Benzimidazoles as potent and orally active mGlu5 receptor antagonists with an improved pk profile

Article abstract of DOI:10.1021/ml100215b

A focused chemical optimization effort of compound 1 based on metabolite elucidation is described, resulting in 15i, a highly potent and selective mGlu5 receptor antagonist with an improved pharmacokinetic profile compared to 1. Characterization of 15i in

Full text of DOI:10.1021/ml100215b

pubs.acs.org/acsmedchemlett
Benzimidazoles as Potent and Orally Active mGlu5
Receptor Antagonists with an Improved PK Profile
David Carcache,† Ivo Vranesic,‡ Joachim Blanz,† Sandrine Desrayaud,†
Markus Fendt,‡ and Ralf Glatthar*,†
†Global Discovery Chemistry and ‡Neuroscience, Novartis Institutes for BioMedical Research, Basel CH-4002 Switzerland
ABSTRACT A focused chemical optimization effort of compound 1 based on
metabolite elucidation is described, resulting in 15i, a highly potent and selective
mGlu5 receptor antagonist with an improved pharmacokinetic profile compared
to 1. Characterization of 15i in vivo in the fear-potentiated startle (FPS) paradigm
revealed a robust reduction of conditioned fear behavior. This effect nicely
correlates with the rat brain pharmacokinetics.
KEYWORDS mGlu5, benzimidazoles, pharmacokinetics
-Glutamate serves as the neurotransmitter at the ma-
jority of excitatory synapses in the mammalian central
nervous system (CNS) and as such is involved in a
We recently disclosed the piperidyl amide-based selec-
tive mGluR5 antagonist 1 (Figure 1) that shows robust
anxiolytic-like activity in different animal models of fear
and anxiety.16
L
variety of physiological and pathophysiological functions. The
existence of neuromodulatory glutamate receptors, called
metabotropic glutamate receptors (mGluRs), provides a me-
chanism by which glutamate can modulate cell excitability and
synaptic transmission. Metabotropic glutamate receptors are
G-protein-coupled receptors linked to multiple second messen-
ger systems modulating for example ion channel functions in
the neurons.1-3 Eight different types of metabotropic gluta-
mate receptors are known (mGluR1-mGluR8), which are
divided into three groups according to their sequence
homology, effector coupling and pharmacology. Group I mGlu
receptors (mGluR1 and mGluR5) are positively coupled to
phospholipase C; group II mGlu receptors (mGluR2 and
mGluR3) and group III mGlu receptors (mGluR4, mGluR6,
mGluR7 and mGluR8) are negatively coupled to adenylate
cyclase. mGluRs are broadly distributed throughout the CNS
and are specifically localized at discrete synaptic and extra-
synaptic sites.
mGlu5 receptor expression is especially high in brain
regions thought to mediate and modulate emotions like fear
and anxiety (amygdala, prefrontal cortex, hippocampus and
basal ganglia).4-6 mGlu5 receptors are predominantly post-
synaptically located and have been shown to play a role in
regulating glutamatergic transmission via potentiation of
NMDA receptor activity. Excessive glutamatergic transmis-
sion has been proposed to play a role in psychiatric diseases
like anxiety disorders and depression.7,8 Additionally, mGlu5
receptor antagonism has been proposed as a potential target
for the treatment of pain,9 obesity,10 Parkinson's disease,11
drug abuse,12 migraine,13 gastroesophageal reflux disease
(GERD)14 and fragile X syndrome (FXS).15 Therefore, the
development of potent and selective mGlu5 receptor antago-
nists as potential therapeutic agents has been the focus of
significant research in our laboratories.
The moderate metabolic stability of compound 1, based on
in vivo plasma clearance in rat and in vitro microsomal
clearance data in rat and human required further optimization.
In the present letter, the chemical derivation effort of 1 to
an improved compound with a superior pharmacokinetic
profile and increased potency in vivo will be discussed.
As a result of the moderate metabolic stability of 1, the
biotransformation of this compound was studied in human
and rat liver microsomes as well as in vivo in rat after
intravenous or oral administration (Scheme 1).
In microsomal incubations, six phase I metabolites were
found and characterized by means of mass spectrometry.
Metabolites m1, m2 and m3 were oxidation products of the
2-ethylpiperidin-1-yl moiety. Further oxidation and ring-
opening led to metabolite m4, whereas oxidation of the
methyl group in the 6-methylpyridin-3-ylamino moiety led
to metabolite m5. H/D exchangeindicated N-oxidation at the
5-chloro-6-(6-methylpyridin-3-ylamino)pyridin-3-yl moiety
in the case of metabolite m6. In vivo, the metabolites
m1-m3 were also detected in plasma besides the parent
compound.
The formation of phase II metabolites was not observed in
any of the in vitro or in vivo samples. The abundances of 1
and its metabolites in plasma suggested that the elimination
of 1 may predominantly occur via oxidation of the 2-ethyl-
piperidin-1-yl moiety (m1-m3), with major metabolite
being m1 in vivo and m5 in vitro.18
With the piperidyl moiety identified as the major site of
metabolism in compound 1, the initial derivation efforts
Received Date: September 16, 2010
Accepted Date: October 21, 2010
Published on Web Date: November 02, 2010
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Scheme 2a
Figure 1. mGluR5 antagonist. ahmGluR5: Ca2þ flux using glutamate
and phosphoinositol turnover using quisqualate as agonist;17 data
are geometric means of n g 2, IC50 (nM). bSolubility (mg/L) measured
c
in a dissolution template potentiometric titration approach. HLM/
RLM: human and rat liver microsomal intrinsic clearance ( μL/min/mg
e
microsomal protein). dn= 3 Sprague-Dawley rats/group. MRT =
mean residence time (h).
Scheme 1. Proposed Chemical Structures of Metabolites of 1
Detected after Incubation with Rat and Human Liver Microsomes
as Well as in Vivo in Rat Plasma
a Reagents and conditions: for X = Br, (a) NaH, THF, 70 °C, 14 h; (c) BuLi,
B(OiPr)3, THF, -78 °C to rt, 3 h; (e) Pd(PPh3)4, Na2CO3, C6H6, MeOH,
120 °C, 40 min; for X = CN, (b) Pd(OAc)2, rac-2,20-bis(diphenylphosphino)-
1,10-binaphthyl (rac-BINAP), K2CO3, toluene, 100 °C, 16 h; (d) NaOMe,
NH4Cl, MeOH, 65 °C, 2 h; (f) (i) NH4OH, THF, H2O, 80 °C, 5 h-18 h; (ii) R1I,
K2CO3, DMF, rt, 18 h.
aimed at exploring bioisosteric replacements for the left-
hand fragment in 1. Compound 2 was used as a reference for
the optimization of this series, which contains the central
and right-hand-side fragments that led to most potent
derivatives described in our preceding letter, combined with
the piperidyl amide left-hand-side fragment of 1.16 Several
5-ring heterocyclic bioisosteres such as oxadiazole, triazole,
and isoxazole were investigated and shown to be inactive.
Only the imidazole system proved to be a suitable bioisos-
teric replacemement for the piperidyl amide, albeit with
reduced potency.
Scheme 3a
The imidazole derivatives were prepared by the methods
depicted in Scheme 2, whereas the benzimidazole analo-
gues were synthesized according to Scheme 3. The synthesis
started by reacting pyridine 3 with 4-chloroaniline to afford
4, followed by boronic acid formation to give 5. Suzuki
coupling with 6 provided 7a.19 The synthesis of 7b-e
commenced with a Buchwald reaction between pyridine 8
and 4-chloroaniline, leading to 9, followed by amination of
the nitrile group to give the amidine 10. Condensation of 10
with R-chloroketones and subsequent N-alkylation of the
imidazole ring afforded 7b-e.
The preparation of the benzimidazole derivatives started
with the 2-chloronicotinic acid 11 or ester 12, which were
reacted with an aromatic amine to provided 13a and 14. In
the case of 14, the ester group was first hydrolyzed to 13b,
and the carboxylic acid intermediates were then engaged in
condensation reactions with phenylenediamine reagents to
give rise to the benzimidazole compounds 15a-e, 16 and
17. For compounds 16 and 17 (W = Cl), final N-alkylation,
followed by separation of the regioisomers, led to 15f-i.
The bicyclic derivative 7a, which can be seen as a cyclized
version of compound 2, served as a lead for our optimization.
The SAR evaluation around the imidazole moiety started with
a Reagents and conditions: for R = H, (a) AcOH, 150 °C, 75 min; for R =
Me, (b) Pd(dba)3, rac-BINAP, K2CO3, toluene, 160 °C, 2 h; (c) 1 M
NaOH, MeOH, rt, 1 h; (d) Polyphosphoric acid (PPA), 180-210 °C,
0.1-18 h; (e) R1I, NaH, DMF, rt, o/n.
structurally simpler derivative, where the fused 6-membered
ring of 7a was opened. The 1,5-dimethylimidazole derivative
7b displayed only marginal activity, whereas the 1,4-dimethyl
analogue 7c exhibited micromolar potency. Exploration of the
N-alkyl substituent permitted identification of the potent
n-propyl derivative 7d. Further exploration of the 4- and
5-positions led to compound 7e, and subsequent bridging of
those two positions into a benzimidazole moiety resulted in
15a, the most active derivatives of this subset (Tabl e 1 ).
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Table 1. Bioisosteric Replacement of the Piperidyl Amide Frag-
ment: In Vitro hmGluR5 Antagonism
Table 2. Optimization of Benzimidazole Fragment: In Vitro hmGluR5
Antagonism
compd
R1
R3
R2
IC50 [μM]a
compd R1 4-W 5-W 6-W 7-W IC50 [μM]a HLMb RLMb
2
0.064
1.2
7a
7b
7c
7d
7e
15a
-(CH2)4-
H
15b
15c
15d
15e
15f
Pr
Et
H
H
H
H
Cl
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
Cl
0.10
0.11
0.36
0.11
0.20
83
120
347
55
158
118
279
826
69
Me
Me
Pr
Me
H
H
48% @ 10
2.9
Me
Me
Me
Bu
iBu
Pr
Pr
Pr
Pr
H
0.23
Pr
Me
0.25
74
Pr
-phenyl-b
0.18
15g
15h
15i
>0.5
400
56
34
a hmGluR5 Ca2þ flux7 using glutamate as agonist; data are geometric
0.13
33
means of n g 2. b Benzimidazole.
0.024
83
112
a hmGluR5 Ca2þ flux7 using glutamate as agonist; data are geometric
means of n g 2. b HLM: human liver microsomes. RLM: rat liver
microsomes. CLint (intrinsic clearance) reported in μL/min/mg protein.
Having identified the benzimidazole moiety as a suitable
bioisostere for the former piperidyl amide, the 4-chloroani-
line right-hand fragment was exchanged for the less lipophi-
lic and more soluble 3-amino-6-methylpyridyl group as in 1,
leading to compound 15b.
Table 3. Pharmacokinetic Parametersa of 15ib and 1c
Further exploration of the SAR around the benzimidazole
moiety revealed that structural variation of the N-substituent
was tolerated with minimal impact on the potency, but
significant difference in terms of human microsomal stabi-
lity (15c-e). Subsequently, a “chloro screen” on the benzi-
midazole moiety (15f-i) permitted identification of the
7-chlorobenzimidazole 15i as the most potent analogue of
this series (Table 2). Due to its improved potency, 15i was
selected for further profiling in vitro and in vivo despite a
similarly high intrinsic clearance in liver microsomes com-
pared to original compound 1.
15i
1
plasmaCL (mL/min/kg)
MRT (h)
15
37
2.2
1.9
0.5
60
0.9
0.9
0.25
41
t
1/2 (h)
max (h)
T
Cmaxd (pmol/mL/μmol/kg)
AUCpod (pmol h/mL/μmol/kg)
F (%)
449
41
241
54
brain/plasma-AUCpo ratio
1.67
1.36
a n = 3 Sprague-Dawley rats/group. b 3.6 μmol/kg po, 2.4 μmol/kg
In vitro radioligand displacement assays utilizing [3H]-
ABP68820 showed that 15i is a high-affinity ligand at the
previously characterized allosteric binding site located in the
membrane-spanning region of mGlu5 receptors21 with a Ki
at the human recombinant receptor of 2.4 nM.22 In addition,
15i showed a high degree of selectivity in functional assays
over representatives of group I, II and III metabotropic
glutamate receptor subtypes (IC50 > 10 μM for hmGluR1,
-2, -7) and ionotropic glutamate receptors (IC50>10 μM).23
Single-dose pharmacokinetic studies of 15i in rats showed
that this compound is superior to 1 in terms of lower plasma
clearance, prolonged mean residence time and apparent
elimination half-life. This could not be expected based on the
similar intrinsic clearance of both compounds in liver micro-
somes, but might be explained by the higher plasma protein
binding of 15i,24 resulting in a protective effect in vivo. Brain
penetration and bioavailability were similar (Table 3).
As in a preceding study16 with 1, we assessed efficacy and
potency of 15i in vivo by determining the compound's effect
on the expression of fear-potentiated startle (FPS) in rats.25,26
In the present study, rats were orally treated with 0.3, 1, and
3 mg/kg of 15i, or vehicle 60 min prior to the behavioral
test (group size n = 10). The FPS response was reduced in a
iv. c 30 μmol/kg po, 10 μmol/kg iv. d Dose-normalized value.
Figure 2. (A) Expression of fear-potentiated and baseline startle
response (0.3-3 mg/kg compound 15i). Mean startle magnitudes
and SEMs of startle stimulus alone and CS-startle stimulus trials,
respectively, as well as the difference. Multifactorial ANOVA: inter-
action treatment ꢀ trial type: F3,36=7.49, p < 0.001, p=0.001;
**p < 0.01 (Dunnett post hoc test vs 0 mg/kg). (B) Brain concen-
tration (mean ( SEM) of compound 15i in the different treatment
groups as well as the mean percent startle difference ((SEM).
Compound 15i brain levels are negatively correlated with the
expression of fear-potentiated startle. For experimental details see
the Supporting Information.
dose-dependent manner; the effects caused by 1 and 3 mg/kg
were statistically significant vs the vehicle control (Figure 2). The
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minimal effective dose of 15i is thus considered to be 1 mg/kg po
in this paradigm, whereas we found 3 mg/kg for 1.25,26
(7)
Phan, K. L.; Fitzgerald, D. A.; Cortese, B. M.; Seraji-Bozorgzad,
N.; Tancer, M. E.; Moore, G. J. Anterior cingulate neurochem-
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The amount of FPS reduction induced by 15i was posi-
tively correlated with the compound's plasma and brain
concentrations, measured in a subset (n = 4) of the animals
that underwent the FPS test (samples collected at 90 min
postdose). Both plasma and brain concentrations were
proportional to the applied doses (0.3 to 3 mg/kg); the
brain/plasma ratio varied between 1.2 and 2.0.
In conclusion, biotransformation investigations with com-
pound 1 identified structural liabilities that guided a focused
chemical optimization effort resulting in 15i, a selective,
orally bioavailable mGluR5 antagonist of similar potency
and affinity in vitro and superior metabolic stability com-
pared to 1. In vivo characterization of 15i in the FPS model
revealed a robust anxiolytic-like effect with a minimal effec-
tive dose of 1 mg/kg po and positive correlation with brain
and plasma exposures. In view of this promising profile,
compound 15i was considered for further development.
(8)
(9)
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King, C.; Tehrani, L.; Cosford, N. D. P.; Anderson, J.; Varney,
M. A.; Strack, A. M. Metabotropic Glutamate Receptor mGlu5
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SUPPORTING INFORMATION AVAILABLE Experimental
details. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author: *E-mail: ralf.glatthar@novartis.com.
ACKNOWLEDGMENT The excellent technical assistance of
€
Christian Boesch, Ralf Boesch, Hugo Burki, Stefan Imobersteg, Martin
Gunzenhauser, Nicole Reymann, Patrick Seitzer, Christine Stierlin,
Peter Wipfli, Francis Risser, Pierrette Guntz and Valerie Cordier,
Philippe Ramstein, Werner Gertsch is gratefully acknowledged.
(15) Bear, M. F.; Huber, K. M.; Warren, S. T. The mGluR theory of
fragile X mental retardation. Trends Neurosci. 2004, 27, 370.
(16) Spanka, C.; Glatthar, R.; Desrayaud, S.; Fendt, M.; Orain, D.;
Troxler, T.; Vranesic, I. Piperidyl amides as novel, potent and
orally active mGlu5 receptor antagonists with anxiolytic-like
activity. Bioorg. Med. Chem. Lett. 2010, 20, 184.
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