Substituent effects of N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamides on positive allosteric modulation of the metabotropic glutamate-5 receptor in rat cortical astrocytes

Article abstract of DOI:10.1021/jm051252j

CDPPB [3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide] was recently described as the first centrally active, positive allosteric modulator of rat and human metabotropic glutamate receptor (mGluR) mGluR₅ subtype. We explored the structural requirements for potentiation of glutamate-induced calcium release in naturally expressed mGluR5 in cultured rat astrocytes and increasing affinity for the allosteric antagonist binding site by evaluating 50 analogues of CDPPB. In the fluorometric calcium assay, CDPPB exhibited an EC₅₀ value of 77 ± 15 nM in potentiating mGluR ₅-mediated responses in cortical astrocytes and a K_i value of 3760 ± 430 nM in displacing [³H]methoxyPEPy binding in membranes of cultured HEK-293 cells expressing rat mGluR₅. The structure-activity relationships showed that electronegative aromatic substituents in the para-position of the benzamide moiety of CDPPB increase potency. Both binding and functional activities were further increased with a halogen atom in the ortho-position of the 1-phenyl ring. These effects of substitution do not match those of either aromatic ring of MPEP [2-methyl-6-(phenylethynyl)-pyridine] for the antagonist allosteric binding site. Combination of the optimal substituents and aromatic positions resulted in 4-nitro-N-(1-(2-fluorophenyl)-3-phenyl-1H-pyrazol-5-yl)benzamide (VU-1545) showing K_i = 156 ± 29 nM and EC₅₀ = 9.6 ± 1.9 nM in the binding and functional assays, respectively.

Full text of DOI:10.1021/jm051252j

3332
J. Med. Chem. 2006, 49, 3332-3344
Substituent Effects of N-(1,3-Diphenyl-1H-pyrazol-5-yl)benzamides on Positive Allosteric
Modulation of the Metabotropic Glutamate-5 Receptor in Rat Cortical Astrocytes
Tomas de Paulis,*,† Kamondanai Hemstapat,† Yelin Chen,† Yongqin Zhang,† Samir Saleh,† David Alagille,‡
Ronald M. Baldwin,‡ Gilles D. Tamagnan,‡ and P. Jeffrey Conn†,§
Department of Pharmacology, Vanderbilt UniVersity School of Medicine, NashVille, Tennessee 37232, Department of Psychiatry,
Yale UniVersity and Veterans Administration, West HaVen, Connecticut 06516, and Program in Drug DiscoVery,
Vanderbilt Institute for Chemical Biology, NashVille, Tennessee 37232
ReceiVed December 15, 2005
CDPPB [3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide] was recently described as the first centrally
active, positive allosteric modulator of rat and human metabotropic glutamate receptor (mGluR) mGluR5
subtype. We explored the structural requirements for potentiation of glutamate-induced calcium release in
naturally expressed mGluR5 in cultured rat astrocytes and increasing affinity for the allosteric antagonist
binding site by evaluating 50 analogues of CDPPB. In the fluorometric calcium assay, CDPPB exhibited an
EC50 value of 77 ( 15 nM in potentiating mGluR5-mediated responses in cortical astrocytes and a Ki value
of 3760 ( 430 nM in displacing [3H]methoxyPEPy binding in membranes of cultured HEK-293 cells
expressing rat mGluR5. The structure-activity relationships showed that electronegative aromatic substituents
in the para-position of the benzamide moiety of CDPPB increase potency. Both binding and functional
activities were further increased with a halogen atom in the ortho-position of the 1-phenyl ring. These
effects of substitution do not match those of either aromatic ring of MPEP [2-methyl-6-(phenylethynyl)-
pyridine] for the antagonist allosteric binding site. Combination of the optimal substituents and aromatic
positions resulted in 4-nitro-N-(1-(2-fluorophenyl)-3-phenyl-1H-pyrazol-5-yl)benzamide (VU-1545) showing
Ki ) 156 ( 29 nM and EC50 ) 9.6 ( 1.9 nM in the binding and functional assays, respectively.
Introduction
Metabotropic glutamate receptors (mGluRs) play an important
role in controlling neuronal excitability and synaptic transmis-
sion in the central nervous system (CNS) of the mammalian
brain.1 As a result, mGluRs are potential targets for therapeutic
intervention in a variety of neurological and psychiatric ill-
nesses.1,2 In particular, agonists at the mGluR5 subtype could
be useful as antipsychotic agents because of their ability to
facilitate NMDA receptor function without causing NMDA-
mediated excitoxicity.3,4 Direct acting orthosteric mGluR5
agonists, such as 3,5-DHPG [3,5-dihydroxyglycine], have poor
subtype selectivity and may cause unwanted side effects.5 In
contrast, indirect agonists that act via allosteric potentiation of
receptor function are predicted to be less problematic.4 In
recombinant systems, these agents do not activate mGluR5 when
added alone but shift the concentration-response for glutamate
to the left. Furthermore, electrophysiological studies have shown
Figure 1. Structures of the positive allosteric modulators ADX-47273
that such agents cause an increase in the effect of threshold
(1) and CDPPB (2) and the negative modulators MPEP(3), pyrazole
4a, and pyrrole 4b of mGluR5.
agonist concentrations in neurons in the hippocampus and
subthalamic nucleus without inducing an effect by themselves.6
glutamate binding site but each potentiates the response of
mGluR5 to activation by glutamate.
Discovery of selective positive allosteric modulators of mGluR5
raises the exiting possibility that such compounds will provide
a novel approach for developing therapeutic agents that activate
this glutamate receptor subtype. We have now reported discov-
ery of three distinct series of allosteric potentiators of mGluR5.6-10
A fourth structural series with similar properties, represented
by ADX-47273 [(S)-1-(4-fluorophenyl)-3-(3-(4-fluorophenyl)-
[1,2,4]oxadiazol-5-yl)piperidine, 1], was recently presented.11,12
None of these compounds has intrinsic activity at the orthosteric
To date, one of the promising positive allosteric modulators
of mGluR5 is CDPPB [3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-
yl)benzamide, 2]. This compound is centrally active and induces
an 8-fold maximal potentiation of glutamate-induced calcium
release in cultured CHO cells expressing human mGluR5 with
an EC50 value of 27 nM.9 Further, CDPPB reverses amphetamine-
induced locomotor activity and impairment of acoustic prepulse
inhibition in the rat,10 two behavioral models in which currently
used antipsychotic agents are effective.2-4 However, the phar-
macological and physicochemical properties of CDPPB are
probably not optimal, considering that CDPPB was selected
from a limited number of structural analogues.9,13 In addition,
* Corresponding author. Phone: 615-390-6977. E-mail: tomas.depaulis@
vanderbilt.edu.
† Vanderbilt University School of Medicine.
‡ Yale University and Veterans Administration.
§ Vanderbilt Institute for Chemical Biology.
10.1021/jm051252j CCC: $33.50 © 2006 American Chemical Society
Published on Web 05/02/2006

PositiVe Allosteric Modulation of mGluR5
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11 3333
Figure 2. Structures of compounds 20-50 (Table 1) and byproduct A (51) and B (52) (Table 2).
Scheme 1
CDPPB lacks ideal selectivity because it is a weak antagonist
at the mGluR8 receptor.10 CDPPB exhibits weak affinity for
the antagonist MPEP [2-methyl-6-(phenylethynyl)pyridine, 3]
allosteric binding site of mGluR5, as evidenced by inhibiting
the binding of [3H]methoxyPEPy14 [3-methoxy-5-(pyridin-2-
ylethynyl)pyridine] with a Ki of 2.7 µM.10 To take advantage
of the structure-activity relationships of the MPEP series, we
wanted to establish whether the aromatic rings of CDPPB could
mimic the aryl groups of MPEP and its congeners. It has already
been shown that five-membered heterocyclic rings can substitute
for the triple bond of MPEP.15 For example, the 1,3-diarylpyra-
zole 4a (Figure 1) binds to the MPEP binding site with an
affinity of 530 nM.16 Replacing the pyrazole with a pyrrole ring
and adding a m-fluorine atom to become compound 4b increased
affinity 100-fold.17 Other allosteric mGluR5 antagonists have
demonstrated that allosteric action can be altered from negative
to positive modulation with aromatic substitution.7 If the relevant
binding site of CDPPB is identical to that of MPEP, it would
be possible to control the type of modulation by simply applying
the appropriate structural modifications. For example, shifting
the 6-methyl group of MPEP to the adjacent 5-position
(5-MPEP)18 completely abolishes the negative allosteric modu-
lation and creates a neutral ligand, while retaining binding
affinity for the MPEP site.19 Here, we present the results of
Scheme 2
structural modifications of CDPPB in the search for a more
potent candidate for future development into a pharmacological
tool or therapeutic agent. Part of this study was presented at
the Fifth International Symposium on the Metabotropic Glutamate
Receptor (Taormina, Italy, Sept 18-23, 2005).20
Chemistry
We first investigated the effect of aromatic substituents in
the benzamide moiety of CDPPB (Figure 2). In the previous
series, Lindsley et al.9 demonstrated a detrimental effect of
having a substituent in the ortho-position of the benzamide
phenyl ring and little variation in activity with substituents in
the meta- or para-position. Alkyl groups and particularly halogen
atoms, such as fluoro or chloro atoms, in either the aromatic 3-
or 4-positions of the benzamide seemed to cause a 10-fold
increase in activity.9 We expanded the scope of substituents in
these positions to include groups with both strong electroneg-
ative and electropositive properties. Reacting 5-amino-1,3-
phenyl-1H-pyrazole with the appropriately substituted benzoic
acids via their acid chlorides gave the desired benzamides 20-
29 (Scheme 1). Acid chlorides that were unavailable were
prepared from the corresponding carboxylic acids using thionyl
chloride. The corresponding 4-carboxamide and 4-carboxylic
acid derivatives 30 and 31 were prepared by using terephthaloyl
dichloride in excess, followed by quenching of the monosub-
stituted 4-chlorocarboxybenzamide with ammonium hydroxide
or methanol, respectively (Scheme 2). Basic hydrolysis of the
resulting methyl ester gave compound 31. The facile preparation
of the pyrazole scaffold21 allowed us to explore the substitution
effect on all three phenyl groups of CDPPB. Aryl-substituted
5-amino-1,3-diphenyl-1H-pyrazoles (37-50), having a halogen
atom, methoxy, or cyano group in one of the phenyl groups of
CDPPB, were prepared by reacting substituted phenylhydra-
zines22 with substituted 1-aminocinnamonitriles according to the
method of Grandberg21 (Scheme 1). The latter were obtained
by reacting the appropriate arylnitriles in base-catalyzed con-
densation with acetonitrile.23 Synthesis of 5-amino-1-(2-pyridyl)-
3-phenyl-1H-pyrazole (16) has been reported starting from
benzoylacetonitrile24 in ethanol or hydrochloric acid25 or from
3-phenylpropynylnitrile.26 We obtained compound 16 in good
yield using the method of Grandberg.21
In the case of benzamides with a cyano or nitro substituent,
the amide condensation resulted in a mixture of three products,
one of which was the desired compound. Isolation and

3334 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11
de Paulis et al.
Scheme 3
Scheme 4
Figure 3. Structures of compounds 53-60, 64-66, 69-71, 73, and
74-76 (Table 2).
characterization of the individual byproducts showed that they
contained two benzoyl moieties in asymmetrical (A) and
symmetrical (B) positions, respectively, as examplified by the
4-cyano compounds 51 and 52 (Figure 2). The presence of
dimeric byproducts A and B in the reaction mixture was detected
by proton NMR as the chemical shift of the pyrazole 4-CH
singlet appearing at 6.6-6.7 ppm compared to 7.1-7.2 ppm
for the monosubstituted compounds. The formation of byproduct
A is the result of a second benzoyl group reacting with the
nitrogen atom in the pyrazole 2-position, followed by rear-
rangement of the pyrazole double bonds. This side reaction has
been observed in the acylation of 5-amino-1-phenyl-3-methyl-
1H-pyrazole.27 Formation of byproduct B is the result of a
second benzoyl group replacing the amide hydrogen atom. A
similar diacylation reaction in substituted pyrazoles has been
seen in the acylation with 2-nitrobenzoyl chloride.28 Use of less
than 1 equiv of acid chloride resulted in difficulties removing
unreacted diphenylpyrazole from the product. The formation
of byproduct B could be avoided by using the corresponding
benzoic acids, instead of acid chlorides and 1,1′-cyclohexyl-
carbodiimide as water scavenger, but byproduct A was still
formed, and overall yields were not improved.
Compounds 53-60 (Figure 3) were prepared from 5-amino-
1,3-diphenyl-1H-pyrazole by the same methods. Permutation
of the pyrazole nitrogen atoms was explored using 4-amino-
1,3-diphenyl-1H-pyrazole (63) and 5-amino-1,4-diphenyl-1H-
pyrazole (68) as starting materials. These pyrazoles were
obtained by reacting phenylhydrazine with 2′-phthalimidoac-
etophenone29 (Scheme 3) or 1-formyl-1-phenylacetonitrile30
(Scheme 4), respectively. The latter reaction has erroneously
been reported to result in 3-amino-1,4-diphenyl-1H-pyrazole
with melting point 137-138 °C.30 In our hands, the resulting
compound was identified as 68 with melting point 139-140
°C,31,32 and the structure was confirmed by crystal crystal-
lographic data (see Supporting Information). Condensation of
these aminopyrazoles with substituted benzoyl chlorides as
Scheme 5
described for CDPPB gave the corresponding substituted
N-pyrazolylbenzamides 64-66 and 69-71 in good yields. The
triazolylpyrazole 73 was prepared by reacting phenylacetylene
with the corresponding 5-azidopyrazole 72 (Scheme 5A). An
annelated phenylpyrazole was explored using 3-amino-2-phe-
nylimidazo[1,2-a]pyridine (74) (Scheme 5B), easily obtained
from 2-aminopyridine and 2′-bromoacetophenone using the
Tschitschibabin reaction as described for the corresponding 2-(4-
methylsulfonylphenyl) derivative,33 followed by nitrosation.34
Condensation with the appropriate benzoyl chloride gave
compounds 7535 and 76, respectively.
Pharmacology
The results of displacement of the potent MPEP analogue
[3H]methoxyPEPy13 binding to membranes of cultured HEK-

PositiVe Allosteric Modulation of mGluR5
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11 3335
Table 1. Displacement of [3H]MethoxyPEPy Binding in HEK Cells, Expressing Rat mGluR5 Receptors, and Potentiation of Glutamate-Induced Calcium
Flux in Cultured Rat Astrocytes
compound
R1
R2
R3
3-CN
Ki (nM)a
EC50 (nM)b
potent.c
log Pd
CDPPB (2)
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
3760 ( 430 (3)e
4180 ( 200 (3)
1290 ( 130 (3)
1650 ( 310 (5)
2220 ( 310 (4)
780 ( 230 (3)
420 ( 160 (3)
265 ( 63 (3)
990 ( 130 (3)
1560 ( 210 (4)
250 ( 85 (3)
>10000
77 ( 15 (7)
203 ( 35 (4)
102 ( 21 (4)
228 ( 39 (7)
39 ( 18 (5)
64 ( 18 (7)
54 ( 9 (6)
4.0
4.4
3.8
4.0
5.1
5.0
4.2
4.6
3.7
5.2
5.0
5.2
4.0
3.9
5.0
4.8
4.1
3.9
5.3
6.5
5.3
4.6
5.0
5.0
4.3
3.8
3.9
4.1
4.7
6.2
4.0
5.0
4.4
4.7
4.8
4.9
3.8
6.0
4.9
5.2
5.2
5.3
5.6
3.1
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
H
3-OCH3
3-CF3
3-NO2
4-CH3
4-OCH3
4-OCF3
4-CF3
4-CN
4-NO2
4-CONH2
4-COOH
3,5-NO2
3,4-OCH3
3,5-OCH3
3,4-CH3
3,4-Cl
H
H
H
H
H
H
H
H
H
18 ( 6 (6)
38 ( 11 (6)
75 ( 13 (7)
10.7 ( 4.7 (6)
inactive
>10000
inactive
NTe
65 ( 27 (3)
3530 ( 1310 (3)
2040 ( 350 (3)
20 ( 4 (7)
3.3
3.7
5.9
4.0
5.2
5.2
5.9
3.4
3.9
4.3
>10000
>10000
430 ( 80 (4)
160 ( 40 (4)
>10000
3200 ( 490 (3)
2700 ( 430 (3)
500 ( 210 (3)
2600 ( 700 (3)
>10000
>10000
>10000
2100 ( 520 (3)
160 ( 30 (3)
110 ( 20 (3)
120 ( 30 (5)
230 ( 80 (3)
230 ( 70 (4)
NTg
13 ( 5 (6)
2-Cl
3-OCH3
2290 ( 620 (2)
600 ( 100 (3)
104 ( 12 (3)
62 ( 8 (3)
H
H
H
H
H
H
H
H
H
H
H
H
2-F
2-Cl
2-Br
(2-py)f
3-Br
3-CN
3-OCH3
2-F
200 ( 67 (3)
inactive
400 ( 150 (3)
117 ( 12 (4)
570 ( 34 (4)
9.6 ( 1.9 (3)
40 ( 12 (3)
61 ( 8 (3)
68 ( 24 (3)
50 ( 10 (3)
170 ( 60 (2)
4.2
5.4
6.7
4.8
3.3
4.4
3.9
3.5
3.8
4-NO2
4-NO2
4-NO2
3-NO2
3-CN
2-Cl
2-Br
2-Br
2-Br
ADX-47273 (1)
a Inhibition constant for displacing [3H]methoxyPEPy binding and standard error of the mean (SEM) from n number of experiments. b Effective concentration
and SEM to reach 50% of the maximum potentiation from 200 nM glutamate. c Maximum increase in glutamate-induced calcium release. d Calculated
lipophilicity using SciLogP v1.5 (Scivision, Burlington, MA). e Not tested. f N-(1-(2-Pyridinyl)-3-phenyl-1H-pyrazol-1-yl)benzamide. g Reported IC50 value
3700 ( 400 nM for displacing [3H]MPEP binding in rat cortical membranes.11
Table 2. Displacement of [3H]MethoxyPEPy Binding in HEK Cells, Expressing Rat mGluR5 Receptors, and Potentiation of Glutamate-Induced Calcium
Flux in Cultured Rat Astrocytes
compd
RCONH-pyrazolea
Ki (nM)b
EC50 (nM)c
potent.d
log Pe
51
52
53
54
55
56
57
58
59
60
64
65
66
69
70
71
73
75
76
4-CN-phenyl dimer A
4-CN-phenyl dimer B
2-NO2-phenyl
3-pyridyl-4-CH3
4-pyridyl
cyclohex-1-enyl
cyclohexyl
cyclopentyl
5230 ( 2000 (3)f
3050 ( 1480 (2)
>10000
43 ( 6 (4)
3.6
3.3
7.4
6.8
5.9
5.0
4.8
4.8
4.2
4.3
4.3
4.0
4.0
3.6
2.9
3.8
3.6
4.3
7.9
3.0
4.3
340 ( 70 (3)
>10000
3480 ( 3 (2)
>10000
280 ( 100 (3)
2500 ( 250 (3)
3300 ( 2200 (3)
>10000
3410 ( 1240 (3)
350 (1)
560 ( 46 (3)
>10000
>10000
>10000
antagonist
antagonist
>10000
2600 ( 290 (3)
antagonist
antagonist
4.4
4.0
3.8
4470 ( 910 (4)
10140 (1)
11600 ( 3400 (4)
3.7
1.8
3.5
cyclopentylmethyl
4-NO2-cinnamoyl
phenylg
>10000
1810 ( 470 (3)
>10000
3-CN-phenylg
4-NO2-phenylg
phenylh
>10000
>10000
1170 ( 440 (4)
1200 ( 900 (3)
>10000
3-CN-phenylh
4-NO2-phenylh
5880 ( 4400 (3)
3920 ( 350 (2)
1990 ( 200 (4)
2.7
phenyli
3-CN-phenyli
a N-(1,3-Diphenyl-1H-pyrazol-5-yl)benzamide unless otherwise noted. b Inhibition constant for displacing [3H]methoxyPEPy binding and standard error
of the mean (SEM) from n number of experiments. c Effective concentration and SEM to reach 50% of the maximum potentiation from 200 nM glutamate.
d Maximum increase in glutamate-induced calcium release. e Calculated lipophilicity using SciLogP v1.5 (Scivision, Burlington, MA). f Number of independent
experiments. g N-(1,3-Diphenyl-1H-pyrazol-4-yl)benzamide. h N-(1,4-Diphenyl-1H-pyrazol-5-yl)benzamide. i N-(2-Phenylimidazo[1,2-a]pyridin-3-yl)benza-
mide.
293 cells expressing rat mGluR5 by the CDPPB analogues are
shown in Tables 1 and 2. In this assay, CDPPB (2) had a Ki of
3.8 ( 0.4 µM in concordance with the previously reported value
of 2.6 ( 0.5 µM in CHO cells expressing the human mGluR5.10
Monosubstituents in either the 3- or 4-position of the benzamide
moiety, regardless of their electronic nature, increased binding
activity, except for CONH2 and COOH groups in the 4-position
(Table 1). Comparison with the unsubstituted analogue (20)
shows that the increase in binding activity by a 3-CN group
was only 11%, while having the cyano group in the para-position
(28) increased binding activity by 270%. A 4-NO2 substitution
(29) resulted in the highest activity. Additional increases were

3336 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11
de Paulis et al.
seen with a halogen atom in the ortho-position of the 1-phenyl
ring. The 2′-chloro-4-nitro analogue 47 had Ki ) 113 ( 24 nM.
In contrast, the 1-(2-pyridyl) analogue 42 was completely devoid
of activity. Attempts to correlate the binding data with various
QSAR models resulted in the nitro compounds being clustered
as outliers. Compound 54 with a 3-pyridinyl group replacing
the benzamide ring combined with a 4-methyl group showed
activity similar to having a phenyl ring with no substituents
(20). The same result was reported for the corresponding
2-pyridinyl analogue.9 However, the 4-pyridinyl analogue 55
displayed no binding activity (Table 2). Thus, all tested
compounds with an ortho-substituent in the benzamide moiety
and all pyridinylcarboxamides proved to have weak or no
activity. With the exception for halogen atoms in the ortho-
position of the 1-phenyl ring, all substitutions in the pyrazole
phenyl rings proved detrimental. This is in sharp contrast to
the effects of aromatic substitution in the MPEP series of
negative allosteric modulators, where meta-substituents increase
binding activity to the MPEP site.14-18 For instance, in the
MPEP series the binding activity increases with the introduction
of a pyridyl ring in either aryl group,15,16 while decreasing
drastically with substitution in the para-position.18 In the CDPPB
series, introducing a m-cyano group (2 vs 20, Table 1) only
marginally increased binding affinity, while a para-substituent
(28) resulted in a substantial increase. Therefore, none of the
aromatic rings of CDPPB seem to mimic the aromatic rings of
MPEP, clearly showing that the structural requirement of the
binding site of CDPPB is different from that of MPEP or
methoxyPEPy.
Figure 4. Comparison of predicted versus observed functional activities
for the active compounds of Table 1, using HQSAR (Tripos, St. Louis,
MO). Correlation coefficient r ) 0.87 (n ) 29).
diphenyl-1H-pyrazol-5-yl analogues 69 and 70 showed antago-
nist properties at the highest concentration, rather than poten-
tiation. Confining the amide moiety in a triazole ring (73)
resulted in decreased activity and produced only 3-fold potentia-
tion (Table 2). No potentiation but weak antagonism was seen
with the annelated benzopyrazoles 75 and 76. This suggests that
the 1,3-disubstituted pyrazole ring system plays an important
role in interacting with the positive allosteric site. Highest
functional potency was seen with the 2′-fluoro-4-nitro analogue
46 with an EC50 of 9.6 ( 1.9 nM. Thus, compound 46 had 8
times the functional potency of CDPPB (Table 1). Like CDPPB,
the novel allosteric potentiator ADX-47273 (1) has shown
pharmacokinetic properties that makes it suitable for in vivo
studies.12 In a direct comparison with compound 1, compound
46 was 10 times more potent in potentiating glutamate-induced
calcium release in rat astrocytes (Table 1). The calculated
lipophilicity of compound 46 is log P ) 4.9 versus log P ) 5.2
for CDPPB (Table 1). Considering that CDPPB is reported to
be 10 times more active at the human mGluR5 than at the rat
receptor,10 further studies of 46 are warranted.
Attempts to find the optimal combination of aromatic
substituents, using Hansch 2D QSAR analysis with standard
parameters, proved less successful in explaining the high
potencies of the nitro analogues 23, 29, 46-49, as well as the
poor activities of the dimethoxy analogues 33 and 34. However,
HQSAR modeling of the functional activities of the CDPPB
analogues presented in Table 1 was able to accurately predict
their activities. Comparison of the observed and predicted
activities is shown in Figure 4. HQSAR utilizes structural
fragments (molecular fingerprints) of varying lengths as descrip-
tors together with partial least-squares (PLS) analysis and cross-
validation. In the best model (r2 ) 0.774, q2 ) 0.047, SE )
0.314), the corresponding 3,4-dinitro compound was predicted
to be the most active compound with a calculated EC50 value
of 3 nM. However, such a compound is not synthetically
feasible, but the positional 3,5-dinitro isomer 32 was prepared
and found to retain half the functional activity of the 4-nitro
compound (29), as predicted (Figure 4).
The results of evaluating the analogues of CDPPB for their
ability to potentiate glutamate-induced calcium release are
shown in Tables 1 and 2. The activity of CDPPB at the rat
mGluR5 was 77 nM, compared to 27 nM seen at the human
mGluR5.10 This is in concordance with the reported differences
between rat and human mGluR5.9,10 Having no substituent in
either meta- or para-position of the benzamide moiety (20)
produced slightly less activity than that of CDPPB, as demon-
strated by Lindsley et al.9 With the exception of having a
m-trifluoromethyl group (22), electron-donating substituents
tended to increase activity. Highest activity was obtained with
a nitro group, particularly in the para-position (29), while a nitro
group in the ortho-position of the benzamide moiety (53)
completely abolished activity. In fact, having a nitro group in
either meta- (23) or para-position (29) proved to be the most
beneficial substitution. The electronically similar 4-carboxylic
acid and 4-carboxamide analogues 30 and 31 were inactive.
Interestingly, a single electropositive substituent, such as a
methoxy group, in either meta- (21) or para-position (26)
bestowed increased activity relative to that of CDPPB. However,
two methoxy groups in either 3,4- (33) or 3,5-positions (34)
reduced functional activity 20- and 15-fold, respectively, relative
to having no substituent (20). In contrast, having two methyl
groups (35) or chloro atoms (36) in the 3,4-positions of the
benzamide resulted in 10- and 18-fold increases, respectively.
Lindsley et al. found the same result with two fluoro atoms in
these positions.9 Replacing the aromatic benzamide ring with
an aliphatic ring decreased activity. The cyclohex-1-enyl-,
cyclohexyl-, and cyclopentyl carboxamides 56-58 were 10
times less active on the average than the benzoyl analogue 20.
The exception was cyclopentylmethylcarboxamide 59, which
retained functional activity. Extending the position of the
4-nitrophenyl group by two carbon atoms (60) reduced activity
10-fold. Varying the positions of the pyrazole nitrogen atoms
produced inactive analogues (64-66, 69-71). In fact, the 1,4-
All compounds in this study were considerably less active
than MPEP (Ki ) 3.4 nM)18 at the [3H]methoxyPEPy binding
site and showed higher functional than binding activity. For the
CDPPB analogues in Table 1 an average ratio of 16 was
obtained between the EC50 for potentation of glutamate-induced
calcium release and the Ki binding affinity to the antagonist
allosteric MPEP site. Although the more potent compounds in

PositiVe Allosteric Modulation of mGluR5
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11 3337
as described.36 Proton and carbon NMR spectra were obtained in
CDCl3 on a Bruker instrument operating at 300 and 75 MHz,
respectively, with tetramethylsilane as internal standard. Mass
spectra were obtained on a triple-quadrupole instrument (Finnigan
TSQ-7000, Thermo Electron Corp, San Jose, CA) equipped with
an electrospray ionization source and operating in either positive
or negative ion mode. High-resolution mass spectra (HRMS) were
obtained using electron ionization on a 8 kV 70-VSE instrument
at the Mass Spectrometry Laboratory, School of Chemical Sciences,
University of Illinios at Urbana-Champaign. X-ray crystallographic
structure of compound 69 was obtained from graphite Mo KR
radiation on a Nonius Kappa CCD diffractometer at Yale University
Chemical Instrumentation Center. Melting points were recorded on
an OptiMelt automated instrument (Stanford Research Systems,
Sunnyvale, CA) operating at a rate of 2 °C/min. Elemental analyses
were performed by Atlantic Microlab (Norcross, GA) and were
within 0.4% of theory unless otherwise noted.
the functional assay, e.g. 26, 35, 36, 46, and 47, were also more
active at the MPEP binding site, there were several exceptions.
For instance, compounds 43, 44, and 59 displayed glutamate
potentiation with virtually no affinity for the MPEP binding
site, while the opposite was seen for compound 57. The 1,4-
diphenylpyrazol-5-ylbenzamides 69 and 70 and the phenylimi-
dazopyridines 75 and 76 displaced [3H]methoxyPEPy binding
in the low micromolar range, while showing antagonstic
acitivities in the functional assay. Although, it is possible that
CDPPB exerts its positive modulation of mGluR5 by binding
to the MPEP site, other factors may contribute to its functional
activity. The existence of a distinct site, at which positive
allosteric modulators of mGluR5 can act, is supported by the
observation that CPPHA [N-(4-chloro-2-((1,3-dioxo-1,3-dihy-
dro-2H-isoindol-2-yl)methyl)phenyl)-2-hydroxybenzamide],8 a
structurally unrelated positive allosteric modulator of mGluR5,
has no affinity for the MPEP site. However, recent studies have
demonstrated that a neutral ligand at the MPEP site, the
corresponding 5-methyl isomer 5-MPEP,18 can inhibit CDPPB-
induced calcium release in cultured astrocytes of the rat.19
Therefore, the binding site responsible for positive modulation
of mGluR5 by CDPPB and its congeners is likely situated in
close proximity to the antagonist MPEP binding site. Definitive
determination of the role of binding to the MPEP site in
mediating allosteric potentiator activity of specific compounds
in this series must await further studies.
In conclusion, we found that only the 5-benzamido-1,3-
diphenylpyrazole scaffold produced positive allosteric modula-
tors. Replacing the m-cyano group of CDPPB (2) with a p-nitro
group (29) resulted in a 20-fold increase in binding activity and
an 8-fold increase in functional activity. Introducing a halogen
atom in the ortho-position of the phenyl ring in the 1-pyrazole
position further increased the activity. In particular, the 1-(2-
fluorophenyl)-4-nitrobenzamide 46 (VU-1545) displayed phar-
macological properties in the low nanomolar range using the
rat mGluR5, which warrants further studies, preferably with
human mGlu receptors. Compound 46 is only half as lipophilic
as CDPPB, which should increase its brain availability. The
apparent correlation between binding affinities and functional
activities20 raises the possibility that activity at the MPEP site
may be important for positive allosteric modulation. However,
there was a 16-fold discrepancy in the average potencies at these
two measures and several obvious outliers. Thus, it is possible
that the apparent correlation between these two measures is a
consequence of coincidental SAR requirements of the aromatic
substituents for binding to the MPEP site and that binding to
this site is not responsible for functional activity. Whether
occupation of the MPEP site is a prerequisite for glutamate
potentiation remains to be determined.
1-Amino-1-phenylacrylonitrile (8). Benzonitrile (5) (10 g, 97
mmol) was dissolved in benzene (250 mL). Acetonitrile (8.0 g,
195 mmol) was added, followed by portions of potassium tert-
butoxide (25 g, 417 mmol). The mixture was stirred for 24 h at 20
°C. Diethyl ether (200 mL) was added, followed by 2% NaHCO3
(100 mL). Separation of the organic layer, washing with 5 N NaCl
(100 mL), drying (Na2SO4), and evaporation of the solvent gave
10.5 g (75%), after recrystallization from i-Pr2O (100 mL). Mp:
1
85-87 °C. Lit.37 mp: 86 °C. H NMR (CDCl3): 7.46 (m, 5H),
7.40 (m, 5H), 5.05 (b, 2H), 4.21 (s, 1H). 13C NMR (CDCl3): 161.3,
135.1, 130.7, 128.7 (2 × CH), 125.7 (2 × CH), 119.3, 63.3.
1-Amino-1-(2-chlorophenyl)acrylonitrile (9) was prepared from
acetonitrile and 2-chlorobenzonitrile (6) (2.75 g, 20 mmol) as
described for compound 8. Recrystallization from i-Pr2O (10 mL)
gave 1.95 g (55%). Mp: 104-106 °C. Lit.38 mp: 106-107 °C.
1H NMR (CDCl3): 7.43 (m, 1H), 7.39 (dt, 2H), 7.34 (m, 1H), 4.98
(b, 2H), 4.06 (s, 1H). 13C NMR (CDCl3): 159.4, 134.6, 131.4, 130.8
(CH), 130.1 (CH), 129.6 (CH), 126.9 (CH), 118.3, 66.5 (CH).
1-Amino-1-(3-methoxyphenyl)acrylonitrile (10) was prepared
from acetonitrile and 3-methoxybenzonitrile (7) (5.3 g, 40 mmol)
as described for compound 8. Recrystallization from i-Pr2O (20
mL) gave 4.8 g (69%). Mp: 64-66 °C. Lit.39 mp: 61-63 °C. 1H
NMR (CDCl3): 7.34 (dd, 1H), 7.08 (d, 1H), 7.01 (s, 1H), 6.99 (m,
1H), 4.98 (b, 2H), 4.24 (s, 1H), 3.84 (s, 3H). 13C NMR (CDCl3):
161.2, 159.6, 136.5, 129.8, 119.2, 118.0, 116.0, 111.4, 63.4, 55.1.
5-Amino-1-phenyl-3-(2-chlorophenyl)-1H-pyrazole (11). 2-
Amino-2-(2-chlorophenyl)acrylonitrile (9) (1.8 g, 10 mmol) was
suspended in 2.5 N HCl (10 mL, 25 mmol) and heated to 50 °C.
Phenylhydrazine (1.2 g. 11 mmol) was added, followed by 12 N
HCl (5 mL, 60 mmol), and the mixture was heated to 110 °C for
20 min. After cooling by adding pieces of ice, 14 N NH4OH was
added dropwise until the solution became basic (7 mL, 100 mmol).
Extraction with EtOAc (2 × 150 mL), washing of the combined
organic layer with water (100 mL), drying (Na2SO4), and evapora-
tion of the solvent gave crude 11. Recrystallization from i-Pr2O
(10 mL) gave 1.02 g (39%). Mp: 139-141 °C. Lit.38 mp: 137 °C.
1H NMR (CDCl3): 7.88 (dd, 1H), 7.64 (d, 2H), 7.49 (t, 2H), 7.43
(dd, 1H), 7.36 (t, 1H), 7.24 (dq, 2H), 6.20 (s, 1H), 3.85 (b, 2H).
13C NMR (CDCl3): 149.0, 144.6, 138.2, 132.2, 131.9, 130.3, 129.9,
129.2 (2 × CH), 128.5, 127.2, 126.5, 123.8 (2 × CH), 92.0.
5-Amino-1-phenyl-3-(3-methoxyphenyl)-1H-pyrazole (12) was
prepared from compound 10 (1.74 g, 10 mmol) and phenylhydrazine
(1.19 g, 11 mmol) as described for compound 11. Recrystallization
Experimental Section
Reagents and starting materials were obtained from Sigma-
Aldrich (Milwaukee, WI) unless otherwise noted. ADX-47273 [(S)-
1-(4-fluorophenyl)-3-(3-(4-fluorophenyl)[1,2,4]oxadiazol-5-yl)-
piperidine, 1] was prepared from (S)-1-BOC-nipecotic acid and
4-fluorobenzonitrile as described.11 MPEP (2-methyl-6-(phenyl-
ethynyl)pyridine hydrochloride, 3) and L-glutamic acid were
obtained from Tocris (Ellisville, MO). CDPPB (2), N-(1,3-diphenyl-
1H-pyrazol-5-yl)benzamide (20), 4-methyl-N-(1,3-diphenyl-1H-
pyrazol-5-yl)benzamide (24), and 4-trifluoromethyl-N-(1,3-diphenyl-
1H-pyrazol-5-yl)benzamide (27) were prepared from the cor-
responding substituted benzoyl chlorides and 5-amino-1,3-diphenyl-
1H-pyrazole (Alfa-Aesar, Ward Hill, MA) as described.9,13 [3H]-
MethoxyPEPy [3-[3H]methoxy-5-(pyridin-2-ylethynyl)pyridine] was
custom radiolabeled (American Radiolabeled Chemicals, St Louis,
MO) by O-alkylation of the corresponding phenol with [3H]CH3I
1
from i-Pr2O (15 mL) gave 1.35 g (51%). Mp: 110-112 °C. H
NMR (CDCl3): 7.64 (d, 2H), 7.49 (t, 2H), 7.38 (m, 3H), 7.30 (t,
1H), 6.86 (d, 2H), 5.94 (s, 1H), 3.86 (b, 1H), 3.84 (s, 3H). 13C
NMR (CDCl3): 159.5, 151.1, 145.5, 138.3, 134.6, 129.2 (2 × CH),
127.2, 123.9 (2 × CH), 118.0, 113.6, 110.3, 88.0, 55.0. Anal.
(C16H14N3O) C, H, N.
5-Amino-1-(2-fluorophenyl)-3-phenyl-1H-pyrazole (13) was
prepared from 1-amino-1-phenylacrylonitrile (8) (1.44 g, 10 mmol)
and 2-fluorophenylhydrazine HCl (1.63 g, 10 mmol) as described
for compound 11. Recrystallization from i-Pr2O (15 mL) gave 0.79
g (31%) as an oil. 1H NMR (CDCl3): 7.80 (d, 2H), 7.61 (dt, 1H),

3338 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11
de Paulis et al.
7.38 (m 3H), 7.30 (m, 3H), 5.98 (s, 1H),3.81 (b, 2H). 13C NMR
(CDCl3): 157.8, 154.5, 146.7, 133.0, 129.8, 128.9, 128.2 (2 × CH),
127.7, 125.4 (2 × CH), 124.9, 116.4 (d), 88.1.
137.6, 136.3, 134.3, 129.7 (2 × CH), 128.3 (2 × CH), 127.9, 125.3
(2 × CH), 124.5 (2 × CH), 118.4, 118.2, 112.3, 95.4, 55.2. Anal.
(C23H19N3O2) C, H, N.
5-Amino-1-(2-chlorophenyl)-3-phenyl-1H-pyrazole (14) was
prepared from 1-amino-1-phenylacrylonitrile (8) (1.44 g, 10 mmol)
and 2-chlorophenylhydrazine (1.79 g, 10 mmol) as described for
compound 11. Recrystallization from i-Pr2O (10 mL) gave 1.35 g
3-Trifluoromethyl-N-(1,3-diphenyl-1H-pyrazol-5-yl)benz-
amide (22) was prepared from 3-trifluorobenzoyl chloride (1.0 g,
4.8 mmol) as described for compound 21. Recrystallization from
1
i-Pr2O (40 mL) gave 0.59 g (34%). Mp: 155-156 °C. H NMR
1
(52%). Mp: 107-109 °C. H NMR (CDCl3): 7.80 (d, 2H), 7.54
(CDCl3): 8.09 (b, NH), 8.04 (s, 1H), 7.90 (m, 3H), 7.81 (m, 1H),
7.60 (m, 4H), 7.50 (m, 2H), 7.41 (dt, 2H), 7.37 (m, 1H), 7.19 (s,
1H). 13C NMR (CDCl3): 162.0. 151.8, 137.5, 135.8, 133.7, 131.1,
130.5, 129.8 (2 × CH), 129.4, 128.9, 128.6, 128.3 (2 × CH), 128.0,
125.5 (2 × CH), 124.4 (2 × CH), 124.0, 96.1. Anal. (C23H16F3N3O·
0.5H2O) H, N; C: calcd, 66.34; found, 67.01.
(m, 2H), 7.39 (m, 4H), 7.28 (t, 1H), 5.96 (s, 1H), 3.69 (b, 2H). 13
C
NMR (CDCl3): 162.1, 151.9, 146.7, 135.6, 133.1, 131.9, 130.3,
130.1, 128.2 (2 × CH), 127.8, 127.6, 125.4 (2 × CH), 87.3. Anal.
(C15H12ClN3) C, H, N.
5-Amino-1-(2-bromophenyl)-3-phenyl-1H-pyrazole (15) was
prepared from 1-amino-1-phenylacrylonitrile (8) (1.44 g, 10 mmol)
and 2-bromophenylhydrazine HCl (2.24 g, 10 mmol) as described
for compound 11. Column chromatoraphy on silica gel in CH2Cl2
gave fractions with Rf ) 0.16 in CH2Cl2. Recrystallization from
3-Nitro-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (23) was
prepared from 3-nitrobenzoyl chloride (0.5 g, 2.7 mmol) as
described for compound 21. The reaction mixture showed three
compounds according to TLC in CH2Cl2. The product was purified
on 100 g of silica gel (Merck 70-230 mesh, 60 A) in CH2Cl2.
Elution with CH2Cl2 gave byproducts A and B (not isolated).
Fractions showing the third spot (Rf ) 0.15) were collected, and
the solvent was removed. Recrystallization from EtOH (10 mL)
gave 0.38 g (24%). Mp: 192-194 °C. 1H NMR (CDCl3): 8.56 (s,
1H), 8.39 (d, 1H, J ) 8.5 Hz), 8.18 (b, NH), 8.07 (d, 1H, J ) 8.5
Hz), 7.88 (d, 2H), 7.67 (t, 1H), 7.58 (m, 4H), 7.47 (q, 1H), 7.39
(m, 4H), 7.14 (s, 1H). MS: 385 (M + H) 100. 13C NMR (CDCl3):
151.8, 148.0, 137.4, 135.5, 134.5, 132.8, 132.4, 130.0, 129.9 (2 ×
CH), 128.6, 128.4 (2 × CH), 128.0, 126.6, 125.5 (2 × CH), 124.4
(2 × CH), 121.8, 96.4. Anal. (C22H16N4O3) C, H, N.
1
i-Pr2O (20 mL) gave 0.62 g (20%). Mp: 111-114 °C. H NMR
(CDCl3): 7.81 (d, 2H), 7.74 (d, 1H), 7.55 (dd, 1H), 7.48 (dt, 1H),
7.37 (m, 3H), 7.31 (t, 1H), 5.99 (s, 1H), 3.67 (b, 2H). 13C NMR
(CDCl3): 1.51.7, 146.4, 137.2, 133.4, 133.1, 130.6, 130.3, 128.4,
128.2 (2 × CH), 127.6, 125.4 (2 × CH), 122.1, 87.3. Anal. (C15H12-
BrN3) C, H, N.
5-Amino-1-(2-pyridyl)-3-phenyl-1H-pyrazole (16) was pre-
pared from 1-amino-1-phenylacrylonitrile (8) (1.44 g, 10 mmol)
and 2-pyridylhydrazine·2HCl (1.82 g, 10 mmol) as described for
compound 11. Recrystallization from EtOH (20 mL) gave 0.45 g
(19%). Mp: 144-147 °C. Lit.24 mp: 159-160 °C. 1H NMR
(CDCl3):25 8.34 (s, 1H), 8.11 (d, 1H), 7.85 (m, 3H), 7.40 (m, 3H),
7.09 (s, 1H), 6.00 (b, 2H), 5.86 (s, 1H). 13C NMR (CDCl3): 162.0,
160.3, 149.3, 146.2, 138.3, 132.9, 128.2 (2 × CH), 127.9, 125.5 (2
× CH), 119.3, 113.5, 86.9.
4-Methoxy-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (25)
was prepared from 4-methoxybenzoyl chloride (0.5 g, 2.9 mmol)
as described for compound 21. Recrystallization from EtOH (20
1
mL) gave 0.94 g (61%). Mp: 155-156 °C. H NMR (CDCl3):
5-Amino-1-(3-bromophenyl)-3-phenyl-1H-pyrazole (17) was
prepared from 1-amino-1-phenylacrylonitrile and 3-bromophenyl-
hydrazine as described for compound 11. Extraction with EtOAc
8.07 (s, 1H), 7.89 (d, 2H), 7.56 (m, 4H), 7.45 (m, 3H), 7.34 (m,
1H), 7.15 (s, 1H), 6.92 (d, 2H), 3.82 (s, OCH3). 13C NMR
(CDCl3): 163.1, 162.7, 151.6, 137.7, 136.6, 133.7, 132.7, 129.7
(2 × CH), 128.8 (2 × CH), 128.3 (2 × CH), 127.8, 125.5 (2 ×
CH), 125.0, 124.5 (2 × CH), 113.9 (2 × CH), 95.8, 60.1. Anal.
(C23H19N3O2) C, H, N.
1
(2 × 100 mL) gave 1.76 g (56%) of an oil. H NMR (CDCl3):
7.87 (t, 1H), 7.81 (d, 2H), 7.61 (dt, 1H), 7.48 (dq, 1H), 7.39 (t,
2H), 7.34 (m, 2H), 5.96 (s, 1H), 3.86 (b, 2H). 13C NMR (CDCl3):
151.7, 145.6, 139.7, 132.9, 130.4, 129.9, 128.2 (2 × CH), 127.8,
126.6, 125.4 (2 × CH), 122.7, 121.8, 88.5.
4-Trifluoromethoxy-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzam-
ide (26) was prepared from 4-trifluoromethoxybenzoyl chloride (1.0
g, 4.8 mmol) as described for compound 21. Recrystallization from
5-Amino-1-(3-cyanophenyl)-3-phenyl-1H-pyrazole (18) was
prepared from 1-amino-1-phenylacrylonitrile (8) (1.0 g, 6.9 mmol)
and 3-cyanophenylhydrazine40 (1.0 g, 7.5 mmol) as described for
compound 11. Recrystallization from i-Pr2O (10 mL) of the residue
from combined chromatography fractions gave 0.32 g (18%). Mp:
131-132 °C. 1H NMR (CDCl3): 8.07 (q, 1H), 7.99 (dq, 1H), 7.80
(d, 2H), 7.59 (m, 2H), 7.41 (d, 1H), 7.38 (d, 1H), 7.33 (t, 1H),
6.01 (s, 1H), 3.86 (b, 2H). 13C NMR (CDCl3): 151.9, 145.4, 139.3,
132.4, 129.9, 129.7, 128.1 (2 × CH), 127.8, 126.9, 126.0, 125.2 (2
× CH), 117.6, 113.1, 89.4. Anal. (C16H12N4) C, H, N.
1
i-Pr2O (30 mL) gave 0.40 g (22%). Mp: 180-181 °C. H NMR
(CDCl3): 8.09 (b, NH), 7.90 (d, 2H), 7.80 (d, 2H), 7.59 (m 4H),
7.48 (m, 1H), 7.43 (t, 2H), 7.35 (d, 1H), 7.30 (d, 2H), 7.19 (s, 1H).
13C NMR (CDCl3): (2 × CH only) 129.8, 128.8, 128.5, 125.5,
124.5, 120.6. Anal. (C23H16F3N3O2) C, H, N.
4-Cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (28) was
prepared from 4-cyanobenzoyl chloride (0.8 g, 4.8 mmol) as
described for compound 21. TLC of the reaction mixture in CH2-
Cl2 showed three spots with Rf of 0.28 (byproduct A, 51), 0.21,
(byproduct B, 52), and 0.12, respectively. Collection of fractions
with Rf ) 0.12 in CH2Cl2 and evaporation of the solvent gave 0.47
g of solid. Recrystallization from i-Pr2O (30 mL) gave 0.31 g (24%).
Mp: 207-208 °C. 1H NMR (CDCl3): 8.05 (b, NH), 7.91 (dd, 2H),
7.84 (d, 2H), 7.76 (d, 2H), 7.59 (d, 4H), 7.51 (q, 1H), 7.43 (t, 2H),
7.35 (m, 1H), 7.21 (s, 1H). 13C NMR (CDCl3): 151.6, 137.2, 136.4,
135.4, 132.3 (2 × CH), 132.2, 129.7 (2 × CH), 128.4, 128.2 (2 ×
CH), 127.8, 127.2 (2 × CH), 125.3 (2 × CH), 124.3 (2 × CH),
117.1, 115.6, 95.7. Anal. (C23H16N4O) H, N; C: calcd, 75.81; found,
75.07.
5-Amino-1-(3-methoxyphenyl)-3-phenyl-1H-pyrazole (19) was
prepared from 1-amino-1-phenylacrylonitrile (8) (1.44 g, 10 mmol)
and 3-methoxyphenylhydrazine41 (1.38 g, 10 mmol) as described
for compound 11. Recrystallization from i-Pr2O (12 mL) gave 0.36
g (36%) as an oil. 1H NMR (CDCl3): 7.81 (dt, 2H), 7.38 (dd, 3H),
7.32 (dt, 1H), 7.19 (m, 2H), 6.90 (ddd, 1H), 5.95 (s, 1H), 4.09 (s,
3H), 3.85 (b, 2H).
3-Methoxy-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (21).
3-Methoxybenzoyl chloride (1.0 g, 5.9 mmol) in CH2Cl2 (5 mL)
was slowly added to a stirred mixture of N,N-diisopropylethylamine
(1.0 g, 7.9 mmol) and 5-amino-1,3-diphenyl-1H-pyrazole (Alfa-
Aesar) (1.0 g, 4.2 mmol) in CH2Cl2 (20 mL) at 4 °C (ice-water).
After 3 h at 20 °C, the solvents were evaporated, water (100 mL)
was added, and the residue was extracted with ethyl acetate (2 ×
100 mL). Washing of the combined organic layer with 1 N HCl,
then 1 N NaOH, and then water (25 mL each), drying (Na2SO4),
and evaporation of the solvent gave a crystallizing residue.
Recrystallization from EtOH (20 mL) gave 1.39 g (91%). Mp:
4-Nitro-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (29) was
prepared from 4-nitrobenzoyl chloride (0.41 g) as described for
compound 21. TLC of the reaction mixture in CH2Cl2 showed three
spots with Rf of 0.45 (byproduct A), 0.32 (byproduct B), and 0.18,
respectively. Separation on 100 g of silica gel (Merck 70-230 mesh,
60 A) in CH2Cl2 gave 0.42 g (26%) with Rf ) 0.18. Mp: 221-
1
223 °C. H NMR (CDCl3): 8.32 (d, 2H, J ) 8.7 Hz),), 8.09 (b,
NH), 7.92 (m, 4H), 7.60 (d, 2H, J ) 4.5 Hz), 7.51 (q, 1H), 7.43 (t,
1H), 7.37 (t, 1H), 7.23 (s, 1H). 13C NMR (CDCl3): 163.5, 162.0,
150.1, 138.5, 138.3, 136.1, 132.3, 128.8 (2 × CH), 128.6 (2 ×
CH), 128.1 (2 × CH), 127.5, 126.9, 124.7 (2 × CH), 123.0 (2 ×
1
177-179 °C. H NMR (CDCl3): 8.08 (b, 1H), 7.92 (d, 2H), 7.60
(m, 4H), 7.49 (t, 1H), 7.3-7.4 (m, 6H), 7.21 (s, 1H), 7.09 (dd,
1H), 3.84 (s, 3H). 13C NMR (CDCl3): 163.0, 162.0, 159.8, 151.7,

PositiVe Allosteric Modulation of mGluR5
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11 3339
CH), 122.9 (2 × CH), 100.4. MS: 385 (M + H) 100. Anal.
(C22H16N4O3·0.5H2O) C, H, N.
× CH), 127.9, 125.5 (2 × CH), 124.5 (2 × CH), 104.7 (2 × CH),
104.0, 95.7, 55.3 (2 × OCH3). Anal. (C24H21N3O3) C, H, N.
3,4-Dimethyl-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (35)
was prepared from 3,4-dimethylbenzoic acid as described for
compound 33. Recrystallization from EtOH (15 mL) gave 0.39 g
4-Amincarboxyl-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzam-
ide (30). Terephthaloyl dichloride (2.0 g, 10 mmol) in CH2Cl2 (15
mL) was slowly added to a stirred mixture of N,N-diisopropyl-
ethylamine (1.0 g, 7.9 mmol) and 5-amino-1,3-diphenyl-1H-
pyrazole (1.0 g, 4.2 mmol) in CH2Cl2 (20 mL) at 4 °C (ice-water)
to produce 5-(4-chlorocarbonylbenzamido)-1,3-diphenyl-1H-pyra-
zole. After 1 h, 14 N NH4OH (10 mL) was added and the mixture
stirred for 1 h at 20 °C. Water was added and the product was
extracted with EtOAc (2 × 200 mL). Insoluble materials, containing
a mixture of product and terephthalic acid according to TLC, were
removed by filtration. Drying and evaporation of the solvent gave
0.83 g. Separation on silica gel in CH2Cl2-MeOH (10:1) and
recrystallization from MeOH gave 0.21 g (23%). Mp: 244-247
°C. 1H NMR (CD3OD): 7.93 (m, 6H), 7.63 (d, 2H), 7.52 (m, 2H),
7.45 (m, 3H), 7.38 (m, 1H), 6.92 (s, 1H), 5.93 (s, 2H + MeOH).
1H NMR (CDCl3, 100 scans): 8.70 (s, 1H), 7.93 (m, 6H), 7.63 (d,
2H), 7.55 (t, 2H), 7.43 (m, 3H), 7.38 (m, 1H), 7.15 (s, 1H), 6.78
(b, 1H), 5.89 (b, 1H). Anal. (C23H18N4O2) C, H, N.
1
(38%). Mp: 163-165 °C. H NMR (CDCl3): 8.05 (b, 1H), 7.90
(dt, 2H), 7.61 (m, 2H), 7.54 (m, 3H), 7.47 (m, 1H), 7.40 (m, 3H),
7.33 (t, 1H), 7.20 (s, 1H), 7.19 (d, 1H), 2.30 (s, 3H), 2.29 (s, 3H).
13C NMR (CDCl3): 163.1, 151.3, 141.2, 137.2, 136.8, 136.1, 132.3,
129.9, 129.3, 129.2 (2 × CH), 127.8 (2 × CH), 127.7, 127.3, 125.0
(2 × CH), 124.0 (2 × CH), 123.5, 95.0, 19.2, 19.1. Anal.
(C24H21N3O) C, H, N.
3,4-Dichloro-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (36)
was prepared from 3,4-dichlorobenzoic acid as described for
compound 33. Recrystallization from EtOH (20 mL) gave 1.05 g
1
(61%). Mp: 180-181 °C. H NMR (CDCl3): 7.95 (b, NH), 7.91
(t, 1H), 7.88 (dd, 2H), 7.58 (d, 4H), 7.52 (m, 2H), 7.47 (m, 1H),
7.41 (d, 2H), 7.35 (m, 1H), 7.17 (s, 1H). Anal. (C22H15Cl2N3O) C,
H, N.
N-(1-Phenyl-3-(2-chlorophenyl)-1H-pyrazol-3-yl)benzamide
(37) was prepared from benzoyl chloride (0.30 g, 2.1 mmol) and
diphenylpyrazole 11 (0.52 g, 2.0 mmol) as described for compound
21. Recrystallization from i-Pr2O (12 mL) gave 0.31 g (41%). Mp:
165-168 °C. 1H NMR (CDCl3): 8.05 (b, 1H), 7.87 (dd, 1H), 7.75
(d, 2H), 7.62 (m, 3H), 7.55 (d, 2H),7.45 (dd, 4H), 7.37 (s, 1H),
7.30 m, 2H). 13C NMR (CDCl3): 162.0, 137.6, 135.4, 132.2, 130.4,
130.0, 129.7 (2 × CH), 128.9, 128.7 (2 × CH), 128.5, 126.8 (2 ×
CH), 126.6, 124.5 (2 × CH). Anal. (C22H16ClN3O) C, H, N.
N-(1-Phenyl-3-(3-methoxyphenyl)-1H-pyrazol-3-yl)benzam-
ide (38) was prepared from benzoyl chloride (0.28 g, 2 mmol) and
diphenylpyrazole 12 (0.52 g, 2 mmol) as described for compound
21. Recrystallization from i-Pr2O (25 mL) gave 0.38 g (51%). Mp:
4-Carboxyl-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (31).
The acid chloride intermediate was prepared as described for
compound 30. After 1 h at 20 °C, the solvents were evaporated,
and the residue was dissolved in DMF (10 mL) and treated with
MeOH (10 mL) for 30 min at 50 °C. Extraction of the product
with EtOAc as described for compound 21, followed by column
chromatography on 100 g of silica gel (Merck 70-230 mesh, 60
A) in CH2Cl2, gave pure fractions. Crystallization from i-Pr2O gave
0.93 g (59%) of 4-methoxycarboxyl-N-(1,3-diphenyl-1H-pyrazol-
1
5-yl)benzamide. Mp: 163-165 °C. H NMR (CDCl3): 8.14 (b,
1H), 8.11 (d, 2H), 7.90, d, 2H), 7.79, (d, 2H), 7.58 (m, 4H), 7.49
(m, 1H), 7.42 (t, 2H), 7.36, (m, 1H, 7.19, (s, 1H), 3.95 (s, 3H). 13
C
1
140-142 °C. H NMR (CDCl3): 8.06 (b, 1H), 7.75 (d, 2H), 7.60
NMR (CDCl3): 165.7, 151.8, 137.5, 136.6, 136.0, 133.3, 132.5,
129.9 (2 × CH), 129.8 (2 × CH), 128.5, 128.3 (2 × CH), 128.2,
127.9, 126.9 (2 × CH), 125.5 (2 × CH), 124.5 (2 × CH), 95.9,
52.3. The methyl ester (0.80 g, 2.0 mmol) was dissolved in MeOH
(20 mL) and 2 N NaOH (10 mL) was added. The mixture was
stirred for 1 h at 60 °C, followed by addition of ice and washing
of the aqueous layer with ether (25 mL), and neutralization with
12 N HCl gave 0.54 g (70%). Mp: 233-235 °C. 1H NMR
(CDCl3): 9.66 (b, 1H), 8.13 (d, 2H), 8.01 (d, 2H), 7.91 (d, 2H),
7.68 (d, 2H), 7.51 (t, 2H), 7.42 (m, 3H), 7.32 (t, 1H), 7.03 (s, 1H).
Anal. (C23H17N3O3) C, H, N.
(m, 5H), 7.50 (m, 5H), 7.34 (t, 1H), 7.23 (s, 1H), 6.90 (dd, 1H).
13C NMR (CDCl3): 159.6, 151.7, 137.6, 136.3, 134.1, 132.8, 132.3,
129.8 (2 × CH), 129.3, 128.7 (2 × CH), 128.4, 126.8 (2 × CH),
124.6 (2 × CH), 118.1, 114.3, 110.2, 95.6, 55.1. Anal. (C23H19N3O2·
0.5H2O) H, N; C: calcd, 73.00; found, 73.51.
N-(1-(2-Fluorophenyl)-3-phenyl-1H-pyrazol-3-yl)benzamide
(39) was prepared from benzoyl chloride (0.60 g, 4.2 mmol) and
diphenylpyrazole 13 (1.0 g, 4.2 mmol) as described for compound
21. Recrystallization from EtOH (8 mL) gave 0.70 g (50%). Mp:
1
171-172 °C. H NMR (CDCl3): 7.99 (b, 1H), 7.89 (d, 2H), 7.75
(d, 2H), 7.67 (dt, 1H), 7.54 (t, 1H), 7.46 (m, 6H), 7.36 (td, 2H),
7.15 (s, 1H). 13C NMR (CDCl3): 163.9, 157.2, 152.8, 137.5, 133.0,
132.5, 132.2, 130.5 (d), 129.1 (2 × CH), 128.7 (2 × CH), 128.3,
128.0, 126.8 (2 × CH), 125.5 (2 × CH), 125.4, 116.5 (d), 96.4.
Anal. (C22H16FN3O) C, H, N.
3,5-Dinitro-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (32)
was prepared from 3,5-dinitrobenzoyl chloride (0.50 g, 2.3 mmol)
as described for compound 21 to yield 0.75 g (50%) from EtOH.
1
Mp: 244-246 °C. H NMR (CDCl3): 9.21 (s, 1H), 8.93 (s, 2H),
8.18 (b, 1H), 7.92 (d, 2H), 7.62 (m, 4H), 7.45 (m, 1H), 7.40 (m,
3H), 7.21 (s, 1H). Anal. (C22H15N5O5) C, H, N.
N-(1-(2-Chlorophenyl)-3-phenyl-1H-pyrazol-3-yl)benzam-
ide (40) was prepared from benzoyl chloride (0.30 g, 2.1 mmol)
and diphenylpyrazole 14 (0.50 g, 2.0 mmol) as described for
compound 21. Recrystallization from i-Pr2O (15 mL) gave 0.41 g
3,4-Dimethoxy-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (33)
was prepared from 3,4-dimethoxybenzoic acid (1.0 g, 6.4 mmol)
by treatment with thionyl chloride (1.0 mL, 13 mmol) in toluene
(20 mL), containing 3 drops of DMF as catalyst, for 2 h at 70 °C.
The solvent was evaporated and the resulting 3,4-dimethoxybenzoyl
chloride was diluted with CH2Cl2 (15 mL) and reacted with
5-amino-1,3-diphenyl-1H-pyrazole as described for compound 21.
Recrystallization from i-PrO2 gave 0.82 g (65%). Mp: 184-185
1
(55%). Mp: 171-173 °C. H NMR (CDCl3): 7.90 (d, 2H), 7.79
(b, 1H), 7.71 (d, 2H), 7.63 (dd, 2H), 7.57 (t, 1H), 7.48 (m, 4H),
7.41 (d, 2H), 7.34 (m, 1H), 7.18 (s, 1H). 13C NMR (CDCl3): 152.7,
137.9, 135.3, 133.2, 132.9, 132.5, 131.3, 131.1, 130.6, 130.5, 129.0
(2 × CH), 128.6 (2 × CH), 128.5, 128.2, 127.1 (2 × CH), 125.8
(2 × CH), 95.7. Anal. (C22H16ClN3O) C, H, N.
1
°C. H NMR (CDCl3): 8.05 (b, 1H), 7.91 (d, 2H), 7.58 (m, 4H),
N-(1-(2-Bromophenyl)-3-phenyl)-1H-pyrazol-3-yl)benzam-
ide (41) was prepared from benzoyl chloride (0.5 g, 3.6 mmol)
and diphenylpyrazole 15 (1.0 g, 3.2 mmol) as described for
compound 21. Recrystallization from EtOH (15 mL) gave 0.45 g
7.46 (m, 4H), 7.36 (t, 1H), 7.19 (s, 1H), 7.17 (dd, 1H), 6.86 (d,
1H), 3.93 (s, 3H), 3.91 (s, 3H). 13C NMR (CDCl3): 162.1, 151.5,
150.9, 148.3, 136.9, 135.8, 131.9, 128.9 (2 × CH), 127.5, 127.5 (2
× CH), 127.1, 124.7 (2 × CH), 123.7 (2 × CH), 118.3, 109.6,
109.3, 94.6, 55.0, 54.9. Anal. (C24H21N3O3) C, H, N.
1
(34%). Mp: 162-163 °C. H NMR (CDCl3): 8.45 (d, 2H), 7.80
(dd, 1H), 7.74 (b, 1H), 7.71 (d, 2H), 7.61 (dd, 1H), 7.4-7.5 (m,
5H), 7.36 (t, 1H), 7.18 (s, 1H). 13C NMR (CDCl3): 163.6, 161.8,
152.3, 137.5, 136.6, 133.6, 133.0, 132.6, 132.2, 131.2, 130.5, 128.8,
128.7 (2 × CH), 128.3 (2 × CH), 127.9, 126.8 (2 × CH), 125.6 (2
× CH), 121.2, 95.3. Anal. (C22H16BrN3O) C, H, N.
3,5-Dimethoxy-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (34)
was prepared from 3,5-dimethoxybenzoic acid (0.50 g, 3.2 mmol)
as described for compound 33. Recrystallization from i-PrO2 (5
1
mL) gave 0.21 g (50%). Mp: 144-146 °C. H NMR (CDCl3):
8.03 (b, 1H), 7.90 (d, 2H), 7.58 (m, 4H), 7.42 (m, 4H), 7.19 (s,
1H), 6.85 (s, 2H), 6.61 (d, 1H), 3.80 (s, 6H). 13C NMR (CDCl3):
160.8, 151.2, 137.6, 136.3, 135.0, 132.7, 129.7 (2 × CH), 128.4 (2
N-(1-(2-Pyridyl)-3-phenyl-1H-pyrazol-3-yl)benzamide (42) was
prepared from benzoyl chloride (0.31 g, 2.1 mmol) and pyridylphe-
nylpyrazole 16 (0.47 g, 2.0 mmol) as described for compound 21.

3340 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11
de Paulis et al.
Recrystallization from EtOH (15 mL) gave 0.23 g (68%). Mp:
152-154 °C. 1H NMR (CDCl3): 8.45 (dt, 1H), 8.27 (d, 1H), 8.05
(d, 2H), 7.98 (d, 2H), 7.91 (tt, 1H), 7.58 (m, 3H), 7.46 (m, 3H),
7.38 (t, 1H), 7.21 (dd, 1H). 13C NMR (CDCl3): 163.2, 162.0, 154.4,
145.8, 140.9, 139.3, 133.6, 132.4, 132.0, 128.6 (2 × CH), 128.3 (2
× CH), 127.1 (2 × CH), 125.8 (2 × CH), 120.1, 114.0, 94.7. Anal.
(C22H16N4O) C, H, N.
3-Nitro-N-(1-(2-bromophenyl)-3-phenyl-1H-pyrazol-5-yl)benz-
amide (49) was prepared from 3-nitrobenzoyl chloride (0.33 g, 1.8
mmol) and diphenylpyrazole 15 (0.50 g, 1.6 mmol) as described
for compound 21. Recrystallization from EtOH (10 mL) gave 0.31
g (42%). Mp: 172-176 °C. 1H NMR (CDCl3): 8.53 (s, 1H), 8.37
(d, 1H), 8.04 (d, 1H), 7.89 (m, 3H), 7.80 (dd, 1H), 7.62 (m, 2H),
7.52 (t, 1H), 7.42 (m, 4H), 7.10 (s, 1H). 13C NMR (CDCl3): 161.4,
152.3, 148.0, 136.7, 136.5, 134.6, 133.6, 132.7, 132.3, 131.3, 131.3,
130.4, 130.0, 128.9, 128.4 (2 × CH), 128.1, 126.6, 125.5 (2 ×
CH), 121.9, 120.9, 96.3. Anal. (C22H15BrN4O3) C, H, N.
N-(1-(3-Bromophenyl)-3-phenyl-1H-pyrazol-3-yl)benzam-
ide (43) was prepared from benzoyl chloride (0.31 g, 2.2 mmol)
and diphenylpyrazole 17 (0.62 g, 2.0 mmol) as described for
compound 21. Recrystallization from i-Pr2O (10 mL) gave 0.13 g
3-Cyano-N-(1-(2-bromophenyl)-3-phenyl-1H-pyrazol-5-yl)-
benzamide (50) was prepared from 3-cyanobenzoyl chloride (0.20
g, 1.2 mmol) and diphenylpyrazole 15 (0.32 g, 1.0 mmol) as
described for compound 21. Yield 0.29 g (66%). Mp: 180-181
1
(16%). Mp: 180-181 °C. H NMR (CDCl3): 8.05 (b, NH), 7.88
(dt, 2H), 7.83 (t, 1H), 7.77 (d, 2H), 7.55 (m, 2H), 7.50, (m, 3H),
7.46 q, 1H), 7.41 (d, 2H), 7.35 (m, 1H), 7.14 (s, 1H). 13C NMR
(CDCl3): 163.8, 162.0, 152.2, 138.9, 136.3, 132.7, 132.4, 131.2,
130.8, 128.8 (2 × CH), 128.4 (2 × CH), 128.1, 127.6, 126.8 (2 ×
CH), 125.5 (2 × CH), 123.2, 122.4, 96.8. Anal. (C22H16BrN3O) C,
H, N.
1
°C. H NMR (CDCl3): 7.99 (s, 1H), 7.91 (m, 3H), 7.82 (dt, 2H),
7.75 (b, 1H), 7.61 (m, 2H), 7.54 (dd, 1H), 7.43 (m, 3H), 7.34 (t,
1H), 7.13 (s, 1H). 13C NMR (CDCl3): 162.0, 152.1, 147.3, 136.7,
136.5, 135.2, 133.1, 132.4, 131.4, 130.9, 130.6, 130.5, 129.7, 128.9,
128.4 (2 × CH), 128.1, 125.5 (2 × CH), 117.3, 113.2, 96.1.
HRMS: obsd, 442.0431; calcd, 442.0429. Anal. (C23H15BrN4O)
C, H, N.
5-(4-Cyanobenzoyl)imino-2-(4-cyanobenzoyl)-1,3-diphenyl-
1,2-dihydropyrazole (51) was isolated as byproduct A in the
preparation of compound 28 (yield 0.20 g, 19%). TLC on silica
with CH2Cl2 showed Rf ) 0.27. Mp: 161-163 °C. 1H NMR
(CDCl3): 8.37 (d, 2H), 7.99 (d, 2H), 7.94 (d, 2H), 7.74 (m, 4H),
7.65 (dd, 2H), 7.53 (t, 2H), 7.44 (t, 1H), 7.33 (m, 3H), 6.57 (s,
1H). 13C NMR (CDCl3): 162.0, 160.6, 151.4, 146.7, 141.3, 138.7,
135.2, 132.8 (2 × CH), 132.4 (2 × CH), 130.9, 130.6 (2 × CH),
128.5 (2 × CH), 128.4 (2 × CH), 128.1, 124.7, 125.2 (2 × CH),
124.7 (2 × CH), 118.2, 117.6, 117.1, 115.3, 96.5. Anal. (C31H19N5O2)
C, H, N
N-(1-(3-Cyanophenyl)-3-phenyl-1H-pyrazol-3-yl)benzamide
(44) was prepared from benzoyl chloride (0.31 g, 2.2 mmol) and
diphenylpyrazole 18 (0.52 g, 2.0 mmol) as described for compound
21. Recrystallization from i-Pr2O (10 mL) gave 0.31 g (43%). Mp:
202-204 °C. 1H NMR (CDCl3): 7.97 (t, 1H), 7.87 (m, 4H), 7.78
(d, 2H), 7.68 (dt, 1H), 7.64 (d, 1H), 7.59 (m, 1H), 7.3-7.5 (m,
5H), 7.07 (s, 1H). 13C NMR (CDCl3): 151.4, 137.8, 135.0, 131.5,
131.3, 130.9, 130.0, 129.2, 127.7 (2 × CH), 127.3 (2 × CH), 127.1,
126.6, 126.2, 125.7 (2 × CH), 124.3 (2 × CH), 116.1, 112.6, 97.6.
Anal. (C23H16N4O) C, H, N.
N-(1-(3-Methoxyphenyl)-3-phenyl-1H-pyrazol-3-yl)benzam-
ide (45) was prepared from benzoyl chloride (0.31 g, 2.2 mmol)
and diphenylpyrazole 19 (0.53 g, 2.0 mmol) as described for
compound 21. Recrystallization from EtOH (7 mL) gave 0.17 g
1
(23%). Mp: 161-163 °C. H NMR (CDCl3): 8.18 (b, 1H), 7.92
N,N-Bis(4-cyanobenzoyl)-1,3-diphenyl-5-amino-1H-pyra-
zole (52) was isolated as byproduct B in the preparation of 28 (yield
0.34 g, 43%). TLC in CH2Cl2 showed Rf ) 0.32. Mp: 179-181
(dt, 2H), 7.76 (d, 2H), 7.57 (m, 1H), 7.45 (m, 5H), 7.34 (m, 1H),
7.24 (s, 1H), 7.15 (m, 2H), 7.01 (dq, 1H). 13C NMR (CDCl3): 163.0,
160.5, 151.5, 138.4, 136.2, 132.7, 132.4, 132.0, 130.2, 128.5 (2 ×
CH), 128.1 (2 × CH), 127.7, 126.6 (2 × CH), 125.3 (2 × CH),
115.7, 114.5, 110.0, 95.1, 55.1. Anal. (C23H19N3O2) C, H, N.
4-Nitro-N-(1-(2-fluorophenyl)-3-phenyl-1H-pyrazol-5-yl)benz-
amide (46) was prepared from 4-nitrobenzoyl chloride (0.78 g, 4.2
mmol) and diphenylpyrazole 13 (1.0 g, 4.0 mmol) as described for
compound 21. TLC of the reaction mixture in CH2Cl2 showed three
spots, corresponding to the N,2- and N,N-diacylated byproducts (A
and B) and the desired product, respectively. Separation on 100 g
of silica gel (Merck 70-230 mesh, 60 A) in CH2Cl2 gave fractions
with Rf ) 0.12. Recrystallization from EtOH (15 mL) gave 0.38 g
1
°C. H NMR (CDCl3): 7.81 (dd, 2H), 7.63 (d, 4H), 7.61 (d, 4H),
7.41 (m, 6H), 7.79 (m, 2H), 6.76 (s, 1H). Anal. (C31H19N5O2) C,
H, N.
2-Nitro-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (53) was
prepared from 2-nitrobenzoyl chloride (0.50 g, 2.3 mmol) as
described for compound 21. Yield 0.14 g (16%) after recrystalli-
zation from EtOH. Mp: 226-229 °C. Lit.28 mp: 230-232 °C. 1H
NMR (CDCl3): 8.13 (d, 1H), 7.91 (d, 2H), 7.65 (m, 2H), 7.56 (m,
4H), 7.44 (t, 2H), 7.37 (q, 1H), 7.23 (s, 1H).
N-(6-Methyl-3-pyridinoyl)-1,3-diphenyl-5-amino-1H-pyra-
zole (54) was prepared from 6-methylnicotinic acid (0.50 g, 3.6
mmol) and thionyl chloride as described for compound 33 with 5
equiv of N,N-diisopropylethylamine. Extraction of the product with
EtOAc followed by column chromatography on 50 g of silica gel
(Merck 70-230 mesh, 60 A) in CH2Cl2 gave pure 54 after
crystallization from i-Pr2O (yield 0.17 g, 18%). Mp: 172-175 °C.
1H NMR (CDCl3): 8.77 (d, 1H), 8.13 (b, 1H), 8.02 (dd, 1H), 7.89
(d, 2H), 7.58 (dd, 4H), 7.44 (m, 3H), 7.34 (tt, 1H), 7.25 (d, 1H),
7.18 (s, 1H), 2.60 (s, 3H). 13C NMR (CDCl3): 162.7, 161.9, 151.7,
146.8, 137.5, 135.9, 135.5, 132.6, 129.8 (2 × CH), 128.5, 128.3 (2
× CH), 127.9, 126.0, 125.5 (2 × CH), 124.5 (2 × CH), 123.3,
95.9, 24.4. Anal. (C22H18N4O) C, H, N.
N-Isonicotinoyl-1,3-diphenyl-5-amino-1H-pyrazole (55) was
prepared from 4-pyridylcarboxyl chloride hydrochloride (0.48 g,
2.7 mmol) as described for compound 21 with 5 equiv of N,N-
diisopropylethylamine. The yield was 0.40 g (28%) from EtOH.
Mp: 185-187 °C. 1H NMR (CDCl3): 8.79 (d, 2H), 8.12 (s, NH),
7.91 (d, 2H), 7.59 (m, 6H), 7.52 (dd, 1H), 7.44 (t, 2H), 7.36 (m,
1H), 7.24 (s, 1H). Anal. (C21H16N4O) C, H, N.
N-Cyclohex-1-enecarboxyl-1,3-diphenyl-5-amino-1H-pyra-
zole (56). Cyclohex-1-enecarboxylic acid chloride (0.48 g, 1.2
mmol) was added to 1,3-diphenylpyrazole (0.24 g, 1.0 mmol) as
described for compound 21. Mp: 196-198 °C. 1H NMR
(CDCl3): 7.89 (m, 2H), 7.66 (b, 1H), 7.57 (s, 4H), 7.3-7.5 (m,
4H), 7.15 (s, 1H), 6.71 (m, 1H), 2.21 (m, 4H), 1.65 (m, 4H). 13C
NMR (CDCl3): 164.1, 151.7, 137.7, 136.6, 132.8, 132.2, 129.7 (2
1
(34%). Mp: 222-224 °C. H NMR (CDCl3): 8.32 (d, 2H), 7.98
(b, NH), 7.91 (m, 4H), 7.71 (t, 1H), 7.3-7.5 (m, 6H), 7.16 (s, 1H).
13C NMR (CDCl3): 154.4, 153.5, 153.1, 150.4, 147.5, 139.1, 137.4,
133.7, 133.0, 131.2 (2 × CH), 130.5 (2 × CH), 129.6 (2 × CH),
129.1, 128.9, 128.8, 128.4, 126.3, 126.2, 126.1, 125.7 (2 × CH),
124.5, 117.4 (2 × CH), 117.1 (2 × CH), 98.0, 88.8. Anal. (C22H15-
FN4O3) C, H, N.
4-Nitro-N-(1-(2-chlorophenyl)-3-phenyl-1H-pyrazol-5-yl)benz-
amide (47) was prepared from 4-nitrobenzoyl chloride (0.35 g, 1.8
mmol) and diphenylpyrazole 14 (0.50 g, 1.8 mmol) as described
for compound 21. Separation on 50 g of silica gel (Merck 70-230
mesh, 60 A) in CH2Cl2 gave fractions with Rf ) 0.12. Recrystal-
lization from EtOH (10 mL) gave 0.19 g (28%). Mp: 176-179
1
°C. H NMR (CDCl3): 8.31 (d, 2H), 7.89 (m, 4H), 7.80 (m, 1H),
7.64 (dd, 2H), 7.51 (dd, 2H), 7.46 (m, 2H), 7.36 (t, 1H), 7.17 (s,
1H). Anal. (C22H15ClN4O3·0.5H2O) C, H, N.
4-Nitro-N-(1-(2-bromophenyl)-3-phenyl-1H-pyrazol-5-yl)benz-
amide (48) was prepared from 4-nitrobenzoyl chloride (0.37 g, 2.0
mmol) and diphenylpyrazole 15 (0.62 g, 2.0 mmol) as described
for compound 21. Separation on 50 g of silica gel (Merck 70-230
mesh, 60 A) in CH2Cl2 gave fractions with Rf ) 0.14. Recrystal-
lization from EtOH (10 mL) gave 0.14 g (18%). Mp: 251-253
1
°C. H NMR (CDCl3): 8.32 (d, 2H), 7.90 (t, 4H), 7.82 (dd, 1H),
7.74 (b, 1H), 7.63 (dd, 1H), 7.55 (dt, 1H), 7.45 (m, 3H), 7.36 (d,
1H), 7.18 (s, 1H). Anal. (C22H15BrN4O3) C, H, N.

PositiVe Allosteric Modulation of mGluR5
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11 3341
× CH), 128.3 (2 × CH), 127.8, 125.5 (2 × CH), 124.5 (2 × CH),
95.0, 25.3, 23.8, 21.6, 21.0. Anal. (C22H21N3O) C, H, N.
N-Cyclohexanecarboxyl-1,3-diphenyl-5-amino-1H-pyrazole (57)
was prepared from cyclohexanecarboxylic acid chloride (0.18 g,
1.2 mmol) as described for compound 21. Chromatography on silica
with hexanes-EtOAc (1:4) gave 0.17 g (49%). Mp: 185-186 °C.
1H NMR (CDCl3): 7.88 (m, 2H), 7.47 (m, 1H), 7.42 (m, 3H), 7.34
(t, 1H), 7.06 (s, 1H), 2.20 (m, 1H), 1.90 (m, 2H), 1.82 (m, 2H),
1.71 (m, 1H), 1.47 (m, 2H), 1.24 (m, 3H). 13C NMR (CDCl3):
173.1, 152.2, 138.3, 137.0, 133.4, 130.3 (2 × CH), 128.9 (2 ×
CH), 128.4 (2 × CH), 126.1 (2 × CH), 125.8 (2 × CH), 96.3,
45.9, 29.7, 25.9 (2 × CH2), 25.8 (2 × CH2). HRMS (C22H23N3O):
obsd, 345.1826; calcd, 345.1830. Anal. (C22H23N3O) C, H, N.
N-Cyclopentanecarboxyl-1,3-diphenyl-5-amino-1H-pyra-
zole (58) was prepared from cyclopentanecarboxylic acid (1.1 g,
10 mmol) as described for compound 33 (yield 15%). TLC in CH2-
Cl2 showed Rf ) 0.16. Mp: 162-164 °C. 1H NMR (CDCl3): 7.87
(d, 2H), 7.54 (m, 4H), 7.47 (m, 1H), 7.40 (t, 2H), 7.33 (d, 1H),
7.30 (b, 1H), 7.07 (s, 1H), 2.63 (q, 1H), 1.80 (m, 6H), 1.60 (m,
2H). 13C NMR (CDCl3): 137.2, 129.6 (2 × CH), 128.3 (2 × CH),
127.8, 125.4 (2 × CH), 124.6 (2 × CH), 95.4, 30.0 (2 × CH2),
29.7, 25.6 (2 × CH2). Anal. (C21H21N3O) C, H, N.
N-(1,3-Diphenyl-1H-pyrazol-4-yl)benzamide (64) was prepared
from benzoyl chloride (0.32 g, 2.1 mmol) and diphenylpyrazole
63 (0.48 g, 2.0 mmol) as described for compound 21 to give 0.39
g (26%) after recrystallization from EtOH (10 mL). Mp: 169-
1
171 °C. H NMR (CDCl3): 8.46 (s, 5-CH), 7.76 (d, 2H), 7.70 (b,
NH), 7.50 (t, 1H), 7.42 (m, 6H), 7.29 (m, 6H). 13C NMR (CDCl3):
164.8, 139.4, 134.4, 133.7, 131.6, 129.1 (4xCH), 128.7, 128.6 (2
× CH), 128.4 (2 × CH), 128.3, 127.0, 126.7 (2 × CH), 124.3 (2
× CH), 120.2. MS: 338 (M-) 100. Anal. (C22H17N3O) C, H, N.
3-Cyano-N-(1,3-diphenyl-1H-pyrazol-4-yl)benzamide (65) was
prepared from 3-cyanobenzoyl chloride (0.39 g, 2 mmol) and
diphenylpyrazole 63 (0.48 g, 2 mmol) as described for compound
21. Recrystallization from EtOH (12 mL) gave 0.17 g (23%). Mp:
217-219 °C. MS: 363 (M-) 100. 1H NMR (CDCl3): 8.73 (b, NH),
7.90 (s, 1H), 7.86 (d, 1H), 7.71 (d, 1H), 7.60 (s, 1H), 7.43 (t. 1H),
7.34 (m, 5H), 7.28 (m, 5H). 13C NMR (CDCl3): 165.4, 138.8, 138.0,
135.1, 133.7, 131.5, 130.9, 130.3, 129.5, 129.0 (2 × CH), 128.6 (2
× CH), 128.1, 127.0, 126.7 (2 × CH), 123.9 (2 × CH), 119.7,
117.5, 112.6. Anal. (C23H16N4O) C, H, N.
4-Nitro-N-(1,3-diphenyl-1H-pyrazol-4-yl)benzamide (66) was
prepared from 4-nitrobenzoyl chloride (0.39 g, 2.1 mmol) and
diphenylpyrazole 63 (0.47 g, 2.0 mmol) as described for compound
21. Separation on 50 g of silica gel (Merck 70-230 mesh, 60 A)
in CH2Cl2 gave fractions with Rf ) 0.11. Recrystallization from
N-(Cyclopentylmethyl)carboxyl-1,3-diphenyl-5-amino-1H-
pyrazole (59) was prepared from cyclopentylacetic acid (0.5 g, 3.9
mmol) as described for compound 33 (yield 0.25 g, 32%). Mp:
168-170 °C. 1H NMR (CDCl3): 7.87 (dt, 2H), 7.53 (s, 4H), 7.44
(m, 3H), 7.34 (m, 2H) 7.05 (s, 1H), 2.32 (d, 2H), 2.20 m, 1H),
1.84 (m, 2H), 1.59 (m, 4H), 1.53 (m, 2H). Anal. (C22H23N3O) C,
H, N.
1
EtOH (20 mL) gave 0.13 g (17%). Mp: 174-175 °C. H NMR
(CDCl3): 8.20 (d, 2H), 8.02 (b, 1H), 7.82 (d, 2H), 7.79 (s, 1H),
7.39 (m, 9H), 7.29 (t, 1H). 13C NMR (CDCl3): 129.0, 128.7, 128.4,
126.8, 124.0, 123.8 (2 × CH only). Anal. (C22H16N4O3) C, H, N.
1-Formyl-1-phenylacetonitrile (67). Sodium methoxide (5.4 g,
104 mmol) was added to a stirred solution of benzyl cyanide (11.7
g, 100 mmol) in benzene (200 mL) at 20 °C. Ethyl formate (8.2 g,
110 mmol) was slowly added and the mixture was heated to 40 °C
for 30 min and then allowed to reach room temperature for 1 h.
Water (200 mL) was added and the aqueous layer was collected.
Neutralization with 6 N HCl (18 mL), cooling to 0 °C, filtering,
and washing of the precipitation with water (20 mL) gave 7.50 g
(48%). Mp: 158-160 °C. Lit.31 mp: 159-160 °C.
N-(4-Nitrocinnamoyl)-1,3-diphenyl-5-amino-1H-pyrazole (60)
was prepared from 4-nitrocinnamic acid (0.64 g, 3.3 mmol) and
1,3-diphenyl-1H-pyrazole (0.78 g, 3.3 mmol) as described for
1
compound 33 (yield 0.31 g, 23%). Mp: 195-197 °C. H NMR
(CDCl3): 8.22 (d, 2H), 7.89 (d, 2H), 7.81 (d, J ) 15.5 Hz, 1H),
7.64 (m, 3H), 7.57 (d, 4H), 7.49 (m, 1H), 7.42 (t, 2H), 7.37 (m,
1H), 7.19 (s, 1H), 6.54 (d, J ) 15.4 Hz, 1H). Anal. (C24H18N4O3)
C, H, N (accounted 88 wt %).
N-(2-Phenyl-2-oxoethyl)phthalimide (61). 2′-Bromoacetophe-
none (10 g, 50 mmol) was dissolved in anhydrous DMF (40 mL).
Potassium phthalimide (10 g, 54 mmol) was added in portions at
20 °C. After stirring for 1 h at 20 °C, CHCl3 (60 mL) was added
followed by water (200 mL). Extraction with chloroform (2 × 60
mL), washing of the combined organic layer with 0.5 N NaOH
(50 mL) and then water (50 mL), drying (Na2SO4), and evaporation
gave 10.6 g (80%) of crystalline residue. Mp: 164-166 °C. Lit.42
mp: 165-167 °C. 1H NMR (CDCl3): 8.00 (d, 2H), 7.90 (dd, 2H),
7.75 (dd, 2H), 7.64 (tt, 1H), 7.52 (t, 2H), 5.14 (s, 2H). 13C NMR
(CDCl3): 190.7, 167.6, 134.1, 133.8, 133.7 (2 × CH), 131.9, 128.6
(2 × CH), 127.9 (2 × CH), 123.3 (2 × CH), 43.9.
5-Amino-1,4-diphenyl-1H-pyrazole (68). Acetic acid (2.5 g, 42
mmol) was added to a mixture of compound 67 (2.9 g, 20 mmol)
and phenylhydrazine (2.8 g, 26 mmol) in benzene (25 mL) and the
mixture was heated to reflux temperature for 5 h. Water was
removed azeotropically using a Dean-Stark water trap. After
cooling, 5 mL of 6 N HCl (30 mmol) was added, followed by
neutralization with 14 N NH4OH. Extraction with EtOAc (2 × 50
mL), washing of the combined organic layer with water (2 × 50
mL), drying (Na2SO4), and evaporation of the solvents gave 2.88
g (61%) after crystallization from i-Pr2O (10 mL). Mp: 139-140
1
°C. Lit.32 mp: 136-137 °C. Lit.33 mp: 140-141 °C. H NMR
(CDCl3): 7.66 (s, 1H), 7.62 (d, 2H), 7.52 (d, 2H), 7.48 (m, 1H),
7.44 (m, 4H), 7.39 (m, 1H), 7.24 (m, 1H). 13C NMR (CDCl3):
140.4, 138.3, 137.6, 128.7 (2 × CH), 128.2 (2 × CH), 126.7, 125.4
(2 × CH), 124.9, 123.1 (2 × CH), 105.1.
4-Phthalimido-1,3-diphenyl-1H-pyrazole (62). Compound 61
(5.3 g, 20 mmol) was suspended in N,N-dimethyldimethoxyfor-
mamide (3.8 g, 48 mmol) and heated to 110 °C for 16 h.
Phenylhydrazine hydrochloride (3.2 g, 22 mmol) was added
followed by 90% aqueous EtOH (100 mL). The mixture was stirred
at 90 °C for 2 h. After cooling the precipitated product was collected
by filtration and washed with EtOH (2 × 10 mL) (yield 6.84 g,
92%). Mp: 232-234 °C. Lit.29 mp: 233-235 °C. 1H NMR
(CDCl3): 7.89 (dd, 2H), 7.81 (s, 1H), 7.75 (dd, 2H), 7.30 (s, 5H),
7.27 (m, 2H), 7.22 (m, 3H). 13C NMR (CDCl3): 167.4, 140.4, 139.4,
138.5, 134.1 (2 × CH), 131.5 129.0 (2 × CH), 128.7 (2 × CH),
128.6 (2 × CH), 128.4 (2 × CH), 127.8, 127.4, 124.7 (2 × CH),
123.6 (2 × CH), 112.6.
N-(1,4-Diphenyl-1H-pyrazol-5-yl)benzamide (69) was prepared
by dropwise addition of benzoyl chloride (0.17 g, 1.2 mmol) to
diphenylpyrazole 68 (0.23 g, 1.0 mmol) in a mixture of N,N-
diisopropylethylamine (0.64 g, 4 mmol) and THF (10 mL) at 20
°C. After 2 h, water (2 mL) was added and the product extracted
with EtOAc (3 × 20 mL). Purification on silica with hexane-
EtOAc (1:4), followed by conversion to the hydrochloride, gave
0.20 g (53%). Mp: 143-145 °C. 1H NMR (CDCl3): 7.08 (t, 1H),
7.14 (t, 1H), 7.18 (t, 2H), 7.23 (t, 2H), 7.33 (t, 2H), 7.39 (d, 2H),
7.54-7.47 (m, 3H), 7.86 (s, 1H), 7.98 (d, 2H). 13C NMR (CDCl3):
112.7, 123.4 (2 × CH), 126.6 (2 × CH), 127.3, 127.9, 128.1, 129.3
(2 × CH), 129.4 (2 × CH), 129.7 (2 × CH), 130.9 (2 × CH),
131.3, 135.0, 138.4, 139.2, 141.1, 163.4. HRMS (C22H17N3O):
obsd, 339.1372; calcd, 339.1371. Anal. (C22H17N3O·0.5HCl) C, H,
N.
4-Amino-1,3-diphenyl-1H-pyrazole (63). Compound 62 (3.7 g,
10 mmol) was suspended in EtOH (50 mL), 50% hydrazine hydrate
(1.5 g, 30 mmol) was added, and the mixture was stirred at 60 °C
for 1.5 h. After cooling to 20 °C the precipitated phthaloyl hydrazide
was removed by filtration, and the mother liquors were reduced to
15 mL by evaporation. Addition of i-Pr2O (15 mL) and cooling
gave 1.68 g (71%). Lit.29 yield: 90% (no data). Mp: 126-128 °C.
1H NMR (CDCl3): 7.47 (s, 1H), 7.38 (d, 1H), 7.33 (m, 2H), 7.24
(m, 7H).
3-Cyano-N-(1,4-diphenyl-1H-pyrazol-5-yl)benzamide (70) was
prepared from 3-cyanobenzoyl chloride (0.16 g, 1.0 mmol) and
diphenylpyrazole 68 as described for compound 21. Chromatog-
raphy on silica with hexane-EtOAc (1:4) yielded 68% of an off-

3342 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11
de Paulis et al.
1
white solid. Mp: 214-215 °C. H NMR (CDCl3): 7.04 (s, 1H),
Anal. (C20H15N3O·HCl·0.3H2O) C, H, N. HRMS (C20H15N3O):
obsd, 313.1214; calcd, 313.1215.
7.75-7.38 (m, 9H), 7.92 (d, 2H), 8.01 (d, 1H), 8.02 (d, 1H), 8.33
(s, 1H), 10.7 (s, 1H). 13C NMR (CDCl3): 101.7, 112.1, 118.5, 123.7
(2 × CH), 125.5 (2 × CH), 127.9, 128.5, 129.1 (2 × CH), 129.6
(2 × CH), 130.4, 131.7, 132.8, 133.0, 134.5, 136.0, 137.0, 139.1,
150.6, 164.6. HRMS (C23H16N4O): obsd, 364.1326; calcd, 364.1324.
Anal. (C23H16N4O) C, H, N.
4-Nitro-N-(1,4-diphenyl-1H-pyrazol-5-yl)benzamide (71) was
prepared from 4-nitrobenzoyl chloride (1.0 g, 5.4 mmol) and
diphenylpyrazole 68 (1.0 g, 4.2 mmol) as described for compound
21. Recrystallization from EtOH (20 mL) gave 0.82 g (51%). Mp:
3-Cyano-N-(2-phenylimidazo[1,2-a]pyridin-3-yl)benzamide (76)
was prepared from 3-cyanobenzoyl chloride (0.16 g, 1 mmol) and
compound 74 (0.22 g, 1 mmol) as described for compound 21.
Chromatography on silica with hexane-EtOAc (1:4). Conversion
to the hydrochloride yielded 48% of an off-white solid. Mp: >260
°C. 1H NMR (CDCl3): 6.83 (t, 1H), 7.23 (d, 2H), 7.31-7.28
(m, 2H), 7.62-7.56 (m, 2H), 7.83-7.78 (m, 2H), 8.45 (d, 1H),
8.53 (s, 1H), 10.9 (s, 1H). 13C NMR (CDCl3): 113.2, 113.4, 115.5,
116.2, 117.0, 118.5, 123.9, 125.2, 126.7, 127.6 (2 × CH), 128.7,
129.0 (2 × CH), 130.2, 132.6, 133.2, 134.1, 135.7, 165.5. HRMS
(C21H14N4O): obsd, 338.1162; calcd, 338.1167. Anal. (C21H14N4O·
HCl·0.7H2O) C, H; N: calcd, 14.46; found, 13.72.
QSAR Modeling. Prediction of the activities of novel analogues
of CDPPB was performed using HQSAR (holographic quantitative
structure-activity relationships) modeling (Tripos, St Louis MO).
This method uses multiple fragments of varying length of the
connection table of each compound to generate overlapping
permutations, encoded into the frequency of occurrence, which serve
as molecular descriptors in the analysis. This technique allows for
rapid modeling independent of geometry optimization or position-
ing. Molecular fragment descriptors using four to eight atom lengths
of the test set were obtained from Sybyl line notation of their 2D
representations (ChemDraw 9.0). The HQSAR program cuts each
molecular fingerprint into strings that are set at the length of 12
prime numbers ranging from 53 to 401. The numerical representa-
tion of the test set was then subjected to partial least-squares analysis
of the binding and functional activities, respectively. Except for
compound 33, compounds with Ki or EC50 values >10 000 nM
were excluded from the analysis.
Competition Binding Assay. Membranes were prepared from
stable human embryonic kidney (HEK) cells, expressing rat
mGluR5a. The cells were homogenized (Polytron) and stored at -80
°C until used. After thawing, membranes were resuspended
(Dounce) in ice-cold buffer containing 50 mM Tris HCl and 0.9%
saline at pH 7.4. Displacement of [3H]methoxyPEPy binding was
performed as described.10,14 Test compounds were dissolved in
100% DMSO and then serially diluted in assay buffer to 5× stock
solution at 0.63% DMSO. The stock solution was added in triplicate
to 96-well plates (0.1 mL) to yield final concentrations of 0.13%
DMSO, 2 nM [3H]methoxyPEPy, and 0.1 mg of cell protein/mL
of buffer. The plates were incubated for 60 min at room temperature
with gentle shaking.43 Bound radioligand was separated from free
ligand by rapid filtration using a Brandel 96-well harvester (Brandel,
Gaithersburg MD) on GF/B filters (Unifilter-96, PerkinElmer Life
Sciences, Boston MA). Scintillation fluid (Microscint, PerkinElmer
Life Sciences, Boston, MA) (30 µL) was added to each filter, and
membrane-bound radioactivity was determined in a microplate
scintillation counter (TopCount NXT, PerkinElmer Life Sciences,
Downers Grove, IL). Nonspecific binding was defined by binding
in the presence of 5 µM MPEP. Inhibition constants (Ki) were
calculated from IC50 values using GraphPad Prism v. 4.01 nonlinear
curve fit and are presented as the average result of three separate
experiments ( standard error of the mean (SEM).
Cell Culture. Secondary cortical astrocytes from 2-4-day-old
Sprague Dawley rats were dissected and grown for 1 week under
5% carbon dioxide at 37 °C in Dulbecco’s modified Eagle medium
(DMEM), containing 10% fetal bovine serum (FBS), 2 mM
glutamine, 20 mM N-(hydroxyethyl)piperazine-N′-(2-ethanesulfonic
acid) (HEPES), and antibiotics and antimycotics (Invitrogen,
Carlsbad CA) as described.4 The cells were trypsinized on day 0
and plated onto poly-D-lysine-coated 96-well plates. On day 1, the
culture medium was changed to fresh medium, supplemented with
G-5 growth factor (1:100) (Invitrogen, Carlsbad, CA), and incubated
for 2 days. On day 3, medium was switched to glutamine-free
DMEM containing 10% dialyzed FBS. Assay was performed on
day 4.
1
224-226 °C. H NMR (CDCl3): 8.20 (d, 2H), 8.02 (b, 1H), 7.83
(d, 2H), 7.79 (s, 1H), 7.38 (m, 9H), 7.29 (t, 1H). 13C NMR
(CDCl3): 129.0 (2 × CH), 128.7 (2 × CH), 128.4 (2 × CH), 126.8
(2 × CH), 124.0 (2 × CH) 123.8 (2 × CH). Anal. (C22H15N4O3)
C, H, N.
5-Azido-1,3-diphenyl-1H-pyrazole (72). 1,3-Diphenyl-1H-pyra-
zol-5-ylamine (0.50 g, 2.1 mmol) was dissolved in 5 mL of
trifluoroacetic acid and a solution of NaNO2 (0.66 g, 9.5 mmol) in
1 mL of H2O was added at 0 °C. After stirring for 10 min, a solution
of NaN3 (1.38 g, 21 mmol) in 4 mL of H2O was added, and the
mixture was stirred for 30 min at 20 °C, subjected to extraction
with EtOAc (3 × 30 mL), washed with H2O, dried (Na2SO4),
evaporated, and purified by silica gel chromatography in hexanes-
EtOAc (7:3) to give 0.47 g (86%) of the azido derivative as a solid.
1H NMR (CDCl3): 6.29 (s, 1H), 7.21-7.16 (m, 2H), 7.30-7.24
(m, 4H), 7.54 (d, 2H), 7.70 (d, 2H). 13C NMR (CDCl3): 152.1,
139.1, 138.7, 133.0, 129.4 (2 × CH), 129.1 (2 × CH), 128.8, 127.8,
126.0 (2 × CH), 123.7 (2 × CH), 93.7.
5-(4-Phenyl-1,2,3-triazol-1-yl)-1,3-diphenyl-1H-pyrazole (73).
The azido derivative 72 (0.32 g, 0.9 mmol) was suspended in 50%
aqueous tert-butyl alcohol, and phenylacethylene (0.14 mL, 0.9
mmol), sodium ascorbate (0.1 mg, 0.09 mmol), and CuSO4 (3 mg,
9 umol) were added. After stirring for 18 h at 20 °C, the reaction
was quenched with H2O (5 mL), extracted with EtOAc (3 × 10
mL), and dried over Na2SO4, and the solvent was evaporated, and
the residue was purified by silica gel chromatography with
hexanes-EtOAc (7:3). Collection of the fractions showing a single
spot on TLC gave 0.28 g (52%) of product. Mp: 120-121 °C. 1H
NMR (CDCl3): 6.84 (s, 1H), 7.22-7.14 (m, 6H), 7.30-7.24 (m,
5H), 7.66 (s, 1H), 7.67 (d, 2H), 7.77 (d, 2H). 13C NMR (CDCl3):
152.1, 148.4, 138.0, 135.8, 132.4, 129.8 (2 × CH), 129.4 (2 ×
CH), 129.3 (2 × CH), 129.2, 129.0 (2 × CH), 126.3 (2 × CH),
126.2 (2 × CH), 124.1 (2 × CH), 122.3, 102.7. Anal. (C23H17N5·
0.2H2O) C, H, N.
3-Amino-2-phenylimidazo[1,2-a]pyridine (74). 2-Phenylimid-
azo[1,2-a]pyridine35 (0.20 g) was dissolved in AcOH glacial (5 mL)
and then a saturated solution of NaNO2 was added dropwise until
a heavy green precipitate was formed and filtered off. The
precipitated nitroso derivative was added to a suspension of Zn
(0.40 g) in 5 mL of AcOH-EtOH (1:1), and the resulting mixture
was stirred for 2 h at 20 °C. Addition of 3 N NaOH until pH 12,
extraction with EtOAc, drying over Na2SO4, evaporation, and
purification by silica gel chromatography (EtOAc) provided the
desired compound as a solid in 95% yield. Mp: 210-212 °C. Lit.37
mp: 210-212 °C. 1H NMR (CDCl3): 6.63 (td, 1H), 6.97 (td, 1H),
7.08 (t, 1H), 7.19 (t, 2H), 7.45 (d, 1H), 7.61 (d, 2H), 7.89 (d, 1H).
13C NMR (CDCl3): 176.5, 138.9, 131.2, 129.0 (2 × CH), 128.0,
127.1 (2 × CH), 126.4, 124.7, 123.0, 115.0, 113.6.
N-(2-Phenylimidazo[1,2-a]pyridin-3-yl)benzamide (75) was
prepared from benzoyl chloride (0.16 g, 1.1 mmol) and 3-amino-
2-phenylimidazo[1,2-a]pyridine (74, 0.23 g, 1.0 mmol) as described
by Holzwart.37 Chromatography on silica with hexane-EtOAc (1:
4) gave pure product according to TLC. Conversion to the
hydrochloride gave 0.17 g (52%). Mp: >240 °C. Lit.37 mp: 240-
1
242 °C. H NMR (CDCl3): 6.65 (t, 1H), 7.06 (t, 1H), 7.19-7.11
(m, 3H), 7.42-7.38 (m, 3H), 7.52 (t, 1H), 7.58 (d, 1H), 7.68 (d,
2H), 7.91 (d, 2H), 8.65 (s, 1H). 13C NMR (CDCl3): 112.7, 115.3,
117.6, 123.5, 124.5, 125.6, 127.5 (2 × CH), 128.1 (2 × CH), 128.3,
128.9 (2 × CH), 129.3 (2 × CH), 131.3, 131.9, 132.9, 133.1, 165.1.
Calcium Mobilization Assay. Measurement of calcium tran-
sients in cultured cortical rat astrocytes was performed as de-
scribed.19 On the day of assay, indicator dye45 (Calcium-3 Kit,

PositiVe Allosteric Modulation of mGluR5
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11 3343
(12) Epping-Jordan, M. P.; Nayak, S.; Derouet, F.; Dominguez, H.; Bessis,
A.-S.; Le Poul, E.; Ludwig, B. L.; Mutel, V.; Poli, S. M.; Rocher, J.
P. In vivo characterization of mGluR5 positive allosteric modulators
as novel treatments for schizophrenia and cognitive dysfunction.
Neuropharmacol. 2005, 49, 231-279. Abstr. 42.
(13) Lindsley, C. W.; Wisnoski, D. D. Pyrazole modulators of metabo-
tropic glutamate receptors. PCT Pat. Appl. WO 2005/030128,
2005.
(14) Cosford N. D. P.; Roppe, J.; Therani, L.; Schweiger, E.; Seiders, T.
J.; Chaudary, A.; Rao, S.; Varney, M. A. [3H]-methoxymethyl-MTEP
and [3H]-methoxy-PEPy: Potent and selective radioligands for the
metabotropic glutamate subtype 5 (mGlu5) receptor. Bioorg. Med.
Chem. Lett. 2003, 13, 351-354.
(15) Poon, S. F.; Eastman, B. W.; Chapman, D. F.; Chung, J.; Cramer,
M.; Holtz, G.; Cosford, N. D. P.; Smith, N. D. 3-[3-Fluoro-5-(5-
pyridin-2-yl-2H-tetrazol-2-yl)phenyl]-4-methylpyridine: A highly
potent and orally bioavailable metabotropic glutamate subtype 5
(mGlu5) receptor antagonist. Bioorg. Med. Chem. Lett. 2004, 14,
5477-5480.
Molecular Devices, Sunnyvale CA) was added to the cells, which
incubated for 1 h at 37 °C under 5% carbon dioxide. The dye was
washed once and replaced with Hank’s balanced salt solution
(HBSS) (Invitrogen, Carlsbad CA) containing 20 mM HEPES and
2.5 mM probenecid, adjusted to pH 7.4. Test compounds were
dissolved in 100% dimethyl sulfoxide (DMSO) and serially diluted
in assay buffer to a 5× stock solution containing 0.63% DMSO.
The stock solution was added to each well to 0.13% final DMSO
concentration. Test compounds were added 5 min before the
addition of an EC20 concentration of glutamate (200-300 nM).
Time-dependent calcium flux was measured in a fluorometric
imaging plate reader operating at 384 nm (FlexStation II, Molecular
Devices, Sunnyvale, CA). Effective concentrations (EC50) were
calculated using GraphPad Prism v. 4.01 nonlinear curve-fit and
presented as the average result of three separate experiments (
SEM.
(16) Roppe, J.; Smith, N. D.; Huang, D.; Tehrani, L.;, Wang, B.; Anderson,
J.; Brodkin, J.; Chung, J.; Jiang, X.; King, C.; Munoz, B.; Varney,
M. A.; Prasit, P.; Cosford, N. D. P. Discovery of novel heteroary-
lazoles that are metabotropic glutamate subtype 5 receptor antago-
nists with anxiolytic activity. J. Med. Chem. 2004, 47, 4645-
4648.
(17) Tehrani, L. R.; Smith, N. D.; Huang, D.; Poon, S. F.; Roppe, J. R.;
Seiders, T. J.; Chapman, D. F.; Chung, J.; Cramer, M.; Cosford, N.
D. P. 3-[Substituted]-5-(5-pyridin-2-yl-2H-tetrazole-2-yl)benzoni-
triles: Identification of highly potent and selective metabotropic
glutamate subtype 5 receptor antagonists. Bioorg. Med. Chem. Lett.
2005, 15, 5061-5064.
Acknowledgment. Vanderbilt University is a site in the
NIH-supported Molecular Libraries Screening Center Network
(MLSCN). This work was supported with grants from NINDS,
NIMH, NARSAD, and the Stanley Foundation. We thank Drs.
Ratna Prasad and Carrie Jones for providing test results for
compound 32 and ADX-47273, respectively. Assistance in the
QSAR modeling by Dr. Eric Dawson, Vanderbilt Center for
Structural Biology, is gratefully acknowledged.
Supporting Information Available: Results from elemen-
tal analyses of new compounds and X-ray crystallography of
compound 69. This material is available free of charge via the
Internet at http://pubs.acs.org.
(18) Alagille, D.; Baldwin, R. M.; Roth, B. L.; Wroblewski, J. T.;
Grajkowska, E.; Tamagnan, G. D. Synthesis and receptor assay of
aromatic-ethynyl-aromatic derivatives with potent mGluR5 antagonist
activity. Bioorg. Med. Chem. 2005, 13, 197-209.
(19) Rodriguez, A. L.; Nong, Y.; Sekaran, N. K.; Tamagnan, G.; Conn,
P. J. A close structural analogue of MPEP acts as a neutral allosteric
site ligand on metabotropic glutamate receptor subtype 5 and blocks
the effects of multiple allosteric modulators. Mol. Pharmacol. 2005,
68, 1793-1802.
(20) Chen, Y.; de Paulis, T.; Hemstapat, K.; Conn, P. J. Novel allosteric
potentiators of mGluR5 act through binding to the same site as that
of MPEP, a related allosteric antagonist. Neuropharmacol. 2005, 49,
231-279. Abstr. 25.
(21) Grandberg, I. I.; Ting. W.-P.; Kost, A. N. Pyrazoles. XX. Synthesis
of 5-aminopyrazoles and their sulfonamide derivatives. Zh. Obshchei
Khimii 1961, 31, 2311-2315 (Chem. Abstr. 1962, 56, 25079).
(22) Hunsberger, I. M.; Shaw, E. R.; Fugger, J.; Ketcham, R.; Lednicer,
D. The preparation of substituted hydrazines. IV. Arylhydrazines via
conventional methods. J. Org. Chem. 1956, 21, 394-399.
(23) Yamagouchi, Y.; Katsuyama, I.; Funabiki, K.; Matsu, M.; Shibata,
K. A new expedient route to 2,6-diaryl-3-cyano-4-(trifluoromethyl)-
pyridines. J. Heterocycl. Chem. 1998, 35, 805-810.
(24) Snyder, H. R. 1H-Pyrazolo[3,4-b]pyridines. J. Heterocycl. Chem.
1975, 12, 1303-1304.
(25) Wang, P.; Xie, Z.; Hong, Z.; Tang, J.; Wong, O.; Lee, C.-S.; Wong,
N.; Lee, S. Synthesis, photoluminescence of new 1H-pyrazolo-
[3,4-b]quinoxaline derivatives. J. Mater. Chem. 2003, 13, 1894-
1899.
(26) Fomum, Z. T.; Ifeadike, P. N. Heterocycles of biological importance.
Part 2. Novel synthesis and pharmacological evaluation of 5-amino-
3-alkyl-1-(2-pyridyl)pyrazoles and 5-amino-3-phenyl-1-(2-pyridyl)-
pyrazole from allenic or acetylenic nitriles and 2-hydrazinopyridine.
J. Heterocycl. Chem. 1985, 22, 1611-1614.
(27) Daidone, G.; Plescia, S. Studies on the synthesis of heterocyclic
compounds. Part VI. The action of methyl salicylate on some
5-aminopyrazoles. J. Heterocycl. Chem. 1981, 18, 747-750.
(28) Plescia, S.; Daidone, G.; Sprio, V.; Aiello, E.; Dattolo, G.; Cirrincione,
G. Studies on the synthesis of heterocyclic compounds. Part I. The
Pschorr reaction in the pyrazole series. J. Heterocycl. Chem. 1978,
15, 1287-1290.
References
(1) Conn, P. J.; Pin, J. P. Pharmacology and functions of metabotropic
glutamate receptors. Annu. ReV. Pharmacol. Toxicol. 1997, 37, 205-
237.
(2) Ritzen, A.; Mathiesen, J. M.; Thomsen, C. Molecular pharmacology
and therapeutic prospects of metabotropic glutamate receptor allos-
teric modulators. Basic Clin. Pharmacol. Toxicol. 2005, 97, 202-
213.
(3) Marino, M. J.; Conn, P. J. Direct and indirect modulation of the
N-methyl D-aspartate receptor. Curr. Drug Targets CNS Neurol.
Disord. 2002, 1, 1-16.
(4) Javitt, D. C. Glutamate as a therapeutic target in psychiatric disorders.
Mol. Psychiat. 2004, 9, 984-997.
(5) Cartmell, J.; Schoepp, D. D. Regulation of neurotransmitter release
by metabotropic glutamate receptors. J. Neurochem. 2000, 75, 889-
907.
(6) O′Brien, J. A.; Lemaire, W.; Chen, T. B.; Chang, R. S.; Jacobson,
M. A.; Ha, S. N.; Lindsley, C. W.; Schaffhauser, H. J.; Sur, C.;
Pettibone, D. J. A family of highly selective allosteric modulators
of the metabotropic glutamate receptor subtype 5 (mGluR5). Mol.
Pharmacol. 2003, 64, 731-740.
(7) Williams, D. L.; Lindsley, C. W. Discovery of positive allosteric
modulators of metabotropic glutamate receptor subtype 5 (mGluR5).
Curr. Top. Med. Chem. 2005, 5, 825-846.
(8) O’Brien, J. A.; Lemaire, W.; Wittman, M.; Jacobson, M. A.; Ha, S.
N.; Wisnoski, D. D.; Lindsley, C. W.; Schaffhauser, H. J.; Rowe,
B.; Sur, C. A novel selective allosteric modulator potentiates
the activity of native metabotropic glutamate receptor subtype 5
(mGluR5) in rat forebrain. J. Pharmacol. Exp. Ther. 2004, 309, 568-
577.
(9) Lindsley, C. W.; Wisnoski, D. D.; Duggan, M. E.; Leister, W. H.;
Hartman, G. D.; Huff, J. R.; Williams, D. L.; O’Brien, J. A.; Lemaire,
W.; Sur, C. Discovery of positive allosteric modulators for the
metabotropic glutamate receptor subtype 5 (mGluR5) that poten-
tiate receptor function in vivo. J. Med. Chem. 2004, 47, 5825-
5828.
(29) Chen, C.; Wilcoxen, K.; McCarthy, J. R. A convenient one-pot
synthesis of 4-amino-3-arylpyrazoles from alpha-phthaloylaminoac-
etophenones. Tetrahedron Lett. 1998, 39, 8229-8232.
(30) Anderson, E. L.; Casey, J. E.; Greene, L. C.; Lafferty, J. J.; Reiff,
H. E. Synthesis and muscle relaxant properties of 3-amino-4-
arylpyrazoles. J. Med. Chem. 1964, 7, 259-268.
(10) Kinney, G. G.; O’Brien, J. A.; Lemaire, W.; Burno, M.; Bickel, D.
J.; Clements, M. K.; Chen, T.-B.; Wisnoski, D. D.; Lindsley, C. W.;
Tiller, P. R.; Smith, S.; Jacobson, M. A.; Sur, C.; Duggan, M. E.;
Pettibone, D. J.; Conn, P. J.; Williams, D. L. A novel selective
positive allosteric modulator of metabotropic glutamate receptor
subtype 5 has in vivo activity and antipsychotic-like effects in
rat behavioral models. J. Pharmacol. Exp. Ther. 2005, 313, 199-
206.
(31) Gagnon, P. E.; Boivin, J. L.; Treblay, M. Contribution to the study
of pyrazolones. Can. J. Chem. 1953, 31, 673-684.
(32) Alberti, C. Preparation and properties of 3-alkyl-5-amino-4-phen-
ylpyrazoles. Gazz. Chim. Ital. 1959, 89, 1017-1032 (Chem. Abstr.
1960, 54, 118214).
(11) Bessis, A.-S.; Bonnet, B.; Le Poul, E.; Rocher, J.-P.; Epping-Jordan,
M. P. Allosteric modulators of metabotropic glutamate receptors. PCT
Pat. Appl., WO 2005/044797, 2005.

3344 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11
de Paulis et al.
(33) Almirante, L.; Polo, L.; Mugnaini, A.; Provinciali, E.; Rugarli, P.;
Biancotti, A.; Gamba, A.; Murmann, W. Derivatives of imidazole.
I. Synthesis and reactions of imidazo[1,2-a]pyridines with analgesic,
antiinflammatory, antipyretic, and anticonvulsive activity. J. Med.
Chem. 1965, 8, 305-312.
(40) Acheson, R. M.; Vernon, J. M. Addition reactions of heterocyclic
compounds. Part X. Products from 1-alkylpyrroles and dimethyl
acetylenedicarboxylate, and the synthesis of some indolecarboxylic
esters. J. Chem. Soc. 1962, 1148-1157.
(41) Szmuszkovicz, J.; Glenn, E. M.; Heinzelman, R. V.; Hester, J. B.;
Youngdale, G. A. Synthesis and antiinflammatory activity of 2,3-
bis(p-methoxyphenyl)indole and related compounds. J. Med. Chem.
1966, 9, 527-536.
(34) Buu-Hoi, N. P.; Jacquignon, P.; Xuong, N. D.; Lavit, D. 2-Arylpyr-
rocolines and 2-arylpyrimidazoles. J. Org. Chem. 1954, 19, 1370-
1375.
(35) Chavignon, O.; Raihane, M.; Deplat, P.; Chabard, J. L.; Gueiffer,
A.; Blache, Y.; Dauphin, G.; Teulade, J. C. Effective strategy for
the synthesis of pyrido[1′,2′:1,2]imidazo[5,4-c]isoquinioline system.
Heterocycles 1995, 41, 2019-2026.
(36) Cosford, N. D. P.; Govek, S. P.; Hamill, T. G.; Kamenecka, T.;
Roppe, J. R.; Seiders, T. J. Alkyne derivatives as tracers for
metabotropic glutamate receptor binding. Int. Pat. Appl. WO 038374,
2004. Example 11, 42-43.
(37) Holzwart, R. Beitrage zur kenntniss der polymerisation von Nitrilen.
Ueber dimolekulares Cyanmethyl. J. Prakt. Chem. 1889, 39, 230-
245.
(38) Von Meyer, E. Untersuchungen aus dem organ.-chem. Laboratorium
der Technischen Hochschule zu Dresden. CXII. Zur Kenntnis der
dimolekularen Nitrile. J. Prakt. Chem. 1915, 92, 174-193.
(39) Hackler, R. E. Improvements in and relating to (3-aryl-5-isothiazolyl)-
ureas. Eur. Pat. Appl. 0129407, 1984.
(42) Sheehan, J. C.; Bolhofer, W. A. An improved procedure for the
condensation of potassium phthalimide with organic halides. J. Am.
Chem. Soc. 1950, 72, 2786-2789.
(43) Patel, S.; Krause, S. M.; Hamill, T.; Chaudhary, A.; Burns, H. D.;
Gibson, R. E. In vitro characterization of [3H]MethoxyPyEP, an
mGluR5 selective radioligand. Life Sci. 2003, 73, 371-379.
(44) Peavy, R. D.; Chang, M. S. S.; Sanders-Bush, E.; Conn, P. J.
Metabotropic glutamate receptor 5-induced phosphorylation of ex-
tracellular signal-regulated kinase in astrocytes depends on trans
activation of the epidermal growth factor receptor. J. Neurosci. 2001,
21, 9619-9628.
(45) Zhang, Y.; Kowal, D.; Kramer, A.; Dunlop, J. Evaluation of FLIPR
calcium 3 assay kitsA new no-wash fluorescence calcium indicator
reagent. J. Biomol. Screen 2003, 8, 571-577.
JM051252J