Cyclohexylmethylpiperidinyltriphenylpropioamide: A selective muscarinic M3 antagonist discriminating against the other receptor subtypes

Article abstract of DOI:10.1021/jm010480k

To discover a highly selective M₃ antagonist, a combinatorial library was prepared. The library was designed to identify a novel structural class of M₃ antagonists by exploring the spatial arrangement of the pharmacophores in known M

Full text of DOI:10.1021/jm010480k

984
J . Med. Chem. 2002, 45, 984-987
Brief Articles
Cycloh exylm eth ylp ip er id in yltr ip h en ylp r op ioa m id e: A Selective Mu sca r in ic M3
An ta gon ist Discr im in a tin g a ga in st th e Oth er Recep tor Su btyp es
Yufu Sagara, Takeshi Sagara, Toshiaki Mase, Toshifumi Kimura, Tomoshige Numazawa, Toru Fujikawa,
Kazuhito Noguchi, and Norikazu Ohtake*
Banyu Tsukuba Research Institute in collaboration with Merck Research Laboratories, Okubo-3, Tsukuba 300-2611,
Ibaraki, J apan
Received October 18, 2001
To discover a highly selective M3 antagonist, a combinatorial library was prepared. The library
was designed to identify a novel structural class of M3 antagonists by exploring the spatial
arrangement of the pharmacophores in known M3 antagonists. After the evaluation of 1000
library members, a potent M3 antagonist, 14a (Ki ) 0.31 nM), with novel structural features
was identified. Compound 14a showed high selectivity for M3 receptors over the other muscarinic
receptor subtypes (M1/M3 ) 380-fold, M2/M3 ) 98-fold, M4/M3 ) 45-fold, M5/M3 ) 120-fold).
In tr od u ction
Acetylcholine (ACh) plays a number of pharmacologi-
cal roles that are mediated by nicotinic and muscarinic
receptors in central and peripheral nervous systems.
Five muscarinic receptors (m1-m5) have been identified
so far that mediate muscarinic functions.1 These mus-
carinic receptors are homologous across receptor sub-
F igu r e 1. Known M2-sparing M3 antagonists.
types as well as across mammalian species2 and are
expressed predominantly in the parasympathetic ner-
design, we paid attention to spatial orientation and
vous system. Although these receptors, with the excep-
arrangement of the pharmacophores for M3 antagonists.
tion of m5, have been proposed to participate in a
An increase in the flexibility of the spatial arrangement
number of physiologic functions, the roles of each
of the molecules may lead to more specific recognition
receptor subtype in the specific muscarinic actions of
of the binding site of M3 receptors over those of the other
ACh remain to be elucidated. For further characteriza-
receptor subtypes. Here, we report the design and
tion of the functions of each receptor, specific antago-
synthesis using combinatorial chemistry and identify a
nists or agonists would be useful.
new structure class of M3 receptor antagonists. Subse-
We have been interested in M3 receptors from their
quent derivation of this class provided a cyclohexyl-
pharmacological properties: the M3 receptor subtype is
methylpiperidinyltriphenylpropioamide (CPTP, 14a ),
which showed potent binding affinity (Ki ) 0.31 nM) to
M3 receptors and was 380-, 98-, 45-, and 120-fold more
homologous to other subtypes, M1, M2, M4, and M5,3 and
has been postulated to facilitate the parasympathetic
stimulation of smooth muscle contraction and glandular
selective for M3 over the M1, M2, M4, and M5 receptors,
secretion in the peripheral system.4 It is widely ex-
respectively.
pressed in the brain;5 however, the physiologic roles
remain unknown. A recent report demonstrated that
M3-deficient mice were hypophagic and lean, suggesting
a role of central M3 receptors in orexigenic activity.6 To
investigate the roles of peripheral and central M3
receptors, selective and effective M3 antagonists were
explored.
Although extensive synthetic efforts have been de-
voted to discover selective M3 antagonists, none of them
have identified any compounds with sufficient selectivity
toward M3 receptors.2,7 To identify a potent and selective
M3 receptor antagonist, we adopted a strategy using
combinatorial chemistry that would effectively yield a
series of structurally diverse antagonists. For the library
Design a n d Syn th esis
Analysis of the structural features of the known
nonselective muscarinic antagonists and M2-sparing M3
antagonists such as 1, 2, and darifenacin8 (Figure 1)
suggested that a bulky aromatic cluster, a cationic
nitrogen-containing part, and a hydrophobic part neigh-
boring the nitrogen atom as the N-substituent are
essential pharmacophores for M3 potency. We postu-
lated that new structure class of muscarinic antagonists
with an increase in the molecular size and flexibility of
the spatial arrangement of the known antagonists could
lead to more specific recognition of the binding site of
M3 receptors over those of the other receptor subtypes.
On the basis of this prediction, we designed muscarinic
antagonists by incorporating a diverse spacer group
between the aromatic cluster and the cationic site
* To whom correspondence should be addressed: Banyu Tsukuba
Research Institute, Okubo-3, Tsukuba, 300-2611, Ibaraki, J apan. Tel:
+81-298-77-2000. Fax: +81-298-77-2029. E-mail: ootakenr@banyu.co.jp.
10.1021/jm010480k CCC: $22.00 © 2002 American Chemical Society
Published on Web 01/18/2002

Brief Articles
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4 985
Sch em e 2. Library Synthesisa
F igu r e 2. Design of a new class of muscarinic antagonists.
Sch em e 1. Design of a Combinatorial Librarya
a
Reagents and conditions: (a) NaBH(OAc)3, DCM; (b) PyBOP,
DIEA, DMF; (c) 20% piperidine-DMF; (d) WSC, HOBt, DMF; (e)
1% H2O-19% DCM-TFA.
Sch em e 3. General Synthetic Method of the Selected
Compoundsa
a
R3COOH: structures of all 25 carboxylic acids are available
a
Reagents: (a) (1) 10% HCl-MeOH, rt, (2) Ph3CCH2CO2H,
in Supporting Information. bGABA: 4-aminobutyric acid. cAcp:
6-aminohexanoic acid.
WSC, HOBt, NEt3, CHCl3, rt; (b) 4 N NaOH, MeOH, CHCl3, rt;
(c) (3R)-3-aminomethyl-1-tert-butoxycarbonylpiperidine, WSC,
HOBt, CHCl3, rt; (d) TFA, CHCl3, rt; (e) aldehyde, NaBCNH3-
ZnCl2, MeOH, rt.
through a combinatorial chemistry approach. Simulta-
neously, optimization of the aromatic cluster was per-
formed (Figure 2).
was introduced by the same procedure. Finally each
group of seven 6 was further split into 25 subgroups
and coupled with 25 carboxylic acids using a WSC-HOBt
method. The number of mixtures (six mixtures per vial)
was chosen after several attempts for ease of product
identification in ESI-MS spectra and for reducing the
risk of producing false positive results in the binding
assay. Finally, the products were cleaved from the resin
by treatment with 80% TFA/DCM (containing 1% H2O).
In total, 175 vials and 1050 compounds (6 mixtures ×
175 vials) were synthesized, and all samples were
subjected to HPLC and mass spectral analysis. In most
cases, the target compounds were produced in more
than 85% purity, and all exhibited major peaks corre-
sponding to the correct molecular ions in the ESI mass
spectra.
A library was constructed as follows (Scheme 1): Two
variable sites were defined (AA1 and AA2) as the spacer
moiety, and commercially available amino acids were
incorporated into each site. Natural and unnatural
amino acids with various sizes, including cyclic amino
acids such as proline, were selected to increase the
diverse display of conformations. A combination of these
amino acids varied the size of the spacer widely, from
three atoms (composed of null and glycine) to 14 atoms
(composed of double 6-aminohexanoic acids).
The cationic tertiary amine core including a hydro-
phobic N-substituent was tentatively fixed to a racemic
1-cyclohexylmethyl-3-aminomethylpiperidine. With re-
spect to the terminal acyl moiety, 25 commercially avail-
able carboxylic acids including aromatic, heteroaromat-
ic, cycloalkyl, and acyclic carboxylic acids were selected.9
The construction of the library was performed using
a (4-formyl-3-methoxyphenoxy)alkyl resin 3,10 which has
been reported to give products with excellent purity11
(Scheme 2). First, an aminomethyl piperidine core was
introduced on the o-formyl resin by reductive alkyla-
tion,12 and then spacer parts were condensed in order
by the following two steps: The resultant amine 4 was
acylated with the first spacer group (six Fmoc amino
acid derivatives) by a standard PyBOP-DIEA method13
to give six pools of 5. After the six pools were mixed
and split into seven groups, their Fmoc groups were
removed by a standard protocol, and a second spacer
group (Fmoc AA2-OH; six amino acids and one deletion)
The general synthetic method of the selected com-
pounds (13a -20a ) is highlighted in Scheme 3. An
aminoester derived from 7 was condensed with 3,3,3-
triphenylpropionic acid under the usual conditions to
produce a diamide 8. Hydrolysis of the ester moiety in
8 and subsequent coupling with an optically active
1-Boc-3-aminomethylpiperidine14 yielded a triamide 10.
Finally, deprotection of the Boc group followed by
reductive alkylation using an appropriate aldehyde and
15
NaBCNH3-ZnCl2 produced a target product 12.16
Resu lts a n d Discu ssion
The primary screening of the library was performed
by examining inhibition of [3H]-NMS binding (1 µM) to

986 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4
Brief Articles
Ta ble 1. Binding Affinities of the Selected Compounds to
Ta ble 2. In Vivo Selectivity of 14a in Rats
Human Muscarinic Receptor Subtypes
selectivity
ED50 (µg/kg, iv)
binding affinity (Ki, nM)a
selectivity
m1/ m2/ m4/ m5/
m3 m1 m2 m4 m5 m3 m3 m3 m3
broncho-
pressor
no. AA2
AA1
Acp
constriction bradycardia response
M1/
M3
M2/
M3
13a null
14a Gly
15a Gly
1.0 110 72
â-Ala 0.31 120 30
30
14
210 110 72 30 200
37 380 98 45 120
1100 90
30 48
M3
M2
M1
14a
Atropine
15
4.3
1550
5.2
>3000
>200 100
3.7 1.2
GABA 3.3 300 270 74
82 22 320
58 41 40
8.2 29 95
5.7 19 890
16
16a â-Ala â-Ala 0.76 36
17a GABA â-Ala 2.1 77
18a L-Pro Gly
19a L-Pro â-Ala 0.27 150 12
20a L-Pro D-Pro 0.25 14
44
17
31
60
43
11
200 37
2000 66
58
2.2 150 13
that cyclohexylmethyl gave the best result from the
viewpoint of both m3 affinity and the selectivities.18
Although the detailed interaction of the compounds
of this class and muscarinic receptors is not clarified,
we speculate that the newly introduced spacer moieties
may allow the acyl part, triphenylpropionamide, to
interact with muscarinic receptors in a significantly
different binding mode from those of the known M3
antagonists.
Compound 14a was subjected to evaluation of its
functional selectivity using an in vitro system with rat
tissues.7e In the isolated rat trachea, this compound
effectively antagonized the acetylcholine (ACh)-induced
responses, with a KB value of 2.3 nM.19 In the isolated
rat atria, this compound showed less potent inhibition
of carbachol-induced bradycardia, with a KB value of 160
nM. These results indicated that the functional selectiv-
ity of this compound was 70-fold greater for tracheal
M3 receptors over cardiac M2 receptors in rat, which was
consistent with the selectivity observed in the binding
assay.
The in vivo selectivity of 14a was next examined in
rat assay systems (Table 2). Intravenous administration
of 14a dose-dependently inhibited ACh-induced bron-
choconstriction, with an ED50 value of 0.015 mg/kg in
rats. In contrast, intravenous administration of 14a up
to 3 mg/kg did not affect the McN A-343-induced pressor
response that is thought to be mediated by M1 recep-
tors.20 Furthermore, 14a inhibited ACh-induced brady-
cardia (M2), with an ED50 value of 1.55 mg/kg after
intravenous administration. These results may suggest
that 14a show greater than 200- and 100-fold selectivity
for M3 over M1 and M2 receptors in these assay systems,
although its ADME properties were not elucidated.
550 45 42 210
3.3 9.0 580
0.82 2.3 150 54
Atropine
Darifenacin
0.50 0.25 1.5 0.34 0.54 0.96 2.9 1.3 3.3
0.84 5.5 47 8.6 2.3 6.5 56 10 2.7
a
Values are the mean of two or more independent assays.
human m3 receptors expressed in CHO cells. Then the
selected compounds were examined for their binding
affinities (Ki values) to each of the five receptor subtypes
(m1-m5).7e After evaluation of whole library members,
only the vials derived from triphenylpropionic acid in
R3 position showed remarkable inhibition (>70% inhibi-
tion).9 The hit vials were further examined by iterative
deconvolution process including chiral resolution in the
3-aminomethylpiperidine moiety and proline spacers
with respect to their affinity for m3 receptors and
selectivity over the other receptor subtypes.17
As a result, (3R)-3-aminomethylpiperidine derivatives
with particular combination of amino acid spacers were
found to show highly potent M3 binding affinities and
moderate-to-high selectivities for m3 over m1 and m2
receptors (Table 1). Among them, compounds 13a , 14a ,
and 19a showed greater than 100-fold m1/m3 selectiv-
ity, which was the highest m1/m3 selectivity reported
to date. Especially, 14a showed sub-nanomolar binding
affinity for m3 receptors and excellent subtype selectiv-
ity over all the other receptor subtypes.
As shown in Table 1, the spacer structures of this
class of compounds were found to significantly affect
their selectivities for m3 over m1 and m2 receptors. For
example, deletion of the amide moiety (13a ) in the
middle of the spacer group in 14a resulted in a 3-fold
decrease in both the m3 binding affinity and m1/m3
selectivity as compared to 14a , while 13a showed almost
similar m2/m3 selectivity. Replacement of the glycyl
moiety in 14a with a â-alanyl group (16a ) led to
moderate m2/m3 selectivity, while replacement with a
γ-aminobutyryl group (17a ) resulted in almost complete
loss of m2/m3 selectivity. Replacement of the â-alanyl
group in 14a with a γ-aminobutyryl group (15a ) also
resulted in a 10-fold reduction in the m3 binding affinity
with a 4-fold decrease in m1/m3 selectivity, suggesting
that the amide moiety in the middle of the spacer moiety
in 14a played an important role in enhancing m3
affinity and m1/m3 selectivity. In contrast, replacement
of the glycyl moiety with a L-prolyl group (19a ) produced
enhanced m1/m3 selectivity (550-fold), despite decreased
m2/m3 selectivity (45-fold), when compared with 14a .
Con clu sion
We designed diverse classes of muscarinic antagonists
using a combinatorial library approach based on the
hypothesis that increases in the molecular size and
conformational flexibility of the molecules produced
more specific binding with m3 receptors than with the
other receptor subtypes. We have found that 3,3,3-
triphenylpropionamide derivatives with one or two
amino acid residues between the triphenylpropionic acid
moiety and the piperidinylmethylamine moiety showed
potent binding affinities for m3 receptors with moder-
ate-to-high subtype selectivities. Particularly, 14a showed
sub-nanomolar m3 affinity and high selectivity over the
other receptor subtypes. Compound 14a also showed
high subtype selectivity (70-fold) for tracheal M3 over
cardiac M2 receptors in the isolated rat tissues. Fur-
thermore, 14a showed greater than 100-fold in vivo
As the piperidine N-substituent was tentatively fixed
to the cyclohexylmethyl moiety in the library, we
examined the effect of the substituent on 14a to find

Brief Articles
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4 987
(3) For example, the human M3 receptor is 76-79% homologous to
the other subtypes, M1, M2, M4, and M5, in the transmembrane
region. (a) Bourdon, H.; Trumpp-Kallmeyer, S.; Schreuder, H.;
Hoflack, J .; Hibert, M.; Wermuth, C.-G. Modelling of the binding
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selectivity for M3 over M1 and M2 receptors in the above
rat assay systems. Therefore, 14a would be useful as a
pharmacological tool to clarify the roles of peripheral
and central M3 receptors. Elucidating the roles of central
M3 receptors that might be involved in food intake would
be of particular interest.
Exp er im en ta l Section
K.; IJ zerman, A. P.; Edvardsen, Ø. TinyGRAP database:
a
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N-(2-[3-([(3R)-1-(Cycloh exylm eth yl)-3-piper idin yl]m eth -
yla m in o)-3-oxop r op yl]a m in o-2-oxoeth yl)-3,3,3-tr ip h en yl-
p r op ioa m id e 14a . To a solution of 14b18 (270 mg, 0.513
mmol) and cyclohexanecarbaldehyde (75 µL, 0.616 mmol) in
MeOH (3 mL) was added NaBH3CN-ZnCl2 (0.3 mol/L, 3.1
mL),15 and the mixture was stirred at room temperature for
40 min. The reaction was quenched by adding saturated
aqueous NaHCO3 solution and extracted with EtOAc. The
organic phase was washed with brine, dried (Na2SO4), and
evaporated. The residue was purified by silica gel column
chromatography (CHCl3-MeOH, 20:2-20:3 elution) to give
14a (261 mg, 0.419 mmol, 82%) as a white solid: mp 143.5-
145.0 °C (Et2O-CHCl3); HPLC tR ) 23.7 min (system A), 17.2
min (system B); 1H NMR (CDCl3) δ 0.81-1.80 (m, 17H), 1.97-
2.08 (m, 3H), 2.28-2.32 (m, 2H), 2.59-2.65 (m, 2H), 3.12-
3.17 (m, 2H), 3.37-3.46 (m, 2H), 3.51-3.55 (m, 2H), 3.64 (s,
2H), 5.60-5.64 (m, 1H), 6.10-6.20 (m, 1H), 6.26-6.30 (m, 1H),
7.19-7.33(m, 15H). HRMS Calcd for C39H51N4O3 (M + H)+:
623.3961. Found: 623.3967. Anal. Calcd for C39H50N4O3: C,
75.21; H, 8.09; N, 9.00. Found: C, 74.96; H, 8.26; N, 8.92.
Bin d in g Assa y. According to the reported method,7e the
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In Vivo Assa y. According to the reported method,7e bron-
choconstriction and bradycardia were evaluated. See Support-
ing Information for the method of pressor evaluation.
Ack n ow led gm en t. We are grateful to Ms. Nami
Sakaizumi, Ms. Ikuko Nishimura, and Mr. Hirokazu
Ohsawa for technical support and Mr. Dan J ohnson and
Ms. Kimberley Marcopul, Merck & Co., Inc., for their
valuable assistance in preparation of the manuscript.
We thank Dr. Mitsuaki Yoshida for helpful discussions
regarding this work.
(9) See Supporting Information.
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screening of the library, and a table of the effect of piperidine
substituents on 14a . This material is available free of charge
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and mass spectra.
(17) The detailed discussion on the deconvolution process will be
reported elsewhere.
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tylmethyl, and no substituent were examined. (See Supporting
Information.)
(19) The pA2 value of 14a in the rat trachea was 8.46.
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because McN A-343 is a partial agonist.
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J M010480K