Design and synthesis of substituted N-(1,3-diphenyl-1H-pyrazol-5-yl) benzamides as positive allosteric modulators of the metabotropic glutamate receptor subtype 5

Article abstract of DOI:10.1016/j.bmcl.2010.12.110

Based on SAR in the alkyne class of mGlu5 receptor negative allosteric modulators and a set of amide-based positive allosteric modulators, optimized substitution of the aryl bring was used to create substituted N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamides.

Full text of DOI:10.1016/j.bmcl.2010.12.110

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2650–2654
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of substituted N-(1,3-diphenyl-1H-pyrazol-5-yl)-
benzamides as positive allosteric modulators of the metabotropic
glutamate receptor subtype 5
Mu-Fa Zou a, Jianjing Cao a, Alice L. Rodriguez b, P. Jeffrey Conn b, Amy Hauck Newman a,
⇑
a Medicinal Chemistry Section, National Institute on Drug Abuse-Intramural Research Program, National Institutes of Health, 333 Cassell Drive Baltimore, MD 21224, United States
b Department of Pharmacology, Vanderbilt Program in Drug Discovery, Vanderbilt University Medical Center, I215D Light Hall, 2215B Garland Avenue,
Nashville, TN 37232-057, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on SAR in the alkyne class of mGlu5 receptor negative allosteric modulators and a set of amide-
based positive allosteric modulators, optimized substitution of the aryl ‘b’ ring was used to create substi-
tuted N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamides. Results from an mGlu5 receptor functional assay,
using calcium fluorescence, revealed varying efficacies and potencies that provide evidence that subtle
changes in compounds within a close structural class can have marked effects on functional activity
including switches in modes of efficacy (i.e., negative to positive allosteric modulation).
Published by Elsevier Ltd.
Received 17 September 2010
Revised 17 December 2010
Accepted 21 December 2010
Available online 28 December 2010
This manuscript is dedicated to Professor
James M. Cook on the occasion of his 65th
birthday
Keywords:
mGluR5
MPEP
CDPPB
Allosteric modulators
Glutamate (L-Glutamic acid) is the major excitatory neurotrans-
mitter within the mammalian central nervous system and regu-
compounds 2-methyl-6-(phenylethynyl)pyridine (MPEP, 1, Fig. 1)
and 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP). How-
ever, cross-target activity and in vivo metabolism may limit further
development of these alkynes as medications.10 In our attempts to
design nonalkynyl mGlu5 receptor antagonists (negative allosteric
modulators), several moderately active diarylamides were discov-
ered (e.g., 2b, in Fig. 1).11 Based on the binding and functional data
for amide-linked derivatives, we previously considered that the
marked differences in functional potency as compared to binding
affinities might be due to different conformational states of the
mGlu5 receptor.11a The discovery of amide-linked positive alloste-
ric modulators of mGlu5 receptor, such as 3-cyano-N-(1,3-
diphenyl-1H-pyrazol-5-yl)benzamide12 (CDPPB; 3, Fig. 1),
provided further support of this idea. Thus, we hypothesized that
the ‘b’ ring in our amide series primarily influences affinity at the
mGlu5 receptor, whereas the ‘a’ ring determines efficacy that leads
to potentiation rather than inhibition of glutamate stimulation.
In this study, we explore (i) the substituent effects at the
6-position of the pyridyl ‘a’ ring of the 3-CN, 5-F phenyl ‘b’ ring
amide (Scheme 1, Table 1), and (ii) structural modifications of
the ‘b’ ring of CDPPB by incorporating previously described11b,c
and optimized substitutions of the ‘b’ ring (Schemes 2 and 3,
Table 2). 1-(2-Chlorophenyl)-3-phenyl-1H-pyrazol-5-yl was used
to replace the ‘a’ ring of CDPPB since a chloro substitution at
lates
glutamate receptors and metabotropic glutamate receptors (mGlu
receptors).1 mGlu receptors are
protein-coupled receptors
a
variety of neuronal activities through ionotropic
G
(GPCRs) that have been cloned, sequenced, and classified into
Group I (mGlu1 and mGlu5 receptors), Group II (mGlu2 and mGlu3
receptors), or Group III (mGlu4, 6, 7, 8 receptors) based on se-
quence homology, pharmacology and 2nd messenger coupling.1
The mGlu5 receptor is primarily located postsynaptically and is
coupled with phospholipase C. Activation of mGlu5 receptor stim-
ulates phospholipase C, which results in the hydrolysis of phospho-
inositide and increases intracellular calcium concentrations.1,2 The
mGlu5 receptors have been targeted in the development of drugs
to treat a variety of neurological and psychiatric illnesses, includ-
ing anxiety, depression, pain, Parkinson’s disease, schizophrenia
and Fragile X syndrome.2–8 Preclinical studies suggest that mGlu5
receptors may also play a role in drug abuse and addiction.9 A large
number of potent noncompetitive antagonists for mGlu5 receptor
have been developed based on the structure of the leading
⇑
Corresponding author.
E-mail address: anewman@intra.nida.nih.gov (A.H. Newman).
0960-894X/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2010.12.110

M.-F. Zou et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2650–2654
2651
a
O
a
O
CN
a
N
H3C
N
N
H3C
N
N
N
b
H
b
H
X
b
R
CN
2a; X = C, R = H
2b; X = C, R = Ph
2c; X = C, R = 3'-FPh
2d; X = C, R = 4'-FPh
2e; X = C, R = 1-naphth
2f; X = N, R = 3,5-diFPh
1, MPEP
3, CDPPB
Figure 1. Structures of mGlu5 receptor allosteric modulators.
R5
R4
R5
R4
O
HOOC
CN
ClOC
CN
N
NH2
CN
a
6a-e
N
N
H
b
F
F
F
5
4
7a; R4=Me, R5=H
7b; R4=Et, R5=H
7c; R4=n-Pr, R5=H
7d; R4=H, R5=Me
7e; R4=n-Bu, R5=H
Scheme 1. Synthesis of (6-substituted pyridine-2-yl)benzamides. Reagents and conditions: (a) SOCl2, reflux, 2 h; (b) 5- or 6-substituted 2-aminopyridines (6a–e), Et3N,
CH2Cl2, rt, 1 h.
Table 1
In vitro data for amide-linked mGlu5 receptor antagonists
R5
O
R1
R4
N
N
H
X
R2
R3
Compd
X
R1
R2
R3
R4
R5
c Log Pf
mGluR5 binding (Ki, nM)a
mGluR5 function IC50 (nM) (Ca+2 flux)
2ad,e
2be
2ce
C
C
C
C
C
N
C
C
C
C
C
—
CN
CN
CN
CN
CN
H
CN
CN
CN
CN
CN
—
H
H
H
H
H
H
H
F
F
F
F
F
Me
Me
Me
Me
Me
Me
Me
Et
n-Pr
H
n-Bu
—
H
H
H
H
H
H
H
H
H
2.1
4.0
4.1
4.1
5.1
3.8
2.2
2.8
3.3
2.2
3.8
3.8
330 20c,d
490 94
13.7 2.54
25.3 1.90
4.57 0.38
640 32.2
98.1 18.9
21.7 5.41
169 21.7
165 21.7
4170 528
1550 146
3.54 1.39
b
Ph
9.8 2.1
b
30FPh
22 5.3
2de
2ee
2fe
40FPh
134 31c
72 12c
1-Naphth
3,5-DiFPh
43 10
7a
7b
7c
7d
H
H
H
H
H
—
65.5 20c
762 177c
325 62c
2831 567c
1054 262c
Me
H
—
7e
1, MPEPc
—
13
1
a
b
c
d
e
f
Data provided by NIMH-PDSP.
Cloned.15
Rat brain (http://pdsp.med.unc.edu).
Compound previously reported.11a
Compound and data reported previously.11b,c
Determined using Sybyl 7.2.3, Tripos Inc.
that position was reported to increase mGlu5 receptor binding
affinity of CDPPB.13
preparation of 1-(2-chlorophenyl)-3-phenyl-5-amino-1H-pyrazol
10 was achieved according to a literature procedure.13 Benzylic
protection of both the OH and COOH groups in compound 11 gave
intermediate 12. Cyanation of 12 with Zn(CN)2 in the presence of
Pd(PPh3)4 catalyst gave intermediate 13. Hydrolysis of 13 under ba-
sic conditions resulted in selective deprotection to give the carbox-
ylic acid 14. The free acid 14 was protected as the ethyl ester 15 via
its acid chloride. Pd/C catalyzed hydrogenation successfully depro-
tected 15, followed by treatment with trifluoromethanesulfonic
Synthesis of the N-(5- or 6-substituted pyridin-2-yl)-3-cyano-5-
fluorobenzamides 7 started from 3-cyano-5-fluorobenzoic acid 4
(Scheme 1). The acid 4 was first converted to the corresponding
acid chloride 5 followed by reaction with 2-aminopyridines 6 to
give the benzamides 7(a–e) in good yields.
Synthesis of substituted N-[1-(2-chlorophenyl)-3-phenyl-1H-
pyrazol-5-yl]benzs amide 19 is shown in Scheme 2. The

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M.-F. Zou et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2650–2654
NH2
CN
a
b
NH2
Cl
CN
N
N
8
9
10
Br
Br
CN
HO
BnO
R2
BnO
BnO
R2
BnO
d
e
h
c
CO2H
CO2Bn
CO2Bn
11
CN
12
13
CN
CN
g
TfO
f
CO2H
CO2Et
CO2Et
14
15
16
CN
CN
O
j
i
CN
R2
N
NH
Cl
N
CO2Et
CO2H
17(a-d)
18(a-d)
19a; R2 = Ph
19b; R2 = 3-FPh
19c; R2 = 4-FPh
19d; R2 = 1-naphth
Scheme 2. Synthesis of substituted N-(1,3-diphenyl-1H-pyrazol-5yl]benzamides. Reagents and conditions: (a) KOBut, CH3CN, C6H6; (b) p-ClC6H4NHNH2, HCl; (c) BnBr, K2CO3,
acetone; (d) Zn(CN)2, Pd(PPh3)4, DMF; (e) NaOH(aq), H2O/EtOH; (f) (i) SOCl2, DMF(cat.), CH2Cl2; (ii) EtOH, Et3N, CH2Cl2; (g) (i) H2, Pd/C, EtOH; (ii) Tf2O, pyridine, CH2Cl2; (h)
R2B(OH)2, Pd(PPh3)4, KFꢀ2H2O, DME/H2O; (i) NaOH(aq),H2O/EtOH; (j) (i) SOCl2, DMF(cat.), CH2Cl2; (ii) 10, Et3N, CH2Cl2.
F
O
O
HO2C
Br
Br
F
a
b
N
H
N
N
H
N
N
N
N
N
N
Cl
Cl
22
20
21
Scheme 3. Synthesis of substituted N-(1,3-diphenyl-1H-pyrazol-5yl]pyridylamide 22. Reagents and conditions: (a) (i) SOCl2, DMF(cat.), CH2Cl2; (ii) 10, Et3N, CH2Cl2, 60%; (b)
3,4-difluorophenylboronic acid, Pd(PPh3)4, Na2CO3, DME/H2O, 80 °C, 78%.
anhydride to give triflate 16, which was reacted with various aryl-
boronic acids under Suzuki-coupling conditions to give the desired
ethyl (3-cyano-4-aryl)benzoates 17(a–d). Hydrolysis of the esters
17(a–d) under basic conditions gave the free acids 18(a–d). Finally,
18(a–d) were converted to the amides 19(a–d) as shown.
Synthesis of substituted N-[1-(2-chlorophenyl)-3-phenyl-1H-
pyrazol-5yl]pyridyl-amides 22 is depicted in Scheme 3. 4-Brom-
opicolinic acid 20 was converted to amide 21 by refluxing with
SOCl2 in CH2Cl2 to form the acid chloride, followed by treatment
of the acid chloride with pyrazolamine 10. Suzuki-coupling of 21
with 3,5-difluorophenyl-boronic acid in the presence of Pd(PPh3)4
catalyst gave the product 22.
as the radioligand in rat brain membranes or HEK293-T cells trans-
fected with cloned rat mGlu5 receptor cDNA. An assay utilizing cal-
cium fluorescence was employed to test functional activity of
compounds by measuring receptor-induced intracellular release
of calcium utilizing a kinetic imaging plate reader that makes
simultaneous measurements of calcium levels in each well of a
384-well plate. Briefly, either vehicle or a test compound was
added to cells expressing rat mGluR5 that were loaded with cal-
cium-sensitive fluorescent dye. After a 2.5 min incubation period,
an EC20 concentration of glutamate was added followed by an
EC80 concentration added 1 min later. The methods for this triple
add protocol, which allows simultaneous testing of antagonists
and potentiators, are described in detail.14 The results of these
in vitro tests for the amides are in Tables 1 and 2.
All the compounds synthesized were assessed in a radioligand
displacement binding assay for mGlu5 receptor, using [3H]MPEP

M.-F. Zou et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2650–2654
2653
Table 2
In vitro data for amide-linked mGlu5 receptor potentiators
O
R1
R2
N
NH
N
X
Cl
Compd
X
R1
R2
c Log Pb
mGluR5 binding (Ki, nM)a
37
2060 520
23
>10,000
344 63
3670 430
mGluR5 function EC50 (nM) (Ca+2 flux)
% Glu max
19a
19b
19c
19d
C
C
C
C
N
CN
CN
CN
CN
H
Ph
7.5
7.7
7.7
8.7
7.5
4.8
9
4140 807
>30,000
2430 481
2170 1000
646 242c
77 15
61.84 3.48
30FPh
40FPh
5
67.98 1.9
50.23 12.62
67.6 7.28
1-Naphth
3,5-DiFPh
22
3, CDPPBd
a
b
c
Data provided by NIMH-PDSP using rat brain (http://pdsp.med.unc.edu).
Determined using Sybyl 7.2.3, Tripos Inc.
Agonist-potentiator.
d
Compound and data reported previously.13
We previously optimized the substitution of the phenyl ‘b’ ring
of a series of N-(6-methylpyridin-2-yl)benzamides and found that
a CN at the 3-position and an aromatic substituent at the 4-posi-
tion resulted in compounds with high binding affinity at the mGlu5
receptor.11 In addition, the 3-CN, 5-F substitution in the amide ‘b’
ring demonstrated high affinity at mGlu5 receptor (e.g., 7a) and
was chosen for exploration of other substitutions in the pyridyl
‘a’ ring. If the substitutions were well-tolerated, the 3-CN, 5-F sub-
stituent would also be utilized to synthesize CDPPB analogues,
with reduced lipophilicity.
Several amides were synthesized with substitutions larger than
methyl (7b, 7c, 7e) or at another position of the pyridyl ring (e.g.,
7d). mGlu5 receptor binding results showed a 5–43-fold decrease
in affinities compared to the parent compound 7a, and functional
test results demonstrated a 8–192-fold decrease. This trend was
especially noted with compound 7d, which has a methyl group
at the 5-position of the pyridyl ‘a’ ring and showed the lowest
potencies in both mGlu5 receptor binding affinity and functional
assays. The results clearly showed that pyridyl ‘a’ ring substitutions
in the amide series, other than the 6-methyl group, were not well-
tolerated at mGlu5 receptor and hence the 3-CN, 5-F substituent
was not chosen for the CDPPB series of compounds.
Figure 2. 3D superimposition of 2b and 19a.
mines efficacy and may be stabilizing a protein conformation that
leads to potentiation rather than inhibition of glutamate
stimulation.
All the CDPPB analogues, except 19d, showed relatively high
mGlu5 receptor binding affinities, especially 19a and 19c that were
99- and 169-fold higher than CDPPB, respectively. However, all the
analogues showed much lower potency in the functional assay
than CDPPB and their relatively high c Log P values render these
compounds poorly water soluble with predicted poor
bioavailability.
This affinity-potency disconnect has been previously observed
and may be explained by differing degrees of positive cooperativity
between the potentiator and glutamate.12a It is also possible these
potentiators do not act entirely through the MPEP binding site and
instead bind to an overlapping or distinct allosteric binding site (in
the case of 19d) as is the case for mGlu5 receptor potentiator
CPPHA.12b
In summary, a series of N-(6-methylpyridin-2-yl)benzamide
analogues (7a–e) were synthesized to explore substitution on the
pyridyl ‘a’ ring of the N-(6-methylpyridin-2-yl)benzamides. In vitro
testing showed that a 6-methyl substituent in the pyridyl ‘a’ ring is
optimal. Incorporating the previously discovered optimal Ar-sub-
stitution into the phenyl ‘b’ ring of CDPPB (19a–d, 22) revealed
varying efficacies and potencies in the calcium fluorescence mGlu5
receptor functional assay suggesting a difference in intrinsic
activity that may reflect distinct binding modes at the protein, as
In the CDPPB analogue series, 19a (R2 = Ph) and 19c (R2 = 4-FPh)
showed high binding affinities, while 19d (R2 = naphth) did not (Ki
>10,000). Moreover, all the CDPPB analogues showed lower mGlu5
receptor binding affinities than their corresponding N-(6-methyl-
pyridin-2-yl)benzamides except the 4-fluorophenyl substituted
compound 19c, which showed a 5.8-fold increase in binding affin-
ity. Several N-(6-methylpyridin-2-yl)benzamide analogs were po-
tent antagonists in the functional assay. However, all the CDPPB
analogs showed lower potency in the functional assay, especially
19b, which was completely inactive. In addition, their intrinsic
activities differed from the benzamide series and included mGluR5
pure potentiators (e.g., 19a, 19c, 19d) and agonist-potentiator (22),
which induced a small response when added alone (EC50 >10 lM)
and potentiated the response to an EC20 concentration of
glutamate.
In superimposing 2b and 19a (Fig. 2), the two molecules fit
quite well on the ‘b’ ring side, and showed similar mGlu5 receptor
binding affinities. On the other hand, there were big differences on
the ‘a’ ring side of the two molecules. These differences may con-
tribute to their different intrinsic activities corresponding to our
initial hypothesis that the ‘b’ ring in the amide series primarily
influences affinity at mGlu5 receptor, whereas the ‘a’ ring deter-
reported recently for
a series of mGlu5 receptor alkynyl
analogues.16

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M.-F. Zou et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2650–2654
6. (a) Marino, M. J.; William, D. L., Jr.; O’Brien, J. A.; Valenti, O.; McDonald, T. P.;
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This work was funded by the NIDA-Intramural Research Pro-
gram and an NRSA Fellowship F32 NS049865 awarded by NINDS
to A.L.R. mGlu5 receptor binding data were provided by the Na-
tional Institute of Mental Health’s Psychoactive Drug Screening
Program, Contract no. NO1MH32004 (NIMH PDSP). We thank Dr.
Tom Keck for helpful comments on an earlier version of this
manuscript.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.12.110.
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