Identification of novel muscarinic M(3) selective antagonists with a conformationally restricted Hyp-Pro spacer.

Article abstract of DOI:10.1016/S0960-894X(02)00843-0

The identification of potent and selective muscarinic M(3) antagonists that are based on the recently discovered triphenylpropioamide derivative, 1, and have a unique amino acid spacer group is described. The introduction of a hydroxyproline-proline group to the spacer site and the use of a propyl or cyclopropylmethyl group as the piperidine N-substituent led to the discovery of the novel M(3) selective antagonists [8c, 8g; K(i)<2 nM (M(3)), M(1)/M(3)>700-fold, M(2)/M(3)>180-fold], which have a more rigid structure than 1.

Full text of DOI:10.1016/S0960-894X(02)00843-0

Bioorganic & Medicinal Chemistry Letters 13 (2003) 57–60
Identification of Novel Muscarinic M3 Selective Antagonists with
a Conformationally Restricted Hyp-Pro Spacer
Yufu Sagara,* Toshifumi Kimura, Toru Fujikawa, Kazuhito Noguchi and
Norikazu Ohtake
Banyu Tsukuba Research Institute in collaboration with Merck Research Laboratories, Okubo-3, Tsukuba 300-2611, Ibaraki, Japan
Received 3 September 2002; accepted 3 October 2002
Abstract—The identification of potent and selective muscarinic M3 antagonists that are based on the recently discovered triphe-
nylpropioamide derivative, 1, and have a unique amino acid spacer group is described. The introduction of a hydroxyproline-pro-
line group to the spacer site and the use of a propyl or cyclopropylmethyl group as the piperidine N-substituent led to the discovery
of the novel M3 selective antagonists [8c, 8g; Ki<2 nM (M3), M1/M3>700-fold, M2/M3>180-fold], which have a more rigid
structure than 1.
# 2002 Elsevier Science Ltd. All rights reserved.
There are five muscarinic acetylcholine receptor sub-
types (M1–M5) known to date.1À5 These receptor sub-
types play a number of pharmacological roles both
centrally and peripherally.6,7 For example, the M1
receptor is located at the postganglionic cholinergic
nerve terminals and glands, which facilitate neuro-
transmission and gastric secretion, respectively. The
neuronal M2 receptor provides functional negative
feedback modulation of acetylcholine (ACh) release in
addition to its role as a cardiac M2 receptor, which reg-
ulates heart rate. The M3 receptor is located in smooth
muscle and mucosal glands, which mediate contraction
and mucus secretion, respectively.8
types has focused on the exploration of M3 selective
antagonists.
As a part of our research in developing a muscarinic M3
receptor antagonist, we discovered the novel M3 selec-
tive antagonist, 1, using a rationally designed combina-
torial library.10 Compound 1 possesses a novel structure
that is distinct from the existing muscarinic antagonists,
and it exhibits excellent M3 binding affinity and selec-
tivity profile as shown in Table 1. A structure activity
relationship(SAR) analysis of analogues of 1 revealed
that amino acid moiety plays a major role in the M3
selectivity toward the other receptor subtypes and that
particular combinations of amino acids show a high
M1/M3 selectivity (37–550-fold), which would not be
attainable with known M3 antagonists.
Orally active muscarinic antagonists such as oxybutynin
have been used for the treatment of urinary tract dis-
orders, including urinary incontinence (UI), via block-
ade of the M3 receptor. However, their subtype non-
selective profiles may cause adverse effects such as a dry
mouth, blurred vision, constipation, and tachycardia,
which limit their clinical utility.9 M3 selective antago-
nists may reduce these adverse effects, but their advan-
tages over the non-selective antagonists remains to be
elucidated because of lack of M3 selective antagonists.
Therefore, pharmaceutical research into therapeutic
agents that are selective for muscarinic receptor sub-
To developa better pharmacological tool for clarifying
the role of the M3 receptor, further improvements in the
subtype selectivity of this novel class of M3 antagonist
were planned. However, the highly flexible structural
feature of 1 might interrupt the understanding of the
SAR and the binding mode in this class of compounds.
Among the novel M3 antagonists identified from the
library, 2a possess a conformationally restricted spacer
(l-Pro-d-Pro) while compound 1 has a flexible spacer
structure. The number of possible conformers of 2a was
predicted to be less than 0.07% of that of 1.11 However,
2a had low subtype selectivity toward the other receptor
subtypes, especially toward the M2 receptor (Table 1).
*Corresponding author. Tel.: +81-298-77-2000; fax: +81-298-77-
2029; e-mail: oowadayf@banyu.co.jp
0960-894X/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00843-0

58
Y. Sagara et al. / Bioorg. Med. Chem. Lett. 13 (2003) 57–60
Based on the above observations, we assumed that 2a
might be an alternative starting point for further chem-
ical modifications to identify more subtype-selective
compounds, if we could find the strategy to improve its
subtype selectivity. Therefore, we attempted to identify
compounds that are analogues of 2a but have compar-
able subtype selectivity with 1.
SAR study around 2a. l-Pro and d-Pro in 2a were
initially replaced with commercially available trans-l-4-
hydroxyproline and trans-d-4-hydroxyproline, respec-
tively (Table 2).
The substitution of l-Pro with l-hydroxyproline (9)
maintained the M3 binding affinity. Interestingly, 9
showed significantly improved selectivity for the M3
receptor toward the M1, M2, and M5 receptors in com-
parison with 2a, while the M3 selectivity toward the M4
receptor was comparable. Replacement of d-Pro with
d-hydroxyproline (10) resulted in a 14-fold reduced
affinity to the M3 receptor and lowered selectivity
toward the M1, M2, and M5 receptor subtypes com-
pared with those of 9. The combination of l-hydroxy-
proline and d-hydroxyproline (11) resulted in a 4-fold
decrease in M3 receptor affinity, while 11 was more
selective toward the M1 and M2, and M5 receptors, than
was 9. These results suggest that the hydroxy groupon
the pyrrolidine ring of l-Pro played an important role
in M1/M3, M2/M3, M5/M3 selectivity, but that the
Here, we report the synthesis and SARs of the ana-
logues of 2a and the identification of the hydroxyproline
derivatives, 8c and 8g, which have significantly
improved M1/M3, M2/M3, and M5/M3 selectivity com-
pared with 1.
The synthesis of the representative compounds, 8, is
shown in Scheme 1. Methyl esterification of commer-
cially available d-benzyloxycarbonylproline 3 was fol-
lowed by deprotection and subsequent amidation with
N-a-benzyloxycarbonyl-O-tert-butyl-l-4-trans-hydroxy-
proline to yield 4. This monoamide 4 was transformed
to diamide 5 by deprotection and condensation with
3,3,3-triphenylpropionic acid. Hydrolysis of 5 and sub-
sequent coupling with (3R)-3-aminomethyl-1-tert-
butoxycarbonylpiperidine afforded triamide 6. Depro-
tection of both tert-butoxycarbonyl and tert-butyl of 6
by treatment with TFA afforded 7. Finally, either
reductive amination or alkylation of 7 provided the
target compounds 8.
hydroxy groupdid not affect the M /M3 selectivity.
4
Considering their M3 affinity and selectivity, 9 was sub-
jected to further modifications. In order to examine the
role of the hydroxy groupon the pyrrolidine, the ( S)-
hydroxy- (8a), (R)-hydroxy- (12), (S)-amino- (13), and
(R)-amino (14) analogues were prepared and assayed. In
these analogues, an optically active 3-(3R)-amino-
methyl-1-cyclohexylmethyl piperidine core was used in
place of the racemic one. The (S)-hydroxy analogue, 8a,
was 2- to 3-fold more M3 selective than the (R)-hydroxy
analogue 12 toward the M1, M2, M5 receptors. The (S)-
The binding affinities of the synthesized compounds
were evaluated using cloned human M1–M5 receptors
according to a method10 described previously, and the
selectivity for M3 toward the other receptor subtypes
was examined.
In the previously reported library,10 only a set of l-Pro
and d-Pro were tested with a racemic 3-aminomethyl-1-
cyclohexylmethyl piperidine core. Because additional
interactions between the hydroxy group(s) on the pyr-
rolidine ring in 2a and the M3 receptor were expected, a
set of hydroxyprolines were combined with the same
racemic piperidine template as in the first step of the
Table 1. M3 antagonists from the former library10
Compd
Binding affinity (Ki, nM)a
M3 M1 M2 M4 M5
Selectivity
M1/ M2/ M4/ M5/
M3 M3 M3 M3
Scheme 1. General synthesis of hydroxyproline derivative 8. Reagents
and conditions: (a) MeOH, DMAP, WSC, CHCl3; (b) H2, Pd(OH)2,
MeOH; (c) N-a-carbobenzoxy-O-tert-butyl-l-4-hydroxyproline, WSC,
HOBt, CHCl3; (d) 3,3,3-triphenylpropionic acid, WSC, HOBt, CHCl3;
(e) aq NaOH, MeOH; (f) (3R)-3-aminomethyl-1-tert-butoxycarbonyl-
piperidine, WSC, HOBt, CHCl3; (g) TFA (neat); (h) aldehyde,
NaBCNH3–ZnCl2, MeOH, rt; or RX, K2CO3, CH3CN, heat.
1
2a
0.31 120
0.25 14
30
14
0.82 2.3 150
37
390
56
97 45 120
3.3 9.2 600
Atropine 0.50
0.25 1.5
0.34
0.54
0.50 3.0 0.68
1.1
aValues are the mean of two or more independent assays.

Y. Sagara et al. / Bioorg. Med. Chem. Lett. 13 (2003) 57–60
59
amino analogue (13), and (R)-amino analogue (14)
showed potent M3 affinity comparable to that of 8a. 13
had higher M1/M3, M2/M3, and M5/M3 selectivity than
did 14. A similar trend was observed for the hydroxy
derivatives, 8a and 12.
36-fold, M2/M3; 2000-fold) among the three com-
pounds; however, its M3 affinity was moderate.
The SAR in Table 4 suggests that selective binding of
these ligands to the M3 receptor was affected by both
the size of piperidine N-substituents and the hydroxy
groupat l-Pro. When the piperidine N-substituent was
small, the resulting ligand possibly did not fit into the
pockets of the receptor subtypes other than the M3
receptor. Independently, the results of the increased
selectivity when the hydroxy groupwas introduced to
l-Pro in 2a indicate that there was a counterpart within
the M3 receptor pocket that interacted with the hydroxy
grouppresumably by forming a hydrogen bond.
These results indicate that the regio- and stereo chem-
istry of the hydroxy or amine groupwere important for
the M3 affinity and selectivity toward the M1, M2, and
M5 receptors and suggest that an additional hydrogen
bonding interaction between these analogues and the
M3 receptor resulted in their improved binding affinity
and selectivity (Table 3).
Since the M1/M3 selectivity of 13 was a half that of 8a,
the trans-l-4-hydroxyproline analogue, 8a, was selected
for further modification.
The representative compounds, 8c and 8g, were sub-
jected to evaluation of their functional antagonism using
an in vitro system with rat tissues.12 In the isolated rat
trachea, 8c and 8g antagonized the acetylcholine (ACh)-
induced muscle contractile responses effectively, with a
KB value of 1.6 and 2.7 nM, respectively. Therefore, both
compound 8c and 8g showed an antagonistic activity
comparable to their binding affinity to the M3 receptor.
The effects of the piperidine substituents in both 2a
(spacer: l-Pro-d-Pro) and 8a (spacer: l-hydroxyproline-
d-Pro) on the M3 affinity and selectivity were also
explored, because our previous SAR study on the ana-
logues of 1 revealed that a substituent on the piperidine
nitrogen substantially affected the M3 affinity and
selectivity.10
In conclusion, the SAR studies of the new triphenyl-
propioamide class of M3 antagonists that were con-
ducted to identify more subtype-selective and
conformationally restricted compounds compared with
1 led to the identification of 8c and 8g, which have a
spacer composed of l-hydroxyproline and d-Pro.
Replacement of the cyclohexylmethyl group in 2a with
ethyl (2b), n-propyl (2c) and n-butyl (2d) improved the
M1/M3, M2/M3, and M4/M3 selectivity but resulted in a
greater than 10-fold reduction in the M3 affinity (Table
4). A similar trend was observed for the analogues of 8a.
After the introduction of ethyl, n-propyl, n-butyl, and
n-hexyl onto the nitrogen of piperidine, subtype selectivity
greatly improved (M1/M3; 300–870-fold, M2/M3; 35–
410-fold, M4/M3; 13–38-fold); and M3 affinity ranged
from 0.12 to 8.3 nM. Among the linear alkyl deriva-
tives, 8c, which has an n-propyl group, was the most
balanced compound [Ki (M3); 1.5 nM, M1/M3; 870-fold,
M2/M3; 180-fold, M4/M3; 38-fold, M5/M3; 2300-fold].
In particular, 8c showed a Ki value of 1.5 nM for the M3
receptor with excellent M3 selectivity (M1/M3; 870-fold,
M2/M3; 180-fold, M4/M3; 38-fold, and M5/M3; 2300-
fold), and 8c was 2-fold more selective than 1 in terms
of the M1/M3 and M2/M3 selectivity. Furthermore,
since 8c possesses a decreased conformational flexibility
as mentioned above, this compound may be a more
useful template for understanding of the binding mode
of this triphenylpropioamide class of compounds. Fur-
ther SAR studies around the unique hydroxyproline-
proline analogues are in progress.
Trends in selectivity were similar between the linear
alkyl derivatives and the cycloalkylmethyl derivatives.
The cyclopropylmethyl analogue, 8g, showed the best
selectivity (M1/M3; 700-fold, M2/M3; 190-fold, M4/M3;
Table 3. Stereochemistry of hydroxy and amine groups at l-proline
Table 2. Effects of the introduction of a hydroxy groupto the
prolinesa
No. R1
Binding affinity (Ki, nM)a
Selectivity
M3
M1
M2
M4
M5 M1/ M2/ M4/ M5/
M3 M3 M3 M3
No. R1 R2
Binding affinity (Ki, nM)b
M3 M1 M2 M4
Selectivity
8a
12
13
14
0.13 18
0.34 27
2.1
1.5
0.81 130 140 16
6.2 1000
M5 M1/ M2/ M4/ M5/
M3 M3 M3 M3
3.6
1.3
230
130
82
79 4.4 11
73 15 10
53 2.9 11
680
1000
480
9
10
OH
H
H
OH 2.4
0.17
48 2.7
170 11
1.4
29
9.6 1700 350 28 13 2300
220 280 16
8.2 1300
0.13
0.17
9.5 1.9
1600 71 4.6 12
670
11 OH OH 0.75 260 21
9.0 0.50 1.9
aCompounds 9–11 are racemic at 3-aminomethyl-1-cyclohexylmethyl
piperidine.
bValues are the mean of two or more independent assays.
aValues are the mean of two or more independent assays.

60
Y. Sagara et al. / Bioorg. Med. Chem. Lett. 13 (2003) 57–60
Table 4. Effects of piperidine N-substituent on receptor binding and selectivity
No.
R1
R3
Binding affinity (Ki, nM)a
Selectivity
M3
M1
M2
M4
M5
M1/M3
M2/M3
M4/M3
M5/M3
15
2b
2c
2d
2a
H
H
H
H
H
H
Ethyl
n-Propyl
99
37
6.9
2.6
0.25
2500
>2500
800
3100
2600
150
26
1600
970
220
61
5000
>5100
4500
1500
150
25
>68
120
77
31
70
22
10
16
26
32
23
9.2
51
>140
650
580
600
n-Butyl
Cyclohexylmethyl
200
14
0.82
2.3
56
3.3
7
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
Ethyl
n-Propyl
n-Butyl
n-Hexyl
n-Octyl
23
8.3
1.5
0.60
0.12
0.34
2.0
0.13
0.13
>2500
>2500
1300
290
44
27
1400
18
6300
3400
270
45
4.2
1.5
380
740
270
57
>5000
>5000
3500
1000
150
>110
>300
870
480
370
79
700
140
61
270
410
180
75
35
4.4
190
16
6.3
32
33
38
22
13
11
36
6.2
2.3
>220
>600
2300
1700
1300
680
2000
1000
280
8b
8c
8d
8e
8f
8g
8a
8h
13
1.5
3.6
72
0.81
0.30
230
Cyclopropylmethyl
Cyclohexylmethyl
Cyclooctylmethyl
4000
130
37
2.1
0.82
7.9
aValues are the mean of two or more independent assays.
8. The precise physiological roles of the M4 and M5 receptors
remain to be elucidated. See: (a) Gomeza, J.; Zhang, L.; Kos-
tenis, E.; Felder, C.; Bymaster, F.; Brodkin, J.; Shannon, H.;
Xia, B.; Deng, C.; Wess, J. Proc. Natl. Acad. Sci. U.S.A. 1999,
96, 10483. (b) Gomeza, J.; Zhang, L.; Kostenis, E.; Felder,
C. C.; Bymaster, F. P.; Brodkin, J.; Shannon, H.; Xia, B.;
Duttaroy, A.; Deng, C.; Wess, J. Life Sci. 2001, 68, 2457. (c)
Bymaster, F. P.; Carter, P. A.; Zhang, L.; Falcone, J. F.;
Stengel, P. W.; Cohen, M. L.; Shannon, H. E.; Gomeza, J.;
Wess, J.; Felder, C. C. Life Sci. 2001, 68, 2473. (d) Eglen,
R. M.; Nahorski, S. R. Br. J. Pharmacol. 2000, 130, 13.
9. Eglen, R. M.; Choppin, A.; Dillon, M. P.; Hegde, S. Curr.
Opin. Chem. Biol. 1999, 3, 426.
Acknowledgements
We are grateful to Ms. Nami Sakaizumi, Ms. Sachie
Arai-Otsuki, and Ms. Minaho Uchiyama for technical
support and Ms. Jocelyn Winward, Merck & Co., Inc.,
for her assistance in preparation of the manuscript.
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