Synthesis and optimization of novel and selective muscarinic M3 receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2007.06.081

A series of constrained piperidine analogues were synthesized as novel muscarinic M₃ receptor antagonists. Evaluation of these compounds in binding assays revealed that they not only have high affinity for the M₃ receptor but also have high selectivity over the M₂ receptor.

Full text of DOI:10.1016/j.bmcl.2007.06.081

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5256–5260
Synthesis and optimization of novel and selective muscarinic
M3 receptor antagonistsq
Naresh Kumar,a,* Kirandeep Kaur,a Shelly Aeron,a Sankaranarayanan Dharmarajan,a
Arun D. V. Silamkoti,a Anita Mehta,a Suman Gupta,b Anita Chugh,b
Jang B. Gupta,b Mohammad Salman,a Venkata P. Pallea and Ian A. Cliffea
aRanbaxy Research Laboratories, New Drug Discovery Research, Department of Medicinal Chemistry,
Gurgaon, Haryana 122 001, India
bRanbaxy Research Laboratories, New Drug Discovery Research, Department of Pharmacology,
Gurgaon, Haryana 122 001, India
Received 17 May 2007; revised 17 May 2007; accepted 25 June 2007
Available online 30 June 2007
Abstract—A series of constrained piperidine analogues were synthesized as novel muscarinic M3 receptor antagonists. Evaluation of
these compounds in binding assays revealed that they not only have high affinity for the M3 receptor but also have high selectivity
over the M2 receptor.
Ó 2007 Elsevier Ltd. All rights reserved.
Overactive bladder (OAB) arises from the uncontrolled
spontaneous activity of the detrusor muscle during blad-
der filling leading to the symptoms of urinary urgency and
increased frequency of micturition with or without incon-
tinence. Urinary bladder contraction is predominantly
under the control of the parasympathetic system, where
the primary neuronal input is via cholinergic muscarinic
receptors. Five distinct muscarinic subtypes are known
to exist (M1–M5) and at least two of them are involved
in normal and disturbed bladder contraction. In patients
with an overactive bladder, blocking the muscarinic
receptors in the detrusor muscle will cause less frequent
and less forceful bladder contractions allowing improved
bladder filling and reduced urge incontinence. However,
since the muscarinic receptors are widely distributed
throughout the body, an antimuscarinic action at organ
systems other than the urinary bladder can lead to several
adverse effects including dry mouth, constipation, blurred
vision, headache, somnolence and tachycardia. It is
believed that achieving subtype selectivity may lead to
organ selectivity. The human urinary bladder smooth
muscle contains a mixed population of muscarinic M2
and M3 receptor subtypes. Although the M2 receptors
predominate in number, the M3 receptors are mainly
responsible for normal micturition contraction because
they mediate direct contraction of the detrusor muscle.1
The M2 receptor contributes by reversing the relaxation
produced by b-adrenoceptor stimulation. However,
blockade of the M2 receptor may lead to adverse cardiac
effects, owing to the presence of functional M2 receptors
in the heart. Thus, a dual M3/M2 receptor antagonist
would produce the required relaxation of the bladder
but might also induce cardiac toxicity. Indeed, the impor-
tance of selectivity is reflected in the recently launched
drugs darifenacin and solefenacin, which have been
reported2 to have M3/M2 selectivity of 59- and 12-fold,
respectively. There are few reports of compounds having
high M3 receptor selectivity,3 and the present study high-
lights the high selectivity seen in a novel class of
azabicyclohexanes.
Recently,4 we disclosed compounds of general structures
1 and 2 as having high affinity for the M3 receptor but
moderate selectivity over the M2 receptor. With a quest
to achieve higher affinity and selectivity, we designed a
new series of compounds 3 based on series 2 in which
the acetylenic group has been replaced by the more
rigid bicyclic moiety, (1a,5a,6a)-6 amino-3-azabicy-
clo[3.1.0]hexane. As with other M3 receptor antagonists,
Keywords: M3 selective muscarinic antagonists; Azabicyclohexanes.
q
Abstract published in 229th ACS meeting, MEDI-61, presented as
poster in National Symposium in Chemistry-9, Delhi (India).
*
Corresponding author. Tel.: +91 124 419 5158; fax: +91 124 234
3544; e-mail: n.kumar@ranbaxy.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.06.081

N. Kumar et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5256–5260
5257
many of the key pharmacophoric features such as a
bulky hydrophobic group, a hydroxyl group, a basic
nitrogen atom and a set distance between the carbonyl
group and the basic nitrogen were maintained. This de-
sign is also supported by reports5,6 that piperidine and
piperazine may be used as the required amine portion.
(NMM) gave 7a–c (Scheme 1). Compound 7d was ob-
tained by coupling rac-mandelic acid with amine 6.
Compounds 7a–c were debenzylated in quantitative
yields under hydrogenation conditions using Pd/C as
catalyst in methanol and the crude secondary amines
R1
OR4
N
R2
H
R
OH
O
H
OH
R3
N
R2
H
N
X
N
H3C
R1
H
H
O
( )n
2
1
3
A number of compounds were synthesized with the
general structure 3, and the present work discusses the
optimization of the groups R1 and R2.
converted to compounds 8a–c by reductive amination
with acetophenone (8a) or alkylation with 1-bromo-eth-
ylbenzene (8b–c) (Scheme 2). The products were diaste-
reomeric mixtures.
rac-2-Cycloalkyl-2-phenyl-2-hydroxy acetic acids 5a–c
were prepared by the addition of cycloalkyl magnesium
bromides to ethyl benzoylformate followed by basic
hydrolysis using literature precedent.7 The amidic
condensation of amine 68 with the acids (5a–c) in the
presence of N-ethyl-N0-(3-dimethylaminopropyl)-N0-
ethylcarbodiimide hydrochloride (EDC), 1-hydrox-
ybenzotriazole (HOBT) and N-methylmorpholine
Compounds 8d–m in which R1 was fixed as cyclopentyl
were obtained from compound 7a by hydrogenation
followed by reductive amination reactions with 2-thio-
phene carboxaldehyde (8d), 4-methylacetophenone (8e)
and 3-phenylpropanal (8l). Compounds 8f–k and 8m
were obtained by hydrogenation of 7a followed by alkyl-
ation with various bromides under alkaline conditions
O
O
OH
OH
a
b
O
R1
O
OH
R1
O
O
Ethyl benzoylformate
4a, R1 = cyclo-C5H9
4b, R1 = cyclo-C6H11
4c, R1 = cyclo-C7H13
5a, R1 = cyclo-C5H9
5b, R1 = cyclo-C6H11
5c, R1 = cyclo-C7H13
H
H
N
H
H2N
OH
Ph
H
N
N
H
6
R1
O
c
7a, R1 = cyclo-C5H9
7b, R1 = cyclo-C6H11
7c, R1 = cyclo-C7H13
Scheme 1. Reagent and conditions: (a) R1MgBr, THF, 0 °C to rt, 20–60%; (b) KOH, MeOH, rt, 70–95%; (c) 6, EDC, HOBT, NMM, DMF, 0 °C to
rt, 70–80%.
H
H
H
H
Me
OH
OH
H
N
H
N
N
N
a, b
R1
R1
O
O
8a-c
7a-c
Scheme 2. Reagent and conditions: (a) H2, Pd/C, MeOH, rt, 80–90%; (b) PhCOMe, Na(OAc)3BH, HOAc, THF, rt, 16% (8a) or PhCH(Br)Me,
K2CO3, KI, CH3CN, reflux, 83% (8b) and 30% (8c).

5258
N. Kumar et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5256–5260
(Scheme 3). The R and S isomers of compound 8m and
the R isomer of 8i were prepared from the enantiomers
of 5a.9
compounds 8k and 8l shows that optimum M3 receptor
affinity and selectivity arises when the phenyl ring is one
atom or preferably two atoms away from the tertiary
nitrogen atom. Branching of the methylene linker pro-
duces a small increase in M3 receptor affinity and M3/
M2 receptor selectivity (compare compound 7a with
compounds 8a and 8e), but this small increase is accom-
panied with more complexity in the molecule i.e. an
additional chiral centre.
The muscarinic M3 receptor binding affinity of com-
pounds 7a–d and 8a–c indicates a preference for bulkier
groups at R1 (Table 1). When R2 is benzyl, M3/M2 selec-
tivity increases with increasing bulk at R1, but interest-
ingly no such an effect is observed when R2 is a-
methylbenzyl.
Keeping in mind the preference for R2 to be an aryl-
ethyl group, we decided to study the effect of substitu-
tion on the phenyl ring of the R2 group (compounds
8f–i). Unfortunately, in all cases, substitution by elec-
tron-donating and electron-withdrawing or alkyl
groups results in decreased M3 receptor affinity and
decreased M3/M2 receptor selectivity. When the R2
A comparison of compounds in which the R1 group was
initially maintained as a cyclopentyl group (Table 2)
reveals that the thiophenyl compound 8d has lower mus-
carinic M3 receptor affinity than the phenyl analogue 7a,
indicating in this case that thiophene does not behave as
bioisostere for benzene. Comparing compound 7a with
H
H
OH
H
R2
OH
H
N
N
N
N
a, b
H
O
H
O
8d-m
7a
Scheme 3. Reagent and conditions: (a) H2, Pd/C, MeOH, rt, 87%; (b) aldehyde or ketone, Na(OAc)3BH, HOAc, THF, rt, 15–20% or alkyl bromide,
K2CO3, KI, CH3CN, reflux, 30–70%.
Table 1. Variation at R1 and R2
H
OH
R2
H
N
N
R1
H
O
Compound
R1
R2
na
Ki SEM (nM)10
M3/M2
M3
M2
CH2
CH2
CH2
CH2
7a
7b
7c
7d
8a
8b
8c
Cyclo-C5H9
Cyclo-C6H11
Cyclo-C7H13
H
3
3
3
1
3
3
3
96 20.7
1422.3 127.3
943.3 149.2
349 65.6
>1000
15
30
51
NA
25
26
22
31.7 8.4
6.8 0.5
>1000
CH(Me)
CH(Me)
CH(Me)
Cyclo-C5H9
Cyclo-C6H11
Cyclo-C7H13
25.1 7.6
5.9 1.4
1.8 0.2
630 59
154 12
39 6.5
a n is the number of experiments.

N. Kumar et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5256–5260
5259
Table 2. Variation at R2
H
OH
R2
H
N
1
N
2
H
O
na
Compound
R2
Ki SEM (nM)10
M2
M3/M2
M3
524
S
CH2
8d
8e
1
3981
8
Me
CH
3
58.9 16
1230 148
21
Me
Me
CH2
8f
2
43.6 13
31.3 4.4
812 27
19
Me
CH2
8g
8h
3
2
298.3 59.5
537 38
10
12
Cl
CH2
43.6
7
MeO
O
O
O
CH2
8i
8j
3
3
20 3.8
277 60
14
19
CH2
42
2
778.3 140.8
CH2
8k
3
1
3
4
1
9.3 0.2
776
335.3 14.4
2630
36
3
CH2
8l
CH2
8m
33 11.7
12.4 2.5
71
1370 277
564 110
1139
41
46
16
CH2
CH2
(2R)-8m
(2S)-8m
O
O
CH2
(2R)-8i
4
12.3 2.9
646 113
52
Darifenacin
Solefenacin
—
—
5
5
2.5 0.45
40.5 16.5
63 4.4
273 50
25
7
a n is number of experiments.
group is an arylmethyl group, it is found that the
naphthylmethyl analogue 8j has higher M3 receptor
affinity and higher M3/M2 receptor selectivity than
the phenylmethyl compound 7a, indicating a lack of
steric hindrance at the receptor close to the basic nitro-
gen atom.

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N. Kumar et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5256–5260
We have shown previously when R1 is a cycloalkyl
group, that the R enantiomer is favoured.4 For example,
when R1 is cyclopentyl and R2 is 2-methyl-2-pentenyl,
(2R)-8m has a higher M3 receptor affinity than the race-
mate. This trend is followed in the current (1a,5a,6a)-6
amino-3-azabicyclo[3.1.0]hexane series: the 2R isomer of
the benzodioxol 8i has not only a higher M3 receptor
affinity but also a higher M3/M2 receptor selectivity than
the racemate.
References and notes
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In conclusion, we have discovered a novel class of com-
pounds with high affinity for the M3 receptor and high
selectivity over the M2 receptor. Whilst we have
described the SAR of compounds in which the R1 group
is cyclopentyl, compounds having improved in vitro
profiles might be produced when the R1 group is the
more bulky cycloheptyl. In addition, highly potent
antagonists might result from the separation of the
compounds into their homochiral forms and such com-
pounds might be developed as selective M3 muscarinic
receptor antagonists for the treatment of OAB.
7. Shacklett, C. D.; Smith, H. A. J. Am. Chem. Soc. 1953, 75,
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Acknowledgment
8. Brighty, K. E.; Castaldi, M. J. Synlett 1996, 1097.
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We thank Dr. Kona Srinivas and his Analytical Chem-
istry group for providing analytical support.
10. The affinity of test compounds for the M2 and M3
muscarinic receptor subtypes was determined by [3H]-N-
methylscopolamine binding studies using rat heart and
submandibular gland, respectively, as previously described
with minor modifications. (a) Moriya, H.; Takagi, Y.;
Nakanishi, T.; Hayashi, M.; Tani, T.; Hirotsu, I. Life Sci.
1999, 64, 235; (b) Cheng, Y.; Prusoff, W. H. Biochem.
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Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.
2007.06.081.