no exception. This section is concerned with
the synthesis, resolution, and examination of the toxicological
properties of organophosphorus esters containing two asymmetric centers,
In particular, work was conducted to determine the effect of simultan-
eous chirality at both the phosphorus atom and the a-carbon atom of
the alkoxy moiety in phosphonothioate esters on the inhibition of
cholinesterase from three different sources. Additional experiments
were performed to determine whether these effects were reflected in
the toxicity of the chiral organophosphorus esters to insects and
mice and in their plant systemic insecticidal effectiveness.
Synthesis and Resolution - The following reactions were used for the
preparation and resolution of the organophosphorus compounds used in
this study. D- and L-isomers of 2-butanol were purchased from Norse
Laboratories, Santa Barbara, CA. Chiral centers are indicated by
asterisks. The o-phenylethylamine salt (XVII) was used for resolution
of the 4 diastereomer salts which were subsequently converted to the
resolved esters of XVIII by treatment with 2-chlorodiethyl sulfide.
The absolute configuration of the two asymmetric centers (C and P) in
XVIII and the magnitude of rotations are given in Table 4.
122
-------
Table 4. OPTICAL ROTATION IN DEGREES OF ARC AND ABSOLUTE
CONFIGURATION OF THE VARIOUS ISOMERS OF XVIII
Isomer
XVIIIa
XVIIIb
XVIIIc
XVIIId
XVIIIe
XVIIIf
Configuration at
C P
^
a D
S
s
R
R
R
racemic
R
S
racemic
R
-33.371
+11.072
+60.098
-60.276
-14.698
+33.780
123
-------
C H, .S C H S a-phenyl- C H. 0
25\f -^-* \* ethylamine^ V HfcHC.H,
a. / \ H2°" * / \ -v / \ 6 5
C^HrfiHO Cl J. C0HCCHO OH C0HrCHO S"
2 5j dioxane 2 5j
/1TT /TLI
CH3 CH3
" /
XV XVI „.- / XVII
(resolved at the
phosphorus atom)
p ^ i. t. f. j -^
* / \ * / \ .
cfii4Pucriu PUP pn c MO
ov.* n*^n.rt oL/ —H- u _ n-Litiu o IN a.
XVIII
Totally racemic XVIII was treated with m-chloroperbenzoic acid to give
the corresponding sulfoxide (XIX) and with potassium permanganate to give
the sulfone (XX).
Cholinesterase Inhibition - Bimolecular inhibition constants (k.) were
determined for cholinesterase from three different sources. Anticholin-
esterase data (k.) for the various isomers of XVIII against house fly
head (HFAChE), bovine erythrocyte (BAChE), and horse serum (HSChE)
cholinesterases are presented in Table 5. Wide variability was observed
in the inhibitory behavior of the various isomers to each enzyme and in
the behavior of each isomer to the different enzymes. For example, the
enantiomers XVIIIc and XVIIId gave inhibition constants of 1.35 X 103 M-1
min"1 and 1.69 X 106 M"1 min"1, respectively, against HFAChE, a rate
difference of about 1250-fold. Similar and consistent, though smaller,
rate differences were observed between the enantiomers of XVIII in their
inhibition of the other two cholinesterases. The largest rate differences
were caused by chirality at the phosphorus atom. The (S)-phosphorus
isomers consistently gave larger inhibition constants than the corres-
ponding (R)-phosphorus isomers; i.e., compare XVIIIa with XVIIIc and
XVIIId with XVIIIf.
The effect of chirality at the cy-carbon (2-butyl) on cholinesterase
inhibition was dependent upon chirality at the phosphorus atom. In the
two isomers with the (S)-phosphorus atom, the (S)-a-carbon compound (XVIIIa)
gave a larger k^ than the (R)-a-carbon compound (XVIIId). However, in the
124
-------
Table 5. ANTICHOLINESTERASE PROPERTIES OF XVIII, ITS RESOLVED
ISOMERS, XIX, AND XX AGAINST HOUSE FLY-HEAD ACETYLCHOLIN-
ESTERASE (HFAChE), BOVINE ERYTHROCYTE ACETYLCHOLIN-
ESTERASE (BAChE), AND HORSE SERUM CHOLINESTERASE (HSChE)
No.
XVIII
XVIIIa
XVIIIc
XVI I Id
XVIIIf
XIX
XX
Config.
C P
racemic
S S
S R
R S
R R
racemic
racemic
HFAChE
k£ (957= CI)
M"1 min"1 X 10~3
969
(29)
1710
(50)
1.35
(0.02)
1690
(110)
6.62
(0.30
556
(21)
696
(39)
BAChE
k.^ (95% CI)
M"1 rain"1 X 10"3
31.8
(0.9)
65.3
(2.1)
0.629
(0.020)
54.5
(2.2)
1.45
(0.07)
43.9
(1-4)
126
(8)
HSChE
k (95% CI)
M"1 min"1 X 10"3
5.95
(0.21)
17.3
(0.9)
0.483
(0.042)
6.85
(0.30)
1.01
(0.18)
3.67
(0.17)
15.1
(0.6)
125
-------
other two isomers with the (R)-phosphorus atom, the (R)-a-carbon
compound (XVIIIf) gave a larger k.^ than the (S)-a-carbon compound
(XVIIIc). Thus, in all three cholinesterases, the order of rate
constants (k^) for the four fully resolved isomers of XVIII is
XVIIIa > XVIIId » XVIIIf > XVIIIc. The consistency of these results
indicates that these asymmetric inhibitors are interacting with the
active site of each enzyme in a similar manner and, therefore, the
stereochemistry involved at the active site of the three enzymes is
probably similar.
Several additional points of interest emerge from the k^ data in Table
5. It can be seen that among the compounds listed, HFAChE is more
sensitive to inhibition than BAChE, which in turn is more sensitive
than HSChE. Comparing, for example, the relative rates of inhibition
of the three enzymes by XVIIIa, HFAChE is 26-fold more sensitive to
inhibition than BAChE which, in turn, is 3.8-fold more sensitive than
HSChE. It can also be seen that the largest rate differences are pro-
duced by the more effective levarotatory (S)-phosphorus inhibitors
(XVIIIa, XVIIId). The dextrorotatory (R)-phosphorus compounds (XVIIIc,
XVIIIf) showed rather low and approximately equal activity against
the three enzymes, although XVIIIf was moderately active against HFAChE.
In contrast, anticholinesterase activity increased dramatically for the
(S)-phosphorus isomers (XVIIIa, XVIIId) in proceeding from HSChE to
BAChE to HFAChE.
As expected, k^ for racemic XVIII against each of the three cholin-
esterases closely equalled the weighted sum of k. values of the
individual resolved inhibitors (XVIIIa, XVIIIc, XVIIId, XVIIIf) after
adjustment for concentration. Against BAChE, the weighted sum of the
k^ values for the four resolved isomers is 3.06 X 10^ M min"-"-, com-
pared to an observed kj_ of 3.18 X 1(A M"1 min-1 for XVIII. These
results indicate that the individual isomers in the racemic mixture
are acting independently of each other.
Against all of the enzymes used, k. for the sulfone (XX) was larger
than that for the sulfoxide (XIX) probably because of the greater
reactivity of XX. Against BAChE and HSChE, k^ of XIX was larger than
that for XVIII; however, against HFAChE, k± for both XIX and XX was
smaller than that for XVIII.
Toxicity - In general, toxicity to either insects or to mice paralleled
anticholinesterase activity, i.e., the stronger inhibitors proved to
be more effective toxicants to each test animal (Table 6). The (R)-
phosphorus isomers (XVIIIc,f) were nontoxic at the maximum dosages used
to all of the insects tested and only moderately toxic to mice. The
(S)-phosphorus isomers (XVIIIa,d) were highly toxic to susceptible
house flies and mice and moderately toxic to resistant house flies and
mosquito larvae. Small differences in toxicity owing to chirality at
the 2-butyl carbon atom also were noted.
126
-------
Table 6. TOXICITY OF XVIII, ITS ISOMERS, XIX AND XX, TO HOUSE FLIES
(TOPICAL, ADULT, FEMALE), MOSQUITO LARVAE
AND WHITE MICE (ORAL)
Musca domestica
No.
XVIII
XVIIIa
XVI lib
XVIIIc
XVI I Id
XVII le
XVIIIf
XIX
XX
NAIDM
(LD50 |_ig/g)
10.8
6.7
9.4
>500
6.9
12.1
>500
39
27
SC
(LD50 ng/g)
78
33
84
>500
42
122
>500
310
>500
Larvae
(LC5Q ppm)
0.65
0.25
0.66
>1
0.15
0.57
>1
>1
>1
Mouse ?
(LD50 iag/g)
3.1
3.2
-
110
2.8
-
125
-
_
127
-------
Because the (R)-phosphorus isomers were nontoxic to insects at the
maximum dosages used, it is not possible to determine whether a
direct correlation exists between in vitro anticholinesterase
activity of the four isomers of XVIII and insecticidal activity.
For example, against HFAChE, k± (M'1 min"1) for XVIIIa and XVIIIc
are 1.71 X 106 and 1.35 X 103, respectively, a difference of about
1270-fold. In comparison, the W^Q values (|ag/g) of XVIIIa and
XVIIIc to house flies are 6.7 and >500, respectively, a difference
of >75-fold.
A similar comparison may be made between inhibition of BAChE and. mouse
toxicity. Owing to the appreciable toxicity of the (R)-phosphorus
enantiomers, calculation of finite values for the ratio of toxicities
was possible. Against BAChE, XVIIIa (k.^ 5.53 x 10^ M'1 min"1) was
104-fold more effective as an inhibitor than XVIIIc (^ 6.29 X 102
M"1 min ) and 34-fold more toxic to the white mouse (3.2 and 110 mg/kg,
respectively). Similarly, XVIIId was 45-fold more effective as an
anticholinesterase than XVIIIf and 37-fold more toxic to the white
mouse. Considering that biological systems are involved, the relation-
ship between inhibition of a mammalian acetylcholinesterase, BAChE,
and toxicity to the white mouse is quite good arid suggests that other
factors involved in intoxication (metabolism, translocation, etc.) are
similar for the different isomers in the mouse.
Plant Systemic Activity - Data for plant systemic activity in cotton
plants against 4 pest species are presented in Table 7. Indicated
are the number of weeks in which >50% mortality of the test species
were obtained following granular treatment of the soil or topical
stem application of young cotton plants. The longer the mortality
is sustained at a high level, the better the toxicant is as a plant
systemic insecticide. Similar data for aldicarb, a well-known plant
systemic insecticide, are included for comparison.
The dextrorotatory (R)-phosphorus isomers (XVIIIc,f) were completely
inactive as plant systemic insecticides. It is not certain whether
the absence of activity is attributable to decreased rates of absorption,
translocation, and perhaps metabolism in the plant owing to stereo-
isomerism or whether the translocated isomers (or their metabolites)
themselves are nontoxic owing to their poor anticholinesterase proper-
ties. In contrast, the levorotatory (S)-phosphorus isomers (XVIIIa,d)
were highly active, a finding which is consistent with their high
anticholinesterase activity toward HFAChE. Since it is unlikely that
the physical properties of the individual isomers which affect move-
ment in plants vary to any large extent, the poor systemic activity
of XVIIIc and XVIIIf probably is attributable to poor anticholinesterase
activity.
128
-------
Table 7. PLANT SYSTEMIC ACTIVITY, THE NUMBER OF WEEKS WITH A
50 PER CENT MORTALITY OF THE TEST ARTHROPODS. THE
ARTHROPODS ARE TETRANYCHUS CINNABARINUS (MITE),
APHIS. GOSSYPII (APHID), BUCCULATRLX
THURBERIELLA (PERFORATOR), AND ESTIGMENE ACREA
(SALT MARSH). 4 = 400 mg SOIL TREATMENT, 8 = 800 mg
SOIL TREATMENT, S = STEM TREATMENT
Compound
XVIII
XVIIIa
XVIIIc
XVIIId
XVIIIf
Aldicarb
Treatment
4
8
S
4
8
Sc
4,8,S
4
8
S
4,8, S
4
8
S
Mite Aphid Perforator Salt Marsh
4
4
4
12a
12
3
All
I4a
14
3
All
10
10
3
4
5
4
15
16
3
completely
15
15
5a
completely
14
15
7
4 0
4 3
4 1
12 2b
12 4a
3 2
inactive
12a 1
12 1
2 1
inactive
15 3
16 4
14 0
Variable, but with at least half of the points 90 per cent plus,
'Maximum to only 60 percent mortality.
"Phytotoxic, plant dead in 3 weeks.
No mortality for first 3 weeks.
o
Average mortality about 40 per cent for some 14 weeks.
129
-------
Little difference in the duration of activity between the two differ-
ent dosages of 800 and 400 mg was found for any of the compounds,
although the activity of the 800-mg treatment occasionally lasted an
additional week. The activities of XVIIIa and XVIIId, the (S)-
phosphorus isomers, are similar, with XVIIIa slightly more effective
against salt marsh caterpillars and XVIIId more effective against
mites. The striking observation here is that persistence of systemic
activity following treatment with XVIIIa or XVIIIc was approximately
three times longer than after treatment with racemic XVIII at 800 mg,
even though the combined amount of XVIIIa and XVIIIc in XVIII at the
higher dosage is equal to 400 mg. As soil insecticides, XVIIIa and
XVIIId are superior to aldicarb against mites and aphids in these tests
but slightly inferior to aldicarb against perforators and salt marsh
caterpillars.
Affinity and Phosphonylation Constants - Studies on the behavior of
organophosphorus esters containing two asymmetric centers were
extended to the isomers of 0-2-butyl S-2-(dimethylammonium)ethyl
ethylphosphonothioate hydrogen oxalate (XXI). The various isomers
were prepared in a manner analogous to the preparation of XVIII except
0-2-butyl ethylphosphonothioic acid was reacted with 2-(dimethylamino)-
chloroethane according to the equation below.
C-H-CHO
2 5|
CH,,
C1CH2CH2N(CH3)2
SNa
oxalic
acid
C0Hr
2 5
C.HLCHO
2 5|
cm
0
\
SCH2CH2fe(CH3)2
oxalate
XXI
The absolute configuration of the C and P atoms of the individual
isomers and magnitude of rotations are given in Table 8.
The enzymes studied were bovine erythrocyte (BAChE) and house fly-head
(HFAChE) acetylcholinesterase, and horse serum pseudocholinesterase
(HSChE). The kinetic treatment of the inhibition reaction was similar
to that of Main and Iverson^ based on the following inhibition
scheme.
(El)
R
(El)
130
-------
Table 8. OPTICAL ROTATION IN DEGREES ARC AND ABSOLUTE
CONFIGURATION OF RACEMIC (XXI) AND RESOLVED
(XXIa-d) COMPOUNDS
No.
XXI
XXIa
XX Ib
XXIc
XXId
Configuration
C P (9 of arc)
racemic
£(+) S(-) - 9.86
S/+) R(+) +48.96
R(-) £(-) -49.36
R(-) R(+) +10.12
a The concentration was approximately 0.01 g/ml in absolute ethanol.
The standard deviations of the observed rotations averaged 0.005°.
131
-------
n
Free enzyme is E, I is inhibitor, (El) is a reversible enzyme-
inhibitor complex, and (El) is irreversibly phosphorylated enzyme.
The individual rate constants are k , k , and the phosphorylation
rate constant, k . The dissociation or affinity equilibrium constant,
Ka, is equal to k.^/k, and the bimolecular inhibition constant, k^, is
equal to k /K . The inhibition constants Ka, k_., and k. , can be
Del 1 O C
evaluated by using the equation below1J, where i is inhibitor concen-
tration and At/AlnV is the reciprocal of the pseudo first-order rate
of enzyme inhibition at inhibitor concentration i.
iAt = i
AlnV k
The kinetic constants, K , k , and k^, for the inhibition of the three
cholinesterases by racemic XXI and its four optical isomers (XVIa-d)
are presented in Table 9. Also included are the ranges of inhibitor
concentrations and the number of individual points used in each final
weighted regression.
In four cases, i.e., in the inhibition of BAChE and HFAChE by XXI or
XXIc, a nonlinear relationship was obtained in plots of iAt/AlnV against
i. These plots typically showed an apparent upward curvature when the
regression line approached the vertical axis. These points were dis-
carded in the final regressions. A similar upward curvature has been
reported by Main-*-^ for the inhibition of HSChE by amiton [0,0-diethyl
j>-2- (diethylamino) ethyl phosphorothioate] and by Iverson^-S for dimethyl-
carbamoyl choline inhibition of electric eel acetylcholinesterase in
the presence of tetramethylammonium ion.
The data in Table 9 show that large variations exist in the bimolecular
inhibition constants (k.) determined for the various isomers for each
enzyme and in the inhibitory activity of each isomer to the various
enzymes. For example, against HFAChE, k. for XXId is 1.60 X 103 M'1
min"1 and that of its enantiomer (XXIa) is 1.65 X 107 M"1 min"1, a
rate difference of 10,300-fold. Similar, though smaller, rate differ-
ences were observed between phosphorus enantiomers against the other
cholinesterases. The magnitude of the k. values for each isomer
against all of the enzymes, with the exception of XXIa and XXIb against
HSChE follows the order XXIa > XXIc » XXIb > XXId. The ^-phosphorus
isomers gave larger rate constants than the R-phosphorus isomers and,
within each phosphorus enantiomer, the S-secondary butyl isomers gave
slightly larger rate constants than the Rp-isomers. Against BAChE and
HFAChE the R-phosphorus inhibitors gave rather small and approximately
equal rate constants, e.g., the k. values for XXId against BAChE and
HFAChE are 1.74 X 103 M"1 min'1 and 1.60 X 103 M"1 min'1, respectively.
The same compound (XXId) gave a 13-fold larger kj_ value (2.15 X 10^ M"1
132
-------
min) against HSChE than against HFAChE. In contrast, the house
fly enzyme was considerably more sensitive to inhibition by the ;S-
phosphorus isomers than was the bovine enzyme, e.g., the k. values for
XXIc against HFAChE and BAChE are 1.46 X 107 M"1 rain'1 and 2.25 X 106
M min"1, respectively, a difference of 6.5-fold. HSChE was even less
sensitive to inhibition by XXIc (k± - 8.63 X 105 M"1 min"1) than was the
bovine enzyme. Overall rate differences (k. for XXIc vs k.^ for XXId)
within each cholinesterase are 9120-fold for HFAChE, 1630-fold for BAChE
and only 40-fold for HSChE. Thus, it appears that differences in anti-
cholinesterase activity attributable to chirality at the phosphorus
atom are much greater with acetylcholinesterase (BAChE and HFAChE) than
with pseudo cholinesterase (HSChE).
The k^ values for the inhibition of BAChE and HFAChE by racemic XXI
closely equal the weighted sums of the k^ values of the individual
isomers. Against BAChE the weighted sum of the k^ values for the
four resolved isomers is 3.03 X 10 M"1 min , compared to an observed
value of 3.50 X 106 M"1 min'1 for XXI. These results indicate that the
individual isomers in the racemic mixture are inhibiting acetylcholin-
esterase independently of each other.
The rather large differences in k^ between phosphorus enantiomers
against HFAChE and BAChE are attributable mainly to Ka, the differences
in affinity averaging around 220 fold. However, corresponding differ-
ences in kp, averaging around 15 fold, also contributed. For example,
comparison of XXIa and XXId for the inhibition of HFAChE shows that
their respective affinity constants (Ka) are 6.70 X 10~6 M and 1.95 X
10"3 M, a difference of 290 fold, and their respective phosphonylation
constants (k ) are 111 min"1 and 3.13 min"1, a difference of 35 fold.
These differences in affinity and phosphonylation constants combine
to give a difference in k^ of 10,340 fold. Further, against BAChE
Ka for XXIa and XXId is, respectively, 9.44 X 10'6 M and 2.98 X 10"3 M,
a difference of 306 fold, and k is 87.4 min'1 and 5.02 min-1, a differ-
ence of 17 fold. Combination or the affinity and phosphonylation con-
stants yields a difference in k^ of 5,330 fold. Compared to BAChE, the
approximately 2-fold larger values for k. for the inhibition of HFAChE
by the ^-phosphorus isomers (XXIa, XXIc) is mainly attributable to
larger phosphonylation constants.
Analogous comparisons with the horse serum enzyme, such as those made
above between the fly and bovine enzymes, are complicated by the
apparent separation of HSChE into different kinetic forms upon inhibi-
tion by compounds XXI, XXIa, and XXIb. A comparison of the results
for XXIc and XXId, inhibitors toward which HSChE behaved as one kinetic
form, reveals a pattern similar to that found in the acetylcholin-
esterases, but to a smaller degree. The difference of 40-fold in k^
between XXIc and XXId against HSChE is attributable mainly to the
16.4-fold difference in K . The greater anticholinesterase activity
(k.^) observed for the R-phosphorus isomer (XXId) against HSChE, com-
pared to BAChE and HFAChE, is attributable to affinity since their
133
-------
respective phosphonylation constants (kp) are rather similar. On the
other hand, the lower anticholinesterase activity of the ^-phosphorus
isomer (XXIc) against HSChE, compared to the acetylcholinesterases,
is attributable mainly to differences in k .
The effect of asymmetry at the 2-butyl carbon atom on anticholinester-
ase activity is small and variable. Comparison of XXIb and XXId against
HFAChE shows that both Ka and k contribute equally to the 4-fold
difference in k^. For the same compounds against BAChE, the 4-fold
difference in kj_ is attributable mainly to the 3-fold difference in
kp. For HSChE, the observed rate differences attributable to
asymmetry at carbon cannot be determined accurately oxving to the
separation of the enzyme into different kinetic forms. Overall,
the differences in kinetic parameters observed between carbon isomers
are in agreement with those reported by Chiu and Dauterman-'-" for
the inhibition of BAChE by the enantiomers of malaoxon, where asymmetry
resides on the a-carbon of the diethyl mercaptosuccinate moiety. In
this case, a 4-fold difference in k^ was observed, attributable to
2-fold differences in both Ka and k_ .
a p
Based on pseudo first-order plots, the inhibition of HSChE by XXI,
XXIa, and XXIb presents a special case in that the compounds appeared
to separate the enzyme into different kinetic forms, designated as A, B,
and C. It is apparent from the constants in Table 9 that the differ-
ences in the rates in which these kinetic forms are inhibited are
primarily attributable to differences in phosphonylation constants,
with the faster forms having larger phosphonylation constants than
the slower forms. The magnitude of the differences in k- for the
inhibition of the kinetic forms by XXI and XXIa, however, is reduced to
some extent by the Ka values. For example, even though kp for the
inhibition of A by XXIa is 105-fold larger than for C, the 13-fold
larger value of Ka reduces the difference in k^ between A and C to
about 8 fold. The Ka values for the inhibition of A, B, and C by
XXIb are approximately the same and, therefore, k^ for each kinetic
form is directly proportional to the k values. Racemic XXI separated
the enzyme into two forms instead of three.
Comparable results were reported by Main^ who also observed that the
relative magnitude of the bimolecular inhibition constants for inhibi-
tion of the various kinetic forms of HSChE were controlled primarily
by k . In addition, the horse serum enzyme was separated into four
kinetic forms at 5°C, and only three forms with a possibility of a
fourth at 25°C . This agrees with the three forms found for XXIa
and XXIb in this study at 30°C.
Although the various kinetic forms of HSChE are designated as A, B,
and C in order of descending k. values, this is not to imply that
the forms identified by a particular symbol are identical to each
other for all three compounds (XXI, XXIa, or XXIb). The symbols are
134
-------
used merely to distinguish between the kinetic forms separable by
each compound and it is possible that a form identified by one
symbol for a given compound is not the same as a form identified by
the same symbol for another compound. From the estimated values
for the relative amounts of each form identified for XXIa and
XXIb (cf Table 9), it appears that form A for XXIa (43%) is the same
as form C for XXIb (41%). This may help to account for the rather
anomalous division of HSChE into only two kinetic forms by XXI. XXI
is an equimolar mixture of all four chiral isomers used and, therefore,
eight independent reactions between the various isomers of XXI and
the enzyme are expected, two of the isomers (XXIa and XXIb) reacting
at different rates with the three kinetic forms and two isomers (XXIc
and XXId) reacting at their respective rates with the enzyme as a
whole. In the early stages of the reaction between XXI and HSChE,
the rate which was observed (XXI-form A) is probably attributable pri-
marily to the reaction between XXIa and its kinetic form A, since k^ .
for this pair of reactants is substantially larger than that for any
other pair of reactants. The fraction of the total enzyme that is
included in XXI-form A (61%) is larger than the fraction in XXIa-
form A (43%) because although the early portion of the reaction
between XXI and HSChE is probably dominated by XXIa-form A, it is a
sum of 4 linear additive reactions (XXIa-form A, XXIb-form C, and XXIc
and XXId against the total enzyme). The remaining portion of the
enzyme (XXI-form B) represents a sum of 6 reactions, (XXIa-forms B and
C, XXIb-forms A and B, and XXIc and XXId against the total remaining
enzyme). Probably XXI-form B shows linear first-order behavior
because it is a sum of several inhibition reactions.
Reproducibility of results obtained by the spectrophotometric technique
used, and the significance of the error terms, is illustrated by the
three independent replicates of the inhibition of BAChE by the R,-j3p
isomer (XXIc). Two gave inhibition constants that were very similar
to each other with small standard errors. The third replicate, covering
a smaller concentration range, deviated slightly from the other two and
had much larger standard errors.
A minimum estimate of the optical purity of these resolved inhibitors
may be obtained by comparison of overall rate differences. The S^S^
inhibitor (XXIa) inactivates the house fly enzyme approximately
10,300-fold faster than the RJL, compound (XXId). If the R isomers
(XXIb, XXId) are assumed to be completely inactive as inhibitors of
cholinesterase, then any inhibitory activity observed in experiments
using FL, isomers should be caused by small contaminations by the ^p
isomers (XXIa, XXIc). From the observed rate difference of 10,300-fold,
calculations indicate that there is less than 0.01 per cent contamina-
tion of £ isomers in the R preparations.
Conclusion - Chirality in the organophosphorus ester 0-2-butyl j>-2-
(ethylthio)ethyl ethylphosphonothioate evidently has a profound effect
135
-------
Table 9. AFFINITY, EQUILIBRIUM, PHOSPHORYLATION RATE, AND BIMOLECULAR RATE CONSTANTS FOR THE INHIBITION
OF THREE CHOLINESTERASES BY RACEMIC (XXI) AND I7S OPTICAL ISOMERS AT 30°C, pH 7.55
OJ
No. Config.
Form % of Total Ka (SE) lcp (SE)
enzyme (M X 105) (min"1)
(M"1 min'1 X 10"5)
I
(M range)
No. of
points
Bovine Erythrocyte Acetylcholinesterase
XXI racemic
XXIa S^p
XXIb SJL
XXIc RS
XXIc RS
XXIc ^>SP
XXId S^p
2.57 (0.37) 89.9 (9.9)
0.944 (0.065 87.4 (4.0)
234 (25) 15.5 (0.5)
2.00 (0.13) 56.7 (2.6)
1.98 (0.07) 55.8 (1.4)
2.97 (0.64) 66.9 (12.2)
298 (9) 5.02 (0.05)
35.0
92.6
0.0648
28.4
28.2
22.5
'0.0174
1 X 10'6-1 X 10"5
1 X 10'7-1 X 10"6
5 X 10"4-1 X 10"2
1 X 10"6-1 X 10"5
1 X 10"6-1 X 10"5
1 X 10"6-8 X 10"6
1 X 10"3-1 X 10"2
6
9
8
7
' 7
7
7
Housefly-Head Acetylcholinesterase
XXI racemic
XXIa ^Sp
XXIb S^
XXIc RcJ3p
XXId RJ
-------
Tableg. cont'd
No. Config.
XXI racemlc
racemlc
XXIa S^p
S^p
^P
XXIb RR
S^
^P
CXIC jyLp
CXId JL-&,
Form
A
B
A
B
C
A
B
C
7. of Total
enzyme
61
39
4"
31
26
20
39
41
Ka
Horse Serum
25.9 (2.2)
0.670 (0.137)
1.9
3.55 (0.49)
0.146 (0.012)
3.44 (1.05)
6.90 (1.31)
2.25 (0.32)
1.08 (0.14)
17.8 (2.1)
k
P
Cholinesterase
212 (15)
2.14 (0.11)
220
103 (12)
2.10 (0.18)
4?.. 9 (4.9)
13.5 (1.2)
1.61 (0.07)
9.33 (0.87)
3.81 (0.10)
ki
8.18
3.19
120
28.9
14.4
12.5
1.96
0.714
8.63
0.215
I
5 X 10~6-5 X 10"5
5 X 10"6-5 X 10~5
io"6-io"5
10"6-10"5
io"6-io"5
io-5-io-4
io"5-io"4
io"5-io"4
5 X 10"7-5 X 10"6
-3 -2
10 -10
No. of
points
7
7
4
6
7
. 7
7
7
6
7
-------
on its toxicological properties. Of the two asymmetric centers in
this molecule, the effect of chirality on anticholinesterase activity
and toxicity was much more pronounced at the. phosphorus atom than at
the 2-butyl carbon atom. On an overall basis, toxicological activity,
i.e., toxicity to insects and mice, and plant systemic activity of
the individual isomers of XVII appeared to be related to their
ability to inhibit stereoselectively the target enzyme, acetylcholin-
esterase, suggesting that other factors involved in intoxication and
detoxication of the individual isomers are similar. As plant systemic
insecticides, however, the more toxic (S)-phosphorus isomers were far
more effective as translocated insect toxicants than predictable from
the activity of the racemic mixture alone. This enhancement of
systemic activity, produced by resolution of the isomers, may have
practical implications and deserves further study.
The kinetic data presented for compounds XXIa-d must be rationalized
on the basis of steric grounds since chemical reactivity arguments are
not applicable to rate differences observed between chiral isomers.
With the acetylcholinesterases (BAChE and HFAChE), the stereochemical
qualities of the j> isomers which are responsible for high affinity
(K ) apparently are also responsible for rapid phosphonylation (kp).
The converse appears to hold true for the Rp isomers since, in general,
these gave unfavorable affinity and correspondingly low phosphonylation
constants.
On an overall basis, the results indicate that the stereochemical
requirements for complex formation and for subsequent phosphonylation
are significantly more rigid with AChE than with pseudo ChE. Further,
for the two AChE's, stereochemical effects are more noticeable in the
inhibition of HFAChE than with BAChE. In spite of these specific
differences in the behavior of the three cholinesterases to inhibition
by chiral isomers, in general, the order in which the three enzymes
are inhibited by the four isomeric inhibitors are similar. This sug-
gests that the stereochemistry associated with the active site of all
three enzymes, while different in detail; is, on the whole, similar.
The effect of small, but systematic variation in the size and shape
of organophosphorus esters on the inhibition of AChE often has been
attributed to the effect of structure modification on the affinity
of the inhibitor for the enzyme. On the other hand, the effect
of change in the reactivity of the ester on inhibition has been associ-
ated with the phosphorylation step. From this study, it appears that
both affinity (Ka) and phosphonylation (k ) are significantly affected
by the stereochemical properties of the cniral phosphorus ester.
138
-------
4. Absolute Configuration of Chiral 0-2-Butyl Ethylphosphonothioic
Acid.
Introduction - In the preceding section, the synthesis, resolution,
anticholinesterase activity and toxicological properties of the chiral
isomers of 0-2-butyl £5-2-(ethylthio)ethyl and 0-2-butyl £-2-(dimethyl-
ammonium) ethyl ethylphosphonothioate are discussed. Each of the
chiral isomers of these organophosphorus esters was prepared directly
from the four corresponding isomers of 0-2-butyl ethylphosphonothioic
acid, resolved via the crphenylethylammonium salt. In light of the
profound differences in anticholinesterase and insecticidal activity
observed between chiral isomers of these esters, it was of fundamental
importance to establish the absolute configuration of the asymmetric
centers in the esters. Since both XXIII and XXI were prepared by
chemical reactions which did not affect the chiral centers, i.e., the
2-butyl carbon or phosphorus atoms, it was possible to relate absolute
configuration of the isomers of these compounds with those of the
intermediate acid (XVI). This report is concerned with establishment
of the absolute configuration of (-)-0-2-butyl (-)-ethylphosphonothioic
acid in the form of its a-phenylethylammonium salt (XVIId) by X-ray
crystallographic analysis.
X-Ray Analysis - (-)-0-2-Butyl (-"> -ethylphosphonothioic acid (XVIId)
bp 80-1° (0.03 mm), ng5 1.4784, aj6 -33.967° (neat), [a]*6 -9.128°
(ethanol) was treated with (-)-cy-phenylethylamine to afford the corres-
ponding ammonium salt, XVIId. Appropriate crystals of XVIId were obtained
as colorless needles, mp 149-149.5°, [a]^ -13.718° (ethanol) by slow
evaporation of a pe'ntyl acetate solution. Anal. Calcd for C, .H^NO-PS:
C, 55.42; H, 8.64. Found: C, 55.25; H, 8.48. Weissenburg and
precession photographs showed monoclinic symmetry and space group P2^.
The unit cell constants, a = 11.553(12), b = 6.700(6), c = 11.723(12) A,
and (3 = 107.03(2)°, were determined from a least-square fit of 12
reflections measured on a Picker automatic diffractometer (Mo&Y, X =
0.71069 A). The density of the crystal measured by flotation was 1.15
g cm"3, which agrees with the value of 1.161 g cm"3 calculated for two
molecules of C,,H0,NO-PS in a unit cell.
14- 2o 2
Intensity data were calculated on the above diffractor, using MoK#
radiation. Reflections having 29 values up to 36° (638 unique
reflections) were collected by the 29-9 scan technique at a scan rate
of 1° per minute and a scan range of 1.9° Background counts of 10
sec were made at each end of the scan.
The structure was solved by the heavy-atom method and refined full
matrix least-squares calculations, using 569 reflections which were
greater than 1.5 a. The phosphorus and sulfur atoms were treated
anisotropically. The refinement converged to a final residue R of
139
-------
9.6%. All hydrogen atoms were observed on the difference electron-
density map, but not included in the refinement. Final atomic
coordinates are given in Table 10.
A structural view of XVIId is shown in Figure 8. The quaternary
ammonium protons are hydrogen bonded to three neighboring ions, one
on the sulfur and two on the oxygen atoms of two different organo-
phosphorus molecules. One of the N-H 0 bonds is shown in Figure
8. The absolute configuration of (-)~0-2-butyl (-)-ethylphosphono-
thioate moiety was determined as RpS-j i.e., R for carbon and j> for
phosphorus, by relating to the known configurations of the S-(-)-a-
phenylethylammonium ion and R-(-)-2-butyl alcohol.
Based on the assignment R^Sp for XVIId, the absolute configurations
of the 2-butyl carbon atom and phosphorus atom in (-)-2-butyl S.-2-
(ethylthio)ethyl (-)-ethylphosphonothioate (XVIIId) and (-)-0-2-butyl
J3-2-(dimethylammonium)ethyl (-)-ethylphosphonothioate (XXIc) are also
assigned the configurations R^Sp- The configuration of the remaining
chiral isomers may be deduced from their respective optical rotations.
Conclusion - The absolute configuration of the C and P atoms in the
(-)-0-2-butyl (-)-ethylphosphonothioate moiety was established by
X-ray diffraction analysis as RpSp- These results were used to assign
the absolute configurations of the C and P atoms in the different
chiral isomers of the esters XVIII and XXI.
140
-------
Table 10. FINAL POSITIONAL PARAMETERS IN J5- (-) -cv-PHENYLETHYLAMMONIUM
SALT OF R-(-) -0-2-BUTYL S_-(-)-ETHYLPHOSPHONOTHIOATE
Atom
S
P
01
02
N
Cl
C2
C3
C4
C5
C6
C7
C8
C9
.CIO
Cll
C12
C13
C14
HC7
HC10
aStandard
digits
X
-0.0053( 7)
-0.0377( 7)
-0.0119(12)
-0.1762(14)
-0.0861(15)
-0.2956(24)
-0.2583(19)
-0.3315(24)
-0.4418(23)
-0.4777(24)
-0.3991(23)
-0.2209(20)
-0.2501(20)
-0.3424(24)
-0.2401(22)
-0.2955(28)
-0.3516(38)
0.0394(23)
0.1765(22)
-0.243
-0.195
deviations given
y
1.0000( 0)
0.7145(15)
0.6464(24)
0.6592(28)
0.2702(27)
0.3879(46)
0.4577(42)
0.6013(44)
0.6542(49)
0.5858(47)
0.4527(43)
0.2308(46)
0.0208(46)
0.8630(60)
0.7090(52)
0.4787(63)
0.5146(59)
0.5439(46)
0.5628(44)
0.238
0.769
in parentheses are in
z
0.2046( 6)
0.2328( 6)
0.3627(13)
0.1758(14)
0.4025(14)
0.3961(23)
0.5092(20)
0.5534(23)
0.4694(25)
0.3551(25)
0.3181(21)
0.3519(19)
0.3938(19)
0.0557(25)
0.0471(23)
0.0003(30)
-0.1156(41)
0.1524(21)
0.2092(21)
0.263
-0.005
least significant
141
-------
M
'3 ^' v ^^v H
9
14 13
Figure 8. ABSOLUTE CONFIGURATION OF S_-(-)-a-PHENYLETHYLAMMONIUM (R) - (-)-0-2-BUTYL
(S)-(-)-ETHYLPHOSPHONOTHIOATE (XVIId). THE NUMBERING OF THE ATOMS CORRESPONDS
TO THE NUMBERING OF THE ATOMIC COORDINATES GIVEN IN TABLE 10
-------
5. References
1. Reiner, E. and W. N. Aldridge. Effect of pH on Inhibition and
Spontaneous Reactivation of Acetylcholinesterase Treated with Esters
of Phosphorus Acids and Carbamic Acids. Biochem. J^. 105:171-9 (1967).
2. Ellman, G. L., K. D. Courtney, V. Andres, Jr., and R. M.
Featherstone. A New and Rapid Colorimetric Determination of Acetyl-
cholinesterase Activity. Biochem. Pharmacol. 7:88-95 (1961).
3. Berman, M. and M. F. Weiss. SAAM Manual USPHS publication 1703,
U.S. Printing Office, Washington, DC (1967).
4. Berman, M. and M. F. Weiss. SAAM Manual Unpublished (1972).
5. Bergmann, F., S. Rimon, and R. Regal. Effect of pH on the Activity
of Eel Esterase Towards Different Substrates. Biochem. J. 68:493-9
'(1958).
6. Fukuto, T. R., M. A. H. Fahmy, and R. L. Metcalf. Alkaline Hydroly-
sis, Anticholinesterase and Insecticidal Properties of Some Nitro-
Substituted Phenyl Carbamates. J. Agr. Food Chem. 15:273-81 (1967).
7. Dittert, L. W. and T. Higuchi. Rates of Hydrolysis of Carbamate
and Carbonate Esters in Alkaline Solution. J. Pharmacol. Sci. 52:852-7
(1963).
8. Bruice, T. C., A. Donzel, R. W. Huffman, and A. R. Butler. Aminol-
ysis of Phenyl Acetates in Aqueous Solutions. VII. Observations on
the Influence of Salts, Amine Structure, and Base Strength. J_. Amer.
Chem. Soc. 89:2106-21 (1967).
9. Milstein, S. and T. H. Fife. The Hydrolysis of ^3-Aryl Phosphoro-
thioates. J. Amer. Chem. Soc. 89:5820-6 (1967).
10. Wilson, I. B. and J. Alexander. Acetylcholinesterase: Reversible
Inhibitors, Substrate Inhibition. J. Biol. Chem. 237:1323-6 (1962).
11. Post, L. C. Inhibition of Cholinesterase by Carbamates. A New
Kinetic Approach. Biochim. Biophys. Acta 250:121-30 (1971).
12. Iverson, F. and A. R. Main. Effect of Charge on the Carbamylation
and Binding Constants of Eel Acetylcholinesterase in Reaction with
Neostigmine and Related Carbamates. Biochemistry 8:1889-95 (1969).
13. Main, A. R. and F. Iverson. Measurement of the Affinity and
Phosphorylation Constants Governing Irreversible Inhibition of Cholin-
esterases by Di-isopropyl Phosphorofluoridate. Biochem. J_. 100:525-31
(1966).
143
-------
14. Main, A. R. Kinetic Evidence of Multiple Reversible Cholinester-
ases Based on Inhibition by Orga nophosphates. J^. B iol. Chem. 244:
829-40 (1969).
15. Iverson. F. The Influence of Tetramethylarnmonium Ion on the
Reaction Between Acetylcholinesterase and Selected Inhibitors.
Mol. Pha.ni]- 7:129-35 (1971).
16. Chiu, Y. C. and W. C. Dauterman. The Affinity and Phosphorylation
Constants of the Optical Isomers of 0,0-Diethyl Malaoxon and the
Geometrical Isomers of Phosdrin with Acetylcholinesterase. Biochem.
Pharmacol. 18:359-64 (1969).
144
-------
E. STRUCTURE - ACTIVITY RELATIONSHIPS IN INSECTICIDES
Work on the relationship between chemical structure and biological
activity was conducted in a variety of areas. This part of the report
summarizes our accomplishments in structure-activity studies of DDT
analogs, oxime carbamates and phosphates, S-aryl phosphoramidothioates
and (),S_-dialkyl phosphoramidothioates.
1. Structure - Activity Correlations in DDT analogs
Introduction - In connection with our studies on the design of
selectively toxic and biodegradable insecticides, a number of silicon-
containing analogs of DDT were synthesized and evaluated as insecticides.
The structure of some of these compounds in which silicon occupies
a key position in the molecule is given below.
H
CH -Si-CH
I
GEL
The rationale behind the examination of silicon DDT analogs as bio-
degradable insecticides resides in the greater instability of the
C-Si bond compared to the C-C bond to ionic and radical reactions.
To our disappointment, virtually all of the approximately 20 silicon
analogs, including those above, were ineffective against houseflies,
showing little or no toxicity at 500 ng/g, alone or in combination
with the synergist piperonyl butoxide. The inactivity of these
silicon DDT analogs was somewhat surprising, particularly since many
of the compounds were direct isosteres of active compounds.
A possible explanation for the ineffectiveness of the silicon analogs
may be that these compounds are inherently inactive owing to poor
fit at the site of action. Silicon is approximately 50% larger in
size than carbon and the Si-C bond (1.86 A) is considerably longer
than the C-C bond (1.48 A). The effect of the increase in bond
length may be seen by comparing the average van der Waal's radii
of 2.79 A and 3.3 A for the t-butyl and trimethylsilyl moieties,
respectively. Therefore, the substitution of a silicon atom in
a central position in the DDT analog would cause an increase in the
overall size of the molecule in all tetrahedral directions and it
145
-------
is possible that this increase in size prevents interaction of the
molecule with the DDT receptor site. Although this may be a plausible
explanation for the inactivity of silicon DDT analogs, actually there
is little theoretical basis for its support. For this reason, it
was decided that an analysis of the relationship between structure
of DDT analogs and insecticidal activity was needed for purposes
of correlation and prediction of activity.
Theory - The working model that we have adopted for the mode of
action of DDT and related compounds is essentially a modification
of the model proposed by Holan.^ The basic premise is that DDT
and its analogs fit in a receptor site of a macromolecule, possibly
a protein or lipoprotein in a nerve membrane. The receptor site may
be visualized as a cavity or a pouch with a limited amount of flexi-
bility and is by no means a rigid structure. Maximum effect occurs
when there is maximum interaction between the DDT molecule and the
receptor site, particularly with respect to the four key substituents
X, Y, L, and Z (structure below) through van der Waal's forces.
For maximum interaction, the overall size of the DDT molecule,
i.e., summation of the size of X, Y, L, and Z, is critical and any
deviation from this size results in reduced interaction, hence
reduced activity. The concept of maximum overall size is illustrated
in Figure 1.
Figure 1. Hypothetical models showing the fit of DDT analogs
containing different size ring substituents with the DDT receptor
site.
U is a substituent approximately the size of Cl, § is smaller than
the ^1 and £ is larger than M.. The point to be made is that good fit
with the receptor site is not restricted to symmetrical molecules
but also may be obtained with unsymmetrical analogs, as long as the
overall size of the molecule remains within the flexible framework
of the receptor site. This concept differs from the model of Holan^
146
-------
where the size of each substituent is assumed to be independent
of each other and greater reliance is placed on shape symmetry.
Basically, our model informs us that as the summation of the size
of X, Y, L, and Z increases, interaction with the receptor increases,
eventually reaching a maximum from which point increase in substi-
tuent size results in decreased interaction. Assuming that interac-
tion or fit with the hypothetical receptor site is directly related
to insect toxicity, it is possible now to correlate insecticidal
activity with free energy parameters on a quantitative basis.
Although several different parameters may be used to estimate the
size of a substituent, e.g., van der Waal's radii, etc., we chose
to use Taft's steric substituent parameter^ Es owing to the belief
that fit at the receptor site should be governed promarily by steric
effects. Keeping in mind that receptor interaction should approach
a maximum and then decrease with increase in the steric parameter
Es, the most reasonable approximation between toxicity and Es
should be parabolic in nature. Therefore, the mathematical model
to express toxicity in terms of Es with change in a single substi-
tuent X may be expressed by the following equation where a, (3,
Log LD5Q =a + 0 E* + V [E*]2 (1)
and Y are constants. A generalized equation for changes in more
than one substituent would be represented by equation 2 where
or1, P', and -y' are a new set of constants and i denotes substituent
i i 2
Log LD_n = a1 + P» Z E + Y' Z [E ] (2)
D\J S S
X, Y, L, and Z.
The data presented in Tables 1 and 2 were subjected to multiple
regression analysis. In accordance with our model, the variables £ E8
and £ Es were forced into the regression equation and other variables
(cf Tables 1 and 2) which might contribute significantly to the
regression sums of squares at the 0.05 level of probability were
entered into the analysis. The data also were analyzed by a program
which allowed the stepwise addition of significant variables but
in no case did any variable or appropriate combination contribute
more significantly to the regression equation than £ Es + £ Es , as
anticipated by the model. Besides Es the free energy parameters
used in the analysis were Hansch's TT constant for hydrophobic
bonding,^ Taft's polar substituent constant^ a* and the resonance
(R) and field constants (F) of Swain and Luptori^.
147
-------
Table 1. EFFECT OF VARIATIONS IN THE X AND Y POSITIONS ON THE INSECTICIDAL ACTIVITY OF DDT ANALOGS
oo
Com- X Y
pound
1 H H
IFF
4 Cl Cl
£ Br Br
411
& CH3 CH3
2 C2H5 C2H5
& OCH3 OCH3
3. OC2H5 OC2H5
IS CHJ H
11 Cl CH3
12 F CH3
13 Br CH3
14 I CH3
15 F OCH_
«.*
0
-0.46
-1.16
-1.36
-1.72
-1.24
-1.62
-1.08
-1.17
-1.24
-1.16
-0.46
-1.36
-1.72
-0.46
Y
0
-0.46
-1.16
-1.36
-1.72
.-1.24
-1.62
-1.08
-1.17
0
-1.24
-1.24
-1.24
-1.24
-1.08
X
IT
0
0.15
0.70
1.02
1.27
0.52
1.00
-0.04
0.50
0.52
0.70
0.15
1.02
1.27
0.15
Y
IT
0
0.15
0.70
1.02
1.27
0.52
1.00
-0.04
0.50
0
0.52
0.52
0.52
0.52
-0.04
Y-/^~\\
\=J
RX
0
-0.34
-0.16
-0.18
-0.20
-0.14
-0.11
-0.50
-0.44
-0 . 14
-0.16
-0.34
-0.18
-0.20
-0.34
RY
0
-0.34
-0.16
-0.18
-0.20
-0.14
-0.11
-0.50
-0.44
0
-0.14
-0.14
-0.14
-0.14
-0.50
-Y
F*
0
0.71
0.69
0.73
0.67
-0.05
-0.06
0.41
0.36
-0.05
0.69
0.71
0.73
0.67
0.71
Y UD50 (Mg/g). LC50 (PPm)
F Housefly Housefly Culex fatigans
(alone) (+ P.B.)
0
0.71
0.69
0.73
0.67
-0.05
-0.06
0.41
0.36
0
-0.05
-0.05
-0.05
-0.05
0.41
2900
30.5
14
27
35
100
70
45
7.0
>500
62.5
250
32.5
120
47
575
17.0
5.5
10.5
14
17.5
29.0
3.5
1.75
33.0
6.5
25
1.75
22.2
11.5
1.1
0.074
0.07
0.074
1.4
0.081
0.18
0.067
0.04
0.51
0.031
0.059
0.04
0.044
0.47
-------
Table 1 continued
16, Cl OCH -1.16 -1.08 0.70 -0.04 -0.16 -0.50 0.69 0.41 41.5 ' 7.0 0.058
U. CH3 OCH3 -1.24 -1.08 0.52 -0.04 -0.14 -0.50 -0.05 0.41 23.5 4.9 0.085
IS CH OC2H -1.24 -1.17 0.52 0.50 -0.14 -0.44 -0.05 0.36 9 1.7 0.13
12 OCH, OC2H -1.08 -1.17 -0.04 0.50 -0.50 -0.44 0.41 0.36 16 3.7 0.039
20. SCH SCH_ -1.29 -1.29 0.62 0.62 -0.19 -0.19 0.33 0.33 225 17.0 0.25
21 SCH OCH3 -1.29 -1.08 0.62 -0.04 -0.19 -0.50 0.33 0.41 32 4.0 0.11
22 SCH OC H -1.29 -1.17 0.62 0.50 -0.19 -0.44 0.33 0.36 32 2.8 0.7
23 CH, CnHc -1.24 -1.62 0.52 1.00 -0.14 -0.11 -0.05 -0.06 11 3.0 0.08
*x 3 25
2& OCH CH(CH ) -1.08 -2.32 -0.04 1.30 -0.50 -0.14 0.41 -0.10 160 61.5 0.32
25. Cl CHCCH-), -1.16 -2.32 0.70 1.30 -0.16 -0.14 0.69 -0.10 215 93.5 0.15
-------
Table 2. EFFECT OF VARIATION IN THE L AND Z POSITIONS ON THE INSECTICIDAL ACTIVITY OF DDT ANALOGS
Compound
4
2k
21
2&
23.
4Q
4i
22
42
44
45
46
4Z
4S
L
H
H
H
H
H
H
F
F
F
F
F
F
F
F
Z
cci3
CBr3
CF3
CHC12
CHF2
CHC1F
cci3
CHC12
CHClBr
CHC1F
CHBr2
CHBrF
" CHBrCH
CHF2
0
0
0
0
0
0
-0.46
-0.46
-0.46
-0.46
-0.46
-0.46
-0.46
-0.46
EZ
-3.30
-3.67
-2.40
-2.78
-1.91
-2.35
-3.30
-2.78
-2.94
-2.34
-3.00
-2.51
-2.41
-1.91
• L
ir
0
0
0
0
0
0
-0.17
-0.17
-0.17
-0.17
-0.17
-0.17
-0.17
-0.17
Z
nZ o*L
1.70
2.32
0
1.32
0.18
0.85
1.70
1.32
1.52
0.85
1.72
0.93
1.64
0.18
0
0
0
0
0
0
1.10
1.10
1.10
1.10
1.10
1.10
1.10
1.10
11
Z
0*
1.00
1.00
0.92
0.70
0.70
0.70
1.00
0.70
0.70
0.70
0.70
0.70
0.30
0.70
LD50 (W?/g>
Housef lies
2.0
>500
>500
.20
>500
155
125
4.1
2.8
220
7.0
70
9.0
>500
LCcn (ppm)
jU
Culex fatigans
0
0
0
0
0
0
0
0
0
0
0
0
0
0
.070
.550
.250
.038
.950
.120
.092
.024
.021
.110
.022
.085 •
.070
.810
-------
2.8
2.4
2.0
00.8
0.4
0.4 0.8 1.2 1.6 2.0
LOO LOgo(CALC'D)
2.4
2.8
Figure 2. Correlation between observed synergized housefly tox-
icity and toxicity calculated according to eq 4 for variation in X
and Y positions of DDT analogs.
A composite of data developed in two different laboratories over a
period of about 15 years-*'" was used in the analysis of mosquito
toxicity. For the 25 compounds in Table 1, stepwise selection of
variables by significance did not provide any significant correlation
between toxicity and any of the parameters. Forcing E E_ and EE_ in
the regression analysis resulted in equation 5.
Log LC5Q = 0.99 (± 0.29)
Forcing E E and E E
s s
E E + 0.46 (± 0.14)
E E
(5)
n_
25
r_
0.5?5
0.4
However, when the analysis was restricted to the compounds evaluated in
the early study^, i.e. compounds !_-!!_ and the compound where X is Cl and
Y is H, a satisfactory correlation was obtained (equation 6). The
relationship between observed LC5Q and LC5Q calculated according to
equation 6 is shown graphically in Figure 3.
Log LCcr, = 1.63 (± 0.21) E E_ + 0.93 (± 0.12) E E_2 (6)
'50
s
n
12
^
0.933
s
0.21
151
-------
Effect of Variation in X and Y Positions For the series of 25
symmetrical and unsymmetrical DDT analogs in which X and Y are varied
(Table 1), the following equations relating toxicity (1059) against
houseflies, with and without the synergist piperonyl butoxide,
with Es were formulated. Without synergist: ,„,
Log LD = 3.24 (± 0.35) + 1.52 (± 0.22) Z E + 0.65 (± 0.14) £ E 2
DU S 8
n jr £
24 0.736 0.41
With synergist: ,,,.
Log LD5Q = 2.69 (db 0.25) + 1.85 (± 0.22) Z Eg + 0.85 (± 0.11) Z E
2
s
25 0.874 0.31
n is the number of compounds in the analysis, j: is the correlation
coefficient and j3 is the standard error.
Comparison of equations 3 and 4 shows that the use of piperonyl
butoxide significantly improves the correlation between housefly
toxicity and the steric substituent constant Es. Since piperonyl
butoxide is regarded as an inhibitor of microsomal mixed function
oxidase, the improved correlation between synergized toxicity and
Es serves to point out the importance of the effect of oxidative
metabolism on the insecticidal activity of DDT analogs. The
relationship between synergized housefly toxicity and Es is presented
graphically (Figure 2) by plotting log LD5Q observed against log
LD5Q calculated according to equation 4. Several of the compounds
notably £, 2, J>2> -14. and 2Q, appear to be considerably less toxic
than expected from the equation, while a few (,9_, J.3., and .!§) are
more toxic. Since enzymatic dehydrochlorination of DDT and related
compounds is not blocked by piperonyl butoxide, it is possible
that these compounds deviate from the calculated fit because of
differences in their susceptibility to dehydrochlorination by DDT
dehydrochlorinase. However, it should be pointed out that the de-
viation of these points from the line is not as serious as may
appear from casual observation of the figure since most of the
deviations are in the region where high toxicity is expected.
For example, the largest deviation occurs with compounds with a
calculated LD50 in the range of 4-6.5 |ag/g (log LD5Q 0.3-1.2).
Considering the inherent variability usually found in toxicity data
and the number of compounds in the analysis, the fit of the points
to the regression line is quite good.
152
-------
0.2
-O.2
-0.4
-0.6
g-0.6
-1.0
-1.4
-1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0 0.2
LOG L09Q (CALC'D)
Figure 3. Correlation between observed mosquito larvae toxicity
and toxicity calculated according to eq 6 for variation in X and
Y positions in DDT analogs.
On the other hand, analysis of the mosquito toxicity data provided in
the more recent study of DDT analogs,^ i.e., compounds .12-^5, gave poor
correlations between toxicity and Es (_r = 0.433). The basis for the
poor correlation, in light of the highly satisfactory correlation
obtained for the other compounds, is not known. It is noteworthy that
].-_9 are symmetrical compounds while the remaining, except for ^0, are
unsymmetrical DDT analogs. Whether asymmetry in the molecule is respon-
sible for the poor correlation is conjectural at this time. It should
be pointed out, however, that asymmetry did not affect the correlation
on houseflies.
In general, correlation of toxicity with the steric substituent constant
ES was reasonably satisfactory with variation in the X and Y positions
when oxidative metabolism was minimized by piperonyl butoxide. The
successful use of the sum of the steric effects in the X and Y position
in correlating insecticidal activity suggests that the DDT receptor site
is quite flexible and is not as rigid as depicted by Holan.l This
model adequately explains why DDT analogs which contain a substituent
smaller than chlorine in the X position and a substituent larger than
chlorine in the Y position are toxic. Evidently, the degree of inter-
action between the substituents X and Y with the receptor site largely
depends upon the sum of the steric effects produced by both X and Y as
suggested in Figure 1. A graphic illustration of this concept is
presented in Figure 4 where synergized log LD^Q against houseflies is
153
-------
plotted against Eg for different substituents in one position (X) and
the rest of the molecule is held constant (Y = methyl). With the
exception of the point representing the symmetrical dimethyl analog
(X = Y = methyl), the plot is strikingly similar to the well known
potential energy diagram for diatomic molecules. The figure shows that
there is a region where maximum interaction occurs between the compound
and the receptor site and interaction decreases as the sum of Es for X
and Y is above or below this region.
1.6
o
-" 1.2
0.8
0.4
Me
Cl /
Br OEt
-1.6 -1.2 -0.8 -0.4
Figure 4. Relationship between observed synergized housefly
toxicity and ER for substituent X for 1,1,1-trichloro-p-methyl-p'-
X-diphenylethanes.
Effect of Variation in L and Z Positions - The importance of the size of
the downward group Z on the insecticidal activity of DDT analogs is well
known. Holan has indicated that the downward projection of DDT and the
corresponding dichlorocyclopropane analogs are similar and of optimum
size (6.0-6.3 A). Although downward projection into a membrane cavity
to account for the effect of Z on toxicity has been useful in correlating
structure with insecticidal activity, this type of projection analysis
has ignored the effect of the simultaneous presence of the group in the
L position. To illustrate this point, the replacement of the benzylic
hydrogen in DDT by fluorine (3J.) does not effectively change the down-
ward projection of the trichloromethyl moiety; however, housefly
toxicity drops by a factor of about 63. On the other hand, replacement
of the benzylic hydrogen in DDT (£8) with fluorine resulted in a com-
pound (3Z) which was only two-fold less toxic than DDT. From these
examples, it is evident that the effect of substituents on the a-carbon
should be examined together and not separately.
154
-------
Toxicological properties and the various parameters for DDT
analogs where L and Z are varied are given in Table 2. Analysis
of the data provided equation 7 for unsyriergized housefly toxicity.
Log LD = 14.14 (±4.0) + 9.36 (±3.0) E E + 1.12 (±0.47) IE2 (7)
3U S S
+ 4.31 (±1.37) T.a*
n r. £
9 0.856 0.31
For mosquito larvae, equations 8 and 9 were obtained.
Log LC_n = 5.29 (±1.06) + 4.90 (±0.92) E E + 0.76 (±0.16) E E 2 (8)
_)U S S
+ 1.76 (±0.43) E a*
ri _r £
14 0.889 0.29
Log LC = 7.27 (±0.72) + 6.03 (±0.57) EE + 0.76 (±0.09) E E 2 (9)
DU S S
+ 2.27 (±0.26) E a* + 0.37 (±0.08) E it2
14 0.970 0.16
While significant correlation was not obtained when each of the
parameters was used alone, the combination of Es and a* was highly
significant and indicated that steric and polar effects contributed
to interaction of L and Z with the receptor site in both houseflies
and mosquito larvae. Against mosquito larvae, significant improve-
ment in correlation was obtained by the introduction of a ir term
(equation 9). Although the coefficient for this term (0.37) is
small, it does point out the importance of the lipophilic character
of L and Z on toxicity to mosquito larvae.
For groups on the a-carbon atom, equations 7, 8, and 9 predict
that higher log LD5Q or log LC5Q (lower toxicity) will be obtained
for substituents with approximately equal Es values but larger o*
values. This is quite surprising since virtually all of the more
toxic DDT analogs contain polar groups in the Z (or L) position,
155
-------
i.e., groups with reasonably large positive a* constants. However,
of the compounds examined, a* for the Z substituent resides in a
rather narrow range compared to the corresponding Es values and,
therefore, any conclusions concerning the effect of o* on toxicity
must be tempered with caution. It is possible that there is also
a value of a* where maximum toxicity effects would be observed,
similar to that for Es. Unfortunately, however, data was not
available for DDT analogs with a diverse range of a* values for the
Z substituent, particularly those with negative values.
Conclusion - Overall, the steric substituent constant Es appears
to be the single most important parameter for the correlation of
housefly and mosquito larvae toxicity when substituents are varied
in the X, Y, L, and Z positions in DDT analogs. The successful use
of £ Es and £ Es in correlating insecticidal activity with structure
strongly idicates that there is a reasonable amount of flexibility
in the DDT receptor site. The receptor site may be visualized as
a flexible cavity which can accommodate DDT analogs of varying
dimensions as long as the overall size of the molecule does not de-
viate substantially from that of DDT. Compounds possessing the same
geometry as that of DDT but fall outside the range of maximum inter-
action because of their overall size, i.e., smaller or larger,
therefore are not expected to be toxic. The organosilicon analogs
of DDT, mentioned earlier, probably fall into this catagory.
There is evidence to indicate that the effect of ring substituents
is not independent of that of substituents on the a-carbon atom.
For example, the toxicity of Perthane [l,l-dichloro-2,2-bis-(p-
ethylphenyl(ethane] to houseflies synergized with piperonyl butoxide
is approximately twice that of synergized DDT. In comparison, the
toxicity of synergized l,l,l-trichloro-2,2-bis_-(£-ethylphenyl)
ethane, the £-ethyl analog of DDT, is only I/6th that of synergized
DDT. This suggests that when group X or Y or both are altered,
insecticidal activity may be maintained by appropriate alteration
of L or Z. The Es values for the chloro and ethyl moieties are
-1.16 and -1.62, respectively. Changing the ring substituent from
chloro to ethyl, therefore, can be compensated for by substituting
dichloromethyl (Es = -2.78) for trichloromethyl (Es = -3.30) in the
or-position.
It is obvious that a large number of new DDT analogs still remain
to be synthesized and examined for insecticidal activity. The model
that we have developed in predicting the insecticidal activity of
DDT analogs is by no means infallible but it provides a more system-
atic approach to the design of new compounds.
156
-------
2. Structure. Reactivity, and Biological Activity of 0-(Methyl-
carbamoyljoximes of Substituted Benzaldehydes
Introduction - Interest in oxime carbamates originated from the
outstanding insecticidal activity demonstrated by such compounds
as 2-methyl-2-(methylthio)propionaldehyde O-(methylcarbamoyl)oxime
(aldicarb), exo-3-chloro-endo-6-cyano-2-norbornanone 0-(methylcar-
bamoyl)oxime (Tranid^ Union Carbide), 2-oximino-l,3-dithiolane
methylcarbamate (American Cyanamid) and other oxime carbamates
and phosphates. Owing to the wide variation in structure of the
above oxime carbamates, a systematic examination of these compounds
was initiated in an attempt to establish the relationship between
structure and reactivity with anticholinesterase and insecticidal
activity. This part of the report presents results from a study
of a series of 0-(methylcarbamoyl)oximes of substituted benzalde-
hydes of general structure shown below.
0
II
-CH=NOCNHCH0
Synthesis - All substituted benzaldoximes were prepared in the
conventional manner from the appropriately substituted benzaldehyde
and hydroxylamine. Methylcarbamates were prepared by reaction
between the benzaldoxime and methylisocyanate using triethylamine
as catalyst.
Alkaline Hydrolysis - Table 3 provides data for the pseudo Ist-order
hydrolysis constants (kfc) in phosphate buffer at pH 11.95, anti-
cholinesterase activity (ISQ)> and toxicity to the housefly of the
carbamate alone and synergized with 1:5 parts piperonyl butoxide.
Linear regression analysis of kjj values for the meta- and para-
substituted oxime carbamates produced the equation 10 where r is
log 10 l = 0.9080 + 0.271 (r = 0.98) (10)
the correlation coefficient. Use of the regression equation to
calculate new sigma constants gave values of 0.61 and 0.92 for
m-nitro and p_-nitro, respectively. These values are in excellent
agreement with the sigma values of 0.67 and 0.92 for m-nitro and
£-nitro substituents calculated by linear regression analysis
(equation 11) of data obtained by Brady and Goldstein7 for the
ionization constants of substituted benzaldoximes:
pK =-0.774a+10.66 (r « 0.99) (11)
3.
157
-------
Table 3. PHYSICAL AND BIOLOGICAL PROPERTIES OF C)-(METHYLCARBAMOYL)OXIMES OF SUBSTITUTED
BENZALDEHYDES AND RELATED KETONES
t-n
00
0
Iv II
s C=NOCNHCH,
R2
»
40
41
42
43
44
45
46
47
48
49
50
51
«i
phenyl
jp_-me t hy Ipheny 1
o-ethylphenyl
o-isopropylphenyl
m-isopropylphenyl
p-isopropylphenyl
o-t-butylphenyl
2,4, 6-trimethylphenyl
o-bromophenyl
_p_-bromophenyl
£-nitrophenyl
m-nitrophenyl
_p_-nitrophenyl
Oxime
R£ isomer
H syn
H syn
H
H
H
H syn
H
H syn
H syn
H syn
H
H syn
H syn
(min"-*-) I^Q
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
1.
175
131
188
222
167
133
226
185
596
332
-
622
190
4.
2.
1.
7.
3.
4.
5.
3.
3.
7.
8.
1.
6
6
5
2
7
5
5
0
8
1
3
4
(M)
X
-
X
X
X
X
X
X
X
X
X
X
X
10
10
10
10
10
10
10
10
10
10
10
10
-5
-5
-5
-6
-5
-5
-5
-5
-5
-5
-6
-5
LD50 M-S/8
Alone 1:5 P.B.
>500
-
>500
>500
>500
>500
>500
>500
>500
>1000
>500
>1000
>1000
>50
-
50
40
>50
>100
>50
>50
>100
>100
>100
40
>100
-------
Table 3 continued.
_52
_53
_54
55
56
57
58
59
60
61
62
o-methoxyphenyl
o-ethoxyphenyl
o-propoxyphenyl
o-methylthiophenyl
o-CF_-phenyl
3 , 4-methylenedioxy-
phenyl
2-CH-S-4 , 5-methylene-
dioxyphenyl
phenyl
£-f luorophenyl
£-f luorophenyl
thienyl
H syn
H syn
H
H syn
H
H syn
H
thienyl -
cyclo- -
butyl
cyclo- -
propyl
cyclo- -
propyl
0.119 3.
0.111 3,
0.151 9.
0.325 4.
-
0.114 8.
"** -L •
2.
1.
6.
1.
1
7
1
9
1
7
8
7
8
5
X
X
X
X
-
X
X
X
X
X
X
10
10
10
10
10
10
10
10
10
10
-5
-5
-6
-6
_5
_/
-5
-5
-6
-6
>500
>1000
>500
>500
>500
>500
>500
>500
>500
>500
75
>50
>50
>50
>50
>50
>50
>50
20
40
12.5
10
-------
The values of 0.67 and 0.92 for m-nitro and £-nitro imply that field
effects are somewhat less and resonance effects somewhat greater
in substituted benzaldoximes than in benzoic acids. Unfortunately,
there were not enough meta- and para-substituents examined to subject
the kb values to correlation with the field and resonance constants
of Swain and Lupton^.
The ortho-substituted compounds were not included in the analysis
above because of steric or "ortho" effects • However, kb values
for these compounds were well correlated with the ao constants
of Tribble obtained from nmr spectra of ortho-substituted phenols.
Linear regression analysis of the data provided the equation 12
below. The value of the reaction constant p is 0.758 for the ortho-
log 10 k = 0.758 a + 0.355 (r = 0.95) (12)
bo
substituted oxime carbamates compared to 0.906 for the meta- and
para-substituted compounds. For the meta- and para-substituted
series all of the compounds except the m-isopropyl compound were
in the svn configuration and, therefore, the p value is essentially
that of the syn isomers. However, in the case of the ortho series
the configuration of the ethyl, isopropyl, and t^-butyl derivatives
was not known with certainty and, therefore, it is possible that
p in this series was obtained from hydrolysis data involving syn-
anti pairs. The difference in p constants does not necessarily
imply a different mechanism for hydrolysis since the differences
in kb values for ortho- and para-substituents are small enough to
be accounted for entirely by steric inhibition of resonance by
substituents in the ortho position.
Cholinesterase Inhibition - Anticholinesterase data in Table 3
show that all of the benzaldoxime methylcarbamates were amazingly
similar in their ability to inactivate fly-head cholinesterase,
i.e. 150 values in the neighborhood of 10"^ M with a range between
9 X 10-5 to 5 X 10~6 M. This is in direct contrast to the sub-
stituted phenyl N-methylcarbamates which have been found to vary
as much as 10-> in their 159 values , depending on the nature
and position of the substituent. Closer examination of the data
reveals an approximate but direct relationship between anticholi-
nesterase activity and alkaline hydrolysis rates for the meta-
and para-substituted compounds. Aside from the m-isopropyl deriva-
tive which is a much stronger inhibitor than predicted by its hy-
drolysis rate, a fair correlation between log kb and -log 150 is
obtained. The direct relationship between anticholinesterase activity
and reactivity as measured by alkaline hydrolysis rates is analogous
to results obtained in a previous study of 0-(methylcarbamoyl)oximes
of ring-substituted acetophenones •
160
-------
The narrow range of anticholinesterase activity cited above and
the direct correlation between anticholinesterase activity and k^
of the benzaldoxime methylcarbamates suggest that steric properties
of the molecule and interaction of the carbamate and enzyme to form
the complex are of lesser importance in the inhibition process than
the carbamylation reaction. Thus, the situation appears to be
similar to cholinesterase inhibition by organophosphorus esters but
unlike substituted phenyl methylcarbamates where it has been demon-
strated that variation in inhibition is more a function of degree
of complex formation rather than carbamylation*•*-.
No relationship between anticholinesterase activity and hydrolysis
rates or any other parameter was evident with the ortho-substituted
£-(methylcarbamoyl)-benzaldoximes. The order of inhibition for the
ortho alkyl substituents is isopropyl > ethyl > t^-butyl - methyl,
where methyl is for the 2,4,6-trimethyl compound C461. Although
the range in 150 values between the isopropyl (42) and 2,4,6-trimethyl
compounds is small (150 1.5 X 10~5 to 5.5 X IQ~5), the lower activity
of the t.-butyl (45_) derivative may be due to steric effects, possibly
to inhibition of rotation. The order of inhibition with the ortho-
alkoxy analogs was propoxy > ethoxy - methoxy but again difference
in activity was slight.
Toxicity - None of the ring substituted £-(methylcarbamoyl)benzald-
oximes were toxic to house flies when used alone. Cotreatment with
5 parts of piperonyl butoxide to 1 part of carbamate increased
toxicity to house flies in some cases but unusual examples of syn-
ergism were not achieved. The m-nitro- C5Q) and the £-isopropyl-
benzaldoxime C&2J carbamates were the only compounds which showed
significant activity against house flies with piperonyl butoxide.
Overall, the poor insecticidal activity of these compounds was dis-
appointing but not entirely unexpected owing to the generally weak
activity previously found for the J3-(methylcarbamoyl)oximes of sub-
stituted acetophenones.
Four ()-(methylcarbamoyl)ketoximes also are listed in Table 3 as a
continuation of previous work with these compounds , The cyclo-
propyl thienyl ketoxime carbamate was the only compound in the entire
series which proved to be significantly toxic without piperonyl
butoxide. All of the ketoxime carbamates, however, were significant-
ly synergized by means of piperonyl butoxide and these compounds
were included in this paper to point out the variety of organic
groups which may be incorporated into an oxime carbamate.
Conclusion - This series of substituted ()-(methylcarbamoyl)benzal-
doximes is less toxic to insects and slightly less active as inhibi-
tors of cholinesterase than the phenyl N-methylcarbamates. However,
they are more stable to alkaline hydrolysis. The structure-activity
study indicates that electron-withdrawing substituents increase
anticholinesterase activity which is opposite to the relationship
161
-------
found with the phenyl N-methylcarbamates. This would be logical if
there existed an optimum reactivity for the reaction with cholinesterase;
the multi-step reaction sequence for the inhibition process lends
itself to such a theory.
3. Substituted Acetophenone 0-(diethylphosphoryl)0ximes, Thermal
Rearrangement and Biological Activity.
Introduction - In an early study concerned with the relation between
structure of a series of ()-(diethyl phosphoryl)oximes of ring-substituted
acetophenones and biological activity, preliminary evidence was present-
ed which indicated that oxime phosphate esters containing electron-
donating substituents on the benzene ring were unstable and rearranged
to the imidoyl phosphate as exemplified below with the p_-methoxy
derivative (£3).
0
:N°OEt)2
Al 64
-D
-------
this material gave a mixture of p_-methoxy-acetophenone and p_-methoxy-
acetanilide as the organosoluble products, indicating that the compound
previously examined as J&i for toxicity and anticholinesterase activity
was actually a mixture of 6£ and (&.
For the preparation of j6Jt, the general procedure of Atherton £t_ ill. 12
was used by reacting equimolar amounts of silver diethyl phosphate
and £-methoxyphenylacetimidoyl chloride. Structures were confirmed
by pmr spectroscopy.
Thermal Rearrangement - Pmr analysis was the principal method used
for the study of the rearrangement of &3. The rate of rearrangement
was monitored by estimating the appearance of the C-methyl proton
of the imidoyl phosphate at 6 2.03 relative to the disappearance of
the acetophenone C-methyl protons at 6 2.30.
When a sample of pure j6^3_ was heated in a sealed tube under nitrogen
at 90° for 15 hr, the singlets at 6 2.30 and
-------
The thermal rearrangement of .6J3 was examined also in the presence
of a nucleophilic solvent, ethyl alcohol, at 70°C. The reaction was
examined spectrophotometrically every 10 min for 150 min and the
spectral changes are shown in Fig. 5. ^2 in ethyl alcohol shows
an absorption maximum at 265 nm which upon standing at 70° gradually
disappeared with the simultaneous appearance of a new maximum at
237 nm. The reaction was essentially complete after 150 min since
no further change in the spectrum occurred after another 2.5 hr.
The spectrum at this time was identical to the spectrum obtained
from equimolar amounts of ethyl N-p_-methoxyphenylacetimidate and
diethyl phosphoric acid in ethyl alcohol. The results indicate
that rearrangement of ,63_ and solvolysis of the intermediate
carbonium ion to give ethyl N-p_-methoxyphenylacetimidate takes place
according to the following equations.
0
II
(EtO)2PO"
(EtO) r02H'
"\
-N=C-
EtOH
OEt
CH3
(EtO)2PO~
In the absence of nucleophilic solvent the imidoyl phosphate,6j4_ is
formed.
Diethyl H-Acetyl-M-Substituted-Phenylphosphoramidates - During
the course of this study on the thermal rearrangement of substituted
acetophenone 0_-(diethyl phosphoryl)oximes to the substituted N-
phenylacetimidoyl diethyl phosphate, it occurred to us that further
rearrangement of the latter to diethyl N-acetyl-N-phenylphosphor-
amidates was possible according to the equation below. The
C=NOP(OEt),
A
164
-------
0
200
250 300 350
WAVELENGTH , millimicrons
400
Figure 5. Absorption spectra showing the rearrangement and solvolysis of p-methoxyacetophenone
0-(diethyl phosphoryl)oxime in absolute ethanol at 70°.
-------
phosphoramidate £ having the same empirical formula as a or b. would
give the same elemental analysis and alkaline hydrolysis conceivably
would give acetanilide by cleavage of the P-N bond. In order to
check out this possibility, several key diethyl N-acetyl-N-phenyl-
phosphoramidates were prepared and compared by pmr and infrared
analysis with the products obtained from the thermal rearrangement
of the oxime phosphate. The methyl protons in all cases appeared as
singlets at 6 1.98-2.00 and this coupled with infrared spectral data
indicated that £ was not obtained upon heating .a..
Examination of diethyl N-acetyl-N-phenylphosphoramidate and N-acetyl-N-
jD-methoxyphenylphosphoramidate, however, showed that each was moder-
ately toxic to house flies and effective as an anticholinesterase.
The unexpected biological activity, therefore, stimulated further
examination of these compounds and a series of diethyl N-acetyl-
N-phenylphosphoramidates were synthesized and assessed for insecti-
cidal and anticholinesterase activity. Biological data for the
various compounds are given in Table 4.
The data shows that all of the N-acetylphosphoramidates listed
in the table are moderately good inhibitors of house fly-head
cholinesterase with bimolecular inhibition constants in the region
of 10^ M~l min"!. The most potent inhibitor in the series was the
unsubstituted amidate (£5) which had a k^ value of 5.1 X KF M~l
min'l. Examination of the inhibition data showed no obvious correla-
tion with any of the reactivity parameters. Compounds containing
electron-withdrawing substituents such as the halogen in 2$), 2^.,
22.) 23. and 2J± were all less effective anticholinesterases than .6J5.
Compound j66_ which contains the p_-thiomethyl moiety (sigma constant
of zero) was almost equal to 6% in activity.
The toxicity data show that several of the diethyl N-acetyl-N-
phenylphosphoramidates were toxic to the house fly and toxicity was
enhanced by the use of piperonyl butoxide. As in the case of anti-
cholinesterase activity, ,6J> was most effective with an LDso value of
60.5 l-ig/g. Again, no obvious correlation between toxicity and
reactivity parameters was evident although it is noteworthy that the
two poorest anticholinesterases CZ1 and 2J&) were least toxic to
house flies.
Conclusion - The results show that ()-(diethyl phosphoryl)oxime of
ring-substituted acetophenones, particularly those with electron-
donating substituents, are thermally unstable and readily undergo
rearrangement to give the substituted N-phenylacetimidoyl diethyl
phosphate. The rearrangement product is extremely unstable to traces
of moisture and rapidly decomposes to the substituted acetanilide
and diethyl phosphoric acid. p_-Methoxyacetophenone 0-(diethyl
phosphoryl)oxime C43) may be prepared free from any rearranged
material by carefully avoiding heat during any part of the reaction
or work-up. Heat also must be avoided in preparing the corresponding
16.6
-------
Table 4. TOXICOLOGICAL PROPERTIES OF 0,0-DIETHYL N-ACETYL-N-PHENYLPHOSPHORAMIDATES
0
(/ \\-N-P-(OC2H5)2
Compound
£5 H
66 £-CH3S
£ -T _ /-ITT
O / p— UH-
68 £-CH30
(69 £-CH 0
70 £-Cl
Zi 2rcl
72_ m-Cl
73 £-Br
Zl £-F
75 o-iPr
I M
2.40 X 10"7
3.40 X 10"7
1.40 X 10"6
9.40 X 10~7
1.20 X 10~6
1.32 X 10~6
7.20 X 10~5
1.15 X 10~6
2.04 X 10"6
1.60 X 10~6
4.40 X 10~5
L
k, M"1 min"1
i
5.1 X 104
4.8 X 104
3.2 X 104
2.5 X 104
3.8 X 104
2.9 X 104
3.3 X 103
1.9 X 104
1.5 X 104
1.4 X 104
8.3 X 103
Musca
LD5Q ^
60.5
425
100
140
420
180
>500
180
200
145
>500
domestica Culex piplens
+ P.B. (1:5)
LD |ig/g LCcn PPm
j() j(J
35 4.7
56.5 >10
55 >10
59 >10
180 >10
60 >10
245 >10
47 >10
75 >10
60 >10
170 >10
-------
p_-methyl and unsubstituted analogs. The rather low Ea value (8.2
Kcal) for the thermal rearrangement of j6J3 provides a measure for
the instability of 0-(diethyl phosphoryl)oximes of ring-substituted
acetophenones containing electron-donating substituents. Overall,
the results show that care must be taken in avoiding heat when com-
pounds of this type are prepared.
The compound reported previously as .63. was unusually potent as
an inhibitor of fly-head cholinesterase, the molar concentration
to give 50% inhibition of enzyme (150) was 7.2 X 10"^ and moderately
toxic to house flies, LD5Q 27 ng/g/fly. However, j6£ prepared with-
out application of any heat, was virtually nontoxic to the house
fly (LD5Q > 500 ^tg/g) and was about 500-fold less effective in
inhibiting fly-head cholinesterase (150 3.45 X 10"^). Thus, it
appears that the strong anticholinesterase activity and house fly
toxicity reported earlier for .6J3 was caused by the rearranged product
.6Jt rather than ;63_. Although £4 undoubtedly is formed when 6^ is
heated, it is possible also that tetraethyl pyrophosphate (TEPP)
also may be formed in the presence of small amounts of moisture
according to the equations below:
0 0
64 +HO 1 (EtO)0POH + CH.O-V V-NHCCH.
—— L 2. j \— / j
00 0
/O II II /7~\ II
64+(EtO)0Px > (EtO)0POP(OEt)0 + CH-O-/7 N)-NHCCHQ
The reaction between dialkyl imidoyl phosphate and dialkyl phosphoric
acid to give the corresponding pyrophosphate has been established
by Atherton et al. ^. Therefore, the possibility exists that
TEPP also may be contributing to the toxicological properties of
impure .6J3.
Comparison of pmr and infrared spectra showed that diethyl N-
acetyl-N-phenylphosphoramidates are not produced from the thermal
rearrangement of the corresponding 0_-(diethyl phosphoryl)oximes of
substituted acetophenone. The N-acetyl-N-phenylphosphoramidates
065~-Z5), however, were of considerable interest owing to their
unexpected anticholinesterase activity and toxicity to insects.
These compounds are unusual in that the most likely bond to break
when cholinesterase enzyme is phosphorylated in the inhibition
process is the P-N linkage and apparently the acetanilide anion
is the leaving group. Compounds of this type deserve further
examination as possible insecticides.
168
-------
4. Toxicologocal and Hydrolytic Properties of S-Aryl Phosphora-
midothioates
13
Introduction - Previous studies concerning the relationship
between structure, reactivity and insecticidal activity of ()-alkyl
;S-alkyl phosphoramidothioates revealed that several of the compounds
were exceptionally toxic to the housefly, although they were rela-
tively weak inhibitors of fly-head AchE. One of these compounds,
Monitor or 0,^-dimethyl phosphoramidothioate has been developed
as a useful insecticide. The outstanding insecticidal properties
of compounds of this type, combined with the limited amount of
information available on the chemistry and mode of action of phos-
phoramidothioate esters, prompted us to extend our investigations
to include phosphoramidothioates containing aryl moieties. This
section is concerned with the chemical, biochemical, and toxicological
Rl ,°
^T//
properties of 0-ethyl £-aryl phosphoramidothioates and related
esters of the general structure indicated (A) where RI is NH2,
NHCH3 or N(CH3)2, and R2 is C2H5 or OC2H5.
Synthesis - 0,0-Diethyl substituted S^-phenyl phosphorothioates were
prepared by established procedures from the condensation of the
substituted sodium benzenethiolate with diethyl phosphorochloridate
or by the reaction of triethyl phosphite with the appropriate
benzenesulfonyl chloride. The ()-ethyl S^-phenyl phosphorochlorido-
thioates were prepared from the corresponding (),()-diethyl S^-phenyl
phosphorothioates by the action of phosphorus pentachloride according
to the procedure used to convert phosphonates to phosphonochloridates.
Evidently, this procedure has not been applied to phosphate or
phosphonate esters containing an £-phenyl moiety but the reaction
was quite successful, as yields up to 64% were obtained. The
()-ethyl S^-phenyl phosphoramidothioates were prepared from the
corresponding chloridothioates by reaction with ammonia or the
appropriate amine.
Hydrolysis - Data for the alkaline hydrolysis and toxicological
properties of the various S^-aryl phosphoramidothioates and phos-
phonamidothioates are given in Table 5. Alkaline hydrolysis rates
were determined spectrophotometrically by estimating directly the
amount of arylthiolate ion formed under pseudo-first-order conditions
of excess sodium hydroxide. Excellent first-order plots were obtained
in all cases, indicating that hydrolysis occurred essentially by
P-S bond cleavage.
For the various 0-ethyl substituted S_-phenyl phosphoramidothioates
in Table 5, a linear relationship was observed between the logarithm
169
-------
of the calculated second-order hydrolysis, constants, k2, and Hammett's
a constants derived from substituted benzoid acids. From the line
fitting the data (correlation coefficient r_ =* 0.99), the value of 1.27
for the reaction constant, p, was calculated. This value is very
similar to the p values of 1.03 and 1,32 calculated for the hydrolysis
of (),()-diethyl substituted jS-phenyl phosphorothioates-^ and diethyl
substituted phenyl phosphates,. respectively. The close similarity of p
values in these cases suggests that the hydrolytic reaction in these
three cases takes place by a common mechanism.
Comparison of .78, .84, and jJJj, compounds of identical structure except
for the number of methyl groups on the amido nitrogen, shows that
sequential replacement of hydrogen by a methyl group results in marked
increase in hydrolytic stability, e.g., k2 (M"1 min"1) is 56.0 for J&,
1.1 for .§£, and 4.97 x 10~3 for .&5. Similarly, the k2 value of 0.63 for
the ttf-methylphosphonamidothioate, _&§, is approximately 11-fold greater
than k2 of 0.050 for the N,ll-dimethyl analog, M- The difference in
this case, however, is much smaller than the 200-fold difference between
the analogous phosphoramidothioates, j£4, and j$%.
±
Values for the energy (Ea) and entropy (As_ ) of activation for 7&> .84,
^5, ;8J>, and §2 are given in Table 5. The data show that the range in
Ea for the five compounds is small (14.0-15.7 kcal/mol). In comparison,
the change in AS_- is much more substantial, becoming uniformly more
negative with each replacement of hydrogen by a methyl group. On this
basis it appears that the decrease in k2 with nitrogen substitution is
caused more by steric restraints than by electronic effects.
The rate constant, k2, for the hydrolysis of .84, a phosphoramido-
thioate, is almost two-fold larger than k2 for the corresponding
phosphonamidothioate, .86. The significant but faster rate of hydrol-
ysis of .84 compared to .86 was unexpected, since phosphonate esters
generally are less stable to alkali than phosphate esters. From
the values of Eg and AS* for .84 and .86, the free energy of activation
Al?* was calculated to be 17.8 and 18.1 kcal/mol, respectively,
values which are consistent with the second-order hydrolysis constants
(k2).
Cholinesterase Inhibition and Toxicity - The data in Table 5 for
anticholinesterase activity show that all of the primary phosphor-
amidothioates (Z6,-.IL3) are moderately strong inhibitors of fly-head
cholinesterase with k^ values (M~ min ) ranging from approximately
1 x 10^ to 5.9 x 10->. Attempts to correlate rates of cholinesterase
inhibition with k2 or any of-- the free energy parameters for ring
substituents (cf, ir,ir2 ), or combination of these parameters, proved
to be unfruitful. The failure to obtain a suitable relationship
between k^ and the various parameters was disappointing, since an
excellent correlation between k and a combination of TT and hydrolysis
170
-------
rates previously was demonstrated for a series of methyl substituted
phenyl N-methylphosphoramidates.
Progressive substitution of the hydrogens on the amido nitrogen by a
methyl group resulted in expected decrease in anticholinesterase
activity. Within this limited series of compounds a reasonably good
linear relationship was obtained between log k^ and log k£, indicating
that anticholinesterase activity is primarily a function of the
reactivity of the ester.
The primary phosphoramidothioates in Table 5 showed wide variability in
their toxicity to houseflies compared to their activity as anticholin-
esterases. In general, these esters were substantially less toxic than
the primary (),j>-dialkyl phosphoramidothioates^. For example, 0_,S_-
dimethyl phosphoramidothioate (Monitor) with an LD^Q to houseflies of
1.3 (ig/g is approximately six-fold more effective than (J-ethyl _§_-£-
chlorophenyl phosphoramidothioate (.78), the most toxic compound in this
series. Compared to Monitor (k± = 9.2 x 102 M min"1), however, 78
is about 450-fold more effective in inhibiting fly-head cholinesterase.
It appears that anticholinesterase activity is not a useful guide for
the prediction of the insecticidal activity of phosphoramidothioates.
As in the case of cholinesterase inhibition, progressive replacement of
hydrogens on the amido nitrogen by methyl groups resulted in a decrease
in housefly toxicity. For example, the housefly LD5Q (p,g/g) of the
primary amidate C7J5) was 8.2, the monomethylamidate (84) was 13, and
dimethylamidate was 83. The effect of sequential methyl substitution
on the reduction of toxicity, however, is much less in comparison to
the £,j3-dialkyl phosphoramidothioate analogs, e.g., the toxicity of the
IJ-methyl derivative of Monitor to houseflies (1050, |j,g/g) was 49 and of
lJ,_N-dimethyl was >500.
The virtual absence of toxicity demonstrated by £-p_-chlorophenyl N-
methyl-P-ethylphosphonamidothioate (86) toward houseflies (LD^Q >500
pg/g) was surprising since measurable toxicity was found for the
corresponding K[,tJ-dimethyl analog (LD^Q 75 p,g/g) . Unfortunately,
comparison with the primary phosphonamidothioate was not possible since
all attempts to synthesize this compound were unsuccessful. Further,
the corresponding N-methylphosphoramidothioate analog (84) with k^
and k2 values similar to those of_86 also was quite toxic to flies.
The toxicity of _§£, however, was pronouncedly synergized when applied
together with 5:1 parts piperonyl butoxide (LD5Q 17 p,g/g) . The level
of synergism of this magnitude, although quite common for methyl-
carbamate esters, is unusual for organophosphorus esters and suggests
that_86, for some reason, is peculiarly susceptible to oxidative
detoxication.
In limited studies using the white mouse, an LD5Q value of 5-8 mg/kg
was obtained forJ&j This compound, therefore, is approximately
171
-------
of equal toxicity to houseflies and mice.
Conclusion - The excellent correlation obtained between a and log k2
for the primary phosphoramidothioates CZ&-.&3) , combined with the
similarity in the magnitude of p calculated for this series and for
substituted phenyl diethyl phosphates and phosphorothioates, indicates
that substituent effects are transmitted from the ring to the phos-
phorus atom with equal facility through sulfur and oxygen. The alkaline
hydrolysis of diethyl phenyl phosphates and diethyl !3-phenyl phos-
phorothioates undoubtedly takes place by nucleophilic attack of
hydroxide ion on the phosphoryl center. In light of the similarity
in P values for the three series of esters, it appears that S^-phenyl
phosphoramidothioates also hydrolyze by a direct displacement reaction
on phosphorus, as shown below.
HO
R,R' = methyl or hydrogen
The values for Ea and AS* for 2&, £4 and §£, and for £§. and 82.
also are consistent with a direct displacement mechanism. As indicat-
ed earlier, there is a small but significant increase in Ea, accomp-
anied by a larger but uniform decrease in AS^ with each replacement
of hydrogen by a methyl group. The decrease in k2, therefore, can
be accounted for primarily on the basis of polar and steric effects
created by each succeeding methyl group.
It is apparent from the toxicological data that the insecticidal
activity of the various phosphoramidothioates and phosphonamido-
thioates in Table 5 can not be anticipated from the structure nor
from any of the reactivity parameters. jS-Aryl phosphoramidothioates
generally are less toxic to insects than the simple j3-alkyl phos-
phoramidothioates, although they are substantially more effective
as anticholinesterases. However, because of the unpredictable
variability in their insecticidal activity, other esters of related
structure deserve to be examined.
5. Physical and Chemical Basis for Systemic Movement of Organo-
phosphorus Esters in Plants
Introduction - Relatively little information is available on the
relationship between chemical structure and the ability of the
172
-------
Table 5. TOXICOLOGICAL AND HYDROLYTIC PROPERTIES OF S-PHENYL PHOSPHORAMIDOTHIOATES OF GENERAL STRUCTURE A
Rl
26 NH2
ZZ NH2
2!L NH2
IS. m2
.80 NH_
£.
Si NH2
w -§2 NH,
£.
-83. NH2
84 NHCHC
D
J5 N(CH3)2
M NHCH-
J
J7 N(CH )
R2 X
ClC1 H H
OC-H, F
OC2H5 cl
OC2H5 Br
OC2H5 CH3
°C2H5 C2H5
OC2H5 (CH3)2CH
OC2H5 (CH3)3C
OC2H5 Cl
AP U PI
OO.H Cl
CW PI
? S
CTT pi
25
Fly-head ChE
k.' X 10~5
(M'1 min-1)
37.5°
2.10
0.961
4.32
5.60
5.27
5.86
4.37
2.60
0.262
0.0924
0.545
b
Muse a
domes tica
LD50 (jig/g)
22.0
27.0
8.2
11.0
12.5
55.0
32.0
100.0
13.0
83.0
>500 (17)a
75.0
Culex
pipiens k,
LC5Q (ppm)
0.096
0.012
0.15
0.024
0.044
0.34
>1.0
' >1.0
0.92
>1.0
0.92
0.93
2nd order
. (M"1 min'1)
29.5° E (Kcal/mole) AS~ (e.u.)
25.8
31.6
56.0 +14.0 -6.3
55.7
17.3
17.1
17.4
16.9
1.10 +14.4 -12.8
0.00497 +15.6 -19.6
0.632 +15.5 -10.2
0.0551 +15.7 -14.3
vlith piperonyl butoxide 5:1 (w/w).
A linear pseudo-first-order inhibition rate could not be obtained.
-------
compound to be translocated in a plant. Since a wide variety of
organic insecticides of different physical and chemical properties
have been demonstrated to move systemically in plants, the question,
what are the critical factors which govern systemic in movement,
has often been raised. The following discussion presents results
of a systematic analysis of the relation between systemic movement
in the cotton plant of a series of phosphor midothioate esters and
their octanol-water partitioning properties. These compounds were
selected because of the known systemic insecticidal activity and
stability in plants of 0,Jv-dimethyl phosphoramidothioate (Monitor),
the parent compound of the series.
Synthesis and Method of Analysis - 0,5^-Dialkyl phosphoramidothioates
were prepared in an earlier study.13 The 0,0-dimethyl N-alkylphos-
phoramidothioates were prepared according to conventional methods
by reaction of the desired amine with dimethyl phosphorochlorido-
thionate in anhydrous ether. Liquid products were purified by
distillation at reduced pressure and solid products by recrystalli-
zation from n-hexane. The (),^-dimethyl N-alkylphosphoramidothioates
were isomerized to (),S^dimethyl N-alkylphosphoramidothioates by
heating the thionate in a several fold excess of methyl iodide.
Octanol-water partition coefficients were determined according to
Fujita et al.^' Analysis of each phase was conducted by gas-liquid
chromatography (glc) or UV spectrophotometry. Glc also was the
principal means for the estimation of translocated materials in
leaf tissue. Excised cotton leaves placed in water were treated
on the petiole 2" below the leaf blade. The amount of material
moved from the petiole surface into the blade was analyzed after
appropriate time intervals.
Octanol-Water Partition - Relevant data concerning octanol-water
partitioning properties of the various phosphoramidothioate esters
and miscellaneious organophosphorus insecticides are presented in
Table 6. For the Q,S^-dimethyl N-alkylphosphoramidothioate analogs
C§5~£6) a gradual increase in the octanol-water partition coefficient
(P) was observed as the N-alkyl chain size was increased, although
there appeared to be an abrupt increase between the N-ethyl and N-
propyl derivatives (compare f$% and 5fi)• Branching in the alkyl
group of the N-butyl analogs CSi and JJ£) did not result in any
significant change in partition coefficients. The partition coeffi-
cients for the limited number of ()>.§.-dialkyl phosphoramidothioates
G£Z~.25) > as expected, also increased with lengthening of the alkyl
chain on oxygen and sulfur but the relative increase appeared to
be slightly greater than with the N-alkyl derivatives.
Water solubility, in the absence of significant inductive or polar
effects, should be inversely related to octanol-water partition
coefficients. As expected, the water solubilities of the analogous
series of (),£>-dimethyl N-alkylphosphoramidothioates decreased as
174
-------
Table 6. PHYSICAL AND CHEMICAL PROPERTIES OF O.S-DIALKYL N-ALKYLPHOSPHORAMIDOTHIOATES
AND OTHER ORGANOPHOSPHORUS INSECTICIDES
RO ^0
/<
T?Q M— B '
IVO 0! IV
1
H
% Distribution Partition
R R'
o Q fti r*i3
jfta 3 3
Q Q f^tl /"* U
,£52 ^"2 25
on r*u « F u
-« ^-"3 H'^Q^y
Ql r*H n ••("* H
Q o r*H c — (* H
52 CH3 iso-C4H9
56 CH3 t-C4H9
95 CH n— C H
96 CH3 a-C8H17
31 CH3 H
3& C2H5 H
OQ « r1 13 u
ZZ n-C-jH^ it
100_ demeton thiol
101^ dimethoate
iQZ. methyl paraoxon
octanol
phase
46.22
54.21
81.75
89.71
89.85
90.37
90.02
92.79
97.00
18.00
58.50
94.50
98.85
76.25
95.00
water
phase
53.78
45.79
18.25
10.29
10.15
9.63
9.98
7.21
3.00
82.00
41.50
5.50
1.15
23.75
5.00
coefficient
121
0.86
1.18
4.48
8.71
8.85
9.39
9.03
12.87
32.33
0.22
1.41
17.20
85.96
3.21
19.00
Log P
-0.07
0.07
0.65
0.94
0.95
0.97
0.96
1.11
1.51
-0.66
0.15
1.23
1.93
0.50
1.28
0.59
0.73
1.31
1.60
1.61
1.63
1.62
1.77
2.17
0.00
-
-
— ,
Water
solubility
(mg/ml, 25"C)
>1000.00
> 1000. 00
. 176.10
58.61
67.78
-
78.88
4.92
0.36
>1000.00
, >200.00
-
2.003
25.00a
6.43
E
— s
-1.24
-1.31
-1.60
-1.63
-2.37
-2.17
-2.78
-1.60
-1.57
-
-
-
-
-
.
-------
Table 6 continued.
103 paraoxon 97.50 2.50 38.84 1.59 - 2.4
104 naled 96.00 4.00 24.00 1.38
-------
the length of the alkyl chain was increased and the n-octyl analog
C9JD was found to be the least soluble compound in the series.
The octanol-water partition coefficients of the other organophos-
phorus esters were in the order: demeton thiol > paraoxon > naled >
methyl paraoxon > dimethoate. The literature values for the water
solubilities of these compounds were found to be in the reverse order,
except for naled for which a water solubility value was not available.
Systemic Movement - Data for the movement of the various organo-
phosphorus esters from the leaf petiole surface into the leaf blade
are given in Table 7. From the relative amounts translocated at the
indicated time intervals, the values for the rate of trans location
(ktr) were calculated. Examination of the data for the 0^,8^- dimethyl
N-n-alkylphosphoramidothioate series C§fi~Si» J9J>, .9J>) reveals the
existence of a parabolic relationship between the rate of translocation
and octanol-water partition coefficients and optimum rate was found
with the N-propyl derivative 0£Q) • The parabolic relationship
which is obtained is shown graphically in Figure 6 where log ktr
is plotted against log P. A similar plot was obtained when log
ktr was plotted against ir. This is to be expected since for an
analogous series of compounds ir is directly proportional to log P.
The values of ktr and IT for the 0_,S^-dimethyl N-n-alkylphosphor-
amidothioates were subjected to multiple regression analysis. By
linearly combining ir and TT^ terms the following regression equation
13 was obtained (r_ = correlation coefficient, n = number of compounds).
Log k = -0.753 + 2.318ir - 0.974ir2 (13)
r = 0.999 n = 6
The excellent value for the correlation coefficient (IT) indicates
good fit of the data to the equation.
For the limited number of 0_,£5-dialkyl phosphoramidothioates examined
(32, 9& and 22), a similar parabolic relationship, as with the
N-n-alkyl derivatives, was observed between ktr and IT (or log P).
The replacement of the methyl groups in $2 with ethyl (.9J3) resulted
in increased lipophilicity as well as in systemic movement. However,
substitution with propyl evidently resulted in excessive lipophilicity
and ktr for the di-n-propyl derivative (9_2) was substantially less
than that for .9JJ.
Although the (D,S^dialkyl phosphoramidothioates and 0_,S/-dimethyl
N-n-alkylphosphoramidothioates are structurally closely related to
each other, it appears that the two series of compounds follow a
177
-------
Table 7. TRANSLOCATION DATA FOR THE VARIOUS O.S-DIALKYL N-ALKYLPHOSPHOR-
AMIDOTHIOATES AND
OTHER
ORGANOPHOSPHORUS INSECTICIDES
R°\p*°
RSX XN-R'
1
H
Rate of
% of Applied amount
moved
Time
R R' i
S& CH-j CH3
83. CHj ^2^5
22 CH n-CQH7
j ~ j f
Q 1 pu n — f1 H
QO PT4 K™f H
53 CH3 lso-C4H9
"^^** 3 ^" & 9
oc pu ««.f" H
26 CH3 n-CgH17
S2 CH3 H
3& C2H5 H
22 n-C3Hy H
J.BQ demeton thiol
iQi dimethoate
Jj£2 methyl paraoxon
±Q2. paraoxon
iSA naled
hrs) 2
3.31
3.
3.
7.
3.
1.
0.
0.
0.
3.
2.
2.
11.
0.
0.
2.
-
29
12
04
21
80
12
22
68
85
44
25
45
00
30
39
4
5.59
8.
11.
14.
10.
. 7.
0.
7.
0.
9.
12.
10.
18.
3.
0.
8.
-
63
13
00
21
50
28
26
91
47
90
86
12
00
84
25
8
15.97
21.
25.
30.
22.
13.
0.
15.
3.
22.
25.
33.
21.
9.
3.
20.
-
37
70
90
60
85
67
30
18
00
90
15
82
50
02
86
ii
21.35
29.
40.
39.
27.
15.
0.
23.
4.
37.
59.
42.
16.
27.
9.
34.
-
23
81
90
30
10
68
28
80
13
10
55
83
35
39
90
movement
in
2
r
petiole for
1
2
3
3
2
0
0
2
0
3
5
3
2
3
1
3
1
.97
.57
.71
.24
.13
.95
.05
.00
.48
.45
.77
.92
.61
.04
.07
.33
.77b
slope
0.967
0.981
0.999
0.970
0.937
0.869
0.768
0.999
0.991
0.998
0.940
0.944
i.oooa
0.932
0.926
0.999
-
Regression analysis for three points only. Rate of movement for first 4 hrs only.
178
-------
0.8
0.6
0.4
- 0.2
H—
_*:
o 0.0
-0.2
-0.4
-0.6
98
,-Q.
j_
O96
-0.8 -0.4
0.0
1.4
LOG P
0.8
1.2
1.6
Fig. 6. Relationship between log ktr and log P for phosphoramidothioate
esters; solid line is for (),^-dimethyl IJ-n-alkylphosphorainidothioates
and dotted line is for (),jS-dialkyl phosphoramidothioates.
179
-------
0.6
0.3
0.0
v- -0.3
H—
CD
O
-1 -0.6
-0.9
-1.2
-1.5
SO
x " " "" ^ v"
12 x'
- ^
53
""•
... 1 1 1
\
. \
0 95
\
\
\
•' \
\
\
^
3
1 1
0.5
1.0 1.5
n
2.0 2.5
Fig. 7. Relationship between log ktr and IT for £,jv-dimethyl N-
alkylphosphoramidothioates; dotted line is for n-alkyl esters and
solid line is for branched butyl esters.
180
-------
separate parabolic relationship. This is evident from the dotted
curve in Figure 6 and from regression analysis when data for 3&
and .9J) were included with those of the N-n-alkylphosphoramidothioates.
In this case, regression equation 14 with a correlation coefficient
of 0.844, was obtained.
Log k = -0.736 + 2.401ir -0.992ir2 (14)
r = 0.844 n = 8
The significantly poorer correlation coefficient obtained compared
to the (),JS-dimethyl N-n-alkylphosphoramidothioate series (cf equation
13) indicates that the N-n-alkyl and unsubstituted phosphoramidothio-
ates should be treated separately. Evidently, alkyl substitution
on the amido nitrogen atom results in compounds with reduced rates
of systemic movement in the cotton plant, even though individual
N-substituted and unsubstituted phosphoramidothioates may have
similar octanol-water partitioning properties.
Profound changes in systemic movement were observed when branching
was introduced into the N-alkyl chain. For the various isomeric
N-butyl derivatives (.9J., %£, %£ and 24_), marked reduction in ktr
values occurred with increase in branching and translocation was
minimal with the t>butyl derivative C24J. In spite of rather similar
values of TT for the four different butyl isomers (1.60-1.63), there
was approximately a 65-fold difference in ktr between the n-butyl
and Jt-butyl derivatives with the values of the other two isomers
ranging in between. The relation between ktr and for the four
isomeric butyl derivatives is shown graphically in Figure 7 by the
solid line. These results indicate that within the series of nine
^),£-dimethyl N-alkylphosphoramidothioates, solvent partitioning
properties alone, i.e., TT Or log P, do not totally account for
systemic movement.
The reduction in systemic movement with increase in branching in
the N-butyl chain suggests that steric effects also should be
considered in seeking correlation of the sytemic data. For the (),.S-
dimethyl N-alkylphosphoramidothioates C8J3-.9J>), including the branched
butyl derivatives, the following regression equation relating ktr
and if was obtained. The values for F (F-statistics) and standard
error (s) are included.
Log k = 0.307 + 0.273* - 0.267*2 (15)
r = 0.372; n = 9; s_ = 0.636; F_ = 0.483
181
-------
Use of Taft's steric substituent constant (Eg) alone in the analysis
resulted in equation 16.
Log ktr » 1.498 + 0.782E (16)
s
r = 0.680; n = 9; s. = 0.465; F = 6.033
From the magnitude of the correlation coefficients for equation 15
and equation 16, evidently neither Es alone nor the combination of
IT and IT alone are able to provide significant correlation with the
translocation data. On the other hand, linear combination of TT,
9
ir^, and Es resulted in substantial improvement in correlation, in
accordance with equation 17 below.
Log k = - 0.196 + 4.187TT - 1.551TT2 + 1.209E (17)
tr s
r = 0.863; n = 9; s = 0.379; F = 4.865
The F-statistics show significant correlation at the 10% level.
These results provide quantitative evidence that steric effects, in
addition to solvent partitioning properties, are important in
predicting systemic movement.
In addition to octanol-water partitioning properties, water
solubilities of several representative phosphoramidothioates were
determined (Table 6). The results indicate that, within the limits
of the compounds examined, water solubility is not a critical factor
in regulating the degree of systemic movement. For example, compounds
.8J5 and j§£ with solubilities of >1000 mg/ml were decidedly poorer in
their ability to penetrate and move through the petiole than was
51, a compound of much lower water solubility. In contrast, the
highly water soluble (),£-dimethyl phosphoramidothioate (52) trans-
located rapidly and the poorly soluble n-octyl derivative (56)
quite slowly. Similarly, the systemic movement of the other organo-
phosphorus esters QfiQ-iOJt) evidently was not related to their
water solubilities.
Conclusion - The foregoing results clearly indicate that even within
a relatively restricted series of compounds, systemic movement in
the cotton plant is not directly correlated with any single chemical
or physical parameter. This is not at all surprising since penetration
and translocation of foreign materials in a plant are doubtlessly
complex processes and a number of different factors must be involved.
182
-------
The development of TT as a useful parameter originated from an
attempt to correlate plant growth promoting properties with the
structure of a series of substituted phenoxyacetic acids.18 In
this classic study TT was developed to account for substituent effects
on the rate of penetration of an externally applied acid to the site
of action. Penetration to the site of action was visualized as a
random walk process involving multiple partitions between lipophilic
and hydrophilic phases in the plant and the addition of TT to the
regression equations provided highly significant improvement to the
correlation. Therefore, the failure of if (or log P) to correlate
satisfactorily the rate of systemic movement of the phosphoramido-
thioates (compounds £§-.£5) was unexpected. Only in the limited
case of the N-n-alkyl derivatives was satisfactory correlation obtained
with IT and ir- alone.
There is little doubt that steric effects play a significant role
in determining the rate in which the N-alkylphosphoramidothioates
penetrate and move in the cotton leaf petiole. The poor ability of
the isobutyl (j?3J or _t-butyl (%£) derivatives to move systemically
may be explained purely on the basis of the bulkiness of the branched
butyl moieties which may impede the movement of the molecule through
plant cuticle and membranes. Another possible explanation resides
in hydration effects associated with the amido moiety. The effect
of alkyl substituents in limiting the extent which water is solvated
to amines is well known and the possibility remains that hydration
of the amido moiety, or its absence owing to steric effects, may
drastically affect rates of systemic movement. For example, in the
case of 0%, the bulky _t-butyl group prevents solvation by water to
occur, thus rendering the molecule more hydrophobic and increasing
the possibility of retention by lipophilic phases in the plant. On
the other hand, compounds without alkyl substituents on the amido
proton probably are highly sovated by water and hydrophobic inter-
action with lipophilic plant constituents which reduces penetration
and systemic movement is minimized. The greater than expected sys-
temic movement of the 0,8^-dialkyl phosphoramidothioates C22-.9J))
is consistent with this kind of interpretation.
6. References
1. Holan, G. New Halocyclopropane Insecticides and the Mode of
Action of DDT. Nature 221:1025-9, 1969.
2. Taft, R. W., Jr. Separation of Polar, Steric, and Resonance
Effects in Reactivity, in Steric Effects in Organic Chemistry,
M. S. Newman, Ed., Wiley, New York. 1956. pp. 556-676.
3. Hansch, C., and T. Fujita. A Method for the Correlation of
Biological Activity and Chemical Structure. J. Amer., Chem. Soc.
86:1616-1626, 1964.
183
-------
4. Swain, C. G., and E. C. Lupton. Field and Resonance Compo-
nents of Substituent effects. J. Amer. Chem. Soc. 90:4328-37,
1968.
5. Metcalf, R. L., and T. R. Fukuto. The Comparative Toxicity
of DDT and Analogues to Susceptible and Resistant Houseflies
and Mosquitoes. Bull. Wld. Hlth. Org. 38:633-47, 1968.
6. Metcalf, R. L., I. P. Kapoor, and A. S. Hirwe. Biodegradable
Analogues of DDT. Bull. Wld. Hlth. Org. 44:363-73, 1971.
7. Brady, 0. L., and R. F. Goldstein. The Isomerism of the Oximes.
Part XXV. The Dissociation Constants of Some Isomeric Aldoximes.
J. Chem. Soc. 1918-24, 1926.
8. Tribble, M. T. Nmr Studies of Ortho-Substituted Phenols in
DMSO Solutions: Polar Effects of Ortho-Substituents. Ph.D.
Dissertation, Louisiana State University, Baton Rouge, La.,
1968.
9. Metcalf, R. L., and T. R. Fukuto. Effects of Chemical Structure
on Intoxication and Detoxication of Phenyl N-Methylcarbamates
in Insects. J. Agr. Food Chem. 13:220-31, 1965.
10. Fukuto, T. R., R. L. Metcalf, R. L. Jones, and R. 0. Myers.
Structure, Reactivity, and Biological Activity of 0-(Dialkyl
phosphoryl)oximes and 0_-(Methylcarbamoyl)oximes of Substituted
Acetophenones and o/-Substituted Benzaldehydes. J. Agr. Food
Chem. 17:923-30, 1969.
11. O'Brien, R. D., and B. D. Hilton. The Reaction of Carbamates
with Cholinesterase. Mol. Pharmacol. 2:593-605, 1966.
12. Atherton, F. R., R. J. M. Cremlyn, A. L. Morrison, Sir A. Todd,
and R. F. Webb. The Use of Imidoyl Phosphates as Intermediates
in the Synthesis of Pyrophosphates. Chem. Indust. 1183-5, 1955.
13. Quistad, G. B., T. R. Fukuto, and R. L. Metcalf. Insecticidal,
Anticholinesterase and Hydrolytic Properties of Phosphoramido-
thiolates. J. Agr. Food. Chem. 18:189-94, 1970.
14. Murdock, L. L., and T. L. Hopkins. Insecticidal, Anticholin-
esterase, and Hydrolytic Properties of 0_,p_-Dialkyl-S-Aryl
Phosphorothiolates in Relation to Structure. J. Agr. Food
Chem. 16:954-8, 1968.
15. Fukuto, T. R., and R. L. Metcalf. Structure and Reactivity
of Some Diethyl Substituted Phenyl Phosphates. J. Agr. Food
Chem. 4:930-5, 1956.
184
-------
16. Neely, W. B., and W. K. Whitney. Statistical Analysis of
Insecticidal Activity in a Series of Phosphoramidates. J.
Agr. Food. Chem. 16:571-3, 1968.
17. Fujita, T., J. Iwasa, and C. Hansch. A New Substituent
Constant, , Derived from Partition Coefficients. J. Amer.
Chem. Soc. 86:5175-80, 1964.
18. Hansch, C., P. M. Muir, T. Fujita, P. P. Maloney, *C. F.
Geiger, and M. J. Streich. The Correlation of Biological
Activity of Plant Growth Regulation and Chloromyatin Deriva-
tives with Hammett Constants and Partition Coefficients.
J. Amer. Chem. Soc. 85:2817-24, 1963.
185
-------
F. STUDIES ON THE OXIDATIVE CONVERSION OF PS TO PO ESTERS
The oxidation of phosphorothionate (PS) to the corresponding phos-
phate (PO) ester in plants and animals is a well established phenom-
enon. Since the toxic action of phosphorothionate insecticides
(generally poor inhibitors of the cholinesterase enzymes) is attri-
buted to the anticholinesterase action of the related phosphate
ester produced metabolically, the conversion of PS to PO esters
is considered an activation reaction. PS to PO conversion, therefore,
is an essential step in the intoxication of animals exposed to
phosphorothionate esters. This part of the report is concerned
with our studies on some of the fundamental aspects of this important
reaction.
1. Oxidative Rearrangement of 0-Ethyl S-Phenyl Ethylphosphonodithioate
(Dyfonate)
Introduction - In an earlier communication McBain et al. reported
that an oxygenated product was obtained from 0-ethyl S^-phenyl
ethylphosphonodithioate (Dyfonate) was treated with m-chloroper-
benzoic acid. A product with similar chromatographic properties
also was occasionally obtained when Dyfonate was incubated with
liver microsomes in the presence of NADPH2- On the basis of analytical,
spectral, and chemical evidence, the structure of this oxygenated
unknown was suggested to be a resonance hybrid or rapidly equili-
brating tautomer mixture of the structures la and Ib shown below.
EtO ^S-T y EtO'
la ^ - ' Ib
In connection with our investigation of the stereochemistry of the
oxidation of Dyfonate to its oxon we have determined that this
oxygenated material is phenyl ethoxy(ethyl)phosphinyl disulfide (II)
of structure given below. Proof for this conclusion is presented
as follows.
EtO' XS
II
Structure Analysis - The oxygenated product (II) was obtained accord-
ing to the procedure of McBain et al.l by treatment of Dyfonate
with m-chloroperbenzoic acid and purification by column chromato-
graph. II also was prepared by reaction between benzenesulfenyl
186
-------
chloride and 0-ethyl ethylphosphonothioic acid (ETP) following
the general procedure of Schrader and Lorenz^. Comparison of the
two samples of II obtained from the m-chloroperbenzoic acid oxidation
of Dyfonate and the reaction of benzenesulfenyl chloride with ETP
were identical. Proofs of identity were identical Rf values in four
tic systems, identical Rf values for the products (mainly ETP and
diphenyl disulfide) formed by storage decomposition, and superim-
posibility of all spectra. The mass spectra provided the clearest
evidence for the structure of compound II, and its identity with
the m-chloroperbenzoic acid oxidation product of Dyfonate. Parti-
cularly significant is the presence of ions at m/e 141 (Pb^"1")
(Fig. 1). Comparison of these spectra with the spectra of benzene-
thiol and diphenyl disulfide explains part of the intensity of the
ion at m/e 65 and the ions at m/e 77, 78, 109 and 110. Comparison
of intensities also indicates that the ions at m/e 141 and 142 are
not caused by the presence of a trace impurity of diphenyl disulfide.
This latter compound decomposed initially by loss of both PhS- and
PhS2- in analogy to compound II.
The ir spectra of sulfides and disulfides are in general very similar,
e.g. the spectra of diphenyl sulfide and diphenyl disulfide. An
even closer analogy is the near identity of the ir spectra of aryl
diethoxyphosphinothioyl disulfides and S^-aryl 0,0-diethyl phosphoro-
dithioates. It is, therefore, reasonable that compound II and the
Dyfonate oxon would have similar ir spectra.
31
The P nmr spectra of Dyfonate, the oxygenated Dyfonate and Dyfonate
oxon show upfield shifts of 6.5, 20, and 58 ppm with respect to
phosphorus oxide.1 The ^lp chemical shifts are dependent on both
the polarizability of the atoms attached to phosphorus and the double
bond order. Shielding decreases as the polarizability of the atoms
attached to phosphorus increases and as the percentage of the double
bond character decreases. It is not clear why a combination of
these factors should place the ->lp mm- shift of structures such as
la, Ib, etc. intermediate between that of Dyfonate and Dyfonate oxon.
However, on these arguments the 31p chemical shift of compound
II would be expected to be downfield from that of Dyfonate oxon.
The hydrolysis of the oxygenated Dyfonate (II) parallels the hydrolysis
of alkyl and aryl dialkoxyphosphinyl disulfides because in these .
compounds the disulfide bond is cleaved by a variety of reagents.
From this analogy, the observed decomposition of compound II predom-
inantly to ETP by disulfide bond cleavage rather than to f)-etnyl
ethylphosphonic acid (EOP) by phosphorus-sulfur bond cleavage (below)
is readily predictable. Bis-(dialkoxyphosphinyl) disulfides also
cleave in a smilar manner. These compounds and bis-(dialkoxyphos-
phinothioyl) disulfides readily lose sulfur in the presence of a
sulfur acceptor. Elemental sulfur was detected in the perbenzoic
acid oxidation of Dyfonate.*
187
-------
ETO
oo
oo
xoo -
*
s
50 -
40 -
30 -
20 -
10 -
^
M/e 65
•
»
^
J
x
-cs t
O?
77 78
Fragment POjH^' CgHs* CjHj*'
C5H5+
o.td«i« 36'° "•* 15'2
Eel. Inc. Z
Capd II 35.1 12.0 14.0
_^S (
u
M
\
\
1
- n,*«>s-
r\ -Phss-
V
-s -^KO)-
f
1
-PhS4 + H
'«
|,
' /\^^-^^
93 97 109 110 121 141 142 154
C2H6P02+ PS02H2*P PhS* PhSH*' It-?'0* PhSS* PbSSH*' E£?iPOStft"
100 6.0 22.4 12.1 36.4 9.2 4.4 4.8
100 5.4 21.6 10.8 36.0 8
.3 3.5 4.4
' M tH-l M+2
Fig. 1. Mass spectrum of compound II. Fragmentation pattern and relative intensities (average
of all spectra) shown for the m-chloroperbenzoic acid oxidation product of Dyfonate and for
compound II synthesized from benzenesulfenyl chloride and 0-ethyl ethylphosphonothioic acid.
-------
EtO
S-S-
(II)
disulfide cleavage
Et
phosphorus-sulfur cleavage
EtO SH
(ETP)
(EOF)
The failure to isolate Dyfonate oxon from the "oxygenated Dyfonate"
is as expected for compound II but is not explained by structures
such as la and Ib.
Conclusion - The proposed structure (II) for the m-chloroperbenzoic
acid oxidation product is, therefore, consistent with all of the
spectral and chemical evidence reported by McBain et al. for their
oxygenated Dyfonate. It appears likely that the oxygenated Dyfonate
(II) is not a significant enzymatically-formed intermediate in
microsomal systems. The *-"o labeling data from microsomal experi-
ments of McBain et al. on Dyfonate and similar data of Ptashne
et al. on parathion support this conclusion since the oxygen in
oxidatively formed ETP and diethyl phosphorothioic acid comes from
water. Caution must be thus exercised in drawing too close an analogy
between peracid oxidation models and microsomal systems.
2. Oxidative Rearrangement of H-(Dimethoxyphosphinothioyl)carbamate
Esters
Introduction - In connection with our studies on the comparative
metabolism of 2,2-dimethyl-2,3-dihydrobenzofuranyl-7 N-methyl-
N-(dimethoxyphosphinothioyl)carbamate (III) (see part B-3 ) in
mammals and insects it became necessary to synthesize the corresponding
N-dimethoxyphosphinyl derivative IV.
0
OCN:
0
OCN
III
IV
Attempts to synthesize IV by the condensation reaction used for III
failed and, therefore, it was decided to prepare IV by peracid oxida-
189
-------
tion of III. Treatment of III with m-chloroperbenzoic acid gave IV
in about 40% yield but also gave an unexpected rearrangement product
which subsequently was identified as the N-dimethoxyphosphinylthio
derivative V. This section reports results leading to the identifica-
tion of V as well as other products obtained from the oxidation of III.
(carbofuran)
Structure Analysis - Treatment of III with m-chloroperbenzoic acid
afforded IV and V as the principal products along with lesser
amounts of VI (carbofuran) and starting material (III) . Sulfur
also was isolated. VI was identical to material synthesized inde-
pendently from methyl isocyanate and the corresponding phenol.
Attempts to synthesize IV by the method used for III failed and its
structure was established from the following spectra data: IR,
1725 cm"1 (C=0) and 1260 cm"1 (P=0) ; NMR (6, 60 MHz, CDC13) 3.28
(doublet, J = 8 Hz, 3H, -NCH3) , and 6.8-7.2 (multiplet, 3H, aromatic
protons); MS, m/e 329 (M+, 8.8%), 166 (31.4%), 109 (100%), and 164
(10.8%). The structure of V was established from the following spec-
tral data: IR, 1725 cm"1 (C=0) and 1260 cm"1 (P=0) ; NMR (6, 60 MHz,
CDC13), 3.45 (singlet, 3H, -NCH3) , 3.95 (doublet, J = 13 Hz, 6H,
OCH3) and relevant absorptions similar to IV; MS, m/e 361 (M+, 10.6%),
198 (62.3%), 141 (100%) and 109 (34%). The downfield shift and
the change to a singlet for N-CHjj absorption for V is consistent with
the displacement of this moiety another atom away from phosphorus.
The value of m/e 198 (62.3%) in the MS of V is consistent with
[0=£N(CH3SP(0)(OCH3)2], m/e 166 ("1.4%) for IV with [0=*N(CH3)P
(0)(OCH3)2l and m/e 182 (29.4%) for III with [0=fN(CH3)P(S) (OCH3)2] .
Further fragmentation of each of these ions by the loss of methyl
isocyanate produced: m/e 141 (100%) [SP(0) (OCH3)2l+ for V which
then lost sulfur to give m/e 109 (34%) [OP(OCH3)2] ; m/e 109 (100%)
[OP(OCH3)2]+ for IV; and m/e 125 (100%) [SP(OCH3)2] for III.
Further evidence for the structure of V was obtained from its
decomposition on silica gel thin-layer plates. V evidently is
unstable to prolonged atomspheric exposure on silica gel and de-
composes mainly to a disulfide VII, the carbamate VI, and sulfur,
presumably by the following scheme.
190
-------
„ atmospheric
moisture
+ HOP(OCH3)2
VII
VII, mp 131-3°; NMR (6, 60 MHz, CDC13), 3.45 (singlet, 6H, -NCHj),
3.05 (singlet, 4H, -CH2), 1.25 (singlet, 12H, -CH3), and 6.8-7.2
(multiplet, 6H, aromatic protons); MS m/e 504 (M^, 5.4%), 447
(2.1%), 390 (6.2%), 164 (27%), 163 (100%), 149 (10%), 135 (27%),
and 107 (14.5%).
14 14
Oxidation of C-labeled II (N CH3) with m-chloroperbenzoic acid
also was carried out for quantitative determination of the products
by TLC. The results are presented in the table below.
Table 1. YIELDS OF PRODUCTS FROM THE OXIDATION OF III.
Oxidation products of III'
Decomposition products of
Compound
Radioactivity Compound
% Radioactivity
III
VI
V
IV
0.56
0.34
0.15
0.08
11.5
13.7
35.0
39.8
VI
IVC
VII
0.34
0.08
0.60
31.4
6.4
62.2
fBased on 92% total recovery.
Based on 977o total recovery.
It is uncertain whether IV is formed from V or whether this small
amount was present as a contaminant in V prior to TLC exposure.
Conclusion - The above information clearly reveals that oxidation
of III produces a rearrangement product (V) similar to that obtained
from the oxidation of Dyfonate. The results may be explained on
the basis that oxidation of III proceeds through an unstable S-oxide
intermediate^ which may be written in the following resonance and
tautomeric forms.
191
-------
0 0 S
s s f
M 1+ '
p- -p±
This intermediate explains the formation of IV and V as follows.
o »s- oo
»-
• M 7
ArOCN- P- (OCH3)2 - > ArOCN - P- (OCH3)2 + S
CH3 CH3
0 ,Sr-0 00
I (\V ,| ||
ArOCN- P-(OCH,)7 - > ArOCN-S-P-(OCH-),
I J * I J *
3. The Resolution and Determination of the Absolute Configuration
of Dyfonate and Related Compounds
Introduction - In order to study the stereochemistry of the PS to PO
activation reaction of thionate esters it became necessary to
resolve the isomers of Dyfonate and determine their absolute config-
urations. The results from this investigation are presented as
follows.
Synthesis and Resolution - The starting material in the synthesis
of resolved Dyfonate was 0-ethyl ethylphosphonic acid as shown below.
C H S a-phenyl- C H /S +
2 5Np'/ ethylamine 3 p^ _ H N-CHC H
/ \ ----- \ n u A n O I)
C2H50 OH 7 L2 5
(resolved) IX
PCI
W~ C2
(resolved Dyfonate) XI X
192
-------
Resolution of the (+)- and (-)- isomers of VIII was achieved by
fractional cyrstallization of its a-phenylethylamine salt IX. The
free resolved acid generated from IX was treated with phosphorus
pentachloride to afford the chloride X which was subsequently
reacted with benzenethiolate to give resolved Dyfonate (XI). The
last two reactions took place with minimum racemization. Synthesis
of resolved Dyfonate oxon (XII) was obtained by reaction of resolved
VIII with phenyldiazonium chloride.
Determination of Absolute Configuration - The (-)-rotating isomer
of IX was subjected to X-ray analysis. The crystal used for X-ray
analysis had dimensions 0.10 X 0.45 X 0.10 mm. Precession and
Weissenberg photographs showed mono-clinic symmetry and systematic
extinctions OkO with k = 2n + 1. Since the molecule is chiral,
the space group is uniquely determined as P2,.
The cell constants, a = 11.500 (13), b = 6.691(7), c = 11.327(13) A,
and (3 = 115.08(3)°, were determined from a least-squares fit of 12
carefully centered relections measured on a Picker automatic dif-
fractometer (MoKo?, \ = 0.71069 A). The density, 1.15 g cm'3,
measured by flotation in hexane/carbon tetrachloride, agrees with
the value 1.158 g cm"3 calculated for two molecules of C^2H22N02PS
in the unit cell.
Three-dimensional intensity data were collected on the above dif-
fractometer, MoK 6? radiation mode monochromatic by Bragg reflection
from a graphite crystal, scanning reflections in the 29-9 mode at a
scan rate of l°/min and a scan range of 2.0°, and taking background
counts of 10 sec at each end of the scan. Of 688 unique reflections
having 29 values less than 37°, 632 remained after rejection of those
with intensities less than 1.5o. These were put on a common scale
using the intensities of three standards (collected every 75 reflec-
tions) and corrected for Lorentz-polarization effects.
The structure was solved by the heavy-atom method, successive cycles
of Fourier synthesis, and subsequently full-matrix least-squares
refinement. Sulfur and phosphorus atoms were refined anisotropically,
whereas other atoms were refined isotropically. The final residue
R was 10.57o. All hydrogen atoms were observed on the difference
Fourier map, but not included in the refinement.
The absolute configuration of the (-)-O-ethyl ethylphosphonothioate
moiety was established as S^ by relation to the known configuration
of the §_-(-) -a-phenylethylammonium ion. A stereoscopic view of the
molecule (both cation and anion) is shown in Fig. 2. The quaternary
ammonium protons form hydrogen bonds to three neighboring anions,
one to the sulfur (3.29 A) and two to the charge-bearing oxygens
(2.78 and 2.77 A) of two separate anions.
193
-------
Fig. 2. Stereoscopic view of a-phenylethylammonium salt of 0-ethyl
ethylphosphonothioic acid. The double line indicates the hydrogen
bond.
Because Dyfonate is a liquid at room temperature, it was necessary
to obtain an analog which was a crystalline solid for X-ray analysis.
Fortunately, 0-ethyl 55-p-bromophenyl ethylphosphonodithioate (XIII)
also turned out to be a solid, albeit of rather low melting point
(31.5°C). Colorless crystals of the (-)-enantiomer of XIII were
obtained by slow evaporation of a water-methanol solution, and
subjected to X-ray analysis. Preliminary X-ray examination of the
crystals of XIII (performed at 19°C) revealed a triclinic symmetry
and the space group was determined as PI. Assumption of two mole-
cules of XIII per unit cell was in complete agreement with the
density both calculated from X-ray data and that obtained by the
flotation method (in CC1.-HCC1 ).
Although the two independent molecules in the unit cell have the
same chirality, they are apparently related by a pseudo center-of-
symmetry (excluding the ethyl and ethoxyl moieties in XIII). If
the two molecules were perfectly related by the pseudo center-of-
symmetry in the crystal, it would be impossible to determine the
absolute configuration. Fortunately the nature of the molecular
arrangement in the crystal is such that the overall geometry makes
the heavy-atoms in the two molecules in the unit cell slightly off
from being centrosymmetrical and this phenomenon made the determina-
tion of the absolute configuration of XIII possible.
The structures were solved by the heavy-atom method, through successive
cycles of full-matrix least-squares calculations. Bromine, sulfur
and phosphorus atoms were refined anisotropically whereas others
were refined isotropically. The absolute configuration of the chiral
194
-------
phosphorus center of the (-)-rotomer of XIII determined via the
anomalous dispersion effects of the bromine, sulfur and phosphorus
was eventurally assigned as S_. A stereoscopic view of one of the two
molecules in the unit cell is shown in Figure 3. The other molecule
is not included in the figure because the two molecules have identical
configuration and chirality.
Fig. 3. Stereoscopic view of one of the two molecules S_(-)-0-ethyl
S_-p-bromophenyl ethylphosphonodithioate ester (XIII) in the unit cell.
The absolute configuration of the isomers of Dyfonate oxon (XII) was
established by nucleophilic substitution reaction of the sodium
salt of (-)-0-ethyl ethylphosphonothioic acid (VIII) with phenyl-
diazonium chloride. Because bonds attached to the phosphorus atom
in the resolved acid are not affected by this reaction, the absolute
configuration of XII is identical to that of VIII.
Conclusion - The absolute configuration of the chiral isomers of
0-ethyl ethylphosphonothioic acid and 0-ethyl p-bromophenyl ethyl-
phosphonodithioate has been established by X-ray crystallography.
Since the stereochemistry of the reactions leading to Dyfonate and
the p-bromo analog are the same, the absolute configuration of
Dyfonate also was established. The absolute configuration of chiral
Dyfonate oxon was determined from its relationship to chiral 0-ethyl
ethylphosphonothioic acid.
The assignment of absolute configuration of the individual chiral
isomers of these compounds now makes it possible to study the
mechanism of the PS to PO activation reaction in biological and
model oxidation systems. This work is currently in progress.
4. References
1. McBain, J. B., J. Yamamoto, and J. E. Casida. Oxygenated Inter-
mediate in Peracid and Microsomal Oxidations of the Organo-
phosphonothionate Insecticide Dyfonate®. Life Sciences 10:
1311-9, 1971.
195
-------
2. Schrader, G., and W. Lorenz. Ger. Pat. 1,103,324 (October
20, 1959). Sulfenylthiophosphate and -thiophosphonate esters.
Chem. Abs. 56:5888c, 1962.
3. McBain, J. B., I. Yamamoto, J. E. Casida. Mechanism of Activation
And Deactivation of Dyfonate® (0-Ethyl S_-phenyl Ethyl phosphono-
dithioate) by Rat Liver Microsomes. Life Sciences 10:947-54, 1971.
4. Ptoshne, K. A., R. H. Wolcott, and R. A. Neal. Oxygen-18
Studies on the Chemical Mechanisms of the Mixed Function Oxidase
Catalyzed Desulfuration and Dearylation Reactions of Parathion.
J. Pharmocol. Exp. Therap. 179:380-5, 1971.
5. Herriott, A. W. Peroxy Acid Oxidation of Phosphinothioates,
a Reversal of Stereochemistry. J. Amer. Chem. Soc. 93:3304-5,
1971.
196
-------
G. STUDIES OF INSECTICIDE SYNERGISM AND INSECT GROWTH REGULATORS
During the course of our studies on insecticide synergists we dis-
covered that some of the intermediates which were synthesized on
route to substituted 1,3-benzodioxoles were not only effective
synergists but also were effective in preventing the normal develop-
ment of mosquito larvae into adults. For this reason, the results
obtained on insecticide synergism and insect growth regulators
are combined.
1. Carbaryl Synergism by Substituted 1.3-Benzodioxoles and Related
Compounds and Inhibition of Rat liver Mixed Function Oxidase
Introduction - Several years ago Hansch , by means of multiple
regression analysis of data on the synergism of the toxicity of
carbaryl to houseflies by a series of ring-substituted 1,3-benzo-
dioxole derivatives, showed that synergistic activity may be correlated
with the parameters TT and a- for the substituents according to the
equation below.
log SR5 = -0.195 TT2 + 0.670 TT + 1.316 o- + 1.612
n = 13, r = 0.929, s = 0.171
In this equation, SR^ is the degree of synergism obtained using 5
parts of synergist to 1 part carbaryl, IT is essentially a "hydro-
phobic binding" constant, a- is a parameter similar to Hammett's
a constant obtained from the radical phenylation of substituted
benzene derivative, n is the number of compounds, £ is the correlation
coefficient, and £ is the standard deviation from the regression.
From this regression equation, several 1,3-benzodioxole derivatives,
including those given below, were predicted as potential synergists.
This section is concerned with the synthesis of compounds of this
type, and evaluation of their synergistic activity and inhibition
of rat liver microsomal oxidase.
Toxicological Data - All compounds indicated in Tables 1-3 were
synthesized by literature methods or purchased from commercial
sources. Structures of all compounds were confirmed by pmr and
usually mass spectroscopy.
197
-------
The synergists were evaluated by the topical application of a 5:1
(w/w) combination of synergist and carbaryl (1-naphthyl N-methyl-
carbaraate) in acetone to the thoraces of 3-day-old female NAIDM
houseflies, Musea domestica. All synergists were nontoxic at maximum
concentrations employed in this work. Log SR5 data in the tables
are based on an LD5Q value for unsynergized carbaryl of 50 ng/female
fly. Aldrin epoxidation was used as the index of microsomal
oxidase activity, and female rat liver was the enzyme source.
Inhibitor potencies of the various compounds were determined by
measuring per cent inhibition of epoxidation at 5 X lO'^l. 150 values
were determined by using 3 or 4 different concentrations.
Data for the synergism of carbaryl and the in vitro inhibition
of female rat liver microsomal oxidase activity by compounds closely
related to those specifically suggested as potentially effective syner-
gists by Hansch* are given in Table 1. These compounds are methylene-
dioxy derivatives of 6-chloro-acridine Cl and £), anthraquinone C3J,
anthracene 04.), and phenazine (j>) • Four of the five compounds in
Table 1 have high SR^ values, particularly when their relatively
high molecular weights are taken into account. Semiquantitatively,
the data support the prediction by Hansch that methylenedioxyaryls
of the type represented by J.-J> should be effective carbamate syner-
gists.
The ease with which acridines, phenazines, anthracenes, and anthra-
quinones form radical species is well known and, therefore, compounds
J.-4 can be expected to have high a- values. In addition, since
phenothiazine, dibenzofuran, and methylacridine have high log P
values , i.e., in the vicinity of 4±0.1, and in light of their close
structural relationships it is not unreasonable to assume similar
high log P values for compounds 4-5- Thus, based on equation 1,
compounds J.-4 are expected to have high synergistic activity.
On route to the synthesis of compounds j., £, and j> and the attempted
synthesis of some related phenothiazine analogs, a number of 4,5-
disubstituted 1,3-benzodioxoles whose structures are indicated
in Table 2 were prepared as intermediates. Since compounds of this..
general type were used in the structure-activity analysis by Hansch ,
the intermediates listed in Table 2 were examined for synergistic
activity. Several of the compounds, particularly JJ., J^, and J.4,,
proved to be exceptionally effective in synergizing the toxicity
of carbaryl to houseflies. Unfortunately, data were not available
to calculate o- for the various X and Y substitutents (exception
of NC>2) and it was not possible to compare calculated (from equation
1) log SRc values with experimental values. Known chemistry suggests
high a* values for most of these substituents, particularly the
()-phenyl and S^-phenyl moieties where additional resonance structure
for the substituted 1,3-benzodioxole radical may be envisioned. IT
values for ()-phenyl and S^-phenyl substituents also are not known but
they undoubtedly are favorable for synergism. Moderately large
changes in synergistic activity accompanying small changes in
198
-------
Table 1. TOXICOLOGICAL AND OXIDASE INHIBITION DATA FOR FUSED
POLYCYCLIC 1,3-BENZODIOXOLES
No.
Compound
HP °C
LDSO mg/kg
^
Log SR5
7. Microsomai
oxidase inhibition
at 5 X 10"4 M
178-80 34
>300 >48
1.87
13 * 11
3 i 3
258-60
10
!.4
22.25 ±
130.5-132 9.4
2.43
27 ^ 3
5 I
220
32.1
1.89
25 ± 7
199
-------
Table 2. TOXICOLOGICAL AND OXIDASE INHIBITION DATA FOR 3,4-DISUBSTITUTED 1,3-DIOXOLES
M
O
O
No.
io
14
15
>
NO
SEt
MP °C
LD_Q carbaryl
(mg/kg 5:1) Log SRj
N02 137.5-38 18.1
N02 181-82 70.1
N02 161.5-62.5 54.2
N02 259-61 25.1
N02 125-26 70.0
N02 125-26 5.2
N02 140.5-41.5 5.04
N02 134-35 12.9
NHAc 103.5-10A.5 4.90
NHAc 161-2 30.9
2.14
1.S5
1.67
2.00
1.55
2.68
2.70
2.29
2.71
1.91
% Oxidase inhibition
at 5 x 10"4 M
15 ± 9
30 ± 10
22 ± 10
-5 ± 9
49.5
95.5 ± 0.5
5 ± 1.5
71.0 ± 2
53 i 7
84 i 4
-------
Table 2 continued
r-o
o
13
22
NHAc
NHAc
137.5-38.5
128.5-29.5
21.1
33.1
NHCOEt 70.5-71.5 11.5
132-133.5 40.1
NHj-HCl 158-61
NH2'HC1 156-59
NH,-HC1 163-65
NH2'HC1 126-29
2.07
1.88
2.34
1.79
64.6 * 7
7 ± 2
78.2 ± 4.8
80 + 1
83.5 ± 1.3
90 + 1.2
76.5 Jb 6
65 ± 5.5
24 0-
NHj-HCl 146-50
-------
Table 3. OXIDASE INHIBITION DATA FOR SUBSTITUTED 1,3-BENZODIOXOLES
AND THEIR CORRESPONDING CATECHOLS
No. Compound
25 (°TtN°2
ijf\ MO
ti\J w .- "^^jX" ""^ o
26_ YTl
y~— » KO ^^
/ 0
"SsV^^sY^^Sst^*^sl
3 yyu .
HQ.^1^
s. uy^
/°yysO
0 ^^"^ N02
H0^-N-xS'^~^
28 JklC
— HO ^^^NO.
( VrSEt
il \ oAANo,
2
HO -.,,-x^ SEt
29_ J^Jk
2
0 O/^)
12 („ YlT
% Inhibition
at 5 X 10"4 M I50 Log SR$
-6 ± 6 >2 X 10"3 2.40
.3
31 ^ 6 >2 X 10 J
22. 25 ±2. 5 1.18±0.38 X 10"3 2.4
_o
13 i 3 1.3110.31 X 10 J
15 i 9 3.6iO X 10"4 2.14
91.5 ^ 1 1.08±0.1 X 10"4
45.5 .+ 0.5 3.9±0.9 X lo"5 2.68
-4
48 ± 5 7.4±1.4 X 10 *
5 ± 1.5 >2 X 10"3 2.70
"•NO-
°
88.9 it 11.5 3.63±0.5 X 10
-4
202
-------
Table 3 continued
Cl
31
32
Cl
Cl
3 i 2 >2 X 10
"3
2.36
54.0 i 9 4.15±1.1 X 10
"4
33
49 i 0 4.99 X 10"4 2.44
34
Br
94.8 i 1.2 3.7±0.6 X 10"5
35
36
0
HO
HO'
44.5 ± 4.75 5.0±0.3 X 10"4 1.88
71.6 i 2.0 2.8±0 X 10
-4
37
49.9 i 1.9 6.75±1.75 X 10"4 2.64
38
HO.
HO'
61.25 ± 1.75 3.25+0.1 X 10
-4
25 i 7 . 8.75±1.25 X 10"4 1.89
39
52.5 Jr 4 3.0i0.4 X 10
-4
203
-------
structure within each sub-series, e.g., compare J.4, and JJ5, £ and 2>
etc., however, are difficult to explain.
Inhibition of Microsomal Oxidase - Available evidence indicates that
the 1,3-benzodioxoles prevent oxidative metabolism by acting as
alternative substrates and/or binding with the microsomal enzyme
system. Examination of the data in Tables 1 and 2 shows that there
is little relationship, if any, between in vitro inhibition of rat
liver microsomal aldrin epoxidation and in vivo synergistic activity.
For example, compound J.2,, one of the most potent synergists of the
series (log SR5 2.70), produced very little inhibition of aldrin
epoxidation at the concentration of 5 X 10~^M. Similarly, 2, a
moderately good synergist, was virtually inactive as an inhibitor
at the same concentration and in some of the replicated measurements
appeared to cause enhancement of the rate of aldrin epoxidation by
the enzyme. The most effective inhibitor among the compounds in
Tables 1 and 2 was JJ., a material which also happened to be a very
effective synergist.
Although a number of factors above may contribute to the discrepancy
between synergistic activity and inhibition, the possibility remains
that the 1,3-benzodioxole derivatives alone are not totally respon-
sible for synergism. Microsomal oxidases are known to catalyze the
demethylenation of the methyhenedioxy moiety in 1,3-benzodioxoles
to yield formic acid and catechols ' and the suggestion has been
made that the catechols also may be involved in preventing detoxi-
cation of an insecticide . Experimental evidence for this possi-
bility, however, has not been provided.
Therefore, a series of methylenedioxyphenyl compounds and their
corresponding catechols were examined as inhibitors of microsomal
aldrin epoxidation in an attempt to define the nature of the structural
features upon which inhibitory action depends. In Table 3 are listed
the values for the per cent inhibition at a concentration of 5 X lO'^M
test compound and the molar 159 (molar concentration to inhibit
5070 of a fixed amount of oxidase) for each of the substances examined.
For the pairs represented by .2J> and .2J>, ,3_ and .££, ,3Jj and .3J>, 32. and
.3J3, the methylenedioxyphenyl compound and corresponding catechol were
each approximately equal in their effectiveness in inhibiting aldrin
epoxidation. The methylenedioxyphenyl compounds £ and .Ji were more
potent than their respective catechols, Z& and 2£, but in four cases
C5 and 3%, 12 and .3J), .3J. and ^2., .3J3 and _3Jt) the catechol was the
more potent inhibitor. The most effective inhibitors tested were
4-nitro-5-ethylthio-l,3-benzodioxole Q,!) and 4,5-dibromocatechol
C34J with I5Q values of 3.9 X 1(T5M and 3.7 X 10-5M, respectively.
The compounds varied considerably in aqueous solubility which made
interpretation of small differences in inhibition values of question-
able validity. However, this does not obscure the interesting finding
that certain of the cataechols are as potent, or even more potent, as
inhibitors of aldrin epoxidation than the corresponding methylene-
204
-------
dioxyphenyl compound. It should be pointed out that none of the
catechols were active in synergizing the toxicity of carbaryl to
houseflies. This, of course, is not surprising since these materials
are expected to be transient in biological systems.
Conclusion - The synergism data in Table 1 for several of the
polyaromatic 1,3-dioxole derivatives confirmed predictions made
earlier by Hansch. Several of the 4,5-disubstituted-l,3-benzodioxoles
were outstanding in their effectiveness in synergizing the toxicity
of carbaryl. A direct relationship between synergistic activity
and inhibition of microsomal aldrin epoxidation was not observed.
However, data on inhibition of microsomal oxidase revealed that
the corresponding catechols are often more potent inhibitors than
the related 1,3-benzodioxoles and it is possible that in some cases
the catechol may contribute to synergistic activity.
2. The Effect of 1,3-Benzodioxoles. Catechols. and Quinones in
Mosquito Larvae
Introduction - Apparent juvenile hormone /JH) effects have been demon-
strated in houseflies and other insects ' after treatment with
1,3-ben.^odioxole synergists. These synergists are known to inhibit
insect mixed function oxidase systems, insect tyrosinase systems,
and oxidative phosphorylation. Various oxidative processes involved
in insect metamorphoses show differing activities at different stages
of development ' . Interference with any of these could well result
in apparent JH activity. Further, as inhibitors of oxidative proces-
ses, materials such as MON-0585, 2,6-di-t>butyl-4-(a,a-dimethylbenzyl)
phenol, and other radical inhibitors may affect the titre of natural
JH by interfering with the degradation of the hormone even though
the principal metabolites of externally applied JH are hydrolytic in
nature-^. Because of the apparent JH activity of MON-0585, it was
decided to examine various substituted 1,3-benzodioxoles discussed
in the preceding section. This report is concerned with the effect
on mosquito larvae of three classes of inhibitors of oxidauive
processes, i.e., substituted 1,3-benzodioxoles, catechols, and quinones,
Toxicoloaical Data - All compounds were synthesized according to
literature methods or purchased from commercial sources. Several
of the compounds were available from the project described in the
preceding section. Structures were confirmed by pmr and usually
by mass spectroscopy. The method of mosquito larvae (Culex pipiens
quinqi.'g ca_s_ciatus) bioassay was essentially that of Sacher-*-1 for
continuous exposure except that a larger volume of water was used
for reasons discussed later.
Results were tabulated for % 24-hour larval mortality, % total
larval mortality, % non-emerging adults and % emerging adults.
Larvae that had not progressed to the pupal stage after 21 days
were classified as dead.
205
-------
Table 4. TOXICOLOGICAL DATA FOR SUBSTITUTED 1,3-BENZODIOXOLES AGAINST
3RD-INSTAR CULEX PIPIENS LARVAE.
Rl
/°'\fxix^R2
R4
Cone.
No. Rl R2 R3 R4 ppm
AQ H N02 H H 1
4i Cl Cl Cl Cl 1
0.1
42, H t-Bu H H .1
43. H N02 SET H 1
.1
44 H SC,H, NO. H "1
^^ D J t.
.1
45 H OC,H, NO, H 1
** 65 2
.1
46 H Br NO- H 1
42 H OC6H4£-Br N02 H 1
.1
48 H SC6H4£-Mc N02 H 1
42 H Bf Bf H 1
.1
50 H S(0)C,H, NO- H 1
24 Hr
kill
0
0
0
0
100
0
100
0
5
0
0
25
40
40
100
0
0
% Dead
larvae
25
100
25
30
100
90
100
5
75
85
40
95
50
50
100
5
15
% Dead
pupae
5
0
0
0
0
0
0
5
5
0
0
0
5
25
0
0
0
°L Dead
adults
15
0
0
0
0
0
0
5
20
0
0
0
0
0
0
5
5
% Live
adults
55
0
75
70
0
10
0
85
0
15
60
5
45
25
0
90
80
206
-------
Table 5. TOXICOLOGICAL DATA FOR SUBSTITUTED BENZCATECHOLS AGAINST 3RD-
INSTAR CULEX PIPIENS LARVAE.
No. Rl R2
51 H N02
53, ci ci
52. H OC,HC
D J
5& H H
55. H N02
5& H N02
SL H Br
5& H t-Bu
.52 Br H
60 OH H
;$:;
Cone. 24 Hr
R3 R4 ppm kill
SET H 1 100
0.1 0
Cl Cl 1 100
.1 0
N02 H 1 100
v.l 100
.05 100
.02 50
H H 1 35
H H 1 0
Br H 1 10
Br H .1 0
H H 1 0
.1 0
£-Bu H .1 0
H H 1 0
% Dead
larvae
100
15
100
10
100
100
100
60
80
45
15
30
85
25
15
85
% Dead
pupae
0
0
0
0
0
0
0
0
0
5
0
0
0
0
0
5
% Dead
adults
0
5
0
10
0
0
0
0
5
5
0
5
10
5
5
0
°L Live
adults
0
80
0
80
0
0
0
40
15
45
85
75
5
70.
80
10
207
-------
Table 6. TOXICOLOGICAL DATA FOR SUBSTITUTED 1.4-NAPHTHAQUINONES AND
2,3-NAPHTHALENEDIOLS AGAINST 3RD-INSTAR CULEX PIPIENS LARVAE.
0
^^^ II Y
0
Cone. 24 Hr
No. X Y ppm kill
£1 Cl Cl 0.1 60
.05 10
j62 CH_ H 1 45
3
£3 OH H 1 0
64 SCH, H 1 100
j
.1 0
65 OCH_ OC!I 1 0
•*K> ••»•»••« ^
66 SC,HC SC,H_ 1 0
65 65
O6
t
KL H H .1 0
j&g Cl Cl .1 5
63. Br Br .1 0
.02 0
% Dead
larvae
100
85
75
35
100
0
0
10
^.OH
5
30
55
0
% Dead
pupae
0
5
0
0
0
0
0
0
0
0
45
15
% Dead
adults
0
0
0
0
0
0
0
40
5
0
0
5
'% Live
adults
0
10
75
65
0
100
100
50
90
70
0
80
208
-------
Table 7. TOXICOLOGICAL DATA FOR MISCELLANEOUS RADICAL INHIBITORS
AGAINST 3RD-INSTAR CULEX PIPIENS LARVAE
No . Compound
0
jl if
CH,0 'X^X
3 "
0
0
fiir °6H5
^ C6H5S -V
0
H
Cone. 24 Hr 7. Dead 7. Dead 7. Dead 7, Live
ppm kill larvae pupae adults adults
1 5 95 0 0 5
1 0 00 0 100
75
20
OH
•* Z\
CH3
?> OH
x\x-k,oH
^\^
0
0 9~~"\
•j II 0
fu
0 OH
>\xWOH
Du
9~\
~^^J^ 0
••sX^v^
1 0
0.5 5
.1 100
.02 0
1 100
.1 5
.1 100
.02 0
1 100
.1 10
85
35
0
20
0
20
0
45
0
0
10
5
0
10
0
5
0
5
0
0
0
5
0
0
0
0
0
0
0
5
5
50
0
70
0
75
0
50
0
85
209
-------
Table 7 continued.
No.
Compound
Cone. 24 Hr % Dead % Dead % Dead % Live
ppm kill larvae pupae adults adults
16
12 IN.
Br
>r-7v 9 /T~\
HO-// \YcHy';Ny-CH-
-------
In other experiments the volume of the aqueous test solution was
varied, but the concentration of the compound was kept constant.
Conditions employed were 20 larvae in 40 ml of water in a 50 ml glass
beaker and 20 larvae in 200 ml of water in a 400 ml glass beaker. In
some cases 4th-instar larvae also were used.
All classes of compounds investigated showed larvicidal activity
although the type of activity varied. Mortality data against the
later 3rd-instar of Culex pipiens are recorded in Tables 4-7. For
these larvae ecdysis occurred for over 50% of the control larvae
within 12 hours following treatment. Mortality was higher when early
3rd- or 2nd-instar larvae were used, but lower when 4th-instar larvae
were treated. MON-0585 was routinely used as a reference compound
in treating larvae obtained from any single batch. Since results
obtained in long term toxicological studies of inhibitors of oxida-
tive systems depend so much upon the conditions employed (cf below),
it is suggested that the use of MON-0585 as a reference compound be
continued.
Mortality often was high within the 24-hour period following treat-
ment. For example, larvae treated with ,43_> -Z4> and 2& at 0«1 PPm
visibly responded to the test compound within 30 minutes. The larvae
became agitated and frequently collided with each other.
Larvae that failed to respond to stimuli of light, sound and motion
after 24 hours were often normal after 48 hours. Most of the
catechols and quinones (Tables 5 and 6) showed this type of response.
As an example, 7 out of 20 larvae treated with catechol (Jj4J at 1 ppm
were moribund and considered "dead" at 24 hours, but only 2 were dead
at 48 and 72 hours after treatment. However, 80% of the larvae were
dead before the end of the bioassay period (21 days). A similar
effect has been observed for phenothiazine^ a compound which also
is a free radical inhibitor.
Apart from 24-hour mortality, most larval deaths occurred immediately
after ecdysis or after an unduly long larval duration. Larvae
treated with catechols and 2,3-naphthalenediols (Tables 5 and 6)
often remained attached to the terminal appendages of the moult
from the previous instar. These larvae drowned slowly. Death in
the pupal stages also commonly occurred immediately after pupation
or in the late pharate adult stage. The latter were identified by
their "sooty black" color.
Adults frequently failed to emerge fully after ecdysis. The degree
of emergence varied from a simple split along the middle line of
the cephalo-thorax to emergence of all parts except the abdomen
and wing tips. Partially emerged adults remained alive but were
unable to escape.
The results described above parallel results obtained from derivatives
21 1
-------
13 11
of farnesoic acid and from MON-0585 . It appears that inhibitors
of oxidative systems in general produce apparent JH effects. This
may be attributed to interference of the metabolism of endogenous
JH, but interference with other oxidative processes is also possible.
Several of the compounds tested were significantly more effective
as mosquito larvicides than MON-0585. The most effective compounds
were £3_, £5, &, £1, J&* 1&> &Q» 81, and ££. These materials were
equal to or more effective than MON-0585 under similar test conditions.
It is noteworthy that each of the various classes of compounds studied
is represented by at least one compound with high activity. Of
some significance is the fact that two of the more active compounds,
3J& and 2&> are of plant origin. The most effective compound was
4-nitro-5-phenoxycatechol (.5J3) where significant activity was ob-
served as low as 0.02 ppm. The corresponding methylenedioxy deriva-
tive C4J5) also was effective. The latter, as described in the
preceding section, were found to be one of the most powerful carbamate
synergists ever examined in this laboratory.
It should be noted that the toxicity obtained repeatedly in this work
for MON-0585 is below the LD50 value of 0.05 ppm quoted for the
3rd-instar Aedes aegypti larvae by Sacher . In our study treatment
of 20 Culex pipiens larvae in 100 ml of water at a concentration of
0.05 ppm resulted in only 25% mortality (average of 16 determinations).
However, treatment of 20 larvae in 40 ml of water at a concentration
of 0.05 ppm resulted in 86% mortality (average of 16 determinations).
Emergence of controls at the two different volumes of 40 and 100 ml
were identical. It appears, therefore, that the effect of MON-0585
is increased by a "crowding" factor. This result emphasizes the
importance of using a reference compound in all long-term toxicologi-
cal studies of mosquito larvae.
Conclusion - A variety of substituted 1,3-benzodioxoles, catechols,
and quinones, all materials which affect free radical processes,
were quite effective larvicides, showing activity comparable to
or greater than that demonstrated by MON-0585. These compounds
appeared to inhibit the development of larvae into pupae or adults.
Symptoms of intoxication paralleled those obtained from the treatment
of mosquito larvae with farnesoic acid (a JH mimic) or MON-0585.
3. References
1. Hansch, C. The Use of Homolytic, Steric, and Hydrophobic
Constants in a Structure-Activity Study of 1,3-Benzodioxole
Synergists. J. Med. Chem. 11:920-24, 1968.
2. Brooks, G. T. The Metabolism of Diene-Organochlorine (Cyclodiene)
Insecticides. Residue Rev. 27:81-138, 1969.
212
-------
3. Casida, J. E., J. L. Engel. E. G. Essac, F. X. Kamienski, and
S. Kuwatsuka. Methylene-C1 -Dioxyphenyl Compounds: Metabolism
in Relation to Their Synergistic Action. Science 153:1130-3,
1966.
4. Kuwatsuka, S. Biochemical Toxicology of Insecticides., R. D.
O'Brien and I. Yamamoto [Eds.] Academic Press, New York pp.
131-44, 1969.
5. Mitlin, N. Inhibition of Development in the House Fly by 3,4-
Methylenedioxyphenyl Compounds. J. Econ. Entomol. 49:683-84,
1956.
6. Bowers, W. S. Juvenile Hormone: Activity of Natural and Synthe-
tic Synergists. Science 161:895-97, 1968.
7. Bowers, W. S. Juvenile Hormone: Activity of Aromatic Terpenoid
Ethers. Science 164:323-25, 1969.
8. Krieger, R. I., and C. F. Wilkinson. Microsomal Mixed-Function
Oxidases in Insects. Biochem. Pharmacol. 18:1403-15, 1969.
9. Krieger, R. I., M. D. Gilbert, and C. F. Wilkinson. Microsomal
Mixed-Function Oxidase Activity in Macremphytus varianus. J.
Econ. Entomol. 63:1322-23, 1970.
10. Slade, M., and C. H. Zibitt. Insect Juvenile Hormones, pp.
155-76. J. J. Menn and M. Beroza [ed.]. Academic Press, New York.
341 p.
11. Sacher, R. M. A Mosquito Larvicide with Favorable Environmental
Properties. Mosquito News 13:8910-12, 1971.
12. Yates, W. W. Time Required for Aedes vexans and A. lateralis
Larvae to Obtain a Lethal Dose of Several Larvicides. J. Econ.
Entomol. 39:468-71, 1946.
13. Spielman, A., and V. Skaff. Inhibition of Metamorphosis and of
Ecdysis in Mosquitoes. Insect Physiol. 13:1087-95, 1967.
213
-------
H. STUDIES ON CHEMICAL REACTIONS INVOLVING CARBAMATE AND ORGANO-
PHOSPHORUS ESTERS
During the project period several investigations were conducted on
some of the fundamental aspects of reactions involving phosphoramid-
othioate and carbamate esters. The results are summarized below.
1. Alkaline Hydrolysis of Phosphoramidothioate Esters
Introduction - 0,S_-Dimethyl phosphoramidothioate (Monitor or J.)
is a relatively simple organophosphorus ester which has proved to be
a highly useful insecticide. In an earlier investigation-*- on the
mode of action of J. and related esters it was assumed that the
alkylthiolate moiety was released when the cholinesterase enzyme
was inhibited by the phosphoramidothioate ester. Subsequently,
however, examination of the reaction between JL and hydroxide ion
showed that methylthiolate ion was not always the major product but
methoxide also was liberated, the relative amounts depending on the
conditions of the reaction. Because of the possible connection between
alkaline hydrolysis rates and anticholinesterase activity, an examina-
tion of the alkaline hydrolysis of J., its N-methyl (£) and N,N-dimethyl
Q) analogs was conducted. Product and kinetic analysis were under-
taken to sort out the various individual reactions and to assess
quantitatively their relative importance in the overall hydrolysis
reaction. Particular attention was given to the effect of sequential
substitution of methyl groups on the nitrogen atom and of solvent
on the specific rates of P-0 and P-S bond cleavage.
Products of Alkaline Hydrolysis - Pmr spectra of the products obtained
from the hydrolysis of 0-methyl S_-methyl phosphoramidothioate
Q.) with equimolar amounts of potassium hydroxide in water and in
50% aqueous acetone showed that two monoanionic products were obtained,
one by PrS cleavage giving 0-methyl phosphoramidate anion C5) and the
other by P-0 cleavage giving S_-methyl phosphoramidothioate anion
(ji). In water the major product obtained was £ since analysis of
the pmr integrals for P-OCH., protons and P-SCH_ protons showed a
ratio of ,5_/£ of 1/4.5. In 50% aqueous acetone, however, .5, was the
major product, and the ratio of 4>A6 in this case was 5.1/1. Support
for the ratio of products obtained by integration of pmr spectra
was provided by glc analysis after remethylation of the mixture of
4 and £ with diazomethane. Remethylation of j} and £ gave dimethyl
214
-------
phosphoramidate (.4J , retention time 1.75 rain, and J., retention
time 3.50 min, respectively, and the ratio of these products was
virtually identical to the ratio of %/6 obtained by proton integration.
Confirmation of product ratios by glc, therefore, allowed the use
of pmr as the major means of product analysis.
Data for product analysis by pmr after alkaline hydrolysis of J.
in a variety of solvent systems are given in Table 1. The results
indicate that the solvent strongly influences the relative percent-
ages of 4 and j6 produced and, in the case of the methanol-water system,
increasingly larger amounts of P-0 bond cleavage were obtained with
increasing amounts of water; e.g. 4/£ in absolute methanol was 93/7
compared to 18/82 in water.
When J. was treated with an equimolar amount of potassium hydroxide
in absolute ethanol or propanol, the major product was neither 4> nor
6 but was the potassium salt of 0-ethyl and 0-propyl phosphoramidic
acid, respectively. Further, distillation of the reaction mixture
containing n-propanol or ethanol as a solvent gave a low boiling
fraction (37-4(f ) which was identified by glc and pmr as dimethyl
sulfide. Dimethyl sulfide also was isolated by the same procedure
in varying quantities after the treatment of JL, ^-methyl S^-methyl N-
methylphosphoramidothioate (.2,) and 0-methyl ^-methyl N,N-dimethyl-
phosphoramidothioate Q) with potassium hydroxide in water (Table 2).
Dimethyl sulfide apparently is formed from the reaction between
methylthiolate anion, produced by P-S bond cleavage, and the starting
ester. Therefore, the reactions involved in the decomposition of
(J.) and related esters by hydroxide ion may be depicted as follows.
CHoS^ ,0 . -2o CHS 0
^?' + OH > 3 P + CHJDH (2)
CH 0'
VNRR' 3
CH.S ,0 -2s "0 ^0
3 ^P + 20H~ > P + CH-S (3)
CH30X ^NRR1 CE3°'- NRR'
CH-S ,0 -2c CH3Sx ,0
^XPX + CH.S- - > 3 P + CH SCH (4)
CRO' NNRR' ~0' S'
1^ R,R' = H
2^R = H.R1 = CH
"** J
3_ R,R' = CILj
k,, , k« and k0 are the specific second-order rate constants for
— /o —2s — zc
215
-------
Table 1. SALT DISTRIBUTION IN ALKALINE HYDROLYSIS OF 0
PHOSPHORAMIDOTHIOATE IN VARIOUS ORGANIC
SOLVENT MIXTURES AT 23°
METHYL S -METHYL
WATER
% Organic Solvent in
aqueous mixture (v/v)
methanol
acetone
mesityloxide 10, ethanol
propionaldehyde 50, ethanol
formaldehyde 30
acetcphenone 20, ethanol 60
acetonitrile 50
2-butanone 45, ethanol 10
ethanol 100
propanol 100
benzaldehyde 25, ethanol 20
100
95
90
85
80
75
65
50
0
50
30
25
% Salt
P-OCH3
93
86
79
76
67
63
50
40
18
83
33
80
100
25
23
60
7.5
P-OC_H
10
P-OC.H
83
Containing
7
14
21
24
33
37
50
60
82
17
67
20
-
75
77
40
12.5
5 80%
22.5
7
17
Extensive polymerization occurred.
216
-------
Table 2. PRODUCT DISTRIBUTION AFTER HYDROLYSIS OF PHOSPHORAMIDO-
THIOATES BY AQUEOUS POTASSIUM HYDROXIDE IN A SEALED AMPOULE
R
H
H
CH3
CH3
Temp
R1 °C
H 23
CH3 23
CH3 23
CH3 96
RR'N 0
m Q r\ru
vjti-o ui>n_
Reaction
time % Salt containing
(hr) P-OCH3 P-SCH3 % MeOH
4 18 82 75
16 52 48 30
96 50 50 13-153
14 42 58 24
7 CH S H
10
19
_b
32
o
Reaction mixture was distilled to obtain methanol in high concentra-
tion for glc.
Reaction at atmospheric pressure, dimethylsulfide was not determined.
217
-------
P-0, P-S and C-0 bond cleavage, respectively.
Evidently, £5-methyl phosphoramidothioate anion (£) may be produced
in two ways, from replacement by hydroxide ion according to equation
(2) or from 0_-demethylation by methylthiolate ion according to equation
(4) . The extent of 6, (or its N-methyl and N,N-dimethyl equivalent)
formed by replacement of methoxide by hydroxide ion was determined
by glc analysis for methanol in the reaction mixture. Quantitative
data showing the relative amounts of the various products produced
after treatment of J., £, and J3 by equimolar aqueous potassium hydroxide
are presented in Table 2. The results indicate that the ()-demethyla-
tion reaction to produce .6 or its N-alkyl derivatives becomes increasing-
ly important with sequential methylation of nitrogen.
Finally, in order to determine whether potassium hydroxide treatment
of j. produced methanol by P=0 or C-0 bond cleavage, the reaction
was carried out in water enriched with I^O^. The liberated methanol
was examined by mass spectrometry and no significant (^"-incorporation
was found, indicating that hydrolysis occurred by P-0 bond cleavage.
Kinetics of Hydrolysis of Q-Methyl ^.-Methyl Phosphoramidothioate -
Under conditions of high concentrations of J. compared to hydroxide,
the value for the overall pseudo first-order rate constant k^ were
obtained by following hydroxide ion consumption from estimation of
the change in pH. Under these conditions equation 4 may be omitted
in the kinetic analysis of J. since hydroxide ion is not involved
in this equation. Also, kj^ = k^o + aki g where k;io an(i kls are
the pseudo first-order constants~for P-TJ and P-S cleavage7 respec-
tively. The value of the coefficient a depends on the pH of the
solution, i.e. a is 2 at pH substantially greater than the pKa of
methanethiol (10.7) and a is 1 at pH lower than 10.7. From the
slope of a plot between pH and time, the value for the overall
pseudo first-order constant k^ at 27° was calculated to be 0.96
and 9.6 M~l min"* (J. was 0.1 M) , respectively.
The value of k20, the second-order constant for P-0 bond cleavage
was determined Directly by following methanol production under
second-order conditions of J. and hydroxide ion. The plot of the
data was reasonably linear up to about 65% reaction and JC2O was cal
culated to be 8.4 M"1 min"1 at 27°. From the relationship"^ =_,
= ~* "1 ~*
+ 2k_2s = 9.6 M~ min", kj?s was calculated to be 0.6M~ min
VaTues for k_ and k_ are given in Table 3.
~£o_ ~*-&.
Effect of Acetone on Hydrolysis of 1. - The effect of acetone on
the values of the pseudo first-order constants k' io and k'is under
conditions of constant pH for P-0 and P-S cleavage~~is shown~~in Table
4. Because of interference by acetone in the glc analysis of methanol,
it was not possible to determine k'io directly in solvents containing
acetone. Therefore, k'i0 was calculated from the values of k'l (over-
all pseudo first-order constant at constant pH) and k' determined
J-S
218
-------
Table 3. SECOND-ORDER RATE CONSTANTS FOR P-0 (k. ) AND P-S (k. )
—/£ ~*-§.
CLEAVAGE OF PHOSPHORAMIDOTHIOATES IN AQUEOUS
POTASSIUM HYDROXIDE AT 27°
k0 (M min )
—zo ~
k_ (M min )
(CH30)(CH3S)P(0)NH2 8.4
(CH30)(CH3S)P(0)NHCH 1.0 X 10
(CH30)(CH3S)P(0)N(CH3)2
-3
0.6
4.4 X 10
-2
1.5 X 10
-4
Table 4. PSEUDO FIRST-ORDER RATE CONSTANTS IN AQUEOUS ACETONE FOR
THE ALKALINE HYDROLYSIS OF 0-METHYL ^-METHYL PHOS-
PHORAMIDOTHIOATE AT pH 10, |i = 0.2 M
7o Acetone
(v/v)
k' X 10'
- Is.
(min"1)
k'. X 10
- lo
(min"1)
3
Calcd from
Calcd from
HO"
CH SH
release (t%) consumption
0
10
20
30
40
3.0
1.6
3.1
4.2
6.2
5.0
5.5
5.9
6.0
6.9
8.6
10.6
12.5
10.1
12.2
219
-------
at pH 10.0 iri aqueous solvents containing 0-40% acetone and sodium
carbonate as the buffer. k'ls was calculated from the relationship
below by following methanethiol formation where [A ] is the initial
[CH.,SH] -IB , ,
= (1 -e- i) (5)
-k11
concentration of J.. A plot of [CH3SH]/AO against (1 -e —]_ ) gave
a linear relationship and k'is was calculated from the slope. The
value of k'i was determined fr"om the amount of base needed to main-
tain a pH of 10.0 using a micrometer driven syringe containing stan-
dardized aqueous potassium hydroxide, k1^ also was calculated from
the relationship tj. = In 2/k1^ which follows from equation 5 where
tj. is the time released to liberate one-half of the total amount
or methanethiol released. The data in Table 4 show good agreement
between the values of k'i obtained by estimation of potassium hydroxide
consumption and that calculated from the tj, value for methanethiol
release.
Hydrolysis of Q-Methyl ^.-Methyl M-Methvlphosphoramidothioate (2) -
The overall rate of reaction between 0-methyl ^-methyl N-methylphos-
phoramidothioate (.£) and hydroxide ion determined by estimating
change in pH was approximately 110-fold slower than J.» with a pseudo
first-order rate (excess of 2) constant ki of 4.45 X 10"^ min"-*- and
^ / 1 1
a second-order rate constant k2 of 8.9 X 10 M min"1. Because of
the slow rate of reaction between Z. an{* hydroxide ion, loss of Z
by dealkylation (equation 3) was significant and it was possible to
determine k]_c (the pseudo first-order rate constant for C-0 cleavage)
as well as k/[o and k^s. Kinetic analysis was accomplished by im-
posing again,~as in tEe case of 4> pseudo first-order conditions,
i.e. high concentrations of £ relative to hydroxide ion. Under
these conditions, equations 2, 3, and 4 may be designated as follows.
-lo
A + B > C (6)
ki
—Is
A + B ^ D (7)
-lc
A + D > G (8)
220
-------
where A = £, B = hydroxide ion, C = methanol, D = methylthiolate
ion, and G = dimethyl sulfide. From equations 6, 7, and 8, and since
A is constant, 9 and 10 may be obtained.
-d[B]
klQ[B] + 2klg[B] (9)
d[D]
- k[D] (10)
l£
-k t
Since B = B0 e — i , where Bo is the initial hydroxide ion concentra-
tion, equation 10 becomes 11.
d[D]
- = k. B e -I* - V [D]
dt -^ ° -Ic
This first-order differential equation has the following solution.
ki CB ]
— lsL oj
k, - k.
— 1£ —1
When k. is small compared to k1 , integration of equation 11 results
• 10 JLC JLS
in 13. — —
[D] ^ -k t
- = - (1 - e V) (13)
tV *1
This equation is similar to equation 5 where B0 (initial hydroxide
ion concentration) is substituted for AQ (phosphoramidothioate
concentration) .
The plot of [CH3S~]/[OH~] vs_ (1 - e~-l ) gave the expected linearity
over the major portion of the reaction, from which k^s at 27 was
calculated as 2.20 X 10~2 min'1. From the relation kj = kio +
2klg = 4.45 X 10"2 min'1 the value of kio was calculated as~5 X 10"
min"!. The corresponding second-order rate constants, k20 and k2s
given in Table 3 were obtained by dividing the values of Ttio and ~~
y °-5 M (initial concn of £) . ~
The value of k^c was calculated by using equation 12 with the aid
of a computer. HBy setting the value of k^ to 4.45 X 10~2 min~l,
estimated values of k^s and kic were substituted in equation 12
until the calculated values ofTmethylthiolate ion concentration
at different time intervals coincided with the experimental values.
Best fit of the data was obtained when k. was 2.2 X 10~2 min-1 and
—Is
221
-------
-3 -1
was 3.6 X 10 min
Hydrolysis of 0-Methyl S-Methyl N.N-Dimethylphosphoramidothioate (3) -
The rate of hydrolysis of £ was much slower than £ under identical
first-order conditions of excess phosphoramidothioate over base
with a pseudo first-order constant k^ for the disappearance of hydroxide
ion of 1.4 X 10~4 min-1. No methanethiol was detectable at any time
during the course of the reaction, indicating that the rate of reaction
between methanethiolate ion and £ to form dimethyl sulfide was con-
siderably faster than the initial reaction between hydroxide ion
and JJ. k_ic> the pseudo first-order rate constant for methyl-oxygen
bond cleavage for £ and ,3_ under the same condition should be similar,
and based on the value of k^c = 3.6 X 10"^ min~l obtained for £, the
dealkylation reaction for .3. should be approximately 25-fold faster
than the initial reaction.
Analysis of products after reaction between .3, and potassium hydrox-
ide showed that P-0 cleavage was much slower than P-S cleavage
(23°) and this coupled with the above information gives the order
k_lc ^iSls >;>klO' By making the assumption that kl£ is negligibly
small oomparell to kjs, the kinetics of the reaction may be approxi-
mated to a second-orHer situation with the provision that 2 moles
of 3 are consumed (one mole by P-S cleavage in the rate determining
step and one mole by a rapid dealkylation reaction) with 2 moles
of hydroxide ion (one mole for P-S cleavage and 1 mole for rapid
neutralization of the resulting phosphoramidic acid) according
to the following equations.
CH,S 0 "0 ,0
3 XP* • + 20H" v* P' + SCH> (14)
CHS. X0 CH3S ^0
3 >f + -SCH ——-* *^* + CHSCH3 (15)
By using equimolar amounts of ^ and hydroxide ion the value for the
second-order rate constant k£s for P-S cleavage may be determined
from equation 16 where [OH~T and [OHO] are the concentrations of
hydroxide ion at time t and time zero, respectively. A plot of
~ ~ + 2kt (16)
1/[OH ] against time from data obtained in a separate experiment
using equimolar concentrations of 4 and hydroxide ion gave an excel-
lent straight line over a 45% reaction range, a finding which
222
-------
provided support for the assumptions made in the kinetic approach.
From the plot, k2s was found to be 1.5 X 10'^ M"1 min'1 (27 ).
-4 -1
The value of k^ (1.4 X 10 min ) obtained under the pseudo first-
order condition of excess phosphoramidothioate (J3 was 0.5 M) also
is equal to 2k^s since k-^o is negligible. From this relationship
also k2s may be""calculate3 as 1.4 X 10-4 M~l min"-'-, in good agreement
with the1 value obtained under second-order conditions.
Analysis of Data - Because of the known greater lability of the P-S
bond, the hydrolysis of ^ in aqueous potassium hydroxide by predom-
inantly P-0 bond rather than P-S bond cleavage was unexpected. The
ionizing capacity of the solvent evidently plays a dominant role
in establishing the direction in which j. is hydrolyzed by potassium
hydroxide. The influence of solvent on the reaction is illustrated
in Figure 1 which shows the relation between Grunwald-Winstein Y
values^ for water-methanol and the log of the ratio of & to ,$• Al-
though the product ratio in two parallel second-order reactions at
any time is equal to the ratio of rate constants, the value of
£/4 may only be considered as an approximation of the relative rate
constants for P-0 and P-S cleavage since the same salt is obtained
by £-demethylation and P-0 cleavage. Figure 1 shows, however, that
alkaline hydrolysis of J. by P-0 bond cleavage is favored in solvents
of greater ionizing capacity. In less polar solvents P-S bond cleav-
age predominates, suggesting that the two competing reactions (P-0
and P-S cleavage) occur by different mechanisms.
Table 4 shows the effect of increasing amounts of acetone on the
pseudo first-order rate constants for P-0 (k' ^o) and P-S (k']_s)
cleavage of J. at pH 10.0. Figure 2 gives the "relation between
these constants and Grunwald-Winstein Y values for the relevant
acetone-water mixtures. Although the relationship is clearly not
linear, it does focus on the far greater dependency of k1is on
solvent polarity, e.g. k'is increases almost 4-fold from W% to
407o acetone while k'^0 remains virtually constant. Thus, in increasing
a polar solvent P-S cleavage becomes increasingly important, result-
ing eventually in a changeover in ratio of products.
The values for the specific second-order rate constants for P-0
(k2o) and P-S (k£s) bond cleavage for j., £, and J3 in aqueous potassium
hydroxide at 27^ Tn Table 3 show that there is a marked decrease
in both k2o and k2s with sequential substitution of amido protons
by methyl." In addition, the relative rates for P-0 and P-S cleavage
also decreased, e.g. k2o/k2s was 14 for J., 0.023 for 2. and presumably
much smaller for J3. Thus, J was hydrolyzed predominantly by P-0
cleavage while P-S cleavage predominated with .2, and J3.
A plausible mechanism in which two different processes are taking
place simultaneously may be suggested from the data, process
223
-------
4,0
3.0
2.0
1.0
.8
.6
10
* .4
o
H .3
cc
.2
.1
.08
.06
.04
-2
O
O
O
O
O
_L
J.
0 I
Y VALUES
4
Figure 1. Relation between Grunwald-Winstein Y values and the log
of the ratio of potassium _S-methyl phosphoramidothioate
(6) to ()-methyl phosphoramidate (5).
224
-------
10
8
6
i
.c 4
E
X
-^ 2
\
-
-
° 0
0
©
©
®
1 1 1 1 1 1
2 34
Y values
Figure 2. Relationship between k, (O) and k (O) and Grunwald-Winstein
~ _ ~~^-2.
Y values for the alkaline hydrolysis of ^-methyl ^-methyl
phosphoramidothioate in aqueous acetone.
225
-------
which involves hydroxide ion attack on the amido proton, leading to
the phosphoramidothioate anion which decomposes in a rate detemining
step to give P-S cleavage, and process (£) which involves attack
of hydroxide ion on the phosphorus atoa, eventually resulting in
P-0 cleavage.
CK3°x
X
I
R
u o
"2"
CH S
R = E or CH-
(b)
. -OCH
oCH.
' '' "^5--
or
OH
CH3S KR
CHS SER
Process ^ leading to P-S cleavage and phosphoriridate formation
is analogous to the nechanisn proposed for base catalyzed hydrolysis
of phosphoranidic chlorides. Direct support for this nechanisn is
found in results obtained iron the analysis of products after base
catalyzed hydrolysis of _i in absolute ethanoi and propanol. The
observation that 4 reacts with potassiin hydroxide in absolute ethanoi
or propanol to give as sajor produces dimethyl sulfide and the potas-
sium salt of ethyl and propyl phcsphcrani die acid, respectively,
strongly indicates that this reaction proceeds through a -€taphos-
phorinidate. In ethanoi or propanol the intermediate is sclvolyzed
to give ethyl or propyl methyl phosphoramidate which is in turn
denethylatec by methylthiolate anion to produce the respective
products as shown below.
-------
CHS
3
OH
NH
RC>
CH3S"
CH SCH3
•HN=P
OCTL
ROH, R = C2H5, C3H?
RO
The finding that P-0 cleavage predominates in highly polar solvents
suggests that this reaction proceeds by a process involving hydroxide
ion attack on the phosphorus atom, either by a concerted SN2(P) or
by an addition-elimination reaction. Although a concerted 5^2 reac-
tion may lead to P-0 cleavage, it does not account for the preponderance
of P-0 over P-S cleavage, particularly since methylthiolate ion
is a superior leaving group compared to methoxide ion. Thus, process
b. leading to P-0 cleavage probably involves, at least in the case
of j. and £, the addition of the hydroxide ion to the phosphoramido-
thioate followed by a rapid elimination of the alkoxy group rather
than a concerted SN2 type reaction. The addition step is expected
to lead to a trigonal bipyramidal intermediate and from preference
rules^ the methoxy and hydroxy groups should occupy apical positions.
With the methoxy group in an apical position, its departure may be
assisted by the nitrogen proton either directly or indirectly by
the intervention of a water molecule as indicated in the mechanism
given above. The addition-elimination mechanism explains why P-0
cleavage takes place with J. and 2. but does not occur with £ where
the nitrogen atom is fully substituted with methyl groups. The
exclusive cleavage of the P-S bond in j3 probably occurs by a concerted
Sfl2 mechanism in which the best leaving group, i.e. methylthiolate,
is displaced by hydroxide ion. This is the usual type of substitution
reaction with most triesters of phosphoric acid. The fact that the
hydrolysis of J3 by P-S cleavage is approximately 3 X 10^ times
slower than that of J. suggests different mechanisms for the hydrolysis
of 4 and .3.
Conclusion - The results indicate that solvent polarity has a pro-
found effect on the direction in which phosphoramidothioate esters
hydrolyze under alkaline conditions. In aqueous potassium hydroxide
0,j3-dimethyl phosphoramidothioate is hydrolyzed by P-0 cleavage as
the major route while in less polar solvents P-S cleavage predominates.
Both rate constants and the relative rates of P-O/P-S bond cleavage
decreased markedly with sequential substitution of the amido protons
227
-------
with methyl groups, and the N,N-dimethyl derivative hydrolyzed
virtually exclusively by P-S bond cleavage but at a rate some
10^ times slower than P-S cleavage in unsubstituted phosphoramido-
thioate. Exclusive P-S bond cleavage in the N,N -dime thy Iphosphoramido-
thioate evidently occurs by a normal concerted 8^2 reaction in which
the best leaving group departs. In phosphoramidothioates containing
at least one amido proton the results are rationalized in terms
of two competing preocesses, an addition-elimination reaction on
phosphorus leading to P-0 bond cleavage and an elimination reaction
involving the amido proton to give P-S bond cleavage.
The question remains, what is the leaving group (methoxide or
thiomethylate) , when cholinesterase is inhibited by Monitor or a
related phosphoramidothioate ester? The fact that P-0 cleavage
predominates in an aqueous environment would suggest that methoxide
is the leaving group in the inhibition process. However, because of
the presence of lipophilic sites on the enzyme it is not possible
to rule out thiomethylate as the leaving group. The chemistry and
mode of action of Monitor and related phosphoramidothioate esters
deserve further study.
2. Reaction of Trialkyl Phosphites with Haloamides
Introduction - Synthetic routes to insecticidally active (),S_-dialkyl
N-acylphosphoramidothioates are multistep and often result in poor
yields. Because of the need to prepare a radiolabel of the insecti-
cide Orthene (0, ^-dimethyl N-actylphosphoramidothioate) for metabolism
studies, alternative methods for a convenient synthesis of this com-
pound were investigated. In particular, the reaction between N-
bromoacetamide and trimethyl phosphorothioate was examined. In
all cases, no Orthene could be isolated although the starting mat-
erials were consumed and alkyl halides were evolved. In an attempt
to understand this reaction, the reaction between triethyl phosphite
and N-haloamides was investigated.
Product Analysis - The reaction between trialkyl phosphites and the
following N-haloamides was examined:
N-bromoacetamide Q) , N-chlorobenzamide (£) , N-chlorosuccimide
N-bromosuccimide (iQ) , N-bromo-2-pyrrolidinone Oii) ,
N-chloroacetamide CX2J , N-chloro-N-methylacetamide
and N-bromobenzamide
The cyclic haloamides C2-11) reacted with 1 equivalent of triethyl
phosphite to give ethyl halide and the expected Arbuzov products
45 or J.&. The product 15, which was the same whether prepared from
228
-------
N-P(OC2H5)2
15_ 16
3. or }£, was identical to that reported previously.
The acyclic primary haloamides (2, &> -i^> and .14) did not give the
expected Arbuzov products but instead reacted to give products
(Table 5) consistent with Scheme I.
Scheme I (Square brackets are used to indicate intermediates that
were never isolated)
(EtO) P + R'CONHX
* (KtO)^PO +
C-NH
(I)
C"NH
•> R'CH + HX
(2)
(EtO)3P
HX
(EtO)2P(0)H + EtX
(3)
(EtO)2P(0)H
R'CONHX
(EtO)2P(0)X + R'CONH2
(4)
R1 - CH-, Cg
X - Cl, Br
When N-chloro-N-methylacetamide (£3) and trialkyl phosphite were
reacted, the only products were the trialkyl phosphate and N-methyl-
acetimidoyl chloride, analogous to step 1 in the scheme. For the
primary haloamides, Scheme I is supported by the following evidence.
1. Reaction of one equivalent of primary haloamide with one equiva-
lent of triethyl phosphite led to the formation of approximately
one-half equivalent of ethyl halide, nitrile, amide, triethyl
phosphate, and diethyl halophosphate (cf Table 5).
2. The reaction was exothermic until almost two equivalents of tri-
ethyl phosphite had been added. At this point, no triethyl
phosphite could be isolated when it was introduced rapidly.
229
-------
Table 5. PRODUCTS OBTAINED FROM THE REACTION BETWEEN N-HALOAMIDES AND TRIETHYL PHOSPHITE AT 25°
Haloamide
7
8
N> 9
u> ~
o
1£
11
12
H
14
Liquid
vehicle
toluene
CCIA
ether
benzene
benzene
CCIA
CCIA
ether
Moles of
Haloamide
0.5
0.5
1.0
1.0
1.0
0.5
1.0
1.0
reagents
Phosphite
1.0
i.o'
1.0
1.0
1.0
1.0
1.0
1.0
Moles of products
RCN
0.46
0.44
0.34
0.41
0.47
0.48
0.48
0.44
RCONH2
<0.04
0.02
0.42
0.48
0.48
<0.02
0.44
0.46
EtX
0.45
0.25
0.48
0.50
0.50
0.50
0.37
(EtO)2P(0)X
=0
0.07
0.32
0.36
0.42
0.03
0.37
0.43
(EtO)2P(0)H
0.36
0.32
0.02
<0.01
= 0
0.36
0.05
0.06
(EtO)3P(0)
0.41
0.45
0.47
0.41
0.36
0.36
0.46
0.43
-------
3. Addition of two equivalents of triethyl phosphite to one equiva-
lent of primary haloamide gave in good yields the products
indicated in step 5 (5 is the summation of steps 1-3). Small
amounts of amide and diethyl halophosphate (the products of
step 4) also were isolated.
2(EtO)3P + R'CONHX }• (EtO>2P(0)H + (EtO^PO + R'CN + EtX (5)
4. When one equivalent of triethyl phosphite was reacted with one
equivalent of 2. in tne presence of pyridine, the ratio of
acetonitrile to ethyl bromide increased and pyridinium hydro-
bromide was isolated (cf Table 6). Pyridine, by acting as a
scavenger for HBr formed in step 2, decreased the availability
of HBr to react with triethyl phosphite (step 3).
5. When one equivalent of triethyl phosphite was reacted with one
equivalent of 2 i-n tne presence of one equivalent of HBr, the
ratio of ethyl bromide to acetonitrile increased, indicating
that HBr can compete with 2 f°r triethyl phosphite under the
conditions of the reaction (cf Table 5).
The first step in the reaction sequence (step 1) for acyclic N-
haloamides probably occurs through the imidoyl phosphonium halide
as shown below. The formation of the imidoyl phosphonium halide
(RO)3PO + * ^C-NR" (6)
X
R - CH3, C2H5, n-C^
R1- Cl^, C6H5
R"- H, CHj
X - Cl, Br
is required by the isolation of N-methylacetimidoyl chloride from
.12, and the isolation of the respective nitriles from 2> .§» .!£» and j^.
The reaction between trimethyl phosphorothioite and Q was reinvesti-
gated in the light of above information. One equivalent of trimethyl
phosphorothioite reacted with one equivalent of J5 to give approximately
0.3 equivalents each of benzamide, benzonitrile, and methyl chloride
together with other products that were not identified. These products
in large part explain the failure to isolate any dimethyl N-benzoyl-
phosphoramidothioate from this reaction.
231
-------
Table 6. EFFECT OF HER ON THE REACTION BETWEEN N-BROMOACETAMIDE
AND TRIETHYL PHOSPHITE IN BENZENE
Moles of reagents Moles of volatile products
Haloamide Phosphite Other EtBr CH CN
1.0 1.0 - 0.37 0.48
1.0 1.0 HBr - 1.0 0.74 0.08
1.0 1.0 pyridine - 1.0 0.26 0.68
232
-------
Conclusion - The reaction between trialkyl phosphites and acyclic
primary halomides did not give the desired Arbuzov products but
instead gave the variety of products indicated in Scheme I. Except
for the cyclic haloamides, this procedure holds little promise
for the synthesis of insecticidal 0,8^-dialkyl N-acylphosphoramido-
thioates.
3. Synthesis of Hydroxymethylcarbamates
Introduction - During the course of our investigations on the
synthesis and evaluation of N-derivatized methylcarbamate esters
as selective insecticides, we discovered a convenient, high-yield
method for the preparation of hydroxymethylcarbamates. These
compounds are of considerable importance since they have often
been found as metabolites in plants and animals treated with methyl-
carbamate esters.
Synthesis - The method described below is typical and is applicable
to different methylcarbamte esters. The example given is for the
preparation of 1-naphthyl hydroxymethylcarbamate (hydroxymethyl-
carbaryl).
A mixture of 5.6 g of 1-naphthyl methylcarbamate (0.03 mol), 40 ml
of THF, 10 ml of water, 1.2g of paraformaldehyde (0.04 mol), and
1.0 ml of concentrated hydrochloric acid was stirred and heated
gently to 50-60°C until a clear solution was obtained. The mixture
was maintained at this temperature for 1 hr, then allowed to stand
at room temperature for another hour. Dilution of the mixture with
100 ml of water produced an oil which was separated and the aqueous
layer was extracted with three 50-ml portions of ether and the ex-
tracts were combined with the oil. The ether solution was washed
with water, dried over anhydrous sodium sulfate, and the ether was
removed to give an oil which solidified when concentrated under
vacuum. Recrystallization from benzene and hexane gave 3.8g of
1-naphthyl hydroxymethylcarbamate (597» yield), mp 134-6°C. Pmr
spectrum in cig-dimethylsulfoxide with tetramethylsilane (TMS) as
an internal standard showed a 2H doublet centered at 6 4.7 (J. =
7 Hz) for NCH20 protons and a 7H multiplet at 6 7.25-8.22 for the
aromatic protons. Infrared spectrum in Nujol oil showed a band
for NH, OH at 3300 cm"1, and a carbonyl band at 1705 cm"1.
Similar procedures were used for the synthesis of hydroxymethyl-
carbofuran and phenyl hydroxymethylcarbamate. In these cases the
reaction mixture was neutralized with sodium carbonate at the end
of the heating period and the THF solvent was removed under vacuum.
The above reaction was not applicable to the preparation of the hy-
droxymethyl derivative of aldicarb, an oxime carbamate. In this
case the bis adduct of structure below was obtained.
233
-------
CH, 0
I 3 II
(CH0 SCCH=NOCNHCH0).0
3 22
4. References
1. Quistad, G. B., T. R.iFukuto, and R. L. Metcalf. Insecticidal,
Anticholinesterase and Hydrolytic Properties of Phosphoramidothio-
lates. J. Agr. Food Chem. 18:189-94, 1970.
2. Greenwald, E., and S. Winstein. The Correlation of Solvolysis
Rates. J. Amer. Chem. Soc. 70:846-54, 1948.
3. Westheimer, F. H. Pseudo-Rotation in the Hydrolysis of Phosphate
Esters. Accounts Chem. Res. 1:70-69, 1968.
4. Tolis, A. K., W. E. McEwen, and C. A. VanderWerf. Reaction of
Compounds of Trivalent Phosphorus with N-Haloamides. Tetrahedron
Lett. 3217-21, 1964.
234
-------
I. INSECTICIDE CYCLIC NUCLEOTIDE INTERACTIONS
The fundamental discovery, that when certain hormones bind to their
target cells there is an intracellular increase in cyclic nucleotides
which then act as "second messengers" for these hormones, was originally
made with the elucidation of the role of cyclic AMP (adenosine 3",5'-
cyclic monophosphate) as the intracellular mediator of the hepatic
glycogenolytic effect of epinephrine and glucagon.1 Extensive subse-
quent studies of cyclic AMP have provided new insights into our under-
standing of the secretion of neurotransmitters, hormones, and other
bioactive molecules, their actions at receptor sites, and the resultant
regulatory mechanisms.2 Most studies have been carried out on cyclic
AMP and mammalian systems, and much less attention has been directed to
cyclic GMP (guanosine 3',5'-cyclic monophosphate) or to invertebrate
systems. Evidence continues to accumulate concerning the fundamental
role of cyclic nucleotides in insects in morphogenesis and development,3"5
nervous system activity,7>^ flight muscle activity,9»-° glandular
secretion,11'12 and control of cuticular tanning.13'1^ These studies
have also primarily been on cyclic AMP, and surprising little attention
has been directed to cyclic GMP even though it was first reported as
early as 196915 that the cricket contained more cyclic GMP than cyclic
AMP, the reverse of the situation reported for mammals. In 1971
several insect tissues were shown to have high cyclic GMP-dependent
protein kinase activities,^° and the authors concluded that "it is to
be expected that further studies with arthropod material will be
valuable for elucidating the respective roles of cyclic AMP and cyclic
GMP in biological regulation." Yet arthropod tissues still essentially
remain to be exploited in this regard.
Our interests in the cyclic nucleotides are multiple. There is the
basic interest in characterization of these systems in insects. Our
first studies17'1^ were investigations of the characteristics, previously
unevaluated, of insect adenyl cyclase and phosphodiesterase, the enzymes
responsible respectively for synthesizing and degrading cyclic AMP. Both
enzymes were shown to be present in various tissues of the Madagascar
cockroach, Gromphadorhina portentosa, and to have many of the same
characteristics reported for the mammalian enzymes. We propose, as one
future project, to investigate in the same manner the characteristics of
guanyl cyclase and phosphodiesterase in insects. Secondly, we are
interested in the basic involvement of cyclic nucleotides in insects as
model systems for the further elucidation of neurotransmitter and
hormone action at the target membrane, cellular, and systems levels.
Thirdly, we are interested in the effects of drugs and foreign compounds
on such systems. Cyclic nucleotide interactions are clearly involved in
many biochemical phenomena in inter- and intra-cellular communication
and differentiation and growth. Therefore, cyclic nucleotide actions
should be relevant to the acute and chronic toxic and stress effects of
foreign compounds in disrupting normal and effecting abnormal communi-
cation and regulation.
235
-------
Several neurosecretory factors are proposed or known to be released from
the insect CNS on insecticide poisoning.19?20 Possible mechanisms for
and consequences of increased Malpighian tubule secretion on insecticide
poisoning in Rhodnius prolixus have been proposed involving the release
of bound diuretic hormone by paralytic doses of a variety of insecticides,
regardless of their primary modes of action on the CNS, and cyclic AMP
as a second messenger.21 Insecticide induced release of the hormone
controlling integument elasticity in Rhodnius,22 and the hyperglycaemic
and adipokinetic hormones in Schistocerca gregaria23 have also been
reported.
In mammals, it has been reported that the incubation of rat renal
cortices with DDT, chlordane, endrin, dieldrin, and heptachlor resulted
in significant increases of cyclic AMP and that oral administration of
these insecticides to rats resulted in an increase in endogenous cyclic
AMP and the basal fluoride-stimulated activities of liver and kidney
adenyl cyclase.21* »25 Recently, it has also been shown in in vitro and
in vivo experiments with rats that dichlorvos, Dursban, and diazinon
depressed endogenous corticosterone synthesis and blocked cortico-
steroidogenesis in response to ACTH and cyclic AMP stimulation of a
suspension of adrenal cells.2^
Studies of the possible interactions between insecticidal activity and
the cyclic nucleotides as second messengers have potentially important
implications to our more complete understanding of acute toxicity and
particularly of chronic toxicity, which is essentially unresolved.
Further, they may provide evidence for the actual systemic lesions
which are responsible for the deaths of insects from insecticidal
poisoning, also an area which is as yet not clearly elucidated, and of
clues for the development of new toxicants with greater specificity.
Initially our studies2^7 involved a survey of the direct effects of tepp,
methyl paraoxon, DDT, dieldrin, aldicarb, dimetilan, rotenone, allethrin,
and oxythioquinox on cockroach brain adenyl cyclase and phosphodiesterase
±n_ vitro. The results with cockroach brain adenyl cyclase in vitro
appear in Table 1. In general they are highly variable and in no case
do they show a classical inhibition curve. In all cases, except DDT,
the high standard deviations and/or inconsistencies of action as
inhibitor or activator with change in concentration lead to the conclu-
sion that these compounds have essentially no direct effects on adenyl
cyclase in vitro. With DDT, there is a relatively consistent inhibition
at all concentrations, suggesting that DDT may be an inhibitor of adenyl
cyclase. The absence of increased effect by DDT with increasing con-
centration may be due to solubility limitations.
The results with cockroach brain phosphodiesterase in vitro appear in
Table 2. Dimetilan, rotenone, allethrin, and particularly methyl
paraoxon show a general relationship of increasing inhibition with
increasing concentration of insecticide, though effective concentrations
are relatively high. On the other hand, DDT and particularly dieldrin
236
-------
Table 1. DIRECT EFFECTS OF INSECTICIDES-ACARICIDES ON COCKROACH BRAIN ADENYL CYCLASE
Average protein concentration in each assay was 145 io,g. Each value is the
mean and standard deviation of f.hree determinations.
Compound
Tepp
Methyl
paraoxon
DDT
Dieldrin
Aldicarb
Dimetilan
Rotenone
Allethrin
Oxythioquinox
Percent effect on cockroach brain adenyl cyclase activity in comparison with controls
10~6M
—
—
—
—
—
—
—
—
-8.110.9
5 x 10~6M
—
—
—
—
—
—
—
—
-26.0±2.2
10~5M
—
—
—
-28.0±1.1
+16.4133.6
—
+42.3110.2
-18.2147.2
+27.8135.6
5 x 10~5M
-3.4114.4
+16.5±25.7
-60.4±5.3
-48.915.8
+14.1122.0
-4.0117.7
+19.2123.1
-32.2116.7
-25.419.9
10~4M
+36.214.9
-7.7126.6
-44.416.2
+24.4112. 4
+57.0143.6
-42.1115.1
+41.2167.6
-10.2130.3
+2.4138.2
5 x lO^M
+13.213.5
+0.9110.9
-62.513.6
+16.0148.2
+28.615.0
-27.7123.4
-20.713.3
-55.0111.7
—
10~3M
-4.117.2
-57.811.9
-63.218.9
-44.118.6
+2.411.2
-9.6116.4
-25.314.5
+15.217.4
—
5 x 10~3M
+48.4149.6
-29.3141.0
-70. 13.6
—
—
-23.6121.6
—
—
—
CO
-------
Table 2. DIRECT EFFECTS OF INSECTICIDES-ACARICIDES ON COCKROACH BRAIN PHOSPHODIESTERASE
Average protein concentration in each assay was 6.7 ^g. Each value is the
mean and standard deviation for three determinations.
Compound
Tepp
Methyl
paraoxon
DDT
Dieldrin
Aldicarb
Dimetilan
Rotenone
Allethrin
Oxythioquinox
Percent effect on cockroach brain phospbodiesterase activity in comparison with controls
10"6M
—
—
—
—
—
—
—
—
-89.7±2.9
5 x 10~6M
—
—
—
—
—
—
—
—
-87.3±1.7
10~5M
—
—
—
+0.5±6.0
+8.0+7.0
—
-22.5+1.1
-3.4±6.5
-85.8±1.7
_5
5 x 10 M
+13.3±7.1
+1.2±8.0
+28.8±5.6
+45.0±2.7
+10.5±3.0
-6.1+6.9
-28.9+2.0
-2.5+7.1
-83.8+4.2
10~4M
-3.3+1.2
-28.9±1.8
+25.4±14.0
+54.1±4.4
+15.2±5.6
-12.0+2.9
-33.9±0.7
-32.0±32,7
-81.7±1.3
5 x 10~4M
+5.3±7.2
-34.7+8.1
+28.U7.6
+75.7±6.0
-5.4±7.7
-21.5+13.3
-52.0+2.9
-37.0±9.8
—
10~3M
-12.5+0.1
-80.6±2.1
+5.3+6.7
+64.0+5.3
-14.2±1.8
-34.5±1.4
-50.3±2.0
-36.7±7.2
—
5 x 10~3M
-22.1+2.9
-86.5±3.5
+7.6+7.0
—
—
-40.3±4.2
—
—
—
to
U)
OO
-------
appear to be activators of phosphodiesterase. The inconsistencies of
action as inhibitor or activator with increasing incubation concentra-
tions suggest that tepp and aldicarb have no direct effect on phospho-
diesterase in vitro. The most striking and potentially significant
result of this exploratory survey was the observation that oxythio-
quinox is a potent inhibitor of cockroach brain phosphodiesterase,
giving over 80% inhibition with an incubation concentration of 1 (j>l.
The limited solubility of oxythioquinox in the buffer may explain
similar levels of inhibition with increasing concentrations. In
comparison, using identical assay techniques, 1000-fold greater con-
centrations (1 mM) of aminophylline and theophylline, the most widely
used phosphodiesterase inhibitors in adenyl cyclase assays, inhibited
83.2% and 73.8%, respectively. This observation prompted us to obtain
and evaluate the following series of quinoxalinedithiol derivatives
(Fig. 1) as phosphodiesterase inhibitors.
c-o
OXYTHIOOUINOX
SAS 1946
SAS 2501
H»C.
C=*0
SAS 2079
Figure 1. Chemical structures of quinoxalinedithiol derivatives.
239
-------
The relative inhibition of cockroach brain phosphodiesterase by the
various quinoxalinedithiol derivatives may be compared from their I
values given in Table 3.
50
Table 3. INHIBITION OF COCKROACH BRAIN PHOSPHODIESTERASE
BY QUINOXALINEDITHIOL DERIVATIVES.
Assays contained an average of 6.48 ^g protein.
Inhibition concentrations ranging from 10~^ to
10 M were evaluated for each compound. Regres-
sion lines were calculated from at least three
points per line and three replications per point.
Compound
i50
Oxythioquinox
SAS 1948
SAS 2061 (QDSH)
SAS 2079
SAS 2185
SAS 2501
SAS 2551
8.6 x 10
3.2 x 10
-7
1.0 x 10
-7
1.6 x 10
-7
1.5 x 10
-6
4.2 x 10
-6
1.4 x 10
-7
A typical linear regression of log of inhibitor concentration versus
mean percent inhibition is shown in Fig. 2 for SAS 2079.
3 40
X
-BO
IO'T I0~*
SAS 2079 CONCENTRATION (M)
IO-"
Figure 2. Inhibition of cockroach brain phosphodiesterase by SAS 2079.
Each assay contained 6.3 y,g protein. Each point is the mean of three
determinations; brackets indicate standard deviation. Regression
line is calculated from first four points.
240
-------
Normally with increase in concentration of the quinoxaline inhibitors,
inhibition reached limiting values of 70-90 percent, probably due to
limits in solubility of the inhibitors in the incubation buffer at
the higher concentrations. All of the quinoxalinedithiol derivatives
examined were potent inhibtors of cockroach brain phosphodiesterase.
The inhibitory effects of caffeine, aminophylline, SQ 65,442, and the
quinoxalinedithiol derivatives on cockroach brain, rat brain, and beef
heart phosphodiesterases are compared in Table 4.
Table 4. THE EFFECTS OF METHYLXANTHINES, SQ 65,442, AND
QUINOXALINEDITHIOL DERIVATIVES UPON THE ACTIVITY
OF PHOSPHODIESTERASES FROM VARIOUS SOURCES.
Protein concentrations in the assays were as
follows: cockroach: 6.53 p,g; rat: 6.57 |j,g;
beef heart: 5.52 p,g. The incubation concen-
tration of the methylxanthines was 1 mM, that
of SQ 65,442 and quinoxalinedithiol derivatives
was 1 |j,M. Each value is the mean and standard
deviation of three determinations.
Percent inhibition of phosphodiesterases from
Compound
Caffeine
Aminophyll ine
SQ 65,442
Oxythioquinox
SAS 1948
SAS 2061 (QDSH)
SAS 2079
SAS 2185
SAS 2501
SAS 2551
. .
Cockroach brain
45.6 2.5
83.7 4.5
48.5 3.7
75.4 2.1
78.8 2.4
69.7 4.1
80.3 1.9
56.2 3.5
51.3 3.3
70.2 4.1
Rat brain
56.0 3.7
85.7 1.8
66.9 1.6
69.7 4.8
70.8 2.7
69.3 1.7
78.2 1.4
60.9 3.0
51.7 10.7
69.3 1.9
Beef heart
60.3 1.7
90.7 1.3
13.0 3.5
62.2 4.1
60.0 3.9
46.1 7.8
76.4 0.8
41.2 9.6
34.3 4.3
63.4 3.8
241
-------
Generally the inhibitory activities of the quinoxalines and SQ 65,442,
at 1000-fold less in concentration, were equal to or greater than
caffeine and slightly less than aminophylline on all sources of
phosphodiesterase studied. However, with the beef heart enzyme, the
inhibitory activity of SQ 65,442 was markedly less than with the
enzymes from cockroach and rat brains, and it was also somewhat lower
with the beef heart enzyme for quinoxaline inhibitors. The methyl-
xanthines were relatively equally inhibitory to the phosphodiesterases
from all three sources. Overall the most potent inhibitor of phospho-
diesterases from all three sources was SAS 2079. The inhibition of
rat brain phosphodiesterase as a function of concentration of SAS 2079,
SQ 65,442, and aminophylline is shown in Fig. 3.
100 -
I0~* IO'5 10"*
INHIBITOR CONCENTRATION (M)
ID'3
Figure 3. Inhibition of rat brain phosphodiesterase by SAS 2079,
SQ 65,442, and aminophylline. Each assay contained 6.1 ^,g protein.
With SAS 2079, inhibition reached a limiting value of about 70% at
concentrations above 10~6 ^, whereas inhibition by SQ 65,442 and amino-
phylline increased linearly across the entire range of concentrations
examined. Although the quinoxalinedithiol derivatives are generally
more active inhibitors of phosphodiesterases at lower concentrations
than are SQ 65,442 and the methylxanthines, it appears that solubility
deficiencies limit their effectiveness, with inhibition reaching
limiting values of about 70-90 percent as concentrations are increased.
Cheung28 reported that bovine brain phosphodiesterase is inhibited by
£-hydroxymercuribenzoate and the inhibition is reversible by 2-
mercaptoethanol, indicating that the enzyme possesses sulfhydryl groups
and that at least some of them are necessary for activity. This
evidence, together with that of Carlson and DuBois29 that oxythioquinox
and QDSH are inhibitors of sulfhydryl enzymes of intermediate carbo-
hydrate metabolism, suggests that the quinoxalinedithiol derivatives
are also acting as sulfhydryl inhibitors of phosphodiesterase.
242
-------
Oxythioquinox and other quinoxalinedithiol derivatives have been demon-
strated to be potent in vitro inhibitors of phosphodiesterases from
cockroach brain, rat brain, and beef heart, probably by acting as
sulfhydryl inhibitors. However, any direct relationship of this activity
to their mode of toxic action remains to be determined. On the basis of
the broad distribution of phosphodiesterases in the animal kingdom, it
seems unlikely that phosphodiesterase inhibition is a direct cause of
their selective acaricidal activity. Although the quinoxalinedithiol
inhibitors appear to be more active than any previously reported
phosphodiesterase inhibitors,30 their low water solubilities appear to
cause inhibition to reach limiting values of about 70-90 percent as
concentrations are increased in in vitro aqueous systems. Their
evaluation as phosphodiesterase inhibitors to enhance the accumulation
of cyclic AMP in adenyl cyclase assays of crude mitochondrial fraction
of mouse brain did not demonstrate any significant advantage over the
use of aminophylline, a broadly used standard inhibitor for this purpose.
Although direct j.n vitro inhibition of adenyl cyclase or phosphodiesterase
was not generally shown by the insecticides in the above survey, it is
possible that indirect rather than direct effects may be involved in
vivo and that the insecticides may directly affect some other part of
the system (release of a bioactive factor such as diuretic hormone,
for example) which may then in turn affect cyclic nucleotide titers.
Our subsequent work this year has been involved with examining this
hypothesis by evaluating ^n vivo levels of both cyclic AMP and cyclic
GMP in various cockroach tissues under normal and drug or insecticide
challenged conditions.
The initial part of the work was directed toward familiarization and
substantiation of bio-analytical methodology31' for determining in
situ levels of cyclic AMP and cyclic GMP in tissues of the Madagascar
cockroach. Control levels have now been determined in brain, terminal
abdominal ganglion, Malpighian tubules, rectum, and gastric caecae as
summarized in the following table.
Table 5. NORMAL (CONTROL) LEVELS OF CYCLIC NUCLEOTIDES IN
VARIOUS TISSUES OF THE MADAGASCAR COCKROACH
Tissue
Brain
Terminal abdominal ganglion
Malpighian tubules
Rectum
Gastric caecae
Cyclic nucleotides (pmole/mg protein)
Cyclic AMP
6.71 ± 0.48a
2.74 ± 1.92
8.28 ± 1.23
3.10 ± 1.14
2.42 ± 0.45
Cyclic GMP
17.43 ± 1.41
20.74 ± 15.93
29.06 ± 20.61
4.17 ± 2.22
4.11 ± 2.47
Average of three control determinations ± standard deviation.
243
-------
The Malpighian tubules were found to have the highest control levels of
both nucleotides. The CNS tissues, brain and terminal abdominal ganglion,
were intermediate and the gut tissues, gastric caecae and rectum, were
lowest. The high levels in Malpighian tubules, which are somewhat
greater than those usually observed in mammalian tissues, suggest a
probable extensive involvement of cyclic nucleotides in tubule function.
Additionally, the higher levels of cyclic GMP than of cyclic AMP in all
of the cockroach tissues is the reverse of that normally found in
mammalian tissues and confirms the original observation from the cricket}5
noted above. These results also suggest that the Madagascar cockroach
may have considerable potential for further studies on elucidating the
respective roles of cyclic AMP and cyclic GMP in biological regulation.
We anticipate undertaking an initial study in this regard to characterize
guanyl cyclase and cyclic GMP phosphodiesterase, neither of which has
been evaluated in insects, in comparison with our previous characteri-
zations of the two cyclic AMP enzymes.
It has recently been reported33 that both exposure to cold and
administration of aminophylline (an inhibitor of phosphodiesterase)
result in rapid increases in cyclic AMP i.n vivo in the rat adrenal
medulla and adrenal cortex. In order to determine whether or not levels
of cyclic AMP and cyclic GMP could be similarly affected in cockroach
tissues by challenge with a known active drug, four male Madagascar
cockroaches were injected with 200 pmole aminophylline/g body weight,
sacrificed after 30 and 60 min, and the cyclic nucleotide levels deter-
mined in the pooled excised tissues: brain, terminal abdominal ganglion,
Malpighian tubules, rectum, and gastric caecae, as summarized in the
following table.
Table 6. LEVELS OF CYCLIC NUCLEOTIDES IN VARIOUS TISSUES OF
THE MADAGASCAR COCKROACH 30 AND 60 MINUTES FOLLOWING
INJECTION OF 200 pmole AMINOPHYLLINE/g BODY WEIGHT
Tissue
Cyclic nucleotide (pmole/me protein)
Cyclic AMP
Brain
Terminal abdominal ganglion
Malpighian tubules
Rectum
Gastric caecae
Cyclic GMP
Brain
Terminal abdominal ganglion
Malpighian tubules
Rectum
Gastric caecae
Control
6.18
1.46
9.17
2.77
2.03
0
0
13.85
1.74
2.13
30 min
8.81
2.74
15.95
4.40
4.39
0
0
23.98
10.83
3.41
60 min
7.06
1.95
14.28
3.08
1.73
0
6.81
67.50
4.10
2.83
244
-------
The results show that inhibition of phosphodiesterase by injected
aminophylline causes a marked increase in tissue levels of both cyclic
nucleotides in 30 min, though the levels may then either increase or
decrease by 60 min. They confirm a comparable response to aminophylline
challenge in the cockroach to that found for rat adrenal medulla and
adrenal cortex and suggest that this methodology might be appropriate
for evaluating a challenge by insecticide poisoning, which then was
our next experiment.
Sixteen male Madagascar cockroaches were injected with 40 p,g of
parathion in 4 y£. of acetone, four cockroaches sacrificed at each of
four different stages of poisoning symptoms, and the cyclic nucleotides
determined in the pooled excised tissues: brain, terminal abdominal
ganglion, Malpighian tubules, rectum, and gastric caecae. The four
stages of poisoning symptoms are described in the following list. The
times after injections shown are approximate averages, since the cock-
roaches were sacrificed by symptomology, not time.
Stage
1. Control
2. Normal
3. Active
Time after
injection (hr)
- 4.5
~ 0.5
4. Incoordinated
5. Paralyzed
Description of symptomology
No signs of effects from injection
of 4 ]i& of acetone.
Antennae and palpi moving;
normal exploration movements
in cage.
Rapid movements; spontaneous
startle reaction and hissing
behavior; abdomen shows tremors,
flexing, and rotation; leg
tremors.
Hissing behavior and leg and body
tremors accentuated; positional
instability—insect falls on back
with difficulty in righting, if
righted again falls on back.
On back; antennae in tetany;
occasional leg and body tremors
and flexing; abdomen bloated with
secretion from anus.
The cyclic nucleotide levels found in the various stages are shown in
Table 7.
245
-------
Table 7. LEVELS OF CYCLIC NUCLEOTIDES IN VARIOUS TISSUES OF
THE MADAGASCAR COCKROACH AT VARIOUS STAGES OF SYMPTOMOLOGY
FOLLOWING INJECTION OF 40 \i% OF PARATHION PER COCKROACH
Tissue
Cyclic AMP
Brain
Terminal abdominal
ganglion
Malpighian tubules
Rectum
Gastric caecae
Cyclic GMP
Brain
Terminal abdominal
ganglion
Malpighian tubules
Rectum
Gastric caecae
Cyclic nucleotides (pmole/mg) protein
Control
7.13
4.95
6.88
2.15
2.91
18.42
9.47
20.82
4.69
6.88
Normal
15.22
6,39
10.81
4.89
5,39
9.18
7.36
25.17
5.40
4.48
Active
10.88
4.14
19.03
3.35
8.75
9.25
7.53
41.85
5.84
29.34
Incoordinated
10.48
3.96
9.34
3.21
4.26
5.52
8.08
26.45
3.99
108.82
Paralyzed
5.04
0
11.49
2.08
5.77
8.00
0
35.43
6.26
98.78
Although the implications of these results are not yet understood, it is
obvious that a number of interesting differential changes of cyclic
nucleotide levels occurred. In the challenge with aminophylline there
was an increase in level of both cyclic nucleotides in all tissues in 30
min. This was to be expected if aminophylline as a phosphodiesterase
inhibitor protected the cyclic nucleotides being produced at a normal
basal rate from normal degradative loss by enzymatic hydrolytic mechanisms.
In the challenge with parathion, however, the changes in levels of cyclic
nucleotides are much more specific and differential. Marked changes in
levels occurred for both nucleotides for brain, Malpighian tubules and
gastric caecae, but changes in levels were minimal for terminal abdominal
ganglion and rectum. Differences in time sequence of changes in levels
are also of potential significance. The maximum increase in cyclic AMP
in the brain occurred in the normal stage, whereas in Malpighian tubules
and gastric caecae it occurred in the active stage. The cyclic GMP
levels decreased rather than increased in the brain, and in the Mal-
pighian tubules they reached their maximum level in the active stage,
whereas in the gastric caecae this occurred in the incoordinated stage.
These differences suggest a time sequence of events in the different
tissues and different involvement patterns for the two cyclic nucleo-
tides. Obviously further investigation will be necessary for elucidation
of these observations. They do, however, indicate a basic involvement
of the cyclic nucleotides in the intoxication process. The elucidation
of this involvement may hold keys to our more complete understanding of
insecticidal intoxication and its consequences in both insects and
mammals and particularly in relation to chronic toxicity.
246
-------
References
1. Sutherland, E. W., and T. W. Rail. The Relation of Adenosine 3',5'-
Phosphate and Phosphorylase to the Actions of Catecholamines and
Other Hormones. Pharm. Rev. 12:265-299, 1960.
2. Robison, G. A., R. W. Butcher, and E. W. Sutherland. Cyclic AMP.
Academic Press, N. Y., 1971. 531 p.
3. Vedeckis, W. V. and L. I. Gilbert. Production of Cyclic AMP and
Adenosine by the Brain and Prothoracic Glands of Manduca sexta.
J. Insect Physiol. 19:2445-2457, 1973.
4. Vedeckis, W. V., W. E. Bollenbacher, and L. I. Gilbert. Cyclic AMP
as a Possible Mediator of Prothoracic Gland Activation. Zool.
Jahrb., Abt. Allg. Zool. Physiol. Tiere ^78:440-448, 1974.
5. Morishima, I. Cyclic AMP Phosphodiesterase Activity During the
Development of the Silkworm, Bombyx mori. J. Insect Physiol. 19:
2261-2265, 1973.
6. DeReggi, M. L., and H. L. Cailla. Physiological Cyclic AMP in
Drosophila. FEBS Lett. 4^6:293-295, 1974.
7. Nathanson, J. A., and P. Greengard. Serotonin-Sensitive Adenylate
Cyclase in Neural Tissue and its Similarity to the Serotonin
Receptor: A Possible Site of Action of Lysergic Acid Diethylamide.
Proc. Nat. Acad. Sci. U.S.A. 2!= 797-801, 1974.
8. Tsuzuki, J., and R. W. Newburgh. Cyclic Nucleotide Binding and
Protein Kinase in the Central Nervous System of Galleria mellonella.
Biochem. Biophys. Acta 354:101-106, 1974.
9. Achazi, R. K. Activity and Subcellular Localization of Adenyl
Cyclase in the Flying Muscle of Locusta migratoria. Z. Naturforsch.
Teil C ^9: 451-452, 1974.
10. Bockbreder, C. Einfluss von electrischer Stimulation auf Phosphylase-
Aktivitat und cAMP-Gehalt in Insektenflugmuskeln im Locusta migra-
toria. Experientia _30:1290-1292, 1974.
11. Berridge, M. J. Effects of Derivatives of Adenosine 3',5'-
Monophosphate on Fluid Secretion by the Salivary Glands of
Calliphora. J. Exp. Biol. j>9:595-606, 1973.
12. Prince, W. T., H. Rasmussen, and M. J. Berridge. Role of Calcium
in Fly Salivary Gland Secretion Analyzed with the lonophore
A-23187. Biochem. Biophys. Acta 329:98-107, 1973.
247
-------
13. Fuchs, M. S., and D. A. Schlaeger. Stimulation of Dopa Decarboxy-
lase Activity by Ecdysone and its Enhancement by Cyclic AMP in
Adult Mosquitoes. Biochem. Biophys. Res. Commun. 54_: 784-789, 1973.
14. Vandenberg, R. D., and R. R. Mills. Hormonal Control of Tanning by
the American Cockroach. Cyclic AMP as a Probable Intermediate.
J. Insect Physiol. 20:623-627, 1974.
15. Ishikawa, E., S. Ishikawa, J. W. Davis, and E. W. Sutherland.
Determination of Guanosine 3',5'-Monophosphate in Tissues and of
Guanyl Cyclase in Rat Intestine. J. Biol. Chem. j!44_: 6371-6376, 1969.
16. Kuo, J. F., G. R. Wyatt, and P. Greengard. Cyclic Nucleotide-
Dependent Protein Kinases. IX. Partial Purification and Some
Properties of Guanosine 3',5'-Monophosphate-Dependent and
Adenosine 3',5'-Monophosphate-Dependent Protein Kinases from
Various Tissues and Species of Arthropoda. J. Biol. Chem. 246;
7159-7167, 1971.
17. Rojakovick, A. S., and R. B. March. The Activation and Inhibition
of Adenyl Cyclase from the Brain of the Madagascar Cockroach
(Gromphadorhina portentosa). Comp. Biochem. Physiol. 43B;209-215,
1972.
18. Rojakovick, A. S., and R. B. March. Characteristics of Cyclic 3',5'-
Nucleotide Phosphodiesterase from the Brain of the Madagascar
Cockroach (Gromphadorhina portentosa). Comp. Biochem. Physiol. 47B:
189-199, 1974.
19. Roan, C. C., and T. L. Hopkins. Mode of Action of Insecticides.
Ann. Rev. Entomol. _6:333-346, 1961.
20. Sternburg, J. Autointoxication and Some Stress Phenomena. Ann. Rev.
Entomol. j3:19-38, 1963.
21. Casida, J. E., and S. H. P. Maddrell. Diuretic Hormone Release on
Poisoning Rhodnius with Insecticide Chemicals. Pest. Biochem.
Physiol. _!:71-83, 1971.
22. Maddrell, S. H. P., and S. E. Reynolds. Release of Hormones in
Insects After Poisoning with Insecticides. Nature (London) 236;
404, 1972. -
23. Samaranayaka, M. Insecticide-Induced Release of Hyperglycaemic and
Adipokinetic Hormones of Schistocerca gregaria. Gen. Comp.
Endocrinol. ^4:424-436, 1973.
24. Kacew, S., and R. 0. Singhal. Stimulation of Kidney Cortex Cyclic
3',5'-Adenosine Monophosphate-H^ Formation by DDT and Related
Insecticides. Fed. Proc. 32:802, 1973.
248
-------
25. Kacew, S., and R. L. Singhal. Influence of p,p'-DDT, a-Chlordan,
Heptachlor, and Endrin in Hepatic and Renal Carbohydrate Metabolism
and Cyclic AMP-Adenyl Cyclase System. Life Sci. 13j1363-1371, 1973.
26. Given, M., and C. B. Brown. Effect of Organophosphate Insecticides
on Adrenal Corticosterone Formation. Pestic. Biochem. Physiol. 4_:
254-259, 1974. ~
27. Rojakovick, A. S., and R. B. March. Insecticide Cyclic Nucleotide
Interactions. I. Quinoxalinedithiol Derivatives: A New Group of
Potent Phosphodiesterase Inhibitors. Pestic. Biochem. Physiol.
(in press).
28. Cheung, W. Y. Cyclic Nucleotide Phosphodiesterase, In: Advances
in Biochemical Psychopharmacology, Greengard, P., and E. Costa
(Eds.). Raven Press, N. Y., 1970. Vol. 3, p. 54.
29. Carlson, G. P., and K. P. DuBois. Studies of the Toxicity and
Biochemical Mechanisms of Action of 6-Methyl-2,3-quinoxalinedithiol
Cyclic Carbonate (Morestan). J. Pharmacol. Exp. Ther. 173:60-70,
1970.
30. Amer, M. S., and W. E. Kreighbaum. Cyclic Nucleotide Phosphodi-
esterases: Properties, Activators, Inhibitors, Structure-Activity
Relationships and Possible Role in Drug Development. J. Pharm.
Sci. (in press).
31. Oilman, A. C. A Protein Binding Assay for Adenosine 3':5'-
Cyclic Monophosphate. Proc. Nat. Acad. Sci. U.S.A. 67^:305-312, 1970.
32. Murad, F., V. Manganiello, and M. Vaughan. A Simple, Sensitive
Protein-binding Assay for Guanosine 3',5'-Monophosphate. Proc.
Nat. Acad. Sci. U.S.A. 68_: 736-739, 1971.
33. Byus, C. V., and D. H. Russell. Ornithine Decarboxylase Activity:
Control by Cyclic Nucleotides. Science 187:650-652, 1975.
249
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J. INSECTICIDE PENETRATION AND ITS SPECIFICITY AS A RESISTANCE MECHANISM
Penetration of foreign compounds into the organism, together with
partitioning and distribution factors within the organism, is probably
the most inadequately elucidated aspect of toxicology. It is the
generally accepted view that the integument or skin is directly
penetrated by the foreign compound, and that once penetrated the
foreign compound partitions into and is carried by the hemolymph or
blood to be partitioned to the site of action in the tissues. In the
past few years renewed interest in the penetration of contact insecti-
cides into the insect body has been stimulated by a new hypothesis by
P. Gerolt1 that contact insecticides do not reach the site of action
via the hemolymph, but follow a lateral route through the integument
and tracheae to the cellular level. Although it is increasingly
apparent that Gerolt's hypothesis is incorrect,2'3 additional interest
in penetration has been stimulated by the mounting evidence of the
role of decreased penetration as a mechanism of insect resistance to
insecticides.2»4'5
On the supposition that a comparison of insecticide absorption into
susceptible and insecticide-resistant insects might provide a viable
model system for studying factors of insecticide penetration, a survey
was undertaken of the penetration of the insecticides used in resistance
selection into susceptible and various house fly strains selected over
the last twenty years. Penetration was determined by applying the
insecticides to the cuticle, rinsing the treated insects with ethanol
at various time intervals, and determining the amount of insecticide in
the rinse by GLC analysis. Comparison of the penetration of 11 insecti-
cides was made at the dosage levels (0.2 and 1.0 pg/fly) at 0, 1, 2,
and 4 hours on two susceptible strains (NAIDM and WHO) and nine resistant
strains [Super Pollard, MIPC (m-isopropylphenyl N-methylcarbamate),
Ronnel, Ronnel II, DPEP (0-(2,4-dichlorophenyl) 0-ethyl phosphoradmido-
thioate), Fenthion, Parathion, Dimethoate, and Chlorthion].
A plot of |j,g absorbed/female fly versus time for ronnel, the most com-
pletely absorbed of the compounds studied, is shown in Figure 1. This
plot is typical of most of the insecticides and strains studied. The
greatest differences in absorption between strains were at 1 hour,
relatively equal amounts being absorbed by 4 hours. The initial rate of
absorption was greater for the susceptible than for the resistant strains.
Greater actual amounts were absorbed at the higher than the lower
dosage.
250
-------
T~^n
Figure 1. Absorption of ronnel by various house fly strains.
251
-------
A similar plot for dimethoate (also typical of dieldrin, DDT, and
fenoxon) is shown in Figure 2. Note that here less of the higher
dosage is absorbed initially than of the lower dosage by the resistant
strains. Such an inverse relationship has not been reported previously,
and this is one observation of this initial survey which merits further
verification and elucidation.
Figure 2. Absorption of dimethoate by various house fly strains.
The initial survey disclosed that reduced penetration is primarily
operative within the first few hours following application, for by four
hours penetration of a given insecticide is nearly equal in all strains.
A summary of the absorption data one hour after application, at which
maximum differences were observed, is shown in Table 1.
252
-------
Table 1. PERCENT INSECTICIDE ABSORBED IN THE HOUSE FLY
ONE HOUR AFTER TOPICAL APPLICATION OF 0.2 OR 1.0 ug/FEMALE FLY.
House fly strains selected for resistance with each insecticide underlined.
xs
CH 0) P'
Strain
G.P.b
p.c.c
NAIDM
WHO
S. Pollard
MIPC
Ronnel
Ronnel II
DPEP
Parathion
Fenthion
Chlorthion
Dimethoate
Strain
G.P.
P.C.
WHO
NAIDM
S. Pollard
MIPC
Fenthion
Ronnel
Ronnel II
Parathion
Chlorthion
Dimethoate
DPEP
l^iiify
0.2
100
72
95
,
95±1
95
DPEP
0.2
18
46
80
78
66±4
66
74
59
57
72
60
66
60
1.0
100
80
99
99 d
99±1°
99
99
99
99
99
99
99
99
1.0
5
10
62
61
52±4
48
43
43
39
37
36
34±10
26±6
Strain
G.P.
P.C.
WHO
NAIDM
Parathion
Fenthion
MIPC
Dimethoate
Ronnel II
Chlorthion
Ronnel
S. Pollard
DPEP
Strain
G.P.
P.C.
NAIDM
WHO
MIPC
Ronnel II
Ronnel
Chlorthion
DPEP
Fenthion
Parathion
S. Pollard
Dimethoate
Ronnel
0.2
62
38
89
81
77
75
74
71
75
65
70
63±2
79
Parathion
0.2
24
20
81
81
70
74
70
58
76
74
67
55±12
58
1.0
15
35
81
74
67
67
67
67
64
63
62
57±11
42±18
1.0
0
5
61
57
46
36
35
32
30
30
28
26±11
25±7
253
-------
Table 1 (continued). PERCENT INSECTICIDE ABSORBED IN THE HOUSE FLY
Fenthion
Strain
G.P.
P.C.
NAIDM
WHO
MIPC
Fenthion
Ronnel II
S. Pollard
DPEP
Parathion
Chlorthion
Dimethoate
Ronnel
0.2
22
20
79
79
58
59
56
46±13
59
56
41
52
56
1.0
0
0
61
58
34
33
33
33±7
32
29
27
23
22
Monitor
0.2
G.P.
P.C.
WHO
MIPC
DPEP
NAIDM
S. Pollard
Ronnel II
Fenthion
Dimethoate
Ronnel
Parathion
Chlorthion
1.0
46
36
84
80
58
74
84±1
66
60
60
44
66
44
5
30
60
58
52
47
46±2
46
-
-
46
40
36
Chlorthion
Strain
G.P.
P.C.
NAIDM
WHO
MIPC
Fenthion
S. Pollard
Parathion
Ronnel II
Ronnel
Dimethoate
Chlorthion
DPEP
0.2
0
16
84
87
77
62
64±5
69
64
69
68
71
72
1.0
0
0
58
57
32
29
24 ±4
23
23
19
19
18
16±13
Dieldrin
Strain
G. P.
P.C.
WHO
NAIDM
MIPC
DPEP
S. Pollard
Ronnel
Dimethoate
Fenthion
Chlorthion
Ronnel II
Parathion
0.2.
24
52
85
71
61
70
50±5
68
72
52
54
60
63
0
0
54
52
30
26
25±4
21
19±8
14
13
12
5
254
-------
Table 1 (continued). PERCENT INSECTICIDE ABSORBED IN THE HOUSE FLY
Strain
G.P.
P.C.
WHO
NAIDM
S. Pollard
Ronnel II
Ronnel
MIPC
Parathion
Dimethoate
DPEP
Chlorthion
Fenthion
Strain
G.P.
P.C.
NAIDM
MIPC
Ronnel
WHO
Ronnel II
S. Pollard
Fenthion
Chlorthion
Parathion
Dimethoate
DPEP
Fenoxon
0.2
12
12
68
73
76+3
70
64
67
57
66
70
60
47
Dimethoate
0.2
4
38
72
64
71
74
60
64 ±10
72
68
60
74
63
1.0
0
15
44
42
36
34
28
18
15
13
12
8
3
1.0
0
25
28±1
27
25
20±8
20
17 ±8
14 ±6
8 ±12
6+2
5±1
2 ±8
Strain
G.P.
P.C.
WHO
NAIDM
MIPC
Fenthion
Ronnel
DPEP
Chlorthion
Parathion
Dimethoate
S. Pollard
Ronnel II
DDT
0.2
44
42
57
52
51
40
48
47
44
57
48
34+6
36
1.0
0
10
39
29
22
13
12
12+4
12
10+4
9
9±7
6
*Lost from cuticular surface.
G.P. = Glass pipet treated with insecticide in ethanol (0.5 yl)
containing extractable cuticular components of 25 house flies/ml.
"P.C. = Empty NAIDM house fly puparial case treated with insecticide in
j ethanol (0.5 ul).
Average ± S.D.
255
-------
These data disclose two striking results for further study. As noted
above (Fig. 2), with dimethoate, DDT, fenoxon, and dieldrin, one or
more of the most penetration-resistant strains absorb less actual
insecticide for the higher than the lower dosage at 1 hour, whereas
the reverse and expected pattern is shown at later times and by the
other strains and for other insecticides. Delayed penetration has
previously been characterized as non-specific and has generally been
considered to be related to physical properties such as solubility.
A much more specific pattern appears to emerge from this initial survey
which is suggestive of structure-activity rather than physical property
relationships. This is suggested by the changing pattern of relative
absorption by the different strains for the various insecticides and
particularly by the fact that the strain selected with a particular
insecticide is among the least absorptive of that insecticide as
summarized in Table 2 showing the rank order of house fly strains in
decreasing order of absorption of insecticides at 1.0 ^g/fly dose.
Table 2. RANK ORDER OF HOUSE FLY STRAINS IN DECREASING ORDER OF
ABSORPTION OF INSECTICIDES AT 1.0 |j,g/FLY DOSE (FROM TABLE 1)
Insecticide
Ronnel
DPEP
Parathion
Fenthion
Monitor
Chlorthion
Dieldrin
Fenoxon
DDT
Dimethoate
Ave . Rank
Rank order of strains (1
WHO
1
1
2
2
1
2
1
1
1
4
1.6
NAIDM
2
2
1
1
4
1
2
2
2
1
1.8
MIPC
5
4
3
3
2
3
3
6
3
2
3.4
Fenthion
4
5
8
4a
7
4
8
11
4
7
6.2
= most absorptive)
S. Pollard
10
3
10
6
5
5
5
3
10
6
6.3
Ronnel II
7
7
4
5
6
7
10
4
11
5
6.6
Ronnel Parathion DPEP
Ronnel
DPEP
Parathion
Fenthion
Monitor
Chlorthion
Dieldrin
Fenoxon
DDT
Dimethoate
Ave. Rank
9
6
5
11
9
8
6
5
5
3
6.7
3
8
9
8
10
6
11
7
8
9
7.9
11
11
7
7
3
11
4
9
6
11
8.0
Chlorthion
8
9
6
9
11
1Q
9
10
7
8
8.7
Dimethoate
6
10
11
10
8
9
7
8
9
ifi
8.8
Rank order of strain for insecticide it was selected with underlined.
256
-------
This is also supported by the fact that no direct relationship between
water solubility or partitioning behavior of the insecticides and their
degrees of absorption in the susceptible NAIDM strain was found, as
shown in Table 3.
Table 3. WATER SOLUBILITY, PARTITIONING COEFFICIENTS, AND
CUTICULAR ABSORPTION OF VARIOUS INSECTICIDES
IN THE NAIDM STRAIN OF THE HOUSE FLY
Insecticide
Water Partitioning
solubility coefficient*
% of applied dose absorbed
DDT
Dieldrin
DPEP
Parathion
Chlorthion
Ronnel
Fenthion
Fenoxon
Dimethoate
CHO 0
P
PPM
0.0034
0.25
5
24
40
44
55
5500
ca. 20,000
Isooctane:
80% acetone
0.93
0.88
0.76
0.56
1
0.2 ^
52
71
78
81
84
81
79
73
72
hour
ig 1.0 yg
29
52
61
61
58
74
61
42
28
4
0.2 p,
80
91
95
93
99+
95
94
94
84
hours
g 1.0 pg
59
74
80
79
83
86
83
68
52
soluble
74
47
88
67
A third piece of evidence that the penetration-delaying mechanism (and
thus perhaps the penetration mechanism itself) may be more specific than
non-specific is provided by a study of the absorption of and resistance
to tributyltin chloride (TBTCl) in the house fly strains.
TBTC1 has been utilized as a diagnostic toxicant in identifying the
absorption delaying mechanisms for resistance controlled by gene(s) on
chromosome III in the house fly.4'5 The resistant house fly strains used
in these studies show relative resistances of 10- to 32-fold to TBTCl
when compared at the LD5Q level to the susceptible NAIDM strain (see
Table 4). With the thought that evaluation of absorption of TBTCl in
comparison to resistance to TBTCl in the house fly strains might assist
in interpreting the data on absorption patterns of other insecticides
in the strains, a GLC method for TBTCl was worked out. Conditions were
as follows: 2 ft 4 mm ID glass U-column packed with 5% OV101 on Gas
Chrom Q 80/100 mesh; temperatures: column oven 158°, flash heater 290°,
detector 250°; gas flows (ml/min): H 33, He 40, air 330; FID detector,
minimum quantitatively detectable TBTCl 250 ng. When absorption of
TBTCl into the various strains was determined, no relationship of
absorption to resistance level was found as shown in Table 4. There
257
-------
is also no relationship between resistance ratio as an indicator of
level of delayed absorption as a resistance mechanism and the average
rank order of absorption of the various strains as shown in Table 2.
Although these results were thus of little help in interpreting the
other absorption data, they raise a number of questions for further
investigations on the role and mechanisms of TBTC1 resistance.
Table 4. TOXICITY AND RESISTANCE RATIOS FOR TRIBUTYLIN CHLORIDE
IN THE HOUSE FLY IN COMPARISON WITH PER CENT ABSORPTION3
1 HOUR AFTER TOPICAL APPLICATION OF 10 ^/FEMALE FLY
Strain
G.P.b
p.c.c
NAIDM
WHO
S. Pollard
Dimethoate
Parathion
MIPC
Chlorthion
Ronnel
DPEP
Fenthion
24-hour LDso
^g/female fly
0.59
0.62
5.9
8.4
8.4
9.1
10.0
12.3
18.0
19.0
Resistance ratio
LDso strain X
LD50 NAIDM
1
1
10
14
14
15
17
21
30
32
% TBTC1 absorbed
(lost) in 1 hour
24
33
51
62
64
64
59
54
60
60
68
58
rLoss from cuticular surface.
G.P. = Glass pipet treated with insecticide in ethanol (0.5 yl)
containing extractable cuticular components of 25 house flies/ml.
P.C. = Empty NAIDM house fly puparial case treated with insecticide in
ethanol (0.5 pi).
This initial survey suggested a number of potential entries for further
investigation of the fundamental aspects of insecticide penetration and
its interrelationships with metabolism, toxicity, and resistance. But
before attempting more critical and fundamental extensions of the work,
our attention was directed toward further elucidation and interpretation
of these initial studies.
For example, there was the question of significance of differences
between the individual absorption figures. It was not difficult to
258
-------
accept the large differences between the most and least absorbing strains
as being significant since these differences were normally 20% or more,
but was a difference of 5% or even 10% significant? Since the logistics
of making all determinations in sufficient replications to assess
variability was insurmountable, the expedient of making replicate (4)
determinations of absorption for all insecticides with a single strain
and for a few selected insecticides with other strains was resorted to.
A summary of the absorption data, including the replicate averages and
their standard deviations, 1 hour after application is found in Table 1.
The data for 1 hour are utilized for comparison since the differences
in absorption between strains were greatest at this time. Greater
differences are also apparent at the higher than lower dosage. These
observations suggest that the initial rate of absorption is the most
critical difference factor and that the delayed absorption mechanism is
more operative at the higher than lower dose. Thus, further studies
of absorption rates within the first hour after application of insecti-
cide and the effects of dosage level merit further consideration. The
replicate averages and their standard deviations give some confidence
that there are significant differences in absorption between the two
susceptible strains and the nine resistant strains, but the significance
of differences between the resistant strains is much less clear,
although in a number of cases there appear at least to be significant
differences between the most and least absorbing of the resistant strains.
However, the question of non-specificity or specificity of absorption
mechanisms and of delayed absorption as a resistance mechanism remains
unresolved since many of the differences in absorption between the
resistant strains do not appear to be significant. If the mechanisms
were non-specific and related only to physical properties such as
solubility or partitioning properties, one would expect that the strains
would fall into a common rank order for all of the insecticides in
relation to the degree of development of the delayed penetration
resistance mechanism. It seems reasonably apparent from the rank orders
of absorption presented in Table 2 that the two susceptible strains
quite uniformly absorb the most insecticide and that the MIPC resistant
strain does the same among the resistant strains. There is considerable
non-uniformity of rank among the remaining resistant strains, however.
Perhaps most striking is the observation that the rank order of a strain
for the insecticide it was selected with is normally very high, which
suggests that there may be some specificity for delayed absorption of
the selecting insecticide. Although this order appears to be something
more than random, it is apparent that additional studies will be required
to assess the question of non-specificity of absorption as related to
physical properties such as solubility or partitioning or of specificity
of absorption as related to chemical structure-activity properties such
as binding to cuticular components or carrier molecules.
In the initial survey, there did not appear to be any direct relationship
between solubility or partitioning properties and absorption. Since
i
259
-------
losses might occur in the methods used by volatilization as well as
absorption, it was decided to investigate relative volatility of the
various insecticides so that losses from this source might be corrected.
It was soon observed that a number of the insecticides volatilized from
a glass surface, but this did not seem to be a reasonable comparison to
the house fly cuticle, since cuticular components might be expected to
have retarding effects on volatility. Therefore, measurements were
made of losses from glass (disposable glass pipets) by insecticides
applied in ethanol (0.5 yl) containing the extractable cuticular compo-
nents of 25 house flies/ml. The cuticular components had a deterring
effect on volatilization. Measurements of losses were also made by
applying the insecticides in ethanol to empty house fly puparial cases
(NAIDM) with the reasoning that these might closely approximate the
adult cuticular surface, but that the effects of absorption (the insect
pupa is a notably insecticide-tolerant stage usually attributed to poor
absorption) and the metabolism might be bypassed. The data for these
measurements are also shown in Table 1 under the headings G.P. (glass
pipet) and P.C. (puparial case). It is apparent that they do not
provide a completely adequate pseudo-fly for measuring volatility
corrections, but primarily serve to point out the complicated inter-
actions of the many factors involved—the effects of dose on volatility
and absorption, of cuticular components on volatility and extractability,
of partitioning from the cuticle to other tissues, of metabolism, of
excretion, etc. If either of the two methods were to be used in further
studies, it appears that the glass pipet method might be most representa-
tive of volatility losses per se. They also point out that further
studies on the relationship of absorption to solubility and partitioning
properties must contain an estimate of losses due to volatility.
References
1. Gerolt, P. Mode of Entry of Contact Insecticides. J. Insect
Physiol. 15:563-580, 1969.
2. Ebeling, W. Permeability of Insect Cuticle. In: The Physiology of
Insecta, Rockstein, M. (Ed.). New York, Academic Press, Inc., 1974.
Vol. VI, 271-343.
3. Olson, W. P. Dieldrin Transport in the Insect. Examination of
Gerolt's Hypothesis. Pestic. Biochem. Physiol. _3:384-392, 1973.
4. Plapp, F. W., and R. F. Hoyer. Insecticide Resistance in the House
Fly: Decreased Rate of Absorption as the Mechanism of Action of a
Gene that Acts as an Intensifier of Resistance, J, Econ. Entomol.
_63:1298-1303, 1968.
5. Sawick, R. M., and K. A. Lord. Properties of a Mechanism Delaying
Penetration of Insecticides into Houseflies. Pestic. Sci. !_:
213-217, 1970.
260
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6. Beroza, M., M. N, Inscol, and M. C, Bowman, Distribution of
Pesticides in Immiscible Binary Solvent Systems for Cleanup and
Identification and its Application in the Extraction of Pesticides
from Milk. Residue Reviews 30:1-61, 1969,
261
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K- GAS CHROMATOGRAPHY OF INSECTICIDES ON MODIFIED SUPPORTS
The direct gas chromatography of most carbamate and many phosphate
insecticides has presented difficulties because of on-column pyrolysis
or retention on reactive sites. Thus we have been activity investi-
gating GLC methods to improve our capabilities in this regard. Recently,
the development of a novel gas chromatographic phase, obtained by heat
treatment and exhaustive methanol extraction of a 6% Carbowax 20M
packing was reported.1 This new phase allowed fast, efficient, and
selective separations of several types of polar compounds with little
indication of decomposition of labile compounds. The new methodology
showed characteristics of the more desirable features of both gas-solid
and gas-liquid chromatography—low bleed, short retention times, high
selectivity, and excellent peak symmetry and resolution of highly
polar compounds. Further studies have demonstrated that the methodology
is not limited to Carbowax 20M as a coating phase.2'3 The application
of the modified Carbowax 20M support to the direct gas chromatography of
carbamate insecticides has also been demonstrated,^ but this novel
methodology has not been extended to other pesticides or the modification
of other support phases for the gas chromatography of pesticides.
We have now investigated and extended the use of this new support
methodology to the gas chromatography of a number of carbamates and
phosphates, which we have previously been unsuccessful in chromatograph-
ing, as well as in chromatographing a number of chlorinated hydrocarbon
insecticides. Our initial studies were with a modified Carbowax 20M
column. The packing was prepared by coating HCl-washed Chromosorb-W
80/100 with 6% Carbowax 20M. The coated support was conditioned over-
night at 280° and then exhaustively extracted with methanol, producing
a solid support presumably surface-modified with a monomolecular layer
of Carbowax 20M. The GLC conditions utilizing a 6 ft 2 mm I.D. glass
U-column were as follows: carbamates and phosphates: Rb2SO^ AFID,
H 23 ml/min, He 30 ml/min, air 234 ml/min, and oven temperatures 135-
185°; chlorinated hydrocarbons: EC detector, Argon (95)-methane (5)
30 ml/min, and oven temperatures 165-225°C. This new methodology
appears most promising in permitting us to utilize GLC analysis for a
number of previously quantitatively unchromatographable compounds in our
studies on insecticide absorption, as shown in Table 1. Further, the
modified Carbowax 20M column almost appears to be a universal column
for multi-classes of insecticides. A limiting characteristic of this
column, however, was the failure to achieve separation of phosphoro-
thionates and their oxygen analogs, which would limit its applicability
to many types of uses.
262
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Table 1. RETENTION TIME IN MINUTES FOR VARIOUS CARBAMATE,
PHOSPHATE, PHOSPHOROTHIONATE, AND CHLORINATED HYDROCARBON
INSECTICIDES ON A CARBOWAX 20M-SURFACE-MODIFIED GLC COLUMN
Insecticide Retention time in
135° 145° 155°
Temik 4.4 2.8
Methomyl 10.5 6.4 3.9
MIPC 2.4
Propoxur 3 . 0
Zectran 6.7
Pyrolan 10.2
Carbaryl
Monitor 1.7
Dyfonate 2.3
Orthene 4.6
Dimethoxon
Dasanit
Fenoxon
Paraoxon
Chloroxon
Ciodrin
Phenkapton
Lindane
Aldrin
Dieldrin
Kepone
Mir ex
DDT
min at following temperatures
165° 170° 185° 205° 225°
1.5
2.0
4.4 1.8
6.8 2.6
12.5 3.5
1.5
2.0
3.0
4.5
5.0
7.0
7.5 3.0
11.0 4.0
15.0 6.4
12.0
1.2 0.6
1.5 ' 0.8 0.4
4.6 2.2 1.1
2.9,6.0 1.3,5.6
2.4 1.3
5.0 2.2 1.0
263
-------
Therefore, we have investigated modified supports prepared with a number
of other gas chromatography phases: SE30, stabilized DECS, OV101,
QF1, Dexil 300, and Silar 9CP. Modified supports prepared with the
first five phases above showed variable and limited usefulness in
comparison with the modified Carbowax 20M column. However, our most
recent trials with a modified Silar 9CP column have shown excellent
chromatography and separations of phosphorothionates and their oxygen
analogs as shown in Table 2. This column has provided greater
separation of phosphorothionates and their oxygen analogs than any
column we have ever examined. Further, it is the first column on
which we have successfully chromatographed compound 8579. This study
is being continued with the preparation of additional modified supports
and the examination of chromatography of additional pesticides.
264
-------
Table 2. RETENTION TIME IN MINUTES FOR VARIOUS PHOSPHOROTHIONATE
AND PHOSPHATE INSECTICIDES ON A SILAR 9CP-SURFACE-MODIFIED GLC COLUMN.
Insecticide
140°
Bidrin 1.6
Dyfonate 3.2
Dioxathion
Fenthion
Fenoxon
8125a
8579b
Chlorthion
Chloroxon
Methamidphos
Acephate
Parathion
Paraoxon
Dimethoate
Dimethoxon
Carbophenothion
Rueline
Dasanit
Retention time in rain at following temperatures
155° 170° 185° 200° 215° 230° 245° 260°
1.0 0.8 0.6
1.7 1.1
1.4 0.9
2.0 1.2
5.8
2.5 1.4
2.1
2.8 1.6
4.5
2.8 1.7
2.0
6.8
2.8
2.8
0.7
1.0
3.3 1.8
1.0
1.2 0.9
1.1
2.5 1.4
1.2
2.2 1.3 0.9
1.3 0.9
3.7 2.0
1.7 1.1
3.9 2.1 1.3
1.7 1.1
2.6 1.9 1.1
3.7 2.4 1.4
a
0-(2,4-dichlorophenyl) ()-ethyl phosphoramidothioate
2,4-dichlorophenyl ethyl phosphoramidate
265
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References
1. Aue, W. A., C. R. Hastings, and S. Kapila, On the Unexpected
Behavior of a Common Gas Chromatographic Phase. J. Chromatog.
77^:299-307, 1973.
2. Hastings, C. R., J. M. Augl, S. Kapila, and W. A. Aue. Non-
Extractable Polymer Coatings (Modified Supports) for Chromatography.
J. Chromatog. 8]^:49-55, 1973.
3. Aue, W. A., C, R. Hastings, and K. 0, Gerhardt. Gas Chromatography
on Modified Supports. J. Chromatog. ^9:45-49, 1974.
4. Lorah, E. J., and D. D. Hemphill. Direct Chromatography of Some
N-Methyl Carbamate Pesticides. J.A.O.A.C. .57^:570-575, 1974.
266
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L. NEUROPHYSIOLOGICAL STUDIES ON THE MODE OF ACTION OF INSECTICIDES
Studies on the action of insecticides on insects were carried out at two
levels. First, the poisoning process of known insecticides was studied
by recording motor units from walking and flying house flies before,
during, and after insecticide treatment. Over the course of the grant
period this preparation became more sophisticated as the central
nervous system was isolated. Second, the actions of neurally active
compounds were measured at discrete tissues or subcellular elements.
This usually involved the insect neuromuscular junction as a model
system. Recently, the second half of the project has been expanded
in two ways: To do a structure-activity study of antagonists of
inhibitory synaptic transmission and to evaluate neurally active
compounds for insect and mammalian convulsant properties. These
latter approaches were deliberately designed to gain more information
about the selective toxicity of newer types of chemicals.
1. Synaptic Preparations
Visceral neuromuscular junction - Many, but not all, visceral muscles
in insects are innervated by neurosecretory axons. The neurosecretory
junctions within the heart muscle of the American cockroach, Periplaneta
americana, show ultrastructural and electrophysiological evidence of
chemically transmitting synapses and cytochemical evidence for the
presence of monoamines. Electron microscopy of nerve terminals shows
that synaptic vesicles may be formed directly from electron-dense
"neurosecretory" granules.
The alary muscles of the cockroach, P_. americana, are striated with an
A-band of 3.0 to 3.5 pm long. Each muscle fiber was 10 to 12 |jm in
diameter, and Z-lines appeared as small discrete units staggered through-
out the sarcoplasm. Mitochondria were conspicuously located near the
Z-line areas and were absent from the middle portion of the sarcomere.
A transverse membrane system was present which formed dyad structures
with a relatively sparse sarcoplasmic reticulum. Cockroach alary
muscles were innervated by axons containing electron-dense granules of
near 100 nm in diameter. These are thought to be typical of
"neurosecretory" axons based on their ultrastructural appearance.
The hyperneural muscle of P_. americana is striated with an A-band at
least 2 ^m long. Z-bands were discrete units, but arranged with some
order in the myoplasm. The sarcoplasmic reticulum was reduced and 1
thick was surrounded by 10 to 12 thin myofilaments. The muscle is
innervated from the median nerves by axons containing electron-dense
granules which may be opaque near the neuromuscular junction amid
numerous synaptic vesicles. Depolarizing intracellular current
injection produces an ohmic voltage response of the membrane potential,
and neurally evoked contraction is effected by summated excitatory
postsynaptic potentials. All contractal activity ceases when the
innervation is removed. The muscle appears to be electrically
267
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inexcitable and to be under obligatory control of central motor units.
By virtue of attachments along the ventral nerve cord in the abdomen,
the hyperneural muscle, when activated, moves the nerve cord uni-
directionally. In effect, the hyperneural muscle is called upon by the
central nervous system to move the ventral nerve cord presumably into
a greater mix with the hemolymph in response to as yet unknown stimuli.
A new transducer was designed and built to measure the extremely fine
movements of the hyperneural muscle during bioassays. The hyperneural
muscle reponds with sustained contracture to applications of L-glutamic
acid at near 10~^ 11. D-glutamic acid was much less active. The
responses to glutamate are usually extremely consistent and highly
reproducible. However, some preparations showed no response to
L-glutamic acid even at 10~^ M, while neurally evoked responses were
normal. High magnesium or low calcium salines perfused onto the
preparations blocked neurally evoked contractions. The glutamate re-
sponse was blocked reversibly in low calcium solutions, suggesting that
the glutamate effect, when present, was presynaptic.
Dopamine, acetylcholine, 5-hydroxytryptamine, synephrine, Rogitine^
strychnine, pentobarbital, and picrotoxin, all suspected to varying
degrees of some action on insect central or peripheral synaptic trans-
mission, had no effect on the patterns of neurally evoked contracture
of the hyperneural muscle.
Suspected neurotransmitter and neurohormone chemicals have been assayed
on the hyperneural muscle, including: 19 of the ordinary amino acids,
acetylcholine, gamma aminobutyric acid, tryptamine, 5-hydroxytryptamine
(5-HT), dopamine, octopamine, synephrine, adrenaline, adenosine-
triphosphate (ATP), oxytocin, and vasopressin. Besides these compounds,
several blocking agents were assayed, including: strychnine, Rogitine
(which contains phentolamine), and the Wellcome Research 5-HT blocker,
501 c 67 (related to xylamidine), which is shown in Figure 1. Of these,
only glutamate and aspartate had any significant effect on neurally
evoked contractions, and then only at 10~4 M or greater.
CH0 NH
i3 i2
0-CH-CH2-NH-C-CH2-NH
•HC1
Figure 1.
Reserpine caused a gradual increase in heartbeat to 10% over control rate
3 minutes after drop-on assay of 10~^ M. When hearts were bathed in a
continuous, uniform flow of 10"^ M aminophylline while preparation was
maintained at a constant temperature, no pronounced changes were
evident after 1% hours. Dose-response curves of 5-HT on cockroach hearts
268
-------
which were pretreated with 10 M aminophylline were not appreciably
different from control assays. It was concluded that if 5-HT were
acting on adenyl cyclase in the myocardium, aminophylline did not
change the 5-HT effect as might be expected.
Tyramine, tryptamine, octopamine, synephrine, 5-HT, and dopamine all
acted specifically on the myocardium and not on the cardiac neurons of
the cockroach heart. Threshold concentrations ranged from 5 x 10~° to
5 x 10 M in drop-on assays. All of the compounds listed showed
similar qualitative responses, and it was assumed that they were
acting at a similar site on the myocardium.
A known 5-HT antagonist (Wellcome Research Laboratories 501 c 67, Fig. 1)
applied to the myocardium in a range of concentrations (10~^ M -
10"^ M) with increasing doses of 5-HT produced the parallel dose-
response curves characteristic of competitive inhibition. Identical
assays using this antagonist with the catecholamine, octopamine,
revealed a similar pattern of reduced response. Dose ratios for
antagonist - 5-HT and antagonist - octopamine were also shown to be
similar, suggesting that the two antagonists act on the same receptors.
Cyclic AMP and cyclic GMP showed no obvious immediate response on
myocardium or cardiac neurons, but poor solubility in saline was a
problem.
L-glutamic acid, picrotoxin, reserpine, theophylline, aminophylline,
prostaglandin Fl, F2, and El were all assayed on cockroach, house fly,
and noctuid moth heartbeat, but produced no appreciable immediate
responses as saturated solutions or at concentrations of at least
10~3 M in the appropriate saline.
Heart preparations bathed in a continuous, uniform flow of 10~^ M
dibutyryl cyclic AMP in saline while monitoring a constant temperature
over a 1-hour period showed no pronounced rate change. When hearts
were treated with 10~" M 5-HT following 1 hour of exposure to cAMP,
response was typical of hearts with no prior exposure to cAMP.
Inhibitory neuromuscular transmission - Gamma aminobutyric acid (GABA)
is thought to be the inhibitory neuromuscular transmitter in arthropods.
Picrotoxin and bicuculline have been found to block inhibitory neuro-
muscular transmission at rather low concentrations.
Compounds interfering with inhibitory synaptic transmission would be
ideal models around which to develop insecticides. Blockage of
inhibitory influences would free many motor pathways to fire in unison,
which would be expressed as convulsions. It is known from work reported
below that hyperactivity in the nervous system, if maintained a
sufficient length of time, leads to central nervous damage evidenced by
tissue alterations.
269
-------
When applied to the isolated thoracic ganglion of house fly, picrotoxin
caused characteristic convulsions in 6 min at 10~^ M. However, when
applied on the desheathed ganglion, picrotoxin produced the same
convulsions in 1 min at 10~H M. By way of comparison, carbofuran, a
carbamate cholinesterase inhibitor, caused similar convulsions in the
desheathed ganglion after 33 min at 10'^ ^. Clearly, picrotoxin has
exceptionally high intrinsic toxicity to the insect central nervous
system compared to carbofuran whose LD5Q to whole house flies (6.7 pg/g)
is sufficient for this to be a commercial insecticide (Furadan®).
On the other hand, picrotoxin is rather unimpressive on the isolated
thoracic ganglion and even less impressive on the whole fly. When
applied topically at 50 pg (equivalent to a dose of 2500 pg/g),
picrotoxin was nontoxic to the house fly. However, when applied with
piperonyl butoxide (1:5), picrotoxin showed an LD5Q of 75 pg/g.
Although this is an order of magnitude less toxic than carbofuran, it
does reflect the extremely high toxicity inherent in picrotoxin and
suggests that the compound is metabolized at a fairly high rate by
the intact insect.
Commercial picrotoxin is a mixture of picrotin and picrotoxinin, the
latter of which is thought to be the toxic component. The structure of
picrotoxinin (Fig. 2a) is rather complex, with two lactone rings and an
epoxide moiety. Picrotin is similar except for a hydrated double bond
(Fig. 2b). Tutin (Fig. 2c) resembles picrotoxinin near the position of
the double bond.
A
i—OH
2a
Figure 2
2b
2c
270
-------
Tutin was also examined for convulsant properties on the house fly
thoracic ganglion. Ten ^ tutin caused an initial decrease in activity,
then convulsions after 52 rain. 0.1 p,M tutin produced convulsions in
the desheathed ganglion in 35 to 40 min.
The GABA antagonist drug bicuculline shows a slow but significant
hydrolysis into the pharmacologically less active substance bicucine in
solutions of physiological (neutral) pH. Spectral analysis reveals that
the lactone moiety of bicuculline is reversibly cleaved with a half-time
of 53 min at 23° upon adjusting a stock solution at pH 3 to a pH value
of 7.6 with 10 mM Tris-HCl. This occurs very rapidly, however, at 37°.
Therefore, caution must be exercised in experiments using the drug in
solutions at physiological pH values, especially at elevated tempera-
tures. Solutions of bicuculline prepared at neutral pH should not be
kept for any unnecessary period of time before experimentation, since
misleading negative results could occur.
Bicuculline produced convulsions in the house fly thoracic ganglion at
100 p,M, bicucine at 250 ^M, and the other analogs were inactive at
500 |j>I concentrations. The evidence suggested that bicuculline was
relatively less specific than picrotoxin in blocking inhibitory
transmission.
On the cockroach inhibitory neuromuscular junction, bicuculline caused
reversible block at 100 ^M, or roughly 100 times the concentration of
picrotoxin producing the same effect. Five p,M bicuculline (Fig. 3a) had
no effect and 50 p£I was threshold, causing decreased inhibitory
postsynaptic potential amplitude.
The bicuculline analogs bicucine methyl ester (Fig. 3c) and bicuculline
methiodide (Fig. 3d) blocked inhibitory transmission reversibly at or
near 80 ^ with 40 ptf thresholds, but bicucine (Fig. 3b) was inactive
at 100 oJ-L
COOH
Figure 3a
3b
271
-------
N (CH3)2 I
-OCR.
Figure 3c
3d
Excitatory neuromuscular junction - Excitatory transmission was examined
at the insect neuromuscular junction and developed as an ultrasensitive
assay for certain chemicals. A certain amount of basic work clarified
the process of spontaneous release of neurotransmitter molecules, the
so-called "quantal" release as measured by miniature postsynaptic
potentials.
In the retractor unguis muscles of 1?. americana and Blaberus giganteus
the rate of spontaneously occurring miniature potentials from nerve-
muscle synapses associated with the "white" muscle fibers was greater
than that observed in the "red" muscle fibers. In 5% of the intra-
cellular recordings, particularly from the "white" fibers, short
"bursts" of relatively high (20-100 sec ) rates of m.e.p.p.s. occurred
infrequently in an unpredictable manner. This bursting phenomenon was
not observed in extracellular recordings of the miniature potential
discharge.
Rigorous statistical analyses indicated that the spontaneous mode of
transmitter release at cockroach retractor unguis muscle fibers was
predictable by the negative binominal theorem with a 90% reliability.
Perfusion of nerve-muscle preparations with high Mg^+ salines resulted
in low spontaneous discharge rates which were characterized by a
negative binomial distribution. The fit of the data to the negative
binomial theorem implied some mutual interaction between those
processes responsible for the spontaneous release of transmitter from
cockroach nerve terminals.
Neurotomy of motor axons to skeletal muscles in insects lead to
aggregation and clumping of synaptic vesicles after 48 hours. Treatment
of in vitro nerve-muscle preparations with various respiratory poisons
caused aggregation similar to that developed in neurotomized animals.
This suggested that vesicle aggregation in both cases may have resulted
from a decrease in available adenosine triphosphate in the nerve
terminal with subsequent alteration in the normal charge density which
272
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supports a repulsive force between the vesicles.
The application of metabolic inhibitors to nerve-muscle synapses on
"white" and "red" fibers in the retractor unguis muscles of £.
americana and IJ. giganteus resulted in a dramatic increase in the
spontaneous miniature potential discharge and was accompanied by a
summation of the miniature potentials to form "composite" potentials.
Axon terminals associated with "white" muscle fibers responded faster
to metabolic inhibitors than those axon terminals associated with "red"
muscle fibers. Correlated ultrastructural and electrophysiological
studies inferred that a tentative relationship existed between the
miniature potential activities and synaptic vesicle distributions of
the nerve-muscle synapses during the phases of metabolic inhibition.
Experimentation with known metabolic inhibitors such as antimycin,
oligomycin, rotenone, and salicyl anilide produced a change in the
nature of the "spontaneous" miniature excitatory postsynaptic potentials
recorded from the isolated metathoracic retractor unguis muscle of the
cockroach, I?, americana (Table 1). These changes consisted of an initial
"bursting" of the miniature potentials, eventually cumulating to a
frequency three times that in "control" or "normal" preparations.
Finally, the miniature potentials decreased in frequency and ceased,
with the muscle becoming electrically silent and nonresponses to
electrical stimulation. This indicates that the site of action of these
metabolic inhibitors is probably presynaptic.
Table 1. EFFECTS OF RESPIRATORY INHIBITORS ON SPONTANEOUS
DISCHARGE OF MINIATURE POSTSYNAPTIC POTENTIALS IN
NEUROMUSCULAR JUNCTION OF P. AMERICANA RETRACTOR UNGUIS
Effect transmission
Compound
Cone.
(M)
Latency
(min)
Failure
(min)
ir Value
I. Antimycin A^ 10
II. Antimycin AI 10
III. Antimycin A3 10
IV. Rotenone 10
V. 1,4-Napthoquinone 10
VI. 4-N02~methylene- 10"
dioxy benzene
VII. 4-N02~catechol 10"
VIII. l,2-dihydroxy-9, 10
10-anthroquinone
-10
-6
-6
20
60
15
10
120
60
45
15
120
425
55
50
240
540
210
75
high
high
high
high
medium
medium
low
a
ir value is relative octanol/water partition coefficient.
273
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Compounds were specifically designed to interfere with the electron
transport mechanism and oxidative phosphorylation processes of the
mitochondrial systems of the peripheral nerve terminals. It was found
that certain catechols (Table 1, VII.) were very effective at low
concentrations (10~" M) in bringing about neuromuscular transmission
failure, whereas the methylenedioxy-type compounds (Table 1, VI.)
produced a reduced effect. Contrary to the latter observation, experi-
mentation with two good methylenedioxy synergists produced neuromuscular
transmission failure, while their "presumed" dihydroxy metabolite forms
were even more effective. Speculation was raised as to the possible
consequences of synergistic action beyond inhibition of mixed-function
oxidases.
Another preparation was designed to examine the action of compounds at
the cholinergic synapse in the cockroach sixth abdominal ganglion.
Neurally evoked excitatory postsynaptic potentials (EPSP's) were
recorded from the cereal-giant synapse in the sixth abdominal ganglion
of the cockroach by the sucrose gap method. Dieldrin at 10~5 M caused
an amplitude increase and prolongation of the EPSP in 30 minutes, which
suggested that synaptic electrogenic currents were altered or the amount
of neurotransmitter release per shock was increased.
Some controversy has arisen over the site of action of dieldrin. These
results suggest that its central effects in insects may involve the
presynaptic terminal of sensory axons.
2. Neurophysiological Basis for Response to Insecticides
Locomotion - Behavior of house flies to poisoning was initially measured
by recording locomotion. Locomotory activity of adult female house
flies, Musca domestica L., was recorded under various conditions of light
and temperature and in response to DDT application. House flies were
more active at room temperature (22°C) than in the cold (16°C), as
expected. Pronounced circadian rhythm was exhibited at 22°C in the
laboratory associated with normal day-night conditions. No circadian
rhythm patterns were evident in the cold in absence of light.
Response to lethal doses of DDT occasionally showed two peaks of
locomotory activity sometimes separated by a period of depressed
locomotion. Extracellular recordings of dorsal longitudinal flight
muscle activity of Musca during DDT poisoning also showed two peaks of
activity, in this case always separated by a phase of markedly depressed
activity. The poisoning process in general was defined in four stages
in terms of motor responses. The final stage of poisoning was redefined
as tetany rather than prostration due to intensive motor unit activity
which lasted for several hours, long after the cessation of convulsive
movement.
274
-------
The flight motor preparation - To observe a more sophisticated type of
central nervous activity during poisoning, the output of two flight
motor neurons was recorded from whole intact and tethered house flies
during poisoning.
Muscle potentials recorded extracellularly from one pair of fibers of
the dorsal longitudinal flight muscle in the house fly, M. domestica,
show a very regular pattern with both pairs driven in alternating
sequence (Fig. 4). House flies with chronically implanted electrodes
activated the flight muscles without externally observed motion, either
walking or in flight, in apparent shivering responses. The frequency
of activation of the flight muscle in shivering was temperature-
dependent below 25° C with 0 of 2.57. No shiver was recorded above
26°C. 10
JJ
Calibration:
20 MV, vertical;
1 sec, horizontal
Figure 4. Control of the activity of the flight muscles of Musca.
During insecticide poisoning, both gross locomotory activity and flight
muscle activation first increased, then decreased slightly, then
gradually increased again to a condition of tetany. After treatment
with lethal doses of peripheral nerve poisons such as DDT and pyrethrin
analogs (Fig. 5), the flight muscle activity pattern gradually increased
in tetany and was constantly active for several hours. When poisoned
with central nervous poisons such as Lindane,® dieldrin, endrin, and
organophosphorus and carbamate anticholinesterase poisons (Figs. 6 and
7), the post-tetanic period of poisoning was characterized by discrete
bursts of motor unit activation interrupted by inactivity. The high-
frequency spasms of flight muscle activity during poisoning resembled
hyperactivity obtained due to o erheating of house flies.
275
-------
It
Figure 5. Flight muscle potentials 2% hours after poisoning by 5
trans-Barthrin, 6-chloropiperonyl-(+)-trans-chrysanthemate.
Calibration same as Fig. 4.
Figure 6. Flight muscle activity 10 min after treatment with 5 p,g
carbamate, 2,3-dihydro-2,2-dimethyl-7-benzofuranyl-N-methyl-N-
thio-p_-t-butylphenylcarbamate. (Arrow shows "uncoupling of motor
units). Calibration same as Fig. 4.
Figure 7. Flight muscle activity 6 hours after treatment with 0.2 ^g of
ronnel (0,()-dimethyl ()-2,4,5-trichlorophenyl phosphorothioate).
(Arrow shows "uncoupling" of motor units). Calibration same as Fig. 4.
Other motor units - It became obvious that when other motor units were
recorded during poisoning, each class of insecticide was acting in a
unique manner in terms of motor output (compare, for example, Fig. 5
with Figs. 6 and 7.).
The average heartbeat rate of female adult M. domestica was near 250
beats/min jLn vivo at 23°C. However, standard deviation values ranged
from ±35 to ±60, depending on the individual house fly. Heartbeat rates
in tethered house flies fluctuated between cessation to over 300 beats/
min. The heartbeat rate was temperature-dependent with a (Jig °f 2.3.
Either a bite by the Lynx spider, Peucetia vividans (Hentz), or sever-
ing the abdomen from the thorax caused the heartbeat to become extremely
276
-------
steady at near 300 beats/min, which gradually decreased over several
minutes.
Application of lethal doses of Monitor® or Lindane to the house fly
caused thoracic temperature to increase by at most 3°C in conjunction
with increased convulsive activity and increased average heartbeat rate.
In late stages of poisoning, the heartbeat was relatively uniform,
indicating a disruption in cardioregulatory nervous activity. Response
of the house fly to carbofuran or its N-thiomethyl analog was similar
to that of Monitor and Lindane except in late stages of poisoning
where the heartbeat continued to exhibit large variations in average
rate.
Speed of action of carbamate versus organophosphate insecticides - The
accumulation of lifC-labeled carbofuran, leptophos, and Monitor into the
thoracic ganglion of the house fly, M. domestica, was measured following
topical application on the abdomen or perfusion of the ganglion by the
compounds in saline solution.
14
The penetration of C insecticide into the thoracic ganglion was
correlated with a disruption in characteristic coupling between units
of the flight motor, termed uncoupling (cf. Figs. 6 and 7). An accumu-
lation of about 25 pg of carbofuran per thoracic ganglion was correlated
with uncoupling (Fig. 8), while leptophos or Monitor did not produce
uncoupling until 1000 pg had accumulated in the thoracic ganglion.
60
40
20
10 20
M I N
Figure 8. Amounts of radiolabel incorporated into thoracic ganglion at
various times following direct contact with a wax block 9.6 ng
carbofuran/g parafin. CNS = thoracic ganglion. Arrow indicates
relative time to uncouple flight motor under these conditions.
It was noted that carbamate insecticides are generally fast acting as a
class compared to organophosphorus insecticides whether treated topically
on the whole house fly or perfused directly on the isolated thoracic
ganglion. It was concluded that this general property was due to an
inherent difference in the interaction of these classes of insecticide
with the central nervous system.
277
-------
Difference between organophosphorus and carbamate insecticide poisoning
Three criteria, latency to convulsions, still period, and recovery,
were defined and used to distinguish between carbamate and phosphate
insecticide poisoning in house flies, M. domestica. Because of shorter
latencies with carbamate-treated flies, the central nervous system was
considered more sensitive to the presence of carbamates than phosphates.
Recovery from carbamate-induced tetany was considered correlated with
reactivation of carbamylated cholinesterase. There appeared to be a
fundamental difference between the insecticidal actions of carbamates
and phosphates not wholly explainable by cholinesterase inhibition.
Tethered or held house flies were less susceptible to poisoning than
unrestrained flies.
3. Histopathology of Insecticides in House Flies
The cause of death from insecticide poisoning has not been established.
It was decided to examine the thoracic ganglion of house flies and
correlate tissue changes with house fly poisoning symptoms.
SNAIDM ? house flies were treated with LD5Q (6.7 p,g/g) of carbofuran on
the tip of the abdomen. After 24 hours, 5% of the flies behaved
normally, 45% abnormally, and 50% were immobile. The abnormal flies
were classified into one of the six categories: (a) unable to fly,
(b) crossed front legs, (c) curled tarsi, (d) unable to walk or climb,
(e) legs tucked under the thorax, or (f) rear legs extended and
dragging.
All flies with abnormal behavior showed vacuolization in the neuropile
of the thoracic ganglion. Thirty-three percent of the (a) flies showed
vacuolization at the base of or in the dorsal nerve (flight motor
nerve). The (b) flies showed vacuoles in the prothoracic region,
whereas the (f) flies had lesions in the metathoracic neuromere of the
thoracic ganglion.
In all abnormal flies, vacuolization was found only in the neuropile
area. In the immobile (dead) flies vacuolization was extensive in all
areas of the neuropile. Thus a good correlation existed between
central lesions produced by carbofuran and ultimate behavior of the
house flies.
Lindane, dieldrin, pyrethrins, and DDT were also treated on the S A
strain at LD5Q values. Thirty percent of the Lindane-treated
flies survived, and of these two-thirds showed no lesions in the
thoracic ganglion and no abnormal behavior. The remaining one-third
were unable to fly and had vacuolization in the glial or perineurial
layer of the thoracic ganglion. All of the Lindane-poisoned flies
which were classed as dead had normal-appearing neuropile with general
lesions in the perineurium.
278
-------
Those flies treated with IJ>5Q of dieldrin which showed abnormal
behavior after 24 hours had vacuolization in all areas of the thoracic
ganglion plus lesions in the perineurial area of the brain. Of the
apparently normal survivors, one-third showed slight vacuolization in
glia and perineurium of the thoracic ganglion.
Pyrethrins caused general vacuolization in thoracic ganglia of house
flies which did not survive. All 24-hour survivors had little
vacuolization in the thoracic ganglia despite stereotyped abnormal
behavior including inability to fly, rear legs contracted under the
thorax, and improper landing from flight. Of these survivors, some
vacuolization of the optic ganglion was noted.
4. References
1. Gerschenfeld, H. M. Chemical Transmission in Invertebrate Central
Nervous Systems and Neuromuscular Junctions. Physiol. Rev. 53;
1-119, 1973.
279
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M- INSECT RESISTANCE TO CHEMICALS: RESISTANCE TO INSECT GROWTH
REGULATORS
1. Cross Resistance to Juvenile Hormone Analogs in Insecticide-
Resistant Strains of the House Fly
Introduction - It is now well known that juvenile hormones (JH) play an
important role in insect growth and development. It is also generally
acknowledged that synthetic juvenile hormone analogs (JHA) hold promise
as insect control agents. One attractive aspect of these compounds was
the suggestion by Williams1 that insects could not develop resistance
to them. However, the results described below, 2'^ on the presence of
cross resistance to JHAs in insecticide-resistant strains of the house
fly have introduced an element of caution in these predictions.
Materials and Methods - The strains tested comprised one susceptible
(NAIDM-S) , one organochlorine-resistant (DDT/lindane-R) , a carbamate-
resistant (OMS-15-R, selected by m-isopropylphenyl methylcarbamate) ,
a field strain (SK-R) colonized in 1971, and five organophosphorus-
resistant [parathion-R; dimethoate-R; fenthion-R; Chlorthion-R, selected
by (),0-dimethyl ()-(3-chloro-4-nitrophneyl) phosphorothioate; and OMS-
12-R, selected by 0-ethyl ()-(2,4-dichlorophenyl) phosphoramidothioate].
With the exception of NAIDM-S and SK-R strains, all have been under
specific selection pressure in the laboratory for more than 10 years,
and each is believed to have attained maximal resistance to the select-
ing insecticide. The levels of resistance in the various strains
towards their selecting compounds are: DDT/lindane-R >1000x; parathion-
R >1643x; dimethoate-R 60x; fenthion-R 435x; Chlorthion-R >430x; OMS-12-
R 88x; and OMS-15-R >100x. In all instances, the resistance is highest
towards the selecting compound, although each strain demonstrates vari-
ous levels of cross resistance to other compounds as well. The SK-R
strain was reported to possess the following levels of resistance at the
LDgs to organophosphorus insecticides: diazinon, 580x; ronnel, 737x;
malathion, >100x; fenthion, 43x; naled, 26x; and dimethoate,
The chemical structures of the compounds tested were as follows:
I. Methoprene (Altosid®) : isopropyl ll-methoxy-3,7,ll-trimethyldo-
deca-2,4-dienoate, a mixture of isomers, supplied by Zoecon
Corporation, Palo Alto, Ca.
II. Ro 7-9767: 6,7-epoxy-3,7-diethyl-l-(3,4-(methylenedioxy)phenoxy)
2-cis/trans-octene, a mixture of isomers, supplied by Hoffmann-
LaRoche Inc., Nutley, New Jersey.
III. R-20458: trans l-(4'-ethylphenoxy)-6,7-epoxy-3,7-dimethyl-2-
octene, supplied by Stauffer Chemical Co., Mountain View, Ca.
IV. NIA 23509: 10,ll-epoxy-N-ethyl-3,7,ll-trimethyl-2,6-dodecadiena-
mide, a mixture of isomers supplied by FMC Corp., Princeton,
New Jersey.
280
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V. Sesamex: acetaldehyde 2-(2-ethoxy-ethoxy) ethyl 3,4-methylene
dioxyphenyl acetal, supplied by the World Health Organization.
Acetone was used in making all weight/volume dilutions. All chemicals,
both neat and diluted, were stored at 0°C in the dark. Solutions were
kept for one month at most and were remade if further tests were required.
White prepupae, a stage lasting at most 1 h at 24°C and known to be the
most sensitive to exogenous JH, were treated by the application of
0.5 (jl of JHA solution. Treated prepupae in batches of ten were placed
in unwaxed 8 oz squat paper cups. The cups were covered with nylon
netting and stored under a 12:12 h photoperiod at 27°C. Relative
humidity was 60% during photophase and 95% during scotophase. Effects
were assessed 7 days after treatment.
Activity of the compounds was based on the number of flies successfully
completing emergence from the puparium. Eclosed adults failing to free
themselves completely from the puparium were scored as affected. Such
flies showed the presence of varying degrees of morphogenetic damage
characteristic of juvenile hormone action: deformation of one or
both wings, or lack of wing expansion were common defects; the
genitalia of both sexes were poorly developed and in some males they
failed to complete the 180° rotation; misshaped ventral arches of the
fifth and sixth abdominal segments, and abnormally formed primary
forceps, were also found in some males.
Each replicate consisted of ten insects. In most instances, a total of
ten replicates/dose were run, each on a different day. Acetone-treated
controls were maintained for each test. Cross resistance determinations
were performed mostly at two dose levels, chosen to produce 60% and
90% effect against the S strain. Duncan's multiple range test was used
to rank the mean responses of the strains to the JHAs at the 95%
confidence level. Probit analysis was used in determining dose-response
regression lines for the responses of the S and certain R strains
towards the compounds.
Results - Dose-response data summarized in Table 1 indicate that the
JHAs tests varied considerably in their activity toward S house flies.
Methoprene was by far the most active compound tested (EDso: 0.0033 ygj
prepupa; EDgs: 0.00634 pg/prepupa), producing its effect over a narrow
range of doses, as shown by the relatively high slope value (b=5.8).
The other compounds were much less active and the slope of the regression
lines became increasingly lower with loss of activity. The least
active compound tested, NIA 23509, was 10,200 times less active than
methoprene at EDso.
281
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Table 1. COMPARATIVE ACTIVITY OF VARIOUS JUVENILE HORMONE
ANALOGUES ON THE NAIDM SUSCEPTIBLE STRAIN OF HOUSE FLY
Compound
Methoprene
Ro 7-9767
R-20458
Sesamex
NIA 23509
(pg/prepupa)
0.0033
(0.00302-0.0036)
0.325
(0.142-0.581)
5.57
(4.63-6.69)
33.5
(28.0-40.2)
33.6
(20.7-65.1)
Slope
5.8
2.0
1.2
1.5
1.2
Fold decrease
in activity
at ED5Q
1
98
1,690
10,200
10,200
for 50% effect.
Upper and lower fiducial limits.
The ranking of cross resistance of the nine house fly strains to metho-
prene was obtained by treatment with 0.025 |j,g/prepupa of the JHA, a
dose approximately 2.7 times the minimum required to produce complete
suppression of adult emergence in the NAIDM-S strain. The dimethoate-R
strain was the most resistant one tested (Table 2). OMS-15-R and
OMS-12-R strains showed a high level of cross resistance, while the
fenthion-R strain was intermediate. The Chlorthion-R strain had only
weak resistance to methoprene and the SK-R, parathion-R, and DDT/lindane-R
strains showed minimal resistance.
Subsequent tests were run to examine in greater detail the susceptibility
of the dimethoate-R, fenthion-R, and SK-R strains to methoprene. The
results in Table 3 show an increased homogeneity of response (_b=2.8) and
a relatively low level of cross resistance to methoprene (2.55x at ED50)
in the SK-R field strain. Greater homogeneity of response (_b=1.9) was
found in the two laboratory-stressed strains along with increased resist-
ance levels, i.e. 5.5x in fenthion-R and 39.4x in dimethoate-R. The
resistance of these strains is fully evident when the relative resistance
ratios are examined at the practical control level of ED 95, viz. SK-R 5.1,
fenthion-R 17.5, and dimethoate-R 150.8
282
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Table 2. RESPONSE OF VARIOUS STRAINS OF THE HOUSE FLY
TO TREATMENT WITH METHOPRENE AT 0.025 ug/PREPUPA
Strain
NAIDM-S
DDT/lindane-R
Parathion-R
SK-R
Chlorthion-R
Fenthion-R
OMS-15-R
OMS-12-R
Dimethoate-R
Prepupae
treated
100
100
100
100
100
100
100
100
100
Percentage
mortality3
100. Oa
97.0 ±1.53a
ab
91.0 ±3.48
ab
89.3 ±4.38
80. 3± 5.79b
56.4±4.23°
28.0±4.16d
27.2 ±5. 08d
12.0 + 4.426
Any two means having the same superscript are not significantly
different at the 5% level as determined by Duncan's multiple range test,
Table 3. RESPONSE OF FOUR STRAINS OF THE HOUSE FLY TOWARD METHOPRENE
Strain
NAIDM-S
SK-R
Fenthion-R
Dimethoate-R
EDso3
yg/prepupa
0.0033
0.0034
0.018
0.13
ED953
yg/prepupa
0.0063
0.032
0.11
0.95
Slope
(b)
5.8
2.8
1.9
1.9
Resistance
at ED50
2.5
5.5
39.4
Ratiob
at ED 9 5
5.1
17.5
150.8
Dose for 50% or 95% effect.
3ED of R strainTED of S strain.
283
-------
The cross resistance data obtained on the various strains utilizing
compounds Ro 7-9767, R-20458, Sesamex, and NIA 23509 are given in
Table 4. The overall picture emerging from all tests was as follows:
the dimethoate-R and OMS-15-R strains appeared to exhibit a generally
high level of resistance to all the JHAs tested. The fenthion-R strain
showed high resistance levels to all the compounds but methoprene,
towards which it exhibited an intermediate level of 17.5x. Similarly,
the DDT/lindane-R strain manifested only negligible tolerance to
methoprene, but an intermediate response to all the other compounds.
The OMS-12-R strain demonstrated intermediate to high levels of cross
resistance to all JHAs tested, and the SK-R, parathion-R, and Chlorthion-
R strains showed, for the most part, a relatively low to intermediate
level of resistance.
Discussion - The varying degrees of juvenile hormone activity of the
compounds tested against the susceptible NAIDM flies are most likely due
to differences in the intrinsic juvenile hormone activity of the chemi-
cals and their metabolic stability. Increased intrinsic activity could
result from a sterochemical structure better suited for function at the
target site of the natural JH.
Metabolic stability may also be an imporant factor: it has been
suggested that not all cells in the insect become sensitive to JH at the
same time so that the longer a JHA remains intact, the more likely it
is to contact each cell during the sensitive period.5
The metabolic stability of methoprene may possibly explain its relative-
ly high activity on susceptible flies as compared to the other compounds.
Studies of the inactivation of the natural juvenile hormones in various
species have established that metabolism proceeds primarily by esterase
attack at the carboxymethyl moiety and by epoxide hydration.6~8 Metho-
prene lacks the epoxide moiety, and a recent study suggests that the
terminal isopropyl group may provide protection from carboxyesterase
degradation.9 The other compounds, possessing epoxy groups or unpro-
tected esters, may more easily be inactivated.
Finding cross resistance to JHAs was not entirely unexpected. Inactiva-
tion sites, present in these compounds7"9 are likely targets for the
degradative metabolic systems of insecticide-resistant strains.
The varying spectrum of cross resistance to the JHAs in each strain
would suggest that several degradative mechanisms may be involved in
resistance. In light of the large variability in the ranking responses
of the organophosphorus-resistant strains, phosphotriesterase activity,
common in OP-resistant insects, may be of minor importance in cross
resistance to JHAs.
It appears that mixed function oxidases play an important role in the
deactivation of JHAs. The OMS-15-R (carbamate-resistant) strain, known
284
-------
Table 4. RESPONSE OF VARIOUS STRAINS OF THE HOUSE FLY TO TREATMENT OF PREPUPAE WITH JHAs
Strains
NAIDM-S
Chlorthion-R
SK-R
Parathion-R
DDT/Lindane-R
OMS-15-R
OMS-12-R
Fenthion-R
Dimethoate-R
NAIDM-S
Chlorthion-R
SK-R
Parathion-R
DDT/Lindane-R
OMS-15-R
OMS-12-R
Fenthion-R
Dimethoate-R
Percentage mortality at doses indicated3
Ro 7-9767
0.5 (jg/prepupa
52. 8± 7.69a
15. 8± 6.25^
12. 1± 3.26?
16.2+ 4.73?
14.0+ 6.53?
6.3± 2.38°
4.1± 3.08?
11. 0± 2.77°
5.1± 2.26
2 p,g/prepupa
97. 0± 2.13a
65. 6 ± 7.04?
64. 4 ±4. 03°
60. 3 ±4. 01
38.0 ±4. 90°
13. 4 ±3. 70*:
12.7±4.70d
12. 0± 2.00d
7.1 ±4. 70
R-20458
10 (jg/prepupa
Sesamex
50 p,g/prepupa
62.1±5.40a 1 65.9±6.83a
52.5±5.65ab ! 40. 5± 10. 19
53.7±3.54a°
34.4+ 7.39?°
35.5 + 4.57
5.0± 1.67e
29. 0± 5.07°
de
12. 4 ± 5.68"
8.2± 5.18
100 pg/prepupa
97. 0± 2.14a
90. 7 ± 3.60a
94. 3± 2.57a
89. 5 ± 3.18a
74.6±5.03a°
35. 5 ± 5.42d
no
61. 5 ± 2.83°^
52.4 ± 5.83 .
44.0 ±4. 01
46. 3 ± 5.11b
36. 9 ± 8.68^
34. 2± 5.36
12. 5± 4.71°
8.2+2.03°
c
13. 3 ± 2.20
15. 5± 4.18°
200 |ig/prepupa
85. 9± 2.69a
45. 2 ±8. 26°
71. 1± 4.59°
67. 8± 6.85
51. 6± 5.22°
24.5±4.89^
15.0 ±4. 28°
29. 3 ± 2.91d
25. 6 ± 3.06
NIA 23509
50 ^/prepupa
59. 0± 6.40a
36. 1± 7.667°
37. 9± 7.26^°
23.2 + 4.42^°d
35.0+ 6.54°°
11. 3 ± 3.547
41. 5 ±6. 34°
20. 6 ± 3.53° ,
27. 9± 6.19
200 p,g/prepupa
72. 0± 3.59a
56.1±8.75°
55. 1± 5.36°°
51. 8 ± 5.10°°
54.0±4.27°°
44.3±5.26b°d
53. 7 ± 3.17°°
34.9±3.76d
40.1 ±3. 60
to
oo
Average percentage mortality± S% based on 100 prepupae/dose after correction for control mortality
by Abbott's formula. Any two means having the same superscript are not significantly different
at the 5% level as determined by Duncan's multiple range test.
-------
to metabolize carbaraates by MFO action, showed high levels of cross
resistance to all the JHAs. This is in agreement with the results of
Terriere and Yu9 that methoprene metabolism in a housefly strain
possessing a high oxidase content proceeds at more than twice the rate
found in a susceptible strain with a low oxidase content. Likewise, a
study by Vinson and Plapp1^ showed that cross resistance to certain
JHAs in the house fly is controlled by genetic factor(s) in linkage
group II, which also controls high levels of oxidase activity.
2. Cross Resistance to Chitin Synthesis Inhibitors in Insecticide-
Resistant Strains of the House Fly
Introduction - Among various synthetic insect growth regulators
investigated in recent years are included the benzoylphenylureas whose
action resides in their ability to inhibit the synthesis and deposition
of chitin, thereby causing death of developing insects.1 Insecticidal
activity has been reported against a large number of insect species.
One major problem facing every potential insecticide is the possibility
of development of resistance to it by the target pest. We have therefore
investigated the presence of cross resistance to the most potent known
member of this class of compounds, Dimilin® [l-(4-chlorophenyl)-3-
(2,6-difluorobenzoyl)urea] in certain insecticide-resistant strains
of the house fly.13
Materials and Methods - The strains utilized and the methods of tests
were the same as described in Section 1, with the exception of the use
of tetrahydrofuran as solvent due to the low solubility of Dimilin
in acetone.
Results - The visible response of the treated insects was failure to
emerge from the puparium, ranging from minimal leg attachment to
complete lack of emergence.
Base-line data for the NAIDM-S strain showed a modest ED50 of 0.265 |jjg/
prepupa and a relatively high ED95 of 70 ^g/prepupa. The low slope of
the regression line (b=0.68) may indicate a slow rate of penetration
through the cuticular layer.
Various levels of cross resistance to Dimilin by the eight insecticide-
resistant strains of the house fly were revealed by the application of
the compound at two dosage levels, e.g. 1 and 10 ^/prepupa (Table 5).
The low dose provided ranking separation only between NAIDM-S and the
resistant strains. But 10 ^/prepupa provided significant separation
of the strains into three groups: the parathion-R and Chlorthion-R
strains were the least resistant, the DDT/lindane-R, fenthion-R, OMS-15-
R, dimethoate-R, and SK-R strains were intermediate, while the OMS-12-R
was the most resistant of all strains tested.
286
-------
Table 5. SUSCEPTIBILITY OF VARIOUS STRAINS OF THE HOUSE FLY
TO DIMILIN AT DOSES OF 1 g AND 10 g PER PREPUPA3
Strain
NAIDM-S
Parathion-R
Chlorthion-R
DDT/Lindane-R
Fenthion-R
OMS-15-Rb
Dimethoate-R
SK-R
OMS-12-R0
1 Ltg/prepupa
% mortal.
60
32.3
28.3
21
20.9
25
18.5
17.3
15.2
S-
X
5.96a
6.39b
7.93b
5.18b
6.01b
6.19b
3.84b
5.27b
4.98b
10 ^g/prepupa
% mortal.
87
63.3
58.6
42
35.6
32.4
32.3
28.1
18.5
Sx
4.73a
3.48b
5.37b
6.38°
6.62°
5.28Cd
6.18cd
4.12cd
4.95d
Based on 100 insects per test. Any two means having the same super-
script letter are not significantly different at the 5% level by
, Duncan's multiple range test.
m-Isopropylphenyl methylcarbamate.
c—
'()-Ethyl 0-(2,4-dichlorophenyl)phosphoramidothioate.
Previous reports have indicated that the benzoylphenylureas act only as
stomach poisons. ' However, by topically applying Dimilin in
tetrahydrofuran to white prepupae, we have demonstrated that entry via
the stomach route is not an essential requirement for toxicity.
Discussion - The relatively high levels of cross resistance toward
Dimilin demonstrated by this study may be disconcerting. Extrapolation
of susceptibility data for the strain with the lowest resistance,
parathion-R, reveals the presence of tolerance of approximately ten-fold
at the ED5Q. Resistance in the OMS-12-R strain is obviously several
fold greater. It is especially significant that the field strain, SK-R,
is among the most resistant toward Dimilin. The detection of high
levels of resistance in the house fly serves to emphasize the need for
judicious use of new chemicals against presently susceptible populations,
under conditions which minimize the degree of selection pressure.
3. Genetic Basis of Cross Resistance to Juvenile Hormone Analogs
Introduction - Since the above studies (Section 1) have indicated a
relationship between resistance to certain insecticides and cross
resistance to JHAs, a study was undertaken to establish the genetic
287
-------
basis of this relationship. If linkage between factors responsible for
insecticide resistance and JHA cross resistance could be demonstrated,
this would enhance the hypothesis for co-identity of these phenomena.
Materials and Methods - Dose-response assays were carried out with
methoprene on a ronnel-selected strain (R), a susceptible marker strain
(boaa), and on "sister" strains in which single pairs of chromosomes
of the R strain had been incorporated. The genetic method utilized and
the properties of these strains have been described.
16
Results and Discussion - The regression lines obtained with each
population are given in Figure 1, and the calculated resistance ratios
are presented in Table 6. Despite the low resistance present in the
parental strains (e.g. 6.3x), it is clear that the greatest share of
resistance is contributed by factors on chromosome 2. Chromosome 3 by
itself does not contribute detectable resistance, but in combination
with chromosome 2 it evidently acts as an enhancer of resistance. This
is in accord with our earlier investigations which established that
resistance to ronnel is conferred mainly by chromosome 2 and that the
effects of chromosomes 2 and 3 are synergistic.
Table 6. GENETIC ANALYSIS OF RESISTANCE TO METHOPRENE IN A
RONNEL-SELECTED STRAIN OF HOUSE FLY
Strain
Parental strains
Susceptible (boaa)
Resistant (ronnel)
Derivative strains
R chromosome II
" III
" II + III
«5>°
.46 x 10~4
2.9 x 10~4
-4
1.8 x 10
-4
.3 x 10
-4
2.9 x 10
LC95
2.0 x 10~4
8.3 x 10~4
-4
5.6 x 10
-4
1.2 x 10
-4
10 x 10
o
Resistance ratio at
LC50
6.3
3.9
.7
6.3
LC95
4.2
2.8
.6
5.0
Resistance ratio =
of R or derivative strain
of S strain
288
-------
.00001
.0001
.001
CONCENTRATION (%ALTOSID)
c 3
Figure 1. Dose-mortality relationships of R, S, and derivative strains
(with chromosomes II, III, and II + III) established by exposing late
4th instar larvae to methoprene-treated media.
289
-------
4. Development of Resistance by Specific Selection with Methoprene
and Characteristics of Resistance
Introduction - Although the presence of cross resistance to JHAs in
insecticide-resistant strains was clearly demonstrated, the possibility
of development of resistance by specific selection by these materials
remained unknown. It was, therefore, considered desirable to initiate
selections by a representative compound, methoprene, and to subsequently
carry out a comprehensive study of the resistance which might result.
Materials and Methods - The study involved treatment of successive
generations of two strains (Dimethoate-R and field SK) by partially
lethal doses of methoprene. A sensitive selection method was developed
involving exposure of late 4th instar larvae to a pupation medium
impregnated with the required amount of methoprene. Data were obtained
on the extent of pupal eclosion, adult fecundity, and egg fertility. A
second series of selections (Series II) on the same parental strains was
also carried out in order to test the results obtained in Series I.
Results and Discussion - In conjunction with selection for resistance
the comparative biological and biotic effects of methoprene on house
flies were investigated. The effects of topical treatment of prepupae
included non-emergence or partial emergence of adults, deformed and
non-functional wings, incomplete development of genitalia, incomplete
rotation of male genitalia, and "fatal drunkeness." Longevity, fecundity
and fertility were assayed quantitatively following treatment of
dimethoate-R and NAIDM-S strains at the "maximum sublethal" and ED50
levels. No effects were noted at the sublethal doses, but at the ED50
the post-eclosion biotic potential of both strains as expressed by
fertility and fecundity was severely impaired.
Selection of the two strains by methoprene produced a rapid increase in
resistance to this compound (Table 7, Figure 2). In the dimethoate-R
strain, resistance rose from an initial level of 39.4-fold to 1188-fold
at the completion of 30 generations of selection in Series I and
6060-fold at the completion of 18 generations of selection in Series II.
Similar selection of strain SK resulted in an increase in resistance
from an initial level of 2.5-fold to 1273-fold in Series I and 112-fold
in Series II (Table 7). Substantially high resistance was also
evident when susceptibility to methoprene was determined by exposure
of 4th instar larvae to treated media, instead of by topical applica-
tion to white prepupae (Table 8, Figure 3).
The above large increases in resistance were also accompanied by a
substantial reduction in oogenetic disturbances in the adult insect
following exposure to the chemical.
The effect of development of resistance to methoprene on cross resistance
toward other JHAs was also determined. The strains under investigation
showed increases in cross resistance toward the JHAs Ro 7-9767 and
290
-------
^ 7
tr
o
.001
.01 ~l
DOSAGE (ALTOSID
Figure 2, Development of resistance to methoprene (Altosid) in
dimethoate-R (top) and SK (bottom) strains of the house fly in two
series of selections, I and II. Assays performed by topical application
to white prepupae.
291
-------
>-
I-
a:
o
DIMETHOATE-RX SER j F
V '
DIMETHOATE-R STRAIN
BY ALTOSID
SER.n,F,7
SER.I,F3o
SELECTION OF SK STRAIN
BY ALTOSID
.0001
.001
CONCENTRATION (%ALTOSID)
GO
O
or
CL
Figure 3. Development of resistance to methoprene (Altosid) in
dimethoate-R (top) and SK (bottom) strains of the house fly in two
series of selections I and II, Assays performed by exposure of 4th
instar larvae to treated media.
292
-------
Table 7. EFFECT OF METHOPRENE SELECTION PRESSURE ON CROSS
RESISTANCE TO INSECTICIDES IN THE HOUSE FLY
S-NAIDM
EDso
Compound y/PrePuPa
Methoprene . 0033
Dimethoate .011
OMS-12 .072
Parathion .024
Fenthion . 043
OMS-15 1.3
Aldrin .058
Dieldrin .008
a
Resistance ratio
SK-R
P
2.5
3.8
6.0
59.2
10.9
3.1
5.0
4.3
Ser. I
F30
1273.
6.0
15.7
118.3
29.0
(30%)
1.1
6.5
Ser. II
F17
112.
8.5
26.4
-
-
(20%)
1.9
—
Dimethoate-R
P
39.4
103.6
18.5
16.7
93.0
(0%)
_ .
12.5
Ser. I
F30
1188.
23.6
15.3
63.8
50.7
(2.5%)
2.1
10.9
Ser. II
FIB
6060.
40.
18.1
-
-
(2%)
2.1
—
Resistance ratio = Efo °ff selected
EDsg of parental
strain
strain
Table 8. DEVELOPMENT OF RESISTANCE TO METHOPRENE BY SELECTION
OF A DIMETHOATE-RESISTANT AND A FIELD (SK) STRAIN OF HOUSE FLY
Strain
NAIDM
Parental SK
Ser. I, F30
Ser. II, Fi7
Parental Dimethoate
Ser. I, F30
Ser. II, F18
EC50
(%)
.54 x 10~4
3.1 x 10~4
16 x 10"4
11 x 10~4
12 x 10~4
72 x 10~4
18 x 10~4
EC95
(%)
2.2 x 10~4
9.5 x 10~4
28 x 10~3
32 x 10~4
23 x 10~4
.57
1.17
Resistance ratio at
EC50
5.2
3.5
6.0
15.0
EC95
29.5
3.4
247.8
508.7
Resistance ratio =
EC of selected strain
EC of parental strain
293
-------
R-20458. In general the magnitude of increase was positively correlated
with the intrinsic activity of the compounds against the susceptible
strain. Thus the degree of change in strain SK for methoprene, Ro 7-
9767 and R-20458 (ED50 0.017, 2.6 and 49 pg/pupa, respectively) was
6060x, 1545x, and 12.3x, respectively.
Further studies concerned the effect of selection by methoprene on the
insecticide-resistance levels of the above strains (Table 7). In
general only small changes in the cross resistance spectrum (less than
3x) were noted, although resistance to methoprene itself had increased
to a 1273x level in strain SK and 6060x level in strain dimethoate-R.
The most pronounced chance in cross resistance was toward fenthion,
which rose from an initial level of 10.9x before methoprene selection
in strain SK to 29x after 30 generations of pressure. In the same
strain there were small increases in cross resistance toward dimethoate
(1.6x), OMS-12 (2.6x), dieldrin (1.5x), and parathion (2.Ox). In strain
dimethoate-R there was an increase in cross resistance to parathion
(3.8x), but small reductions in cross resistance to dimethoate (4.4x),
OMS-12 (1.2x), fenthion (1.8x), and dieldrin (l.lx). These results
indicate a random drift in the cross resistance pattern of the strains
and suggest that the induced resistance toward the juvenile hormone
analog may be due to factors unrelated to insecticide resistance.
The possibility of reduced penetration as a mechanism of resistance to
JHAs was examined by the use of radiolabeled R-20458. Sublethal doses
of this JHA were applied topically to white prepupae and allowed to
penetrate for various time intervals between 0 and 720 minutes, and
the radioactivity was determined at the end of each period. The results
revealed that the JHA penetrated somewhat more slowly in susceptible
than in resistant pupae, especially during the 90-360 minute period.
Thus a reduction in the rate of penetration, as is usually observed in
cases of insecticide resistance, was not found to be a factor in
resistance to the JHA.
In view of the similarity in the results of selection in the two
series, only the strains in Series I have been retained for further
study on the mechanisms of resistance. Beyond generation 30, resistance
appears stable and is being maintained by treatment of pupation medium
at a concentration of 200 ppm methoprene. Such exposure provides
approximately 60% pupal mortality and 73% egg sterility, i.e. a total
pressure of 89%.
5. Selection of House Flies for Resistance to Dimilin
Introduction - In view of our earlier finding that insecticide-resistant
house flies possess various degrees of cross resistance to the chitin
synthesis inhibitor Dimilin,13 a strain (dimethoate, methoprene-
resistant) was placed under selection pressure by Dimilin in order to
294
-------
enhance and concentrate the existing resistance factors for further
study. Interest in Dimilin as a potential insecticide has been enhanced
by the demonstration of activity of a high order against a variety of
pest species in several insect families and orders.
Materials and Methods - The selection procedure developed for this
purpose involves the incorporation of Dimilin in the larval medium,
beginning with the first instar of larval growth, and assessment of
results on the basis of adult emergence, fecundity, and fertility.
Results and Discussion - Following selection for 4 generations at
2 ppm Dimilin and of 6 additional generations at 3 ppm, suppression of
adult emergence declined from 96.4% in FI to 87.6% in FIO- Concurrently,
egg fertility increased from 58.2% in FI to 72.2% in FIo. In the F20
selected generation the strain could tolerate 0.125 ppm Dimilin in the
rearing medium, suffering a reduction in adult yield of approximately
75%. This effect is equivalent to approximately 8x increase in
resistance over the level present in the parental (Dimethoate-R) strain.
6. References
1. Williams, C. M. Third generation pesticides. Scientific Amer. 217;
13-17, January 1969.
2. Cerf, D. C., and G. P. Georghiou. Evidence of cross-resistance to a
juvenile hormone analogue in some insecticide-resistant houseflies.
Nature (London) 239^:401-2, 1972.
3. Cerf, D. C., and G. P. Georghiou. Cross resistance to juvenile
hormone analogues in insecticide-resistant strains of Musea domestica
L. Pestic. Sci. (London) _5:759-67, 1974.
4. Georghiou, G. P., M. K. Hawley, E. C. Loomis, and M. F. Coombs.
Insecticide resistance in houseflies in California. Calif. Agric.
_26:4-6, September 1972.
5. Reddy, G., and A. Krishnakumaran. Relationship between morphogenetic
activity and metabolic stability of insect juvenile hormone analogues.
J. Insect Physiol. (London) 18:2019-28, 1972.
6. Ajami, A. M., and L. M. Riddiford. Comparative metabolism of the
Cecropia juvenile hormone. J. Insect Physiol. (London) 19;635-45,
1973.
7. White, A. F. Metabolism of the juvenile hormone analogue methyl
farnesoate 10,11-epoxide in two insect species. Life Sci. 11 (2):
201-10, 1972.
295
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8. Slade, M., and C. Zibbitt. Metabolism of Cecropia juvenile
hormone in insects and mammals. In: Insect Juvenile Hormones:
Chemistry and Action, Menn, J. J., and M. Beroza (Eds.). New York,
Academic Press, 1972. 155-76.
9. Terriere, L. C., and S. J. Yu. Insect juvenile hormones: induc-
tion of detoxifying enzymes in the housefly and detoxication by
housefly enzymes. Pest. Biochem. Physiol. 3^96-107, 1973.
10. Vinson, S. B., and F. W, Plapp, Jr. Third generation pesticides:
the potential for development of resistance by insects. J. Agric.
Food Chem. 22:356-60, 1974.
11. van Daalen, J. J., J. Meltzer, R. Mulder, and K. Wellinga.
Naturwissenschaften _59:312, 1972.
12. Pest, L. C., and W. R. Vincent. A new insecticide inhibiting
chitin synthesis. Naturwissenschaften ^0:431-2, 1973.
13. Cerf, D. C., and G. P. Georghiou. Cross-resistance to an inhibitor
of chitin synthesis, TH 60-40, in insecticide-resistant strains of
the housefly. J. Agric. Food Chem. ^2_(6) :1145-6, Nov./Dec. 1974.
14. Mulder, R., and M. J. Gijswijt. The laboratory evaluation of two
promising new insecticides which interfere with cuticle deposition.
Pestic. Sci. 4_:737, 1973.
15. Wellinga, K., R. Mulder, and J. J. van Daalen. Synthesis and
laboratory evaluation of l-(2,6-disubstituted benzoyl)-3-
phenylureas, a new class of insecticides. I. l-(2,6-dichloro-
benzoyl)-3-phenylureas. J. Agric. Food Chem. ^:348, 1973.
16. Georghiou, G. P. Isolation, characterization and re-synthesis of
insecticide resistance factors in the housefly, Musca domestica.
Proc. 2nd Int. Congr. Pestic. Chem. 2^77-94, 1971.
296
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N. INSECT RESISTANCE TO CHEMICALS: INSECTICIDE RESISTANCE IN MOSQUITOES
1. Resistance to Organophosphates and Carbamates in Anopheles
albimanus Based on Reduced Sensitivity of Acetylcholinesterase
Introduction - One of the outstanding cases of resistance in recent
years has been that of Anopheles albimanus, the principal vector of
malaria in Central America, which has become resistant to a large variety
of organophosphate and carbamate insecticides.1'2 Work in our labora-
tory had shown that resistance was of unusually high level (parathion
84x, propoxur >1000x),3 that it could not be significantly reduced by
known synergirts, ** and was not due to enhanced metabolism of the
insecticides.5
Although in most cases studied to-date resistance to OP and carbamate
insecticides is dependent primarily on the biochemical alteration of the
toxicant to non-toxic or less toxic products, in a small number of cases
an apparently far more efficient mechanism of resistance has arisen
consisting of insensitivity of the target enzyme, acetylcholinesterase
(AChE), to inhibition by the toxicant. This property was known to
exist only in acarines6"8 but has recently been reported also in
two species of insects, namely Nephotettix cincticepsg and Musca
domestica. Our results indicate that this is also the case with
Anopheles albimanus.
Materials and Methods - The parental strain (P) was collected in El
Salvador in 1970 when it displayed low levels of tolerance to parathion,
methyl parathion, and malathion. Part of P was subsequently selected
in the larval stage by propoxur for 8 generations (Carb.-L strain),
developing moderate to high resistance to carbamate, OP, DDT, and
dieldrin insecticides.1* A homozygous resistant strain (OP-R) was
obtained by selecting Carb.-L with ethyl parathion for 17 generations.
A second homozygous resistant strain (Carb.-R) was obtained by selection
of P in the adult stage by propoxur, after outcrossing it to a 1972
collection from El Salvador. The susceptible strain (S) had been
collected in Haiti in 1965, and was of normal susceptibility to OP,
carbamate, and DDT insecticides.
The homogenates used for enzyme assays were made by grinding 1000 4th
instar larvae in 0.1 M phosphate buffer at pH 7.5 in an ice-cold all
glass homogenizer. The grinding ratio was 100 larvae per ml of buffer.
The homogenate was centrifuged at 10,000 rpm for 1 hour and the super-
natant used as the source of the enzyme.
AChE activity was determined by the method described by Ellman et al.H
with acetylthiocholine iodide as the substrate. The incubation mixture
consisted of 0.2 ml of enzyme pipetted into a 15 ml conical centrifuge
tube which was maintained at 30°±1°C in a water bath. The enzyme was
allowed to equilibrate for up to 2 minutes, then 50 |j,l of the inhibitor
297
-------
was forced under the surface of the preparation. The cholinesterase
inhibition was stopped after different time intervals by diluting the
mixture with 3 ml buffer solution containing 3.75 x 10~3 M acetyl-
thiocholine iodide and 2.0 x 10~4 M DTNB. The amount of residual
cholinesterase activity was measured at 412 m^, on a Unicam Sp-800
spectrophotometer. The reference cuvette contained 3 ml of the same
buffer mixture that was used to quench the inhibition of the enzyme in
order to cancel any spontaneous hydrolysis of the substrate.
The bimolecular rate constant (k^) was measured for the following
equation, in which IS is the enzyme, I_ the inhibitor, and P_ the products.
E + I
\_
El
El' -
/
P + E
12
The method of Aldridge was used to calculate (k^) values.
of the plots were obtained by linear regression analysis.
The slopes
Results and Discussion - The increases in resistance toward parathion,
paraoxon, and propoxur in larvae of strain OP-R and Carb.-R as a result
of their selection by parathion and propoxur, respectively, are indi-
cated in Table 1. The resistance level of OP-R rose to 84x for parathion
and 46x for paraoxon. The Carb.-R strain showed a 51.3x increase in
cross resistance toward parathion. Both strains were almost immune to
propoxur, their extrapolated LC5g values having increased more than
2000x. However, the strains showed only a low level of tolerance
toward fenthion, not exceeding 1.6x.
Table 1. COMPARATIVE LEVELS OF RESISTANCE IN A. ALBIMANUS
SELECTED BY PARATHION (OP-R) AND PROPOXUR (CARB.-R)
Compound
Parathion
Paraoxon
Fenthion
Propoxur
S
LC50
(ppm)
.008
.11
.054
.54
OP-R
LC50
(ppm)
.67
5.1
.082
30%b
Carb . -R
LC50
(ppm)
.41
-
.084
36%b
RRa
OP-R/S
84
46
1.5
>2000
Carb.-R.
51.3
-
1.6
>2000
Resistance ratio = •**n————~
, LC50
% kill with 1000 ppm propoxur
298
-------
The values of the bimolecular rate constants (k^) of the 4 strains are
indicated in Table 2. Both OP-R and Carb.-R strains showed extremely
high insensitivity of AChE to inhibition by propoxur amounting to
25,300x and 12,600x, respectively vis a vis the P strain. A pronounced
decrease in sensitivity of AChE toward parathion in the OP-R and Carb.-R
strains, amounting to 371x and 459x, respectively, is also evident.
Table 2. K. VALUES FOR LARVAE OF FOUR STRAINS OF A. ALBIMANUS
Strains
Parathion-selected
(OP-R)
Propoxur-selected
(Carb.-R)
Parental (P)
Susceptible (S)
Ratios:
P/OP-R
S/OP-R
P/ Carb.-R
S/Carb.-R
Paraoxon
2.1 x 103
1.7 x 103b
1.1 x 106
7.8 x 105
524
371
647
459
Propoxur
.19 x 102
.38 x 102
4.8 x 105
2.95 x 105
25.3 x 103
15.5 x HX:
12.6 x 10^
7.8 x 10
Fenoxon
1.5 x 103
4.6 x 103
3.1
-
.Average of 4 replications, expressed as M min
Average of 3 replications.
-1
An apparent anomaly in the relative sensitivity of AChE of the resistant
strains was noted: a higher k^ value (i.e. higher sensitivity) was
found in the OP-R than in the Carb.-R strain when paraoxon was used as
enzyme inhibitor, and the opposite occurred when propoxur was used as
the inhibitor. However, the k^ data are generally in agreement with
the bioassay results showing high resistance to parathion and nearly
complete immunity to propoxur in both strains.
As might have been expected, only a small difference in k^ values was
observed when fenoxon was used as the inhibitor since the levels of
tolerance of the resistant strains toward fenthion are minimal.
The results in Table 3 indicate that the affinity of AChE of the R
strain for acetylthiocholine, as represented by kjj values, was 3.1x
and 3.4x "poorer" than that of the S and P enzymes. These data are
in agreement with results reported with house flies and mites.
However, there was no significant difference in the affinity for
acetylthiocholine among AChE of the Carb.-R, P, and S strains.
299
-------
Table 3. MICHAELIS CONSTANTS (Km) AND MAXIMUM VELOCITY (Vmax)
FOR HYDROLYSIS OF ACETYLTHIOCHOLINE BY LARVAL AChE OF FOUR
STRAINS OF A. ALBIMANUS AT 30 ± 1°C, pH 7.5
Strains
R
C
S
P
Km (M)a
16.7 x 10~4a
5.6x 10~4b
5.3 x 10~4b
4.9 x 10~4b
a
Vmax (p, moles/L. per min)
13. 3a
15. 5b
25.4°
29. 5d
Any two means having the same superscript are not significantly
different at 5% level as determined by Duncan's multiple range test,
The data for maximum velocity of hydrolysis of acetylthiocholine (Vmax)
of the 4 strains showed significant differences. In general, Vmax
values of the 2 R strains were almost half those of the P and S strains.
These results are in agreement with similar data obtained on ticks8
and mites,6 and suggest that the AChE's in the 4 strains may be
different isozymes. 3
The data obtained suggest that the selection process resulted in the
preponderance of individuals possessing an alteration in the active
site of AChE which is involved in binding carbamate and OP insecticides.
As a result, certain OP and carbamate compounds are no longer able to
bind effectively to the altered site. Such alteration, however, does
not confer insensitivity to all OP insecticides, as has been clearly
demonstrated with fenoxon. It may be assumed that the S strains consist
mostly of individuals with "sensitive" AChE. In the presence of indi-
viduals with "insensitive" AChE, the selective process is believed to
proceed rapidly, since data obtained in our laboratory indicate that
OP and carbamate resistance in this species manifests a high degree
of dominance.
In view of the decisiveness of OP and carbamate resistance conferred
on A_. albimanus by AChE insensitivity, it is feared that such
resistance may be a strong obstacle in the control of the species by
these insecticides. However, the demonstration that insensitivity does
not extend to fenoxon indicates that this property is at present
specific for certain compounds or groups rather than generic.
300
-------
2. Characteristics of Multiresistance in Culex quinquefasciatus
Introduction - Despite the development of high resistance to organophos-
phates in field populations of Aedes nigromaculis1'* and Culex tarsalis15
in California during the past several years, the common house mosquito
Culex quinquefasciatus has remained largely susceptible to these insect-
icides. However, reports were received of failures of chlorpyrifos to
give adequate control of the species in the San Joaquin Valley in 1974.
In view of the importance of £. quinquefasciatus as a vector of human
disease worldwide, we initiated a study of the character of the emerging
resistance and of the possibility of its suppression by synergists or
alternative insecticides.16
Materials and Methods - Larvae in excess of 10,000 were collected in
September, 1974, from dairy waste drains (Camara Dairy) approximately
4 miles from Hanford, California. These had been treated with various
OP's for several years and with chlorpyrifos during 1973 and 1974.
Additionally, it may be assumed that the mosquito population was
indirectly exposed to chemicals applied to crops in the area.
The rearing and testing methods have been already described.17When
synergism was tested, 0.5 ml of synergist was applied 4 hours in advance
of the application of 0.5 ml of insecticide. When only insecticide was
being tested, 1 ml volumes of the solutions were applied per 100 ml of
water. All tests were performed on F^ generation larvae.
Results and Discussion - Spectrum and Levels of Resistance - It is
apparent from the data in Table 4 that the population of £. £. quin-
quefasciatus near Hanford has developed a wide spectrum of OP multi-
resistance. This comprises the main OP's used up to now in mosquito
control in California, namely Abate®, at a level of 116.7x the normal
LC5Q, chlorpyrifos 52.2x, fenthion 48.9x, methyl parathion 24x, malathion
16.4x, and parathion 12.9x. Cross resistance obviously extends to
related materials as indicated by a 12.4x increase in LC50 for fenitro-
thion and 83.3x increase for chlorpyrifos-methyl. Neither of these
chemicals has been employed for mosquito or agricultural pest control
in the area.
The very limited cross resistance toward propoxur (2.7x) is noteworthy.
Obviously the mechanisms of OP multiresistance in this strain do not
significantly enhance carbamate metabolism. Absence of carbamate cross
resistance was also observed in £. tarsalis from the Coachella Valley,
whose multiresistance included chlorpyrifos-methyl 18.9x, fenthion
16.Ix, chlorpyrifos 8x, fenitrothion 7.3x, methyl parathion 7.Ox, Abate
3.9x, and malathion 3.3x.18
The resistance observed is believed to be of relatively recent origin,
since the population had been found to be susceptible to OP's as
recently as 1970 (G. P. Georghiou, unpublished). Furthermore, the
301
-------
Table 4. RESISTANCE SPECTRA OF CULEX P. QUINQUEFASCIATUS AT
HANFORD, CALIFORNIA, 1974
Insecticide
Abate
Chlorpyrifos
Chlorpyrifos methyl
Parathion
Fenthion
Methyl parathion
Fenitrothion
Malathion
Propoxur
Carbofuran
DDT
Susceptible
lab, strain
LC50
(ppm)
.0018
.0023
.003
.0041
.0047
.005
.017
.11
.35
.052
.065
slope
(b)
13.6
7.0
7.0
24.0
10.8
19.1
10.1
4.9
7.8
3.8
5.6
Resistant field strain
LC50
(ppm)
.21
.12
.25
.053
.23
.12
.21
1.8
.93
.14
.39
slope
(b)
3.2
6.1
3.6
4.8
6.1
4.0
4.9
3.3
5.3
4.6
1.3
RR3
116.7
52.2
83.3
12.9
48.9
24.0
12.4
16.4
2.7
2.7
6.2
Resistance ratio = LCso resistant strain T LCso. susceptible strain.
302
-------
levels of resistance at Camara are significantly higher than at a
neighboring dairy 6 miles distant, suggesting that the populations have
not as yet had adequate time to fully intermingle.
Results and Discussion - Synergism and Resistance - In order to gain an
insight into the mechanisms of resistance a number of OP's were tested
with the synergists piperonyl butoxide (p.b.), ^,jS,j3-tributyl
phosphorotrithioate (DBF), and triphenyl phosphate (TPP). The synergists
were used at maximal sublethal concentrations of 5 ppm for p.b. and 2.5
ppm for DBF and TPP, irrespective of the dosage of insecticide tested.
At higher concentrations DBF was approximately equitoxic to the two
strains, the LC^Q levels being 29 ppm in the Camara and 31 ppm in the
S strain, with similar slope values (b=2.4). Table 5 indicates the LC5Q
values, synergism ratios and resistance ratios obtained. P.b. exhibited
practically no synergism (or antagonism) of methyl parathion, methyl
paraoxon, and chlorpyrifos in either the S or resistant (R) strain, the
synergism ratios ranging from 1.0 to 1.3. In contrast, DBF exhibited a
remarkable degree of synergism of OP's in the R strain, resulting in
almost complete elimination of resistance. In all cases, DBF reduced
the LC5Q values in the R strain to near the level of the unsynergized
insecticides in the S strain. This effect was most pronounced in the
case of chlorpyrifos.
Absence of synergism by p.b., a known inhibitor of the mixed function
oxidase (MFO) system, would suggest that MFO detoxication enzymes are
not involved in resistance in the strain studied. This would also
explain, at least in part, the absence of carbamate resistance, since
in a propoxur-selected strain of this species, MFO enzymes were shown
to be primarily responsible for a higher rate of metabolism of
propoxur, carbaryl, aldicarb, and carbofuran.
Synergism of OP's by DBF and TPP has been reported earlier. Plapp et
al. observed strong synergism of malathion in C^. tarsalis and suggest-
ed that the phenomenon was due to inhibition of the carboxyesterase
detoxication enzyme. But subsequent observations of synergism of other
OP's lacking carboxyester linkage22'23 suggests that the action of DBF
is broader, probably involving inhibition of a wider range of hydrolytic
esterases. In this connection, Jao and Casida have demonstrated that
synergism of resmethrin and tetramethrin by DBF in various insects is
due to inhibition of pyrethroid-hydrolyzing esterases. Hydrolytic
enzymes were also found to be primarily responsible for metabolism of
fenthion in C^. _p_. quinquefasciatus from Burma, made 8-fold resistant by
fenthion pressure in the laboratory. '
Results and Discussion - Alternative Chemicals - Table 6 presents the
cross resistance spectrum of the Camara strain toward 12 new or experi-
mental chemicals representing OP's, carbamates, organochlorines,
pyrethroids, and insect growth regulators.
303
-------
Table 5. SYNERGISM OF ORGANOPHOSPHATES BY PIPERONYL BUTOXIDE, DEF& AND TPP AGAINST
SUSCEPTIBLE AND OP MULTIRESISTANTC CULEX P. QUINQUEFASCIATUS LARVAE
Methyl parathion
Methyl paraoxon
Chlorpyrif os
Methyl parathion
Methyl paraoxon
Parathion
Chlorpyrifos
Malathion
S Strain
Insecticide
alone
(ppm)
.005
.025
.0023
.005
.025
.0041
.0023
.11
Plus
synergist
LC50
(ppm)
.0048
.024
.0020
.0012
.0057
.0012
.0024
.10
Synergismd
ratio
Synergist:
1.0
1.0
1.2
Syn
4.2
4.4
3.4
1.0
Syn
1.1
R Strain
Insecticide
alone
(ppm)
Piperonyl B
.12
.4
.12
ergist: DEF
.12
.4
.053
.12
ergist : TPP
1.8
Plus
synergist
LC50
(ppm)
utoxide
.12
.31
.094
.0016
.007
.0028
.0013
.14
Synergism^
ratio
1.0
1.3
1.3
75.0
57.1
18.9
92.3
12.9
Resistance ratio
Insecticide
alone
24.0
16.0
52.2
24.0
16.0
12.9
52.2
16.4
Plus
synergist
25.0
12.9
47.0
1.3
1.2
2.3
0.54
1.4
OJ
o
-p-
v.S , S , S-tr ibutyl phosphorotr ithioate
Triphenyl phosphate
Camara strain, 1974
Synergism ratio
insecticide alone T LCso insecticide in presence of synergist,
-------
Table 6. RELATIVE TOXICITY OF NEW CHEMICALS AGAINST SUSCEPTIBLE
AND OP-MULTIRESISTANT STRAINS OF CULEX P. QUINQUEFASCIATUS
Insecticide
Susceptible strain
Resistant strain
LC
(ppm)
Slope
LC Slope
(ppm (b)
RR
Chlorphoxim
Cyanox0 ,
Celamerck S-2957
Ciba-Geigy 188096
.0035
.017
.0013
.042
7.6
11.1
10.9
7.9
.043
.29
.048
.15
4.5
3.9
3.9
6.3
12.3
17.1
37.7
3.6
CRC 11786
CRC 117838
.002
.013
10.4
6.4
.0037 3.8
.018 4.7
1.9
1.4
R. L. Metcalf XVI
.054
6.1
.039 4.9
.7
Cismethrin
Biopennethrin
Methoprene
Dimilin1
.0016
.0037
.00029
.0021
5.3
6.3
2.4
3.6
.0016
.0030
.0015
.0031
4.1
5.0
2.3
3.5
1.0
.81
4.9
1.5
{•Resistance ratio, see footnote a Table 4
0_-chlorophenyl glyoxylonitril oxime 0-ester with £,()-diethylphosphoro-
thioate.
Q
,0-p_-cyanophenyl 0_,^-dimethyl phosphorothioate
0-(2,5-dichloro-4-methylthiophenyl) (),()-diethylphosphorothioate
e?-(6-chloro-oxazolo(4,5b)pyridine-2(3H)-0-ylmethyl) 0,0-dimethyl
,: phosphorothioate.
2,2-dimethyl-2,3-dihydrobenzofuranyl-7 methyl(4-tert-butyl-2-methyl-
phenylthio)carbamate.
rO-isopropoxyphenyl methyl(4-tert-butyl-2-methylphenylthio)carbamate.
2-(£-tolyl)-2-(£-ethoxyphenyl) 1,1,1-trichloroethane (Metcalf, et al.,
± 1971).
(5-benzyl-3-furyl)methyl (+)-£i^-2,2-dimethyl-3-(2-methyl-l-propenyl)-
cyclopropane-1-carboxylate
3-phenoxybenzyl j-trans-2,2-dimethyl-3-(2,2,-dichlorovinyl)cyclopropane-
, carboxylate.
,isopropyl ll-methoxy-3,7,ll-trimethyldodeca-2,4-dienoate
1-(4-chlorophenyl)-3-(2,6-difluorobenzoy1)-urea
305
-------
It is apparent that the broad spectrum of OP resistance observed (Table
4) extends also at relatively high levels to new experimental OP's such
as Chlorphoxim, Cyanox, and Celamerck S-2957. Compound Ciba-Geigy 18809
is suspected to act as a carbamate after cleavage of the phosphate
moiety, as described for Itf-phosphorothioylcarbamate esters by Fahmy
et al.,27 thus the relatively low resistance to it of 3.6x. Similarly,
CRC 11786 and CRC 11783 are N-sulfenylated derivatives of the carbamates
carbofuran and propoxur, respectively.28 In the absence of carbamate
resistance they are nearly as toxic to the R as to the S strain. Deriva-
tization has resulted in a pronounced increase in larvicidal activity
(approximately 26x over the parent compounds), thus placing them within
the activity range of other common larvicides.
The OP-resistant strain is susceptible to RLM XVI, a biodegradable
analogue of DDT,29 despite its 5.7x resistance to DDT (Table 4). Of much
interest is the absence of cross resistance to the synthetic pyrethroids
cismethrin and biopermethrin. Unlike natural pyrethrins and related
synthetic pyrethroids, biopermethrin is photostable30 and thus holds
promise as a larvicide against R strains.
Insect growth regulators are attracting much interest in mosquito control
in view of their high activity, unique mode of action, and relatively
low mammalian toxicity. Both methoprene and Dimilin manifest outstand-
ing activity against both S and R strains. However, the cross resistance
ratio of 4.9x toward methoprene should be noted, in view of the success-
ful selection of C^. tarsalis,31 C^. j>. pipiens,32 and Musca domestica
(page 290, this report) for higher tolerance toward this compound.
3. References
1. Georghiou, G. P., V. Ariaratnam, and S. B. Breeland. Anopheles
albimanus: development of carbamate and organophosphorus resist-
and in nature. Bull. W.H.O. (Geneva) 46^:551-4, 1971.
2. Georghiou, G. P. Studies on resistance to carbamate and organophos-
phate insecticides in Anopheles albimanus. Amer. J. Trop. Med. &
Hyg. 21:795-806, 1972.
3. Ayad, H., and G. P. Georghiou. Resistance to organophosphates and
carbamates in Anopheles albimanus based on reduced sensitivity of
acetylcholinesterase. J. Econ. Entomol. j>i8(3) :295-7, 1975.
4. Ariaratnam, V., and G. P. Georghiou. Selection for resistance to
carbamate and organophosphorus insecticides in Anopheles albimanus.
Nature (London) 232:642-4, 1971.
306
-------
5. Ariaratnam, V., and G, P, Georghiou. Carhamate resistance in
Anopheles albimanus; penetration and metabolism of carbaryl in
propoxur-selected larvae, Bull. W,H,0, (Geneva), in press.
6. Smissaert, H. R, Cholinesterase inhibition in spider mites suscep-
tible and resistant to organophosphate. Science 143;129-31, 1964.
7. Voss, G., and F. Matsumura. Resistance to organophosphorus com-
pounds in the two-spotted spider mite: two different mechanisms of
resistance. Nature (London) 202;319-20, 1964.
8. Roulston, W. J., H. J. Schnitzerling, and C. A. Schuntner. Acetyl-
cholinesterase insensitivity in the Biarra strain of the cattle
tick Boophilus microplus, as a cause of resistance to organophos-
phorus and carbamate acaricides. Aust. J, Biol. Sci. 21;759-67,
1968.
9. Hama, H., and T. Iwata. Insensitive cholinesterase in the Nakaga-
wara strain of the green rice leafhopper, Nephottetix cincticeps
Uhler (Hemipter: Cicadellidae), as a cause of resistance to carba-
mate insecticides. Appl. Ent. Zool. j6:183-91, 1971.
10. Tripathi, R. K., and R. D. O'Brien. Insensitivity of acetylcholin-
esterase as a factor in resistance of houseflies to the organophos-
phate rabon. Pest. Biochem. Physiol. _3:495-8, 1972.
11. Ellman, G. L., K. D. Courtney, V. Andres, Jr., and R. M. Feather-
stone. A new and rapid colorimetric determination of acetylcholin-
esterase activity. Biochem. Pharmacol. ^:88-95, 1961.
12. Aldridge, W. N. Some properties of specific cholinesterase with
particular reference to the mechanism of inhibition of diethyl
£-nitrophenyl thiophosphate (E605) and analogues. Biochem. J.
46:451-60, 1950.
13. Eldefrawi, M. E., R. K. Tripathi, and R. D. O'Brien. Acetylcholin-
esterase isozymes from the housefly brain. Biochem. Biophys. Acta.
^2:308-14, 1970.
14. Brown, A. W. A., L. L. Lewallen, and P. A. Gillies. Organophosphor-
us resistance in Aedes nigromaculis in California. Mosquito News
23:341-5, 1963.
15. Georghiou, G. P., P. A, Gillies, and D. J. Womeldorf. Culex
tarsalis Coquillett: Detection of resistance to parathion, methyl
parathion, fenthion, Dursban,® and Abate® in a malathion-resistant
population. Calif. Vector Views 16:115-8, 1969.
307
-------
16, Georghiou, G. P., V, Ariaratnam, M, E, Pasternak, and C. S. Lin,
Organophosphorus multiresis.tance in Culex pipjens quinquefasciatus
in California, J. Econ. Entomol,, 1975, in press.
17. Georghiou, G. P., R. L. Metcalf, and F, E. Gidden. Carbamate
resistance in mosquitoes, I. Selection of Culex pipiens quinque-
fasciatus Say for resistance to Baygon, Bull. WHO _35_: 691-708, 1966.
18. Apperson, C. S., and G. P. Georghiou, Comparative resistance to
insecticides in populations of three sympatric species of mosqui-
toes in the Coachella Valley of California. J. Med. Entomol. 11:
573-6, 1974.
19. Shrivastava, S. P., G. P. Georghiou, and T. R. Fukuto. Metabolism
of IJ-methylcarbamate insecticides by mosquito larval enzyme system
requiring NADPH2. Entomol. Exp. Appl. 1.4:333-48, 1971.
20. Shrivastava, S. P., G. P. Georghiou, R. L. Metcalf, and T. R.
Fukuto. Carbamate resistance in mosquitoes. II. The metabolism
of propoxur by susceptible and resistant Culex pipiens fatigans
Wied. Bull. WHO 42:932-42, 1970.
21. Plapp, F. W., Jr., W. S. Bigley, G. A. Chapman, and G. W. Eddy.
Synergism of malathion against resistant house flies and mosquit-
oes. J. Econ. Entomol. 5^6:643-9, 1963.
22. Bell, J. D. , and J. R. Busvine. Synergism of organophosphates in
Musca domestica and Chrysomya putoria. Entomol. Exp. Appl. 10:
263-9, 1967.
23. Apperson, C. S., and G. P. Georghiou. Mechanisms of resistance to
organophosphorus insecticides in Culex tarsalis. J. Econ. Entomol.
1975, in press.
24. Jao, L. T., and J. E. Casida. Insect pyrethroid-hydrolyzing
esterases. Pestic. Biochem. Physiol. 4^465-72, 1974.
25. Stone, B. F., and A. W. A. Brown. Mechanisms of resistance to
fenthion in Culex pipiens fatigans Wied. Bull. WHO 4(): 401-8, 1969.
26. Stone, B. F. Metabolism of fenthion by the southern house mosquito.
J. Econ. Entomol. ^2:977-81, 1969.
27. Fahmy, M. A. H., T. R. Fukuto, R. 0, Myers, and R. B. March. The
selective toxicity of new N-phosphorothioylcarbamate esters. J.
Agr. Food Chem. 18:793-6, 1970.
308
-------
28. Black, A. L., V. C. Chiu, M. A. H, Fahmy, and T. R. Fukuto.
Selective toxicity of tfl-sulfenylated derivatives of insecticidal
methylcarbamate esters. J. Agr. Food Chem. 21^:7^7, 1973.
29. Metcalf, R. L., I. P. Kapoor, and A. S. Hirwe. Biodegradable
analogues of DDT. Bull. WHO 44:363-74, 1971.
30. Elliott, M., A. W. Farnham, N. F, Janes, P. H, Needham, D. A.
Pulmar, and J. H. Stevenson. A photostable pyrethroids. Nature
(London) ^46:169, 1973.
31. Georghiou, G. P., C. S. Lin, and M. E. Pasternak. Assessment of
potentiality of Culex tarsalis for development of resistance to
carbamate insecticides and insect growth regulators. Proc.
and Papers, Calif. Mosquito Control Assoc. 42^117-8, 1974.
32. Brown, T. M., and A. W. A. Brown. Experimental induction of
resistance to a juvenile hormone mimic. J. Econ. Entomol. 67:
799-801, 1974.
309
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0. JOINT ACTION OF HERBICIDES AND INSECTICIDES
1, Joint Action of Bipyridilium Herbicides with Propoxur and Fenthion
Introduction - While many investigators have shown the presence of
interactions between herbicides and insecticides with respect to their
toxicity to plants,1'4 the effect of such combinations against insects
does not appear to have been examined. Interactions in plants are most
commonly synergistic due to inhibition of herbicide degradation in the
presence of the insecticide or due to inhibition of insecticide
metabolism in the presence of the herbicide.8
The bipyridilium herbicides diquat and paraquat are broad spectrum herbi-
cides which act by interfering with photosynthetic electron trans-
port.9'10 The herbicides undergo an oxygen-reversed one-electron reduc-
tion to form stable free radicals at fixed potentials (Eo).11
Phenazine methosulfate (Eo = +0.08v at pH 7.0),12 which is well known to
participate in biological redox reactions13 has been shown to form a
stable free radical on reductionlt+ and is a potent inhibitor of microso-
mal oxidations (R. I. Krieger, unpublished). For a series of diquater-
nary compounds, inhibitory potency is dependent on the reduction poten-
tial (Eo) of the salts suggesting that inhibition is due to disruption
of microsomal electron transport.
Diquat and paraquat find common usage in aquatic weed control.9 At high
concentrations (275 ppm), diquat enhances the in vivo toxicity to
mosquito fish of carbaryl and Zectran® (4-dimethylamino-3,5-xylyl
methylcarbamate) but does not affect the acute toxicity of aldrin, DDT,
or parathion. This effect is associated with inhibition of the micro-
somal mixed function oxidase system of the animal and thus with impair-
ment of its metabolic oxidative capability.
Because the development of effective insecticide synergists is of great
interest in studies of insecticide metabolism as well as in insect pest
control, we investigated the possibility that quaternary compounds
capable of interrupting microsomal electron transport could potentiate
the toxicity of the insecticides propoxur and fenthion to mosquito
larvae.16 Insecticide-susceptible as well as resistant strains of
mosquitoes were employed since it has been established that the higher
insecticide tolerance of at least one of the resistant strains is largely
due to enhanced mixed function oxidase activity.17
Materials and Methods - Diquat [6,7-dihydro(l,2-a:2',l'-c)pyrazinium
dibromide (Compound III)] and its analogues [3,4-benzo-6,7-dihydrodi-
pyrido-(l,2-a:2',l'-c)pyrazinium dibromide (II) and 7,8-dihydro-6H-
dipyrido(l,2-a:2,l-c)-(l,4)diazepinium dibromide (V)] were synthesized
by established methods.11'18 Paraquat [l,l-dimethyl-4,4-bipyridylium
dichloride (IV)] was obtained pure by crystallization of technical
310
-------
material, phenazine methosulfate (I) was purchased from the Aldrich
Chemical Co. and crystallized before use.
Most tests were performed on a strain of Culex pipiens fatigans (Wied.)
of normal susceptibility to insecticides, Strains of _C, £. fatigans
resistant to propoxur (resistance level 30-fold) or fenthion (resistance
level 30-fold) and a susceptible strain of Anopheles albimanus (Wied.)
were also used but to a smaller extent, All bioassay tests were per-
formed by established procedures.
Results - Table 1 shows toxicity of the quaternary salts against
insecticide-susceptible (S) strains of Culex and Anopheles. The com-
pounds manifest extremely low toxicity to mosquito larvae, the concen-
trations required for kill far exceeding those being applied in practice
for weed control, i.e., 0.5-5 ppm. The LC5Q values obtained contrast
sharply with the corresponding values for common mosquito larvicides
such as malathion, fenthion, and chlorpyrifos (LCcQ for C^. £. fatigans
0.08, 0.0077, and 0.0022 ppm, respectively),19 The propoxur-resistant (R)
and fenthion-R strains of Culex were less susceptible to the herbicides
by a factor of ca. 2-fold.
Tables 2 and 3 show the effect of pretreatment of larvae by the maximum
sublethal dose of the quaternary compound, followed 2 hours later by
application of propoxur. Against Culex, diquat at 200 ppm and paraquat
and phenazine methosulfate at 100 ppm caused a definite increase in the
toxicity of propoxur (Table 2). Diquat was the strongest synergist,
producing a 2.2-fold increase in toxicity. The same compound was also
strongly synergistic of propoxur against larvae of Anopheles (Table 3).
Paraquat was much less synergistic against this species, and phenazine
methosulfate showed no effect at a dose of 60 ppm. The degree of
synergism demonstrated by the salts does not appear to be correlated with
their relative toxicity to the larvae. However, synergism was dose
dependent. Thus, sublethal concentrations of 100, 200, and 400 ppm
diquat, when followed by application of 0.3 ppm propoxur, caused 75,
83.3, and 98.3% mortality in Culex larvae.
Diquat also was strongly synergistic with propoxur against the propoxur-
R strain of Culex. Concentrations of 0.5, 1, and 1.5 ppm propoxur,
producing <2.5% kill, resulted in 70, 77, and 100% kill when preceded by
application of 1000 ppm diquat.
A limited number of tests with fenthion and diquat on fenthion-S and R
strains of Culex also revealed a strong level of synergistic interaction
by these compounds. Thus, against S larvae, fenthion at 0.006 ppm
produced 23.8% mortality when used alone and 81.3% when preceded by 200
ppm diquat. Against R larvae, 0,05 ppm fenthion produced 1.2% alone
and 86.2% when preceded by 400 ppm diquat.
311
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Table 1. TOXICITY OF HERBICIDES TO 4TH INSTARS OF INSECTICIDE SUSCEPTIBLE
STRAINS OF CULEX P. FATIGANS AND ANOPHELES ALBIMANUS
Compound
Phenazine methosulfate (I)
Compound II
Diquat (III)
Paraquat (IV)
Compound V
Eo(v)a
+0.08
-0.18
-0.35
-0.45
-0.55
Culex p. fatigans
LC50 (ppm)
385
1,000
690
275
1,000
Max. sublethal
(ppm)
100
200
200
50
1,000
Anopheles albimanus
LC50 (ppm)
212
705
475
1,000
750
Max. sublethal
(ppm)
60
80
100
100
100
10
I-"
t-0
Oxidation reduction potential (volts).
-------
Table 2. JOINT ACTION OF PROPOXUR WITH SUBLETHAL CONCENTRATIONS OF HERBICIDES
AGAINST 4TH INSTARS OF INSECTICIDE-SUSCEPTIBLE CULEX P. FATIGANS
Propoxur (ppm)
0
.1
.15
.2
.3
.4
.5
.7
LC50 (ppm)
SRa
Propoxur alone
0
0
0
4
18.3
36.7
63.3
88.3
0.44
%
+ Compound II
200 ppm
0
0
15
33
66
86
100
-
0.25
1.8
Mortality
Propoxur + He
+ Diquat
200 ppm
0
-
-
48.3
81.7
95
-
-
0.2
2.2
rbicide
+ Paraquat
100 ppm
0
-
-
3.3
36.7
-
80
-
0.35
1.3
+ Phenazine
Methosulf ate
100 ppm
1.7
-
-
20
45
-
81.7
96.7
0.31
1.4
00
SR (synergism ratio) =
propoxur
propoxur + herbicide
-------
Table 3. JOINT ACTION OF PROPOXUR WITH SUBLETHAL CONCENTRATIONS
OF HERBICIDES AGAINST 4TH INSTARS OF INSECTICIDE-
SUSCEPTIBLE ANOPHELES ALBIMANUS
Propoxur
(ppm)
0
0.1
0.2
0.3
0.4
0.6
% Mortality
Propoxur
alone
0
0
0
0
1.7
8.3
Propoxur + Herbicide
+ Diquat
100 ppm
3.3
5.0
20
43.3
91.7
100
+ Paraquat
100 ppm
0
-
+ Phenazine
Methosulfate
60 ppm
0
-
0 ; 5
5
10
36.7
10
5
3.3
314
-------
Discussion - The diquaternary salts and phenazine methosulfate exhibit
only low to moderate toxicity to the mosquito larvae. The ionic nature
of the salts could be expected to greatly restrict their absorption by
the larvae and undoubtedly influences their low toxicity. In each case,
the maximum sublethal concentrations are considerably below the estab-
lished LC50 values.
The detection of synergistic activity by these compounds, albeit at high
concentrations, is significant in that it does support the hypothesis
that compounds capable of blocking the microsomal electron flow i£ vitro
can potentiate the toxicity of certain insecticides, possibly by inhibi-
tion of microsomal detoxication processes.
The compounds tested were selected to cover a range of redox potentials.
Since diquat is the most effective synergist against both species of
mosquito larvae, these results indicate that the mixed function oxidase
activity of the larvae is susceptible to inhibition by electron transfer
agents with redox potentials near that of diquat (Eo = -0.349V.).11
This potential is well below that of other compounds such as quinones
known to participate in microsomal electron transfer and suggests that
other compounds more lipophilic than diquat with similar redox properties
should be examined for synergistic activity.
2. References
1. Hacskaylo, J., J. K. Walker, Jr., and E. G. Pires. Response of
cotton seedlings to combinations of pre-emergence herbicides and
systemic insecticides. Weeds 12^:288-91, 1974.
2. Nash, R. G. Phytotoxic pesticide interactions in soil. Agron. J.
59^:227-30, 1967.
3. Nash, R. G. Synergistic phytotoxicities of herbicide-insecticide
combinations in soil. Weed Sci. 3.6^:74-7, 1968.
4. Bowling, C. C., and H. R. Hudgins. The effect of insecticides on
the selectivity of propanil on rice. Weeds _14_:94-5, 1966.
5. Matsunaka, S. Propanil hydrolysis: inhibition in rice plants by
insecticides. Science 160:1360-1, 1968.
6. Swanson, C. R., and H. R. Swanson. Inhibition of degradation of
monuron in cotton leaf tissue by carbamate insecticides. Weed Sci.
1£: 481-4, 1968.
7. Chang, F.-Y., L. W. Smith, and G. R. Stephenson. Insecticide
inhibition of herbicide metabolism in leaf tissues. J. Agric. Food
Chem. 19:1183-6, 1971a.
315
-------
8. Chang, F.-Y., G. R. Stephenson, and L. W. Smith, Influence of
herbicides on insecticide metabolism in leaf tissues. J, Agric.
Food Chem. 19_:1187-90, 1971h,
9. Akhavein, A, A,, and D. L. Linscott, The dipyridylium herbicides
paraquat and diquat. Residue Rev. ^3:97-145, 1968,
10. Black, C. C., Jr., and L. Myers, Some biochemical aspects of the
mechanisms of herbicidal activity. Weeds ^.4_:331-8, 1966.
11. Homer, R. F., and T. E. Tomlinson. The stereochemistry of the
bridged quaternary salts of 2,2'-bipyridyl. J. Chem. Soc. 1960
(II):2498-503, 1960.
12. Albert, A, Heterocyclic Chemistry. London, University of London,
Althone Press, 1968, 547 p.
13. Swan, G. A., and D. G. I. Gelton, Phenazines. New York, Inter-
science, 1957, 693 p.
14. White, J. R., and H. H. Dearman. Generation of free radicals from
phenazine methosulfate, streptonigrin and rubiflavin in bacterial
suspensions. Proc. Natl. Acad. Sci. U.S.A. :54_:887, 1965.
15. Krieger, R. L., and P. W. Lee. Inhibition of in vivo and in vitro
epoxidation of aldrin and potentiation of toxicity of various
insecticide chemicals by diquat in two species of fish. Arch.
Environ. Contam. Toxicol. 1^:112-21, 1973.
16. Georghiou, G. P., A. L. Black, R. I. Krieger, and T. R. Fukuto.
Joint action of diquat and related one-electron transfer agents with
propoxur and fenthion against mosquito larvae. J. Econ. Entomol.
67(2):184-6, 1974.
17. Shrivastava, S. P., G. P. Georghiou, R. L. Metcalf, and T. R.
Fukuto. Carbamate resistance in mosquitoes. II. The metabolism
of propoxur by susceptible and resistant Culex pipiens fatigans
Wied. Bull. WHO 42^:932-42, 1970.
18. Black, A. L., and L. A. Summers. One-electron transfer properties
of diquaternary salts of 2-(2-pyridyl)-quinoline. J. Chem. Soc.
Sect. C Org. Chem. 1971:2271-3, 1971.
19. Georghiou, G. P., R. L. Metcalf, and F, E, Gidden. Carbamate
resistance in mosquitoes. I. Selection of Culex pipiens quinque-
fasciatus Say for resistance to Baygon. Bull. WHO _35:691-708, 1966.
316
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P. LIST OF INVENTIONS OR PUBLICATIONS
1. Selective toxiclty of Phoxim (phenylglyoxylonitrile oxime £,0-
diethyl phosphorothioate), J. H, Vinopal and T. R. Fukuto,
Pestic. Biochem, Physiol. 1: 44-60, 1971.
2. Metabolism of insecticides and fungicides, T. R. Fukuto and J. J.
Sims, In Pesticides in the Environment, R. White-Stevens, ed.,
Marcel Dekker, New York, 1971, pp. 145-236.
3. Relationship between the structure of organophosphorus compounds
and their activity as acetylcholinesterase inhibitors, T. R.
Fukuto, Bull. Wld. Hlth. Org, 44: 31-42, 1971.
4. Intracellular potential characteristics of some orthopteroid
insect hearts, T. A. Miller, Comp. Biochem. Physiol. 40A: 761-9,
1971.
5. The effect of light, temperature, and DDT poisoning on housefly
locomotion and flight muscle activity, T. A. Miller, L. J. Brunner,
and T. R. Fukuto, Pestic. Biochem. Physiol. 1: 483-91, 1971.
6. Studies of cardio-regulation in the cockroach Periplaneta americana,
T. Miller and P. N. R. Usherwood, J. Exp. Biol. 54: 329-48, 1971.
7. Metabolism of N-methylcarbamate insecticides by mosquito larval
enzyme system requiring NADPl^, S. P. Shrivastava, G. P. Georghiou,
and T. R. Fukuto, Ent. Exp. et Appl. 14: 333-348, 1971.
8. Insecticide resistance resulting from sequential selection of house-
flies in the field by organophosphates, G. P. Georghiou and M. K.
Hawley, Bull. Wld. Hlth. Org. 45: 43-51, 1971.
9. Isolation, characterization and resynthesis of insecticide resist-
and factors in the housefly, Musca domestica L., G. P. Georghiou,
Proceedings 2nd Int. Congr. Pestic. Chem. 2: 77-94, 1971.
10. Resistance of insects and mites to insecticides and acaricides and
the future of pesticide chemicals, G. P. Georghiou, In Agricultural
Chemicals—Harmony or Discord for Food, People and the Environment,
J. E. Swift, Ed., Univ. of California, Div. of Agric. Sci., 1971,
pp. 112-24.
11. Convenient method for synthesis of hydroxymethylcarbamates, M. A. H.
Fahmy and T. R. Fukuto, J, Agr. Food Chem. 20: 168-9, 1972.
12. Metabolism of carbamate insecticides, T. R. Fukuto, Drug Metab. Rev.
1: 117-51, 1972.
317
-------
13. Structure, reactivity, and biological activity of ()-(methylcarba-
moyl)oximes of substituted benzaldehydes, R. L. Jones, T. R.
Fukuto, and R. L, Metcalf, J. Econ. Entomol, 65: 28-32, 1972.
14. Alkaline hydrolysis of phosphoramidothioate esters, M, A. H.
Fahmy, A. Khasawinah, and T. R, Fukuto, J. Org. Chem. 37, 617-25,
1972.
15. Structure for the oxygenated product of peracid oxidation of
Dyfonate insecticide (0-ethyl j[-phenyl ethylphosphonothioate),
D. A. Wustner, J. M. Desraarchelier, Life Sciences 11: 585-8, 1972.
16. Sterilization of the beet leafhopper: Induction of sterility and
evaluation of biotic effects with a model sterilant (OM-53139) and
°^Co irradiation, R. V. W. E, Ameresekere and G. P. Georghiou,
J. Econ. Entomol. 64: 1074-80, 1971,
17. Histopathological studies on irradiated and chemosterilized beet
leafhopper, Circulifer tenellus, R. V. W. E. Amereskere, G. P.
Georghiou, and V. Sevacherian, Ann. Entomol. Soc. Amer. 64:
1971.
18. Selection for resistance to carbamate and organophosphorus insecti-
cides in Anopheles albimanus, Nature (London) 232: 642-644, 1971.
19. Metabolism of 2-[methoxy(methylthio)phosphinyl]-3-ethyl-5-methyl-
1,3-oxazolidine in the cotton plant and house flies, T. R. Fukuto,
S. P. Shrivastava, and A. L. Black, Pestic. Biochem. Physiol. 2:
162-9, 1972.
20. The metabolism of ABATE in mosquito larvae and house flies, J. G.
Leesch and T. R. Fukuto, Pestic. Biochem. Physiol. 2: 162-9, 1972.
21. Insecticidal, anticholinesterase and hydrolytic properties of
^-aryl phosphoramidothioates, J. R. Sanborn and T. R. Fukuto,
J. Agr. Food Chem. 20: 926-30, 1972.
22. Thermal rearrangement of substituted acetophenone 0-(diethyl
phosphoryl)oxime and synthesis and biological studies of a series
of related phosphoramidates, E. M. Bellett and T. R. Fukuto,
J. Agr. Food Chem. 20: 931-9, 1972,
23. Reaction of trialkyl phosphites and haloamides, J. M. Desmarchelier
and T. R. Fukuto, J, Org, Chem, 37: 4218-20, 1972.
24. Oxidative rearrangement of N-(dimethoxyphosphinothioyl)carbamate
esters, M. A. H. Fahmy and T. R. Fukuto, Tetrahedron Lett. 41:
4245-8, 1972.
318
-------
25. Flight motor activity of houseflies as affected by temperature
and insecticides, T. A. Miller and J, M. Kennedy, Pestic. Biochem.
Physiol, 2: 206-22, 1972.
26. The evoluation of resistance to pesticides, G. P. Georghiou,
Ann, Rev. Ecol, Systematics 3; 133-68, 1972.
27. Evidence of cross-resistance to a juvenile hormone analogue in
some insecticide-resistant houseflies, D. C. Cerf and G. P.
Georghiou, Nature (London) 239: 401-2, 1972.
28. Morphology of the tymbal organ of the beet leafhopper, Circulifer
tenellus, J. W. Smith, Jr., and G. P, Georghiou, Ann. Entomol.
Soc. Amer. 65: 221-26, 1972,
29. Studies on resistance to carbamate and organophosphorus insecti-
cides in Anopheles albimanus, G. P. Georghiou, Amer. J. Trop. Med.
& Hyg. 3: 133-168, 1972.
30. A useful device for cleaning mosquito-rearing pans, C. S.
Apperson and G. P. Georghiou, Mosquito News 32(3): 457, 1972.
31. The activation and inhibition of adenyl cyclases from the brain of
the Madagascar cockroach (Gromphadorhina portentosa), A. S.
Rojakovick and R. B. March, Comp. Biochem. Physiol. 43B: 209-15,
1972.
32. Recent developments in insecticide resistance in houseflies in
California, G. P. Georghiou, M. K. Hawley, E. C. Loomis, and
M. F. Coombs, Calif. Agric. 26: 4-6, 1972.
33. Aryl IJ-hydroxy- and LJ-methoxy-N-methylcarbamates as potent reversi-
ble inhibitors of acetylcholinesterase, Y. C. Chiu, M. A. H. Fahmy,
and T. R. Fukuto, Pestic. Biochem. Physiol. 3: 1-6, 1973.
34. Carbaryl synergism of substituted 1,3-benzodioxoles and related
compounds and inhibition of mixed function oxidase, J. M.
Desmarchelier, R. I. Krieger, P. W. Lee, and T. R. Fukuto, J. Econ.
Entomol. 66: 631-8, 1973.
35. Structure-activity correlations in DDT analogs, M. A. H. Fahmy,
T. R. Fukuto, R. L, Metcalf, and R. L. Holmstead, J. Agr. Food
Chem. 21, 585-91, 1973,
36. The reactivation of carbamate-inhibited cholinesterase, W. D. Reed
and T. R, Fukuto, Pestic. Biochem. Physiol, 3: 120-30, 1973.
37. The metabolism of ()-(4-bromo-2,5-dichlorophenyl) 0-methyl
phenylphosphonothioate (leptophos) in the white mouse and on
cotton plants, R. L. Holmstead, T. R. Fukuto, and R. B. March,
319
-------
Arch. Environ, Contain, Toxicol, 1: 133-47, 1973'."
38. Selective toxicity of; N-sulfenylated derivatives of insecticidal
methylcarbamate esters, A. L. Black, Y. C. Chiu, M, A. H. Fahmy,
and T. R. Fukuto, J, Agr, Food Chem, 21(5): 747-51, 1973.
39, Steroselectivity in cholinesterase inhibition, toxicity, and plant
systemic activity by the optical isomers of 0-2-butyl j[-2-(ethyl-
thio)ethyl ethylphosphonothioate, D. A. Wustner and T. R. Fukuto,
J, Agr. Food Chem. 21: 756-61, 1973.
40. Metabolism of 2,2-dimethyl-2,3-dihydrobenzofuranyl-7-N-methyl-N-
(2-toluenesulfenyl)carbamate in the housefly and white mouse,
A. L. Black, Y. C. Chiu, T, R. Fukuto, and T. A. Miller, Pestic.
Biochem. Physiol. 3: 435-46, 1973,
41. In vivo measurement of house fly temperature, flight muscle
potentials, heartbeat and locomotion during insecticide
poisoning, T. A. Miller and J. M. Kennedy, Pestic. Biochem.
Physiol. 3: 370-83, 1973.
42. An examination of temporal differences in the action of carbamate
and organophosphorus insecticides on houseflies, T. A. Miller,
J. M. Kennedy, C. Collins, and T. R. Fukuto, Pestic. Biochem.
Physiol. 3: 447-55, 1973.
43. Excitatory transmission in insect neuromuscular junctions,
T. Miller and D. Rees, Am. Zool. 13: 299-313, 1973.
44. Measurement of insect heartbeat by impedance conversion. T. Miller,
Physiol. Teacher 2(1): 1-3, 1973.
45. Fine structure of the alary muscles of the American cockroach,
M. E. Adams, T. Miller, W. W. Thomson, J. Insect Physiol. 19:
2199-208, 1973.
46. Regulation of the heartbeat of the American cockroach, T. A. Miller,
In Neurobiology of Invertebrates, J, Salanki, Ed., Akademiai
Kiado, Budapest, 1973, pp. 195-212.
47. A time saving device for adult mosquito bioassays, C. S. Apperson
and G. P, Georghiou, Mosquito News 33(1); 112, 1973,
48. Seasonal escalation of organophosphorus and carbamate resistance
in Anopheles albimanus by agricultural sprays, G, P, Georghiou,
S, G. Breeland, and V, Ariaratnam, Environ, Entomol, 2(3): 369-374,
1973.
320
-------
49. Selective toxicity of N-substituted bis-carbamoyl sulfides,
M, A. H, Fahmy, Y, C, Chiu, and T, R. Fukuto, J. Agr. Food Chem.
22; 59-62, 1974.
50. Joint action of diquat and related one-electron transfer agents
with propoxur and fenthion against mosquito larvae, G. P. Georghiou,
A. L, Black, R. I. Krieger, and T, R. Fukuto, J. Econ. Entomol.
67: 184-6, 1974.
51. Toxicological effects produced by some 1,3-benzodioxoles, catechols,
and quinones in Culex mosquito larvae, J. M. Desmarchelier and
T. R. Fukuto, J. Econ. Entomol. 67: 153-8, 1974.
52, Physical and chemical basis for systemic movement of organophos-
phorus esters in the cotton plant, M. Hussain, T. R. Fukuto,
and H. T. Reynolds, J. Agr. Food Chem. 22; 225-30, 1974.
53. Electrophysiology of the insect heart, T. A. Miller, In The
Physiology of Insecta, M. Rockstein, Ed., 2nd Edition, Academic
Press, New York, 1974, pp. 169-200.
54. Cross-resistance to an inhibitor of chitin synthesis, TH 60-40,
in insecticide-resistant strains of the house fly, D. C. Cerf
and G. P. Georghiou, J. Agr. Food Chem. 22, 1145-6, 1974.
55. Affinity and phosphonylation constants for the inhibition of
cholinesterases by the optical isomers of 0-2-butyl £>-2-
(dimethylammonium)ethyl ethylphosphonothioate hydrogen oxalate,
D. A. Wustner and T. R. Fukuto, Pestic. Biochem. Physiol. 4:
365-76, 1974.
56. Absolute configuration of chiral ()-2-butyl ethylphosphonothioic
acid, G. H. Y. Lin, D. A, Wustner, T. R. Fukuto, and R. M. Wing,
J. Agr. Food Chem. 22: 1134-5, 1974.
57. Oxidative rearrangement of organophosphorus thionate esters,
D. A. Wustner, M. A. H. Fahmy, and T. R. Fukuto, Residue Revs.
53: 53-65, 1974.
58. Characteristics of cyclic 3',5'-nucleotide phosphodiesterase from
the brain of the Madagascar cockroach (Gromphadorhina portentosa),
A. S. Rojakovick and R. B, March, Comp, Biochem. Physiol, 47B:
189-99, 1974.
59. Cross resistance to juvenile hormone analogs in insecticide-
resistant strains of Musca domestica L., D, C, Cerf and G. P.
Georghiou, Pesticide Science 5: 759-67, 1974.
321
-------
60. infrastructure and electrical properties of the hyperneural muscle
of Periplaneta americana, T, Miller and M, E. Adams, J, Insect
Physiol, 20; 1925-36, 1974,
61, Seasonal and sptatial distributions of three mosquito species in
the Coachella Valley of California and their influence on exposure
to insecticidal selection, C, S, Apperson, G. P, Georghiou, and
Leonard Moore, Mosquito News 34: 91-7, 1974,
62, Time-sequence response of Culex tarsails following exposure to
insect growth regulators, G, P, Georghiou and Chi S. Lin,
Proc. & Papers, Calif. Mosq, Control, Assoc. 42: 165-166, 1974.
63. Assessment of potentiality of Culex tarsalis for development of
resistance to carbamate insecticides and insect growth regulators,
G. P. Georghiou, Chi S, Lin, and M, E, Pasternak, Proc. & Papers,
Calif. Mosq. Control Assoc,: 42; 117-18, 1974.
64. The spontaneous release of transmitter from insect nerve terminals
as predicted by the negative binomial therom, D. Rees, J. Physiol.
236: 129-42, 1974.
65. The effect of metabolic inhibitors on the cockroach nerve-muscle
synapse, D. Rees, J. Exp. Biol. 61: 331-43, 1974.
66. Anopheles albimanus; resistance to organophosphates and carbamates
based on reduced sensitivity of acetylcholinesterase, H. Ayad
and G. P. Georghiou, J. Econ. Entomol. 68: 295-297, 1975.
67. Neurosecretion and the control of visceral organs in insect, T.
Miller, Ann. Rev. Entomol, 20: 133-149, 1975.
68. Insect visceral muscle, In Insect Muscle, P. N. R. Usherwood, Ed.,
Academic Press, London, 1974, pp, 545-606.
69. Thiolysis as an activation process in N-sulfenylated derivatives of
methylcarbamate esters, Y. C. Chiu, A. L. Black, and T. R. Fukuto,
Pestic. Biochem. Physiol. 5: 359-66, 1975.
70. Organophosphorus multiresistance in Culex pipiens quinquefasciatus
in California, G, P« Georghiou, V< Ariaratnam, M. E. Pasternak, and
C, S. Lin, J, Econ. Entomol, 68(4); 461-7, 1975,
71. Studies on the neuropharmacological activity of bicuculline and
related compounds, R, W, Olsen, M, Ban, and T, Miller, Brain
Res., in press.
322
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72. Carbamate insecticides, T. R. Fukuto, Proc. Rockefeller Foundation
International Conf. on Insecticides for the Future: Needs and
Prospects, Bellagio, Italy, April 22-27, 1974. Wiley Interscience,
in press.
73. Insecticide cyclic nucleotide interactions. I. Quinoxalinedithiol
derivatives: a new group of potent phosphodiesterase inhibitors,
A. S. Rojakovick and R. B. March, Pestic. Biochem. Physiol, in
press.
75. Physicochemical aspects of insecticidal action, T. R. Fukuto,
In Insecticide Biochemistry and Physiology, C. F. Wilkinson,
Ed., Plenum Publishing Corp. In press.
76. Metabolism of 2,2-dimethyl-2,3-dihydrobenzofuranyl-7-N-
dimethoxyphosphinothioyl-N-methylcarbamate in the house fly,
rat, and mouse, R. I. Krieger, P. W, Lee, M. A. H. Fahmy, M.
Chen, and T. R. Fukuto, Pestic. Biochem. Physiol., in press.
77. Penetration and comparative metabolism of leptophos in susceptible
and resistant house flies, P. W, Lee and T. R. Fukuto, Arch.
Environ. Contam. Toxicol., in press.
78. Alteration of £,0-dimethyl j>-[a-(carboethoxy) benzyl] phosphoro-
dithioate (phenthoate) in citrus, water, and upon exposure to air
and sunlight, D. Y. Takade, M. S. Seo, and T. R. Fukuto, Arch.
Environ. Contam. Toxicol., in press.
79. Distinguishing between carbamate and organophosphate insecticide
poisoning in house flies by symptomology, T. Miller, Pestic.
Biochem. Physiol, in press.
80. Chemical instability of the GABA antagonist bicuculline under
physiological conditions, R. W. Olsen, M. Ban, T. A. Miller, and
G. A. R. Johnston, Brain Res., in press.
81. Selective toxicity of derivatized aromatic and heterocyclic
methylcarbamates, T. R. Fukuto, A. L. Black, Y. C. Chiu, and
M. A. H. Fahmy, Environ. Quality and Safety, in press.
323
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TECHNICAL REPORT DATA
it'lcuse read Instructions on the reverse before completing)
1. REPORT NO. 2.
EPA-600/1-76-004
4. TITLE AND SUBTITLE
3. RECIPIENT'S ACCESSION-NO.
5. REPORT DATE
January 1976
CHEMISTRY AND MODE OF ACTION OF INSECTICIDES 6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
T. R. Fukuto
9. PERFORMING ORGANIZATION NAMP. AND ADDRESS
Department of Entomology
University of California
Riverside, California 92502
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, N.C. 27711
8. PERFORMING ORGANIZATION REPORT NO.
10. PROGRAM ELEMENT NO.
1EA078
11. eeWT-RACT/GRANT NO.
R801837
13. TYPE OF REPORT AND PERIOD COVERED
January, 1971 thru Sept., 1975
14. SPONSORING AGENCY CODE
EPA-ORD
15. SUPPLEMENTARY NOTES
16. ABSTRACT
This report summarizes research accomplishments for the period January 1, 1971
to September 1, 1975. The study is concerned with the intoxication and detoxication
processes which take place when an animal or plant is exposed to different organic
insecticides. Progress in the following general areas is reported.
1. Insecticide selectivity.
2. Insecticide metabolism.
3. Inhibition of the cholinesterase enzymes.
4. Structure- activity relationships in insecticides.
5. Oxidative conversion of PS to PO esters.
6. Insecticide synergism and insect growth regulators.
7. Chemical reaction involving carbamate and organophosphorus esters.
8. Insecticide cyclic nucleotide interactions.
9. Insecticide penetration and its relation to resistance.
10. Gas chromatography of insecticides.
11. Neurophysiological studies on insecticide mode of action.
12. Insecticide resistance...
13. Joint action of insecticides and herbicides.
17. KEY WORDS AND DOCUMENT ANALYSIS
a. DESCRIPTORS
Insecticides
Toxicity
Detoxification
Metabolism
13. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
b. IDENTIFIERS/OPEN ENDED TERMS c. COSATI Held/Group
Synergism 06 , A
07 ,C
19. SECURITY CLASS (This Report) 21. NO. OF PAGES
UNCLASSIFIED 333
20. SECURITY CLASS (This page) 22. PRICE
UNCLASSIFIED
EPA Form 2220-1 (9-73)
325
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