FEDERAL WATER QUALITY ADMINISTRATION • 16020DHV07/70
AQUATIC PLANT CHEMISTRY.
ITS APPLICATION TO
WATER POLLUTION CONTROL
U.S. DEPARTMENT OF THE INTERIOR
WASHINGTON, D. C. 20242
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WATER QUALITY RESEARCH SERIES
The Water Quality Research Reports describe the results and
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Washington, D. C. 20242.
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AQUATIC PLANT CHEMISTRY
ITS APPLICATION TO WATER POLLUTION CONTROL
Robert T. LaLonde
Chemistry Department
State University College of Forestry at Syracuse University
Syracuse, New York 13210
For the
FEDERAL WATER QUALITY ADMINISTRATION
DEPARTMENT OF THE INTERIOR
16020DHV07/70
July, 1970
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FWPCA Review Notice
This report has been reviewed by the Federal Water Quality
Administration and approved for publication. Approval does
not signify that the contents necessarily reflect the views
and policies of the Federal Water Q iality Administration, nor
does mention of trade names or commercial products constitute
endorsement or recommendation for use.
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J Acres’ inn VUThbC, 21 Subject
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New York State University of; College of Forestry at Syracuse University
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AQUATIC PLANT CHEMISTRY. ITS APPLICATION TO WATER POLLUTION CONTROL
ioJ Ah 1th 5)
LaLonde, Robert T.
Date Pages Contract Number
July 1, 1970 921141
Project Number Note
16O2ODHVO7/7O I
22 Citation
---A
Water Resources Research Catalog Vol. 5, p. 1-484 Dec. 1969.
23 best lipt on (Starred First) -
Bacteria*, Antibiotics”, Chemistry*, Rooted Aquatic Plants”, Aquatic Bacteria,
Hydrophytes, Chemical Properties, Chemical Analysis, Ecology
25 tdcntu frees (Starred First)
Alkaloids*, Nuphar*, Nuphar Alkaloids, Chemical Ecology
27 ’ Abstract
__J
Possibly aquatic plants can play a role in aquatic ecology through the production
and release of biologically active agents. The research objective was to determine
the presence, chemical nature and biological activity of plant-produced agents.
Nuphar luteum subsp. variegatum and macrophyllum produce both C-l5 alkaloids and
C-3O, sulfur-containing alkaloids. Two of the latter type were isolated. Both
were determined as stereoisomeric biscarbinolamines incorporated into two
deoxynupharidine moieties linked together at C-7 through a thiaspirane ring. Two
new C-l5 alkaloids, 7-epideoxynupharidine and 3-epinuphamine, were discovered in
N. luteuzn subsp. variegatu n. Both C-3O, sulfur-containing alkaloids were active
against Corynetacterluin michiganense. The more abundant of the two C—30 alkaloids
was tested further against five additional phytopathogenic bacteria and was active
against four of these. The same alkaloid was inactive against three bacteria taken
from the site where the plant material was harvested. C-l5 alkaloids, nupharidine
and deoxynupharidine, were inactive against C. michiganense, the bacteria most
sensitive to the sulfur-containing alkaloids. There is now sufficient indication
that Nuphar produces agents which are active against some phytopathogenic bacteria.
The extent of the release of Nuphar-produced agents into the surrounding aquatic
environment should be studied.
This report was submitted in fulfillment of project 16O2ODHVO7/70, Contract 921141
under the sponsorship of the Federal Water Pollution Control Administration.
Ab stractor
tnsuutution
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fl 10 1 IREV OCT iSili P qy (NT OF THE INTERIOR
R SIC A5,4INeTON 0 C 10240
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CONTENTS
Section P ge
Abstract
List of Figures
List of Tables
INTRODUCTION i
Scope, Purpose and Relation to Water Quality Control 1
General Background and a General Description of
Various Phases of the Project 1
2 APPROACH AND METHODS 2
Isolation 2
Identification and Structure Determination 3
Screening for Antibacterial Properties 3
Plant ? terial 4
3 RESULTS AND DISCUSSION 4
Alkaloids isolated by the “Methylenebis(salicylic)
Acid Method 4
Alkaloids WPC 45.0 and VPM 7415 from Rhizomes of
Nuphar luteum subsp. macrophylluin 4
a. The Nature of WPC 45.1 and CFW 225.1 5
b. The Nature of WPM 74.15 11
The Structure of WPC 451 and VPM 74.15 12
Alkaloids Isolated Through pH Adjustment of a 10%
Aqueous Acetic Solution 12
7-Epideoxynupharidine 14
Deoxynupharidine. Relative and Absolute
Configuration 14
Nuphamine 15
3-Epinuphainine 15
Nupharidine. Studies of the Stereochemistry and
the Course of the Polonovsky Reaction 16
Correlation Studies 21
Conversion of Nupharidine to Nupharanilne 22
Attempted Conversion of Nupharidirie to Nuphainine 24
Starting from Diol 25
Starting from Forniamido-Ketone 26
Antibacterial Properties of WPC 45.1, VPM 74.15 and
CFW 225.1 30
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Section Page
4 CONCLUSIONS 30
5 BECOMMEND TIONS 33
6 REFERENCES 34
7 ACKNOWLEDGI€NTS 36
LIST OF PUBLICATIONS 37
9 GLOSSARY OF ABBREVIATIONS AND SYIVIBOLS 38
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FIGURES
Page
1. Possible Mass Spectral Fragmentation Routes of
WPC 45.1 13
TABLES
1. The Mass Spectrum of WPC 45.1. Principal Peaks, %
Relative Intensity and Possible Assignments 7
2. Principal Peaks, % Relative Intensity and Possible
Assignments. The Mass Spectrum of WPC 45.1, Hydride
Reduction Products and Neothiobinupbaridine (NTBN) 9a
3. C 1 and C 7 Methyl Resonance, CDC1 3 C 6 H (rel. to TMS) 18
4. Shifts of the Principal Peaks (We) .n the Mass Spectra
Ôf Deuterated Deoxynupharidine and A°-Dehydrodeox.ynuphar-
idine 20a
5. Cumulative Results of Inhibition, Bacteria Nuphar
Alkaloids 31
6. Results of Disc Plate Tests. Total Diametera f Zone
of InhihLtion in cm for the weight in g Given for
WPC 45.1, VPM 74.15 and CFW 225.1 32
7. Results of Serial Dilution—Turbidimetric Tests Given
in ttKletttt Units of Transmission. Inhibition by WPC
45.1 33
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SECTION 1
INTRODUCTION
Scope. Purpose and Relation to Water Quality Control
The several reports that some aquatic plants produce
protistostatic materials suggested an ecological relationship,
based on chemical interaction, between these aquatic plants and
other water organisms. Such an ecological relationship could effect
water quality. Thus an infestation of antibacterial-producing
plants could have an adverse effect on water quality through the
poisoning of bacteria which ordinarily consume organic material in
the water. On the other hand, such aquatic plants could be introduced
and managed in some areas to eliminate undesirable organisms which in
high populations are making water unsuitable for consumption,
recreation or support of wildlife.
While the biological action of these reported protistostatic
materials seemed secure, their chemical nature has remained obscure.
The primary objectives of the research described herein was to isolate
the new and previously described antibacterial materials in their
pure form, verify their antibacterial activity, determine their
chemical structures and determine their distribution in aquatic plants.
General Background and a General Description of Various Phases of the Project
Preparation of the roots of the aquatic species Nuphar luteuzn - the
yellow water lily - have been used in folklore medicine for the cure of
any number of ailments, among them diseases now known to be of bacterial
origin. Likely these reports served as the impetus which led a group of
Russian workers to investigate the antibiotic properties of various
species of the aquatic family Ny]nphaeaceae. A 1962 British pat nt (1),
in the name of three Russian workers, described the isolation of a
C 30 H O 5 N 2 alkaloid from Nuphar which was active against fungi and gram
positive bacteria. The reported physical properties helpful in
identifying this substance are meager and further studies dealing with
the nature of the C 30 H O 5 N 2 alkaloid have not appeared in the chemical
literature. However a member of this same group of workers subsequently
disclosed (2) the isolation and partial structure determination of a
C 3 OH OLN 2 S alkaloid from Nuphar but antibacterial properties were not
revealed. About the same time, a second group of Russians described
(3) the isolation of a crystalline substance named nupharine which was
active against twenty—three of forty-five phytopathogenic bacteria.
Corynebacteria were especially sensitive; xanthomonals were less sensitive.
Unfortunately, no physical and chemical properties of this crystalline
material were disclosed.
Thus it seemed quite clear that antibacterials were being produced
by Nuphar luteum of east European origin but the chemical nature of the
specific agent, or agents, responsible for this activity had not been
elucidated nor were the physical properties adequately described so that
the substances could be subsequently identified with certainty when
isolated from other species of Nuphar . Therefore our first concern was
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to correlate well characterized substances with specific antibacterial
activity. Involvement in this area constituted the initial phase of
research which has been conducted during the grant period January 1,
1968 - December 31, 1969.
SECTION 2
APPROACH AND METHODS
Isolation
Since various antibacterials had been isolated by the Russian
workers, the most expeditious treatment of the problem seemed to be
an attempt to repeat this work using the reported isolation procedures.
Many of these procedures were similar and we picked the one which had
been used to isolate the C 30 H O N 2 alkaloid. The procedure involved
the following steps. Dried, powdered Nuphar luteum was treated with
10% aqueous ammonia. The resulting mixture was shaken a number of
times for periods of one to two hours with dichioroethane and the
separate extracts were treated with 10% sulfuric acid. The combined
acid extracts were made alkaline with aqueous ammonia and the
resulting precipitate was dissolved in ethanol and treated with 15%
alcoholic niethylenebissalicyclic acid. The precipitated alkaloid
methylenebissalicylate was basified with aqueous ammonia and the
solution was extracted with ether. The extract was dried and
chromatographed on alumina. Repeated column chromatography, monitored
by thin layer chromatography, was used to isolate pure alkaloids.
Later when the presence of the carbinol amine functional group
in these alkaloids had been established, the methylenebissalicyclic
acid precipitation step and the use of alcohol or ether solvents was
abandoned. It seemed likely that the c rbinol amine ethyl ethers
isolated were really artifacts, being formed by facile etherification
of the carbinol amine by the ethanol used in the precipitation step.
Accordingly in later extractions using dichloroethane, the extract
was concentrated and treated with 5% aqueous sulfuric acid. The
aqueous solution was basified with aqueous ammonia (pH 9) and
extracted with chloroform. The chloroform extract yielded a mixture
of the free carbinol amine alkaloids which were separated by column
chromatography. Methylene chloride could be used as the extracting
solvent, in place of dichloroethane with no apparent modification in
the array of alkaloids. The dichloroethane and methylene chloride
extraction procedures were used for extracting solvents for the
greens of Nuphar luteurri subsp. variegatum and rhizomes of N. luteum
subsp. macrophyllum .
In addition to using the rather specific extraction procedures
described above, a more general isolation procedure was employed.
As is revealed in a later section of this report, the two different
isolation procedures furnished different alkaloids. Extraction of
plant material with methylene chloride or dichloroethane afforded
the C 0 , sulfur-containing alkaloids, deoxynupharidine and
7-epiaeoxynupharidine. The last two alkaloids belong to the C 15
class. The second isolation procedure, described immediately below,
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produced the C 15 quinolizidine and piperidine type alkaloids only.
The second procedure involved the following steps. Dried powdered
Nuphar luteum was extracted several times with methanol. The combined
methanol extracts were concentrated at reduced pressure at 350• The
concentrate was treated with 10% aqueous acetic acid solution and
suspended solids were removed by filtration. The clear acetic acid
solution was extracted with hexane, benzene and rnethylene chloride.
Each of these extracts was dried, concentrated and set aside for
investigation. The acetic acid solution was brought to pH 7,
extracted with chloroform, then brought to pH 12 and again extracted
with chloroform. pH 7 and pH 12 chloroform extracts were set aside
for purification. Isolation of pure alkaloids was achieved through
column chromatography on alumina. Chroinatographic fractions were
monitored by thin layer chromatography (tic).
Identification and Structure Determination
Known pure alkaloids were identified by tic Hf values, optical
rotation, the melting points and optical rotations of solid
derivatives, comparison spectra and chemical correlation with other
known alkaloids.
The structures of new alkaloids were elucidated largely through
spectral evidence and chemical correlation. The specific methods
employed, information obtained and conclusions are covered in Section
3 (Results and Discussion) and Section 4 (Conclusions).
Mass spectra were obtained on a RMtJ6-Perkin-Elmer-Hitachj mass
spectrometer with an all glass heated inlet, and determined at 70eV
and a chamber temperature 160-165°. Nuclear magnetic resonance
spectra were obtained on Varian A-60 and HR 100 spectrometers and
a Joelco HNM—4H 100 spectrometer. Spectra were determined in
chloroform and carbon tetrachloride solution, 2% TNS (lOT).
Infrared spectra were obtained on a Perkin—Elmer 137. Melting
points reported were determined on a K Sf1er micro-hot stage and
are uncorrected. Optical rotations were obtained from a Perkin-
Elmer 141 polariineter. The elemental analyses were performed by
Galbraith Laboratories. Ultraviolet spectra were obtained on a
Cary 14.
Screening for Antibacterial Properties
Two routine methods for determining antibacterial susceptibility
were used. The disc plate method was employed for semi-quantitative
determinations and preliminary tests, while the serial dilution—
turbidimetric method was used for more quantitative studies.
The phytopathogens chosen for study were Corynebacterhun
inichiganense, C. flaccumfaciens, Xanthomonas phaseoli, X. vesicatoria ,
and Erwinia caratovora and were obtained from Dr. Robert Dickey,
Department of Plant Pathology, Cornell University. Arthobacter ( TH—1 )
was also tested and was obtained from Dr. Robert Bauni, Chemistry
Department, SUN! College of Forestry. A number of bacteria were
isolated from the water and mud of the southeastern corner of Green
Lake, Tully, New York, the same area from which Nuphar luteum subsp.
variegatuin was collected. Three of these bacteria were tested for
sensitivity to WPC 45.1.
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All bacteria were grown and maintained at 3000 in shake
flasks and cultures were transferred directly from these. Disc
plate cultures were grown for 48—72 hours and the serial dilution-
turbidimetric cultures were grown for 24 hours. All organisms
were tested first by the disc plate method. In the case of .
inichiganense and Arthrobacter (TH-l) further studies were
carried out by the serial dilution—turbidimetric method. The
number of organisms tested by the second method was limited
primarily by the amount of alkaloid available.
Nupharidine and dexoynupharidine were dissolved in distilled
water and WPC 45.1, VPN 74.15 and CFW 225.1 in 0.O1M acetic acid.
Plant Material
Nuphar luteum is widely distributed throughout the northern
hemisphere. Subspecies obtained for study and their origin are
as follows:
1. Nuphar luteuni . subsp. variegatuni (Beal)
Green Lake, Tully, Onondaga County, New York
2. N. luteum subsp. macrophyllum (Beal)
Tidal flats along the lower Hudson River near Columbianville,
New York
3. ! . luteum subsp. ozarkanum (Beal)
South central Missouri
4. N. luteum
The Netherlands
Greens and rhizomes of the first listed subspecies and rhizomes
of the second listed subspecies have been examined to date. Plant
materials were air dried then powdered prior to extraction.
SECTION 3
RESULTS AND DISCUSSION
Research in aquatic plant chemistry carried out at the
College of Forestry have been supported by FWPCA (#16O2ODHV) and
the Nclntire-Stennis Cooperative Forestry Research Program of
the USDA. Research in the different programs occasionally was
closely related, particularily in the case of the C 15 Nuphar
alkaloids. Results reported herein are largely those obtained
under the FWPCA program but also include results from the USDA
program whic1 are closely related and assist in the development
of a meaningful discussion.
Alkaloids Isolated by the “Methylenebis(saljcyeljc) Acid Method ”
Alkaloids WPC 45.0 and VPM 74.15 from Rhizomnes of Nuphar
luteuin subsp. macrophylluni .
The dried powdered rhizomes of the sub-title species were
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treated by the “inethylenebis(salicyiic) acid procedure” in the
usual manner. Thereby, an ether extract was obtained which when
concentrated under vacuum gave a thick paste representing 2.5—5%
of the weight of the dry plant material. A preliminary survey
of alkaloid content by tic (thin layer chromatography), using
several solvent systems, indicated that the crude mixture contained
deoxynupharidifle and 7-epideoxynupharidine. The identification
is based on the coincidence of Rf values with Rf values of
authentic samples. In addition, these preliminary tic studies
showed the presence of no less than three additional alkaloids
whose Rf values were always lower than those of deoxyrrnpharidine
and 7 -epideoxynupharidine.
Cc (column chromatography) on alumina, using ether-hexane,
was carried out to first remove deoxynupharidine and 7-epideoxy-
nupharidine. The mixture remaining then was chromatographed
repeatedly; the more mobile fractions were recombined after
each chromatogram and from them was isolated alkaloid VPM 74.15
giving a single spot on tic. In the same manner the less mobile
fractions were recombined and from them was isolated lkaloid
WPC 45.1, a liquid giving a single spot on tlc: + 86.9
(c 19mg/cc, NeOH). No attempt has been made to isolate the
alkaloids of intermediate mobility.
The Nature of WPC 45.1 and CFW 225.1
Analysis for nitrogen gave values of 4.97 and 4.73%
(N calc’d for C 3 LHSON 2 O 4 S: 4.80). Analysis for sulfur gave
a value of 7.27 (% S calc ’d for C 4 H 50 N 2 0 4 S: 5.50) which was
unsatisfactory for the molecular ISormula consistent with the
nitrogen analysis and the ins (mass spectrum) but nevertheless
established the presence of sulfur. The ir (infrared spectrum)
showed bands at 6.67 and ll.4 8 p. thus indicating the presence
of a furan ring. Bohlmann bands, and bands characteristic of
carbonyl and hydroxyl groups were absent. WPC 45.1 was
recovered unchanged after shaking with palladium on charcoal under
an atmosphere of hydrogen. The uv (ultraviolet spectrum)
showed only end absorption beyond 215 iun. The 100 MHz rimr
(nuclear magnetic resonance spectrum) confirmed the presence
of a furan ring (or rings) in showing a doublet at 7.27 and
a singlet at 6.35s whose integrated intensities were in
the ratio of 2:1. In addition the rnnr showed a) a band
of overlapping multiplets in the region of 3.36-3.95 which
indicated protons alpha to nitrogen or oxiygen, b) a band of
overlapping multipiets in the 2.0—2.6s region which suggested
protons alpha to nitrogen or sulfur and c) a band between
0.9 and 2.05 corresponding to methyl, methylene and methylnyl
protons. The integrated intensities of peaks in the 7.27—
6.485, 3.36—2.05, and 2.0-0.95 regions was 6:8:36 assuming
the presence of two furan rings. Of special interest was
the appearance of a methyl doublet at 0.95 (J=5.5Hz) and a
methyl triplet at 1.125 (J=7Hz) both of which were imposed
on the envelope of other protons. The 1.125 methyl triplet,
in conjuction with a methylene quartet at 3.355 (J7Hz),
indicated the presence of one or more ethoxy groups.
The principal peaks of the mass spectrum and their
possible assignment are given in Table 1. Possible modes of
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fragmentation follow from a gross structure, , assigned on the basis
of studies on the biscarbinolaimine diethyl ether and the parent
biscarbinolanilne described below. Plausible fragmentation routes to
ions a-i are given in Figure 1.
The mass spectrum of WPC 45.1 was of interest in the following
respects. The parent ion at 582 indicated a molecular forna ila
consistent with the number of hydrogens suggested by the runr studies
and the percentage nitrogen. The presence of ethoxy groups was
indicated by in/e 554, 538, 510 and 509. The loss of ethylene from
ethyl ethers is a well known mode of fragmentation (4). The
generation of nile 538 ion through the loss of C 2 H 4 0 was established
by the high resolution ins study of this ion: ni/e 538 calculated
for C 32 H 4 60 3 SN 2 ; 538.3229; ni/e 538 observed, 538.3144 ( 0.0085).
The presence of nile 230 strongly suggested that WPC 45.1 was
composed of two deoxynupharidine moities. The presence of peaks
corresponding to ions strengthened this suggestion. Further-
more, the appearance of this latter group of peaks suggested a
diineric deoxynupharidine connected through ring B, a structural
feature which was reinforced by the absence of a peak at ni/e 98.
ss spectral studies of deoxynupharidine—6,7-d 2 carried out in
cur laboratory have shown that nile 98 originates from ring B.
Therefore, if WPC 45.2 contained two deoxynupharidine moities
linked through ring A, the presence of a peak at ni/e 98 might be
expected. Thus the appearance of the group of peaks and the
absence of ni/e 98 suggested that the gross structural framework
of rings A and A’ of WPC 45.1 was the same as in deoxyiiupharidine.
This meant the ethoxy substituents must be in ring B and B’ and
considering the likely identity of m/e 222 with ion , the ethoxy
groups could be located specifically at carbons 6 and 6’.
The only remaining structural feature not yet accounted for
was the place of sulfur. This was incorporated into the
structure as a thiaspirane involving two niethylenes (carbons 17
and 18) and linking the two quinolizidine ring systems through
carbons 7 and 7’. At this point in the structure determination,
the evidence for the thiaspirane linkage was by no means
compelling. However, the appearance of We 230 was consistent as
was the lack of any singlet methyl groups in the nmr. Since the
mnr revealed only split methyl resonance, the C 7 methyls of both
deoxynupharidine components must be incorporatea into the structure
in a higher oxidation state. The involvement of the thiaspirane
linkage was substantiated in the work which followed.
Treatment of WPC 45.1 in anhydrous ether with a 50:50
solution of 70% perchioric acid in ethanol gave a diperchiorate
salt, mp (melting point) 233-234°, which analyzed for C 0 H 38 N 2
0 2 S 2 HC1O 4 . The ins, which did not show a parent pea1 , was
very much the same as the ins of °-dehydrodeoxynupharidine, thus
again suggesting the symmetrical juxtaposition of two deoxy—
nupharidine components possessing unrearranged carbon skeltons.
The ir showed a strong band at 6.0 .i, due to the presence of
> C = , and furan bands at 6.65 and ll.43 . Strong
broad adsorption in the 8 .5-9.5i region was attributed to the
perchlorate ion. The uv showed Xmax 208 ( € 12,000) and
291 nm (€ 2,300).
The conversion of WPC 45.1 to the diperchiorate salt with
concomitant loss of two ethoxy groups confirmed the presence of
the biscarbjnolamjne ether function. Still further confirmation
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Table 1
The Mass Spectrum of WPC 45.1
Principal Peaks, Relative Intensity and Possible Assignment
% Rel.
mt. M±rn/e Possible Assignnlenta
582 5.5 0 M , C 34 H 50 0 4 N 2 S
554 12.5 28 N -C 2 H 4 , Ion a
538 100 44 Mt (C 2 H 4 ÷0), Ion b
510 50 72 Mt(C 2 H 4 x2±O)
509 17 73 M -(C 2 H 5 ÷C 2 H 4 +0)
491 28 91
490 17 92
462 7 120 M -(C 2 H 5 Ox2+CH 3 x2)
230 50 Ion c
222 27 Ion d
178 7 lone
176 11 Ion f
164 12 Ion g
107 8 Ionh
94 4 loni
a
Structures of possible ion fragments ( -.i) corresponding to irVe are
given in Figure 1.
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7 ’-
3F =
, m/e 16li c, m/e 230
S - (
3f OC 2 A(
f, rn/e 176 d, m/e 222
Figure 1. Possible Mass Spectral Fraginentabion Routes of WPC 145.1.
e m/e 178
• h, m/e 107 1, nile 914
b, m/e 538
a, nile 5514
‘I ,
-A
\
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was furnished by the conversion of WPC 45.1, with sodium borohydride
in methanol, to an amorphous solid whose ms showed at n /e 494.
Reduction with sodium borodeuteride gave a product having M 496.
Principal peaks in the ms are correlated, along with the reported ins
of neothiobinupharidine, in Table 2. Interestingly, the ins of the
product of hydride reduction was virtually identical with the
published (5) ins of neothiobinupharidine, an alkaloid having the
structure shown in a and whose gross structure differs from 1
through the lack of ethoxy groups at 6 and 6’. The structure a was
determined by x-ray analysis (6) and is to date the only structure
of the thiobinupharidine class of alkaloids which is secure. Although
the ins of the two materials were the same, other physical properties,
notably the runr, were not. Therefore, the reduction product of WPC
45.1 and neothiobinupharidine possessed the same skeletal framework
but were stereoisomers. Noteworthy in connection with the mention
of the ninr of the reduction product, is the two proton, AB pattern
at 2.32 . Its presence substantiated the presence of the CH 2 —S group
proposed as part of the thiaspirane linkage.
At this point in the investigation, it seemed likely that the
true natural product was really the parent biscarbaniolainine. The
biscarbinolaaine had undergone ether formation with the ethanol
employed in the precipitation step. Therefore, the extraction was
repeated but the methylenebissalicylic acid step was omitted as was
the use of ethanol as a solvent. Under these conditions was
isolated liquid CFt4 225,1 ( [ a] S - - 44.5, c 1.2, CH 2 C1 2 ), the free
biscarbinolamine corresponding to the ethyl ether form - WPC 45.1, 1.
The ir of CFW 225.1 showed an OH band at 2.9i.i., furan bands at
6.64 and ll.43 i. No BohJ .mann bands were observed. The ms displayed
a parent peak at n /e 526 and peaks at 509, 508, 492, 491 and 490 which
corresponded to the loss of OH, H 2 0 and the combination of any two of
these fragments. The uv showed X 1 208 nm (12,200) when netural but
an additional Xmax at 291 (1850) when acidified; this new band disappeared
when the solution was made basic. The rmir (60 and lOO MHz) displayed
resonance bands as follows: O.92 (d, 5.5Hz, 6H, CH 3 CH), 4.33 (d,
5Hz, 1H, HC 6 OH), 4.1 br in, 1H, C 6 ’ OH), 3.74 (q, 1H, HC 4 ), 3.61 (q,
1H, iiC 1 ’), 2.49 (ABq, 2H, CH 2 S), 6.50 (in, 2H, furan 7.48 (m, hE,
furan H). The presence of a single methyl resonance indicates both
methyl groups are to be found in the same type of environment. Judging
from the chemical shift and the magnitude of the coupling constant,
both methyl groups are equatorial. Based on earlier studies in the
deoxynupharidine series (7) the presence of an axial methyl group
would have given a signal at a somewhat lower field (— l.lô) with a
larger coupling constant ( ) 5.5Hz).
2
9
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Table 2
Principal Peaks, % Relative Intensity and Possible Assignments.
The MS of WPC 45.1, Hydride Reduction Products and Neothiobi—
nupharidine (NTBN).
% Relative Intensity
NaBH 4 BD 4
Redn. NTBN Redn. Assignment
496 35 M +d 2
494 19.5 39.2
463 2.4 (M ÷ d 2 )-SH
461 1.6 2.9 M - SR
449 2.4 (M + d 2 )-SCH 3
447 1.6 2.6 - SCH
361 12.6
359 5.9 10.2
248 2.1 (M + d 2 )/2
247 1.2 1.6 M /2
231 34.5
230 46.6 40.6
3f 1 ’W’O)
179 100
‘4-
178 100 100
136 4.6 7.2 7.8
107 11.1 7.3 21
94 10 24.6 19.2
9a
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The appearance of the 2.49o LB quartet clearly revealed the
involvement of sulfur with one of two methylenes. Since the
quartet was not split further nor were there any more than two
hydrogens a to sulfur, the involvement of sulfur in a five-membered
thiaspirane which linked carbons 7 with 7’ was beyond doubt.
That this thiaspirane linked the two quinolizidine systems in a
symmetrical fashion was indicated by the following nmr features.
1) Only single kinds of a- and -furan protons were observed.
2) The chemical shifts of protons substituted on carbons 4 and 4t
were very nearly the same. 3) A single kind of split methyl
resonance was observed. That the resonande of protons at carbons
6 and 6’ are so dissimilar is due very likely to one proton being
axial and the other being equatorial. Thus the overall shape of
the structure was envisioned as possessing a pseudo element of
syizunetry-i.e., pseudo C 2 , pseudo c i, etc.— but lacking local
symmetry in the region of the 0H 2 —S group and, therefore, lacking
true symmetry throughout.
Biscarbinolamine CFW 225.1 was converted to a diperchlorate
salt identical in all respects with the diperchlorate obtained
from the biscarbinolamine ethyl ether, WPC 45.1.
As expected, reduction of CFW 225.1, with sodium bor ydride
in methanol, gave the dehydroxy counterpart, a liquid [ a] + 7 8
(c 18 mg/cc, MeOH). The uv (X x 215 nm, 13,000) remained unchanged
on addition of acid or base.
In addition to single kinds of a and furan protons, the most
significant features of the mnr were a single methyl resonance at
0.93 (br d, 6H, Cij CH), the -01125— LB quartet at 2.32 and a 2.7—3.l
four proton multiplet. The last named signal was assigned to the two
protons at carbons 4 and 4’ plus two equatorial protons at carbons 6
and 6’. Other work (7) has shown that the 06 equatorial proton of
deoxynupharidine is at 2.7b while the 06 axial proton is located
considerably upfield, near l.8o. The nxnr of the 06 protons is of
considerable importance as it relates to the nmr of the product
obtained from sodium borodeuteride reduction of OEM 225.1. The ninr
of the latter product showed only three protons in the 2.7-3.lb
region and diminished intensity in the l.75b region. This result
could only mean that deuterium entered equatorially at one center
but axially at the other. We have interpreted this result in terms
of sulfur participation in reduction and it is depicted in the
reaction equation below.
%% %fl
I I __ 17 ’ 7L
0 / 1
10
-------
Considering the various shapes of all the possible stereoisomers
of , there is no reason why the reducing agent should approach C6
and CE’ from opposite directions unless some agent shields the axial
approach at C 6 but not C 6 ’. The proximity of the sulfur atom to C 6
and the energetic and entropy factors more favorable to three-membered
ring participation than four-membered ring participation ( ) leads us
to postulate an episulfonium ion intermediate which acts as the
shielding agent and prevents C 6 axial attack. As shown in the
reaction equations above, the episulfoniuni ion possibly may be formed
subsequent to the formation of an immonium ion. Deuteride displacement
proceeding with inversion leads to C 6 equatorial substitution. In
terms of the episulfonium ion intermediate, it follows that the C 7
sulfur atom must be equatorial to account for equatorial deterium
substitution.
The Nature of VPM 74.15
The ir spectrum of VPM 74.15 was similar to that of WPC 45.1,
there being no significant difference in the position of bands. The
uv in neutral solution showed only end absorption beyond 210 ma but
in acid a new band appeared at 295 nm C €. 1,000). The Ins was
similar to that of WPC 45.1. Although there were no peaks at m/e
554 and 526, corresponding to partially ethylated biscarbinolamine,
the peak at 564 (5 2-H 2 0) suggested that perhaps this material was not
completely ethylated, a suggestion which was reinforced by the nxnr
spectrum which revealed 5 protons in the region 3.22-4.25o. Judging
from the mar of the biscarbinolamine, which displayed three protons
in the same region, a fully ethylated biscarbinolanline should have
contained 7 protons in the 3.22—4.25 region.
Thus the isolation of the partially ethylated biscarbinolamine
was ample demonstration of the disadvantages of using the Russian
isolation procedure. Treatment of WPM 74.15 with dilute aqueous
hydrochloric acid then 2 aqueou$ base produced the free liquid,
biscarbinolainine: [ a]D 5 — 69° (c 10 jag/cc, CH 2 C1 2 ). Its nxnr was
significantly different from that of the free biscarbinolainine
(CFtJ 225.1) corresponding to WPC 45.1, in a number of respects.
Firstly the former showed two kinds of furan rings as evidenced
by a(I 4 B ) signals at 7.37 and 7.27b and and (2H) signals at 6.47
and 6.27b. Secondly, the chemical shifts of protons attached to
C 1 and C 4 t were widely separated -‘ = 0.7 ppm) whereas the same
protons of the free biscarbinolainine CF1 1 225.1 were found at nearly
the same field strength (A2f= 0.13). Thirdly, the chemical shifts
of protons attached to C 6 and C ’ were found at 4,10 Cd, 5Hz) and 3.92b
(d of m, 10Hz) whereas the counEerparts in biscarbinolandne CFW 225.1
were observed at 4.10 (xn,Wl/2h 3Hz) and 4.33 (d 5Hz). The first
two of these features showed that the furan rings, and the carbon
atoms to which they are attached, C 1 and C 4 1 , must be in different
environments in biscarbinolamine VPM 74.15 but in nearly the same
environment in biscarbinolainine CFTA 225.1. The third significant
difference in rnnr properties strongly suggested that the stereo-
chemistry at C6 and C 6 ’ of biscarbinolaimtne VPM 74.15 was the same
but the stereochemistry at these two centers in CFW 225.1 was
different.
VPN 74.15 was treated with sodium borohydride in methanol to
obtain the C 4 , C 4 ’ dehydroxyamine. The ms, ir and uv were
consistent with the same gross structure possessed by neothiobinuphar-
idine, a. The ninr showed: 1) two kinds of furan signals for a protons
(2H, 7.31 and 7.22b). 2) a total of four protons in the 2.42-3.15
region corresponding to two protons at C 4 and C 4 ’ and two equatorial
protons at C 6 and C 6 ’ and 3) an AB quartet at 2.33b corresponding
11
-------
to—CH -S. The latter signal and the corresponding signal in the mnr
of de?iydroxylated CFW 225.1 are found at the same field strength
which is higher than that (2.69b) observed for the CH 9 S group of
neothiobinupharidine, a. Applying the same rule for aistinguishing
axial from equatorial quinolizidine methyls to CH 2 S groups, the
2.69o signal is down field and would correspond to axial substitution
of the niethylene at C 7 ’ in neothiobinupharidine. The 2.33 signal of
dehydroxy CFW 225.1 and VPM 74.15 are upfield and correspondingly
would mean equatorial substitution of the methylene at C 7 ’.
Soditun borodeuteride reduction of VPM 74.15 produced the
deuterated analog of the dehydeoxy VPM 74.15. Interestingly the
deuterated analog showed simplified but undiminished intensity of
the signals in the equatorial C 6 and C 6 ’ region. However, the signals
in the axial C 6 and C 6 ’ region were of diminish intensity. This
result must mean that deuterium atoms at both C 6 and C 6 ’ are axial.
As described earlier biscarbinolamine CFW 225.1 gave equatorial
and axial deuterium at C 6 and C 6 ’ respectively. This was ascribed
to participation in reduction by equatorially substituted sulfur
at C 7 . Using the same rationale, the observation of only axial
deuterium at C 6 and C 6 ’ in the product from biscarbinolainine VPM
74.15 must mean the sulfur at C, 7 is axially substituted. It should
be noted that both VPM 74.15 and WPC 45.1 give at least one axial
deuteriuni. This finding means that without sulfur participation
axial attack is the normal mode.
The Structure of WPC 45.1 and VPM 74.15
Results disclosed in previous sub-sections establish that WPC
45.1 and VPM 74.15 are 6,6’ biscarbinolaniine ethyl ethers. These
have been converted by hydrolysis to the free 6,6t_biscarbinolainines.
The nmr studies clearly showed that the biscarbinolanilnes contain
secondary hydroxyl groups adjacent to fully substituted carbon.
The biscarbinolanilnes and/or ethyl ethers are reduced by sodium
borohydride to two different bisdeoxynupharidine thiaspiranes
neither of which is identical to neothiobinupharidine. Sodium
borodeuteride reduction of WPC 45.1 produced one equatorial and
one axial deuterium while the same treatment of VPM 74.15 produced
two axial deuteriunis. This result has been interpreted in terms
of sulfur atom participation in the course of reduction. Thus the
deuteride attacks at C 6 from the side opposite the sulfur atom.
This must mean that the C 7 sulfur is axial and equatorial in
reduction products of WPC 45.1 and VPM 74.15 respectively.
The ninr of the two reduced products show the CH 2 -S-signal at
higher field than the signal for the same group in neothiobinupharidine,
2. Therefore, the CH 2 -S-group is attached equatorially to the second
quinolizidine ring system. Thus on the basis of the evidence obtained
to date, the reduction product of WPC 45.1 (CFW 225.1) is assigned
structure while the reduction product of VPM 74.15 is assigned
structure .. These are tentative assignments of stereostructure.
The gross structural type can be assigned with confidence. Also
included for comparison with structure a, and • is the fourth
possible stereostructure . The structure of WPC 45.1 (CFW 225.1)
is related to its reduction product and the structure of VPM
74.15 is related to its reduction product .
Alkaloids Isolated Through pH Adjustment of a 10% Aqueous Acetic
Acid Solution .
Most of the work which would be reported under this sub—heading
12
-------
2
•R4
R 4
3
4
S
R 4
5
13
-------
already has been described in publications which have recently appeared
or are now in press and will appear shortly. Since these publications
treat the work in detail, only a brief description will be given here.
The unpublished portions are treated in more detail and objectives and
results are included.
7 -Epide oxynupharidine
7-Epideoxynupharidine, , was isolated for the first time from
natural sources (9). This alkaloid was obtained from a methylene
/1
chloride extract of a 10% aqueous acetic acid solution containing
the crude mixture from N. luteuxn subsp. variegatum (rhizoines).
Shortly before its isolation from natural sources, the alkaloid had
been obtained in our laboratory by synthesis. Thus treatinent 6 of
nupharidine 2 under Polonovsky reaction conditions produced -dehydro-
deoxynupharidine, , which on catalytic hydrogenation gave a 7:1
mixture of deoxynupharidine, 9, and 7-epideoxynupharidine, 6. This
two step reaction sequence is shown in the reaction equation below.
The Polonovsky step involved treating 2. in chloroform solution with
ctL
acetic anhydride and fused potassium acetate at room temperature
(11).
Deoxynupharidine. Relative and Absolute Configuration.
Deoxynupharidine is ubiquitous in Nuphar and its presence in
the same methylene chloride extract along with 7—epideoxynupharidine
was not unexpected. Although the gross structure and relative
configuration of deoxynupharidine had been convincingly established
a number of years ago, we wished to obtain corroborative evidence of
7
14
-------
the relative stereochemistry of the two methyl groups based on 100 MHz
ninr studies. These studies focused on the C 6 -methylene group and
thereby the axial disposition of the C 7 methyl was confirmed (7).
Results from these studies have been helpful in the interpretation of
the more complex mnr of the sulfur-bearing C 30 alkaloids.
In recent times, the previously assigned (ii) absolute configuration
of deoxynupharidine has been questioned (12). Since the absolute
configuration of several other Nuphar alkaloids of both the
quinolizidine and piperidine type had been related to deoxynupharidine,
the absolute configuration was an important point to establish. The
correlation between (-)-deoxynupharidine and (—)-(R)-cL-methyladipic
acid, carried out in our laboratory (7) revee.led that (—)-deoxynupharidine
belonged to the enantiomeric series. The correct absolute configuration
of deoxynupharidine and correlated Nuphar alkaloids is given in the
structures appearing in this report.
Nuphandne
Nuphamine, Q, had been isolated earlier by the Japanese workers
from II. iaponicum D. C. (13).
. luteum subep. variegatum also produces this alkaloid. In our
laboratory it was isolated from the pH 12-chloroform extract after
elution chromatography on alumina. Fractions removed with ether—
hexane 5:95 and benzene contained deoxynupharidine, 7-epideoxy—
nupharidine and other unknown components. Ether elution gays
nuphainine: {a] 5 —55.4°(c, 1.22, CHC1 . ), reported (13) [ a]? —60.4.°
The ir, rmir and ins were consistent with the structure. Treatment
of a sample of nuphamine with excess methyl iodide and sodium
bicarbonate in methanol afforded the N-methyl methiodide of nupheanine:
mp 164-164.5, reported (13) 164°.
3-Epinuphan ilne
This new alkaloid was isolated from the pH 7—chloroform extract
after repeated elution chromatography with cyclohexane-methylene
chloride-methanol. The structure of 3—epinuphamine, U, was assigned
on the basis of nmr, ir and mass spectra and correlation with nuphenine,
(14), as shown in the reaction equation below. The trans stereochemistry
IN I , , ‘
OH
15
-------
of the allylic side chain was established by ninr studies of the
allylic alcohol and uv studies of the corresponding aldehyde
obtained on manganese dioxide oxidation of the allylic alcohol,
U.
Nupharidine. Studies of Stereochemistry and the Course of the
Polonovsky Reaction.
Nupharidine, , is a well known, naturally occurring, N-oxide
related to deoxynupharidine. Possibly this alkaloid is the biogenetic
link between quinolizidine, piperidine and the sulfur-bearing C. 0
alkaloids. This proposal, though highly speculative at this po1 nt,
nevertheless has some basis in 6 the clean and facile Polonovsky type
conversion of nupharidine to -dehydrodeoxynu haridine, (see Section
3). Moreover with the solitary exception of &-dehydrodeoxynupharidine
(15) all other Nuphar alkaloids, including the C 0 —sulfur—bearing
alkaloids possess a ring B elaborated quinolizidine or a ring B-cleaved
quinolizidine, i.e., a piperidine Nuphar alkaloid. Therefore the
Po1o aovsky type transforinatj.on may be analogous to what is happening
in nature. The resulting °-dehydrodeoxynupharidine, a ring B enamine,
would be susceptible to enamine oxidation or substitution and as such
would be a key intermediate in the transformation of quinolizidine
to piperidines and the C 30 -sulfur alkaloids.
Because of the speculated importance of nupharidine in biogenesis
and also because of the realized synthetic entry to the less abundant
piperidine alkaloids through nupharidine, a study of the course of
the Polonovsky reaction was initiated.
The original rationale for employing nupharidine to obtain a
functionalized ring—B quinolizidine was based chiefly on the postulate
of Wenkert (16) which treated the Polonovsky reaction as if it were
cis-eliinination. Thus if nupharidine were to have a trans-quinolizidine
N—oxide ring system as is shown in structure , then the only
proton cis to the N—oxide is the CAd proton and Polonovsky elimination
would be forced into ring B througI the immoniuni form, , of the
enamine, .
‘1,
76
16
-------
On the other hand, if nupharidine possessed a cis-fused quinolizidine
N-oxide system, cis elimination could possibly remove hydrogens at
C4 C 6 and C 10 and thereby produce a 3 inixture of three enamines.
However, of the three possible, the A —enainine seemed unlikely
since models showed its formation would involve introduction of an
extremely strained double bond between nitrogen and C 4 . In any case,
a cis Polonovsky elimination from a fused quinolizidine N-oxide
was not envisioned as giving a single product. However, rans
elimination from a cis-fused system could give only the A enamine
as the C 6 a hydrogen now is the only hydrogen trans to the N-oxide.
6
As results substantiated, nupharidine was converted to A —dehydro-
deoxynupharidine in 84% yield under Polonovsky conditions. Nupharidine
used in these and other studies was isolated conveniently as its
crystalline acetate salt by concentration of the original methylene
chloride extract previously described in the isolation section. The
A 6 -enamine next was converted by catalytic addition of deuterium to
deoxynupharidine-6 , 7 —d 2 which in its turn was oxidized with
hydrogen peroxide in acetone to the deuterium labelled nupharidine,
. The stereochemistry of the deuteriuin atom was assigned 6 on the
basis of preferred cis hydrogenation of the face of the A —enamine
8 as evidenced by the formation of deoxynupharidine 7-epideoxynupharidine
in a ratio of 7 to 1. Repeating the Polonovsky reaction on the
deuterium labelled nupharidine gave a A 6 -dehydrodeoxynupharidine whose
nip’ showed no vinyl hydrogen but whose ir and ms were consistent with
A°-dehydrodeoxynupharidine-6-d 1 (10). Thus it was clear that the
Polonovsky elimination was stereospecific, being either a cis
elimination from a trans quinolizidine N—oxide or a trans elimination
from a cis quinolizidine N—oxide. Since the stereochemistry of the
N-oxide had never been determined it was not possible at this point
to make a conclusive distinction between the two stereocheinical modes
of elimination. However, there was some reason to believe that
N—oxidation of deoxynupharidine was equatorial, resulting in the cis
fused quinolizidine N—oxide. Thus Fodor has demonstrated the
equatorial quarternization of some tropanes (17) and studies by
M hrle (18) and Katritzky (19) have shown that the substituted
quinolizidines, such as , undergo predominantly equatorial
quaternization. Considering an N-oxide to have at least the steric
/5 -
requirements of N-alkyl, it would seem reasonable that nupharidine is
a cis quinolizidine N-oxide. It an attempt to gain some evidence on
this point, the ninr solvent-induced-shifts of equatorial and axial
methyls were compared in deoxynupharidine and 7-epideoxynupharidine
with shifts in nupharidine and 7—epinupharidine. As the data in
Table 3 illustrate, equatorial methyl groups undergo an upheld
shift of 3-5 Hz on changing the solvent from chloroform to benzene.
The single axial methyl (C 7 -CH. , deoxynupharidine) undergoes a
downfield shift of 4.2 Hz. Both methyls of 7-epinupharidine and
nupharidine undergo upfield shifts and this result would indicate
that these methyl groups are equatorial. To accommodate two
equatorial methyl groups in nupharidine, rings A and B must be
0, ,
C/ (a I 1)
7?
cb (eqi atcrial)
17
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Table 3
C 1 -C 7 Methyl Resonance, CIJC1 3 C 6 H 6 (Rel To TMS)
C 1 —CH 3 C 7 -CH 3
Cpd. Solvent Conf . Hz A Conf . Hz A
Deoxy— CDC1 3 54.0 59.8
C 6 H 6 (e) 49.0 +5.0 a(a) 64.0 —4.2
7-Epi- CDC1 3 55.0 44.0
C 6 H 6 (e) 52.0 +3.0 (e) 41.0 +3.0
7-Epinuph. CDC 1 3 55.5 47.8
C 6 H 6 (e) 43.5 +12.0 3(e) 37.0 +10.8
Nuph. CDC1 3 59.6 50.6
C 6 H 6 (e) 44.0 +15.6 44.0 +6.6
18
-------
cis—fused. This being the case, the Polonovsky elimination is
occurring stereospecifically trans.
The weak point in the above described nmr based studies of
nupharidine stereochemistry is the assumption that the magnetic
influence of N-oxide oxygen and the nitrogen non—bonding electron
pair is the same. This assumption might not be warranted.
Therefore, an x-ray analysis of nupharidine hydrobromide has been
initiated in order to put the stereochemistry on a firm basis and
settle the question of the stereochemical course of the Polonovsky
reaction. The x-ray studies are being done in collaboration with
Miss Jean Ohrt of the Center for Crystallographic Research, Roswell
Park, Buffalo, New York.
The deuteriuin labelled compounds already mentioned in the
paragraphs above, were also useful in confirming nmr assignments
of deoxynupharidine ang assessing the ins fragmentation routes of
deoxynupharidine and -dehydrodeoxynupharidine. Thus the 2.7
quartet (J12.5, 2.5 Hz) assigned to C 6 a—H (equatorial) in
deoxynupharidine was reduced to a broad singlet in deoxynupharidine
-6p, 7t3—d 9 . The l.8& quartet (J=12.5, 2.5 Hz), of which only the
lower nerd half was clearly visible, was assigned to the C 6 —H
(axial) in deoxynupharidine. In the labelled deoxynupharidine, the
l.88b signal was absent.
Correlations of principal ins fragmentation peaks generated by
deoxynupharidine, deoxynupharidine-6 , 7 -d 2 , 1 °-dehydrodeoxynupharidine
and dehydrodeoxynupharidine-6-d 1 are given in Table 4. The ins of
deoxynupharidine always displays intense peaks at nile 94, 97, 98, 136
and 233. The last peak corresponds to the parent ion which is shifted
two nile units higher in the labelled compound. Neither nile 94 nor 136
is shifted on labelling. Therefore nile 94 and 136 does not include
C 6 and 07. Possibly the generation of these two fragments may result
as shown in 16 below. It is not surprising that nile 136 is at best a
minor peak in the ins of 6 -dehydrodeoxynupharidine and nuphenine,
a piperidine type nuphar alkaloid. The complement of the nile 136 mode
of the cleavage would generate ni/e 97 and 98, but now charge would be
located on nitrogen as opposed to oxygen as when ni/e 136 is formed.
Accordingly, ni/e 97 is shifted to m/e 95, a strong peak, in the ins
of A 6 —dehydrodeoxynupharidine.
Peaks which correspond to M — 15, 29, 43, 57, 71 and 85 are
of moderate to weak intensity peaks and seem to be generated through
the loss of the alkyl groups CH 3 —C 6 H 13 . All of the peaks are
shifted two mass units higher in the ms of labelled deoxynupharidine.
The most intense peak of this type is nile 190. Possibly generation
96
19
-------
of this peak corresponds to the loss of the C 1 —methyl, 01, 02 and
an additional hydrogen from the charged fragment. The genesis of
the nile 107, 108 pair of peaks probably involves fragmentation of
ring A, as does the genesis of ni/e 136. Significantly, the 107,
108 pair also are found as intense peaks in the ms of nuphenine,
. The ni/e 176-178 group of peaks is shifted to 178—180 in the
spectrum of the labelled deoxynupharidine and must therefore,
include C 6 and C 7 . Additional ins studies with deoxynupharidine
labelled at other positions must be carried out before a distinction
can be made among the various possible routes consistent with
labelling results already obtained.
Significant information relevant to the ms fragmentation
could be obtained if ring A of deoxunupharidine could be labelled.
Several atten ts have been made in our laboratory to achieve this
goal. These atten ts have focused on the anticipated reactivity of
the hydrogen atom at C 4 . At first glance, this hydrogen might be
activated by the adjacent furan ring and nitrogen atom. This being
the case, a t 3 —enamnine, or the corresponding innnonium ion, would be
formed. To these intermediates could be added deuterium, catalytically
to introduce two deuterium atoms, or reductively with sodium boro-
deuteride to introduce one deterium atom.
/
/iil
20
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Table 4
Shifts of the Principal Peaks (m/e) in the Mass Spectra of
Deutera ted Deoxynupharidine and t 6 -Dehydrodeoxynupharidine.
6
Deoxy- -Dehydrodeoxy-
Peak nupharidine-d 0 - 6 ,7 -d 2 nupharidine-d 0 - 6-d 1
M 233 235 231 232
Mt .l 232 234 230 231
M -l5 218 220 216 217
Mt29 204 206 204(202) 203
190 192 188 189
M -55 178 180 176 177
M -56 177 179
M -57 176 178 174 175
Mt71 162 164 160 161
M -85 148 150(50%) 146(148) 149
M -97 136 136 136 136
Mt-125 108 108 108 108
Mt126 107 107 107 107
M -135 98 100
M -136 97 99 95 96
Mt 139 94 94 94 94
20a
-------
However, participation of the nitrogen non-bonding electron
pair and furan it electrons in stablizing the intermediate, whether
a carboniuin ion or free radical, would necessarily involve a planar
framework. That is, the furan ring, C 3 , C 4 , N, C and C 10 would be
coplanar. Models show that such a structure is sEerically hindered
since a and/or furanyl protons would be forced into the region
occupied by the equatorial proton at C 6 . This analysis tends to be
substantiated by experiments which show that the C 1 proton is inert.
Dehydrogenation of deoxynupharidine was attempted ilsing dichioro-
dicyanoquinone, chloranil and selenium dioxide under standard
conditions used for the dehydrogenation of steriodal ketones. In
all three cases, unconverted deoxynupharidine was recovered.
Treatment of deoxynupharidine with bromine in aqueous solution at
pH 5 resulted in electrophilic substitution of the furari ring.
Mercuric acetate oxidation has been used in quinolizidine systems
for selective introduction of an enainine double bond (20). The
selectivity is believed (21) to stem from a preferential loss of a
hydrogen anticoplanar to the nitrogen non—bonding electron pair.
In deoxynupharidine there are three hydrogens with the preferred
steric requirement for mercuric acetate oxidation. Oxidation at C 1
would be prohibited for the steric reasons already discussed. How ver,
judging from models, the formation of either iromoniuni ions 17 or 14
could be achieved without introduction of excessive strain. Presumably,
it is the latter innnonium ion which is formed as an intermediate in
the Polonovsky transformation of nupharidine to Ł? 6 -dehydrodeoxynupharidine.
Prom the actual treatment of deoxynupharidine with mercuric acetate in
acetic acid solution, only unconverted deoxyriupharidine was detected.
Car1 nion oxidation was also tried as a method to introduce
functionality into deoxynupharidine. Deoxynupharidine was treated with
potassium t—butoxide in dimethyl sulf oxide and t-butyl alcohol solution
in the presence of oxygen. Uncoverted deoxynupharidine was recovered
in 0%.
Correlation Studies
Various transformations and interconversions of piperidine and
quinolizidine type alkaloids were attempted in order to: 1) correlate
structure, 2) obtain minor alkaloids in sufficient quantity for
biological screening and 3) obtain intermediates which could be
labelled and used subsequently in ms fragmentation studies.
,1/
H
G
/7
///
21
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Conversion of Nupharidine to Nupharainine.
Nupharamine, a piperidine nuphar alkaloid, has been isolated
by the Japenese workers from j. Japonicum (22) but has not been
isolated to date from any plant material examined in our laboratory.
Furthermore, all of the other piperidine alkaloids were isolated
only in small amounts. Consequently, a synthesis approach to
these less available alkaloids was atten ted.
The basis for choosing nupharidine as the starting material
was its ample supply and the stereochemmical considerations which
suggested a singular potential in nupharidine for functionalizing
ring B of the quinolizidine ring system by way of the Polonovsky
reaction. The latter aspect has been discussed already ip this
Section, as has the •actual conversion of nupharidine to °-dehydro-
deoxynupharidine, Generation of the 6-(3—furanyl)-piperidine
skeleton was achieved through oxidation. Sodium dichromate in
acetic acid—acet 9ne at 00 for two hours produced the formamido—ketone
, [ a] 5 —154.6(c 20 mg/cc ethanol), directly in 15% yield. A
satisfactory analysis was obtained for C 15 H 21 NO. 3 . The ms gave a
parent peak at m/e 263 and prominent peaks aE 234 and 192 which
L
8
OIY
t8
22
-------
corresponded to the loss of CHO and the four carbon keto side chain.
The ir showed carbonyl bands at 5.83 (keto) and 6 .O3p. (ainido), methyl
group baz ids at 7.29 and 7.38I and furan peaks at 6.66 and ll.45ii.
The nznr revealed a doublet methyl at l.06b, a keto singlet methyl at
2.0Th, broad tx iplet pairs centered at 3.18 and 4.l5 and a pair of
broad multiplets at 4.7 and 5.55b. These pairs of triplets and broad
multiplets were assigned to C 1 and C 6 protons of respectively. Also
observed were a pair of -furanyl protons at 6.3 and 6.42b, two
a-furanyl protons at 7.37o and a pair of formyl protons at 8.02 and
8.6 . The four signals appearing as pairs were attributed to
restricted rotation of the formyl groups and the consequent differences
in magnetic envirorunents of the four different protons. The
restricted rotation of amides is well known (23)!and has been
encountered previously for acylated piperidines (24).
Other methods of oxidative cleavage of the enamine double bond
were explored. Air oxidation in the presence of cuprous chloride at
room temperature produced a complex mixture containing only a small
amount of the formamido—ketone as evide iced by the relative intensity
of 5.8 and 6 .Op bands in the ir. A two step o idation sequence was
explored. Osmium tetroxide oxidation of the °-enainine was carried
out in ether solution at 25°. The osmate ester was decomposed with
mannitol. Elution chromatography produced the diol . The ir of
this material showed strong OH absorption at 2.87p., furan bands at
6.7 and ll.48p. but no Bohlinann bands in the region of 3. 6 ji. The nmr
revealed furan signals at 7.35 and 6.36 , a methyl doublet at O.98b,
a methyl singlet at i.06o and a C 4 —H quartet at 3.6Th. In addition
the rnnr showed an OH doublet (J=5.3Hz) at 2.37o and an OH singlet
at 3.08o, both of hich disappeared on addition of D 2 O.
Corresponding to the OH doublet was an a—proton doublet (J=5.3Hz)
at 4.0Th which was reduced to a singlet on addition of D 2 O. Cleavage
of the dial to the formainido-ketone, , was achieved with sodium
metaperiodatg in aqueous tert-butyl alcohol at 25°. The overall
yield from -enainine was 38%. An attempt was made to obtain the
d .o1 without using expensive osmium tetroxide. Treatment of the
i °-enainine with -chloroperbenzoic acid at room temperature in
chloroform solution produced a crude product mixture whose ir and
ninr showed no furan signals. In another attempt, rhuthenium
tetroxide in carbon tetrachioride solution at 0° produced material
whose nmr showed deminished furan signals. Since it appeared that
the furan ring had been oxidized in both rhuthenium tetroxide and
peroxide procedures, further attempts along these lines were
abandoned. Finally, a quantitative conversion of enainine to
formamido-ketone was achieved in one step with osmium tetroxide,
in small amounts, and sodium metaperiodate in dioxane containing
a few drops of pyridine.
The conversion of the formamido-ketone to nupharamine, ag,
was accomplished in one step with a large excess of methyl magnesium
iodide. Purification of the product mixture by elution chromatography
gave two fractions of pure nupharamine, [ a] 5 - 39.5°(c 18 mg/cc,
ethanol), which represented a 50% conversion. Other fractions also
contained nupharaniine. The ms of the synthetic material gave a
parent peak at ln/e 251. The ir and nmr were identical with the
spectra of an authentic sample of nupharamine generated from the
perchlorate salt of nupharamine. The latter was kindly provided
by Dr. I. Kawasaki, of Osaka University, who had isolated nupharamine
from N. japonicum . The synthetic nupharainine was converted to its
perchiorate salt; mp 163-166°, mixture mp 163-166°.
23
-------
Attempted Conversion of Nupharidine to Nuphamine.
Presently, conversion of quinolizidi e to piperidine type nuphar
alkaloids through the employment of the °-enamine, and its oxidized
transformation products, cannot be claimed as a general procedure.
While conversion of nupharidine to nupharamine was achieved in over—all
good yield, extending the use of the enamine, , diol , the
formainido-ketone or the further elaboration of the isopentyl side
chain skeleton of nupharamine has met only with failure in preparing
nuphanilne, 21.
21
Initially, three schemes involving the use of diol 18,
forniamido-ketone 19 and nupharainine, 20, were contemplated. In
time, there appeared a report (25) which described the conversion
of enainines to a-acetoxy ketones with thallium ace ate. This
report lead us to consider the direct use of the —dehydrodeoxy—
nupharidine, .
A synthesis starting with the forniamido-ketone has the advantage
that the secondary nitrogen is already protected by the forniamido
group. However, a carbon atom must be added in order to complete the
isopentyl side chain and coincidentally allow the future development
of primary allyl alcohol. The use of the diol as a starting point
has the advantages of possessing all the requisite number of carbon
atoms in elevated oxidation states in the right location for
subsequent conversion to the primary allyl alcohol side chain.
Another appealing feature was a synthesis starting from enamine
and proceeding through oxidation to diol, ig, hydrolysis of the
resulting carbinol amine functions reduction of the a].dehyde form -
22, and finally dehydration seemed closer to a possible biogenetic
transformation of quinolizidine to piperidine type nuphar alkaloids.
0
24
-------
0
aa
-
- 4 qoh’
The thallium acetate oxidation of the 6 -enainine seemed like a
very direct route to an appropriately functionalized isopentyl side
chain. However, expections for success were not high since the
oxidation procedure does not seem to be generalj.y applicable to all
enaniines but rather only those which are substituted at the a—position.
The use of nupharamine in theory seemed straightforward. Protection
of piperidine nitrogen, dehydration and allylic oxidation at the vinyl
methyl group were envisioned as the three basic steps.
Work in our laboratory has proceeded along all four of the
principal routes discussed above. The results are treated separately
in each of the sub—sections below.
Starting from Diol
Although the route from the diol seemed for a number of reasons
the most appealing, work proceeding along these lines was met with the
earliest failures. Attempts to detect any of the aldehyde form in
equilibrium with the carbinol amine form were carried out in a number
of solvents of varying polarity. In no case could an aldehydic proton
be found in the nmr nor an aldehyde band observed in the ir. Also,
oxidation of the carbinol amine with ch omic anhydride in the
presence of pyridine gave not the desired carboxylic acid, .but the
formamido-ketone j9 . Similar results have already been described
Oh’
25
-------
elsewhere in this report for the chronic anhydride oxidation in acid
solution.
Starting with Formamido-Ketone
Cyanohydrin Formation and Dehydration.
Generation of the isopentyl side chain containing the requisite
carbons in an elevated oxidative state was atten ,ted through HG ! ’ !
addition to the formainido—ketone, .
1 - I /C A l
Conditions listed below were employed:
1) Acetone cyanohydrin, neat or in ethanol solution for 15 hr
at 25° with and without added ammonia.
2) Acetone cyanohydrin in refluxing methanol for 4 hr.
3) Acetone cyanohydrin in diethylamine solution at 25° for
15 hr.
4) Sodium cyanide in sulfuric acid ethanol at 25° for 24 1-irs.
In no case could the transformation of formainido—ketone to cyanohydrin,
be detected. Starting formanido—ketone could be recovered in nearly
quantitative amounts.
23
‘ 9
21
26
-------
Carbon framework extension by ylide addition.
The formation of an epoxide at the side chain, as depicted bela z,
was attempted by treating the forniainido-ketone with the ylide generated
from trimethylsulfoxonium iodide. Two products were obtained, each in
0—
C4±SC/ x
/ 9
- cb =Af / X
15% yield. One of them contained neither ketone nor furan ring as
evidenced by the ir. The other contained a furan ring but no ketone
group and was suspected as the desired epoxide 5.. However, insufficient
quantity prevented further studies necessary to confirm the structure.
Since the yield of the desired epoxide was poor at best, and there was
indication that the furan ring as well as the ketone group reacted with
the ylide, the search for better conditions was not attempted.
Instead, another Wittig extention reaction was tried. The formamido—
ketone was treated with triphenyiphosphonium methylide in refluxing
ether solution far 10 hr in an attempt to prepare . Only unconverted
formainido—ketone was obtained.
It is clear from the observations described above that the
carbonyl of the 3—ketobutyl side chain is extremely inert to nucleophiles,
even the highly nucleophilic dimethylsulfoxonium methylide. In line
with this lack of reactivity is the finding that the treatment of
N-acetyl-2- [ 2-ethanal]-piperidine, , with triphenyiphosphonium
isopropylide produced no Wittig product.
2ff
2G
27
-------
+/ / =c(ci- X
Thallium acetate oxidation of t 6 —dehydrodeoxynupharidine.
Treatment of t 6 -dehydrodeoxynupharidine, , with thallium acetate
in acetic acid solution at room temperature afforded two products. The
first gave an ir which showed no carbonyl, olefinic double bonds-other
than furan, or hydroxyl group. The ins showed principal peaks at m/e
478 (ivit), 463, 450, 435, 313, 272, 231, 232 which suggested the
n terial possessed a deoxynupharidine dimer structure.
The second product gave an ir which showed a carbonyl band and an
ins having M 1 at We 230. These products are under continued investi-
gation at this time.
Elaboration of the isopentyl side chain of
nupharamine.
Nupharamine, Q, was treated with thionyl chloride in refluxing
benzene according to the procedure of Arata (26). Although Arata
reports only the formation of a , ‘ —dimethylallyl side chain, the
product obtained in our laboratory was a mixture of olefins, , as
evidenced by the mnr.
0
28
-------
A Ci
SOC/a ,
EQ V )
A
The olefin mixture, , was acetylated with acetyl chloride in
triethylamine to give the acetamide which in turn was treated with
selenium dioxide in refluxing methanol for 3 hr. Regardless of the
location of the double bond in the side chain of , a single allyl
alcohol was expected on the tasis that selenium dioxide of dipentene,
Q, in acetic anhyd ide at 500 gave the single allyl acetate j.
The ir of the product mixture obtained from the ainide derivative of
the olefin mixture displayed a hydroxyl band at 2.8k, a single
amide carbonyl band at 6.l , and bands at 6.67 and li.45p.. The tic
30
AcO
N
29
29
-------
sha zed the product mixture to contain four components, no one of
which corresponded to the Rf value of N-acetyl nuphainine. The
authentic sample of N-acetyl nuphainine was prepared from nuphamine
isolated from N. luteum subsp. variegatum.
Antibacterial Properties of WPC 45.1. VPM 7L.l5 and CFW 225.1
The cumulative results of the inhibition studies are given
in Table •. Specific information about the degree of sensitivity
is presented in Tables and 7. The sensitivities of the selected
phytopathogens to alkaloids WPC 45.1, VPM 7I .l5 and CFW 225.1 as
determined by the disc plate test are given in Table . The
sensitivities of C. imichiganense and Arthrobacter (TH-l) to Wpc
45.1 as determined by the serial dilution-turbidimetric method are
given in Table . Since the available quantity of pure VPM 74.15
was small, inhibition studies with this alkaloid were carried out
on only a limited scale.
Although Q. michiganense was particularily sensitive to the
sulfur-bearing alkaloids VFW 225.1, WPC 45.1 and VPM 74.15, this
bacteria was insensitive to the non—sulfur, C 15 —alkaloids
deoxynupharidine and nupharidine. The lack of activity by the
N-oxide alkaloid nupharidine was somewhat surprising since a number
of synthetic N—oxides have been employed as bactericides. Possibly
screening nupharidine against a broad spectrum of bacteria would
show some selective sensitivity.
Section 4
CONCLUSIONS
Two sulfur—containing C 3 —a1ka1oids have been isolated in
pure form from the rhizomes oI’ Nuphar luteum subep. macrophyllum
by using a modified procedure of the Russian workers (1). The same
two alkaloids are present in Nuphar luteuin subsp. variegatum . One
of these alkaloids is CFW 225.1, the free biscarbinolamnine form of
the diethyl ether WPC 45.1 which was first isolated by using the
Russian procedure precisely as reported.
Alkaloid WPC 45.1 was active against four of six phytopathogenic
bacteria. The second alkaloid was VPM 75.15 which was about one—half
as active against the same sensitive bacteria. Three other unidentified
bacteria were obtained from the region where . luteum subsp.
variegatum was harvested; these bacteria were insensitive to WPC 45.1.
While the sulfur-containing C 39 —alkaloids were active against some
phytopathogens, two C alkaloids, nupharidine and deoxynupharidine,
were not. Thus not ail alkaloids of Nuphar are active and not all
all bacteria tested are sensitive.
Studies directed toward the structure of the two alkaloids showed
that both contain the thiaspirane, deoxynupharidine structural frame-
work of structure . Reduction of both alkaloids produced the
dehydroxylated counterparts, that from WPC 45.1 has tentatively been
assigned stereostructure h. The dehydroxylated counterpart of VPM
75.15 has been assigned structure . These assignments are based
on the stereochemistry of sodium borodeuteride reduction of VPM 75.15
and WPC 45.1 and n.m.r. results. The stereostructures also assume
that both deoxynupharidine moieties are the same and belong to the
30
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Table 5
Cumulative Results of Inhibition. Bacteria Nuphar Alkaloids
WPC VPN CFW
Bacteria 45.1 74.15 225 j Deoxynupharidine Nupharidine
Corynebacterium michigariense pos. p08. p08. neg. neg.
Q.. flaccumfaciens - pos. pos.
C. insidiosum pos. p0 5.
Xanthomonas phaseoli pos. pos.
. vesicatoria neg.
Erwinia caratovora neg.
Arthrobacter (TH—l) pos.
Green Lake Tully #1 neg. neg. neg.
Green Lake Tully #4 neg. neg. neg.
Green Lake Tully #5 neg. neg. neg.
-------
Table 6
Results of Diac Plate Tests. Total Diaznetera of Zone Inhibition in cm for the Weight in p g Given
for WPC 45.1, VPN 74.15 and CF ’I 225.1
WPC 45.1 VPM 74.15 CFW 225.1
Bacteria 1 j.g 10 ij.g 1 ig 10 jig 3 jig 4 j.ig 10 jig
Corynebacterium imichiganese 1.6 3 1.4 2.2 0.0 2.2
C. flaccumfaciens 3 1.6 2.8
C. insidiosum 3 0.0 1.8
X. phaseoli 2.5 0.0 2.1—2.3
a
The diameter of the disc employed was 1.25 cm. The zone inhibition diameter is the total diameter.
-------
Table 7
Results of Serial Dilution-Turbidimetric Tests Given In “KJ.ett’ t
Units of Transmission. Inhibition by WPC 45.1.
Concentration of WPC 45.1 in ij.gImJ.
31
Bacteria 200 Q 10 j_ 1 Blank
C. michiganense 11 11 11 205 315 325 410 420
11 13 32 22 310 56 435 450
12 12 42 40 76 315 360
11 11 37 40 240 280 450
Arthrobacter (TH—l) 34 84 450 520 510
22 86 72 495 500
same enantiomeric series as deoxynupharidine, the absolute configuration
of which was determined as a part of this study.
Work with the C 15 class of Nuphar alkaloids resulted in the
discovery of 7-epideoxynupharidine and 3-epinuphamine, both new
alkaloids. The absolute configuration of deoxynupharidine was
established arid a method of converting quinolizidine to piperidine
type C 15 alkaloids was found.
Section 5
RECOMMENDATIONS
The antibacterial activity of Nuphar now can be linked to the
presence of sulfur—containing alkaloids. Therefore, it would be
desirable to extend the antibacterial studies to a greater range of
bacteria, especially a far greater number of those present in the
soil and water in which Nuphar grow. It would also be desirable
to determine the activity of the alkaloids against other aquatic
protista, the most important of which would seem to be fungi.
The release of the antibacterial alkaloids by the plants into
water in control tanks should be studied. In connection with this
area of investigation, methods for determining the alkaloid concentra-
tions in water must be found. Once these analytical problems have
been solved and the water content of plant-produced alkaloid has been
determined, then the biological significance of growing Nuphar should
33
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be established.
Within the area of chemistry, the most important problem
remaining is the determination of the stereochemistry of alkaloids
WPC 45.1 and 11PM 74.15.
Section 6
REFERENCES
1. Brit. 968,042; . 4., , 15939 (1964).
2. T. N. Il’inskaya, A. D. Kuzovkov and T. G. Monakhova, Khim.
Prir. Soedin. , , 178 (1967).
3. K. C. Bel’t rukova and L. T. Pastushenko, Mikrobiol . .,
Akgd. Nauk RSR, , 36 (1963); C. A., , 5536 (1963).
4. H. Budzikiewicz, C. Djarassi and D. H. Williams, “ kss
Spectrometry of Organic Compounds,” Holden- [ y, Inc., San
Francisco, 1967, Chapt. 6.
5. 0. .Achmatowicz, H. Banaszek, Gh Spiteller and J. T. Wrobel,
Tetrahedron Letters , 927 (1964).
6. G. I. Birnbaum, ibid., 4149 (1965).
7. C. F. Wong, E. Auer and R. T. LaLonde, . Q g. Chem., ,
517 (1970).
8. B. Capon, Quart . ., , 45 (1964).
9. C. F. Wong and R. T. LaLonde, Phirtochem. , 2 659 (1970).
10. E. Auer and R. T. LaLonde, Abstr . Paiers, 157th Meeting
of the American Chemical Society , Minneapolis, Minn., April
1969, ORGN 150.
11. (a) N. Kotake, I. Kawasaki, T. Okamoto, S Natustani, S.
Kusumoto and T. Kaneko, Bull. Chem . ., , 1335
(196 ); (b) Y. Arata and T. Iwai, Kanazawa Doigaku
Yakugakuba Kenkyu Nempo , , 39 (1962).,
12. D. C. Aidridge, J. J. Armstrong, R. N. Speake and W. B.
Turner, . Chem . 1667 (1967).
13. Y. Arata and T. Ohashi, Chein. Pharm . Bull., , 1247 (1965).
14. R. Barchet and T. P. Forrest, Tetrahedron Letters , 4229
(1965).
34
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15. Y. Arata, Chem. Pharm. Bull . (Tokyo), , 907 (1965).
16. E. Wenkert, Experientia , 10, 346 (1954).
17. G. Fodor, Abstr . of Papers, 159th Meeting of the American
Chemical Society , Houston, Texas, February 1970, ORGN 87.
18. H. M hr1e, C. Karl and V. Scheidegger, Tetrahedron , , 6813
(1968).
19. (a) T. M. Moynehan, K. Schofield, R. A, Y. Jones, and A. R.
Katritzky, J. Chem . 2637 (1962); b) C. D. Johnson, R.
A. Y. Jones, A. Ft. Katritzky, C. R. Palmer, K. Schofield, and
R. T. Wells, ibid., 6797 (1965).
20. L. W. Haynes in tiEnaminesti, A. G. Cook, Ed., } rce1 Dekkar,
New York, 1969, Chapt. 2.
21. N. J. Leonard, A. S. Hay, R. W. Fu.lmer and V. W. Gash, J.
Amer. Chem . Soc., , 1552 (1955).
22. Y. Arata and T. Ohashi, . Pharin . ., Japan , , 792 (1957).
23. R. A. Johnson, 1. Q g. Chem., , 3627 (1968).
24. (a) W. D. Phillips, . Chem. Phys . , 1363 (1955), (b) H. S.
Gutowsky and C. H. Hoim, ibid., , 1228 (1956).
25. M. E. Kuehne and T. J. Giacobbe, . Q g. Cheiu., , 3359 (1968).
26. Y. Arata, . Pharm . Japan , , 729, 734 (1959).
35
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Section 7
ACKNOWLEDGMENTS
The Principal Investigator acknowledges the able laboratory
assistance of Drs. E. Auer, C. F. Wong, W. P. Cullen, V. P.
Muralidharan, F. Stretton and Mr. J. Woolever, all of whom
participated in various aspects of the chemical investigations
described in this report. The principal investigator also
acknowledges the advice given by Dr. It. H. Baum in matters
pertaining to antibacterial testing and to Mr. G. Cinq—Mars
for executing the antibacterial testing. Determination of mass
spectra by Mrs. Hazel Jennison and nuclear magnetic resonance
spectra by Miss Mary Lou Hull is gratefully acknowledged.
36
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Section 8
LIST OF PUBLICATIONS
1. “The Application of Polonoveky Reaction Conditions to
Nupharidine.” E. Auer and R. T. LaLonde, Abstr . of Papers,
157th Meeting of the American Chemical Society , Minneapolis,
Minn., April 1969, ORGN 150.
2. “The Stereochemistry of (—)-Deoxynupharidine. The Synthesis
of (—)-(R)—a—Methyladipic Acid,” C. F. Wong, E. Auer and R. T.
LaLonde, . Q g. Chein., , 517 (1970).
3. “7-Epideoxynupharidine from Nuphar luteum subsp. Variegatum, ”
C. F. Wong and R. T. LaLonde, Phytochem. , 2 659 (1970).
4. “The Structure of 3—Epinuphamine, A New Alkaloid from Nuphar
luteum subsp. Variegatum, ” C. F. Wong and R. T. LaLonde,
Phytochem. , in press.
5. “Sesquiterpenic Alkaloids of Nuphar luteum subsp. Variegatum, ”
C. F. Wong and R. T. LaLonde, Phirtochem. , in press.
37
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Section 9
GLOSSARY OF. ABBRE 1IATTONS AND SYMBOLS
br in broad inultiplet fluclear magnetic resonance signal
br d broad doublet nuclear magnetic resonance signal
c concentration
Cc column chromatography
cc cubic centimers
C 2 symmetry designation, a two fold axis of symmetry
CFW 225.1 free biscarbinolainine form of the diether biscarbinol-
amine WPC 45.1; see WPC 45.1
CHC1 3 chloroform
CH 2 C1 2 methylene chloride -
d doublet
eV electron Volt
Hz magnetic field strength in Hertz
ir infrared spectrum (spectra)
J coupling constant
in a multiplet nuclear magnetic resonance signal
M+ molecular ion or parent peak in the mass spectrum
ni /e mass divided by charge
milligrams
mHz magnetic field strength in mega Hertz
nip melting point
ins mass spectrum (spectra)
38
-------
MeOH methanol
nm wavelength in nanometers
rnnr nuclear magnetic resonance spectrum (spectra)
NTBN neothiobinupharidine
q a quartet nuclear magnetic resonance signal
(R) absolute configuration designation
Rf chroniatographic retention
s singlet
subsp. subspecies
t a triplet nuclear magnetic resonance signal
tic thin layer chromatography
TMS tetramethylsilane
uv ultraviolet spectrum (spectra)
VPM 74.15 one of two pure alkaloids isolated from .
luteum subsp. variegatuin
WPC 45.1 one of two pure alkaloids isolated from N.
luteum subsp. variegatuin
[ a] specific optical rotation measured at temperature
t and at the sodium D line
b see’r
Zr frequency difference, but magnetic field strength
difference when applied to ninr.
C molecular extinction coefficient
Xmax wavelength corresponding to the ultraviolet
absorption maximum
wavelength in microns
p.g micrograms
symmetry designation, a plane of symmetry
39
-------
10-b, where { H samp1e I1_reference)/
H—reference] x 10 , H—sample is the magnetic
field strength f or resonance of any given
proton in the sample and H—reference is the
magnetic field strength for resonance of a
reference proton
(—) levorotatory optical rotation
40
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