Synthesis of some new amide-linked bipyrazoles and their evaluation as anti-inflammatory and analgesic agents

Article abstract of DOI:10.3109/14756366.2015.1094469

Four series of new bipyrazoles comprising the N-phenylpyrazole scaffold linked to polysubstituted pyrazoles or to antipyrine moiety through different amide linkages were synthesized. The synthesized compounds were evaluated for their anti-inflammatory and

Full text of DOI:10.3109/14756366.2015.1094469

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Synthesis of some new amide-linked bipyrazoles
and their evaluation as anti-inflammatory and
analgesic agents
Wissam H. Faour, Mohamed Mroueh, Costatantine F. Daher, Rasha Y.
Elbayaa, Hanan M. Ragab, Asser I. Ghoneim, Ahmed I. El-mallah & Hayam M.
A. Ashour
To cite this article: Wissam H. Faour, Mohamed Mroueh, Costatantine F. Daher, Rasha Y.
Elbayaa, Hanan M. Ragab, Asser I. Ghoneim, Ahmed I. El-mallah & Hayam M. A. Ashour
(2015): Synthesis of some new amide-linked bipyrazoles and their evaluation as anti-
inflammatory and analgesic agents, Journal of Enzyme Inhibition and Medicinal Chemistry,
DOI: 10.3109/14756366.2015.1094469
To link to this article: http://dx.doi.org/10.3109/14756366.2015.1094469
Published online: 20 Oct 2015.
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J Enzyme Inhib Med Chem, Early Online: 1–16
2015 Taylor & Francis. DOI: 10.3109/14756366.2015.1094469
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RESEARCH ARTICLE
Synthesis of some new amide-linked bipyrazoles and their evaluation as
anti-inflammatory and analgesic agents*
Wissam H. Faour1, Mohamed Mroueh2, Costatantine F. Daher3, Rasha Y. Elbayaa4,5, Hanan M. Ragab4,
Asser I. Ghoneim6,7, Ahmed I. El-mallah8, and Hayam M. A. Ashour4
2
1School of Medicine, Lebanese American University, Byblos, Lebanon, Department of Pharmaceutical Sciences, School of Pharmacy, Lebanese
3
American University, Byblos, Lebanon, Department of Natural Sciences, School of Arts and Sciences, Lebanese American University, Byblos,
4
Lebanon, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Alexandria University, Alexandria, Egypt, Department of Analytical &
5
6
Pharmaceutical Chemistry, Faculty of Pharmacy & Drug Manufacturing, Pharos University in Alexandria, Alexandria, Egypt, Department of
7
Pharmacology and Therapeutics, Faculty of Pharmacy, Beirut Arab University, Beirut, Lebanon, Department of Pharmacology and Toxicology,
Faculty of Pharmacy, Damanhour University, Damanhour, Egypt, and 8Department of Pharmacology, Faculty of Pharmacy & Drug Manufacturing,
Pharos University in Alexandria, Alexandria, Egypt
Abstract
Keywords
Four series of new bipyrazoles comprising the N-phenylpyrazole scaffold linked to
polysubstituted pyrazoles or to antipyrine moiety through different amide linkages were
synthesized. The synthesized compounds were evaluated for their anti-inflammatory and
analgesic activities. In vitro COX-1/COX-2 inhibition study revealed that compound 16b
possessed the lowest IC50 value against both COX-1 and COX-2. Moreover, the effect of the
most promising compounds on inducible nitric oxide synthase (iNOS) and cyclooxygenase
(COX-2) protein expression in lipopolysaccharide (LPS)-activated rat monocytes was also
investigated. The results revealed that some of the synthesized compounds showed anti-
inflammatory and/or analgesic activity with less ulcerogenic potential than the reference drug
diclofenac sodium and are well tolerated by experimental animals. Moreover, they significantly
inhibited iNOS and COX-2 protein expression induced by LPS stimulation. Compounds 16b and
18 were proved to display anti-inflammatory activity superior to diclofenac sodium and
analgesic activity equivalent to it with minimal ulcerogenic potential.
Anti-inflammatory, analgesic, COX-2, iNOS
History
Received 9 June 2015
Revised 19 July 2015
Accepted 5 August 2015
Published online 13 October 2015
Introduction
synthesis using stronger anti-inflammatory drugs such as
glucocorticoids.
Inflammation is a complex biological response of body tissues
that arises in response to traumatic, infectious, post-ischaemic,
toxic, or autoimmune injury. The process normally leads to
recovery from infection and to healing. However, if targeted
destruction and assisted repair are not properly phased, inflam-
mation can lead to persistent tissue damage by infiltrating
leukocytes and lymphocytes or damaged collagen1. Anti-
inflammatory pharmacotherapy targeting cyclooxygenase (COX)
enzymes is a well-established strategy to treat inflammation.
Modalities of inhibiting COX enzyme-associated prostanoid
synthesis fall within two categories, the use of non-steroidal
anti-inflammatory drugs (NSAIDs)2 or blocking COX protein
NSAIDs are the most widely prescribed therapeutics world-
wide, primarily for the treatment of pain and inflammation.
The major mechanism by which NSAIDs exert their anti-inflam-
matory activity is the suppression of prostaglandin biosynthesis
from arachidonic acid by inhibiting the COX enzyme which exists
in three distinct isoforms: COX-1, COX-2, and COX-33–5. The
constitutive COX-1 isoform provides cytoprotection in the
gastrointestinal (GI) tract and controls renal function in the
kidneys, whereas the inducible COX-2 is upregulated by pro-
inflammatory mediators such as endotoxins, mitogens, or cyto-
kines including tumor necrosis factor a (TNF-a) and interleukins
(ILs) such as IL-6 and IL-16,7. Furthermore, COX-3 which is a
variant of COX-1 presents in central nervous system and has been
proposed to be another target for anti-inflammatory agents8. GI
injury is one of the major hallmarks of NSAIDs and
is contraindicated in patients with history of GI bleeding or
distress9. Moreover, administering additional gastroprotective
treatment in conjunction with NSAIDs raises the issues of
polypharmacy and drug-induced organ injury in high-risk patients
notably elderly and infants10. Consequently, a new generation of
COX-2 selective inhibitors has been clinically used with the hope
that they would exhibit a reduced risk in GI events. However, the
*A part of this research was presented in abstract form at the
3rd International Conference of Organic Chemistry (ICOC-2014)
‘‘Organic Synthesis – Driving Force of Life Development’’, 25–26
September
WWW.CHEMISTRY.GE\CONFERENCES\ICOC-2014.
2014,
Tbilisi,
Georgia.
Available
at:
Address for correspondence: Rasha Y. Elbayaa, Department of
Pharmaceutical Chemistry, Faculty of Pharmacy, Alexandria University,
Alexandria, Egypt. Tel: +2 (03) 4871317. Fax: +2 (03) 4873273. E-mail:
rashabayaa72@gmail.com

2
W. H. Faour et al.
J Enzyme Inhib Med Chem, Early Online: 1–16
Figure 1. Chemical structure of previously
synthesized anti-inflammatory pyrazoles and
bipyrazoles (A–D) and the newly synthesized
compounds (E–H).
NHC H
R
6
5
S
N
F C
N
CN
3
N
N
N
N
N
N
R
SO NH
2
SO NH
2
SO NH
2 2
2
2
Celecoxib; R = CH
SC-558; R = Br
(A)
(B)
3
NHC H
6
5
N
N
S
N
N
N
N
O
COCH
3
N
N
N
N
SO NH
2
SO NH
2
(2C) 2
(D)
R
H CS
3
CN
H CS
CN
N
3
O
O
CN
R
NH
N
N
N
N
H
N
N
H
N
H N
H
2
N
(F)
(E)
R = C H , 4-OCH C H , 4-ClC H
R = C H , 4-OCH C H , 4-ClC H
6
5
3
6
4
6
4
6
5
3
6
4
6
4
1
R
H CS
CN
CN
H CS
3
3
O
O
CN
N
N
N
N
N
N
NH
H
N
H
X
NH
H N
2
O
H C
3
N
N
(H)H C
3
(G)
1
R = H, SCH , -NHC H , -NH-
X = N, CH, -C-NHC H , -C-NH-C H (4-Cl),
3
6 5
6
5
6 4
C H (4-Cl)
-C-SCH
6
4
3
increased cardiovascular risk associated with COX-2-selective
In the course of a research program aiming at finding out new
inhibitors led to reconsideration of their appropriate use11. Thus, lead structures that would act as potent anti-inflammatory agents,
the development of safe and effective therapy for treating we have reported the synthesis and anti-inflammatory activities
inflammatory conditions still presents a major challenge to the of some lead compounds comprising mainly the pyrazole
medical community.
counterpart substituted with various functionalities and
Pyrazoles represent a key structural motif in heterocyclic attached to different heterocyclic ring systems through various
chemistry and occupy a significant position in medicinal chem- linkages27–31. In particular, potential anti-inflammatory activity
istry because of their capability to exhibit a wide range of was displayed by the pyrazolyl derivatives (A–C; Figure 1)28,30
bioactivities. They were found to display anti-inflammatory12–14 and the bipyrazolyl lead structure (D; Figure 1)27.
and analgesic activities15,16 in addition to COX-2 inhibitory
In view of the above-mentioned facts, we report herein the
activity17,18. The pyrazole unit is one of the core structures in a synthesis, anti-inflammatory, and analgesic activities of some new
number of selective COX-2 inhibitors such as celecoxib and amide-linked bipyrazoles. The designed compounds comprise
SC-558 (Figure 1)19–21. Moreover, extensive studies have been the N-phenylpyrazole scaffold linked either to polysubstituted
devoted to N-aryl pyrazole derivatives as dual anti-inflamma- pyrazoles or to antipyrine moiety through different amide
tory and antimicrobial agents22–24. On the other hand, great linkages and have the general formulae E-H (Figure 1). The
attention has been focused on pyrazoline derivatives as a substitution pattern of the synthesized compounds was carefully
potent class of anti-inflammatory, analgesic, and antipyretic selected so as to confer different electronic and lipophilic
agents13,25,26 since the development of antipyrine, the first properties to the molecules. In addition to the targeted in vivo
pyrazoline derivative used in the management of pain, inflam- anti-inflammatory and analgesic activities, the in vitro COX1/
mation, and fever.
COX2 inhibitory activity of the synthesized compounds and their

DOI: 10.3109/14756366.2015.1094469
Synthesis and evaluation of amide-linked bipyrazoles
3
effect on inducible nitric oxide synthase (iNOS) and COX-2 appropriate aldehyde (10 mmol) was added. The reaction mixture,
protein expression in lipopolysaccharide (LPS)-activated rat was heated under reflux for 5 h. The solid products formed upon
monocytes were also investigated.
cooling was collected by filtration and crystallized from dioxane/
water (3:1).
Methods and materials
2-Cyano-N-(4-cyano-3-(methylsulfanyl)-1-phenyl-1H-pyrazol-5-
yl)-3-phenylacrylamide 4a
Chemistry
All reagents and solvents were purchased from commercial
suppliers and, when necessary, were purified and dried by
standard protocols. Melting points were determined in open
glass capillaries using Stuart capillary melting point apparatus
(Stuart Scientific Stone, Staffordshire, UK) and are uncorrected.
Infrared (IR) spectra were recorded on Perkin-Elmer 1430
infrared spectrophotometer (PerkinElmer, Beaconsfield, UK) and
measured by ꢀ cmꢀ1 scale using KBr cell. NMR spectra were
scanned on Varian Mercury VX-300 or Bruker-400 MHz spec-
trometer using tetramethylsilane (TMS) as internal standard and
dimethyl sulfoxide d6 (DMSO-d6) as solvent (chemical shifts are
given in ꢁ ppm). Splitting patterns were designated as follows: s:
singlet; d: doublet; m: multiplet. Mass spectra were run on a gas
chromatograph/mass spectrometer Shimadzu GCMS-QP 2010
Plus (70 eV). Microanalyses were performed at the Regional
Center for Mycology and Biotechnology, Al-Azhar University,
Cairo, Egypt and the found values were within 0.4% of the
theoretical values. Follow-up of the reactions and checking
the purity of the compounds was made by thin layer chroma-
tography (TLC) on silica gel-precoated aluminum sheets
(Type 60 GF254, Merck, Darmstadt, Germany) and the spots
were detected by exposure to UV lamp at ꢂ 254 nm for
few seconds.
Pale yellow crystals (2.77 g, 72%), mp: 205–207 ꢁC. IR (KBr,
cmꢀ1): 3300 (NH), 3056, 2925, 2853 (CH), 2239 (CN), 1698
(C¼O), 1622 (C¼N), 1580, 1496, 1443 (C¼C), 1306, 1075 (C-S-
1
C). H-NMR (300 MHz, DMSO-d6, ꢁ ppm): 2.57, 2.60 (2s, each
3H, 2SCH3, E and Z isomers), 7.01–8.11 (m, 22H, aromatic-H
and ¼ CH, E and Z isomers), 14.16, 14.35 (2s, each 1H, 2NH,
D2O exchangeable,
E and Z isomers). Anal. Calcd for
C21H15N5OS (385.44): C, 65.44; H, 3.92; N, 18.17. Found: C,
65.18; H, 3.63; N, 18.53.
3–(4-Chlorophenyl)-2-cyano-N-(4-cyano-3-(methylsulfanyl)-1-
phenyl-1H-pyrazol-5-yl)acrylamide 4b
White crystals (3.1 g, 74%), mp: 225–227 ꢁC. IR (KBr, cmꢀ1):
3289 (NH), 3057, 2925 (CH), 2236 (CN), 1605 (C¼N), 1581,
1497, 1445 (C¼C), 1307, 1107 (C-S-C). 1H-NMR (300 MHz,
DMSO-d6, ꢁ ppm): 2.59, 2.62 (2s, each 3H, 2SCH3, E and
Z isomers), 7.03–8.12 (m, 20H, aromatic-H and ¼ CH, E and
Z isomers), 14.24, 14.43 (2s, each 1H, 2NH, D2O exchangeable,
E and Z isomers). Anal. Calcd for C21H14ClN5OS (419.89):
C, 60.07; H, 3.36; N, 16.68. Found: C, 60.22; H, 3.31; N, 16.76.
2-Cyano-N-(4-cyano-3-(methylsulfanyl)-1-phenyl-1H-pyrazol-5-
yl)-3–(4-methoxyphenyl)acrylamide 4c
5-Amino-3-methylsulfanyl-1H-pyrazole-4-carbonitrile 2
White crystals (3.0 g, 72.2%), mp: 218–219 ꢁC. IR (KBr, cmꢀ1):
3290 (NH), 3056, 2924, 2856 (CH), 2230 (CN), 1700 (C¼O),
1620 (C¼N), 1580, 1488, 1456 (C¼C), 1302, 1078 (C-S-C),
1266, 1024 (C-O-C). 1H-NMR (300 MHz, DMSO-d6, ꢁ ppm):
2.57, 2.60 (2s, each 3H, 2SCH3, E and Z isomers), 3.82, 3.84 (2s,
each 3H, 2OCH3, E and Z isomers), 7.01–8.14 (m, 20H, aromatic-
H and ¼ CH, E and Z isomers), 14.19, 14.33 (2s, each 1H, 2NH,
A solution of 2-bis(methylthio)methylene)malononitrile (17 g,
100 mmol) and phenyl hydrazine (10.8 g, 100 mmol) in methanol
(200 mL) was heated under reflux for 3 h. The reaction mixture
was concentrated under reduced pressure and the separated solid
product was filtered and recrystallized from methanol to give
colorless needles (22 g, 96%), mp; 141–142 ꢁC; reported mp:
136 ꢁC32.
D2O exchangeable,
E and Z isomers). Anal. Calcd for
C22H17N5O2S (415.46): C, 63.60; H, 4.12; N, 16.86. Found: C,
63.35; H, 3.86; N, 16.55.
2-Cyano-N-(4-cyano-3-(methylsulfanyl)-1-phenyl-1H-pyrazol-5-
yl)acetamide 3
The aminopyrazole 2 (4.6 g, 20 mmol) was added to a mixture of
cyanoacetic acid (2.55 g, 30 mmol) and acetic anhydride (30 mL)
and heated at 50 ꢁC for 5 h. The mixture was allowed to cool then
poured into ice-cold water. The formed precipitate was filtered,
washed with water, dried, and crystallized from methanol.
Colorless needles (4.4 g, 74%), mp: 186–187 ꢁC. IR (KBr,
cmꢀ1): 3253 (NH), 3048, 2950, 2907 (CH), 2224 (CN), 1704
(C¼O), 1594 (C¼N), 1563, 1524, 1459 (C¼C), 1319, 1075 (C-S-
General procedure for the synthesis of 3-Amino-N-(4-cyano-3-
(methylsulfanyl)-1-phenyl-1H-pyrazol-5-yl)-5-substituted-1H-pyr-
azole-4-carboxamides 5
A mixture of compounds 4a–c (10 mmol) and hydrazine hydrate
98% (0.50 g, 10 mmol), in ethanol (25 mL) was heated under
reflux for 6 h. The solid products formed upon cooling
were filtered, washed with ethanol, and crystallized from
dioxane/water (4:1).
1
C). H-NMR (300 MHz, DMSO-d6, ꢁ ppm): 2.59 (s, 3H, SCH3),
3.99 (s, 2H, CH2), 7.46-7.60 (m, 5H, phenyl-H), 11.17 (s, 1H, NH,
D2O exchangeable). 13C-NMR (75 MHz, ꢁ ppm): 14.09 (CH3),
26.46 (CH2), 89.10 (pyrazole C4), 112.24, 115.58 (CN), 124.54
(phenyl C2,6), 129.35 (phenyl C4), 130.03 (phenyl C3,5), 137.41
(phenyl C1), 141.21 (pyrazole C3), 150.77 (pyrazole C5), 162.65
(C¼O). Anal. Calcd for C14H11N5OS (297.34): C, 56.55; H, 3.73;
N, 23.55. Found: C, 56.68; H, 3.72; N, 23.74.
3-Amino-N-(4-cyano-3-(methylsulfanyl)-1-phenyl-1H-pyrazol-5-
yl)-5-phenyl-1H-pyrazole-4-carboxamide 5a
White crystals (2.9 g, 70%), mp: 233–234 ꢁC . IR (KBr, cmꢀ1):
3451, 3327, 3187 (NH), 3060, 2923 (CH), 2227 (CN), 1673
(C¼O), 1625 (C¼N), 1580, 1500, 1463 (C¼C), 1311, 1108 (C-S-
1
C). H-NMR (300 MHz, DMSO-d6, ꢁ ppm): 2.62 (s, 3H, SCH3),
5.42 (s, 2H, NH2, D2O exchangeable), 6.86-7.54 (m, 8H,
aromatic-H), 8.01 (d, J ¼ 7.8 Hz, 2H, phenyl C2,6-H), 10.60,
11.90 (2s, each 1H, 2NH, D2O exchangeable). 13C-NMR
(75 MHz, ꢁ ppm): 14.99 (CH3), 99.28, 99.39 (pyrazole C4),
116.14 (CN), 120.73, 120.79 (phenyl C2,6), 126.41 (aminopyr-
azole C5), 128.27, 128.55 (phenyl C4), 129.56, 129.62 (phenyl
General procedure for the synthesis of 2-Cyano-N-(4-cyano-3-
(methylsulfanyl)-1-phenyl-1H-pyrazol-5-yl)-3-substituted acryl-
amides 4
To a solution of the cyanoacetamide 3 (2.97 g, 10 mmol) in
absolute ethanol (25 mL) containing few drops of piperidine, the

4
W. H. Faour et al.
J Enzyme Inhib Med Chem, Early Online: 1–16
C3,5), 139.01, 139.17 (phenyl C1), 142.21 (cyanopyrazole C3), 35.29 (NCH3) 96.52 (pyrazole C4), 99.68 (pyrazolinone C4),
155.22 (cyanopyrazole C5), 158.27 (aminopyrazole C3), 165.47 106.21 (acrylamide C2), 113.40, 117.99 (CN), 125.39, 125.72
(C¼O). MS (m/z, %): 359 (M+.– C3H6N), 77 (100). Anal. Calcd (phenyl C2,6), 127.56, 127.77 (phenyl C4), 129.07 (pyrazolinone
for C21H17N7OS (415.47): C, 60.71; H, 4.12; N, 23.60. Found: C, C5), 129.22, 129.55 (phenyl C3,5), 133.60, 139.62 (phenyl C1),
60.87; H, 4.19; N, 23.72.
147.91 (pyrazole C3), 150.24 (pyrazole C5), 156.63, 157.38
(C¼O). MS (m/z, %): 374 (M+ – C6H7N3O, 3.87), 56 (100). Anal.
Calcd for C25H21N9O2S (511.56): C, 58.70; H, 4.14; N, 24.64.
Found: C, 58.87; H, 4.17; N, 24.82.
3-Amino-5-(4-chlorophenyl)-N-(4-cyano-3-(methylsulfanyl)-1-
phenyl-1H-pyrazol-5-yl)-1H-pyrazole-4-carboxamide 5b
White crystals (3.15 g, 70%), mp: 263–264 ꢁC. IR (KBr, cmꢀ1):
3432, 3298, 3150 (NH), 3071, 2908 (CH), 2228 (CN), 1670
(C¼O), 1643 (C¼N), 1582, 1500 (C¼C), 1315, 1097 (C-S-C).
1H-NMR (300 MHz, DMSO-d6, ꢁ ppm): 2.51 (s, 3H, SCH3), 5.41
(s, 2H, NH2, D2O exchangeable), 7.0–7.55 (m, 7H, aromatic-H),
7.95 (d, J ¼ 7.8 Hz, 2H, chlorophenyl C2,6-H), 10.65, 11.92 (2s,
each 1H, 2NH, D2O exchangeable). 13C-NMR (75 MHz, ꢁ ppm):
14.49 (CH3), 98.62 (two pyrazole C4), 116.15 (CN), 120.14
(phenyl C2,6), 125.77 (aminopyrazole C5), 127.67 (phenyl C4),
128.87 (chlorophenyl C2,3,5,6), 128.89 (phenyl C3,5), 129.65
(chlorophenyl C1), 137.69 (chlorophenyl C4), 138.25 (phenyl
C1), 141.51 (cyanopyrazole C3), 154.43 (cyanopyrazole C5),
157.53 (aminopyrazole C3), 164.45 (C¼O). MS (m/z, %): 393
(M+.– C3H6N, 22.1), 64 (100). Anal. Calcd for C21H16ClN7OS
(449.92): C, 56.06; H, 3.58; N, 21.79. Found: C, 56.18; H, 3.62;
N, 21.95.
General procedure for the synthesis of 2-cyano-N-(4-cyano-3-
(methylsulfanyl)-1-phenyl-1H-pyrazol-5-yl)-3-substituted acryla-
mides 7
To a solution of compound 3 (2.97 g, 10 mmol) in absolute
ethanol (15 mL) containing three drops of sodium hydroxide
(10%), the appropriate 3-aryl-1-phenyl-1H-pyrazole-4-carboxal-
dehydes (10 mmol) was added. The reaction mixture was heated
under reflux for 2 h and then allowed to cool. The obtained
precipitate was collected by filtration, washed with ethanol, dried,
and recrystallized from a mixture of DMF/ethanol (1:1).
2-Cyano-N-(4-cyano-3-(methylsulfanyl)-1-phenyl-1H-pyrazol-5-
yl)-3-(1,3-diphenyl-1H-pyrazol-4-yl)acrylamide 7a
Yellowish white crystals (3.9 g, 74%), mp: 278–279 ꢁC. IR (KBr,
cmꢀ1): 3373 (NH), 3061, 2926 (CH), 2228 (CN), 1697 (C¼O),
1630 (C¼N), 1582, 1523, 1498, 1458 (C¼C), 1297, 1067 (C-S-
1
C). H-NMR (300 MHz, DMSO-d6, ꢁ ppm): 2.49 (s, 3H, SCH3),
3-Amino-N-(4-cyano-3-(methylsulfanyl)-1-phenyl-1H-pyrazol-5-
yl)-5-(4-methoxyphenyl)-1H-pyrazole-4-carboxamide 5c
7.46-7.64 (m, 13H, aromatic-H), 7.93 (d, J ¼ 8.1 Hz, 2H, phenyl
C2,6-H), 8.1 (s, 1H, methine-H), 9.21(s, 1H, pyrazole C5-H), 11.12
(s, 1H, NH, D2O exchangeable). 13C-NMR (75 MHz, ꢁ ppm):
14.09 (SCH3), 90.32, 102.45 (pyrazole C4), 112.30 (acrylamide
C2), 114.73, 116.55 (CN), 120.20, 124.17 (phenyl C2,6), 128.63,
129.38 (phenyl C4), 129.43 (phenyl C2,6), 129.52 (phenyl C4),
129.87, 130.0, 130.30 (phenyl C3,5), 130.36 (diphenylpyrazole
C5), 131.10, 137.59, 138.98 (phenyl C1), 141.82, 144.79 (pyrazole
C3), 150.77 (cyanopyrazole C5), 155.55 (acrylamide C3), 161.39
(C¼O). MS (m/z, %): 527 (M+.,8.4), 298 (100). Anal. Calcd for
C30H21N7OS (527.6): C, 68.29; H, 4.01; N, 18.58. Found: C,
68.52; H, 4.02; N, 18.65.
White crystals (3.0 g, 68%) mp: 254–256 ꢁC. IR (KBr, cmꢀ1):
3444, 3309, 3168 (NH), 3059, 2924, 2856 (CH), 2227 (CN), 1675
(C¼O), 1628 (C¼N), 1586, 1525, 1468 (C¼C), 1310, 1112 (C-S-
C), 1262, 1229 (C-O-C). 1H-NMR (300 MHz, DMSO-d6, ꢁ ppm):
2.55 (s, 3H, SCH3), 3.89 (s, 3H, OCH3), 5.46 (s, 2H, NH2, D2O
exchangeable), 7.01–7.58 (m, 7H, aromatic-H), 8.0 (d, J ¼ 8.0 Hz,
2H, methoxyphenyl C2,6-H), 10.58, 11.90 (2s, each 1H, 2NH, D2O
exchangeable). 13C-NMR (75 MHz, ꢁ ppm): 14.76 (CH3), 98.46,
99.24 (pyrazole C4), 113.99 (methoxyphenyl C3,5), 116.18 (CN),
120.58 (phenyl C2,6), 121.68 (methoxyphenyl C1), 126.43
(aminopyrazole C5), 128.34 (phenyl C4), 129.40 (phenyl C3,5),
131.03 (methoxyphenyl C2,6), 139.04 (phenyl C1), 142.26
(cyanopyrazole C3), 155.40 (cyanopyrazole C5), 157.88 (amino-
pyrazole C3), 160.12 (methoxyphenyl C4), 165.48 (C¼O). Anal.
Calcd for C22H19N7O2S (445.49): C, 59.31; H, 4.30; N, 22.01.
Found: C, 59.67; H, 4.19; N, 22.32.
3-(3–(4-Chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-2-cyano-N-(4-
cyano-3-methylsulfanyl-1-phenyl-1H-pyrazol-5-yl)acrylamide 7b
Yellowish white crystals (4.38 g, 78%), mp: 248–249 ꢁC . IR
(KBr, cmꢀ1): 3376 (NH), 3058, 2926 (CH), 2232 (CN), 1704
(C¼O), 1631 (C¼N), 1587, 1501, 1458 (C¼C), 1297, 1095 (C-
S-C). 1H-NMR (300 MHz, DMSO-d6, ꢁ ppm): 2.52 (s, 3H,
SCH3), 7.48-7.65 (m, 12H, aromatic-H), 7.95 (d, J ¼ 8.1 Hz, 2H,
phenyl C2,6-H), 8.22 (s, 1H, methine-H), 9.21(s, 1H, pyrazole
C5-H), 11.18 (s, 1H, NH, D2O exchangeable). 13C-NMR
(75 MHz, ꢁ ppm): 14.16 (SCH3), 90.42, 102.63 (pyrazole C4),
112.36 (CN), 114.84 (acrylamide C2), 116.56 (CN), 120.18,
124.24 (phenyl C2,6), 128.69, 129.41 (phenyl C4), 129.45
(chlorophenyl C2,6), 129.76, 129.90 (phenyl C3,5), 130.08
(chlorophenyl C3,5), 130.49 (diphenylpyrazole C5), 131.53
(chlorophenyl C1), 134.65 (chlorophenyl C4), 138.68, 139.23
(phenyl C1), 141.94, 145.02 (pyrazole C3), 150.79 (cyanopyr-
azole C5), 155.64 (acrylamide C3), 161.43 (C¼O). MS (m/z, %):
563 (M+. + 2, 3.8), 561 (M+., 9.2), 332 (100). Anal. Calcd for
C30H20ClN7OS (562.04): C, 64.11; H, 3.59; N, 17.44. Found: C,
64.18; H, 3.54; N, 17.71.
2-Cyano-N-[4-cyano-3-(methylsulfanyl)-1-phenyl-1H-pyrazol-5-
yl]-2-[(2,3-dihydro-1,5-dimethyl-3-oxo-2-phenyl–1H-pyrazol-4-
yl)hydrazono]acetamide 6
A well-stirred solution of 4-aminoantipyrine (1.02 g, 5 mmol) in
2 N HCl (1.5 mL) was cooled in ice salt bath and diazotized with
1 N NaNO2 solution (0.35 g, 5 mmol) in 2 mL water. The resulting
cold solution of the diazonium salt was then added to a cold
solution of the cyanoacetamide 3 (1.48 g, 5 mmol) in ethanol
(20 mL) in the presence of sodium acetate (0.43 g, 5.2 mmol). The
reaction mixture was then stirred at room temperature for 1 h and
the separated product was filtered, washed with water then
ethanol, dried, and crystallized from dimethylformamide (DMF)/
ethanol (2:1). Red crystals (1.86 g, 73%), mp: 139–140 ꢁC. IR
(KBr, cmꢀ1): 3308 (NH), 3062, 2924 (CH), 2215 (CN), 1652
(C¼O), 1592 (C¼N), 1496, 1456 (C¼C), 1294, 1045 (C-S-C).
1H-NMR (300 MHz, DMSO-d6, ꢁ ppm): 2.49 (s, 3H, SCH3), 2.56
(s, 3H, C-CH3), 3.23 (s, 3H, N-CH3), 7.30–7.57 (m, 10H,
aromatic-H), 10.61, 13.90 (2s, each 1H, 2NH, D2O exchange-
able). 13C-NMR (75 MHz, ꢁ ppm): 10.60 (C-CH3), 14.49 (SCH3),
2-Cyano-N-(4-cyano-3-methylsulfanyl-1-phenyl-1H-pyrazol-5-yl)-
3-(3–(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)acrylamide 7c
Yellowish white needles (4.18 g, 75%), mp: 270–272 ꢁC. IR (KBr,
cmꢀ1): 3368 (NH), 3064, 2928 (CH), 2230 (CN), 1700 (C¼O),

DOI: 10.3109/14756366.2015.1094469
Synthesis and evaluation of amide-linked bipyrazoles
5
1630 (C¼N), 1584, 1511, 1488, 1453 (C¼C), 1295, 1075 (C-S- 2-Cyano-N-(4-cyano-3-(methylsulfanyl)-1-phenyl-1H-pyrazol-5-
C), 1260, 1025 (C-O-C). 1H-NMR (300 MHz, DMSO-d6, ꢁ ppm): yl)-3-(2,3-dihydro-1,5-dimethyl-3-oxo-2-phenyl-1H-pyrazol-4-
2.49 (s, 3H, SCH3), 3.80 (s, 3H, OCH3), 7.45-7.67 (m, 12H, ylamino)acrylamide 11
aromatic-H), 7.93 (d, J ¼ 8.1 Hz, 2H, phenyl C2,6-H), 8.15 (s, 1H,
Method I: A mixture of 9 (1.76 g; 5 mmol) and 4-aminoantipyrine
(1.0 g; 5 mmol) was fused at 170 ꢁC in an oil bath for 1 h.
The reaction mixture was left to cool and the obtained solid
residue was triturated with hot ethanol (20 mL) and filtered, then
crystallized from DMF/ethanol (4:1). Grayish crystals (1.91 g,
75%), mp: 261–262 ꢁC.
methine-H), 9.20 (s, 1H, pyrazole C5-H), 11.15 (s, 1H, NH, D2O
exchangeable). 13C-NMR (75 MHz, ꢁ ppm): 14.11 (SCH3), 55.63
(OCH3), 90.38, 102.51 (pyrazole C4), 112.32 (acrylamide C2),
114.13 (methoxyphenyl C3,5), 114.68, 116.46 (CN), 120.19
(phenyl C2,6), 121.89 (methoxyphenyl C1), 123.18 (phenyl C2,6),
126.68, 126.84 (phenyl C4), 129.96, 130.18 (phenyl C3,5), 130.33
(diphenylpyrazole C5), 130.96 (methoxyphenyl C2,6), 139.73,
139.78 (phenyl C1), 141.88, 145.02 (pyrazole C3), 150.79
(cyanopyrazole C5), 155.61 (acrylamide C3), 160.12 (methox-
yphenyl C4), 161.33 (C¼O). Anal. Calcd for C31H23N7O2S
(557.62): C, 66.77; H, 4.16; N, 17.58. Found: C, 66.43; H, 4.06;
N, 17.78.
Method II: A mixture of the ethoxyacrylamide 12 (1.06 g;
3 mmol) and 4-amino antipyrine (0.61 g; 3 mmol) was fused at
170 ꢁC in an oil bath for 1 h. The reaction mixture was left to cool
and the obtained solid residue was triturated with hot ethanol
(20 mL) and filtered, then crystallized from DMF/ethanol (4:1).
Grayish crystals (1.12 g, 73.2%). IR (KBr, cmꢀ1): 3339, 3287
(NH), 3045, 2922 (CH), 2227 (CN), 1666, 1631 (C¼O), 1589
1
(C¼N), 1560, 1497, 1465 (C¼C), 1309, 1066 (C-S-C). H-NMR
(400 MHz, DMSO-d6, ꢁ ppm): 2.43 (s, 3H, C-CH3), 2.49 (s, 3H,
SCH3), 3.17 (s, 3H, N-CH3), 7.39-7.57 (m, 11H, aromatic-H
and ¼ CH), 11.23, 12.74 (2s, each 1H, 2NH, D2O exchangeable).
13C-NMR (100 MHz, ꢁ ppm): 10.36 (C-CH3), 12.97 (SCH3),
35.83 (NCH3), 94.67 (pyrazole C4), 99.17 (acrylamide C2),
109.95 (pyrazolinone C4), 116.85 (two CN), 124.20, 125.75
(phenyl C2,6), 127.98, 128.26 (phenyl C4), 129.65, 129.75 (phenyl
C3,5), 134.65 (pyrazolinone C5), 137.50, 138.49 (phenyl C1),
143.06 (pyrazole C3), 145.98 (pyrazole C5), 150.85 (acrylamide
C3), 151.22, 160.55 (C¼O). MS (m/z, %): 510 (M+. – C4H2, 3.8),
56 (100). Anal. Calcd for C26H22N8O2S (510.57): C, 61.16; H,
4.34; N, 21.95. Found: C, 61.34; H, 4.31; N, 22.17.
2-Cyano-N-(4-cyano-3-(methylsulfanyl)-1-phenyl-1H-pyrazol-5-
yl)-3-(dimethylamino)acrylamide 9
A mixture of 3 (1.5 g, 5 mmol), DMF dimethyl acetal (6.55 g,
7.36 mL, 5.5 mmol) in dry dioxane (25 mL) was heated under
reflux for 4 h. The separated solid product obtained on standing at
room temperature was collected by filtration, washed with
ethanol, and recrystallized from DMF/ethanol (3:1). White
needles (1.51 g, 85%), mp: 216–218 ꢁC. IR (KBr, cmꢀ1): 3236
(NH), 3011, 2927 (CH), 2222, 2196 (CN), 1682 (C¼O), 1612
(C¼N), 1569, 1532, 1495, 1458 (C¼C), 1274, 1072 (C-S-C). 1H-
NMR (300 MHz, DMSO-d6, ꢁ ppm): 2.49 (s, 3H, SCH3), 3.14,
3.21 (2s, each 3H, N(CH3)2), 7.38-7.52 (m, 5H, phenyl-H), 7.68
(s, 1H, ¼CH), 9.72 (s, 1H, NH, D2O exchangeable). Anal. Calcd
for C17H16N6OS (352.41): C, 57.94; H, 4.58; N, 23.85. Found: C,
57.61; H, 4.63; N, 23.53.
2-Cyano-N-(4-cyano-3-(methylsulfanyl)-1-phenyl-1H-pyrazol-5-
yl)-3-ethoxyacrylamide 12
A mixture of the cyanoacetamide 3 (2.97 g, 10 mmol), triethyl
orthoformate (1.48 g, 1.66 mL, 10 mmol) in dry dioxane (5 mL)
containing few drops of piperidine was heated under reflux for 5
h. The reaction mixture was left to attain room temperature then
poured into ice-cold water and stirred for 30 min. The separated
solid product was filtered, washed with water, dried, and
crystallized from ethanol. White needles (2.3 g, 65%), mp: 237–
238 ꢁC. IR (KBr, cmꢀ1): 3197 (NH), 3079, 3036, 2931 (CH),
2226 (CN), 1685 (C¼O), 1593 (C¼N), 1527, 1463, 1430 (C¼C),
5-Amino-N-(4-cyano-3-(methylsulfanyl)-1-phenyl-1H-pyrazol-5-
yl)-1H-pyrazole-4-carboxamide 10
Method I: A mixture of the enaminonitrile 9 (3.52 g, 10 mmol)
and hydrazine hydrate 98% (0.50 g, 10 mmol) in dry dioxane
(20 mL) was heated under reflux for 5 h. The reaction mixture was
concentrated and poured into ice-cold water. The separated solid
product was collected by filtration, washed with ethanol, and
crystallized from dioxane/ethanol (2:1). Beige crystals (2.3 g,
68%), mp: 195–196 ꢁC.
Method II: A mixture of the ethoxyacrylamide 12 (1.0 g,
2.8 mmol) and hydrazine hydrate 98% (0.14 g, 2.8 mmol) in
absolute ethanol (20 mL) was heated under reflux for 5 h. The
reaction mixture was cooled to room temperature and the
separated solid product was collected by filtration, washed
with ethanol, and crystallized from dioxane/ethanol (2:1).
Beige crystals (0.69 g, 72%). IR (KBr, cmꢀ1): 3328, 3201
(NH), 3022, 2924 (CH), 2223 (CN), 1650 (C¼O), 1612
1
1316, 1072 (C-S-C), 1264, 1024 (C-O-C). H-NMR (400 MHz,
DMSO-d6, ꢁ ppm): 1.23 (t, J ¼ 6.9 Hz, 3H, CH3), 2.59 (s, 3H,
SCH3), 3.99 (q, J ¼ 6.9 Hz, 2H, CH2), 7.49-7.56 (m, 5H, phenyl-
H), 7.94 (s, 1H, ¼CH), 10.58 (s, 1H, NH, D2O exchangeable).
Anal. Calcd for C17H15N5O2S (353.4): C, 57.78; H, 4.28; N,
19.82. Found: C, 57.41; H, 4.53; N, 19.49.
N1-(4-Cyano-3-(methylsulfanyl)-1-phenyl-1H-pyrazol-5-yl)malo-
namide 13
(C¼N), 1548, 1504, 1460 (C¼C), 1301, 1113 (C-S-C). This compound was obtained as a major product in the first trial
1H-NMR (400 MHz, DMSO-d6, ꢁ ppm): 2.49 (s, 3H, SCH3), to prepare compounds 14a, b upon treatment of the cyanoace-
6.09 (s, 2H, NH2, D2O exchangeable), 7.38-7.58 (m, 5H, tamide 3 (1.19 g; 4 mmol) with the selected isothiocyanate
phenyl-H), 7.78 (s, 1H, pyrazole C3-H), 10.10, 11.92 (2s, each (4 mmol) in DMF (8 mL) containing finely divided KOH
1H, 2NH, D2O exchangeable). 13C-NMR (100 MHz, ꢁ ppm): (0.22 g; 4 mmol) at room temperature followed by the addition
14.11 (SCH3), 90.46 (cyanopyrazole C4), 112.73 (amino of dimethyl sulfate (0.5 g, 0.38 mL, 4 mmol) and stirring for an
pyrazole C4), 114.49 (CN), 124.0 (phenyl C2,6), 129.95 overnight at room temperature. It was crystallized from DMF/
(phenyl C4), 130.29 (phenyl C3,5), 137.77 (phenyl C1), ethanol (3:1). White crystals (1.07 g, 85%), mp: 259–260 ꢁC. IR
143.01 (cyanopyrazole C3), 149.37 (aminopyrazole C3), (KBr, cmꢀ1): 3433, 3328 (NH), 3066, 2984, 2924 (CH), 2213
150.39 (cyanopyrazole C5), 152.80 (aminopyrazole C5), (CN), 1638 (C¼O), 1592 (C¼N), 1502, 1463, 1434 (C¼C), 1316,
1
163.06 (C¼O). MS (m/z, %): 339 (M+., 1.6), 64 (100). 1115 (C-S-C). H-NMR (400 MHz, DMSO-d6, ꢁ ppm): 2.68 (s,
Anal. Calcd for C15H13N7OS (339.38): C, 53.09; H, 3.86; N, 3H, SCH3), 3.98 (s, 2H, CH2), 7.15 (s, 2H, NH2, D2O
28.89. Found: C, 53.21; H, 3.63; N, 28.53.
exchangeable), 7.33 (t, J ¼ 8.2 Hz, 1H, phenyl C4-H), 7.53

6
W. H. Faour et al.
J Enzyme Inhib Med Chem, Early Online: 1–16
(t, J ¼ 7.6 Hz, 2H, phenyl C3,5-H), 8.13 (dd, J ¼ 8.2 Hz, 1. 0 Hz, 2H, NH2, D2O exchangeable), 6.76 (t, J ¼ 7.8 Hz, 1H, phenyla-
2H, phenyl C2,6-H), 11.4 (s, 1H, NH, D2O-exchangeable). 13C- mino C4-H), 7.05-7.59 (m, 9H, aromatic-H), 8.05, 9.31, 11.30 (3s,
NMR (100 MHz, ꢁ ppm): 15.73 (CH3), 54.89 (CH2), 100.64 each 1H, 3NH, D2O exchangeable). 13C-NMR (75 MHz, DMSO-
(pyrazole C4), 115.50 (CN), 120.91 (phenyl C2,6), 126.59 (phenyl d6) d 14.16 (CH3), 87.10, 88.87 (pyrazole C4), 112.94 (CN),
C4), 129.59 (phenyl C3,5), 138.96 (phenyl C1), 142.07 (pyrazole 116.60 (phenylamino C2,6), 119.86 (phenylamino C4), 124.34
C3), 150.74 (pyrazole C5), 154.07, 166.55 (C¼O). Anal. Calcd for (phenyl C2,6), 129.19 (phenyl C4), 129.21 (phenyl C3,5), 129.92
C14H13N5O2S (315.35): C, 53.32; H, 4.16; N, 22.21. Found: C, (phenylamino C3,5), 137.51 (phenyl C1), 142.65 (phenylamino
53.41; H, 4.26; N, 22.49.
C1), 143.53, 149.92 (pyrazole C3), 150.51, 150.69 (pyrazole C5),
162.62 (C¼O). MS (m/z, %): 256 (M+. – C9H10N4, 2.8), 80 (100).
Anal. Calcd for C21H18N8OS (430.49): C, 58.59; H, 4.21; N,
26.03. Found: C, 58.77; H, 4.18; N, 26.28.
General procedure for the synthesis of 2-Cyano-N-(4-cyano-3-
(methylsulfanyl)-1-phenyl-1H-pyrazol-5-yl)-3-(methylsulfanyl)-3-
(substituted amino) acrylamides 14
3–(4-Chlorophenylamino)-5-amino-N-(4-cyano-3-(methylsulfa-
nyl)-1-phenyl-1H-pyrazol-5-yl)-1H-pyrazole-4-carboxamide 15b
To a well-stirred ice-cold suspension of finely divided KOH
(0.22 g; 4 mmol) in dry DMF (8 mL) was added the cyanoaceta-
mide derivative 3 (1.19 g; 4 mmol) followed by phenyl; or 4-
chlorophenyl isothiocyanate (4 mmol). The mixture was stirred at
0–5 ꢁC for 2 h, left to attain room temperature then stirring is
continued for an overnight. Dimethyl sulfate (0.5 g, 0.38 mL,
4 mmol) was then added and stirring was maintained for 24 h. The
reaction mixture was poured into ice-cold water and the separated
product was filtered, washed with ethanol, dried, and crystallized
from DMF/ethanol (2:1).
White needles (1.63 g, 70.1%), mp: 215–216 ꢁC . IR (KBr,
cmꢀ1): 3347, 3220, 3166 (NH), 3062, 2956 (CH), 2224 (CN),
1686 (C¼O), 1621 (C¼N), 1567, 1517, 1490 (C¼C), 1313, 1077
1
(C-S-C). H-NMR (300 MHz, DMSO-d6) ꢁ 2.60 (s, 3H, SCH3),
6.20 (s, 2H, NH2, D2O exchangeable), 7.19-7.55 (m, 9H,
aromatic-H), 8.38, 9.40, 11.44 (3s, each 1H, 3NH, D2O
exchangeable). 13C-NMR (75 MHz, DMSO-d6) ꢁ 13.69 (CH3),
86.55, 88.40 (pyrazole C4), 112.29 (CN), 117.43 (chlorophenyl
C2,6), 122.62 (chlorophenyl C4), 123.65 (phenyl C2,6), 123.90
(phenyl C4), 128.21 (chlorophenyl C3,5), 129.17 (phenyl C3,5),
136.84 (phenyl C1), 141.74 (chlorophenyl C1), 141.96, 149.34
(pyrazole C3), 149.60, 149.80 (pyrazole C5), 161.89 (C¼O). MS
(m/z, %): 464 (M+ 0.13), 208 (100). Anal. Calcd for
C21H17ClN8OS (464.93): C, 54.25; H, 3.69; N, 24.10. Found:
C, 54.41; H, 3.63; N, 24.49.
2-Cyano-N-(4-cyano-3-(methylsulfanyl)-1-phenyl-1H-pyrazol-5-
yl)-3-(methylsulfanyl)-3-(phenylamino)acrylamide 14a
Yellowish crystals (1.0 g, 56%), mp: 197–198 ꢁC. IR (KBr, cmꢀ1):
3215, 3195 (NH), 3062, 2926 (CH), 2226, 2195 (CN), 1630
(C¼O), 1597 (C¼N), 1542, 1502, 1448 (C¼C), 1275, 1245, 1086
1
(C-S-C). H-NMR (300 MHz, DMSO-d6, ꢁ ppm): 2.34, 2.59 (2s,
each 3H, 2SCH3), 6.78-7.55 (m, 10H, aromatic-H), 8.79, 11.43
(2s, each1H, 2NH, D2O exchangeable). Anal. Calcd for
C22H18N6OS2 (446.55): C, 59.17; H, 4.06; N, 18.82. Found: C,
59.28; H, 4.12; N, 18.95.
General procedure for the synthesis of 2-Cyano-N-(4-cyano-3-
(methylsulfanyl)-1-phenyl-1H-pyrazol-5-yl)-3-(2,3-dihydro-1,5-
dimethyl-3-oxo-2-phenyl-1H-pyrazol-4-ylamino)-3-(substituted
amino)acrylamides 16
3–(4-Chlorophenylamino)-2-cyano-N-(4-cyano-3-(methylsulfa-
nyl)-1-phenyl-1H-pyrazol-5-yl)-3-(methylsulfanyl)acrylamide 14b
A mixture of the acrylamide 14 (5 mmol) and 4-aminoantipyrine
(1 g; 5 mmol) was fused at 170–180 ꢁC in oil bath for 2 h. Ethanol
(20 mL) was added and the reaction mixture was refluxed for
further 2 h then concentrated and left to stand for an overnight.
The separated solid product was filtered, washed with ethanol,
dried, and crystallized from ethanol–DMF (4:1).
Beige crystals (1.25 g, 65%), mp: 204–206 ꢁC . IR (KBr, cmꢀ1):
3224, 3197 (NH), 3059, 2925 (CH), 2226, 2196 (CN), 1630
(C¼O), 1591 (C¼N), 1540, 1501, 1448 (C¼C), 1280, 1245, 1088
1
(C-S-C). H-NMR (300 MHz, DMSO-d6, ꢁ ppm): 2.24, 2.60 (2s,
each 3H, 2SCH3), 7.33-7.51 (m, 5H, phenyl-H), 7.55, 7.56 (2d,
J ¼ 7.8 Hz, each 2H, chlorophenyl-H), 9.25, 11.48 (2s, each1H,
2NH, D2O exchangeable). Anal. Calcd for C22H17ClN6OS2
(480.99): C, 54.94; H, 3.56; N, 17.47. Found: C, 55.02; H,
3.59; N, 17.56.
2-Cyano-N-(4-cyano-3-(methylsulfanyl)-1-phenyl-1H-pyrazol-5-
yl)-3-(2,3-dihydro-1,5-dimethyl-3-oxo-2-phenyl-1H-pyrazol-4-
ylamino)-3-(phenylamino)acrylamide 16a
Light brown crystals (2.1 g, 70%), mp: 279–280 ꢁC. IR (KBr,
cmꢀ1): 3376, 3312, 3187 (NH), 3038, 2921 (CH), 2225 (CN),
1662, 1631 (C¼O), 1590 (C¼N), 1560, 1498 (C¼C), 1310, 1067
General procedure for the synthesis of 5-Amino-N-(4-cyano-3-
(methylsulfanyl)-1-phenyl-1H-pyrazol-5-yl)-3-(substituted
amino)-1H-pyrazole-4-carboxamides 15
1
(C-S-C). H-NMR (300 MHz, DMSO-d6, ꢁ ppm): 2.39 (s, 3H, C-
CH3), 2.51 (s, 3H, SCH3), 3.15 (s, 3H, N-CH3), 7.33-7.57 (m,
15 H, aromatic-H), 7.69, 11.20, 12.40 (3s, each 1H, 3NH, D2O
exchangeable). 13C-NMR (75 MHz, ꢁ ppm): 13.65 (C-CH3), 14.95
(SCH3), 35.84 (N-CH3), 97.51 (pyrazole C4), 99.15 (acrylamide
C2), 99.55 (pyrazolinone C4), 112.80, 116.84 (CN), 120.36
(pyrazolinone phenyl C2,6), 123.73 (phenylamino C2,6), 124.23
(phenylamino C4), 125.73 (pyrazolinone phenyl C4), 127.21
(phenyl C4), 128.01 (pyrazolinone C5), 129.13 (phenyl C2,6),
129.40 (pyrazolinone phenyl C3,5), 129.65 (phenyl C3,5), 129.80
(phenylamino C3,5), 139.07 (pyrazolinone phenyl C1), 139.43
(phenyl C1), 142.26 (phenylamino C1), 148.69 (pyrazole C3),
A mixture of the acrylamide 14 (5 mmol) and hydrazine hydrate
98% (0.25 g; 5 mmol) was heated on a water bath for 2 h. Then,
ethanol (20 mL) was added and the reaction mixture was refluxed
for further 2 h. The reaction mixture was left to stand overnight
and the separated solid product was filtered, washed with ethanol,
dried, and crystallized from ethanol.
5-Amino-N-(4-cyano-3-(methylsulfanyl)-1-phenyl-1H-pyrazol-5-
yl)-3-(phenylamino)-1H-pyrazole-4-carboxamide 15a
White needles (1.46 g, 68%), mp: 199–200 ꢁC. IR (KBr, cmꢀ1): 150.21 (pyrazole C5), 155.39, 157.48 (C¼O), 161.50 (acrylamide
3373, 3225, 3163 (NH), 3068, 2926 (CH), 2222 (CN), 1681 C3). MS (m/z, %): 460 (M+. -C10H7N, 3.7), 56 (100). Anal. Calcd
(C¼O), 1626 (C¼N), 1570, 1515, 1488 (C¼C), 1275, 1078 (C-S- for C32H27N9O2S (601.68): C, 63.88; H, 4.52; N, 20.95. Found: C,
C). 1H-NMR (300 MHz, DMSO-d6) d 2.59 (s, 3H, SCH3), 6.12 (s, 63.97; H, 4.61; N, 21.17.

DOI: 10.3109/14756366.2015.1094469
Synthesis and evaluation of amide-linked bipyrazoles
7
3-(Chlorophenylamino)-2-cyano-N-(4-cyano-3-(methylsulfanyl)-
1-phenyl-1H-pyrazol-5-yl)-3-(2,3-dihydro-1,5-dimethyl-3-oxo-2-
phenyl-1H-pyrazol-4-ylamino)acrylamide 16b
2-Cyano-N-(4-cyano-3-(methylsulfanyl)-1-phenyl-1H-pyrazol-5-
yl)-3-(2,3-dihydro-1,5-dimethyl-3-oxo-2-phenyl-1H-pyrazol-4-
ylamino)- 3-(methylsulfanyl)acrylamide 19
Light brown crystals (2.35 g, 74%), mp: 294–295 ꢁC. IR (KBr, A mixture of 17 (2 g; 5 mmol) and 4-aminoantipyrine (1 g;
cmꢀ1): 3376, 3312, 3187 (NH), 3041, 2923 (CH), 2224 (CN), 5 mmol) was fused at 140 ꢁC in an oil bath for 1 h. The reaction
1662, 1631 (C¼O), 1590 (C¼N), 1561, 1496 (C¼C), 1308, 1093 mixture was left to cool and the obtained solid residue
(C-S-C). 1H-NMR (300 MHz, DMSO-d6, ꢁ ppm): 2.38 (s, 3H, C- was triturated with ethanol (20 mL) and filtered, then
CH3), 2.52 (s, 3H, S-CH3), 3.17 (s, 3H, N-CH3), 7.35-7.59 (m, crystallized from dioxane. Brown fine crystals (1.9 g, 68.6%),
14 H, aromatic-H), 7.82, 11.30, 12.43 (3s, each 1H, 3NH, D2O mp: 221–222 ꢁC. IR (KBr, cmꢀ1): 3334, 3286 (NH), 3055, 2926
exchangeable). 13C-NMR (75 MHz, ꢁ ppm): 13.58 (C-CH3), 14.94 (CH), 2227 (CN), 1665, 1630 (C¼O), 1590 (C¼N), 1550, 1498,
1
(SCH3), 35.88 (N-CH3), 97.56 (pyrazole C4), 99.25 (acrylamide 1450 (C¼C), 1310, 1023 (C-S-C). H-NMR (300 MHz, DMSO-
C2), 99.73 (pyrazolinone C4), 113.04, 116.92 (CN), 120.56 d6, ꢁ ppm): 2.59, 2.65, 2.69 (3s, each 3H, 2SCH3, and C-CH3),
(pyrazolinone phenyl C2,6), 123.94 (chlorophenylamino C2,6), 3.15 (s, 3H, N-CH3), 7.30–7.56 (m, 10H, aromatic-H), 11.20,
124.38 (pyrazolinone phenyl C4), 125.73 (phenyl C4), 127.71 12.68 (2s, each 1H, 2NH, D2O exchangeable). 13C-NMR
(phenyl C2,6), 128.13 (Chlorophenylamino C4), 129.24 (pyrazo- (75 MHz, ꢁ ppm): 11.29 (C-CH3), 13.14 (SCH3), 35.78 (NCH3),
linone C5), 129.54 (pyrazolinone phenyl C3,5), 129.83 (phenyl 92.01 (pyrazole C4), 99.14 (acrylamide C2), 109.58 (pyrazolinone
C3,5), 129.89 (phenylamino C3,5), 139.14 (pyrazolinonephenyl C4), 113.16, 116.89 (CN), 123.99 (phenyl C2,6), 125.46 (phenyl
C1), 139.69 (phenyl C1), 142.45 (phenylamino C1), 149.02 C4), 127.88 (phenyl C2,6), 128.29 (phenyl C4), 129.54, 129.83
(pyrazole C3), 150.28 (pyrazole C5), 155.44, 157.57 (C¼O), (phenyl C3,5), 129.95 (pyrazolinone C5), 137.58, 138.64 (phenyl
161.53 (acrylamide C3). MS (m/z, %): 460 (M+. – C10H6ClN, C1), 142.98 (pyrazole C3), 146.02 (pyrazole C5), 155.20, 157.63
11.3), 56 (100). Anal. Calcd for C32H26ClN9O2S (636.13): C, (C¼O), 163.06 (acrylamide C3). MS (m/z, %): 459 (M+. – C5H5S,
60.42; H, 4.12; N, 19.82. Found: C, 60.19; H, 4.33; N, 19.59.
11.3), 56 (100). Anal. Calcd for C27H24N8O2S2 (556.66): C,
58.26; H, 4.35; N, 20.13. Found: C, 58.41; H, 4.63; N, 20.49.
2-Cyano-N-(4-cyano-3-(methylsulfanyl)-1-phenyl-1H-pyrazol-5-
yl)-3,3-bis(methylsulfanyl)acrylamide 17
Biological screening
To a well-stirred ice-cold suspension of 3 (2.97 g, 10 mmol) and Carrageenan-induced mouse paw edema
finely powdered potassium hydroxide (0.56 g, 10 mmol) in dry
Swiss albino mice of both sexes, weighing 20–35 g, were used
dimethyformamide (10 mL), carbon disulfide (0.91 g, 0.72 mL,
12 mmol) was gradually added over a period of 1 h. The reaction
mixture was stirred at room temperature for 3 h, then cooled again
to 0 ꢁC and treated with dimethyl sulfate (1.5 g, 1.14 mL,
12 mmol) dropwise then stirred at room temperature for an
additional 6 h. The reaction mixture was poured into ice/water and
the resulting precipitate was filtered off, dried, and crystallized
from dioxane. Yellowish crystals (3.0 g, 75%), mp: 230–232 ꢁC .
IR (KBr, cmꢀ1): 3338 (NH), 3038, 2995, 2925, 2855 (CH), 2206
(CN), 1660, (C¼O), 1599 (C¼N), 1567, 1515, 1455 (C¼C),
1303, 1118 (C-S-C). 1H-NMR (300 MHz, DMSO-d6, ꢁ ppm):
2.60, 2.62, 2.71 (3s, each 3H, 3SCH3), 7.45-7.59 (m, 3H, phenyl
C3,4,5-H), 7.65 (d, J ¼ 7.2 Hz, 2H, phenyl C2,6-H), 11.81(s, 1H,
NH, D2O exchangeable). Anal. Calcd for C17H15N5OS3
(401.53): C, 50.85; H, 3.77; N, 17.44. Found: C, 51.04; H,
3.72; N, 17.61.
throughout the work. The mice were kept in an animal house
under standard conditions of light and temperature with free
access to food and water. The animals were randomly divided into
groups of six mice each. In the control group, only the vehicle
(polyethylene glycol (PEG), acetone, and DMSO) was adminis-
tered. Positive control groups were treated with diclofenac Na
(50 mg/kg body weight) as the reference drug. The tested
compounds were given in equimolar doses to that of diclofenac.
All drugs and the vehicle were administered intraperitoneally.
After 1 h, the mice were challenged with subcutaneous injection
of 0.025 mL of 2% w/v solution of carrageenan into the plantar
side of the right hind paw. The volume of the injected paw was
determined plethysmographically using a pressure transducer
transforming the volume being increased by immersion of the
paw into a proportional voltage recorded on a computer. The paw
volume was measured immediately after carrageenan injection
(zero time) and 4 h later33. The difference between the two
readings gave the actual edema volumes to calculate the
percentage protection. The anti-inflammatory activity of the test
compounds relative to that of diclofenac was also calculated.
The percentage protection against inflammation was calculated
as follows:
5-Amino-N-(4-cyano-3-(methylsulfanyl)-1-phenyl-1H-pyrazol-5-
yl)-3-(methylsulfanyl)-1H-pyrazole-4-carboxamide 18
A mixture of the acrylamide 17 (2 g, 5 mmol) and hydrazine
hydrate 80% (0.25 g, 5 mmol) in ethanol (20 mL) was heated
under reflux for 2 h. The reaction mixture was left to cool and the
precipitated solid product was filtered, dried, and crystallized
from dioxane. Beige crystals (1.34 g, 70%), mp: 235–236 ꢁC. IR
(KBr, cmꢀ1): 3353, 3299, 3208 (NH), 3058, 2922 (CH), 2225
(CN), 1646, (C¼O), 1613 (C¼N), 1548, 1520, 1434 (C¼C),
1289, 1213, 1106, 1031 (C-S-C). 1H-NMR (300 MHz, DMSO-d6,
ꢁ ppm): 2.59, 2.65 (2s, each 3H, 2SCH3), 5.84 (s, 2H, NH2, D2O
exchangeable), 7.30–7.78 (m, 5H, phenyl-H), 9.55, 11.25 (2s,
each 1H, 2NH, D2O exchangeable). 13C-NMR (75 MHz, ꢁ ppm):
14.12 (CH3), 92.10, 94.87 (pyrazole C4), 123.24 (phenyl C2,6),
128.89 (phenyl C4), 129.23 (phenyl C3,5), 138.45(phenyl C1),
141.15(aminopyrazole C5), 141.86 (pyrazole C3), 150.56, 152.69
(pyrazole C5), 162.62 (C¼O). MS (m/z, %): 385 (M+., 3.0), 129
(100). Anal. Calcd for C16H15N7OS2 (385.47): C, 49.85; H, 3.92;
N, 25.44. Found: C, 49.48; H, 4.13; N, 25.29.
Vc ꢀ Vd=Vc ꢂ 100
where, Vc is the increase in paw volume in the absence of test
compound (control) and Vd is the increase in paw volume after
injection of the test compound. Data were expressed as the
mean SD. Significant difference between the control and treated
groups was determined using one-way analysis of variance
(ANOVA) followed by Tukey simultaneous comparison
t-values. The difference in results was considered significant
when p50.01.
Animal care and all experimental procedures used in this study
were performed in accordance with the regulations and guidelines
stipulated by the Institutional Animal Care and Use Guidelines at
Beirut Arab University, Lebanon.

8
W. H. Faour et al.
J Enzyme Inhib Med Chem, Early Online: 1–16
Gastric-ulcerogenic effect
cells/cm2, in a final volume of 2 mL. The cells were allowed to
adhere to plastic dishes overnight at 37 ꢁC, 5% CO2, in complete
RPMI medium (Sigma-Aldrich) supplemented with fetal bovine
serum (10%) (Sigma-Aldrich), penicillin and streptomycin
(Sigma-Aldrich), 100 U/mL and 100 mg/mL respectively. The
next day, by vigorous washes with PBS (three times), the non-
adherent cells (mainly lymphocytes) were removed. Macrophages
obtained in this fashion are greater than 95% pure. Cell viability
was determined by trypan blue exclusion. PMBCs were then
cultured in RPMI serum-free media and stimulated with factors
for 6 h prior to cell lysis and western blot analysis.
After the employment of anti-inflammatory activity experiment,
mice were killed under deep ether anesthesia and the stomach of
each mouse was removed. An opening at the greater curvature was
made and the stomach was cleaned by washing with cold saline
and inspected for any evidence of hyperemia, hemorrhage,
erosion, or ulcer. An arbitrary scale was used to calculate the
ulcer index (UI) which indicates the severity of stomach lesions34.
The UI of each group was calculated as the sum of the following
three figures:
UI ¼ UN + US + [UP/10]
UN ¼ average of number of ulcers per animal
US ¼ average of severity score
Cell stimulation protocol
UP ¼ percentage of animals with ulcers
Monocytes were cultured in a total volume of 2 mL for 6 h in 6-
well plates in the presence of LPS (Sigma-Aldrich) and increasing
concentrations of either of the following compounds 7a, 11, 15a,
16a, 16b, 18, and 19. Macrophages are cultured in RPMI-free
media and incubated with either vehicle alone, or solvent alone
(consist of methanol 1:2 and DMSO 1:2, then PEG 400 is added
1:1), or LPS (100 ng/mL) alone (Sigma-Aldrich), or 25 mM,
50 mM, or 100 mM respectively of each compound for 30 min prior
to stimulation with LPS (100 ng/mL) for 6 h.
Severity was recorded with the following scores:
ꢃ 0 ¼ no ulcer
ꢃ 1 ¼ superficial ulcers
ꢃ 2 ¼ deep ulcers
ꢃ 3 ¼ perforation.
Acute toxicity
Animals employed in the carrageenan-induced paw edema
experiment were observed for morbidity or mortality every 24 h
for 14 d after receiving the tested compounds35.
Preparation of cell extracts and western blotting
Cells in each well were lysed in RIPA Buffer containing 50 mM
Tris-HCL (pH 7.5) (Sigma-Aldrich), 150 mM NaCl (Fisher
Scientific, Pittsburgh, PA), 1% Tween 20 (Himedia), 0.5%
sodium dodecyl sulfate (SDS) (Himedia, Paris, France), and 2%
beta-mercaptoethanol (Sigma-Aldrich). Then 10 mL of the protein
lysate was used to measure the total protein concentration using
NanoDrop 2000c Spectrophotometer (Thermo Scientific,
Pittsburgh, PA). Equal amount of 50 mg of total protein from
each sample was analyzed on 10% SDS–polyacrylamide gel
electrophoresis (PAGE). Protein samples were diluted 1:1 in
2ꢂ Laemmli sample buffer before loading onto 10% SDS-PAGE.
Protein bands in the gel were transferred onto polyvinylidene
fluoride membrane (Sigma-Aldrich). After blocking of the
membrane in 5% skimmed milk in Tris-buffered saline containing
0.05% Tween-20 for 1 h at room temperature, the membranes
were incubated overnight at 4 ꢁC with rabbit polyclonal anti-rat
COX-2 (Abcam, Cambridge, UK; 1:1000 dilution) or rabbit anti-
rat iNOS (Abcam; 1:1000 dilution) in TTBS (1X) containing 5%
skimmed milk. The second goat polyclonal anti-rabbit antibody-
horseradish peroxidase conjugate (Abcam, 1:2000 dilution) was
subsequently incubated with membranes for 1 h at room
temperature. Membranes are then washed extensively for 1–2 h
with TTBS (1X). Following incubation with ECL chemilumines-
cence reagent (Clarity Western ECL Substrate, Bio-Rad,
Hercules, CA), protein bands on the membrane were visualized
by ChemiDocTM MP imaging system (Bio-Rad). Data for the in
vitro analysis were statistically analyzed using one-way ANOVA
followed by Tukey simultaneous comparison t-values using SPSS.
Values denote mean (n ¼ 3) and statistical differences were
considered significant at *p50.05 (Figures S3–S10 in
Supplementary Material).
In vitro COX study
The inhibitory COX activity of the tested compounds and the
reference was assayed using Cayman colorimetric COX (ovine)
inhibitor screening assay kit (Catalog No. 760111, Cayman
Chemicals, Ann Arbor, MI) according to the manufacturer’s
instructions36. Aliquots (170 mL) of the assay buffer (0.1 N Tris-
HCl, pH 8.0), heme, and enzyme ovine (COX-1 or COX-2) were
placed in a 96-well plate. The test compounds were added to the
aliquots. The plate was carefully shaken for a few seconds and
then incubated for 5 min at 25 ꢁC. The colorimetric substrate
N,N,N0,N0-tetramethyl-p-phenylenediamine (20 mL, TMPD) and
arachidonic acid (20 mL) were added to the aliquots. The plate was
carefully shaken for a few seconds and then incubated for 5 min at
25 ꢁC. The absorbance was measured at 590 nm using a 96-well
Tecan Safire plate reader. The mixture of 160 mL of assay buffer
and 10 mL of heme served as a background control. The mixture
of 150 mL of assay buffer, 10 mL of heme, and 10 mL of ovine
COX-1 or COX-2 showed 100% initial activity as a control
experiment in the absence of inhibitor. Diclofenac and celecoxib
were used as reference standards in the study. The assays
were performed in triplicate and the IC50 values were calculated
from the concentration curves by means of GraphPad software
PRISM.
Cell isolation and culture
Fresh peripheral blood mononuclear cells (PBMCs), consisting of
lymphocytes and monocytes, were isolated from EDTA (Sigma
Chemical Co., St. Louis, MO) treated blood samples of healthy
young male Sprague-Dawley rats of 6 weeks age weighing around
250 g. All procedures on rats were done according to the IRB
guidelines of the Lebanese American University. Blood samples
were mixed with EDTA, to a final concentration of 4 mM to
prevent coagulation, diluted 1/2 with sterile and warm phosphate-
buffered saline (PBS) (1X) (Sigma-Aldrich, St. Louis, MO), and
centrifuged over Ficoll (Histopaque-1077, Sigma-Aldrich).
PBMCs were washed twice in sterile warm PBS (1X) before
seeding. The mononuclear cells were seeded in 6-well culture
plates (Corning Costar) at a density of approximately 4 ꢂ 105
Analgesy-meter induced force in rats
Analgesic activity was tested in rats using an analgesy-meter
according to a previously described procedure37. Male albino rats
weighing 120–150 g were used throughout this assay. They were
kept in the animal house under standard conditions of light and
temperature with free access to food and water. The animals were
randomly divided into groups each of six rats. One group of six
rats was kept as a control and another group received the standard

DOI: 10.3109/14756366.2015.1094469
Synthesis and evaluation of amide-linked bipyrazoles
9
drug diclofenac sodium (at a dose of 50 mg/kg body weight i.p.). the weight of force at which the animal tries to free itself before
The tested compounds were administrated intraperitoneally at a administration of the test compound.
dose equimolar to that of diclofenac sodium. The rats were gently
placed between the plinth and plunger. Mechanical pain was
Statistical analysis
induced onto the paw of the rats by the use of analgesy-meter
(Ugo Basile, Varese, Italy) which exerted force at a constant rate.
The rats were restrained while their tails were placed between the
plinth and the plunger. The force was continuously monitored by a
pointer moving along a linear scale. Pain response was taken to be
the point at which the rat struggles to set itself free or its paw slips
off the plinth of the instrument. The weight causing pain before
treatment (0) and then 4 h after treatment to each rat in all the
groups was recorded. Percentage pain inhibition (increase in pain
threshold) produced relative to the control group was calculated
for each group over the period of study using the formula:
Data were statistically analyzed using one-way ANOVA followed
by Tukey simultaneous comparison t-values. Statistical differ-
ences were considered to be significant at p50.01.
Results and discussion
Chemistry
The synthetic strategies adopted for the synthesis of the
intermediate and target compounds are depicted in Schemes
1–3. In Scheme 1, the starting 5-amino-3-methylsulfanyl-1H-
pyrazole-4-carbonitrile 2 was prepared as previously described32.
Synthesis of the cyanoacetamide derivative 3 was achieved by
pouring the pyrazolecarbonitrile 2 into a solution of the
cyanoacetylating reagent (cyanoacetic acid and acetic anhydride)
Ft ꢀ Fo=Ft ꢂ 100
where Ft is the weight of the induced force at which the animal
tries to free itself after administration of the test compound, Fo is
H CS
CN
3
N
SCH
C
3
NH
2
N
i
CN
C
H CS
3
CN
2
1
ii
H CS
CN
CN
3
H CS
H CS
CN
O
3
3
O
O
CN
R
CN
CN
R
N
N
N
N
N
N
N
H
N
N
iii
H
vi
H
N
H
H
N
7a-c
4a-c
3
X
vii
iv
v
N
N
CN
H CS
CN
R
H CS
H CS
CN
H N
3
3
3
2
O
O
O
CN
N
N
N
N
NH
N
N
N
N
N
N
H
H
N
NH
H
N
NH
R
H N
2
O
H C
3
N
N
H C
5a-c
8a-c
6
3
For 4, 5, 7 and 8 : a, R = C H ; b, R = 4-ClC H ; c, R = 4-OCH C H
3 6 4
6
5
6
4
Scheme 1. Synthesis of compounds 2–7. Reagents and conditions: (i) phenylhydrazine/ethanol/reflux; (ii) cyanoacetic acid/acetic anhydride/50 ꢁC;
(iii) the appropriate aldehyde/ethanol/piperidine/reflux; (iv) N2H4 98%/ethanol/reflux; (v) 4-amino antipyrine/NaNO2/ethanol/sodium acetate/r.t.; (vi)
3-aryl-1-phenyl-1H-pyrazole-4-carboxaldehyde/ethanol/NaOH/reflux; (vii) N2H4 98%/ethanol/reflux. For 4, 5, 7 and 8: a, R ¼C6H5; b, R ¼ 4-ClC6H4;
c, R ¼ 4-OCH3C6H4.

10 W. H. Faour et al.
J Enzyme Inhib Med Chem, Early Online: 1–16
H CS
CN
3
O
CN
N
N
N
N
N
H
N(CH )
3 2
H
9
iii
ii
H CS
CN
H N
2
H CS
3
CN
3
O
O
CN
i
NH
N
N
N
N
N
N
N
H
H
NH
H CS
CN
3
H
O
CH
CH
3
CN
O
N
N
N
N
H
3
3
11
10
iv
vi
v
CN
H CS
3
O
CN
N
N
H
OCH CH
2
3
H
12
Scheme 2. Synthesis of compounds 9–12. Reagents and conditions: (i) DMF-DMA/dry dioxane/reflux; (ii) N2H4 98%/dry dioxane/reflux; (iii) 4-
aminoantipyrine/oil bath/170 ꢁC; (iv) triethyl orthoformate/piperidine/dioxane/reflux; (v) N2H4 98%/ethanol/reflux; (vi) 4-aminoantipyrine/oil bath/
ꢁ
170 C.
1
followed by heating the mixture at 50 ꢁC for 5 h modifying the decomposition product separated out as a white solid. H-NMR
reaction conditions previously reported for synthesis of allied of the separated decomposition products confirmed their struc-
compounds38. It should be noted here that heating the reaction tures as the corresponding 3-aryl-1-phenyl-1H-pyrazole-4-
mixture at 95 ꢁC for 30 min. as previously reported38 resulted in a carboxaldehydes. In Scheme 2, reaction of the cyanoacetamide
black tar which was very difficult to work up or purify, whereas, 3 with DMF dimethyl acetal in dry dioxane resulted in the
heating at 50 ꢁC for a longer time gave a pure product in a good corresponding enaminonitrile 9 in a good yield. Refluxing the
yield. Condensation of 3 with aromatic aldehydes in boiling enaminonitrile 9 with hydrazine hydrate in ethanol as previously
ethanol containing a catalytic amount of piperidine afforded described for the synthesis of analogous compounds42 gave rise to
the corresponding acrylamides 4a–c. 1H-NMR of the latter the corresponding 5-aminopyrazole 10 (Method I). Inspection
compounds revealed presence of double the number of the of the 1H-NMR spectrum enabled establishing structure 10 as
expected protons indicating presence of these compounds as a 5-aminopyrazole derivative since the pyrazole C3-H appeared as a
mixture of E and Z isomers. Reaction of compounds 4a–c with singlet at 7.78 ppm which is characterized from the tautomeric
hydrazine hydrate following reaction conditions39 described 3-aminopyrazole that would reveal pyrazole C5-H as a doublet43.
for synthesis of analogous compounds yielded the respective Fusion of 9 with 4-aminoantipyrine in an oil bath at 170 ꢁC gave
3-aminopyrazoles 5a–c in a moderate yield. The reaction rise to the corresponding pyrazole derivative 11 (Method I).
mechanism involves b-attack on the –CO–C¼C moiety in 4 Synthesis of the ethoxyacrylamide derivative 12 was achieved by
with subsequent 1,5-intramolecular dipolar cyclization and con- heating 3 with triethyl orthoformate in dioxane containing few
comitant aromatization39. Microanalysis and spectral data of 5a–c drops of piperidine. At this stage, it seemed of interest to
were fully consistent with the proposed structures. On the investigate the possibility of using compound 12 as a precursor for
other hand, coupling of compound 3 with the diazonium salt of the alternative synthesis of compounds 10 and 11 (Method II).
4-aminoantipyrine in ethanol buffered with sodium acetate Accordingly, heating 12 with hydrazine hydrate in ethanol
adopting
antipyrinyl hydrazone derivative
a
published procedure40 gave the corresponding furnished the corresponding 5-aminopyrazole 10 whereas,
in
good yield. fusion of 12 with 4-aminoantipyrine at 170 ꢁC yielded the
6
a
Condensation of 3 with 3-aryl-1-phenyl-1H-pyrazole-4-carbox- corresponding pyrazole derivative 11. Compounds 10 and 11
aldehydes41 in ethanolic NaOH 10% afforded the corresponding from both methods gave identical Rf value in TLC, melting point
acrylamides 7a–c. Trials to cyclize compounds 7a–c into the and IR spectra.
corresponding 3-aminopyrazoles 8a–c using hydrazine hydrate
In Scheme 3, trials to prepare the ketene N, S-acetals 14a, b
were unsuccessful. In fact, follow-up of the reaction by TLC through treatment of the cyanoacetamide 3 with aryl isothiocyan-
revealed complex reaction mixture and extensive degradation. ates in DMF containing potassium hydroxide under cooling till
However, on cooling the reaction mixture, the major complete addition of the isothiocyanate as previously described44

DOI: 10.3109/14756366.2015.1094469
Synthesis and evaluation of amide-linked bipyrazoles 11
CN
H CS
3
H CS
O
CN
H N
CN
H CS
3
2
3
O
O
CN
CN
iv
iii
N
NH
N
N
N
N
N
N
H
N
N
NH
N
H
H
SCH
RHN
3
O
RHN
14a,b
ii
RHN
H C
3
N
N
15a,b
16a,b
H C
3
H C
3
CN
CN
H CS
3
H CS
3
O
O
CN
CONH
2
i
N
N
N
H
N
N
N
H
13
3
v
H CS
H N
2
CN
H CS
CN
H CS
CN
3
3
3
O
O
O
CN
CN
NH
vi
vii
N
N
N
N
H
N
H
N
N
N
N
N
H
NH
SCH
3
H CS
H CS
H CS
3
3
3
O
H C
3
N
19
N
17
18
H C
3
For 14-16: a, R = C H ; b, R = ClC H
6 4
6
5
ꢁ
Scheme 3. Synthesis of compounds 13–19. Reagents and conditions: (i) RNCS/KOH/DMF/r.t.; (ii) RNCS/KOH/DMF/0 C; (iii) N2H4 98%/ethanol/
reflux; (iv) 4-aminoantipyrine/oil bath/170 ꢁC; (v) CS2/KOH/DMF/0 ꢁC then (CH3)2SO4/r.t.; (vi) N2H4 80%/ethanol/reflux; (vii) 4-aminoantipyrine/oil
bath/140 ꢁC. For 14–16: a, R ¼C6H5; b, R ¼ClC6H4.
were unsuccessful and the corresponding malonamide 13 was 5-aminopyrazole isomer is lower than the isomeric 3-aminopyr-
separated instead. The chemical structure of 13 was confirmed on azole by 1.6 kcal/mol. On the other hand, fusion of 14a, b with 4-
the basis of 1H-NMR which portrayed a D2O exchangeable singlet aminoantipyrine at 170 ꢁC resulted in the corresponding pyrazoles
at 7.15 ppm attributed to the CONH2 protons. Additionally, its 16a, b. Treatment of 3 with carbon disulfide in DMF containing
13C-NMR showed two signals at 154.07 and 166.55 ppm charac- potassium hydroxide at 0–5 ꢁC, followed by methylation using
teristic for two C¼O groups. To prevent hydrolysis of the CN dimethyl sulfate at room temperature gave rise to the bis(methyl-
group to the corresponding CONH2, the reaction mixture was sulfanyl) acrylamide derivative 17 which upon condensation with
cooled at 0–5 ꢁC during addition of the isothiocyanate and cooling hydrazine hydrate furnished the 5-aminopyrazole derivative 18 as
was maintained for further 2 h then the reaction mixture was left described for the synthesis of analogous compounds47. Finally,
at room temperature over night till complete reaction with aryl fusion of 17 with 4-aminoantipyrine at 140 ꢁC gave rise to the
isothiocyanates followed by addition of dimethyl sulfate to corresponding derivative 19 in a good yield. Spectral data of
achieve methylation. In this case, the ketene N,S acetals 14a, b compounds 18 and 19 were in agreement with their assigned
were separated in a good yield and purity. Microanalysis and structures.
spectral data of 14a, b were in agreement with their proposed
structures. Reaction of 14a, b with hydrazine hydrate in ethanol Biological evaluation
yielded the requisite 5-aminopyrazoles 15a, b. It should be noted
here that a literature survey revealed that some authors reported
Anti-inflammatory activity
the formation of the isomeric 3-aminopyrazoles under the same The anti-inflammatory activity of the synthesized compounds was
reaction conditions44, while others claimed the separated products evaluated utilizing the carrageenan-induced paw edema bioassay
to be the isomeric 5-aminopyrazoles45. In our study, the in mouse using diclofenac sodium (50 mg/kg) as a reference
preferential formation of the isomeric 5-amino pyrazoles (15a, standard anti-inflammatory agent33. The data obtained are
b) was supported from molecular orbital calculations utilizing presented in Table 1 and expressed as means SD. Statistical
semi-empirical PM6 method implemented in Gaussian 09 differences of control and test groups were carried out using
program46, which revealed that the heat of formation of the one-way ANOVA followed by Tukey simultaneous comparison

12 W. H. Faour et al.
J Enzyme Inhib Med Chem, Early Online: 1–16
Table 1. Anti-inflammatory activity of the synthesized compounds in carrageenan-induced mouse paw edema bioassay.
Compounda
Increase in paw volume (mL)b
% Protection
Activity relative to diclofenac
ED50 mg/kg (mmol/Kg)
Control
Diclofenac
5a
5b
5c
6
7a
7b
7c
0.120 0.013c
0.057 0.008d
0.095 0.019c,d
0.098 0.010c,d
0.088 0.019c,d
0.095 0.010c,d
0.050 0.009d
0.075 0.006c,d
0.053 0.006d
0.097 0.010c,d
0.060 0.012d
0.053 0.005d
0.085 0.017c,d
0.060 0.014d
0.026 0.006c,d
0.030 0.008c,d
0.065 0.013d
0
53
21
18
27
21
58
38
56
19
50
56
29
50
78
75
46
0.0
1.0
0.4
0.3
0.5
0.4
1.1
0.7
1.1
0.4
0.9
1.1
0.6
0.9
1.5
1.4
0.9
47.25 (148.52)
72.13 (136.71)
78.22 (139.17)
10
11
80.25 (157.17)
62.0 (144.02)
15a
15b
16a
16b
18
94.56 (157.17)
62.52 (98.28)
41.88 (108.64)
96.46 (173.28)
19
aCompounds were administered intraperitoneally to mice in doses equimolar to 50 mg/kg diclofenac sodium.
bValues are presented as means SD of 6 mice.
cDenotes significant difference from diclofenac sodium value at p50.01.
dDenotes significant difference from control value at p50.01.
t-values. The difference in results was considered significant diclofenac sodium. Substitution on the phenyl ring with a
when p50.01.
methoxy group (compound 7c) did not significantly alter the
activity, whereas, substitution with a chloro group (compound 7b)
markedly decreased the activity. Regarding the 5-amino-3-
substituted pyrazole derivatives (series G), the 5-amino-3-
(un)substituted pyrazolyl derivative (compound 10) was found
to be the least active. Substitution at position 3 with phenylamino,
4-chlorophenyl amino or methylsulfanyl group greatly improved
the activity in the order methylsulfanyl ꢄ phenylamino ꢄ 4-
chlorophenylamino. In this context, compound 15a comprising
the phenylamino moiety displayed activity equivalent to diclofe-
nac sodium, whereas, compound 18 with a methylsulfanyl moiety
was superior to the reference drug (% protection ¼ 75). In series H
comprising the antipyrinyl moiety, the anti-inflammatory activity
appears to be closely related to the nature of the (X) group. When
X was a nitrogen atom (compound 6), the activity was weak.
Replacement of the nitrogen atom with CH group (compound 11)
increased the activity to approach that of diclofenac sodium.
Substitution of the hydrogen atom of the CH group with
phenylamino or methylsulfanyl moieties (compounds 16a and
19 respectively) did not result in a significant change in activity,
whereas, substitution with 4-chlorophenylamino group (com-
pound 16b) noticeably improved the activity to surpass that of
diclofenac sodium. In this view, compound 16b emerged with the
highest activity in this study (% protection ¼ 78).
Carrageenan-induced paw edema bioassay
In this inflammatory model, each test compound was adminis-
tered intraperitoneally (at a dose equimolar to that of diclofenac
sodium) 1 h prior to the induction of inflammation by
carrageenan injection. Diclofenac sodium was utilized as a
reference drug at a dose of 50 mg/kg, i.p. Preliminary study of
the time response curve for the synthesized compounds revealed
that the maximum activity was observed after 4 h, therefore the
anti-inflammatory activity was calculated after 4 h and presented
in Table 1 as the mean paw volume (mL) in addition to the
percentage of anti-inflammatory activity (AI%). The anti-inflam-
matory activity of the test compounds relative to that of
diclofenac was also calculated.
A comparative study of the anti-inflammatory activity of the
test compounds relative to the reference drug after 4 h (Table 1)
revealed that compounds 16b and 18 showed anti-inflammatory
activity (% protection ¼ 78% and 75% respectively) superior to
that of diclofenac sodium (% protection ¼ 53%), whereas com-
pounds 7a, 7c, 11, 15a, and 16a were nearly equivalent to the
reference drug in inhibiting paw edema with percentage activity
of (58–50%). The other compounds showed moderate to weak
activity.
A deep insight in the structures of the synthesized compounds
revealed that they represent four different series of compounds
(E-H; Figure 1); series E (compounds 5a–c) comprising the
3-amino-5-substituted pyrazole moiety linked to the N-phenyl
pyrazole through amide link, series F (compounds 7a–c)
comprising the 1-phenyl-3-substituted pyrazole linked to the
N-phenylpyrazole scaffold through 2-cyano acrylamide moiety,
series G (compounds 10, 15a, 15b, and 18) comprising the
5-amino-3-substituted pyrazole attached to the N-phenylpyrazole
through amide bridge and series H (compounds 6, 11, 16a, 16b,
and 19) comprising the 4-aminoantipyrinyl moiety attached to the
N-phenylpyrazole through -X ¼C(CN)-CONH- spacer. The pre-
liminary anti-inflammatory results revealed that the 3-aminopyr-
azolyl derivatives (5a–c, series E) did not show significant
anti-inflammatory activity. Among compounds 7a–c (series F),
the phenyl derivative 7a displayed nearly equipotent activity to
The most active compounds (7a, 7c, 11, 15a, 16a, 16b, and 18)
were further tested at different doses in order to determine their
ED50 values after 4 h interval (Table 1). In terms of ED50,
compounds 7a, 7c, 16b, and 18 (ED50 ¼ 98.28–139.17 mmol/Kg)
were more active than diclofenac sodium (ED50 ¼ 148.52 mmol/
Kg), however, compounds 16b and 18 were found to be the most
potent (ED50 ¼ 98.28 and 108.64 respectively, Table 1).
Ulcerogenic activity
All the synthesized compounds were further evaluated for their
gastric ulcerogenic potential in mice using diclofenac sodium as
reference drug34. Results (Table 2) revealed that the tested
compounds were less ulcerogenic than diclofenac sodium except
for compounds 6, 7b, and 7c which possessed nearly the same
ulcerogenic potential.

DOI: 10.3109/14756366.2015.1094469
Synthesis and evaluation of amide-linked bipyrazoles 13
Table 2. Ulcerogenic activity of the tested compounds and diclofenac Table 3. In vitro COX-2 selectivity index of the synthesized compounds.
sodium in mice.
IC50/mMa
% Animals with
ulcers/10
Average number
of ulcers/animal
Average
severity
Ulcer
index
Compounds
COX-1
COX-2
COX-1/COX-2b
Compound
c
5a
5b
5c
6
7a
7b
7c
10
11
15a
15b
16a
16b
18
19
4100
4100
4100
4100
33.6
27.9
38.8
4100
46.6
37.4
4100
4100
4100
4100
13.4
23.7
33.8
4100
24.8
35.8
4100
20.8
11.6
18.5
22.8
–
Control
Diclofenac
5a
5b
5c
6
7a
7b
7c
1
9
7
5
8
9
3
8
9
6
4
7
7
7
3
4
5
1
5
3
2
3
4
1
4
4
2
1
3
2
2
1
1
2
1
2
1
1
2
2
1
2
2
2
1
1
1
1
1
1
1
3
16
11
8
13
15
5
14
15
10
6
11
10
10
5
–
–
–
2.5
1.17
1.14
–
1.87
1.04
–
1.6
1.75
1.59
1.72
0.27
407
10
11
4100
33.3
20.3
15a
15b
16a
16b
18
29.4
39.2
0.06
Diclofenac
Celecoxib
0.22
0.07
6
8
28.5
19
aValues are means of three determinations acquired using an ovine COX-
1/COX-2 assay kit (catalog no. 760111, Cayman Chemicals, Ann Arbor,
MI) and the deviation from the mean is 510% of the mean value.
bIn vitro COX-2 selectivity index (COX-1 IC50/COX-2 IC50).
cNot determined.
Toxicity
No morbidity or mortality was observed in animals that received
the tested compounds. All mice showed no toxicity after being
observed every 24 h for 14 d after receiving the tested compounds
at the doses used for anti-inflammatory activity35.
COX-2 expression would hold tremendous potential in advancing
the treatment of inflammatory or chronic immune disorders.
Hence, the most promising compounds were evaluated for their
molecular anti-inflammatory effect on the level of protein
expression of iNOS and COX-2 (Figure 2A) in LPS-activated
rat PBMCs. Additionally, the inhibitory potency of the
compounds on COX-2 protein upregulation was compared to
the anti-inflammatory glucocorticoid dexamethasone (Figure 2B).
The results in Figure 2(A) revealed that iNOS and COX-2 proteins
were slightly detectable in unstimulated cells. After treatment
with LPS for 6 h, the protein expression of iNOS and COX-2 was
markedly increased. Co-treatment with the test compounds dose-
dependently (25, 50, and 100 mM) inhibited iNOS and COX-2
protein expression induced by LPS. All the tested compounds
significantly suppressed COX-2 expression at a concentration of
100 mM, and five of them (11, 15a, 16b, 18, and 19) reduced
COX-2 protein expression to levels comparable to control
unstimulated cells (Figures S3–S10 in Supplementary Material).
However, at a concentration of 50 mM, compounds 7a, 11, 15a,
16b, and 18 reduced COX-2 protein expression to levels
comparable to control (Figures S3–S10 in Supplementary
Material). When a concentration of 25 mM was used, all
compounds showed detectable inhibitory effect on COX-2 protein
expression, and three of them (7a, 15a, and 19) inhibited COX-2
expression by at least 80% (Figures S3–S10 in Supplementary
Material) when compared to cells treated with LPS alone. On the
other hand, all compounds at 100 mM, completely reduced iNOS
protein expression to levels similar to control non-stimulated
cells. Same results were obtained with these compounds when
used at 50 mM except for compounds 11 and 16b. However, when
the compounds were used at 25 mM, only compounds 7a, 16a,
15a, and 19 were able to strongly inhibit iNOS expression.
Finally, it could be concluded that the most active compounds
might have exerted their anti-inflammatory effect in part through
inhibition of COX-2 and iNOS proteins expression. However,
further investigation in order to rule out the detailed mechanisms
by which these compounds block COX-2 and iNOS protein
translation is in progress.
In vitro COX-1/COX-2 inhibition assay
The synthesized compounds were further evaluated for their
ability to inhibit COX-1 and COX-2 isoenzymes using the in vitro
colorimetric COX (ovine) inhibitor assay method36, which utilizes
the peroxidase component of cyclooxygenase. The peroxidase
activity is assayed colorimetrically by monitoring the appearance
of oxidized TMPD, which is produced during the reduction of
PGG2 to PGH2, at 590 nm. The efficacies of the tested compounds
were determined as the concentration causing 50% enzyme
inhibition (IC50) and recorded in Table 3. The data revealed that
some of the test compounds inhibit COX isoenzymes in a high
concentration range compared to celecoxib. The results also
showed that compounds 7b, 7c, 15a were non selective, while
compounds 7a, 11, 16a, 16b, 18, and 19 showed relatively slight
selectivity toward COX-2 than COX-1. Among the tested
compounds, compound 16b demonstrated the highest inhibitory
activity as it showed IC50 value of 20.3 mM for COX-1 and
11.6 mM for COX-2. These results indicate that further investiga-
tion is needed in order to gain insight into the mechanism of
action of the examined compounds.
Amide-linked bipyrazoles reduced LPS-induced iNOS and COX-2
protein expression
iNOS and COX-2 are key proinflammatory enzymes induced by
LPS or cytokines and were found to work in concert in a number
of similar pathophysiological activities and inflammatory dis-
ease48,49. Under basal condition, the products of iNOS and COX-
2, including nitric oxide (NO) and prostaglandins (PGs), are
involved in modulation of cellular functions and homeostasis.
However, during inflammation, NO and PGs are released
simultaneously in large amounts up to micromolar concentra-
tion50. Previous study has shown that NO directly increases COXs
activity and leads to a remarkable sevenfold increase in PGE2
formation49; further studies suggest that there is a considerable
cross talk between NO and PGs biosynthetic pathways51,42
Therefore, a compound with dual inhibitory effect on iNOS and
.

14 W. H. Faour et al.
J Enzyme Inhib Med Chem, Early Online: 1–16
Table 4. Analgesic activity of the synthesized compounds using the
analgesy-meter-induced pain in rats.
(A)
Weight causing
pain (g)b
Activity relative
to diclofenac
Compounda
% Protection
COX-2
iNOS
7a
Control
Diclofenac
5a
5b
5c
6
7a
7b
7c
92.50 6.14c
193.00 4.81d
128.5 2.46c,d
104.75 3.42c,d
143.00 4.26c,d
162.25 3.06c,d
159.00 5.78c,d
145.75 2.59c,d
175.75 3.65c,d
106.25 4.26c,d
177.5 6.18c,d
185.75 4.49d
106.00 1.47c,d
177.25 2.17c,d
199.50 2.85d
193.25 3.63d
175.50 2.46c,d
0
52
28
12
35
43
42
36
47
13
48
50
13
48
54
52
47
0.0
1.0
0.54
0.23
0.67
0.82
0.81
0.69
0.90
0.25
0.92
0.96
0.25
0.92
1.0
COX-2
iNOS
15a
COX-2
iNOS
11
10
11
15a
15b
16a
16b
18
COX-2
iNOS
16a
1.0
0.90
COX-2
iNOS
16b
18
19
aCompounds were administered intraperitoneally to rats in doses
equimolar to diclofenac sodium.
COX-2
iNOS
bValues are presented as means SEM of 6 rats.
cDenotes significant difference from diclofenac sodium value at p50.01.
dDenotes significant difference from control value at p50.01.
COX-2
iNOS
19
reference drug (% protection ¼ 52). The other compounds showed
moderate to weak activity.
(B)
Further interpretation of the results revealed that the 3-
aminopyrazolyl derivatives (5a–c, series E) did not display
considerable analgesic activity. Among compounds 7a–c (series
F), the phenyl derivative 7a displayed moderate activity (%
protection ¼ 42). Substitution on the phenyl ring with chloro
group (compound 7b) decreased the activity, whereas, introduc-
tion of a methoxy group (compound 7c) slightly increased the
activity to approach that of diclofenac sodium. Regarding the 5-
amino-3-substituted pyrazolyl derivatives (series G), the unsub-
stituted 5-aminopyrazolyl derivative (compound 10) was the least
active (% protection ¼ 13). However, substitution at position 3
with phenylamino (compound 15a) or methylsulfanyl group
(compound 18) greatly improved the activity to be close to that
of diclofenac sodium whereas substitution with 4-chlorophenyl
amino group (compound 15b) did not alter the activity. In series
H comprising the antipyrinyl moiety, the anti-inflammatory
activity appears to be closely related to the nature of the (X)
group. When X was a nitrogen atom (compound 6), the activity
was moderate (% protection ¼ 43). Replacement of the nitrogen
atom with CH group (compound 11) improved the activity (%
protection ¼ 48) to approach that of diclofenac sodium. On the
other hand, substitution of the hydrogen atom of the CH moiety
with phenylamino or methylsulfanyl groups (compounds 16a and
19) did not alter the activity, whereas, substitution with a 4-
chlorophenylamino moiety (compound 16b) slightly improved the
activity.
Figure 2. Amide-linked bipyrazoles blocked LPS-induced COX-2 and
iNOS protein expression in rat monocytes. In (A), PMBCs were incubated
with vehicle alone, or with solvent alone, or with LPS (100 ng/mL) alone,
or with 25 mm, 50 mm or 100 mm of each of the test compounds
respectively for 30 min prior to stimulation with LPS (100 ng/mL) for 6 h
as indicated. Cell lysates were resolved by SDS-PAGE and immuno-
blotted with anti-COX-2 and anti-iNOS antibodies. In (B), PBMCs
were incubated with vehicle alone, or LPS (100 ng/mL) alone, or
LPS (100 ng/mL) + dexamethasone (40 mM), or LPS (100 ng/mL) + 7a
(50 mM), or LPS (100 ng/mL) + 16b (50 mM), or LPS (100 ng/mL) +
11(50 mM),
or
LPS
(100 ng/mL) + 16a
(50 mM),
or
LPS
(100 ng/mL) + 15a (50 mM), or LPS (100 ng/mL) + 19 (50 mM), or LPS
(100 ng/mL) + 18 (50 mM). Expression levels were normalized to total
amount of protein loaded onto the gel and assessed by densitometric
analysis (n ¼ 3). *p50.05 versus vehicle control.
Analgesic activity
The analgesic activity of the synthesized compounds was
evaluated in rats using the analgesy-meter-induced pain method
and utilizing diclofenac sodium (50 mg/kg, i.p.) as reference
drug36. The results were recorded as the pre and post treatment
(4 h) weight causing pain for each rat and the percentage
protection. The results were presented in Table 4 and expressed
as means SEM. Statistical differences of control and test groups
were carried out as described under section ‘‘Anti-inflammatory
activity’’.
A comparative study of the analgesic activity of the test
compounds relative to the reference drug revealed that most of the
tested compounds exhibited significant analgesic activity; how-
ever, none of them was superior to diclofenac sodium. Seven
compounds namely; 7c, 11, 15a, 16a, 16b, 18, and 19 displayed
analgesic activity (% protection ¼ 47–54) nearly equivalent to the
Conclusion
The aim of the present study was to synthesize and investigate the
anti-inflammatory and analgesic activities of new amide-linked
bipyrazoles with the hope of discovering new lead structures
devoid of the GI side effects associated with conventional
NSAIDs. The obtained results clearly revealed that compounds
7a, 11, 15a, 16a, 16b, and 18 showed anti-inflammatory activity
similar to or even higher than that of diclofenac sodium. In vitro
COX-1/COX-2 inhibition study revealed that among the

DOI: 10.3109/14756366.2015.1094469
Synthesis and evaluation of amide-linked bipyrazoles 15
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Acknowledgements
The authors are deeply grateful to Professor Dr Ahmed Khodair, Prof. of
Organic Chemistry, Department of Chemistry, Faculty of Science, Kafr
El-Sheikh University, Egypt, for his appreciable help in confirming the
chemical structure of compounds 15 utilizing PM6 method implemented
in Gaussian 09 program. The authors would like also to thank Sirine
Zaatari for technical support.
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