Identification and synthesis of novel alkaloids from the root system of Nicotiana tabacum: Affinity for neuronal nicotinic acetylcholine receptors

Article abstract of DOI:10.1016/j.lfs.2005.09.009

A novel pyridine derivative, 3,5-bis-(1-methyl-pyrrolidin-2-yl)-pyridine, and a pair of diastereomers of 1,1′-dimethyl-[2,3′]bipyrrolidinyl were isolated from the root of Nicotiana tabacum plants and identified as novel alkaloids by GC-MS analysis. The structures of these new alkaloids were confirmed by total synthesis. The affinities of these novel alkaloids, and other structurally related compounds for α4β2*, α7* neuronal nicotinic acetylcholine receptors (nAChRs), and for nAChRs mediating nicotine-evoked dopamine release from rat striatum were also assessed. The results indicate that these compounds do not interact with α7* nAChRs, but inhibit [³H]nicotine binding to the α4β 2* nAChR subtype. The results also demonstrate that these compounds act as antagonists at nAChRs mediating nicotine-evoked dopamine release from rat striatum.

Full text of DOI:10.1016/j.lfs.2005.09.009

Life Sciences 78 (2005) 495 – 505
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Identification and synthesis of novel alkaloids from the root system of
Nicotiana tabacum: Affinity for neuronal nicotinic acetylcholine receptors
Xiaochen Wei a, Sangeetha P. Sumithran a, A. Gabriela Deaciuc a, Harold R. Burton b,
Lowell P. Bush b, Linda P. Dwoskin a, Peter A. Crooks a,
*
a
Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536, United States
b
Agronomy Department, College of Agriculture, University of Kentucky, Lexington, KY 40536, United States
Abstract
A novel pyridine derivative, 3,5-bis-(1-methyl-pyrrolidin-2-yl)-pyridine, and a pair of diastereomers of 1,1V-dimethyl-[2,3V]bipyrrolidinyl were
isolated from the root of Nicotiana tabacum plants and identified as novel alkaloids by GC-MS analysis. The structures of these new alkaloids
were confirmed by total synthesis. The affinities of these novel alkaloids, and other structurally related compounds for a4h2*, a7* neuronal
nicotinic acetylcholine receptors (nAChRs), and for nAChRs mediating nicotine-evoked dopamine release from rat striatum were also assessed.
The results indicate that these compounds do not interact with a7* nAChRs, but inhibit [3H]nicotine binding to the a4h2* nAChR subtype. The
results also demonstrate that these compounds act as antagonists at nAChRs mediating nicotine-evoked dopamine release from rat striatum.
D 2005 Elsevier Inc. All rights reserved.
Keywords: Alkaloids; Nicotine; Nicotiana tabacum; Neuronal nicotinic acetylcholine receptors
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 495
Experimental . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 497
Chemicals. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 497
Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 497
Isolation and identification of minor alkaloids from tobacco samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 498
Total synthesis of the minor tobacco alkaloids from tobacco root extracts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499
Synthesis of 1,1V-dimethyl-[2,3V]bipyrrolidinyl diastereomers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499
Synthesis of 3,5-bis-(1-methyl-pyrrolidin-2-yl)pyridine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 500
Dimethyl pyridine-3,5-dicarboxylate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 500
3,5-Bis-(1-methyl-pyrrolidin-2-yl)pyridine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 500
[3H]Dopamine release assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 501
[3H]Nicotine and [3H]methyllycaconitine binding assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 501
Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 501
Acknowledgements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 505
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 505
Introduction
Over four thousand different compounds have been isolated
and identified in Nicotiana tabacum plants (Weeks, 1999),
which are the commercial source for different tobacco
products. Alkaloids constitute an important part of these
* Corresponding author. Tel.: +1 859 257 1718; fax: +1 859 257 7585.
E-mail address: pcrooks@email.uky.edu (P.A. Crooks).
0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.lfs.2005.09.009

496
X. Wei et al. / Life Sciences 78 (2005) 495–505
H
2'
2'
2'
2'
N
N
N
H
N
H
CH3
H
N
N
N
N
S-(-)-Nicotine
Nornicotine
Anabasine
Anatabine
Fig. 1. Structures of the four major tobacco alkaloids.
components, because of their biological and pharmacological
activities.
The five-membered 2-pyrrolidinyl and 1-methyl-2-pyrroli-
dinyl moieties in nornicotine and nicotine are formed from
arginine and/or ornithine through putrescine, 1-methylputres-
cine, and 1-methyl-1,2-pyrrolinium ion via decarboxylation,
methylation and oxidation reactions in the plant root system
(see Fig. 2). The intermediate 1-methyl-1,2-pyrrolinium ion
undergoes condensation with 3,6-dihydronicotinic acid at the
3-position to afford the S-(À)-nicotine molecule, which then is
transported from the root to the other parts of the plant (Fig. 3).
Nornicotine is a terminal alkaloidal product, and is produced as
an N-demethylated metabolite of nicotine in fresh tobacco
plants or in cured tobacco leaves.
The principal tobacco alkaloids are nicotine, and three other
structurally related compounds: nornicotine, anabasine and
anatabine. These four alkaloids all have a 3-pyridyl moiety in
their structure, which is connected to a 1-methyl-2-pyrrolidinyl
group to afford nicotine, a 2-pyrrolidinyl group to afford
nornicotine, a 2-piperidinyl group to afford anabasine, and a 2-
piperidin-4-enyl group to afford anatabine (Fig. 1). The N.
tabacum plant biosynthesizes nicotine in only the 2V-S-chiral
form; however, the other three major alkaloids are present in
both the 2V-S- and 2V-R-chiralities in various enantiomeric
ratios.
The 1-methyl-1,2-pyrrolinium ion is also the precursor of
other pyrrolidino alkaloids, such as hygrine and related natural
products, which have also been isolated from numerous plants
(Massiot and Delaude, 1986). Some of these alkaloids have
very important biological and pharmacological activities
(Dewick, 1997).
The 3-pyridyl moiety in these alkaloids is formed bio-
synthetically from aspartic acid and glyceraldehyde-3-phos-
phate through quinolinic acid, then nicotinic acid, and finally
3,6-dihydronicotinic acid (Bush et al., 1999). The six-mem-
bered piperidine and piperidin-4-ene ring moieties of anabasine
and anatabine, respectively, are biosynthesized from different
sources: i.e. lysine is the precursor for the 2-piperidinyl moiety
in the anabasine molecule, whereas both the 3-pyridyl and 2-
piperidin-4-enyl moieties in the anatabine molecule are
biosynthesized from 3,6-dihydronicotinic acid (Bush et al.,
1999).
The formation of 1-methyl-1,2-pyrrolinium ion is regulated
through arginine decarboxylase and/or ornithine decarboxyl-
ase, enzymes located in the tobacco root system (Bush et al.,
1999). 1-Methyl-1,2-pyrrolinium ion can dimerize at neutral
pH in vitro, and after reduction or oxidation of this dimer, 1,1V-
dimethyl-[2,3V]bipyrrolidinyl and/or 1-methyl-3-(1V-methylpyr-
NH
Arginine
decarboxylase
NH
N
H
N
H 2N
H OOC
NH2
NH2
NH2
H
CO2
Agmatine
Arginine
Ornithine
decarboxylase
O
N
H
H 2
N
NH2
H 2
N
NH2
NH2
NH2
HOOC
CO2
1-Carbamylputrescine
Ornithine
Putrescine
Putrescine N-methyl-transferase
S-adenosyl-methionine
(CH3 donor)
+
N
CHO
Methylputrescine
oxidase
NHCH3
H 2N
NHCH3
CH3
1-Methylputrescine
4-Methylamino
butanal
1-Methyl-1,2-
pyrrolinium ion
Fig. 2. Biosynthesis of N-methyl-1, 2-pyrrolinium ion in N. tabacum.

X. Wei et al. / Life Sciences 78 (2005) 495–505
497
H
H
H
+
COOH
[H]
COOH
CO2
N
N
N
[O]
CH3
CH3
CH3
N
N
N
N
N
H
Nicotinic
acid
3,6-Dihydro-
nicotinic acid
3,6-Dihydronicotine
(S)-(-)-Nicotine
Fig. 3. Biosynthesis of S-(À)-nicotine in N. tabacum.
rolidin-2-yl)pyrrole is formed (Swan and Wilcock, 1974; Leete
et al., 1988). Since both reduction and oxidation conditions can
exist inside plant cells, these two dimeric products can be
formed if the 1-methyl-1,2-pyrrolinium ion pool is concentrat-
ed enough in the tobacco root system.
nauer and Gretillat, 1989). 3,5-Di-pyrrolidin-2-yl-pyridine
(DINORNIC) was obtained from sodium borohydride reduc-
tion of the above product (Wei et al., unpublished data).
All other chemicals and solvents were obtained from
commercial sources and used without further purification.
In order to identify new, minor alkaloid products in the
tobacco plant, GC-MS analysis was employed. This analytical
method is a very powerful and sensitive structural identification
tool, especially when utilized in the selective ion monitoring
(SIM) mode. In this study, a simple GC-MS analysis procedure
was developed to identify minor alkaloids from N. tabacum
root systems. The structures of these minor alkaloids were
confirmed by total synthesis, and their binding activities at
different neuronal nicotinic acetylcholine receptor (nAChR)
subtypes were evaluated.
Instrumentation
The analytical instrumentation comprised an Agilent 6890
gas chromatograph, an Agilent 7683 autosampler and an
Agilent 5973 mass-selective detector (MSD) (Agilent Co.,
Willmington, DE). An HP-5MS GC column (30 mÂ0.25 mm
I.D.Â0.25 Am film thickness) (Agilent Co., Willmington, DE)
was used for the analyses, and ultra pure helium was used as
carrier gas at a flow rate of 1.0 ml/min. The temperature at the
injection port, MS interface, electron impact source and Mass
filter was set constant at 280 -C, 280 -C, 230 -C, and 150 -C,
respectively. The analytical sample (1 Al) was injected in the
split mode. The initial temperature of the GC oven was held at
120 -C for 2 min; then it was heated to 160 -C at a rate of 10
-C/min. After holding the oven temperature at 160 -C for 1
min, the oven was heated to 275 -C at a rate of 60 -C/min and
held at this temperature for 10 min in order to elute possible
interfering high boiling point impurities.
Experimental
Chemicals
3,5-Bis-(4,6-dihydro-3H-pyrrol-2-yl)-pyridine (DM 3,5)
was prepared from the condensation of dimethyl 3,5-dinicoti-
nate and 1-vinylpyrrolidin-2-one followed by treatment with
acid, and then alkaline hydrolysis of the resulting condensation
product, utilizing a modification of the reported method for the
preparation of the corresponding 2,6-substituted isomer (Ber-
NMR spectra were recorded in CDCl3 on a Varian 300 MHz
instrument and are reported in ppm relative to TMS as the
Abundance
TIC: 1402RT01.D
2000000
1800000
1600000
1400000
1200000
1000000
800000
600000
400000
200000
9.20 min
7.77 min
7.89 min
14.72 min
0
4.00
6.00
8.00
10.00
12.00
14.00
16.00
18.00
Time-->
Fig. 4. Total ion chromatogram of an alkaloidal fraction from a tobacco root sample.

498
X. Wei et al. / Life Sciences 78 (2005) 495–505
internal standard. High resolution mass spectra were recorded
on a JEOL JMS-700T MStation unit.
over anhydrous K2CO3, filtered, and evaporated to dryness
under vacuum, and the residue was reconstituted in 300 Al of
chloroform. One microliter of the final chloroform solution was
injected onto the Agilent 6890/5973 GC-MS system for
analysis. A total ion chromatogram obtained from a represen-
tative tobacco root sample is illustrated in Fig. 4. Nicotine
appears at a retention time of 9.2 min. The mass spectra of two
previously unreported alkaloidal components eluting at reten-
tion times of 7.77 and 7.89 min in this total ion chromatogram
are shown in Fig. 5.
Isolation and identification of minor alkaloids from tobacco
samples
Dried and ground tobacco samples (N. tabacum roots, 250
mg) were passed through a 40-mesh wire screen, and were then
shaken in 10 ml of 0.5 N HCl for 5 min in a linear shaker. After
filtering off the remaining particulates, the filtrate was extracted
with 10 ml of chloroform to remove the acidic and neutral
interfering components. The aqueous phase was then basified
to pH 10 with 10 N NaOH solution, and extracted with 2Â10
ml of chloroform. The combined organic phases were dried
The mass fragmentation patterns of these two minor
alkaloids were identical, suggesting that they may be isomeric
in nature. The molecular ion for both compounds appears at
m/z 168 and the base peaks for each alkaloid was observed at
Abundance
Scan 909 (7.771 min): 1402RT01.D
84
32000
30000
28000
26000
24000
22000
20000
18000
16000
14000
12000
10000
8000
42
168
110
96
6000
136
124
4000
67
55
2000
153
150
207
210
0
40
50
60
70
80
90
100
110
120
130
140
160
170
180
190
200
m/z-->
Abundance
Scan 929 (7.888 min): 1402RT01.D
84
4500
4000
3500
3000
2500
2000
1500
1000
500
42
168
110
96
124
67
55
137
0
40
50
60
70
80
90
100
110
120
130
140
150
160
170
m/z-->
Fig. 5. Mass spectra of two alkaloidal components (retention time 7.77, top; retention time 7.89, bottom) isolated from tobacco root.

X. Wei et al. / Life Sciences 78 (2005) 495–505
499
m/z 84, which corresponds to a 1-methyl-1,2-pyrrolinium ion.
An ion at m/z 153 ion indicated the loss of one methyl group
from the molecule. Other major ions appeared at m/z 136, 124,
110, 96, 82, 67 and 42. The above molecular weight and
fragmentation data suggest that these novel isomeric alkaloids
may be dimeric 1-methypyrrolidino compounds resulting from
the self-condensation of 1-methyl-1,2-pyrrolinium ion in the
root, followed by reduction, to afford the two possible
diastereomeric products.
Total synthesis of the minor tobacco alkaloids from tobacco
root extracts
Confirmation of the above tentatively identified minor
alkaloids from N. tobacum root was obtained by total synthesis
of the proposed structures of these alkaloids (see below).
Synthesis of 1,1V-dimethyl-[2,3V]bipyrrolidinyl diastereomers
A third novel alkaloidal component eluting with a retention
time of 14.72 min in the total ion chromatogram of the basic
extract from the tobacco root sample was also investigated. The
mass spectrum of this component is illustrated in Fig. 6.
The third alkaloidal component exhibited a molecular ion
at m/z 245, and a base ion at m/z 84, with other major ions at
m/z 230, 216, 189, 161, 132, and 42. This component could
also be detected as a very minor alkaloid in tobacco leaf
extracts, but was present in extremely small amounts relative
to nicotine. The base ion at m/z 84 can be attributed to a 1-
methyl-1,2-pyrrolinium ion formed by cleavage of a 1-
methyl-2-pyrrolidinyl moiety from the molecule. The ion at
m/z 161 led us to assume that the alkaloid was a nicotine
derivative, since the loss of a proton from the nicotine
molecule affords the same ion. From the mass of the molecule
(i.e. m/z 245), one possible structure was considered to be a
molecule in which two 1-methyl-2-pyrrolidinyl moieties are
attached to a central pyridine moiety. From biosynthetic
pathway considerations, and the mass spectrum of this
unknown component, the most probable structure for this
compound was considered to be the nicotine-related com-
pound: 3,5-bis-(1-methyl-pyrrolidin-2-yl)-pyridine. Interest-
ingly, no other isomeric form of this alkaloid could be
identified in the root extract.
The two novel minor alkaloids tentatively identified as
diastereomeric bipyrrolidinyl alkaloids in the root sample were
synthesized from 1-methyl-2-pyrrolidone, and obtained as a 1:6
inseparable isomeric mixture from self-condensation of 1-
methyl-2-pyrrolidone in the presence of POCl3, followed by
catalytic hydrogenation of the resulting enone product with Pd/
H2 and subsequent LiAlH4 reduction (Fig. 7) (Matthews,
1985).
The GC retention times and the mass spectra of the two
synthetic diastereomers were identical to the retention times
of the minor isomeric alkaloids isolated from the root extract
1
eluting at 7.77 and 7.89 min. The H-NMR spectrum of the
diastereomeric mixture of the synthetic 1,1V-dimethyl-
[2,3V]bipyrrolidinyl diastereomers afforded: y (CDCl3) 3.03
and 2.80 (1 H, m, diastereomeric H-2 protons in a ratio of
1:6), 2.61 (2 H, m, mixture of diastereomeric H-5 protons,
2.46 (2 H, m, mixture of diastereomeric H-5V protons), 2.35
and 2.34 (3 H, 2Âs, N–CH3 protons in a ratio of 1:6), 2.32
and 2.33 (3 H, 2Âs, N–CH3 protons in a ratio of 1:6), 2.17
(2 H, m, mixture of diastereomeric H-2V protons, 2.01 (1 H,
m, mixture of diastereomeric H-3Va protons), 1.80 (1 H, m,
mixture of diastereomeric H-3Vb protons), 1.64 (4 H, m,
mixture of diastereomeric H-3V, H-4 and H-4V protons) ppm.
HRMS (mixture): m/z 168.16.
Abundance
Scan 2103 (14.723 min): 1402RT01.D
84
13000
12000
11000
10000
9000
8000
7000
6000
5000
4000
3000
2000
1000
0
189
245
42
161
216
202
117
132
145
173
55
65
104
230
93
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240
m/z-->
Fig. 6. Mass spectrum of the third alkaloidal component (retention time 14.72) isolated from tobacco root.

500
X. Wei et al. / Life Sciences 78 (2005) 495–505
N
N
N
CH3
CH3
CH
3 LiAlH4
1. POCl3
H2/Pd
2. hydrolysis
O
N
O
O
N
N
N
CH3
CH3
CH3
CH3
1,1'-Dimethyl-
1,1'-Dimethyl-
(2,3'-bipyrrolidiny)l-
1'-one
2,3'-bipyrrolidinyl
DiPyr
DiPyr Amide
Mixture of two
pairs of
enantiomers
Mixture of two pairs
of enantiomers
Fig. 7. Synthesis of 1,1V-dimethyl-[2,3V]bipyrrolidinyl.
Synthesis of 3,5-bis-(1-methyl-pyrrolidin-2-yl)pyridine
(8.4, molecular ion), 181 (18.9), 164 (40.1), 136 (38.3), 104
(11.9), 77 (18.5), 69 (100), and 50 (24.7).
This new nicotine-like alkaloid was prepared utilizing a
similar synthetic strategy to that described for the synthesis of
nicotine (Spa¨th and Bretschneider, 1928) (see Fig. 8), utilizing
pyridine-3,5-dicarboxylic acid as starting material.
3,5-Bis-(1-methyl-pyrrolidin-2-yl)pyridine
A solution of dimethyl pyridine-3,5-dicarboxylate (2.5 g,
14.8 mmol) and 1-methyl-2-pyrrolidinone (3.7 g, 37 mmol) in
25 ml of anhydrous tetrahydrofuran was added drop-wise,
through a dropping funnel, to a suspension of sodium hydride
(2 g, 50% in mineral oil, 42 mmol, rinsed with benzene) in 100
ml of anhydrous tetrahydrofuran, under a nitrogen atmosphere,
with magnetic stirring. The speed of addition was carefully
controlled such that the reaction temperature never exceeded
ambient temperature. After addition, the reaction mixture was
heated under reflux for 10 h, and after cooling to room
temperature, the tetrahydrofuran was removed by evaporation
under vacuum. To the resulting residue was added a mixture of
5 ml of concentrated hydrochloric acid in 5 g of ice to dissolve
all the solids. Additional concentrated hydrochloric acid (85
ml) was added to the mixture, which then was heated under
reflux for 20 h. The mixture was evaporated to dryness under
vacuum and elevated temperature. The residue was dissolved
in 50 ml of cold water, and the pH of the solution was adjusted
Dimethyl pyridine-3,5-dicarboxylate
Pyridine-3,5-dicarboxylic acid (5 g, 30 mmol) was refluxed
in 25 ml of thionyl chloride for 25 h. After cooling to room
temperature, the excess thionyl chloride was removed by
evaporation under vacuum in a rotary evaporator. Anhydrous
methanol (25 ml) was added to the residue. The clear mixture
was heated at reflux for 10 h. After removing methanol by
evaporation under vacuum, ice-water (20 ml) was added to the
residue and the pH was adjusted to 10 by adding saturated
aqueous Na2CO3 solution. The dimethyl ester product precip-
itated out as a white solid, and was collected by filtration under
suction, washed with cold water until the pH of the filtrate was
neutral, and dried under vacuum at room temperature
overnight. The product (5.1 g, 87.4% yield) melted at 82–84
-C [lit. 83.5–85 -C (Eliel et al., 1953)]; MS: m/z 196 (5.7), 195
O
O
2
O
O
O
O
O
N
H3C
CH3
CH3
O
O
CH3
H3C
N
N
NaH, THF
Refluxed for
10 hours
N
N
1. HCl,Refluxed.
for 20 hours
2. NaOH
NaBH4/H2O
N
N
CH3
N
N
CH3
CH3
CH3
N
N
3,5-bis-(1-methyl-
pyrrolidin-2-yl)-
pyridine
3,5-bis-(1-methyl-
pyrrolidin-2-enyl)-
pyridine
DINIC
Fig. 8. Synthesis of 3,5-bis-(1-methyl-pyrrolidin-2-yl)-pyridine.

X. Wei et al. / Life Sciences 78 (2005) 495–505
501
to 10 with 10% aqueous sodium hydroxide solution under
stirring and cooling in an ice-water bath. Sodium borohydride
(2.5 g, 66 mmol) was added to this alkaline solution with
stirring (magnetic) at room temperature, followed by contin-
uous stirring for 20 h. Methylene chloride (4Â25ml) was then
added to extract the desired product from the reaction mixture.
The combined organic extracts were dried over anhydrous
potassium carbonate and filtered. After removal of the solvent,
the residue was purified through a silica gel column using
methanol in chloroform (10:90, v/v) as eluent. The pale-yellow
oily product (600 mg, 19% overall yield) was obtained as a
single diastereomer, and exhibited the same retention time by
GC-MS analysis as was obtained for the third minor alkaloid
identified in the root extract; it also exhibited an identical mass
production and manufacture of potential new medicinal
products from the tobacco plant. In this study, we analyzed
both field and greenhouse grown tobacco roots with a sensitive
analytical gas chromatographic unit connected to an MS
detector in the SIM mode to identify new tobacco alkaloids,
and to carry out a preliminary examination of their pharma-
cological activities at nicotinic receptors. These studies have
identified a novel pyridine derivative, 3,5-bis-(1-methyl-
pyrrolidin-2-yl)-pyridine, and a pair of previously unreported
diastereomers of 1,1V-dimethyl-[2,3V]bipyrrolidinyl from the
root of N. tabacum plants. The structures of these new
alkaloids have been confirmed by total synthesis.
Although 1-methyl-1,2-pyrrolinium ion has previously been
isolated from N. tobacum root (Feth and Wagner, 1989), there
are no previous reports on the isolation of the diastereomeric
1,1V-dimethyl-[2,3V]bipyrrolidinyls from tobacco root extracts.
1-Methyl-1,2-pyrrolinium ion has previously been synthesized
by several routes: (a) the reduction of 1-methylpyrrolidin-2-one
with either lithium aluminum hydride or sodium bis-(2-
methoxyethoxy) aluminum hydride (Swan and Wilcock,
1974); (b) the oxidation of 1-methylpyrrolidine with mercuric
(II) acetate in aqueous acetic acid solution (Leonard and Cook,
1959); and (c) the hydrolysis of 4-methylaminobutan-1-al
diethyl acetal in acidic solution (Leete et al., 1988). In all three
of these reactions, a dimeric by-product was isolated and
identified as 1-methyl-3-(1V-methyl-2V-pyrrolidinyl)-2,3-pyrro-
line, which has also been reported to be formed during the
reduction of 1-methylpyrrole by zinc in hydrochloric acid
(Lukes et al., 1959). Hydrogenation of this product over PtO2
in dilute HCl afforded a mixture of the diastereomers of 1,1V-
dimethyl-[2,3V]bipyrrolidinyl (Lukes et al., 1959). We prepared
1,1V-dimethyl-[2,3V]bipyrrolidinyl as a mixture of the two
possible diastereomers via the self-condensation of 1-methyl-
pyrrolidin-2-one under acidic conditions (Fig. 7). After
catalytic hydrogenation of the resulting enone product with
H2/Pd, subsequent LiAlH4 reduction afforded 1,1V-dimethyl-
[2,3V]bipyrrolidinyl as a 1:6 mixture of diastereomers. 1-
Methyl-1,2-pyrrolinium ion exists in aqueous solution at
neutral pH in equilibrium with its enamine form: 1-methyl-
2,3-pyrroline, which is formed by the loss of a proton from the
iminium ion. As a nucleophile, this enamine will attack the
more electro-positive 2-position of the 1-methyl-1,2-pyrroli-
nium ion species to afford 1-methyl-3-(1V-methyl-2V-pyrrolidi-
nyl)-1,2-pyrrolinium ion. After losing a proton, 1-methyl-3-(1V-
methyl-2V-pyrrolidinyl)-2,3-pyrroline is formed (Fig. 9). (Note:
the formation of this condensation product in the plant requires
no enzyme).
1
fragmentation pattern. Examination of the H-and 13C-NMR
spectra of the synthetic compound indicated it to be a pure
diastereomer: 1H-NMR y (CDCl3) 8.42 (2 H, d, H-2 and H-6),
7.68 (1 H, dd, H-4), 3.25 (2 H, ‘‘t’’, 2ÂH-2V), 3.09 (2 H, ‘‘t’’,
2ÂH-5a), 2.30 (2 H, dd, 2ÂH-5b), 2.20 (2 H, m, 2ÂH-3a),
2.16 (6 H, s, 2ÂCH3), 1.96 (m, 2ÂH-3b), 1.78 (4 H, m,
2ÂH2-4 ppm ; 13C-NMR (CDCl3) y 22.8, 35.4, 40.7, 57.3,
69.2, 134.0, 138.6, 148.5 ppm. HRMS: m/z 245.19.
[3H]Dopamine release assay
Striatal slices (500 Am thickness, 6–8 mg weight) from
male Sprague–Dawley rats were obtained and [3H]dopamine
release assays were performed as previously described (Grine-
vich et al., 2003).
[3H]Nicotine and [3H]methyllycaconitine binding assays
The new alkaloids were also evaluated in the [3H]nicotine
and [3H]methyllycaconitine binding assays, utilizing previous-
ly reported methods (Zheng et al., 2005).
In all the above assays, concentrations of inhibitor
molecules that produced 50% inhibition (IC50 values) were
determined from the concentration–effect curves via an
iterative curve-fitting program (Prism 3.0; GraphPad Software
Inc., San Diego, CA). Inhibition constants (Ki values) were
determined using the Cheng–Prusoff equation (Cheng and
Prusoff, 1973).
Results and discussion
Most of the previous studies on the isolation and structural
identification of tobacco alkaloids have focused on the leaf
tissues because of the commercial value of this part of the
tobacco plant in the production of tobacco products. Never-
theless, studies on the root system are as important as studies
on leaf tissues, since many of the alkaloidal components in the
tobacco plant, and especially the principal alkaloid, nicotine,
are synthesized in the roots before being transported to other
parts of the plant. The tobacco root can be easily transformed
by Agrobacterium rhizogenes infection to produce Fhairy root_
(Bush et al., 1999). These hairy roots have been utilized in the
elucidation of the biosynthetic pathways for different alkaloidal
components, and also in exploring the possibilities for the
The levels of 1-methyl-1,2-pyrrolinium ion in the tobacco
plant root system have been determined utilizing an HPLC
method (Feth and Wagner, 1989). 1-Methyl-1,2-pyrrolinium
salt was extracted from root samples, and detected and
quantified as 1-methyl-2,3-pyrroline in 0.5 M HCl. The levels
of this alkaloidal component in N. tabacum and N. glutinosa
were found to be 28 Ag/g and 84 Ag/g fresh weight, respectively,
in the root systems from these plants. Since the physiological
pH in growing tobacco plants is around 7.2, both 1-methyl-1,2-
pyrrolinium ion and 1-methyl-2,3-pyrroline should be present in

502
X. Wei et al. / Life Sciences 78 (2005) 495–505
N
N
N
+
N
CH3
CH3
CH3
-H+
+H+
Reduction
-H+
CH3
+
N
+
+H+
N
N
N
N
CH3
CH3
CH3
CH3
CH3
Diastereomers
Fig. 9. Formation of 1,1V-dimethyl-[2,3V]bipyrrolidinyl.
the root systems, thereby providing a significant opportunity for
the condensation of these two species to occur to afford dimeric
2,3V-connected bipyrrolidino alkaloidal species.
two 1-methyl-2-pyrrolidinyl moieties are attached to a central
pyridine moiety. From biosynthetic pathway considerations,
and the mass spectrum of this unknown component, the most
probable structure for this compound was considered to be the
nicotine-related structure: 3,5-bis-(1-methyl-pyrrolidin-2-
yl)pyridine. This compound was synthesized utilizing an
unambiguous synthetic procedure. The proton NMR spectrum
of the synthetic product was consistent with its proposed
structure, and was obtained as a pure diastereomeric product.
This synthesized product has two chiral carbon atoms at the C-
2V-and C-2µ positions of each of the two 1-methyl-2-pyrroli-
dinyl rings. Since there was no regioselective control in the
reduction step with sodium borohydride, the product was
expected to be obtained as a mixture of three possible
stereoisomers: i.e. the cis-isomer (meso form), which is
optically inactive because of symmetry in the molecule, and
a pair of trans-enantiomers. It is likely that the separation of the
cis- and trans-products is possible on an ordinary achiral GC
column under normal GC conditions. Different GC temperature
programs were used to assess the purity and composition of the
synthetic product. Under all programs examined, the product
afforded only one peak in the GC chromatogram.
The total ion chromatogram of an extract from a green house-
grown tobacco root sample is illustrated in Fig. 4. Nicotine
appears at a retention time of 9.2 min. The mass spectra of the
two diastereomers of 1,1V-dimethyl-[2,3V]bipyrrolidinyl eluting
at retention times of 7.77 and 7.89 min in this total ion
chromatogram are shown in Fig. 5. The retention times for
these two alkaloids were identical to the retention times of the
synthetic mixture of 1,1V-dimethyl-[2,3V]bipyrrolidinyl diaster-
eomers (obtained as a mixture of the two possible diastereomers
in a 1:6 molar ratio). Also, the mass spectra of both
diastereomers in the synthetic sample and the two isomeric
components isolated from tobacco root were identical. The base
peaks for both diastereomers were observed at m/z 84, which
corresponds to the 1-methyl-1,2-pyrrolinium ion formed from
the cleavage of the two 1-methylpyrrolidine rings. The
molecular ion appears at m/z 168. Loss of one methyl group
from the molecular ion affords an ion at m/z 153 ion. Other major
ions appear at m/z 136, 124, 110, 96, 82, 67 and 42. Separation of
the synthetic diastereomers was attempted through different
chromatographic techniques; however, none of the approaches
utilized were successful. The N-methyl groups in each diaste-
reomer could be clearly differentiated in the NMR spectrum,
allowing a molar ratio for the two diastereomers to be
determined. However, it was not possible to assign the correct
stereochemistry to each of the diastereomeric products. Since the
GC retention times of the two diastereomeric dipyrrolidino
alkaloids isolated from the tobacco root system were identical to
the two synthetic diastereomeric products under a variety of
different temperature conditions, we conclude that the two
diastereomers of 1,1V-dimethyl-[2.3V]bipyrrolidinyl are identi-
fied as new tobacco alkaloids in the tobacco root system.
A third alkaloidal component eluting with a retention time
of 14.72 min in the total ion chromatogram of the basic extract
from the tobacco root sample was also investigated. The mass
spectrum of this component is illustrated in Fig. 6. The alkaloid
has a base ion at m/z 84 and other major ions at m/z 245, 230,
216, 189, 161, 132, and 42. The ion at m/z 84 is attributable to
the 1-methyl-1,2-pyrrolinium ion formed by cleavage of a 1-
methyl-2-pyrrolidinyl moiety from the molecule. The m/z 161
ion led us to assume that the alkaloid was a nicotine derivative,
since the loss of a proton from nicotine molecule affords the
same ion. From the mass of the molecule (m/z 245), one
possible structure was considered to be a molecule in which
The above unknown alkaloid extracted from the tobacco
root system had an identical mass spectrum and retention time
to the synthetic 3,5-bis-(1-methyl-pyrrolidin-2-yl)pyridine.
However, it is not possible at this time to determine the correct
configuration of this pure synthetic diastereomer until further
stereochemical analyses can be performed. Thus, the chemical
identity of this new alkaloidal component is assigned as a pure
diastereomer of 3,5-bis-(1-methyl-pyrrolidin-2-yl)pyridine.
Since N. tabacum plants can only biosynthesize S-(À)-
nicotine, this new alkaloid is tentatively assigned as the 2V-
S,2µ-S-enantiomer of 3,5-bis-(1-methyl-pyrrolidin-2-yl)pyri-
dine. Studies on the configuration of this new alkaloid are
currently underway.
Two possible biosynthetic pathways in the tobacco plant
may lead to this new alkaloid, i.e. biosynthesis from pyridine
3,5-dicarboxylic acid, or from 3,6-dihydronicotine, a key
intermediate in nicotine biosynthesis. Pyridine 3,5-dicarboxylic
acid has been identified in extracts of tobacco root systems by
esterification with dry hydrochloride in methanol and subse-
quent analysis of the resulting dimethyl ester by GC-MS (Wei,
2000). Another key intermediate, nicotine-5-carboxylic acid
has also been identified as its methyl ester in the same
analytical sample. Although the levels of these two acids are
very low in the root, the presence of these intermediates

X. Wei et al. / Life Sciences 78 (2005) 495–505
503
HOOC
COOH
HOOC
COOH
COOH
CO2
[H]
N
N
N
H
3,5-Dinicotinic acid
1,2-Dihydronicotinic acid
2,5-Dihydro-3,5-dinicotinic
acid
H
H
+
HOOC
HOOC
N
N
N
[O]
[H]
CH3
CH3
CH3
N
N
5-Carboxylnicotine
5-Carboxyl-3,6-dihydronicotine
CO2
+
N
H
H
HOOC
N
CO2
CH3
N
CH3
CH3
N
N
H
5-Carboxyl-2,5-dihydronicotine
1,2-Dihydronicotine
H
H
H
H
N
N
[O]
N
N
CH3
H3C
CH3
H3C
N
N
2,5-Dihydro-3,5-dinicotine
3,5-Dinicotine
Fig. 10. Formation of 3,5-bis-(1-methyl-pyrrolidin-2-yl)pyridine from pyridine 3,5-dicarboxylic acid.
H
H
+
N
N
N
CH3
CH3
CH3
N
N
H
3,6-Dihydronicotine
1,2-Dihydronicotine
H
H
H
H
[O]
N
N
N
N
H3C
CH3
H3C
CH3
N
N
2,5-Dihydro-3,5-dinicotine
3,5-bis-(1-Methyl-pyrrolidin-2-yl)pyridine
DINIC
Fig. 11. Formation of 3,5-bis-(1-methyl-pyrrolidin-2-yl)pyridine from the 1-methyl 1,2-pyrrolinium ion intermediate involved in nicotine biosynthesis.

504
X. Wei et al. / Life Sciences 78 (2005) 495–505
2.5
Table 1
Affinity of minor alkaloids and related compounds for a4h2* and a7* nAChRs
2.0
1.5
1.0
0.5
0.0
Compounda
[3H]Nicotine
[3H] Methyllycaconitine
Ki TSEMb (AM)
Ki TSEM (AM)
DiPyr
15.8T6.17
>100
>100
>100
>100
>100
>100
DiPyr Amide
DM 3,5
DINIC
>100
1.18T0.06
10.7T1.42
DINORNIC
a
For full structures of these compounds, see (Figs. 7, 8 and 12).
b
N =3–4 independent experiments/compound.
control
-8.0
-7.5
-7.0
-6.5
-6.0
log [1,1'-Dimethyl-[2,3']bipyrrolidinyl-2'-one] (M)
constitute the basis for the biosynthetic pathway to 3,5-bis-(1-
methyl-pyrrolidin-2-yl)pyridine shown in Fig. 10. In this
respect, it is also worth noting that when pyridine 3,5-
dicarboxylic acid was fed to green house grown tobacco
plants, higher levels of 3,5-bis-(1-methyl-pyrrolidin-2-yl)pyr-
idine were obtained in both leaf and root samples.
2.0
1.5
1.0
0.5
0.0
3,6-Dihydronicotine is formed as a key intermediate from
the condensation between 3,4-dihydropyridine and 1-methyl-
1,2-pyrrolinium ion. This intermediate can be isomerized to
3,4-dihydronicotine, similar to the formation of 3,4-dihydro-
pyridine from 2,5-dihydropyridine (Bush et al., 1999). It is
feasible that 3,4-dihydronicotine can condense with 1-methyl-
1,2-pyrrolinium ion at the 2-position, to form 2,5-dihydro-3,5-
di-(1-methyl-2-pyrrolidinyl)pyridine, and after further oxida-
tion, 3,5-bis-(1-methyl-pyrrolidin-2-yl)pyridine is obtained
(Fig. 11). However, as yet, there is no substantial proof for
this pathway.
control
-8.0
-7.5
-7.0
-6.5
-6.0
log [1,1'-Dimethyl-[2,3']bipyrrolidinyl] (M)
Fig. 13. Minor tobacco alkaloids and related compounds inhibit nicotine-
evoked [3H]dopamine ([3H]DA) release from superfused rat striatal slices.
(N =4 independent experiments/compound).
We have evaluated the pharmacological properties of these
new alkaloids and three structurally related compounds on
different subtypes of nAChRs. These compounds were
assessed for their ability to inhibit the binding of [3H]nicotine
and [3H]methyllycaconitine to a4h2* and a7* nAChRs using
standard procedures and rat brain homogenates (Table 1).
None of the compounds inhibited [3H]methyllycaconitine
binding, indicating that they do not interact with a7*
nAChRs. With respect to the a4h2* site, several of the
compounds inhibited [3H]nicotine binding to rat brain
membranes. The most potent of the compounds was 3,5-bis-
(1-methyl-pyrrolidin-2-yl)pyridine (Fig. 8) (DINIC), which
exhibited a Ki of 1.18 AM, followed by 3,5-di-pyrrolidin-2-yl-
pyridine (Fig. 12) (DiNORNIC; Ki =10.7 AM) and 1,1V-
dimethyl-[2,3V]bipyrrolidinyl (Fig. 7) (DiPyr; Ki =15.8 AM).
Thus, several of the above compounds demonstrated affinity
at a4h2* nAChR, although this affinity was significantly
lower than that of S-(À)-nicotine for this nicotinic receptor
subtype.
All five of compounds were also assessed for their ability to
inhibit nicotine-evoked [3H]dopamine release from superfused
rat striatal slices. The intrinsic activity (ability of the
compounds to evoke [3H]dopamine release) was also deter-
mined in the same experiments. Initially, the effect of a single
concentration (100 nM) of alkaloid was investigated. None of
the compounds exhibited intrinsic activity at this concentration.
Importantly, four of the five compounds showed inhibition
(36–64% inhibition) of the effect of nicotine in the [3H]dopa-
mine release assay. A greater range of concentrations (10 nM–
1 AM) were examined for two of these compounds. At 10 nM,
the synthetic analogue 1,1-dimethyl-[2,3V]bipyrrolidinyl-2V-one
(Fig. 7) (DiPyr Amide) exhibited 50% inhibition of nicotine-
evoked [3H]dopamine release, and maximal inhibition was
N
N
N
H
N
H
N
N
3,5-Di-pyrrolidin-
2-yl-pyridine
3,5-Bis-(4,6-dihydro-3H-pyrrol-2-yl)-
pyridine
DM 3,5
DINORNIC
Fig. 12. Structure of DINORNIC (3,5-di-pyrrolidin-2-yl-pyridine) and DM 3,5 (3,5-bis-(4,6-dihydro-3H-pyrrol-2-yl)-pyridine.

X. Wei et al. / Life Sciences 78 (2005) 495–505
505
Feth, Friedhelm, Wagner, Karl G., 1989. Determination of ornithine, putrescine,
N-methylputrescine and N-methylpyrroline pools in tobacco tissue by high-
performance liquid chromatography. Physiologia Plantarum 75, 71–74.
Grinevich, V.P., Crooks, P.A., Sumithran, S.P., Haubner, A.J., Ayers, J.T.,
Dwoskin, L.P., 2003. N-Alkylpyridinium analogs, a novel class of nicotinic
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observed at this low concentration (Fig. 13). One-way repeated
measures ANOVA revealed a significant concentration-depen-
dent effect of DiPyr Amide ( F5,2 =3.465, p <0.05). Fig. 13 also
illustrates the concentration response for 1,1V-dimethyl-
[2,3V]bipyrrolidinyl (DiPyr)-induced inhibition of nicotine-
evoked [3H]dopamine release. DiPyr also produced significant
inhibition ( F5,17 =2.832, p <0.05), and concentrations of 300
nM and 1 AM produced 57% and 70% inhibition, respectively
(Fig. 13). Thus, these results suggest that these compounds
appear to act as antagonists at nAChRs mediating nicotine-
evoked dopamine release from rat striatum.
Leete, E., Kim, S.H., Rana, J., 1988. Chemistry of the tropane alkaloids and
related compounds. Part 38. The incorporation of [2-13C, 14C,15N]-1-
methyl-D1-pyrrolinium chloride into cuscohygrine in Erythroxylum coca.
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Leonard, N.J., Cook, A.G., 1959. Unsaturated amines: XIV. The mercuric
acetate oxidation of substituted pyrrolidines. Journal of the American
Chemical Society 81, 5627–5631.
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This project was funded under the NIH grant number U19
DA 017548.
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