Discovery of a muscarinic M3 receptor antagonist with high selectivity for M3 over M2 receptors among 2-[(1S,3S)-3-sulfonylaminocyclopentyl]phenylacetamide derivatives.

Article abstract of DOI:10.1016/S0968-0896(00)00008-0

In the course of developing a metabolically stable M3 receptor antagonist from the prototype antagonist, J-104129 (1), introduction of certain substituents into the cyclopentane ring of 1 was found to be effective not only in improving metabolic stability but also in greatly enhancing the subtype selectivity. Among the cyclopentane analogues, sulfonamide derivatives (10f) and (10g) displayed 160- and 310-fold selectivity for M3 over M2 receptors, and both were significantly more selective than the prototype antagonist (120-fold). Subsequent derivatization of the sulfonamide series led to the highly selective M3 receptor antagonists (10h, 10i and 10j) with >490-fold selectivity for M3 over M2 receptors. Among them, p-nitrophenylsulfonamide (J-107320, 10h) exhibited 1100-fold selectivity for M3 receptors (Ki = 2.5 nM) over M2 receptors (Ki = 2800 nM) in the human muscarinic receptor binding assay using [3H]-NMS as a radio ligand.

Full text of DOI:10.1016/S0968-0896(00)00008-0

Bioorganic & Medicinal Chemistry 8 (2000) 825±832
Discovery of a Muscarinic M3 Receptor Antagonist with
High Selectivity for M3 Over M2 Receptors Among
2-[(1S,3S)-3-Sulfonylaminocyclopentyl]phenylacetamide
Derivatives
Morihiro Mitsuya,* Yoshio Ogino, Kumiko Kawakami, Minaho Uchiyama,
Toshifumi Kimura, Tomoshige Numazawa, Takuro Hasegawa, Norikazu Ohtake,
Kazuhito Noguchi and Toshiaki Mase*
Banyu Tsukuba Research Institute in collaboration with Merck Research Laboratories, Okubo-3, Tsukuba 300-2611, Ibaraki, Japan
Received 5 November 1999; accepted 21 December 1999
AbstractÐIn the course of developing a metabolically stable M3 receptor antagonist from the prototype antagonist, J-104129 (1),
introduction of certain substituents into the cyclopentane ring of 1 was found to be e€ective not only in improving metabolic sta-
bility but also in greatly enhancing the subtype selectivity. Among the cyclopentane analogues, sulfonamide derivatives (10f) and
(10g) displayed 160- and 310-fold selectivity for M3 over M2 receptors, and both were signi®cantly more selective than the proto-
type antagonist (120-fold). Subsequent derivatization of the sulfonamide series led to the highly selective M3 receptor antagonists
(10h, 10i and 10j) with >490-fold selectivity for M3 over M2 receptors. Among them, p-nitrophenylsulfonamide (J-107320, 10h)
exhibited 1100-fold selectivity for M3 receptors (Ki=2.5 nM) over M2 receptors (Ki=2800 nM) in the human muscarinic receptor
binding assay using [3H]-NMS as a radio ligand. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
adverse e€ects. Therefore, it has been hypothesized that
selective blockade of muscarinic M3 receptors may be
therapeutically useful in the treatment of respiratory
disorders such as chronic obstructive pulmonary disease
(COPD), gastrointestinal disorders such as irritable
bowel syndrome (IBS) and urinary tract disorders such
as urinary incontinence (UI).8 Thus, pharmaceutical
research into therapeutic agents selective for muscarinic
receptor subtypes has been recently focused on
exploration of M3 selective antagonist for the above
disorders.
In the muscarinic ®eld, ®ve receptor subtypes have been
identi®ed and cloned.1±5 These are classi®ed as m1, m2,
m3, m4 and m5 with distinct but homologous gene
sequence. Structural and pharmacological criteria have
suggested the presence of at least four subtypes, denoted
M1, M2, M3 and M4 whilst a physiological role for M5
gene product remains to be identi®ed.6,7 M1 receptors
are found in parasympathetic ganglia and in parts of
central nervous system and facilitate neurotransmission.
M2 receptors are localized to the post ganglionic choli-
nergic nerve terminals and provide a functional negative
feedback modulation of acetylcholine (ACh) release.
M3 receptors localized in smooth muscle and mucosal
glands mediate contraction and mucus secretion,
respectively. Subtype selective ligands that recognize the
di€erent localizations and functions of these receptor
subtypes provide the possibility of developing more
ideal drugs since they would avoid the occurrence of
In a previous paper, we reported the identi®cation of
J-104129 (1), a prototype muscarinic M3 receptor
antagonist with 120-fold greater selectivity for M3 over
M2 receptors in a series of 4-acetamidopiperidine deri-
vatives (Fig. 1).9 J-104129 (1) is a potent bronchodilator
in rats, guinea pigs and dogs.10 In rats, oral adminis-
tration of J-104129 (1) showed durable bronchodilatory
action lasting over 10 h with 10 mg/kg. However, a
recent pharmacokinetic study in the same species
revealed its relatively short half life (T1/2=2 h). The dis-
crepancy prompted us to explore active metabolite(s) in
rats. As a result, we identi®ed an active metabolite (5b)
*Corresponding authors. Tel.: +81-298-77-2000; fax: +81-298-77-
2029; e-mail: mituyamr@banyu.co.jp
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00008-0

826
M. Mitsuya et al. / Bioorg. Med. Chem. 8 (2000) 825±832
phenylacetamide series. In this paper, we describe the
identi®cation of the active metabolites of J-104129 (1)
and the SAR of the substituted cyclopentyl derivatives.
Results and Discussion
Chemistry
Figure 1.
General synthetic methods of derivatives (5 and 10) are
highlighted in Scheme 1. Michael addition of an enolate
of dioxolane (2),11 derived from (R)-mandelic acid, with
cyclopentenone gave ketones (3) and (4) in 31 and
34% yields, respectively, after separation by silica gel
column chromatography.12 These compounds were used
as key intermediates in the following derivatization.
Alkaline hydrolysis of 3 followed by coupling with 4-
aminopiperidine produced an amide (5a) in 52% yield.
Reduction of 5a and the subsequent separation of the
diastereomers on the resulting secondary alcohol moiety
a€orded the derivatives (5b) and (5c) in 25 and 35%
yields, respectively. Other congeners (5d), (5e) and (5f)
with a hydroxyl group on the cyclopentane ring of
J-104129 (1). Interestingly, 5b showed not only
improved metabolic stability, but also subtype selectivity
as high as that of the parent compound (1). It should be
noted that 5b was the ®rst example indicating that sub-
stitution on the cyclopentyl moiety might e€ectively
improve both the metabolic stability and subtype selec-
tivity. Thus, we tried to introduce various substituents
into the cyclopentane ring based on the structure of the
metabolite (5b) to further investigate the structure±
activity relationships (SAR) on subtype selectivity and
metabolic stability of the 2-(3-substituted cyclopentyl)-
Scheme 1. Preparation of 5a±f and 10a±j. Conditions: (a) LDA, THF then 2-cyclopenten-1-one; (b) NaOH, H2O-MeOH; (c) 1-(4-methyl-3-pentenyl)-
.
4-aminopiperidine, WSC HCl, HOBt, CHCl3; (d) NaBH4, MeOH; (e) recrystallization from hexane:AcOEt; (f) MsCl, Et3N, AcOEt; (g) NaN3,
DMF; (h) Ph3P, H2O, THF; (i) acid chloride, Et3N, CHCl3; (j) NaH, THF.

M. Mitsuya et al. / Bioorg. Med. Chem. 8 (2000) 825±832
827
were prepared from 4 in 75, 33 and 36% yields, respec-
tively, by the method described above.
in the mandelic acid series involving 1 had so far been
performed only by introducing simple substituents such
as a halogen and alkyl moiety into the benzene ring or
by replacing the cyclopentane ring with other ring-sized
cycloalkanes.
Preparation of derivatives (10) was initiated from 6.
Fractional recrystallization of the diastereomeric mix-
ture, obtained by reduction of 3 with sodium boro-
hydride gave the alcohol (6) in 33% yield. The
stereochemical structure of the cyclopentane moeity in 6
was determined by conversion to 11. Mesylation of 6
and subsequent nucleophilic substitution with sodium
azide gave cyclopentylazide (7) in 82% yield. The
cyclopentylazide (7) was converted to an amide (8) in
92% yield in a manner similar to that described for the
preparation of 5a. The amide (8) was reduced with tri-
phenylphosphine and subsequently treated with various
acid chloride analogues to produce derivatives (10a±10j)
in 48ꢀ94% yields.
With the regio- and stereochemical structures of sub-
stituents on the cyclopentane ring being ®xed in the
(1S,3S)-con®guration identical to that in 5b, derivatives
(10a±10j) were prepared and evaluated in the human
muscarinic binding assay, and metabolic stability was
examined in human microsomes (Table 2).
Conversion of the hydroxyl group in 5b to an acetamide
(10a) or methyl carbamate (10d) resulted in con-
siderably decreased M3 selectivity, but improved meta-
bolic stability. Although the corresponding phenyl
derivatives (10b) and (10e) also showed low M3 selec-
tivity, these exhibited some degree of improved binding
anity.
Biological properties
First, J-104129 (1) was incubated in rat hepatic micro-
somes for the identi®cation of its active metabolites.
Oxidation of the cyclopentane moiety of 1 was predicted
since oxybutynin (12) or its dimethyl analogue (13) was
reported to be metabolized on 3- and 4-position of cy-
clohexyl group to produce metabolites (14) and (15)13,14
(Fig. 2). Therefore, we synthesized the compounds (5b),
(5c), (5e) and (5f) and compared their retention times
and molecular weights by LC/MS with those of the
metabolites in rats for unambiguous determination of
their structures. Simultaneously, the binding anities of
these compounds were determined by inhibition of spe-
ci®c binding of [3H]-NMS using membranes from CHO
cells expressing cloned human m1±m3 receptors9 (see
Table 1). As expected, the hydroxylated compounds
(5b), (5c), (5e) and (5f) were identi®ed as the metabolites
of 1. Among them, 5b with (1S,3S)-con®guration on the
cyclopentane moiety was the most active metabolite,
showing improved metabolic stability and potent M3
binding anity (Ki=18 nM) while maintaining M3
selectivity (130-fold) over M2 receptors. Moreover, oral
administration of 5b inhibited ACh-induced broncho-
constriction in anesthetized rats9 with an ED50 value of
0.33 mg/kg. This interesting result indicated that the
derivatization of J-104129 (1) by introducing certain
substituents into the cyclopentane ring may improve the
M3 selectivity and metabolic stability. This new type of
derivatization further intrigued us in that modi®cation
Next, we tried to introduce certain sulfonamides into
the cyclopentane moiety. In contrast to the amide and
carbamate derivatives, methanesulfonamide (10f) and
benzenesufonamide (10g) displayed 160- and 300-fold
selectivity for M3 over M2 receptors, although they also
bound to M1 receptors with high anity. The signi®cant
improvement in M3 selectivity especially over M2
receptors as compared to that of the prototype J-104129
(1) prompted further modi®cation of this sulfonamide
series. Introduction of a methoxy moiety (10i) as a
representative electron-donating group and
a tri-
¯uoromethyl (10j), an electron-withdrawing group, into
the para-position of the benzene ring further enhanced
M3 selectivity up to 640- and 490-fold, respectively. The
best result was obtained by introducing a nitro group
into the para-position. The resulting p-nitrophe-
nylsulfonamide (10h) exhibited 1100-fold selectivity for
M3 receptors (Ki=2.5 nM) over M2 receptors (Ki=2800
nM). These results indicated that substituted benzene-
sulfonamide on the cyclopentane ring played an impor-
tant role in enhancing selectivity for M3 over M2
receptors. We are now investigating the role of the p-
nitrophenylsulfonamide moiety for sparing M2 recep-
tors by computer modeling.
Conclusion
In the process of developing an M3 receptor antagonist
with improved metabolic stability over the prototype,
J-104129 (1), we found that introduction of a sub-
stituent with the (1S,3S)-con®guration into the 3-posi-
tion of the cyclopentane group in 1 e€ectively improved
both metabolic stability and selectivity for M3 over M2
receptors. In this series, the substituted benzenesulfona-
mide derivatives (10g, 10h, 10i and 10j) showed more
than 300-fold selectivity for M3 over M2 receptors. In
particular, J-107320 (10h) displayed 1100-fold selectivity
for M3 receptors over M2 receptors in the human mus-
carinic binding assay. The SAR on the subtype selectivity
described here will be useful for designing an ideal M3
selective antagonist.
Figure 2.

828
M. Mitsuya et al. / Bioorg. Med. Chem. 8 (2000) 825±832
Table 1. Biological properties of 1 and 5a±f
Binding anity (Ki nM)a
Selectivity
m2/m3
Metabolic stabilityb
% remaining
Compound
R
m3
71
m1
m2
5a
260
9200
130
130
48
NTc
5b
5c
18
88
2300
Rat75
Dog 75
Human 76
480
1100
23,000
NT
5d
360
140
770
1300
640
2200
19
29,000
12,000
30,000
490
81
86
NT
NT
NT
5e
5f
39
J-104129 (1)
4.2
120
Rat 10
Dog 31
Human 54
aValues are the mean of two or more independent assays.
b% Remaining after 30 min incubation in hepatic microsomes (n=3).
cNot tested (for compounds 5c, 5d, 5e, and 5f).
Table 2. Biological properties of 10a±j
Binding anity (Ki nM)a
Selectivity
m2/m3
Metabolic stabilityb
% remaining
Compound
R
m3
m1
m2
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
5b
CH3CONH
PhCH2CONH
PhCOCONH
CH3OCONH
PhCH2OCONH
CH3SO2NH
30
13
4.1
11
5.2
28
6.0
2.5
9.0
6.6
18
60
47
11
34
16
77
3.3
4.6
5.6
11
88
1200
350
260
480
40
27
64
43
85
11
58
75
31
95
31
38
50
19
76
220
41
4500
1800
2800
5700
3200
2300
160
300
1100
640
490
130
PhSO2NH
4-NO2-PhSO2NH
4-CH3O-PhSO2NH
4-CF3-PhSO2NH
OH
aValues are the mean of two or more independent assays.
b% Remaining after 30 min incubation in human microsomes (n>2).

M. Mitsuya et al. / Bioorg. Med. Chem. 8 (2000) 825±832
829
Experimental
temperature. After being stirred for 15 h, the mixture
was concentrated in vacuo. The residue was partitioned
between Et2O and saturated aq NaHCO3. The organic
layer was separated, washed with H2O and brine and
dried over MgSO4. Removal of the solvent in vacuo
gave the crude residue which was puri®ed by pre-
parative TLC (CHCl3:MeOH=9:1) to yield 5a (95 mg,
52%) as an oil: 1H NMR (CDCl3) d 1.28±2.40 (16H, m),
1.60 (3H, s), 1.68 (3H, s), 2.71±2.89 (2H, m), 3.29 (1H,
m), 3.53±3.79 (2H, m, containing OH), 5.06 (1H, m),
6.24 (1H, m), 7.28±7.46 (3H, m), 7.59 (2H, brd, J=8.1
Hz); HRMS calcd for C24H35N2O3 (M+H)+: 399.2648,
found 399.2654; [a]2d0 52.2 (c=1.0, CHCl3).
Melting points (mp) were determined with a Yanaco MP
micromelting point apparatus and were not corrected.
Proton NMR spectra were obtained on a Varian
Gemini-300 with tetramethylsilane as an internal stan-
dard. Mass spectrometry were performed with JEOL
JMS-SX 102A. IR spectra were recorded with Horiba
FT-200 spectrometer. Optical rotations were measured
with Jasco DIP-370 polarimeter. TLC were done with
Merck Kieselgel F254 pre-coated plates. Silica gel (SiO2)
column chromatography was carried out on Wako gel
C-300.
(2R,5R)-2-tert-Butyl-5-[(1S)-3-oxocyclopentyl]-5-phenyl-
1,3-dioxolan-4-one (3) and (2R,5R)-2-tert-butyl-5-[(1R)-
3-oxocyclopentyl]-5-phenyl-1,3-dioxolan-4-one (4). To a
suspension of (2R,5R)-2-tert-butyl-5-phenyl-1,3-dioxo-
lan-4-one11 (2) (3.00 g, 13.6 mmol) in THF (120 mL)
was added a 1.5 M solution of lithium diisopropylamide
mono(tetrahydrofuran) in cyclohexane (10.0 mL, 15.0
mmol) dropwise at 78 ꢁC, and the mixture was stirred
for 30 min at the same temperature. A solution of 2-
cyclopenten-1-one (1.25 g, 15.23 mmol) in THF (10 mL)
was added for 10 min maintaining the temperature
below 70 ꢁC. After being stirred for additional 2 h, the
reaction mixture was quenched by saturated aq NH4Cl
solution, and the mixture was allowed to warm to room
temperature. The mixture was diluted with H2O and
extracted with AcOEt. The organic layer was washed
with brine, dried over anhydrous MgSO4 and con-
centrated in vacuo. The residue was puri®ed by SiO2
column chromatography (hexane:AcOEt=12:1) to give
less polar 3 (1.27 g, 31%) and polar 4 (1.48 g, 36%) as
white crystals. The regiochemical structures of 3-oxocy-
clopentyl group were determined by NOE experiment.
(2R)-N-[1-(4-Methyl-3-pentenyl)piperidin-4-yl]-2-[(1S,3S)-
3-hydroxycyclopentyl]-2-hydroxy-2-phenylacetamide (5b)
and (2R)-N-[1-(4-methyl-3-pentenyl)piperidin-4-yl]-2-
[(1S,3R)-3-hydroxycyclopentyl]-2-hydroxy-2-phenylace-
tamide (5c). To a solution of 5a (63 mg, 0.16 mmol) in
MeOH (3 mL) was added NaBH4 (40 mg, 1.1 mmol) at
0 ꢁC. After 1 h, the mixture was diluted with H2O and
extracted twice with CHCl3. The combined organic
layer was dried over MgSO4 and concentrated in vacuo
to give the crude residue which was puri®ed by pre-
parative TLC (CHCl3:MeOH=9:1) to yield 5b (15 mg,
25%) and 5c (22 mg, 35%) as oils. The stereochemical
structure of 5c was determined by conversion of 6 to 5c
in a similar method described for 5a (70%). 5b: 1H
NMR (CDCl3) d 1.20±1.95 (10H, m), 1.61 (3H, s), 1.69
(3H, s), 2.00±2.21 (4H, m), 2.26±2.39 (2H, m), 2.69±2.88
(2H, m), 3.31 (1H, m), 3.70 (1H, m), 3.75 (1H, brs, OH),
4.43 (1H, m), 5.08 (1H, m), 6.07 (1H, brd, J=7.8 Hz),
7.21±7.42 (3H, m), 7.59 (2H, brd, J=7.2 Hz); MS m/z
401 (M+H)+; [a]d20 20.0 (c=1.0, CHCl3); 5b fumarate
mp 161±162 ꢁC (i-Pr2O/EtOH); anal. calcd for C24H36
N2O3 C4H4O4 0.25H2O: C, 64.53; H, 7.83; N, 5.38.
Found: C, 64.47; H, 7.86; N, 5.40. 5c: 1H NMR
(CDCl3) d 1.35±1.80 (8H, m), 1.60 (3H, s), 1.68 (3H, s),
1.87±2.20 (6H, m), 2.25±2.36 (2H, m), 2.75±2.95 (2H,
m), 3.58 (1H, m), 3.73 (1H, m), 4.35 (1H, m), 5.05 (1H,
m), 5.78 (1H, m), 7.19±7.38 (3H, m), 7.74 (2H, brd,
J=8.3 Hz); MS m/z 401 (M+H)+; [a]d20 +37.6 (c=1.0,
CHCl3); 5c oxalate mp 200±201 ꢁC (i-Pr2O:EtOH); anal.
1
3: H NMR (CDCl3) d 0.92 (9H, s), 1.74±1.84 (2H, m),
2.09 (1H, m), 2.20±2.46 (3H, m), 2.89 (1H, m), 5.39 (1H,
s), 7.30±7.46 (3H, m), 7.67±7.73 (2H, m); MS m/z 303
(M+H)+; mp 86±87 ꢁC (hexane/AcOEt); anal. calcd for
C18H22O4: C, 71.50; H, 7.33. Found: C, 71.56; H, 7.41;
[a]2d0 16.2 (c=1.0, CHCl3). 4: 1H NMR (CDCl3) d 0.93
(9H, s), 1.82±2.00 (2H, m), 2.10±2.28 (3H, m), 2.34 (1H,
m), 2.89 (1H, m), 5.44 (1H, s), 7.26±7.40 (3H, m), 7.62±
7.79 (2H, m); MS m/z 303 (M+H)+; mp 104±105 ꢁC
(hexane/AcOEt); anal. calcd for C18H22O4: C, 71.50;
H, 7.33. Found: C, 71.50; H, 7.27; [a]2d0 +82.8 (c=1.0,
CHCl3).
.
calcd for C24H36N2O3 C2H2O4: C, 63.65; H, 7.81; N,
5.71. Found: C, 63.86; H, 7.83; N, 5.57.
(2R)-N-[1-(4-Methyl-3-pentenyl)piperidin-4-yl]-2-[(1R)-3-
oxocyclopentyl]-2-hydroxy-2-phenylacetamide (5d). Com-
pound 5d was obtained as an oil from 4 in a similar
1
(2R)-N-[1-(4-Methyl-3-pentenyl)piperidin-4-yl]-2-[(1S)-3-
oxocyclopentyl]-2-hydroxy-2-phenylacetamide (5a). To a
solution of 3 (110 mg, 0.36 mmol) in MeOH (1.5 mL)
was added 3 N NaOH (300 mL, 0.90 mmol) at room
temperature. After being stirred for 5 h, the mixture was
diluted with H2O and washed with Et2O. The aqueous
layer was acidi®ed with 1 N HCl and extracted three
times with CHCl3. The combined organic layer was
dried over MgSO4 and concentrated in vacuo. To a
solution of the residual oil (90 mg) and 1-(4-methyl-3-
pentenyl)-4-aminopiperidine9 (84 mg, 0.47 mmol) in
CHCl3 (4 mL) were added HOBt (105 mg, 0.79 mmol)
and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
method described for 5a (75%): H NMR (CDCl3) d
1.30±2.48 (16H, m), 1.60 (3H, s), 1.68 (3H, s), 2.68±2.90
(2H, m), 3.29 (1H, m), 3.63 (1H, brs, OH), 3.73 (1H, m),
5.07 (1H, m), 6.24 (1H, m), 7.24±7.42 (3H, m), 7.54 (2H,
brd, J=8.4 Hz); HRMS calcd for C24H35N2O3 (M+H)+:
399.2648, found 399.2638; [a]2d0 +30.2 (c=1.0, CHCl3).
(2R)-N-[1-(4-Methyl-3-pentenyl)piperidin-4-yl]-2-[(1R,3R)-
3-hydroxycyclopentyl]-2-hydroxy-2-phenylacetamide (5e)
and (2R)-N-[1-(4-methyl-3-pentenyl)piperidin-4-yl]-2-
[(1R,3S)-3-hydroxycyclopentyl]-2-hydroxy-2-phenylace-
tamide (5f). Compound 5e (33%) and 5f (36%) were
obtained as a white crystal and a foam from 5d in a simi-
lar method described for 5b and 5c. The stereochemical
.
hydrochloride (WSC HCl, 118 mg, 0.62 mmol) at room

830
M. Mitsuya et al. / Bioorg. Med. Chem. 8 (2000) 825±832
structure of the cyclopentane moeity in 5f was deter-
1
further puri®cation. To a solution of the mesylate in
DMF (200 mL) was added NaN3 (2.70 g, 41.5 mmol) at
room temperature, and the mixture was heated at 90 ꢁC
for 1 h. The mixture was diluted with H2O and extrac-
ted with AcOEt. The organic layer was washed with
H2O and brine and dried over MgSO4. The solvent was
evaporated to give the crude material which was
puri®ed by SiO2 column chromatography (hexane:
AcOEt=10:1) to yield 7 (9.55 g, 82%) as a white solid:
1H NMR (CDCl3) d 0.92 (9H, s), 1.49±1.70 (3H, m),
1.77±1.89 (2H, m), 1.96 (1H, m), 2.82 (1H, m), 4.01 (1H,
m), 5.42 (1H, s), 7.26±7.43 (3H, m), 7.67 (2H, brd,
J=8.2 Hz); MS m/z 330 (M+H)+; mp 62±64 ꢁC (hex-
ane at 20 ꢁC); anal. calcd for C18H23N3O4: C, 65.63;
H, 7.04; N, 12.76. Found: C, 65.48; H, 7.02; N, 12.81;
[a]2d0 +3.2 (c=1.0, CHCl3).
mined by NOE experiment. 5e: H NMR (CDCl3) d
1.20±220 (14H, m), 1.60 (3H, s), 1.68 (3H, s), 2.26±2.36
(2H, m), 2.68±2.88 (2H, m), 3.30 (1H, m), 3.70 (1H, m),
3.74 (1H, brs, OH), 4.34 (1H, m), 5.06 (1H, m), 6.08
(1H, brd, J=8.4 Hz), 7.23±7.40 (3H, m), 7.57 (2H, brd,
J=7.2 Hz); MS m/z 401 (M+H)+; [a]d20 11.6 (c=1.0,
CHCl3); mp 181±182 ꢁC (hexane:CHCl3); anal. calcd for
C24H36 N2O3 0.25H2O: C, 71.17; H, 9.08; N, 6.92.
Found: C, 71.11; H, 9.03; N, 6.92. 5f: 1H NMR (CDCl3)
d 1.22±2.20 (14H, m), 1.59 (3H, s), 1.67 (3H, s), 2.22±
2.39 (2H, m), 2.79±2.95 (2H, m), 3.52 (1H, m), 3.76 (1H,
m), 4.22 (1H, m), 5.03 (1H, m), 6.10 (1H, m), 7.19±7.37
(3H, m), 7.75 (2H, brd, J=7.2 Hz); MS m/z 401
(M+H)+; [a]d20 +5.8 (c=1.0, CHCl3); anal. calcd for
.
C24H36N2 O3 0.5H2O: C, 70.38; H, 9.11; N, 6.84.
Found: C, 70.59; H, 9.38; N, 6.69.
(2R)-N-[1-(4-Methyl-3-pentenyl)piperidin-4-yl]-2-[(1S,3S)-
3-azidocyclopentyl]-2-hydroxy-2-phenylacetamide (8). To
a solution of 7 (730 mg, 2.2 mmol) in MeOH (10 mL)
was added 3 N NaOH (2 mL, 6.0 mmol), and the mix-
ture was stirred for 14 h at room temperature. After
addition of H2O, the mixture was concentrated in vacuo
to remove MeOH and washed with Et2O. The aqueous
layer was acidi®ed with 1 N HCl and extracted three
times with CHCl3. The combined organic layer was
dried over MgSO4 and concentrated in vacuo. To a
solution of the residual oil (620 mg) and 1-(4-methyl-3-
pentenyl)-4-aminopiperidine9 (470 mg, 2.6 mmol) in
CHCl3 (30 mL) were added HOBt (460 mg, 3.4 mmol)
(2R,5R)-2-tert-Butyl-5-[(1S,3R)-3-hydroxycyclopentyl]-
5-phenyl-1,3-dioxolan-4-one (6). To a solution of 3
(1.1 g, 3.5 mmol) in MeOH (50 mL) was added NaBH4
(700 mg, 19 mmol) at 78 ꢁC in several portions. The
mixture was allowed to warm to 0 ꢁC over 1 h and con-
centrated to a small volume. After addition of H2O, the
mixture was extracted twice with CHCl3. The combined
organic layer was dried over MgSO4 and concentrated
in vacuo to give a mixture of diastereomers (ca. 3.5:1).
This was recystallized twice from hexane:AcOEt to
obtain 6 (350 mg, 33%) as a white crystal. The stereo-
chemical structure of 6 was determined by conversion to
1
.
11. 6: H NMR (CDCl3) d 0.92 (9H, s), 1.34 (1H, m),
and WSC HCl (560 mg, 2.9 mmol) at room temperature.
1.50±1.79 (3H, m), 1.86 (1H, m), 2.11 (1H, m), 2.67 (1H,
m), 4.29 (1H, m), 5.51 (1H, s), 7.23±7.41 (3H, m), 7.67
(2H, brd, J=7.2 Hz); MS m/z 305 (M+H)+; mp 123±
125 ꢁC (hexane/AcOEt); anal. calcd for C18H24O4: C,
71.03; H, 7.95. Found: C, 71.13; H, 8.07; [a]2d0 +25.8
(c=1.0, CHCl3).
After being stirred for 18 h, the mixture was con-
centrated in vacuo. The residue was partitioned between
Et2O and saturated aq NaHCO3. The organic layer was
separated, washed with brine and dried over MgSO4.
Evaporation of the solvent in vacuo gave the residue
which was puri®ed by SiO2 column chromatography
(CHCl3:MeOH=100:1) to yield 8 (920 mg, 92%) as a
1
(1R,4R,5R)-4-Hydroxy-4-phenyl-2-oxabicyclo[3,2,1]octan-
3-one (11). To a solution of 6 (160 mg, 0.53 mmol) in
THF (8.0 mL) was added NaH (60% in oil, 30 mg, 0.75
mmol) at 0 ꢁC. The mixture was stirred for 1 h at the
same temperature. After addition of saturated aq
NH4Cl, the mixture was extracted with AcOEt. The
organic layer was washed with H2O and brine and dried
over MgSO4. The solvent was evaporated and the
residue was puri®ed by preparative TLC (hexane:
white solid: H NMR (CDCl3) d 1.28±1.97 (10H, m),
1.62 (3H, s), 1.69 (3H, s), 2.04±2.23 (4H, m), 2.27±2.39
(2H, m), 2.70±2.90 (2H, m), 3.20 (1H, m), 3.71 (1H, m),
3.85 (1H, brs, OH), 5.08 (1H, m), 5.99 (1H, m), 7.27±
7.43 (3H, m), 7.55 (2H, brd, J=7.2 Hz); MS m/z 426
(M+H)+; mp 137±139 ꢁC (hexane:AcOEt); anal. calcd
for C24H35N5O2: C, 67.73; H, 8.29; N, 16.46. Found: C,
67.82; H, 8.42; N, 16.36; [a]2d0 29.2 (c=1.0, CHCl3).
1
AcOEt=4:1) to yield 11 (70 mg, 61%) as an oil: H
(2R)-N-[1-(4-Methyl-3-pentenyl)piperidin-4-yl]-2-[(1S,3S)-
3-aminocyclopentyl]-2-hydroxy-2-phenylacetamide
NMR (CDCl3) d 1.55 (1H, m), 1.81±2.01 (2H, m), 2.06±
2.24 (2H, m), 2.49 (1H, m), 2.75 (1H, m), 3.38 (1H, s,
OH), 4.93 (1H, m), 7.25±7.41 (5H, m); HRMS calcd for
C13H14O3Na (M+Na)+: 241.0841, found 241.0827;
[a]2d0 50.0 (c=1.0, CHCl3).
(9).
To a solution of 8 (330 mg, 0.78 mmol) in THF (6 mL)
were added H2O (1 mL) and Ph3P (260 mg, 0.99 mmol),
and the mixture was heated at 75 ꢁC for 6 h. After eva-
poration of THF, the mixture was acidi®ed with 1 N
HCl and washed with CHCl3. The aqueous layer was
basi®ed with 1 N NaOH and extracted three times with
CHCl3. The combined extract was dried over MgSO4
and concentrated in vacuo to produce the crude amine
(290 mg), which was used in the next step without
further puri®cation: 1H NMR (CDCl3) d 1.27±2.23
(14H, m), 1.61 (3H, s), 1.69 (3H, s), 2.26±2.38 (2H, m),
2.70±2.90 (2H, m), 3.28 (1H, m), 3.69 (1H, m), 5.08 (1H,
m), 7.25±7.40 (3H, m), 7.53±7.63 (2H, m); MS m/z 426
(M+H)+.
(2R,5R)-2-tert-Butyl-5-[(1S,3S)-3-azidocyclopentyl]-5-
phenyl-1,3-dioxolan-4-one (7). To a solution of 6 (10.5
g, 34.5 mmol) and Et3N (7.20 mL, 51.7 mmol) in AcOEt
(350 mL) was added MsCl (3.00 mL, 38.8 mmol) at 0 ꢁC
and the mixture was stirred for 1 h. After addition of
saturated aq NaHCO3, the organic layer was separated,
washed with H2O and brine and dried over MgSO4. The
solvent was evaporated to dryness and the resulting
mesylate (13.5 g) was used in the next step without

M. Mitsuya et al. / Bioorg. Med. Chem. 8 (2000) 825±832
831
(2R)-N-[1-(4-Methyl-3-pentenyl)piperidin-4-yl]-2-[(1S,3S)-
3-acetylaminocyclopentyl]-2-hydroxy-2-phenylacetamide
(10a). To a solution of 9 (40 mg, 0.10 mmol) and Et3N
(30 mL, 0.22mmol) in CHCl3 (2 mL) was added acetyl
chloride (15 mL, 0.21 mmol) at 0 ꢁC, and the mixture
was stirred at the same temperature for 2 h. The excess
amount of acetyl chloride was quenched with 3 N
NaOH. The organic layer was separated, washed with
brine and dried over MgSO4. Removal of the solvent in
vacuo gave the crude residue, which was puri®ed by
preparative TLC (CHCl3:MeOH=9:1) to yield 10a (27
1H NMR (CDCl3) d 1.19±2.20 (14H, m), 1.60 (3H, s), 1.68
(3H, s), 2.25±2.40 (2H, m), 2.72±2.90 (2H, m), 3.26 (1H,
m), 3.69 (1H, m), 4.06 (1H, m), 4.87 (1H, m), 5.06±5.17
(3H, m+s), 6.49 (1H, m), 7.21±7.41 (8H, m), 7.56 (2H,
brd, J=7.2 Hz); HRMS calcd for C32H44N3O4 (M+H)+:
534.3332, found 534.3312; [a]2d0 9.4 (c=1.0, CHCl3).
(2R)-N-[1-(4-Methyl-3-pentenyl)piperidin-4-yl]-2-[(1S,3S)-
3-methanesulfonylaminocyclopentyl]-2-hydroxy-2-phenyl-
acetamide (10f). Compound 10f was obtained from 9
and methanesulfonyl chloride (48%): 1H NMR (CDCl3)
d 1.25±1.88 (8H, m), 1.60 (3H, s), 1.68 (3H, s), 1.95±2.19
(6H, m), 2.25±2.34 (2H, m), 2.70±2.93 (2H, m), 2.98
(3H, s), 3.25 (1H, m), 3.52 (1H, brs, OH), 3.68 (1H, m),
3.90 (1H, m), 4.49 (1H,m), 5.06 (1H, m), 6.58 (1H, brd,
J=8.1 Hz), 7.23±7.40 (3H, m), 7.54 (2H, brd, J=7.5
Hz); MS m/z 478 (M+H)+; mp 211±212 ꢁC (AcOEt);
anal. calcd for C25H39N3O4S: C, 62.86; H, 8.23; N, 8.80;
S, 6.71. Found: C, 62.89; H, 8.55; N, 8.79; S, 6.92; [a]d20
14.4 (c=1.0, CHCl3).
1
mg, 61%) as a white solid: H NMR (CDCl3) d 1.18±
2.20 (14H, m), 1.60 (3H, s), 1.68 (3H, s), 1.96 (3H, s),
2.28±2.39 (2H, m), 2.72±2.90 (2H, m), 3.29 (1H, m), 3.37
(1H, brs, OH), 3.69 (1H, m), 4.28 (1H, m), 5.07 (1H, m),
5.54 (1H, brd, J=7.5 Hz), 6.58 (1H, brd, J=8.4 Hz),
7.22±7.40 (3H, m), 7.58 (2H, brd, J=8.4 Hz); MS m/z
442 (M+H)+; mp 139.5±141 ꢁC (hexane:AcOEt); anal.
calcd for C26H39N3O3: C, 70.71; H, 8.90; N, 9.52.
Found: C, 70.54; H, 9.03; N, 9.50; [a]2d0 +5.0 (c=1.0,
CHCl3). The following compounds (10b±j) were pre-
pared from 9 and the appropriate acid chlorides in a
similar method described for 10a.
(2R)-N-[1-(4-Methyl-3-pentenyl)piperidin-4-yl]-2-[(1S,3S)-
3-phenylsulfonylaminocyclopentyl]-2-hydroxy-2-phenyl-
acetamide (10g). Compound 10g was obtained from 9
and phenylsulfonyl chloride (63%): 1H NMR (CDCl3) d
1.17±1.88 (10H, m), 1.60 (3H, s), 1.68 (3H, s), 2.03±2.21
(4H, m), 2.29±2.40 (2H, m), 2.76±2.93 (2H, m), 3.22 (1H,
m), 3.58±3.68 (2H, m), 5.06 (1H, m), 5.41 (1H, m), 6.38
(1H, brd, J=8.2 Hz), 7.20±7.36 (3H, m), 7.46±7.60 (5H,
m), 7.87±7.95 (2H, m); MS m/z 540 (M+H)+; mp 197.5±
198.5 ꢁC (hexane:AcOEt); anal. calcd for C30H41N3O4S:
C, 66.76; H, 7.66; N, 7.79; S, 5.94. Found: C, 66.65; H,
7.95; N, 7.74; S, 6.11; [a]2d0 19.2 (c=1.0, CHCl3).
(2R)-N-[1-(4-Methyl-3-pentenyl)piperidin-4-yl]-2-[(1S,3S)-
3-phenylacetylaminocyclopentyl]-2-hydroxy-2-phenylace-
tamide (10b). Compound 10b was obtained as an oil
from 9 and phenylacetylchloride (70%): 1H NMR
(CDCl3) d 1.16±2.20 (14H, m), 1.60 (3H, s), 1.68 (3H, s),
2.24±2.39 (2H, m), 2.70±2.90 (2H, m), 3.23 (1H, m), 3.53
(2H, s), 3.69 (1H, m), 4.24 (1H, m), 5.07 (1H, m), 5.56
(1H, brd, J=7.6 Hz), 6.58 (1H, brd, J=8.2 Hz), 7.20±
7.41 (8H, m), 7.55 (2H, brd, J=7.2 Hz); HRMS calcd
for C32H44N3O3 (M+H)+: 518.3383, found 518.3383;
[a]2d0 +13.4 (c=1.0, CHCl3).
(2R)-N-[1-(4-Methyl-3-pentenyl)piperidin-4-yl]-2-[(1S,3S)-
3-(4-nitrophenyl)sulfonylaminocyclopentyl]-2-hydroxy-2-
phenylacetamide (10h). 10h was obtained from 9 and 4-
(2R)-N-[1-(4-Methyl-3-pentenyl)piperidin-4-yl]-2-[(1S,3S)-
3-phenylglyoxylaminocyclopentyl]-2-hydroxy-2-phenyl-
acetamide (10c). Compound 10c was obtained as an oil
from 9 and phenylglyoxyl chloride (94%): 1H NMR
(CDCl3) d 1.30±1.92 (8H, m), 1.60 (3H, s), 1.68 (3H, s),
2.03±2.21 (6H, m), 2.28±2.40 (2H, m), 2.72±2.91 (2H, m),
3.32 (1H, m), 3.70 (1H, m), 4.38 (1H, m), 5.06 (1H, m),
6.38 (1H, m), 7.11 (1H, m), 7.25±7.41 (3H, m), 7.43±7.69
(5H, m), 8.33 (2H, brd, J=7.1 Hz); HRMS calcd for
C32H42N3O4 (M+H)+: 532.3175, found 532.3161; [a]2d0
6.6 (c=1.0, CHCl3).
1
nitrophenylsulfonyl chloride (61%): H NMR (CDCl3)
d 1.18±2.28 (14H, m), 1.62 (3H, s), 1.69 (3H, s), 2.35±
2.48 (2H, m), 2.80±3.00 (2H, m), 3.22 (1H, m), 3.50±3.87
(3H, m), 5.06 (1H, m), 6.39 (1H, m), 7.20±7.39 (3H, m),
7.50 (2H, brd, J=8.3 Hz) 8.10 (2H, brd, J=9.0 Hz) 8.36
(2H, brd, J=9.0 Hz); MS m/z 585 (M+H)+; IR (KBr)
3250, 1650, 1543, 1525, 1352, 1336, 1157 cm 1; mp 208±
210 ꢁC (hexane:AcOEt); anal. calcd for C30H40N4
.
O6S 0.25H2O: C, 61.15; H, 6.93; N, 9.51; S, 5.44.
Found: C, 61.25; H, 7.03; N, 9.44; S, 5.04; [a]2d0 13.4
(c=1.0, CHCl3).
(2R)-N-[1-(4-Methyl-3-pentenyl)piperidin-4-yl]-2-[(1S,3S)-
3-methoxycarbonylaminocyclopentyl]-2-hydroxy-2-phenyl-
acetamide (10d). Compound 10d was obtained as an oil
(2R)-N-[1-(4-Methyl-3-pentenyl)piperidin-4-yl]-2-[(1S,3S)-
3-(4-methoxyphenyl)sulfonylaminocyclopentyl]-2-hydroxy-
2-phenylacetamide (10i). 10i was obtained as an oil from
9 and 4-methoxyphenylsulfonyl chloride (84%): 1H NMR
(CDCl3) d 1.19±2.24 (14H, m), 1.61 (3H, s), 1.69 (3H, s),
2.28±2.41 (2H, m), 2.71±2.92 (2H, m), 3.20 (1H, m),
3.58±3.82 (3H, m), 3.87 (3H, s), 4.91 (1H, m, NH), 5.06
(1H, m), 6.25 (1H, brd, J=8.1), 6.98 (2H, d, J=9.0),
7.20±7.38 (3H, m), 7.51 (2H, brd, J=7.2 Hz) 7.82 (2H, d,
J=9.0 Hz); HRMS calcd for C31H44N3O5S (M+H)+:
570.3002, found 570.3012; [a]2d0 20.8 (c=1.0, CHCl3).
1
from 9 and methoxycarbonyl chloride (57%): H NMR
(CDCl3) d 1.20±2.20 (14H, m), 1.60 (3H, s), 1.68 (3H, s),
2.26±2.38 (2H, m), 2.71±2.90 (2H, m), 3.25 (1H, m), 3.65
(3H, s), 3.68 (1H, m), 4.03 (1H, m), 4.72 (1H, m), 5.07
(1H, m), 6.44 (1H, m), 7.21±7.40 (3H, m), 7.56 (2H, brd,
J=7.2 Hz); HRMS calcd for C26H40N3O4 (M+H)+:
458.3019, found 458.2996; [a]2d0 13.4 (c=1.0, CHCl3).
(2R)-N-[1-(4-Methyl-3-pentenyl)piperidin-4-yl]-2-[(1S,3S)-
3-benzyloxylcarbonylaminocyclopentyl]-2-hydroxy-2-
phenylacetamide (10e). Compound 10e was obtained as
an oil from 9 and benzyloxycarbonyl chloride (72%):
(2R)-N-[1-(4-Methyl-3-pentenyl)piperidin-4-yl]-2-[(1S,3S)-
3-(4-tri¯uoromethylphenyl)sulfonylaminocyclopentyl]-2-

832
M. Mitsuya et al. / Bioorg. Med. Chem. 8 (2000) 825±832
hydroxy-2-phenylacetamide (10j). Compound 10j was
obtained as an oil from 9 and 4-tri¯uorophenylsulfonyl
chloride (68%): 1H NMR (CDCl3) d 1.18±2.29 (14H, m),
1.60 (3H, s), 1.68 (3H, s), 2.33±2.45 (2H, m), 2.80±3.00
(2H, m), 3.22 (1H, m), 3.58±3.85 (3H, m), 5.05 (1H, m),
6.40 (1H, brd, J=8.4), 7.20±7.39 (3H, m), 7.50 (2H, brd,
J=7.0 Hz) 7.78 (2H, d, J=8.5 Hz) 8.03 (2H, d, J=8.5
Hz); HRMS calcd for C31H41N3O4F3S (M+H)+:
608.2770, found 608.2751; [a]2d0 13.0 (c=1.0, CHCl3).
synthesized standard samples (tR: 5b 11.5 min, 5c 14.7
min, 5e 11.2 min, 5f 14.2 min; Capcell pak C18 UG-120
4.6Â250 mm, eluent CH3CN:H2O:100 mM AcONH4
(25:65:10±55:35:10 for 20 min), ¯ow rate=0.5 mL/min,
oven temperature 40 ꢁC, UV detection 220 nm).
Acknowledgements
We are grateful to Ms. T. Ahmed and Ms. A. Dobbins,
Merck & Co., for critical reading of this manuscript.
We also acknowledge Mr. S. Abe and Ms. A. Shimizu
for analytical support.
Binding assay. According to the reported method,9 the
binding anities were determined by inhibition of spe-
ci®c binding of [3H]-NMS using membranes from CHO
cells expressing cloned human m1±m3 receptors.
References
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rats and female dogs. Human liver microsomes were
obtained from Pennsylvania Regional Tissue Bank via
KAC. The test compound (10 mM) was incubated at
37 ꢁC for 30 min with the hepatic microsomes (1 mg
protein/mL) in the presence of an NADPH-generating
system. The reaction was terminated by mixing 4
volumes of EtOH to the medium. After centrifugation,
the supernatant was analyzed for concentration of
the test compound by HPLC (GL-Science Inertsil ODS-
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taining 10 mM AcONH4, ¯ow rate=0.1 mL/min, oven
temperature 40 ꢁC, electrochemical detection 950 mV).
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Identi®cation of the metabolites of 1 in rat microsomes.
Compound 1 (100 mM) was incubated at 37 ꢁC for 2 h
with rat hepatic microsomes (3 mg protein/mL) in the
presence of an NADPH-generating system. The reac-
tion was terminated by adding 4 volumes of EtOH. The
supernatant was obtained after the centrifugation of the
mixture and concentrated to dryness under N2-¯ow.
The residue was dissolved in a small volume of
CH3CN:H2O (55:45) containing 10 mM AcONH4 and
®ltered. The ®ltrate was analyzed by LC/MS/MS (GL-
Science Inertsil ODS-3.5 mm, 1.5Â150 mm, eluent
CH3CN:H2O (20:80±70:30) containing 10 mM
AcONH4, ¯ow rate=0.1 mL/min, oven temperature
40 ꢁC, tandem mass Quattro 2 Micromass, ES+).
Hydroxylated compounds (m/z 401 (M+H)+) were
detected. Among these, 5b, 5c, 5e and 5f were identi®ed
by comparison of their retention times with those of the