Muscarinic acetylcholine receptor antagonists: SAR and optimization of tyrosine ureas

Article abstract of DOI:10.1016/j.bmcl.2008.09.020

SAR exploration of multiple regions of a tyrosine urea template led to the identification of very potent muscarinic acetylcholine receptor antagonists such as 10b with good subtype selectivity for M₃ over M₁. The structure-activity r

Full text of DOI:10.1016/j.bmcl.2008.09.020

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5481–5486
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Muscarinic acetylcholine receptor antagonists: SAR and optimization
of tyrosine ureas
,
*
*
Jian Jin , Yonghui Wang , Dongchuan Shi, Feng Wang, Wei Fu, Roderick S. Davis, Qi Jin,
James J. Foley, Henry M. Sarau, Dwight M. Morrow, Michael L. Moore, Ralph A. Rivero,
Michael Palovich, Michael Salmon, Kristen E. Belmonte, Jakob Busch-Petersen
Centers of Excellence for Drug Discovery and Molecular Discovery Research, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA 19406, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
SAR exploration of multiple regions of a tyrosine urea template led to the identification of very potent
muscarinic acetylcholine receptor antagonists such as 10b with good subtype selectivity for M3 over
M1. The structure–activity relationships (SAR) and optimization of the tyrosine urea series are described.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 13 August 2008
Revised 4 September 2008
Accepted 5 September 2008
Available online 10 September 2008
Keywords:
Muscarinic acetylcholine receptor
antagonists
M3 mAChR
SAR
Subtype selectivity
COPD
Asthma
Tyrosine ureas
Five muscarinic acetylcholine receptor (mAChR) subtypes, M1–
M5, are knownto date.1–3 These seven-transmembrane (7TM)recep-
tors share a common orthosteric ligand-binding site with an extre-
mely high sequence homology, which explains why it has been
difficult historically to identify subtype-selective ligands.3 M1–M5
mAChRs are widely distributed in mammalian organs and the cen-
tral and peripheral nerve system where they mediate important
neuronal and autocrine functions.4,5 In the mammalian lung, only
M1, M2, and M3 mAChRs have been recognized as playing important
and functional roles.6 M3 is predominantly expressed on airway
smooth muscle and mediates smooth muscle contraction.7 M2 is pri-
marily found on postganglionic nerve termini and functions to limit
acetylcholine release from parasympathetic nerves.8 M1 is found in
parasympathetic ganglia and facilitates neurotransmission through
ganglia thus enhancing cholinergic reflexes.9
mediated by M2 on parasympathetic nerves, causing excess acetyl-
choline reflexes10 which result in airway hyperreactivity and
hyperresponsiveness mediated by increased acetylcholine release
and thus excess stimulation of M3. Therefore, potent mAChR antag-
onists, particularly directed toward the M3 subtype, would be use-
ful as therapeutics in these mAChRs-mediated disease states.
Inhaled delivery could potentially reduce side effects mediated
by peripheral and/or central M1, M2, or M3 antagonism5 by avoid-
ing substantial systemic exposure. We previously reported a novel
muscarinic acetylcholine receptor antagonist series exemplified by
2a with high potency, outstanding selectivity, and excellent in vivo
efficacy and long duration of action in the bronchoconstriction
model in mice via inhaled delivery (Fig. 1).11 Herein, we describe
SAR and optimization of this tyrosine urea series from high-
throughput screen (HTS) hit 1a to the identification of lead com-
pounds such as 10b and 2a.
In chronic obstructive pulmonary disease (COPD) and asthma,
inflammatory conditions lead to loss of neuronal inhibitory activity
The HTS hit 1a,11 a mixture of two diastereoisomers, was an
antagonist in a M3 fluorometric imaging plate reader (FLIPR) as-
say12 with a pIC50 of 7.7 and was about 10-fold selective for M3
over M2 and 100-fold selective for M3 over M1 (Fig. 1).13,14 On
the basis of its good potency and subtype selectivity, 1a was con-
sidered an acceptable starting point for our lead optimization pro-
gram aimed at improving potency via SAR exploration.
* Corresponding authors. Tel.: +1 919 843 1645, fax: +1 919 966 0204 (J.J); tel.: +1
610 917 6678; fax: +1 610 917 7391 (Y.W).
E-mail addresses: jianjin@unc.edu (J. Jin), yonghui.2.wang@gsk.com (Y. Wang).
Present address: Center for Integrative Chemical Biology and Drug Discovery,
Division of Medicinal Chemistry and Natural Products, Eshelman School of Pharmacy,
The University of North Carolina at Chapel Hill, CB# 7360, Beard Hall, Chapel Hill, NC
27599, USA.
A robust solid-phase synthesis was developed to efficiently
explore multiple regions of the series in parallel (Scheme 1).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.09.020

5482
J. Jin et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5481–5486
OH
O
O
H
N
H
N
H
N
H
N
N+
N
N
H
N
H
OH
O
O
EtO
O
2a
O
O
1a
M3 FLIPR pA2 = 9.9
M2 FLIPR pA2 = 9.0
M1 FLIPR pA2 = 7.8
M3 FLIPR pIC50 = 7.7
M2 FLIPR pIC50 = 6.8
M1 FLIPR pIC50 = 5.7
HO
HO
Figure 1. In vitro profile of HTS hit 1a and structure of lead compound 2a.
Resin-bound nosyl-protected diamines 311 were coupled with
Fmoc-protected -amino acids, followed by Fmoc removal, to pro-
diamine and amino acid, and left-hand side (LHS) ester and amide
moieties were explored in parallel.
L
duce resin-bound amines 4. Urea formation from intermediates
4, nosyl-group removal and subsequent reductive amination affor-
ded resin-bound intermediates 5, which were then cleaved from
resin to produce the targeted tertiary amines 1 in good overall
yields.15 Alkylation of tertiary amines 5, followed by resin cleavage
afforded the desired quaternary ammonium salts 2. Intermediates
5 were also converted to amides 6 in good yields via hydrolysis of
the ester, amide formation from the resulting acid, and resin cleav-
age. Using this robust solid-phase synthesis, the right-hand side
(RHS) N-capping group, quaternary ammonium salt, central
Modifications to the RHS N-capping group were well tolerated
(Table 1). Substitution at the para- (1a and 1c), meta- (1b and
1d), or ortho-position (1e) of the benzyl group gave similar M3
FLIPR potency. 3,4-Disubstituted compounds such as 1f had similar
potency as mono-substituted benzyl analogs. A wide range of sub-
stituents on the benzyl ring was tolerated—from electron-donating
groups such as hydroxyl (1b) and methoxy (1d) to hydrogen (1j)
and electron-withdrawing groups such as chloro (1h), cyano (1g),
and trifluoromethyl (1i). While a small alkyl group such as ethyl
(1k) was significantly less potent, a larger alkyl group such as
cyclopropylmethyl (1m) was about equipotent compared to benzyl
analogs. Modifications to this region did not have substantial im-
pact on sub-type selectivity—the compounds were 10-fold selec-
tive for M3 over M2 and 100-fold selective for M3 over M1 in
general.
To avoid substantial systemic exposure thus reduce potential
side effects mediated by peripheral and/or central M1, M2, or M3
antagonism via minimizing cell membrane permeability, we at-
tempted to convert tertiary amines to quaternary ammonium salts
and were pleased to find that quaternary ammonium salts such as
2b were slightly more potent and had similar subtype selectivity
compared to the corresponding tertiary amine 1n (Fig. 2).16 Intro-
ducing the quaternary ammonium moiety indeed reduced mem-
brane permeability and thereby oral bioavailability significantly.
For example, compound 2a had extremely low artificial membrane
permeability (less than 3 nm/s) and no oral bioavailability.11
O
n N
n N
a, b
R1
H2N
N
Nosyl
Nosyl
HN
DMHB
n = 1, 2
DMHB
4
3
O
H
R3
H
N
n N
N
c, d, e
N
O
O
R2
DMHB
O
5
R1
O
R3
H
H
n N
N
N
f
N
H
Table 1
5
O
O
R2
SAR of the RHS N-capping group
1
O
O
H
N
H
N
R1
N
R
N
H
O
EtO
O
H
R3
H
n N+
N
N
N
H
O
g, f
O
O
R2
-
5
CF3COO
HO
O
a
2
Compound
R
FLIPR pIC50
M2
R1
M3
M1
5.7
6.1
6.3
5.6
6.0
6.0
6.6
5.9
<5.5
5.7
1a
1b
1c
1d
1e
1f
1g
1h
1i
4-Hydroxyphenyl
3-Hydroxyphenyl
4-Methoxyphenyl
3-Methoxyphenyl
2-Methoxyphenyl
3,4-Methylenedioxyphenyl
3-Cyanophenyl
3-Chlorophenyl
3-Trifluoromethylphenyl
Phenyl
7.7
7.7
7.7
7.6
7.3
7.5
8.2
8.1
7.8
7.8
6.1
7.6
6.8
6.8
6.6
6.5
6.8
6.7
7.0
6.9
6.2
6.7
5.6
7.0
O
H
R3
H
N
n N
N
R5
N
h, i, f
N
H
5
O
R4
6
O
R1
Scheme 1. Reagents and conditions: (a) Fmoc amino acids, DIC, HOAt, NMP, rt; (b)
20% of piperidine in NMP, rt; (c) 4-isocyanatobenzoates, DCE, rt; or 4-aminobenzo-
ates, 1,10-(oxomethanediyl)bis-1H-pyrrole, DIEA, DCE, rt; (d) K2CO3, PhSH, NMP, rt;
(e) R3CHO, Na(OAc)3BH, 10% of HOAc in NMP, rt; (f) 50% of TFA in DCE, rt; (g) MeI,
CH3CN, rt; (h) potassium trimethylsilanolate, THF, rt; (i) R4R5NH, PyBOP, NMP, rt.
1j
1k
1m
Methyl
Cyclopropyl
<5.5
6.2
a
Means of at least two determinations with standard deviation of < 0.3.

J. Jin et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5481–5486
5483
OH
OH
O
O
H
N
H
N
N+
H
N
H
N
N
N
H
N
H
EtO
O
EtO
O
O
1n
O
2b
M3 FLIPR pIC50 = 7.8
M2 FLIPR pIC50 = 6.9
M1 FLIPR pIC50 = 6.0
M3 FLIPR pIC50 = 8.2
M2 FLIPR pIC50 = 7.4
M1 FLIPR pIC50 = 6.6
HO
HO
Figure 2. Potency comparison of tertiary amine 1n with quaternary ammonium salt 2b.
Table 2
Table 3
SAR of the central tyrosine moiety
SAR of the LHS ester moiety
O
OH
O
O
N+
H
N
H
H
N
H
N
N
N
O
N
N
H
H
EtO
O
O
O
R
O
O
R
a
HO
Compound
R
FLIPR pIC50
M2
a
M3
M1
Compound
R
FLIPR pIC50
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2q
4-Hydroxy
4-Methoxy
4-Amino
4-Methyl
4-Fluoro
4-Chloro
4-Bromo
4-Cyano
4-Trifluoromethyl
4-Phenyl
4-Phenylcarbonyl
3-Chloro
8.2
7.4
7.8
7.8
7.8
8.0
7.9
7.3
7.5
7.8
7.9
8.1
6.9
7.6
5.9
7.4
8.3
7.8
8.0
8.0
8.2
8.4
7.9
8.0
8.3
8.6
8.4
7.0
7.9
7.3
6.8
7.1
5.9
7.0
7.0
7.4
7.5
6.6
7.3
7.9
8.0
7.6
5.7
7.4
6.2
M3
M2
M1
1p
1a
1q
1r
1s
1t
Methyl
Ethyl
Propyl
Isopropyl
Isobutyl
Cyclopropylmethyl
7.6
7.7
8.0
8.3
6.6
7.0
6.5
6.8
7.0
7.1
5.9
6.4
6.0
5.7
6.0
6.0
<5.5
6.0
a
Means of at least two determinations with standard deviation of < 0.3.
2-Chloro
3,4-Dichloro
3,4-Dimethoxy
SAR of esters, N-isopropyl (6d) was the most potent in the M3 FLIPR
assay. Small alkyl groups such as N-methyl (6a) and large alkyl
groups such as N-phenyl (6f) and N-benzyl (6g) were significantly
less potent. A tertiary amide was tolerated but did not provide any
potency enhancement (6h vs 6b). The morpholine analog (6i) was
also less potent. The amides exemplified by 6d had similar subtype
a
Means of at least two determinations with standard deviation of < 0.3.
A number of substituents on the central phenyl ring was ex-
plored (Table 2). In general, substituents on the phenyl ring did
not have substantial impact on M3 potency. Electron-donating
groups such as hydroxyl (2c), methoxy (2d), amino (2e), and
methyl (2f); halo groups such as fluoro (2g), chloro (2h), and bro-
mo (2i); and electron-withdrawing groups such as cyano (2j) and
trifluoromethyl (2k) all had good potency in the M3 FLIPR assay.
While moving the chloro group from the para-position (2h) to
the meta-position (2n) maintained M3 potency, the ortho-chloro
group (2o) resulted in about 10-fold potency loss. 3,4-Disubsti-
tuted compounds such as 2p and 2q were less potent compared
to the corresponding mono-substituted analogs. In contrast to
the modest impact on M3 potency, the substituents had a signifi-
cant impact on mAChR subtype selectivity. Except for the 4-hydro-
xy (2c), other substituents (2d–q) resulted in equi- or more potent
compounds against M2 compared to M3. In most cases, subtype
selectivity for M3 over M1 was also not as good as that of com-
pound 2c. On the basis of both M3 potency and subtype selectivity,
4-hydroxy (2c) was optimal.
Table 4
SAR of the LHS amide moiety
CN
O
H
N
H
N
N
R2
N
N
H
O
R1
O
HO
Compound
NR1R2
FLIPR pIC50
M2
a
M3
M1
6a
6b
6c
6d
6e
6f
6g
6h
6i
N-Methylamino
N-Ethylamino
5.6
6.5
6.3
7.0
6.8
6.3
5.8
6.4
6.0
<5.5
<5.5
<5.5
5.8
<5.5
<5.5
<5.5
<5.5
<5.5
<5.5
<5.5
<5.5
<5.5
N-Propylamino
N-Isopropylamino
N-Cyclopropylamino
N-Phenylamino
N-Benzylamino
N,N-Diethylamino
4-Morpholino
For the LHS ester moiety (Table 3), isopropyl (1r) was optimal
among the six groups examined. Compound 1r had a pIC50 of 8.3
in the M3 FLIPR assay, 4-fold more potent than the HTS hit 1a,
and good subtype selectivity—greater than 10-fold selective for
M3 over M2 and 100-fold selective for M3 over M1. Amides, bioisos-
teres of the esters, were also investigated (Table 4). Similar to the
5.8
<5.5
<5.5
<5.5
<5.5
a
Means of at least two determinations with standard deviation of < 0.3.

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J. Jin et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5481–5486
F
F
O
O
N+
H
N
H
H
N
H
N
N+
N
N
H
N
H
EtO
O
EtO
O
O
2s
O
2r
M3 FLIPR pIC50 = 8.7
M2 FLIPR pIC50 = 8.6
M1 FLIPR pIC50 = 7.0
M3 FLIPR pIC50 = 8.2
M2 FLIPR pIC50 = 7.4
M1 FLIPR pIC50 = 6.7
HO
HO
Figure 3. Potency comparison of pyrrolidinium salt 2r with piperidinium salt 2s.
Table 5
O
Further optimization of the LHS moiety
H2N
N
N
Nosyl
O
H
N
H
N
N+
DMHB
11
R
N
H
OH
O
t-BuO
a, b, c
HO
O
N
H
H
N
a
Compound
R
FLIPR pA2
M2
N
N
R
OH
M3
M1
7.8
O
DMHB
2a
7
9.9
9.0
O
12
t-BuO
O
O
O
N
H
N
9.2
8.7
8.4
8.2
7.1
6.8
6.9
7.5
8.6
O
H
H
N
N+
N
R
N
OH
d, e
8
N
H
O
-
N
CF3COO
9
8.7
8.2
N
7-9, 10a-b
HO
O
NH2
O
10a
10.0
10.7
9.7
S
f, g, h
N
O
O
CH3
NC
O
N
10b
10.3
13
S
O
NH2
NO2
a
f, i, h
Means of at least two determinations with standard deviation of < 0.3.
N
NC
N
O
14
selectivity as esters. In addition, the central diamine moiety was
briefly examined. As shown in Figure 3, piperidinium salt 2s was
slightly more potent than pyrrolidinium salt 2r in the M3 FLIPR as-
say. 2s had similar subtype selectivity for M3 over M1 but was less
subtype selective for M3 over M2 compared to 2r.
Having identified the preferred structural motifs in each of the
five regions examined, we then combined the best SAR elements
into single molecules and further optimized two of these regions.
As expected, combining the isopropyl ester, 3(S)-amino piperidine
and quaternary ammonium salt into a single molecule (2a)
resulted in excellent M3 potency (pA2 = 9.9) (Table 5).17 2a also
had good subtype selectivity—about 10-fold selective for M3 over
M2 and 100-fold selective for M3 over M1. Compared to ester 2a,
isopropyl amide 7 was less potent, but still possessed high M3
potency with a pA2 of 9.2 and good subtype selectivity for M3 over
M2 and M1. Other ester bioisosteres such as oxadiazole were
explored. Oxadiazoles 8 and 9 had good M3 potency and were
j, k
NH2
NO2
O
HO
S
R'
S
O
O
15a, R' = i-Pr
15b, R' = c-hexyl
Scheme 2. Reagents and conditions: (a) anilines, CDI, DIEA, DCE, rt; (b) K2CO3,
PhSH, NMP, rt; (c) 3-hydroxybenzaldehyde, Na(OAc)3BH, 10% of HOAc in NMP, rt;
(d) MeI, CH3CN, rt; (e) 20% of TFA in DCE, rt; (f) NH2OH, EtOH, reflux; (g) p-NO2–
PhCOCl, pyridine, heating; (h) Na2S, dioxane/H2O (1:1), heating; (i) MeCOCl,
pyridine, heating; (j) (COCl)2, DCM, DMAP, TEA, isopropanol or cyclohexanol, rt;
(k) Pd/C, H2 (15 psi), EtOH, rt.
subtype-selective for M3 over M2 and M1, but were significantly
less potent than ester 2a. The LHS phenyl group could be replaced
by a thiophene group. Isopropyl thiophenecarboxylate 10a had

J. Jin et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5481–5486
5485
1) a, b, or c
O
O
2) d
H
N
H
N
H
N
H
N
N+
R2
NH
R1
N
N
H
O
O
O
O
DMHB
O
O
t-BuO
HO
16
17
Scheme 3. Reagents and conditions: (a) i—allyl bromide, CH3CN, rt; ii—3-hydroxybenzaldehyde, Na(OAc)3BH, 10% of HOAc in NMP, rt; (b) i—cyclopropylcarboxyaldehyde,
Na(OAc)3BH, 10% of HOAc in NMP, rt; ii—cyclopropylmethylbromide, CH3CN, 75 °C; (c) 1,6-hexyldibromide, CH3CN, 75 °C; (d) 50% of TFA in DCE, rt.
Table 6
We then further explored the quaternary ammonium salt moi-
ety. In addition to N-methyl quaternary ammonium salt 2a, other
quaternary ammonium salts exemplified by N-allyl ammonium
salt 16a had excellent M3 potency (pA2 = 10.0) and good subtype
selectivity (greater than 10-fold selective for M3 over M2 and
100-fold selective for M3 over M1) (Table 6). Symmetrical quater-
nary ammonium salts such as N,N-dicyclopropylmethyl compound
16b also possessed high M3 potency and good subtype selectivity
for M3 over M2 and M1, but were less potent compared to 2a and
16a. In addition, compound 16c, which possesses a spiro quater-
nary ammonium center, was less potent and less selective for M3
over M2 and M1 compared to 16b, but still showed good M3 po-
tency with a pA2 of 8.5. Symmetrical quaternary ammonium salts
exemplified by 16b and 16c eliminated the chiral center at the
quaternary ammonium nitrogen. Synthesis of compounds 16a,
16b, and 16c is outlined in Scheme 3. Resin-bound piperidine 17
was prepared according to Scheme 1. Reductive amination of inter-
mediate 17, alkylation of the resulting tertiary amines and subse-
quent resin cleavage and simultaneous removal of the tert-butyl
protecting group afforded the desired compounds 16a and 16b.
Compound 16c was prepared via alkylation of 17 with 1,6-hexyl-
dibromide, resin cleavage and protecting group removal.
Further optimization of the quaternary ammonium salt moiety
O
H
N
H
N
N+
R2
R1
N
H
O
O
O
HO
a
Compound
FLIPR pA2
M2
N+
R2
M3
M1
R1
N+
N+
2a
16a
16b
9.9
9.0
8.8
8.3
8.3
7.8
7.8
7.4
7.1
OH
10.0
9.2
OH
In summary, SAR exploration of multiple regions of the HTS hit
1a led to the identification of key structural motifs necessary for
achieving high M3 potency and good subtype selectivity. Further
optimization of this series resulted in highly potent M3 antagonists
such as 2a and 10b with greater than 100-fold subtype selectivity
for M3 over M1.
N+
N+
16c
8.5
Acknowledgments
a
Means of at least two determinations with standard deviation of < 0.3.
We thank Bing Wang for NMR, Qian Jin for LC/MS, and Carl
Bennett for purification.
similar M3 potency to isopropyl benzoate 2a. Further optimization
of the ester group resulted in cyclohexyl ester 10b, which was the
most potent M3 antagonist to date with a pA2 of 10.7. Compared to
2a, compounds 10a and 10b were slightly less selective for M3 over
M2, but maintained good subtype selectivity for M3 over M1.
Synthesis of compounds 7, 8, 9, 10a, and 10b is outlined in Scheme
2. Urea formation from resin-bound primary amine 11, prepared
according to Scheme 1, and commercially available 4-amino-N-iso-
propyl benzamide and anilines 13, 14, 15a, and 15b (vide infra),
followed by nosyl removal and reductive amination, afforded
resin-bound intermediates 12. Alkylation of tertiary amines 12,
followed by resin cleavage and simultaneous removal of the
tert-butyl protecting group, produced the desired quaternary
ammonium salts 7, 8, 9, 10a, and 10b. Anilines 13 and 14 were
synthesized in a 3-step sequence—formation of amidoximes from
nitriles and hydroxylamine,18 cyclization of amidoximes with acid
chlorides to form oxadiazoles,19 and reduction of the nitro group.20
2-Amino-5-thiophenecarboxylates 15a and 15b were prepared
from commercially available 2-nitro-5-thiophenecarboxylic acid
under standard ester formation and hydrogenation conditions.
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a mean of at least two 17. The pIC50 limit of the M3 FLIPR assay was about 9.0. pA2 was determined and
13. The biological assay results in the paper are
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diastereoisomer was the most preferred, see Ref. 11 for details.