Novel N-substituted benzimidazolones as potent, selective, CNS-penetrant, and orally active M1 mAChR agonists

Article abstract of DOI:10.1021/ml100105x

Virtual screening of the corporate compound collection yielded compound 1 as a subtype selective muscarinic M₁ receptor agonist hit. Initial optimization of the N-capping group of the central piperidine ring resulted in compounds 2 and 3 with s

Full text of DOI:10.1021/ml100105x

pubs.acs.org/acsmedchemlett
Novel N-Substituted Benzimidazolones as Potent, Selective,
CNS-Penetrant, and Orally Active M1 mAChR Agonists
Brian Budzik,† Vincenzo Garzya,† Dongchuan Shi, Graham Walker, Marie Woolley-Roberts,
Joanne Pardoe, Adam Lucas, Ben Tehan, Ralph A. Rivero, Christopher J. Langmead,
Jeannette Watson, Zining Wu, Ian T. Forbes, and Jian Jin*
GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, Pennsylvania 19426
ABSTRACT Virtual screening of the corporate compound collection yielded
compound 1 as a subtype selective muscarinic M1 receptor agonist hit. Initial
optimization of the N-capping group of the central piperidine ring resulted in
compounds 2 and 3 with significantly improved potency and selectivity. Subse-
quent optimization of substituents on the phenyl ring of the benzimidazolone
moiety led to the discovery of novel muscarinic M1 receptor agonists 4 and 5 with
excellent potency, general and subtype selectivity, and pharmacokinetic (PK)
properties including good central nervous system (CNS) penetration and oral
bioavailability. Compound 5 showed robust in vivo activities in animal models of
cognition enhancement. The combination of high potency, excellent selectivity,
and good PK properties makes compounds 4 and 5 valuable tool compounds for
investigating and validating potential therapeutic benefits resulting from selective
M1 activation.
KEYWORDS M1 muscarinic acetylcholine receptor, M1 mAChR, CNS-penetrant
and orally active M1 mAChR agonists, subtype selective, benzimidazolones,
1-(N-substituted piperidin-4-yl)benzimidazolones
allosteric binding site.11 , 1 2 A number of subtype selective M1
he muscarinic acetylcholine receptors (mAChRs) be-
agonists were subsequently reported including TBPB and its
analogues13-15 and our 20 biaryl amides.16,17 In this letter,
we describe the identification, synthesis, and evaluation of a
series of novel 1-(N-substituted piperidin-4-yl)benzimidazo-
lones, which were first disclosed in our published patent appli-
cations,18,19 in a number of in vitro and in vivo biological and
pharmacokinetic (PK) studies. Optimization of this benzimi-
dazolone series resulted in the discovery of compounds 4 and
5as potent, selective, central nervous system (CNS)-penetrant,
and orally active M1 mAChR agonists.
Virtual screening of the corporate compound collection
against the allosteric binding site11,12 of M1 mAChR using an
M1 mAChR homology model, which was built on the basis of
the crystal structure of bovine rhodopsin,20 yielded about
1000 putative hits. Evaluation of these compounds in our
human M1 fluorometric imaging plate reader (FLIPR) assay,
which measures compound induced Ca2þ mobilization in
Chinese hamster ovary (CHO) cells that stably expressed
human M1 mAChR, led to the identification of compound 1
as an M1 mAChR agonist with moderate potency21 (Figure 1).
Compound 1 was subsequently evaluated in M2-M5 FLIPR
assays and found to have good subtype selectivity for M1
long to the seven-transmembrane receptors (7TMRs,
T
also known as G-protein coupled receptors (GPCRs))
family A and are activated by their native ligand neurotrans-
mitter acetylcholine.1-4 Five muscarinic mAChR subtypes,
M1-M5, are known to date.2-4 M1-M5 mAChRs are widely
distributed in mammalian organs and the central and peri-
pheral nerve system where they mediate important neuro-
nal and autocrine functions including memory and attention
mechanisms, motor control, and nociception.5,6 In particu-
lar, selective M1 agonism has been suggested as a therapeu-
tic approach in dementia including Alzheimer's disease and
age-associated memory impairment or cognitive impair-
ment associated with schizophrenia.7 Furthermore, recent
clinical results from pan mAChR agonists have resulted in
severe GI and CV side effects that further demonstrate the
need for selective M1 agonists.8,9 In addition, the activation
of M1 mAChR has been reported to play a role in protecting
neurons from apoptosis induced by oxidative stress.10 How-
ever, M1-M5 mAChRs 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.4
Recently, an allosteric binding site unique to M1 mAChR
has been identified.11 , 1 2 This breakthrough led to the discovery
of AC-42, the first subtype selective M1 agonist, which achieved
the high degree of subtype selectivity via binding to this unique
Received Date: May 9, 2010
Accepted Date: June 2, 2010
Published on Web Date: June 08, 2010
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Figure 2. Profile of compound 2.
Figure 1. Virtual screening hit 1.
over M2-M4 and modest subtype selectivity for M1 over M5.
It is worth noting that compound 1 was also identified as a hit
via high throughput screening (HTS) of the corporate com-
pound collection that was carried out later. It was gratifying
that both hit generation approaches (virtual screening and
HTS) yielded several common hits such as compound 1 and
the virtual screening strategy provided our drug discovery
program a tractable hit ahead of the HTS campaign. How-
ever, compound 1 showed high affinity to a number of
aminergic 7TMRs including 5HT1A, 5HT2A, D2, and D3
(Figure 1). In addition, compound 1 had high binding affinity
to hERG (the human Ether-a-go-go Related Gene). To im-
prove potency and selectivity and reduce the potential hERG
liability, we initially focused our medicinal chemistry effort
on optimizing the N-capping group of the central piperidine
ring of compound 1.
Figure 3. Profile of compound 3.
Scheme 1. Synthesis of Compounds 1, 2, and 3a
Compounds with various N-capping groups were synthe-
sized from commercially available 1-(piperidin-4-yl)benzimi-
dazolone (6) (Scheme 1). Alkylation of 6 with 1-chloro-3-(p-
fluorophenoxy)propane under microwave heating conditions
afforded compound 1. Reductive alkylation of 6 with cyclo-
hexanoneor4-tetrahydropyranoneyieldedcompounds2and
3 respectively.
a (a) 1-Chloro-3-(p-fluorophenoxy)propane, KI, DIEA, CH3CN, μW
210 °C, 2 min, 56%; (b) cyclohexanone, Na(OAc)3BH, AcOH, DCE, rt,
16 h, 44%; (c) 4-tetrahydropyranone, Na(OAc)3BH, AcOH, DCE, rt,
16 h, 42%.
Initial optimization of the right-hand-side N-capping group
of compound 1 led to the discovery of compound 2, which
possesses a cyclohexyl group instead of the floppy 3-(4-
fluorophenoxy)propoxy group in compound 1. Compound 2
was a significantly higher potency M1 mAChR agonist than
compound 1 (Figure 2). In addition, compound 2 showed
excellent selectivity for M1 over M3, M5, 5HT1A, 5HT2A, D2,
and D3 in a very significant improvement over compound 1.
In hERG binding assay, compound 2 showed drastic impro-
vement (about 1000-fold) over compound 1. However, com-
pound 2 suffered from poor selectivity for M1 over M2 and M4.
Additionally, in in vitro metabolic stability studies using rat
and human liver microsomes, compound 2 had high intrinsic
clearance (CLi) for rat and moderate CLi for human.
To improve metabolic stability of this series, the cyclohexyl
ring of compound 2 was replaced with the tetrahydropyran
(THP) group in compound 3. This modification resulted in
improved in vitro metabolic stability (CLi e 0.5 mL/min/g for
both rat and human) while maintaining good M1 mAChR
agonist activity, good selectivity for M1 over M3 and M5, and
good hERG profile. However, selectivity for M1 over M2 and M4
was not improved. In addition, in an in vivo rat PK study,
compound 3 showed high in vivo clearance (CLb = 68 mL/
min/kg) and short half-life (T1/2 = 0.5 h) after intravenous (iv)
administration at 1 mg/kg (Figure 3).
To improve subtype selectivity for M1 over M2 and M4 and
in vivo rat PK properties of this series, we explored substi-
tuents on the phenyl ring of the benzimidazolone moiety.
Optimization of this region led to the discovery of com-
pounds 4 and 5. Compound 4 that contains a 6-methyl group
on the benzimidazolone ring had excellent M1 mAChR
agonist activity (pEC50 = 8.3) and selectivity against all 4
subtypes (>100-fold selective for M1 over M2-M5) (Figure 4).
Compound 4 also had good in vitro metabolic stability versus
rat and human liver microsomes and was >5,000-fold selec-
tive for M1 mAChR over hERG channel. Importantly, in an in
vivo rat PK study, compound 4 appeared to show significantly
reduced in vivo clearance (estimated CLb = 25 mL/min/kg)22
and moderate half-life (T1/2=2.6 h), oral bioavailability (esti-
mated F=55%),22 and CNS penetration (brain/blood ratio =
0.6) after oral (po) administration at 3 mg/kg. To assess
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Scheme 2. Synthesis of Compounds 4 and 5a
Figure 4. Profile of compound 4.
a (a) KI, Na2CO3, DMF, 50 °C, 15 h, 84%; (b) H2, 5% Pd/C, MeOH, 100%;
(c) N,N0-carbonyldiimidazole (CDI), CH3CN, 40 °C, 36 h, 76%; (d) 2.5 M
aqueous NaOH, reflux 15 h, 100%; (e) 4-tetrahydropyranone, Na-
(OAc)3BH, AcOH, DCE, rt, 16 h, 51%; (f) DIEA, DMF, 80 °C, 38 h,
64%; (g) Raney nickel, N2H2 H2O, EtOH, 45 °C, 50 min, 92%; (h) CDI,
3
Figure 5. Profile of compound 5.
THF, 45 °C, 80 min, 77%; (i) 20% TFA/DCM, 1 h, rt, 94%; (j) 4-tetra-
hydropyranone, Ti(O-i-Pr)4, MeOH, 1.5 h, then NaBH3CN, 1.5 h, 42%.
general selectivity, compound 4 was evaluated in the CEREP
selectivity panel, which consists of a broad range of 7TMRs (or
GPCRs), ion channels, and transporters, and found to be clean
(less than 30% inhibitions at 1 μM) against all targets in the
panel except hitting κ-opioid receptor (κOR) at 40% inhibition
at 1 μM and, as expected, the mAChRs in the panel.
Similar to compound 4, compound 5, which has a 5-fluoro-
6-methyl benzimidazolone moiety at the left-hand side,
potently activated M1 mAChR, had excellent subtype and
general selectivity, and possessed good in vitro and in vivo
PK properties (Figure 5). The combination of these excellent
properties makes compounds 4 and 5 outstanding tool
compounds for in vivo animal studies.
Synthesis of compounds 4 and 5 is outlined in Scheme 2.
Aromatic nucleophilic substitution of aryl fluoride 7 with
protected 4-aminopiperidine 8 yielded nitroaniline 9, which
was quantitatively hydrogenated to 10. Benzimidazolone
formation via CDI gave 11, followed by quantitative depro-
tection of the ethyl carbamate by alkaline hydrolysis to give
12. Reductive alkylation of 12 with 4-tetrahydropyranone
furnished compound 4. Analogous steps for the synthesis of
compound 5 began with aryl fluoride 13 undergoing dis-
placement with N-Boc-4-aminopiperidine 14 to give nitro-
aniline 15, which was reduced via transfer hydrogenation
with Raney nickel and hydrazine to afford 16 which was
cyclized by CDI to yield benzimidazolone 17. Boc deprotec-
tion to 18 followed by Ti(O-i-Pr)4 facilitated reductive alkyla-
tion with 4-tetrahydropyranone produced compound 5.
The effects of compound 5 on cell firing rate in the CA1
region of the rat hippocampus were evaluated next. Intra-
venous administration of 5 (1 mg/kg) significantly enhanced
cell firing rate in the hippocampal CA1 region (Figure 6).
Over a 3-fold enhancement (P<0.01, repeated measures
ANOVA) of neuronal activity was observed. A lower dose of
5 (0.3 mg/kg, iv) failed to produce a significant increase
in cell firing rate. Pretreatment of the mAChR antagonist
scopolamine (3 mg/kg, ip) 15 min prior to the iv adminis-
tration of 5 (1 mg/kg) abolished the enhancement in cell
firing rate induced by 5. These studies confirmed that
compound 5 activated native mAchRs in vivo.
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campal CA1 cell firing and passive avoidance experiments. fpKi
overview. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author: *To whom correspondence should be
addressed. Current address: Center for Integrative Chemical Biol-
ogy and Drug Discovery, Division of Medicinal chemistry & Natural
Products, Eshelman School of Pharmacy, The University of North
Carolina at Chapel Hill, Chapel Hill, NC 27599. Phone: 919-843-
8459. Fax: 919-843-8465. E-mail: jianjin@unc.edu.
Figure 6. Effect of iv administration of compound 5 on cell firing
rate in the CA1 region of the rat hippocampus.
Author Contributions: † These authors contributed equally to
this work.
ACKNOWLEDGMENT We thank Minghui Wang and Bing Wang
for NMR, Bill Leavens for HRMS, and Andrew Foley and Claire Barry
for in vivo passive avoidance support.
ABBREVIATIONS mAChRs, muscarinic acetylcholine re-
ceptors; 7TMRs, seven-transmembrane receptors; GPCRs,
G-protein coupled receptors; PK, pharmacokinetic; CNS,
central nervous system; FLIPR, fluorometric imaging plate
reader; CHO cells, Chinese hamster ovary cells; HTS, high
throughput screening; hERG, the human Ether-a-go-go Re-
lated Gene; IA, intrinsic activity; fpKi, functional pKi; CLi,
intrinsic clearance; THP, tetrahydropyran; κOR, κ-opioid
receptor.
Figure 7. Effect of oral administration of compound 5 on scopol-
amine-induced amnesia in rats.
In addition, compound 5 was evaluated in a passive
avoidance rat model to assess its ability of enhancing cogni-
tion and memory. As shown in Figure 7, scopolamine
rendered the animals amnesic in the passive avoidance task
(P < 0.05 cf. vehicle treated controls). Oral administration of
compound 5 reversed the scopolamine-induced amnesia in
a dose-dependent manner with significant effects observed
following 3 and 10 mg/kg doses (P < 0.05 cf. scopolamine in
both cases). Taken together, these results suggest that selec-
tive M1 mAChR agonists such as compound 5 are effective
cognition enhancers and could provide a potentially valuable
treatment option for schizophrenia.
In conclusion, a series of novel 1-(N-substituted piperidin-
4-yl)benzimidazolones was identified as M1 mAChR agonists
via virtual screening. Optimization of this benzimidazolone
series resulted in the discovery of compounds 4 and 5 as
potent, CNS-penetrant, orally active M1 mAChR agonists
with excellent subtype and general selectivity. Compound
5 significantly enhanced cell firing rate in rat hippocampal
CA1 region and reversed the scopolamine-induced amnesia
in a dose-dependent manner, demonstrating robust in vivo
efficacy in preclinical schizophrenia models. The combina-
tion of outstanding potency, excellent selectivity, and good
PK properties makes these compounds valuable tools for
the biomedical research community to further investigate
and validate potential therapeutic benefits resulting from
selective M1 agonism.
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