1-(1,2,5-thiadiazol-4-yl)-4-azatricyclo[2.2.1.02,6]heptanes as new potent muscarinic M1 agonists: Structure-activity relationship for 3-aryl- 2-propyn-1-yloxy and 3-aryl-2-propyn-1-ylthio derivatives

Article abstract of DOI:10.1021/jm9910019

Two new series of 1-(1,2,5-thiadiazol-4-yl)-4- azatricyclo[2.2.1.0^2,6]heptanes were synthesized and evaluated for their in vitro activity in cell lines transfected with either the human M₁ or M₂ receptor. 3-Phenyl-2-propyn-1-yloxy and -1-ylthio analogues substituted with halogen in the meta position showed high functional potency, efficacy, and selectivity toward the M₁ receptor subtype. A quite unique functional M₁ receptor selectivity was observed for compounds 8b, 8d, 8f, 9b, 9d, and 9f. Bioavailability studies in rats indicated an oral bioavailability of about 20-30%, with the N-oxide as the only detected metabolite.

Full text of DOI:10.1021/jm9910019

J . Med. Chem. 1999, 42, 1999-2006
1999
1-(1,2,5-Th ia d ia zol-4-yl)-4-a za tr icyclo[2.2.1.02,6]h ep ta n es a s New P oten t
Mu sca r in ic M1 Agon ists: Str u ctu r e-Activity Rela tion sh ip for
3-Ar yl-2-p r op yn -1-yloxy a n d 3-Ar yl-2-p r op yn -1-ylth io Der iva tives
Lone J eppesen,* Preben H. Olesen, Lena Hansen, Malcolm J . Sheardown, Christian Thomsen,
Thøger Rasmussen, Anders Fink J ensen, Michael S. Christensen, Karin Rimvall, J ohn S. Ward,†
Celia Whitesitt,† David O. Calligaro,† Frank P. Bymaster,† Neil W. Delapp,† Christian C. Felder,†
Harlan E. Shannon,† and Per Sauerberg
Novo Nordisk A/ S, Health Care Discovery, Novo Nordisk Park, DK-2760 Ma˚løv, Denmark, and
The Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, Indiana 46285
Received J anuary 7, 1999
Two new series of 1-(1,2,5-thiadiazol-4-yl)-4-azatricyclo[2.2.1.02,6]heptanes were synthesized
and evaluated for their in vitro activity in cell lines transfected with either the human M1 or
M2 receptor. 3-Phenyl-2-propyn-1-yloxy and -1-ylthio analogues substituted with halogen in
the meta position showed high functional potency, efficacy, and selectivity toward the M1
receptor subtype. A quite unique functional M1 receptor selectivity was observed for compounds
8b, 8d , 8f, 9b, 9d , and 9f. Bioavailability studies in rats indicated an oral bioavailability of
about 20-30%, with the N-oxide as the only detected metabolite.
Ch a r t 1
In tr od u ction
The observation that presynaptic cholinergic termi-
nals degenerate whereas the postsynaptic M1 receptors
are preserved in Alzheimer’s disease brain tissue led
to the hypothesis that M1-selective agonists would be
beneficial in the treatment of Alzheimer’s disease.1 This
hypothesis was supported by our findings that the
muscarinic M1 agonist xanomeline (3-(3-hexyloxy-1,2,5-
thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine)
(Chart 1), in a phase II clinical trial, showed improve-
ments in cognitive parameters in Alzheimer’s disease
patients.2,3 Furthermore, behavioral symptoms associ-
ated with Alzheimer’s disease, such as hallucinations,
delusions, and vocal outburst, were significantly de-
creased with xanomeline treatment.2,3 Xanomeline has
been shown to be a partial agonist at all muscarinic
receptor subtypes (M1-M5), but with the highest efficacy
and potency at M1 and M4 receptors. In an attempt to
identify a new drug candidate for Alzheimer’s disease,
the main focus was on the improvement of the M1
receptor selectivity as well as of the oral bioavailability.
An enhancement of the M1 selectivity was expected to
further increase the cognitive improvements, while
minimizing peripheral side effects associated with ac-
tivation of M2 and M3 receptors. Xanomeline has been
shown to be completely absorbed after oral administra-
tion, but the compound undergoes extensive biotrans-
formation in rat and human. In rats the oral bioavail-
ability was estimated to be 3-7%.3 The primary site of
metabolism of xanomeline was in the hexyloxy side
chain, which undergoes â-oxidation yielding hydroxy,
keto, and shorter side chain carboxylic acid analogues.4,5
The secondary site is the tetrahydropyridine ring, which
undergoes N-oxidation and N-demethylation. The thia-
diazole ring appears, however, to be metabolically
stable.6 Extensive structure-activity relationship (SAR)
work has previously been carried out around xano-
meline7-11 showing that the hexyloxy side chain is
crucial for the biological profile.7
In our search for a functionally equivalent but more
stable side chain, we discovered that the 3-phenyl-2-
propyn-1-yloxy group was quite unique to replace the
hexyloxy group, giving a compound (12) with a very
robust M1 response.12 This finding is in agreement with
a previous report showing the utility of the 3-aryl-2-
propynyl chain in the muscarinic agonist (-)-(R)-(Z)-3-
(3-(3-methoxyphenyl)-2-propynyloxyimino)-1-azabicyclo-
[2.2.1]heptane, PD 15183213 (Chart 1). To prevent the
metabolic demethylation as observed with xanomeline,
the N-methyl group has been incorporated in a ring
giving various azabicyclic and tricyclic ring systems.12
Some of these more conformationally constricted aza-
cycles, with various phenylpropargyloxy/thio side chains,
also showed a high efficacious M1 receptor response.12
We describe here two of these series (8 and 9), in
which the N-methyltetrahydropyridine ring of xanome-
line has been replaced with the [2.2.1.02,6]azatricycle.
Variations of the substitution pattern of the aryl group
were examined (8a -h and 9a -h ), revealing compounds
with a pronounced functional M1 receptor selectivity.
† Lilly Corp. Center.
10.1021/jm9910019 CCC: $18.00 © 1999 American Chemical Society
Published on Web 05/08/1999

2000 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11
J eppesen et al.
Sch em e 1a
a
1b-d ,f,g: X ) Br; (i) (Ph3P)2PdCl2, CuI, (i-Pr)2NH, Et3N. 1e,h : X ) I; (i) (Ph3P)4Pd, CuI, (i-Pr)2NH. 2a -e: (ii) Et3N, CH2Cl2, CH3SO2Cl.
*Commercially available.
Sch em e 2a
a
(i) DIBAL, THF; (ii) KCN, H2O, NH4Cl, NH3; (iii) S2Cl2, DMF; (iv) NaHS, DMF, CH3(CH2)2Br, K2CO3; (v) oxone, HCl, H2O; (vi) NaH,
THF, 1a -h ; (vii) NaHS, DMF, 2a -e; (viii) NaHS, THF, (C6H5)3P, 1f-h , C2H5O2CNdNCO2C2H5.
Ch em istr y
ylformamide gave the 3-chloro-1,2,5-thiadiazole 5. To
obtain a better leaving group than chlorine, 5 was con-
verted into the propylthio intermediate 6 by treating 5
with sodium hydrogen sulfide monohydrate, followed by
alkylation with propyl bromide. Oxidation of 6 with an
equimolar amount of oxone gave the corresponding
sulfone 7. Excess of oxone gave rise to some impurity
of the corresponding N-oxide 10 (Chart 2), which was
formed during the workup process. Further investiga-
tion showed that the pH in the reaction medium was
very crucial for the N-oxide formation, with a pH
optimum around pH 7. No amine oxidation was detected
under very basic or acidic conditions. Reduction of 10
with iron powder in glacial acetic acid gave compound
The syntheses of the azatricyclic thiadiazoles 8a -h
and 9a -h began with the known 1-cyano-4-azatricyclo-
[2.2.1.02,6]heptane14 (Scheme 2), which is obtained by
reaction of 1-azabicyclo[2.2.1]heptan-3-one15,16 with po-
tassium cyanide to give 3-cyano-3-hydroxy-1-azabicyclo-
[2.2.1]heptane, followed by a ring closure with diethyl-
aminosulfur trifluoride.17 Reduction of the tricyclic ni-
trile, with diisobutylaluminum hydride, gave the al-
dehyde 3. Aminonitrile 4 was obtained from 3 under
Strecker conditions or by stepwise conversion of 3 to a
cyanohydrin followed by treatment with aqueous am-
monium chloride under basic conditions. Cyclization of
the aminonitrile 4 with sulfur monochloride in dimeth-

Thiadiazolylazatricycloheptanes as New M1 Agonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11 2001
Ch a r t 2
and a SAR analysis around various saturated carbon
chain analogues showed that the hexyloxy group is
optimal for functional M1 selectivity.12 On the other
hand, the carbon chain underwent extensive biotrans-
formation, and therefore an exchange of this metaboli-
cally labile hexyloxy group with a more stable side chain
was essential for the design and synthesis of new
analogues. This SAR work led to the discovery of 12,
which possesses the same M1 potency and an even
higher efficacy than xanomeline. In comparison with PD
151832, which contains a similar side chain, 12 was
clearly far more efficacious and at least 50 times more
potent in the A9L-M1 cell line. On the basis of these
findings, two series of 3-aryl-2-propyn-1-yloxy and -1-
ylthio analogues, where the tetrahydropyridine ring was
exchanged with the azatricycle (8a -h , 9a -h ), were
synthesized. The rationale for choosing this very con-
strained heterocycle was primarily to prevent the
metabolic demethylation observed with xanomeline, but
also to attempt to enhance the muscarinic M1 receptor
subtype selectivity. A prevention of the conformational
flexibility of a molecule can, in theory, change receptor
selectivity, due to possible differences in the structural
requirements for activation of the receptor subtypes.
The change in the pKa for the nitrogen in the azacycle,
in this case a reduction due to the increased ring strain
in the tricycle,17 could also have an important influence
on the selectivity.
7. The target compounds 8a -h were obtained by react-
ing 7 with an appropriate alcohol (1a -h ) and a base,
e.g., sodium hydride. 1a is commercially available,
whereas 1b-h were synthesized by an arylalkyne cross-
coupling reaction employing a Pd catalyst such as Pd-
(PPh3)4 or PdCl2(PPh3)2 and a catalytic amount of
copper(I) iodide. An organic amine base, such as diiso-
propylamine, was used both as the reagent as well as
the solvent, and no cosolvent was needed (Scheme 1).
The sulfur analogues 9a -e were obtained by reacting
the chloro intermediate 5 with sodium hydrogen sulfide
monohydrate followed by a reaction with the mesylates
2a -e (Scheme 1). Conversion of the chloro intermediate
5 to the thiolate with sodium hydrogen sulfide mono-
hydrate followed by a Mitsunobu reaction with the
appropriate alcohol (1f-h ) gave 9f-h .
Compound 12, the aryl-2-propyn-1-yloxy analogue of
xanomeline, was synthesized via a method analogous
to that which has been previously reported for xanome-
line,7 using 3-(3-chloro-1,2,5-oxadiazol-4-yl)pyridine as
starting material.
Compound 12, the direct tricyclic analogue 8a , and
the corresponding thio analogue 9a all had similar
biological profiles (Table 1). The M2/M1 selectivity ratios
were, however, improved for 8a and 9a (11 and 14,
respectively), compared to 12 and xanomeline (2 and 3,
respectively).
Biologica l Eva lu a tion
In vitro receptor binding studies using tritiated oxo-
tremorine-M ([3H]Oxo-M) were used to determine the
affinity for the muscarinic agonist conformational state.
Second-messenger changes in cell lines transfected with
human muscarinic receptor subtypes were used to
determine functional potency and efficacy. The ability
of each compound to stimulate phosphoinositol (PI)
hydrolysis in the A9L-M1 cell line was determined up
to a concentration of 100 µM, and the efficacy of the
compound was expressed as a percentage of that pro-
duced by 100 µM carbachol. The EC50 for PI hydrolysis
was determined for each compound that produced at
least 25% increase in hydrolysis. The EC50’s for less
efficacious compounds could not be determined with
sufficient accuracy due to the inherent variability in the
assay. Potency at M2 receptors was expressed as the
EC50 for the compound to inhibit forskolin-induced
cAMP formation by 50% in CHO cells transiently
expressing the M2 muscarinic receptor, and the efficacy
was expressed as the percent of the maximal inhibition
produced by carbachol.
Substitution on the phenyl ring with various halogens
gave rise to some very interesting findings. In the
oxygen series 8, the 4-fluoro analogue 8c had the same
profile as the unsubstituted 8a , whereas the 4-chloro
compound 8e was markedly less efficacious in the M1
cell line. In contrast, substitution in one of the meta
positions with either fluorine (8b) or chlorine (8d )
enhanced the M1 receptor potency approximately 10-
fold. In addition to the increased M1 potency, 8b and
8d both showed a distinct M1 selectivity, with a M2/M1
ratio of 139 and 217, respectively. The same observation
was made with the 3,5-difluoro analogue 8f, which was
a very potent full M1 agonist with the same remarkable
functional M1 selectivity. The M2/M1 ratio for 8f was
calculated to be 133. The mixed 3-chloro-5-fluoro com-
pound 8g was less potent than the unsubstituted 8a ,
and with a 3,5-dichoro substitution (8h ) a major loss in
potency was observed in the two cell lines.
In the sulfur series (9), there was the same trend in
the influence of the substitution as described above for
the oxygen series. The 4-fluoro compound 9c was
greater than 10-fold more potent in both cell lines than
the unsubstituted analogue 9a . The 4-chloro compound
9e was, as observed for 8e, less efficacious in the M1
cell line. 9b and 9d , with a fluorine or a chlorine in the
meta position, respectively, were more potent in the M1
cell line and had a very favorable M2/M1 ratio (362 and
74, respectively). This high ratio was also observed for
the 3,5-difluoro analogue 9f, where the M2/M1 ratio was
218. The 3-chloro-5-fluoro analogue 9g was equipotent
The receptor reserve differs for the two cell lines,
making comparison of potency and efficacy difficult. The
reported M2/M1 selectivity ratios (Table 1) are therefore
not absolute values, but rather relative numbers.
Resu lts a n d Discu ssion
The main goal of this project was to synthesize a M1-
selective agonist with good oral bioavailability. The
hexyloxy side chain in xanomeline has earlier been
shown to be very crucial for the pharmacological profile,

2002 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11
J eppesen et al.
Ta ble 1. Receptor Binding and Functional M1 and M2 Profiles for the Halogen-Substituted
1-(1,2,5-Thiadiazol-4-yl)-4-azatricyclo[2.2.1.02,6]heptanes
[3H]Oxo-M receptor binding
to rat brain membranes
PI hydrolysis
in A9L-M1 cells
c-AMP formation
in CHO-M2 cells
IC50
(nM, (SEM)
% max
((SEM)
EC50
(nM, (SEM)
% max
((SEM)
EC50
(nM, (SEM)
ratio EC50(M2)/
EC50(M1)
compd
X
R1
R2
R3
8a
8b
8c
8d
8e
8f
8g
8h
9a
9b
9c
9d
9e
9f
O
O
O
O
O
O
O
O
S
S
S
S
S
H
F
H
Cl
H
F
H
H
F
H
H
H
H
H
F
Cl
Cl
H
H
H
H
H
F
16 ( 2.5
21 ( 5.1
10 ( 1.6
47 ( 5.6
63 ( 8.3
30 ( 11
92 ( 4.7
1688 ( 99
27 ( 53
26 ( 7.2
20 ( 4.6
49 ( 7.6
242 ( 56
28 ( 9.1
142 ( 11
788 ( 117
34 ( 8.2
16 ( 2.3
150 ( 11
14 ( 2.4
83.7 ( 17.8
91.0 ( 2.6
101.0 ( 7.3
98.5 ( 1.5
60.5 ( 3.8
107.3 ( 3.5
102.8 ( 11.0
87.5 ( 1.8
91.9 ( 5.2
102.0 ( 6.2
97.5 ( 9.5
67.5 ( 6.4
52.9 ( 14.6
85.5 ( 4.1
101.0 ( 9.9
57.7 ( 7.9
99 ( 2.3
70.5 ( 0.1
4.9 ( 3.4
71.0 ( 17.9
10.5 ( 0.8
nd
20.2 ( 4.2
110.0 ( 41.7
1200 ( 900
61.9 ( 4.4
2.5 ( 0.5
4.5 ( 0.7
9.0 ( 5.0
107.0 ( 4.5
7.6 ( 3.3
46.0 ( 5.4
1810 ( 210
4600 ( 830
134.5 ( 32.5
7121 ( 567
110.9 ( 64.0
99 ( 9
104 ( 5
109 ( 5
101 ( 14
102 ( 13
68 ( 14
76 ( 17
23 ( 12
106 ( 1
106 ( 6
88 ( 1
779 ( 272
683 ( 131
791 ( 40
2280 ( 720
1210 ( 55
2690 ( 189
1480 ( 101
nd
853 ( 103
904 ( 121
83 ( 13
673 ( 167
80 ( 28
1655 ( 391
763 ( 42
528 ( 208
700 ( 85
398 ( 79
26700 ( 3300
241 ( 45
11
139
11
217
nd
133
13
nd
14
362
18
74
1
H
Cl
H
H
H
H
H
F
H
Cl
H
H
H
F
Cl
H
F
H
Cl
H
F
96 ( 2
76 ( 12
79 ( 16
90 ( 9
84 ( 17
100 ( 3
82 ( 8
S
S
S
218
17
0.3
0.2
3
9g
9h
F
Cl
Cl
Cl
carbacol
xanomeline
PD 151832
12
65.6 ( 14.7
37.3 ( 13.8
111.6 ( 4.2
89 ( 11
115 ( 12
4
2
with 9a in the cell lines, whereas a major loss in efficacy
as well as potency in the M1 cell line was observed for
the dichloro analogue 9h .
prominent tremors). Compounds 8b and 9d produced
profuse salivation and received a score of 2. 8b also
induced tremor with a score of 1.4. Compounds 8d , 8f,
9b, and 9f, on the other hand, did not cause any
salivation or tremor.
These results clearly show that the substitution
pattern in the phenyl ring is very important for the
biological profile. A fluorine or a chlorine in the meta
position in both the oxygen and sulfur series gave rise,
in both the oxygen and sulfur series, to compounds with
very favorable M2/M1 ratios, and all compounds were
at the same time more potent and efficacious than
xanomeline in the M1 cell line. The same was true for
the 3,5-difluoro analogues, whereas decreased potencies
were observed with the disubstituted 3-chloro-5-fluoro
analogues 8g and 9g. This was even more pronounced
with the 3,5-dichloro analogues 8h and 9h , indicating
a limitation in steric tolerance for receptor activation.
A fluorine in the para position was tolerated in both
series, whereas the more bulky chlorine caused a large
loss of the efficacy in the M1 cell line.
Due to their very favorable M2/M1 ratio, compounds
8b, 8d , 8f, 9b, 9d , and 9f were taken into further
investigations. It is known that nonselective muscarinic
agonists produce parasympathomimetic side effects, and
a number of these effects are produced by activation of
M2 or M3 receptors.3,18,19 In our paradigm, compounds
that induced salivation or tremor in mice were consid-
ered nonselective and were excluded in our search for
a selective M1 agonist. The side effects were determined
in mice 30 min after drug administration (10 mg/kg)
according to a previously described method.19 Briefly,
salivation was scored on a scale by observation accord-
ing to severity (0 for no salivation, 1 for slight salivation,
2 for profuse salivation). Tremor was also scored obser-
vationally on a scale of 0-2 (0 for no tremor, up to 2 for
Among compounds 8d , 8f, 9b, and 9f, compound 8f
was selected for further investigation due to the promis-
ing profile of being a fully efficacious M1 agonist with
only partial efficacy at the M2 receptor subtype while
causing no tremor or salivation. Oral bioavailability
studies with 8f in rats indicated an oral bioavailability
of about 20-30%. In the HPLC traces, an additional
peak with a longer retention time than that of 8f was
observed in samples from both intravenous and oral
administration. This peak was more pronounced follow-
ing oral administration, probably due to first-pass
metabolism, and no other peaks were observed. To
identify this metabolite, 11 was synthesized. 11 coeluted
with the unknown peak indicating that the detected
metabolite was N-oxidized 8f. This was further con-
firmed by LC/MS/MS where the peak eluting later than
8f showed the same characteristic fragment ion as 11.
This N-oxidation of the azatricycle was to some extent
expected, as the increased ring strain in the azatricycle
relative to the azacycle in xanomeline resulted in a
substantial reduction in pKa.17 The assumed N-oxide
was the only detected metabolite, indicating that the
propargyl group as well as the halogen substitution
results in an effective shielding of the side chain from
metabolic attack. This is probably the explanation of the
pronounced improvement of the bioavailability. Further
pharmacological and metabolism studies will show
whether this series of compounds will provide a second-
generation M1 agonist.

Thiadiazolylazatricycloheptanes as New M1 Agonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11 2003
3-(3,5-Diflu or op h en yl)p r op -2-yn -1-ol (1f): yield 30%; 1H
NMR (CDCl3, 200 MHz) δ 1.88 (bs, 1 H), 4.50 (s, 2 H), 6.70-
6.85 (m, 1 H), 6.85-7.00 (m, 2 H).
Su m m a r y
This study showed that 3-aryl-2-propyn-1-yloxy and
-1-ylthio derivatives of 1-(1,2,5-thiadiazol-4-yl)-4-azatri-
cyclo[2.2.1.02,6]heptane are very potent and efficacious
M1 agonists. The substitution pattern in the phenyl ring
in the side chain moiety had a major effect on the M1
receptor selectivity. The m-fluoro, m-chloro, or m-di-
fluoro compounds (8b, 8d , 8f, 9b, 9d , and 9f) were all
very selective with M2/M1 ratios of around 100 or more,
whereas for xanomeline and its 3-phenyl-2-propyn-1-
yloxy analogue 12 this ratio was 2-3. By comparing 12
with 8a , the effect of exchanging the tetrahydropyridine
ring with the tricyclic amine was an improvement in
the selectivity as well. The 3,5-dichloro or 4-chloro
analogues were, on the other hand, all markedly less
efficacious, which was interpreted as being due to a
limitation in the steric tolerance of the receptor. 8d , 8f,
9b, and 9f did not cause any salivation or tremor which
indicates a selective M1 profile. Preclinical analysis in
rats demonstrated a pronounced improvement of the
oral bioavailability of 8f (20-30%) compared to xanome-
line (3-7%), with the N-oxide as the only detected
metabolite.
3-(3-Ch lor o-5-flu or op h en yl)p r op -2-yn -1-ol (1g): yield
1
55%; H NMR (CDCl3, 200 MHz) δ 1.80 (bs, 1 H), 4.50 (s, 2
H), 6.98-7.12 (m, 2 H), 7.22 (s, 1 H).
P r ep a r a t ion of Com p ou n d s 1e,h . To a solution of the
appropriate chloro-substituted iodobenzene (30.0 mmol) in
diisopropylamine (100 mL) under a nitrogen atmosphere were
added copper(I) iodide (100 mg, 0.5 mmol) and tetrakis-
(triphenylphosphine)palladium (300 mg, 0.25 mmol). After the
mixture stirred for 1 h, a solution of 3-propynol (1.85 g, 33.0
mmol) in diisopropylamine (50 mL) was added. After stirring
under a nitrogen atmosphere at room temperature for 16 h,
the reaction mixture was filtered and the filtrate evaporated
to dryness. The product was purified by flash chromatography
using CH2Cl2 as eluent to give the product as a viscous oil.
3-(4-Ch lor op h en yl)p r op -2-yn -1-ol (1e): yield 96%; 1H
NMR (CDCl3, 200 MHz) δ 1.75 (bs, 1 H), 4.50 (s, 2 H), 7.22-
7.40 (m, 4 H).
3-(3,5-Dich lor op h en yl)p r op -2-yn -1-ol (1h ): yield 87%; 1H
NMR (CDCl3, 200 MHz) δ 1.80 (t, J ) 6 Hz, 1 H), 4.50 (d, J )
6 Hz, 2 H), 7.28-7.35 (m, 3 H).
P r ep a r a tion of Com p ou n d s 2a -e. A solution of the
appropriate alcohol (1a -e) (20.0 mmol) in CH2Cl2 and NEt3
(2.43 g, 24 mmol) was cooled to - 20 °C. Methanesulfonyl
chloride (2.25 g, 22.0 mmol) was added dropwise over 10 min
under a nitrogen atmosphere. The reaction mixture was stirred
for 1.5 h at - 20 °C, diluted with CH2Cl2 (20 mL), and washed
with H2O (3 × 20 mL). The organic phase was dried (MgS04),
evaporated, and if needed submitted to flash chromatography
using CH2Cl2 as eluent to give the pure product.
Meth a n esu lfon ic a cid 3-p h en ylp r op -2-yn yl ester (2a ):
yield 99%; 1H NMR (CDCl3, 200 MHz) δ 3.15 (s, 3 H), 5.08 (s,
2 H), 7.34-7.40 (m, 3 H), 7.40-7.50 (m, 2 H).
Met h a n esu lfon ic a cid 3-(3-flu or op h en yl)p r op -2-yn yl
ester (2b): yield 78%; 1H NMR (CDCl3, 200 MHz) δ 3.15 (s, 3
H), 5.08 (s, 2 H), 7.04-7.40 (m, 4 H).
Met h a n esu lfon ic a cid 3-(4-flu or op h en yl)p r op -2-yn yl
ester (2c): yield 49%; 1H NMR (CDCl3, 200 MHz) δ 3.15 (s, 3
H), 5.08 (s, 2 H), 6.96-7.10 (m, 2 H), 7.40-7.52 (m, 2 H).
Meth a n esu lfon ic a cid 3-(3-ch lor op h en yl)p r op -2-yn yl
ester (2d ): yield 46%; 1H NMR (CDCl3, 200 MHz) δ 3.15 (s, 3
H), 5.08 (s, 2 H), 7.24-7.48 (m, 4 H).
Meth a n esu lfon ic a cid 3-(4-ch lor op h en yl)p r op -2-yn yl
ester (2e): yield 80%; 1H NMR (CDCl3, 200 MHz) δ 3.15 (s, 3
H), 5.08 (s, 2 H), 7.30-7.44 (m, 4 H).
Exp er im en ta l Section
Gen er a l Ch em istr y. Melting points were determined with
a Bu¨chi capillary melting apparatus and are uncorrected. The
200-MHz 1H NMR spectra were recorded on a Bru¨ker AC-200
MHz FT spectrometer. The 400-MHz 1H NMR spectra were
recorded on a Brucker AMX2-400 MHz spectrometer. Chemical
shifts are given in ppm (δ) relative to Me4Si, and coupling
constants are in Hz. FAB/MS were recorded with a UG
AutoSpec Ultima sector mass spectrometer (EBE-geometry)
interfaced to a data processing system. Cesium fast ions
generated with 33 kV and a 2 µA emission current were used
for bombardment. Reactions were followed by either thin-layer
chromatography performed on silica gel 60 F254 (Merck) TLC
aluminum sheets and/or by HPLC performed on a Merck
Hitachi system, containing a L-6200A Intelligen pump and a
L-4000A UV detector with a Resource RPC column and/or by
gas chromatography (GC) on a Chrompack CP 9001 chromato-
graph using a CP-Sil 5 CB, 25-m column. Flash chromatog-
raphy was performed on silica gel 60 (230-400 mesh ASTM,
Merck). Preparative HPLC was performed on a Dan Process
system using a Knauer UV detector, with a HO50700 column
containing Source 15RPC. Elemental analyses were performed
by Novo Nordisk, Microanalytical Laboratory, Denmark. Freeze-
drying was performed on a Hetosicc apparatus, using 0.1-0.2
mmHg vacuum and a temperature graduated from -20 °C to
room temperature over 3-4 days.
1-F or m yl-4-a za tr icyclo[2.2.1.02,6]h ep ta n e (3). 1-Cyano-
4-azatricyclo[2.2.1.02,6]heptane13 (5.73 g, 47.6 mmol) in dry
THF (100 mL) was treated with diisobutylaluminum hydride
(78.3 mL, 20% in toluene, 95.2 mmol) at 0 °C under a nitrogen
atmosphere. After 3 h the reaction mixture was allowed to
warm to room temperature and quenched with 2-propanol (10
mL) and 4 N HCl (pH 2-3), followed by Rochelle salt (25 g).
The organic solvent was removed in vacuo and the residue
extracted with Et2O (2 × discarded). The aqueous phase was
made alkaline with 6 N NaOH (pH 10-11) and the mixture
extracted with CH2Cl2/2-propanol (9:1) (6×). The organic
extracts were dried (MgSO4) and filtered. Evaporation of the
solvent gave 4.15 g (71%) of the title compound: 1H NMR
(CDCl3, 200 MHz) δ 2.27 (s, 2 H), 2.58 (d, J gem ) 10.5 Hz, 2
H), 2.66 (d, J gem ) 10.5 Hz, 2 H), 2.68 (s, 2 H), 9.50 (s, 1 H).
2-Am in o-2-(4-a za t r icyclo[2.2.1.02,6]h ep t -1-yl)a cet on i-
tr ile (4). To an ice-cooled solution of 1-formyl-4-azatricyclo-
[2.2.1.02,6]heptane (3) (3.56 g, 29 mmol) in H2O (5 mL) was
added a solution of potassium cyanide (2.08 g, 31.9 mmol) in
H2O (5 mL) over a period of 30 min. After an additional 1 h,
cooling was removed and the reaction stirred at room tem-
perature for 16 h. To the crude cyanohydrin product were then
added 25% NH3 (4.0 mL, 52 mmol) and ammonium chloride
(7.76 g, 145 mmol). The reaction mixture was stirred at room
temperature for 18 h and then extracted with 2-propanol/
CH2Cl2 (1:10) (10 × 75 mL). The organic phases were dried
(MgSO4), and the solvent was evaporated to give the title
P r ep a r a tion of Com p ou n d s 1b-d ,f,g. To a mixture of
copper(I) iodide (100 mg, 0.5 mmol) and bis(triphenylphos-
phine)palladium(II) chloride (210 mg, 0.3 mmol) in diisopro-
pylamine (100 mL) and NEt3 (20 mL) under a nitrogen
atmosphere was added the appropriate fluoro- or chloro-sub-
stituted bromobenzene (30 mmol). After the mixture stirred
at room temperature for 30 min, 3-propynol (1.85 g, 33 mmol)
was added and the reaction mixture heated at 60 °C for 24 h.
The reaction mixture was filtered and the filtrate evaporated
to dryness. The product was purified by flash chromatography
using CH2Cl2 as eluent to give the product as a viscous oil.
3-(3-F lu or op h en yl)p r op -2-yn -1-ol (1b): yield 72%; 1H
NMR (CDCl3, 200 MHz) δ 1.88 (t, J ) 6 Hz, 1 H), 4.48 (d, J )
6 Hz, 2 H), 7.00-7.35 (m, 4 H).
3-(4-F lu or op h en yl)p r op -2-yn -1-ol (1c): yield 23%; 1H
NMR (CDCl3, 200 MHz) δ 2.33 (t, J ) 6 Hz, 1 H), 4.48 (d, J )
6 Hz, 2 H), 6.93-7.05 (m, 2 H), 7.35-7.47 (m, 2 H).
3-(3-Ch lor op h en yl)p r op -2-yn -1-ol (1d ): yield 75%; 1H
NMR (CDCl3, 200 MHz) δ 1.80 (t, J ) 6 Hz, 1 H), 4.48 (d, J )
6 Hz, 2 H).

2004 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11
J eppesen et al.
compound as an oil (2.5 g, 58%): 1H NMR (CDCl3, 200 MHz)
δ 1.40 (s, 1 H), 1.72 (s, 2 H), 2.15-2.30 (m, 8 H). The compound
was sufficiently pure for the next reaction step.
H2O) C: calcd, 54.67; found, 55.02. H: calcd, 4.07; found, 4.25.
N: calcd, 10.07; found, 9.98.
1-[3-[3-(3-F lu or op h en yl)-2-p r op yn -1-yloxy]-1,2,5-th ia -
d ia zol-4-yl]-4-a za tr icyclo[2.2.1.02,6]h ep ta n e oxa la te (8b):
1-(3-Ch lor o-1,2,5-th iadiazol-4-yl)-4-azatr icyclo[2.2.1.02,6]-
h ep ta n e (5). To a solution of sulfur monochloride (3.2 mL,
40.2 mmol) in DMF (5 mL) was added a solution of crude
2-amino-2-(4-azatricyclo[2.2.1.02,6]hept-1-yl)acetonitrile (4) (3.0
g, 20.1 mmol) in DMF (5 mL) at 5-10 °C over a period of 30
min. The reaction mixture was stirred for an additional 1 h
at 5-10 °C, after which ice-water (12 mL) was added to the
reaction. The mixture was filtered, and the filtrate was made
alkaline (pH ) 10) with 4 N NaOH. The product was extracted
with CH2Cl2 (4×). The organic phases were dried (MgSO4), and
the solvent was evaporated. The residue was purified by
column chromatography on silica gel using CH2Cl2 graduated
to CH2Cl2/MeOH (9:1) as eluent. The title compound was
obtained as an oil in 2.1 g (48%) yield: 1H NMR (CDCl3, 200
MHz) δ 2.12 (s, 1 H), 2.55 (d, J gem ) 10.5 Hz, 2 H), 2.65 (d,
J gem ) 10.5 Hz, 2 H), 2.70 (s, 2 H); MS m/z 213 (M + 1).
1-(3-P r op ylt h io-1,2,5-t h ia d ia zol-4-yl)-4-a za t r icyclo-
[2.2.1.02,6]h ep ta n e (6). A solution of 1-(3-chloro-1,2,5-thia-
diazol-4-yl)-4-azatricyclo[2.2.1.02,6]heptane (5) (700 mg, 3.3
mmol) and sodium hydrogen sulfide monohydrate (730 mg, 9.9
mmol) in DMF (20 mL) was stirred at room temperature under
a nitrogen atmosphere for 1 h. 1-Propyl bromide (900 µL, 9.9
mmol) and potassium carbonate (4.6 g, 33 mmol) was added,
and the reaction mixture was stirred at room temperature for
30 min. pH was adjusted with 4 N HCl (pH 2) and the mixture
washed with Et2O (2× discarded) The aqueous phase was
made alkaline with 2 N NaOH (pH 10-11) and the mixture
extracted with Et2O (3×). The Et2O phases were dried (MgSO4)
and evaporated. Crystallization of the residue with oxalic acid
from acetone gave the title compound as the oxalate salt in
935 mg (82%) yield: mp 151-154 °C; 1H NMR (CDCl3, 200
MHz) δ 1.05 (t, J ) 8 Hz, 3 H), 1.80 (sextet, J ) 8 Hz, 2 H),
2.13 (s, 2 H), 2.62 (d, J gem ) 10.5 Hz, 2 H), 2.80 (d, J gem ) 10.5
Hz, 2 H), 2.88 (s, 2 H), 3.25 (t, J ) 8 Hz, 2 H). Anal.
(C13H17N3O4S2) C, H, N.
1
yield 64%; mp 177-179 °C; H NMR (DMSO-d6, 400 MHz) δ
2.52 (s, 2 H), 3.05 (d, J gem ) 10.5 Hz, 2 H), 3.12 (d, J gem ) 10.5
Hz, 2 H), 3.35 (s, 2 H), 5.35 (s, 2 H), 7.32 (m, 3 H), 7.45 (m, 1
H). Anal. (C19H16FN3O5S) C, H, N.
1-[3-[3-(4-F lu or op h en yl)-2-p r op yn -1-yloxy]-1,2,5-th ia -
d ia zol-4-yl]-4-a za tr icyclo[2.2.1.02,6]h ep ta n e oxa la te (8c):
1
yield 80%; mp 185-187 °C; H NMR (DMSO-d6, 400 MHz) δ
2.52 (s, 2 H), 3.05 (d, J gem ) 10.5 Hz, 2 H), 3.12 (d, J gem ) 10.5
Hz, 2 H), 3.32 (s, 2H), 5.32 (s, 2 H), 7.25 (t, 2 H), 7.55 (m, 2
H). Anal. (C19H16FN3O5S‚1.5H2O) C: calcd, 51.35; found, 51.31.
H: calcd, 4.50; found, 4.68. N: calcd, 9.45; found, 9.17.
1-[3-[3-(3-Ch lor op h en yl)-2-p r op yn -1-yloxy]-1,2,5-th ia -
d ia zol-4-yl]-4-a za tr icyclo[2.2.1.02,6]h ep ta n e oxa la te (8d ):
1
yield 66%; mp 160-162 °C; H NMR (DMSO-d6, 400 MHz) δ
2.52 (s, 2 H), 3.05 (d, J gem ) 10.5 Hz, 2 H), 3.12 (d, J gem ) 10.5
Hz, 2 H), 3.35 (s, 2H), 5.35 (s, 2 H), 7.44 (m, 2 H), 7.52 (m, 2
H). Anal. (C19H16ClN3O5S‚0.5H2O) C: calcd, 51.46; found,
51.69. H: calcd, 3.83; found, 3.73. N: calcd, 9.48; found, 9.11.
1-[3-[3-(4-Ch lor op h en yl)-2-p r op yn -1-yloxy]-1,2,5-th ia -
d ia zol-4-yl]-4-a za tr icyclo[2.2.1.02,6]h ep ta n e oxa la te (8e):
1
yield 60%; mp 172-173.5 °C; H NMR (DMSO-d6, 400 MHz)
δ 2.52 (s, 2 H), 3.05 (d, J gem ) 10.5 Hz, 2 H), 3.12 (d, J gem
)
10.5 Hz, 2 H), 3.35 (s, 2 H), 5.35 (s, 2 H), 7.48 (m, 4 H). Anal.
(C19H16ClN3O5S) C, H, N.
1-[3-[3-(3,5-Diflu or op h en yl)-2-p r op yn -1-yloxy]-1,2,5-
t h ia d ia zol-4-yl]-4-a za t r icyclo[2.2.1.02,6]h ep t a n e oxa la t e
(8f): yield 48%; mp 173-175 °C; 1H NMR (DMSO-d6, 400 MHz)
δ 2.52 (s, 2 H), 3.05 (d, J gem ) 10.5 Hz, 2 H), 3.12 (d, J gem
)
10.5 Hz, 2 H), 3.35 (s, 2H), 5.35 (s, 2 H), 7.25 (m, 2 H), 7.38
(m, 1 H). Anal. (C19H15F2N3O5S‚3H2O) C: calcd, 46.62; found,
47.01. H: calcd, 3.06; found, 3.17. N: calcd, 8.58; found, 8.44.
1-[3-[3-(3-Ch lor o-5-flu or op h en yl)-2-p r op yn -1-yloxy]-
1,2,5-th ia d ia zol-4-yl]-4-a za tr icyclo[2.2.1.02,6]h ep ta n e ox-
1
a la te (8g): yield 78%; mp 155-156 °C; H NMR (DMSO-d6,
400 MHz) δ 2.52 (s, 2 H), 3.05 (d, J gem ) 10.5 Hz, 2 H), 3.12 (d,
J gem ) 10.5 Hz, 2 H), 3.35 (s, 2 H), 5.35 (s, 2 H), 7.38 (d, J )
9.0 Hz, 1 H), 7.44 (s, 1 H), 7.55 (d, J ) 9.0 Hz, 1 H). Anal.
(C19H15ClFN3O5S‚0.5H2O) C: calcd, 49.52; found, 49.65. H:
calcd, 3.50; found, 3.30. N: calcd, 9.12; found, 8.95.
1-[3-[3-(3,5-Dich lor op h en yl)-2-p r op yn -1-yloxy]-1,2,5-
t h ia d ia zol-4-yl]-4-a za t r icyclo[2.2.1.02,6]h ep t a n e oxa la t e
(8h ): yield 22%; mp 121-124 °C; 1H NMR (DMSO-d6, 400
MHz) δ 2.52 (s, 2 H), 3.05 (d, J gem ) 10.5 Hz, 2 H), 3.12 (d,
J gem ) 10.5 Hz, 2 H), 3.35 (s, 2 H), 5.35 (s, 2 H), 7.55 (s, 2 H),
7.75 (m, 1 H). Anal. (C19H15Cl2N3O5S) C, H, N.
P r ep a r a tion of Com p ou n d s 9a -e. A solution of 1-(3-
chloro-1,2,5-thiadiazol-4-yl)-4-azatricyclo[2.2.1.02,6]heptane (5)
(3.0 g, 14 mmol) and sodium hydrogen sulfide monohydrate
(3.14 g, 42 mmol) in dry DMF (80 mL) was stirred at room
temperature under a nitrogen atmosphere for 1 h. The solvent
was evaporated. H2O was added and the pH adjusted by
addition of 4 N HCl (pH 9). The mixture was cooled on ice,
and 4-(4-azatricyclo[2.2.1.02,6]hept-1-yl)-1,2,5-thiadiazole-3-
thiol (1.7 g, 58%) was isolated by filtration: mp 205-207 °C.
A mixture of 4-(4-azatricyclo[2.2.1.02,6]hept-1-yl)-1,2,5-thia-
diazole-3-thiol (106 mg, 0.5 mmol) and potassium carbonate
(207 mg, 1.5 mmol) in THF (10 mL) was cooled on ice. A
solution of the appropriate methanesulfonate (2a -e) (0.5
mmol) in THF (3 mL) was added. The reaction mixture was
stirred overnight starting at 0 °C and then allowed to warm
to room temperature. Ice water was added and pH adjusted
by addition of 4 N HCl (pH 2) (9d and 9e precipitated as the
HCl salt and were isolated by filtration). THF was removed
by evaporation and the residue washed with Et2O (3× dis-
carded). The aqueous phase was made alkaline with 25%
NH3 (pH 10-11) and the mixture extracted with Et2O (3×).
The extracts were dried (MgSO4), and the solvent was evapo-
rated. The residue was taken up in acetone and precipitated
with oxalic acid in acetone to give the product as the oxalate
salt.
1-(3-P r op ylsu lfon yl-1,2,5-th ia d ia zol-4-yl)-4-a za tr icyclo-
[2.2.1.02,6]h ep ta n e (7). 1-(3-Propylthio-1,2,5-thiadiazol-4-yl)-
4-azatricyclo[2.2.1.02,6]heptane (6) (585 mg, 1.7 mmol) was
dissolved in H2O (20 mL) and 1 N HCl (2 mL) and the solution
cooled in ice-water. Oxone (1.57 g, 2.55 mmol) in H2O (10 mL)
was added under stirring. Cooling was removed and the
reaction stirred for 2.5 h. The reaction was made alkaline (pH
10-11) with 5 N NaOH and then extracted with Et2O (6×).
The organic phases were dried (MgSO4), and the solvent was
evaporated. Crystallization of the residue with oxalic acid from
acetone gave the title compound as the oxalate salt in 545 mg
(83%) yield: mp 187-191 °C; 1H NMR (CDCl3, 200 MHz) δ
1.13 (t, J ) 8 Hz, 3 H), 1.95 (sextet, J ) 8 Hz, 2 H), 2.20 (s, 2
H), 2.62 (d, J gem ) 10.5 Hz, 2 H), 2.86 (d, J gem ) 10.5 Hz, 2 H),
2.96 (s, 2 H), 3.56 (t, J ) 8 Hz, 2 H). Anal. (C13H17N3O6S2) C,
H, N.
P r ep a r a tion of Com p ou n d s 8a -h . To a solution of the
appropriate alcohol (1a -h ) (2.1 mmol) in dry THF (10 mL)
was slowly added sodium hydride (80%) (80 mg, 2.7 mmol)
under a nitrogen atmosphere. After 1 h 1-(3-propylsulfonyl-
1,2,5-thiadiazol-4-yl)-4-azatricyclo[2.2.1.02,6]heptane (7) (200
mg, 0.7 mmol) in dry THF (3 mL) was added. Stirring was
continued for 5 days. The reaction was quenched by addition
of 6 N HCl (pH 2) and the THF evaporated. The residue was
washed with Et2O (3× discarded). The aqueous phase was
made basic (pH 10-11) with 25% NH3 and extracted with Et2O
(3×). The extracts were dried (MgSO4), filtered, and concen-
trated. The residue was taken up in acetone and precipitated
with oxalic acid in acetone to give the product as the oxalate
salt.
1-[3-(3-P h en yl-2-p r op yn -1-yloxy)-1,2,5-th ia d ia zol-4-yl]-
4-a za tr icyclo[2.2.1.02,6]h ep ta n e oxa la te (8a ): yield 76%; mp
1
182-185 °C; H NMR (DMSO-d6, 400 MHz) δ 2.52 (s, 2 H),
3.05 (d, J gem ) 10.5 Hz, 2 H), 3.12 (d, J gem ) 10.5 Hz, 2 H),
3.30 (s, 2H), 5.30 (s, 2 H), 7.45 (m, 5 H). Anal. (C19H17N3O5S‚

Thiadiazolylazatricycloheptanes as New M1 Agonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11 2005
1-[3-(3-P h en yl-2-p r op yn -1-ylth io)-1,2,5-th ia d ia zol-4-yl]-
from 5% to 12% as eluent. The title compound was isolated
4-a za tr icyclo[2.2.1.02,6]h ep ta n e oxa la te (9a ): yield 73%; mp
after freeze-drying: MS FAB+ m/z 302 (M + H)+.
1
154-157 °C; H NMR (DMSO-d6, 400 MHz) δ 2.45 (s, 2 H),
1-[3-[3-(3-Ch lor op h en yl)-2-p r op yn -1-yloxy]-1,2,5-th ia -
d ia zol-4-yl]-4-a za tr icyclo[2.2.1.02,6]h ep ta n e 4-Oxid e (11).
To a solution of 8d (105 mg, 0.24 mmol) in EtOH (50 mL) and
H2O (50 mL) was added a solution of oxone (162 mg, 0.26
mmol) in H2O (25 mL). pH was adjusted to pH 7 with 1 N
NaOH until the reaction was completed after 1.5 h. The
solution was acidified with 1 N HCl. By removing EtOH in
vacuo the product precipitated and was isolated by filtration
to give 60 mg (70%) of the title compound: MS FAB+ m/z 360
(M + H)+.
2.95 (d, J gem ) 10.5 Hz, 2 H), 3.08 (d, J gem ) 10.5 Hz, 2 H),
3.20 (s, 2 H), 4.40 (s, 2 H), 7.38 (s, 5 H). Anal. (C19H17N3O4S2)
C, H, N.
1-[3-[3-(3-F lu or op h en yl)-2-p r op yn -1-ylth io]-1,2,5-th ia -
d ia zol-4-yl]-4-a za tr icyclo[2.2.1.02,6]h ep ta n e oxa la te (9b):
1
yield 56%; mp 148-149 °C; H NMR (DMSO-d6, 400 MHz) δ
2.40 (s, 2 H), 2.90 (d, J gem ) 10.5 Hz, 2 H), 3.02 (d, J gem ) 10.5
Hz, 2 H), 3.15 (s, 2 H), 4.40 (s, 2 H), 7.25 (m, 3 H), 7.40 (m, 1
H). Anal. (C19H16FN3O4S2) C, H, N.
1-[3-[3-(4-F lu or op h en yl)-2-p r op yn -1-ylth io]-1,2,5-th ia -
3-[3-(3-P h en yl-2-p r op yn -1-yloxy)-1,2,5-th ia d ia zol-4-yl]-
1,2,5,6-tetr a h yd r o-1-m eth ylp yr id in e (12). Under a nitrogen
atmosphere, sodium hydride (80%) (300 mg, 10.0 mmol) was
added to an ice-cooled solution of 3-phenyl-2-propyn-1-ol (1.32
g, 10.0 mmol) in THF (30 mL). After the mixture stirred 1 h
at room temperature, a solution of 3-(3-chloro-1,2,5-thiadiazol-
4-yl)pyridine7 (980 mg, 5.0 mmol) in THF (15 mL) was added.
The reaction mixture was stirred at 50 °C for 2 h and
evaporated. The residue was dissolved in H2O (50 mL) and
extracted with Et2O (3 × 100 mL). The combined organic
phases were dried and evaporated to give crude 3-[3-(3-phenyl-
2-propyn-1-yloxy)-1,2,5-thiadiazol-4-yl]pyridine. The product
was redissolved in acetone (20 mL) and treated with methyl
iodide (1 mL, 15.0 mmol). The reaction mixture was stirred at
room temperature for 18 h and evaporated. The residue was
dissolved in MeOH (40 mL), and sodium borohydride (570 mg,
15.0 mmol) was added under a nitrogen atmosphere at 0 °C.
The reaction mixture was stirred for 15 min and evaporated.
The residue was dissolved in H2O (40 mL) and the product
extracted with Et2O (3 × 200 mL). The dried Et2O phases were
evaporated, and the residue was purified by column chroma-
tography on silica gel using EtOAc/MeOH (4:1) as eluent. The
product was crystallized as the oxalate salt from acetone to
give 930 mg (60% overall yield) of the title compound: mp
d ia zol-4-yl]-4-a za tr icyclo[2.2.1.02,6]h ep ta n e oxa la te (9c):
1
yield 62%; mp 149-150 °C; H NMR (DMSO-d6, 400 MHz) δ
2.45 (s, 2 H), 2.95 (d, J gem ) 10.5 Hz, 2 H), 3.08 (d, J gem ) 10.5
Hz, 2 H), 3.20 (s, 2 H), 4.40 (s, 2 H), 7.20 (m, 2 H), 7.42 (m, 2
H). Anal. (C19H16FN3O4S2) C, H, N.
1-[3-[3-(3-Ch lor op h en yl)-2-p r op yn -1-ylth io]-1,2,5-th ia -
d ia zol-4-yl]-4-a za t r icyclo[2.2.1.02,6]h ep t a n e h yd r och lo-
r id e (9d ): yield 66%; mp 175-177 °C; 1H NMR (DMSO-d6,
400 MHz) δ 2.77 (s, 2 H), 3.43 (d, J gem ) 10.5 Hz, 2 H), 3.53 (d,
J gem ) 10.5 Hz, 2 H), 3.70 (s, 2 H), 4.40 (s, 2 H), 7.40 (m, 4 H),
11.45 (bs, 1 H). Anal. (C17H15Cl2N3S2) C, H, N.
1-[3-[3-(4-Ch lor op h en yl)-2-p r op yn -1-ylth io]-1,2,5-th ia -
d ia zol-4-yl]-4-a za t r icyclo[2.2.1.02,6]h ep t a n e h yd r och lo-
r id e (9e): yield 72%; mp 154-156 °C; 1H NMR (DMSO-d6, 400
MHz) δ 2.77 (s, 2 H), 3.43 (d, J gem ) 10.5 Hz, 2 H), 3.53 (d,
J gem ) 10.5 Hz, 2 H), 3.70 (s, 2 H), 4.40 (s, 2 H), 7.40 (dd, J )
8.0 Hz, 4 H), 11.45 (bs, 1 H). Anal. (C17H15 Cl2N3S2‚1H2O) C:
calcd, 49.27; found, 49.31. H: calcd, 4.11; found, 3.98. N: calcd,
10.14; found, 9.69.
P r ep a r a tion of Com p ou n d s 9f-h . To a solution of 4-(4-
azatricyclo[2.2.1.02,6]hept-1-yl)-1,2,5-thiadiazole-3-thiol (211
mg, 1.0 mmol) (prepared as described under 9a -e) in dry THF
(10 mL) was added triphenylphosphine (262 mg, 1.0 mmol),
and the mixture was stirred under a nitrogen atmosphere. The
mixture was cooled on ice, and the appropriate alcohol (1f,g,h )
(1.5 mmol) dissolved in dry THF (5 mL) was added followed
by diethyl azodicarboxylate (174 mg, 1.0 mmol). The mixture
was stirred for 16 h starting at 0 °C and then allowed to warm
to room temperature. Ice water was added and pH adjusted
with 4 N HCl to pH 2.0; THF was removed in vacuo. By
addition of Et2O the product precipitated and was isolated by
filtration.
1
150-151 °C; H NMR (DMSO-d6, 200 MHz) δ 2.62 (m, 2 H),
2.82 (s, 3 H), 3.1 (m, 2 H), 4.00 (s, 2 H), 5.44 (s, 2 H), 7.14 (m,
1 H), 7.45 (m, 5 H). Anal. (C19H19N3O5S‚0.5H2O).
Ra d ioliga n d Bin d in g Assa ys. Fresh or frozen rat brain
tissue (cortex) was homogenized in assay buffer (20 mM Hepes,
pH 7.4) 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]oxotremorine-M
(Oxo-M; NEN, NET-671; final concentration 1 nM) and test
drug. The tubes were incubated at room temperature for 60
min. Unbound ligand was separated from bound ligand by
vacuum filtration through GF/C filters presoaked in 0.5% poly-
(ethylenimine). Filters were washed once with 10 mL of wash
buffer and transferred to vials; 4 mL of scintillation fluid was
added, and the radioactivity was measured by scintillation
counting. Nonspecific binding was measured with 10 µM
arecoline.
1-[3-[3-(3,5-Diflu or op h en yl)-2-p r op yn -1-ylt h io]-1,2,5-
t h ia d ia zol-4-yl]-4-a za t r icyclo[2.2.1.02,6]h ep t a n e h yd r o-
1
ch lor id e (9f): yield 88%; mp 194-196 °C; H NMR (DMSO-
d6, 400 MHz) δ 2.77 (s, 2 H), 3.43 (d, J gem ) 10.5 Hz, 2 H),
3.53 (d, J gem ) 10.5 Hz, 2 H), 3.70 (s, 2 H), 4.42 (s, 2 H), 7.15
(m, 2 H), 7.35 (m, 1 H), 11.50 (bs, 1 H). Anal. (C17H14F2ClN3S2‚
0.33H2O) C: calcd, 50.50; found, 50.52. H: calcd, 3.47; found,
3.54. N: calcd, 10.40; found, 10.04.
1-[3-[3-(3-Ch lor o-5-flu or op h en yl)-2-p r op yn -1-ylt h io]-
1,2,5-th ia d ia zol-4-yl]-4-a za tr icyclo[2.2.1.02,6]h ep ta n e h y-
d r och lor id e (9g): yield 59%; mp 201-203 °C; 1H NMR
(DMSO-d6, 400 MHz) δ 2.77 (s, 2 H), 3.43 (d, J gem ) 10.5 Hz,
2 H), 3.53 (d, J gem ) 10.5 Hz, 2 H), 3.70 (s, 2 H), 4.42 (s, 2 H),
7.25 (d, J ) 9.0 Hz, 1 H), 7.30 (s, 1 H), 7.50 (d, J ) 8 Hz),
11.70 (bs, 1 H). Anal. (C17H14FCl2N3S2) C, H, N.
1-[3-[3-(3,5-Dich lor op h en yl)-2-p r op yn -1-ylt h io]-1,2,5-
t h ia d ia zol-4-yl]-4-a za t r icyclo[2.2.1.02,6]h ep t a n e h yd r o-
ch lor id e (9h ): yield 60%; mp 184-187 °C; 1H NMR (DMSO-
d6, 400 MHz) δ 2.77 (s, 2 H), 3.43 (d, J gem ) 10.5 Hz, 2 H),
3.53 (d, J gem ) 10.5 Hz, 2 H), 3.70 (s, 2 H), 4.42 (s, 2 H), 7.43
(s, 2 H), 7.65 (s, 1 H), 11.75 (bs, 1 H). Anal. (C17H14 Cl3N3S2)
C, H, N.
1-(3-P r op ylsu lfon yl-1,2,5-th ia d ia zol-4-yl)-4-a za tr icyclo-
[2.2.1.02,6]h ep ta n e 4-Oxid e (10). To a suspension of 7 (150
mg, 0.5 mmol) in H2O (5 mL) was added a solution of oxone
(360 mg, 0.58 mmol) in H2O. pH was adjusted to pH 7 with 1
N NaOH until the reaction was completed. The solution was
acidified with 1 N HCl to pH 2, and the product was purified
by HPLC using 0.1% TFA in H2O and acetonitrile graduated
Stim u la tion of P h osp h oin ositol Hyd r olysis in A9L-M1
Cells. A mouse fibroblast cell line (A9L) stably expressing the
M1 receptor subtype was obtained from Mark Brann (Univer-
sity of Vermont). A9L cells were incubated at 37 °C in a
humidified atmosphere (5% CO2) as a monolayer culture in
DMEM supplemented with 10% fetal calf serum (Gibco, Grand
Island, NY), 100 units/mL each of penicillin G and strepto-
mycin, and 4 mM glutamine (M.A. Bioproducts, Walkersville,
MD). A9L cells were grown to confluence in the presence of
1.3 µCi/mL [3H]inositol for 48 h prior to assaying. On the day
of inositol phosphate determination, cells were detached by
rinsing with Ca2+-free Dulbecco’s phosphate-buffered saline
and then incubated for 5 min in 0.25% trypsin. The detached
cells were collected by centrifugation (300g for 2 min) and
resuspended in oxygenated HEPES/LiCl buffer (Hepes 30 mM,
NaCl 142 mM, KCl 5.6 mM, CaCl2 2.2 mM, NaHCO3 1.8 mM,
MgCl2 1.0 mM, glucose 5.6 mM, and LiCl 10 mM, pH 7.4 at
37 °C). Cells (0.5-1.0 mg of protein) were incubated at 37 °C
for 45 min in the presence of 100 µM carbachol or increasing
concentrations of drugs. The total water-soluble inositol
phosphate fraction was separated from [3H]inositol by ion-

2006 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11
J eppesen et al.
exchange chromatography as previously described.20 [3H]IP
was eluded directly into scintillation vials for counting with 4
mL of 0.1 ammonium formate/0.01 mM formic acid/5 mM
sodium borate. Data are expressed as the percent of total [3H]-
IP stimulated in the presence of 100 µM carbachol stimulation,
approximately 8000 dpm. All drugs were dissolved in distilled
water and added to the incubation mixture in a 50-µL aliquot.
Basal inositol phosphate hydrolysis was the amount isolated
in the presence of 50 µL of distilled water, between 600 and
800 dpm. EC50 values for dose-response curves were deter-
mined using a four-parameter logistic model (GraphPAD,
California). Affinity constants were determined using linear
regression.
Mea su r em en ts of cAMP F or m a tion in Ch in ese Ha m -
ster Ova r y (CHO) Cells Exp r essin g th e Hu m a n Mu sca -
r in ic M2 Recep tor . Plasmids containing the human M2
receptor were transfected into CHO cells which were cultured
in 150-mm Petri dishes with DMEM supplemented with 10%
fetal bovine serum, 2 mM glutamine, 0.1 mg/mL streptomycin,
0.1 mU/mL penicillin, 1 µM atropine, and 1 µM methotrexate
in an incubator at 37 °C (95% air, 5% CO2). One day before
the experiment, cells were washed twice with 20 mL of growth
media without atropine and left in this medium overnight. On
the day of the assay, cells were washed twice with phosphate-
buffered saline, pH 7.4, and incubated in phosphate-buffered
saline + 1 mM EDTA, pH 7.4, for 5 min at 37 °C. The detached
cells were transferred to 50-mL vials and washed twice with
assay buffer (composition: DMEM + 20 mM HEPES (pH 7.4)
+ 0.5 mM 3-isobutyl-1-methylxanthine) by centrifugation
(1000g, 5 min). Cells were resuspended in ice-cold assay buffer
and preincubated for 30 min at 0 °C. Test substances, buffer,
and cells were added to test tubes in a volume of 450 µL, and
the incubation was started by adding 50 µL of forskolin (10
µM final concentration). After the tubes were incubated for
10 min at 37 °C, ice-cold ethanol (1 mL/tube, 96%) was added
and the cells were transferred to an ice bath. Samples were
centrifuged (4500 rpm, 4 °C, 15 min), and the supernatant was
transferred to fresh tubes. The ethanol was evaporated under
a stream of nitrogen at 70 °C, and pellets were dissolved for
measurements of cAMP using a radioimmunoassay kit (Am-
ersham, U.K.). EC50 values were calculated from dose-
response curves (5 points minimum) by a nonlinear regression
analysis using the GraphPad Prism program (GraphPad
Software).
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Electrospray MS and MS/MS for Metabolite Identification in
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rats. Following intravenous and oral administration of 8f,
blood samples were taken and plasma was separated. 8f was
extracted from plasma using solid-phase extraction (SPEC
PLUS, C8/SCX) and quantified by HPLC with UV detection
(282 nm). The stationary phase was a C18, 250 × 4.5-mm
column (Symmetry, Waters) and the mobile phase acetonitrile:
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