Pyrazole derivatives as potent inhibitors of c-Jun N-terminal kinase: Synthesis and SAR studies

Article abstract of DOI:10.1016/j.bmc.2014.08.028

Mitogen activated protein kinases including c-Jun N-terminal kinase play an indispensable role in inflammatory diseases. Investigation of reported JNK-1 inhibitors indicated that diverse heterocyclic compounds bearing an amide group rendered potent JNK-1 inhibitory activity which prompted us to synthesize new JNK-1 inhibitors containing a pyrazole heterocyclic group. A DABCO mediated 1,3-dipolar cycloaddition reaction in neat resulted in pyrazole carboxylic acid which was converted to desired amides. Upon confirmation of the structures, all the compounds were screened for JNK-1 inhibitory activity and in vivo anti-inflammatory activity. Several synthesized analogues have exhibited JNK-1 inhibitory activity less than 10 μM, in particular compounds 9c, 10a and 10d were found to be potent among all the compounds.

Full text of DOI:10.1016/j.bmc.2014.08.028

Bioorganic & Medicinal Chemistry 22 (2014) 6209–6219
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Pyrazole derivatives as potent inhibitors of c-Jun N-terminal kinase:
Synthesis and SAR studies
Anuradha Doma a, Ravindra Kulkarni a, , Radhakrishna Palakodety b, G. Narahari Sastry c,
Janardhan Sridhara c, Achaiah Garlapati a,
⇑
a University College of Pharmaceutical Sciences, Kakatiya University, Warangal 506009, Andhra Pradesh, India
b D-206/B, Discovery Laboratory, Organic Chemistry Division-III, Indian Institute of Chemical Technology, Hyderabad 50067, India
c Centre for Molecular Modeling, Indian Institute of Chemical Technology, Hyderabad 50067, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 30 May 2014
Revised 21 July 2014
Accepted 21 August 2014
Available online 6 September 2014
Mitogen activated protein kinases including c-Jun N-terminal kinase play an indispensable role in
inflammatory diseases. Investigation of reported JNK-1 inhibitors indicated that diverse heterocyclic
compounds bearing an amide group rendered potent JNK-1 inhibitory activity which prompted us to syn-
thesize new JNK-1 inhibitors containing a pyrazole heterocyclic group. A DABCO mediated 1,3-dipolar
cycloaddition reaction in neat resulted in pyrazole carboxylic acid which was converted to desired
amides. Upon confirmation of the structures, all the compounds were screened for JNK-1 inhibitory activ-
ity and in vivo anti-inflammatory activity. Several synthesized analogues have exhibited JNK-1 inhibitory
Keywords:
JNK-1
Inflammation
Pyrazoles
IC50
activity less than 10
compounds.
l
M, in particular compounds 9c, 10a and 10d were found to be potent among all the
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
activate downstream substrates including numerous transcription
factors. JNKs are well known for their ability to activate
The c-Jun N-terminal kinases (JNK), a group of conserved family
transcriptional factor c-Jun by phosphorylation at both Ser-63
and Ser-73 within the transactivation domain of the transcription
factor c-Jun.12 c-Jun, is a component of activator protein-1(AP-1)
complex, which is an important transcription factor that is
activated by JNK mediated phosphorylation.13 The other
transcription factors that are activated by JNK are ATF2 and
Elk-1.14 Activated ATF2 heterodimerizes with c-Jun and stimulates
the expression of c-Jun gene where as activated Elk-1 is involved
in induction of the c-fos gene that induces heterodimer formation
of AP-1 with c-Jun.15 Activation of ATF2 and c-Jun transcription
factors activate the promoters of matrix metallo proteinase
genes and c-Jun itself resulting in expression of matrix metallo
proteinases that degrade type II collagen in rheumatoid arthritis.
Thus JNKs play a key role in autoimmune and inflammatory
diseases including arthritis, neurological, metabolic diseases and
cancer.16,17
Numerous compounds with different heterocyclic rings have
been reported to possess JNK-1 inhibitory activity including
pyrazole, indazole, pyridine and benzotriazole scaffold18,19 and
few of the reported molecules are in advanced stage of clinical
trials Figure 1a. Our group is actively involved in design and devel-
opment of p38 MAP kinase inhibitors, now intended to expand the
p38 kinase drug discovery to JNK-1 which is also a known
validated target for inflammatory diseases.20–25
of serine/threonine mitogen activated protein kinases, are involved
in vital regular physiological functions in human life. Three distinct
genes Jnk1, Jnk2, and Jnk3 have been identified which encode for
ten different JNKs with varying size from 46 kDa to 55 kDa and
show more than 80% sequence similarity.1,2 JNK-1 and 2 isoforms
expressed ubiquitously while JNK-3 expressed in brain and testis.3
JNKs are activated by proinflammatory cytokines such as TNF-
a
and IL-Ib as well as environmental stress, such as anisomycin, UV
irradiation, hypoxia, and osmotic shock.4,5 Members of the JNK
family (JNK-1, 2 and 3) act as an integrated unit for multiple intra-
cellular biochemical signals governing a wide variety of cellular
processes such as proliferation, apoptosis, migration and transcrip-
tional regulation.6 Like other MAP kinases, JNKs are activated by
dual specific serine/threonine mitogen activated protein kinase
kinase-4 and 7 (MAPKK4 and MAPKK7) by phosphorylation at
Thr-183 and Tyr-185. Both, MAPKK-4 and 7 are further activated
by MAPKKK including ASK1 and TAK-13, depending up on nature
of stimulus and the cell lineage.7–11 Phosphorylated JNKs further
⇑
Corresponding author. Tel.: +91 9440601054; fax: +91 870 2453508.
E-mail addresses: achaiah_g@yahoo.co.in, achaiah1960@gmail.com (A. Garlapati).
Current address: SVERI’s College of Pharmacy, Gopalpur-Ranaji Road, Gopalpur,
Pandharpur 413304, Maharashtra, India.
http://dx.doi.org/10.1016/j.bmc.2014.08.028
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

6210
A. Doma et al. / Bioorg. Med. Chem. 22 (2014) 6209–6219
N
O
O
Cl
Ph
N
N
H
O
O
O
N
H
N
N
N
H
Figure 1a. JNK-1 inhibitors bearing indazole, pyridine and Indole scaffold.
Compound D1, 3-(4-pyridyl)-indazole was identified as novel
2.1. Synthetic methodology
scaffold for JNK inhibition with pIC50 value 5.8 from high through
put screening,26 compound D2 resulted from modification of ATP
competitive inhibitors and retained the activity. Compound D3
was discovered as a new class of potent and selective inhibitor of
JNK3.27 An X-ray crystal structure of D4, a potent JNK inhibitor
bound to the ATP binding site of JNK-128,29 revealed that the phen-
oxy group binds in the pocket occupied by the ribose of the com-
petitive binder ATP. The amide present in D4 is acting as a H-
2.1.1. Procedure for preparation of ethyl 5-phenylpyrazole-3-
carboxylate 3
To a mixture of phenyl acetylene 1 (0.1 g, 1 mmol) and ethyl
diazoacetate
2 (0.114 g) was added DABCO (1,4-diazabicy-
clo[2.2.2]octane) (0.011 g, 0.1 mmol) and stirred for 6 h at ambient
temperature. Upon completion of the reaction which was moni-
tored by TLC, the crude reaction product was purified by passing
through Silica gel using 20% ethyl acetate in pet. ether to afford
compound 3.
bond acceptor making a key interaction with the hinge-region
0
through Met111 (2.8 ÅA). In view of the above reports, we designed
substituted pyrazoles (the general structure, 8a–i, 9a–e and 10a–
h) with phenyl pyrazole and various alkyl, aryl, aralkyl and heteryl
moieties attached via amide linker as shown in Figure 1b. This
manuscript aimed to present synthesis, JNK-1 inhibitory and
anti-inflammatory activity of substituted pyrazoles.
2.1.2. Procedure for preparation of 5-phenylpyrazole-3-
carboxylic acid 4
A solution of the ester 3 (0.1 g, 0.46 mmol) in MeOH (4 mL) was
treated with 4 N NaOH (4 mL) and stirred at rt for 30 min. Later
methanol was removed in vacuo and adjusted to pH 2–3 with aq
1 N HCl and extracted with EtOAc. The organic layer was dried over
anhydrous sodium sulfate and evaporated to get acid 4.
2. Material and methods
All the solvents were purchased from local vendors and are
purified before being used and chemicals were purchased from
M/s Sigma Aldrich, S. D. Fine chemicals and Loba. Pre-coated silica
gel F254 (Merck) plates were employed for thin layer chromatog-
raphy and column chromatography was performed using silica
gel 60–120 mesh. Melting points were recorded in open glass cap-
illaries using Polmon melting point apparatus and are uncorrected.
Infrared spectra were recorded on Shimadzu FT IR spectrophotom-
eter in KBr pellet. Mass spectra obtained on VG-7070H mass
spectrometer. 1H NMR spectra were recorded at 300 MHz on a
Bruker Avance NMR spectrometer in CDCl3 (d 7.26) or DMSO-d6
(d 2.49).
2.1.3. General procedure for preparation of N-substituted 5-
phenylpyrazole-3-carboxamides 8a–i, 9a–e and 10a–h
A solution of acid 4 (0.09 g, 0.47 mmol) in dry DMF (2 mL) trea-
ted sequentially with amine (5a–i, 6a–e and 7a–h; 0.517 mmol)
and triethylamine (0.94 mmol) was stirred under a N2 atmosphere
for 15 min, later TBTU (0.56 mmol) was added and reaction mix-
ture refluxed for 4–10 h. The reaction mixture was quenched with
aq satd NH4Cl solution (10 mL). After 10 min, it was diluted with
CHCl3 (2 ꢀ 10 mL) and washed with water (10 mL), NaHCO3 solu-
tion (10 mL) and brine (10 mL). The organic layers were dried over
anhydrous sodium sulfate, evaporated and the residue purified by
NH
N
N
O
Cl
N
N
N
N
N
H
H
N
H
O
D3
D1
(CH2)n
Ar/Het
N
N
H
8a-i, 9a-e
and 10a-h
O
Cl
N
H
MeO
O
O
N
H
N
N
O
NH
D4
D2
Figure 1b. The design of pyrazole amides 8a–i, 9a–e and 10a–h.

A. Doma et al. / Bioorg. Med. Chem. 22 (2014) 6209–6219
6211
column chromatography using 30% ethyl acetate in pet. ether to
afford corresponding amides 8a–i, 9a–e and 10a–h.
The prepared protein structure (2GMX) was used for grid genera-
tion using the default value of protein atom scaling (1.0) within a
cubic box centered on the co-crystal ligand. The constraints in
hinge region with carbonyl backbone in Glu109, –NH backbone
moiety in Met111 were imposed and extra precision (XP) flexible
docking of ligands was carried out with default value of ligand
atom scaling (0.8). The post docking minimization has been carried
out and maximum of 10 poses per ligand was saved. The best pri-
oritized docked complex structure (10a) was used for grid genera-
tion using the above discussed default parameters and subjected to
XP refinement docking for all the series of Phenyl pyrazole amide
compounds. The obtained docked complex structures were ana-
lyzed and the compounds were prioritized by using docking score,
interactions with active site residues.
2.2. Pharmacological screening methodology
2.2.1. JNK-1 Inhibitory activity
A cell line based in vitro method was followed to screen the
compounds for JNK-1 inhibitory activity. COS cells upon transfec-
tion with FLAG-tagged JNK-1 were exposed to 0.5 M sorbitol for
30 min to elicit maximal JNK-1 activation and then FLAG-JNK
was immune precipitated with GST-ATF2. The immune precipitate
was pretreated by test compound and incubated in buffer B. During
this incubation period phosphorylation takes place and this phos-
phorylated substrate is separated by SDS–PAGE, visualized by
autoradiography and quantitated by Cerenkov counting method
using the kinase profiler service according to the manufacturer
procedure.
2.4. Compound characterization
2.4.1. Ethyl 5-phenyl-3-pyrazole carboxylate 3
2.2.2. Anti-inflammatory activity
IR (KBr) (m
, cmꢁ1): 3141 (NAH str.), 2990 (CAH aromatic str.),
The method described by Winter et al., was followed to screen
the anti-inflammatory activity of synthesized compounds. Suspen-
sion of test compounds were administered orally one hour later 1%
carrageenan was injected into subplantar region of right paw of
rats in all groups and paw volume was monitored at different time
intervals using digital plethysmometer (UGO Basil, Italy). The per-
centage inhibition of paw volume for each test group is calculated
using following equation.30–32
2928 (CH2-str.), 1722 (C@O str.), 1571 (NAH bnd.); 1H NMR
(300 MHz, CDCl3): d 7.57 (d, 2H, Ar-H, J = 7.9 Hz), 7.40–7.22 (br
m, 3H, Ar-H), 7.10 (s, 1H, Ar-H), 4.34 (q, 2H, CH2, J = 7.1 Hz), 1.2–
1.4 (t, 3H, CH3, J = 6.9 Hz); ESI-MS (m/z): 217 [M+H]+.
2.4.2. 5-Phenyl-3-pyrazole carboxylic acid 4
IR (KBr) (m
, cmꢁ1): 3332 (OH str.), 3137 (CAH aromatic str.),
2923 (CH2 str.), 1730 (C@O str.), 1568 (NAH bnd.); 1H NMR
(300 MHz, CDCl3): d 7.56 (d, 1H, Ar-H), 7.50–7.40 (m, 3H, Ar-H),
7.10 (d, 2H, Ar-H), 2.10 (br s, 1H, OH); ESI-MS (m/z): 188 [M+H]+.
Percentageof inhibitionð%Þ ¼ ½1 ꢁ VolumeinmLðtestcompoundÞ
=VolumeinmLðcontrolÞꢂ ꢀ 100:
2.4.3. N-Phenyl-5-phenyl-3-pyrazole carboxamide 8a
IR (KBr) (m
, cmꢁ1): 3375 (NAH str.), 1742 (C@O-str.), 1659 (NAH
2.3. Molecular docking studies
bnd.); 1H NMR (300 MHz, CDCl3): d 13.30 (br s, 1H, NH), 9.21 (br s,
1H, NH) 8.10–7.70 (m, 4H, Ar-H), 7.62–7.21 (m, 6H, Ar-H), 7.10 (s,
1H, Ar-H); ESI-MS (m/z): 264 [M+H]+.
2.3.1. Dataset preparation
A dataset comprising of 22 phenyl pyrazolyl amides were
drawn with all possible tautomers and were minimized with
OPLS-2005 force field using water as solvent in the GB/SA contin-
uum solvation model.33 The extended cutoff includes contribution
of van der Waals interaction (8.0), electrostatic (20), and hydrogen
bond (4.0). The minimization of molecules was carried out using
Polak–Ribiere Conjugate Gradient (PRCG) method with maximum
of 5000 iterations. The extensive conformational search was car-
ried out with Mixed torsional/Low-mode sampling method with
the use of 100 steps per rotatable bond, maximum number of steps
1000, energy window for saving structures with 5.02 kcal/mol,
eliminate the redundant conformers with maximum atom devia-
tion cut-off 0.5 Å and saved 100 structures for each search. The
generated conformers (1177) further subjected to selection of spe-
cific tautomeric states (554) that are fit to hinge region of JNK1 and
other tautomeric states were removed. The selected conformers
were used for molecular docking study.
2.4.4. N-(2,5-Dichlorophenyl)-5-phenyl-3-pyrazole carboxamide
8b
IR (KBr) (m
, cmꢁ1): 3300 (NAH str.), 1665 (C@O-str.), 1580 (NAH
bnd.); 1H NMR (500 MHz, CDCl3, 298 K): d 13.5 (br s, 1H, NH), 11.2
(br s, 1H, NH), 10.1 (br s, 1H, NH), 7.7 (d, 2H, Ar-H, J = 6.9 Hz), 7.5–
7.3 (br m, 3H, Ar-H), 6.7 (s, 1H, CH@C), 2.5 (s, 2H, NH), 1.3 (s, 9H, t-
butyl); MS (ESI) MS (ESI) m/z: 355 [M+Na]+.
2.4.5. N-(4-Nitrophenyl)-5-phenyl-1H-pyrazole-3-carboxamide
8c
IR (KBr) (m
, cmꢁ1): 3421 (NAH str.), 3070 (CAH-aromatic str.),
1790 (C@O str.), 1610 (NAH bnd.); 1H NMR (400 MHz, CDCl3,
298 K): d 7.80–7.90 (m, 4H, Ar-H), 7.50–7.20 (m, 5H, Ar-H), 7.0
(s, 1H, Ar-H); MS (ESI) m/z: 309 [M+H]+.
2.4.6. N-(4-Methoxyphenyl)-5-phenyl-3-pyrazolecarboxamide
8d
2.3.2. Protein preparation
IR (KBr) (m
, cmꢁ1): 3287 (NAH str.), 3005 (CAH-aromatic str.),
The crystal structure of human JNK1 (PDB ID: 2GMX) has been
selected for the docking studies. The 2GMX structure was prepared
by adjusting bond orders, tautomers and adding hydrogen atoms
using protein preparation wizard of Schrödinger software graphi-
cal user interface Maestro v9.3.34 Further the protein was mini-
2928 (CH3 str.), 1606 (C@O-str.), 1507 (NAH bnd.); 1H NMR
(400 MHz, CDCl3, 298 K): d 13.51 (s, 1H, NH), 9.4 (s, 1H, NH),
7.72 (d, 2H, Ar-H, J = 7.3 Hz), 7.70–7.21 (m, 5H, Ar-H), 6.82 (d,
3H, Ar = H, J = 8.7 Hz), 3.9 (s, 3H, OCH3); MS (ESI) m/z: 294
[M+H]+; Anal. Calcd for C17H15N3O2: C, 69.61; H, 5.15. Found: C,
69.50; H, 5.02.
mized by OPLS-2005 force field with converge heavy atoms to
0
RMSD 0.3 ÅA relative to original protein structure.
2.4.7. N-(4-Hydroxyphenyl)-5-phenyl-3-pyrazole carboxamide
2.3.3. Docking studies
8e
In the first step, the docking study has been carried out with
most potent compound (10a) and its conformers with prepared
2GMX crystal structure using Glide module of Schrödinger suite.35
IR (KBr) (
m
, cmꢁ1): 3408 (NAH str.), 3192 (CAH aromatic, str.),
1730 (C@O str.), 1582 (NAH bnd.); 1H NMR (400 MHz, CDCl3,

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A. Doma et al. / Bioorg. Med. Chem. 22 (2014) 6209–6219
298 K): d 7.5 (d, 2H, Ar-H), 7.4–7.3 (m, 3H, Ar-H), 7 (s, 1H, CH@C),
6.7 (br m, 1H, NH), 2.8 (m, 1H, CH), 0.8 (m, 2H, CH2), 0.6 (m, 2H,
CH2); 13C NMR (75 MHz, CDCl3, 278 K): d 128.3 (3C), 127.8 (2C),
125, 102, 40, 21.7, 5.9 (2C); MS (ESI) m/z 280 [M+H]+.
Ar-H), 7.80–7.60 (m, 3H, Ar-H), 7.50–7.20 (m, 4H, Ar-H), 7.15 (s,
1H, Ar-H), 2.60 (s, 3H, CH3); MS (ESI) m/z: 328 [M+Na]+.
2.4.12. N-Benzyl-5-phenyl-3-pyrazole carboxamide 9a
IR (KBr) (m
, cmꢁ1): 3449 (NAH str.), 3053 (CAH aromatic), 2926
2.4.8. N-(4-Bromophenyl)-5-phenyl-3-pyrazole carboxamide 8f
(CH, aliphatic, str.), 1665 (C@O-str.), 1614 (NAH bnd.); 1H NMR
(500 MHz, CDCl3, 298 K); d 8.1 (d, 2H, Ar-H, J = 8.3 Hz), 7.62 (d,
2H, Ar-H, J = 8.3 Hz), 7.52 (t, 3H, Ar-H, J = 7.5 Hz), 7.4–7.32 (m,
3H, Ar-H), 7.2 (s, 1H, NH), 2.35 (s, 1H, CH2); MS (ESI) m/z: 300
[M+Na]+; Anal. Calcd C17H15N3O: C, 73.63; H, 5.45. Found: C,
73.51; H, 5.37.
IR (KBr) (m
, cmꢁ1): 3360 (N–H str.), 3189 (CAH-aromatic, str.),
1602 (C@O-str.), 1547 (NAH bnd.); 1H NMR (400 MHz, CDCl3,
298 K): d 8.1 (d, 1H, Ar-H, J = 8.3 Hz), 7.8 (d, 1H, Ar-H, J = 7.5 Hz),
7.7 (d, 1H, Ar-H, J = 8.3 Hz), 7.5 (d, 3H, Ar-H, J = 6.7 Hz), 7.4–7.2
(br m, 7H, Ar-H), 7.1 (d, 1H, NH), 6.9 (s, 1H, CH@C), 6 (q, 1H, CH,
J = 7.3 Hz), 1.7 (d, 3H, CH3, J = 6.7 Hz); 13C NMR (75 MHz, CDCl3,
278 K): d 160.9, 145.9, 138.1, 133.8, 130.9, 128.9 (3C), 128.6 (3C),
128.2, 126.4, 125.7, 125.4, 125.1, 123.2, 122.5, 103.1, 44.6, 21;
MS (ESI) m/z: 342 [M]+, 344 [M+H]+.
2.4.13. N-(3,4-Dichlorobenzyl)-5-phenyl-3-pyrazole
carboxamide 9b
IR (KBr) (
1755 (C@O-str.), 1611 (NH bnd.); 1H NMR (400 MHz, CDCl3,
298 K): 7.95 (d, 1H, Ar-H, J = 1.5 Hz), 7.86 (d, 1H, Ar-H,
m
, cmꢁ1): 3410 (NAH str.), 2900 (CH, aliphatic, str.),
d
2.4.9. N-(3-Chloro-4-fluorophenyl)-5-phenyl-3-pyrazole
J = 1.7 Hz), 7.71–7.60 (dd, 1H, Ar-H, J = 1.7 Hz), 7.62 (s, 1H, Ar-H),
7.50 (t, 1H, Ar-H, J = 1.8 Hz), 7.46 (s, 1H, NH), 7.40 (s, 1H, Ar-H),
7.40–7.30 (br m, 2H, Ar-H), 7.10 (dd, 1H, Ar-H, J = 2 Hz), 4.70 (s,
2H, CH2); MS (ESI) m/z: 347 [M+H]+; Anal. Calcd for C17H13Cl2N3O:
C, 58.98; H, 3.78. Found: C, 58.89; H, 3.67.
carboxamide 8g
IR (KBr) (m
, cmꢁ1): 3360 (NAH str.), 3189 (CAH-aromatic, str.),
2922 (CH2 str.), 1663 (C = 0 str.), 1528 (NAH bnd.); 1H NMR
(400 MHz, CDCl3, 298 K): d 13.3 (s, 1H, NH), 9.6 (s, 1H, NH), 7.9
(d, 1H, Ar-H, J = 1.5 Hz), 7.7 (d, 2H, Ar-H, J = 8 Hz), 7.6 (br d, 1H,
Ar-H, J = 7.1 Hz), 7.3 (t, 2H, Ar-H, J = 8 Hz), 7.2 (d, 1H, Ar-H,
J = 7.1 Hz), 7.12 (t, 2H, Ar-H, J = 8.9 Hz); MS (ESI) m/z: 337
[M+Na]+; Anal. Calcd for C16H11ClFN3O: C, 60.87; H, 3.51. Found:
C, 60.77; H, 3.40.
2.4.14. N-(4-Fluorobenzyl)-5-phenyl-3-pyrazole carboxamide 9c
IR (KBr) (m
, cmꢁ1): 3325 (NAH str.), 3127 (CAH-aromatic, str.),
2928 (CH2 str.), 1663 (C@O-str.), 1586 (NAH bnd.); 1H NMR
(400 MHz, CDCl3, 298 K): d 8.10 (m, 1H, Ar-H), 7.82 (m, 1H, Ar-
H), 7.81 (m, 1H, Ar-H), 7.82–7.62 (m, 3H, Ar-H), 7.53–7.42 (m,
3H, Ar-H), 6.40 (s, 1H, Ar-H); MS (ESI) m/z: 318 [M+Na]+.
2.4.10. N-Mesityl-5-phenyl-3-pyrazole carboxamide 8h
IR (KBr) (m
, cmꢁ1): 3360 (NAH str.), 3189 (CAH-aromatic, str.),
2922 (CH3, str.), 1658 (C@O-str.), 1568 (NAH bnd.); 1H NMR
(400 MHz, CDCl3, 298 K): d 8.16 (m, 1H, Ar-H), 8.14 (m, 1H, Ar-
H), 7.50 (m, 2H, Ar-H), 7.42 (m, 1H, Ar-H), 6.81 (m, 2H, Ar-H), 6.7
2 (s, 1H, Ar-H), 2.40 (s, 3H, CH3), 2.30 (s, 6H, 2. CH3); MS (ESI) m/
z: 306 [M+H]+.
2.4.15. N-(4-Methoxybenzyl)-5-phenyl-3-pyrazole carboxamide
9d
IR (KBr) (m
, cmꢁ1): 3341 (NAH str.), 3215 (CAH aromatic, str.),
2923 (CH2 str.), 1636 (C@O str.), 1539 (NAH bnd.); 1H NMR
(400 MHz, CDCl3, 298 K): d 9.12 (t, 1H, NH, J = 5.2 Hz), 8.80 (t, 1H,
NH, J = 6 Hz), 7.80 (t, 2H, Ar-H, J = 7.3 Hz), 7.50–7.20 (m, 4H, Ar-
H), 7.0 (s, 1H, Ar-H), 6.91 (s, 2H, Ar-H), 6.82 (d, 1H, Ar-H,
J = 8.3 Hz), 4.42 (dd, 2H, CH2, J = 5.8, 6.4 Hz), 3.71 (s, 3H, OCH3);
MS (ESI) m/z: 308 [M+H]+; Anal. Calcd for C18H17N3O2: C, 70.34;
H, 5.58. Found: C, 70.23; H, 5.47.
2.4.11. N-(3-Acetylphenyl)-5-phenyl-3-pyrazole carboxamide 8i
IR (KBr) (m
, cmꢁ1): 3316 (NAH str.), 3193 (CAH-aromatic, str.),
2924 (CH, aliphatic, str.), 1686 (C@O-str.), 1604 (NAH bnd.); 1H
NMR (400 MHz, CDCl3, 298 K): d 8.10 (m, 1H, Ar-H), 8.0 (m, 1H,
R4
R5
O
H
N
R3
R2
n
Aromatic
COOEt
i
N
-
a
5
COOH
N
R1
a
b
N
Amines
Benzylamines
e
-
H
a
6
N
1
c
N
H
+
n=0, 8a-i
n=1, 9a-e
N
H
7
a
COOEt
-
h
:
H
N2
e
t
O
-
amine
3
NHR
4
2
N
N
H
10a-h
7a-h: Het-amines
R=Alkyl or Aryl Group
6a-e: substiuted benzylamines
a: benzylamine
5a-i substituted anilines
a: aniline
a: 5-methyl-furan-2-yl amine
Reagents and conditions:
b:2,5-dichloro aniline
c: 4-nitroaniline
b:3,4-dichloro benzylamine
c: 4-fluoro benzylamine
d: 3-methoxybenzylamine
e: 3,4-dimethoxy benzylamine
b: 5-(4-methoxy)phenyl-isoxazol-5-yl amine
c: 5-t-butyl-isoxazol-5-yl amine
d: 2-mercaptobenzimidazol-5-yl amine
e: 2-thiazolyl amine
(a) DABCO
d: 4-methoxyaniline
e: 4-hydroxyaniline
f: 4-bromoaniline
g: 4-chloroaniline
h: 2,4,6-trimenthyl anilie
i: 3-acetyl aniline
(b) NaOH, MeOH, rt, 30 min
(c) RNH2, Et3N, cat. TBTU,
dry DMF, reflux. N2
f: cyclopropyl amine
g: 1-(2-naphthyl) ethyl amine
h: 2-pyridinyl amine
Scheme 1. Synthesis of pyrazole containing JNK-1 inhibitors.

A. Doma et al. / Bioorg. Med. Chem. 22 (2014) 6209–6219
6213
2.4.16. N-(3,4-Dimethoxybenzyl)-5-phenyl-3-pyrazole
Ar-H), 7.50–7.20 (br m, 5H, Ar-H), 6.91–6.70 (br m, 2H, Ar-H);
MS (ESI) m/z: 335 [M+H]+; Anal. Calcd for C16H13N5O5: C, 59.43;
H, 4.05. Found: C, 59.33; H, 4.0.
carboxamide 9e
IR (KBr) (m
, cmꢁ1): 3221 (NAH str.), 3027 (CAH-aromatic, str.),
2967 (CH, aliphatic, str.), 1646 (C@O-str.), 1595 (NAH bnd.); 1H
NMR (400 MHz, CDCl3, 298 K): d 9.0 (t, 1H, NH, J = 5.2 Hz), 8.70
(t, 1H, NH, J = 6 Hz), 7.80 (t, 2H, Ar-H, J = 7.3 Hz), 7.50–7.20 (m,
3H, Ar-H), 7.0 (s, 1H, Ar-H), 6.90 (s, 2H, Ar-H), 6.80 (d, 1H, Ar-H,
J = 8.3 Hz), 4.40 (dd, 2H, Ar-CH2, J = 5.8, 6.4 Hz), 3.70 (s, 3H,
OCH3); MS (ESI) m/z: 360 [M+Na]+.
2.4.21. 5-Phenyl-N-(thiazol-2-yl)-3-pyrazole carboxamide 10e
IR (KBr) (m
, cmꢁ1): 3376 (NAH str.), 3216 (CAH aromatic, str.),
1730 (C@O-str.), 1680 (NAH bnd.); 1H NMR (400 MHz, CDCl3,
298 K): d 9 (t, 1H, NH, J = 5.2 Hz), 8.7 (t, 1H, NH, J = 6 Hz), 7.8 (t,
2H, Ar-H, J = 7.3 Hz), 7.5–7.2 (m, 4H, Ar-H), 7 (s, 1H, Ar-H); MS
(ESI) m/z: 271 [M+H]+.
2.4.17. N-(Furan-2-yl)methyl-5-phenyl-3-pyrazole carboxamide
10a
2.4.22. N-Cyclopropyl-5-phenyl-3-pyrazole carboxamide 10f
IR (KBr) (
m
, cmꢁ1): 3420 (NAH str.), 3129 (CAH aromatic str.),
IR (KBr) (m
, cmꢁ1): 3400 (NAH str.), 3127 (CAH-aromatic, str.),
2925 (CH2-str.), 1640 (C@O str.), 1546 (NAH bnd.); 1H NMR
(300 MHz, CDCl3, 298 K): d 7.58 (d, 2H, Ar-H, J = 7.1 Hz), 7.45–
7.29 (br m, 3H, Ar-H), 7.21 (1H, Ar-H), 7.0 (s, 1H, Ar-H), 6.30 (dd,
2H, Ar-H, J = 4.1 Hz), 2.0 (br s, 1H, NH); MS (ESI) m/z: 268
[M+H]+, 290 [M+Na]+; Anal. Calcd for C15H13N3O2: C, 67.40; H,
4.90. Found: C, 67.30; H, 4.70.
2921 (CH2 str.), 1648 (C@O str.), 1539 (NAH bnd.); 1H NMR
(400 MHz, CDCl3, 298 K): d 7.68 (d, 2H, Ar-H), 7.44–7.32 (m, 3H,
Ar-H), 7.0 (s, 1H, Ar-H), 6.71 (br m, 1H, NH), 2.88 (m, 1H, CH),
0.89 (m, 2H, CH2), 0.65 (m, 2H, CH2); 13C NMR (75 MHz, CDCl3,
278 K): d 128.3 (3C), 127.8 (2C), 125, 102, 40, 21.7, 5.9 (2C); MS
(ESI) m/z: 250 [M+Na]+; Anal. Calcd for C13H13N3O: C, 68.70; H,
5.77. Found: C, 68.59; H, 5.67.
2.4.18. N-(4-(4-Methoxyphenyl)isoxazol-3-yl)-5-phenyl-1H-
pyrazole-3 10b
Table 2
IR (KBr)(m
, cmꢁ1): 3320 (NAH str.), 3039 (CAH aromatic, str.),
Inhibition results for compounds 8a–i and 9a–e against JNK-1
2829 (CH3 str.), 1695 (C@O-str.), 1642 (NAH bnd.); ESI-MS (m/z):
361 [M+H]+.
R5
R4
O
2.4.19. N-(5-t-Butyl-2,5-dihydroisoxazol-3-yl)-5-phenyl-3-
R3
n
NH
pyrazole carboxamide 10c
IR (KBr) (m
, cmꢁ1): 3375 (NAH str.), 3124 (CAH aromatic, str.),
R1
R2
2923 (CH2 str.), 1742 (C@O str.), 1595 (NAH bnd.); 1H NMR
(500 MHz, CDCl3, 298 K): d 13.82 (br s, 1H, NH), 11.46 (br s, 1H,
NH), 10.21 (br s, 1H, NH), 7.70 (d, 2H, Ar-H, J = 6.9 Hz), 7.53–7.31
(br m, 4H, Ar-H), 6.78 (s, 1H, Ar-H), 2.5 (s, 1H, CH), 1.3 (s, 9H,
t-butyl); MS (ESI) m/z: 311 [M]+.
N
N
H
Compound
n
R1
R2
R3
R4
R5
IC50a l
M
8a
8b
8c
8d
8e
8f
8g
8h
8i
9a
9b
9c
9d
9e
0
0
0
0
0
0
0
0
0
1
1
1
1
1
H
H
H
H
H
H
H
CH3
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
H
Cl
H
H
H
H
H
CH3
H
H
9.6
6.2
9.8
8.1
NT
5.9
3.1
11.4
6.9
9.1
5.8
2.9
6.1
5.8
NO2
OCH3
OH
Br
2.4.20. N-(2-Mercaptobenzimidazol-5-yl)-5-phenyl-3-pyrazole
carboxamide 10d
IR (KBr) (m
, cmꢁ1): 3285 (NAH str.), 3052 (CAH aromatic str.),
1739 (C@O str.), 1649 (NAH bnd.); 1H NMR (400 MHz, CDCl3,
298 K): d 9.10 (br s, 1H, NH), 8.71 (br s, 1H, NH), 7.71 (br t, 2H,
Cl
CH3
H
H
H
H
Cl
H
H
COCH3
H
H
H
H
H
H
Cl
F
H
H
H
H
Table 1
Physical properties of pyrazole compounds
OCH3
OCH3
OCH3
Compd
no
Mol. for.
Mol.
wt.
Yield
(%)
Mp (°C)
Rf
cꢃLogPa
a
IC50 in lM, SEM 0.2, NT—Not tested against the isolated JNK-1.
8a
8b
8c
8d
8e
8f
8g
8h
8i
9a
9b
9c
9d
C16H13N3O
263.30
332.19
308.30
293.33
279.30
342.20
315.74
305.37
305.34
277.33
346.22
295.32
307.36
337.38
267.29
360.12
312.37
335.39
270.31
227.27
341.42
264.29
78.5
46.2
76.0
48.5
86.4
92.4
67.5
54.9
77.6
91.2
79.3
68.6
72.4
66.8
72.4
58.0
65.0
92.0
91.6
89.0
61.0
89.0
134–136
152–154
122–124
130–132
132–134
210–212
190–192
174–176
111–113
161–163
198–200
182–184
115–117
242–244
201–203
245–247
190–192
240–242
202–204
128–130
136–138
198–200
0.32
0.34
0.47
0.42
0.46
0.36
0.38
0.45
0.44
0.41
0.35
0.54
0.41
0.34
0.36
0.43
0.40
0.58
0.56
0.33
0.35
0.38
3.715
4.412
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
16H11Cl2N3O
16H12N4O3
17H15N3O2
16H13N3O2
16H12BrN3O
16H11ClFN3O
18H17N3O
3.6512
3.68874
3.048
4.66816
4.69272
4.063
3.31822
3.894
5.2
4.037
3.813
3.552
3.07
Table 3
Inhibition results for compounds 10a–h against JNK-1
O
R1
N
H
18H15N3O2
17H15N3O
N
N
H
17H13Cl2N3O
17H14FN3O
18H17N3O2
19H19N3O3
15H13N3O2
2OH16N4O3
17H20N4O2
17H13N5OS
13H10N4OS
13H13N3O
a
Compd
R1
Furan-2yl methyl
5-(4-Methoxyphenyl)isoxazol-3-yl
5-t-Butylisoxazol-3-yl
2-Mercaptobenzimidazol-5-yl
2-Thiazolyl
Cyclopropyl
1-(1-Naphthyl)ethyl
2-Pyridinyl
IC50
9e
10a
10b
10c
10d
10e
10f
2.8
3.2
NTb
2.9
6.8
6.2
NTb
8.9
10a
10b
10c
10d
10e
10f
10g
10h
3.428
3.09148
4.2042
2.51174
2.51
5.377
2.6482
10g
10h
22H19N3O
15H12N4O
a
IC50 in lM, SEM 0.2.
NT—Not tested against the isolated JNK-1.
a
b
Calculated using ChemBio 3D Ultra.

6214
A. Doma et al. / Bioorg. Med. Chem. 22 (2014) 6209–6219
Table 4
Anti-inflammatory activity of compounds 8a–i
R4
R3
R5
R2
O
N
H
R1
N
N
H
Compd no
R1
R2
R3
R4
R5
Mean paw volume (mL) SEM and % inhibition
1 h
2 h
3 h
4 h
8a
8b
8c
8d
8e
8f
H
H
H
H
H
H
H
H
H
F
H
0.52 0.059
32.48**
0.45 0.051**
35.02
0.35 0.042*
45.64
0.26 0.032*
30.58
H
Cl
H
Cl
H
0.52 0.065*
31.02
0.51 0.057**
40.16
0.51 0.050**
45.47
0.38 0.033**
18.90
H
H
NO2
OCH3
OH
Br
0.50 0.069*
89.24
0.42 0.054*
94.31
0.36 0.041*
92.56
0.22 0.025*
68.70
H
H
H
0.56 0.057**
33.3
0.18 0.042*
79.54
0.14 0.027*
81.08
0.07 0.011*
66.44
H
H
H
0.17 0.035**
22.11
0.42 0.038*
43.24
0.055 0.012**
23.28
0.063 0.016**
16.64
H
H
H
0.53 0.045
0.45 0.054*
46.31
0.52 0.035*
51.4
0.36 0.022*
38.22
42.16*
8g
8h
8i
H
H
Cl
H
0.29 0.037*
68.81
0.20 0.028*
72.7
0.073 0.018*
90.54
0.054 0.011*
64.22
CH3
H
H
CH3
H
H
H
CH3
H
0.40 0.047*
53.65
0.47 0.027*
45.6
0.43 0.035*
44.24
0.28 0.016*
26.8
COCH3
0.55 0.063*
41.3
0.54 0.057**
56.7
0.56 0.053*
74.32
0.39 0.032*
56.8
NT—Not determined.
*
p <0.01.
p <0.05.
**
Table 5
Anti-inflammatory activity of compounds 9a–e
R5
R4
O
R3
N
H
R1
R2
N
N
H
Compd no
R1
R2
R3
R4
R5
Mean paw volume (mL) SEM and % inhibition
1 h
2 h
3 h
4 h
9a
9b
9c
9d
9e
H
H
H
H
H
H
H
H
H
H
H
Cl
F
H
Cl
H
H
H
H
H
H
0.60 0.079**
35.48
0.41 0.050*
53.4
0.38 0.045*
48.64
0.22 0.018*
25.66
0.51 0.047*
45.16
0.49 0.098*
44.31
0.54 0.033*
27.02
0.31 0.014*
10.11
0.55 0.038*
67.86
0.23 0.019*
70.27
0.21 0.020*
75.27
0.14 0.010*
54.66
H
OCH3
OCH3
0.54 0.067*
41.93
0.53 0.058**
39.77
0.50 0.050**
32.43
0.28 0.032**
16.46
OCH3
0.42 0.054*
54.83
0.47 0.027*
46.59
0.42 0.038*
43.24
0.26 0.019*
24.80
NT—Not determined.
*
p <0.01.
p <0.05.
**
2.4.23. N-(1-(Naphthalen-2-yl)ethyl)-5-phenyl-3-pyrazole
8H, Ar-H), 7.10 (d, 1H, NH), 6.90 (s, 1H, CH@C), 6.20 (q, 1H, CH,
J = 7.3 Hz), 1.72 (d, 3H, CH3, J = 6.7 Hz);13C NMR (75 MHz, CDCl3,
278 K): d 160.9, 145.9, 138.1, 133.8, 130.9, 128.9 (3C), 128.6 (3C),
128.2, 126.4, 125.7, 125.4, 125.1, 123.2, 122.5, 103.1, 44.6, 21;
MS (ESI) m/z: 364 [M+Na]+; HRMS (ESI+): m/z [M+Na]+; calcd for
carboxamide 10g
IR (KBr) (m
, cmꢁ1): 3400 (NAH str.), 3127 (CAH aromatic, str.),
2921 (CH2 str.), 1646 (C@O str.), 1539 (NAH bnd.); 1H NMR
(400 MHz, CDCl3, 298 K): d 8.40 (d, 1H, Ar-H, J = 8.3 Hz), 7.56 (d,
1H, Ar-H, J = 8.3 Hz), 7.54 (d, 1H, Ar-H, J = 7.5 Hz), 7.40–7.20 (m,
C22H19N3O: C, 77.40; H, 5.61. Found: C, 77.29; H, 5.55.

A. Doma et al. / Bioorg. Med. Chem. 22 (2014) 6209–6219
6215
2.4.24. 5-Phenyl-N-(pyridin-2-yl)-3-pyrazole carboxamide 10h
at 1.3 ppm is accounted for methyl protons. The molecular ion
peak [m/z] at 216.10 in mass spectrum of compound 3 found to
be in conformity with the molecular formula of the assigned
structure.
IR (KBr) (m
, cmꢁ1): 3376 (NAH str.), 3024 (CAH Aromatic str.),
1730 (C@O-str.), 1664 (NAH bnd.); 1H NMR (400 MHz, CDCl3,
298 K): d 8.2 (m, 1H, Ar-H), 8.2 (m, 1H, Ar-H), 7.9–7.6 (m, 4H, Ar-
H), 7.1 (m, 2H, Ar-H, NH), 7.5–7.2 (m, 2H, Ar-H), 9.2 (br s, 1H,
NH); MS (ESI) m/z: 265 [M+Na]+.
Hydrolysis of compound 3 led to formation of 3-pyrazole car-
boxylic acid derivative 4 which showed presence of a broad IR
absorption band peaking at 3433 cmꢁ1 accounted for OH group
of carboxylic acid apart from a band at 1704 cmꢁ1 for carbonyl
group stretching. 1H NMR of compound 4 also confirmed the
hydrolysis of 3 by the appearance of a broad singlet at 2.1 ppm
due to OH proton. The absence of IR frequencies for methyl and
methylene group deduced completion of hydrolysis and this was
also supported by the absence of corresponding peaks in 1H
NMR. IR spectrum of all the amides demonstrated presence of a
medium intense band at ꢄ3200 cmꢁ1 due to stretching vibration
of amide NH. 1H NMR spectra of these amide derivatives supported
the formation of amide bond by showing a signal between 8.0–9.0
for the amide proton and by the absence of the broad peak due to
carboxylic acid OH at 2.1 ppm. Structures assigned for the synthe-
sized compounds were further ascertained based on the m/z values
of individual compounds and elemental analysis (C, H and N) data.
3. Results and discussions
3.1. Chemistry
The title compounds were synthesized by adopting appropriate
synthetic methodologies as shown in Scheme 1 and their physical
properties are listed in Table 1. The key intermediate 3 was synthe-
sized by the 1,3-dipolar cycloaddition reaction of phenylacetylene
(dipolarophile) and ethyldiazoacetate (dipole). This reaction was
mediated by solid catalyst DABCO resulting in compound 3 in a
neat reaction. Despite a high yielding reaction, the product was
purified over silica using 20% ethyl acetate. Compound 3 upon
alkaline hydrolysis and subsequent acidification resulted in corre-
sponding acid 4. Further, compound 4 was treated with various
amines (5a–h, 6a–i and 7a–e) in presence of coupling agent and
base yielded expected amides (8a–i, 9a–e and 10a–h), which were
purified by passing through silica gel using 30% ethyl acetate in
petroleum ether as eluent.
3.2. JNK-1 inhibitory activity
All the twenty two molecules (8a–i, 9a–e and 10a–h) were
screened for in vitro JNK-1 inhibitory activity as listed in Tables 2
and 3. Compound 10a was found to be the most potent among
all the synthesized compounds with IC50 value of 2.8 lM. Three
compounds 8e, 10c and 10g have not been tested for JNK-1 inhib-
IR spectrum of compound 3 (KBr) divulged intense absorption
band at 2950 cmꢁ1 accounting for methyl group, band for carbonyl
group of ester appeared at 1664 cmꢁ1 and
a
peak for NH
was observed at 3230 cmꢁ1
.
1H NMR spectrum of compound 3
itory activity and one compound 8h showed mild JNK-1 inhibitory
confirmed the cyclization of phenylacetylene with diazonium com-
pound, all the aromatic protons of 5-phenyl group resonated in the
range of d 7.7–7.2 as one doublet at d 7.7 and another at d 7.2 as
triplet. C4 proton of pyrazole was observed little downfield at
7.0 ppm and a broad singlet at d 7.6 is resultant of NH resonance.
A quartet peak at d 4.3 is due to CH2 protons and a triplet found
activity with IC50 value of 11.9
inhibited JNK-1 with IC50 values less than 10
phenyl compound 8a demonstrated significant inhibitory activity
with IC50 of 9.6 M and substitution at R3 with groups like
halogens helped in improving the activity as compound 8f with
l
M where as, rest of the compounds
lM. Unsubstituted
l
Table 6
Anti-inflammatory activity of compounds 10a–h
O
R1
N
H
N
N
H
Compd
R1
Mean paw volume (mL) SEM and % inhibition
1 h
2 h
3 h
4 h
10a
10b
10c
10d
10e
10f
2-Methyl furanyl
3-(4-Methoxy phenyl)isoxazolyl
5-t-Isoxazolyl
0.25 0.042*
73.11
0.16 0.27*
81.81
0.065 0.026*
91.89
0.033 0.016*
66.33
0.29 0.037*
68.81
0.20 0.028*
72.72
0.073 0.018*
90.54
0.058 0.012*
71.66
0.27 0.019*
68.60
0.25 0.027*
70.60
0.42 0.024*
52.8
0.51 0.031*
41.7
2-Mercapto benzimidazol-5-yl
2-Thiazolyl
0.05 0.037*
94.62
0.04 0.0032*
95.45
0.062 0.021*
91.62
0.045 0.015*
74.15
0.21 0.026*
45.31
0.23 0.017*
51.06
0.068 0.084*
54.65
0.041 0.044*
31.40
Cyclopropyl
0.28 0.028*
69.89
0.13 0.023*
85.22
0.11 0.028*
88.13
0.06 0.008*
55.87
10g
10h
1-(l-Naphthyl) ethyl
2-Pyridinyl
0.36 0.020*
61.29
0.23 0.019*
73.86
0.19 0.021*
74.32
0.11 0.014*
50.64
0.56 0.057**
33.3
0.18 0.042*
79.54
0.14 0.027*
81.08
0.08 0.01*
64.26
Diclofenac sodium (50 mg/kg)
NT—Not determined.
23**
36**
47**
51**
*
p <0.01.
p <0.05.
**

6216
A. Doma et al. / Bioorg. Med. Chem. 22 (2014) 6209–6219
bromine group showed IC50 value of 5.9
l
M and compound 8g
meta substituted compound 8i (R2 = COCH3) was found to show
though contains fluoro group at R4 along with chloro at R3 was
the most potent among the series (8a–h). Surprisingly methoxy
substituted compound 8d was found to be more potent than 8a
this indicates that not only steric property but also electronic prop-
erties may also contribute towards JNK-1 inhibitory activity. Lone
superior activity over unsubstituted analog 8a and similarly 2,5-
dichloro analog 8b demonstrated enhanced activity over 8a.
In order to ascertain influence of a linker/spacer (ACH2A)
between the amide nitrogen and substituted phenyl group, a series
of N-substituted benzyl 9a–e (n = 1) were synthesized and
screened for JNK-1inhibitory activity. Among this series, com-
pound 9c with fluoro group at R3 was found to be the most potent
with IC50 value of 2.9
found to be least active in the series with IC50 of 9.1
it is more potent than 8a. Dichloro and dimethoxy analogs (9b and
9e) showed less activity (IC50 = 5.8 M) when compared to 9c.
l
M and unsubstituted compound 9a was
Table 7
lM, however
Docking parameters (kcal/mol) and physicochemical properties of phenyl pyrazola-
mide series
l
S. no
gscore
evdw
ecoul
Energy
HBA
HBD
RB
Overall substitutions over benzyl ring improved activity.
8a
8b
8c
8d
8e
8f
8g
8h
8i
9a
9b
9c
9d
ꢁ7.97
ꢁ7.27
ꢁ6.69
ꢁ8.04
ꢁ8.58
ꢁ6.37
ꢁ8.4
ꢁ36.65
ꢁ23.36
ꢁ15.78
ꢁ39.19
ꢁ37.33
ꢁ38.08
ꢁ38.96
ꢁ18.05
ꢁ40.29
ꢁ40.08
ꢁ44.58
ꢁ40.08
ꢁ41.33
ꢁ43.04
ꢁ38.47
ꢁ39.46
ꢁ38.41
ꢁ42.43
ꢁ38.28
ꢁ35.09
ꢁ46.61
ꢁ37.28
ꢁ4.22
ꢁ2.62
ꢁ2.58
ꢁ3.41
ꢁ6.14
ꢁ3.04
ꢁ3.2
ꢁ40.86
ꢁ25.98
ꢁ18.36
ꢁ42.6
2
2
2
3
3
2
2
2
3
2
2
2
3
4
3
5
4
2
3
2
2
3
2
2
2
2
3
2
2
2
2
2
2
2
2
2
2
2
2
4
2
2
2
2
3
3
4
4
3
3
3
3
4
4
4
4
5
6
4
5
4
3
3
3
4
3
Further, compounds with N-heterocyclic rings (10a–h) were
synthesized and evaluated for the JNK-1 inhibitory activity with
an expectation of improved potency. Compound 10a with N-
methyl-2-furanyl substitution exhibited maximum potency
ꢁ43.47
ꢁ41.12
ꢁ42.16
ꢁ22.61
ꢁ44.16
ꢁ44.87
ꢁ48.98
ꢁ45.37
ꢁ46.03
ꢁ48.31
ꢁ44.01
ꢁ43.99
ꢁ43.04
ꢁ46.76
ꢁ42.89
ꢁ39.19
ꢁ50.6
against JNK-1 inhibition with IC50 of 2.8
2-mercaptobenzimidazol-5-yl ring showed equipotent activity
(IC50 = 2.9 M) when compared to 9c. The rest four 10b, 10e, 10f
lM. Compound 10d with
ꢁ7.91
ꢁ8.24
ꢁ8.92
ꢁ9.41
ꢁ8.94
ꢁ9.15
ꢁ9.23
ꢁ8.73
ꢁ7.24
ꢁ8.09
ꢁ8.29
ꢁ7.56
ꢁ8.2
ꢁ4.55
ꢁ3.87
ꢁ4.79
ꢁ4.4
l
and 10h demonstrated superior activity over the phenyl and ben-
zyl analogs (8a and 9a). The 4-methoxyphenylisoxazol-3-yl analog
10b showed significant JNK-1 inhibitory activity (IC50 = 3.2 lM),
where as substitution with cyclopropyl and thiazole rings (10e
and 10f) resulted in drastic decrease in potency with IC50 values
of 6.2 and 6.8 lM, respectively. A lone compound with six member
heterocyclic ring, that is, 2-pyridyl analog (10h) was found to be
slightly better than 8a and 9a.
ꢁ5.3
ꢁ4.7
9e
ꢁ5.27
ꢁ5.53
ꢁ4.53
ꢁ4.63
ꢁ4.33
ꢁ4.61
ꢁ4.1
10a
10b
10c
10d
10e
10f
10g
10h
3.3. In vivo antiinflammatory activity
ꢁ9.25
ꢁ7.85
ꢁ3.98
ꢁ4.47
ꢁ41.75
Encouraging results of in vitro JNK-1 inhibitory activity of all
synthesized compounds prompted us to evaluate them for
MW, molecular weight; RB, rotatable bond; HBA, hydrogen bond acceptor; HBD,
hydrogen bond donor.
Figure 2. Binding modes all 22 phenyl pyrazoloamide series of compounds (cyan) in the active site of JNK1 and the hydrogen bond interactions are shown in red color dotted
lines, the important residues are shown in green color and protein cartoon is represented in wheat color.

A. Doma et al. / Bioorg. Med. Chem. 22 (2014) 6209–6219
6217
Figure 3. The probable binding mode of most potent compound (10a, cyan) in the active site of JNK1 and the hydrogen bond interactions are shown in red color dotted lines,
the important residues are shown in green color and protein cartoon is represented in wheat color.
in vivo antiinflammatory activity in carrageenan induced rat paw
edema and the results are listed in Tables 4–6. Nearly all com-
pounds exhibited significant antiinflammatory activity and peak
paw protection was observed at the 3rd hour of carrageenan chal-
lenge. For compounds 8c, 8e and 9a the peak in vivo activity was
observed at the 2nd hour of carrageenan injection and rest of the
compounds 8h, 9b, 9d and 9e the apex of the activity was noticed
at the 1st hour and activity gradually decreased with respect to
time duration. Compounds 8c and 10d were found to be the most
potent among all the tested compounds. Compound 10d showed
95.45% paw protection at the second hour of carrageenan challenge
which could be attributed to its JNK-1 inhibitory activity. Interest-
ingly, compound 8c with lesser JNK-1 inhibitory activity also
exhibited almost equal potency (94.31% protection) at the 2nd
hour when compared with 10d. In similar fashion, most potent
compound in the in vitro activity 10a demonstrated 91.89% activity
at the 3rd hour. Substitution on aromatic ring as in 8a–i resulted in
augmentation of activity in maximum compounds, with exception
8b and 8e demonstrated further inferior activity when compared
to unsubstituted compound 8a. Compound 8c, which showed
much less in vitro JNK-1 inhibition (IC50 = 9.8 lM), exhibited
potent activity in in vivo assay with 92.56% inhibition of rat paw
edema at 3rd hour. N-Benzyl derivatives 9a–e were found to be
less potent when compared with substituted N-phenyl analogs

6218
A. Doma et al. / Bioorg. Med. Chem. 22 (2014) 6209–6219
Figure 4. The probable binding mode of least potent compound (10g, cyan) in the active site of JNK1 and the hydrogen bond interactions are shown in red color dotted lines,
the important residues are shown in green color and protein cartoon is represented in wheat color.
8a–i. Compound 9c the most potent JNK-1 inhibitor (IC50 = 2.9
l
M)
and 10d have also exhibited potent antiinflammatory activity with
greater than 90% paw edema inhibition at the 3rd hour of carra-
geenan administration and even 10b showed similar activity profile.
among all the compounds, showed only moderate in vivo activity
(75.27% inhibition at 3rd hour). However this compound 9c was
the most potent among the N-benzyl derivatives (9a–i).
In order to explore further influence of substituents, N-heterocy-
clic compounds 10a–h have also been subjected for in vivo activity.
This seriesof compoundswere proved to besuperiorin in vitroactiv-
ity among the three series. Two of the most active compounds 10a
3.4. Molecular docking
All the twenty two compounds have been docked into human
JNK1 target. With understanding of available JNK-1 inhibitors

A. Doma et al. / Bioorg. Med. Chem. 22 (2014) 6209–6219
6219
profile, the pyrazole derivatives 8a–i, 9a–e and 10a–h seem to be
References and notes
ATP competitive inhibitors. The compounds were docked into
hinge region that connected both N-terminal lobe and C-terminal
region. The active site was divided into hinge region (Glu109,
Leu110, Met111), gate keeper (Met108), catalytic triad region
(Lys55, Asp169, Glu73), G-loop region (Gly33, Ser34, Gly35), and
DFG-loop region (Asp169, Phe170, Gly171). An attempt has been
made to analyze the probable binding modes, key active site inter-
actions for the most potent (10a), least active (10g), compound
with best docked score (9b) and compound with least docked score
(8f). All the docked compounds found to retain hinge backbone
interactions with Glu109 and Met111 in which the pyrazole nitro-
gen showed hydrogen bond interaction with carbonyl backbone
Glu109 and carbonyl moiety of amide showed hydrogen bond
interaction with NH of Met111 backbone residue. The phenyl ring
in compound 10a accommodated near the catalytic triad residues
Lys55, Asp169, Glu73 and showed hydrophobic interactions with
Val40, Leu168. The pyrazole ring accommodated in close proximity
to hydrophobic residues Ile32, Leu110, Val158. The docking scores
have been listed in Table 7 and docking poses of all the molecules,
most active and least active have been portrayed in Figures 2–4.
1. Kyriakis, J. M.; Brautigan, D. L.; Ingebristen, T. S.; Avruch, J. J. Biol. Chem. 1991,
266, 10043.
2. Barr, R. K.; Bogoyevitch, M. A. Int. J. Biochem. Cell Biol. 2001, 33, 1047.
3. Davis, R. J. Cell 2000, 103, 239.
4. Karin, M.; Gallagher, E. IUBMB Life 2005, 57, 283.
5. Dong, C.; Davis, R. J.; Flavell, R. A. Annu. Rev. Immunol. 2002, 20, 55.
6. (a) Du, L.; Lyle, C. S.; Obey, T. B.; Gaarde, W. A.; Muir, J. A.; Bennett, B. L. J. Biol.
Chem. 2004, 279, 11957; (b) Higuchi, H.; Grambihler, A.; Canbay, A.; Bronk, S.
F.; Gores, G. J. J. Biol. Chem. 2004, 279, 51.
7. Fleming, Y.; Armstrong, C. G.; Morrice, N.; Paterson, A.; Goedert, M.; Cohen, P.
Biochem. J. 2000, 352, 145.
8. Tibbles, L. A.; Ing, Y. L.; Kiefer, F. N.; Woodgett, J. R.; Lassam, N. EMBO J. Biol.
Chem. 1996, 15, 7026.
9. Ichijo, H.; Nishida, E.; Irie, K.; Dijke, P.; Saitoh, M.; Morguchi, T. Science 1997,
275, 90.
10. Bogoyevitch, M. A.; Boehm, I.; Oakley, A.; Ketterman, A. J.; Barr, R. K. Biochim.
Biophys. Acta. 2004, 1696, 89.
11. Badrinarayan, P.; Sastry, G. N. Curr. Pharm. Des. 2013, 19, 4714.
12. Kallunki, T.; Deng, T.; Hibi, M.; Karin, M. Cell 1996, 87, 929.
13. Pulverer, B. J.; Kyriakis, J. M.; Avruch, J.; Nikolakaki, E.; Woodgett, J. R. Nature
1991, 353, 670.
14. Cavigelli, M.; Dolfi, F.; Claret, F. X.; Karin, M. EMBO J. 1995, 14, 5957.
15. Dam, H. V.; Wilhelm, D.; Herr, I.; Steffen, A.; Herrlich, P.; Angel, P. EMBO J. 1995,
14, 1798.
16. Manning, A. M.; Davis, R. J. Nat. Rev. Drug Disc. 2003, 2, 554.
17. LoGrasso, P.; Kamenecka, T. Mini Rev. Med. Chem. 2008, 8, 755.
18. Stocks, S. J.; Barber, S.; Ford, R.; Leroux, F.; St-Gallay, S.; Teaguea, S.; Xuec, Y.
Bioorg. Med. Chem. Lett. 2005, 15, 3459.
4. Conclusions
19. Palmer, W. S.; Alam, M.; Arzeno, H. B.; Chang, K. C.; Dunn, J. P.; Goldstein, D. M.;
Gong, L.; Goyal, B.; Hermann, J. C.; Hogg, J. H.; Hsieh, G.; Jahangir, A.; Janson, C.;
Jin, S.; Kammlott, R. U.; Kuglstatter, A.; Lukacs, C.; Michoud, C.; Niu, L.; Reuter,
D. C.; Shao, A.; Silva, T.; Trejo-Martin, T. A.; Stein, K.; Tan, Y. C.; Tivitmahaisoon,
P.; Tran, P.; Wagner, P.; Weller, P.; Wua, S. Y. Bioorg. Med. Chem. Lett. 2013, 23,
1486.
20. Ravindra, G. K.; Achaiah, G.; Sastry, G. N. J. Comp. Aided Mol. Des. 2007, 4, 155.
21. Kulkarni, R. G.; Achaiah, G.; Sastry, G. N. Curr. Pharm. Des. 2006, 12, 2437.
22. Kulkarni, R. G.; Achaiah, G.; Laufer, S.; Chandrashekar, V. M. Med. Chem. 2013, 9,
213.
Twenty two new pyrazole heterocyclic group containing com-
pounds have been synthesized and screened against JNK-1 in the
current study. In a neat 1,3-dipolar cycloaddition reaction, phenyl-
acetylene was reacted with ethyldiazoacetate in presence DABCO,
the ester that resulted was hydrolyzed and converted into desired
amides using suitable primary amines. All compounds, except
three showed JNK-1 inhibitory activity with IC50 of less than
23. Kulkarni, R. G.; Achaiah, G.; Laufer, S.; Chandrashekar, V. M. Med. Chem. 2013, 9,
90.
10 lM. Among the N-phenyl and N-benzyl derivatives halogen
substitution enhanced the JNK-1 inhibitory activity as witnessed
in compounds 9c and 8g. Further different heterocyclic amines
were used to synthesize compounds 10a–h, compounds 10a, 10b
and 10d were found to possess potent JNK-1 inhibitory activity.
In the in vivo antiinflammatory activity, compounds 8c and 8g
demonstrated potent activity at the third hour of the observation
and compounds 10a, 10b, 10f and 10h showed over 80% paw
edema protection.
24. Badrinarayanan, P.; Sastry, G. N. J. Chem. Inf. Model. 2011, 51, 115.
25. Badrinarayanan, P.; Sastry, G. N. J. Mol. Graph. Mod. 2012, 34, 89.
26. King, S.; Teague, S.; Xue, Y.; Swahn, B. Chem. Abstr. 2003, 139, 53019.
27. Romain, N.; Youseung, S.; Xinyi, S.; Yuanjun, H.; Marcel, K.; Weimin, C.; Yuan,
Y. L.; Li, L.; Claudia, H. R.; Phil, L.; Michael, D. C.; Derek, R. D.; Theodore, M. K.
Bioorg. Med. Chem. Lett. 2011, 21, 2732.
28. Dong, C.; Davis, R. J.; Flavell, R. A. Annu. Rev. Immunol. 2002, 20, 55.
29. Bogoyevitch, M. A.; Kobe, B. Microbiol. Mol. Biol. Rev. 2006, 70, 1061.
30. Winter, C. A.; Risley, E. A.; Nuss, G. W. Exp. Biol. Med. 1962, 111, 544.
31. Rosa, D.; Willoughby, M. D. A. J. Pharm. Pharmacol. 1971, 23, 297.
32. Kumar, A.; Ilavarasan, R.; Jayachandran, T.; Deecaraman, M.; Kumar, R. M.;
Aravindan, P.; Padmanabhan, N.; Krishan, M. R. Afr. J. Biotech. 2008, 7, 941.
33. Still, W. C.; Tempczyk, A.; Hawley, R. C.; Hendrickson, T. J. Am. Chem. Soc. 1990,
112, 6127.
Acknowledgments
34. Maestro, version 9.3, Glide version 2.8, Schrödinger, LLC, New York, NY 2013.
35. Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicic, J. J.; Mainz, D. T.;
Repasky, M. P.; Knoll, E. H.; Shelley, M.; Perry, J. K.; Shaw, D. E.; Francis, P.;
Shenkin, P. S. J. Med. Chem. 2004, 47, 1739.
The author A. D. thanks AICTE, India for providing fellowship
under quality improvement program and also wish to thank the
Director IICT for permitting to carry planned research project in
IICT. Author A.G. thanks AICTE India, for granting funds under
modernization of laboratory facilities (Letter No. 8023/RID/BOR/
MOD-111./2007–08, dated June 20, 2008).