A new series of M3 muscarinic antagonists based on the 4-amino-piperidine scaffold

Article abstract of DOI:10.1016/S0960-894X(02)00487-0

A series of 4-amino-piperidine containing molecules have been synthesized and structure-affinity relationship toward the M3-muscarinic receptor has been investigated. Chemical modulations provided molecules with K_i for the human M3-R up to 1 nM with variable selectivity (3- to 40-fold) over the human M2-R. Compounds 2 (pA₂=8.3, 8.6) demonstrates in vitro on guinea pig bladder and ileal strips potent anticholinergic properties and tissue selectivity.

Full text of DOI:10.1016/S0960-894X(02)00487-0

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2535–2539
A New Series of M3 Muscarinic Antagonists Based on the
4-Amino-piperidine Scaffold
O. Diouf,a S. Gadeau,a F. Chelle,a M. Gelbcke,a P. Talaga,b B. Christophe,b M. Gillard,b
R. Massinghamb and M. Guyauxb,*
a
´
Laboratoire de Chimie Pharmaceutique Organique, Universite Libre de Bruxelles, Institut de Pharmacie,
Campus Plaine CP205/5, Boulevard du Triomphe, B-1050 Bruxelles, Belgium
bUCB S.A., Pharma Sector, Preclinical Research, Chemin du Foriest, B-1420 Braine-l’Alleud, Belgium
Received 11 April 2002; revised 6 June 2002; accepted 27 June 2002
Abstract—A series of 4-amino-piperidine containing molecules have been synthesized and structure–affinity relationship toward the
M3-muscarinic receptor has been investigated. Chemical modulations provided molecules with Ki for the humanM3-R up to 1 nM
with variable selectivity (3- to 40-fold) over the humanM2-R. Compounds 2 (pA2=8.3, 8.6) demonstrates in vitro on guinea pig
bladder and ileal strips potent anticholinergic properties and tissue selectivity. # 2002 Elsevier Science Ltd. All rights reserved.
The M3 muscarinic receptor (M3-R) plays a key role in
the contraction of smooth muscle in a variety of organs.
Ability to block the effect of the parasympathetic system
and acetylcholine on post-junctional M3-R explains the
therapeutic efficacy of anticholinergic drugs used to
treat diseases associated with altered smooth muscle
contractility such as, urinary urge incontinence and
irritable bowel syndrome.1 Unfortunately, the useful-
ness of current anticholinergic medication is limited by
side effects such as tachycardia, dry mouth, constipation
and blurred vision.2 Selectivity towards M3 over M2
muscarinic receptors minimizes the risk of cardiovas-
cular effects due to known involvement of M2-R in the
regulationof cardiac rhythm. Nevertheless, requirement
of M3-subtype selective antagonism remains con-
troversial. Firstly, non cardiac systemic side effects (e.g.,
dry mouth) are prominent and are all M3-R mediated.
Secondly, M2-R predominate in number over M3-R in
smooth muscle and are thought to assist contraction by
abrogating muscle relaxation induced by activation of
the sympathetic system.1,3,4 Thus, there is a great inter-
est in finding muscarinic antagonists displaying tissue
selectivity for the targeted organ.
Screening of our in house library identified compound 1
which was selected for further chemical modulation.
These compounds were tested for muscarinic affinity
using recombinant human muscarinic m3 and m2
receptors, and were thereafter tested for functional
muscarinic receptor subtype or tissue selectivity in
guinea pig urinary bladder, ileal smooth muscle (M3)
and atrial muscle (M2) preparations.
Chemistry
The synthetic route5 to the 4-substituted amino-piperi-
dine derivatives is outlined in Scheme 1. Briefly, N-ben-
zylated piperidone and the corresponding amine are
dissolved inCH 2Cl2 inthe presence of titanium chloride
in catalytic amounts, to give the imine derivatives which
are thenreduced using sodium borohydride inMeOH
to the corresponding secondary amines. Compounds 1–
25 are obtained by acylation using the corresponding
acyl chlorides in the presence of potassium carbonate.
Results and Discussion
Inthis communication, we describe the invitro pharma-
cological profile of new N,N-disubstituted 4-amino-1-
benzylpiperidine derivatives as muscarinic antagonists.
The synthesis of 1 (CAS No. 95869-86-6) and some
related 4-amino-piperidines analogues has been pub-
lished previously,6 mainly as synthetic intermediates for
the preparation of various potential therapeutic agents
such as antiarrhytmic benzopyran derivatives,7 anti-
asthma benzimidazolone neurokinin 1 antagonists8 and
*Corresponding author. Tel.: +32-2-386-2608; fax: +32-2-386-3133;
e-mail: michel.guyaux@ucb-group.com
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00487-0

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O. Diouf et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2535–2539
contrary to what was noted for the R1 position. More-
over, the substitution of the phenyl or thienyl ring at the
R2 positionwith NO (respectively, 21 vs 1, 22 vs 3),
2
induces a decrease in affinity for the M3-R (pKi=6.1 vs
7.6 and 8.1 vs 8.7, respectively).
Introduction of a hydroxyl (6, pKi=8.6) or a OMe
group (7, pKi=8.4) at the para positionof the phenyl
ring in R1 leads to compounds with slightly less affinity
for the M3-R than the corresponding halogenated deri-
vatives, but still more potent than the unsubstituted
compound 1. Compound 8, bearing a 3,4-diCl aryl
group inR1 displayed almost the same affinity as the
corresponding 4-Cl derivative 4. Incontrast, 9, bearing
a 2,6-diCl aryl group inR1 is 150-fold less potent than
compound 5, the isosteric 2-thienyl derivative bearing a
4-Cl aryl group at the same position.
Scheme 1. Procedure for the synthesis of the series of 4-amino-piper-
idine derivatives: (a) R1-NH2, CH2Cl2, TiCl4, 0 ꢁC; (b) NaBH4,
MeOH; (c) CHCl3, K2CO3, R2COCl.
therapeutic claims related to analgesia.9 Thus, to the
best of our knowledge, no publication has reported on
the antimuscarinic properties of such compounds.
As seen in Table 1, the unsubstituted compound 1
(R1=R2=Ph) had an affinity of 25 nM (pKi 7.6) for the
M3-R. The introduction of a bromine (2) or a chlorine
(4) atom at the para position of the phenyl ring in R1
increased the affinity for the M3-R almost 25-fold. The
same trend was observed with a 2-thienyl moiety (3 and
5 vs 16) inR2. Whereas the phenyl ring inR2 could be
replaced by a 2-thienyl moiety (1 vs 16), a slight 4-fold
decrease inaffinity for the M3-R was observed using a
furyl moiety (17 vs 1; pKi=7.0 vs 7.6) as the replacing
aromatic isosteric group.
The replacement of the aryl moiety in R1 with a cyclo-
butyl group reduced at least 400-fold the affinity for the
M3-R inboth phenyl ( 10 vs 1) and 2-thienyl (11 vs 16)
families. Moreover, a 30-fold reductionof affiinty
occurs when replacing the phenyl group in R1 by a 3-
pyridyl moiety (18; pKi=7.5 vs 12; pKi=6.0).
Interestingly, the replacement of the aryl moiety by a
cyclo-butyl group inR2 ( 13 vs 1, 14 vs 2) did not change
the affinity for the M3-R. In contrast, replacing the
cyclo-alkyl group inR2 by a methyl group ( 15 vs 2,
pKi=6.8 vs 9.0) induces a considerable reduction (150-
fold) inaffinity.
Interestingly, the 4-Br/4-Cl-aryl substitutions in R2 (18,
19, 20) did not increase the affinity for the M3-R
Table 1. The affinities for M2-R and M3-R were measured by the
ability of the compounds to displace [3H] N-methylscopolamine using
membrane preparations from CHO-K1 cells expressing the human
recombinant m2 and m3 muscarinic receptors10
Compounds bearing a (CH2)n–Ph (n=1,2) group in
R1 (25) or R2 (23, 24) were 3- to 10-fold less potent
than 1.
R1
R2
M3 M2 Bladder Ileum Atria
7.6 6.5
In summary, the following structure–affinity relationships
toward the M3-R canbe outlined:
1
2
3
4
5
6
7
8
Ph
4-Br-Ph
4-Br-Ph
4-Cl-Ph
4-Cl-Ph
4-OH-Ph
4-OMe-Ph
3,4-diCl-Ph
2,6-diCl-Ph
cButyl
Ph
Ph
2-Thienyl
Ph
2-Thienyl
Ph
Ph
Ph
2-Thienyl
Ph
2-Thienyl
4-Br-Ph
cButyl
cButyl
Me
2-Thienyl
2-Furyl
4-Br-Ph
4-Cl-Ph
4-Cl-Ph
4-NO2-Ph
9.0 8.3
8.7 7.6
8.9 8.1
8.4 7.3
8.6 7.1
8.4 6.9
8.7 7.7
6.2 5.7
++ ++
8.3
7.5
7.9
7.4
7.6
7.5
7.8
—
8.6
8.0
8.1
7.5
7.8
7.6
7.9
—
7.7
6.8
7.9
7.0
6.8
7.0
6.9
—
ꢀ the R1 group inthe 4-amino-piperidine scaffold
is preferably anaromatic ring.
ꢀ Substituting the para positionof the aryl group in
the R1 positionby halogenatoms (Br, Cl) or elec-
tron donating group (OMe) considerably increases
the M3-R affinity whereas no change or even a
decrease inaffinity is observed whensubstituting
the corresponding meta and ortho positions.
ꢀ Substituting the para positionof the aryl group
in R2 by halogens does not change the affinity
for the M3-R contrary to what was observed in
R1 position but the introduction of an electron-
withdrawing group like NO2 decreases the affi-
nity for the M3-R.
ꢀ Only the R2 position allows the replacement of
the aromatic ring by cyclo alkyl (e.g., cButyl)
groups.
ꢀ Increasing the length of the spacer between the
nitrogen atom of the amide link and the aryl
groups in R1 and R2 significantly decreases affi-
nity the M3-R.
9
10
11
12
13
—
—
—
—
—
—
cButyl
3-Py
Ph
+
+
6.0 ++
7.8 6.6
8.8 7.7
6.8 6.4
7.6 6.7
7.0 6.1
7.5 6.2
7.6 6.2
9.1 7.7
6.1 5.4
—
—
7.9
—
—
—
—
—
—
—
7.9
—
—
—
—
—
—
—
7.5
—
—
—
—
—
14 4-Br-Ph
15 4-Br-Ph
16
17
18
19
Ph
Ph
Ph
Ph
20 4-Br-Ph
21 Ph
22 4-Br-Ph 5-NO2-2-thienyl 8.1 6.5
7.4
—
7.6
—
6.9
—
6.0
—
—
—
6.7
—
—
—
5.7
—
—
—
23
24
Ph
Ph
Ph-CH2
Ph-CH2-CH2
Ph
7.6 6.5
7.1 6.1
6.8 6.2
25 Ph-CH2
Results are expressed as pKi or inhibition (+ <20%; ++ 20–50%) of
the radioligand specific binding by 10 mM of the test compounds.
Antagonism of M3-R and M2-R was measured by the ability of com-
pounds to displace (pA2) the concentration–response curve of carbachol
onisolated guinea-pig urinary bladder, ileum 11 and paced left atria.12
ꢀ Replacement of the piperidinic N-benzyl group
(results not shown) by H or Me groups induces a
400- to 1000-fold loss of affinity for the M3-R.

O. Diouf et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2535–2539
2537
Thus, the 4-amino-piperidine moiety can be considered
as a good alternative to other reported M3-R musca-
rinic antagonist scaffolds.13ꢂ17 Indeed, the main phar-
macophoric elements allowing a good affinity for the
M3-R canbe defiend as two aromatic groups (oen
replaceable by a cyclo-alkyl group) linked to a basic
nitrogen via a piperidine containing spacer (Fig. 1). The
basic nitrogen being generally linked to a lipophilic
group (e.g., benzyl or isoprenyl). The SARs (structure–
affinity relationships) related to these scaffolds are close
to what has beenpublished inthis article for the
4-amino-piperidine scaffold.
It thus becomes clear that the functional as well as the
tissue selective activity will definitely help to better
define the therapeutic potential of such compounds.
As illustrated inFigure 2A, most of the present com-
pounds have at least 3-fold greater affinity for the human
M3-R over the M2-R. The most selective compounds are
22 (40 fold selectivity), 6 and 7 (32-fold selectivity). These
compounds displayed a rather selective affinity for the
muscarinic receptor, similar to that documented for the
selective M3 antagonist Darifenacine.18ꢂ20
The functional antagonism of carbachol responses in
guinea pig urinary bladder, ileal and atrial preparations
Figure 1. Structures of 2 and of other M3 antagonists with a 1,4-sub-
stituted piperidine in their backbone: a ,13 b ,14 c ,15 d ,16 e.17
Figure 2. Analysis of data from Table 1. Values of Tolterodine (T), Darifenacine (D) and Oxybutynin (O) are from Newgreen et al.,18 Gilberg et
al.19 and Wallis et al.,20 respectively.

2538
O. Diouf et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2535–2539
(Table 1 and Fig. 2) was evaluated for compounds dis-
playing a pKi value greater than8.0 for the M3-R.
Increasing concentrations of these compounds induced
a parallel shift to the right of the concentration–
response curve to carbachol on all three organs, indi-
cating the competitive nature of the antagonism on both
M3 and M2 muscarinic receptors.
compounds. These results indicate that lead compounds
derived from the 4-amido- or 4-sulfonamido-N-benzyl
piperidine scaffold may have therapeutic potential for
the treatment of urinary incontinence and/or irritable
bowel syndrome.
Acknowledgement
Compound 2 was the most potent anticontractile agent
onboth the ileum (p A2=8.6) and the bladder
(pA2=8.3). Anticontractile potency of compound 2 on
the bladder lies betweenthe reported p A2 values18,20 of
Darifenacine (pA2=8.7), Tolterodine (pA2 blad-
der=7.8) and Oxybutynin (pA2 bladder=7.4), which
are drugs under development or already used for the
treatment of irritable bowel syndrome and urinary
incontinence.
We are grateful to Dr E. Differding for commenting on
the manuscript.
References and Notes
1. Eglen, R. M.; Choppin, A.; Watson, N. Trends Pharmacol.
Sci. 2001, 22, 409.
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Compounds 2, 8, 6, 7 were 3- to 5-fold more potent on
the bladder thanonthe atria (Fig. 2B). They behave in
vitro as slightly more selective (bladder vs atria) than
Tolterodine (pA2 atria=7.7, non-selective) and Oxybu-
tynin (pA2 atria=7.1; 2-fold selectivity) but far less
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5. General synthesis procedure for 1–26. Intermediate amines:
the primary amine (3 equiv) and N-benzylpiperidin-4-one (1
equiv) are stirred in100 mL of dry CH 2Cl2. The mixture is
cooled to 5 ꢁC, and then a solution of 0.6 equiv of TiCl4 in20
mL of dry CH2Cl2 is added dropwise. The reactionis followed
by TLC. When the ketone derivative is completely consumed
MeOH is added followed by 4 equiv of NaBH4. Stirring is
maintained between 0 and 5 ꢁC for at least 2 h. Solvent is
evaporated invacuo, and the residue triturated inwater (100
mL). The resulting precipitate is filtered, and the filtrate
extracted with AcOEt. The organic layer is extracted by a 10%
aqueous solutionof AcOH. The aqueous layer is alkalinized
with K2CO3 up to pH 7–8. The product is thenextracted with
chloroform, the organic layer being dried with CaCl2. The
hydrochloride derivative solubilized indry acetone is formed
by adding Hcl dissolved in diethyl ether. The precipitate is fil-
tered, dried, and recrystallized from acetone.
selective thanDarifeancien (p
A2 Atria=7.3; 25-fold
selectivity). Compounds 22, 6 and 7 were thus func-
tionally far less selective (2-, 6-, 3-fold, respectively)
than expected from their binding profile. This dis-
crepancy might be due to the experimental conditions
(human versus guinea pig preparations, membrane ver-
sus tissue extract), which could affect molecular diffu-
sion properties, thus changing compound distribution at
the membrane M3-R vs the tissue M3-R. Most com-
pounds were equiactive on the bladder and the ileum
(selectivity ratio <3; Fig. 2C) with the exceptionof
compound 22 which was 5-fold more potent on the
ileum thanonthe urinary bladder.
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chloride (1 equiv) and K2CO3 (4 equiv) are heated under reflux
indry chloroform for 30 min. Then3 equiv of acyl chloride
diluted inchloroform are added dropwise. After 20 h of stir-
ring at room temperature, the solvent is evaporated in vacuo.
The residue dissolved in50 mL of water and extracted with
CHCl3. The organic layer is washed with a 10% aqueous
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CHCl3 layer is washed with anaqueous solutionof K 2CO3
and water, dried with CaCl2 and evaporated in vacuo. The
hydrochloride derivative is formed by adding HCl in diethyl
ether to the amine solution in dried acetone. The precipitate is
filtered, dried, and recrystallized from a mixture of acetone/
diethyl ether (1:3). N-(1-benzylpiperidin-4-yl)-N-(4-bromophe-
The most potent compound 2 failed (results not shown)
to compete with radioactive ligands specific to the
adenosine A1, A2, adrenergic a1, a2, an db, sero-
toninergic 5-HT1ꢂ7, dopaminergic D1, D2 and histami-
nergic H1, H2, H3 receptors at 10 mM indicating that it
binds in a relatively selective manner to muscarinic
receptors. Furthermore, onisolated rat aorta pre-
contracted with 100 mM KCl,21 compound 2 inhibited
KCl induced contractions (pD02=4.2 and 4.1, respec-
tively) at a concentration range far exceeding that
required for muscarinic mediated anticontractile effects
inthe bladder or ileum. This indicates that 2 is unlikely
to possess calcium antagonist potential at relevant
pharmacological doses.
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.
Mp 241.5 ꢁC. IR (KBr), n (cmꢂ1): 1639 (CO); 1H NMR
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4H, N(CH2)2), 4.2 (s, 2H, CH2f), 4.8 (m, 1H, CH), 7.0–7.6
(m, 14H, aromatics), 10.8 (s, 1H, NH+). 13C NMR (DMSO-
d6), d (ppm): 26.4 (C(CH2)2), 50.1–50.3 (N(CH2)2+CH), 58.4
(CH2f), 120.4–137.7 (C aromatics), 120.4, 129.4, 136.1, 137.7
(Cq), 169.2 (C¼O). MS (ESI) MH+=449 (100%); 451 (99%).
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displayed onthe atria. Thus compound 2 is as potent
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