Muscarinic analgesics with potent and selective effects on the gastrointestinal tract: Potential application for the treatment of irritable bowel syndrome

Article abstract of DOI:10.1021/jm9602470

Irritable bowel syndrome (IBS) is a pathophysiolocal condition characterized by abnormal bowel habits that are frequently accompanied by abdominal pain. Current therapy based on reducing high-amplitude GI contractions with nonselective muscarinic antagoni

Full text of DOI:10.1021/jm9602470

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538
J . Med. Chem. 1997, 40, 538-546
Mu sca r in ic An a lgesics w ith P oten t a n d Selective Effects on th e Ga str oin testin a l
Tr a ct: P oten tia l Ap p lica tion for th e Tr ea tm en t of Ir r ita ble Bow el Syn d r om e
Charles H. Mitch,*,† Thomas J . Brown,† Frank P. Bymaster,† David O. Calligaro,† Donna Dieckman,†
Leander Merrit,† Steven C. Peters,† Steven J . Quimby,† Harlan E. Shannon,† Lisa A. Shipley,† J ohn S. Ward,†
Kristian Hansen,‡ Preben H. Olesen,‡ Per Sauerberg,‡ Malcolm J . Sheardown,‡ Michael D. B. Swedberg,‡
Peter Suzdak,‡ and Beverley Greenwood*,†,§
Neuroscience Research, Lilly Research Laboratories, a Division of Eli Lilly and Company, Lilly Corporate,
Indianapolis, Indiana 46285, and Health Care Discovery, Novo Nordisk, Malov, Denmark
Received March 28, 1996X
Irritable bowel syndrome (IBS) is a pathopysiolocal condition characterized by abnormal bowel
habits that are frequently accompanied by abdominal pain. Current therapy based on reducing
high-amplitude GI contractions with nonselective muscarinic antagonists is limited in efficacy
due to typical muscarinic side effects and provides no pain relief. We have previously found
potent antinociceptive agents acting through muscarinic receptors. In the present work, new
1,2,5-thiadiazole-based structures with muscarinic activity have been evaluated both for activity
as analgesics in the mouse withing assay and for activity in normalizing spontaneous cluster
contractions in ferret jejunum as a model of IBS in humans. (5R,6R)-exo-6-[4-[(4,4,4-
Trifluorobutyl)thio]-1,2,5-thiadiazol-3-yl]-1-azabicyclo[3.2.1]octane (35, LY316108/NNC11-2192)
was found to offer an exceptional profile combining analgesic potency in mouse writhing (ED50
) 0.1 mg/kg) along with potency for normalization of GI motility (ED50 ) 0.17 mg/kg). This
combination of GI and analgesic potency suggests 35 as an excellent candidate for evaluation
as a potential treatment of IBS.
In tr od u ction
used to prevent gastroesophageal reflux because they
increase pressure within the lower esophageal sphinc-
ter.13 Furthermore, atropine-like muscarinic antago-
nists act as antispasmodics by reducing gastrointestinal
hypermotility in patients with IBS; however, the efficacy
of currently available muscarinic compounds is limited
because of typical anticholinergic side effects which
result from a lack of muscarinic receptor selectivity.14-16
IBS patients have been observed to have discrete
clusters of jejunal contractility concurrent with the
sensation of abdominal pain.5 A remarkably similiar
pattern of spontaneous jejunal motility has been found
in the anesthetized ferret.17 On the basis of this
similarity, the ferret has been used here as a preclinical
model of IBS for the testing of new compounds based
on their ability to inhibit the pattern of spontaneous
cluster contractions.
Abdominal pain is perhaps the most troublesome
symptom of IBS. However, currently available analge-
sic therapy does not provide relief because of the
undesirable side effect profiles of opioid or nonsteroidal
antiinflammatory drugs.18,19 Increasing evidence is
accumulating to suggest that muscarinic agents can
possess analgesic activity.20 We have previously found
muscarinic ligands based on the azacycle 1,2,5-thiadia-
zole moiety to possess potent antinociceptive activity
with a wide safety margin relative to typical muscarinic
side effects.21,22 These muscarinic analgesics would
appear to provide a good starting point for the search
for compounds that could treat both pain and gas-
trointestinal hypermotility. In the present study our
goal was to discover a potent nonopioid analgesic that
would inhibit gastrointestinal hypercontractility with-
out major side effects.
Irritable bowel syndrome (IBS) is a pathophysiological
condition of the gastrointestinal tract, defined by a
characteristic symptomatology of altered bowel habits,
such as diarrhea, constipation, or alternating episodes
of both, frequently accompanied by abdominal pain.1
Approximately 3% of the United States population has
been diagnosed with symptoms of IBS, comprising
almost 3.5 million physician visits yearly.2 In addition,
it is estimated that as much as one-third of the United
States population experiences IBS-like symptoms with-
out consulting a physcian.3 This syndrome clearly has
a major impact on health care resources and the
economy due to lost productivity.
Recent evidence suggests that the major symptoms
of IBS are due, at least in part, to disturbed gastrointes-
tinal motility, characterized by hypercontractility.4-6 In
patients with IBS the relationship between abnormal
gastrointestinal motility and the cause of abdominal
pain is unresolved. Although the etiology and patho-
physiology of IBS are unknown, it is well established
that neurons within the enteric nervous system of the
gut wall contain acetylcholine and that muscarinic
receptors are located on these enteric neurons as well
as smooth muscle cells and mucosal enterocytes.7-10 The
relative importance of cholinergic neurotransmission in
the regulation of gastrointestinal function under physi-
ological and pathophysiological conditions is unknown;
however, muscarinic agonists and antagonists pro-
foundly influence gastrointestinal tract function.11,12 In
humans, muscarinic agonists such as bethanechol are
* To whom correspondence should be addressed.
† Lilly Research Laboratories.
‡ Novo Nordisk.
Ch em istr y
§ Current address: Oklahoma Foundation for Digestive Research,
Presbyterian Hospital, Oklahoma City, OK 73104.
X Abstract published in Advance ACS Abstracts, J anuary 15, 1997.
We have previously disclosed the usefulness of 1,2,5-
thiadiazoles and pyrazines in the design of new mus-
S0022-2623(96)00247-6 CCC: $14.00 © 1997 American Chemical Society

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Muscarinic Analgesics for Treatment Of IBS
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4 539
Sch em e 1a
Sch em e 2a
a
(a) Ethyl cyanoacetate, AcONH4, AcOH; (b) H2, Pd/C; (c)
isoamyl nitrite, Na; (d) S2Cl2; (e) H2, Pd/C; (f) Na2S, BrBu; (g)
D-tartaric acid.
a
(a) KCN, NH4Cl; (b) S2Cl2; (c) NaOR; or Na2S, RBr; (d) MeI or
EtI; (e) NaBH4; (f) vinyl chloroformate; (g) HCl.
lation with bromobutane gave 10, which was resolved
crystallographically with D-tartaric acid to give (+)-11.
Preparation of the azabicyclo[3.2.1]octanes com-
menced with ethyl nipecotate (12)29 (Scheme 3). Alky-
lation with ethyl bromoacetate followed by Claisen
condensation and decarboxylation afforded racemic 13.
Resolved material was prepared at this stage by crystal-
lization with D- or L-tartaric acid. Knoevenagel con-
densation with ethyl cyanoacetate and hydrogenation
of the unsaturated intermediate gave cyano ester 14.
Conversion to an intermediate oxime with isoamyl
nitrite, heterocyclization with S2Cl2, and hydrogenolysis
of the resulting R-chloro group on the azabicyclo[3.2.1]-
octane ring afforded 15. Thioetherification with Na2S
and alkyl bromide gave 16, typically as a 9:1 mixture
favoring the endo diastereomer. Equilibration of 16
with KO-t-Bu resulted in conversion to a 9:1 mixture
now favoring the exo isomer 17.
carinic agonists.22-24 The SAR disclosed herein is based
on varying the azacycle attached to these useful het-
erocycles. Tetrahydropyridyl, quinuclidyl, and 1-aza-
bicyclo[3.2.1]octyl groups have been found to be par-
ticularly effective in our labs and by others.25-27 The
alternative use of the 1-azabicyclo[2.2.1]heptyl group
has been previously reported to result in extremely
potent but nonselective muscarinic drugs.21
Tetrahydropyridines were prepared by the method
outlined in Scheme 1.22 Strecker reaction with pyridi-
necarboxaldehyde (1) afforded an intermediate amino-
nitrile which was cyclized to the 1,2,5-thiadiazole 2 by
treatment with S2Cl2. Alkoxy side chains were substi-
tuted onto the 1,2,5-thiadiazole by displacement with
the corresponding sodium or potassium alkoxide. Al-
ternatively, alkylthio side chains were incorporated by
a two-step, one-pot process involving initial displace-
ment with Na2S followed by alkylation with the corre-
sponding alkyl bromide. Pyridine 3a or 3b was then
alkylated with MeI or EtI and reduced with NaBH4 to
give the desired tetrahydropyridines 4a or 4b. In some
cases the N-methyltetrahydropyridines were further
transformed by N-demethylation with vinyl chlorofor-
mate followed by acid hydrolysis to give N-hydridotet-
rahydropyridine 5.
The preparation of trifluoro-substituted side chains
necessitated a modification of the above route due to
instability to the basic conditions employed for diaste-
reomer equilibration. As an alternative, 17 (R1 ) butyl),
was oxidized with oxone to the sulfone 18. The sulfone
was then displaced with Na2S and followed by alkylation
with the desired trifluoroalkyl bromide to afford 19.
P h a r m a cology
Preparation of thiadiazole-substitued quinuclidine 11
is shown in Scheme 2.22 Knoevenagel condensation of
quinuclidinone (6) and ethyl cyanoacetate afforded an
unsaturated cyanoester intermediate which was hydro-
genated to 7. Heterocyclization of 7 to the R-chloro-
chlorothiadiazole 8 was achieved through reaction with
isoamyl nitrite to give an intermediate cyano oxime
followed by treatment with S2Cl2. Atmospheric hydro-
genation with Pd/C resulted in selective hydrogenolysis
of the quinuclidinyl chloride to give 9. Displacement
of the thiadiazole chloride with Na2S followed by alky-
Discrete clusters of jejunal contractility separated by
periods of quiescence have been observed in IBS pa-
tients concurrently with the sensation of abdominal
pain.5 The anesthetized ferret displays a similar pat-
tern of spontaneous jejunal motility.17 Given the close
resemblence in jejunal activity to the clinical disease
state, the anesthetized ferret has been used in the
present work as an in vivo model for IBS. Test
compounds were evaluated for their ability to inhibit
this characteristic pattern of spontaneous jejunal motil-

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540 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4
Mitch et al.
Sch em e 3a
were evaluated to determine a dose of a compound that
inhibited spontaneous motility by 50% (ED50). Through-
out the SAR, transmural potential difference was ex-
pressed as the dose of compound that stimulated an
increase in transmural potential difference of greater
than 1 mV (ED1mV).
Standard compounds were evaluated for their effect
in the above animal models. Atropine is a muscarinic
antagonist with well-characterized antispasmodic activ-
ity, including inhibition of spontaneous clusters of
gastrointestinal motility in the ferret. In the ferret
model above, atropine had no effect on transmural
potential difference. Atropine was devoid of antinoce-
ciptive activity in the mouse writhing assay. In the
ferret, muscarinic agonists such as acetylcholine in-
creased intestinal motility and increased transmural
potential difference in the direction of lumen negativity.
Affinity for muscarinic receptors was determined
using [3H] oxotremorine-M in radioligand displacement
experiments in rat cortex. The use of oxotremorine-M
for receptor binding studies is considered representative
of binding to the agonist conformational state of mus-
carinic receptors without regard to selectivity for sub-
types of muscarinic receptors.34
Resu lts a n d Discu ssion
On the basis of the similarity of the pattern of discrete
clusters of jejunal motility between IBS patients and
the anesthetized ferret, we sought to find compounds
that would potently inhibit this response. We also
wanted the compounds to show a wide separation of
dose for these desired effects relative to doses causing
an increase in transmural potential difference across
the lumen of the intestinal wall, as a marker of
undesired intestinal secretory activity. We also desired
the compounds to be potent analgesics in the mouse
writhing assay in order to treat the abdominal pain
associated with abnormal contractility in IBS patients
Compounds acting at muscarinic receptors would ap-
pear to offer a novel method for simultaneously treating
both the intestinal hypercontractility and pain associ-
ated with this disorder with a single pharmacologic
agent. Compounds were selected for evaluation in the
ferret model primarily based on affinity for muscarinity
receptor binding and activity in the mouse writhing
assay. However, the pharmacological diversity of com-
pounds tested was broadened to include some with good
affinity for muscarinic receptors, as measured by dis-
placement of [3H] oxotremorine-M, even though lacking
analgesic activity. Results are shown in Table 1.
The structure activity profile of these compounds
clearly varies between the different assays. We have
previously reported on the muscarinic analgesic activity
of a series of azacycle-substitued 1,2,5-thiadiazoles
acting at muscarinic receptors.21,22 A notable structure-
activity feature of that series is the optimal analgesic
activity that was observed for alkoxy and thioether side
chains of three and four carbons in chain length. This
optimum analgesic activity residing at chain lenghts of
three and four carbons was also found for the present
series of compounds reported in Table 1. Among the
tetrahydropyridines, 20 and 21 are reasonably potent
analgesics, but each of these compounds lacked separa-
tion between effects on motility and transmural poten-
tial difference. Also, 20 was 10-fold more potent than
a
(a) BrCH2CO2Et; (b) KOtBu; (c) HCl; (d) resolution with D- or
L-tartaric acid; (e) ethyl cyanoacetate; (f) H2, Pd/C; (g) isoamyl
nitrite, NaOEt; (h) S2Cl2; (i) H2, Pd/C; (j) Na2S, RBr; (k) NaOEt;
(1) KHSO5; (m) Na2S, RBr.
ity. Transmural potential difference (PD) between
lumen and serosa in the jejunum was measured as an
on-line marker of intestinal secretory activity.10,30,31
While the exact relationship between PD and diarrhea
or constipation remains speculative, minimizing the
effect on PD was considered desirable to reduce the
liklihood of unwanted gastrointestinal side effects.32 In
order to maximize the therapeutic-safety ratio, com-
pounds were sought with the widest possible separation
between the dose required for inhibition of spontaneous
jejunal motility and the dose producing an increase in
PD.
Full details of the anesthetized ferret assay have been
published elsewhere.9,10 Briefly, intestinal motility was
measured manometrically using catheters inserted into
the jejunum. Potential difference was measured using
a pair of agar-KCl electrodes, with one placed in the
lumen, the other in contact with serosal fluid, and both
connected via calomel half cells to an electrometer.
Analgesic activity was assessed in vivo using the
abdominal constriction response in mice to the admin-
istration of 0.5% acetic acid, ip, the so-called mouse
writhing assay.33 Compounds with a potency equal to
or better than morphine were desired. Under the test
conditions used here, morphine had an ED50 of 0.3 mg/
kg, sc.
The in vivo SAR in the ferret model was developed
based on the threshold dose required to inhibit spon-
tanous jejunal motility. A select number of compounds

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J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4 541
Ta ble 1. Tetrahydropyridine Analogs
a
b
Minimum effective dose to inhibit jejunal motility. Number of determinations given in parentheses; SEM reported when replicate
values differed from each other. c Dose required to produce a 1 mV increase in transmural potential difference. See ref 23 for synthesis.
d
Ta ble 2. Quinuclidine Analogs
a
b
Minimum effective dose to inhibit jejunal motility. Number of determinations given in parentheses; SEM reported when replicate
values differed from each other. c Dose required to produce a 1 mV increase in transmural potential difference. See ref 26 for synthesis.
d
21 at inhibiting motility while being 3-fold less potent
in the mouse writhing assay. Compound 22 is a very
potent and functionally selective m1 agonist; however
it was only moderately potent in the mouse writhing
assay, consistent with previous resports that analgesia
is not mediated by m1 receptors.35,36 Compound 22 also
had very poor activity for inhibiting motility in the ferret
jejunum. Compound 23 showed less affinity for mus-
carinic receptor binding in the oxotremorine-M assay
than any other compound reported in this paper and
was inactive in mouse writhing and in the ferret
jejunum at the highest doses tested (10 mg/kg, sc in the
mouse, and 30 µg/kg, iv in the ferret). In line with
observations about optimal chain length, compounds 24
and 25 with propylphenyl and propylthiophene side
chains, respectively, were of only limited activity in both
the mouse writhing and ferret models. No tetrahydro-
pyridyl compounds were found with both good analgesic
potency and good separation between effects on motility
and potential difference.
with potent analgesic activity, was found by combining
a quinuclidine with a thiobutylthiadiazole, as in 26 (see
Table 2). We, and others, have previously reported the
bioisosteric replacement of the pyrazine heterocycle for
the 1,2,5-thiadiazole system with favorable results.24-26
The thiobutylpyrazine substituted with a quinuclidine,
27, showed 10-fold separation between motility and
potential difference effects, but unfortunately the com-
pound was only weakly active as an analgesic. Increas-
ing the length of the pyrazine side chain to thiohexyl,
as in compound 28, resulted in completely abolishing
both motility and potential difference effects and also
diminished the compound’s affinity for binding musca-
rinic receptors.
Most of the azabicyclo[3.2.1]octane compounds exam-
ined were quite potent, both as analgesics and as
inhibiters of motility (see Table 3). For example,
compound 29 was the most potent analgesic in the series
and was also very potent at inhibition of motility.
However, 29 was also equipotent at increasing trans-
mural potential difference. The resolved (+)-enantiomer
of this compound, 30, was also tested and found to be
The first compound in the series with some separation
between motility and potential difference effects, along

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Mitch et al.
Ta ble 3. 1-Azabicyclo[3.2.1]octane Analogs
a
b
Minimum effective dose to inhibit jejunal motility. Number of determinations given in parentheses; SEM reported when replicate
values differed from each other. c Dose required to produce a 1 mV increase in transmural potential difference.
Ta ble 4. Resolved Isomers
a
Dose to produce a 50% reduction in spontaneous motility. b Dose required to produce a 1 mV increase in transmural potential difference.
equivalent in GI activity to its racemate, 29. The one
exception to the analgesic potency of the [3.2.1]azabi-
cyclics was 31, substituted with a thioisohexyl side
chain, resulting in diminished analgesic potency and a
reversal of the desired selectivity, with greater potency
for increasing transmural potential difference than for
inhibition of motility.
On the basis of the superior attributes of 33 in
meeting the desired pharmacological profile, the com-
pound was resolved into its constituent enantiomers (see
Table 4). For comparison purposes, the corresponding
isomers with endo configuration were also evaluated,
37 and 38. Each of the endo isomers is much less active
than the associated exo enantiomers in both mouse
writhing and ferret GI assays. Between the enanti-
omers with exo stereochemistry, the (5R,6R) isomer, 35
(LY316108/NNC 11-2192), was clearly superior. Com-
pound 35 was 3-fold more potent as an analgesic than
its enantiomer, 36. Also, 35 was 88-fold more selective
for inhibition of motility in comparison to its effect on
transmural potential difference. In contrast, 36 was
actually more selective for increasing the potential
difference than for inhibition of motility.
Incorporation of a trifluoro functionality at the end
of the thiadiazole side chain afforded compounds with
the desired profile of analgesic potency combined with
selectivity for inhibition of motility versus increasing
transmural potential difference. In particular, 33 with
a 1-thio-4,4,4-trifluorobutyl side chain showed 30-fold
separation between motility and potential difference
effects along with very potent activity in the mouse
writhing assay. The analogous compounds with tri-
fluoropropyl and trifluoropentyl side chains, 32 and 34,
respectively, were similarly potent as analgesics but
were somewhat less selective between motility inhibi-
tion and potential difference effects.
The trifluoro functionality also imparted enhanced
bioavailability characteristics. In rats, plasma concen-
trations of 35 were nearly 40-fold higher than those seen
with a comparable dose of 30, the congener to 35

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J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4 543
ridine22 (0.86 g, 2.4 mmol) in 1,2-dichloroethane (20 mL) was
added a solution of 1-chloroethyl chloroformate (0.35 g, 2.4
mmol) in 1,2-dichloroethane at 0 °C. The reaction mixture was
heated to 40 °C for 2 h and evaporated. The residue was
dissolved in methanol, heated to reflux for 1 h, and evaporated.
The residue was dissolved in diluted sodium hydroxide and
extracted with ether. The combined ether phases were dried
and evaporated. Crystallization as the oxalate salt from
acetone gave 21: mp 157-159 °C. Anal. (C13H19N3O4S2) C,
H, N.
differing only in the absence of the terminal trifluoro
substituents on the side chain. Mean plasma concen-
trations of 805 and 21 ng/mL were found for 35 and 30,
respectively, measured 1 h following an oral dose of 30
mg/kg of each. Similiarly, for oral administration in
mice, area under the curve (AUC) of parent plasma
concentration plotted against time was over 40-fold
higher for 35 than for 30, after normalizing for dose
(AUC/dose: 193 for 35 vs 4.5 for 30).
3-[4-(5-H exen yloxy)-1,2,5-t h ia d ia zol-3-yl]-1,2,5,6-t et -
r a h yd r o-1-m eth ylp yr id in e Ma lea te Sa lt (22). To a solu-
tion of 5-hexen-1-ol (900 mg, 9 mmol) and sodium hydride (310
mg, 13 mmol) in dry tetrahydrofuran was added a solution of
3-(4-chloro-1,2,5-thiadiazol-3-yl)pyridine23 (590 mg, 3 mmol)
in dry tetrahydrofuran. The reaction mixture was stirred at
room temperature for 1 h. Water was added, and the mixture
was extracted with ether. The ether phase was dried and
evaporated. The residue was taken up in 5 mL of acetone,
methyl iodide (0.5 mL, 7.5 mmol) was added, and the mixture
was stirred at room temperature for 18 h. The resulting
precipitate was collected by filtration and then suspended in
ethanol (20 mL). Sodium borohydride (150 mg, 4 mmol) was
added to this solution and the reaction mixture was stirred at
-10 °C for 0.5 h. After evaporation, the residue was dissolved
in water and extracted with ethyl acetate. The dried organic
phases were evaporated, and the residue was purified by
column chromatography (SiO2, eluent: ethyl acetate/methanol
(4:1)). Compound 22 was crystallized as the maleate salt from
acetone to yield 250 mg. Anal. (C18H25N3O5S) C, H, N.
3-[4-(Hexylth io)-1,2,5-th iadiazol-3-yl]-1,2,5,6-tetr ah ydr o-
Hepatic metabolism studies in isolated perfused rat
liver suggest that the increased plasma levels of 35 may
be due to enhanced metabolic stability for 35 relative
to 30. Analysis of rat liver perfusate for parent and
putative metabolites was carried out using HPLC/mass
spectrometry. It was found that nonfluorinated ali-
phatic side chains, as in 30, underwent extensive
oxidation to hydroxy- and keto-substituted side chains.
In contrast, no side chain oxidation was found for 35,
demonstrating that the terminal trifluoro group ef-
fectively blocked side chain oxidation by the liver in this
model. The substantially higher plasma concentrations
of parent 35 observed in vivo may be a result of reduced
first pass hepatic metabolism imparted by the trifluoro
substituents.
Compound 35 showed an outstanding pharmacologi-
cal profile with very high affinity for muscarinic recep-
tors as measured by displacement of [3H]oxotremorine-
M and is a highly potent analgesic in the mouse
writhing assay. Compound 35 also potently inhibited
the characteristic discrete clusters of jejunal contrac-
tions in the ferret. Much higher doses of compound
were required before an increase in transmural poten-
tial difference was observed, indicating minimal effect
on fluid and electrolyte secretion. It is hoped that this
high potency for analgesia and motility inhibition will
result in a compound with unique therapeutic efficacy
for the treatment of IBS. Similarly, it is hoped that the
low potency for increasing transmural potential differ-
ence will be predictive of a lack of GI side effects. On
oral administration, compound 35 also showed enhanced
plasma levels of parent relative to those found for the
nonfluorinated analog 30. This suggests a highly desir-
able profile of biological activity for 35, suggesting its
potential for clinical evaluation in the treatment of IBS.
1-eth ylp yr id in e Oxa la te (23). A solution of 3-[4-(hexylthio)-
23
1,2,5-thiadiazol-3-yl]pyridine
(1.2 g, 4.3 mmol) and ethyl
iodide (2.0g, 13 mmol) in acetone (4 mL) was stirred at 40 °C
for 16 h. The precipitate was collected by filtration and
suspended in ethanol (10 mL). Sodium borohydride (410 mg,
10.8 mmol) was added to the solution, and the mixture was
stirred for 1 h at 0 °C. Water was added, and the mixture
was extracted with ethyl acetate. After drying, the ethyl
acetate phase was evaporated and the residue purified by
column chromatography (eluent: ethyl acetate/methanol (4:
1)). Crystallization with oxalic acid from acetone gave 23; mp
134-135 °C. Anal. (C17H27N3O4S2) C, H, N.
3-[4-[(3-P h en ylpr opyl)th io]-1,2,5-th iadiazol-3-yl]-1,2,5,6-
tetr a h yd r o-1-m eth ylp yr id in e Oxa la te (24). Sodium hy-
drogen sulfide monohydrate (0.25 g, 3.3 mmol) was added to
a solution of 3-(3-chloro-1,2,5-thiadiazol-4-yl)pyridine23 (0.59
g, 3.0 mmol) in DMF (20 mL) at room temperature, and the
reaction mixture was stirred for 1 h. Potassium carbonate
(1.24 g, 9 mmol) and 1-bromo-3-phenylpropane (0.90 g, 4.5
mmol) were added, and the reaction mixture was stirred for
an additional 24 h. Water (50 mL) was added and the mixture
extracted with ether. The combined ether phases were dried
and evaporated. The residue was taken up in acetone (5 mL),
and methyl iodide was added. The reaction was stirred at
room temperature for 20 h, and then evaporated. The residue
was taken up in ethanol (20 mL), sodium borohydride (290
mg, 7.5 mmol) was added, and the reaction mixture was stirred
at -10 °C for 1 h. After evaporation the residue was dissolved
in water and extracted with ethyl acetate. The dried organic
phases were evaporated, and the residue was purified by
column chromatography (SiO2, eluent: ethyl acetate/methanol
(4:1)). Compound 24 was crystallized as the oxalate salt from
acetone: mp: 110-110.5 °C. Anal. (C19H23N3O4S2) C, H, N.
3-[4-[(3-(2-Th ien yl)-1-p r op yl)t h io]-1,2,5-t h ia d ia zol-3-
yl]-1,2,5,6-tetr a h yd r o-1-m eth ylp yr id in e Oxa la te (25). A
solution of 3-(4-chloro-1,2,5-thiadiazol-3-yl)pyridine23 (2.0 g,
10.1 mmol) in DMF (10 mL) was cooled to 5 °C, and potassium
carbonate (2.8 g, 20.2 mmol) and sodium hydrosulfide mono-
hydrate (1.5 g, 20.2 mmol) were added to the reaction. After
the mixture was stirred for 1 h, additional potassium carbonate
(1.4 g, 10.1 mmol) and a solution of 3-(2-thienyl)-1-chloropro-
pane (1.8 g, 11.2 mmol) in DMF (5 mL) were added, and the
solution was stirred for 1 h at room temperature. The reaction
was quenched with water, and then the mixture was extracted
with CH2Cl2 (3 × 75 mL). The organic phase was dried over
Exp er im en ta l Section
All compounds were prepared as racemates except were
specifically indicated otherwise.
Melting points were determined with a Mel-Temp apparatus
and are uncorrected. Proton and carbon magnetic resonance
spectra were recorded on a GE QE-300 spectrometer at 300
and 75 MHz, respectively, and are reported in ppm on a δ scale
from internal tetramethylsilane. Microanalyses, mass spectral
measurements, and X-ray crystal structures were determined
by the Structural and Organic Chemistry Research Depart-
ment of the Lilly Research Laboratories. Optical rotations
were obtained on a Perkin-Elmer Model 248 automatic pola-
rimeter. Gas chromotagraphy was performed on a Hewlett-
Packard 5890 instrument with an HP-1 megabore capillary
GC column and flame-ionization detection. Preparative chro-
matography was performed on a Waters Prep 2000 system.
When necessary, solvents and reagents were dried prior to
use. Diethyl ether and tetrahydrofuran were distilled from
sodium metal/benzophenone ketyl. All other reagents were
used as received from Aldrich Chemical Co., Milwaukee, WI.
3-[4-(Bu t ylt h io)-1,2,5-t h ia d ia zol-3-yl]-1,2,5,6-t et r a h y-
d r op yr id in e Oxa la te Sa lt (21). To a solution of 3-[4-
(butylthio)-1,2,5-thiadiazol-3-yl]-1,5,6-tetrahydro-1-methylpy-

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544 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4
Mitch et al.
NaCl/Na2SO4 and then evaporated under vacuum. The resi-
due was purified by flash chromatography, eluting with 1:1
ethyl acetate/hexane. The resulting oil was taken up in
acetone (30 mL) and methyl iodide (2.0 g, 14 mmol) added.
The reaction mixture was stirred for 16 h at room temperature
and evaporated under vacuum. The residue was suspended
in ethanol (15 mL) and NaBH4 (0.53 g, 14 mmol) added. After
being stirred for 0.5 h, the solution was evaporated under
vaccum and the residue was dissolved in water and extracted
with ethyl acetate. The organic phases were dried over NaCl/
Na2SO4 and evaporated under vacuum. The residue was
purified by flash chromotagraphy, eluting with 5% EtOH, 0.5%
NH4OH, and CHCl3. Crystallization as the oxalate salt gave
350 mg of 25: mp 98-100 °C. Anal. (C17H21N3O4S3) C, H, N.
(S)-3-[4-(Bu tylth io)-1,2,5-th ia d ia zol-3-yl]-1-a za bicyclo-
[2.2.2]octa n e Ta r tr a te (26). A mixture of 3-quinuclidinone
hydrochloride, 7 (100 g, 620 mmol), ethyl cyanoacetate (140
g, 1240 mmol) and triethylamine (93.5 g, 930 mmol) was
heated at 80 °C for 2 h. After the mixture was cooled to room
temperature, toluene (1.5 L) was added. The organic phase
was washed with H2O (2 × 100 mL), and concentrated HCl
was added to pH ) 4.0. After azeotropic removal of H2O, the
precipitated crystals were filtered and washed with cold
acetone. The crystals were suspended in absolute EtOH (1000
mL) and hydrogentated over Pd/C (3 g, 10%) at room temper-
ature and atmospheric pressure. The catalyst was filtered and
the filtrate evaporated to half volume. Upon addition of Et2O
and cooling, a solid precipitate separated. The precipitate was
added to a solution of Na metal (11.5 g, 500 mmol) dissolved
in an EtOH/MeOH mixture (1:1, 500 mL), and the reaction
mixture was stirred at room temperature for 30 min. The
reaction mixture was cooled to 5 °C, and isoamyl nitrite (64.45
g, 650 mmol) was added at such a rate that the temperature
was kept below 10 °C. The reaction mixture was then
evaporated at reduced pressure. Traces of alcohols were
removed by azeotropic evaporation with toluene. The residue
was dissolved in DMF (250 mL) and slowly added to a solution
of S2Cl2 (203 g, 1500 mmol) in DMF (75 mL). The temperature
of the reaction mixture was kept just below 0 °C during the
addition. After addition, the reaction mixture was allowed to
warm to room temperature and stirred at this temperature
for 42 h. Ice water (500 mL) was carefully added, and the
suspension was heated to 70 °C to redissolve precipitated
compound. Sulfur was filtered off and the filtrate made
alkaline with 4 N NaOH. The alkaline suspension was
extracted with toluene (2 × 500 mL). The organic extracts
were dried over MgSO4 and concentrated under vacuum. The
crude compound was redissolved in EtOH (250 mL) and then
precipitated with concentrated HCl. This compound was taken
up in absolute EtOH (500 mL) and hydrogenated over Pd/C
(8 g, 5%) at room temperature and atmospheric pressure. The
catalyst was filtered off and the filtrate evaporated at reduced
ammonium acetate (1.25 g), and ethyl cyanoacetate (37 g, 0.33
mol) in toluene (500 mL) was refluxed with a Dean-Stark trap
for 40 h. The water phase was made basic with 28% am-
monium hydroxide solution and extracted with ether (4 × 200
mL). The organic phases were dried over magnesium sulfate
and evaporated. The residue was purified by column chro-
matography (eluent CH2Cl2/MeOH (9:1)). The resulting prod-
uct (41 g, 0.19 mol) in absolute ethanol (500 mL) was treated
with 10% Pd/C (5 g) and hydrogen in a Parr shaker at 30 psi
for 5 h. Filtration and evaporation yielded 14, 36 g (0.16 mol).
The crude product was taken up in absolute ethanol (100 mL)
and added to a solution of sodium (4 g, 0.21 mol) in absolute
ethanol (100 mL). Isoamyl nitrite (25 mL, 0.19 mol) was added
over 0.5 h, and the mixture was heated at 50 °C for 4 h.
Evaporation of the reaction mixture gave a crude sodium salt,
which was used without further purification. A solution of the
crude salt in DMF (150 mL) was added to a solution of S2Cl2
(50 mL, 0.68 mol) in DMF (100 mL) at 0 °C over 1 h. The
reaction mixture was stirred overnight, and ice water (500 mL)
was added. The mixture was filtered and the filter cake
washed with 1 N HCl (3 × 100 mL). The water solution was
extracted with ether (2 × 200 mL) and then basified with a
28% NH4OH solution and extracted with ether (4 × 200 mL).
The combined ether extracts from the last extraction were
dried and evaporated. The residue was purified by column
chromatography (eluent: CH2Cl2/MeOH (9:1)) to give 11 g of
product. A solution of the product (1.3 g, 5 mmol) in absolute
ethanol (100 mL) was treated with 10% Pd/C (300 mg) in a
Parr shaker at 20 psi for 4 h. The solution was filtered and
evaporated. The residue was purified by column chromatog-
raphy (CH2Cl2/MeOH/TEA (9:1:0.25)). The first fractions
contained the exo isomer, and the later fractions contained
the endo isomer. To a solution of the exo isomer (229 mg, 1.0
mmol) in DMF (20 mL) was added sodium hydrogen sulfide
monohydrate (230 mg, 3.0 mmol). The reaction mixture was
stirred at room temperature for 1 h. Potassium carbonate
(1.38 g, 10 mmol) and 1-butyl bromide (343 mg, 2.5 mmol) were
added, and the mixture was stirred for 1 h. A solution of 1 N
HCl was added and the mixture extracted with ether (2 × 50
mL). The aqueous solution was basified with a 28% NH4OH
and extracted with ether (2 × 50 mL). The ether phase was
dried and evaporated. The residue was crystallized as the
oxalate salt from acetone/ether to give 200 mg of 29: mp 143-
145 °C. Anal. (C15H23N3O4S2) C, H, N.
exo-(5S ,6S )-6-[4-(Bu t ylt h io)-1,2,5-t h ia d ia zol-3-yl]-1-
a za bicyclo[3.2.1]octa n e (-)-(D)-Ta r tr a te (30). To a solu-
tion of (+)-29 free base (5.5 g, 19.43 mmol) in ethanol (50 mL)
was added a solution of (-)-D-tartaric acid (2.9 g, 19.4 mmol)
in water (10 mL). Ether (200 mL) was added to the solution
to give a slightly cloudy solution. Compound 30 was precipi-
tated overnight, and the crystals were collected by filtration
(3.05 g). Recrystallizaiton twice from elthanol/ether gave 1.90
g of 30: mp 106-108 °C; (Free base) [R] ) +14.94° (c ) 4.09/
MeOH). Anal. as the tartrate salt (C17H27N3O6S2) C, H, N.
(+)-exo-6-[4-[4-Met h ylp en t yl)t h io]-1,2,5-t h ia d ia zol-3-
yl]-1-a za bicyclo[3.2.1]octa n e Oxa la te (31). The same pro-
cedure as for compound 29 was used, but with 1-bromo-4-
methylpentane as the alkyl bromide. Anal. as the oxalate salt
(C17H27N3O4S2) C, H, N.
(5S,6S)-exo-6-[4-[3,3,3-Tr iflu or op r op yl)th io]-1,2,5-th ia -
d ia zol-3-yl]-1-a za bicyclo[3.2.1]octa n e Oxa la te (32). To a
solution of compound 30 (2.8 g, 10 mmol), 1 N HCl (10 mL),
and water (23 mL) at 0 °C was added Oxone (9.2 g, 15 mmol)
in water (45 mL). The reaction was stirred overnight at room
termperature. The pH of the reaction mixture was adjusted
to above 9, and then the reaction mixture was extracted with
CHCl3 (3 × 100 mL). The organic extracts were dried over
NaCl/Na2SO4 and then evaporated. To a solution of the
resulting sulfone (0.5 g, 1.6 mmol) in DMF (10 mL) was added
Na2S(H2O)9 (0.5 g, 2 mmol). The reaction was stirred at 100
°C for 3 h, whereupon 1-iodo-3,3,3-trifluoropropane (448 mg,
2 mmol) in DMF (5 mL) was added to the reaction. After 30
min, heating was discontinued, and the reaction mixture was
stirred at room temperature for 16 h. The reaction mixture
was quenched with water (20 mL) and then extracted with
ethyl acetate (3 × 50 mL). The organic extracts were dried
1
pressure to /10 volume. The residue was suspended in Et2O
and made alkaline with 4 N NaOH, followed by extraction with
Et2O (2 × 500 mL) and evaporation under vacuum of the
combined ethereal extracts. To a solution of this residue in
DMF (500 mL) were added K2CO3 (38 g, 274 mmol) and
NaHS‚H2O (22.3 g, 301 mmol). The reaction mixture was
stirred at rooom temperature for 2 h and then cooled to 0 °C.
1-Bromobutane (41.3 g, 301mmol) was added. The reaction
mixture was stirred overnight, diluted with H2O (2700 mL),
and extracted with toluene (2 × 800 mL). The combined
toluene extracts were dried over MgSO4 and evaporated under
vacuum (81.9 g). The residue was taken up in absolute EtOH
(250 mL), D-tartaric acid (43.4 g, 290 mmol) in THF (280 mL)
was added. The mixture was heated at 50 °C until a
homogeneous solution was obtained. The mixture was slowly
cooled to 5 °C over 2-3 h. The precipitated crystals were
filtered, washed with THF (25 mL), and dried. The crude
material was recrystallized from a 1:1 mixture of EtOH and
THF giving 42.1 g of 26 as the (+)-tartrate salt: mp 106-108
°C; [R]D free base ) +14.94° (c ) 4.09, MeOH). Anal. for
tartrate salt (C17H27N3O6S2) C, H, N.
(+)-exo-6-[4-(Bu t ylt h io)-1,2,5-t h ia d ia zol-3-yl]-1-a za -
bicyclo[3.2.1]octan e Oxalate (29). A solution of 1-azabicyclo-
[3.2.1]octane-6-one, 1329 (41.25 g, 0.33 mol), acetic acid (2 mL),

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Muscarinic Analgesics for Treatment Of IBS
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4 545
over NaCl/Na2SO4 then evaporated. The resulting oil was
purified by flash chromatography over silica gel (96.7% CHCl3,
3% EtOH, 0.3% NH4OH) to yield 32 (210 mg, 0.65 mmol).
Crystallization with oxalic acid in acetone gave a salt with mp
93-96 °C. Anal. (C14H18N3O4S2F3) C, H, N.
(+)-exo-6-[4-[4,4,4-Tr iflu or obu tyl)th io]-1,2,5-th ia d ia zol-
3-yl]-1-a za bicyclo[3.2.1]octa n e Oxa la te (33). The same
procedure as for compound 29 was used, but with 1-bromo-
4,4,4-trifluorobutane as the alkyl bromide: mp 101-105 °C.
Anal. as the oxalate salt (C15H20N3O4S2F3) C, H, N.
(5S,6S)-exo-6-[4-[(5,5,5-Tr iflu or op en tyl)th io]-1,2,5-th ia -
d ia zol-3-yl]-1-a za bicyclo[3.2.1]octa n e Oxa la te (34). The
same procedure as for compound 32 was used, but with
1-bromo-5,5,5-trifluoropentane as the alkyl halide: mp 125-
127 °C. Anal. as the oxalate salt (C16H22N3O4S2F3) C, H, N.
(5R,6R)-exo-6-[4-[(4,4,4-Tr iflu or obu tyl)th io]-1,2,5-th ia -
d ia zol-3-yl]-1-a za bicyclo[3.2.1]oct a n e Oxa la t e (35) a n d
(5R,6S)-en d o-6-[4-[(4,4,4-Tr iflu or ob u t yl)t h io]-1,2,5-t h ia -
d ia zol-3-yl]-1-a za bicyclo[3.2.1]octa n e Oxa la te (37). To a
solution of (+)-1-azabicyclo[3.2.1]octan-6-one29 (124 g, 1000
mmol) in EtOH (100 mL) was added a solution of (-)-
camphorsulfonic acid (232 g, 1000 mmol) in EtOH (200 mL).
The mixture was heated to 70 °C and slowly cooled over 2 h
to 5 °C. The precipitated crystals were collected by filtration
and washed with cold EtOH (3 × 40 mL). The crude compound
was crystallized from EtOH (150 mL) (57.3 g). The resolved
material was then converted to 35 and 37 by the same
procedure as for 29, using 1-bromo-4,4,4-trifluorobutane as the
alkyl halide. Crystallization with oxalic acid in acetone of the
exo and endo fractions gave [5R-(exo)]-6-[4-[(4,4,4-Trifluoro-
butyl)thio]-1,2,5-thiadiazol-3-yl]-1-azabicyclo[3.2.1]octane
oxalate (35) (mp 153-154 °C) and [5R-(endo)]-6-[4-[(4,4,4-
trifluorobutyl)thio]-1,2,5-thiadiazol-3-yl]-1-azabicyclo[3.2.1]-
octane oxalate (37) (mp 70 °C). Anal. for 35 (C15H20F3N3O4S2)
C, H, N. Anal. for 37 (C15H20F3N3O4S2) C, H, N.
intraluminal manometry catheters into the jejunum for motil-
ity measurements. Transmural potential difference was de-
termined using calomel half-cells connected by salt bridges;
one salt bridge was inserted into the lumenal fluid of the
jejunum and the other via a wick electrode into the serosal
cavity fluid. Each animal served as its own control for
assessment of drug effect.
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(5S,6S)-exo-6-[4-[(4,4,4-Tr iflu or obu tyl)th io]-1,2,5-th ia -
d ia zol-3-yl]-1-a za bicyclo[3.2.1]octa n e Oxa la te (36). The
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for 29, using 1-bromo-4,4,4-trifluorobutane as the alkyl halide.
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-
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