Download Synthesis, Molecular Docking, and Biological Evaluation of Some

Chem Biol Drug Des 2014; 84: 473–488
Research Article
Synthesis, Molecular Docking, and Biological
Evaluation of Some Novel Hydrazones and Pyrazole
Derivatives as Anti-inflammatory Agents
Khaled O. Mohammed1 and Yassin M. Nissan2,*
1
Pharmaceutical Organic Chemistry Department, Faculty
of Pharmacy, Cairo University, Kasr Elini Street, Cairo
11562, Egypt
2
Pharmaceutical Chemistry Department, Faculty of
Pharmacy, Cairo University, Kasr Elini Street, Cairo 11562,
Egypt
*Corresponding author: Yassin M. Nissan,
[email protected]
2-Hydrazinyl-N-(4-sulfamoylphenyl)acetamide 3 was
the key intermediate for the synthesis of novel hydrazones 4–10 and pyrazole derivatives 11–17. All compounds were tested for their in vivo anti-inflammatory
activity and their ability to inhibit the production of
PGE2 in serum samples of rats. IC50 values for the
most active compounds for inhibition of COX-1 and
COX-2 enzymes were determined in vitro, and they
were also tested for their ulcerogenic effect. Molecular
docking was performed on the active site of COX-2 to
predict their mode of binding to the amino acids. Most
of the synthesized compounds showed good antiinflammatory activity especially compounds 3, 4, 8, 9,
15, and 17 which showed better activity than diclofenac as the reference drug. Compounds 3, 8, 9, 13, and
15–17 were less ulcerogenic than indomethacine as
the reference drug. Most of the synthesized compounds interacted with Tyr 385 and Ser 530 in molecular docking study with additional hydrogen bond for
compound 17. Compound 17 showed good selectivity
index value of 11.1 for COX-1/COX-2 inhibition in vitro.
Key words: 4-benzenesulfonamide, anti-inflammatory,
hydrazone, pyrazole
Received 22 February 2014, revised 26 March 2014 and
accepted for publication 3 April 2014
Although of the high effectiveness of the steroidal antiinflammatory drugs, their serious side-effects limit their use
in common inflammation and as pain killers (1). The search
for safer drugs launched with the development of nonsteroidal anti-inflammatory drugs. In spite of their great
safety over the steroidal drugs, they still have some sideeffects, the most common one is the development of peptic and duodenal ulcer on long-term use (2). Moreover,
ª 2014 John Wiley & Sons A/S. doi: 10.1111/cbdd.12336
some kidney and liver malfunctions were observed (2). The
rise of selective COX-2 inhibitors reduced the risk of some
of these side-effects (3), but other side-effects were also
observed, and the most serious one was the capability of
some drugs of causing cardiac arrest in some patients (4).
But although high selectivity is demanded to reduce the
gastrointestinal side-effects, it was discussed recently that
COX-2 high selectivity could be the major cause of cardiac
and renal problems (5,6), and hence, moderate selectivity
could reduce the risk of both gastrointestinal and cardiac
side-effects (7).
Therefore, seeking for new molecules with anti-inflammatory activity and diminished side-effects is still a goal for
medicinal chemists all over the world.
In our seek for novel anti-inflammatory agents with less
side-effects and better activity, we have focused in the
current research of merging more than one active moiety
known for their presence in several anti-inflammatory
agents. The first moiety was phenylacetamide, a wellknown moiety in several pain killers especially in paracetamol and phenacetin (8–10). Literature describes the usage
of phenylacetamide in the synthesis of new anti-inflammatory agents (11–14). The second moiety was sulfamoyl
moiety in para position of phenylacetamide. The antiinflammatory effect of sulfonamides was reported in many
researches (14, 15). The presence of 4-benezensulfonamide in the structure of celecoxib, one of the highly selective anti-inflammatory drugs (16), and valdecoxib is also
encouraging to use such moiety in our design for new
anti-inflammatory agents.
H2 NO 2S
H 2NO2 S
N
N
O
CF3
Celecoxib
N
Valdecoxib
473
Mohammed and Nissan
On the other hand, several hydrazone derivatives were
synthesized and evaluated for their anti-inflammatory activity as well as COX-2 selectivity (17–19). It was interesting
also to synthesize a series of pyrazole derivatives as pyrazole moiety is still a target for many researchers in their seek
for new and selective anti-inflammatory agents (20–23).
Based on the aforementioned and in our seek to develop
new anti-inflammatory agents, we have designed and synthesized novel hydrazones and pyrazole derivatives merging
phenylacetamide, sulfamoyl and hydrazone or pyrazole moieties in two series of compounds (A & B) hoping to reach
new agents with better activity and less side-effects. All the
synthesized compounds were subjected for in vivo biological evaluation through rat paw edema, in vitro assay for both
COX-1 and COX-2 inhibitions, evaluating their ulcerogenic
activity and their ability to inhibit PGE2 production in rat
serum samples. Moreover, molecular docking on the active
site of COX-2 was performed to predict their binding mode
to the amino acids of the active site of the enzyme.
H
N
H 2NO2 S
N
H
O
A
N
H
N
Ar
H2 NO2 S
N
N
Ph
O
Ar
B
Experimental
General chemistry
Melting points were determined on Electro thermal Stuart
5MP3 digital melting point apparatus and were uncorrected. Elemental microanalyses were performed at the
micro analytical centre, Al-Azhar University, Cairo, Egypt.
IR spectra were recorded on a Bruker Fourier transform
(FT)-IR spectrophotometer as KBr disks. NMR spectra (in
DMSO-d6) were recorded on Bruker AC-300 Ultra Shield
NMR spectrometer (Bruker, Flawil, Switzerland, d ppm) at
400 MHz using TMS as internal standard and peak multiplicities are designed as follows: s, singlet; d, doublet; t,
triplet; and m, multiplet. Silica gel used for column chromatography was obtained from Fluka, and 70—230 mesh
thin layer chromatography was carried out on silica gel
TLC plates with fluorescence indicator (F254).
2-Hydrazinyl-N-(4-sulfamoylphenyl)acetamide 3
A mixture of the chloro derivative 2 (0.01 mol, 2.48 g) with
hydrazine hydrate (0.01 mol, 0.5 g) was refluxed in absolute ethanol (20 mL) in the presence of catalytic amount of
anhydrous sodium acetate (0.5 g) for 3 h. The precipitate
formed was filtered, dried, washed with hot ethanol, and
crystallized from DMF/H2O to give white powder of 3.
Yield: 80%; mp 208–210 °C; IR (KBr) (cm1): 3358–3332
(2NH2), 3280–3196 (2NH), 3100–3052 (arom.CH), 2924
(aliph.CH), 1685(C=O); 1HNMR(400 MHz, DMSOd6) d
ppm: 3.43(s, 2H, NH2, D2O exchangeable), 4.37(s, 2H,
474
CH2),7.266 (s, 2H, SO2NH2, D2O exchangeable), 7.74–
7.87 (m, 4H, Ar H), 7.89 (s, 1H, NH, D2O exchangeable),
10.39 (s, 1H, NH, D2O exchangeable), 13CNMR
(100.63 MHz, DMSO-d6) d ppm: 64.18(1), 119.52(2),
127.12(2), 138.86(1), 142.22(1), 169.72(1); Anal. Calcd. for
C8H12N4O3S: C, 39.34; H, 4.95; N, 22.94; Found: C,
39.41; H, 4.97; N, 23.01.
General procedure for preparation of compounds
4-10
To a solution of the hydrazino derivative 3 (0.01 mol,
2.44 g) in absolute ethanol (20 mL), the appropriate aromatic aldehyde was added (0.01 mol) and the solution
was refluxed for 3–4 h, cooled and the formed precipitate
was filtered off, dried, and crystallized from ethanol to give
the corresponding hydrazone derivatives 4–10, respectively.
2-(2-Benzylidenehydrazinyl)-N-(4-sulfamoylphenyl)
acetamide 4
Yield: 72%; mp 198–200 °C; IR (KBr) (cm1): 3373–3332
(NH2), 3250–3194 (2NH) 3131–3057 (arom. CH), 2954
(aliph. CH), 1689 (C=O); 1HNMR (400 MHz, DMSO-d6) d
ppm: 4.46 (s, 2H, CH2), 6.93–7.22 (m, 3H, ArH), 7.27 (s,
2H, SO2NH2, D2O exchangeable),7.28–7.32 (m, 2H,
ArH),7.47–7.85(m, 4H, ArH),7.88 (s, 1H, NH, D2O
exchangeable), 8.72 (s, 1H, N=CH), 10.89 (s, 1H, NH,
D2O exchangeable); 13CNMR (100.63 MHz, DMSO-d6) d
ppm: 58.98(1), 119.39(2), 125.91(1), 127.32(2), 128.83(1),
129.00(1), 129.40(1), 131.59(1), 131.85(1), 136.40(1),
139.22(1), 141.95(1), 169.86(1); Anal. Calcd. for
C15H16N4O3S: C, 54.20; H, 4.85; N, 16.86; Found: C,
54.28; H, 4.90; N, 16.94.
2-(2-(2-Fluorobenzylidene)hydrazinyl)-N-(4sulfamoylphenyl)acetamide 5
Yield: 70%; mp 202–204 °C; IR (KBr) (cm1): 3338–3329
(NH2), 3267–3225 (2NH), 3107–3051 (arom. CH), 2924
(aliph. CH), 1701 (C=O); 1HNMR (400 MHz, DMSO-d6) d
ppm: 4.45 (s, 2H, CH2), 7.11–7.13 (m, 2H, ArH) 7.15–7.23
(m, 2H, ArH),7.27 (s, 2H, SO2NH2,D2O exchangeable),
7.51–7.83 (m, 4H, ArH), 7.84 (s, 1H, NH, D2O exchangeable),8.85 (s, 1H, N=CH), 10.83 (s, 1H, NH, D2O
exchangeable); 13CNMR (100.63 MHz, DMSO-d6) d ppm:
58.94(1), 115.81(1), 116.03(1), 119.38(2), 127.31(2),
127.68(1), 127.76(1), 130.49(1), 133.07(1), 139.22(1),
141.94(1), 160.82(1), 169.80(1); Anal. Calcd for
C15H15FN4O3S: C, 51.42; H, 4.32; N, 15.99; Found: C,
51.49; H, 4.30; N, 16.17.
2-(2-(4-Bromobenzylidene)hydrazinyl)-N-(4sulfamoylphenyl)acetamide 6
Yield: 65%; mp 188–190 °C; IR (KBr) (cm1): 3404–3354
(NH2), 3261–3186 (2NH), 3101–3049 (arom. CH), 2924
Chem Biol Drug Des 2014; 84: 473–488
Novel Hydrazones and Pyrazole Derivatives
(aliph. CH), 1689 (C=O); 1HNMR (400 MHz, DMSO-d6) d
ppm: 4.46 (s, 2H, CH2), 7.15–7.23 (m, 2H, ArH),7.27 (s,
2H, SO2NH2, D2O exchangeable), 7.43–7.50 (m, 2H,
ArH),7.78–7.81 (m, 4H, ArH), 7.82 (s, 1H, NH, D2O
exchangeable), 8.70 (s, 1H, N=CH), 10.81 (s, 1H, NH,
D2O exchangeable); 13CNMR (100.63 MHz, DMSO-d6) d
ppm: 58.90(1), 119.381(2), 120.71(1), 127.31(4), 127.75
(1), 130.17(1), 131.92(1), 135.79(1), 139.24(1), 141.92(1),
169.62(1); Anal. Calcd for C15H15BrN4O3S: C, 43.81; H,
3.68; N, 13.62; Found: C, 43.39; H, 3.65; N, 13.71.
2-(2-(4-Chlorobenzylidene)hydrazinyl)-N-(4sulfamoylphenyl)acetamide 7
Yield: 70%; mp 192–194 °C; IR (KBr) (cm1): 3404–3354
(NH2), 3261–3186 (2NH), 3097–3049 (arom CH), 2954 (aliphatic CH), 1689 (C=O); 1HNMR (400 MHz, DMSO-d6) d
ppm: 4.46 (s, 2H, CH2), 7.21 (s, 2H, SO2NH2, D2O exchangeable), 7.34–7.36 (m, 2H, ArH),7.49–7.52 (m, 2H,
ArH), 7.79–7.81(m, 4H, ArH), 7.82 (s, 1H, NH, D2O
exchangeable), 8.74 (s, 1H, N=CH), 10.82 (s, 1H, NH,
D2O exchangeable); 13CNMR (100.63 MHz, DMSO-d6) d
ppm: 58.92(1), 119.39(2), 127.31(4), 127.44(1), 129.02(1),
130.11(1), 132.14(1), 135.44(1), 139.22(1), 141.96(1),
169.65(1); Anal. Calcd for C15H15ClN4O3S: C, 49.11; H,
4.12; N, 15.27; Found: C, 49.22; H, 4.14; N, 15.38.
C15H15FN4O3S: C, 51.42; H, 4.32; N, 15.99; Found: C,
51.58; H, 4.31; N, 16.12.
2-(2-(4-Methoxybenzylidene)hydrazinyl)-N-(4sulfamoylphenyl)acetamide 10
Yield: 60%; mp 178–180 °C; IR (KBr) (cm1): 3442–3352
(NH2), 3263–3205 (2NH), 3105–3049 (arom. CH), 30082954 (aliph. CH), 1693 (C=O); 1HNMR (400 MHz, DMSOd6) d ppm: 3.73 (s, 3H, OCH3), 4.41(s, 2H, CH2) 6.86–
6.89 (m, 2H, ArH), 7.27 (s, 2H, SO2NH2, D2O exchangeable), 7.41–7.44 (m, 2H, ArH), 7.79–7.821(m, 4H, ArH),
7.83 (s, 1H, NH, D2O exchangeable), 8.65 (s, 1H, N=CH),
10.80 (s, 1H, NH, D2O exchangeable); 13CNMR
(100.63 MHz, DMSO-d6) d ppm: 55.58(1), 59.07(1),
116.43(1), 114.49(2), 119.37(2), 127.31(4), 129.13(1),
139.21(1), 141.95(1), 159.44(1), 170.05(1); Anal. Calcd for
C16H18N4O4S: C, 53.03; H, 5.01; N, 15.46; Found: C,
53.17; H, 5.04; N, 15.53.
General procedure for preparation of compounds
11–17
To a solution of the hydrazino derivative 3 (0.01 mol, 2.44 g)
in glacial acetic acid (20 mL), the appropriate aromatic chalcone derivative (0.01 mol) was added and the solution was
refluxed in oil bath for 6–8 h. The formed precipitate was filtered, washed, dried, and crystallized from DMF/H2O to give
the pyrazole derivatives 11-17, respectively.
2-(2-(4-(Dimethylamino)benzylidene)hydrazinyl)-N(4-sulfamoylphenyl)acetamide 8
Yield: 75%; mp 162–164 °C; IR (KBr) (cm1): 3367–3329
(NH2), 3246–3178 (2NH), 3097–3039 (arom. CH), 3005–
2920 (aliph. CH), 1701 (C=O); 1HNMR (400 MHz, DMSOd6) d ppm: 2.88 (s, 6H, 2CH3), 4.37 (s, 2H, CH2),
6.64–6.66 (m, 2H, ArH), 7.27 (s, 2H, SO2NH2, D2O exchangeable), 7.30–7.32 (m, 2H, ArH), 7.79–7.83 (m, 4H,
ArH), 7.85 (s, 1H, NH, D2O exchangeable), 8.50 (s, 1H,
N=CH), 10.80 (s, 1H, NH, D2O exchangeable); 13CNMR
(100.63 MHz, DMSO-d6) d ppm: 40.20(2), 59.24(1),
112.48(1), 119.36(2), 124.37(1), 127.11(1), 127.31(4),
133.14(1), 139.19(1), 141.97(1), 150.52(1), 170.29(1); Anal.
Calcd for C17H21N5O3S: C, 54.38; H, 5.64; N, 18.65;
Found: C, 54.48; H, 5.69; N, 18.79.
2-(3,5-Diphenyl-1H-pyrazol-1-yl)-N-(4sulfamoylphenyl)acetamide 11
Yield: 50%; mp 223–225 dec°C; IR (KBr) (cm1): 3355–3331
(NH2), 3276 (NH), 3103–3085 (arom. CH), 2856 (aliph. CH),
1687 (C=O); 1HNMR (400 MHz, DMSO-d6) d ppm: 4.57 (s,
2H, CH2), 6.62 (s, 1H, C4 pyrazole), 7.21 (s, 2H, SO2NH2,
D2O exchangeable), 7.28–7.30 (m, 4H, ArH), 7.72–7.74 (m,
2H, ArH), 7.81–7.85 (m, 8H, ArH), 10.79(s, 1H, NH, D2O
exchangeable); 13CNMR (100.63 MHz, DMSO-d6) d ppm:
54.60(1), 119.37(2), 119.43(4), 127.00(2), 127.35(4), 138.53
(2), 139.32(2), 141.82(2), 142.34(2), 162.86(1), 168.25(1);
Anal. Calcd for C23H20N4O3S: C, 63.87; H, 4.66; N, 12.95;
Found C, 63.64; H, 5.18; N, 13.11.
2-(2-(4-Fluorobenzylidene)hydrazinyl)-N-(4sulfamoylphenyl)acetamide 9
Yield: 80%; mp 185–187 °C; IR (KBr) (cm1): 3356–3320
(NH2), 3263–3220 (2NH), 3105–3051 (arom. CH), 2954
(aliph. CH), 1687 (C=O); 1HNMR (400 MHz, DMSO-d6) d
ppm: 4.45 (s, 2H, CH2) 7.11–7.23 (m, 2H, ArH), 7.26 (s, 2H,
SO2NH2, D2O exchangeable), 7.51–7.55 (m, 2H, ArH),
7.72–7.82 (m, 4H, ArH), 7.85 (s, 1H, NH, D2O exchangeable), 8.70 (s, 1H, N=CH), 10.81 (s, 1H, NH, D2O exchangeable); 13CNMR (100.63 MHz, DMSO-d6) d ppm: 58.94(1),
116.43(1), 119.38(2), 127.31(4), 127.76(1), 130.49(1),
133.03(1), 139.22(1), 141.94(1), 160.83(1), 169.79(1);
2-(5-(2-Fluorophenyl)-3-phenyl-1H-pyrazol-1-yl)-N(4-sulfamoylphenyl)acetamide 12
Yield: 43%; mp 217–219 °C; IR (KBr) (cm1): 3353–3325
(NH2), 3278 (NH), 3105–3055 (arom. CH), 2954 (aliph.
CH), 1685 (C=O); 1HNMR (400 MHz, DMSO-d6) d ppm:
4.57 (s, 2H, CH2), 6.62 (s, 1H, C4 pyrazole), 7.21 (s, 2H,
SO2NH2, D2O exchangeable),7.28–7.30 (m, 3H, ArH),
7.72–7.74 (m, 2H, ArH), 7.81–7.85 (m, 8H, ArH), 10.77(s,
1H, NH, D2O exchangeable); 13CNMR (100.63 MHz,
DMSO-d6) d ppm: 58.72(1), 119.39(2), 119.45(4), 123.51
(1), 127.02(2), 127.36(4), 138.54(1), 139.32(2), 141.82(2),
142.33(1), 162.87(2), 168.32(1); Anal. Calcd for
Chem Biol Drug Des 2014; 84: 473–488
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Mohammed and Nissan
C23H19FN4O3S: C, 61.32; H, 4.25; N, 12.44; Found: C,
61.17; H, 4.66; N, 12.49.
2-(5-(4-Bromophenyl)-3-phenyl-1H-pyrazol-1-yl)-N(4-sulfamoylphenyl)acetamide 13
Yield: 55%; mp 223–225 °C; IR (KBr) (cm1): 3354–3332
(NH2), 3278 (NH), 3103–3050 (arom. CH), 2955 (aliph.
CH), 1685 (C=O); 1HNMR (400 MHz, DMSO-d6) d ppm:
4.57 (s, 2H, CH2), 6.63 (s, 1H, C4 pyrazole), 7.27 (s, 2H,
SO2NH2, D2O exchangeable),7.28–7.32 (m, 3H, ArH),
7.72–7.88 (m, 10H, ArH), 10.80 (s, 1H, NH, D2O
exchangeable); 13CNMR (100.63 MHz, DMSO-d6) d ppm:
54.58(1), 119.23(2), 119.27(2), 119.44(4), 126.96(1),
127.24(4), 127.35(2), 138.53(2), 142.11(2), 163.50(2),
169.67(1); Anal. Calcd for C23H19BrN4O3S: C, 54.02; H,
3.74; N, 10.96; Found: C, 53.93; H, 4.17; N, 11.05.
2-(5-(4-Chlorophenyl)-3-phenyl-1H-pyrazol-1-yl)-N(4-sulfamoylphenyl)acetamide 14
Yield: 80%; mp 208–210 °C; IR (KBr) (cm1): 3356–3332
(NH2), 3280 (NH), 3104–3053 (arom. CH), 2956 (aliph.
CH), 1684 (C=O); 1HNMR (400 MHz, DMSO-d6) d ppm:
4.58 (s, 2H, CH2), 6.62 (s, 1H, C4 pyrazole), 7.22 (s, 2H,
SO2NH2, D2O exchangeable), 7.25–7.32 (m, 3H, ArH),
7.72–7.86 (m, 10H, ArH), 10.82 (s, 1H, NH, D2O
exchangeable); 13CNMR (100.63 MHz, DMSO-d6) d ppm:
59.65(1), 119.38(2), 119.44(4), 123.49(1), 127.01(2),
127.35(4), 138.53(1), 139.31(2), 141.84(2), 142.34(1),
162.87(2), 168.31(1); Anal. Calcd for C23H19ClN4O3S: C,
59.16; H, 4.10; N, 12.00; Found: C, 58.98; H, 4.49; N,
12.03.
2-(5-(4-(Dimethylamino)phenyl)-3-phenyl-1Hpyrazol-1-yl)-N-(4-sulfamoylphenyl)acetamide 15
Yield: 58%; mp 168–170 °C; IR (KBr) (cm1): 3356–3334
(NH2), 3273 (NH), 3103–3059 (arom. CH), 2924 (aliph.
CH), 1697 (C=O); 1HNMR (400 MHz, DMSO-d6) d ppm:
2.88 (s, 6H, 2CH3), 4.37 (s, 2H, CH2), 6.64 (s, 1H, C4 pyrazole), 7.22 (s, 2H, SO2NH2, D2O exchangeable),7.27–
7.32 (m, 3H, ArH), 7.68–7.89 (m, 10H, ArH), 10.80 (s, 1H,
NH, D2O exchangeable); 13CNMR (100.63 MHz, DMSOd6) d ppm: 40.40(2), 59.25(1), 111.54(1), 112.15(1), 112.48
(1), 119.37(1), 122.00(4), 124.36(1), 126.96(1), 127.12(1),
127.24(4), 129.96(1), 133.14(1), 139.06(1), 142.05(1),
150.52(1), 163.51(1), 168.29(1); Anal. Calcd for
C25H25N5O3S: C, 63.14; H, 5.30; N, 14.73; Found: C,
62.96; H, 5.74; N, 14.72.
2-(5-(4-Fluorophenyl)-3-phenyl-1H-pyrazol-1-yl)-N(4-sulfamoylphenyl)acetamide 16
Yield: 55%; mp 224–226 °C; IR (KBr) (cm1): 3357–3332
(NH2), 3280 (NH), 3107–3054 (arom. CH), 2950 (aliph. CH),
1687 (C=O); 1HNMR (400 MHz, DMSO-d6) d ppm: 4.57 (s,
2H, CH2), 6.63 (s, 1H, C4 pyrazole), 7.22 (s, 2H, SO2NH2,
476
D2O exchangeable), 7.26–7.31 (m, 3H, ArH), 7.75–7.82 (m,
10H, ArH), 10.79 (s, 1H, NH, D2O exchangeable); 13CNMR
(100.63 MHz, DMSO-d6) d ppm: 58.77(1), 119.39(2),
119.45(3), 123.51(1), 127.01(2), 127.36(4), 138.54(2),
139.33(2), 141.82(2), 142.33(1),162.87(2), 168.32(1); Anal.
Calcd for C23H19FN4O3S: C, 61.32; H, 4.25; N, 12.44;
Found: C, 61.13; H, 4.75; N, 12.39.
2-(5-(4-Methoxyphenyl)-3-phenyl-1H-pyrazol-1-yl)N-(4-sulfamoylphenyl)acetamide 17
Yield: 52%; mp 225–227 °C; IR (KBr) (cm1): 3358–3332
(NH2), 3278 (NH), 3099–3055 (arom. CH), 2954 (aliph.
CH), 1685 (C=O); 1HNMR (400 MHz, DMSO-d6) d ppm:
3.60 (s, 3H, OCH3), 4.57(s, 2H, CH2), 6.92 (s, 1H, C4 pyrazole), 7.21 (s, 2H, SO2NH2, D2O exchangeable), 7.23–
7.26 (m, 3H, ArH), 7.71–7.88 (m, 10H, ArH), 10.29 (s, 1H,
NH, D2O exchangeable); 13CNMR (100.63 MHz, DMSOd6) d ppm: 59.25(1), 64.20(1), 119.22(2), 119.49(4), 127.09
(2), 127.15(4), 127.24(2), 138.60(1), 138.92(2), 142.10(2),
142.30(1), 162.50(1), 169.67(1); Anal. Calcd for
C24H22N4O4S: C, 62.32; H, 4.79; N, 12.11; Found C,
62.17; H, 5.27; N, 12.15.
Molecular docking
All the molecular modeling studies were carried out on an
Intel Pentium 1.6 GHz processor, 512 MB memory with
Windows XP operating system using Molecular Operating
Environment (MOE, 10.2008) software. All the minimizations were performed with MOE until a RMSD gradient of
0.05 kcal/mol Ao1 with MMFF94X force field and the partial charges were automatically calculated. The X-ray crystallographic structure of COX-2 enzyme with its cocrystallized ligand (Diclofenac) in the file (PDB ID: 1PXX)
was obtained from the Protein Data Bank. The enzyme
was prepared for docking studies where: (i) Ligand molecule was removed from the enzyme active site; (ii) hydrogen atoms were added to the structure with their standard
geometry; (iii) MOE Alpha Site Finder was used for the
active sites search in the enzyme structure, and dummy
atoms were created from the obtained alpha spheres; (iv)
the obtained model was then used in predicting the
ligand–enzyme interactions at the active site.
Biological screening
Carrageenan-induced rat paw edema assay
Male Wistar rats, each weighing 120–180 g, were purchased from the animal breeding unit of the National Ophthalmology Institute, Egypt. They were housed under
appropriate conditions of controlled humidity, temperature,
and light. The animals were allowed free access to water
and were fed a standard pellet rat diet. The animals were
kept at an ambient temperature of 22 °C 2 °C and a
humidity of 65–70%. The study was conducted according
to the guidelines for animal experiments set by Faculty of
Chem Biol Drug Des 2014; 84: 473–488
Novel Hydrazones and Pyrazole Derivatives
Pharmacy, Cairo University, in accordance with the international guidelines.
A 1% suspension of carrageenan was prepared by sprinkling 1 g carrageenan powder in small amounts over the
surface of 100 mL saline (NaCl 0.9% w/v), and the particles allowed to soak between additions. The suspension
was then left at 37 °C for 2–3 h before use. Right paw is
marked with ink at the level of lateral malleolus; basal paw
volume is measured plethysmographically by volume displacement method using plethysmometer (UGO Basile
7140) by immersing the paw till the level of lateral malleolus. 0.05 mL of this suspension was injected into the subplantar surface of the right hind paw of the rat. The paw
volume is measured again at 1, 2, 3, & 4 h after challenge.
The increase in paw volume is calculated as percentage
compared with the basal volume. The animals were randomized and divided into groups consists of six animals
per group. The tested compounds and the reference drug
(diclofenac) were given orally (5 mg/kg) 30 min prior to the
intraplatar injection of carrageenan. The difference of average values between treated animals and control group is
calculated for each time interval and evaluated statistically.
The percent inhibition is calculated using the formula as
follows:
%edema inhibition ¼ ½1 ðVt=VcÞ 100
Vt and Vc are edema volume in the drug-treated and control groups, respectively (24).
PGE2 inhibition in rat serum samples
All synthesized compounds as well as diclofenac as the
reference drug were given orally to male albino rats, and
blood samples were taken after 2 h of administration.
Serum samples were prepared by centrifugation where
PGE2 concentrations were estimated using rat-specific
immunoassay kit (25) using diclofenac as the reference
drug. The assay was performed following the instructions
in the leaflet of the kit (26).
In vitro COX-1 and COX-2 inhibition assay
COX-1 and COX-2 inhibitory activity was determined using
COX (ovine) inhibitor screening assay enzyme-immuno
assay (EIA) kit according to manufacturer’s instructions
(27). COX catalyzes the first step in the biosynthesis of
arachidonic acid to PGH2. The PGF2a produced from
PGH2 by reduction with stannous chloride is measured by
EIA. The compounds were dissolved in dimethylsulfoxide
(DMSO). The enzymes COX-1 and COX-2 (10 lL), heme
(10 lL), and samples (20 lL) were added to the supplied
reaction buffer solution (950 lL, 0.1 M Tris–HCl, pH 8 containing 5 mM ethylenediaminetetraacetate (EDTA), and
2 mM phenol). The mixture of these solutions were incubated for a period of 10 min at 37 °C, after that COX
reactions were initiated by adding arachidonic acid (10 lL,
Chem Biol Drug Des 2014; 84: 473–488
making final concentration 100 lM) solution. The COX
reactions were stopped by addition of HCl (1 M, 50 lL)
after 2 min, and then saturated stannous chloride (100 lL)
was added and again incubated for 5 min at room temperature. The PGF2a formed in the samples by COX reactions was quantified by EIA. The precoated 96-well plate
was incubated with samples for 18 h at room temperature.
After incubation, the plate was washed to remove any
unbound reagent, and then Ellman’s reagent (200 lL),
which contains substrate to acetylcholinesterase, was
added and incubated for 60–90 min (until the absorbance
of Bo well is in the range 0.3–0.8 A.U.) at room temperature. The plate was then read by an ELISA plate reader at
410 nm. The IC50 of inhibition of COX-1 and COX-2 was
calculated by the comparison of the sample-treated incubations with control incubations. Celecoxib was used as
reference standard in the study.
Ulcerogenic effect
Male albino rats (100–120 g) were fasted for 12 h prior to
the administration of the compounds. The animals were
divided into groups, each of six animals. The control
group received 1% Tween-80 orally. Other groups
received indomethacin or the tested compounds orally in
two equal doses at 0 and 12 h for three successive days
at a dose of 20 mg/kg per day. Animals were killed by
diethyl ether 6 h after the last dose, and the stomach
was removed. An opening at the greater curvature was
made, and the stomach was cleaned by washing with
cold saline and inspected with a 3 A magnifying lens for
any evidence of hyperemia, hemorrhage, definite hemorrhagic erosion, or ulcer. An arbitrary scale was used to
calculate the ulcer index which indicates the severity of
the stomach lesions. Ulcers were classified into levels:
level I, in which the ulcer area is <1 mm2; level II, in which
the ulcer area is in the range from 1 to 3 mm2; and level
III, in which the ulcer area equals 3 mm2. The ulcer index
was calculated as 1 9 (number of ulcers of level
I) + 2 9 (number of ulcers of level II) + 3 9 (number of
ulcers of level III) (28).
Results and discussion
Chemistry
Sulfanilamide 1 (CAS No. 63-74-1) was reacted with choloroacetylchloride in DMF in the presence of catalytic
amounts of anhydrous sodium acetate to afford 2-chloroN-(4-sulfamoylphenyl)acetamide 2. Structure of compound
2 was verified by its reported melting point = 217 °C (29).
Compound 2 was then reacted with hydrazine hydrate in
absolute ethanol in the presence of catalytic amounts of
anhydrous sodium acetate to obtain 2-hydrazinyl-N-(4-sulfamoylphenyl)acetamide 3. The structure of compound 3
was confirmed by elemental and spectral analyses. IR of
compound 3 revealed bands at 3358 and 3280 cm1
corresponding to NH–NH2 groups and 1685 cm1 for
477
Mohammed and Nissan
C=O. 1H-NMR spectrum in (DMSO-d6) of 3 exhibited a
singlet signal at 3.43 ppm exchangeable by D2O corresponding to NH2 and another singlet at 7.89 ppm
exchangeable by D2O corresponding to NH group. Condensation of compound 3 with different aromatic aldehydes in absolute ethanol led to the formation of the
corresponding hydrazone derivatives 4–10. The structures
of compounds 4–10 were confirmed by spectral and elemental analyses. 1H-NMR spectra in (DMSO-d6) of compounds 4–10 revealed singlet signals in the range of
8.50–8.74 ppm corresponding to N=CH proton, which is
the result of the condensation reactions. The 1H-NMR
spectra of compounds 4–10 were devoid of the singlet
signals at 3.43 for the reacted NH2, and instead singlet
signals appeared in the range of 7.82–7.88 ppm which
was exchangeable by D2O for NH groups. Compounds
11–17 were synthesized through the reaction between the
hydrazinyl derivative 3 with different aromatic chalcones in
glacial acetic acid. The aromatic pyrazole derivatives were
obtained rather than the pyrazoline derivatives as the
reaction was conducted in glacial acetic acid not in absolute ethanol (30). The structures of compounds 11–17
were confirmed by elemental and spectral analyses. 1HNMR spectra in (DMSO-d6) of compounds 11–17
revealed singlet signals in the range of 6.62–6.92 ppm
corresponding to CH at C4 of pyrazole ring. The spectra
also lack the signals which correspond to NH–NH2
groups. There was no doublet or triplet signals in 1HNMR spectra in (DMSO-d6) of compounds 11–17, confirming the formation of aromatic pyrazole cyclization
rather than pyrazoline formation (Scheme 1).
Molecular docking
In 2003, it was discovered that a series of anti-inflammatory drugs inhibits PGE2 synthesis by interacting with
Tyr 385 and Ser 530 in the active site of COX-2
enzyme and compounds that could interact with such
amino acids can exhibit anti-inflammatory activity (31).
Interactions with Arg 120 may play also a role in the
inhibitory activity (31). Tyr-385 is essential for the cyclooxygenase activity of PGH synthase and that nitration of
this residue can be prevented by indomethacin. We
conclude that Tyr 385 is at or near the cyclooxygenase
active site of PGH synthase and the tyrosine residue
could be involved in the first step of the cyclooxygenase
reaction, which is the abstraction of the 13-proS hydrogen from arachidonate (32). Acetylation of Ser 530 of
sheep prostaglandin endoperoxide (PGG/H) synthase by
aspirin causes irreversible inactivation of cyclooxygenase
enzyme. Thus, Ser 530 does lie in a highly conserved
region, probably involved in cyclooxygenase catalysis
(33). Arg 120 is located near the mouth of the hydrophobic channel that forms the cyclooxygenase active
site of prostaglandin endoperoxide H synthases
(PGHSs)-1 and -2. Arg 120 forms an ionic bond with
the carboxylate group of arachidonate, and this interaction is an important contributor to the overall strength of
478
arachidonate binding to PGHS-1(34). The structure of
arachidonic acid (substrate) co-crystallized with the active
site of COX-2 is displayed in (Figures 1 and 2).
In our attempts to predict the mechanism of action of the
synthesized compounds as well as to evaluate their binding mode to the amino acids of the active site of COX-2
enzyme, molecular docking of all of the synthesized compounds was performed on the active site of COX-2
enzyme. The Protein Data Bank file with the code 1PXX
was used for this purpose. The file contains COX-2
enzyme co-crystallized with diclofenac. All docking procedures were achieved by MOE (Molecular Operating Environment) software 10.2008 provided by chemical
computing group, Canada.
To perform accurate validation of the docking protocol,
docking of the co-crystallized ligand (Diclofenac) should be
carried out to study the scoring energy (S), root mean
standard deviation (rmsd), and amino acid interactions.
Docking was performed using London dG force, and
refinement of the results was carried out using force-field
energy. Diclofenac is fitted in the active site pocket with
S = 11.9408 Kcal/mol and rsmd= 0.3682. Diclofenac
interacts with the two amino acids Tyr 385 and Ser 530
by three hydrogen bonds, the bond length of the two
hydrogen bonds with Ser 530 was 2.7 Ao and 2.9 A while
that with Tyr 385 was 2.7 Ao as displayed in (Figures 3
and 4).
Following the aforementioned docking protocol, all the
synthesized compounds were docked on the active site of
COX-2 enzyme. All the synthesized compounds were fit in
the active site of the enzyme. Docking scores, amino acid
interactions as well as hydrogen bond lengths were summarized in Table 1.
All compounds were fit on the active site of COX-2
enzyme with an energy score ranging between 11.8629
and 13.6435 kcal/mol comparable with that of diclofenac. All of the synthesized compounds interacted with Ser
530 and/or Tyr 385 with one or more hydrogen bonds
except for compounds 6, 7, and 11. The sulfonamide
group which represents an acidic moiety for interaction
was the most interacting group in all compounds except
for compounds 6, 7, and 11. Compounds 9 and 17
showed the best docking score values with 13.4546 and
13.6435 Kcal/mol, respectively, while compound 11
showed the worst docking score value with 11.8629
Kcal/mol.
Regarding the amino acid interactions between the synthesized compounds and the amino acids of the active
site of COX-2 enzyme, the sulfonamide moiety interacted
with Tyr 385 by one hydrogen bond in the case of
compounds 3, 4, 8, 9, 10, 12, 13, and 16, while compounds 5, 14, 15, and 17 interacted with the same
amino acid by two hydrogen bonds. Studying the interChem Biol Drug Des 2014; 84: 473–488
Novel Hydrazones and Pyrazole Derivatives
Scheme 1: Reagents and solvents (a) anhydrous sodium acetate, absolute ethanol. (b) anhydrous sodium acetate, hydrazine hydrate,
absolute ethanol. (c) absolute ethanol. (d) glacial acetic acid.
action of the synthesized compounds with Ser 530, it
was observed that compounds 4, 8, 10, 12, 15, 16,
and 17 interacted with Ser 530 by one hydrogen bond
while compounds 5 and 9 interacted with Ser 530 by
two hydrogen bonds. Arg 120 interactions were
observed in case of compounds 3 with two hydrogen
bonds, compounds 6 and 7 with one hydrogen bond.
Compound 11 was the only compound to interact with Tyr
355 with one hydrogen bond. Finally, an additional hydrogen bond was observed in case of compound 17 between
the p-methoxy group and Tyr 115.
Based on the pattern of diclofenac interactions with the
amino acids of the active site of COX-2, good to moderate
anti-inflammatory activity could be observed with compounds 3–17 except for compounds 6, 7, and 11. This
conclusion could be postulated on the fact that the sulfonamide group which is the acidic moiety replacing carboxylChem Biol Drug Des 2014; 84: 473–488
ate in diclofenac should interact with the amino acids Tyr
385 and Ser 530, and as this is achieved in all compounds except 6, 7, and 11, weak or no anti-inflammatory
activity could be observed for these compounds. On the
other hand, good anti-inflammatory activity could be
observed with compounds 5 and 17 as they interact with
the amino acids of the active site of COX-2 enzyme with
four and five hydrogen bonds, respectively. The 2D and
3D interaction descriptions of compounds 5 and 17 with
the active site amino acids of COX-2 enzyme are displayed in (Figures 5–8), respectively.
Biological screening
In vivo anti-inflammatory activity
All synthesized compounds 3–17 were evaluated for their
in vivo anti-inflammatory activity by carrageenan-induced
rat paw edema method (24). The protocol of animal exper479
Mohammed and Nissan
involves prostaglandins as the mediators for the inflammation, any anti-inflammatory activity in the second phase of
this biphasic event can be attributed to the inhibition of
prostaglandin synthesis (24).
All the synthesized compounds showed good to moderate
anti-inflammatory activity especially after 4 h of administration except compounds 6, 7, and 11 with edema inhibition
percentage values of 31%, 44%, and 25%, respectively.
Compounds 5, 10, and 16 showed comparable activity with
that of diclofenac with edema inhibition percentage values
of 60%, 60%, and 61%, respectively. Compounds 12, 13,
and 14 were slightly less active than diclofenac with edema
inhibition percentage values of 57%, 50%, and 57%,
respectively. On the other hand, compounds 3, 4, 8, 9, 15,
and 17 were more active than diclofenac with edema inhibition percentage values of 68%, 71%, 67%, 84%, 68%, and
81%, respectively.
Figure 1: Arachidonic acid 2D interaction description with active
site of COX-2.
iments was approved by ethical committee. Each test
compound was dosed orally (5 mg/kg body weight)
30 min prior to induction of inflammation by carrageenan
injection. Diclofenac was used as a reference anti-inflammatory drug, and anti-inflammatory activity was calculated
at hourly intervals up to 4 h after injection and presented
in Table 2 as the mean paw volume (mL) SE as well as
the percentage edema inhibition. Considering the fact that
carrageenan-induced paw edema assay is a biphasic
event (25) involving the release of histamine and serotonin
as the mediators of inflammation in the first phase which
normally lasts for about 2 h after the carrageenan injection
followed by the second phase which generally operates
between 2 and 4 h after the carrageenan injection and
It was obvious from the above results that the adapted
design for the synthesis of novel anti-inflammatory agents
has succeeded in the light of the biological results for the
in vivo evaluation of anti-inflammatory activity for the synthesized compounds.
The starting hydrazinyl derivative 3 showed better antiinflammatory activity than that of diclofenac with rising
edema inhibition percentage value from 62% for diclofenac to 68% for the hydrazino derivative 3. Upon condensation with different aromatic aldehydes, the formed
hydrazone derivatives 4–10 gave also active compounds,
especially for the unsubstituted derivative 4 with edema
inhibition percentage value 71%, p-N,N-dimethyl derivative 8 with edema inhibition percentage value of 67%,
and the p-flouro derivative 9 with edema inhibition percentage value of 84% which was the best activity
observed in this series. However, less active compounds
Figure 2: Arachidonic acid 3D
interaction description with active
site of COX-2.
480
Chem Biol Drug Des 2014; 84: 473–488
Novel Hydrazones and Pyrazole Derivatives
Figure 3: Diclofenac 2D
interaction description with active
site of COX-2.
Figure 4: Diclofenac 2D
interaction description with active
site of COX-2.
were achieved in case of p-bromo derivative 6 and pchloro derivative 7 with edema inhibition percentage values of 31% and 44%, respectively, while the o-flouro
derivative 5 and the p-methoxy derivative 10 showed
comparable activity with that of diclofenac with edema
inhibition percentage value of 60%.
The order of anti-inflammatory activity for hydrazone derivatives with its starting hydrazino derivative 3 could be
summarized in Table 3.
Chem Biol Drug Des 2014; 84: 473–488
The second series in this research was the pyrazole derivatives 11–17. In this series, the observed anti-inflammatory
activities were good to moderate activities except for the unsubstituted derivative 11 with edema inhibition percentage
value of 25%. Moderate activity was observed for o-flouro
derivative 12, p-bromo derivative 13, and the p-chloro derivative 14 with edema inhibition percentage values of 57%,
50%, and 57%, respectively. The p-flouro derivative 16
showed comparable activity with that of diclofenc with
edema inhibition percentage value of 61%, while the p-N,N481
Mohammed and Nissan
Table 1: Binding scores and amino acid interactions of the
docked compounds on the active site of COX-2 enzyme
Compound
No.
S
Kcal/Mol
Amino acid
interactions
Interacting
groups
H bond
length Ao
3
12.5870
4
12.7512
5
12.1160
6
7
8
11.9050
12.0361
13.2910
9
13.4546
10
12.5816
11
12
11.8629
12.5105
13
14
15
11.9204
12.0730
12.8598
16
12.2798
17
13.6435
Tyr 385
Arg 120
Tyr 385
Ser 530
Tyr 385
Ser 530
Arg 120
Arg 120
Tyr 385
Ser 530
Tyr 385
Ser 530
Tyr 385
Ser 530
Tyr 355
Tyr 385
Ser 530
Tyr 385
Tyr 385
Tyr 385
Ser 530
Tyr 385
Ser 530
Tyr 385
Ser 530
Tyr 115
SO2NH2
NH, NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
C=NH
C=NH
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
C=O
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
OCH3
2.95
2.92,
2.67
2.98
2.65,
1.48,
2.88
2.75
2.39
1.50
2.28
2.10,
2.47
1.51
3.00
1.96
2.34
1.90
2.36,
2.78,
3.12
2.88
1.79
2.38,
2.23
2.82
3.02
2.72
2.98
2.35
2.74
2.29
2.98
dimethyl derivative 15 and p-methoxy derivative 17 showed
better activity than diclofenac with edema inhibition percentage values of 68% and 81%, respectively.
The order of anti-inflammatory activity for the pyrazole
derivatives could be summarized in Table 4.
Comparing the anti-inflammatory activity of hydrazone
derivatives 4–10 and their pyrazole analogs 11–17, it was
obvious that the anti-inflammatory activity of the p-bromo
pyrazole derivative 13 and p-chloro pyrazole derivative 14
was much better than that of their hydrazone derivative
analogs 6 and 7 as the edema inhibition percentage values reach 57% and 50% for 13 and 14, respectively,
instead of 31% and 44% for 6 and 7, respectively.
In case of p-methoxy pyrazole derivative 17, the
anti-inflammatory was remarkably increased with edema inhibition percentage of 81% which was better than that of diclofenac instead of 60% for its hydrazone derivative analog 10.
However, this was not the case for the rest of pyrazole
derivatives compared with their hydrazone derivative analogs as the activity decreased especially for the unsubstituted pyrazole derivative 11 with edema inhibition
percentage value of 25% instead of 71% for its hydrazone
derivative analog 4.
482
Evaluation of PGE2 inhibition in rat serum samples
After in vivo evaluation of anti-inflammatory activity, it
was interesting to measure the inhibition of PGE2 production that could contribute to the inhibition of COX-1
and COX-2 enzymes (25). The assay employs the quantitative sandwich enzyme immunoassay technique. A
monoclonal antibody (specific for rat PGE2) has been
precoated into a micro plate. Standards, controls, and
samples are pipetted into the wells, and any rat PGE2
present in the solutions is bound by the immobilized antibody. After washing away any unbound substances, an
enzyme-linked monoclonal antibody specific for PGE2
[PGE2 conjugate] is added to the wells. Following a wash
to remove any unbound antibody-enzyme reagent, a
substrate solution is added to the wells. The enzyme
reaction yields a blue product that turns yellow when the
stop solution is added. The intensity of the color measured is in proportion to the amount of rat PGE2 bound
in the initial step. The sample values are then read off
the standard curve.
All synthesized compounds 3–17 were given orally to male
albino rats, and blood samples were taken after 2 h of
administration. Serum samples were prepared by centrifugation where PGE2 concentrations were estimated using
rat-specific immunoassay kit (26) using diclofenac as the
reference drug. Results of PGE2 concentrations SE as
well as percentage of inhibition are listed in Table 5.
Compounds 9 and 17 showed the highest inhibition percentage of PGE2 production in serum samples with inhibition percentage values of 78.12% and 76.49%,
respectively. These compounds were also the most active
compounds in rat paw edema evaluation with edema inhibition percentage values of 84% and 81%, respectively.
On the other hand, the least active compounds in rate
paw edema test such as compounds 6, 7, and 11
showed low percentage inhibition of PGE2 production with
values of 16.12%, 37.22%, and 17.85%, respectively.
From these results, we can assume that COX enzyme inhibition could be the mechanism of action for the antiinflammatory activity of the newly synthesized compounds.
In vitro COX-1 and COX-2 inhibition assay
To study the mechanism of action of the newly synthesized
compounds and whether their anti-inflammatory effect is
due to COX enzyme inhibition, in vitro assay for COX-1 and
COX-2 enzymes inhibition was conducted. The most active
compounds were screened using EIA kit (Cayman Chemical Company, Ann Arbor, MI, USA). The COX-1 and
COX-2 inhibitory activities of compounds were evaluated
by method as described by Gautam et al. (35). IC50 values
for the most active compounds were determined using
celecoxib as the reference drug. Furthermore, selectivity
index for COX-1/COX-2 inhibition was calculated for each
compound. IC50 values for COX-1 and COX-2 inhibitions
as well as selectivity index were listed in Table 6.
Chem Biol Drug Des 2014; 84: 473–488
Novel Hydrazones and Pyrazole Derivatives
Figure 5: Compound 5 2D
interaction description with active
site of COX-2.
Figure 6: Compound 5 3D
interaction description with active
site of COX-2.
Chem Biol Drug Des 2014; 84: 473–488
483
Mohammed and Nissan
Figure 7: Compound 17 2D
interaction description with active
site of COX-2.
Figure 8: Compound 17 3D
interaction description with active
site of COX-2.
All the tested compounds showed inhibitory effect on both
COX-1 and COX-2 enzymes with IC50 values in the range
of 13.26–47.96 lM for COX-1 and in the range of 1.73–
11.74 lM with selectivity index (SI) ranging between 4.0
and 11.1.
By studying the results in Table 6, we can notice that the
selectivity index (SI) for the hydrazone derivatives 5, 8, 9,
and 10 were 4.9, 8.7, 7.6, and 7.0, respectively. These val484
ues have improved with their pyrazole analogs 12, 15, 16,
and 17 to rise to 6.5, 8.8, 9.4, and 11.1, respectively.
The selectivity index of the active synthesized compounds
cannot be compared with that of the reference drug (celecoxib), but although high selectivity is demanded to
reduce the gastrointestinal side-effects, it was discussed
recently that COX-2 high selectivity could be the major
cause of cardiac and renal problems (5, 6) and hence
Chem Biol Drug Des 2014; 84: 473–488
Novel Hydrazones and Pyrazole Derivatives
Table 2: Rat paw edema volume and percentage of inhibition
Volume of edema (mL)
1h
2h
Compound
(Edema inhibition %)
Control
Diclofenac
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
35.26
35.03
30.19
9.653*,**
28.91
31.22
31.07
22.1*,**
19.07*,**
18.86*,**
41.33
39.3
26.19
17.02*,**
24.49*,**
20.5*,**
9.925*,**
2.707
1.809 (1)
3.141 (14)
3.337 (72)
2.168 (18)
2.63 (11)
2.671 (12)
1.262 (37)
2.019 (45)
1.041 (46)
1.168 (0)
3.181 (0)
3.395 (25)
2.3 (51)
2.533 (30)
1.477 (41)
2.574 (71)
3h
66.3
53.29*
36.37*,**
14.48*,**
44.6*
49.45*
37.73*,**
37.55*,**
37.21***
25*,**
59.2
40.84*,**
32.63*,**
21.7*,**
23.71*,**
24.4*,**
14.99*,**
2.357
1.835 (20)
2.476 (45)
3.192 (78)
4.377 (33)
3.667 (25)
1.695 (43)
1.826 (43)
3.446 (44)
1.587 (62)
1.514 (11)
1.89 (38)
3.106 (50)
2.356 (67)
1.522 (64)
1.022 (63)
2.084 (77)
4h
83.51
50.21*
44.1*
32.57*,**
47.97*
54.6*
59.57*
36.61*,**
26.52*,**
44.88*
58.28*
45.35*
53.63*
26.78*,**
28.68*,**
44.14*
19.48*,**
Values were expressed as mean SEM of six rats. Statistical analysis was performed by
*Significantly different from control at p < 0.05.
**Significantly different from diclofenac at p < 0.05.
ANOVA
2.523
3.206 (40)
2.389 (47)
3.524 (60)
4.824 (43)
3.423 (35)
1.483 (29)
1.814 (56)
5.994 (68)
2.219 (46)
3.589 (30)
2.41 (45)
3.049 (36)
2.509 (67)
2.235 (65)
1.111 (47)
1.519 (76)
91.37
34.28*
29.24*
25.95*
36.96*
62.82*,**
51.12*,**
30.25*
14.33***
37.16*
68.65*,**
39.55*
45.52*,**
39.63*
28.42*
34.97*
16.75*,**
3.092
1.617 (62)
3.21 (68)
2.967 (71)
3.325 (60)
4.507 (31)
1.701 (44)
1.6 (67)
1.948 (84)
1.665 (60)
1.115 (25)
2.759 (57)
3.255 (50)
2.474 (57)
2.519 (68)
2.065 (61)
2.82 (81)
followed by Tukey’s post hoc test.
Table 3: The anti-inflammatory activity of compounds 3–10 in descending order
Compound
No.
H
N
N
H
O
H 2NO2 S
N
H
H 2NO2 S
O
N
H
N
moderate selectivity could reduce the risk of both gastrointestinal and cardiac side-effects (7).
Ulcerogenic effect
The most active compounds 3-5, 8, 9, and 12–17 were
evaluated for their ulcerogenic effects in rats, and indoChem Biol Drug Des 2014; 84: 473–488
4-F
84
4
H
71
3
R
H
N
9
NH 2
O
H2 NO 2S
Inhibition %
R
N
H
N
R
8
5
10
7
6
68
4-N(CH3)2
2-F
4-OCH3
4-Cl
4-Br
67
60
60
44
31
methacin was used as the reference standard. The results
were expressed by the number of ulcers SE as well as
ulcer index SE (28) as shown in Table 7. All of the tested
compounds were less ulcerative than indomethacin. Compounds 4, 5, 10, 12, and 14 were ulcerative in a comparable manner with indomethacin, while compounds 3, 8, 9,
13, 14, 15, 16, and 17 were much less ulcerative than
485
Mohammed and Nissan
Table 4: The anti-inflammatory activity of compounds 11–17 in descending order
Compound
H
N
H 2NO2 S
N
N
Ph
O
No.
R
Inhibition %
17
15
16
12
14
13
11
4-OCH3
4-N(CH3)2
4-F
2-F
4-Cl
4-Br
H
81
68
61
57
57
50
25
R
Table 5: Concentration and inhibition percentage of PGE2 in
serum samples
Group
PGE2 (Pg/mL)
Normal control (1% Tween-80)
Carrageenan (1%Tween-80)
Diclofenac
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
1.98
49.81*,**
11.8*,**
22.31*,**
23.91*,**
30.21*,**
41.78*,**
31.27*,**
25.78*,**
10.9*,**
28.97*,**
40.92*
30.21*,**
29.35*,**
31.75*,**
21.32*,**
24.19*,**
11.71*,**
0.50
2.58
1.81
2.97
2.98
3.51
3.51
3.09
3.18
1.93
3.21
4.15
3.25
2.99
3.02
2.27
1.99
1.18
IC50 (lM)
Inhibition (%)
76.31
55.21
52.00
39.35
16.12
37.22
48.24
78.12
41.84
17.85
39.35
41.08
36.26
57.20
51.44
76.49
Values were expressed as mean SEM of six rats. Statistical
analysis was performed by ANOVA followed by Tukey’s post hoc
test.
*Significantly different from normal control at p < 0.05.
**Significantly different from carrageenan at p < 0.05.
indomethacin. These less ulcerative compounds showed
good selectivity index (SI) of COX -1/COX-2 inhibition with
values of 7.4, 8.7, 7.6, 8.3, 7.0, 8.8, 9.4, and 11.1,
respectively.
Comparing the ulcerative effect of the tested hydrazone
derivative compounds 5, 8–10 with the ulcerative effect of
their pyrazole analogs 12, 15–17 may led to the conclusion that cyclization to pyrazole did not much decrease
the ulcerative effect except for compound 10 with an ulcer
index value of 12.4 which has markedly decrease in case
of its pyrazole analog 17 as the ulcer index was
decreased to 2.79.
486
Table 6: COX-1 and COX-2 inhibition IC50 and selectivity index
(SI)
Compound
COX-1
COX-2
Selectivity
index
3
4
5
8
9
10
12
13
14
15
16
17
Celecoxib
15.89
24.06
28.34
19.91
13.26
18.70
24.01
20.59
47.69
14.86
21.09
19.61
>30
2.12
3.51
5.68
2.26
1.73
2.65
3.64
2.46
11.74
1.68
2.22
1.76
0.22
7.4
6.8
4.9
8.7
7.6
7.0
6.5
8.3
4.0
8.8
9.4
11.1
>200
Conclusion
In the light of biological results, we can conclude that the
hydrazino derivative 3 synthesized hydrazone derivatives
4–10 with the exception of 5 & 6 and the synthesized
pyrazole derivatives 12–17 could represent good candidates for the search for novel anti-inflammatory agents
urged by their good anti-inflammatory activity in vivo and
in vitro assays and the low ulcerative effect of most of
the synthesized compounds. Compound 17 has edema
inhibition percentage value of 81% better than that of
diclofenac, percentage of inhibition of PGE2 production
value of 76.49% with slight increase than that of diclofenac, the best selectivity index among the synthesized
compounds with a value of 11.1 and much less ulcerative effect with ulcer index value of 2.79 compared with
12.82 for indomethacin. Molecular docking study for
compound 17 showed good energy score and interactions with Tyr 385 and Ser 530 by three hydrogen bonds
and additional hydrogen bond with Tyr 115 with the
methoxy group.
Chem Biol Drug Des 2014; 84: 473–488
Novel Hydrazones and Pyrazole Derivatives
Table 7: Ulcer numbers and index for the most active compounds
Compound
Normal control
(1% Tween-80)
Ulcer control
(indomethacin
20 mg/kg)
3
4
5
8
9
10
12
13
14
15
16
17
Ulcer number
(mean SE)
U.I. (mean SE)
0.00 0.00
0.00 0.00
8.62* 0.50
12.82* 0.29
3.56*,**
8.29*
8.35*
1.88**
1.92**
8.22*
8.3*
3.60*,**
8.12*
1.85*,**
3.65*,**
2.47*,**
0.31
0.81
0.73
0.29
0.21
0.31
0.56
0.33
0.59
0.19
0.32
0.43
3.7*,**
12.27*
12.31*
3.39*,**
3.45*,**
12.4*
12.29*
3.71*,**
12.3*
3.85*,**
3.69*,**
2.79*,**
0.27
0.99
1.03
0.31
0.37
0.70
1.1
0.24
0.47
0.31
0.33
0.29
Values were expressed as mean SEM of six rats. Statistical
analysis was performed by ANOVA followed by Tukey’s post hoc
test.
*Significantly different from normal control at p < 0.05.
**Significantly different from ulcer control (indomethacin 20 mg/kg)
at p < 0.05.
Acknowledgments
The authors thank Dr. Aymen El-Sahar, assistant lecturer
of Pharmacology, Faculty of Pharmacy, Cairo University,
for his efforts in performing in vivo biological screening and
Dr. Waleed Ali, associate professor of Biochemistry, Faculty of Medicine, Cairo University, for his work in performing in vitro assays.
Conflict of interest
The authors declare that they have no conflict of interest.
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Figure S1. IR spectrum of compound 4.
Figure S2. 1H-NMR spectrum of compound 4.
Figure S3.
13
C-NMR spectrum of compound 4.
Figure S4. IR spectrum of compound 3.
Figure S5. 1H-NMR spectrum of compound 3.
Figure S6.
13
C-NMR spectrum of compound 3.
Figure S7. IR spectrum of compound 6.
Figure S8. 1H-NMR spectrum of compound 6.
Figure S9.
13
C-NMR spectrum of compound 6.
Figure S10. IR spectrum of compound 9.
Figure S11. 1H-NMR spectrum of compound 9.
Figure S12.
13
C-NMR spectrum of compound 9.
Figure S13. 1H-NMR spectrum of compound 12.
Figure S14.
13
C-NMR spectrum of compound 12.
Figure S15. 1H-NMR spectrum of compound 14.
Figure S16. IR spectrum of compound 16.
Figure S17. 1H-NMR spectrum of compound 16.
Figure S18.
13
C-NMR spectrum of compound 16.
Figure S19. IR spectrum of compound 14.
Chem Biol Drug Des 2014; 84: 473–488