Discovery of novel and long acting muscarinic acetylcholine receptor antagonists

Article abstract of DOI:10.1021/jm800634k

High throughput screening and subsequent optimization led to the discovery of novel quaternary ammonium salts as highly potent muscarinic acetylcholine receptor antagonists with excellent selectivity. Compounds 8a, 13a, and 13b showed excellent inhibitory

Full text of DOI:10.1021/jm800634k

4866
J. Med. Chem. 2008, 51, 4866–4869
Discovery of Novel and Long Acting
Muscarinic Acetylcholine Receptor
Antagonists
Jian Jin,* Yonghui Wang, Dongchuan Shi, Feng Wang,
Roderick S. Davis, Qi Jin, Wei Fu, James J. Foley,
Edward F. Webb, Chris J. Dehaas, Manuela Berlanga,
Miriam Burman, Henry M. Sarau, Dwight M. Morrow,
Parvathi Rao, Lorena A. Kallal, Michael L. Moore,
Ralph A. Rivero, Michael Palovich, Michael Salmon,
Kristen E. Belmonte, and Jakob Busch-Petersen*
Figure 1. In vitro profile of HTS hit 1.
result in airway hyperreactivity and hyperresponsiveness medi-
ated by increased acetylcholine release and thus excess stimula-
tion of M3. Therefore, potent mAChR antagonists, particularly
directed toward the M3 subtype, would be useful as therapeutics
in these mAChRs-mediated disease states. Inhaled delivery of
such antagonists could potentially prevent side effects mediated
by peripheral and/or central M1, M2, or M3 antagonism5 by
avoiding substantial systemic exposure.
High throughput screening (HTS) of the corporate compound
collection using a fluorometric imaging plate reader (FLIPR)
assay (measuring inhibition of acetylcholine-mediated [Ca2+]i-
mobilization in Chinese hamster ovary (CHO) cells stably
expressing human recombinant M3 receptor) led to the identi-
fication of pyrrolidine 1, a mixture of two diastereoisomers, as
an antagonist with a pIC50 of 7.7 (Figure 1).11,12 The compound
was subsequently tested in M2 and M1 FLIPR assays and found
to be about 10-fold selective for M3 over M2 and 100-fold
selective for M3 over M1.
To explore this novel HTS hit, an efficient and robust solid-
phase synthesis was developed (Scheme 1). Commercially
available Boc-protected 3-aminopyrrolidine and piperidine (2)
were converted to nosyl-protected diamines 3 in two steps. The
diamines 3 were loaded onto commercially available 2,6-
dimethoxy-4-polystyrenebenzyloxybenzaldehyde resin (DMHB
resin)13 via reductive amination to afford resin-bound amines
4. Amines 4 were coupled with Fmoc-protected tert-butylty-
rosine, followed by Fmoc removal, to produce resin-bound
amines 5. Urea formation from intermediates 5, nosyl-group
removal, and subsequent reductive amination afforded resin-
bound intermediates 6. Resin cleavage and simultaneous removal
of the tert-butyl group of 6 produced the targeted tertiary amines
7 in good yields and purity. Alkylation of tertiary amines 6,
followed by resin cleavage and tert-butyl group removal
afforded the desired quaternary ammonium salts 8.
The preferred stereochemistry was determined by preparing
all four possible diastereoisomers starting from optically pure
3-aminopyrrolidine and protected tyrosine. As shown in Table
1, 7b, the (3S,3′S) diastereoisomer, was the most potent
diastereoisomer, 100-fold more potent than the other three
diastereoisomers. Compound 7b also had the best subtype
selectivity, about 10-fold selective for M3 over M2 and 80-fold
selective for M3 over M1.
GlaxoSmithKline, 709 Swedeland Road,
King of Prussia, PennsylVania 19406
ReceiVed May 27, 2008
Abstract: High throughput screening and subsequent optimization led
to the discovery of novel quaternary ammonium salts as highly potent
muscarinic acetylcholine receptor antagonists with excellent selectivity.
Compounds 8a, 13a, and 13b showed excellent inhibitory activity and
long duration of action in bronchoconstriction in vivo models in two
species via intranasal or intratracheal administration. The novel inhaled
muscarinic receptor antagonists are potentially useful therapeutic agents
for the treatment of chronic obstructive pulmonary disease and other
bronchoconstriction disorders.
Muscarinic acetylcholine receptors (mAChRsa) belong to the
family A A2 subfamily of seven-transmembrane (7TM) recep-
tors. Five distinct subtypes, denoted as M1, M2, M3, M4, and
M5 mAChRs, have been cloned from several species including
human and mouse, exhibiting a very high sequence homology
across species.1-3 The five subtypes share a common orthosteric
ligand-binding site with an extremely high sequence homology,
which explains why it has been difficult historically to identify
subtype selective ligands.3 M1-M5 mAChRs are widely dis-
tributed in mammalian organs 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 Blockade of M3 on airway
smooth muscle reduces excess airway smooth muscle contrac-
tion. M2 is primarily found on postganglionic nerve termini and
functions to limit acetylcholine release from parasympathetic
nerves.8 Blockade of the M2 function would be expected to
enhance bronchoconstriction. M1 is found in parasympathetic
ganglia and facilitates neurotransmission through ganglia, thus
enhancing cholinergic reflexes.9 Blockade of M1 may help to
reduce bronchoconstriction. In chronic obstructive pulmonary
disease (COPD) and asthma, inflammatory conditions lead to
loss of neuronal inhibitory activity mediated by M2 on para-
sympathetic nerves, causing excess acetylcholine reflexes10 that
Lead optimization of this series led to the identification of
quaternary ammonium salt 8a with excellent potency in the M3
FLIPR assay (pA2 ) 9.9) and affinity in a M3 binding assay
(pKi ) 9.5) (Table 2). In a kinetics studies using the M3 FLIPR
assay, 8a was found to be a competitive antagonist with a pKB
of 10.1, consistent with its binding affinity. 8a was also a potent
M2 and M1 antagonist and maintained the same level of subtype
selectivity (10-fold selective for M3 over M2 and 100-fold
selective for M3 over M1) compared with 1 and 7b. Compound
* To whom correspondence should be addressed. For J.J.: current address,
Center for Integrative Chemical Biology and Drug Discovery, Eshelman
School of Pharmacy, The University of North Carolina at Chapel Hill,
Campus Box 7360, Chapel Hill, NC 27599; phone, 919-843-0766; fax, 919-
966-0204; e-mail, jianjin@unc.edu. For J.B.-P.: phone, 610-270-7831; fax,
610-270-4451; e-mail, Jakob.2.Busch-Petersen@gsk.com.
a Abbreviations: mAChRs, muscarinic acetylcholine receptors; 7TM,
seven-transmembrane; COPD, chronic obstructive pulmonary disease; HTS,
high throughput screening; CYP450, cytochrome P450; PK, pharmacoki-
netic; Penh, enhanced pause.
10.1021/jm800634k CCC: $40.75
2008 American Chemical Society
Published on Web 08/05/2008

Letters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 4867
Table 2. Profiles of 3-Aminopiperidinium Salt 8a
Scheme 1. Synthesis of Pyrrolidine/Piperidine-Based Tertiary
Amines and Quaternary Ammonium Saltsa
a Rat PK parameters are from an iv/po study in Sprague-Dawley rats
dosed at 2.2 mg/kg (iv) and 4.8 mg/kg (oral).
two targets in the panel. The compound was an antagonist of
two ligand-gated ion channels, R1 nicotinic acetylcholine
receptor and R3 nicotinic acetylcholine receptor, with pIC50
values of 6.1. In addition to the excellent general selectivity,
8a had good developability properties. For example, 8a was
more than 10,000-fold selective for M3 over five common
cytochrome P450 (CYP450) isozymes (pIC50 ) 5.4 or less) and
hERG (binding pIC50 ) 5.6). In a rat iv/po pharmacokinetic
(PK) studies, 8a had low-moderate clearance (Cl ) 15 (mL/
min)/kg), short half-life (T1/2 ) 1.1 h), and no appreciable oral
bioavailabilityssuitable for inhaled delivery. The low oral
bioavailability of 8a was consistent with its extremely low
artificial membrane permeability (less than 3 nm/s), which was
suitable for targeting membrane-bound receptors such as
mAChRs.
a (a) 2-nitrobenzenesulfonyl chloride, pyridine, CH2Cl2, 0 °C to room
temp; (b) 4 M HCl in 1,4-dioxane, MeOH, room temp; (c) 2,6-dimethoxy-
4-polystyrenebenzyloxybenzaldehyde (DMHB-resin), Na(OAc)3BH, DIEA,
1% of HOAc in NMP, room temp; (d) Fmoc-Tyr(tBu)-OH, DIC, HOAt,
NMP, room temp; (e) 20% of piperidine in NMP, room temp; (f)
4-isocyanatobenzoates, DCE, room temp; or 4-aminobenzoates, 1,1′-
(oxomethanediyl)bis-1H-pyrrole, DIEA, DCE, room temp; (g) K2CO3,
PhSH, NMP, room temp; (h) various benzaldehydes, Na(OAc)3BH, 10%
of HOAc in NMP, room temp; (i) 50% of TFA in DCE, room temp; (j)
MeI, CH3CN, room temp.
Compound 8a was a mixture of (1S,3S,3′S) and (1R,3S,3′S)
diastereoiosmers as a result of forming a new chiral center: the
quaternary ammonium nitrogen. It was determined that the ratio
of the two diastereoiosmers was about 8:1 with the (1S,3S,3′S)
diastereoisomer being the major, using extensive 2D and NOE
NMR analysis. Separation of the two diastereoisomers via
chromotagraphy was difficult. In the course of further lead
optimization of the series to reduce the number of chiral centers,
imidazothiazole based quaternary ammonium salts 13a and 13b
were identified (Table 3). Compound 13a had outstanding
potency with a pA2 of 10.6 in the M3 FLIPR assay and showed
about 60-fold selective for M3 over M2, while the selectivity
for M3 over M1 was 30-fold. Similarly, 13b had a pA2 of 10.9
in the M3 FLIPR assay and was about 15-fold selective for M3
over M2 and 25-fold selective for M3 over M1. In addition, 13b
showed excellent general selectivity in CEREP (74% inhibition
at 1 µM against NK2 receptor, less than 25% inhibition against
other targets in the panel except muscarinic receptors).
Imidazothiazolium salts 13 were synthesized according to the
route outlined in Scheme 2. The imidazothiazole methylamine
10 was prepared by condensation of 2-amino-5-methylthiazole
with 1,3-dichloroacetone to give the imidazothiazole methyl-
chloride 9, followed by displacement of the chloride by azide
and reduction to give the primary amine. Amide formation of
10 with N-Fmoc-O-tBu-Tyr and subsequent Fmoc deprotection
Table 1. Preferred Stereochemistry
8a was profiled in more than 100 in-house 7TM, ion channel,
enzyme, transporter, and nuclear hormone receptor selectivity
assays and was found to have excellent general selectivity
displaying less than a 30,000-fold selectivity margin against only

4868 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Table 3. Potency of Imidazothiazolium Salts 13a and 13b
Letters
Figure 2. Effect of intranasal administration of 8a and 13a on
methacholine-induced bronchoconstriction in conscious mice.
Scheme 2. Synthesis of Imidazothiazole-based Quaternary
Ammonium Saltsa
Figure 3. Effect of intratracheal administration of 13b on acetylcholine-
induced bronchoconstriction in conscious guinea pigs at 4 h.
Figure 4. Effect of intratracheal administration of 13b on acetylcholine-
induced bronchoconstriction in conscious guinea pigs at 24 h.
ylchloroformate mediated urea formation from the corresponding
arylamine. The former method was used for the preparation of
13a. The final compounds 13 were then prepared by quater-
nization with the appropriate halide (in the case of 13a,
2-bromomethylnaphthalene) followed by cleavage of the tert-
butyl ether under acidic conditions. Alternatively, the amine 10
could be coupled with N-Boc-O-tBu-Tyr followed by quater-
nization and acid mediated deprotection to give 14. The final
compounds 13 were then obtained using the above-mentioned
procedures for urea formation. This method was used for the
preparation of 13b. The cyclohexyl 5-amino-2-thiophenecar-
boxylate used in the preparation of 13b was prepared from
2-nitrothiophene-5-carboxylic acid ester formation with cyclo-
hexanol and subsequent reduction of the nitro group under
standard hydrogenation conditions.
a (a) (i) NaBr, 1,3-dichloroacetone, EtOAc; (ii) AcOH, heat; (b) NaN3,
DMSO; (c) H2, Pd/C, MeOH; (d) Fmoc-Tyr(tBu)-OH, HATU, Hunig’s base,
DMF; (e) piperidine, DMF; (f) R-NdCdO, EtOAc, dichlorobenzene; (g)
R1-X, MeCN/CHCl3, heat; (h) TFA; (i) Boc-Tyr(tBu)-OH, HATU, Hunig’s
base, DMF; (j) R-NH2, 4-NO2-PhOCOCl, DIEA, CH2Cl2.
Compounds 8a and 13a were evaluated in a methacholine-
induced bronchoconstriction model in conscious mice. As shown
in Figure 2, intranasal administration of 8a and 13a at a single
dose (5 µg/animal)14 significantly inhibited methacholine-
induced bronchoconstriction at 15 min and 5 h after dosing.
Excellent inhibitory activity (greater than 80%) was maintained
at 24 h. Even at 48 h after the single low dose, 8a and 13a still
yielded the primary amine 11. Elaboration resulting in ureas
12 was affected by reaction with isocyantes or by p-nitrophen-

Letters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 4869
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(11) The biological assay results in the paper are a mean of at least two
determinations with standard deviation of less than (0.3 unless
otherwise noted.
exhibited over 30% and 60% of bronchoprotection, respectively,
thus demonstrating excellent in vivo efficacy and duration of
action.
In addition, 13b was evaluated in an acetylcholine-induced
bronchoconstriction model in conscious guinea pigs, measuring
enhanced pause (Penh), an indicator of bronchoconstriction,15
using barometric plethysmography (Figures 3 and 4). 13b
showed significant inhibitory activity at 4 h (93% inhibition)
and 24 h (88% inhibition) in the guinea pig model following a
single low dose (2.5 µg/animal, intratracheal dosing). The
excellent in vivo efficacy and duration of action results of 13b
were similar to those of 8a and 13a in the in vivo mouse model,
demonstrating that these novel muscarinic receptor antagonists
are potentially useful therapeutic agents for the treatment of
COPD and other bronchoconstriction disorders.
In conclusion, novel, very potent, and highly selective
mAChR antagonists were discovered. Quaternary ammonium
salts 8a, 13a, and 13b with 10- to 100-fold subtype selectivity
for M3 over M2 and M1 significantly inhibited methacholine/
acetylcholine-induced bronchoconstriction in two species with
excellent duration of action. Full accounts on this novel series
including detailed SAR will be the subject of future publications.
(12) (a) Busch-Petersen, J.; Fu, W.; Jin, J.; Moore, M. L.; Rivero, R. A.;
Shi, D.; Wang, F.; Wang, Y. Novel M3 Muscarinic Acetylcholine
Receptor Antagonists. WO 018508, 2007. (b) Busch-Petersen, J.; Fu,
W.; Jin, J.; Moore, M. L.; Rivero, R. A.; Shi, D.; Wang, F.; Wang, Y.
Novel M3 Muscarinic Acetylcholine Receptor Antagonists. WO
018514, 2007.
Acknowledgment. We thank Bing Wang and Minghui
Wang for NMR, Qian Jin for LC/MS, Carl Bennett and Lili
Zhu for purification, and Maite De Los Frailes for assay support.
(13) (a) Jin, J.; Graybill, T. L.; Wang, M. A.; Davis, L. D.; Moore,
M. L. Convenient preparation of 4-formyl-3,5-dimethoxyphenol and
its incorporation into linkers and resins for solid-phase synthesis.
J. Comb. Chem. 2001, 3, 97–101. (b) Available from Polymer
Laboratories, part number 1466-6689, 150-300 µm, 1.5 mmol/g
loading.
(14) Foster, W. M.; Walters, D. M.; Longphre, M.; Macri, K.; Miller, L. M.
Methodology for the measurement of mucociliary function in the
mouse by scintigraphy. J. Appl. Physiol. 2001, 90, 1111–1118.
(15) Hamelmann, E.; Schwarze, J.; Takeda, K.; Oshiba, A.; Larsen, G. L.;
Irvin, C. G.; Gelfand, E. W. Noninvasive measurement of airway
responsiveness in allergic mice using barometric plethysmography.
Am. J. Respir. Crit. Care Med 1997, 156, 766–775.
Supporting Information Available: Synthetic procedures and
characterization data for all compounds, procedures for M3, M2,
and M1 FLIPR and binding assays, in vivo bronchoconstriction
mouse and guinea pig models, and specta of compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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JM800634K