Synthesis and pharmacological characterization of novel analogues of the nicotinic acetylcholine receptor agonist (±)-UB-165

Article abstract of DOI:10.1021/jm020814l

(±)-UB-165 (1) is a potent neuronal nicotinic acetylcholine receptor (nAChR) ligand, which displays functional selectivity between nAChR subtypes. Using UB-165 as a lead structure, two classes of racemic ligands were synthesized and assessed in binding assays for three major nAChR subtypes (α4β2*, α3β4, and α7). The first class of compounds comprises the three pyridine isomers 4-6, corresponding to the 3-, 2-, and 4-substituted pyridine isomers, respectively. Deschloro UB-165 (4) displayed a 2-3-fold decrease in affinity at α4β2* and α3β4 nAChR subtypes, as compared with (±)-UB-165, while at the α7 subtype a 31-fold increase in affinity was observed. At each of the nAChR subtypes, high affinity binding was dependent on the presence of a 3-substituted pyridine, and the other isomers, 5 and 6, resulted in marked decreases in binding affinities. The second class of compounds is based on replacing the pyridyl unit of 1 with a diazine moiety, giving pyridazine (7), pyrimidine (8), and pyrazine (9), which retain the 3-pyridyl substructure. Modest reductions in binding affinity were observed for all of the diazine ligands at all nAChR subtypes, with the exception of 7, which retained potency comparable to that of 4 in binding to α7 nAChR. In functional assays at the α3β4 nAChR, all analogues 4-9 were less potent, as compared with 1, and the rank order of functional potencies correlated with that of binding potencies. Computational studies indicate that the 3-substituted pyridine 4 and 2-substituted pyridine 5, as well as the diazine analogues 7-9, all conform to a distance-based pharmacophore model recently proposed for the α4β2* receptor. However, the nicotinic potencies of these ligands vary considerably and because 5 lacks appreciable nicotinic activity, it is clear that further refinements of this model are necessary in order to describe adequately the structural and electronic demands associated with this nAChR subtype. This rational series of compounds based on UB-165 presents a systematic approach to defining subtype specific pharmacophores.

Full text of DOI:10.1021/jm020814l

J . Med. Chem. 2002, 45, 3235-3245
3235
Syn th esis a n d P h a r m a cologica l Ch a r a cter iza tion of Novel An a logu es of th e
Nicotin ic Acetylch olin e Recep tor Agon ist (()-UB-165
Christopher G. V. Sharples,† Gunter Karig,‡ Graham L. Simpson,‡ J ames A. Spencer,‡ Emma Wright,‡
Neil S. Millar,§ Susan Wonnacott,*,† and Timothy Gallagher‡
Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, United Kingdom, School of Chemistry,
University of Bristol, Bristol BS8 1HS, United Kingdom, and Department of Pharmacology, University College London,
Gower Street, London WC1E 6BT, United Kingdom
Received J anuary 7, 2002
(()-UB-165 (1) is a potent neuronal nicotinic acetylcholine receptor (nAChR) ligand, which
displays functional selectivity between nAChR subtypes. Using UB-165 as a lead structure,
two classes of racemic ligands were synthesized and assessed in binding assays for three major
nAChR subtypes (R4â2*, R3â4, and R7). The first class of compounds comprises the three
pyridine isomers 4-6, corresponding to the 3-, 2-, and 4-substituted pyridine isomers,
respectively. Deschloro UB-165 (4) displayed a 2-3-fold decrease in affinity at R4â2* and R3â4
nAChR subtypes, as compared with (()-UB-165, while at the R7 subtype a 31-fold increase in
affinity was observed. At each of the nAChR subtypes, high affinity binding was dependent on
the presence of a 3-substituted pyridine, and the other isomers, 5 and 6, resulted in marked
decreases in binding affinities. The second class of compounds is based on replacing the pyridyl
unit of 1 with a diazine moiety, giving pyridazine (7), pyrimidine (8), and pyrazine (9), which
retain the “3-pyridyl” substructure. Modest reductions in binding affinity were observed for
all of the diazine ligands at all nAChR subtypes, with the exception of 7, which retained potency
comparable to that of 4 in binding to R7 nAChR. In functional assays at the R3â4 nAChR, all
analogues 4-9 were less potent, as compared with 1, and the rank order of functional potencies
correlated with that of binding potencies. Computational studies indicate that the 3-substituted
pyridine 4 and 2-substituted pyridine 5, as well as the diazine analogues 7-9, all conform to
a distance-based pharmacophore model recently proposed for the R4â2* receptor. However,
the nicotinic potencies of these ligands vary considerably and because 5 lacks appreciable
nicotinic activity, it is clear that further refinements of this model are necessary in order to
describe adequately the structural and electronic demands associated with this nAChR subtype.
This rational series of compounds based on UB-165 presents a systematic approach to defining
subtype specific pharmacophores.
In tr od u ction
nAChR subtype;10 these nAChRs bind [3H]epibatidine
(but not [3H]nicotine) with relatively high affinity.5,11
Neuronal nicotinic acetylcholine receptors (nAChRs)
are pentameric ligand-gated cation channels, which are
comprised of various combinations of R and â subunits
(R2-10, â2-4).1-3 Consequently, there is potential for
huge diversity of nAChR subtypes and defining the
subunit composition of native nAChRs is a considerable
challenge. However, a few subtypes of nAChR predomi-
nate; notably, R4â2* and R7 subtypes are widespread
in the vertebrate central nervous system (CNS).4,5 The
R4â2* nAChR binds nicotine with high affinity under
equilibrium binding conditions and accounts for the
majority of [3H]nicotine binding sites in rodent brain.6
The R7 nAChR is considered to exist as a homopen-
tamer7,8 and is defined by high affinity binding of the
specific ligands R-bungarotoxin (RBgt)4 and methylly-
caconitine (MLA).9 In autonomic ganglia of the periph-
eral nervous system (and neuroblastoma cell lines
derived from them), R3â4* nAChRs constitute the major
Neuronal nAChRs are implicated in diverse CNS
functions, including neuronal development, cognitive
processing, and reward,12 and have been linked to
various clinical states including Alzheimer’s and Par-
kinson’s diseases, schizophrenia, and nociception.13
Hence, neuronal nAChRs constitute a major target for
drug discovery. The generation of subtype selective
ligands to specifically target relevant nAChRs with a
minimum of side effects, including those mediated
through nAChRs expressed in the peripheral nervous
system, is a goal. Understanding the molecular features
of nicotinic drugs that define particular nAChR sub-
types, and hence identification of nAChR subtype
specific pharmacophores, would facilitate rational drug
design.
Early nicotinic pharmacophore models were based on
distances between pharmacophoric elements in the
classical agonists, typically the distance between a basic
nitrogen (which is protonated under physiological condi-
tions) and a hydrogen bond-accepting moiety, typically
a nitrogen (or carbonyl oxygen). N-N distances of 5.9
Å14 or ranging from 4.4 to 5.0 Å15 have been proposed,
although the tolerance of the nAChR for the gross steric
* To whom correspondence should be addressed. Tel.: (44-1225)-
826391. Fax: (44-1225)826779. E-mail: bsssw@bath.ac.uk.
† University of Bath.
‡ University of Bristol.
§ University College London.
10.1021/jm020814l CCC: $22.00 © 2002 American Chemical Society
Published on Web 06/14/2002

3236 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15
Sharples et al.
F igu r e 1. Structures of UB-165 (1), anatoxin-a (2), and
epibatidine (3).
bulk of the ligand was not considered. Indeed, more
recent studies of high affinity binding to R4â2* nAChR
have indicated a more complex mode of action, which
may implicate cationic π system interactions between
ligand and receptor and vice versa.16 This would be
consistent with the presence of aromatic amino acids
in the nAChR agonist binding site.17
F igu r e 2. Novel pyridine and diazine analogues.
the 9-azabicyclo[4.2.1]nonene unit, in contrast to the
smaller asymmmetric, but less dissymmetric, 7-azabicy-
clo[2.2.1]heptane ring present in epibatidine.23,24 The
high potency with which (()-UB-165 binds to brain
nAChR points toward this ligand as both a useful lead
structure and a valuable probe for nAChRs, with
potential as a tool to dissect the roles of individual
nAChRs in physiological processes. In functional assays,
(()-UB-165 was found to be a potent but inefficacious
partial agonist at R4â2* nAChR in rat thalamic synap-
tosomes and at human R4â2 nAChR expressed in
Xenopus oocytes, and this property was exploited to
implicate R4â2* nAChR in the presynaptic modulation
of [3H]dopamine release from isolated nerve terminals
from rat striatum.25
UB-165 is a useful candidate for structure-activity
studies as it is readily amenable to structural modifica-
tion and possesses a semirigid molecular skeleton,
which restricts the number of stable conformations and
hence eases mapping of a pharmacophore. Modifiying
UB-165 may lead to the generation of novel ligands,
which are able to further discriminate between nAChR
subtypes. The chloro moiety of UB-165 does not appear
to contribute significantly toward biological potency (see
below), and for the purposes of synthetic accessibility,
this component has not been retained. Our initial
investigations into structural variants of UB-165 have
focused on the generation of two groups of molecules in
which the heteroaryl (pyridyl) component of UB-165 has
been varied (Figure 2).
Class 1 comprises the three pyridyl isomers: 4 (the
deschloro analogue of UB-165) and the 2-pyrido and
4-pyrido analogues 5 and 6, respectively. Class 2
explores further modification of the heteroaryl compo-
nent by incorporation of an additional nitrogen (i.e.,
diazines rather than pyridines), and we have synthe-
sized the pyridazine, pyrimidine, and pyrazine ana-
logues 7-9, respectively. It should be noted that while
there are other diazine isomers possible, we have
restricted the isomers prepared to those that retain the
“3-pyridyl” substructure associated with 1 and 4. The
reasons for this restriction are discussed in more detail
below. Both classes of compounds have been examined
for binding affinity at the R4â2* and R7 nAChRs
expressed in rat brain and at the rat R3â4 nAChR
expressed in a stably transfected cell line. In addition,
The early pharmacophore models were largely based
on knowledge of the interaction of nicotinic ligands with
nAChRs in skeletal muscle and were developed before
the extent of the heterogeneity of their neuronal coun-
terparts was appreciated. Subtly different pharmacoph-
ores must exist not only for different nAChR subtypes
but also for different states of a particular nAChR. A
high affinity desensitized state is favored in radioligand
binding assays whereas an activated state is measured
in assays of nAChR function. Different molecular fea-
tures may preferentially favor the stabilization of one
state over the others. A complete subtype specific
pharmacophore would aim to describe the molecular
features that determine high affinity binding, receptor
activation, and selectivity between nAChR subtypes.
Indeed, the recent pharmacophore studies have focused
on a specific nAChR (R4â2*): while the basis of the new
models involves distance parameters, these are not
based simply on the geometry (N-N distance) of the
ligand.16,18 The multidisciplinary combination of rational
chemical synthesis of novel drugs and their subsequent
evaluation in assays of biological activity enables us to
address the key issues associated with developing and
improving subtype specific neuronal pharmacophores.
UB-165 (1) is a rationally designed hybrid molecule
consisting of a 9-azabicyclo[4.2.1]nonene ring coupled
to a chloropyridine via C(3) of the pyridyl unit. The two
major structural componentssthe azabicycle and the
chloropyridinesare present in the molecular structures
of the potent nicotinic agonists anatoxin-a (2) and
epibatidine (3), respectively (Figure 1).
Compound 1 was conceived to probe the molecular
basis of the enantioselectivity shown by nAChR.19 The
natural enantiomer (+)-anatoxin-a is 2 orders of mag-
nitude more potent than (-)-anatoxin-a at muscle
nAChR20 and neuronal nAChR.21 In contrast, the enan-
tiomers of epibatidine are essentially equipotent.22 UB-
165 was found to preserve the enantioselectivity asso-
ciated with anatoxin-a at rat brain nAChR.19 This
suggested that the observed enantioselectivity arises
from the higher degree of dissymmetry associated with

Nicotinic Acetylcholine Receptor Agonist
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15 3237
Sch em e 1a
a
Reagents: (a) ArZnX, Pd(PPh3)4, THF. (b) HCl, H2O. (a) The overall yield for step a (Pd(0) cross coupling) and step b (N deprotection)
in Scheme 1, respectively. Ligands 4-9 were isolated as the corresponding hydrochloride salts. (b) This represents the overall yield for
the formation of 6 for Pd-mediated coupling,26 reduction of the intermediate 3-bromo residue, and N-Voc cleavage.
functional potency at the R3â4 nAChR was assessed
use of a (3-bromo-4-pyridyl)zinc halide (see Scheme 1).26
Once the Negishi coupling step was complete, the bromo
directing group was removed by selective reduction to
provide 11c, which then underwent cleavage of the
N-vinyloxycarbonyl moiety to give 6 (Scheme 1).
using a calcium fluorescence assay.
Resu lts a n d Discu ssion
Ch em istr y. (()-UB-165 (1) has provided an initial
lead, and we now describe the synthesis of two classes
of analogues based on (i) positional isomers of the
pyridyl moiety (class 1) and (ii) selected diazine variants
(class 2). The synthetic strategy employed for both
classes of ligands is shown in Scheme 1 and is based on
the route developed originally for 1.
Bin d in g Assa ys. Compound 1 and the two classes
of analogues based on UB-165 were tested for their
binding affinities at the major nAChR subtypes present
in the CNS and peripheral nervous system. These
comprised the rat brain R4â2* and R7 nAChRs, which
were labeled by [3H]nicotine and [3H]MLA, respectively,
and the rat R3â4 nAChR expressed in a stably trans-
fected cell line27 and labeled by [3H]epibatidine. The
binding affinities of (()-UB-165 (1) at R4â2* and R3â4
nAChR (Ki ) 0.27 and 6.5 nM, respectively) were
intermediate between those of (()-anatoxin-a (2) and
(()-epibatidine (3), with the potency order 3 > 1 > 2.
At the R7 nAChR, the potency order was 3 > 2 > 1,
with a Ki value of 2760 nM for 1 (Table 1).
In this process, the key step is a Negishi Pd(0)-
mediated cross-coupling of vinyl triflate 10 with the
appropriate heteroaryl zinc halide (ArZnX) using Pd-
(PPh3)4 as the catalyst to provide coupled adducts 11a -
f. Following isolation and purification, the N-vinyloxy-
carbonyl protecting group of 11 underwent essentially
quantitative acidic cleavage, and final products 4-9
were isolated and screened as the corresponding hydro-
chloride salts. This approach provided all of the ana-
logues described in this paper, with the exception of the
4-pyridyl derivative 6. In this case, we were unsuccess-
ful in generating the requisite 4-pyridylzinc halide
needed, and indeed vinyl triflate 10 also failed to couple
to a variety of other 4-pyridyl organometallic reagents
based on Suzuki and Stille conditions. As a result, a
procedure has been developed, which represents a new
method for the synthesis of 4-substituted pyridines,
including the 4-pyridyl analogue 6. This is based on the
To facilitate future chemical modification of the
heteroaryl component, the deschloro analogue 4 of 1 was
synthesized and evaluated. This modification resulted
in a 2-3-fold decrease in binding affinity of 4 at the
R4â2* and R3â4 nAChRs, as compared with 1. Thus,
the chlorine atom is not crucial for nicotinic activity and
its presence has little influence on the binding potency
of 1 at these nAChR subtypes. Similarly, the analogous
modification of epibatidine did not change binding
affinity at rat brain R4â2* nAChR with respect to the

3238 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15
Ta ble 1. Binding Affinities Values of Compounds at nAChR Subtypes
Sharples et al.
nAChR subtype
Ki (nM)a (affinity ratio)b
R3â4
compd
R4â2*
R7
(()-UB-165 (1)
0.27 ( 0.05 (1)
1.25 ( 0.2 (1)
0.021 ( 0.005 (1)
0.43 ( 0.03 (1)
1540 ( 280 (1)
10 950 ( 1225 (1)
2.6 ( 0.2 (1)
6.5 ( 2.3 (24.1)
25.4 ( 1.9 (20.3)
0.077 ( 0.008 (3.7)
22.5 ( 5.5 (52)
10 500 ( 1650 (6.8)
22 000 ( 4770 (2)
306 ( 51 (20)
236 ( 30 (96)
2846 ( 484 (49)
2760 ( 473 (10 222)
1140 ( 124 (1472)
101 ( 16 (4810)
89 ( 35 (207)
(()-anatoxin-a (2)
epibatidine (3)
4
5
6
7
8
9
47 500 ( 11 050 (31)
>50 000 (-)
68.4 ( 16.2 (118)
1706 ( 223 (696)
5773 ( 484 (100)
2.45 ( 0.37 (1)
58 ( 13 (1)
a
b
Results are the mean of at least three independent determinations ( SEM. Affinity ratio is the potency relative to that at the R4â2*
nAChR.
parent molecule.22 Moreover, chlorination of nicotine
also had a minimal influence on binding affinity at rat
brain R4â2* nAChR.23,28 However, at the R7 nAChR, a
31-fold increase in affinity for 4, as compared with 1,
was observed, suggesting that at this nAChR subtype
a small advantage arises from the loss of the halogen.
at R4â2*, R3â4, and R7 nAChR. At the R4â2* subtype,
a similar 5-fold decrease in binding potency was re-
ported for a pyrimidinyl derivative of epibatidine.32
Pyrazine 9 resulted in greater decreases in potency than
7 or 8 at all nAChR subtypes relative to 4, with
decreases in binding affinity of 135-, 32-, and 257-fold
at R4â2*, R3â4, and R7 nAChR, respectively (Table 1).
Pyridazine 7, like pyrimidine 8, was slightly less potent
at R4â2* and R3â4 nAChRs as compared with 4, with
respective decreases in binding potency of 6- and 13.6-
fold. These results indicate that the presence of a
diazine will generally decrease binding affinity with the
greatest impact being exerted by an addition of a
nitrogen at the 6′ position, as in pyrazine 9. However,
7 differed from 8 and 9 in retaining activity at the R7
nAChR: it was the most potent of all of the analogues
examined in this study at the R7 nAChR (Table 1),
binding with a potency comparable to that of 4.
Compounds 1-9 had the following rank orders of
potency for binding to each nAChR subtype: R4â2* 3
> 1 > 4 > 2 > 7 ) 8 > 9 . 5 . 6; R3â4 3 > 1 > 4 ) 2
> 8 > 7 > 9 . 5 . 6; and R7 7, 4 > 3 > 2 > 8 > 1 > 9
. 5 . 6. The rank order profiles of compound potencies
at R4â2* and R3â4 nAChRs are very similar whereas
the profile at R7 nAChR is more divergent. This implies
that R7 nAChR possesses a pharmacophore that is
distinct from that of R4â2* and R3â4 nAChRs.
n ACh R Su btyp e Selectivity. The binding affinities
of compounds 1-9 were compared to examine subtype
selective interactions (Table 1). (()-UB-165 (1) was more
selective for R4â2* nAChR over R3â4 and R7 nAChR
than (()-anatoxin-a (2) and (()-epibatidine (3). Relative
to R4â2*, (()-UB-165 had a 24- and 10 222-fold differ-
ence in potency at R3â4 and R7 nAChR, respectively,
as compared to (()-anatoxin-a, which had 20- and 1472-
fold differences, and (()-epibatidine, which had 3.7- and
4810-fold differences, respectively. This selectivity for
the R4â2* nAChR vs the R7 nAChR is decreased in 4 to
207- vs 10 222-fold. However, 4 exhibits increased
selectivity with respect to R3â4 nAChR (52- vs 24-fold).
nAChR subtype selectivity was greatly decreased in the
pyridyl isomers 5 and 6. Pyrimidine 8 was more selec-
tive for the R4â2* nAChR over R7 and R3â4 nAChR
than 4 (respective potency differences of 696- and 96-
fold). In contrast, pyridazine 7 and pyrazine 9 had a
poorer selectivity profile for the R4â2* nAChR: 7 had
20- and 118-fold differences in potency, and 9 had 49-
and 100-fold differences in potency at R7 and R3â4
nAChRs, respectively.
Deschloro UB-165 (4) was compared with the two
other pyridyl isomers, comprising our first class of
analogues, to explore the effect of translocation of the
pyridyl nitrogen, which is proposed to act as a hydrogen
bond acceptor in models of the nicotinic pharmaco-
phore.14-16,29 As compared to the 3-substituted pyridines
(represented by UB-165 and the deschloro analogue 4),
the 2-pyrido analogue 5 and the 4-pyrido analogue 6
represent structures in which the distance between the
cationic nitrogen and the pyridyl nitrogen has been
respectively decreased and increased. For both 5 and
6, substantially diminished binding affinities at all
nAChR subtypes examined were observed and this
points toward the importance of a nitrogen atom in the
3′ position (as in 4). As compared with 4, the 2-pyridyl
variant 5 was 3581-fold less potent at the R4â2* nAChR
with smaller decreases in potency of 467- and 534-fold
at the R3â4 and R7 nAChR, respectively. The 4-pyridyl
derivative 6 resulted in even greater reductions in
affinity at each of these nAChR subtypes, and as for 5,
the greatest decrease was at the R4â2* nAChR with a
25 419-fold loss of potency. Smaller decreases of 978 and
>562-fold were observed for 6 at the R3â4 and R7
nAChRs, respectively (Table 1).
The second class of compounds incorporates a diazine
moiety, but the design had certain constraints imposed.
Given that a 3-pyridyl unit seems to be a requisite for
high affinity binding, we have restricted the diazine
class to those that retain this feature. We have synthe-
sized and evaluated three isomeric diazine analogues:
pyridazine 7, pyrimidine 8, and pyrazine 9. The pres-
ence of an additional nitrogen atom produces a more
π-electron deficient aromatic ring, and this exerts a
significant effect on the pKa of the nitrogen atom
representing the hydrogen bond acceptor associated
with the ligand-receptor interaction.30 Pyridine (pKa
5.22) is significantly more basic than pyridazine (pKa
2.33), pyrimidine (pKa 1.31), and pyrazine (pKa 0.65),
and the value of this type of structural variation in
medicinal chemistry has been recognized as a useful
tool.31 The pyrimidine analogue 8 was slightly less
potent than 4 at all nAChR subtypes, with respective
decreases in binding affinity of 6-, 10.5-, and 19.2-fold

Nicotinic Acetylcholine Receptor Agonist
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15 3239
Ta ble 2. Activation Potencies of Compounds at the R3â4
NAChR
compd
EC50 (µM)a
(EC50/Ki)b
(()-UB-165 (1)
0.31 ( 0.07
2.29 ( 0.69
0.014 ( 0.002
1.59 ( 0.31
>100
48
90
182
71
(()-anatoxin-a (2)
(()-epibatidine (3)
4
5
6
7
8
9
>100
4.19 ( 0.99
3.89 ( 0.20
>100
14
16
a
EC50 is the concentration required to achieve a half-maximal
activation. Results are the mean of at least three independent
b
determinations ( SEM. Ki values are taken from Table 2.
F u n ction a l Stu d iessActiva tion of r3â4 n ACh R.
(()-UB-165 (1) and the analogues described in this
study were also tested for their functional potency at
the R3â4 nAChR, stably expressed by the LR3â4 cell
line.27 This assay was performed in order to confirm that
occupation of the agonist binding site, as shown in
competition radioligand binding assays, leads to recep-
tor activation. The functional assay monitored increases
in the fluorescence of a calcium sensitive dye, fluo-3,
upon agonist stimulation. (()-UB-165 (1) evoked in-
creases in intracellular calcium with an EC50 value of
0.31 µM, intermediate between that of (()-epibatidine
(3) and that of (()-anatoxin-a (2), which gave EC50
values of 0.014 and 2.29 µM, respectively (Table 2,
Figure 3). Compound 4 had a slightly reduced EC50
value of 1.59 µM while 5 and 6 were essentially inactive
over the concentration range examined, with EC50
values >100 µM. Given the small amounts of compound
available, it was not practical to extend the dose-
response curves to determine full functional dose-
response relationships. However, these results do indi-
cate a critical role for the positioning of the pyridyl
nitrogen and support a role for internitrogen distances
in determining functional potency. Similar results were
also observed by Spang and colleagues on R3â4 nAChR
expressed in Xenopus oocytes: the (+) and (-) enanti-
omers of the deschloro epibatidine analogue 2PABH (cf.
5 in the present study) were 406- and 2952-fold,
respectively, less potent than deschloro epibatidine.33
The pyridazinyl and pyrimidinyl derivatives 7 and 8
had only slightly reduced potencies when compared to
4 (respective EC50 values of 4.19 and 3.89 µM), while
pyrazine 9 had a greatly reduced potency (EC50 value
> 100 µM). EC50 values are about 2 orders of magnitude
greater than corresponding Ki values for each com-
pound, as greater concentrations of agonist are required
to stabilize the activated state of the nAChR (repre-
sented by the EC50 value) than the high affinity
desensitized state, which is represented by the Ki
value.34 The ratio of EC50/Ki for 7 and 8 (16- and 14-
fold, respectively) was decreased as compared to 4 (71-
fold). Thus, the presence of a second nitrogen (diazine
vs pyridine) may decrease the difference in affinities of
ligand interaction with the activated and desensitized
receptor states, which are measured in functional and
binding assays, respectively.
F igu r e 3. Dose-response relationships for agonist-induced
increases in intracellular calcium, as measured by fluo-3
fluorescence, evoked by (A) (()-UB-165 (1), (()-anatoxin-a (2),
and (()-epibatidine (3); (B) 4-6; (C) 4 and 7-9 at the R3â4
nAChR expressed in LR3â4 cells. Functional responses are
expressed as the percentage of a maximally efficacious con-
centration of (-)-nicotine (100 µM). Vertical bars indicate the
SEM from at least three independent determinations.
Com p u ta tion a l Stu d ies. As alluded to earlier, a
compelling role for structure-activity relationships is
to define and develop new pharmacophore models with
specificity for individual receptor subtypes. While this

3240 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15
Sharples et al.
Ta ble 3. Pharmacophore Parameters for Compounds 4-6
goal is still some way off, it is instructive to compare
the molecules discussed in this paper against current
pharmacophore models. Internitrogen (N-N) distances
have been linked to nicotinic activity, with a value of
4.8 ( 0.3 Å derived for the classic Beers-Reich (and
later Sheridan) models.14,15 Glennon and co-workers
have shown that for epibatidine (one of the most potent
agonists known) the N-N distance (5.51 Å) exceeds this
value.23,35 However, the N-N distance of 3 (and indeed
most other ligands) is dependent on the conformation
selected for measurement, and others have recognized
that alternative low energy conformations of 3 with
N-N distances ranging from 4.7 to 5.5 Å may neverthe-
less be significant in terms of bioactivity.36 Furthermore,
Danish workers have suggested that the N-N distance
in isolation is not the essential (or unique) pharma-
cophore component.18,36 Rather, a refined series of
distance relationships have been proposed, which are
associated with the putative ligand-receptor complex.
This has led to an improved pharmacophore model for
the R4â2* nAChR.18 This model consists of (i) a point
corresponding to a protonated nitrogen atom, (ii) a point
corresponding to an electronegative atom capable of
forming a hydrogen bond, and (iii) the center of a
heteroaromatic ring or a CdO bond. These elements
were related by the following parameters: an (a-b)
distance of 7.3-8.0 Å, an (a-c) distance of 6.5-7.4 Å,
and an angle of 30.4-35.8° between the two distance
vectors (∆bac). Given the ongoing debate, we have
examined the structural properties of ligands 4-6 in
terms of these alternative pharmacophore concepts, and
the key structural parameters are summarized in Table
3.
Figure 4a,b shows, respectively, the conformational
profiles (based on the C(1)-C(2)-C(3)-C(2′) torsion
anglessee also footnote b, Table 3) of the pyridine
isomers 4 (deschloro UB-165, the 3-substituted isomer)
and 5 (the 2-substituted isomer); the N-N distances
associated with the low energy conformer populations
are indicated.
In the case of 4, the N-N distances range from 4.84
to 5.81 Å (if conformers up to 5 kJ mol-1 above the local
minima are also recognized). This certainly encompasses
the Beers-Reich distance (4.8 ( 0.3 Å) but at the higher
end of the range, and conformations with larger N-N
distances are also easily accessible.
For the 2-substituted pyridine analogue 5, the N-N
distances are generally shorter, ranging from 3.8 to 4.8
Å, and the variation in conformational minima (as
compared to 4) is more dramatic, which presumably
reflects destablizing interactions associated with the
pyridine nitrogen lone pair and the azabicycle. For the
4-substituted pyridine 6, the N-N distance (6.1 Å) is
essentially independent of torsion angle changes associ-
ated with rotation of the heteroaryl unit. It is interesting
to recall that unlike 4, both 5 and 6 are weak nicotinic
ligands.
The (a-b) distance and the bac angle (∆bac) associ-
ated with the Tønder pharmacophore for the R4â2*
nAChR have been shown to correlate well to nicotinic
binding potency.18 We have calculated the (a-b) and
∆bac parameters for compounds 4-6 (Table 3).
N-N
torsionb distancec (a-b)d ∆bacd
energya (kJ )
(deg)
(Å)
(Å)
(deg)
Compound 4a
point 1 +5
29.5
55
5.1
5.1
5.5
5.7
5.9
5.9
5.5
5.4
7.0
7.35
9.5
36.1
35.4
26.8
20.0
9.2
15.1
31.6
34.2
point 2 global minimum
point 3 +1.8
122.5
150
215
238
308
332
point 4 + 6.8
point 5 +8.2
10.3
10.8
10.6
8.6
point 6 +3.2
point 7 +1.8
point 8 +6.8
7.8
Compound 5a
point 1 +6
51
138
229
311
338
3.8
4.4
4.8
4.3
4.1
3.2
7.4
8.7
6.0
4.6
25.6
34.8
30.8
36.8
35.2
point 2 +4.6
point 3 +2.7
point 4 global minimum
point 5 +5
Compound 6e
6.0
10.1
25.4
a
Conformational and distance properties measured at points
shown in see Figure 4a for deschloro UB-165 (4) and Figure 4b
for analogue 5, including at values associated with conformations
5 kJ higher in energy than the local minima. Energy values shown
b
are relative to the global minimum position. Torsion angle is
defined by C(1)-C(2)-C(3′)-C(2′) as illustrated in structure 1. In
the case of compound 5 (incorporating a 2-substituted pyridine),
the equivalent dihedral corresponds to C(1)-C(2)-C(2′)-N(1′).
c Beer-Reich internitrogen (N-N) distance. (a-b) and ∆bac
d
parameters corresponding to the R4â2* pharmacophore model.18
Point a was derived as described in the literature using the N(9)
lone pair, which is formally (given that N(9) is assumed to be
protonated when bound to the receptor) anti to the larger four
carbon ring of the 9-azabicyclo[4.2.1]non-2-ene moiety. e Compound
6 clearly has a wide range of conformational states available and
displays a profile that is very similar to that shown in Figure 4a
for compound 4. However, the symmetry of the 1,4-disubstituted
pyridine unit of 6 means that the pharmacophore parameters
(N-N distance, as well as (a-b) and ∆bac) remain constant and
independent of rotation about the key torsion angle, which is now
C(1)-C(2)-C(4′)-C(3′).
Figure 4a) are 7.35 Å and 35.4°, respectively: these
values lie within the optimal range for the R4â2*
nAChR pharmacophore (see above).18 Distance and
angle values for other low energy conformations do not,
however, fall within the accepted range.
The (a-b) distances determined for 5 range from 3.2
to 8.7 Å, with ∆bac values of 25.6-36.8°. The optimal
(a-b) distance (which was shown to be the most reliable
parameter for correlating nicotinic potency)18 and ∆bac
value can be achieved by compound 5 (e.g., at point 2
in Figure 4b). However, this ligand shows markedly
reduced affinity (as compared to 4) for the R4â2*
nAChR. This raises two issues. First, the conformation
of 5, which meets the R4â2* parameters, lies well above
(+4.6 kJ ) the global minima. Although this does not of
itself preclude this being of bioactive significance,
conformation populations associated with 5 will never-
theless be heavily biased away from this “preferred”
arrangement. Interestingly, this is in contrast to 4
where the R4â2* pharmacophore parameters are met
by, inter alia, a conformation located at the global
minimium. Second, biological activity is influenced by
binding of the ligand per se and not just by individual,
albeit important, components; with one exception,37
current pharmacophore models do not recognize inter-
actions associated with other structural components of
the ligand (see below).
For the most potent analogue, 4, the (a-b) distance
and ∆bac values at the global minimum (point 2 in

Nicotinic Acetylcholine Receptor Agonist
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15 3241
F igu r e 5. Space-filling representations of compounds 4 and
5 using conformations with (a-b) and ∆bac parameters fitting
the proposed R4â2* nAChR pharmacophore.18
potencies of this group of ligands are markedly different
to that of 4, and the relationship between diazine
structure and hydrogen bond acceptor properties30,31 has
already been highlighted (see also the note added in
proof). Clearly, as the structure-activity relationship
of these and related compounds is developed further,
these data will present an opportunity to refine the
pharmacophore for the R4â2* nAChR, as well as develop
pharmacophore models appropriate to other nicotinic
receptor subtypes.
Additional factors must also be considered in develop-
ing pharmacophore models, and these include other
structural parameters, such as the bulk and the elec-
tronic/hydrophobic properties of the azabicyclo[4.2.1]-
nonene scaffold associated with the ligands reported in
this paper. The interactions associated with this aspect
of ligand structure, while not considered explicitly by
either of the pharmacophore models discussed above
(however, see ref 37), must nonetheless be accom-
modated within the relevant receptor-ligand complex.
Both 4 and 5 can meet the (a-b) and ∆bac demands
associated with the R4â2* nAChR pharmacophore18 but
using quite different torsion angles and therefore dif-
ferent conformations. These conformational changes
result in a “relocation” of the hydrophobic bulk of the
9-azabicyclo[4.2.1]non-2-ene moiety within the active
site (relative to the two nitrogen-based pharmacophore
sites) for 4 vs 5, which is illustrated in Figure 5.
In the case of compound 5, which although able to
meet certain pharmacophore parameters still lacks
appreciable nicotinic activity, the need to accommodate
the sterically demanding molecular scaffold of the
bicyclic moiety within the binding site may, in turn,
inhibit the ligand-receptor interaction. Further refine-
ments of pharmacophore models must consider those
parts of the ligand that, while not directly involved in
the primary binding interaction, may nevertheless exert
a negative (or indeed positive) influence on the acces-
sibility and acceptability of the ligand to the receptor
binding site. One way forward will be to probe the
ability of individual receptors to accommodate ligands
carrying the same array of primary binding components
F igu r e 4. Conformational profiles of compounds 4 (a) and 5
(b).
In the case of compound 6, symmetry dictates that
the N-N distance (measured at 6.00 Å) and (a-b) (10.1
Å) and ∆bac (25.4°) values are independent of the C(1)-
C(2)-C(3′)-C(2′) torsion angle, but in this case, none
of these parameters fall within the range of optimal
values required and 6 interacts with R4â2* nAChR with
diminished potency relative to 4 and 5.
Like 4, the class 2 compounds 7-9 also conform to
the basic pharmacophoric elements for the R4â2* nAChR
because a “3′-pyridyl” moiety is present in each of these
ligands. (The presence of the pyrimidine component
within 8 offers this ligand an entropic (statistical)
advantage over 7 and 9, but the significance, if any, of
this remains to be demonstrated.) However, the nicotinic

3242 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15
Sharples et al.
oxycarbonyl-9-azabicyclo[4.2.1]non-2-one (1.6 g, 7.6 mmol) in
dry tetrahydrofuran (THF, 60 mL) cooled to -78 °C was added
KHMDS (0.5 M in toluene, 16.9 mL, 8.42 mmol), and the
solution was stirred at -78 °C for 1.75 h. 2-[N,N-Bis(trifluo-
romethylsulfonyl)amino]-5-chloropyridine (2.53 g, 7.6 mmol)
in dry THF (30 mL) was then added via cannula. The reaction
mixture was stirred at -78 °C for 4 h and then allowed to
warm to room temperature. The solvents were removed in
vacuo, and the crude material was purified by flash chroma-
tography (eluting with 4:1 hexane:ethyl acetate) to give vinyl
triflate 10 (1.70 g, 65%) as a colorless oil. IR (film) ν (cm-1):
but held within a larger bicyclic framework, and this is
currently under investigation.
Con clu sion s
Chemical modification of 1 has produced a series of
analogues, which have been utilized to probe biological
activity at three major neuronal nAChR subtypes. High
affinity binding of 1 is critically dependent on the
presence of the hydrogen bond-accepting pyridyl nitro-
gen in the 3′ position, as in 1 and 4. Repositioning the
pyridyl nitrogen to the 2′ or 4′ positions (as in 5 and 6,
respectively) leads to either a decrease or an increase
in the distance from the cationic nitrogen present on
the 9-azabicyclo[4.2.0]nonene ring, resulting in substan-
tial decreases in binding affinity with respect to 4.
Replacing the pyridyl unit with a diazine moiety that
retains the “3-substituted pyridine” unit leads to dimin-
ished binding potency, as compared with 4. This was
true for all nAChR subtypes with the exception of the
pyridazine analogue 7, which retained potency at R7
nAChR. For activation of R3â4 nAChR, functional
assays demonstrated a similar potency relationship as
for binding to this nAChR subtype with the rank order
3 > 1 > 4 g 2 > 7 ) 8 (5, 6, and 9 were essentially
inactive at the concentrations tested). Computational
studies involving low energy conformers of compounds
4-6 show agreement with an R4â2* pharmacophore
model in the case of 4 and 5, but 6 was unable to meet
the pharmacophore parameters. Because 5 lacks ap-
preciable nicotinic activity, this indicates that further
refinements of pharmacophore models are necessary. In
summary, 1 has provided a useful framework for the
generation of a series of analogues to informatively
explore the molecular determinants of nAChR drug
interaction.
1
1717, 1238, 1067. H NMR (400 MHz, CDCl3): 7.24-7.15 (1
H, m, Voc), 5.80 (1 H, m, 3-H), 4.88-4.47 (4 H, m, Voc and
1-H, 6-H), 2.31-1.69 (8 H, m). 13C NMR (75.5 MHz, CDCl3):
153.3 (C), 142.2/141.8 (CH), 121.3/121.0 (CH), 116.4 (C), 96.0/
95.8 (CH2), 58.7/58.5 (CH), 55.4/55.3 (CH), 32.0/30.7 (CH2),
30.7/29.8 (CH2), 28.8/27.8 (CH2), 19.8 (CH2) (CF3 was not
observed). MS (70 eV): m/z 341 (M+, 8%), 332 (9), 298 (90), 84
(100). HRMS: calcd for
341.0552.
C12H14F3NO5S, 341.0545; found,
The following procedures were used to prepare all com-
pounds shown in Scheme 1 (with the exception of the 4-pyridyl
variant 6) and are illustrated by the conversion of vinyl triflate
10 via 11b to the 2-pyridyl derivative 5 as a specific example.
Characterization data for individual compounds are then listed
below, and full details for the preparation of 6 are provided
here.
Gen er a l P r oced u r e 1. Pd(0)-mediated cross-coupling of
vinyl triflate 10 to ArZnX was performed to give adducts 11a -
f.
2-(2-P yr id in yl)-9-vin yloxyca r bon yl-9-a za bicyclo[4.2.1]-
n on -2-en e (11b). A solution of 2-bromopyridine (136 mg, 0.855
mmol) in dry THF (7 mL) was cooled to -78 °C under a
nitrogen atmosphere and nBuLi (1.6 M in hexanes, 0.53 mL,
0.86 mmol) was added dropwise. The solution was then stirred
at -78 °C for 20 min. Anhydrous zinc chloride (117 mg, 0.855
mmol) in dry THF (3 mL) was added, and the solution was
allowed to warm to room temperature. Triflate 10 (108 mg,
0.317 mmol) in dry THF (7 mL) was then added via a cannula
followed by a solution of Pd(PPh3)4 (11.6 mg, 0.0095 mmol) in
dry THF (3 mL) also via a cannula. The resulting solution was
heated at reflux for 1 h and then allowed to cool and quenched
with saturated aqueous ammonium chloride solution (30 mL).
The organic layer was separated, and the aqueous layer was
extracted with diethyl ether (3 × 20 mL). The combined
organic phases were dried (Na2SO4), the solvent was removed
in vacuo, and the residue was purified by flash chromatogra-
phy (1:3 ethyl acetate:hexane) to give the title compound 11b
(72 mg, 84%) as a pale yellow oil. IR (film) ν (cm-1): 1713,
Exp er im en ta l Section
Ch em istr y. Gen er a l Com m en ts. Proton and carbon as-
1
signments are based on a combination of H-1H and 1H-13C
COSY and DEPT spectra. For 10 and 11a -f, which incorpo-
rate an N-vinyloxycarbonyl (Voc) moiety, carbamate resonance
resulted in broadened 1H and 13C spectra, which resulted in
significant, though not universal, doubling of lines. Where
doubling of signals was apparent, this has been indicated.
9-Vin yloxyca r bon yl-9-a za bicyclo[4.2.1]n on a n -2-on e. To
a solution of 9-methyl-9-azabicyclo[4.2.1]nonan-2-one in dry
dichloromethane (95 mL) was added anhydrous potassium
carbonate (4.13 g, 29.67 mmol), and the solution was stirred
at room temperature for 1 h. The mixture was cooled to 0 °C,
vinyl chloroformate (1.34 mL, 14.84 mmol) was added slowly,
and the solution was stirred at room temperature for 24 h,
after which time, further vinyl chloroformate (1.34 mL, 14.84
mmol) was added and stirring was continued for another 24
h. Silica gel was added, and the solvent was removed in vacuo.
The residue was purified by flash chromatography (3:1 hexane:
ethyl acetate) to give 9-vinyloxycarbonyl-9-azabicyclo[4.2.1]-
nonan-2-one (1.95 g, 94%) as a colorless oil. IR (film) ν (cm-1):
1
1420, 1147. H NMR (300 MHz, CDCl3): 8.57 (1 H, m, ArH),
7.78-7.62 (1.5 H, m, ArH), 7.46 (0.5 H, d, J ) 8.1 Hz, ArH),
7.25-7.10 (2 H, m, ArH and Voc), 6.50 (0.5 H, m, 3-H), 6.42
(0.5 H, m, 3-H), 5.44 (0.5 H, br d, J ) 9.2 Hz, 1-H), 5.29 (0.5
H, br d, J ) 9.0 Hz, 1-H), 4.78 (0.5 H, d d, J ) 14.0, 1.6 Hz,
Voc), 4.62-4.52 (1 H, m, 6-H), 4.43 (0.5 H, d d, J ) 6.3, 1.5
Hz, Voc), 4.28 (0.5 H, d d, J ) 14.0, 1.3 Hz, Voc), 4.24 (0.5 H,
d d, J ) 6.3, 1.5 Hz, Voc), 2.60-1.65 (8 H, m). MS (CI): m/z
271 (M+ + H, 49), 227 (100). HRMS: calcd for C16H19N2O2
(MH+), 271.1446; found, 271.1442.
2-(3-P yr id in yl)-9-vin yloxyca r bon yl-9-a za bicyclo[4.2.1]-
n on -2-en e (11a ). Colorless oil, isolated in 70% yield. IR (film)
ν (cm-1): 1714, 1422. 1H NMR (400 MHz, CDCl3): 8.63 (0.5
H, d, J ) 2 Hz, 2′-H), 8.61 (0.5 H, d, J ) 2 Hz, 2′-H), 8.49 (1
H, d d d, J ) 7.8, 4.9, 1.5 Hz, 5′-H), 8.13 (0.5 H, d d d, J ) 7.8,
2.0, 2.0 Hz, 4′-H), 7.75 (0.5 H, d d d, J ) 7.8, 2.0, 2.0 Hz, 4′-
H), 7.32-7.19 (2 H, m, 6′-H and Voc), 5.91 (1 H, m), 4.90 (1 H,
m), 4.80 (0.5 H, d d, J ) 14, 1.6 Hz, Voc), 4.58 (1.5 H, m, Voc)
4.47 (0.5 H, d d, J ) 6.4, 1.7 Hz, voc), 4.40 (0.5 H, d d, J ) 6.4,
1.7 Hz, voc), 2.50-168 (8 H, m). 13C NMR (75.5 MHz, CDCl3):
150.7/150.4 (C), 147.9/147.5 (CH), 147.4/147.1 (CH), 146.0/
145.4 (C) 142.3/142.1 (CH), 138.1/137.8 (C), 135.0/133.7 (CH),
130.0/129.8 (CH), 123.35/123.1 (CH), 95.3/95.2 (CH2), 59.1/58.8
(CH), 56.6 (CH) 32.0/31.8 (CH2), 31.2/30.7 (CH2) 29.0/28.3
1
1715, 1648. H NMR (300 MHz, CDCl3): 7.26 (0.4 H, d d, J )
14.0, 6.3 Hz, Voc), 7.18 (0.6 H, d d, J ) 14.0, 6.3 Hz, Voc),
4.88 (0.4 H, d d, J ) 14.0, 1.6 Hz, Voc), 4.77 (0.6 H, d d, J )
14.0, 1.6 Hz, Voc), 4.65-4.61 (1 H, m), 4.51 (0.4 H, d d, J )
6.2, 1.7 Hz, Voc), 4.48-4.42 (1.6 H, m), 2.68-2.56 (1 H, m),
2.49-1.58 (9 H, m). 13C NMR (75 MHz, CDCl3): 214.3 (C),
151.3 (C), 142.1 (CH), 96.0 (CH2), 64.8 (CH), 56.9 (CH), 41.7
(CH2), 33.5 (CH2), 33.0 (CH2), 26.6 (CH2), 19.2 (CH2). MS (60
eV): m/z 209 (M+, 9%), 83 (100). HRMS: calcd for C11H15NO3,
341.0545; found, 341.0552.
2-[(Tr iflu or om eth yl)su lfon yloxy]-9-vin yloxyca r bon yl-
9-a za bicyclo[4.2.1]n on -2-en e (10). To a solution of 9-vinyl-

Nicotinic Acetylcholine Receptor Agonist
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15 3243
(CH2), 24.1/24.0 (CH2). MS (CI): m/z 271 (M+ + 173), 227 (100).
compound 5 in quantitative yield as a colorless glass. 1H NMR
(300 MHz, CD3OD): 8.76 (1 H, m, ArH), 8.59 (1 H, t, J ) 8.0
Hz, ArH), 8.17 (1 H, d, J ) 8.2 Hz, ArH), 7.97 (1 H, m, ArH),
6.95 (1 H, m, 3-H), 4.90 (1 H, d, J ) 8.5 Hz, 1-H), 4.38 (1 H,
m, 6-H), 2.88-2.62 (3 H, m), 2.44-1.94 (5 H, m). 13C NMR
(75.5 MHz, CD3OD): 153.7 (C), 148.2 (CH), 147.7 (CH), 142.5
(CH), 136.8 (C), 127.0 (2 x CH), 61.2 (CH), 59.1 (CH), 32.2
(CH2), 28.7 (CH2), 25.2 (CH2), 24.8 (CH2). MS (70 eV): m/z 200
(M+, 69%), 171 (66), 105 (100). HRMS: calcd for C13H16N2,
200.1313; found, 200.1310.
HRMS: calcd for C16H19N2O2 (MH+), 271.1446; found, 271.1442.
2-(4-P yr id in yl)-9-vin yloxyca r bon yl-9-a za bicyclo[4.2.1]-
n on -2-en e (11c). Freshly cut lithium (106 mg, 15.32 mmol)
and naphthalene (1.99 g, 15.57 mmol) in dry THF (15 mL)
under a nitrogen atmosphere were stirred at room temperature
for 2 h. After this time, 0.95 mL of this solution was removed
and transferred to a clean, dry flask. Anhydrous zinc chloride
(0.5 M in THF, 0.97 mL, 0.485 mmol) was added dropwise over
10 min. 2-(3-Bromo-4-pyridinyl)-9-vinyloxycarbonyl-9-azabicyclo-
[4.2.1]non-2-ene (Spencer et al., 2001.) (85 mg, 0.243 mmol)
in dry THF (4 mL) was added, and the suspension was heated
to reflux for 1 h. The mixture was allowed to cool and quenched
with water (10 mL). The organic phase was separated, and
the aqueous phase was extracted with dichloromethane (2 ×
10 mL) followed by ethyl acetate (3 × 10 mL). The combined
organic residues were dried (Na2SO4), and the solvent was
removed in vacuo. The residue was purified by flash chroma-
tography (1:2 ethyl acetate/hexane) to give the title compound
11c (55 mg, 83%) as a colorless oil. IR (CHCl3) ν (cm-1): 1711,
1425. 1H NMR (270 MHz, CDCl3): 8.55 (2 H, m, ArH), 7.55 (1
H, m, ArH), 7.31 (1 H, m, ArH), 7.24 (0.5 H, d d, J ) 14.2, 6.3
Hz, Voc), 7.18 (0.5 H, d d, J ) 14.2, 6.3 Hz, Voc), 6.10 (1 H, m,
3-H), 4.96 (1 H, m, 1-H), 4.80 (0.5 H, d d, J ) 13.8, 1.6 Hz,
Voc), 4.60 (1 H, m, 6-H), 4.50 (0.5 H, d d, J ) 13.8, 1.6 Hz,
Voc), 4.46 (0.5 H, d d, J ) 6.3, 1.6 Hz, Voc), 4.37 (0.5 H, d d,
J ) 6.3, 1.6 Hz, Voc), 2.56-1.64 (8 H, m, CH2). MS (CI): m/z
271 (MH+, 66%), 227 (100), 199 (30). HRMS: calcd for
2-(3-P yr id in yl)-9-a za b icyclo[4.2.1]n on -2-en e H yd r o-
ch lor id e Sa lt (4). Colorless glass. 1H NMR (400 MHz,
CDCl3): 10.20 (2 H, br, NH), 9.28 (1 H, br, ArH), 8.62 (1 H,
br, ArH), 8.46 (1 H, br, ArH), 7.90 (1 H, br, ArH), 6.41 (1 H,
m, H-3), 5.00 (1 H, m), 4.46 (1 H, m), 2.79-1.50 (8 H, m). 13C
NMR (75.5 MHz, CDCl3): 147.7 (CH), 147.5 (CH), 143.8 (C),
139.8 (C), 133.3 (CH), 130.5 (CH), 123.0 (CH), 65.5 (CH), 62.1
(CH), 32.2 (CH2), 28.4 (CH2), 24.8 (CH2), 24.6 (CH2). MS (CI):
m/z 201 (M+, 100). HRMS: calcd for C13H17N2 (MH+), 201.1392;
found, 201.1384.
2-(4-P yr id in yl)-9-a za b icyclo[4.2.1]n on -2-en e H yd r o-
1
ch lor id e Sa lt (6). Colorless glass. H NMR (270 MHz, CD3-
NO2): 10.47 (1 H, broad s, NH), 9.79 (1 H, broad s, NH), 8.71
(2 H, br s, ArH), 8.04 (2 H, br s, ArH), 6.86 (1 H, m, 3-H), 4.84
(1 H, m, 1-H), 4.42 (1 H, m, 6-H), 2.86-1.91 (8 H, m, CH2).
13C NMR (75.5 MHz, CD3NO2): 150.3 (CH), 145.0 (C), 136.4
(C), 126.8 (CH), 123.8 (CH), 60.9 (CH), 59.2 (CH), 32.1 (CH2),
28.4 (CH2), 25.0 (CH2), 24.8 (CH2). MS (CI): m/z 201 (MH+,
100%). HRMS: calcd for C13H17N2, 201.1392; found, 201.1392.
C
16H19N2O2 (MH+), 271.1446; found, 271.1458.
2-(3-P yr idazin yl)-9-vin yloxycar bon y-9-azabicyclo[4.2.1]-
2-(3-P yr id a zin yl)-9-a za bicyclo[4.2.1]n on -2-en e Hyd r o-
n on -2-en e (11d ). Colorless oil, isolated in 87% yield. IR
(CHCl3) ν (cm-1): 1715, 1430. 1H NMR (270 MHz, CDCl3): 9.05
(1 H, br t, J ) 9 Hz, 6′-H), 7.88 (0.5 H, d d, J ) 9, 1 Hz, 4′-H),
7.68-7.40 (1.5 H, m), 7.22 (1 H, m), 6.40 (1 H, m, 3-H), 5.67
(0.5 H, br d, J ) 9 Hz, 1-H), 5.31 (0.5 H, br d J ) 9 Hz, 1-H),
4.80 (0.5 H, d d, J ) 13.8, 1.6 Hz, Voc), 4.60 (1 H, m, 6-H),
4.50 (0.5 H, d d, J ) 13.8, 1.6 Hz, Voc), 4.46 (0.5 H, d d, J )
6.3, 1.6 Hz, Voc), 4.37 (0.5 H, d d, J ) 6.3, 1.6 Hz, Voc), 2.58-
2.52 (3 H, m), 2.30-2.01 (5 H, m). MS (60 eV): m/z 271 (M+,
100), 228 (95). HRMS: calcd for C15H17N3O2, 271.1321; found,
271.1320.
2-(5-P yr im idin yl)-9-vin yloxycar bon yl-9-azabicyclo[4.2.1]-
n on -2-en e (11e). Colorless solid, isolated in 77% yield; mp
85-86 °C (toluene). IR (CHCl3) ν (cm-1): 1705, 1648, 1424.
1H NMR (CDCl3, 270 MHz): 9.12 (1 H, s, ArH), 8.95 (1 H, s,
ArH), 8.80 (1 H, s, ArH), 7.22 (1 H, m, Voc), 5.97 (1 H, m, 3-H),
4.89 (1 H, m, 1-H), 4.82 (0.5 H, d d, J ) 13.8, 1.6 Hz, Voc),
4.61 (1.5 H, m, Voc and 6-H), 4.48 (0.5 H, d d, J ) 6.6, 1.6 Hz,
Voc), 4.43 (0.5 H, d d, J ) 6.8, 1.6 Hz, Voc), 2.58-1.80 (8 H,
m). MS (CI): m/z 272 (MH+, 65%), 228 (100), 185 (35).
HRMS: calcd for C15H18N3O2 (MH+), 272.1399; found, 272.1391.
1
ch lor id e Sa lt (7). H NMR (300 MHz, CD3OD): 9.07 (1H, d
d, J ) 5, 1.5 Hz, 6′-H), 7.95 (1 H, d d, J ) 8, 1.5 Hz, 4′-H), 7.64
(1H, d d, J ) 8, 5 Hz, 5′-H), 6.88 (1 H, m, 3-H), 5.53 (1 H, br
d, J 9.5 Hz, 1-H), 4.32 (1 H, m, 6-H), 2.70-2.05 (8 H, m). 13C
NMR (75.5 MHz, CD3OD): 160.0, 150.1, 140.5, 138.6, 127.9,
124.5, 63.8 (CH), 58.0 (CH), 30.4 (CH2), 28.1 (CH2), 27.2 (CH2),
23.2 (CH2). MS (60 eV): m/z 201 (M+, 100), 172 (90). HRMS:
calcd for C12H15N3, 201.1266; found, 201.1266.
2-(5-P yr im id in yl)-9-a za bicyclo[4.2.1]n on -2-en e Hyd r o-
ch lor id e Sa lt (8). Colorless glass. δH (270 MHz, CD3NO2):
9.96 (1 H, broad s, NH), 9.70 (1 H, broad s, NH), 9.57 (2 H, s,
ArH), 9.43 (1 H, s, ArH), 6.67 (1 H, m, 3-H), 4.86 (1 H, m,
1-H), 4.46 (1 H, m, 6-H), 2.86-1.92 (8 H, m). 13C NMR (75.5
MHz, CD3NO2): 157.0 (CH), 154.7 (CH), 144.1 (C), 136.0 (C),
131.4 (CH), 60.1 (CH), 59.0 (CH), 30.7 (CH2), 28.0 (CH2), 27.1
(CH2), 23.5 (CH2). MS (CI): m/z 202 (MH+, 100%), 185 (10),
173 (9), 159 (9), 133 (8), 82 (9). HRMS: calcd for C12H16N3
(MH+), 202.1344; found, 202.1346.
2-(2-P yr a zin yl)-9-a za b icyclo[4.2.1]n on -2-en e H yd r o-
1
ch lor id e Sa lt (9). Colorless glass. H NMR (270 MHz, CD3-
OD): 9.14 (1 H, d, J ) 1.6 Hz, 3′-H), 9.09 (1 H, d d, J ) 3, 1.6
Hz, 5′-H), 8.68 (1 H, d, J ) 3 Hz, 6′-H), 7.14 (1 H, m, 3-H),
5.37 (1 H, br d, J ) 9.6 Hz, 1-H), 4.36 (1 H, m, 6-H), 2.75-
2.02 (8 H, m). 13C NMR (75.5 MHz, CD3OD): 155.0 (C), 147.0
(C), 143.8 (CH), 141.9 (CH), 132.9 (CH), 130.0 (CH), 60.8 (CH),
59.0 (CH), 31.6, 29.0, 28.4, 24.7. MS (60 eV): m/z 201 (M+,
100). HRMS: calcd for C12H15N3, 201.1266; found, 201.1261.
2-(2-P yr a zin yl)-9-vin yloxyca r bon yl-9-a za bicyclo[4.2.1]-
n on -2-en e (11f). Colorless oil, isolated in 80% yield. IR
(CHCl3) ν (cm-1): 1707, 1648. 1H NMR (270 MHz, CDCl3): 8.85
(0.5 H, d, J ) 1 Hz, 3′-H), 8.77 (0.5 H, d, J ) 1 Hz, 3′-H), 8.53-
8.39 (2 H, m, 5′-H and 6′-H), 7.20 (1 H, m, Voc), 6.51 (1 H, m,
3-H), 5.48 (1H, m, 1-H), 4.78 (0.5 H, d d, J ) 14, 1.5 Hz, Voc),
4.60 (1 H, m, 6-H), 4.48 (0.5 H, d d, J ) 14, 1.5 Hz, Voc), 4.29
(1 H, m, Voc), 2.75-1.80 (8 H, m). MS (60 eV): m/z 271 (M+,
10), 228 (100). HRMS: calcd for C15H17N3O2, 271.1321; found,
271.1331.
Biology. Cell Cu ltu r e. The mouse fibroblast LR3â4 cell
line stably transfected with the rat R3 and â4 subunit cDNAs
were maintained as previously described.27 Expression of
nAChRs was induced by 1 µM dexamethasone.
Gen er a l P r oced u r e 2. Acid-mediated cleavage of N-
vinyloxycarbonyl group was performed to give compounds 4-9.
Mem br a n e P r ep a r a tion . Rat P2 brain membranes were
prepared as described previously.9 Membranes from LR3â4
cells were prepared as follows: cells were briefly washed once
with phosphate-buffered saline (PBS, 150 mM NaCl, 2 mM
KH2PO4, 8 mM K2HPO4, pH 7.4) and scraped into ice-cold
homogenization buffer (PBS, 10 mM EDTA, 10 mM EGTA, 1
mM PMSF, pH 7.4). Cells were pelleted by centrifugation (500
g, 3 min) and sonicated. The resulting homogenate was
centrifuged (100 000g, 30 min), and the membrane pellet was
resuspended in ice-cold homogenization buffer by use of a hand
held homogenizer. The membranes were stored in aliquots at
-20 °C.
2-(2-P yr id in yl)-9-a za b icyclo[4.2.1]n on -2-en e H yd r o-
ch lor id e Sa lt (5). To a solution of 2-(2-pyridinyl)-9-vinyloxy-
carbonyl-9-azabicyclo[4.2.1]non-2-ene (42 mg, 0.156 mmol) in
dioxane (6 mL) were added water (1 mL) and concentrated
hydrochloric acid (0.2 mL). The mixture was heated at reflux
for 22 h and then allowed to cool. The majority of the dioxane
was evaporated in vacuo, and the residue was diluted with
water (5 mL), cooled to 0 °C, and made basic by the dropwise
addition of 5 M NaOH solution. The basic solution was
extracted with dichloromethane (5 × 10 mL), and the combined
organic phases were evaporated in vacuo to give the title

3244 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15
Sharples et al.
Ra d ioliga n d Com p etition Bin d in g Assa ys. (()-[3H]-
Ep iba tid in e Bin d in g. LR3â4 cell membranes (10 µg of
protein) were diluted into Krebs-Ringer Tris HEPES buffer
(118 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl2, 20 mM HEPES,
20 mM Tris, 0.1 mM PMSF, 0.01% NaN3, pH 7.4) containing
1 nM (()-[3H]epibatidine and competing drugs to a total
volume of 1 mL. Nonspecific binding was determined in the
presence of 1 mM (-)-nicotine. Samples were incubated for 2
h at 37 °C before filtration through Gelman GFA/E filter paper
(presoaked overnight in 0.3% polyethyleneimine in PBS) using
a Brandel Cell Harvester. Filters were added to Optiphase Safe
scintillation cocktail, and samples were counted for 5 min each
in a Packard Tricarb scintillation counter.
(-)-[3H]Nicotin e Bin d in g a n d [3H]MLA Bin d in g. Com-
petition binding assays were performed as previously de-
scribed.9,25 In brief, rat brain P2 brain membranes (250 µg
protein) were incubated with serial dilutions of competing
drugs and 2 nM [3H]MLA for 2.5 h at room temperature or 10
nM [3H]nicotine for 30 min at room temperature plus 1 h at 4
°C, respectively. Bound and free radioligand was separated
by filtration as described above. Nonspecific binding was
determined in the presence of 1 mM (-)-nicotine.
used by these authors was adapted from that previously
reported19 for UB-165. Steitz and co-workers also describe
computational results associated with the atomic electrostatic
potential charges of the nitrogen atoms associated with the
pyridyl and diazine ligands and apply these data to correlate
the binding affinity and the hydrogen bond acceptor capacity
of the heteroaryl moiety.
Ack n ow led gm en t. This study was supported finan-
cially by grants from the BBSRC (Project Grants
7/MOLO4724 and 86/B11785) and a Wellcome Trust/
HEFCE J oint Equipment Initiative (056427). This paper
is dedicated to the memory of Professor Malcolm M.
Campbell (1943-2001).
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