J-104129, a novel muscarinic M3 receptor antagonist with high selectivity for M3 over M2 receptors

Article abstract of DOI:10.1016/S0968-0896(99)00177-7

A new class of 4-acetamidopiperidine derivatives has been synthesized and investigated for human muscarinic receptor subtype selectivity. Introduction of a hydrocarbon chain of appropriate length into the piperidine nitrogen of the racemic N-(piperidin-4-

Full text of DOI:10.1016/S0968-0896(99)00177-7

Bioorganic & Medicinal Chemistry 7 (1999) 2555±2567
J-104129, a Novel Muscarinic M3 Receptor Antagonist with High
Selectivity for M3 over M2 Receptors
Morihiro Mitsuya,* Toshiaki Mase,* Yoshimi Tsuchiya, Kumiko Kawakami,
Hiromi Hattori, Kensuke Kobayashi, Yoshio Ogino, Toru Fujikawa, Akio Satoh,
Toshifumi Kimura, Kazuhito Noguchi, Norikazu Ohtake and Koji Tomimoto
Banyu Tsukuba Research Institute in collaboration with Merck Research Laboratories, Okubo 3, Tsukuba 300-2611, Ibaraki, Japan
Received 22 April 1999; accepted 23 June 1999
AbstractÐA new class of 4-acetamidopiperidine derivatives has been synthesized and investigated for human muscarinic receptor
subtype selectivity. Introduction of a hydrocarbon chain of appropriate length into the piperidine nitrogen of the racemic N-
(piperidin-4-yl)-2-cyclobutyl-2-hydroxy-2-phenylacetamide platform conferred up to 70-fold selectivity for human muscarinic M3
receptors over M2 receptors. Subsequent synthetic derivatizations resulted in highly potent M3 receptor antagonists with selectivity
greater than two orders of magnitude for M3 over M2 receptors, from which the analogue 4r was selected. Preparation of both
enantiomers of 4r led to the identi®cation of (2R)-N-[1-(4-methyl-3-pentenyl)piperidin-4-yl]-2-cyclopentyl-2-hydroxy-2-phenylace-
tamide (J-104129, (R)-4r), which exhibited 120-fold selectivity for M3 receptors (Ki=4.2 nM) over M2 receptors (Ki=490 nM). In
isolated rat trachea, (R)-4r potently and speci®cally antagonized acetylcholine (ACh)-induced responses with a KB value of 3.3 nM.
The highly subtype-selective pro®le was also seen in isolated rat tissue assays (50-fold) and in anesthetized rats (>250-fold). Oral
administration of J-104129 ((R)-4r) antagonized ACh-induced bronchoconstriction with an ED50 value of 0.58 mg/kg in rats. Thus,
J-104129 ((R)-4r) may e€ectively facilitate bronchodilation in the treatment of obstructive airway disease. # 1999 Elsevier Science
Ltd. All rights reserved.
Introduction
suggested the presence of four muscarinic receptors,
denoted M1, M2, M3 and M4; the physiological role(s)
of the m5 gene product remains to be identi®ed.6,7 The
di€erent localizations and functions of these receptor
subtypes raised the possibility of designing antagonists
that selectively interact with distinct subtypes, thus
avoiding the occurrence of adverse e€ects.
Anti-cholinergic drugs have been used for a century in
the treatment of obstructive airway diseases. Although
classical muscarinic antagonists such as atropine are
potent bronchodilators, their clinical utility is limited
due to the high occurrence of both peripheral and cen-
tral adverse e€ects such as tachycardia, blurred vision
and dementia. Subsequent development of the qua-
ternary derivatives of atropine such as ipratropium
bromide resulted in the potential clinical use of mus-
carinic antagonists as aerosol agents. Although they are
better tolerated than parenterally administered atropine,
the quartenary derivatives may be not ideal anti-choli-
nergic bronchodilators because of their short duration
and non-selective pro®le for muscarinic receptor subtypes.
The airways of several species including humans contain
at least three pharmacological muscarinic receptor sub-
types as follows:8,9 neuronal M1 receptors, which facil-
itate parasympathetic neurotransmission; neuronal
presynaptic M2 receptors, which inhibit acetylcholine
(ACh) release as part of a negative feedback mechan-
ism; and smooth muscle M3 receptors, which mediate
contraction and secretion. There are a number of
reports indicating that non-selective muscarinic antago-
nists increase ACh release from isolated human and
guinea pig bronchial tissues and potentiate broncho-
constriction in certain animals by blocking the neuronal
M2 receptors after vagal stimulation.10±13 Furthermore,
we recently demonstrated that the blockade of airway
neuronal M2 receptors stimulates SO2-induced mucus
hypersecretion in a rat bronchitis model.14 Therefore,
In the last decade, ®ve muscarinic receptor subtypes
have been identi®ed and cloned: they are classi®ed as
m1, m2, m3, m4 and m5.1±5 Pharmacological criteria
Key words: Antagonist; bronchodilators; cholinergic activity; receptors.
* Corresponding authors.
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00177-7

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M. Mitsuya et al. / Bioorg. Med. Chem. 7 (1999) 2555±2567
identi®cation of M3 antagonists with far greater selectiv-
ity for M3 over airway neuronal M2 receptors might lead
to an ideal anti-cholinergic bronchodilator with greater
ecacy than the existing non-selective antagonists.8±10,15
A number of studies in the muscarinic ®eld have sug-
gested that binding is initiated by a tight ion±ion inter-
action between the quarternary ammonium moiety of a
ligand and the aspartic residue in the third transmem-
brane of the receptor proteins.16,17 It has been very dif-
®cult to distinguish between receptor subtypes due to a
high degree of sequence homology, especially in the
close circumstance of the binding space around the
aspartic (Asp) residue. In fact, only a few compounds
such as revatropate,10 darifenacin10 and Scios Nova
compounds 118 have been reported to show subtype
selectivity for M3 over M2 receptors. Accordingly, fur-
ther exploration has been desired to develop a new class
of muscarinic antagonists highly selective for M3 over
M2 receptors.
In the course of our program to develop a novel
bronchodilator with fewer muscarinic side e€ects, we
designed a new class of 4-acetamidopiperidine deriva-
tives 4 as the lead structure based on the structural fea-
tures of the following two compounds: the Scios Nova
compounds 1 exhibiting good selectivity for M3 over M1
and M2 receptors in isolated tissues assays18 and the
amide analogue 3 of oxybutynin (2),19,20 which was
reported not only to protect against hydrolysis by liver
esterase but also to reduce several side e€ects, probably
due to di€erential tissue distribution (Fig. 1).21,22 The
synthesized derivatives were tested for their binding
anities for human m1, m2 and m3 receptors expressed in
CHO cells. Further evaluation of the selected compound
was conducted in in vitro and in vivo functional assays.
Figure 1.
with mesylates or halides in the presence of a base
(method A) or was subjected to reductive alkylation
with the corresponding aldehydes (method B).
The synthesis of 4q±t, 14a±c, 15a, 15b and 18 is outlined
in Schemes 2 and 3. Hydroxy or aminopiperidine deri-
vative 12, 13 was prepared from commercially available
4-piperidone hydrochloride (9) and 5-bromo-2-methyl-
2-pentene (10) in two steps involving alkylation and
reduction or reductive amination, respectively. The
coupling reaction of the compounds 12, 13 with acids
5a±g completed the synthesis of the derivatives 4q±t,
14a±c under similar conditions described for the pre-
paration of the compound 7.
Chemistry
The 4-acetamidopiperidine derivatives 4a±k, 4o and 4p
were generally synthesized as racemates by the methods
described in Scheme 1. The usual coupling of racemic
carboxylic acid 5a with 4-amino-1-tert-butyloxy-
carbonylpiperidine (6) followed by acidic deprotection
gave amide 8. Subsequently, the amide 8 was alkylated
Scheme 1. Conditions: (a) WSC±HCl, HOBt, CHCl3; (b) HCl±MeOH; (c) mesylate or halide, K2CO3, CH3CN; (d) aldehyde, NaB(OAc)3H, THF.

M. Mitsuya et al. / Bioorg. Med. Chem. 7 (1999) 2555±2567
2557
Carbamate 15a and urea 15b were prepared from acid
5c. Curtius rearrangement of 5c followed by treatment
of the resulting isocyanate with 12 or 13 gave 15a and
15b, respectively.
iodomethane in the presence of sodium hydride and
tetra-n-butylammonium iodide to yield 17. Deprotec-
tion of the trimethylsilyl and tert-butyloxycarbonyl
groups by acid exposure and subsequent alkylation with
the bromide 10 yielded the desired N-methylamide 18.
N-Methylamide derivative 18 was obtained from the
common intermediate 7, in which the hydroxy group
was protected as trimethylsilylether to give 16. The
amide moiety of 16 was alkylated by treatment with
As for 4r, optically active compounds (R)-4r and (S)-4r
were prepared to determine the biologically active
enantiomer and for use in further biological evaluations.
Scheme 2. Conditions: (a) 5-bromo-2-methyl-2-pentene 10, K2CO3, Kl, DMF; (b) NaBH4, MeOH; (c) AcONH4, NaBCNH3, MeOH; (d) 12 or 13,
WSC±HCl, HOBt; (e) DPPA, toluene then 12 or 13.
Scheme 3. Conditions: (a) TMSCl, imidazole, DMF; (b) NaH, Mel, n-Bu4Nl, THF; (c) HCl±MeOH then 10, K2CO3, Kl, DMF.

2558
M. Mitsuya et al. / Bioorg. Med. Chem. 7 (1999) 2555±2567
Optical resolution of racemic 2-cyclopentyl-2-hydroxy-
2-phenylacetic acid 5e was achieved by repetitive
recrystallization of its cinchonidine salt, avoiding the
use of amphetamine.23 Subsequent treatment with hy-
drochloric acid a€orded the chiral acid ( )-5e in >99%
ee and 24% chemical yield. The absolute con®guration
of the acid ( )-5e was determined to be R by the X-ray
crystallographic analysis of (R)-mandelic acid salt of
(R)-( )-4r.24 The antipodal (S)-4r was obtained by pre-
parative HPLC (98% ee).
pounds shown in Table 1, the derivative 4g was selected
for further optimization of the other parts of the mole-
cule (Table 2).
Removal of the tert-alcohol (14b) and methylation of
the amide (18) or the alcohol (14a) reduced both selec-
tivity and anity, indicating that a hydrogen bonding
interaction contributes to receptor binding and espe-
cially to selectivity for m3 over m2 receptors. On the
other hand, the ester analogue 14c exhibited remarkably
high anity (m3=0.28 nM) but low selectivity (m3/
m2=13). This ®nding coincides with a number of
reports that known ester classes of compounds potently
bind to all receptor subtypes in a non-selective man-
ner.25,26 The carbamate analogue 15a also enhanced
binding anity (m3=3.8 nM) despite a lack of tert-hy-
droxy function, while urea 15b showed lower anity
(m3=86 nM). Among these compounds with a variety
of linkage moieties, 4g with the tert-alcohol and amide
linkage displayed the best selectivity (m3/m2=50).
Results and Discussion
This investigation was prompted by the observation
that the N-benzyl and 1,1-diphenyl-3-piperazinylpro-
panone derivatives (Nova compounds, 1a, 1b) showed
high selectivity for M3 over M2 receptors in isolated
tissues assays.18 We speculated that the substituents on
the piperazine nitrogen and at the 1-position of the
1-phenyl-3-piperadinylpropan-2-one may contribute to
di€erentiation among the receptor subtypes. In our
acetamidopiperidine series 4, an N-substituent (R0) on
the piperidine ring would be one of the most e€ective
moieties for improving the subtype selectivity for M3
over M2 receptors according to the above speculation.
Therefore, extensive investigation of the substituent on
the piperidine nitrogen of acetamidopiperidine series 4
was made a priority, whereas a cyclobutylmandelic acid
moiety was ®xed to interpret the correlation between
Nova's and our compounds. After selecting a favorable
N-substituent, further optimization of the other func-
tional groups, e.g. the cyclobutyl, hydroxyl and amide
moieties, was performed systematically to identify a
compound with the best selectivity.
Subsequently, the cyclobutyl moiety of 4g was opti-
mized by replacing it with di€erent ring sized cycloalk-
yls and a phenyl group. The ring expansion of the
cyclobutyl moiety in 4g gave cyclopentyl and cyclohexyl
derivatives 4r, 4s which exhibited greatly improved
subtype selectivity (4r: m3/m2=120, 4s: m3/m2=110),
whereas the ring size reduction (4q) or replacement with
a phenyl group (4t) greatly reduced the selectivity (m3/
m2=21 or 26). This result indicated that the substituent
on the manderoyl group also played an important role
in the improvement of subtype selectivity. Thus, the
cyclopentyl group at this position was superior with
respect to selectivity and anity (Table 3).
Finally, to determine the absolute con®guration of the
biologically active enantiomer, (R)-4r and (S)-4r were
prepared and compared in binding assays as shown in
Table 4. The (R)-form showed much higher anity than
did the antipode, as found in other antagonists with a
similar structure.21,27 Furthermore, (R)-4r retained high
m3/m2 selectivity (120-fold), while the antipode (S)-4r
showed moderate selectivity (28-fold). Thus, we identi-
®ed (R)-4r as the best compound in the series of 4-acet-
amidopiperidine derivatives.
First, introduction of low alkyl groups such as methyl
and butyl substituents into the piperidine nitrogen gave
compounds 4a, 4b that showed less than 10-fold m3
over m2 selectivity, comparable to that of the corre-
sponding piperazinylpropanone derivatives.18 When a
benzyl group was introduced, the m3/m2 selectivity of
the resulting derivative 4o did not improve despite good
binding anity. As the ®rst example of a compound
exhibiting more than 20-fold m3/m2 selectivity, the
pentyl derivative 4c with a low anity for m3 receptors
(Ki=240 nM) was identi®ed. This result prompted us to
perform further derivatization by introducing various
types of long hydrocarbon chains and branched chains
to improve m3/m2 selectivity. Surprisingly, the 4-
methylpentyl and 4-methylpentenyl derivatives 4e, 4g
and 4h prepared as representatives with a branched
hydrocarbon side chain showed approximately 50-fold
m3 selectivity over m2 receptors. This subtype selectiv-
ity was maximized in the case of the (4R)-4-methylhexyl
derivative 4i (m3/m2=71). In contrast, introduction of
ether (4k), amide (4l), alcohol (4m) and carboxylic acid
(4n) moieties into the side chain resulted in a consider-
able decrease in the binding anity. Thus, we found
that the nature of the N-substituent on the piperidine in
this series signi®cantly in¯uenced not only receptor
subtype selectivity but also anity for m3 receptors.
Based on the selectivities and anities of the com-
Next, we evaluated (R)-4r in isolated tissues assays to
determine whether it is a highly selective M3 receptor
antagonist over M2 receptors. Several functional assays
in isolated tissues have been pharmacologically
well-characterized.27±29 Carbachol-induced contractile
responses in isolated rat trachea are mediated through
M3 receptors. McN A-343-induced inhibition of twitch
contraction in isolated rabbit vas deferens and carba-
chol-induced bradycardia in isolated rat right atria are
mediated through M1 and M2 receptors, respectively.
As shown in Table 5, in isolated rat trachea, (R)-4r
potently and speci®cally antagonized the ACh-induced
responses with a KB value of 3.3 nM. In isolated rat
right atria, (R)-4r showed less potent inhibition of car-
bachol-induced bradycardia with a KB value of 170 nM.
In isolated rabbit vas deferens, (R)-4r antagonized the
inhibition of McN A-343 against the twitch response

M. Mitsuya et al. / Bioorg. Med. Chem. 7 (1999) 2555±2567
Table 1. Binding anity to muscarinic receptors (1)
2559
Compound
R
% Yield (method)
Binding anity (Ki, nM)a
Selectivity
m1
m2
m3
m1/m3
m2/m3
4a
4b
Me
59 (B)
84 (B)
180
1500
160
1.1
1.9
9.0
7.8
2900
12,000
1500
4c
4d
4e
4f
57 (B)
59 (B)
77 (A)
70 (A)
62 (A)
52 (A)
46 (A)
1400
1300
750
270
130
300
690
6500
4400
3700
1900
1300
1600
4800
240
130
73
5.9
10
27
35
51
37
50
54
71
10
52
5.2
4.9
9.7
10
4g
4h
4i
26
30
68
4j
72 (A)
2300
2000
1400
6500
31,000
680
530
500
3.4
3.8
2.9
Ð
10
58
40
Ð
4k
4l
63 (A)
b
20,000
b
b
4m
>3000
>2900
>60,000
>3000
>2800
4n
4o
4p
a
>59,000
35
Ð
Ð
14
4.2
80 (B)
74 (B)
4.8
130
2.1
2.3
2.7
210
50
Values are the mean of two or more independent assays.
See Experimental.
b
Table 2. Binding anity to muscarinic receptors (2)
Compound
R1
R2
% Yield (method)
Binding anity (Ki, nM)a
Selectivity
m1
m2
m3
m1/m3
m2/m3
4g
18
OH
OH
OMe
H
OH
H
CONH
CONMe
CONH
CONH
COO
62 (A)
b
130
79
>3000
990
1300
150
15,000
5100
3.5
110
3000
26
140
>2900
250
4.9
0.6
Ð
4.0
3.5
3.8
4.0
50
1.1
Ð
21
13
30
34
14a
14b
14c
15a
15b
50 (C)
40 (C)
34 (C)
23 (D)
49 (D)
0.97
14
340
0.28
3.8
86
NHCOO
NHCONH
H
a
Values are the mean of two or more independent assays.
See Experimental.
b

2560
M. Mitsuya et al. / Bioorg. Med. Chem. 7 (1999) 2555±2567
Table 3. Binding anity to muscarinic receptors (3)
Compound
R
% Yield (method)
Binding anity (Ki, nM)a
Selectivity
m1
m2
m3
m1/m3
m2/m3
4g
4q
4r
4s
62 (A)
60 (C)
59 (C)
50 (C)
62 (C)
130
670
25
1300
2700
760
26
130
6.5
8.7
67
4.9
5.1
3.8
1.8
2.6
50
21
120
110
26
16
940
4t
173
1700
a
Values are the mean of two or more independent assays.
induced by electrical ®eld stimulation, with a KB value
of 14 nM, exhibiting modest tracheal selectivity over
rabbit vas deferens. Accordingly, (R)-4r is a potent M3
receptor antagonist with 50-fold selectivity for tracheal
M3 over cardiac M2 receptors.
Table 5. The Kb values (nM) of (R)-4r and atropine in functional
assays using isolated tissuesa
Compound
Rabbit vas
deferens (M1)
Rat right
atria (M2)
Rat trachea
(M3)
(R)-4r
Atropine
14
0.11
170
1.5
3.3
0.49
Furthermore, the in vivo selectivity of (R)-4r was
examined in rats (Table 6). Intravenous administration
of (R)-4r dose-dependently inhibited ACh-induced
bronchoconstriction with an ED50 value of 0.012 mg/
kg. In contrast, intravenous administration of (R)-4r up
to 3 mg/kg did not a€ect the ACh-induced bradycardia
that was mediated by M2 receptors. These results indi-
cated that (R)-4r showed >250-fold bronchial selectivity
over heart.
a
Values are the mean of more than three experiments.
Table 6. In vivo activity in rats
Compound
Bronchus (M3)
Heart (M2)
ED50 (iv)
ED50 (iv)a
ED50 (po)a
0.58
(R)-4r fumarate
0.012
(1)c
<3b
(>250)c
0.0037
(0.9)c
Finally, we examined the oral activity of (R)-4r in anes-
thetized rats. Oral administration also potently antag-
onized ACh-induced bronchoconstriction with an ED50
value of 0.58 mg/kg, indicating that (R)-4r is a potent
oral bronchodilator.
Atropine
0.0043
(1)c
0.27
a
ED50 values (mg/kg) are the mean of three experiments.
52.7‹2.2% inhibition at 3 mg/kg, n ˆ 4.
b
c
Values in parentheses represent selectivity for M3 (-fold).
Table 4. Binding anity of (R)-4r and (S)-4r
Conclusion
For the purpose of developing a selective M3 receptor
antagonist as a bronchodilator, a new class of 4-acet-
amidopiperidine derivatives was prepared and evaluated
for anti-muscarinic activities. Among these derivatives,
(2R)-N-[1-(4-methyl-3-pentenyl)piperidin-4-yl]-2-cyclo-
pentyl-2-hydroxy-2-phenylacetamide (R)-4r (J-104129),
displayed 120-fold selectivity for m3 over m2 receptors
with a Ki value of 4.2 nM for anity in the binding
assay. This highly subtype-selective pro®le was also seen
in both in vitro (isolated tissues) and in vivo studies
(antagonism for ACh-induced bronchoconstriction versus
ACh-induced bradycardia in rats). Furthermore, it was
Compound
Binding anity (Ki, nM)a
m1 m2 m3
490
Selectivity
m1/m3
m2/m3
(R)-4r
(S)-4r
4r
19
1700
25
4.2
540
6.5
4.5
3.1
3.8
120
28
120
15,000
760
a
Values are the mean of two or more independent assays.

M. Mitsuya et al. / Bioorg. Med. Chem. 7 (1999) 2555±2567
2561
demonstrated that (R)-4r was an orally active M3 receptor
antagonist in rats. Since (R)-4r (J-104129) with high
selectivity for M3 over airway neuronal M2 receptors
should not induce M2-mediated adverse e€ects such as
tachycardia and paradoxical bronchoconstriction, it may
provide more e€ective anti-cholinergic therapy than do
non-selective muscarinic antagonists in obstructive airway
diseases. Detailed pharmacological and pharmacokinetic
pro®les of this compound will be reported elsewhere.
N-[1-(4-Methyl-4-pentenyl)piperidin-4-yl]-2-cyclobutyl-2-
hydroxy-2-phenylacetamide (4f). To a solution of 4-
methyl-4-pentenol32 (50 mg, 0.50 mmol) in AcOEt (5
mL) were added triethylamine (0.12 mL, 0.86 mmol)
and mesylchloride (0.05 mL, 0.65 mmol) at 0ꢀC. After
stirring for 1 h, the mixture was quenched with sat.
NaHCO3 and further stirred for 1 h at room tempera-
ture. The organic layer was separated, washed with
brine and dried over MgSO4. The solvent was evapo-
rated under reduced pressure to give the crude mesylate
which was used in the next step without puri®cation. To
a suspension of 8 (130 mg, 0.40 mmol), K2CO3 (200 mg,
1.45 mmol) and potassium iodide (10 mg, 0.06 mmol) in
CH3CN was added the crude mesylate, and the mixture
was heated at 75ꢀC for 4 h. After cooling to room tem-
perature, the mixture was diluted with H2O and extrac-
ted twice with CHCl3. The solvent was evaporated
under reduced pressure and the resulting residue was
puri®ed by silica gel column chromatography
(CHCl3:MeOH=100:1) to yield 4f (104 mg, 70%) as an
Experimental
Melting points 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. IR spectra were recorded with Horiba FT-200
spectrometer. Mass spectrometry were performed with
JEOL JMS-SX 102A. Optical rotations were measured
with Jasco DIP-370 polarimeter. TLC were done with
Merck Kieselgel F254 pre-coated plates.
1
oil: H NMR (CDCl3) d 1.30±2.20 (16H, m), 1.71 (3H,
s), 2.24±2.39 (2H, m), 2.69±2.88 (2H, m), 3.29±3.58 (2H,
m), 3.71 (1H, m), 4.66 (1H, brd, J=1.4 Hz), 4.69 (1H,
brd, J=1.4 Hz), 6.15 (1H, brd, J=7.8 Hz), 7.21±7.41
(3H, m), 7.48 (2H, brd, J=8.4 Hz); MS m/z 371
N-(1-tert-Butyloxycarbonylpiperidin-4-yl)-2-cyclobutyl-
2-hydroxy-2-phenylacetamide (7). To a stirred solution
of 2-cyclobutyl-2-hydroxy-2-phenylacetic acid (5a)30
(1.43 g, 6.94 mmol) and 4-amino-1-tert-butyloxy-
carbonylpiperidine (6)31 (1.38 g, 6.90 mmol) in CHCl3
(60 mL) were sequentially added 1-hydroxybenzo-
triazole (1.39 g, 10.29 mmol), 1-(3-dimethylaminopro-
pyl)-3-ethylcarbodiimide (1.50 g, 7.82 mmol) at room
temperature, and the mixture was stirred for 15 h. The
mixture was diluted with Et2O and washed with satd
NaHCO3 and brine, and dried over MgSO4. Evapora-
tion of the solvent gave the crude product, which was
puri®ed by silica gel column chromatography to a€ord
7 (2.10 g,78%) as a white solid: mp 154.5±156ꢀC (from
ꢀ
+
.
(M+H) . 4f fumarate: mp 170±173 C (from i-Pr2O/i-
.
PrOH). Anal. calcd for C23H34N2O2 C4H4O4: C, 66.64;
H, 7.87; N, 5.76. Found: C, 66.44; H, 7.73; N, 5.71.
The following compounds 4e, 4g±k and 19 were pre-
pared from 8 and the appropriate alcohols or bromides
in a similar method described for 4f.
N-[1-(4-Methyl-1-pentyl)piperidin-4-yl]-2-cyclobutyl-2-
hydroxy-2-phenylacetamide (4e). 4e was obtained from
8 and 1-bromo-4-methylpentane (77%): 1H NMR
(CDCl3) d 0.87 (6H, d, J=6.6 Hz), 1.10±1.20 (2H, m),
1.34±1.60 (5H, m), 1.70±2.15 (10H, m), 2.24±2.33 (2H,
m), 2.72±2.86 (2H, m), 3.30±3.60 (2H, m), 3.72 (1H, m),
1
n-hexane/CHCl3); H NMR (CDCl3) d 1.15±1.35 (2H,
m), 1.44 (9H, s), 1.70±2.13 (8H, m), 2.75±2.91 (2H, m),
3.31 (1H, OH, s), 3.38 (1H, m), 3.75±4.07 (3H, m), 6.22
(1H, NH, brd, J=7.8 Hz), 7.20±7.39 (3H, m), 7.49 (2H,
brd, J=8.4 Hz); MS m/z 389 (M+H)+. Anal. calcd for
6.13 (1H, d, J=8.1 Hz), 7.22±7.38 (3H, m), 7.49 (2H,
+
.
brd, J=8.3 Hz); MS m/z 373 (M+H) . 4e fumarate:
mp 188±190ꢀC (from i-Pr2O/i-PrOH). Anal. calcd for
.
.
C23H36N2O2 C4H4O4: C, 66.37; H, 8.25; N, 5.73.
Found: C, 66.43; H, 8.60; N, 5.75.
C22H32N2O4 0.2H2O: C, 67.39; H, 8.33; N, 7.14. Found:
C, 67.19; H, 8.37; N, 7.13.
N-(Piperidin-4-yl)-2-cyclobutyl-2-hydroxy-2-phenylacet-
amide hydrochloride (8). A solution of 7 (2.00 g, 5.15
mmol) in 10% HCl±MeOH (60 mL) was stirred at room
temperature for 15 h and then evaporated to dryness in
vacuo to obtain the crude oily residue 8 (1.68 g) which
was used in the next step without further puri®cation:
1H NMR (CDCl3) d 1.24±1.35 (2H, m), 1.60±2.19 (8H,
m), 2.57±2.71 (2H, m), 2.90±3.09 (2H, m), 3.38 (1H, m),
3.78 (1H, m), 6.38 (1H, m), 7.18±7.40 (3H, m), 7.50 (2H,
brd, J=7.2 Hz); MS m/z 289 (M+H)+. Free base of 8:
mp 153±155ꢀC (from hexane±AcOEt). Anal. calcd for
N-[1-(4-Methyl-3-pentenyl)piperidin-4-yl]-2-cyclobutyl-2-
hydroxy-2-phenylacetamide (4g). 4g was obtained from 8
and 5-bromo-2-methyl-2-pentene (62%): 1H NMR
(CDCl3) d 1.38±1.52 (2H, m), 1.60 (3H, s), 1.68 (3H, s),
1.65±2.22 (12H, m), 2.27±2.38 (2H, m), 2.70±2.88 (2H,
m), 3.59 (1H, m), 3.46 (1H, brs), 3.73 (1H, m), 5.08 (1H,
m), 6.18 (1H, brd, J=8.0 Hz), 7.21±7.40 (3H, m), 7.49
+
.
(2H, brd, J=8.4 Hz); MS m/z 371 (M+H) . 4g fuma-
rate: mp 185±187ꢀC (from i-Pr2O/i-PrOH). Anal. calcd
.
for C23H34N2O2 C4H4O4: C, 66.64; H, 7.87; N, 5.76.
Found: C, 66.24; H, 7.87; N, 5.64.
.
C17H24N2O2 0.2H2O: C, 69.93; H, 8.42; N, 9.52. Found:
C, 69.70; H, 8.62; N, 9.42.
N-[1-(4-Methyl-2-pentenyl)piperidin-4-yl]-2-cyclobutyl-2-
hydroxy-2-phenylacetamide (4h). 4h was obtained from 8
1
and 4-methyl-2-pentenol33 (52%): H NMR (CDCl3) d
General procedure (method A)
0.98 (6H, d, J=6.9 Hz), 1.38±2.18 (12H, m), 2.28 (1H,
m), 2.69±2.86 (2H, m), 2.82 (2H, brd, J=6.6 Hz), 3.29±
3.49 (2H, m), 3.71 (1H, m), 5.40 (1H, m), 5.56 (1H, m),
The experimental procedure of 4f was described as a
representative example.

2562
M. Mitsuya et al. / Bioorg. Med. Chem. 7 (1999) 2555±2567
6.16 (1H, m), 7.20±7.40 (3H, m), 7.49 (2H, brd, J=8.4
+
rated under reduced pressure and the resulting residue
was puri®ed by silica gel column chromatography
(CHCl3:MeOH=100:1) to obtain 4b (64 mg, 84%) as
an oil: H NMR (CDCl3) d 0.90 (3H, t, J=7.4 Hz),
1.20±1.53 (6H, m), 1.55±2.19 (10H, m), 2.24±2.40 (2H,
.
Hz); MS m/z 371 (M+H) . 4h fumarate: mp 175±
176.5ꢀC (from i-Pr2O/i-PrOH). Anal. calcd for
.
C23H34N2O2 C4H4O4: C, 66.64; H, 7.87; N, 5.76.
Found: C, 66.48; H, 7.98; N, 5.73.
1
m), 2.69±2.90 (2H, m), 3.29±3.58 (2H, m), 3.71 (1H, m),
6.14 (1H, brd, J=8.3 Hz), 7.19±7.39 (3H, m), 7.49 (2H,
brd, J=8.4 Hz); MS m/z 345 (M+H) . 4b fumarate:
mp 174±176ꢀC (from i-Pr2O/i-PrOH). Anal. calcd for
.
C21H32N2O2 C4H4O4: C, 65.20; H, 7.88; N, 6.08.
Found: C, 65.04; H, 8.01; N, 6.03.
N-[1-[(4S)-4-Methyl-1-hexyl]piperidin-4-yl]-2-cyclobutyl-
2-hydroxy-2-phenylacetamide (4i). 4i was obtained from
8 and (S)-4-methyl-1-hexanol (46%). 1H NMR (CDCl3)
d 0.79±0.90 (6H, m), 1.00±1.54 (9H, m), 1.68±2.15 (10H,
m), 2.27 (2H, brt, J=7.8 Hz), 2.70±2.85 (2H, m), 3.29±3.51
(2H, m), 3.71 (1H, m), 6.12 (1H, brd, J=8.1 Hz), 7.21±
7.38 (3H, m), 7.44±7.51 (2H, m); MS m/z 387 (M+H)+.
A mixture of diastereomers (tR 11.6 and 14.0 min, Dai-
cel Chiralcel OJ 0.46Â25 cm, n-hexane:AcOEt= 95:5,
¯ow rate=0.5ml/min, UV detection 220 nm).
+
.
The following compounds 4a, 4c, 4d, 4o and 4p were
prepared from 8 and the appropriate aldehydes as
described for 4b.
N-(1-Methylpiperidin-4-yl)-2-cyclobutyl-2-hydroxy-2-
phenylacetamide (4a). 4a was obtained from 8 and par-
aformaldehyde (59%): H NMR (CDCl3) d 1.30±1.51
N-[1-(4-Ethyl-1-hexyl)piperidin-4-yl]-2-cyclobutyl-2-hydr-
oxy-2-phenylacetamide (4j). 4j was obtained from 8 and
4-ethyl-1-hexanol34 (72%): 1H NMR (CDCl3) d 0.82
(6H, s), 1.10±1.54 (10H, m), 1.70±2.17 (11H, m), 2.23±
2.35 (2H, m), 2.70±2.88 (2H, m), 3.62±3.80 (2H, m), 3.71
(1H, m), 6.16 (1H, brd, J=8.1 Hz), 7.21±7.39 (3H, m),
7.49 (2H, brd, J=8.3 Hz); MS m/z 401 (M+H)+.
1
(2H, m), 1.60±2.17 (10H, m), 2.25 (3H, s), 2.60±2.80
(2H, m), 3.36 (1H, m), 3.70 (1H, m), 6.20 (1H, brd,
J=7.8 Hz), 7.20±7.40 (3H, m), 7.49 (2H, brd, J=8.1
+
.
Hz)ꢀ; MS m/z 302 (M+H) . 4a fumarate: mp 193±
.-
194 C (from i-PrOH). Anal. calcd for C18H26N2O2
4j fumarate: mp 190±191ꢀC (from i-Pr2O/i-PrOH).
C4H4O4: C, 63.14; H, 7.23; N, 6.69. Found: C, 63.22; H,
7.38; N, 6.67.
.
.
Anal. calcd for C25H40N2O2 C4H4O4: C, 67.42; H, 8.58;
N, 5.42. Found: C, 67.08; H, 8.73; N, 5.39.
N-(1-Pentylpiperidin-4-yl)-2-cyclobutyl-2-hydroxy-2-
phenylacetamide (4c). 4c was obtained from 8 and
n-pentylaldehyde (57%): 1H NMR (CDCl3) d 0.88
(3H, t, J=6.9 Hz), 1.18±1.55 (8H, m), 1.68±2.18 (10H,
m), 2.21±2.39 (2H, m), 2.70±2.90 (2H, m), 3.29±3.59
(2H, m), 3.70 (1H, m), 6.15 (1H, brd, J=7.8 Hz),
7.20±7.40 (3H, m), 7.48 (2H, brd, J=8.3 Hz); MS m/z
N-[1-(2-Isopropyloxyethyl)piperidin-4-yl]-2-cyclobutyl-2-
hydroxy-2-phenylacetamide (4k). 4k was obtained from
8 and 2-isopropyloxy-1-ethanol (63%): 1H NMR
(CDCl3) d 1.13 (6H, d, J=6.0 Hz), 1.40 (1H, m), 1.56±
2.27 (10H, m), 2.55 (2H, t, J=6.0 Hz), 2.72±2.88 (2H,
m), 3.29±3.78 (6H, m), 6.11 (1H, d, J=7.5 Hz), 7.21±
+
ꢀ
.
7.47 (3H, m), 7.48 (2H, brd, J=8.1ꢀHz); MS m/z 375
359 (M+H) . 4c fumarate: mp 158±160 C (from i-
.
Pr2O/i-PrOH). Anal. calcd for C22H34N2O2 C4H4O4: C,
+
.
(M+H) . 4k fumarate: mp 172±175 C (from i-Pr2O/i-
.
PrOH). Anal. calcd for C22H34N2O3 C4H4O4: C, 63.65;
H, 7.81; N, 5.71. Found: C, 63.63; H, 7.96; N, 5.72.
65.80; H, 8.07; N, 5.90. Found: C, 65.74; H, 8.43; N,
5.87.
N-[1-(3-Ethoxycarbonylpropyl)piperidin-4-yl]-2-cyclobutyl-
2-hydroxy-2-phenylacetamide (19). 19 was obtained
from 8 and ethyl 4-bromobutyrate (90%): 1H NMR
(CDCl3) d 1.24±1.48 (2H, m), 1.26 (3H, t, J=7.2 Hz),
1.60±2.15 (12H, m), 2.21±2.39 (4H, m), 2.66±2.80 (2H,
m), 3.36 (1H, m), 3.46 (1H, brs), 3.70 (1H, m), 4.12 (2H,
d, J=7.2 Hz), 6.13 (1H, brd, J=8.4 Hz), 7.22±7.40 (3H,
m), 7.50 (2H, brd, J=8.4 Hz); HRMS m/z calcd for
C23H34N2O4 (M+H)+: 403.2597. Found: 403.2589.
N-(1-Hexylpiperidin-4-yl)-2-cyclobutyl-2-hydroxy-2-phe-
nylacetamide (4d). 4d was obtained from 8 and n-hex-
ylaldehyde (59%): 1H NMR (CDCl3) d 0.87 (3H, t,
J=6.8 Hz), 1.21±1.50 (8H, m), 1.55±2.12 (12H, m),
2.24±2.31 (2H, m), 2.70±2.82 (2H, m), 3.25±3.60 (2H,m),
3.72 (1H, m), 6.11 (1H, d, J=9.6 Hz), 7.23±7.37 (3H,
m), 7.48 (2H, brd, J=8.4 Hꢀz); MS m/z 373 (M+H)+.
.
4d fumarate: mp 164±166.5 C (from i-Pr2O/i-PrOH).
.
Anal. calcd for C23H36N2O2 C4H4O4: C, 66.37; H, 8.25;
N, 5.73. Found: C, 66.53; H, 8.52; N, 5.75.
General procedure (method B)
N-(1-Benzylpiperidin-4-yl)-2-cyclobutyl-2-hydroxy-2-phe-
nylacetamide (4o). 4o was obtained from 8 and benzal-
dehyde (80%): H NMR (CDCl3) d 1.26±1.50 (2H, m),
The experimental procedure of 4b was described as a
representative example.
1
1.65±2.20 (10H, m), 2.64±2.81 (2H, m), 3.35 (1H, m),
3.46 (2H, s plus 1H, brs), 3.72 (1H, m), 6.08 (1H, brd,
J=8.7 Hz), 7.19±7.41 (8H, m), 7.48 (2H, brd, J=8.3
Hz); MS m/z 379 (M+H)+; mp 183±185ꢀC (from n-
hexane/CHCl3). Anal. calcd for C24H30N2O2: C, 76.16;
H, 7.99; N, 7.40. Found: C, 75.90; H, 8.26; N, 7.39.
N-(1-Butylpiperidin-4-yl)-2-cyclobutyl-2-hydroxy-2-phenyl-
acetamide (4b). To a solution of 8 (72 mg, 0.22 mmol)
and n-butylaldehyde (40 mg, 0.55 mmol) in THF (3 mL)
was added sodium triacetoxyborohydride (100 mg, 0.64
mmol) at room temperature and the mixture was stirred
for 17 h. After the addition of saturated aqueous
sodium bicarbonate soln, the mixture was extracted
with CHCl3. The organic layer was washed with brine
and dried over anhyd Na2SO4. The solvent was evapo-
N-[1-(Phenylethyl)piperidin-4-yl]-2-cyclobutyl-2-hydroxy-
2-phenylacetamide (4p). 4p was obtained from 8 and
1
phenylacetaldehyde (74%): H NMR (CDCl3) d 1.30±

M. Mitsuya et al. / Bioorg. Med. Chem. 7 (1999) 2555±2567
2563
2.24 (12H, m), 2.51±2.62 (2H, m), 2.71±2.92 (4H, m),
3.29±3.48 (2H, m), 3.73 (1H, m), 6.15 (1H, brd, J=7.8
Hz), 7.14±7.39 (8H, m), 7.49 (2H, brd, J=8.3 Hz); MS
m/z 393 (M+H)+; mp 143±145ꢀC (from n-hexane/
CHCl3). Anal. calcd for C25H32N2O2 0.2H2O: C, 75.74;
H, 8.24; N, 7.07. Found: C, 75.73; H, 8.52; N, 7.07.
m), 5.23 (1H, brd, J=8.4 Hz), 7.19±7.39 (5H, m); MS
m/z 355 (M+H)+. Mp 132±133ꢀC (from i-Pr2O/i-
PrOH). Anal. calcd for C23H34N2O: C, 77.92; H, 9.67;
N, 7.90. Found: C, 77.57; H, 9.98; N, 7.89.
.
N-(4-Methyl-3-pentenyl)piperidin-4-yl 2-cyclobutyl-2-hy-
droxy-2-phenylacetate (14c). 14c was obtained from 12
and 2-cyclobutyl-2-hydroxy-2-phenylacetic acid (5a)30
General procedure (method C)
1
(34%): H NMR (CDCl3) d 1.52±2.70 (18H, m), 1.64
(3H, s), 1.71 (1H, s), 3.33 (1H, m), 3.85 (1H, brs), 4.86
The experimental procedure of 4r was described as a
representative example.
(1H, m), 5.10 (1H, m), 7.22±7.41 (3H, m), 7.60 (2H, brd,
+
.
J=8.4 Hz); MS m/z 372 (M+H) . 14c fumarate: mp
N-[1-(4-Methyl-3-pentenyl)piperidin-4-yl]-2-cyclopentyl-
2-hydroxy-2-phenylacetamide (4r). To a solution of 2-
cyclopentyl-2-hydroxy-2-phenylacetic acid (5e)35 (150
mg, 0.68 mmol) and 13 (190 mg, 1.04 mmol) in CHCl3
(20 mL) were added 1-hydroxybenzotriazole (220 mg,
1.63 mmol),1-(3-dimethylaminopropyl)-3-ethylcarbodii-
mide(160 mg, 0.83 mmol) at room temperature. The
mixture was stirred overnight and concentrated under
reduced pressure. The residue was shaken with Et2O
and sat. NaHCO3. The Et2O layer was separated,
washed with H2O and brine, and dried over MgSO4.
After removal of the solvent, the crude product was
puri®ed by silica gel column chromatography
(CHCl3:MeOH=100:1) to give 4r (154 mg, 59%) as an
oil. (R)-4r was prepared by the same method. (S)-4r was
obtained by preparative HPLC (98% ee, tR of (S)-4r:
16.8 min, tR of (R)-4r: 20.3 min, Daicel Chiralcel OD
0.46Â25 cm, eluent n-hexane:EtOH:TFA=92.5:7.5:0.1,
¯ow rate=0.5 ml/min, UV detection 230 nm): 1H NMR
(CDCl3) d 1.13±1.94 (12H, m), 1.60 (3H, s), 1.68 (3H, s),
2.00±2.20 (4H, m), 2.25±2.38 (2H, m), 3.02 (1H, m), 3.14
(1H, brs), 3.70 (1H, m), 5.07 (1H, m), 6.31 (1H, brd,
J=7.9 Hz), 7.21±7.39 (3H, m), 7.59 (2H, brd, J=8.3
Hz); MS m/z 385 (M+H)+; IR (KBr) 3400, 29ꢀ50, 1710,
138±140ꢀC (from i-Pr2O/i-PrOH). Anal. calcd for
.
C23H33NO3 C4H4O4: C, 66.51; H, 7.65; N, 2.87. Found:
C, 66.25; H, 7.70; N, 2.90.
N-[1-(4-Methyl-3-pentenyl)piperidin-4-yl]-2-cyclopropyl-
2-hydroxy-2-phenylacetamide (4q). 4q was obtained
from 13 and 2-cyclopropyl-2-hydroxy-2-phenylacetic
1
acid (5d)30 (60%): H NMR (CDCl3) d 0.44±0.69 (4H,
m), 1.32±1.99 (5H, m), 1.61 (3H, s), 1.68 (3H, s), 2.02±
2.20 (4H, m), 2.25±2.38 (2H, m), 2.70±2.89 (2H, m), 3.39
(1H, m), 3.78 (1H, m), 5.06 (1H, m), 6.03 (1H, m), 7.20±
7.42 (3H, m), 7.61 (2H, brd, J=8.6 Hz); MS m/z 357
+
ꢀ
.
(M+H) . 4q 1/2fumarate: mp 75±78.5 C (from i-Pr2O/
.
.
i-PrOH). Anal. calcd for C22H32N2O2 0.5C4H4O4
1.5H2O: C, 65.28; H, 8.45; N, 6.34. Found: C, 65.31; H,
8.41; N, 6.00.
N-[1-(4-Methyl-3-pentenyl)piperidin-4-yl]-2-cyclohexyl-
2-hydroxy-2-phenylacetamide (4s). 4s was obtained
from 13 and 2-cyclohexyl-2-hydroxy-2-phenylacetic acid
1
(5f)35 (50%): H NMR (CDCl3) d 0.88 (1H, m), 1.01±
1.95 (16H, m), 1.60 (3H, s), 1.68 (3H, s), 2.02±2.50 (4H,
m), 2.25±2.38 (2H, m), 2.70±2.90 (2H, m), 3.71 (1H, m),
5.06 (1H, m), 6.57 (1H, brd, J=8.6 Hz), 7.20±7.40 (3H,
m), 7.60 (2H, brd, J=8.6 Hz); MS m/z 399 (M+H)+.
1
.
1655, 1520 cm . (R)-4r fumarate mp 203±205 C (from
ꢀ
.
i-Pr2O/i-PrOH). Anal. calcd for C24H36N2O2 C4H4O4:
.
4s fumarate: mp 206±210 C (from i-Pr2O/i-PrOH).
.
Anal. calcd for C25H38N2O2 C4H4O4: C, 67.68; H, 8.23;
N, 5.44. Found: C, 67.62; H, 8.52; N, 5.46.
C, 67.18; H, 8.05; N, 5.60. Found: C, 66.98; H, 8.04; N,
5.56; [a]2d0 7.1 (c=1.0, MeOH).
The following compounds 14a±c, 4q, 4s, 4t were pre-
pared from 12 or 13, and the appropriate acids as
described for 4r.
N-[1-(4-Methyl-3-pentenyl)piperidin-4-yl]-2-hydroxy-di-
phenylacetamide (4t). 4t was obtained from 13 and 2,2-
diphenyl-2-hydroxyacetic acid 5g (62%): 1H NMR
(CDCl3) d 1.41±1.80 (4H, m), 1.63 (3H, s), 1.71 (3H, s),
1.93±2.36 (6H, m), 2.69±2.88 (2H, m), 3.87 (1H, m), 5.07
(1H, m), 6.42 (1H, m), 7.26±7.88 (10H, m); MS m/z 393
(M+H) . 4t fumarate: mp 213±216 C (from i-Pr2O/i-
.
PrOH). Anal. calcd for C25H32N2O2 C4H4O4: C, 68.48;
H, 7.13; N, 5.51. Found: C, 68.40; H, 7.11; N, 5.48.
N-[1-(4-Methyl-3-pentenyl)piperidin-4-yl]-2-cyclobutyl-2-
methoxy-2-phenylacetamide (14a). Compound 14a was
obtained from 13 and 2-cyclopentyl-2-methoxy-2-phe-
+
ꢀ
.
1
nylacetic acid (20) (50%): H NMR (CDCl3) d 1.41±
2.27 (13H, m), 1.64 (3H, s), 1.71 (3H, s), 2.30±2.48 (3H,
m), 2.81±2.97 (2H, m), 3.14 (3H, s), 3.27 (1H, m), 3.87
(1H, m), 5.11 (1H, m), 6.81 (1H, brd, J=7.8 Hz), 7.23±
7.44 (5H, m); MS m/z 385 (M+H) . 14a fumarate: mp
144±145ꢀC (from i-Pr2O/i-PrOH). Anal. calcd for
.
C24H36N2O2 C4H4O4: C, 67.18; H, 8.05; N, 5.60.
Found: C, 66.87; H, 8.33; N, 5.58.
N-[1-(2-Dimethylcarbamoyl-1-ethyl)piperidin-4-yl]-2-cyclo-
butyl-2-hydroxy-2-phenylacetamide (4l). A solution of
free base of 8 (51 mg, 0.18 mmol) and N,N-dimethyla-
crylamide (50 mg, 0.50 mmol) in EtOH (3 mL) was
heated at 80ꢀC for 2 h. The mixture was cooled to room
temperature and concentrated. H2O and CHCl3 were
added to the residue. The organic layer was separated,
dried over MgSO4 and concentrated to a€ord the crude
product which was puri®ed by preparative TLC to give
+
.
N-[1-(4-Methyl-3-pentenyl)piperidin-4-yl]-2-cyclobutyl-2-
phenylacetamide (14b). 14b was obtained from 13 and a-
cyclobutylphenylacetic acid (5c)36 (40%): 1H NMR
(CDCl3) d 1.27±1.49 (2H, m), 1.60 (3H, s), 1.68 (3H, s),
1.51±1.98 (8H, m), 2.00±2.39 (6H, m), 2.71±3.06 (3H,
m), 3.27 (1H, d, J=10.8 Hz), 3.76 (1H, m), 5.05 (1H,
1
4l (49 mg, 72%) as a white foam: H NMR (CDCl3) d
1.30±1.50 (2H, m), 1.68±2.24 (10H, m), 2.49 (2H, brt,
J=7.4 Hz), 2.64±2.84 (4H, m), 2.94 (3H, s), 3.01 (3H, s),

2564
M. Mitsuya et al. / Bioorg. Med. Chem. 7 (1999) 2555±2567
3.30±3.60 (2H, m), 3.72 (1H, m), 6.18 (1H, brd, J=8.1
Hz), 7.22±7.40 (3H, m), 7.49 (2H, brd, J=8.1 Hz); MS m/z
methane (0.090 mL, 1.45 mmol) was added. The mix-
ture was allowed to warm to room temperature and
stirred for 19h. The reaction was quenched with satd
NH4Cl solution and the mixture was extracted with
AcOEt. The organic layer was washed with brine, dried
over MgSO4 and concentrated. The residue was puri®ed
by silica gel column chromatography (n-hexane:
ꢀ
Et2O/EtOH). Anal. calcd for C22H33N3O3 HCl: C, 62.32;
+
.
388 (M+H) . 4l hydrochloride: mp 204±206 C (from
.
H, 8.08; N, 9.91. Found: C, 62.16; H, 7.83; N, 9.86.
N-[1-(4-Hydroxy-1-butyl)piperidin-4-yl]-2-cyclobutyl-2-
hydroxy-2-phenylacetamide (4m). To a solution of 19
(60 mg, 0.15 mmol) in THF (3 mL) was added lithium
aluminum hydride (10 mg, 0.26 mmol) at ice-bath tem-
perature. The mixture was stirred at the same tempera-
1
AcOEt=20:1±5:1) to give 17 as a oil (95 mg, 26%): H
NMR (CDCl3) d 0.21 (9H, s), 1.02±2.20 (11H, m), 1.40
(9HÂ1/4, s), 1.44 (9HÂ3/4, s), 2.40 (3HÂ3/4, s), 2.41±
2.54 (2HÂ1/4, m), 2.70 (3HÂ1/4, s), 2.71±2.88 (2HÂ3/4,
m), 3.20 (1H, m), 3.59±3.71 (1HÂ1/4, m), 3.87±4.30
(2H, m), 4.49±4.62 (1Hx3/4, m), 7.21±7.41 (5H, m);
HRMS m/z calcd for C26H43N2O4Si (M+H)+:
475.2992. Found: 475.3023.
.
ture for 2 h. After addition of Na2SO4 10H2O, the
mixture was stirred for an additional 3 h at room tem-
perature. Inorganic salts were ®ltered o€ and washed
with THF and i-PrOH. The ®ltrate was concentrated
and the residual oil was puri®ed by preparative TLC
(CHCl3:MeOH=9:1) to provide 4m as an oil (39 mg,
N-Methyl-N-[1-(4-methyl-3-pentenyl)piperidin-4-yl]-2-cy-
clobutyl-2-hydroxy-2-phenylacetamide (18). 17 (85 mg,
0.18 mmol) was dissolved in 10% HCl±MeOH (2 mL),
and the mixture was stirred for 17 h at room tempera-
ture. The solvent was evaporated to obtain the crude oil
(61 mg) which was used without puri®cation. To a
solution of the above amine hydrochloride in CH3CN (3
mL) were added K2CO3 (80 mg, 0.58 mmol) and 5-
bromo-2-methyl-2-pentene (10) (35 mg, 0.21 mmol) at
room temperature. The mixture was heated for 4h at
80ꢀC and then cooled to room temperature. After dilu-
tion with H2O, the mixture was extracted three times
with CHCl3. The combined organic extract was dried
over MgSO4 and concentrated. Puri®cation by pre-
parative TLC (CHCl3:MeOH=19:1) a€orded 18 (55
mg, 79%) as an oil: 1H NMR (CDCl3) d 1.30±2.67
(17H, m), 1.62 (3H, s), 1.69 (3H, s), 2.49 (3HÂ1/3, brs),
2.81 (3HÂ2/3, brs), 2.98 (1H, m), 3.31 (1H, m), 3.53
(1HÂ2/3, m), 4.48 (1HÂ1/3, m), 5.04 (1H, m), 5.57 (1H,
m), 7.11±7.43 (5H, m); MS m/z 385 (M+H)+.
1
73%): H NMR (CDCl3) d 1.34±2.20 (16H, m), 2.26±
2.44 (2H, m), 2.79±3.00 (2H, m), 3.35 (1H, m), 3.45±3.81
(4H, m), 6.22 (1H, brd, J=7.8 Hz), 7.21±7.40 (3H, m),
7.48 (2H, brd, J=8.3 Hz); MS m/z 361 (M+H)+.
ꢀ
.
4m fumarate: mp 151±153 C (from Et2O/EtOH). Anal.
.
calcd for C21H32N2O3 C4H4O4: C, 63.01; H, 7.61; N,
5.88. Found: C, 62.82; H, 7.83; N, 5.92.
N-[1-(3-Carboxylpropyl)piperidin-4-yl]-2-cyclobutyl-2-hy-
droxy-2-phenylacetamide (4n). 19 (63 mg, 0.16 mmol)
was dissolved in EtOH (2 mL) and 1N NaOH (1 mL)
was added at room temperature. After stirring for 16h,
the mixture was adjusted to pH 4 with 1 N HCl and
concentrated. The residue was puri®ed by preparative
TLC (CHCl3:MeOH=4:1) to obtain 4n (30 mg, 51%)
1
as a white solid: H NMR (CDCl3) d 1.49±2.19 (12H,
m), 2.30±2.79 (6H, m), 3.00±3.21 (2H, m), 3.38 (1H, m),
3.80 (1H, m), 6.54 (1H, brd, J=7.8 Hz), 7.20±7.40 (3H,
m), 7.42±7.57 (2H, m); MS m/z 375 (M+H)+. Mp 98±
102ꢀC (from Et2O/EtOH). Anal. calcd for
ꢀ
.
18 fumarate: mp 81±83 C (from Et2O/EtOH). Anal.
.
.
.
C21H30N2O4 1.5H2O: C, 62.82; H, 8.28; N, 6.98. Found:
C, 63.14; H, 8.15; N, 7.01.
calcd for C24H36N2O2 0.5C4H4O4 0.5H2O: C, 69.15; H,
8.70; N, 6.20. Found: C, 69.12; H, 8.67; N, 6.20.
N-(1-tert-Butyloxycarbonylpiperidin-4-yl)-2-cyclobutyl-
2-trimethylsilyloxy-2-phenylacetamide (16). To a stirred
solution of 7 (330 mg, 0.86 mmol) and imidazole (215
mg, 3.16 mmol) in DMF (8 mL) was added trimethylsi-
lylchloride (0.30 mL, 2.36 mmol) at room temperature,
and the mixture was stirred for 18 h. After addition of
H2O, the mixture was extracted with Et2O. The organic
extract was washed with H2O and brine, and dried over
MgSO4. Evaporation of the solvent gave the crude pro-
duct which was puri®ed by silica gel column chromato-
graphy (n-hexane:AcOEt=4:1) to a€ord 16 (380 mg,
96%) as an oil: 1H NMR (CDCl3) d 0.05(9H, s), 1.31±
1.50 (2H, m), 1.49 (9H, s), 1.66±2.29 (8H, m), 2.88±3.02
(2H, m), 3.31 (1H, m), 3.90±4.11 (3H, m), 7.02(1H, brd,
J=8.4), 7.22±7.48 (5H, m); HRMS m/z calcd for
C25H41N2O4Si (M+H)+: 461.2836. Found: 461.2863.
1-(4-Methyl-3-pentenyl)-4-piperidone (11). To a suspen-
sion of 4-piperidone monohydrate hydrochloride 9 (2.00
g, 14.75 mmol), potassium iodide (80 mg, 0.48 mmol)
and K2CO3 (6.01 g, 43.5 mmol) in CH3CN (100 mL)
was added 5-bromo-2-methyl-2-pentene (10) (2.58 g,
15.8 mmol) at room temperature. The mixture was
heated for 7 h at 80ꢀC and cooled to room temperature.
After dilution with H2O, the mixture was extracted
three times with CHCl3. The combined organic extract
was dried over MgSO4 and concentrated. The residual
oil was puri®ed by silica gel column chromatography
(CHCl3:MeOH=50:1) to obtain 11 (2.60 g, 97%) as an
oil: 1H NMR (CDCl3) d 1.63 (3H, s), 1.71 (3H, s), 2.13±
2.30 (2H, m), 2.39±2.54 (6H, m), 2.77 (4H, brt, J=6.0
Hz), 5.12 (1H, m); HRMS m/z calcd for C11H19NO
(M+): 181.1467. Found: 181.1464.
N-Methyl-N-(1-tert-butyloxycarbonylpiperidin-4-yl)-2-
cyclobutyl-2-trimethylsilyloxy-2-phenylacetamide (17).
To a stirred solution of 16 (350 mg, 0.76 mmol) in
THF (6 mL) were sequentially added sodium hydride
(60% in oil, 50 mg, 1.25 mmol), tetra-n-butylammonium
iodide (18 mg, 0.049 mmol) at 0ꢀC. After 10 min, iodo-
4-Hydroxy-1-(4-methyl-3-pentenyl)piperidine (12). To a
stirred solution of 11 (450 mg, 2.48 mmol) in MeOH (20
mL) was added NaBH4 (150 mg, 3.97 mmol) at 0ꢀC.
The mixture was stirred for 1 h at the same temperature.
After addition of H2O, MeOH was evaporated and the
mixture was extracted twice with CHCl3. The combined

M. Mitsuya et al. / Bioorg. Med. Chem. 7 (1999) 2555±2567
2565
organic extract was dried over MgSO4 and con-
centrated. The residual oil was puri®ed by silica gel col-
umn chromatography (CHCl3:MeOH=50:1±20:1) to
(14H, m), 2.43±2.78 (3H, m), 3.55 (1H, m), 4.08 (1H,
m), 4.36 (1H, dd, J=9.2, 5.6 Hz), 4.57 (1H, m), 5.06
(1H, m), 7.14±7.42 (5H, m); MS m/z 370 (M+H)+. Mp
123±124.5ꢀC (from n-hexane/AcOEt). Anal. calcd for
C23H35N3O: C, 74.75; H, 9.55; N, 11.37. Found: C,
74.59; H, 9.77; N, 11.27.
1
obtain 12 (445 mg, 98%) as an oil: H NMR (CDCl3) d
1.50±1.78 (2H, m), 1.62 (3H, s), 1.69 (3H, s), 1.84±1.98
(2H, m), 2.06±2.39 (6H, m), 2.71±2.88 (2H, m), 3.62±
ꢀ
(from i-Pr2O/i-PrOH). Anal. calcd for C11H21NO
.
3.78 (1H, m), 5.08 (1H, m). 12 fumarate: mp 122±124 C
.-
Methyl 2-cyclobutyl-2-methoxy-2-phenylacetate (20). To
a stirred solution of methyl 2-cyclobutyl-2-hydroxy-2-
phenyl acetate (5a)30 (230 mg, 1.05 mmol) in DMF (8
mL) was added sodium hydride (60% in oil, 75 mg, 1.88
mmol) at 0ꢀC. After 10 min, iodomethane (0.15 mL,
2.41 mmol) was added, and the mixture was stirred for 1
h. The reaction was quenched with satd NH4Cl solution
and the mixture was extracted with AcOEt. The organic
layer was washed with brine, dried over MgSO4 and
concentrated. The residue was puri®ed by silica gel col-
umn chromatography (n-hexane:AcOEt=20:1) to give
C4H4O4: C, 60.18; H, 8.42; N, 4.68. Found: C, 60.28; H,
8.73; N, 4.99.
N-[1-(4-Methyl-3-pentenyl)piperidin-4-yl]-N0 -(ꢀ-cyclo-
butylbenzyl)carbamate (15a). To a solution of 2-cyclo-
butylphenylacetic acid (5c)36 (55 mg, 0.29 mmol) and
Et3N (50 mL, 0.36 mmol) in toluene (2 mL) was added
diphenylphosphoryl azide (98 mg, 0.36 mmol) at room
temperature. The mixture was heated for 40 min at
100ꢀC and cooled to room temperature. Alcohol 12 (57
mg, 0.31 mmol) was added and the resultant mixture
was heated at 100ꢀC for 5 h. After cooling, the mixture
was diluted with AcOEt and washed with satd NaHCO3,
H2O and brine, and dried over MgSO4. The solvent was
evaporated under reduced pressure and the residue was
puri®ed by preparative TLC (CHCl3:MeOH=9:1) to
1
20 (230 mg, 94%) as an oil: H NMR (CDCl3) d 1.51±
1.96 (4H, m), 2.00±2.21 (2H, m), 3.05 (1H, m), 3.27 (3H,
s), 3.82 (3H, s), 7.23±7.41 (5H, m); HRMS m/z calcd for
C14H18O3 (M+): 234.1256. Found: 234.1230.
2-Cyclobutyl-2-methoxy-2-phenylacetic acid (5b). 20
(230 mg, 0.982 mmol) was dissolved in MeOH (6 mL)
and 2N NaOH solution (2 mL) was added at room
temperature. The mixture was heated at 60ꢀC for 4 h
and cooled to room temperature. After evaporation of
MeOH, the mixture was washed with Et2O, acidi®ed
with 1 N HCl and extracted twice with CHCl3. The
combined extract was concentrated to give the acid 5b
(210 mg) as an oil which was used without puri®cation:
1H NMR (CDCl3) d 1.69±1.97 (2H, m), 2.00±2.18 (3H,
m), 2.41 (1H, m), 3.25 (1H, m), 3.23 (3H, s), 7.23±7.41
(5H, m).
1
a€ord 15a (25 mg, 23%) as an oil: H NMR (CDCl3) d
1.35±2.37 (17H, m), 1.61 (3H, s), 1.69 (3H, s), 2.40±2.81
(2H, m), 4.50±4.70 (2H, m), 4.83 (1H, m), 5.06 (1H, m),
+
.
7.10±7.39 (5H, m); MS m/z 371 (M+H) . 15a fuma-
rate: mp 125±128ꢀC (from i-Pr2O/i-PrOH). Anal. calcd
.
for C23H34N2O2 C4H4O4: C, 66.64; H, 7.87; N, 5.76.
Found: C, 66.45; H, 7.96; N, 5.90.
4-Amino-1-(4-methyl-3-pentenyl)piperidine (13). To a
stirred solution of 11 (210 mg, 1.16 mmol) and NH4OAc
(253 mg, 3.28 mmol) in MeOH (10 mL) was added
NaBH3CN (183 mg, 2.91 mmol) at room temperature.
The mixture was stirred for 10 h at the same tempera-
ture. After addition of satd NaHCO3, MeOH was eva-
porated and the mixture was extracted three times with
CHCl3. The combined CHCl3 extract was dried over
MgSO4. The solvent was evaporated to obtain the crude
oil 13 (190 mg) which was used in the next step without
puri®cation: 1H NMR (CDCl3) d 1.30±1.50 (2H, m),
1.62 (3H, s), 1.69 (3H, s), 1.74±1.89 (2H, m), 1.94±2.09
(2H, m), 2.11±2.38 (4H, m), 2.65 (1H, m), 2.80±2.95
(2H, m), 5.09 (1H, m); MS m/z 183 (M+H)+.
Optical resolution of 2-cyclopentyl-2-hydroxy-2-phenyl-
acetic acid (5e). Racemic 2-cyclopentyl-2-hydroxy-2-
phenylacetic acid (5e)35 (77.5 g, 0.352 mol) in toluene
(15 L) was treated with cinchonidine (100.9 g, 0.343
mol). The suspension was dissolved by heating at 130ꢀC
and the solution was gently cooled to 55ꢀC. The formed
precipitate was collected, washed with toluene and
dried. This process was repeated additional two times to
a€ord ( )-5e cinchonidine salt (46.3 g) as a white solid:
1H NMR (CD3OD) d 1.20±1.77 (9H, m), 1.88 (1H, m),
2.00±2.24 (3H, m), 2.72 (1H, m), 3.03 (1H, m), 3.10±3.29
(2H, m), 3.47 (1H, dd, J=13.2 and 10.5 Hz), 3.59 (1H,
m), 4.17 (1H, m), 4.85±5.11 (2H, m), 5.73 (1H, m), 6.03
(1H, brd, J=2.1 Hz), 7.13 (1H, m), 7.17±7.31 (2H, m),
7.61 (1H, m), 7.64±7.80 (4H, m), 8.07 (1H, d, J=8.4
Hz), 8.20 (1H, d, J=8.1 Hz), 8.86 (1H, d, J=4.5 Hz).
Mp 223±225ꢀC (from toluene). [a]2d0 87.4 (c=1.0,
MeOH). This was partitioned with 1 N HCl and AcOEt.
The aq layer was separated, extracted with AcOEt and
the combined AcOEt layers were washed with brine,
dried over Na2SO4 and concentrated to obtain ( )-5e
(18.6 g, 24%, >99% ee): [a]2d0 1.9 (c=3.0, MeOH).
The enantiomeric excess was determined by HPLC
analysis (tR of (+)-acid: 10.2 min, tR of ( )-acid: 11.3
min, Daicel Chiralcel OJ 0.46Â25 cm, eluent n-hex-
ane:EtOH:TFA=7:3:0.1, ¯ow rate=0.5 ml/min, UV
detection 220 nm).
N-(ꢀ-Cyclobutylbenzyl)-N0 -[1-(4-methyl-3-pentenyl)pi-
peridin-4-yl]urea (15b). To a solution of a-cyclobutyl-
phenylacetic acid (5c)36 (148 mg, 0.779 mmol) and Et3N
(140 mL, 1.01 mmol) in toluene (5 mL) was added
diphenylphosphoryl azide (280 mg, 1.02 mmol) at room
temperature. The mixture was heated for 30 min at
100ꢀC and cooled to room temperature. A solution of
the crude amine 13 (190 mg, ca. 1.04 mmol) in toluene
(2 mL) was added and the resultant mixture was heated
at 100ꢀC for 3 h. After cooling, the mixture was diluted
with AcOEt and washed with satd NaHCO3, H2O and
brine, and dried over MgSO4. The solvent was evapo-
rated under reduced pressure and the residue was pur-
i®ed by preparative TLC (CHCl3:MeOH=9:1) to a€ord
15b (140 mg, 49%) as a yellow solid: 1H NMR (CDCl3)
d 1.08±1.41 (2H, m), 1.60 (3H, s), 1.68 (3H, s), 1.49±2.37

2566
M. Mitsuya et al. / Bioorg. Med. Chem. 7 (1999) 2555±2567
Binding assay
were recorded isometrically by a multi-channel poly-
graph (model RMP-6018, Nihon-Kohden). Following
the stabilization of the right atria beat, cumulative con-
centration response curves to carbachol were obtained
before and after addition of the test compounds.
Responses were expressed as a percentage of inhibition
induced by carbachol.
The binding anities (Ki) to ®ve subtypes were deter-
mined by inhibition of speci®c binding of [3H]-NMS
using the human receptor membranes. In competitive
experiments, membranes from CHO cells stably expres-
sing cloned human m1±m5, purchased from Receptor
Biology, Inc., Baltimore, MD were incubated with 0.2
nM [3H]-NMS in the medium consisted of a bu€er
containing 50 mM Tris±HCl, 10 mM MgCl2 and 1 mM
EDTA at pH 7.4 and 25ꢀC for 2 h. Non-speci®c binding
was determined in the presence of 1 mM NMS. Incuba-
tions were terminated by rapid ®ltration over Packard
UniFilter-GF/C, using cell harvester Packard Filterma-
teTM 196. Then, the ®lter was rinsed four times with 1
mL bu€er each. The radioactivity was counted by
Packard TopCountTM liquid scintillation counter. IC50
values were converted to apparent Ki values using the
Cheng±Pruso€ equation.37
Acetylcholine-induced bronchoconstriction in rats
Male Sprague±Dawley rats (380±420 g) were anesthe-
tized (0.75 or 1.0 g/kg, respectively) and a-chloralose
(37.5 or 50 mg/kg, respectively) injected intraper-
itoneally. A tracheal cannula was inserted for ventilated
respiration. A cannula was inserted into the jugular vein
for intravenous administration. In the case of the
experiments for oral activity, the animals were fasted
overnight before oral drug administration. The anes-
thetized animals were paralysed with succinylcholine (10
mg/kg s.c.) and placed in the Plethysmograph-Box and
respired with positive pressure, constant rate and
volume ventilation (6 mL/kg, 90 strokes/min for rats)
from a Harvard rodent respirator. Air ¯ow was mea-
sured by placing a pneumotachograph, which was con-
nected to a Validyn di€erential pressure transducer
(model MP45-14), between the tracheal cannula and the
ventilation. Transpulmonary pressure was measured
with a Validyn di€erential pressure transducer (model
MP45-28).
Functional assay using isolated tissues
The pKB value, as an index of potency, was determined
for each individual curve by the equation
pKB= log{[B]/(concentration ratio 1)}, where con-
centration ratio is the ratio of ED50 values with and
without the antagonist and [B] is the concentration of
the antagonist.
Rat trachea (M3)
Acetylcholine-induced bradycardia in rats
Male Sprague±Dawley rats (250±400 g) were exsangui-
nated. The trachea was prepared free of serosal con-
nective tissue and single open ring preparations were
placed in 5 mL organ baths containing modi®ed Krebs±
Henseleit solution maintained at 32ꢀC and continuously
aerated with 95% O2 and 5% CO2, and connected to
isometric transducers (model TB-651T, Nihon-Kohden)
with sutures. Mechanical responses were recorded iso-
metrically by a multi-channel polygraph (model RMP-
6018, Nihon-Kohden). The tissue was equilibrated for
at least 60 min with initial tension of 1 g, and then con-
tracted by adding 1 mM carbachol twice. The second
contraction with carbachol was taken as a reference.
The concentration±contraction curves for carbachol
were obtained by cumulative addition of carbachol to
the organ bath. The test compound or vehicle was
added 10 min before the addition of carbachol.
Male Sprague±Dawley rats (300±350 g) were used. The
animals were anesthetized with urethane (1.0 g/kg) and
a-chloralose (0.05 g/kg) injected intraperitoneally. The
trachea, carotid artery and jugular vein were cannulated
after a midline neck incision. The animals were pre-
treated with succinylcholine (10 mg/kg s.c.) and then
ventilated with room air at a constant rate and volume
(8 mL/kg, 90 strokes/min) by a Harvard Apparatus
rodent ventilator. Arterial blood pressure was measured
with a pressure transducer (Model 041-500-503, COBE)
and the heart rate was integrated from the blood pres-
sure signal. Output signal from the transducer was
ampli®ed through a blood pressure ampli®er (AP-641G,
Nihon-Kohden) and Heart rate counter (AT-601G,
Nihon-Koden). The above parameters were all recorded
on a heat recorder (Nihon-Koden). Bradycardia was
induced by an administration of acetylcholine (5 mg/kg),
delivered into the saphenous vein, at 5 min intervals.
Once three similar responses (control response) were
obtained, the compound was administered 5 min before
the following acetylcholine administration. The ED50
values for the reversal of acetylcholine-induced brady-
cardia were calculated from dose±response curves with a
probit analysis, after changes of heart rate induced by
acetylcholine at pre- or post-drug-administration were
expressed as the percent control for each dose of the
compound.
Rabbit vas deferens (M1)
Experiments were conducted according to the described
method.29
Rat right atria (M2)
Male Sprague±Dawley rats (250±400 g) were exsangui-
nated and right atria were isolated. Right atria were
placed in 20 mL of organ baths containing modi®ed
Krebs±Henseleit solution maintained at 32ꢀC and con-
tinuously aerated with 95% O2 and 5% CO2, and con-
nected to isometric transducers (model TB-651T,
Nihon-Kohden) with sutures. Mechanical responses
Acknowledgements
We would like to thank Ms. A. Dobbins for critical
reading. We also acknowledge Mr. S. Abe, Mr. H.

M. Mitsuya et al. / Bioorg. Med. Chem. 7 (1999) 2555±2567
2567
Ohsawa, Ms. C. Suzuki and Ms. A. Shimizu for analy-
tical support.
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