Design and synthesis of piperidinyl piperidine analogues as potent and selective M2 muscarinic receptor antagonists

Article abstract of DOI:10.1016/S0960-894X(00)00457-1

Identification of a number of highly potent M₂ receptor antagonists with >100-fold selectivity against the M₁ and M₃ receptor subtypes is described. In the rat microdialysis assay, this series of compounds showed pronounced enhancement of brain acetylcholine release after oral administration. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0960-894X(00)00457-1

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2247±2250
Design and Synthesis of Piperidinyl Piperidine Analogues as
Potent and Selective M2 Muscarinic Receptor Antagonists
Yuguang Wang,* Samuel Chackalamannil, Zhiyong Hu, John W. Clader,
William Greenlee, William Billard, Herbert Binch, III, Gordon Crosby, Vilma Ruperto,
Ruth A. Du€y, Robert McQuade and Jean E. Lachowicz
Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
Received 5 June 2000; accepted 21 July 2000
AbstractÐIdenti®cation of a number of highly potent M2 receptor antagonists with >100-fold selectivity against the M1 and M3
receptor subtypes is described. In the rat microdialysis assay, this series of compounds showed pronounced enhancement of brain
acetylcholine release after oral administration. # 2000 Elsevier Science Ltd. All rights reserved.
Alzheimer's disease (AD), the most common form of
dementia, a€ects the independent living of the elderly
population.1 AD patients show a progressive loss of
memory and cognitive function, which is due in part to
the impairment of the cholinergic system.2 Currently
available cholinergic therapy for AD is based on
increasing acetylcholine levels by inhibiting acetyl-
cholinesterase, the enzyme that hydrolyzes acetyl-
choline.2 Enhancement of acetylcholine levels could also
be achieved by selectively inhibiting presynaptic M2
muscarinic receptors, which regulate acetylcholine
release by an inhibitory feedback mechanism.2 It is
essential that M2 receptor antagonists are selective versus
M1 receptors because the post-synaptic M1 receptors
mediate the acetylcholine e€ect. It has been demon-
strated that M1 agonists improved cognition.3 Addi-
tionally, selectivity versus M3 receptors is also needed
because inhibition of peripheral and central M3 receptors
can also cause side e€ects.4
appreciable selectivity versus other receptor subtypes
(Table 1). We wish to report here and in a subsequent
communication that we have achieved this goal and
identi®ed a number of highly potent and highly selective
M2 antagonists.
Our design strategy was to generate additional sites of
receptor interactions that could potentially serve to dis-
criminate among various receptor subtypes. Toward this
goal, an N-substituted piperidine surrogate that could
provide steric and hydrogen-bonding interactions with
receptor subtypes replaced the cyclohexyl ring of 1 (Fig. 1).
Compounds 6, 7, and 8 (Table 1), representing three
structural subclasses, were synthesized as shown in
Scheme 1. N-Boc protection of commercially available
piperidine derivative 2 followed by displacement of ¯uorine
atom with 4-methoxy thiophenol gave 3. The ketone 3
was transformed to sulfone alkene 4 by sequential
treatment with Tebbe reagent,8 sul®de oxidation and
deprotection.9 Reductive amination of 4 followed by
deprotection gave 5 which was converted to the ®nal targets
by treatment with di€erent reagents such as sulfonyl chlo-
ride, acetyl chloride, and chlorofomate.10
The highly conserved amino acid sequences of muscar-
inic receptor subtypes renders design of a selective M2
antagonist dicult.5 In fact, despite several reports of
potent M2 antagonists,3,6 there have not been any
reports of a potent M2 antagonist with >40-fold selec-
tivity versus the M1 and M3 receptors. In an e€ort to
discover selective M2 antagonists, we selected the vinyl
piperidine derivative 1 as our lead.7 Compound 1 is a
potent M2 antagonist (Ki=0.17 nM), but devoid of
The binding anity of the synthesized antagonists
against cloned human muscarinic receptors were assayed
according to the reported protocol.11
The results of varying N-substituent on the piperidine ring
are presented in Table 1. The unsubstituted compound 5
showed reduced anity toward the M2 receptor, in
*Corresponding author. Tel.: +1-908-740-3508; fax: +1-908-740-4563;
e-mail: yuguang.wang@spcorp.com
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00457-1

2248
Y. Wang et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2247±2250
Table 1. Results of M2 binding anity and selectivity against M1 and M3
Compound
X
R
M2 (Ki, nM)
M1 (Ki, nM)
M3 (Ki, nM)
M1/M2
M3/M2
1
5
6
7
8
C
N
N
N
N
0.17
1.14
0.11
0.38
0.29
2.8
9.1
6.5
42.0
25.7
0.48
3.4
3.7
121.6
10.1
16
8
59
111
89
3
3
34
324
35
H
COOEt
CO-n-C3H7
SO2-n-C3H7
Figure 1. Elongation of 1.
comparison to 1, with little selectivity versus the M1 and
M3 receptors. However, the carbamate derivative 6 and
the propyl sulfonamide 8 showed improved selectivity
versus M1 and M3 receptors, whereas the propyl amide
7 showed excellent selectivity. This enhanced selectivity
is principally due to the decreased M1 and M3 receptor
anity, since the M2 anity remains unchanged.
explored. The results of the amide subseries are in Table
2. As shown, the M2 receptor anity of these compounds
is highly sensitive to the substitutes of the amide moiety.
For example, amides 9, 11, and 12 lowered the M2 recep-
tor anity considerably with concomitant loss of selec-
tivity. On the other hand, amides 7 and 10 showed
excellent M2 anity and M1/M2 and M3/M2 ratios.
In an e€ort to further optimize the binding pro®le of
these compounds, each of the above subseries was further
The sulfonamide series a€orded the most promising com-
pounds in terms of both receptor anity and subtype
Scheme 1. (a) (BOC)2O, 10% NaOH/Et2O, 89%; (b) NaH, DMF, 4-methoxythiophenol, 65 ꢀC, 6 h, 89%; (c) Tebbe reagent, 90%; (d) NaBO3,
HOAc, 83%; (e) 30% TFA/CH2Cl2, 100%; (f) NaBH(AcO)3, 1,2-DCE, 1-t-butoxycarbonyl-4-piperidone, 75%; (g) RCl, Et3N, CH2Cl2.

Y. Wang et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2247±2250
2249
selectivity (Table 3). In this series, the M2 receptor an-
ity was una€ected by the substitutes of the sulfonamides
in most of the cases, whereas the M1 anity and M3
anity were quite sensitive to these substitutions. This
property entailed a number of compounds with excel-
lent M2 potency and M1 and M3 selectivity. For exam-
ple, sulfonamides 13, 14, and 15 have subnanomolar M2
anity and excellent selectivity toward M1 and M3
receptors.
of time through a dialysis membrane probe.12 The
acetylcholinesterase inhibitor neostigmine was perfused
through the probe to produce an acetylcholine level
high enough to activate the M2 receptor inhibitory
feedback mechanism and dampen the acetylcholine
release. A stable baseline level of acetylcholine was
routinely achieved under these conditions. When the M2
receptor antagonist 6 was administered orally to rats,
the level of acetylcholine was increased signi®cantly
over the baseline, as shown in Figure 2. This result is
consistent with the blockade of the M2 receptor by
antagonist 6 and a consequent increased release of acetyl-
choline due to partial reversal of the M2 inhibitory
feedback mechanism. Additionally, the high level of the
acetylcholine release also suggests that the carbamate 6
has good oral bioavailability and blood±brain barrier
penetration.
The data for the carbamate derivatives are presented in
Table 4. Although these derivatives showed uniformly
high anity in the M2 binding assay, they were less
selective toward M1 and M3 receptors than the corre-
sponding amide and sulfonamide derivatives. The
results of the three subseries demonstrated that the
substitution changes in the amide and the sulfonamide
series a€ected the M1 and M3 anity much more than
the carbamate series. As a result, high selectivity of M2
versus M1 and M3 was observed in the amide series and
the sulfonamide series, both of which have lowered M1
and M3 binding anity.
In summary, we have identi®ed several potent and
selective M2 receptor antagonists. For example, com-
pounds 10, 13, and 14 show subnanomolar Ki values for
the M2 receptor and greater than 150-fold selectivity
against the M1 and M3 receptors. Additionally, the repre-
sentative data presented for compound 6 demonstrated
The in vivo e€ect of the M2 antagonist was measured
using a microdialysis paradigm, in which the acetylcho-
line level in the rat striatum was monitored as a function
Table 4. Results of M2 anity and selectivity of the carbamate series
Table 2. Results of M2 anity and selectivity of the amide series
Compound
R
M2 (Ki, nM)
M1/M2
M3/M2
6
Et
Me
0.11
0.99
0.59
0.41
0.35
0.30
0.45
59
61
69
36
28
35
24
34
55
40
n/a
n/a
n/a
n/a
Compound
R
M2 (Ki, nM)
M1/M2
M3/M2
18
19
20
21
22
23
9
7
10
11
12
Et
n-C3H7
Cyclopropyl
i-C3H7
CH2Ph
3.8
16
111
262
53
13
324
260
40
n-C3H7
i-C3H7
n-C4H9
i-C4H9
CH2Ph
0.38
0.26
2.2
2.99
32
48
Table 3. Results of M2 anity and selectivity of the sulfonamide
series
Compound
R
M2 (Ki, nM)
M1/M2
M3/M2
Figure 2. E€ect of 6 on acetylcholine release in rat striatum following
oral administration. Each data point represents a 10-min collection of
microdialysate and constitutes the meanÆSEM of three individual
rats. Perfusion rate was 2 mL/min using Ringer's solution containing
1 mM neostigmine. Arrow indicates time of 6 administration (10 mg/
kg, orally in water). *Signi®cant stimulation over each of three pre-
injection baseline points (p < 0.05, Duncan's mutiple range statistic).
13
8
14
15
16
17
Et
0.46
0.29
0.16
0.38
0.36
1.45
162
89
198
150
104
23
382
35
440
405
558
54
n-C3H7
i-C3H7
n-C4H9
Ph
CH2Ph

2250
Y. Wang et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2247±2250
that the M2 antagonists upon oral administration,
stimulate brain acetylcholine release in functional
microdialysis assay. A detailed structure±activity rela-
tionship study of this class of compounds, as well as their
in vivo ecacy in animal models of cognition, will be
published in the future.
golo, N. Life Sci. 1999, 64, 535. (b) Doller, D.; Chacka-
lamannil, S.; Czarniecki, M.; McQuade, R.; Ruperto, V.
Bioorg. Med. Chem. Lett. 1999, 9, 901. (c) Engel, W. E.;
Eberlein, W. G.; Mihm, G.; Hammer, R.; Trummlitz, G. J.
Med. Chem. 1989, 32, 1718. (d) Malaska, M. J.; Fauq, A. H.;
Kozikowski, A. P.; Aagaard, P. J.; Mckinney, M. Bioorg.
Med. Chem. Lett. 1993, 3, 1247.
7. Billard, W.; Binch, H., III; Bratzler, K.; Chen, L. Y.;
Crosby, G.; Du€y, R. A.; Dugar, S.; Lachowicz, J.; McQuade,
R.; Pushpavanam, P.; Ruperto, V. B.; Taylor, L. A.; Clader, J.
W. submitted together with this letter for publication.
8. Pine, S. H.; Shen, G. S.; Hoang, H. Synthesis 1991, 165.
9. Shimazaki, M.; Takahashi, M.; Komatsu, H.; Ohta, A.;
Kajiii, K.; Kodama, Y. Synthesis 1992, 555.
Acknowledgements
The authors would like to thank Mr. Yao H. Ing and
Dr. P. Das for mass spectral data, and Dr. Murgolo for
molecular modeling studies.
10. All of the target compounds showed satisfactory result in
the analyses of NMR, MS, LC/MS, and HRMS.
11. For radioligand binding analysis, each muscarinic recep-
tor subtype was stably expressed in CHO-K1 cells. Clonal cell
lines were selected which expressed receptors at levels between
1 and 9 pmol/mg protein. The Kd of QNB (l-quinuclidinyl
benzilate) at each receptor subtype was determined by satura-
tion binding using 5±2500 pM [3H]QNB in 10 mM potassium
phosphate bu€er, pH 7.4. Protein concentrations were adjus-
ted for each assay to achieve between 700 and 1500 cpm spe-
ci®c binding. Competition binding experiments were
performed using 180 pM [3H] QNB. All binding experiments
were performed in the presence of 1% DMSO and 0.4%
methylcellulose. Non-speci®c binding was de®ned by 0.5 mM
atropine. After equilibrium was reached (120 min incubation
at room temperature), bound and free radioactivity were
separated by ®ltration using Whatman GF-C ®lters. Investi-
gation of M2 receptor antagonist activity was performed on
related compounds by measuring the e€ects of the compounds
adenylyl cyclase inhibition mediated by oxotremorine M (see
6a for details).
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