Coaxing a pyridine nucleus to give up its aromaticity: Synthesis and pharmacological characterization of novel conformationally restricted analogues of nicotine and anabasine

Article abstract of DOI:10.1021/jm049707c

A series of novel nicotine and anabasine related conformationally restricted compounds including those with π-bonds in the connecting tether were synthesized following the hitherto unprecedented phenylsulfanyl group assisted generation of pyridine o-quino

Full text of DOI:10.1021/jm049707c

J. Med. Chem. 2004, 47, 6691-6701
6691
Coaxing a Pyridine Nucleus To Give Up Its Aromaticity: Synthesis and
Pharmacological Characterization of Novel Conformationally Restricted
Analogues of Nicotine and Anabasine#
Tarun K. Sarkar,*,† Sankar Basak,† Irving Wainer,‡ Ruin Moaddel,‡ Rika Yamaguchi,‡ Krzysztof Jozwiak,‡
Hui-Ting Chen,§ and Chun-Cheng Lin§
Department of Chemistry, Indian Institute of Technology, Kharagpur-721302, India; Gerontology Research Center, National
Institute on Aging, National Institutes of Health, Baltimore, USA; Institute of Chemistry, Academia Sinica, Taipei, Taiwan
Received April 18, 2004
A series of novel nicotine and anabasine related conformationally restricted compounds
including those with π-bonds in the connecting tether were synthesized following the hitherto
unprecedented phenylsulfanyl group assisted generation of pyridine o-quinodimethane inter-
mediates and their trapping by an intramolecular Diels-Alder reaction. Pharmacological
characterization of some of these analogues at activating R3â4 nAChRs was investigated, and
constrained anabasine analogues 35 and 43 as well as constrained nicotine analogue 42 were
found to exhibit moderately potent nicotinic agonist activity. Of special note is the fact that
the pyrrolidinic nitrogen in these compounds is bound to a carbomethoxy group and, therefore,
is not free to be protonated unlike all the known analogues of nicotine and anabasine, specifically
designed as nAChRs agonists/antagonists. The structure-activity relationship studies indicate
that when π-cation interaction is absent, the position of chlorine atom in the pyridine ring
and steric bulk at the connecting tether between the pyridine and pyrrolidine ring of the
constrained nicotinic ligands are important descriptors for their binding affinity at R4â2 and
R3â4 nAChRs as well as the subtype selectivity issue. These findings are likely to improve our
understanding of the structural requirements for selectivity, which, at present, is probably
the most important goal in the field of nicotinic ligands.
Introduction
In the last several decades, pharmacologists and
chemists have focused an immense amount of research
on the nicotinic receptor.1-6 The critical neurotransmis-
sion pathway is found in both mammals and insects,
and, in humans, the nicotinic pathway is associated with
pain7 and other chronic neurological disorders such as
Figure 1. Structures for nicotine and anabasine.
Alzheimer’s disease,8-10 Parkinson’s disease,10-12 Tour-
ette’s syndrome,13 schizophrenia,4 attention-deficit/
hyperactivity disorder,14 and depression.15 Critical limi-
tations for the potential use of nicotine as a therapeutic
agent are its peripheral side effects that are due to the
nonselective profile of nicotine. Compounds that target
specific nAChR subtypes in the CNS may not induce
these adverse effects and yet retain the beneficial effects
of nicotine. One way to achieve this goal is to modify
the parent biomolecule, e.g. 1/2 (Figure 1), in such a way
that its original conformational mobility is severely
limited to one particular conformation.6 In light of this
a variety of constrained nicotine analogues, e.g. 3-18
(Figure 2), have been synthesized.16-31 Among these,
analogues 4, 8, 10, and 14 were evaluated as agonists,
while 7 and 9 were evaluated as antagonists for neu-
ronal nicotinic acetylcholine receptors (nAChRs). In
particular, compound 4 (n ) 1), which selectively
activates human recombinant R2â4 and R4â4 nAChRs,
has been shown to be active in animal models of
Parkinson’s disease and pain.23 In order to develop novel
conformationally restricted analogues of nicotine and
anabasine, we settled on 19 (Figure 3), a reported
constrained analogue of nicotine, because it provides a
reasonable mimic of a trans conformer of nicotine that
is supported by crystal structure data.20,31 It may be
mentioned here that, for the monoprotonated species of
nicotine in aqueous solution, there are four major
conformations present, of which the two approximately
isoenergetic trans rotamers are preferred over their cis
counterparts by >10:1 (Figure 4).32 We envisioned that
the ability to “freeze out” the conformational dynamics
of nicotine at various torsion angles (abcd) maintaining
skew orientation of pyridine and pyrrolidine rings as
in 19 might help to identify conformational factors
which appear to be critical for the interaction with
various nAChR subtypes.
# This paper is dedicated to Professor Tim Gallagher for his
outstanding contribution to the development of new nicotinic agonists.
* Author to whom correspondence should be addressed. E-mail:
tksr@chem.iitkgp.ernet.in. Phone: +91-03222-283330. Fax: +91-3222-
255303.
The binding units or additional substituents which
may be used to restrict the conformational flexibility of
nicotine may themselves interact with the portion of
binding site, either favorably or unfavorably, and in turn
impart some selectivity for one subtype or another.
† Indian Institute of Technology.
‡ National Institutes of Health.
§ Academia Sinica.
10.1021/jm049707c CCC: $27.50 © 2004 American Chemical Society
Published on Web 12/08/2004

6692 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27
Sarkar et al.
Figure 2. Selected conformationally constrained analogues of nicotine and anabasine.
Scheme 1
Figure 3. A mimic of a trans conformer of nicotine.
logues of nicotine and anabasine and their pharmaco-
logical activity for the R4â2 and R3â4 subtypes of the
nicotinic receptor.
Results and Discussion
Figure 4. The major conformational isomers of nicotine in
Chemistry. A simple retrosynthetic analysis for both
conformationally constrained nicotines and anabasines
20 shows that these compounds are also derivable via
the generation of pyridine o-quinodimethane intermedi-
ates 21 through formal imine tautomerization of 22,
available from the corresponding aldehyde 23, and its
subsequent intramolecular trapping (Scheme 1). Al-
though the chemistry of heterocyclic o-quinodimethanes
has registered a burgeoning growth in recent years,35-44
they have found, in general,45-47 and pyridine o-quin-
odimethanes,41 in particular, only limited applications
in the synthesis of natural products.
To investigate the feasibility of this approach, we
synthesized imines 27 and 28 from the dichloro-
substituted aldehyde 2448 (Scheme 2). The choice of
chloro substituents flanking the pyridine nitrogen was
crucial for this study: the C-2 chloro group was selected
for enhanced cis-stereoselectivity at the ring juncture
in the Diels-Alder step, while the C-6 chloro group was
aqueous solution.
Disappointingly, the single synthetic route17,20,21,23,27,28,31
followed over a period of two decades for the ring
systems 4 - 9 does not appear to be viable for the
synthesis of a large diversity of bridged nicotines and
anabasines required to probe the conformation of (S)-
nicotine which induces ion channel opening. Therefore,
development of an alternate route which is easy, high-
yielding, and flexible was desirable. Recently, we dem-
onstrated a route to constrained anabasines (cf. 4, n )
2) involving a domino [4 + 2] cycloaddition/ring open-
ing-elimination sequence of 3-amino-substituted furo-
[3,4-c]pyridines.33 Unfortunately, this approach turned
out to be inefficient in the case of constrained nicotines
(cf. 4, n ) 1).33,34 In this article we describe an alterna-
tive synthesis of both conformationally restricted ana-

Analogues of Nicotine and Anabasine
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27 6693
Scheme 2a
a
i
(a) CH2dCHCH2(CH2)nNH2, benzene; (b) Pr2NEt, ClCO2Me, xylene.
chosen for stronger binding affinity of constrained
nicotines and anabasines toward nAChRs, as will be
apparent from a later discussion. Unfortunately, treat-
ment of 27 or 28 with methyl chloroformate in the
presence of Hu¨nig’s base and refluxing the mixture in
xylene for an extended period49 did not give any traces
of 32 or 33. This failure may be due to (i) the low acidity
of methyl hydrogen which does not lend itself to rapid
abstraction, (ii) recalcitrance of the pyridine nucleus to
give up its aromaticity, or (iii) in situ generated o-
quinodimethane intermediate being not sufficiently
reactive to allow a Diels-Alder reaction to occur. It
should be borne in mind that the aromaticity of pyridine
is of the same order as that of benzene,50 and the
previous generation of indole 2,3-o-quinodimethanes49
(or pyrrole o-quinodimethanes51) via a formal imine
tautomerization protocol necessitated inductive deacti-
vation of the indolic nitrogen lone pair of electrons by
an arylsulfonyl47 or a carboalkoxy group.52 In order to
overcome these problems we deemed it important to
replace one of the hydrogens on the methyl group in the
imine by a substituent which would not only make the
methyl hydrogens more acidic (cf. 22) but also stabilize
the o-quinodimethane intermediates (cf. 21). A phenyl-
sulfanyl group appeared to fulfill these criteria; also it
lends itself to easy reductive removal. In the event, we
prepared imine 29 from 25, an aldehyde which was
prepared from commercially available ethyl 4-chloroac-
etoacetate.53,54 With 29 in hand, we then carried out the
formal imine tautomerization reaction using methyl
chloroformate in the presence of diisopropylethylamine
and were pleased to note that the desired constrained
nicotine 34 was formed as a white crystalline solid (mp
156-157 °C) in 44% yield. The structure and stereo-
chemistry of 34 were assigned on the basis of extensive
NMR measurements and finally confirmed by X-ray
crystallographic study (Figure 5). Interestingly, no other
diastereomers could be traced in the crude reaction
product by NMR spectroscopy.
Figure 5. ORTEP perspective view of 34.
the alternate transition state 39 (endo-E,E) as the latter
is badly strained (Figure 6). The other most likely
transition states, e.g. 40 (exo-Z,E) and 41 (endo-Z,E),
are disfavored due to steric interaction between the
substituents at C-2 and C-8.
In order to explore the scope and limitations of this
methodology we have also examined the formal imine
tautomerization protocol for the synthesis of conforma-
tionally constrained anabasines. Thus, under conditions
similar to those described for 34, treatment of imine 30
with methyl chloroformate in the presence of diisopro-
pylethylamine followed by preparative thin-layer chro-
matography gave an oil in 40% yield which was homo-
geneous on TLC. Proton NMR of this oil showed that it
was a mixture of diastereomers corresponding to the
desired Diels-Alder adducts. The diastereomers do not
resolve on TLC (silica gel), and their relative abundance
(69:25:6) was determined by LC-MS analysis. Finally,
the major diastereomer 35 (mp 154-155 °C) was
separated by preparative HPLC (column: ZORBAX SIL,
4.6 mm × 25 mm, ethyl acetate:hexane ) 1:9). Unlike
34, we failed to grow suitable crystals of 35 for X-ray
analysis. The structure and stereochemistry of 35 were
settled by a combination of 1H and 13C NMR, 1D
homonuclear decoupling experiments, COSY, HMQC,
NOESY, and HRMS studies. The transition state 38
(exo-E,E) is once again favored in this case.
The exclusive formation of 34 is explicable in terms
of transition state 38 (exo-E,E) which is favored over

6694 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27
Sarkar et al.
Figure 6. Plausible transition structures for the formation of 34.
Scheme 3a
Scheme 4
Table 1. Conformational Parameters for Compounds 34 and
42, Obtained from X-ray Study
torsion angle (deg)
N-N distance
compd C11-C10-C9-C6 C11-C10-C9-N2
(Å)
34
42
-156.24
-144.38
86.06
101.21
4.638
a
following the same route developed for 46. In addition,
the carbamate group in 34 can be removed only by
refluxing the mixture of 34 and TMSI in acetonitrile
giving 49 in 70% yield. It may be mentioned here that
49 is a highly acid sensitive compound, and even traces
of acid present in CDCl3, CHCl3, etc. are able to
decompose it. Therefore, further studies with 49 ought
to be carried out very carefully, and acetone, DMSO,
etc. are found suitable solvents for this purpose.
Furthermore, controlled hydrogenation of 42 using
10% Pd-C catalyst gives monochlorinated product 50,
the structural assignment of which was based on the
1H NMR spectroscopic data (Scheme 4).
It may be mentioned here that 34-37, 42-45, 49, and
50 are novel constrained nicotine and anabasine ana-
logues. X-ray crystallographic data indicates that con-
formationally constrained nicotine analogue 4255 has a
somewhat different conformation than 34 (Table 1).
With 42 and 43 in hand, we embarked upon a
program to modify the tether with the hope of generat-
ing a large number of conformationally restricted bio-
molecules having altered torsion angles (abcd). Thus,
bromination of 42 was carried out in dichloromethane
at 0 °C, and the resulting brominated product was found
to be as a mixture of two diastereomers 51 and 52 in a
ratio of 2:1 (NMR), respectively (Scheme 5). Epoxides
54 and 55 were synthesized by a standard synthetic
protocol and isolated as a chromatographically (silica
gel) inseparable mixture (4:1). The diol 53 was obtained
as a pure crystalline solid after chromatographic sepa-
ration from the other minor cis-diol. The ratio of 53 and
the other minor cis-diol was not determined. Semiem-
pirical PM3 calculations showed that the torsion angle
(abcd) in each of these compounds 51-55 is different
from the others.
(a) NaIO4, MeOH-H2O, room temperature; (b) ∆, CH2Cl2; (c)
H2, Pd-C, NaOAc, MeOH; (d) KOH, MeOH; (e) LAH,THF, room
temperature; (f) TMSI, CH3CN.
During the course of our studies, it appeared to us
that better yield of the bridged products may be
achieved by further enhancing the stability of the in situ
generated pyridine o-quinodimethane intermediates.
For this purpose, we have also investigated the chem-
istry of the closely related sulfone-substituted imine 31,
available from 26, which in turn was prepared by
oxidation (RuCl3/NaIO4) of 25. However, an inseparable
(TLC, SiO2) mixture of two diastereomers is formed in
a ratio of 65:35 (42%), which were separated by pre-
parative HPLC to give 36 (mp 213-214 °C) and 37 (mp
210-211 °C). The structure and stereochemistry of 36
and 37 are based on extensive NMR (1H and 13C NMR,
1D homonuclear decoupling experiments, COSY, HSQC,
NOESY) as well as HRMS studies. Obviously, the major
diastereomer 36 is forming via the transition state 38
(exo-E,E) as encountered before. We attribute transition
state 41 (endo-Z,E) for the formation of minor diaster-
eomer 37 where steric interactions presumably twist the
nitrogen atom of the dieneamine segment out of conju-
gation with the diene system which is effectively
stabilized by the phenylsulfonyl group.
The feasibility of our approach for the preparation of
some constrained nicotines of proven bioactivity, e.g.
4623,28 and 48,20 has been shown in Scheme 3. Thus,
NaIO4 oxidation of 34 and thermally induced syn
elimination of the resulting sulfoxide gave 42 (mp 172-
173 °C) in 68% overall yield. One-pot reductive elimina-
tion of chlorine atoms and saturation of the π-bond in
42 followed by deprotection of the carbamate group
under alkaline conditions yielded 46. The compound 44
can be transformed into 48, also of known bioactivity,
in good yield by exposure of 44 to LiAlH4 in Et2O at
room temperature for 3 h. Subsequently, 35 was elabo-
rated into conformationally constrained anabasine 47
Pharmacology. Of the set of novel conformationally
constrained analogues of nicotine and anabasine listed
in this paper, compounds 34, 35, 36, 42, 43, 47, 49, 50,
and 54/55 were tested for their functional activities at

Analogues of Nicotine and Anabasine
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27 6695
Scheme 5a
a
(a) Br2, CH2Cl2, 0 °C; (b) OsO4, Py, dioxane; (c) mCPBA, CH2Cl2.
epibatidine [EC50 ) (28.25 ( 1.63) × 10-3 µM]. Interest-
ingly, constrained anabasine analogue 35 (18.24 ( 3.40
µM) and conformationally constrained nicotine analogue
42 (20.81 ( 2.92 µM) have equivalent EC50 values
relative to nicotine (19.83 ( 1.61 µM). However, in the
case of 49, although the EC50 value was similar to that
of nicotine, the maximum agonist response was 35% of
the response obtained with nicotine, suggesting that the
compound had partial agonist/antagonist properties. A
mixture of epoxides 54 and 55 is found to have a weak
EC50 value (>300 µM), whereas compounds 34, 36, 47,
and 50 show no functional activity at a concentration
of 1 mM and are considered to be ineffective agonists
for R3â4 nAChRs subtype.
Previous pharmacological models based on the R4â2
receptor have suggested that the π-cation interaction
is a key element.1-7 Very recently, using the X-ray
structure of acetylcholine binding protein (AChBP)56 as
template, a structural model of R3â4 neuronal nicotinic
receptor binding domain has been developed.57 In this
model the quaternary ammonium group of nicotine is
found to form a π-cation interaction with RTyr194 and
long-range electrostatic interaction with the RTyr90 side
chain, in accordance with the experimentally character-
ized residues essential for nicotine binding. In addition,
ACh ammonium group as well as cytisine ammonium
group are also bound to the same region as nicotine,
forming a π-cation interaction. However, it should be
noted that the pyrrolidinic nitrogen of 35, 42, and 43 is
bound to a carbomethoxy moiety and, therefore, should
not be protonated. Thus, the π-cation interaction does
not appear to play a role in the activity of these
compounds.
Figure 7. The dose-response curve (EC50) of compound 35
(quadruplicate test of concentrations ranging from 0.279 µM
to 1 mM) for the R3â4 nicotinic receptor was determined by
nonlinear regression of experimental data using sigmoidal
dose-response curve fitting in Graph Pad Prism software. The
stimulation of [86Rb+] efflux was measured and was expressed
as a percentage of total [86Rb+] loaded.
Table 2. Functional Activity for Multiple Compounds Using
the Rubidium Efflux Assay for the R3â4 Subtype of the
Nicotinic Receptor and Maximal Effect Reported Relative to
Nicotine
compd
EC50 (µM)
% maximal response
epibatidine
(28.25 ( 1.63) × 10-3
13.80 ( 2.99
18.24 ( 3.40
19.83 ( 1.61
20.81 ( 2.92
26.36 ( 1.78
>1000
na
75
43
35
100
100
100
35
nicotine
42
49
50
10
36
>1000
10
34
>1000
10
54/55
47
>300
40
>1000
10
To address the issue of binding selectivity among
R4â2 and R3â4 nAChRs subtypes, binding affinity
studies have also been carried out by displacement
chromatographic experiments utilizing immobilized
nAChRs stationary phase; a detailed discussion of the
chromatographic studies is contained in a recent com-
munication.58 Some data from this work are listed in
Table 3. In these studies the ∆ mL (displacement of 3H-
epibatidine) produced by the addition of nicotine or test
ligands is equivalent to the binding affinities of the
ligands. The greater the displacement of epibatidine (∆
mL), the greater the affinity. The chromatographic
R3â4 nAChRs, stably expressed by the KXR3â4R2 cell
line. The nAChRs ligand-stimulated 86Rb+ efflux from
KXR3â4R2 cells was monitored by liquid scintillation
counting. A representative concentration-response curve
for our tested compounds is shown in Figure 7, and EC50
values are also summarized in Table 2. The data from
these studies demonstrate that the constrained ana-
basine analogue 43 has statistically similar potency to
nicotine with regard to activation of R3â4 nAChRs with
EC50 values of 13.8 ( 2.99 µM and 19.83 ( 1.61 µM,
respectively, but it was significantly less potent than

6696 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27
Sarkar et al.
Table 3. Determination of Binding Affinities of Constrained
Nicotine and Anabasine Analogues Using Immobilized R4â2
and R3â4 nAChRs Based Liquid Chromatographic Stationary
Phases
due to the presence of a dominant π-cation interaction.
In the case of 35 replacement of pyrrolidine ring by pip-
eridine ring tolerates bulkiness of the group at the con-
necting tether during its binding to the R3â4 nAChRs
at the activated state; therefore, ring size might be a
factor for functional activity. From this study it is clear
that a steric bulk effect between the pyridine and pyr-
rolidine rings of nicotine may be considered as a para-
meter in refining the nAChRs pharmacophore model.63-65
From the binding studies it appears to us that when
π-cation interaction is absent, tolerability of the above-
mentioned steric bulk at the axial position of the
connecting tether of the ligand by R4â2 nAChRs is
higher than that by R3â4 nAChRs (Table 3). This means
that greater steric bulk due to an axial benzylic sub-
stituent in the connecting tether of the ligand will make
it more R4â2 subtype selective, although it requires
steric balance for affinity. For example, 34 and 36 are
selective for R4â2 subtype over the R3â4 subtype.
However, when steric bulk in the connecting tether is
absent, steric bulk associated with the chloro substitu-
ent at the C-2 position of the pyridine ring makes 50
R4â2 subtype selective. In addition, when both the 2-
and 6-positions of the pyridine ring of the constrained
nicotine are occupied by chloro substituents, then the
effect of the chloro substituent at the C-6 position will
be the dominating factor. This might have a favorable
impact on R3â4 subtype selectivity. For example if we
compare R4â2 and R3â4 chromatographic data of 42 and
nicotine itself, it is clear that 42 shows somewhat more
R3â4 subtype selectivity. Our observations might play
a crucial role for further development of subtype selec-
tive nAChRs ligands.
∆ 3H-epibatidine for
R4â2 nAChRs (mL)
∆ 3H-epibatidine for
R3â4 nAChRs (mL)
sample
3H-epibatidine
42
0.28
0.26
0.23
0.18
0.17
0.13
0.28
0.16
nicotine
44
0.12
50
-0.32
-0.04
-0.12
34
36
studies have demonstrated that various ligands have
the following rank orders of potency for binding to each
R4â2 and R3â4 nAChRs subtype: for R4â2, epibatidine
> 49 > 42 > 54/55 ≈ nicotine > 44 > 43 > 50 > 34 >
36 > 35 > carbachol > 37; for R3â4, epibatidine > 49 >
42 ≈ 54/55 > 43 > nicotine > 44 > 35 > carbachol ≈
37 ≈ 34 > 36 > 50.
These rank order profiles of compound potencies at
R4â2 and R3â4 nAChRs demonstrate an interesting and
significant relationship between structures and activity
(SAR).
Binding data of 49 are fully corroborated with the fact
that π-cation interaction is a primary determinant in
high affinity binding.1-7,59-61 This is also supported by
the observations that many of the constrained ana-
logues, e.g. 34 and 44 where the N-atom of the pyrro-
lidine ring is attached to the electron-withdrawing
CO2Me group, show lower affinity toward nicotinic
acetylcholine receptors. The binding data of these
ligands including that of 42 suggests that when π-cat-
ion interaction is absent, the key factors that control
the activity of the ligands are (i) position of chlorine
atom in the pyridine ring and (ii) steric bulk at the
connecting tether between pyridine and pyrrolidine
rings of our ligands. An in-depth discussion of these
points follows.
The binding data for 42, 44, and 50 for R4â2 nAChRs
are consistent with observations that reported by Casida
et al.62 In that paper they suggested that the position
of chlorine atom in the pyridine ring of the nicotinic
ligand is important. Thus, in 50, loss of binding affinity
for R4â2 nAChRs may be due to the presence of one
chlorine atom at the C-2 position of pyridine ring. But
in the case 44, the C-2 chlorine atom is absent and,
therefore, binding affinity is somewhat better. In 42 the
effect of one chlorine atom at C-6 overwhelms the effect
of the other chlorine atom at C-2 and thereby enhances
its affinity to the R4â2 nAChRs in comparison to that
of 50.
On the other hand, 34 and 36 bind to the receptor
moderately due to the presence of bulky substituents
such as -SPh group and -SO2Ph group at the axial
position (cf. Figure 5) in the middle ring, whereas be-
cause of the presence of a less sterically demanding
connecting tether in 42 it binds to the receptor with high
affinity. It seems, therefore, that lesser steric bulk at
the connecting tether is favorable for affinity. Interest-
ingly, steric bulk at the equatorial position in the middle
ring of constrained nicotines is least favorable for R4â2
nAChRs; this might be the reason why 37 is as inactive
as carbachol. Unlike 34, 49 is active in spite of the pre-
sence of a bulky substituent at the connecting tether
Conclusion
In conclusion, we have demonstrated for the first time
that phenylsulfanyl group assisted generation of pyri-
dine o-quinodimethanes provides an alternative syn-
thesis of constrained nicotine and anabasine analogues.
This route offers new possibilities in drug development
because of its considerable flexibility for the synthesis
of a variety of constrained nicotine and anabasine
analogues. In addition, from a series of novel confor-
mationally restricted analogues, four new nAChRs
agonists have been identified. The constrained ana-
basine analogues 35 and 43 as well as constrained
nicotine analogue 42 are found to elicit moderately
potent nicotinic agonist activity via occupation of R3â4
nAChRs subtype. The structure-activity relationships
of these conformationally restricted analogues where the
pyrrolidinic nitrogen is not free to be protonated should
be useful in further studies aimed at the elucidation of
nAChR pharmacophore. The modified imine tautomer-
ization protocol may also be useful for rapid construction
of the tricyclic skeleton as present in the biologically
significant sceletium alkaloids, e.g. sceletium-A4.66
Experimental Section
Chemistry. All melting points are uncorrected. Unless
otherwise noted, all reactions were carried out under an inert
atmosphere in flame-dried flasks. Solvents and reagents were
dried and purified by distillation before use as follows: Tet-
rahydrofuran, benzene, and Et2O from sodium benzophenone
ketyl; dichloromethane and acetonitrile from P2O5; DMSO and
DMF from CaH2; Et3N, pyridine, and diisopropylamine from
solid KOH; and methanol from Mg. After drying, organic

Analogues of Nicotine and Anabasine
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27 6697
extracts were evaporated under reduced pressure and the
residue was flash chromatographed on silica gel (Acme’s,
particle size 230-400 mesh) using an ethyl acetate-petroleum
ether (60-80 °C) mixture as eluent unless specified otherwise.
TLC was recorded using precoated plates (Merck, silica gel
60 F254) or SiO2 (Glaxo, Sd 60-120 mesh, Acme 100-200
mesh).
155 °C. 1H NMR (500 MHz, CDCl3): δ 7.54 (s, 1H), 7.42-7.30
(m, 5H, phenyl group), 4.92 (bs, 1H), 4.41 (t, 1H, J ) 6.4 Hz),
3.60 (s, 3H), 3.60-3.45 (m, 1H), 3.30-2.98 (m, 1H), 2.36-2.26
(m, 1H), 2.25-2.15 (m, 1H), 1.95-1.85 (m, 1H), 1.85-1.75 (m,
1H), 1.62-1.45 (m, 3H). 13C NMR (125 MHz, CDCl3): δ 155.9,
152.4, 150.3, 148.2, 133.3, 132.9, 129.4, 129.2, 128.6, 124.1,
53.8, 52.7, 45.3, 43.3, 32.9, 31.6, 27.0, 23.5. HRMS (FAB): calcd
for C20H21N2O2SCl2 (M + H) 423.0701, found 423.0698.
5-Benzenesulfonyl-7,9-dichloro-2,3,3a,4,5,9b-hexahydro-
pyrrolo[3,2-h]isoquinoline-1-carboxylic Acid Methyl Es-
ter (36 and 37). A flask is charged with a magnetic stirrer,
CCl4 (2 mL), CH3CN (2 mL), H2O (6 mL), aldehyde 25 (120
mg, 0.4 mmol), and NaIO4 (0.34 g, 1.6 mmol). To this biphasic
solution was added a catalytic amount of RuCl3‚xH2O (∼5-
10 mg), and the resulting mixture was stirred vigorously for
2 h at room temperature. Then CH2Cl2 (∼10 mL) was added,
and the phases were separated. The upper aqueous phase was
extracted three times with CH2Cl2. The combined organic
extracts were dried (Na2SO4) and concentrated in vacuo. The
resulting residue was diluted with ether (15 mL), filtered
through a Celite pad, and concentrated under reduced pres-
sure. The crude product was purified by column chromatog-
raphy (silica gel) and afforded sulfone group bearing aldehyde
26 (80 mg, 60% yield).
Following the standard procedure, the aldehyde 26 (80 mg,
0.24 mmol) was then exposed to but-3-enylamine (30 mg, 0.42
mmol) in dry benzene (5 mL). The crude imine, thus prepared,
was directly treated with methyl chloroformate (0.36 mL, 4.6
mmol) in the presence of diisopropylethylamine (0.08 mL, 0.4
mmol) at 0 °C, and resulting mixture was heated at 148 °C
for 3 h. Concentration and chromatographic purification of the
residue gave a white solid (50 mg, 42%). LC-MS analysis
shows that it is a mixture of two diastereomers in a ratio of
65:35. These two diastereomers were separated by preparative
HPLC (column: ZORBAX SIL, 4.6 mm × 25 mm, ethyl acetate:
hexane 1:9) to give 36 (mp 213-214 °C) and 37 (mp 210-211
°C). Constrained nicotine analogue 36 exhibited the following
properties. Mp: 213-214 °C. 1H NMR (500 MHz, CDCl3): δ
7.85-7.72 (m, 3H), 7.68-7.57 (m, 2H), 7.05 (bs, 1H). 4.84 (d,
1H, J ) 4.5 Hz), 4.27 (dd, 1H, J ) 6 and 4 Hz), 3.51 (s, 3H),
3.60-3.42 (m, 2H), 2.88-2.75 (m, 1H), 2.31 (dt, 1H, J ) 14.5
and 4.5 Hz), 2.21-2.08 (m, 1H), 1.95-1.82 (m, 1H), 1.68 (t,
1H, J ) 10.5 Hz). 13C NMR (100 MHz, CDCl3): 156.8 (s), 155.3
(s), 154.6 (s), 148.4 (s), 136.8 (s), 134.8 (d), 129.7 (d), 129.2 (d),
128.8 (s), 123.6 (d), 63.5 (d), 55.9 (d), 52.4 (q), 45.2 (t), 34.2
(d), 29.1 (t), 24.9 (t). ESI-MS: m/z (relative intensity) 441 [(M
+ H)+, 100], 405 [(M - Cl), 39]. HRMS (FAB): calcd for
C19H19N2O4SCl2 (M + H) 441.0442, found 441.0453.
2,6-Dichloro-4-[(phenylthio)methyl]nicotinaldehyde
(25). To a stirred solution of 2,6-dichloro-4-[(phenylthio)-
methyl]nicotinonitrile (4 g, 13.56 mmol) in CH2Cl2 (60 mL)
cooled to -78 °C was added a 1.0 M solution of DIBAL-H (15
mL) in toluene dropwise over a period of 1 h. The resulting
mixture was allowed to attain room temperature. After 2 h
stirring at room temperature it was quenched with saturated
aqueous NH4Cl at 0 °C, stirred for another 1 h at that
temperature, and then acidified with 3 N HCl. The organic
layer was separated, and the aqueous layer was extracted with
CH2Cl2 (2 × 10 mL). The combined organic fractions were
washed with saturated aqueous NaHCO3 and brine, dried over
anhydrous Na2SO4, and concentrated under reduced pressure.
The crude residue was purified by chromatography (EtOAc:
petroleum ether 1:99) to give 2.5 g (62%) of aldehyde 25 as a
white crystalline solid. Mp: 61-62 °C. IR (KBr): 1688, 1565,
1
1526, 1317 cm-1. H NMR (200 MHz CDCl3:CCl4 7:3): δ 4.37
(s, 2H), 7.00 (s, 1H), 7.28 (bs, 5H), 10.44 (s, 1H). 13C NMR (50
MHz, CDCl3): δ 36.0 (t), 113.9 (s), 124.9 (d), 127.8 (d), 129.0
(d), 131.9 (d), 133.3 (s), 158.3 (s), 153.9 (s), 154.2 (s), 190.0 (d).
Anal. Calcd for C13H9Cl2NOS: C, 52.36; H, 3.03; N, 4.69.
Found: C, 52.23; H, 3.19; N, 4.68.
7,9-Dichloro-5-phenylsulfanyl-2,3,3a,4,5,9b-hexahydro-
pyrrolo[3,2-h]isoquinoline-1-carboxylic Acid Methyl Es-
ter (34). A solution of but-3-enylamine (40 mg, 0.6 mmol) in
dry benzene (1 mL) was added to a stirred solution of aldehyde
25 (100 mg, 0.34 mmol). After stirring for 2 h at room
temperature, anhydrous Na2SO4 was added and stirring
continued for a further 2 h. The mixture was then filtered and
the filtrate allowed to stand over 4 Å molecular sieves
overnight at room temperature. It was then filtered once again,
and the filtrate was concentrated in vacuo. The crude product
was immediately dissolved in dry xylene (15 mL) and cooled
to 10 °C, and diisopropylethylamine (0.1 mL, 0.58 mmol) was
added. The mixture was then treated with methyl chlorofor-
mate (0.5 mL, 6.5 mmol) at 0 °C. After stirring for 10 min, the
mixture was then allowed to reflux for 3 h. After concentration
under reduced pressure, the crude product was purified by
column chromatography (silica gel, EtOAc-petroleum ether
10: 90) to give 34 as a yellowish oil, which on crystallization
from methanol gives a white crystalline solid (70 mg, 44%
1
yield). Mp: 156-157 °C. H NMR (500 MHz, CDCl3): δ 7.45
Constrained nicotine analogue 37 exhibited the following
1
(s, 1H), 7.40-7.26 (m, 5H, phenyl group), 5.07 (d, 1H, J ) 5.6
Hz), 4.25 (dd, 1H, J1 ) 9.6 Hz, J2 ) 4.8 Hz), 3.8-3.65 (m, 1H),
3.57 (s, 3H), 3.43 (td, 1H, J1 ) 10.9 Hz, J2 ) 7.2 Hz), 2.70 (tt,
1H, J1 ) 5.6 Hz, J2 ) 5.6 Hz), 2.26-2.13 (m, 1H), 2.01(ddd,
1H, J1 ) 14 Hz, J2 ) 5.2 Hz, J3 ) 5.2 Hz), 1.91-1.79 (m, 2H).
13C NMR (125 MHz, CDCl3): δ 155.5 (s), 152.5 (s), 152.3 (s),
148.9 (s), 133.8 (s), 131.8 (d), 129.5 (d), 128.8 (s), 128.0 (d),
121.8 (d), 56.3 (d), 52.3 (q), 46.5 (d), 46.5 (t), 35.8 (d), 33.8 (t),
31.7 (t). HRMS (FAB): calcd for C19H19N2O2SCl2 (M + H)
409.0544, found 409.0539.
properties. Mp: 210-211 °C. H NMR (500 MHz, CDCl3): δ
7.58-7.47 (m, 3H), 7.52-7.41 (m, 2H), 7.27 (s, 1H), 4.66-4.53
(bs, 1H), 4.51 (t, J ) 9.2 Hz, 1H), 3.58-3.12 (bm, 5H), 2.39-
2.26 (bm, 1H), 2.26-2.17 (m, 1H), 2.14-2.02 (m, 1H), 1.95-
1.75 (m, 2H). 13C-DEPT 45 (100 MHz, CDCl3): 135.2, 129.6,
129.1, 123.3, 63.6, 55.8, 52.0, 45.0, 31.5, 23.5, 22.6. ESI-MS:
m/z (relative intensity) 441 [(M + H)+, 100], 463 [(M + Na),
83]. HRMS (FAB): calcd for C19H19N2O4SCl2 (M + H) 441.0442,
found 441.0444.
7,9-Dichloro-2,3,3a,9b-tetrahydropyrrolo[3,2-h]isoquin-
oline-1-carboxylic Acid Methyl Ester (42). To a stirred
solution of 34 (180 mg, 0.44 mmol) in methanol (4 mL) and a
small amount of CH2Cl2 (∼0.5 mL) was added a solution of
NaIO4 (0.15 g, 0.70 mmol) in H2O (3 mL) dropwise. After the
solution was stirred for 5-6 days at room temperature, water
and CH2Cl2 were added, the aqueous layer was extracted with
CH2Cl2, and the combined organic fractions were washed with
water, dried (Na2SO4), and concentrated under reduced pres-
sure. The crude residue was purified by chromatography (silica
gel, ethyl acetate/petroleum ether 15:85) to give 42 as a white
crystalline solid (50 mg, 38% yield) and the other unconverted
diastereomeric sulfoxide of 34 (70 mg). This sulfoxide (70 mg)
was then dissolved in CH2Cl2 (6 mL) and was heated at reflux
for 4 h. After concentration under reduced pressure, the crude
product was purified by silica gel chromatography to give
8,10-Dichloro-6-phenylsulfanyl-3,4,4a,5,6,10b-hexahy-
dro-2H-[1,9]-phenanthroline-1-carboxylic Acid Methyl
Ester (35). Similar to the preparation of 34, imine 30 was
prepared first from aldehyde 25 (100 mg, 0.34 mmol) and pent-
4-enylamine (50 mg, 0.58 mmol). The crude imine 30 was then
treated with methyl chloroformate (0.5 mL, 6.5 mmol) in the
presence of diisopropylethylamine (0.1 mL, 0.58 mmol), and
the resulting mixture was heated at reflux for 3 h. The solvent
was evaporated under reduced pressure, and the crude residue
was purified by column chromatography to give a yellow oil
(80 mg, 40% yield). HPLC-MS analysis of the yellow oil shows
that it is a mixture of three diastereomers in a ratio of 69:25:
6. The major diastereomer was purified by preparative HPLC
(column: ZORBAX SIL, 4.6 mm × 25 mm, ethyl acetate:
hexane ) 1:9) to give 35 as a white crystalline solid. Mp: 154-

6698 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27
Sarkar et al.
another crop of 42 (40 mg, 81% yield). The combined yield of
42 from 34 is ∼68%. Constrained nicotine analogue 42
exhibited the following properties. Mp: 172-174 °C. 1H NMR
(200 MHz, CDCl3): δ 6.97 (s, 1H), 6.46 (dd, 1H, J1 ) 2.2 Hz,
J2 ) 9.6 Hz), 6.00 (d, 1H, J ) 9.6 Hz), 4.93 (d, 1H, J ) 5.2 Hz),
3.70 (t, 1H, J ) 9.2 Hz), 3.54 (s, 3H), 3.46-3.10 (m, 2H), 2.42-
2.05 (m, 2H). 13C NMR (50 MHz, CDCl3): δ 155.2 (s), 152.6
(s), 149.5 (s), 144.8 (s), 136.6 (d), 126.1 (d), 124.1 (s), 120.1 (d),
54.2 (d), 52.2 (q), 46.9 (t), 40.7 (d), 29.7 (t). LC-MS: m/z 299
[(M + H)+].
2,3,3a,4,5,9b-Hexahydropyrrolo[3,2-h]isoquinoline-1-
carboxylic Acid Methyl Ester (44). A mixture of 42 (90 mg,
0.3 mmol), fused NaOAc (0.12 g, 1.46 mmol), 10% Pd-C (120
mg), and dry methanol (6 mL) was stirred overnight under a
hydrogen atmosphere at room temperature. The solution was
then filtered through a small pad of Celite and the filtrate
concentrated in vacuo. The resulting residue was purified by
chromatography (silica gel, ethyl acetate:petroleum ether 20:
80) to afford 44 as a colorless oil (60 mg, 85% yield). 1H NMR
(200 MHz, CDCl3): δ 8.73 (bs, 1H), 8.34 (bs, 1H), 6.99 (d, 1H,
J ) 4.6 Hz), 4.99 (bs, 1H), 4.62-4.08 (bm, 1H), 3.76 (s, 3H),
3.68-3.46 (m, 1H), 3.46-3.27 (m, 1H), 2.94-2.51 (m, 3H),
2.22-1.88 (m, 2H), 1.87-1.51 (m, 1H). 13C NMR (50 MHz,
CDCl3): δ 156.8 (s), 150.1 (d), 146.8 (d), 146.0 (s), 133.2 (s),
122.8 (d), 55.7 (d), 52.5 (q), 45.8 (t), 36.6 (d), 28.1 (t), 25.0 (t),
23.2 (t). LC-MS: m/z 233 [(M + H)+].
2,3,3a,4,5,9b-Hexahydro-1H-pyrrolo[3,2-h]isoquino-
line (46). To a stirred solution of 44 (50 mg, 0.21 mmol) in
methanol (1 mL) was added a 40% aqueous solution of KOH
(5 mL) dropwise, and the resulting mixture was heated at
reflux for 3 h. After removal of most of the methanol under
reduced pressure, the mixture was extracted with ether. The
combined ethereal extracts were dried (Na2SO4) and concen-
trated in vacuo. The residue was then purified by column
chromatography (basic alumina, ethyl acetate:petroleum ether
25:75) to give 46 as a colorless oil (30 mg, 80% yield). 1H NMR
(200 MHz, CDCl3): δ 8.57 (s, 1H), 8.30 (d, 1H, J ) 5 Hz), 7.00
(d, 1H, J ) 5 Hz), 4.01 (d, 1H, J ) 6.7 Hz), 3.26-3.02 (m, 1H),
3.02-2.88 (m, 1H), 2.88-1.32 (m, 8H). 13C NMR (50 MHz,
CDCl3): δ 151.1 (d), 147.3 (d), 146.6 (s), 133.0 (s), 123.1 (d),
56.8 (d), 45.6 (t), 37.0 (d), 32.6 (t), 28.2 (t), 25.5 (t).
1-Methyl-2,3,3a,4,5,9b-hexahydro-1H-pyrrolo[3,2-h]iso-
quinoline (48). To a stirred suspension of lithium aluminum
hydride (20 mg, 0.5 mmol) in dry THF (3 mL) was added a
solution of 44 (50 mg, 0.21 mmol) in THF (1 mL) dropwise at
room temperature. After the mixture was stirred for 3 h at
the same temperature, solvent was removed in vacuo and ether
was added. Excess lithium aluminum hydride was then
quenched with saturated aqueous solution of Na2SO4 dropwise
carefully to make a pasty mass (not layer), and solution was
decanted. Further extraction of the pasty mass with ether,
drying of combined ethereal extracts (Na2SO4), and concentra-
tion gave the crude residue, which was purified by column
chromatography (basic alumina, ethyl acetate:petroleum ether
25:75) to afford 48 as a colorless oil (30 mg, 74% yield). 1H
NMR (200 MHz, CDCl3): δ 8.38 (d, 1H, J ) 4.6 Hz), 8.32 (s,
1H), 7.08 (d, 1H, J ) 4.8 Hz), 3.79-3.57 (m, 1H), 3.16-1.38
(m, 12H). 13C NMR (50 MHz, CDCl3): δ 150.2 (d), 149.5 (s),
148.0 (d), 132.2 (s), 123.4 (d), 64.4 (d), 55.6 (t), 40.3 (q), 36.0
(d), 29.7 (t), 28.9 (t) 26.1 (t). EI-MS: m/z (relative intensity)
188 [M+, 96], 187 [(M - H)+, 100], 160 [(M - C2H4)+, 6], 144
[(M - NMe2)+, 23], 130 [(M - N(CH3)C2H5)+, 57], 117 [8], 96
[34], 69 [10], 59 [21]. HRMS (EI): calcd for C12H16N2 188.1313,
found 188.1309.
124.1 (s), 120.4 (d), 52.9 (q), 52.4 (d), 41.0 (t), 36.5 (d), 25.9 (t),
23.8 (t). MSI-MS: m/z (relative intensity) 352 [(M + K + H)+,
37], 330 [(M + H2O)+, 100], 313 [(M + H)+, 22], 277 [(M -
Cl)+, 92].
3,4,4a,5,6,10b-Hexahydro-2H-[1,9]-phenanthroline-1-
carboxylic Acid Methyl Ester (45). Following the procedure
for the synthesis of 44, a sample of 43 (100 mg, 0.32 mmol)
gave 45 as a colorless oil (70 mg, 89% yield). 1H NMR (200
MHz, CDCl3): δ 8.54-8.16 (bs, 1H), 8.28 (s, 1H), 7.02 (d, J )
4.9 Hz, 1H), 5.61-5.22 (m, 1H), 5.05-4.44 (bs, 1H), 4.32-3.82
(m, 2H), 3.66 (s, 3H), 2.98-1.14 (m, 8H).13C NMR (CDCl3, 50
MHz): δ 156.5 (s), 147.9 (d), 146.7 (2C; s and d), 130.6 (s),
123.5 (d), 52.8 (q), 52.1 (d), 39.2 (t), 32.9 (d), 26.4 (t), 25.2 (t),
23.9 (t), 23.7 (t). ESI-MS: m/z (relative intensity) 247 [(M +
H)+, 100]. Stereochemistry at the ring juncture is tentatively
assigned.
1,2,3,4,4a,5,6,10b-Octahydro-[1,9]-phenanthroline (47).
Following the conditions described for the preparation of 46,
a sample of 45 (60 mg, 0.24 mmol) gave 47 as a colorless oil
1
(36 mg, 78% yield). H NMR (200 MHz, CDCl3): δ 8.47 (bs,
1H), 8.31 (d, 1H), 7.00 (d, J ) 4.9 Hz, 1H), 3.83 (d, J ) 3 Hz,
1H), 3.18-1.34 (m, 12 H). 13C NMR (CDCl3, 50 MHz): δ 150.7
(d), 147.5 (d), 146.3 (s), 134.4 (s), 123.7 (d), 54.2 (d), 46.4 (t),
33.2 (d), 29.2 (t), 28.4 (t), 22.1 (t), 21.8 (t). EI-MS: m/z (relative
intensity) 188 [M+, 85], 187 [(M - H)+, 100], 171 [(M - NH3)+,
18], 159 [(M - C2H5)+, 18], 144 [(M - C2H5NH)+, 10], 130 [(M
- C3H7NH)+, 48], 117 [7], 98 [20], 83 [10], 69 [18], 55 [15].
HRMS (EI): calcd for C12H16N2 188.1313, found 188.1312.
7,9-Dichloro-5-phenylsulfanyl-2,3,3a,4,5,9b-hexahydro-
1H- pyrrolo[3,2-h]isoquinoline (49). To a stirred solution
of 34 (100 mg, 0.24 mmol) and NaI (120 mg, 0.80 mmol) in
CH3CN (3 mL) was added freshly distilled trimethylsilyl
chloride (0.70 mL, 0.5 mmol) at room temperature. The
mixture was then heated at reflux for 2 h. MeOH (1 mL) was
added, and after stirring for 15 min at room temperature the
mixture was evaporated to dryness under reduced pressure.
NaOH (2 N) was added and the slurry extracted thoroughly
with ethyl acetate. The combined organic extracts were dried
(Na2SO4) and concentrated in vacuo. The residue was purified
by column chromatography (Al2O3, ethyl acetate:petroleum
ether 25:75) to afford 49 as a yellowish oil (60 mg, 70% yield).
[CAUTION: The compound 49 is highly acid sensitive, even
toward CHCl3, CDCl3, etc.]. 1H NMR (200 MHz, DMSO-d6): δ
7.62-7.31 (m, 5H), 7.21 (s, 1H), 4.81-4.70 (m, 1H), 3.99 (d, J
) 5.7 Hz, 1H), 2.98-2.82 (m, 2H), 2.82-2.59 (bm, 1H), 2.36-
1.39 (m, 5H). 13C NMR (50 MHz, acetone-d6): δ 152.3 (s), 152.1
(s), 147.8 (s), 134.6 (s), 133.8 (d), 130.1 (d), 129.0 (d), 124.3
(d), 56.3 (d), 47.7 (d), 44.9 (t), 33.3 (d), 31.8 (t), 30.2 (t). HRMS
(ESI): calcd for C17H17N2SCl2 (M + H) 351.0484, found
351.04809.
9-Chloro-2,3,3a,4,5,9b-hexahydropyrrolo[3,2-h]isoquin-
oline-1-carboxylic Acid Methyl Ester (50). The compound
42 (50 mg, 0.17 mmol), fused NaOAc (60 mg, 0.73 mmol), and
10% Pd-C (∼50 mg) were stirred in methanol under a
controlled hydrogen atmosphere. The mixture was filtered
through Celite and evaporated under reduced pressure. The
residue was chromatographed (silica gel, ethyl acetate:petro-
leum ether 20:80) to give 50 as a colorless oil (28 mg, 63%
yield). 1H NMR (200 MHz, CDCl3): δ 8.17 (d, J ) 4.8 Hz, 1H),
6.99 (d, J ) 4.8 Hz, 1H), 5.14 (d, J ) 5.8 Hz, 1H), 3.76 (t, J )
9.8 Hz, 1H), 3.54 (s, 3H), 3.53-3.29 (m, 1H), 2.77-1.64 (m,
7H).
4,5-Dibromo-7,9-dichloro-2,3,3a,4,5,9b-hexahydropyr-
rolo[3,2-h]isoquinoline-1-carboxylic Acid Methyl Ester
(51 and 52). Bromine (0.014 mL, 0.28 mmol) in CH2Cl2 (0.7
mL) was added dropwise with stirring to a solution of 42 (80
mg, 0.21 mmol) in CH2Cl2 (4 mL) at 0 °C. After the mixture
was stirred for 30 min at the same temperature, solvent was
removed in vacuo. The residue was purified by preparative
thin-layer chromatography (silica gel, ethyl acetate:petroleum
ether 15:85) to give 51 and 52 in a ratio of 2:1 (62 mg, 51%
yield). 1H NMR (200 MHz, CDCl3): δ 7.41 (s, 1H), 5.46 (d, J )
6 Hz, minor diastereomer 52) and 5.38 (d, J ) 5.3 Hz, major
diastereomer 51) [1H], 5.17 (d, J ) 7 Hz, minor diastereomer
52) and 5.14 (d, J ) 5.9 Hz, major diastereomer 51) [1H], 4.63
8,10-Dichloro-3,4,4a,10b-tetrahydro-2H-[1,9]-phenan-
throline-1-carboxylic Acid Methyl Ester (43). A sample
of 35 (380 mg, 0.89 mmol) under conditions similar to those
described for the preparation of 42 gave 43 as a white
crystalline solid (130 mg, 46% yield). Mp: 132-134 °C. 1H
NMR (200 MHz, CDCl3): δ 6.94 (s, 1H), 6.40 (dd, J ) 9.6 and
5.8 Hz, 1H), 6.29 (d, J ) 9.6 Hz, 1H), 5.70 (d, J ) 6.5 Hz, 1H),
4.32-4.11 (m, 1H), 3.74 (s, 3H), 2.85-2.64 (m, 1H), 2.58-2.36
(m, 1H), 1.84-1.12 (m, 4H). 13C NMR (CDCl3, 50 MHz): δ
155.9 (s), 149.0 (s), 147.9 (s), 147.7 (s), 140.1 (d), 124.4 (d),

Analogues of Nicotine and Anabasine
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27 6699
(dd, J ) 6.4 and 6.2 Hz, minor diastereomer 52) and 4.56 (dd,
J ) 6.6 and 5.5 Hz, major diastereomer 51) [1H], 3.88-3.39
(m, 2H), 3.64 (s, minor diastereomer 52) and 3.63 (s, major
diastereomer 51) [3H], 3.32-3.05 (m, minor diastereomer 52)
and 3.06-2.82 (m, major diastereomer 51) [1H], 2.56-1.82 (m,
2H). 13C NMR (CDCl3, 50 MHz): δ 155.3 (s), 153.5 (s), 149.3
(s), 149.0 (s), 147.7 (s), 126.0 (s), 125.9 (s), 123.8 (d), 55.4 (d),
52.5 (q), 51.8 (d), 51.0 (d), 48.4 (d), 47.2 (d), 46.8 (d), 44.8 (t),
44.4 (t), 40.7 (d), 28.8 (t), 26.7 (t). HRMS (ESI): calcd for
C13H13N2O2Cl2Br2 (M + H) 456.8715, found 456.8716.
(>300000 MW). These plates were then incubated for 48 h at
37 °C to reach greater than 90% confluence. The medium was
then removed, and the cells were incubated with 0.5 mL of
[86Rb+] (2 µCi/well) in growth medium for 4 h at 37 °C. The
medium was then aspirated, and the cells were washed for 2
min with 1 mL of buffer A (15 mM Hepes, 140 mM NaCl, 2
mM KCl, 1 mM MgSO4, 1.8 mM CaCl2, 11 mM glucose, pH
7.4) per well. The process was repeated two additional times,
first with a wash of 2 min and then with a final wash of 7
min. One milliliter solutions of either buffer A (control) or
buffer A with the test compounds (experimental) were then
added to the wells for 2 min, after which they were collected
into scintillation vials and counted for [86Rb+] efflux using
liquid scintillation counting. The EC50 values of the test
compounds (quadruplicate test of concentrations ranging from
0.279 µM to 1 mM) were determined by nonlinear regression
of experimental data using sigmoidal dose-response curve
fitting in Graph Pad Prism software.69
7,9-Dichloro-4,5-dihydroxy-2,3,3a,4,5,9b-hexahydro-
pyrrolo[3,2-h]isoquinoline-1-carboxylic Acid Methyl Es-
ter (53). To a stirred solution of 42 (70 mg, 0.23 mmol) in dry
pyridine (4 mL) was added a solution of OsO4 (70 mg, 0.28
mmol) in dry pyridine (1 mL) dropwise. Stirring was continued
at room temperature in the dark for 12 h. The mixture was
then poured into a saturated solution of NaHSO3, stirred
overnight, and extracted with ethyl acetate. The combined
organic extracts were washed with water, dried (Na2SO4), and
concentrated under reduced pressure. The crude residue was
then purified by preparative thin-layer chromatography (silica
gel, ethyl acetate:petroleum ether 40:60) to give 53 contami-
nated with a minor amount of the other diastereomer as a
white crystalline solid (50 mg, 64% yield). A pure sample of
53 can be obtained by further purification of the mixture of
diastereomers by preparative thin-layer chromatography. The
constrained nicotine analogue 53 exhibited the following
Acknowledgment. Financial support from CSIR,
Government of India, is gratefully acknowledged. S.B.
thanks IIT, Kharagpur, and CSIR, Government of India,
for a research fellowship. We are also thankful to Prof.
H. K. Fun (Malaysia), Dr. S. Djuric (Abbott, Chicago,
IL), Dr. C. Fehr (Firmenich, Geneva, Switzerland), and
Prof. K. Maruoka (Kyoto University, Japan) for continu-
ing help and support.
1
properties. Mp: 210-212 °C. H NMR (200 MHz, CDCl3): δ
7.55 (s, 1H), 5.16 (d, J ) 6.1 Hz, 1H), 4.69 (m, 1H), 4.08 (d, J
) 3 Hz, 1H), 3.94-3.68 (m, 1H), 3.57 (s, 3H), 3.40-3.16 (m,
1H), 2.68-2.54 (m, 1H), 2.40-2.01 (m, 2H). 13C NMR (50 MHz,
acetone-d6): δ 156.5 (s), 154.7 (s), 149.6 (s), 147.4 (s), 128.6
(s), 120.2 (d), 74.7 (d), 69.4 (d), 54.4 (d), 52.2 (q), 47.0 (t), 45.3
(d), 29.3 (t). HRMS (ESI): calcd for C13H15N2O4Cl2 (M + H)
333.0403, found 333.0405.
Supporting Information Available: 1H and 13C NMR
spectra of all compounds; COSY, HMQC/HSQC, HMBC, and
NOESY/TROESY of 34-37 and 51-55 including complete
discussion regarding stereochemical assignments; MS and
HRMS of 34-37, 43, 45, 47-49, and 51-55; LC traces of 35-
37; NOE spectra of 43, 46, 51/52, and 54/55; decoupled spectra
of 43, 44, 51/52, and 54/55; X-ray crystallographic data of 34;
response vs concentration graphs (EC50) of 36, 42, 43, 50, and
54/55. This material is available free of charge via the Internet
at http://pubs.acs.org.
7,9-Dichloro-4,5-epoxy-2,3,3a,4,5,9b-hexahydropyrrolo-
[3,2-h]isoquinoline-1-carboxylic Acid Methyl Ester (54
and 55). m-Chloroperbenzoic acid was first recrystallized by
being dissolved in a mixture of 3 parts of petroleum ether (bp
60-80 °C) and 1 part ether using about 4-5 mL per gram,
seeding, and cooling to -20 °C. The freshly recrystallized
mCPBA (70 mg, 0.4 mmol) was then added to a stirred solution
of 42 (70 mg, 0.23 mmol) in dry dichloromethane (2 mL) at
room temperature overnight. Water was then added, and
organic fractions were separated out. The combined organic
extracts were washed with aqueous sodium hydrogen carbon-
ate and with brine, dried (Na2SO4), and evaporated under
reduced pressure. The crude residue was then purified to give
a diastereomeric mixture of epoxides 54 and 55 as a colorless
oil in a ratio of 4:1 (34 mg, 47% yield). 1H NMR (200 MHz,
CDCl3): δ 7.40 (s, minor diastereomer 55) and 7.36 (s, major
diastereomer 54) [1H]; 5.17 (d, J ) 6.3 Hz, major diastereomer
54) and 5.01 (d, J ) 4.7 Hz, minor diastereomer 55) [1H], 3.91-
3.88 (m, 1H), 3.72 (s, 3H), 3.72-3.57 (m, 1H), 3.38-3.16 (m,
1H), 3.12-2.89 (m, 1H), 2.61-1.45 (m, 3H). HRMS (ESI): calcd
for C13H13N2O3Cl2 (M + H) 315.02977, found 315.02979.
Biology. Minimum essential medium with Earle’s salts and
L-glutamine (MEM), fetal bovine serum (FBS), penicillin-
streptomycin (P/S), and geniticin were purchased from Gibco
(Carlsbad, CA). Poly D-lysine, HEPES, NaCl, KCl, MgSO4,
CaCl2, and glucose were purchased from Sigma Chemical
Company (St.Louis, MO). 86Rubidium chloride ([86Rb+]) was
purchased from Perkin-Elmer (Boston, MA). Twenty-four-well
plates were purchased from Fisher Scientific (Fairlawn, NJ).
Nicotine Stimulated 86Rb+ Efflux Experiments on the
KXr3â4R2 Cells. The KXR3â4R2 cells were provided by Dr.
Kenneth J. Kellar (Georgetown University, Washington, DC)
and were established and maintained as previously de-
scribed.67 The functional studies were carried out on the
nicotinic receptors using the rubidium efflux assay as previ-
ously described.68 Briefly, cells were grown in selection growth
medium (500 mL MEM, 10% FBS, 1% p/s, 350 mg of geniticin).
Once the cells reached greater than 90% confluence, they were
plated (1 mL/well) on 24-well plates coated with poly (D-lysine)
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