P.H. M, F.H. Al-Ostoot, H.K. Vivek et al.
Journal of Molecular Structure 1247 (2022) 131404
MS m/z 339 [M+], 341 [M + 2], 343 [M + 4]. Anal. Calcd. for
C14H12Cl2N3O3(339): C, 49.43; H, 3.26; N, 12.35. Found: C, 49.40;
H, 3.20; N, 12.20%.
Calcd. for C14H12BrN3O3(349): C, 48.02; H, 3.45; N, 12.00. Found:
C, 48.00; H, 3.30; N, 11.98%.
N’(2-(p-tolyloxy)acetyl)nicotinohydrazide (9d)
4.2. Pharmacology
Yield 72%; M.P. 175 °C-178 °C; FT-IR (KBr) νmax (cm-1): 1644
–
(amide, C = O), 3133–3241 (NH NH); 1HNMR (400 MHz, DMSO–
4.2.1. Biological evaluation, animal models and ethics
d6) δ (ppm): 4.66 (s, 2H, OCH2), 6.93–9.00 (m, 8H, Ar-H), 10.30
(s, 1H, NH), 10.62 (s, 1H, NH); 13C NMR (100 MHz, DMSO-
d6) δ (ppm): 167.27, 164.47, 156.98, 150.87, 139.77, 129.66, 125.50,
121.74, 117.03, 66.79, 21.39. LC-MS m/z 286 [M + 1]. Anal. Calcd.
for C15H15N3O3(285): C, 63.15; H, 5.30; N, 14.73. Found: C, 63.10;
H, 5.20; N, 14.60%.
For the entire biological assay, adult male Swiss albino mice
(20–25 g) were used and were housed in controlled conditions at
a temperature of 26 one C and relative humidity of 50–90% with
free access to a standard pellet diet and tap water. Before the ex-
periment, all the animals were housed for a week to adapt to the
experimental environment. All the testing procedures and animal
handling were performed according to the guidelines of the Re-
search Ethical Committee of the Farooqia College of Pharmacy, My-
suru.
N’(2-(4-bromophenoxy)acetyl)nicotinohydrazide (9e)
Yield 68%; M.P. 176 °C-179 °C; FT-IR (KBr) νmax (cm-1): 1648
–
(amide, C = O), 3143–3251 (NH NH); 1HNMR (400 MHz, DMSO–
d6) δ (ppm): 4.66 (s, 2H, OCH2), 7.00–8.74 (m, 8H, Ar-H), 10.34
(s, 1H, NH), 10.72 (s, 1H, NH); 13C NMR (100 MHz, DMSO-
d6) δ (ppm): 167.28, 164.44, 156.95, 150.82, 139.74, 129.67, 125.53,
121.77, 117.03, 66.77. LC-MS m/z 349 [M+], 351 [M + 2]. Anal.
Calcd. for C14H12BrN2O3(349): C, 48.02; H, 3.45; N, 12.00. Found:
C, 48.00; H, 3.34; N, 11.90%.
4.2.2. COX-1 and COX-2 in-vitro inhibitory activity
The efficacy of the title compounds 9(a-e) and 10(a-e) to in-
hibit COX-1, and COX-2 enzymes were determined by adopting the
colorimetric enzyme immune assay [37]. A COX inhibitor screening
assay kit determined the inhibitory efficacy of synthesized com-
pounds against COX-1 and COX-2 [38]. The inhibitory activity of
the compounds was measured by monitoring the production of
N’(2-phenoxyacetyl)isonicotinohydrazide (10a)
Yield 74%; M.P. 171 °C-173 °C; FT-IR (KBr) νmax (cm-1): 1651
–
(amide, C = O), 3146–3251 (NH NH); 1HNMR (400 MHz, DMSO–
ꢀ
ꢀ
oxidized N, N, N , N -tetramethyl-p-phenylenediamine (TMPD) at
590 nm followed by incubation of either COX-1 or COX-2 with
arachidonic acid, respectively. The COX enzyme assay protocol was
performed as per the manufacturer (Cayman Chemical) guidelines.
The enzyme was preincubated independently for 5 min at 25 °C
with the test compound before adding arachidonic acid (final con-
centration 1.1 mM) followed by TMPD. The reaction mixture was
incubated for 5 min at 25 °C, and the percent inhibition was cal-
culated according to the following equation.
d6) δ (ppm): 4.66 (s, 2H, OCH2), 6.93–9.00 (m, 9H, Ar-H), 10.30
(s, 1H, NH), 10.62 (s, 1H, NH); 13C NMR (100 MHz, DMSO-
d6) δ (ppm): 167.54, 164.53, 158.10, 152.89, 148.81, 135.60, 129.86,
128.46, 124.04, 121.68, 115.16, 66.50. LC-MS m/z 271 [M+]. Anal.
Calcd. for C14H13N3O3(271): C, 61.99; H, 4.83; N, 15.49. Found: C,
61.88; H, 4.77; N, 15.45%.
N’(2-(4-chlorophenoxy)acetyl)isonicotinohydrazide (10b)
Yield 76%; M.P. 175 °C-178 °C; FT-IR (KBr) νmax (cm-1): 1649
–
(amide, C = O), 3145–3253 (NH NH); 1HNMR (400 MHz, DMSO–
COX inhibiting activity (%) = [(1- (A1-A2)] × 100
d6) δ (ppm): 4.66 (s, 2H, OCH2), 7.00–8.74 (m, 8H, Ar-H), 10.34
(s, 1H, NH), 10.72 (s, 1H, NH); 13C NMR (100 MHz, DMSO- d6) δ
(ppm): 167.27, 164.47, 156.98, 150.87, 139.77, 129.66, 125.50, 121.74,
117.03, 66.79. LC-MS m/z 306 [M+], 308 [M + 2]. Anal. Calcd. for
C14H12ClN3O3(306): C, 55.00; H, 3.96; N, 13.75. Found: C, 54.98;
H, 3.84; N, 13.60%.
A0 where A0 is the absorbance of the control (without the test
compound), A1 is the absorbance in the presence of the test com-
pound, and A2 is the absorbance sample blank (without TMPD).
The IC50 values were calculated using a calibration curve with dif-
ferent concentrations of samples.
N’(2-(2,4-dichlorophenoxy)acetyl)isonicotinohydrazide (10c)
Yield 75%; M.P. 177 °C-180 °C; FT-IR (KBr) νmax (cm-1):
4.2.3. Anti-inflammatory in vivo activity
The efficacy of the anti-inflammatory activity of the synthesized
compounds 9(a-e) and 10(a-e) was evaluated adopting the in-
vivo formalin-induced rat foot paw edema model [39]. The syn-
thesized compounds 9(a-e) and 10(a-e) (10 mg/kg, b.wt) and the
standard compound diclofenac (10 mg/kg b.wt) were administered
through oral route just previous to the induction of inflammation,
which was achieved using 6% formalin solution as a subcutaneous
injection on the plantar surface of the left hind paw. Then the anti-
inflammatory activity was calculated based on the change in paw
volume using a plethysmometer at 1, 3, and 6 h after injecting for-
malin. Besides, the right hind paw of the animals was served as
a reference for comparison with the opposite limb. The percentage
edema inhibition was expressed as percentage paw-volume change
in milliliters. The percentage inhibition of the inflammatory effect
of the title compounds, compared to control, was calculated using
the following expression.
–
1653 (amide, C = O), 3147–3256 (NH NH); 1HNMR (400 MHz,
DMSO–d6) δ (ppm): 4.65 (s, 2H, OCH2), 7.03–8.83 (m, 7H, Ar-
H), 10.75 (s, 1H, NH), 10.42 (s, 1H, NH); 13C NMR (100 MHz,
DMSO–d6) δ (ppm): 167.32, 164.87, 152.70, 148.72, 148.10, 135.35,
131.47, 130.76, 129.01, 128.04, 125.12, 121.50, 117.12, 65.81. LC-
MS m/z 339 [M+], 341 [M + 2]. 343 [M + 4]. Anal. Calcd. for
C14H12Cl2N3O3(339): C, 49.43; H, 3.26; N, 12.35. Found: C, 49.40;
H, 3.20; N, 12.30%.
N’(2-(p-tolyloxy)acetyl)isonicotinohydrazide (10d)
Yield 77%; M.P. 182 °C-185 °C; FT-IR (KBr) νmax (cm-1): 1651
–
(amide, C = O), 3144–3257 (NH NH); 1HNMR (400 MHz, DMSO–
d6) δ (ppm): 6.67 (s, 3H, CH3), 4.67 (s, 2H, OCH2), 6.93–9.89 (m,
8H, Ar-H), 10.30 (s, 1H, NH), 10.62 (s, 1H, NH); 13C NMR (100 MHz,
DMSO- d6) δ (ppm): 167.27, 164.47, 156.98, 150.87, 139.77, 129.66,
125.50, 121.74, 117.03, 66.79, 21.39. LC-MS m/z 286 [M + 1], Anal.
Calcd. for C15H15N3O3(285): C, 63.15; H, 5.30; N, 14.73. Found: C,
63.10; H, 5.20; N, 14.60%.
% Inhibition = [Degree of inflammation by the (control group-
test group)] × 100
N’(2-(4-bromophenoxy)acetyl)isonicotinohydrazide (10e)
Yield 79%; M.P. 183 °C-186 °C; FT-IR (KBr) νmax (cm-1): 1649
Degree of inflammation by the control group
–
(amide, C = O), 3138–3254 (NH NH); 1HNMR (400 MHz, DMSO–
d6) δ (ppm): 4.66 (s, 2H, OCH2), 7.05–8.74 (m, 8H, Ar-H), 10.34
(s, 1H, NH), 10.72 (s, 1H, NH); 13C NMR (100 MHz, DMSO-
d6) δ (ppm): 167.28, 164.44, 156.95, 150.82, 139.74, 129.67, 125.53,
121.77, 117.03, 66.77. LC-MS m/z 349 [M+], 351 [M + 2]. Anal.
4.2.4. Analgesic activity
4.2.4.1. Acetic acid-induced writhing test. This test was performed
according to the earlier described method [40,41]. In brief, newly
synthesized compounds 9(a-e) and 10(a-e) about 10 mg/kg body
14