Discovery of biphenyl piperazines as novel and long acting muscarinic acetylcholine receptor antagonists

Article abstract of DOI:10.1021/jm800935u

A series of novel biphenyl piperazines was discovered as highly potent muscarinic acetylcholine receptor antagonists via high throughput screening and subsequent optimization. Compound 5c with respective 500- and 20-fold subtype selectivity for M₃

Full text of DOI:10.1021/jm800935u

J. Med. Chem. 2008, 51, 5915–5918
5915
Discovery of Biphenyl Piperazines as Novel
and Long Acting Muscarinic Acetylcholine
Receptor Antagonists
Jian Jin,* Brian Budzik, Yonghui Wang, Dongchuan Shi,
Feng Wang, Haibo Xie, Zehong Wan, Chongye Zhu,
James J. Foley, Edward F. Webb, Manuela Berlanga,
Miriam Burman, Henry M. Sarau, Dwight M. Morrow,
Michael L. Moore, Ralph A. Rivero, Michael Palovich,
Michael Salmon, Kristen E. Belmonte, and
Figure 1. In vitro profile of HTS hit 1.
Scheme 1. General Synthesis of Biphenyl Piperazinesa
Dramane I. Laine´*
GlaxoSmithKline 709 Swedeland Road,
King of Prussia, PennsylVania 19406
ReceiVed July 25, 2008
Abstract: A series of novel biphenyl piperazines was discovered as
highly potent muscarinic acetylcholine receptor antagonists via high
throughput screening and subsequent optimization. Compound 5c with
respective 500- and 20-fold subtype selectivity for M3 over M2 and
M1 exhibited excellent inhibitory activity and long duration of action
in a bronchoconstriction in vivo model in mice via intranasal
administration. The novel inhaled mAChR antagonists are potentially
useful therapeutic agents for the treatment of chronic obstructive
pulmonary disease.
a (a) 2,6-Dimethoxy-4-polystyrenebenzyloxybenzaldehyde (DMHB-
resin), Na(OAc)3BH, DIEA, 10% of HOAc in NMP, rt; (b) various benzoic
acids, DIC, DCE/DMF (1:1), rt; (c) 3-formylphenyl boronic acid, Pd(PPh3)4,
K2CO3 or Cs2CO3, DME, 80 °C; (d) various piperazines, Na(OAc)3BH,
Na2SO4, DCE, rt; (e) TFA, DCE, rt.
(COPD) and asthma, inflammatory conditions lead to loss of
neuronal inhibitory activity mediated by M2 on parasympathetic
nerves, causing excess acetylcholine reflexes,11 which result in
airway hyperreactivity and hyperresponsiveness mediated by
increased stimulation of M3. Therefore, potent mAChR antago-
nists, particularly directed toward the M3 subtype, are useful as
therapeutics for mAChRs-mediated disease states. Besides
preventing any potential M2-mediated bronchoconstriction,
achieving subtype selectivity for M3 over M2 would be desirable
as M2 is found in large numbers on the myocardium and
mediates negative inotropic effects and bradycardia.12 In addi-
tion, inhaled delivery could potentially reduce side effects
mediated by peripheral and/or central M1, M2, or M3 antago-
nism5 by avoiding substantial systemic exposure.
High throughput screening (HTS) of our in-house compound
collection using a fluorometric imaging plate reader (FLIPR)
assay13 resulted in the identification of biphenyl piperazine 1,
as a M3 antagonist with a pIC50 of 7.5 (Figure 1).14–16 In
subsequent evaluation in subtype selectivity assays, compound
1 was found to be more than 100-fold selective for M3 over M2
and about 5-fold selective for M3 over M1. On the basis of its
good potency and subtype selectivity for M3 over M2, 1 was
considered an acceptable starting point for our lead optimization
program aimed at identifying long acting mAChR antagonists.
An efficient and robust solid-phase synthesis was developed
to explore this novel series (Scheme 1). Commercially available
3-bromo benzylamines (2) were loaded onto 2,6-dimethoxy-4-
polystyrenebenzyloxybenzaldehyde resin (DMHB resin)17 via
reductive amination, then coupled with benzoic acids to afford
resin-bound aryl bromides 3. Suzuki coupling of aryl bromides
3 with 3-formylphenyl boronic acid and subsequent reduction
amination of the resulting benzaldehydes with substituted
piperazines, followed by resin cleavage, produced the targeted
biphenyl piperazines 4 in excellent yields and purity.
Five muscarinic acetylcholine receptor (mAChRa) subtypes,
M1-M5, are known to date.1–3 These seven-transmembrane
(7TM) receptors 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 The five subtypes also exhibit a high sequence
homology across species.3 M1-M5 mAChRs are widely dis-
tributed in mammalian organs and the central and peripheral
nerve system where they mediate important neuronal and
autocrine functions.4,5
In the mammalian respiratory system, only M1, M2, and M3
have been recognized as playing important and diverse func-
tional roles.6 M3 is predominately expressed on airway smooth
muscle and mediates smooth muscle contraction and mucus
secretion.7 Blockade of M3 on airway smooth muscle reduces
excess airway smooth muscle contraction. M2 is primarily found
on postganglionic nerve termini, where it inhibits acetylcholine
release from parasympathetic nerves.8 Blockade of the M2
function is 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.
Muscarinic acetylcholine receptor dysfunction in the lungs
has been noted in a variety of different pathophysiological
states.10 In particular, in chronic obstructive pulmonary disease
* To whom correspondence should be addressed. For J.J.: phone, 919-
843-1645; fax, 919-966-0204; E-mail, jianjin@unc.edu; current address:
Center for Integrative Chemical Biology 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.
For D.I.L.: phone, 610-270-7889; fax, 610-270-4451; E-mail,
Dramane.I.Laine@gsk.com.
During the course of the lead optimization, potent M3
antagonists such as 4a, 4b, and 4c, which were single enanti-
omers possessing a (3S)-3-methylpiperazin-1-yl moiety at the
right-hand side (RHS), were identified (Table 1). Methyl ketone
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/jm800935u CCC: $40.75
2008 American Chemical Society
Published on Web 09/18/2008

5916 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19
Table 1. Potency of Compounds 4a, 4b, and 4c
Letters
Table 2. Potency of Compounds 5a, 5b, and 5c
Table 3. Profile of Piperidine 5c
in vitro potency
FLIPR pA2
M3
11.0
M2
8.3
M1
9.7
M3 binding
M3 kinetics
pKi ) 10.0
competitive, pKB ) 10.5
intrinsic clearance
permeability
human: Clint ) 4 mL/min/g liver
rat: Clint >50 mL/min/g liver
Figure 2. Effect of intranasal administration of 1, 4a, and 4b on
methacholine-induced bronchoconstriction in conscious mice.
<3 nm/s
200 µM
4a was more than 100-fold more potent compared to HTS hit
1 in the M3 FLIPR assay with a pA2 of 10.3. The compound
had excellent M2 subtype selectivity, about 2000-fold selective
for M3 over M2, and was also 50-fold selective for M3 over
M1. Ethyl ketone 4b also exhibited excellent M3 potency (pA2
) 10.3) and good subtype selectivity (500-fold selective for
M3 over M2 and 20-fold selective for M3 over M1). In addition
to ketones 4a and 4b, compounds such as 3-cyanobenzamide
4c also had high M3 potency (pA2 ) 9.6) and good subtype
selectivity (1300-fold selective for M3 over M2 and 30-fold
selective for M3 over M1).
solubility
CYP450
hERG binding
pIC50 < 5.0 vs 1A2, 2C19, 2C9, 2D6, and 3A4
pIC50 ) 5.0
subtype selective compared to ketone 4a in general. Most
notably, piperidine 5c showed outstanding M3 potency with a
pA2 of 11.0 - more than 10-fold more potent than piperazine
5a and piperazinium salt 5b. 5c also had good subtype selectivity
(500-fold selective for M3 over M2 and 20-fold selective for
M3 over M1).
In addition to high potency in the M3 FLIPR assay and good
subtype selectivity over M2 and M1, piperidine 5c had excellent
binding affinity to M3 with a pKi of 10.0 (Table 3). In kinetics
studies using the M3 FLIPR assay, 5c was found to be a
competitive M3 antagonist with a pKB of 10.5, consistent with
its FLIPR pA2 and binding pKi. Although 5c showed 70 to 100%
inhibition at 10 µM against R adrenergic, opioid, and serotonin
receptors in the Cerep selectivity screen, the compound exhibited
less than 30% inhibition at 10 µM against enzymes, ion
channels, and transporters in the panel. Most of the potential
7TM liabilities could be mitigated by avoiding substantial
systemic exposure via low membrane permeability and the
relatively low dose administrated by inhaled delivery (vide
infra). In the in vitro human and rat liver microsome stability
studies, 5c showed low to moderate intrinsic clearance vs human
liver microsome (Clint ) 4.3 mL/min/g) but high intrinsic
clearance vs rat enzyme (Clint >50 mL/min/g). 5c had extremely
low artificial membrane permeability21 (less than 3 nm/s),
suitable for inhaled delivery and targeting membrane-bound
receptors such as mAChRs. In addition, 5c had good develop-
ability properties. For example, 5c had high aqueous solubility,
was clean against five common cytochrome P450 (CYP450)
isozymes (pIC50 < 5.0), and was more than 100000-fold
selective for M3 over hERG (binding, pIC50 ) 5.0).
Compounds 1, 4a, and 4b were evaluated in a methacholine-
induced bronchoconstriction model in conscious mice, measur-
ing enhanced pause (Penh), an indicator of bronchoconstric-
tion,18 using barometric plethysmography (Figure 2). Intranasal
administration19 of 4a and 4b at a single dose (5 µg/animal)
significantly inhibited methacholine-induced bronchoconstriction
at 15 min, while HTS hit 1, a more than 100-fold less potent
antagonist, exhibited less inhibition at a higher dose (25 µg/
animal) at 15 min post dosing. However, the excellent inhibitory
activity exhibited by 4a and 4b was not maintained over a 24 h
period. Compounds 4a and 4b showed respective 45% and 20%
of bronchoprotection at 5 h and little bronchoprotection at 24 h
post the single dose.
Further optimization aimed at identifying long acting mAChR
antagonists from the series led to discovery of compounds 5a,
5b, and 5c, which possess an amino or quaternary ammonium
moiety at the left-hand side (LHS), as potent M3 antagonists
(Table 2).20 Similar to 4a, 4b, and 4c, piperazine 5a had
excellent potency with a pA2 of 10.6 in the M3 FLIPR assay
and showed good subtype selectivity (400-fold selective for M3
over M2 and 6-fold selective for M3 over M1). Converting the
secondary piperazine (5a) to the quaternary piperazinium moiety
(5b) maintained the excellent M3 potency (pA2 ) 9.8) but had
lower M2 subtype selectivity (80-fold selective for M3 over M2).
Piperazine 5a and quaternary piperazinium salt 5b were less
Piperazine 5a and piperazinium salt 5b were synthesized
according to the route outlined in Scheme 2. Resin-bound

Letters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19 5917
Scheme 2. Synthesis of Piperazine 5a and Piperazinium Salt 5ba
Figure 3. Effect of intranasal administration of 5b and 5c on
methacholine-induced bronchoconstriction in conscious mice.
3-bromo-4-fluorobenzylamine (2a) produced benzyl amine 9,
which was then converted to the desired piperidine 5c via
coupling with commercially available 3-[(1-Boc-piperidin-4-
yl)methyl] benzoic acid, followed by the removal of the Boc
group.
In the methacholine-induced bronchoconstriction model in
conscious mice, piperazinium salt 5b had excellent inhibitory
activity at 15 min (95% inhibition) and 5 h (85% inhibition)
and showed significant improvement at 5 h compared to ketones
4a and 4b following intranasal administration of a single dose
(5 µg/animal) (Figure 3). However, the bronchoprotective
activity exhibited by 5b was not maintained at 24 h. On the
other hand, intranasal administration of piperidine 5c (5 µg/
animal), which was 15-fold more potent against M3 compared
to piperazinium salt 5b, significantly inhibited methacholine-
induced bronchoconstriction not only at 15 min (96% inhibition)
and 5 h (94% inhibition) but also at 24 h (40% inhibition). Even
at 48 and 72 h post the single low dose, 5c still exhibited over
25% of bronchoprotection, thus demonstrating excellent in vivo
efficacy and long duration of action.
a (a) 2,6-Dimethoxy-4-polystyrenebenzyloxybenzaldehyde (DMHB-
resin), Na(OAc)3BH, DIEA, 10% of HOAc in NMP, rt; (b) 3-formyl benzoic
acid, DIC, DCE/DMF (1:1), rt; (c) N-Boc piperazine or N-methyl piperazine,
Na(OAc)3BH, Na2SO4, DCE, rt; (d) 3-formylphenyl boronic acid, Pd(PPh3)4,
Cs2CO3, DME, 80 °C; (e) (2S)-2-methylpiperazine, Na(OAc)3BH, Na2SO4,
DCE, rt; (f) TFA, DCE, rt; (g) MeI, CH3CN, rt.
Scheme 3. Synthesis of Piperidine 5ca
In conclusion, a series of highly potent and competitive
biphenyl piperazines was discovered as novel mAChR antago-
nists. Piperidine 5c with respective 500- and 20-fold subtype
selectivity for M3 over M2 and M1 exhibited excellent inhibitory
activity and long duration of action in a bronchoconstriction in
vivo model, demonstrating that the novel inhaled mAChR
antagonists are potentially useful therapeutic agents for the
treatment of COPD and other bronchoconstriction disorders.
a (a) n-BuLi, TBDMSCl, (Boc)2O, THF, rt; (b) 3-bromobenzaldehyde,
Na(OAc)3BH, DCM, rt; (c) n-BuLi, B(OMe)3, THF, 78 °C-rt; (d) 2a,
Pd(PPh3)4, K2CO3, dioxane, H2O, 150 °C; (e) 3-[(1-Boc-piperidin-4-
yl)methyl]benzoic acid, EDC, HOBT, DIEA, CHCl3, rt; (f) TFA, DCM,
0 °C - rt.
Acknowledgment. We thank Bing Wang and Minghui
Wang for NMR, Bill Leavens for HRMS, Qian Jin for LC/MS,
Carl Bennett for purification, and Maite De Los Frailes, Parvathi
Rao, and Lorena A. Kallal for assay support.
N-Boc-piperazine 6a and N-methylpiperazine 6b were prepared
from commercially available 3-bromo-4-fluorobenzylamine (2a)
via loading onto DMHB resin, coupling with 3-formyl benzoic
acid and subsequent reductive amination. Suzuki coupling of
6a with 3-formylphenyl boronic acid and subsequent reductive
amination of the resulting benzaldehyde with optically pure (2S)-
2-methylpiperazine, followed by resin cleavage, produced the
desired compound 5a in excellent yield. N-methylpiperazine 6b
was converted to quaternary ammonium salt 7 via Suzuki
coupling and quaternization of the methyl piperazine. Reductive
amination of 7 with optically pure (2S)-2-methylpiperazine and
subsequent resin cleavage produced the desired piperazinium
salt 5b again in good yield. Synthesis of piperidine 5c is outlined
in Scheme 3. Optically pure (2S)-2-methylpiperazine was
selectively protected via a one-pot two-step procedure. Reductive
amination of the resulting amine with 3-bromobenzaldehyde and
subsequent conversion of the corresponding aryl bromide to
boronic acid afforded compound 8. Suzuki coupling of 8 with
Supporting Information Available: Synthetic procedures,
characterization data, and LC-MS spectra for all compounds.
Procedures for M3, M2, and M1 FLIPR and M3 binding assays and
in vivo bronchoconstriction mouse model. This material is available
free of charge via the Internet at http://pubs.acs.org.
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JM800935U