Identification and characterization of m1 selective muscarinic receptor antagonists1

Article abstract of DOI:10.1021/jm980067l

A series of esters of 1,4-disubstituted tetrahydropyridine carboxylic acids (I) has been synthesized and characterized as potential ml selective muscarinic receptor antagonists. The affinity of these compounds for the five human muscarinic receptor subtyp

Full text of DOI:10.1021/jm980067l

356
J . Med. Chem. 1999, 42, 356-363
Id en tifica tion a n d Ch a r a cter iza tion of m 1 Selective Mu sca r in ic Recep tor
An ta gon ists1
Corinne E. Augelli-Szafran,*,† C. J ohn Blankley,† J uan C. J aen,† David W. Moreland,† Carrie B. Nelson,‡
J an R. Penvose-Yi,† Roy D. Schwarz,‡ and Anthony J . Thomas†
Departments of Medicinal Chemistry and Neuroscience Therapeutics, Parke-Davis Pharmaceutical Research,
Division of Warner-Lambert Company, 2800 Plymouth Road, Ann Arbor, Michigan 48105
Received J anuary 28, 1998
A series of esters of 1,4-disubstituted tetrahydropyridine carboxylic acids (I) has been
synthesized and characterized as potential m1 selective muscarinic receptor antagonists. The
affinity of these compounds for the five human muscarinic receptor subtypes (Hm1-Hm5) was
determined by the displacement of [3H]-NMS binding using membranes from transfected
Chinese hamster ovarian cells. One of the most potent and selective compounds of this series
is an analogue of I [11, R1 ) (CH2)5CH3], which has an IC50 value of 27.3 nM at the m1 receptor
and possesses 100-fold (m2), 48-fold (m3), 74-fold (m4), and 19-fold (m5) selectivities at the
other receptors. Thus, this analogue appears to be more selective on the basis of binding than
the prototypical m1 antagonist, pirenzepine. Functional data, such as the inhibition of carbachol-
stimulated phosphatidylinositol hydrolysis, on selected analogues confirmed the muscarinic
antagonistic properties of this chemical series.
In tr od u ction
responsible for the side effects, e.g., sweating, diarrhea,
cramps, excessive salivation, of the currently available
muscarinic agents. Thus, m1 selective muscarinic recep-
tor agonists hold the best promise for selective improve-
ment of cognitive function without the occurrence of side
effects. Therefore, the development of a truly potent and
m1 selective muscarinic receptor antagonist may be the
optimal tool needed for the development of such a
therapeutic agent.
From this series of tetrahydropyridines, several com-
pounds have been identified as m1 selective muscarinic
receptor antagonists and some appear more selective
than pirenzepine, a known m1 selective muscarinic
receptor antagonist. This structure-activity relation-
ship (SAR) study examines the ester functionality of I.
Muscarinic receptors are widely distributed both
centrally and peripherally. These receptors play an
important role in cognitive function, in the central
control of movement, in the peripheral control of gas-
trointestinal functions, and in bronchodilation. Three
muscarinic receptor subtypes (M1, M2, and M3) were
initially characterized pharmacologically by the use of
subtype selective antagonists.2 More recently, five dis-
tinct muscarinic receptor subtypes (m1-m5) have been
characterized at the molecular level.3 Therefore, it is
now possible to specifically target the blockade of one
muscarinic receptor subtype. The advantage of a selec-
tive antagonist for one muscarinic receptor would be the
elimination of the potential side effects that are usually
present with nonselective antagonists. Possible thera-
peutic targets for selective muscarinic antagonists
include an m1 antagonist for the inhibition of gastric
acid secretion in the treatment of peptic ulcers,4 an m2
antagonist as an antibradycardiac drug,5 an m3 agent
as a bronchodilator for pulminary obstructive disorder
and asthma,6 an m2 and/or m3 antagonist for urinary
incontinence,7 and an m4 antagonist to combat Parkin-
sonian movement disorders.8 Also, an m1 antagonist
could be used as a pharmacological tool and aid in the
development of selective m1 agonists which, in turn,
may be useful in the treatment of Alzheimer’s disease.
Alzheimer’s disease is characterized by the progressive
degeneration of cholinergic neurons that project from
the basal forebrain to the cerebral cortex and hippoc-
ampus.9 Cholinomimetic therapy is currently viewed as
the most promising short-term symptomatic treatment
for the disease. The receptors responsible for the central
cognition-enhancing effects of muscarinic cholinomi-
metic agents are generally defined as m1. The activation
of the peripheral m2 and m3 receptors is thought to be
Based on chemical leads from mass screening, it ap-
peared that both the 4-phenyl and the N-ethyl groups
of I were necessary for m1 muscarinic receptor potency
and selectivity. Therefore, this study focuses on varying
R1.
Ch em istr y
All examples shown in Table 1 were prepared by the
synthetic route illustrated in Scheme 1. Treatment of
the protected nicotinic acid derivative, 3-(4,5-dihydro-
4,4-dimethyl-2-oxazolyl)pyridine10 (II), with phenyl-
lithium yielded the 1,4-dihydrophenylpyridine interme-
diate III.11 Aromatization of III in the presence of sulfur
yielded pyridine IV.11 Hydrolysis of the oxazoline ring
of IV with hydrochloric acid gave carboxylic acid V.11
† Department of Medicinal Chemistry.
‡ Department of Neuroscience Therapeutics.
10.1021/jm980067l CCC: $18.00 © 1999 American Chemical Society
Published on Web 01/26/1999

m1 Selective Muscarinic Antagonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3 357
Ta ble 1. Binding Results of 1,4-Disubstituted Tetrahydropyridines
IC50 (nM)b
example
R1
Ia
Hm1
28.2
Hm2
Hm3
Hm4
Hm5
formulac
mp (°C)
pirenzepine
1
968.2
955.6
246.8
340.6 C19H21N5O2
CH3
CH3
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
33117.6 28264.5 88401.3 13042.1 16901.9 C15 19
H
NO2‚C2H2O4
151-154
159-162
162-166
130-135
136-142
135-139
125-129
131-133
130-132
110-112
122-124
59-61
126-128
98-100
f
2
2308.4 1727.9 5523.7 13691.8
2220.1 2763.7 6874.7 10383.0 4169.5 C16 21
888.1 394.8 1689.3 3226.4 1331.2 C16 21
291.2 1668.5 2312.2 3788.8 1127.9 C17H23
278.0
76.8
977.3 C15 19
H
NO2‚C2H2O4
3
CH2CH3
CH2CH3
H
H
NO2‚C2H2O4‚0.15H2O
NO2‚C2H2O4‚0.15H2O
NO2‚C2H2O4‚1.1C2H2O4
H NO2‚C2H2O4d
4
5
(CH2)2CH3
(CH2)2CH3
(CH2)3CH3
(CH2)3CH3
(CH2)4CH3
(CH2)4CH3
(CH2)5CH3
(CH2)5CH3
(CH2)6CH3
(CH2)6CH3
(CH2)7CH3
(CH2)7CH3
(CH2)8CH3
(CH2)8CH3
(CH2)9CH3
(CH2)9CH3
CH2CH(CH3)2
CH2CH(CH3)2
Ph
6
542.3
470.7
684.0 1409.3
602.9 C17 23
264.7 C18 25
254.9 C18 25
374.0 C19 27
341.9 C19 27
508.2 C20 29
138.9 C20 29
537.3 C21 31
194.2 C21 31
400.6 C22 33
57.9 C22 33
328.6 C23 35
85.4 C23 35
7
290.0
535.5
918.0
356.9
656.3
524.0
701.0
432.4
H
NO2‚C2H2O4e
8
67.9 1865.1
84.8 1500.6
63.7
H
NO2‚C2H2O4
9
H
NO2‚C2H2O4‚0.25H2O
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53v
600.7
H NO2‚C2H2O4
27.3 2734.2 1308.0 2030.0
H
NO2‚C2H2O4‚0.76H2O
35.0
161.4
78.0
77.3
22.0
30.4
11.5
484.2 1174.8
106.1 20.5
128.0 1248.2
120.5 427.5
227.0
349.0
367.0
H NO2‚C2H2O4
748.5 1095.5 2196.7
H
NO2‚C2H2O4‚0.25H2O
NO2‚C2H2O4‚0.45H2O
NO2‚C2H2O4‚1.1C2H2O4
NO2‚C2H2O4‚1.8C2H2O4
NO2‚0.5EtOAcg
73.3
773.5
52.5
162.5
675.6 1250.0
86.5 258.8
617.1
H
H
H
f
486.1 1600.8 1577.9
22.2
H
h
201.6
282.3
H
NO2‚0.25EtOAcg
h
983.0 2123.9 1114.8 C24 37
H
NO2‚0.75EtOAcg
h
399.5
844.6
257.5
566.2
896.9
742.6
235.6 C24 37
558.9 C18 25
407.3 C18 25
H
NO2‚0.31EtOAcg
h
H
NO2‚C2H2O4‚1.50H2O‚0.02Et2O
f
H
NO2‚C2H2O4‚0.35H2O‚0.01Et2O 140-142
i
19027.3 14598.2 16753.5 31125.4 31057.6
C
20H21NO2‚C2H2O4j
127-130
185-188
177.5-179
123-124
95-98
Ph
1819.5
13.4
65.4
954.3 4069.7 8428.6 1568.6 C20H21NO2‚C2H2O4
CH2Ph
241.3
450.6
403.6
211.3
146.3
472.8
42.5 C21 23
272.4 C21 23
330.9 C22 25
78.1 C22 25
190.3 C23 27
24.8 C23 27
499.7 C23 27
865.9 C23 27
871.1 4162.1 3565.8 C28 29
2829.7 2573.4 5051.5 7708.4 3108.6 C28 29
59.2 2213.8 1244.3 1132.1 255.8 C23 27
122.0 1035.2 749.3 1160.8 479.4 C23 27
743.1 3323.1 14309.6 7422.0 5000.0 C24 29
69.4 2162.1 1842.0 2775.5 842.0 C24 29
661.1 2046.8 2388.2 1567.9 C25 31
H
NO2‚HClk,l
CH2Ph
H
NO2‚HClk
(CH2)2Ph
(CH2)2Ph
233.6 1480.7 1653.8 1740.6
H
NO2‚C2H2O4‚0.3H2O
NO2‚C2H2O4
9.6
93.5
8.8
243.6
956.4 1491.0 1756.1
136.0 88.3 138.0
734.6 1218.7 2224.5
100.7
69.7
H
134-135.5
100-103
141-143
75-77
(CH2)2Ph-p-CH3
(CH2)2Ph-p-CH3
(CH2)2Ph-p-OCH3
(CH2)2Ph-p-OCH3
CH2CH(Ph)2
CH2CH(Ph)2
(CH2)3Ph
H
NO2‚1.1C2H2O4
NO2‚1.1C2H2O4
NO3‚1.2C2H2O4
NO3‚0.55C2H2O4
H
159.0
H
1941.0 38484.0 52495.5 42312.8
H
h
m
4024.4
475.0
H
NO2‚C2H2O4n
74-80
H
NO2‚C2H2O4
NO2‚HClk
H
NO2‚HClk
h
H
158.5-159.5
112.5-114
230 (d)
116-118
99-101
127-129
118-120
91-93
(CH2)3Ph
o
(CH2)4Ph
H
H
NO2‚C2H2O4
(CH2)4Ph
NO2‚C2H2O4‚0.9H2O
(CH2)5Ph
670.0
316.7
16.9
18.1
54.2
14.5
H NO2‚C2H2O4
(CH2)5Ph
120.7
273.5
255.2
666.3
564.9
447.9
299.3
40.8
109.6
155.8
529.6
114.7
951.2
108.7
62.0
167.6
142.7
836.1
134.7
472.2 C25 31
63.0 C20 27
54.1 C20 27
214.5 C21 29
115.4 C21 29
159.2 C24 33
56.2 C24 33
H
NO2‚C2H2O4‚0.25H2O
NO2‚C2H2O4‚0.25H2O
NO2‚C2H2O4‚0.25H2O
NO2‚C2H2O4‚1.2C2H2O4
C6H11
C6H11
H
H
CH2C6H11
CH2C6H11
(CH2)2C6H11
(CH2)2C6H11
(CH2)3C6H11
(CH2)3C6H11
(CH2)2Ph
H
110-112
94-97
p
H
NO2‚1.6C2H2O4
105.2 1944.1
38.7
H
NO2‚C2H2O4‚0.15H2O
NO2‚C2H2O4‚0.15H2O
NO2‚C2H2O4‚0.25H2O
NO2‚C2H2O4‚0.26H2O
138-142
93-98
238.2
H
963.2 10943.7 24861.7 19581.2 10969.8 C23 33
H
85-88
610.6
62.4
468.0 2801.3 1167.2 1260.5 C23 33
357.1
H
139-141
112-114
78-81
Cq (trans)
Cq (cis)
315.4
145.4
96.8 C22 27
H
NO2‚C2H2O4s
r
(CH2)2Ph
3285.2 4496.8 3175.4 8122.8 4264.0 C22 27
H
NO2‚C2H2O4t
NO2‚C2H2O4u
NO2‚C2H2O4w
H NO2‚C2H2O4
C6H11
C6H
Cq (trans)
Cq (cis)
913.1 4100.3 4514.2 7900.0 2971.4 C20 29
1141.1 1611.2 2551.1 4246.3 1889.3 C20 29
61.4 405.0 220.3 572.7 222.1 C20 31
H
f
H
116-118
11
(CH )5CH3
Cq (cis)
h
2
a
Letters A, B, and C indicate the location of the double bond (i.e., IA: 1,2,3,6-tetrahydropyridine analogue, IB: 1,2,5,6-tetrahydropyridine
b
analogue; and IC: piperidine analogue). The affinity of these compounds for the five human receptor subtypes (m1-m5) was determined
by [3H]-NMS binding using membranes from transfected CHO cells. For compounds tested more than once, the value is given as the
geometric mean. The standard error of the mean (SEM) for all compounds tested ranged from 6-18%. Complete protocol is described by
Dorje et al.3a and Buckley et al.14 c All compounds include 1 mol of oxalic acid and have analytical results within (0.4% of theoretical
values unless otherwise noted. Some difficulty was found in obtaining combustion analysis in the indicated compounds due to the propensity
of these compounds to retain solvents. High mass calcd, 288.1964; found, 288.1953. e High mass calcd, 288.1964; found, 288.1961. f A
d
gum. g Free base. A liquid. High mass calcd, 308.1651; found, 308.1657. j High mass calcd, 308.1651; found, 308.1649. HCl salt. High
h
i
k
l
m
n
mass calcd, 322.1807; found, 322.1793. High mass calcd, 412.2276; found, 412.2277. High mass calcd, 412.2277; found, 412.2282.
o High mass calcd, 364.2277; found, 364.2280. High mass calcd, 328.2277; found, 328.2273. Stereochemistry was determined by proton
p
q
r
s
t
NMR analysis. High mass calcd, 338.2120; found, 338.2116. High mass calcd, 338.2120; found, 338.2111. High mass calcd, 316.2277;
found, 316.2274. High mass calcd, 316.2277; found, 316.2274. v No trans-53 was isolated due to minimal amount of sample. High
u
w
mass calcd, 318.2433; found, 318.2433.

358 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3
Augelli-Szafran et al.
Ta ble 2. Binding Resultsaof 11A, 11B, and 11C (IC50, nM)
Sch em e 1a
example
Hm1
Hm2
Hm3
Hm4
Hm5
11A
11B
11C
30.2
21.1
40.0
1619.5
1304.6
1637.8
457.8
521.6
630.5
505.6
537.6
534.5
162.5
167.0
186.9
a
Complete protocol for determining the affinity of these com-
pounds is referenced in Table 1, footnote b.
Sch em e 2a
a
Reagents and conditions: (a) PhLi, THF, -78 °C; (b) S8, ∆,
PhCH3; (c) conc HCl, D; (d) SOCl2, DMF, D; (e) R1OH, [(CH3)2CH]2-
NEt, CH2Cl2, 0 °C to rt; (f) EtI, CH3CN, D; (g) NaBH4, MeOH/
H2O; (h) C2H2O4, Et2O.
Treatment of V with thionyl chloride, followed by
different alcohols in the presence of N,N-diisopropyl-
ethylamine, gave the corresponding pyridinyl esters
VI.11 Treatment of VI with ethyl iodide yielded quater-
nary salt VII. Reduction of VII with sodium borohydride
afforded 4-substituted tetrahydropyridines IA and IB,
which are separable by chromatography. Catalytic
hydrogenation (5% Pd/C, H2) of 1,2,5,6-tetrahydropy-
ridines (IA) yielded the corresponding piperidines 49-
53 (eq 1). A mixture of primarily cis (IC) versus trans
a
Reagents and conditions: (a) NEt3, EtOH, 25 °C, 30%; (b)
NaH, THF, 23%; (c) C2H4O4/Et2O.
Most final products were converted to the oxalate
salts by treating the free base with 1 mol of oxalic acid
in the presence of diethyl ether. The remaining com-
pounds were converted to the hydrochloric salts using
standard procedures.
The resolution of 11 was performed utilizing di-p-
toluoyltartaric acid. A 95% ethanolic solution of free
base 11 was added to a 95% ethanolic solution of the
appropriate chiral di-p-toluoyltartaric acid. Fractional
recrystallization yielded 11A, 11B, and 11C, respec-
tively (Table 2).
(1)
Synthesis of oxadiazole derivatives12 (54 and 55) of
11 is illustrated in Scheme 2. Hydroxylhexanimidamide
X13 is prepared from hydroxylamine hydrochloride and
hexanonitrile. Treatment of 1 with hydroxylhexanimi-
damide X and sodium hydride yielded oxadiazoles 54
and 55 in a ratio of 1:2. When 2 is treated with X under
the same reaction conditions, 54 and 55 were isolated
in a ratio of 1:14, favoring equilibration to 55.
(ID) piperidine analogues were isolated and separated
by chromatography (cis:trans ratio for 50 and 49, 6:1;
52 and 51, 7.5:1; 53, 8:1). Decoupling experiments were
done to determine the coupling constant between pro-
tons 3 and 4 of IA. For a selected cis analogue, the
observed coupling constant was 3.5 Hz which indicated
that the protons were axial-equatorial (cis) to each
other. Similar experiments were done for the corre-
sponding trans analogue(s).
Resu lts
The purpose of this study was to identify compounds
that have better m1 selectivity than existing muscarinic
antagonists, such as the m1 selective muscarinic recep-

m1 Selective Muscarinic Antagonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3 359
Ta ble 3. Effects of Selected Compounds of IA and IB on PI
Turnover in CHO Hm1 Cells
tor antagonist, pirenzepine. The primary biological
assay that was used to evaluate these compounds is
described by Dorje et al.3a and Buckley et al.14 The
affinity of these compounds for the five human receptor
subtypes (Hm1-Hm5)3c was determined by [3H]-NMS
binding using membranes from transfected Chinese
hamster ovarian (CHO) cells. Activity is expressed as
the micromolar concentration of compound required to
displace [3H]-NMS by 50% (IC50). IC50 values are
calculated using a logit equation.
example
% basal controla
1
2
108
89
11
12
25
26
28
42
98
109
79
99
90
108
a
100 µM of each compound was used. Test run in triplicate.
In this discussion, the IC50 values of Hm1 versus Hm2
are routinely compared. Since there are many side
effects mediated by the m2 receptors, it is desirable to
eliminate affinity at the m2 receptors. This m1 versus
m2 receptor selectivity would add to the attractiveness
of a potent m1 antagonist. In addition to the selectivity
observed for the m1 receptor versus the m2 receptor for
this series of compounds, some of these compounds also
exhibited selectivity for the m1 receptor versus the m3,
m4, and m5 receptors as illustrated in Table 1. From
the first group of compounds in Table 1 (1-22), which
contains various alkyl chains, 11 [R1 ) (CH2)5CH3;
1,2,3,6-tetrahydropyridine analogue] was identified as
one of the most m1 selective muscarinic receptor
antagonists known to date. Both 11 and pirenzepine are
about as equally potent at the m1 receptor. However,
11 is at least 3 times more selective for m1 versus m2
and 8 times more selective for m1 versus m4 compared
to pirenzepine. This is truly an advantage over this
prototypical m1 antagonist. Receptor selectivities at the
m3 and m5 muscarinic receptors are about equal for 11
and pirenzipine. This pronounced selectivity of m1
versus m2 and m4 for 11 was not observed for any other
example shown in Table 1. As the chain length was
shortened (1-10), potency and selectivity were de-
creased. However, when the chain length was length-
ened, (12-20), potency was maintained at the m1
receptor except for C7 (13, IC50 ) 161.4 nM), C8 (15, IC50
) 77.3 nM), and C10 (19, IC50 ) 484.2 nM) derivatives.
Selectivity versus the m2 receptor was either reduced
or lost for all of these compounds when compared to 11.
For many of these examples (1-22), the 1,2,3,6-tetrahy-
dropyridines (IA) are more selective for m1 than m2
than their corresponding 1,2,5,6-analogues (IB) (i.e., 11
versus 12, respectively) but are similar in their m1
affinity.
Ta ble 4. Inhibition of Carbachol Stimulation of PI Turnover in
CHO Hm1 Cells by Selected Compounds of IA and IB
example
% inhibitiona
1
2
0
40
11
12
26
28
42
100
99
100
89
97
a
100 µM concentration, triplicate tubes.
To examine the effect of the double bond of the
tetrahydropyridines on potency and selectivity, pip-
eridines 49-53 were synthesized. Catalytic reduction
of 27, 41, and 11 yielded 49 and 50, 51 and 52, and 53,
respectively. When comparing the affinity and the
selectivity for the m1 receptor of these reduced com-
pounds to the m1 receptor affinity and selectivity of the
corresponding 1,2,3,6- and 1,2,5,6-tetrahydropyridines,
the tetrahydropyridines appear to be the more potent
and, generally, more m1 selective.
The functional activity of a selected group of com-
pounds was determined.15 It is known that the m1, m3,
and m5 muscarinic receptors are preferentially coupled
to the stimulation of phosphoinositide metabolism via
phospholipase C activation whereas the m2 and m4
muscarinic receptors are negatively coupled to adenylate
cyclase.16 Therefore, compounds then can be classified
as receptor agonists or antagonists based on the com-
pounds’ affinities for particular receptor subtypes. The
data illustrated in Tables 3 and 4 indicate that none of
the selected compounds produce any significant stimu-
lation of phosphatidylinositol (PI) hydrolysis at high
concentration (100 µM) in CHO Hm1 cells. These results
indicate that none of the tested compounds are as
efficacious as muscarinic agonists (Table 3). However,
the blockade of carbachol-stimulated PI turnover pro-
duced by selected compounds in CHO Hm1 cells (Table
4) is convincing evidence that the preferred compounds
are indeed muscarinic antagonists. For example, the
weak binding (Table 4) of 1 and 2 (i.e., methyl esters)
correlates well with their weak inhibition of carbachol’s
effects. However, 11, 12, 26, 28, and 42 are potent
antagonists and inhibit the effects of the muscarinic
agonist, carbachol (89-100%).
To further study the structure-activity relationship
of the ester functionality of these tetrahyropyridines,
various benzyl esters were synthesized (23-40). The
most m1 selective muscarinic receptor analogue from
this group of compounds was 35 [R1 ) (CH2)3Ph, Hm1,
IC50 ) 59.2 nM, versus Hm2, IC50 ) 2213.8 nM]. Longer
alkyl tethers [37 (R1 ) (CH2)4Ph) and 39 (R1 ) (CH2)5-
Ph] exhibited a decrease in potency and selectivity.
However, by shortening the alkyl tether (23-28), ad-
ditional potent and m1 selective antagonists ([25, R1 )
CH2Ph, Hm1, IC50 ) 13.4 nM, Hm2, IC50 ) 241.3 nM]
and [28, R1 ) (CH2)2Ph, Hm1, IC50 ) 9.6 nM, Hm2, IC50
) 243.6 nM]) were identified. However, selectivity for
the m1 receptor versus the m2 receptor was decreased
at least 4-fold when compared to 11. Cyclohexyl esters
(41, 42, 44, and 46) were quite potent at the m1 receptor
(IC50 ) 16.9, 18.1, 14.5, 38.7 nM, respectively). However,
none were as selective as 11.
The resolution of 11 was performed with (D)- and (L)-
di-p-toluoyltartaric acid utilizing standard conditions.
Initially, 11 was treated with both the (D)- and (L)-
isomers to give 11A. This racemic salt was synthesized
to determine if the tartaric acid had any effect on the
muscarinic binding results versus free base 11. In
addition, 11 was treated separately with the (D)- and
(L)-tartaric acids to yield enantiomers 11B and 11C.

360 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3
Augelli-Szafran et al.
Ta ble 5. Binding Resultsa of Oxadiazole Analogues (54 and
55) (IC50, nM)
Benzyl ester analogues (23-40) did not improve the
selectivity of 11. For this group of compounds, the
optimal distance between the oxygen atom and the
phenyl ring apparently necessary to maintain reason-
able affinity and selectivity for the m1 receptor is three
carbon atoms (35). To study the effect of a benzyl ester
versus a cyclohexyl ester, 41-48 were examined. Many
of these derivatives (41, 42, 44, 46) were potent m1
muscarinic antagonists, but none were as selective as
11.
The significance of the double bond of I was investi-
gated by studying the piperidines derivatives (49-53).
The most potent and selective piperidine was 53, the
direct analogue of 11. This compound was about 2-fold
less potent and about 16-fold less selective versus the
m2 receptor. These data may indicate that the config-
uration of the 1,2,3,6-tetrahydropyridine contributes to
the m1 receptor affinity and selectivity.
Preliminary functional studies on a select group of
compounds indicated that these compounds possess
muscarinic antagonist properties. The data in Table 3
show that none of the tested compounds produced any
significant stimulation of PI hydrolysis at a high
concentration (100 µM) in CHO Hm1 cells (98% of basal
control). Therefore, these compounds are not efficacious
as m1 selective muscarinic receptor agonists, despite
their affinity for the receptor. The larger esters such as
hexyl, benzyl, and cyclohexyl versus methyl inhibited
carbachol stimulation of PI turnover (Table 4) in CHO
Hm1 cells. These data indicate that these particular
compounds are truly muscarinic antagonists, whereas
the methyl ester is less effective. These data correlate
well with the binding data illustrated in Table 1.
example
Hm1
Hm2
Hm3
Hm4
Hm5
54
55
888.6
1087.7
10788.8
1222.1
6712.1
1927.8
6337.5
8392.3
2075.4
1467.3
a
Complete protocol for determining the affinity of these com-
pounds is referenced in Table 1, footnote b.
HPLC analysis of 11A, 11B, and 11C confirmed 11A
as a racemic mixture and, within experimental error,
confirmed 11B and 11C as the corresponding enanti-
omers. The binding results of (D,L)-(11A), (D)-(11B), and
(L)-(11C) showed no significant difference in their af-
finity for the m1 receptor versus 11. However, selectivity
for the m1 receptor versus the m2 receptor decreased
about 2-fold for each isomer (Table 2).
Muscarinic antagonists, such as scopolamine, have
been shown to alter spontaneous motor activity in
rodents.17 Additionally, scopolamine has been shown
to impair performance in a test of spatial working
memory.18 When 11 was tested in a mouse water maze,
no behavioral activity or gross side effects were observed
with 11, following peripheral administration in rats or
mice at doses up to 178 mg/kg, s.c. These preliminary
in vivo studies15 of 11 indicate that this compound may
be rapidly metabolized in rodents. However, inhibition
of spontaneous motor activity was observed with icv
(intracerebroventricular) injection in mice (30 µg/mouse,
38% inhibition), showing an effect with this compound
when delivered directly to the central nervous system.15
A possible remedy to this speculated metabolism was
to synthesize a bioisostere of the ester group of 11. The
selected bioisostere was an oxadiazole ring. Oxadiazoles
54 and 55 exhibited at least a 30-fold decrease in their
affinity for the m1 receptor versus 11 and 12, respec-
tively (Table 5). With respect to m1 versus m2 selectiv-
ity, at least an 8-fold decrease was observed.
Data obtained from the isomers of 11 (Table 4)
indicate that the racemate is as potent as the (R)- or
the (S)-isomer. Apparently, the configuration at this
chiral center is not important for affinity or selectivity
at the m1 receptor.
Preliminary in vivo studies, i.e., spontaneous motor
activity and spatial working memory, were examined
with 11. When 11 was administered icv, a slight
inhibition of spontaneous motor activity was observed.
If this compound were to get into the brain, in vivo
activity may be observed. However, since no in vivo
activity was noted in the mouse water maze study, it
can only be speculated that the ester group of 11 may
be readily metabolized. Therefore, oxadiazoles 54 and
55, bioisosteres of 11 in which the ester group is masked
by the oxadiazole ring, were synthesized and tested for
m1 affinity and selectivity. Neither compound showed
an improvement over 11, but instead, a 30-fold decrease
in affinity and an 8-fold decrease in selectivity for the
m1 receptor (Table 5) were observed.
In summary, this study has generated 11, a very
potent and m1 selective muscarinic receptor antagonist,
which could be used as a pharmacological tool to assist
in understanding muscarinic receptor subtypes. From
the analogues of I that were studied, the binding site
of the m1 receptor does seem to tolerate other R1
substituents, such as longer chain alkyl esters, benzyl
esters, and cyclohexyl esters, since these analogues
show a strong affinity for the m1 muscarinic receptor.
However, the m1 selectivity of these compounds is not
as pronounced as that of 11. Therefore, it is 11 that
Discu ssion
A structure-activity study which examines the effects
of various R1 groups of I on m1 muscarinic receptor
affinity and selectivity is reported. Approximately 50%
of analogues 1-22, which contain various alkyl chain
lengths (C1-C10), are potent m1 muscarinic receptor
antagonists (IC50 values less than 100 nM). The most
selective m1 muscarinic receptor antagonist identified
from this study is 11, which also showed high affinity
for the m1 muscarinic receptor (11, Hm1: IC50 ) 27
nM). This compound has an m1 receptor affinity similar
to that of the prototypical m1 selective muscarinic
receptor antagonist, pirenzepine (IC50 ) 28.2 nM).
However, 11 is more selective for m1 versus the m2 (3-
fold) and m4 (8-fold) receptors than pirenzepine. There-
fore, 11 could be claimed as the most m1 selective
muscarinic receptor antagonist known to date.

m1 Selective Muscarinic Antagonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3 361
mL, 1.56 equiv), and the reaction mixture was heated to reflux
for 16 h. After the mixture was cooled to room temperature,
the clear liquid was concentrated in vacuo to yield an opaque
white oil. This oil was diluted with diethyl ether and then
concentrated in vacuo two times. This acid chloride was then
diluted in dichloromethane (215 mL) and added dropwise to a
cooled (0 °C) solution of hexanol (29.30 mL, 1.1 equiv) and
diisopropylethylamine (40.65 mL, 1.1 equiv) in dichloromethane
(536 mL). The resulting dark brown solution was allowed to
gradually warm to room temperature and stir for 20 h. The
reaction mixture was then quenched with saturated NaHCO3
(500 mL, pH 8) and extracted two times with dichloromethane.
The layers were separated, and the combined organic layers
were washed with water, dried (MgSO4), filtered, and concen-
trated in vacuo to afford 64 g of crude product. Purification
by flash chromatography (silica gel, 25% EtOAc/hexane)
yielded 34.4 g (57%) of the desired product. 400 MHz NMR
(DMSO-d6): 0.80-0.84 (t, 3H, J ) 7.2 Hz); 0.96-1.01 (m, 2H);
1.07-1.22 (m, 4H); 1.31-1.37 (m, 2H); 4.02-4.06 (t, 2H, J )
6.4 Hz); 7.35-7.38 (m, 2H); 7.45-7.50 (m, 4H); 8.76-8.77 (d,
1H, J ) 5.2 Hz); 8.88 (s, 1H). MS: 284 (MH+).
Step E: P r ep a r a tion of 1-Eth yl-3-h exyloxyca r bon yl-
4-p h en ylp yr id in iu m Iod id e. 4-Phenylnicotinic acid hexyl
ester (34.4 g, 0.121 mol) was combined with ethyl iodide (58.3
mL, 6 equiv) and heated to reflux for 23 h. The reaction
mixture was then cooled to room temperature and concen-
trated in vacuo to yield 52.7 g (99%) of the desired product.
400 MHz NMR (DMSO-d6): 0.11-0.84 (t, 3H, J ) 7.2 Hz);
0.95-1.00 (m, 2H); 1.09-1.27 (m, 4H); 1.34-1.41 (m, 3H);
1.57-1.61 (t, 3H, J ) 7.2 Hz); 4.126-4.158 (t, 2H, J ) 6.4
Hz); 4.693-4.747 (q, 2H, J ) 7.2 Hz); 7.52-7.57 (m, 2H); 7.58-
7.62 (m, 2H); 8.30-8.32 (d, 1H, J ) 6.8 Hz); 9.27-9.29 (m,
1H); 9.50-9.50 (d, 1H, J ) 1.2 Hz). MS: 284 (M+-CH3CH2I).
Step F : P r ep a r a tion of 1-Eth yl-4-p h en yl-1,2,3,6-tetr a -
h ydr opyr idin e-3-car boxylic Acid Hexyl Ester Eth an edio-
a te (11) a n d 1-Eth yl-4-p h en yl-1,2,5,6-tetr a h yd r o-p yr i-
d in e-3-ca r boxylic Acid Hexyl Ester Eth a n ed ioa te (12).
To a cooled solution of 1-ethyl-3-hexyloxycarbonyl-4-phenylpy-
ridinium iodide (52.7 g, 0.12mol) in MeOH (280 mL) was added
water (280 mL), followed by the portionwise addition of NaBH4
(44.0 g, 1.16 mol, 9.7 equiv). This bright orange reaction
mixture was stirred at 0 °C for 1.5 h. The reaction was then
slowly quenched with concentrated HCl (150 mL, pH 1), and
the mixture was stirred at room temperature for 10 min and
neutralized with NH4OH (150 mL, pH 10-11). The mixture
was extracted 3 times with EtOAc, and the combined organic
layers were dried (MgSO4), filtered, and concentrated in vacuo
to afford 33.48 g of crude product, an orange oil with solids.
Purification by flash chromatography (silica gel, 25% EtOAc/
hexane, 2 times) yielded the free bases of 11 (2.83 g, 8%) and
12 (2.51 g, 7%).
possesses m1 affinity and the best m1 selectivity. Even
though 11 is not the most potent compound from this
series, it is indeed the most m1 selective. Detailed
pharmacological studies of 11 will be the topic of future
communications from these laboratories.
Exp er im en ta l Section
High-field nuclear magnetic resonance (NMR) spectra were
recorded in deuteriochloroform (CDCl3) or deuteriomethyl
sulfoxide (DMSO-d6) as solvent on a Varian Unity 400 MHz
spectrometer. All chemical shifts are reported in ppm down-
field from internal tetramethysilane. Infrared spectra were
determined on a Mattson Galaxy FT-IR spectrophotometer.
Elemental analysis for carbon, hydrogen, and nitrogen were
determined on a Control Equipment Corporation CEC440
elemental analyzer and are within 0.4% of theory unless noted
otherwise. Mass spectra were obtained by using a VG Masslab
Trio-2A, Finnigan TSQ-70, or VG Analytical 7070E/HF mass
spectrometer. Melting points were determined on a Thomas-
Hoover melting point apparatus and are uncorrected. All
chemicals and reagents used were of commercial purity unless
otherwise specified.
1-Eth yl-4-p h en yl-1,2,3,6-tetr a h yd r op yr id in e-3-ca r box-
ylic Acid Hexyl Ester Eth a n ed ioa te (11) a n d 1-Eth yl-4-
ph en yl-1,2,5,6-tetr ah ydr opyr idin e-3-car boxylic Acid Hex-
yl Ester Eth a n ed ioa te (12). Step A: P r ep a r a tion of 3-
(4,5-Dih yd r o-4,4-d im eth yl-2-oxa zolyl)-1,4-d ih yd r o-4-p h e-
n ylp yr id in e. To a cooled (-78 °C) solution of 3-(4,5-dihydro-
4,4-dimethyl-2-oxazolylpyridine10 (140.0 g, 0.79 mol) in 500 mL
of dry THF was added phenyllithium (1.42 mol, 1.8 equiv)
under a nitrogen atmosphere until GC analysis indicated no
more starting material present. The reaction mixture was
stirred for 10 min and then quenched with 500 mL water at
-78 °C. After the reaction mixture was warmed to room
temperature with a water bath, 700 mL of ether was added to
the mixture. The resulting solid was filtered, rinsed one time
with chilled methanol, and then dried at 40 °C in a vacuum
oven overnight to yield 166.6 g (0.65 mol, 82%) of the desired
product. 400 MHz NMR (DMSO-d6): 0.96 (s, 3H); 1.10 (s, 3H);
3.74 (s, 2H); 4.42-4.44 (d, 1H, J ) 4.8 Hz); 4.63-4.67 (m, 1H);
6.14-6.17 (m, 1H); 6.94-6.95 (d, 1H, J ) 5.2 Hz); 7.06-7.11
(m, 1H); 7.19-7.24 (m, 4H); 7.98-8.00 (m, 1H, NH). MS: 255
(MH+), 254 (M+).
Step B: P r ep a r a tion of 3-(4,4-Dim eth yl-4,5-d ih yd r oox-
a zol-2-yl)-4-p h en yl-p yr id in e. 3-(4,5-Dihydro-4,4-dimethyl-
2-oxazolyl)-1,4-dihydro-4-phenylpyridine (80.6 g, 0.31 mol) and
sulfur (10.6 g, 0.33 mol, 1.04 equiv) were heated to reflux in
750 mL of toluene for 3.5 h. GC analysis then indicated that
the reaction was complete. The mixture was cooled to room
temperature and filtered. The mother liquor was concentrated
in vacuo and then kept under vacuum overnight at room
temperature. The crude material was distilled (bp 163-166
°C at 2.8-3.2 Torr) to yield 65.3 g (0.25 mol, 81%) of the
desired product. 400 MHz NMR (DMSO-d6): 1.21 (s, 6H); 3.85
(s, 2H); 7.41-7.50 (m, 6H); 8.71-8.72 (d, 1H, J ) 5.2 Hz); 8.78
(s, 1H). MS: 253 (MH+), 252 (M+).
1-Ethyl-4-phenyl-1,2,3,6-tetrahydropyridine-3-carboxylic Acid
Hexyl Ester (11, free base). 400 MHz NMR (DMSO-d6): 0.81-
0.84 (t, 3H, J ) 7.0 Hz); 1.00-1.03 (t, 3H, J ) 7.0 Hz); 1.08-
1.23 (m, 6H); 1.35-1.39 (m, 2H); 2.36-2.46 (m, 3H); 2.53-
2.57 (m, 1H); 2.86-2.92 (m, 1H); 3.10-3.14 (m, 1H); 3.27-
3.32 (m, 1H); 3.79-3.86 (m, 2H); 3.93-3.99 (m, 1H); 6.18-
6.19 (m, 1H); 7.19-7.29 (m, 1H); 7.30-7.35 (m, 2H); 7.37-
7.37 (m, 2H). MS: 316 (MH+). HPLC conditions: Diacel
ChiralPak OD, 0.46 × 250 × 10 µm 200 IPA:700 hexane: 0.1%
Step C: P r ep a r a tion of 4-P h en yl-3-p yr id in e Ca r boxy-
lic Acid Mon oh ydr och lor ide. 3-(4,4-Dimethyl-4,5-dihydroox-
azol-2-yl)-4-phenylpyridine (108.0 g, 0.42 mol) and 220 mL of
concentrated HCl were heated to reflux for 16 h. The mixture
was then cooled to room temperature, filtered, and rinsed with
300 mL of 1 N HCl. The resulting white solid was dried in a
vacuum oven at 40 °C for 6 h to give 95.0 g (0.40 mol, 96%) of
the desired product. 400 MHz NMR (DMSO-d6): 7.47-7.55
(m, 5H); 7.79-7.81 (d, 1H, J ) 5.6 Hz); 8.89-8.90 (d, 1H, J )
5.6 Hz); 9.06 (s, 1H). MS: 200 (MH+), 199 (M+).
DEA; 1.0 mL/min; 20 min. A 1:1 ratio of enantiomers; RT
5.79 and 6.63.
)
1-Ethyl-4-phenyl-1,2,5,6-tetrahydropyridine-3-carboxylic Acid
Hexyl Ester (12, free base). 400 MHz NMR (DMSO-d6): 0.80-
0.88 (m, 3H); 0.90-0.99 (m, 2H); 1.01-1.11 (m, 6H); 1.13-
1.27 (m, 4H); 2.46-2.50 (m, 3H); 2.57-2.60 (m, 2H); 3.33 (s,
2H); 3.74-3.78 (t, 2H, J ) 6.4 Hz); 7.12,-7.14 (m, 2H); 7.26-
7.327(m, 3H). MS: 316 (MH+).
To a solution of each of the free bases of 11 and 12 in diethyl
ether was added 1 equiv of oxalic acid in diethyl ether. Solid
immediately precipitated out of the mixture which was allowed
to stir at room temperature for 16 h. The resulting white solids
were filtered and dried at room temperature to give the oxalate
salts of the corresponding products.
Step D: P r ep a r a tion of 4-P h en yln icotin ic Acid Hexyl
Ester . To a suspension of 4-phenyl-3-pyridinecarboxylic acid
monohydrochloride (50.0 g, 0.212 mol) in thionyl chloride (499
mL, 32.2 equiv) was slowly added dimethylformamide (25.72

362 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3
Augelli-Szafran et al.
1-Ethyl-4-phenyl-1,2,3,6-tetrahydropyridine-3-carboxylic Acid
Hexyl Ester, Ethanedioate (11). Anal. (C20H29NO2‚C2H2O4‚0.76
H2O): C, H, N. 1-Ethyl-4-phenyl-1,2,3,6-tetrahydro-pyridine-
7.05-7.38 (m, 10H). mp: 112-144 °C. High mass calcd,
338.2120; found, 338.2116.
(cis)-1-Ethyl-4-phenylpiperidine-3-carboxylic Acid Phenethyl
Ester Ethanedioate (1:2) (50). 400 MHz NMR (DMSO-d6):
1.18-1.21 (t, 3H, J ) 7.0 Hz); 2.05-2.08 (m, 1H); 2.27-2.29
(m, 1H); 2.49-2.60 (m, 2H); 3.06-3.39 (m, 7H); 3.56-3.61 (m,
1H); 3.95-4.06 (m, 2H); 7.04-7.34 (m, 10H). mp 78-81 °C.
High mass calcd, 338.2120; found, 338.2111.
3-carboxylic Acid Hexyl Ester, Ethanedioate (12). Anal. (C20H29
NO2‚C2H2O4): C, H, N.
-
Hexyl (()-4-P h en yl-1-eth yl-1,2,3,6-tetr a h yd r o-3-p yr id i-
n eca r boxyla te, (()-2,3-Bis[(4-m eth ylben zoyl)oxy]bu ta n e-
d ioic (1:1) (Sa lt) (11A). To a solution of the free base of 11
(200 mg, 0.634 mmol) in 10 mL 95% ethanol was added (D)-
and (L)-di-p-toluoyl-tartaric acid (0.5 equiv of each) in 1 mL
95% ethanol. The white precipitated solid (11A) was filtered
and dried at room temperature. Anal. (C20H29NO2‚C20H18O8‚
0.5 H2O): C, H, N. HPLC conditions: Diacel ChiralPak OD,
0.46 × 250 × 10 µm; 200 IPA:700 hexane:0.1% DEA; 1.0 mL/
min; 20 min. A 1:1 ratio of enantiomers; RT ) 5.79 and 6.62.
(R) or (S) Hexyl-4-p h en yl-1-eth yl-1,2,3,6-tetr a h yd r o-3-
p yr id in eca r boxyla te, [R-(R*,R*)]-2,3-Bis[(4-m eth ylben -
zoyl)oxy]bu ta n ed ioic (1:1) (Sa lt) (11B). The same proce-
dure as that described for 11A was followed, using only (L)-
di-p-toluoyltartaric acid and recrystallized two times. Anal.
(C20H29NO2‚C20H18O8‚0.5H2O): C, H, N. HPLC conditions:
Diacel ChiralPak OD, 0.46 × 250 × 10 µm; 200 IPA:700
hexane:0.1% DEA; 1.0 mL/min; 20 min. RT ) 5.92.
1-E t h yl-3-(3-p e n t yl-[1,2,4]oxa d ia zol-5-yl)-4-p h e n yl-
1,2,3,6-tetr a h yd r op yr id in e Eth a n ed ioa te (1:1) (54) a n d
1-Eth yl-5-(3-p en tyl-[1,2,4]oxa d ia zol-5-yl)-4-p h en yl-1,2,3,6-
tetr a h yd r op yr id in e Eth a n ed ioa te (1:1) (55). Step A: P r e-
p a r a tion of N′-Hyd r oxyh exa n im id a m id e. To a solution of
hydroxylamine hydrochloride (20.0 g, 0.287 mol) and triethy-
lamine (29.04 g, 0.287 mol) in methanol (120 mL) was added
dropwise hexanonitrile (27.96 g, 0.287 mol) in 30 mL methanol.
The reaction mixture was stirred at room temperature for 16
h. The mixture was then concentrated in vacuo, and the
residue was triterated with 250 mL of CHCl3. The white solid
was filtered, and the filtrate was concentrated in vacuo and
triterated with diethyl ether. This white precipitate was
filtered, and the filtrate was concentrated in vacuo to yield
crude product. Purification by chromatography (silica gel, 5%
MeOH/CH2Cl2) yielded N′-hydroxyhexanimidamide (11.0 g
30%). 400 MHz NMR (DMSO-d6): 0.84-0.87 (t, 3H, J ) 7.2
Hz); 1.19-1.32 (m, 4H); 1.43-1.58 (m; 2H); 1.92-1.96 and
2.13-2.19 (m, 2H); 5.37-5.74 (br.s, 1H); 10.3 (br.s, 1H). MS:
131 (MH+); 130 (M+).
(R) or (S) Hexyl-4-p h en yl-1-eth yl-1,2,3,6-tetr a h yd r o-3-
p yr id in eca r boxyla te, [S-(R*,R*)]-2,3-Bis[(4-m eth ylben -
zoyl)oxy]bu ta n ed ioic (1:1) (Sa lt)(11C). The same procedure
as that described for 11A was followed, using only (D)-di-p-
toluoyltartaric acid and recrystallized two times. Anal. (C20H29
-
NO2‚C20H18O8‚H2O): C, H, N. HPLC conditions: Diacel Chiral-
Pak OD, 0.46 × 250 × 10 µm; 200 IPA:700 hexane:0.1% DEA;
1.0 mL/min; 20 min. RT ) 5.69.
Step B: P r ep a r a tion of 1-Eth yl-3-(3-p en tyl-[1,2,4]oxa -
d ia zol-5-yl)-4-p h en yl-1,2,3,6-tetr a h yd r op yr id in e Eth a n e-
d ioa te (1:1) (54) a n d 1-Eth yl-5-(3-p en tyl-[1,2,4]oxa d ia zol-
5-yl)-4-p h en yl-1,2,3,6-tetr a h yd r op yr id in e Eth a n ed ioa te
(1:1) (55).11 A suspension of 1-ethyl-4-phenylpiperidinyl-3-
carboxylic acid methyl ester ethanedioate (1) (2.78 g) in 200
mL of ethyl acetate and 10 mL 1 M K2CO3 was stirred for 15
min. The layers were separated, and the organic layer was
dried (MgSO4), filtered, and concentrated in vacuo to give 2.06
g of the free base of 1. To a green suspension of N′-hydroxy-
hexanimidamide (1.31 g, 0.010 mol, 1.2 equiv) and sodium
hydride (60% dispersion in mineral oil, rinsed with hexane,
1.2 equiv) was added 2.0 g of 4 Å molecular sieves, and the
mixture was heated to reflux for 1 h. After the heating source
was removed, 1-ethyl-4-phenylpiperidinyl-3-carboxylic acid
methyl ester (1, 2.06 g, 0.008 mol) was added, and the reaction
mixture was heated again to reflux for 2.5 h and then at room
temperature for 16 h. The suspension was filtered, and the
filtrate was concentrated in vacuo to give an orange residue.
This residue was diluted with water and extracted 4 times with
dichloromethane. The combined organic layers were dried
(NaSO4), filtered, and concentrated in vacuo to give crude
product. Purification by medium-pressure liquid chromatog-
raphy (silica gel, 20% ethyl acetate/hexane) yielded 0.2 g of
54 and 0.41 g of 55, in the free base forms. To a solution of
free base 54 (0.15 g) in 50 mL of diethyl ether was added oxalic
acid (0.04 g) in 5 mL of diethyl ether, and the mixture was
stirred at room temperature for 16 h. The diethyl ether was
removed, and the solid was filtered, rinsed with additional
ether, and dried to give 1-ethyl-3-(3-pentyl-[1,2,4]oxadiazol-
5-yl)-4-phenyl-1,2,3,6-tetrahydropyridine ethanedioate (1:1)
(54) (0.06 g, 31%). To a solution of free base 55 (0.35 g) in 140
mL of diethyl ether was added oxalic acid (0.97 g) in 10 mL of
diethyl ether, and the mixture was stirred at room tempera-
ture for 16 h. The diethyl ether was removed, and the solid
was filtered, rinsed with additional ether, and dried to give
1-ethyl-5-(3-pentyl-[1,2,4]oxadiazol-5-yl)-4-phenyl-1,2,3,6-tet-
rahydropyridine ethanedioate (1:1) (55) (0.32 g, 72%).
(tr a n s)-1-Eth yl-4-p h en ylp ip er id in e-3-ca r boxylic Acid
P h en eth yl Ester Eth a n ed ioa te (1:2) (49) a n d (cis)-1-
Eth yl-4-p h en ylp ip er id in e-3-ca r boxylic Acid P h en eth yl
Ester Eth a n ed ioa te (1:2) (50). To a solution of 1-ethyl-4-
phenyl-1,2,5,6-tetrahydropyridine-3-carboxylic acid 2-phenyl-
ethyl ester ethanedioate (1:1) (28) (0.75 g, 0.0017 mol) in 75
mL of tetrahydrofuran was added 0.2 g of 5% Pd/C, and the
mixture was shaken under 52 psi H2 for at least 23 h. The
reaction mixture was filtered and concentrated in vacuo to give
crude desired product. To 3 mL of 1 M K2CO3 and 75 mL of
ethyl acetate was added 820 mg of this crude reduced oxalate
salt, and the mixture was stirred. The layers were separated,
and the organic layer was dried (MgSO4), filtered, and
concentrated in vacuo to give a 6:1 ratio of cis:trans isomers.
Purification by medium-pressure liquid chromatography (5%
MeOH/dichloromethane, the trans isomer elutes first) yielded
30 mg of the free base of 49 and 180 mg of the free base of 50,
for an overall yield of 31%.
(trans)-1-Ethyl-4-phenyl-piperidine-3-carboxylic Acid Phen-
ethyl Ester (49). 400 MHz NMR (CDCl3): 0.98-1.06 (m, 1H);
1.17-1.20 (t, 3H, J ) 7.0 Hz); 1.38-1.54 (m, 2H); 1.83-1.86
(m, 1H); 2.46-2.48 (m, 1H); 2.55-2.61 (m, 2H); 2.63-2.65 (m,
1H); 2.67-2.68 (m, 0.5H); 2.89-2.90 (m, 0.5H); 3.00-3.09 (m,
0.5H); 3.10-3.18 (m, 0.5H); 3.34-3.35 (m, 2H); 4.01-4.06 (m,
1H); 4.55-4.59 (m, 1H); 7.06-7.32 (m, 10H). MS: 338 (MH+),
337 (M+).
(cis)-1-Ethyl-4-phenyl-piperidine-3-carboxylic Acid Phen-
ethyl Ester (50). 400 MHz NMR (DMSO-d6): 0.91-0.94 (t, 3H,
J ) 7.0 Hz); 1.71-1.75 (m, 1H); 2.02-2.07 (m, 1H); 2.22-2.33
(m, 3H), 2.40-2.50 (m, 1H); 2.66-2.76 (m, 2H); 2.78-2.81 (m,
1H); 2.82-2.95 (m, 1H); 3.00-3.06 (m, 1H); 3.10-3.19 (m, 1H);
3.99-4.10 (m, 2H); 7.11-7.26 (m, 10H). MS: 338 (MH+), 337
(M+).
Treatment of each isomer separately with 1 mol of oxalic
acid in diethyl ether, followed by filtration, yielded 49 and 50.
1-Ethyl-3-(3-pentyl-[1,2,4]oxadiazol-5-yl)-4-phenyl-1,2,3,6-
tetrahydropyridine Ethanedioate (1:1) (54). 400 MHz NMR
(DMSO-d6): 0.78-0.82 (t, 3H, J ) 7.2 Hz); 1.05-1.28 (m, 7H);
1.51-1.58 (m, 2H); 2.50-2.59 (m, 2H); 2.71-2.76 (m, 2H);
3.12-3.14 (m, 1H); 3.32-3.41 (m, 2H); 3.59-3.63 (m, 1H); 4.78
(br. s., 1H); 6.35 (br. s., 1H); 7.22-7.41 (m, 5 H). Anal.
(C20H27N3O‚0.5H2O4‚C2H2O4): C, H, N.
(trans)-1-Ethyl-4-phenylpiperidine-3-carboxylic Acid Phen-
ethyl Ester Ethanedioate (1:2) (49). 400 MHz NMR (DMSO-
d6): 0.91-0.99 (m, 1H); 1.18-1.25 (m, 3H); 1.33-1.48 (m, 2H);
1.85-1.95 (m, 0.5H); 1.96-2.10 (m, 0.5H); 2.55-2.60 (m, 1H);
2.69-2.71 (m, 1H); 2.88-2.94 (m, 1H); 3.01-3.14 (m, 2H);
3.21-3.39 (m, 1H); 3.42-3.45 (m, 0.5H); 3.50-3.53 (m, 0.5H);
3.79-3.85 (m, 1H); 3.96-4.02 (m, 1H); 4.49-4.53 (m, 1H);

m1 Selective Muscarinic Antagonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3 363
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1-Ethyl-5-(3-pentyl-[1,2,4]oxadiazol-5-yl)-4-phenyl-1,2,3,6-
tetrahydropyridine Ethanedioate (1:1) (55). 400 MHz NMR
(DMSO-d6): 0.82-0.86 (t, 3H, J ) 7.0 Hz); 1.17-1.31 (m, 7H);
1.54-1.61m, 2H); 2.58-2.62 (m, 2H); 2.82 (m, 2H); 3.06-3.11
(m, 2H); 3.24-3.38 (m, 2H); 3.98 (m, 2H); 7.17-7.19 (m, 2 H);
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Biologica l Meth od s. Receptor binding at the five musca-
rinic receptors (Hm1-Hm5) is performed by the methods
described by Dorje et al.3a and Buckley et al.14 with modifica-
tions as described in ref 16. PI turnover assays are performed
by the methods described by Berridge et al.19 with modifica-
tions as also described in ref 16.
Ack n ow led gm en t. We thank Dr. G. McClusky and
staff for spectral and analytical determinations, V.
Hoard, E. Getachew, M. Marlatt, D. J ohnson, N. Colbry,
J . Dodd, and J . McKee for their synthetic assistance of
various intermediates and final compounds, B. Tobias
for special NMR studies, J . Loo and D. DeJ ohn for the
high-resolution mass spectral data, B. Leja for spectral
analysis of 11, and M. Callahan, C. Christofferson, J .
Wiley, and L. Meltzer for the preliminary in vivo
studies.
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