Improving the nicotinic pharmacophore with a series of (isoxazole)methylene-1-azacyclic compounds: Synthesis, structure-activity relationship, and molecular modeling

Article abstract of DOI:10.1021/jm9910627

A series of (isoxazole)methylene-1-azacyclic compounds was prepared. The compounds were tested for affinity to central nicotinic acetylcholine receptors (nAChRs) and central muscarinic receptors. The compounds covered a broad range of affinities for the nAChRs (IC₅₀ = 0.32 to > 1000 nM), with selectivities for the nAChRs over the muscarinic receptors in the range of 3- 183. The high-affinity compound (Z)-26 (3-(4-methyl-5-isoxazolyl)methylene-1- azabicyclo[2.2.2]octane, IC₅₀ = 3.2 nM) having only one energy minimum was used as the reference structure in a computational study. This ligand has enabled definition of an important distance parameter, and the existence of this parameter was supported by showing that other potent nicotinic ligands (for example, nicotine and epibatidine) fit the model.

Full text of DOI:10.1021/jm9910627

4970
J . Med. Chem. 1999, 42, 4970-4980
Im p r ovin g th e Nicotin ic P h a r m a cop h or e w ith a Ser ies of
(Isoxa zole)m eth ylen e-1-a za cyclic Com p ou n d s: Syn th esis, Str u ctu r e-Activity
Rela tion sh ip , a n d Molecu la r Mod elin g
J anne E. Tønder,† J ohn Bondo Hansen,† Mikael Begtrup,‡ Ingrid Pettersson,† Karin Rimvall,†
Birgitte Christensen,† Ulrich Ehrbar,† and Preben H. Olesen*,†
Health Care Discovery, Novo Nordisk A/ S, Novo Nordisk Park, DK-2760 Måløv, Denmark, and Department of Medicinal
Chemistry, Royal Danish School of Pharmacy, Universitetsparken 2, DK-2100 Copenhagen, Denmark
Received April 22, 1999
A series of (isoxazole)methylene-1-azacyclic compounds was prepared. The compounds were
tested for affinity to central nicotinic acetylcholine receptors (nAChRs) and central muscarinic
receptors. The compounds covered a broad range of affinities for the nAChRs (IC50 ) 0.32 to
>1000 nM), with selectivities for the nAChRs over the muscarinic receptors in the range of
3-183. The high-affinity compound (Z)-26 (3-(4-methyl-5-isoxazolyl)methylene-1-azabicyclo-
[2.2.2]octane, IC50 ) 3.2 nM) having only one energy minimum was used as the reference
structure in a computational study. This ligand has enabled definition of an important distance
parameter, and the existence of this parameter was supported by showing that other potent
nicotinic ligands (for example, nicotine and epibatidine) fit the model.
In tr od u ction
ore are (A) a cationic center (e.g., a protonated sp3
nitrogen), (B) an electronegative atom capable of form-
ing a hydrogen bond, and (C) a dummy point or an atom,
which defines a line along which the hydrogen bond may
form. For (S)-nicotine this latter point is considered to
be a point toward the centroid of the pyridine ring. The
optimal distances between the three points were esti-
mated to (A-B) 4.8 ( 0.3 Å; (A-C) 4.0 ( 0.3 Å; (B-C)
1.2 Å.
Since then the interest has primarily been focusing
on the internitrogen (N-N) distance of the ligand’s
supposed binding conformation (Table 1). The main
question underlying the dispute about the correct N-N
distance is: Which low-energy conformation of epiba-
tidine is responsible for the affinity for the nAChRs?
As epibatidine only contains one rotatable bond, its
flexibility is very limited. With the exception of one
report,12 the conformational studies of epibatidine pub-
lished till now5,8,13-15 have all found approximately the
same two low-energy conformers. The actual conformers
found vary a little depending on whether the compounds
under study are protonated or not. The two conformers
are almost equal energetically, they only vary 180° in
rotation of the pyridine ring. Thus, depending on the
epibatidine conformer chosen as reference structure, the
study comes out with an N-N distance close to either
4.6 or 5.5 Å.
Research in the nicotinic area has over the years
demonstrated a variety of diseases or disease states in
which nicotinic therapy would have beneficial effect.
Among these are Alzheimer’s disease, Parkinson’s
disease, Tourette’s syndrome, anxiety, pain, and
depression.1-4 Several compounds with high affinity for
the central nicotinic acetylcholine receptors (nAChRs)
have been described, e.g., epibatidine,4,5 ABT 418,6,7 and
recently a series of pyridyl ethers which include the very
potent compound A85380.8
The structures of epibatidine and ABT 418 suggested
that combining the steric volume close to the sp3
hybridized nitrogen, as in epibatidine, with the isoxazole
from ABT 418 could lead to new structures possessing
affinity for the nAChRs. This led to some novel (isox-
azole)methylene-1-azabicyclic compounds, including (Z)-
14, with nanomolar affinities for the central nAChRs
and possessing agonist properties.9 In the present work,
the series was continued by varying the nature of the
azacycle and the isoxazole substitution pattern, in order
to examine the borders of the nicotinic pharmacophore.
The nicotinic pharmacophore has been developing
since 1970, when Beers and Reich were the first to
suggest two elements essential for nicotinic agonist
activity.10 They proposed that the specific binding of
nicotinic agonists to the nAChRs is mediated by two
elements: (1) a Coulombic interaction involving the
cationic center and (2) a hydrogen bond between the
receptor and an acceptor group in the ligand which is
formed approximately 5.9 Å from the center of the
positive charge.
In this report the synthesis and in vitro binding data
for a large number of compounds in the 3-(isoxazole)-
methylene-1-azacyclic series are presented. Further-
more, the structure-activity relationships based on
molecular modeling of the compounds are thoroughly
discussed. We also report the definition of an important
distance parameter, which predicts affinity for the
nAChRs better than the N-N distance does.
In 1986 Sheridan and co-workers proposed a triangle
as a model of the nicotinic pharmacophore.11 They
suggested that the essential groups in the pharmacoph-
Ch em istr y
Isoxazoles (1-3) were synthesized following a modi-
† Novo Nordisk A/S.
‡ Royal Danish School of Pharmacy.
fied literature procedure.16 Aldehydes were reacted with
10.1021/jm9910627 CCC: $18.00 © 1999 American Chemical Society
Published on Web 11/05/1999

Improving the Nicotinic Pharmacophore
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24 4971
Sch em e 2a
Ta ble 1. Various N-N Distances Proposed over the Years
year
compounds studied
N-N distance (Å)
1986a
nicotine, cytisine, ferruginine
methiodide, muscarone
nicotine, epibatidine
A85380, epibatidine
A85380 analogues, epibatidine
4.8
1994b
1996c
1998d
5.5
4.6, 5.6 or > 6
4.5
a
b
d
Reference 11. Reference 5. c Reference 8. Reference 15.
Sch em e 1a
a
For R1, R2, and R3, see Tables 2 and 3. Reagents and
conditions: (i) n-BuLi or LDA in THF, -78 °C, N2. (ii) m ) 2, n )
1: 1-azabicyclo[2.2.2]octane-3-one, -78 °C, N2. m ) 1, n ) 2:
1-azabicyclo[3.2.1]octane-5-one, -78 °C, N2. (iii) NEt3, EtOH,
SOCl2, -78° f 20 °C, N2.
a
Reagents and conditions: (i) LDA or n-BuLi in THF, -78 °C,
N2. (ii) Azabicyclic ketone, -78 f 20 °C, N2. (iii) Azacyclic ketone,
-78 f 20 °C, N2. (iv) 1-Chloroethyl chloroformate in toluene,
reflux. (v) Reflux in MeOH.
hydroxylamine hydrochloride to form the corresponding
oximes. Subsequently the oximes were R-brominated
with N-bromosuccinimide and then treated with tri-
ethylamine causing elimination of HBr to give the
corresponding nitrile oxides. A 1,3-dipolar cycloaddition
reaction between the nitrile oxides and isopropenyl
acetate produced the 4,5-dihydroisoxazoles, which upon
treatment with hydrochloric acid finally eliminated
acetic acid to give the desired isoxazoles (3-isopropyl-
5-methylisoxazole (1), 3-ethyl-5-methylisoxazole (2), and
3-propyl-5-methylisoxazole (3)).
Sch em e 3a
a
For R2, see Tables 2 and 3. Reagents and conditions: (i)
Dimethyl 2-oxoalkylphosphonate and KOH in water, 0 °C. (ii)
DMF-DMA, 90 °C, N2. (iii) CH3CO2H and NH2OH‚HCl in water,
90 °C.
3-Methoxymethyl-5-methylisoxazole (4) was prepared
from 3-hydroxymethyl-5-methylisoxazole by deprotona-
tion with sodium hydride followed by alkylation with
methyl iodide.
Sch em e 4a
Compounds 5-11 were synthesized in a one-pot
reaction by direct coupling of the azacyclic ketone and
the isoxazole (Scheme 1). The substituted 5-methylisox-
azoles were selectively deprotonated with LDA or n-
BuLi and reacted with an azacyclic ketone to give the
desired C-C bond formation. The formed alcohol was
treated directly with thionyl chloride, triethylamine, and
in some cases ethanol. The chloro intermediate spon-
taneously eliminated HCl to give the desired products
5-11 as a mixture of the (E)- and (Z)-isomers in a total
yield of around 70%. The isomers were separated by
column chromatography and/or fractional crystalliza-
tion, giving the compounds 5-11 in 4.4-29% yield. A
complete separation of the (Z)- and (E)-isomers was not
possible in all cases. The compounds containing a chiral
center ((Z)-11 and (E)-11) were prepared as racemates.
Compounds 13-17 were prepared by a direct coupling
between 3-methyl-5-trimethylsilanylmethylisoxazole
(12)17 and the appropiate azacyclic ketone (Scheme 2).
Treatment of 12 with LDA or n-BuLi generated the
R-silyl carbanion, which then reacted with a ketone,
again with a mixture (approximately 1:1) of the (E)- and
(Z)-isomers in about 60-80% yield as the result. The
isomers were separated using column chromatography
and/or fractional crystallization to give 13-17 in 1.1-
13% yield.
a
Reagents and conditions: (i) t-BuOK, DMF, 0 °C, N2. (ii)
1-Azabicyclo[2.2.2]octan-3-one, 0 f 20 °C. (iii) Acetone oxime,
n-BuLi, N2, 0 °C. (iv) Concd. H2SO4, 20 °C.
The R,â-unsaturated ketones 21-23 were formed as
a mixture of their (E)- and (Z)-isomers by a Horner-
Wadsworth-Emmons reaction between the appropriate
azacyclic ketones and oxoalkyl phosphonates (Scheme
3). After separation of the (Z)- and (E)-enones, the (Z)-
isomers (21-23) were reacted with N,N-dimethylfor-
mamide dimethyl acetal (DMF-DMA) in a modified
Mannich reaction. The intermediate formed was treated
with glacial acetic acid and hydroxylamine to give the
desired (Z)-isoxazoles 24-26 in 18-37% yield.
The R,â-unsaturated fluoro ester 27 was synthesized
by a similar reaction between triethyl-2-fluoro-2-phospho-
noacetate and 1-azabicyclo[2.2.2]octan-3-one (Scheme 4).
Following a modified literature procedure,18 27 was then
treated with deprotonated acetone oxime to induce the
cyclization, whereupon stirring with concentrated sul-
Compounds 15-17 were subsequently dealkylated to
18-20 by treatment with 1-chloroethyl chloroformate
in toluene followed by reflux in methanol.

4972 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24
Tønder et al.
Ta ble 2. In Vitro Binding Data for the 1-Azabicyclo[2.2.2]octan-isoxazolesa
a
b
Values represent mean ( SEM. n g 3, except *n ) 2, and # n )1. Measured by GC.
furic acid caused elimination of water to selectively
produce the (E)-isomer of 28.
is very limited, a conformational search was performed
by rotation around the single bond connecting the two
heterocycles. The more flexible compounds were inves-
tigated using the molecular dynamics option in Sybyl
6.5.22
For the further studies, only low-energy conformers
with a ∆E < 3.0 kcal/mol were used.
As all of the compounds in this series that have been
tested in a functional assay proved to be nicotinic
agonists,9 it is assumed that they interact with the
nAChRs in the same way as nicotine and epibatidine.
The relative configuration of the (Z)- and (E)-
1
compounds was determined on the basis of H NMR
experiments. After the individual protons were assigned
from COSY experiments, NOE effects were used for
determination of the spatial arrangement around the
double bond.
Biology
The oxalate or hydrochloride salts of the compounds
were tested in a binding assay, using the displacement
of [3H]methylcarbamylcholine ([3H]MCC)19,20 from na-
tive cholinergic binding sites in rat brain cortex as a
measure of their affinity for nAChRs. As the ligands for
the nAChRs receptors often display some affinity for the
muscarinic acetylcholine receptors, the affinity for these
was measured using [3H]oxotremorine-M (Oxo-M)21 as
radioligand.
Resu lts a n d Discu ssion
Str u ctu r e-Activity Rela tion sh ip s. In the present
collection of compounds, explorations were concentrated
on alkyl substituents in the isoxazole ring, substituents
of different character on the methylene bridge, and the
nature of the nonaromatic heterocycle.
Th e 1-Aza bicyclo[2.2.2]octa n e Ser ies. Generally,
the (Z)-isomers are more potent than the (E)-isomers
(Table 2). Hence, (E)-14, with an affinity of 504 nM for
the nAChRs, was the most potent of the (E)-compounds.
Com p u ta tion a l Stu d ies
As the conformational freedom in the compounds
containing an azabicycle and an aromatic heterocycle

Improving the Nicotinic Pharmacophore
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24 4973
Ta ble 3. In Vitro Binding Data for the 1-Azabicyclo[3.2.1]octan- and [2.2.1]heptan-isoxazolesa
a
b
Values represent mean ( SEM n g 3, except *n ) 2, and # n ) 1. Measured by GC.
Ta ble 4. In Vitro Binding Data for the 1-Azacyclo-isoxazolesa
a
b
Values represent mean ( SEM n g 3, except *n ) 2. Measured by GC.
In the 1-azabicyclo[2.2.2]octane series different sub-
stitutions within the isoxazole ring show that the
optimum R1 substituent has to be small. Thus, (Z)-14
displayed an affinity of 3.9 nM and (Z)-25 an affinity of
1.7 nM compared to 6.6 nM for (S)-nicotine. There was
a substantial decrease in activity in going from methyl
((Z)-14) to larger R1 substituents. Changing methyl to
ethyl ((Z)-5) led to a 26-fold decrease in affinity and a
further change to propyl ((Z)-7) and isopropyl ((Z)-8)
lowered the affinity 45 and 27 times, respectively. It is
noteworthy that (Z)-6 with the electronically differing
R1 substituent CH2OCH3 had almost the same nicotinic
affinity as (Z)-7.
tinic receptor when compared to the muscarinic recep-
tors, the best compounds showing a selectivity around
150.
Oth er Aza cycles. Interchanging the R1 and R2
substituents from (Z)-13 to (Z)-24, both having the (R/
S)-1-azabicyclo[2.2.1]heptane moiety, enhances the nico-
tinic affinity by a factor 91, as (Z)-24 becomes equipotent
with (()-epibatidine (Table 3). Analogues containing (R/
S)-1-azabicyclo[3.2.1]octane have lower affinities. In-
terestingly, the (E)-isomers (E)-11 and (E)-13 are rela-
tively potent (53 and 23 nM).
Some azamonocycles (piperidine; 17, 20, pyrrolidine;
16, 19, and azetidine; 18) were also tested (Table 4),
but except for (Z)-20 all the compounds had a low
affinity for the nAChR.
Interchanging the R1 and R2 substituents in (Z)-14
gave the equipotent (Z)-26 (3.2 nM). On the methylene
bridge, methyl ((Z)-9) and fluorine (28) were introduced
instead of hydrogen, but with a lowering of affinity as
the result.
Com p u ta tion a l Stu d ies. For the study, 16 com-
pounds from the 3-(isoxazole)methylene-1-azacyclic se-
ries were chosen. (R/S)-Epibatidine, (S)-nicotine, ABT
418, an Anatoxin-a analogue (29)1, Epiboxidine,23
a
All compounds displayed selectivity toward the nico-

4974 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24
Ta ble 5. Results from the Molecular Modelinga
Tønder et al.
a
See Supporting Information for the full results from the conformational search. b Superpositioning with (Z)-26•a using the sp3 nitrogen
(N+), the anionic site point (a), and the hydrogen bond donor site point (b).
which possesses a torsion (τ) of 180° or the pair of
conformers that have a τ close to 180°. In the low-energy
conformer the R2 group is directed away from the
azacycle, thereby avoiding its hydrogen atoms (Table
5, (Z)-26, R2 ) Me).
In the (E)-series the compounds have four ((E)-14) or
two ((R/ S)-(E)-11 and (R/ S)-(E)-13) low-energy confor-
mations with a ∆E < 3.0 kcal/mol. The global minima
all have a τ close to 180°.
Compound 29 is quite rigid due to the integrated
double bond, consequently the conformational search
revealed only three low-energy conformers. UB-165
displays the same symmetry as (S)-nicotine and (R/ S)-
epibatidine, thus the low-energy conformers occur in
pairs differing 180° in rotation of the pyridine ring. The
F igu r e 1. Reference compounds used in the computational
studies. The numbered atoms are the ones employed in
low-energy conformations of 30 are symmetrical around
0°, reflecting the position of the N-methyl group (axial/
equatorial).
measuring the torsional angle (τ).
mixed Anatoxin-a/epibatidine analogue named UB-
165,24 and a rigid nicotine analogue25 (30) were used as
reference compounds (Figure 1).
Su p er p osition in g. For several years it has been
accepted that the presence of a positively charged
nitrogen atom and a hydrogen bond acceptor in the
ligand was important for activity at the nAChRs.10,11
In the present study it is assumed that the sp2 hybrid-
ized nitrogen lone pair interacts with a hydrogen bond
donor in the receptor, and even though the presence of
an anionic site within the nicotinic receptor has been
the matter of some discussion,26-29 it is also assumed,
that such a site is present and interacts with the
protonated nitrogen atom. Thus, the compounds were
superpositioned using the sp3 hybridized nitrogen atom
(N+), its complementary anionic site point (a), and the
hydrogen bond donor site point (b) complementary to
the sp2 nitrogen atom (Figure 2). The site points were
placed 2.9 Å away from the nitrogen atoms. With the
As the sp3 hybridized nitrogen atom probably will be
protonated under physiological conditions, the proto-
nated form of the compounds was chosen for the study.
Con for m a tion a l Sea r ch . The conformational search
of (S)-nicotine gave rise to 20 different low-energy
conformers with a ∆E < 3.0 kcal/mol. The 20 conformers
occurred in pairs of almost equal energy, differing by
approximately 180° in the rotation of the pyridine ring.
For (R)- and (S)-epibatidine, four low-energy conformers
were found (Table 5). These conformers also occurred
in pairs as described for (S)-nicotine.
In the (Z)-series the individual compounds have
differing numbers of low-energy structures, but the
global energy minimum is in every instance the one

Improving the Nicotinic Pharmacophore
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24 4975
potential surface was changed. Hence in 28 there is a
slightly more negative area around the fluorine atom,
which must be poorly tolerated by the receptor (IC50
)
755 nM). Having methyl in the R3 position ((Z)-9) led
to major conformational changes. The global energy
minimum has a τ ) (141°, and the energy gap to the
desirable conformation with τ ) 180° is as large as 4.5
kcal/mol.
F igu r e 2. The position of the elements (N+, a, b) used for
superpositioning the compounds.
The low-energy conformers of the two piperidine
compounds (Z)-20 and (Z)-17 are very much alike when
viewed as models, and at first it is difficult to explain
the marked difference in affinity. Yet, a comparison with
(S)-nicotine and ABT 418 shows that the N-methyl of
(Z)-17 points in the opposite direction to the N-methyl
groups of ABT 418 and the three conformers of (S)-
nicotine, which superimpose (Z)-26•a (Figure 4). The
drastic effect that N-methylation in some cases has on
the affinity has previously been ascribed to the possibil-
ity that the various amine substituents experience
different receptor interactions, due to the varying direc-
tions in which they are oriented.13
The relatively low affinity of (Z)-20 (87 nM), compared
to (Z)-26, is probably due to the changes in electrostatic
potential in the aliphatic area around the sp3 hybridized
nitrogen atom. These changes are apparent when
compared to both (Z)-26 (monoprotonated amine) and
epibatidine (diprotonated amine).
(R)- and (S)-(Z)-11 also have some extra volume in
the aliphatic area close to the N-methyl group of (Z)-
17, but the extra volume must be placed in a more
tolerable area of the receptor, as the affinity of (Z)-11
(130 nM) is not as low as the affinity of (Z)-17 (>1000
nM).
The pyrrolidine and azetidine compounds (Z)-16 and
18 cannot achieve a conformation that superimposes (Z)-
26•a. This explains their low affinities (783 nM and
>1000 nM).
points chosen for superpositioning, all the chiral com-
pounds display symmetrical results for their enanti-
omers, so from now on only the (R)-enantiomers are
mentioned. As the high-affinity compound (Z)-26 has
only one energy minimum, this conformer ((Z)-26•a)
was used as the reference structure in the superposi-
tioning.
Superpositioning (Z)-26•a on the four low-energy
conformers of (R)-epibatidine gave an excellent fit with
the conformer possessing the global energy minimum
(τ ) 7°). The carbon skeleton of (R)-epibatidine follows
the skeleton of (Z)-26•a very closely; the aromatic rings
are coplanar and very close to one another. In addition,
the steric bulk around the sp3 nitrogen atom occupies
the same space (Figure 3).
Of the 20 low-energy conformers found for (S)-
nicotine, three overlapped well with (Z)-26•a (Figure
3). These had conformational energies 1.5, 1.7, and 2.4
kcal/mol above the minimum. As the three energy
minima represent different conformations, it is difficult
to interpret the relation between conformation and
nicotinic activity for (S)-nicotine.
In the case of ABT 418, the overlapping conformation
had an energy 1.5 kcal/mol above the global minimum.
All other reference compounds (29, Epiboxidine, UB-
165, and 30) overlapped well with (Z)-26•a in their
global minimum conformation. Thus the RMS values
were low, and the carbon skeletons of the compounds
followed that of (Z)-26•a very well.
As mentioned above, (E)-11 and (E)-13 are active (53
and 23 nM). Attempts to fit these compounds into the
derived model fail if the points mentioned above (N+,
a, b) are used. However, if the site point complementary
to the isoxazole oxygen (2.9 Å away) is used instead of
the sp2 nitrogen site point, the overlap is much better
(Figure 5). For all the (E)-compounds, local minimum
conformers are used for the superposition, not the global
minima, as these cannot superimpose (Z)-26•a to any
reasonable extent. The local minima are 1.2-2.6 kcal/
mol above their global minimum. Still the superposi-
(Z)-8 was included as an example of the compounds
with large R1 substituents. As expected, the R1 sub-
stituent affected neither the number nor the geometry
of the low-energy conformations found compared to, e.g.,
(Z)-14. Thus the drop in affinity (a factor of 27 compared
to (Z)-14) must be ascribed to unfavorable interactions
between the R1 substituent and the receptor.
The R3 position was examined with 28 and (Z)-9.
Having fluorine in the R3 position of (28) had no effect
on the low-energy conformation; instead the electrostatic
F igu r e 3. Stereoview of (Z)-26•a (orange) superimposed with (R)-epibatidine (magenta) and one of the three overlapping
conformations of (S)-nicotine (τ ) -67°, dark green).

4976 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24
Tønder et al.
molecules in which substituent effects could be ruled
out. The molecules were represented by their supposed
binding conformations and grouped according to their
activity level. It turns out that neither the N-N+
distance nor the (N+-b) distance have optimum values,
which are restricted to the high-affinity compounds (IC50
< 10 nM). Furthermore all high-affinity compounds
from the new series presented in this report have N-N+
distances that exceed the 4.8 ( 0.3 Å proposed by
Sheridan et al.11
Interestingly, all binding conformations of the high-
affinity compounds have (a-b) distances between 7.0
and 8.0 Å, and only two of the other compounds ((Z)-16
and (Z)-13) have an (a-b) distance in that range. On
this basis, it is proposed that the nicotinic agonists
interact with their receptors through one hydrogen bond
and one Coulombic interaction as proposed by Beers and
Reich.10 As the high-affinity compounds all have ap-
proximately the same (a-b) distance, it is very likely
that there is an anionic binding site within the receptor.
The optimum conformation of this anionic site is then
recognized by the compounds possessing an (a-b)
distance of 7.0-8.0 Å.
F igu r e 4. Stereoview of (Z)-17 (lime) superimposed with ABT
418 (blue) and the three overlapping conformations of (S)-
nicotine (red), showing the orientation of the N-methyl groups
(green). All hydrogen atoms, except for those present in the
N-methyl groups, are deleted.
tioning of the (E)-compounds is worse than for the (Z)-
compounds. A possible explanation of the high affinity
possessed by (E)-13 may be the larger energy gain
associated with forming a hydrogen bond to the oxygen
atom instead of to the nitrogen atom.
In support of the model presented in this paper, the
effects of various alkyl substitutions on nicotine itself30
are exactly as would be expected from our model, since
it places the 5- and 6-positions of the pyridine ring in
nicotine at the 4- and 3-positions in the isoxazole ring
in (Z)-26 (Figure 3).
Th e In t er n it r ogen Dist a n ce. Surprisingly (Z)-
26•a, which has an N-N+ distance of 5.6 Å, overlaid
the low-energy conformer of (R)-epibatidine, which has
an N-N+ distance of 4.7 Å. This, combined with the
inconsistency in the reports of the N-N+ distance led
to the idea that the essential parameter might not be
the distance between the nitrogens, but the interaction
sites between the ligands and the receptor.
Con clu sion
The series of structures presented in this report
include compounds with very high affinity for nicotinic
acetylcholine receptors and minimal affinity for mus-
carinic receptors. Computational studies of a large
number of these compounds have clarified that future
modeling of nicotinic agonists should be performed using
the global energy minimum of epibatidine (in which τ
) 7°) as reference structure and that it is important to
use site points when superimposing different molecules.
In addition, when designing novel nicotinic agonists,
attention should be paid to the following features of the
molecule: (a) the (a-b) distance, since there is a strong
indication that the optimum (a-b) distance is between
7 and 8.0 Å, and (b) the aliphatic area around the sp3
nitrogen atom, as the receptor seems to be sensitive to
changes in steric bulk and electrostatic potential.
It is assumed that a hydrogen bond donor is involved
in binding. Thus, if the center of charge is important,10
one would expect the distance between the hydrogen
bond donor site point (b) and the cationic center (N+) to
be essential (Figure 2). Similarly if an anionic binding
site is involved in ligand binding, the distance between
the hydrogen bond donor site point (b) and the assumed
anionic binding site (a) would be essential. Thus the
N-N+ distance, the (N+-b) distance, and the (a-b)
distance were measured and plotted (Figure 6) for the
Exp er im en ta l Section
1H NMR spectra were recorded at 300 MHz on a Bruker
AC-300 MHz FT-NMR instrument, and COESY, NOESY, and
ROESY spectra were recorded at 400 MHz on a Bruker AC-
400 MHz FT-NMR instrument. Mass spectra were recorded
on a Finnigan 5100 mass spectrometer, and melting points
(uncorrected) were determined on a Buchi capillary melting
point apparatus. Column chromatography was performed on
silica gel 60 (70-230 mesh, ASTM, Merck). Elemental analyses
F igu r e 5. Stereoview of (E)-11 (blue) and (E)-13 (green) superimposed with (Z)-26 (orange) using the site point complementary
to the isoxazole oxygen atoms. All hydrogen atoms are deleted.

Improving the Nicotinic Pharmacophore
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24 4977
g, 0.020 mol) in dry THF (40 mL). The sodium salt precipitated
when the reaction mixture was stirred for a few minutes under
N2 at 20 °C. Upon cooling to 0 °C methyl iodide (2.50 g, 0.020
mol) was slowly added. When the addition was complete, the
temperature was raised to 20 °C and stirring continued for 1
h. The reaction was quenched with ethanol (20 mL), where-
upon water (50 mL) and diethyl ether (150 mL) were added.
The phases were separated, and the organic phase was washed
with 1 N HCl (75 mL) and 1 N NaOH (75 mL). Drying (MgSO4)
and evaporation gave 2.40 g (94%) of 3-methoxymethyl-5-
methylisoxazol (4) which was used without further purifica-
tion. 1H NMR (300 MHz, CDCl3): δ 6.01 (s, 1H, Ar-H), 4.48
(s, 2H, Ar-CH2O), 3.40 (s, 3H, OCH3), 2.44 (s, 3H, Ar-CH3)
Meth od B. 3-(3-Eth yl-5-isoxazolyl)m eth ylen e-1-azabicy-
clo[2.2.2]octa n e (5). A solution of 3-ethyl-5-methylisoxazole
(2) (0.43 g, 3.9 mmol) in THF (25 mL) was cooled to -78 °C,
and n-butyllithium (2.5 M in hexanes, 5.5 mmol, 2.2 mL) was
added dropwise under N2. After the mixture was stirred for
15 min at -78 °C, a solution of 1-azabicyclo[2.2.2]octan-3-one
(0.41 g, 3.3 mmol) in THF (8 mL) was slowly added. Stirring
was continued at -78 °C for 90 min, whereupon triethylamine
(0.92 mL, 6.6 mmol), ethanol (0.1 mL), and thionyl chloride
(1.2 mL, 17 mmol) were added. After being stirred for 1 h at
-78 °C, the reaction mixture was allowed to warm to 20 °C
and stirred for 2 h. Addition of diethyl ether (100 mL) and
extraction with 1 N HCl (3 × 50 mL) gave an aqueous layer,
which upon basification (K2CO3) was extracted with dichlo-
romethane (4 × 50 mL). The organic extracts were dried
(MgSO4) and concentrated in vacuo to give 0.65 g of a brown
oil. Column chromatography (ethyl acetate/methanol/ammo-
nium hydroxide, 25% in water: 72/25/3) gave 316 mg (44%) of
(E)-5 ((E)-3-(3-ethyl-5-isoxazolyl)methylene-1-azabicyclo[2.2.2]-
octane) in the first fractions and 63 mg (8.7%) of (Z)-5 ((Z)-3-
(3-ethyl-5-isoxazolyl)methylene-1-azabicyclo[2.2.2]octane) in
the later fractions as colorless oils. None of the compounds
were absolutely free of the other isomer, but crystallization
with HCl in ethyl acetate and subsequent recrystallizations
from acetone gave the almost pure compounds as their
hydrochlorides.
F igu r e 6. Interatomic distances measured in the compound’s
supposed binding conformation. O: N-N+, ×: N+-b, +: a-b.
Activity level A: IC50 < 10 nM; (Z)-26, (Z)-14, (R)-(Z)-24, (Z)-
25, (R)-epibatidine, 29, epiboxidine, and UB-165. Activity level
B: IC50 ) 10-100 nM; (R)-(Z)-13, (R)-(E)-13, (R)-(E)-11, ABT
418, and 30. Activity level C: IC50 ) 100-1000 nM; (Z)-16,
18, and (E)-14.
were performed by Novo Nordisk Microanalytical Laboratory,
Denmark, and were within (0.4% of the calculated values.
The following compounds were prepared according to lit-
erature procedures: 3,4,5-trimethylisoxazole,31,32 N-benzhy-
drylazetidin-3-one,33 1-methylpiperidin-3-one,34 dimethyl-2-
oxobutanephosphonate,35
1-azabicyclo[3.2.1]octan-3-one,36
5-ethyl-3-methylisoxazole,37 ABT 418,6 and (()-epibatidine.38
The Z/ E-purity of the compounds subjected to biological
testing was measured by gas chromatography (Chrompack
CP9001).
Meth od A. 3-Isop r op yl-5-m eth ylisoxa zole (1). To a
solution of isobutyraldehyde (4.5 mL, 0.050 mol) in methanol
(150 mL) was slowly added a mixture of NaHCO3 (8.4 g, 0.10
mol) and hydroxylamine hydrochloride (7.0 g, 0.10 mol) under
N2. The mixture was refluxed for 45 min and then stirred at
20 °C for 30 min, whereupon TLC (dichloromethane/methanol
25:1) showed that all starting material had reacted.
Distillation at 34 °C/0.5 mmHg gave 2.4 g (81%) of isopro-
pyloxime. The compound was used without further purifica-
tion.
A solution of isopropyloxime (2.2 g, 0.25 mol) in dry DMF
(30 mL) was stirred under a stream of N2, and N-bromosuc-
cinimide (4.6 g, 0.026 mol) was slowly added. After the mixture
was stirred for 90 min at 20 °C, TLC (ethyl acetate) showed
that no oxime was left. The mixture was cooled to 0 °C,
isopropenyl acetate (18 mL, 0.25 mol) was added, followed by
dropwise addition of triethylamine (7.0 mL, 0.050 mol) dis-
solved in dry DMF (15 mL). The reaction mixture was stirred
overnight at 20 °C, whereupon 7 N HCl (10 mL, 0.070 mol)
was added, and stirring continued overnight at 20 °C. TLC
showed that the reaction had reached completion. Addition of
water (50 mL), extraction with diethyl ether (3 × 50 mL),
washing of the combined extracts with a saturated solution of
NaHCO3 (2 × 25 mL) and water (25 mL), drying, and
evaporation gave 2.5 g of an orange oil. Distillation at reduced
pressure (0.05 mmHg, 25 °C) gave 1.5 g (48%) of 3-isopropyl-
5-methylisoxazole (1) as a colorless oil.
(Z)-5. Mp: 142-144 °C. MS: m/z 218 (M+). GC: 87% Z. 1H
NMR (300 MHz, CDCl3): δ 6.25-6.15 (m, 1H, CHd), 5.87 (s,
1H, Ar-H), 3.80 (s, 2H, NCH2Cd), 3.22-3.03 (m, 4H, NCH2),
2.69 (quartet, 2H, J ) 8 Hz, Ar-CH2), 2.59-2.49 (m, 1H, CH),
1.93-1.68 (m, 4H, CH2), 1.27 (t, 3H, J ) 8 Hz, CH3). Anal.
1
(C13H18N2O, HCl, /4H2O) C, H, N.
(E)-5. Mp: 194-197 °C. MS: m/z 218 (M+). GC: 93% E. 1H
NMR (300 MHz, CDCl3): δ 6.10-6.00 (m, 1H, CHd), 5.93 (s,
1H, Ar-H), 3.59 (s, 2H, NCH2Cd), 3.52-3.42 (m, 1H, CH),
3.06-2.82 (m, 4H, NCH2), 2.68 (quartet, 2H, J ) 8 Hz, Ar-
CH2), 1.90-1.64 (m, 4H, CH2), 1.27 (t, 3H, J ) 8 Hz, CH3).
1
Anal. (C13H18N2O, HCl, /4H2O) C, H, N.
Compounds 6, 7, 8, 9, 10, and 11 were prepared according
to method B.
Meth od C. (R/S)-3-(3-Meth yl-5-isoxa zolyl)m eth ylen e-
1-a za bicyclo[2.2.1]h ep ta n e (13). To a solution of 3-methyl-
5-trimethylsilanylmethylisoxazole39 (12) (3.38 g, 20.0 mmol)
in THF (100 mL) under N2 was added n-butyllithium (2.5 M
in hexanes, 8.0 mL, 20 mmol) dropwise at -78 °C. After the
mixture was stirred for 60 min at -78 °C, a solution of (R/
S)-1-azabicyclo[2.2.1]heptan-3-one9 in THF (20 mL) was added
dropwise and stirring continued at -78 °C for 60 min,
whereupon concentrated HCl (10 mL) was added, and the
reaction was allowed to warm to 20 °C. The reaction mixture
was poured onto ice and washed with diethyl ether (30 mL).
Basification of the aqueous phase (K2CO3), extraction with
diethyl ether (5 × 20 mL) and dichloromethane (2 × 20 mL),
drying (MgSO4), and evaporation gave 3.1 g (86%) of a yellow
oil. Column chromatography (ethyl acetate/methanol/ammo-
nium hydroxide, 25% in water: 65:33.2) gave 170 mg (4.5%)
of (R/ S)-(Z)-3-(3-methyl-5-isoxazolyl)methylene-1-azabicyclo-
[2.2.1]heptane ((Z)-13), 170 mg (4.5%) of (R/ S)-(E)-3-(3-methyl-
5-isoxazolyl)methylene-1-azabicyclo[2.2.1]heptane ((E)-13), and
2.56 g (67%) of a mixture of the two products. The products
The overall yield was 39% from isobutyraldehyde. The
1
product was used without further purification. H NMR (300
MHz, CDCl3): δ 5.85 (s, 1H, Ar-H), 3.01 (heptet, 1H, J ) 7
Hz, CH-isopropyl), 2.38 (s, 3H, Ar-CH3), 1.26 (d, 6H, J ) 7
Hz, CH3).
Compounds 2 and 3 were prepared following method A.
3-Meth oxym eth yl-5-m eth ylisoxa zol (4). A 55% suspen-
sion of sodium hydride in mineral oil (1.00 g, 0.020 mol) was
added to a solution of 3-hydroxymethyl-5-methylisoxazole (2.30

4978 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24
Tønder et al.
were crystallized with oxalic acid in acetone (1.2 equiv) in
acetone to give the oxalate salts.
for 90 h, then quenched with 1 N HCl (50 mL), rinsed three
times with diethyl ether, made alkaline (K2CO3), and extracted
with dichloromethane (5 × 50 mL). The solvent was removed
after drying (MgSO4), and the crude compound was crystallized
as the hydrochloride salt from ethanol and then recrystallized
from ethanol to give (Z)-3-acetonylidene-1-azabicyclo[2.2.2]-
octane (22) hydrochloride in 21% yield. Mp: 189-190 °C.
MS: m/z 165 (M+). 1H NMR (300 MHz, DMSO): δ 6.38 (s, 1H,
CHd), 4.31 (s, 2H, NCH2Cd), 3.35-3.19 (m, 4H, NCH2), 2.75-
2.66 (m, 1H, CH), 2.20 (s, 3H, CH3), 2.18-1.97 (m, 2H, CH2),
1.85-1.75 (m, 2H, CH2). Anal. (C10H15NO, HCl, CH3CH2OH)
C, H, N.
(Z)-13. Mp: 174-176 °C. MS: m/z 190 (M+). GC: >99% Z.
1H NMR (300 MHz, DMSO): δ 6.62 (s, 1 H, CHd), 6.32 (s, 1
H, Ar-H), 4.26 (d, 1 H, J ) 17 Hz, NCH2Cd), 4.16 (d, 1 H, J )
17 Hz, NCH2Cd), 3.55 (d, 1 H, J ) 5 Hz, CH), 3.44-3.33 (m,
1 H, NCH2), 3.31-3.19 (m, 3 H, NCH2), 2.27-2.18 (m, 1 H,
CH2), 2.22 (s, 3 H, Ar-CH3),1.69-1.62 (m, 1 H, CH2). Anal.
1
(C11H14N2O, C2H2O4, /4H2O) C, H, N.
(E)-13. Mp: 168-170 °C. MS: m/z 190 (M+). GC: >99% E.
1H NMR (300 MHz, DMSO): δ 6.42 (s, 1 H, Ar-H), 6.37 (s, 1
H, CHd), 4.09 (d, 1 H, J ) 17 Hz, NCH2Cd), 3.96 (d, 1 H, J
) 5 Hz, CH), 3.90 (d, 1 H, J ) 17 Hz, NCH2Cd), 3.41 (t, 1 H,
J ) 15 Hz, NCH2), 3.22 (m, 3 H, NCH2), 2.31-2.23 (m, 1 H,
CH2), 2.23 (s, 3 H, Ar-CH3), 1.65-1.58 (m, 1 H, CH2). Anal.
(C11H14N2O, C2H2O4) C, H, N.
Compounds 21 and 23 were prepared according to method
E.
Meth od F . (Z)-3-(5-Isoxa zolyl)m eth ylen e-1-a za bicyclo-
[2.2.2]octa n e (25). (Z)-3-Acetonylidene-1-azabicyclo[2.2.2]-
octane (22) (0.36 g, 2.2 mmol) was dissolved in dimethyl
formamide-dimethyl acetal (DMF-DMA) (3 mL), and the
solution was stirred overnight at 90 °C under N2. The mixture
was evaporated to dryness, and the residue was dissolved in
glacial acetic acid (10 mL). Upon addition of hydroxylammo-
nium chloride (0.50 g, 7.2 mmol), the solution was stirred at
90 °C for 60 min. The brownish mixture was evaporated to
dryness, whereupon the residue was dissolved in water, made
alkaline with K2CO3, and extracted with diethyl ether (2 ×
50 mL). The combined organic layers were dried and concen-
trated in vacuo to a brown oil. Column chromatography
(dichloromethane/methanol/ammonium hydroxide, 25% in wa-
ter: 80:20:0.5) gave 150 mg (37%) of the desired product as a
yellow oil. Crystallization with oxalic acid in acetone/diethyl
ether gave 180 mg of (Z)-3-(5-isoxazolyl)methylene-azabicyclo-
[2.2.2]octane ((Z)-25) oxalate as white crystals. Mp: 122-124
°C. MS: m/z 190 (M+). GC: 87% Z. 1H NMR (300 MHz,
DMSO): δ 8.60 (s, 1 H, Ar-3-H), 6.60 (s, 1 H, CHd), 6.45 (s, 1
H, Ar-4-H), 4.37 (s, 2 H, NCH2Cd), 3.42-3.22 (m, 4 H, NCH2),
2.86 (s, 1 H, CH), 2.11-1.80 (m, 4 H, CH2). Anal. (C11H14N2O,
Compounds 14, 15, and 17 were prepared according to
method C.
1-Met h yl-3-(3-m et h yl-5-isoxa zolyl)m et h ylen ep yr r oli-
d in e (16). A solution of diisopropylamine (0.83 mL, 6.3 mmol)
in THF (15 mL) was cooled to 0 °C, and n-butyllithium (2.36
M in hexanes, 6.3 mmol) was added under N2. After the
mixture was stirred for 60 min at 0 °C, 3-methyl-5-trimeth-
ylsilanylmethylisoxazole39 (12) (0.98 g, 5.8 mmol) was added
dropwise. Stirring was continued for 15 min at 0 °C, followed
by 15 min at -78 °C, during which a dark orange color
appeared. 1-Methylpyrrolidinone40 (0.53 g, 5.3 mmol) was
dissolved in THF (2 mL) and added dropwise to the anion
solution at -78 °C. The reaction mixture was stirred at -78
°C for 1 h, upon which it was allowed to warm to 20 °C and
stirred at that temperature for 2 h. HCl (1 N, 50 mL) and ethyl
acetate (50 mL) were added, and the layers separated. Further
extraction with 1 N HCl (2 × 50 mL), basification (K2CO3) of
the aqueous layers, and extraction of these with dichlo-
romethane (4 × 50 mL) gave a solution, which upon drying
and evaporation yielded 0.63 g of a brown oil. Column
chromatography (eluent: ethyl acetate/methanol/ammonium
hydroxide, 25% in water: 75/25/0.5) followed by crystallization
with oxalic acid in acetone gave 16 mg (1.1%) of (Z)-1-methyl-
3-(3-methyl-5-isoxazolyl)methylenepyrrolidine ((Z)-16) oxalate
and 57 mg (4.0%) of (E)-1-methyl-3-(3-methyl-5-isoxazolyl)-
methylenepyrrolidine ((E)-16) oxalate.
(Z)-16. Mp: 177-179 °C. MS: m/z 178 (M+). GC: >99% Z.
1H NMR (300 MHz, CDCl3): δ 6.23 (s, 1 H, CHd), 5.84 (s, 1
H, Ar-H), 3.50 (s, 2 H, NCH2Cd), 2.69 (s, 4 H, CH2 CH2), 2.46
(s, 3 H, NCH3), 2.28 (s, 3 H, Ar-CH3). Anal. (C10H14N2O,
C2H2O4) C, H, N.
(E)-16. Mp: 170-173 °C. MS: m/z 178 (M+). GC: >99% E.
1H NMR (300 MHz, CDCl3): δ 6.28 (s, 1 H, CHd), 5.90 (s, 1
H, Ar-H), 3.30 (s, 2 H, NCH2Cd), 2.65 (s, 4 H, CH2 CH2), 2.42
(s, 3 H, NCH3), 2.28 (s, 3 H, Ar-CH3). Anal. (C10H14N2O,
C2H2O4) C, H, N.
Meth od D. 3-(3-Meth yl-5-isoxa zolyl)m eth ylen ea zeti-
d in e (18). The free base 1-benzhydryl-3-(3-methyl-5-isox-
azolyl)methyleneazetidine (15) (0.78 g, 2.5 mmol) was dissolved
in toluene (20 mL) and cooled to 0 °C. 1-Chloroethyl chloro-
formate (0.815 g, 5.7 mmol) was added, and the reaction
mixture was stirred for 20 min. The reaction mixture was
evaporated in vacuo, then redissolved in methanol (20 mL),
and heated at reflux for 1 h, and evaporated in vacuo. The
crude material was suspended in diethyl ether and the
precipitated crystals collected by filtration, giving the hydro-
chloride salt of the title compound in 370 mg yield (80%). Mp:
168-169 °C (dec). MS: m/z 150 (M+). 1H NMR (300 MHz,
DMSO): δ 9.88 (s, 2 H, NH2+), 6.56 (s, 1 H, CHd), 6.38 (s, 1
H, Ar-H), 4.87 (s, 2 H, NCH2), 4.76 (s, 2 H, NCH2), 2.23 (s, 3
H, Ar-CH3). Anal. (C8H10N2O, HCl) C, H, N.
1
1.5 C2H2O4, /4C3H6O ) C, H, N.
Compounds 24 and 26 were prepared according to method
F.
(Z/E)-3-(1-Aza bicyclo[2.2.2]oct-3-ylid en e)-3-flu or o-p r o-
p ion ic Acid Eth yl Ester (27). Triethyl-2-fluoro-2-phospho-
noacetate (4.9 g, 20 mmol) was dissolved in dry DMF (20 mL)
at 0 °C under N2, whereupon potassium tert-butoxide (2.5 g,
22 mmol) was added over a period of 5 min. The reaction
mixture was stirred for 1.5 h at 0 °C, upon which 1-azabicyclo-
[2.2.2]octan-3-one (2.5 g, 20 mmol) dissolved in dry DMF (10
mL) was added dropwise. Stirring was continued for 30 min
at 0 °C, followed by 30 min at 20 °C. TLC (dichloromethane/
methanol/ammonium hydroxide, 25% in water: 80/20/0.5)
showed that no starting material was left. The alkaline
reaction mixture was neutralized with glacial acetic acid (1
mL) and concentrated in vacuo. The resulting oil was dissolved
in dichloromethane (100 mL) and washed with 10% NaHCO3
(3 × 100 mL) and water (100 mL). Drying (MgSO4) and
evaporation gave the crude product (4.6 g). Crystallization with
oxalic acid in acetone gave (Z/ E)-3-(1-azabicyclo[2.2.2]oct-3-
ylidene)-3-fluoro-propionic acid ethyl ester (27) as the oxalate
salt (4.8 g, 79%). The product was used without further
purification. 1H NMR (300 MHz, DMSO): δ 4.27 (quartet, 2H,
J ) 7 Hz, OCH2), 4.11 (d, 2H, J ) 3 Hz, NCH2Cd), 3.85-3.75
(m, 1H, CH), 3.38-3.17 (m, 4H, NCH2), 2.09-1.96 (m, 2H,
CH2), 1.90-1.78 (m, 2H, CH2), 1.28 (t, 3H, J ) 7 Hz, CH3).
(E)-3-(F lu or o-(3-m eth yl-5-isoxa zolyl)m eth ylen e)-1-a za -
bicyclo[2.2.2]octa n e (28). To a solution of acetone oxime (590
mg, 8.0 mmol) in dry THF (10 mL) was added n-butyllithium
(1.6 M in hexanes, 10 mL, 16 mmol) under N2 at 0 °C. The
viscous solution was stirred for 45 min at 0 °C, whereupon
(Z/ E)-3-(1-azabicyclo[2.2.2]oct-3-ylidene)-3-fluoro-propionic acid
ethyl ester (27) (1.1 g, 5.0 mmol) in THF (15 mL) was added
dropwise. The mixture was stirred at 0 °C for 5 min, and then
the temperature was kept at 5 °C overnight. The following day
stirring was continued at 50 °C for 3 h, upon which the
temperature was lowered to 0 °C and concentrated sulfuric
acid (6 mL) was added dropwise. After being stirred at 20 °C
Compounds 19 and 20 were prepared according to method
D.
Meth od E. (Z)-3-Aceton ylid en e-1-a za bicyclo[2.2.2]oct-
a n e (22). Dimethyl 2-oxopropylphosphonate (4.9 g, 30 mmol)
was added dropwise to a solution of 1-azabicyclo[2.2.2]octan-
3-one (2.0 g, 16 mmol) and KOH (1.93 g, 30 mmol) in water
(7.7 mL) at -5 °C. The reaction mixture was stirred at -5 °C

Improving the Nicotinic Pharmacophore
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24 4979
for 18 h, the reaction mixture was poured onto ice, and the
solution was made alkaline with NaOH. Dichloromethane (100
mL) was added, and the mixture was filtered through Decalite.
Separation of the layers, extraction of the aqueous layer with
dichloromethane (2 × 100 mL), washing of the combined
organic layers with water (100 mL) and brine (100 mL), drying
(MgSO4), and evaporation gave 900 mg (90%) of a yellow oil.
Column chromatography (dichloromethane/methanol 9:1) gave
300 mg (30%) of (E)-3-(fluoro-(3-methyl-5-isoxazolyl)methyl-
ene)-1-azabicyclo[2.2.2]octane (28). Crystallization with oxalic
acid in acetone gave 410 mg as white crystals. Mp: 162-164
°C. MS: m/z 222 (M+). GC: 78% E. 1H NMR (300 MHz,
DMSO): δ 6.63 (s, 1 H, Ar-H), 4.23 (s, 2 H, NCH2Cd), 3.38 (s,
1 H, CH), 3.34-3.18 (m, 4 H, NCH2), 2.29 (s, 3 H, Ar-CH3),
2.07-1.96 (m, 2 H, CH2), 1.92-1.81 (m, 2 H, CH2). Anal.
(C12H15N2OF, C2H2O4) C, H, N.
Refer en ces
(1) Holladay, M. W.; Dart, M. J .; Lynch, J . K. Neuronal Nicotinic
Acetylcholine Receptors As Targets for Drug Discovery. J . Med.
Chem. 1997, 40, 4169-4194.
(2) Williams, M.; Arneric, S. P. Beyond the Tobacco Debate: Dis-
secting Out the Therapeutic Potential of Nicotine. Exp. Opin.
Invest. Drugs 1996, 5, 1035-1045.
(3) Sal´ın-Pascual, R. J .; Rosas, M.; J imenez-Genchi, A.; Rivera-
Meza, B. L.; Delgado-Parra, V. Antidepressant Effect of Trans-
dermal Nicotine Patches in Nonsmoking Patients With Major
Depression. J . Clin. Psychiatry 1996, 57, 387-389.
(4) Qian, C. G.; Li, T. C.; Shen, T. Y.; Libertine-Garahan, L.;
Eckman, J .; Biftu, T.; Ip, S. Epibatidine Is a Nicotinic Analgesic.
Eur. J . Pharmacol. 1993, 250, R13-R14.
(5) Dukat, M.; Damaj, M. I.; Glassco, W.; Dumas, D.; May, E. L.;
Martin, B. R.; Glennon, R. A. Epibatidine: A Very High Affinity
Nicotine-Receptor Ligand. Med. Chem. Res. 1994, 4, 131-139.
(6) Garvey, D. S.; Wasicak, J . T.; Decker, M. W.; Brioni, J . D.;
Buckley, M. J .; Sullivan, J . P.; Carrera, G. M.; Holladay, M. W.;
Arneric, S. P.; Williams, M. Novel Isoxazoles Which Interact
With Brain Cholinergic Channel Receptors Have Intrinsic
Cognitive Enhancing and Anxiolytic Activities. J . Med. Chem.
1994, 37, 1055-1059.
(7) Arneric, S. P.; Sullivan, J . P.; Briggs, C. A.; Donnelly-Roberts,
D.; Anderson, D. J .; Raszkiewicz, J . L.; Hughes, M. L.; Cadman,
E. D.; Adams, P.; Garvey, D. S.; Wasicak, J . T.; Williams, M.
(S)-3-Methyl-5-(1-Methyl-2-Pyrrolidinyl)Isoxazole (ABT-418): a
Novel Cholinergic Ligand With Cognition-Enhancing and Anxi-
olytic Activities: I. In Vitro Characterization. J . Pharmacol. Exp.
Ther. 1994, 270, 310-318.
(8) Abreo, M. A.; Lin, N. H.; Garvey, D. S.; Gunn, D. E.; Hettinger,
A. M.; Wasicak, J . T.; Pavlik, P. A.; Martin, Y. C.; Donnelly-
Roberts, D. L.; Anderson, D. J .; Sullivan, J . P.; Williams, M.;
Arneric, S. P.; Holladay, M. W. Novel 3-Pyridyl Ethers With
Subnanomolar Affinity for Central Neuronal Nicotinic Acetyl-
choline Receptors. J . Med. Chem. 1996, 39, 817-825.
(9) Olesen, P. H.; Swedberg, M. D. B.; Eskesen, K.; J udge, M. E.;
Egebjerg, J .; Tønder, J . E.; Rasmussen, T.; Sheardown, M. J .;
Rimvall, K. Identification of Novel (Isoxazole)Methylene-1-
Azabicyclic Compounds With High Affinity for the Central
Nicotinic Cholinergic Receptor. Bioorg. Med. Chem. Lett. 1997,
7, 1963-1968.
(10) Beers, W. H.; Reich, E. Structure and Activity of Acetylcholine.
Nature 1970, 228, 917-922.
(11) Sheridan, R. P.; Nilakantan, R.; Dixon, J . S.; Venkataraghavan,
R. The Ensemble Approach to Distance Geometry: Application
to the Nicotinic Pharmacophore. J . Med. Chem. 1986, 29, 899-
906.
(12) Barlocco, D.; Cignarella, G.; Tondi, D.; Vianello, P.; Villa, S.;
Bartolini, A.; Ghelardini, C.; Galeotti, N.; Anderson, D. J .;
Kuntzweiler, T. A.; Colombo, D.; Toma, L. Mono- and Disubsti-
tuted-3,8-Diazabicyclo[3.2.1]Octane Derivatives As Analgesics
Structurally Related to Epibatidine: Synthesis, Activity, and
Modeling. J . Med. Chem. 1998, 41, 674-681.
(13) Glennon, R. A.; Herndon, J . L.; Dukat, M. Epibatidine-Aided
Studies Toward Definition of a Nicotine Receptor Pharmacoph-
ore. Med. Chem. Res. 1994, 4, 461-473.
(14) Manallack, D. T.; Gallagher, T.; Livingstone, D. J . Quantitative
Structure-Activity Relationships of Nicotinic Agonists. In Neu-
ral Networks in QSAR and Drug Design; Devillers, J ., Ed.;
Academic Press: London, 1996; pp 177-208.
(15) Koren, A. O.; Horti, A. G.; Mukhin, A. G.; Gundisch, D.; Kimes,
A. S.; Dannals, R. F.; London, E. D. 2-, 5-, and 6-Halo-3-(2(S)-
Azetidinylmethoxy)Pyridines: Synthesis, Affinity for Nicotinic
Acetylcholine Receptors, and Molecular Modeling. J . Med. Chem.
1998, 41, 3690-3698.
(16) Olesen, P. H.; Seidelmann, D.; Hansen, J . B. Oxazolo[4,5-g]-Beta-
Carbolines and Pyrazino[2,3-g]-Beta-Carbolines - Synthesis of
Modulators of the Gaba(a)/Chloride Channel Complex. Hetero-
cycle 1998, 48, 31-40.
Biologica l Ma ter ia ls a n d Meth od s. (a ) Nicotin ic Re-
cep tor Bin d in g. Fresh or frozen cerebral cortex (male Mol:
WIST rats (Møllegaard, Ll. Skensved, Denmark)) was homog-
enized in assay buffer (50 mM Tris-HCl, pH 7.4, 120 mM NaCl,
5 mM KCl, 2 mM CaCl2, 1 mM MgCl2) and centrifuged for 10
min at 40000g. Pellets were reconstituted in assay buffer, and
an appropriate amount of tissue sample was mixed in tubes
with [3H]methylcarbamylcholine ([3H]MCC; NEN Life Sci-
ences, NET-951; final concentration 2 nM) and test drug. The
tubes were incubated at 0 °C for 60 min. Unbound ligand was
separated from bound ligand by vacuum filtration through
GF/B filters presoaked in 0.5% polyethylenimine. Filters were
washed three times with 5 mL of wash buffer (50 mM Tris-
HCl, pH 7.4) and transferred to vials. Scintillation fluid (4 mL)
was added, and the radioactivity was measured by scintillation
counting. Nonspecific binding was measured with 10 µM
nicotine. IC50 values were determined using nonlinear regres-
sion (InPlot41).
(b) Mu sca r in ic Recep tor Bin d in g. The muscarinic recep-
tor binding was determined following a published procedure.42
Molecu la r Mod elin g. The calculations were performed
using Sybyl 6.5.22 The structures were built by combining and
altering standard fragments from Sybyl. Using Gasteiger-
Hu¨ckel charges, the structures were minimized until a local
minimum was reached. The maximum number of iterations
was set to 1000 and the termination gradient to 0.001.
(a ) Gr id Sea r ch . The conformational search employing
rotation around the single bond connecting the bicycle and the
isoxazole was done using the grid search command, which
contains an energy minimization method that keeps the
torsion angle under examination constant. The torsional scans
were performed from 0 to 360° in 5° intervals, and the inherent
minimization used the TRIPOS force field and Gasteiger-
Hu¨ckel charges. The maximum number of iterations was set
to 1000 and the termination gradient to 0.001. As the
compounds were protonated, the dielectric constant was set
to 4 while keeping the cutoff value at 8 Å.
The resulting low-energy structures were then minimized,
but this time without any constraints.
(b) Molecu la r Dyn a m ics. The remaining compounds were
investigated using the molecular dynamics option in Sybyl.
In the simulation, the compounds were heated to 1000 K
for 100 ps. The step size was 10 fs, and every 500 fs a structure
was selected giving a total of 201 structures. The maximum
number of iterations was set to 10000. Due to the amount of
heat applied, some of the compounds racemized and/or Z/E
interchanged. The 200 structures resulting from a dynamics
run were then minimized.43 The structures were superposi-
tioned using the fit atoms command.
(17) Nesi, R.; Ricci, A.; Taddei, M.; Tedeschi, P. Group IVB Organo-
metallic Derivatives: Synthesis and 13C NMR Spectra of New
Silyl and Stannyl Isoxazoles. J . Organomet. Chem. 1980, 195,
275-283.
(18) He, Y.; Lin, N. H. Studies on Isoxazole Formation From Alkyl
Carboxylic Esters. Synthesis 1994, 989-992.
(19) Abood, L. G.; Grassi, S. [3H]Methylcarbamylcholine, a New
Radioligand for Studying Brain Nicotinic Receptors. Biochem.
Pharmacol. 1986, 35, 4199-4202.
(20) Boksa, P.; Quirion, R. [3H]N-Methyl-Carbamylcholine, a New
Radioligand Specific for Nicotinic Acetylcholine Receptors in
Brain. Eur. J . Pharmacol. 1987, 139, 323-333.
Su p p or tin g In for m a tion Ava ila ble: Full results from
the conformational analyses, experimental procedure for the
syntheses of compounds 2, 3, 6-11, 14, 15, 17, 19-21, 23, 24,
and 26, and characterization of the nicotinic receptor binding
assay. This material is available free of charge via the Internet
at http://pubs.acs.org.
(21) Freedman, S. B.; Harley, E. A.; Iversen, L. L. Relative Affinities
of Drugs Acting at Cholinoceptors in Displacing Agonist and
Antagonist Radioligands: the NMS/Oxo-M Ratio As an Index
of Efficacy at Cortical Muscarinic Receptors. Br. J . Pharmacol.
1988, 93, 437-445.
(22) Tripos Inc., 1699 South Hanley Road, St. Louis, Missouri 63144.

4980 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24
Tønder et al.
(23) Badio, B.; Garraffo, H. M.; Plummer, C. V.; Padgett, W. L.; Daly,
J . W. Synthesis and Nicotinic Activity of Epiboxidine: an
Isoxazole Analogue of Epibatidine. Eur. J . Pharmacol. 1997, 321,
189-194.
(24) Wright, E.; Gallagher, T.; Sharples, C. G. V.; Wonnacott, S.
Synthesis of UB-165: a Novel Nicotinic Ligand and Anatoxin-
a/Epibatidine Hybrid. Bioorg. Med. Chem. Lett. 1997, 7, 2867-
2870.
(25) Dukat, M.; Fiedler, W.; Dumas, D.; Damaj, I.; Martin, B. R.;
Rosecrans, J . A.; J ames, J . R.; Glennon, R. A. Pyrrolidine-
Modified and 6-Substituted Analogues of Nicotine: a Structure-
Affinity Investigation. Eur. J . Med. Chem. 1996, 31, 875-888.
(26) Czajkowski, C.; Karlin, A. Structure of the Nicotinic Receptor
Acetylcholine-Binding Site. Identification of Acidic Residues in
the δ Subunit Within 0.9 nm of the R Subunit-Binding Site
Disulfide. J . Biol. Chem. 1995, 270, 3160-3164.
(27) Nowak, M. W.; Kearney, P. C.; Sampson, J . R.; Saks, M. E.;
Labarca, C. G.; Silverman, S. K.; Zhong, W. G.; Thorson, J .;
Abelson, J . N.; Davidson, N.; Schultz, P. G.; Dougherty, D. A.;
Lester, H. A. Nicotinic Receptor Binding Site Probed With
Unnatural Amino Acid. Incorporation in Intact Cells. Science
1995, 268, 439-442.
(28) Sugiyama, N.; Boyd, A. E.; Taylor, P. Anionic Residue in the
a-Subunit of the Nicotinic Acetylcholine Receptor Contributing
to Subunit Assembly and Ligand Binding. J . Biol. Chem. 1996,
271, 26575-26581.
(29) Karlin, A.; Akabas, M. H. Toward a Structural Basis for the
Function of Nicotinic Acetylcholine Receptors and Their Cousins.
Neuron 1995, 15, 1231-1244.
(30) Wang, D. X.; Booth, H.; Lerner-Marmarosh, N.; Osdene, T. S.;
Abood, L. G. Structure-Activity Relationships for Nicotine
Analogues Comparing Competition for [3H]Nicotine Binding and
Psychotropic Potency. Drug Dev. Res. 1998, 45, 10-16.
(31) Dunstan, W. R.; Dymond, T. S. XLV.-Contributions From the
Research Laboratory of the Pharmaceutical Society of Great
Britain. The Action of Alkalis on the Nitro-Compounds of the
Paraffin Series. Formation of Isoxazoles. J . Chem. Soc. 1891,
59, 410-433.
(32) Micetich, R. G.; Shaw, C. C.; Hall, T. W.; Spevak, P.; Fortier, R.
A.; Wolfert, P.; Foster, B. C.; Bains, B. K. The 5-Alkoxymethyl-,
5-Alkylthiomethyl-, and 5-Dialkylaminomethylisoxazoles. Het-
erocycle 1985, 23, 571-583.
(33) Katritzky, A. R.; Cundy, D. J .; Chen, J . Novel Syntheses of 1,3,3-
Trinitroazetidine. J . Heterocycl. Chem. 1994, 31, 271-275.
(34) Lyle, R. E.; Adel, R. E.; Lyle, G. G. Hydrates of 1-Methyl-3- and
-4-Piperidone Hydrochlorides. J . Org. Chem. 1959, 24, 342-345.
(35) Coppola, G. M. The Chemistry of 2H-3,1-Benzoxazine-2,4(1H)-
Dione (Isatoic Anhydride). 201. Synthesis and Wittig Reactions
of Dimethyl (4-Oxo-1,4-Dihydro-Quinolin-2-yl)Methanephospho-
nates. Synthesis 1988, 81-84.
(36) King, F. D.; Hadley, M. S.; J oiner, K. T.; Martin, R. T.; Sanger,
G. J .; Smith, D. M.; Smith, G. E.; Smith, P.; Turner, D. H.; Watts,
E. A. Substituted Benzamides With Conformationally Restricted
Side Chains. 5. Azabicyclo[x.y.z] Derivatives As 5-HT4 Receptor
Agonists and Gastric Motility Stimulants. J . Med. Chem. 1993,
36, 683-689.
(37) Pe´rez, J . D.; Wunderlin, D. A. Flash Vacuum Pyrolysis of Some
4-Nitroisoxazoles. J . Org. Chem. 1987, 52, 3637-3640.
(38) Clayton, S. C.; Regan, A. C. A Total Synthesis of (()-Epibatidine.
Tetrahedron Lett. 1993, 34, 7493-7496.
(39) Nesi, R.; Ricci, A.; Taddei, M.; Tedeschi, P. Group IVB Organo-
metallic Derivatives: Synthesis and 13C NMR Spectra of New
Silyl and Stannyl Isoxazoles. J . Organomet. Chem. 1980, 195,
275-283.
(40) Prill, E. A.; McElvain, S. M. A Study of the Cyclization of a Series
of ω,ω′-Dicarbethoxydialkylmethylamines Through the Acetoace-
tic Ester Condensation. J . Am. Chem. Soc. 1933, 55, 1233-1241.
(41) GraphPad Software Inc., 5755 Oberlin Drive, #110, San Diego,
CA 92121.
(42) Sauerberg, P.; Kindtler, J . W.; Nielsen, L.; Sheardown, M. J .;
Honore´, T. Muscarinic Cholinergic Agonists and Antagonists of
the 3-(3-Alkyl-1,2,4-Oxadiazol-5-yl)-1,2,5,6-Tetrahydropyridine
Type. Synthesis and Structure-Activity Relationships. J . Med.
Chem. 1991, 34, 687-692.
(43) The macro for Molecular Dynamics data procession was kindly
supplied by Inge Thøger Christensen, Novo Nordisk A/S, Novo
Nordisk Park, 2760 Måløv, Denmark.
J M9910627