S. Emami et al.
Bioorganic Chemistry 112 (2021) 104943
solid; Yield: 84%; m.p.: 319–321 ◦C; IR (KBr, cmꢀ 1): 3458, 3220, 3123,
2942, 1719, 1594, 1087; 1H NMR (400 MHz, DMSO‑d6) δ: 6.95 (d, 1H, J
= 7.6 Hz, H-7), 7.11 (t, 1H, J = 7.6 Hz, H-5), 7.40 (dt, 1H, J = 7.6 and
1.2 Hz, H-6), 7.59 (d, 1H, J = 7.6 Hz, H-4), 7.69 (d, 2H, J = 8.4 Hz, H-3′
and H-5′), 7.90 (d, 2H, J = 8.4 Hz, H-2′ and H-6′), 11.42 (br s, 1H, NH
Isatin), 13.89 (br s, 1H, NH Hydrazone). 13C NMR (100 MHz, DMSO‑d6):
111.72, 120.18, 121.50, 123.25, 129.74, 131.28, 132.38, 138.16,
142.99, 163.48. MS (m/z, %): 299 (M+, 22), 271 (13), 257 (25), 211
(17), 183 (75), 160 (85), 139 (100), 111 (81), 98 (41), 85 (47), 71 (57),
57 (98). Anal. Calcd for C15H10ClN3O2: C, 60.11; H, 3.36; N, 14.02.
Found: C, 60.23; H, 3.37; N, 14.09.
H-5′), 7.87 (d, 1H, J = 7.6 Hz, H-4), 7.97 (d, 2H, J = 8.4 Hz, H-2′ and H-
6′), 14.12 (br s, 1H, NH). 13C NMR (100 MHz, DMSO‑d6): 26.18, 110.54,
119.44, 121.14, 123.79, 129.76, 131.17, 132.30, 138.22, 144.19,
161.62. MS (m/z, %): 313 (M+, 52), 174 (70), 146 (68), 139 (100), 118
(38), 111 (91), 91 (56), 75 (53). Anal. Calcd for C16H12ClN3O2: C, 61.25;
H, 3.86; N, 13.39. Found: C, 61.23; H, 3.92; N, 13.20.
(Z)-N’-(1-methyl-2-oxoindolin-3-ylidene)isonicotinohydrazide
(6e).
Yellow solid; Yield: 83%; m.p.: 223–225 ◦C; IR (KBr, cmꢀ 1): 3434, 3113,
2944, 1699, 1519, 1041; 1H NMR (400 MHz, CDCl3) δ: 3.33 (s, 3H, CH3),
6.94 (d, 1H, J = 7.6 Hz, H-7), 7.20 (t, 1H, J = 7.6 Hz, H-5), 7.46 (t, 1H, J
= 8.0 Hz, H-6), 7.85 (d, 2H, J = 5.2 Hz, H-2′ and H-6′), 7.88 (d, 1H, J =
7.6 Hz, H-4), 8.87 (d, 2H, J = 5.2 Hz, H-3′ and H-5′), 14.23 (s, 1H, NH
Hydrazone). 13C NMR (100 MHz, DMSO‑d6): 26.21, 110.61, 119.28,
121.32, 121.49, 123.86, 132.58, 139.53, 144.38, 151.42, 161.56. MS
(m/z, %): 280 (M+, 28), 174 (56), 146 (97), 117 (45), 91 (100), 78 (97),
51 (95). Anal. Calcd for C15H12N4O2: C, 64.28; H, 4.32; N, 19.99. Found:
C, 64.45; H, 4.36; N, 19.93.
(Z)-4-nitro-N’-(2-oxoindolin-3-ylidene)benzohydrazide (5e). Orange
solid; Yield: 93%; m.p.: 339–341 ◦C; IR (KBr, cmꢀ 1): 3432, 3232, 3113,
2946, 1737, 1598, 1521, 1342, 1143; 1H NMR (400 MHz, DMSO‑d6) δ:
6.92 (d, 1H, J = 8.0 Hz, H-7), 7.09 (t, 1H, J = 7.6 Hz, H-5), 7.42 (dt, 1H,
J = 7.6 and 2.0 Hz, H-6), 7.99 (d, 1H, J = 7.6 Hz, H-4), 8.13 (d, 2H, J =
8.8 Hz, H-3′ and H-5′), 8.44 (d, 2H, J = 8.8 Hz, H-2′ and H-6′), 11.42 (s,
1H, NH Isatin), 14.00 (br s, 1H, NH Hydrazone). 13C NMR (100 MHz,
DMSO‑d6): 111.78, 115.93, 120.05, 121.66, 124.03, 124.72, 127.60,
130.49, 133.68, 138.14, 143.18, 149.85, 163.44. MS (m/z, %): 310 (M+,
23), 282 (13), 264 (16), 236 (26), 211 (15), 183 (23), 160 (100), 132
(95), 120 (24), 104 (95), 92 (30), 76 (67), 57 (87). Anal. Calcd for
4.2. Pharmacology
Pharmacological studies were fulfilled in the Toxicology and Phar-
macology Research Laboratories of the Mazandaran University of
Medical Sciences. All experiments were accomplished in accordance
with the ethical standards and protocols approved by the Committee of
Animal Experimentation of Mazandaran University of Medical Sciences,
Sari, Iran (Ethics Code: IR.MAZUMS.REC.1396.3157). Dual Impedance
Research Stimulator, ear electrodes, and Rotarod device were used for
the anticonvulsant and neurotoxicity evaluations. Male Swiss albino
mice with 20–25 g weight were used for in vivo studies. The animals
were housed at 25 ± 2 ◦C and a 12 h light/dark cycle in standard
Plexiglas cages with access to adequate water and food. The stock so-
lutions of compounds were prepared in DMSO and each compound was
administered as an i.p. injection at the required doses. The negative
control groups were received the vehicle. Normal saline 0.9% was used
to prepare the PTZ solution, and solution was kept at 4 ◦C during testing.
C
15H10N4O4: C, 58.07; H, 3.25; N, 18.06. Found: C, 57.88; H, 3.21; N,
18.12.
(Z)-N’-(1-methyl-2-oxoindolin-3-ylidene)benzohydrazide (6a). Yellow
solid; Yield: 73%; m.p.: 183–185 ◦C; IR (KBr, cmꢀ 1): 3436, 3108, 2956,
1698, 1542, 1043; 1H NMR (400 MHz, CDCl3) δ: 3.34 (s, 3H, CH3), 6.93
(d, 1H, J = 7.6 Hz, H-7), 7.19 (t, 1H, J = 7.6 Hz, H-5), 7.44 (dt, 1H, J =
7.2 and 1.2 Hz, H-6), 7.55 (t, 2H, J = 7.6 Hz, H-3′ and H-5′), 7.63 (t, 1H,
J = 7.2 Hz, H-4′), 7.88 (d, 1H, J = 7.2 Hz, H-4), 8.04 (d, 2H, J = 8.5 Hz,
H-2′ and H-6′), 14.11 (br s, 1H, NH Hydrazone). 13C NMR (100 MHz,
DMSO‑d6): 26.25, 110.63, 115.30, 119.36, 121.33, 123.88, 124.73,
126.47, 129.55, 132.59, 138.08, 144.49, 162.15. MS (m/z, %): 279 (M+,
23), 183 (10), 174 (70), 146 (48), 117 (22), 105 (92), 91 (42), 81 (44),
77 (96), 69 (100). Anal. Calcd for C16H13N3O2: C, 68.81; H, 4.69; N,
15.05. Found: C, 69.02; H, 4.91; N, 15.18.
(Z)-4-hydroxy-N’-(1-methyl-2-oxoindolin-3-ylidene)benzohydrazide
(6b). Yellow solid; Yield: 91%; m.p.: 318–320 ◦C; IR (KBr, cmꢀ 1): 3406,
3179, 3116, 2932, 1685, 1543, 1183, 1042; 1H NMR (400 MHz,
DMSO‑d6) δ: 3.25 (s, 3H, CH3), 6.96 (d, 2H, J = 8.8 Hz, H-3′ and H-5′),
7.17 (d, 1H, J = 8.0 Hz, H-7), 7.18 (t, 1H, J = 6.8 Hz, H-5), 7.48 (dt, 1H,
J = 8.2 and 1.2 Hz, H-6), 7.63 (dd, 1H, J = 8.0 and 1.2 Hz, H-4), 7.79 (d,
2H, J = 8.8 Hz, H-2′ and H-6′), 10.44 (s, 1H, OH), 13.83 (s, 1H, NH
Hydrazone). 13C NMR (100 MHz, DMSO‑d6): 26.15, 110.43, 116.28,
119.68, 120.88, 122.79, 123.70, 130.12, 131.26, 131.87, 136.92,
143.91, 161.71, 162.26. MS (m/z, %): 295 (M+, 37), 267 (10), 173 (81),
146 (68), 117 (59), 93 (100), 77 (30), 65 (84). Anal. Calcd for
4.2.1. MES-induced seizure test
The animals were randomly divided into different treatment groups
then test compounds were administrated i.p. in mice. 30 min later, mice
were restrained for electrical stimulation and electrical seizures were
induced by dual impedance research stimulator (Borj-Sanat, Iran) via
ear clip electrodes, coated with an electrolyte solution. The alternating
current of 50 mA (60 Hz) was delivered for 0.2 s to induce seizure in
mice. After electrical stimulation, animals were instantly moved to a
Plexiglas arena for behavioral observation. The negative control group
was fully associated with the hind-limb tonic extension (HLTE). The
reduction in the incidence of HLTE was recorded in the drug-treated
mice [60].
C
16H13N3O3: C, 65.08; H, 4.44; N, 14.23. Found: C, 65.11; H, 4.27; N,
14.28.
(Z)-4-methyl-N’-(1-methyl-2-oxoindolin-3-ylidene)benzohydrazide
4.2.2. PTZ-induced seizure test
(6c). Yellow solid; Yield: 80%; m.p.: 203–205 ◦C; IR (KBr, cmꢀ 1): 3436,
3120, 2922, 1708, 1534, 1038; 1H NMR (400 MHz, CDCl3) δ: 2.46 (s,
3H, CH3), 3.33 (s, 3H, CH3), 6.92 (d, 1H, J = 8.0 Hz, H-7), 7.18 (t, 1H, J
= 7.6 Hz, H-5), 7.34 (d, 2H, J = 8.0 Hz, H-3′ and H-5′), 7.43 (dt, 1H, J =
7.6 and 1.2 Hz, H-6), 7.89 (d, 1H, J = 7.6 Hz, H-4), 7.94 (d, 2H, J = 8.4
Hz, H-2′ and H-6′), 14.06 (br s, 1H, NH Hydrazone). 13C NMR (100 MHz,
DMSO‑d6): 21.57, 26.18, 110.49, 119.58, 121.03, 123.75, 127.88,
129.57, 130.19, 132.10, 143.78, 144.08, 161.69. MS (m/z, %): 293 (M+,
10), 183 (7), 173 (18), 146 (19), 119 (91), 91 (100), 65 (40), 43 (15).
Anal. Calcd for C17H15N3O2: C, 69.61; H, 5.15; N, 14.33. Found: C,
69.73; H, 5.11; N, 14.06.
At first, each animal group was treated with different doses of
compounds 5a-e and 6a-e. After 30 min, a solution of PTZ (100 mg/kg)
was injected i.p. and animals were kept in distinct cages and monitored
for 0.5 h. The incidence of tonic-clonic convulsion and subsequently
death following it was recorded for each animal in the group [60].
4.2.3. Acute neurotoxicity (Rotarod test)
The selected compound (6e, at the doses of 2.5, 5 and 10 mg/kg),
diazepam (2 mg/kg, as reference drug) and DMSO (as control) were
administered i.p. After administration (0.5, 1, 2, and 4 h), mice were
placed on the rotating rod (6 rpm) and the time that the mice were able
to maintain their balance on the rotating rod was recorded.
(Z)-4-chloro-N’-(1-methyl-2-oxoindolin-3-ylidene)benzohydrazide
(6d). Yellow solid; Yield: 79%; m.p.: 278–280 ◦C; IR (KBr, cmꢀ 1): 3450,
3112, 2944, 1703, 1533, 1013; 1H NMR (400 MHz, CDCl3) δ: 3.33 (s,
3H, CH3), 6.93 (d, 1H, J = 7.6 Hz, H-7), 7.19 (t, 1H, J = 7.6 Hz, H-5),
7.44 (dt, 1H, J = 7.6 and 1.2 Hz, H-6), 7.52 (d, 2H, J = 8.4 Hz, H-3′ and
4.3. Docking simulation
Autodock Vina v1.2 has been utilized to study the binding mode of
9