Bioisosteric replacement of dihydropyrazole of 4S-(-)-3-(4-chlorophenyl)-N-methyl-N′-[(4-chlorophenyl)-sulfonyl]- 4-phenyl-4,5-dihydro-1H-pyrazole-1-caboxamidine (SLV-319) a potent CB1 receptor antagonist by imidazole and oxazole

Article abstract of DOI:10.1016/j.bmcl.2007.12.036

Design, synthesis and conformational analysis of few imidazole and oxazole as bioisosters of 4S-(-)-3-(4-chlorophenyl)-N-methyl-N′-[(4-chlorophenyl)-sulfonyl]- 4-phenyl-4,5-dihydro-1H-pyrazole-1-caboxamidine (SLV-319) 2 is reported. Computer assisted conf

Full text of DOI:10.1016/j.bmcl.2007.12.036

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 963–968
Bioisosteric replacement of dihydropyrazole
of 4S-(ꢀ)-3-(4-chlorophenyl)-N-methyl-N0-
[(4-chlorophenyl)-sulfonyl]-4-phenyl-4,5-dihydro-1H-
pyrazole-1-caboxamidine (SLV-319) a potent CB1 receptor
antagonist by imidazole and oxazoleq
Brijesh Kumar Srivastava,a,* Rina Soni,a Amit Joharapurkar,b
Kalapatapu V. V. M. Sairam,c Jayendra Z. Patel,a Amitgiri Goswami,a
Sandeep A. Shedage,a Sidhartha S. Kar,a Rahul P. Salunke,a Shivaji B. Gugale,a
Amol Dhawas,a Pravin Kadam,a Bhupendra Mishra,a Nisha Sadhwani,d
Vishal B. Unadkat,d Prasenjit Mitra,d Mukul R. Jainb and Pankaj R. Patela
aDepartment of Medicinal Chemistry, Zydus Research Centre, Cadila Healthcare Ltd,
Sarkhej-Bavla N.H. 8A, Moraiya, Ahmedabad 382210, India
bDepartment of Pharmacology, Zydus Research Centre, Cadila Healthcare Ltd,
Sarkhej-Bavla N.H. 8A, Moraiya, Ahmedabad 382210, India
cDepartment of Bio-informatics, Zydus Research Centre, Cadila Healthcare Ltd,
Sarkhej-Bavla N.H. 8A, Moraiya, Ahmedabad 382210, India
dDepartment of Cell-Biology, Zydus Research Centre, Cadila Healthcare Ltd,
Sarkhej-Bavla N.H. 8A, Moraiya, Ahmedabad 382210, India
Received 12 September 2007; revised 27 November 2007; accepted 15 December 2007
Available online 23 December 2007
Abstract—Design, synthesis and conformational analysis of few imidazole and oxazole as bioisosters of 4S-(ꢀ)-3-(4-chlorophenyl)-
N-methyl-N0-[(4-chlorophenyl)-sulfonyl]-4-phenyl-4,5-dihydro-1H-pyrazole-1-caboxamidine (SLV-319) 2 is reported. Computer
assisted conformational analysis gave a direct clue for the loss of CB1 antagonistic activity of the ligands without a fine docking
simulation for the homology model.
Ó 2007 Elsevier Ltd. All rights reserved.
CB1 receptor antagonist is a promising approach to
treat the obesity by reducing appetite and body weight.1
Rimonabant hydrochloride (1) (SR 141716A) (Fig. 1) is
the first therapeutically potent and selective CB1 recep-
tor antagonist, recently approved in Europe as antiobe-
sity drug, which belongs to diaryl pyrazole family.2
tures have been disclosed.3–5 Lange et al. from Solvay
Pharmaceuticals have disclosed the 3,4-diaryl dihydro-
pyrazole 2 (SLV-319) (Fig. 1) as a CB1 antagonist,
which has elicited potent in vitro6 and in vivo7 activities
and are currently in Phase IIB.
Bioisosteric replacement is one of the modest methodolo-
gies to create therapeutically equivalent surrogates. There
are number of reports for the bioisosteric replacement of
pyrazole nucleus of rimonabant 1 by pyrazine,8 imidaz-
ole,9 thiazoles,10 triazoles10 and dihydropyrazoles.11
Since the discovery of Rimonabant, several classes of
CB1 receptor antagonists with diverse chemical struc-
Keywords: Bioisosteric replacement; CB1 receptor antagonist; Imidaz-
ole; Oxazole; Homology model; Conformational analysis.
q
ZRC communication # 235
More recently, we have synthesized several diaryl dihyd-
ropyrazole carboxamide derivatives using bioisosteric
replacement in a rational approach in conjunction with
*
Corresponding author. Tel.: +91 2717 250801; fax: +91 2717
250606; e-mail addresses: brijeshsrivastava@zyduscadila.com;
bksri2000@yahoo.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.12.036

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B. K. Srivastava et al. / Bioorg. Med. Chem. Lett. 18 (2008) 963–968
Table 1. In vitro hCB1 functional assay for assessing cAMP activity
for compounds 3–7
Cl
O
N
N
Compound
Concentration
(lM)
hCB1 (cAMP)a
N
.HCl
pmol/lg protein
H
N
N
DMSO
Forskolin
WIN-55212-2
0.04 0.00
10.12 0.64
1.27 0.04
0.89 0.07
0.91 0.19
1.01 0.17
0.78 0.05
0.95 0.08
4.09 0.27
8.45 1.30
N
N
O
Cl
O
10
100
10
Cl
H
S
N
3
4
5
6
7
2
1
10
10
Cl
Cl
10
10
1
2
Figure 1. Potent CB1 receptor antagonists.
10
10
a Values indicate mean SD performed in duplicate and the results
being representative of at least three independent experiments.
molecular modeling studies.12 Wherein the optimization
of the diaryl dihydropyrazole-3-carboxamide class of
compounds led to the compound as potent CB1 receptor
antagonist with significant antiobesity effect in animal
model and similar interactions of the diaryl dihydropy-
razole-3-carboxamides have been observed in the homol-
ogy models of CB1 receptor as those with 1 and 2.12
Table 2. In vitro hCB1 functional assay for assessing cAMP activity at
lower concentrations of WIN-55212-2 for compounds 3–7
Compound
Concentration
(lM)
hCB1 (cAMP)a
pmol/lg protein
DMSO
Forskolin
WIN-55212-2
0.03 0.00
1.98 0.23
0.69 0.26
1.22 0.08
0.94 0.06
0.55 0.00
0.60 0.31
1.3 0.15
However, no studies have been disclosed towards bio-
isosteric replacement of dihydropyrazole moiety of sul-
2
1
fonyl carboxamidine derivative
heterocycles.
2
by different
3
4
5
6
7
10
10
10
10
10
In continuation of our cannabinoid research,12–17 we se-
lected compound 2 for further modification and the
dihydropyrazole system of 2 was replaced by isosters
such as imidazole and oxazole to afford the compounds
3–7 (Fig. 2).18 Further the compounds 3–7 were studied
for in vitro (Tables 1 and 2), in vivo pharmacological
evaluation in relevant CB1 antagonist models (Table
3) and conformational analyses (Figs. 3 and 4).
a Values indicate mean SD performed in duplicate and the results
being representative of at least three independent experiments.
corresponding imidazole derivatives were synthesized
as described in the literature.9,10,19 Hence, ethyl ester
derivative 8 was directly converted into amide 9 using
trimethylaluminium and ammonium chloride.20 Amide
derivative 9 was converted to nitrile intermediate 10
The compounds 3–7 have been synthesized as depicted
in Scheme 1. The oxazole ethyl ester derivative 8 and
Cl
Cl
Cl
N
N
N
Cl
Cl
Cl
N
O
N
O
N
O
S
O
S
S
O
HN
N
HN
N
HN
N
O
3
5
4
Cl
Cl
Cl
Cl
O
O
N
O
N
O
S
S
O
HN
N
HN
N
O
6
7
Figure 2. Novel Imidazole and Oxazole bioisosters of SLV-319.

B. K. Srivastava et al. / Bioorg. Med. Chem. Lett. 18 (2008) 963–968
965
of triethylamine affording compound 6.23 Employing
the similar set of transformations the compounds 3–5
and 7 were also synthesized.
Table 3. In vivo efficacy of compounds 3–7 in 5% sucrose solution
intake model in female Zucker fa/fa rats at a single oral dose of 10 mg/
kg
Compound
5% Sucrose solution
intake in 4 h in grama
% Inhibition in intake
of 5% sucrose solution
The bioisosters 3–7 have been synthesized and evaluated
in two CB1 antagonist assays.24,25 There are number of
in vitro assay employed to explore the functionality of
CB1 ligands. The cAMP quantification is one of the
most commonly used methods.26 The in vitro screening
of the compounds 3–7 was done in hCB1 (cAMP)
assay24 and to our surprise, the compounds 3–7 did
not response significantly in the forskolin-stimulated
cAMP assay as compared to positive controls 1 and 2
(Table 1). Unlike 1 and 2 none of the compounds res-
cued 100 lM WIN-55212-2 mediated decrease of for-
skolin induced cyclic AMP generation. We further
tested the antagonism of the compounds against 1 lM
WIN-55212-2, where compounds 3 and 7 showed partial
reversal of cyclic AMP decrease induced by 1 lM of the
agonist (Table 2). In order to confirm this loss of respon-
siveness as CB1 antagonist, the same set of molecules
was evaluated against appetite suppression model in
Control
37.9 3.8
32.4* 4.7
37.1 2.9
35.4 3.2
36.3 4.1
37.7 5.2
23.6* 2.7
24.2* 4.2
3
4
5
6
7
2
1
14.4 6.8
2.2 8.5
6.7 7.3
4.3 12.1
0.6 14.1
37.6 5.3
36.1 10.5
a Values indicate Mean SEM for n = 6 rats in 4 h.
* p < 0.05, when compared with the control group, one way ANOVA
followed by Dunnett’s multiple comparison test.
using oxalyl chloride and dimethyl formamide.21 Ami-
dine 11 was conveniently synthesized by reacting nitrile
derivative 10 and methylamine hydrochloride using tri-
methylaluminium.22 Finally, amidine 11 was reacted
with p-chlorobenzenesulfonylchloride in the presence
Figure 3. (a) Energy-minimized structure of compound 2. (b) Energy-minimized structure of compound 3. (c) Energy minimized structure of
compound 6.
Figure 4. (a) Superimposition of molecule 3 (line mode) with 2 (stick mode). (b) Superimposition of molecule 5 (line mode) with 2 (stick mode). (c)
Superimposition of molecule 6 (line mode) with 2 (stick mode).

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B. K. Srivastava et al. / Bioorg. Med. Chem. Lett. 18 (2008) 963–968
Cl
Cl
Cl
(b)
(a)
N
O
N
O
N
O
CN
COOC2H5
CONH2
10
8
9
(c)
Cl
Cl
(d)
Cl
N
N
N
O
O
O
S
O
HN
NH
HN
11
6
Scheme 1. Reagents and conditions: (a) Al(Me)3 (2.0 M sol in toluene), NH4Cl, C6H6, 78–80 °C, 2 h, 68%; (b) (COCl)2, DMF, 0–25 °C, 1 h, 77%; (c)
Al(Me)3 (2.0 M sol in toluene), CH3NH2ÆHCl, C7H8, 108–110 °C, 2 h, 90%; (d) 4-ClC6H4SO2Cl, Ch2Cl2, NEt3, 0–5 °C, 16 h, 45%.
rodents. The CB1 receptor antagonist markedly and
selectively reduces sucrose feeding and drinking in ro-
dents and in obese Zucker fa/fa rats,27,28 thus the
in vivo effects of the compounds 3–7 were evaluated in
5% sucrose solution intake model in female Zucker fa/
fa rats25 (Table 3). Notably, all the compounds 3–7
showed no suppression of sucrose solution consumption
while compounds 1 and 2 induced a significant reduc-
tion in the solution intake. The in vitro and in vivo re-
sults prompted us to further verify the loss in CB1
receptor antagonistic activity. The computer assisted
conformational analysis of the compounds 3–7 was car-
ried out to establish the correlation between the orienta-
tion and biological activity of the molecules.
could well provide the explanation for bioisosters 3–7 did
not show CB1 antagonistic activity.
In summary, the bioisosteric replacement of dihydropy-
razole nucleus of compound 2 by imidazole and oxazole
resulted in the complete loss of required conformation
of the molecules, which is suggested to be necessary
for CB1 receptor binding. Thus, bioisosters 3–7 did
not show any pharmacological effect as CB1 receptor
antagonist. In the absence of crystallized receptor–li-
gand complexes and without performing the molecular
modeling in the homology model our conformational
analysis still gave valuable information on the recep-
tor–ligand interactions.
From the energy-minimized29 structures of compounds
2, imidazole isoster 3 and oxazole isoster 6, large differ-
ences have been observed in the orientation of com-
pounds 3 and 6 as compared to compound 2 (Fig. 3),
which may be attributed for the loss in CB1 receptor
antagonistic activity.
Acknowledgments
We thank both the reviewers for number of excellent
suggestions, management of Zydus Group for encour-
agement and analytical department for support.
A general CB1 inverse agonist pharmacophore model re-
quired for crucial receptor–ligand interaction has been
proposed on the basis of the CB1 receptor modeling.4
Furthermore; conformational analysis29,30 was carried
out on compounds 2, 3, 5 and 6. As the position of nitro-
gen changed, substitution on the central five membered
ring also changed because of the change in the hybridiza-
tion state. As can be seen from Figure 4, orientation of the
p-chlorophenyl in the ligand 3 has become perpendicular
to the p-chlorophenyl in 2. There is also a rotation of the
phenyl ring in the 4th position of the pyrazole ring. Simi-
lar conformational changes have been observed in the
compound 6. As in both compounds 3 and 6, it was ob-
served that the change in nitrogen position altered the po-
sition of phenyl ring and p-chlorophenyl substituent, this
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J = 8.55 Hz, 2H), 7.62–7.59 (m, 3H), 7.55 (d, J = 3.76 Hz,
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1591, 1556 cmꢀ1; ESI-MS: 487.1 [M+H]+.
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24. In vitro cAMP assay: Fatty acid-free BSA, IBMX (isobu-
tyl methyl xanthine), RO20-1724 {4-[(3-butoxy-4-
methoxyphenyl) methyl]-2-imidazololidinone}, forskolin
and DMSO (hybrimax) were purchased from Sigma
Chemical Co. cAMP detection ELISA kit was from Assay
Designs, USA. Tissue culture reagents were purchased
from Sigma and Hi-media. Other reagents used were all of
analytical grade. The cAMP assay was carried out in
Chinese Hamster Ovarian (CHO) cells (CHOK1) stably
expressing human CB1 receptor following the method of
Rinaldi-Carmona et al.26 Cells grown to 80% confluence
were maintained in HAM’S F12 medium containing 10%
heat inactivated dialyzed fetal bovine serum and 0.8 mg/
mL G-418. Cells were seeded at a density of 50,000 cells/
well in 24-well plate, grown for 16–18 h, washed once with
PBS and incubated for 30 min at 37 °C in plain HAM’S
F12 containing 0.25% free fatty acid BSA, IBMX
(0.1 mM) and RO20-1724 (0.1 mM). IBMX, the pan
phosphodiesterase inhibitor and RO20-1724, the specific
phosphodiesterase- 4 inhibitor were added to restore
cAMP up to the detection limit. After 5 min incubation
with the drugs, forskolin was added at a final concentra-
tion of 10 lM and incubation was carried out for another
20 min at 37 °C. The reaction was terminated by washing
once with PBS and adding 200 lL lysis buffer comprising
0.1 N HCl and 0.1% Trition X-100. The lysates were
centrifuged and aliquotes from supernatants were used for
detection of cAMP by ELISA as per the manufacturer’s
protocol.
18. Characterization data for compounds 3–7: 4-Chloro-N-{[2-
(4-chlorophenyl)-1-phenyl-4,5-dihydro-1H-imidazol-4-yl]-
methylamino-methylene} benzenesulfonamide 3: 70%
1
yield; 99.25% purity by HPLC; mp 98–100 °C; H NMR
(300 MHz, DMSO-d6): d 8.34 (d, J = 4.34 Hz, 1H), 7.88–
7.85 (dd, J = 6.72 and 1.83 Hz, 2H), 7.62–7.59 (dd,
J = 6.78 and 1.87 Hz, 2H), 7.55 (d, J = 8.55 Hz, 2H),
7.43 (d, J = 8.56 Hz, 2H), 7.24 (t, J = 7.76 Hz, 2H), 7.08 (t,
J = 7.70 Hz, 1H), 6.91 (d, J = 7.54 Hz, 2H), 5.53–5.46 (dd,
J = 11.81 and 9.48 Hz, 1H), 4.54 (t, J = 11.19 Hz, 1H),
3.86 (t, J = 9.90 Hz, 1H), 2.77 (d, J = 4.65 Hz, 3H); IR
(KBr) 3332, 1581, 1537 cmꢀ1; ESI-MS: 489 [M+H]+.
4-Chloro-N-{[1-(4-chlorophenyl)-2-phenyl-4,5-dihydro-
1H-imidazol-4-yl]-methylamino-methylene}-benzenesul-
fonamide 4: 65% yield; 99.10% purity by HPLC; mp 95–
1
97 °C; H NMR (300 MHz, DMSO-d6): d 7.92–7.89 (dd,
J = 6.75 and 1.83 Hz, 2H), 7.47–7.43 (m, 6H), 7.36–7.31 (t,
J = 7.59 Hz, 2H), 7.16–7.14 (dd, J = 6.78 and 2.0 Hz, 2H),
6.75–6.72 (dd, J = 6.84 and 2.0 Hz, 2H), 5.74 (t,
J = 10.9 Hz, 1H), 4.69 (t, J = 11.23 Hz, 1H), 4.28 (t,
J = 10.32 Hz, 1H), 2.91 (d, J = 4.95 Hz, 3H); IR (KBr)
3328, 1583, 1531 cmꢀ1; ESI-MS: 489.1 [M+H]+.
25. 5% Sucrose Solution Intake in Zucker fa/fa rats: All the
animals used in the study were procured from the
Animal Breeding Facility of Zydus Research Center.
Institutional Animal Ethical Committee approved all the
study protocols. Female Zucker fa/fa rats (age of 10–12
weeks and 300–350 g of weight) were used for in vivo
experiments, compounds were suspended with 0.5%
carboxymethylcellulose sodium salt in distilled water.
The test compounds were administered at the dose of
10 mg/kg and by oral route in a volume of 2 mL/kg
4-Chloro-N-{[1-(4-chlorophenyl)-2-phenyl-1H-imidazol-
4-yl]-methylamino-methylene}-benzenesulfonamide 5: 30%
1
yield; 98.71% purity by HPLC; mp 151–153 °C; H NMR
(300 MHz, DMSO-d6): d 9.09 (d, J = 4.66 Hz, 1H), 8.31 (s,
1H), 7.86 (d, J = 8.50 Hz, 2H), 7.60 (d, J = 8.67 Hz, 4H),
7.40 (d, J = 8.69 Hz, 2H), 7.37 (br s, 5H), 2.87 (d,
J = 4.62 Hz, 3H); IR (KBr) 3340, 1577, 1558 cmꢀ1; ESI-
MS: 486.0 [M+H]+.

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body weight. The obese Zucker fa/fa rats were housed
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F.; Bouaboula, M.; Carayon, P.; Casellas, P.; Le Fur, G.
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29. The energy minimization procedure adopted is smart
minimizer protocol in Discovery Studio 1.6, where 100
cycles of minimization are allowed at steepest descent,
conjugate gradient and Newton–Raphson methods. All
the modeling procedures adopted in the study are molec-
ular mechanics with Charm Force Field.
individually and subjected to training for consuming 5%
sucrose solution over a period of 4 h, by allowing access
to the 5% sucrose solution in the bottles. Food and
water were withdrawn during this time. This training
was given for six consecutive days, at the same time of
the day. On seventh day, the animals were randomized
into groups of six animals each and treated with the test
compounds. After one hour of treatment, the animals
were exposed to the 5% sucrose solution for 4 h as that
of the training schedule. The amount of sucrose solution
consumed by each animal was calculated. Difference
between the control and treatment groups were analyzed
by performing one way ANOVA followed by Dunnett’s
test on sucrose solution consumption using Graph pad
Prism software.
30. All the computations were carried out on Accelrys Inc.
Discovery Studio 1.6. Accelrys Inc., San Diego, CA.