Design, synthesis, characterization and in vitro and in vivo anti-inflammatory evaluation of novel pyrazole-based chalcones

Article abstract of DOI:10.3109/14756366.2013.873037

A series of novel pyrazole-based chalcones have been designed, synthesized from 1-methyl-5-(2,4,6-trimethoxyphenyl)-1H-pyrazole (6). The structures of regioisomers 6 and 7 were determined by 2D ¹H-¹H COSY, ¹H-¹³C HSQC and ¹H-¹³C HMBC experiments. The newly synthesized compounds were tested for their inhibitory activity against COX-1 and COX-2 using an in vitro cyclooxygenase (COX) inhibition assay. Moreover, they were investigated in vivo for their anti-inflammatory activities using carrageenan-induced rat paw edema model for acute inflammation and cotton pellet-induced granuloma model for chronic inflammation. All the synthesized compounds showed potential to demonstrate anti-inflammatory activities, of particular interest compounds 10i, 10e , 10f, and 10h were found to be potent anti-inflammatory agents.

Full text of DOI:10.3109/14756366.2013.873037

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–10
2014 Informa UK Ltd. DOI: 10.3109/14756366.2013.873037
!
RESEARCH ARTICLE
Design, synthesis, characterization and in vitro and in vivo
anti-inflammatory evaluation of novel pyrazole-based chalcones
Hemant V. Chavan1, Laxman K. Adsul1, Amol S. Kotmale1, Valmik D. Dhakane1, Vishnu N. Thakare2, and
Babasaheb P. Bandgar1
1Department of Organic Chemistry, Ratnagiri Sub-Centre, University of Mumbai, Ratnagiri, Maharashtra, India and 2Department of Pharmacology,
Sinhgad Institute of Pharmaceutical Sciences, Kusgaon, Lonavala, Maharashtra, India
Abstract
Keywords
A
series of novel pyrazole-based chalcones have been designed, synthesized from
Anti-inflammatory activity, chalcone,
COSY, HMBC, HSQC, pyrazole
1-methyl-5-(2,4,6-trimethoxyphenyl)-1H-pyrazole (6). The structures of regioisomers 6 and 7
were determined by 2D H–1H COSY, H–13C HSQC and H–13C HMBC experiments. The newly
synthesized compounds were tested for their inhibitory activity against COX-1 and COX-2 using
an in vitro cyclooxygenase (COX) inhibition assay. Moreover, they were investigated in vivo
for their anti-inflammatory activities using carrageenan-induced rat paw edema model
for acute inflammation and cotton pellet-induced granuloma model for chronic inflammation.
All the synthesized compounds showed potential to demonstrate anti-inflammatory activities,
of particular interest compounds 10i, 10e, 10f, and 10h were found to be potent
anti-inflammatory agents.
1
1
1
History
Received 6 October 2013
Revised 25 November 2013
Accepted 28 November 2013
Published online 25 March 2014
Introduction
the classical side effects associated the use of conventional
NSAIDs.
Fast and effective relief of pain and inflammation in human
beings is continued to be a major challenge for medical
researchers. Non-steroidal anti-inflammatory drugs (NSAIDs)
are important therapeutic agents for the alleviation of pain and
inflammation associated with a number of pathological condi-
tions1. A major mechanism of action of NSAIDs is lowering
prostaglandin (PG) production through the inhibition of cycloox-
ygenase (COX); a key enzyme in PGs biosynthesis that catalyses
the conversion of arachidonic acid into PGs and thromboxanes2.
It was discovered that COX exists in two isoforms COX-1 and
COX-2 which are regulated differently. COX-1 is a constitutive
isoform; responsible for the production of cytoprotective prosta-
glandins in the gastrointestinal tract (GI) and proaggregatory
thromboxane in blood platelets. However, COX-2 is an inducible
isoform which is induced in response to the release of several
Pyrazole are an important class of compounds for new drug
development that attracted much attention due to their broad
spectrum of biological activities, such as anti-inflammatory9–14
,
antifungal15, anticancer16–19, and antiviral activities20,21. Pyrazole
derivatives also acting as anti-angiogenic agents22, A3 adenosine
receptor antagonists23, neuropeptide YY5 receptor antagonists24,
kinase inhibitor for the treatment of type 2 diabetes, hyperlipid-
emia, obesity25, and thrombopiotinmimetics26 were reported.
Recently, urea derivatives of pyrazoles have been reported as
potent inhibitors of p38 kinase27.
Among the highly marketed COX-2 inhibitors that comprise
the pyrazole nucleus, celecoxib is the one which is treated as a
safe anti-inflammatory and analgesic agent. It is considered as a
typical model of the diaryl heterocyclic template that is known to
selectively inhibit the COX-2 enzyme. Some other examples of
pyrazole derivatives such as deracoxib, SC-558, mefobutazone,
ramifenazone, famprofazone (Figure 1) have been reported as
potent NSAIDs28–31. On the other hand, the compounds with the
backbone of chalcones have been reported to possess various
biological activities, including anti-inflammatory, antimicrobial,
antifungal, antioxidant, and anticancer activities32,33. Various
methoxylated or hydroxylated chalcones, natural and synthetic,
have already been tested for their possible role as anti-inflam-
matory agents34,35. Recently, we have also reported some simple
methoxylated chalcones and N-phenyl pyrazole chalcones showed
significant anti-inflammatory, antioxidant and antimicrobial
proinflammatory mediators3–5
.
Most of the classical NSAIDs such as aspirin act as
nonselective inhibitors of COX, inhibiting both the COX-1 and
COX-2. The chronic use of NSAIDs to treat pain and inflamma-
tion is often accompanied by side effects such as gastric
ulceration, bleeding and renal impairment. It is believed that
the COX-2 selective inhibitors will greatly reduce these side
effects6–8. Thus, the development of COX-2 selective inhibitors
has led attention to improve the therapeutic potency and to reduce
activities36,37
.
The combination of both the active scaffolds, pyrazole and
chalcone, may provide synergistic effect to improve the anti-
inflammatory activity. These predictions encouraged us to
Address for correspondence: Babasaheb P. Bandgar, Department of
Organic Chemistry, Ratnagiri Sub-Centre, University of Mumbai,
Ratnagiri, Maharashtra, India. E-mail: bandgar_bp@yahoo.com

2
H. V. Chavan et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
H2NO2S
H2NO2S
CF3
H2NO2S
CF3
compound 6 in pure form. The filtrate was then extracted three
times with ethyl acetate. The combined extracts were washed with
water. After drying over anhydrous MgSO4, the solvent was
distilled off under reduced pressure. The resulting residue was
purified by silica gel column chromatography to obtain compound
7 in pure form.
Yield: 82%, MF/FWt: C13H16N2O3/248; m.p.: 147–149 ꢁC; IR
(KBr, cmꢂ1): 3064, 2962, 2943, 2841 (CH), 1612, 1584, 1549,
1473, 1458 (C¼C), 1234 (C¼N), 1161 (C–O); 1H NMR
(500 MHz, CDCl3): ꢀ 3.65 (s, 3H, NCH3), 3.75 (s, 6H,
2 ꢀ OCH3), 3.87 (s, 3H, OCH3), 6.20 (m, 3H, 2 ꢀ ArH, Pyr-H),
7.55 (d, 1H, Pyr-H); 13C NMR (125 MHz, CDCl3): 36.65 (CH3),
55.39 (CH3), 55.74 (CH3), 90.54 (CH), 100.68 (C), 107.44 (CH),
135.51 (C), 137.90 (CH), 159.32 (C), 162.25 (C); 135-DEPT:
36.65 (þ), 55.40 (þ), 55.75 (þ), 90.53 (þ), 107.44 (þ), 137.89
(þ); MS (ESI): m/e 249 (Mþ1).
N
N
N
N
N
N
CHF2
F
H3C
Br
O
Deracoxib
O
Celecoxib
N
SC-558
H3C HN
N
H3C
O
N
N
N
N
N
O
O
Famprofazone
O
Phenylbutazone
Ramifenazone
O
O
O
O
N
N
N
N
N
O
OH
10i
O
N
N
Antipyrine
Morazone
1-Methyl-3-(2,4,6-trimethoxyphenyl)-1H-pyrazole (7)
Yield: 18%, MF/FWt: C13H16N2O3/248; m.p.: 104–106 ꢁC; IR
(KBr, cmꢂ1): 3130, 2992, 2956, 2834 (CH), 1613, 1586, 1505,
1474 (C¼C), 1224 (C¼N), 1161 (C–O); 1H NMR (400 MHz,
CDCl3): ꢀ 3.76 (s, 6H, 2 ꢀ OCH3), 3.85 (s, 3H, OCH3), 3.97 (s,
3H, NCH3), 6.20 (s, 2H, 2 ꢀ ArH), 6.31 (d, J ¼ 2.8 Hz, 1H, Pyr-
H), 7.41 (d, J ¼ 2.8 Hz, 1H, Pyr-H); 13C NMR (100 MHz, CDCl3):
38.94 (CH3), 55.25 (CH3), 55.92 (CH3), 90.92 (CH), 104.42 (C),
107.82 (CH), 130.12 (CH), 144.42 (C), 159.38 (C), 161.08 (C);
135-DEPT: 38.94 (þ), 55.28 (þ), 55.93 (þ), 90.56 (þ), 107.81
(þ), 130.11 (þ); MS (ESI): m/e 249 (M þ 1).
Figure 1. Structures of some known pyrazole NSAIDs and compound
10i.
synthesize hybrid compounds containing arylpyrazole combined
with chalcone scaffold. Based on the earlier findings and in
continuation of our interest in the synthesis of novel bioactive
molecules with potential anti-inflammatory activities38–41, a new
series of pyrazolyl chalcones (10a–n) have been synthesized and
evaluated for their anti-inflammatory potential.
1-[2-Hydroxy-4,6-dimethoxy-3-(2-methyl-2H-pyrazol-3-yl)-
phenyl]-ethanone (8)
Experimental
5-(2,4,6-Trimethoxyphenyl)-1H-pyrazole (5)
Compound 6 (2.48 g, 10 mmol) was dissolved in 25 ml of dry
CH2Cl2 under N2 atmosphere and flask was cooled in an ice bath.
To this solution, BF3.OEt2 (11.36 g, 10.05 ml, 80 mmol) was
added followed by a dropwise addition of acetic anhydride (5.1 g,
4.72 ml, 50 mmol). The resulted solution was then allowed to stir
for 24 h at room temperature. The reaction mixture was diluted
with water, rendered alkaline with Na2CO3 and extracted with
CH2Cl2. Removal of solvent in vacuo gave white solid that was
recrystallized from methanol to obtain compound 8 in pure form.
Yield: 95%; MF/FWt: C14H16N2O4/276; m.p.: 221–223 ꢁC; IR
(KBr, cmꢂ1): 3461 (OH), 2940, 2370 (CH), 1625 (CO), 1597,
1416 (C¼C), 1277 (C¼N), 1133 (C–O); 1H NMR (400 MHz,
CDCl3): ꢀ 2.69 (s, 3H, COCH3), 3.71 (s, 3H, NCH3), 3.88 (s, 3H,
OCH3), 4.02 (s, 3H, OCH3), 6.08 (s, 1H, ArH), 6.26 (d, 1H,
J ¼ 1.6 Hz, C¼CH), 7.59 (d, 1H, J ¼ 1.6 Hz, N¼CH); 13C NMR
(100 MHz, CDCl3): 36.72 (COCH3), 38.80 (NCH3), 56.10
(OCH3), 56.45(OCH3), 90.60 (CH), 101.15 (C), 103.05 (C),
107.55 (CH), 138.22 (CH¼N), 140.20 (C), 162.50 (C), 165.92
(C), 203.50 (CO); MS (ESI): m/e 277 (M þ 1).
Ethyl formate (2.96 g, 3.21 ml, 30 mmol) was added to a
suspension of sodium hydride (60% in oil, 0.8 g, 20 mmol) in
tetrahydrofuran (3 ml) at room temperature. After stirring for
10 min, a solution of 1-(2,4,6-trimethoxyphenyl)-ethanone (3)
(2.1 g, 10 mmol) in THF (15 ml) was added, and the mixture was
stirred for 1 h. To the reaction mixture 1 M HCl (20 ml) was
added, and extracted with diethyl ether (3 ꢀ 25 ml). Organic layer
was concentrated under reduced pressure. Hydrate (5 g, 4.84 ml,
100 mmol) was added to this hydrazine, and the mixture was
stirred for 30 min. The reaction mixture was made alkaline by
adding 6N NaOH (20 ml) and extracted with ethyl acetate. The
organic layer was washed with brine, dried over anhydrous
magnesium sulfate, evaporated in vacuo, and recrystallized from
ethanol to obtain colorless crystals of title compound 5.
Yield: 85%, MF/FWt: C12H14N2O3/234; m.p.: 138–140 ꢁC; IR
(KBr, cmꢂ1): 3293 (NH), 3018, 2969, 2939 (CH), 1607, 1586,
1
1451 (C¼C), 1204(C–O); H NMR (400 MHz, CDCl3): ꢀ 3.85
(s, 3H, OCH3), 3.92 (s, 6H, OCH3), 6.22 (s, 2H, ArH), 6.82
(d, 1H, J ¼ 1.6 Hz, C¼CH), 7.6 (d, 1H, J ¼ 1.6 Hz, N¼CH), 11.38
(bs, 1H, NH); 13C NMR (100 MHz, CDCl3): 55.10 (CH3), 55.66
(CH3), 90.25 (CH), 100.38 (C), 106.98 (CH), 135.05 (C), 137.42
(CH), 158.90 (C), 162.20 (C); MS (ESI): m/e 235 (M þ 1).
General procedure for the preparation of pyrazole-based
chalcones (10a–n)
1-(2-Hydroxy-4,6-dimethoxy-3-(1-methyl-1H-pyrazol-5-yl)phe-
nyl)ethanone (8) (0.276 g, 1 mmol) was dissolved in ethanol
(10 ml) under stirring. To this NaOH (0.12 g; 3 mmol, with a
1-Methyl-5-(2,4,6-trimethoxyphenyl)-1H-pyrazole (6)
5-(2,4,6-Trimethoxyphenyl)-1H-pyrazole 5 (2.34 g, 10 mmol) was minimum of water) was added and stirred for 5 min. To this
dissolved in dry DMF (15 ml) under N2 atmosphere. The flask reaction mixture, aromatic aldehydes 9a–n (1.2 mmol) was then
was cooled in an ice bath and methyl iodide (2.84 g, 1.25 ml, added and stirring continued at room temperature for 24 h.
20 mmol) was added to it. To this solution, sodium hydride (60% Reaction was monitored by TLC. After completion of reaction,
in oil, 0.48 g, 12 mmol) was added in portions and the resulted the mixture was poured over crushed ice and acidified with acetic
solution was then allowed to stir at 0 ꢁC for 15 min. The reaction acid. The separated solid was filtered and washed well with water.
mixture was poured over crushed ice and the resulted solid was Crude product was dried and recrystallized from ethanol to obtain
filtered off, recrystallized from ethanol to afford the title the desired product in pure form.

DOI: 10.3109/14756366.2013.873037
Anti-inflammatory evaluation of pyrazole-based chalcones
3
(E)-1-(2-hydroxy-4,6-dimethoxy-3-(1-methyl-1H-pyrazol-
5-yl)phenyl)-3-phenylprop-2-en-1-one (10a)
(E)-3-(3,4-dimethoxyphenyl)-1-(2-hydroxy-4,6-dimethoxy-
3-(1-methyl-1H-pyrazol-5-yl)phenyl)prop-2-en-1-one (10e)
Yield: 98%; MF/FWt: C21H20N2O4/364; m.p.: 178–180 ꢁC; IR Yield: 98%; MF/FWt: C23H24N2O6/424; m.p.: 220–222 ꢁC; IR
(KBr, cmꢂ1): 3439 (OH), 2986, 2944, 2344 (CH), 1628 (CO), (KBr, cmꢂ1): 3685 (OH), 3020, 2974, 2400 (CH), 1618 (CO),
1
1578, 1556 (C¼C), 1335 (C¼N), 799, 758; H NMR (400 MHz, 1587, 1560, 1511 (C¼C), 1215 (C¼N), 758, 669; 1H NMR
DMSO-d6): ꢀ 3.55 (s, 3H, NCH3), 3.89 (s, 3H, OCH3), 4.08 (s, (400 MHz, DMSO-d6): ꢀ 3.55 (s, 3H, NCH3), 3.82 (s, 6H,
3H, OCH3), 6.15 (s, 1H, ArH), 6.43 (d, 1H, J ¼ 1.6 Hz, Pyr-H), 2 ꢀ OCH3), 3.86 (s, 3H, OCH3), 4.04 (s, 3H, OCH3), 6.14 (s, 1H,
7.41 (d, 1H, J ¼ 1.6 Hz, Pyr-H), 7.47–7.48 (m, 3H, ArH), 7.76– ArH),6.41 (d, 1H, J ¼ 1.7 Hz, Pyr-H), 7.05 (d, 1H, J ¼ 8.7 Hz,
7.77 (m, 3H, 2 ꢀ ArH, CH¼CHCO), 7.91 (d, 1H, J ¼ 15.6 Hz, ArH), 7.32–7.40 (m, 2H, Pyr-H, ArH), 7.51 (s, 1H, ArH), 7.71 (d,
CH¼CHCO), 14.O3 (bs, 1H, OH); 13C NMR (100 MHz, CDCl3): 1H, J ¼ 16 Hz, CH¼CHCO), 7.80 (d, 1H, J ¼ 16 Hz,
36.70 (NCH3), 56.32 (OCH3), 56.60 (OCH3), 91.05 (CH), 102.02 CH¼CHCO), 14.14 (bs, 1H, OH); 13C NMR (100 MHz,
(C), 102.95 (C), 107.64 (CH), 127.50 (COCH), 127.90 (CH), CDCl3): 36.66 (NCH3), 56.05 (OCH3), 56.15 (OCH3), 56.18
128.56 (2 ꢀ CH), 128.65 (2 ꢀ CH), 135.25 (C), 138.28 (CH¼N), (OCH3), 56.60 (OCH3), 90.89 (CH), 102.12 (C), 102.81 (C),
140.33 (C), 145.35 (COCH¼CH), 162.80 (C), 164.44 (C), 166.08 107.64 (CH), 110.67 (CH), 110.92 (CH), 122.55 (CH), 127.33
(C), 193.70 (CO); MS (ESI): m/e 365 (M þ 1)
(CH), 127.67 (COCH), 138.15 (CH¼N), 140.52 (C), 145.30
(COCH¼CH), 150.13 (C), 150.78 (C), 162.68 (C), 164.40 (C),
166.30 (C), 193.80 (CO); MS (ESI): m/e 425 (M þ 1).
(E)-3-(4-fluorophenyl)-1-(2-hydroxy-4,6-dimethoxy-3-(1-methyl-
1H-pyrazol-5-yl)phenyl)prop-2-en-1-one (10b)
(E)-3-(3,4-difluorophenyl)-1-(2-hydroxy-4,6-dimethoxy-
3-(1-methyl-1H-pyrazol-5-yl)phenyl)prop-2-en-1-one (10f)
Yield: 96%; MF/FWt: C21H19N2O4F/382; m.p.: 200–202 ꢁC; IR
(KBr, cmꢂ1): 3447 (OH), 2944, 2372 (CH), 1635 (CO), 1600,
1553, 1479 (C¼C), 1237 (C¼N), 1126 (C–O); 1H NMR Yield: 94%; MF/FWt: C21H18N2O4F2/400; m.p.: 198–200 ꢁC; IR
(400 MHz, DMSO-d6): ꢀ 3.54 (s, 3H, NCH3), 3.86 (s, 3H, (KBr, cmꢂ1): 3685 (OH), 3020, 2400 (CH), 1615 (CO), 1587,
OCH3), 4.07 (s, 3H, OCH3), 6.16 (s, 1H, Ar-H), 6.41 (d, 1515 (C¼C), 1215 (C¼N), 770, 669; 1H NMR (300 MHz,
J ¼ 1.6 Hz, 1H, Pyr-H), 7.25–7.30 (m, 2H, Ar-H), 7.41 (d, CDCl3): ꢀ 3.72 (s, 3H, NCH3), 3.90 (s, 3H, OCH3), 4.10 (s, 3H,
J ¼ 1.6 Hz, Pyr-H), 7.76 (d, J ¼ 16 Hz, 1H, CH¼CHCO), 7.80– OCH3), 6.12 (s, 1H, ArH), 6.28 (s, 1H, Pyr-H), 7.20 (dd, 1H,
7.88 (m, 3H, 2 ꢀ Ar-H, CH¼CHCO), 14.01 (bs, 1H, OH); 13C J ¼ 8 Hz, ArH), 7.30–7.35 (d, 1H, J ¼ 8 Hz, ArH), 7.43 (t, 1H,
NMR (100 MHz, CDCl3): 36.65 (NCH3), 56.02 (OCH3), 56.58 J ¼ 8 Hz, ArH), 7.58 (s, 1H, Pyr-H), 7.69 (d, 1H, J ¼ 16 Hz,
(OCH3), 91.00 (CH), 102.05 (C–CO), 102.96 (C), 107.67 (CH), CH¼CHCO), 7.79 (d, 1H, J ¼ 16 Hz, CH¼CHCO), 14.25 (bs, 1H,
117.58 (2 ꢀ CH), 127.60 (COCH), 130.84 (2 ꢀ CH), 130.95 (C), OH); 13C NMR (100 MHz, CDCl3): 36.66 (NCH3), 56.05 (OCH3),
138.12 (CH¼N), 140.04 (C), 146.43 (COCH¼CH), 147.23 (C), 56.59 (OCH3), 91.01 (CH), 102.06 (C), 102.76 (C), 107.65 (CH),
162.78(C), 164.23 (C), 166.15 (C), 193.76 (CO); MS (ESI): m/e 113.81 (CH), 125.01 (CH), 126.20 (CH), 127.78 (COCH), 133.40
383 (M þ 1)
(C), 138.16 (CH¼N), 140.50 (C), 145.18 (COCH¼CH), 145.92
(C), 146.75 (C), 162.58 (C),164.02 (C), 165.99 (C), 193.62 (CO);
MS (ESI): m/e 401 (M þ 1).
(E)-3-(2,4-difluorophenyl)-1-(2-hydroxy-4,6-dimethoxy-
3-(1-methyl-1H-pyrazol-5-yl)phenyl)prop-2-en-1-one (10c)
(E)-3-(benzo[d][1,3]dioxol-5-yl)-1-(2-hydroxy-4,6-dimethoxy-
3-(1-methyl-1H-pyrazol-5-yl)phenyl)prop-2-en-1-one (10 g)
Yield: 95%; MF/FWt: C21H18N2O4F2/400; m.p.: 198–200 ꢁC; IR
(KBr, cmꢂ1): 3684 (OH), 3020, 2977, 2400 (CH), 1615 (CO),
1587, 1566, 1501 (C¼C), 1215 (C¼N), 767, 669; 1H NMR Yield: 92%; MF/FWt: C22H20N2O6/408; MP: 178–179 ꢁC; IR
(400 MHz, DMSO-d6): ꢀ 3.55 (s, 3H, NCH3), 3.89 (s, 3H, OCH3), (KBr, cmꢂ1): 3430 (OH), 2919 (CH), 1626 (CO), 1599, 1554,
4.01 (s, 3H, OCH3), 6.15 (s, 1H, ArH), 6.43 (d, 1H, J ¼ 1.7 Hz, 1498, 1448 (C¼C), 1248 (C¼N), 1035 (C–O); 1H NMR
Pyr-H), 7.23 (d, 1H, J ¼ 1.7 Hz, Pyr-H), 7.42–7.44 (m, 2H, ArH, (400 MHz, DMSO-d6): ꢀ 3.54 (s, 3H, NCH3), 3.88 (s, 3H,
CH¼CHCO), 7.73 (d, 1H, J ¼ 15.7 Hz, CH¼CHCO), 7.93–7.99 OCH3), 4.06 (s, 3H, OCH3), 6.12 (s, 2H, O–CH2–O), 6.14 (s, 1H,
(m, 2H, ArH), 13.91 (bs,1H, OH); 13C NMR (100 MHz, CDCl3): ArH), 6.41 (d, 1H, J ¼ 1.7 Hz, Pyr-H), 7.01 (d, 1H, J ¼ 8.2 Hz,
36.68 (NCH3), 56.05 (OCH3), 56.68 (OCH3), 91.01 (CH), 102.05 ArH), 7.28 (d, 1H, J ¼ 8 Hz, ArH), 7.39–7.40 (m, 2H, Pyr-H,
(C), 102.97 (C), 107.65 (CH), 113.0 (CH), 115.40 (CH), 118.7 ArH), 7.71 (d, 1H, J ¼ 15.5 Hz, CH¼CHCO), 7.76 (d, 1H,
(C), 122.30 (COCH), 130.08 (CH), 138.47 (CH¼N), 140.66 (C), J ¼ 15.5 Hz, CH¼CHCO); 13C NMR (100 MHz, CDCl3): 36.65
145.28 (COCH¼CH), 148.45 (C), 149.33 (C), 162.81 (C), 164.39 (NCH3), 56.03 (OCH3), 56.07 (OCH3), 91.02 (CH), 100.2
(C), 166.35 (C), 193.75 (CO); MS (ESI): m/e 400 (Mþ).
(OCH2O), 102.15 (C), 102.78 (C), 107.64 (CH), 108.0 (CH),
110.06 (CH), 122.53 (CH), 127.35 (C), 127.68 (COCH), 138.15
(CH¼N), 140.52 (C), 145.32 (COCH¼CH), 150.03 (C), 150.48
(C), 162.56 (C), 164.40 (C), 166.30 (C), 193.69 (CO); MS (ESI):
m/e 409 (M þ 1).
(E)-3-(2,4-dichlorophenyl)-1-(2-hydroxy-4,6-dimethoxy-
3-(1-methyl-1H-pyrazol-5-yl)phenyl)prop-2-en-1-one (10d)
Yield: 97%; MF/FWt: C21H18N2O4Cl2/433; m.p.: 215–217 ꢁC; IR
(KBr, cmꢂ1): 3446 (OH), 2925, 2344 (CH), 1623 (CO), 1583,
1565, 1469(C¼C), 1327 (C¼N), 1123 (C–O), 870; 1H NMR
(400 MHz, DMSO-d6): ꢀ 3.55 (s, 3H, NCH3), 3.86 (s, 3H, OCH3),
(E)-1-(2-hydroxy-4,6-dimethoxy-3-(1-methyl-1H-pyrazol-
5-yl)phenyl)-3-(3-nitrophenyl)prop-2-en-1-one (10 h)
4.01 (s, 3H, OCH3), 6.11 (s, 1H, ArH), 6.33 (d, 1H, J ¼ 1.7 Hz, Yield: 98%; MF/FWt: C21H19N3O6/409; MP: 176–178 ꢁC; IR
Pyr-H), 7.38 (s, 1H, ArH), 7.52 (d, 1H, J ¼ 8.4 Hz, ArH), 7.76 (d, (KBr, cmꢂ1): 3683 (OH), 3020, 2946,2400 (CH), 1618 (CO),
1H, J ¼ 1.7 Hz, Pyr-H), 7.83–7.86 (m, 2H, CH¼CHCO), 7.95 (d, 1587, 1532(C¼C), 1215 (C¼N), 1128 (C–O), 757, 668; 1H NMR
1H, J ¼ 8.4 Hz, ArH); 13C NMR (100 MHz, CDCl3):06 (OCH3), (400 MHz, DMSO-d6): ꢀ 3.55 (s, 3H, NCH3), 3.87 (s, 3H, OCH3),
56.61 (OCH3), 91.02 (CH), 102.11 (C), 102.85 (C), 107.66 (CH), 4.03 (s, 3H, OCH3), 6.13 (s, 1H, ArH), 6.37 (d,1H, J ¼ 1.6 Hz,
121.88 (COCH), 125.08 (C), 126.80 (CH), 129.11 (CH), 130.06 Pyr-H), 7.40 (d, 1H, J ¼ 1.6 Hz, Pyr-H), 7.73–7.77 (m, 2H, ArH,
(CH), 131.18 (C), 137.28 (C), 138.64 (CH¼N), 140.70 (C), CH¼CHCO), 7.95 (d, 1H, J ¼ 15 Hz, CH¼CHCO), 8.22 (d, 1H,
146.01 (COCH¼CH), 162.0 (C), 164.46 (C), 166.83 (C), 193.80 J ¼ 7.3 Hz, ArH), 8.26 (d, 1H, J ¼ 8.2 Hz, ArH), 8.53 (s, 1H,
(CO); MS (ESI): m/e 433 (Mþ).
ArH), 14.1 (bs, 1H, OH); 13C NMR (100 MHz, CDCl3): 36.68

4
H. V. Chavan et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
(NCH3), 56.11 (OCH3), 56.23 (OCH3), 91.17 (CH), 102.15 (C), CH¼CHCO), 13.82 (bs, 1H, OH); 13C NMR (100 MHz, CDCl3):
102.80 (C), 107.68 (CH), 125.07 (CH), 126.10 (CH), 127.81 36.66 (NCH3), 56.12 (OCH3), 56.66 (OCH3), 91.08 (CH), 102.15
(COCH), 130.26 (CH), 135.66 (CH), 138.0 (C), 138.20 (CH¼N), (CH), 102.86 (CH), 107.68 (CH¼N), 122.50 (COCH), 126.84
140.60 (C), 150.08 (C), 162.61 (C), 164.45 (C), 166.32 (C), (CH), 130.02 (2 ꢀ CH), 135.25 (2 ꢀ CH), 137.81 (COCH¼CH),
193.84 (CO); MS (ESI): m/e 410 (M þ 1).
138.11 (CH¼N), 140.10 (CH), 145.37 (COCH¼CH), 162.78 (C),
164.09 (C), 166.21 (C), 193.78 (CO); MS (ESI): m/e 433 (Mþ)
(E)-3-(furan-2-yl)-1-(2-hydroxy-4,6-dimethoxy-3-(1-methyl-
1H-pyrazol-5-yl)phenyl)prop-2-en-1-one (10i)
(E)-3-(2,6-difluorophenyl)-1-(2-hydroxy-4,6-dimethoxy-
3-(1-methyl-1H-pyrazol-5-yl)phenyl)prop-2-en-1-one (10m)
Yield: 93%; MF/FWt: C19H18N2O5/354; MP: 220–222 ꢁC; IR
(KBr, cmꢂ1): 3683 (OH), 3019, 2400 (CH), 1618 (CO), 1551, Yield: 91%; MF/FWt: C21H18N2O4F2/400; MP: 194–196 ꢁC; IR
1
1471, 1413 (C¼C), 1216 (C¼N), 768, 669; H NMR (400 MHz, (KBr, cmꢂ1): 3018, 2979, 2945 (CH), 1619 (CO), 1588, 1469,
DMSO-d6): ꢀ 3.54 (s 3H, NCH3), 3.88 (s, 3H, OCH3), 4.05 (s, 3H, 1412 (C¼C), 1214 (C¼N), 1131 (C–O), 992, 788; 1H NMR
OCH3), 6.14 (s, 1H, Ar-H), 6.42 (d, 1H, J ¼ 1.6 Hz, Pyr-H), 6.70 (300 MHz, CDCl3): ꢀ 3.72 (s, 3H, NCH3), 3.90 (s, 3H, OCH3),
(s, 1H, Furan-H), 7.07 (s, 1H, Furan-H), 7.41 (d, 1H, J ¼ 1.6 Hz, 4.08 (s, 3H, OCH3), 6.12 (s, 1H, ArH), 6.24 (s, 1H, Pyr-H), 6.88
Pyr-H), 7.62 (d, 1H, J ¼ 15.4 Hz, CH¼CHCO), 7.70 (d, 1H, (t, 1H, J ¼ 8 Hz, ArH), 7.28–34 (m, 2H, ArH), 7.60 (s, 1H, Pyr-H),
J ¼ 15.4 Hz, CH¼CHCO), 7.93 (s, 1H, Furan-H), 14.23 (bs, 1H, 7.85 (d, 1H, J ¼ 16 Hz, CH¼CHCO), 8.20 (d, 1H, J ¼ 16 Hz,
OH); 13C NMR (100 MHz, CDCl3): 36.65 (NCH3), 56.02 (OCH3), CH¼CHCO), 14.25 (bs, 1H, OH); 13C NMR (100 MHz, CDCl3):
56.09 (OCH3), 90.96 (CH), 102.10 (C), 102.95 (C), 107.64 (CH), 36.65 (NCH3), 56.06 (OCH3), 56.73 (OCH3), 91.09 (CH), 102.18
113.70 (CH), 115.08 (CH), 122.91 (COCH¼CH), 127.63 (COCH), (C), 102.92 (C), 107.70 (CH), 112.96 (CH), 114.48 (CH), 122.48
138.12 (CH), 140.48 (C), 145.07 (CH), 152.58 (C), 162.48 (C), (COCH), 130.10 (CH), 138.12 (CH¼N), 140.16 (C), 145.63
164.04 (C), 166.0 (C), 193.63 (CO); MS (ESI): m/e 355 (M þ 1). (COCH¼CH), 160.42 (C), 162.75 (C), 165.89 (C), 193.81 (CO);
MS (ESI): m/e 401 (M þ 1).
(E)-1-(2-hydroxy-4,6-dimethoxy-3-(1-methyl-1H-pyrazol-
5-yl)phenyl)-3-(thiophen-2-yl)prop-2-en-1-one (10j)
(E)-1-(2-hydroxy-4,6-dimethoxy-3-(1-methyl-1H-pyrazol-
5-yl)phenyl)-3-(2,4,6-trimethoxyphenyl)prop-2-en-1-one (10n)
Yield: 94%; MF/FWt: C19H18N2O4S/370; MP: 192–194 ꢁC; IR
(KBr, cmꢂ1): 3684 (OH), 3020, 2400 (CH), 1619 (CO), 1586, Yield: 90%; MF/FWt: C24H26N2O7/454; MP: 234–236 ꢁC; IR
1
1561, 1422 (C¼C), 1215 (C¼N), 770, 669; H NMR (400 MHz, (KBr, cmꢂ1): 3684 (OH), 3019, 2942(CH), 1617(CO), 1551, 1467
DMSO-d6): ꢀ 3.55 (s, 3H, NCH3), 3.88 (s, 3H, OCH3), 4.06 (s, 3H, (C¼C), 1216 (C¼N), 1128 (C–O), 769, 669; 1H NMR (400 MHz,
OCH3), 6.14 (s, 1H, Ar-H), 6.42 (d, 1H, J ¼ 1.54 Hz, Pyr-H), 7.20 DMSO-d6): ꢀ 3.54 (s, 3H, NCH3), 3.87 (s, 6H, 2 ꢀ OCH3), 3.92
(dd, 1H, J ¼ 4.6, 3.8 Hz, Thiophene-H), 7.41 (d, 1H, J ¼ 1.54 Hz, (s, 6H, 2 ꢀ OCH3), 4.06 (s, 3H, OCH3), 6.13 (d, 1H, J ¼ 1.6 Hz,
Pyr-H), 7.62–7.68 (m, 2H, CH¼CHCO, Thiophene-H), 7.79 (d, Pyr-H), 6.33 (s, 2H, ArH), 6.39 (s, 1H, ArH), 7.40 (d, 1H,
1H, J ¼ 4.6 Hz, Thiophene-H), 7.95 (d, 1H, J ¼ 15.3 Hz, J ¼ 1.6 Hz, Pyr-H), 8.20 (d, 1H, J ¼ 15.8 Hz, CH¼CHCO), 8.25
CH¼CHCO), 14.16 (bs, 1H, OH); 13C NMR (100 MHz, CDCl3): (d, 1H, J ¼ 15.8 Hz, CH¼CHCO), 14.72 (bs, 1H, OH); 13C NMR
36.65 (NCH3), 56.01 (OCH3), 56.06 (OCH3), 90.95 (CH), 102.08 (100 MHz, CDCl3): 36.63 (NCH3), 56.06 (OCH3), 56.37 (OCH3),
(C), 102.90 (C), 107.64 (CH), 127.68 (COCH), 128.93 (CH), 56.68 (OCH3), 56.73 (OCH3), 90.88 (CH), 92.75 (CH), 102.22
129.55 (CH), 131.01 (CH), 136.72 (COCH¼CH), 138.03 (CH¼N), (C), 102.89 (C), 107.06 (CH), 107.66 (C), 122.50 (COCH),
140.30 (C), 140.58 (CH), 162.45 (C), 163.97 (C), 165.88 (C), 123.42 (COCH¼CH), 138.02 (CH¼N), 140.0 (C), 158.01 (C),
193.60 (CO); MS (ESI): m/e 371 (M þ 1).
159.69 (C), 162.60 (C), 163.87 (C), 165.92 (C), 193.52 (CO); MS
(ESI): m/e 455 (M þ 1).
(E)-3-(4-bromophenyl)-1-(2-hydroxy-4,6-dimethoxy-3-(1-methyl-
1H-pyrazol-5-yl)phenyl)prop-2-en-1-one (10k)
Biological evaluation
Yield: 97%; MF/FWt: C21H19N2O4Br/443; MP: 196–198 ꢁC; IR
(KBr, cmꢂ1): 3446 (OH), 2925 (CH), 1623(CO), 1583, 1565,
1469 (C¼C), 1327 (C¼N), 1123 (C–O); 1H NMR (400 MHz,
DMSO-d6): ꢀ 3.70 (s, 3H, NCH3), 3.88 (s, 3H, OCH3), 4.05 (s,
3H, OCH3), 6.10 (s, 1H, ArH), 6.25 (d, J ¼ 2 Hz, 1H, Pyr-H), 7.45
(d, J ¼ 2 Hz, 1H, Pyr-H), 7.49–7.58 (m, 4H, ArH), 7.72 (d,
J ¼ 16 Hz, 1H, COCH¼CH), 7.87 (d, J ¼ 16 Hz, 1H,
COCH¼CH), 14.26 (s, 1H, OH); 13C NMR (100 MHz, CDCl3):
36.65 (NCH3), 55.95 (OCH3), 56.26 (OCH3), 90.87 (CH), 102.02
(C), 102.90 (C), 107.65 (CH), 123.23 (C), 127.56 (COCH),
128.95 (CH), 132.52 (CH), 135.20 (C), 138.08 (CH¼N), 140.06
(C), 146.35 (COCH¼CH), 162.67 (C), 163.95(C), 165.99 (C),
193.80 (CO); MS (ESI): m/e 444 (M þ 1).
In vitro COX inhibition assay
The assay was performed by using Colorimetric COX (ovine)
inhibitor Screening assay kit (Catalogue No. 760111, Cayman
Chemicals Co., Ann Arbor, MI)42. Briefly, the reaction mixture
contains, 150 ml of assay buffer, 10 ml of heme, 10 ml of enzyme
(either COX-1 or COX-2), and 50 ml of sample (0.1 mM). The
assay utilizes the peroxidase component of the COX catalytic
domain. The peroxidase activity can be assayed colorimetrically
by monitoring the appearance of oxidized N,N,N,N0-
tetramethyl-p-phenylenediamine
Indomethacin (0.1 mM) was used as a standard drug. The percent
COX inhibition was calculated using following equation:
(TMPD)
at
590 nm.
T
COX inhibition activity ð%Þ ¼ 1 ꢂ ꢀ 100:
(E)-3 -(2,6-dichlorophenyl)-1-(2-hydroxy-4,6-dimethoxy-
3-(1-methyl-1H-pyrazol-5-yl)phenyl)prop-2-en-1-one (10l)
C
where T is the absorbance of the inhibitor well at 590 nm, C is the
absorbance of the 100% initial activity without inhibitor well at
590 nm.
Yield: 90%; MF/FWt: C21H18N2O4Cl2/433; MP: 212–214 ꢁC; IR
(KBr, cmꢂ1): 3446 (OH), 2944 (CH), 1623 (CO), 1582, 1567,
1466 (C¼C), 1208 (C¼N), 1123 (C–O), 770; 1H NMR (400 MHz,
DMSO-d6): ꢀ 3.55 (s, 3H, NCH3), 3.83 (s, 3H, OCH3), 4.01 (s,
3H, OCH3), 6.15 (s, 1H, ArH), 6.41 (d, 1H, J ¼ 1.7 Hz, Pyr-H),
7.36–7.46 (m, 2H, ArH, Pyr-H), 7.61 (d, 2H, J ¼ 8 Hz, ArH), 7.76
(d, 1H, J ¼ 16.4 Hz, CH¼CHCO), 8.00 (d, 1H, J ¼ 16.4 Hz,
Animals
Animals were assigned into several groups randomly and each
group consists of six animals. The animals were kept in

DOI: 10.3109/14756366.2013.873037
Anti-inflammatory evaluation of pyrazole-based chalcones
5
appropriate cages at temperature controlled (25 ꢃ 2 ꢁC) rooms, Results and discussion
under a 12 h light and dark cycle and they were fed standard
Chemistry
rodent pellet. All the animals were acclimatized for a week before
the experiment. Experimental protocols were reviewed and Synthesis of target compounds 10a–n was achieved by the
approved by the Institutional Animal Ethics Committee Claisen–Schmidt condensation of 1-(2-hydroxy-4,6-dimethoxy-
(SIPS/IAEC/2011-12/17) and conform to the Indian National 3-(1-methyl-1H-pyrazol-5-yl)phenyl)ethanone 8 with various aro-
Science Academy Guidelines for the use and care of experimental matic/heteroaromatic aldehydes 9a–n in the presence of NaOH
animals in research. The animal house registration number with in ethanol at room temperature in good to excellent
Government of India is 962/c/06/CPCSEA.
yields (Scheme 1). 1-(2-Hydroxy-4,6-dimethoxy-3-(1-methyl-
1H-pyrazol-5-yl)phenyl)ethanone was readily prepared
by the Friedel–Craft acetylation of key intermediate
1-methyl-5-(2,4,6-trimethoxyphenyl)-1H-pyrazole by using
8
Carrageenan induced hind paw edema assay
6
Dose–response study. Rats were divided into various groups
(n ¼ 6) and allowed for free access to water ad libitum. Different
groups of rats administered with indomethacin (100 mg/kg, b.w.)
and various test compounds (100 mg/kg, v) orally. One group of
rats served as a control and administered with gum acacia (1%
(w/v); 10 ml/kg, b.w., p.o.). One hour after the drug administra-
tion, to all rats, hind paw edema was induced by the method of
Winter et al.43 by injecting 0.1 ml of 1% (w/v) solution
of carrageenan subcutaneously into the subplanter region of
hind paw.
acetic anhydride in the presence of boron trifluoride etherate
under nitrogen atmosphere.
The regioselective synthesis of key intermediate
1-methyl-5-(2,4,6-trimethoxyphenyl)-1H-pyrazole 6 was carried
out using the synthetic strategies illustrated in Scheme 2.
Xanthoxyline 2 was synthesized by the Friedel–Craft acetyl-
ation of 1,3,5-trimethoxybenzene 1 by adopting the literature
precedent45, and successive O-methylation of the resulting
xanthoxyline
potassium carbonate in acetone under reflux condition gave
1-(2,4,6-trimethoxyphenyl)ethanone in excellent yield.
2 using dimethyl sulphate and flame-dried
3
Time course study. The hind paw edema volume was measured
by volume displacement method using plethysmometer (UGO,
Besile 7140, Italy) by immersing the paw till the level lateral
malleolus at various time intervals (1, 3 and 6 h) after carrageenan
injection.
2-Formylation of acetophenone 3 with ethyl formate and
sodium hydride gave 3-oxo-3-(2,4,6-trimethoxyphenyl)propanal
4, and without further purification subsequent treatment
of
5-(2,4,6-trimethoxyphenyl)-1H-pyrazole
4
with hydrazine hydrate afforded the corresponding
in 96% yield.
5
Treatment of compound 5 with methyl iodide and sodium
hydride in dry DMF under nitrogen atmosphere afforded a
mixture of 1-methyl-5-(2,4,6-trimethoxyphenyl)-1H-pyrazole 6
and 1-methyl-3-(2,4,6-trimethoxyphenyl)-1H-pyrazole 7, which
were easily seperated by silica gel column chromatography.
The structures of the regioisomers 6 and 7 were determined
using 1D 1H–13C NMR spectra, 135-DEPT and 2D NMR
Cotton pellets induced granuloma assay
Two sterilized cotton pellets (10 mg) were implanted on ventral
regions of rats, procedure described by winter and porter44 and
divided into various four groups (six rats/group). Different groups
of rats administered with indomethacin (100 mg/kg), compound
10e, 10f, 10h, and 10i (100 mg/kg) orally respectively for the
duration of 8 d. Control group received gum acacia (2%, 10 ml/kg,
p.o.). On the day 9, rats were sacrificed with excess ether
anesthesia. The cotton pellets were removed and freed from
extraneous tissue and used for granular tissue formation by
recording wet (immediately) and dry weight of pellets. The
granular tissue formation was studied by drying cotton pellets at
55 ꢁC for 6 h or till the weight of pellets remains constant. The dry
weight was calculated after deducting cotton pellet weight and
taken as a granular tissue formation.
experiments (1H–1H COSY, H–13C HSQC and H–13C HMBC
and NOESY), which were scanned on Bruker AV-400 and
500 MHz instrument (Supporting Data). These two regioisomers
differed only in the position of methyl group on pyrazole nucleus,
1
1
1
therefore, the H and 13C NMR chemical shifts were found to be
almost similar. Thus, the structures of these two regioisomers only
assigned with the help of HMBC, HSQC and COSY correlations.
The most important HMBC and COSY correlations are
summarized in Figure 2 and the complete chemical shift
O
Scheme 1. Reagents and conditions: (a)
Ac2O, BF3.OEt2, 0 ꢁC -rt, N2, 3 h; (b)
NaOH, EtOH, rt, 24 h.
O
O
O
O
O
H
R
a
9
R
O
O
b
O
OH
O
OH
N
N
N
N
N
N
6
8
10a-n
F
Cl
OMe
OMe
F
F
O
R =
O
F
F
Cl
Br
(a)
(h)
(b)
(d)
(e)
(f)
F
(g)
OMe
(c)
Cl
NO2
O
S
Cl
F
MeO
OMe
(l)
(m)
(n)
(i)
(j)
(k)

6
H. V. Chavan et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
Scheme 2. Reagents and conditions:
(a) CH3COCl, AlCl3 (anhydrous), Et2O, N2,
0 ꢁC - rt; (b) Me2SO4, K2CO3, acetone,
reflux, 12 h; (c) HCOOEt, NaH, THF, N2, rt;
(d) NH2NH2.H2O, rt; (e) MeI, NaH, DMF,
N2, 0 ꢁC.
O
O
O
a
b
c
O
OH
O
O
O
O
O
O
1
2
3
O
O
O
O
d
e
+
O
H
O
O
O
O
O
O
O
O
NH
N
N
N
N
N
O
4
5
7
6
(18%)
(82%)
1
Figure 2. HMBC (H to C) and H–1H COSY
correlations.
11
11
O
O
7
7
4
8
9
8
9
6
5
6
5
10
12
12
10
4
O
O
O
O
Symbol Correlations
HMBC
13
3
3
N
N
2
2
1
1
N
N
COSY
13
6
7
1
Table 1. 1H, 13C NMR and 135-DEPT spectral data and H-1H COSY, HSQC and HMBC correlations in compound 6.
Correlations
HSQC
1C 3C
Position
1H NMR (ppm)
13C NMR (ppm)
DEPT
COSY
HMBC
1
2
3
4
7.55 (1H, d)
6.20 (3H, m)
137.90
107.44
135.51
100.68
159.32
90.54
162.25
55.74
55.39
CH
CH
C
C
C
CH
C
CH3
CH3
CH3
2H
1H
–
–
–
–
–
–
–
2C
–
3C, 4C
–
–
–
–
–
–
5, 9
6, 8
7
10, 12
11
13
–
–
4C, 5C, 6C, 7C, 8C, 9C
–
6.20 (3H, m)
–
3.75 (6H, s)
3.87 (3H, s)
3.65 (3H, s)
6C, 8C
–
10C, 12C
11C
13C
5C, 9C
7C
3C
36.65
–
assignment is shown in Tables 1 and 2. According to a
Heteronuclear Multiple Bond Correlation (HMBC) experiment Anti-inflammatory activity
performed with regioisomer 6, the HMBC correlation observed
between H-13 (ꢀH 3.65) and C-3 (ꢀC 135.5) and with regioisomer
In vitro COX inhibition
7, the HMBC correlation observed between H-13 (ꢀH 3.97) and C- All the synthesized compounds (10a–n) were screened for their
1 (ꢀC 130.12) allowing to unambiguous identification of the inhibitory potential against COX-1 and COX-2 enzymes at
regioisomer 6 and 7 (Figures 3 and 4). Additionally, heteronuclear 100 mM using colorimetric COX (ovine) inhibitor screening
single quantum coherence (HSQC) experiment was used to assign assay kit. Indomethacin was used as a reference compound. The
proton signals to the corresponding carbon signals. There is a results were shown in Table 3. The results showed that most of the
strong H–1H COSY correlation between H-1 and H-2.
1
synthesized compounds had an inhibitory profile against both

DOI: 10.3109/14756366.2013.873037
Anti-inflammatory evaluation of pyrazole-based chalcones
7
1
Table 2. 1H, 13C NMR and 135-DEPT spectral data and H-1H COSY, HSQC and HMBC correlations in compound 7.
Correlations
HSQC
1C
2C
–
Position
1H NMR (ppm)
13C NMR (ppm)
DEPT
COSY
HMBC
2C, 3C, 13C
1C, 3C, 4C
–
–
–
4C, 5C, 6C, 7C, 8C, 9C
–
5C, 9C
7C
1C
1
2
3
4
7.41 (1H, d)
6.31 (1H, d)
130.12
107.82
144.42
104.42
159.38
90.92
161.08
55.92
55.25
CH
CH
C
C
C
CH
C
CH3
CH3
CH3
2H
1H
–
–
–
–
–
–
–
–
–
–
–
–
5, 9
6, 8
7
10, 12
11
13
6.20 (2H, s)
–
3.76 (6H, s)
3.85 (3H, s)
3.97 (3H, s)
6C, 8C
–
10C, 12C
11C
13C
38.94
–
Figure 3. HMBC spectrum of compound 6 represents correlation between H-13 and 3C.
COX-1 and COX-2 enzymes, some were found to be selective carrageenan-induced hind paw edema in rats using
against COX-2 (10i, 10f, 10d, 10h and 10e) by a small percentage indomethacin as a reference standard. The results of all the
of inhibition. Compound 10b, 10l and 10c had an inhibitory tested compounds were shown in Table 4. The results
profile against both COX-1 and COX-2. However, all the other revealed that among the all tested compounds, compounds 10i,
compounds showed weak inhibitory activity against both the 10e, 10f, and 10h inhibited the edema formation significantly
isoforms of COX (Table 3). The structure–activity relationship (p50.05) at 3 h and 6 h than other compounds when compared
(SAR) study revealed that, compound carrying furyl heterocycle to carrageenan control rats. Edema formation due to carrageenan
by replacing phenyl ring, 10i, appeared as the most active is a biphasic event. The first phase begins immediately
compound in this series. Moreover, final compounds with –F, after injection and diminishes in 1 h and the second phase
–NO2, and –OCH3 substituents (10f, 10h and 10e) at the meta begins after 1 h. The initial phase of edema is attributed to
position of phenyl ring showed selective inhibitory activity the release of histamine and serotonin and second phase is due
against COX-2 enzyme.
to the release of prostaglandins. All the tested compounds
(10i, 10e, 10f, and 10h) showed greater inhibition of paw edema
formation at 6 h due to the inhibition of prostaglandins release,
which are known to be released at 4–6 h after the carrageenan
injection.
Carrageenan-induced rat paw edema bioassay
Compounds showing significant COX-2 inhibition were
selected for further anti-inflammatory evaluation by using

8
H. V. Chavan et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
Figure 4. HMBC spectrum of compound 7 represents correlation between H-13 and 1C.
Table 3. In vitro COX-1 and COX-2 enzyme inhibitory
activities of pyrazole containing chalcones (10a–n).
Table 4. Anti-inflammatory activity of the target compounds against the
carrageenan-induced paw edema in rats with different time intervals.
% inhibition of COX (100 mM)*
Paw volume (ml)
Compound
COX-1
COX-2
Dose
(mg/kg.p.o.)
Compound
1 h
3 h
6 h
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
10m
10n
Indomethacin
52.42
58.31
37.30
10.05
25.53
48.26
29.73
10.41
41.50
05.86
06.50
40.66
39.82
16.29
70.07
35.43
40.17
35.96
39.64
36.54
50.59
38.59
39.01
52.27
18.06
12.65
37.01
19.64
10.80
43.33
Normal group
Control
Standard*
10a
10b
10c
10d
10e
10f
10g
–
–
0.40 ꢃ 0.04 0.35 ꢃ 0.04
0.94 ꢃ 0.05# 1.17 ꢃ 0.1#
0.38 ꢃ 0.06
1.69 ꢃ 0.08##
100
100
100
100
100
100
100
100
100
100
100
0.84 ꢃ 0.05 0.98 ꢃ 0.07** 0.73 ꢃ 0.05**
0.52 ꢃ 0.04 1.40 ꢃ 0.2
0.51 ꢃ 0.03 1.58 ꢃ 0.3
1.79 ꢃ 0.3
1.72 ꢃ 0.5
1.92 ꢃ 0.4
1.68 ꢃ 0.1
0.68 ꢃ 0.2
1.82 ꢃ 0.07
0.53 ꢃ 0.06 1.42 ꢃ 0.05
0.86 ꢃ 0.17 1.02 ꢃ 0.03* 1.11 ꢃ 0.1*
0.76 ꢃ 0.1
0.75 ꢃ 0.2
0.88 ꢃ 0.1
1.05 ꢃ 0.2*
1.82 ꢃ 0.1
0.98 ꢃ 0.1*
1.12 ꢃ 0.1*
1.98 ꢃ 0.3
1.19 ꢃ 0.1*
10h
10i
10j
0.75 ꢃ 0.04 0.98 ꢃ 0.03* 1.13 ꢃ 0.2*
0.68 ꢃ 0.04 1.54 ꢃ 0.03
1.55 ꢃ 0.09
*Standard: Indomethacin, each value is the mean ꢃ SEM for six rats.
#p50.05, ##p50.01 when compared with normal control group.
**p50.01, *p50.05 when compared with carrageenan control group.
*The determination was performed in duplicate for two
independent experiments.
the exudatory and granulatory phases of inflammation (Table 5).
During the repair process of inflammation, there is a proliferation
Cotton pellet-induced granuloma bioassay
Cotton pellet implantation is the most suitable method for of macrophages, neutrophils, fibroblasts and multiplication of
studying the efficacy of drugs against proliferative phase of small blood vessels, which are the basic sources of forming a
inflammation. Compounds showing promising anti-inflammatory highly vascularized radish mass called as granular tissue (granular
activity in the carrageenan-induced rat paw edema bioassay were tissue formation). Inhibition of this granular phase of inflamma-
further evaluated for their in vivo cotton pellet-induced granuloma tion by 10i, 10e, 10f, and 10h suggested significant anti-
bioassay in rats using indomethacin as a reference standard. In inflammatory property was observed in the experimental animal
this assay, test compounds 10i, 10e, 10f, and 10h inhibited both models.

DOI: 10.3109/14756366.2013.873037
Anti-inflammatory evaluation of pyrazole-based chalcones
9
Table 5. Effects of test compounds on cotton pellet induced
granuloma in rats.
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Weight (mg) Mean ꢃ SEM
Treatment and dose
(100 mg/kg., p.o.)
Wet
Dry
Vehicle control
256 ꢃ 11.2
148 ꢃ 11.5**
189 ꢃ 12.1*
182 ꢃ 8.2*
190 ꢃ 11.8*
178 ꢃ 10.8*
153 ꢃ 9.9
76 ꢃ 4.9**
91 ꢃ 6.3*
79 ꢃ 2.3*
92 ꢃ 4.5*
88 ꢃ 8.4*
Standarda
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10f
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10i
aStandard: Indomethacin.
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**p50.01, *p50.05 when compared with normal control
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Conclusion
In conclusion, we herein report the synthesis, spectral studies, and
biological evaluation of a novel series of pyrazole-based
chalcones. The structures of the two regioisomers 6 and 7 were
assigned with the help of HMBC, HSQC and COSY correlations.
All the newly synthesized compounds (10a–n) were evaluated for
in vitro COX-1/COX-2 inhibition and in vivo anti-inflammatory
activity. Compounds which showed significant COX-2 inhibition
were subjected to anti-inflammatory studies. Some of the
synthesized compounds might constitute initial leads for the
design of new more potent therapeutics against inflammation. The
present study revealed that the compounds 10i, 10e, 10f, and 10h
were effective against both carrageenan-induced paw edema and
cotton pellet-induced granuloma models. This revealed that these
compounds were potent inhibitors of exudative and proliferative
phases of inflammation. Thus, these compounds constitute an
interesting template for the evaluation of new anti-inflammatory
agents and may be helpful for the design of new therapeutic tools
against inflammation.
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molecules: search for potent multidrug resistance modulators.
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Declaration of interest
The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of this article. Author Hemant V.
Chavan is thankful to the Council of Scientific and Industrial Research
(CSIR), New Delhi, Govt. of India, for financial support in the form of
SRF.
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