Material and methods
All chemicals were procured from SD Fine Chem Co. Ltd and used in experiments. The melting point has been identified in the Bouchi oil melting point apparatus and has not been corrected. The microwave-assisted reactions were carried out in an HPL 2300 ET domestic microwave oven: power: 300 W; frequency: 2.45 GHz; temperature range: 60–250 °C [29]. The purity of the compounds was determined by a single spot on the TLC silica gel G plate. IR spectra were recorded on the Shimadzu IR convergence spectrometer using the KBr pellet method. NMR spectra (1H NMR and 13C NMR) are recorded on the Bruker Avance II 500 MHz NMR spectrometer. Mass spectra were performed on the JEOL GC mass spectrometer. The results in the spectrum head show that the molecular mass of the compounds produced was closer to the molecular mass of the expected compounds [30].
Chemistry
The comparative procedure (conventional as well as microwave irradiation method) of pyrazole derivatives by the synthetic route as shown in Scheme 1 is stated below in four steps and is already presented in our previous work with different substituents [28, 29].
Step 1: Synthesis of N'-(1-phenylethylidene) isonicotinohydrazide derivatives 1(A-C).
Whereas R = 4-NO2, 4-OCH3, 4-Br
(Z)-N′-(1-(4-Nitrophenyl) ethylidene) isonicotinohydrazide (1A) Yellow solid (71.6%); m.pt. 226–228 °C; IR (KBr, cm−1): 3280 (N–H str., CONH), 2900 (C-H str.), 1625 (C=C str.), 1590 (C=N str.), 1135 (C–N str.), 1657 (C=O str., CONH); 1H NMR (500 MHz, DMSO) δ (ppm) 9.06 (d, J = 6.3 Hz, 2H, CH, C2 & C6 of 4-pyridine), 7.96 (d, J = 6.3, 2H, CH, C3 & C5 of 4-pyridine), 7.36 (d, 2H, CH of C2 & C6 of 4-nitrophenyl), 8.24(d, 2H, CH of C3 & C5 of 4-nitrophenyl), 7.00 (s, NH of CONH).
(Z)-N'-(1-(4-Methoxyphenyl) ethylidene) isonicotinohydrazide (1B) Yellow solid (72%); m.pt. 193–195 °C; IR (KBr, cm−1): 3396 (N–H str., CONH), 2922 (C–H str.), 1625 (C=C str.), 1585 (C=N str.), 1150 (C–N str.), 1670 (C=O str., CONH); 1H NMR (500 MHz, DMSO) δ (ppm) 9.06 (d, J = 6.3 Hz, 2H, CH, C2 & C6 of 4-pyridine), 7.96 (d, J = 6.3, 2H, CH, C3& C5 of 4-pyridine), 7.00 (s, NH of CONH), 7.5 (d, J = 6.3 Hz, 2H, CH, C2 & C6 of 4-methoxyphenyl), 6.8 (d, J = 5.9 Hz, 2H, CH of C3 & C5 of 4-methoxyphenyl), 3.73 (s, 3H, OCH3).
(Z)-N'-(1-(4-Bromophenyl) ethylidene) isonicotinohydrazide (1C) Brown solid (89%); m.pt. 216–218 °C; IR (KBr, cm−1): 3380 (N–H str., CONH), 2918 (C-H str.), 1620 (C=C str.), 1590 (C=N str.), 1150 (C–N str.), 1690 (C=O str., CONH); 1H NMR (500 MHz, DMSO) δ (ppm) 9.06 (d, J = 6.3 Hz, 2H, CH, C2 & C6 of 4-pyridine), 7.96 (d, J = 6.3, 2H, CH, C3 & C5 of 4-pyridine), 7.00 (s, NH of CONH), 7.45 (d, J = 6.3 Hz, 2H, CH, C2 & C6 of 4-bromophenyl), 6.8 (d, J = 5.9 Hz, 2H, CH of C3 & C5 of 4-bromophenyl).
Step 2: Synthesis of 1-isonicotinoyl-3-phenyl-4,5-dihydro-1
H
-pyrazole-4-carbaldehyde derivatives 2 (A–C)
3-(4-Nitrophenyl)-1-isonicotinoyl-1H-pyrazole-4-carbaldehyde (2A) Yellow solid (70%); m.pt. 230–232 °C; IR (KBr, cm−1): 2900 (C–H str.), 1625 (C=C str.), 1590 (C=N str.), 1135 (C–N str.), 1657 (C=O str., CONH), 1558 (NO2 assym. str.), 1298 (NO2 symm. str.); 1H NMR (500 MHz, DMSO) δ (ppm) 9.06 (d, J = 6.3 Hz, 2H, CH, C2 & C6 of 4-pyridine), 7.96 (d, J = 6.3, 2H, CH, C3 & C5 of 4-pyridine), δ 7.5 (s, 1H, C5 of pyrazole), 7.36 (d, 2H, CH of C2 & C6 of 4-nitrophenyl), 8.24(d, 2H, CH of C3 & C5 of 4-nitrophenyl), 9.61 (s, 1H, CHO).
3-(4-Methoxyphenyl)-1-isonicotinoyl-1H-pyrazole-4-carbaldehyde (2B) Yellow solid (72%); mp 198–200 °C; IR (KBr, cm−1): 2980 (C–H str.), 1638 (C=C str.), 1586 (C=N str.), 1136 (C–N str.), 1670 (C=O str., CONH), 1236 (C–O–C asym. str.), 1095 (C–O–C sym. str.); 1H NMR (500 MHz, DMSO) δ (ppm) 9.06 (d, J = 6.3 Hz, 2H, CH, C2& C6 of 4-pyridine), 7.96 (d, J = 6.3, 2H, CH, C3 & C5 of 4-pyridine), δ 3.29 (s, 3H, OCH3), 7.5 (s, 1H, C-5 of pyrazole), 7.37 (d, 2H, CH of C2 & C6 of 4-methoxyphenyl), 6.83 (d, J = 2H, CH of C3 & C5 of 4-methoxyphenyl), 9.61 (s, 1H, CHO).
3-(4-Bromophenyl)-1-isonicotinoyl-1H-pyrazole-4-carbaldehyde (2C) Brown solid (85%); mp 222–224 °C; IR (KBr, cm−1):2996 (C-H str.), 1648 (C=C str.), 1580 (C=N str.), 1136 (C–N str.), 1672 (C=O str., CONH), 570 (C–Br str.); 1H NMR (500 MHz, DMSO) δ (ppm) 9.06 (d, J = 6.3 Hz, 2H, CH, C2 & C6 of 4-pyridine), 7.96 (d, J = 6.3, 2H, CH, C3 & C5 of 4-pyridine), 7.37 (d, 2H, CH of C2 & C6 of 4-bromophenyl), 7.49 (d, 2H, CH of C-3 & C-5 of 4-bromophenyl), 9.61 (s, 1H, CHO).
Step 3: synthesis of (3-phenyl-4-((phenylimino) methyl)-4,5-dihydropyrazol-1-yl pyridin-4-yl)methanone derivatives/Schiff bases S (1–21).
R1 = H, 4-NO2, 2-NO2, 4-Cl, 2-Cl, 4-OCH3, 2-OCH3, 4-Br.
Step 4: Synthesis of 2-(1-isonicotinoyl-3-phenyl-4,5-dihydro-1H-pyrazol-4-yl)-3-phenyl thiazolidin-4-one derivatives P (1–21).
2-(1-isonicotinoyl-3-(4-nitrophenyl)-1H-pyrazol-4-yl)-3-phenylthiazolidin-4-one (P-1) Yellow solid (89.2%); m.pt- 244–246 °C; IIR (KBr, cm−1): 3200 (C-H str. Ar), 2450 (CH-S str.), 1680 (C = O str.pyrazole), 1730 (C = O str., thiazolidinone), 1361 (C = N str.), 1224 (Ar-N str.), 1157 (R–C-O str.), 1530 (NO2 asymm.str.), 1380 (NO2 symm.str.), 1200 (Ar-N str.), 1350 (C–C str.), 690–710 (Ar. CH-bend, monosubstituted).
2-(1-isonicotinoyl-3-(4-nitrophenyl)-1H-pyrazol-4-yl)-3-(4-nitrophenyl)thiazolidin-4-one (P-2) Yellow solid (87.4%); m.pt.- 266–268 °C; IR (KBr, cm−1): 3220 (C-H str. Ar), 2990 (CH-S str.), 1720 (C=O str., pyrazole),1733 (C=O str., thiazolidinone), 1310 (C=N str.), 1230 (Ar-N str.), 1157 (R–C-O str.), 1510 (NO2 asymm. str.), 1360 (NO2 symm. str.), 1270 (Ar-N str.), 1351 (C–C str.), 690–710 (Ar. CH-bend, monosubstituted).
2-(1-isonicotinoyl-3-(4-nitrophenyl)-1H-pyrazol-4-yl)-3-(2-nitrophenyl)thiazolidin-4-one (P-3) Yellow solid (83.1%); m.pt. 282–284 °C; IR (KBr, cm−1): 3220 (C–H str. Ar), 2990 (CH-S str.), 1720 (C=O str., pyrazole),1733 (C=O str., thiazolidinone), 1310 (C=N str.), 1230 (Ar-N str.), 1157 (R–C–O str.), 1510 (NO2 asymm. str.), 1360 (NO2 symm. str.), 1270 (Ar-N str.), 1351 (C–C str.), 690–710 (Ar. CH-bend, monosubstituted); 1H NMR (500 MHz, DMSO) δ 9.06 (d, J = 6.3 Hz, 2H, CH, C2 & C6 of 4-pyridine), 7.96 (d, J = 6.3 Hz, 2H, CH, C3 & C5 of 4-pyridine), 3.43 (s, J = 5.6 Hz, 2H, C-5 of thiazolidinone), 5.92 (d, 1H, CH, C2 of thiazolidinone), 7.3 (s, 1H, C5 of pyrazole), 7.36 (s, 1H, CH, C-6 of 2-nitrobenzene), 7.70 (s, 1H, CH, C-5 of 2-nitrobenzene), 7.50 (s, 1H, CH, C4 of 2-nitrobenzene), 8.24 (s, 1H, CH, C3 of 4-nitrobenzene).
3-(4-chlorophenyl)-2-(1-isonicotinoyl-3-(4-nitrophenyl)-1H-pyrazol-4-yl)thiazolidin-4-one (P-4) Creamy yellow (80%); m.pt.: 286–288 °C; IR (KBr, cm−1): 3201 (C-H str. Ar), 2928 (CH-S str.), 1680 (C = O str., pyrazole), 1736 (C=O str., thiazolidinone), 1361 (C=N str.), 1224 (Ar–N str.), 1157 (R–C–O str.), 1550 (NO2 asymm. str.), 1386 (NO2 symm. str.), 1298 (Ar–N str.), 1350 (C–C str.), 692 (C–Cl str.); 1H NMR (500 MHz, DMSO) δ 9.06 (d, J = 6.3 Hz, 2H, CH, C2& C6 of 4-pyridine), 7.96 (d, J = 6.3 Hz, 2H, CH, C3 & C5 of 4-pyridine), 3.43 (s, J = 5.6 Hz, 2H, C5 of thiazolidinone), 5.92 (d, 1H, CH, C2 of thiazolidinone), 7.3 (s, 1H, C5 of pyrazole), 4.6 (s, 1H, C1 of thiazolidinone), 7.5 (d, J = 4.5 Hz, 2H, CH of C2 & C6 of 4-chlorophenyl), 6.8 (d, J = 4.6 Hz, 2H, CH of C3 & C5 of 4-chlorophenyl), 7.38 (d, J = 4.8 Hz, 2H, CH, C2 & C6 of 2-nitrophenyl), 8.22 (d, J = 4.8 Hz, 2H, CH, C3 & C5 of 2-nitrophenyl.
3-(3-chlorophenyl)-2-(1-isonicotinoyl-3-(4-nitrophenyl)-1H-pyrazol-4-yl)thiazolidin-4-one (P-5) Creamy yellow (75.8%); m.pt. 274–276 °C; IR (KBr, cm−1): 3201 (C–H str. Ar), 2928 (CH–S str.), 1680 (C=O str., pyrazole), 1736 (C=O str., thiazolidinone), 1361 (C=N str.), 1224 (Ar–N str.), 1157 (R–C–O str.), 1550 (NO2 asymm. str.), 1386 (NO2 symm. str.), 1298 (Ar- N str.), 1350 (C–C str.), 692 (C–Cl str.).
2-(1-isonicotinoyl-3-(4-nitrophenyl)-1H-pyrazol-4-yl)-3-(4-methoxyphenyl)thiazolidin-4-one (P-6) Yellowish brown solid (72.6%); m.pt: 262–264 °C C; IR (KBr, cm−1): 3198 (C–H str. Ar), 2928 (CH–S str.), 1691 (C=O str., pyrazole),1726 (C = O str., thiazolidinone), 1361 (C=N str.), 1224 (Ar-N str.), 1157 (R–C–O str.), 1514 (NO2 asymm.str.), 1384 (NO2 symm.str.), 1296 (Ar-N str.), 1261 (C–O–C asymm. str.), 1076 (C–O–C symm. str.); 1H NMR (500 MHz, DMSO) δ 9.06 (d, J = 6.3 Hz, 2H, CH, C2 & C6 of 4-pyridine), 7.96 (d, J = 6.3 Hz, 2H, CH, C3 & C5 of 4-pyridine), 3.43 (s, J = 5.6 Hz, 2H, C5 of thiazolidinone), 5.92 (d, 1H, CH, C2 of thiazolidinone), 7.3 (s, 1H, C5 of pyrazole), 7.9 (d, J = 4.8 Hz, 2H, CH of C2 & C6 of 4-nitrophenyl), 8.2 (d, J = 4.8 Hz, 2H, CH of C3 & C5 of 4-nitrophenyl), 6.64 (s, 1H, CH, C2 of 4-methoxyphenyl), 6.70 (s, 1H, CH, C4 of 4-methoxyphenyl), 7.22 (s, 1H, CH, C5 of 4-methoxyphenyl), 6.60 (s, 1H, CH, C6 of 4-methoxyphenyl); 13C-NMR (CDCl3, 500 MHz, δppm): 149.8 (4-pyridine C3 & C5), 122.8 (4-pyridine C2 & C6), 167.0 (C=O), 151.8 (pyrazole C3), 46.1 (pyrazole C4), 71.1 (pyrazole C5), 140.0 (4-nitrophenyl C1), 130.0 (4-nitrophenyl C2 & C6), 121.0 (4-nitrophenyl C3 & C5), 60.7 (4-nitrophenyl C4), 47.8 (thiazolidin-4-one C2), 34 (thiazolidin-4-one C5), 170.9 (thiazolidin-4-one C4 (C=O)), 134 (4-methoxyphenyl C1), 122.6 (4-methoxyphenyl C2 & C6), 114 (4-methoxyphenyl C3 & C5), 156.0 (4-methoxyphenyl C4), 55.9 (O-CH3); m/z 503 (M+); Anal.: Calcd. for C25H21N5O5S: C, 59.638; H, 4.28; N, 13.918; S, 6.378; O, 15.898. Found: C, 59.276; H, 4.24; N, 13.886; S, 6.338; O, 15.890.
2-(1-isonicotinoyl-3-(4-nitrophenyl)-1H-pyrazol-4-yl)-3-(2-methoxyphenyl)thiazolidin-4-one (P-7) Yellowish brown solid (93.4%); m.pt.: 250–252 °C; IR (KBr, cm−1): 3198 (C–H str. Ar), 2928 (CH–S str.), 1691 (C=O str., pyrazole),1726 (C=O str., thiazolidinone), 1361 (C=N str.), 1224 (Ar-N str.), 1157 (R–C-O str.), 1514 (NO2 asymm.str.), 1384 (NO2 symm.str.), 1296 (Ar-N str.), 1261 (C–O–C asymm. str.), 1076 (C–O–C symm. str.)
2-(1-isonicotinoyl-3-(4-methoxyphenyl)-1H-pyrazol-4-yl)-3-phenylthiazolidin-4-one (P-8) Yellow solid (82.5%); m.pt. 300–302 °C; IR (KBr, cm−1): 3252 (C–H str. Ar), 2932 (CH–S str.), 1688 (C=O str., pyrazole),1740 (C=O str., thiazolidinone), 1548 (C=N str.), 1226 (Ar–N str.), 1160 (R–C=O str.), 1338 (C=N str.), 1330 (C–O–C asymm. str.), 1128 (C–O–C symm. str.).
2-(1-isonicotinoyl-3-(4-methoxyphenyl)-1H-pyrazol-4-yl)-3-(4-nitrophenyl)thiazolidin-4-one (P-9) Yellow solid (76.3%); m.pt. 308–310 °C; IIR (KBr, cm−1): 3152 (C–H str. Ar), 2928 (CH-S str.), 1681 (C=O str., pyrazole),1733 (C=O str., thiazolidinone), 1649 (C=N str.), 1226 (Ar-N str.), 1157 (R–C=O str.), 1338 (C=N str.), 1514 (NO2 asymm.str.), 1384 (NO2 symm.str.), 1298 (C–O–C asymm. str.), 1108 (C–O–C symm. str.).
2-(1-isonicotinoyl-3-(4-methoxyphenyl)-1H-pyrazol-4-yl)-3-(2-nitrophenyl)thiazolidin-4-one (P-10) Yellow white solid (64.7%); m.pt. 322–324 °C; IR (KBr, cm−1): 3137 (C–H str. Ar), 2962 (CH–S str.), 1670 (C=O str., pyrazole), 1735 (C=O str., thiazolidinone), 1617 (C=N str.), 1284 (Ar-N str.), 1175 (R–C–O str.), 1319, 1411 (C=N str.), 1520 (NO2 asymm. str.), 1350 (NO2 symm. str.), 1310 (C–O–C asymm. str.), 1198 (C–O–C symm. str.); 1H NMR (500 MHz, DMSO) δ 9.06 (d, J = 6.3 Hz, 2H, CH, C2 & C6 of 4-pyridine), 7.96 (d, J = 6.3 Hz, 2H, CH, C3 & C5 of 4-pyridine), 33.43 (s, J = 5.6 Hz, 2H, C5 of thiazolidinone), 5.92 (d, 1H, CH, C2 of thiazolidinone), 7.3 (s, 1H, C5 of pyrazole), 7.9 (d, J = 4.8 Hz, 2H, CH of C2 & C6 of 4-nitrophenyl), 8.2 (d, J = 4.8 Hz, 2H, CH of C3 & C5 of 4-nitrophenyl), 8.24 (s, 1H, CH, C3 of 2-nitrophenyl), 7.50 (s, 1H, CH, C4 of 2-nitrophenyl), 7.70 (s, 1H, CH, C5 of 2-nitrophenyl), 7.36 (s, 1H, CH, C6 of 2-nitrophenyl).
3-(4-chlorophenyl)-2-(1-isonicotinoyl-3-(4-methoxyphenyl)-1H-pyrazol-4-yl)thiazolidin-4-one (P-11) Yellowish white (68.8%); m.pt. 318–320 °C; IR (KBr, cm−1): 3137 (C–H str. Ar), 2959 (CH–S str.), 1681 (C=O str., pyrazole), 1740 (C=O str., thiazolidinone), 1588 (C=N str.), 1221 (Ar-N str.), 1157 (R–C–O str.), 1317, 1415 (C=N str.), 696 (C–Cl str.), 1330 (C–O–C asymm. str.), 1180 (C–O–C symm. str.); 1H NMR (500 MHz, DMSO) δ 9.06 (d, J = 6.3, 2H, CH, C2 & C6 of 4-pyridine), 7.96 (d, J = 6.3, 2H, CH, C3 & C5 of 4-pyridine), 3.43 (s, J = 5.6 Hz, 2H, C5 of thiazolidinone), 5.92 (d, 1H, CH, C2 of thiazolidinone), 7.3 (s, 1H, C5 of pyrazole), 6.64 (s, 1H, CH, C2 of 4-methoxyphenyl), 6.70 (s, 1H, CH, C4 of 4-methoxyphenyl), 7.22 (s, 1H, CH, C5 of 4-methoxyphenyl), 6.60 (s, 1H, CH, C6 of 4-methoxyphenyl),7.04 (d, J = 5.9 Hz, 2H, CH, C2 & C6 of 4-chlorophenyl), 7.32 (d, J = 5.9 Hz, 2H, CH, C3 & C5 of 4-chlorophenyl).
3-(3-chlorophenyl)-2-(1-isonicotinoyl-3-(4-methoxyphenyl)-1H-pyrazol-4-yl)thiazolidin-4-one (P-12) Yellowish white solid (72.4%); m.pt. 338–340 °C; IR (KBr, cm−1): 3189 (C–H str., Ar), 2919 (CH–S str.), 1669 (C=O str., pyrazole), 1733 (C=O str., thiazolidinone), 1556 (C=N str.), 1222 (Ar-N str.), 1140 (R–C–O str.), 678 (C–Cl str.), 1310 (C–O–C asymm. str.), 1198 (C–O–C symm. str.); 1H NMR (500 MHz, DMSO) δ 9.06 (d, J = 6.3 Hz, 2H, CH, C2 & C6 of 4-pyridine), 7.96 (d, J = 6.3 Hz, 2H, CH, C3 & C5 of 4-pyridine), 3.43 (s, J = 5.6 Hz, 2H, C5 of thiazolidinone), 5.92 (d, 1H, CH, C2 of thiazolidinone), 7.3 (s, 1H, C5 of pyrazole), 6.61 (s, 1H, CH, C2 of 4methoxyphenyl), 6.75 (s, 1H, CH, C4 of 4-methoxyphenyl), 7.20 (s, 1H, CH, C5 of 4-methoxyphenyl), 6.66 (s, 1H, CH, C6 of 4-methoxyphenyl), 3.73 (s, 3H, OCH3), 7.11 (s, 1H, CH, C2 of 3-chlorophenyl, 7.25 (d, J = 5.7 Hz, 2H, CH, C4 & C5 of 3-chlorophenyl), 6.98 (s, 1H, CH, C6 of 3-chlorophenyl); 13C-NMR (CDCl3, 500 MHz, δppm): 149.8 (4-pyridine C3 & C5), 122.8 (4-pyridine C2 & C6), 167.0 (C=O), 151.8 (pyrazole C3), 46.1 (pyrazole C4), 71.1 (pyrazole C6), 126.0 (4-methoxyphenyl C1), 130.0 (4-methoxyphenyl C2 & C6), 114.0 (4-methoxyphenyl C3 & C5), 163 (4-methoxyphenyl C4), 55.9 (O-CH3), 47.8 (thiazolidin-4-one C2), 34 (thiazolidin-4-one C5), 170.9 (thiazolidin-4-one C4 (C=O)), 143 (3-chlorophenyl C1), 122.6 (3-chlorophenyl C2 & C6), 134.5 (3-chlorophenyl C3), 124.0 (3-chlorophenyl C4), 130.4 (3-chlorophenyl C5); LC–MS: m/z 490 (M+); Anal.: Calcd. for C25H19ClN4O3S Anal. Calcd.: C, 61.168; H, 3.98; N, 11.418; S, 6.538; Cl, 7.228; O, 9.788. Found: C, 61.068; H, 3.68; N, 11.218; S, 6.508; Cl, 7.218; O, 9.748.
2-(1-isonicotinoyl-3-(4-methoxyphenyl)-1H-pyrazol-4-yl)-3-(4-methoxyphenyl) thiazolidin-4-one (P-13) Yellow solid (76.6%); m.pt. 320–322 °C; IR (KBr, cm−1): 3354 (C–H str. Ar), 2964 (CH–S str.), 1690 (C=O str., pyrazole),1722 (C=O str., thiazolidinone), 1593 (C=N str.), 1259 (Ar-N str.), 1174 (R–C–O str.), 1338, 1417 (C=N str.);1292 (C–O–C asymm. str.), 1126 (C–O–C symm. str.); 1H NMR (500 MHz, DMSO) δ 9.06 (d, J = 6.3 Hz, 2H, CH, C2 & C6 of 4-pyridine), 7.96 (d, J = 6.3 Hz, 2H, CH, C3 & C5 of 4-pyridine), 3.43 (s, J = 5.6 Hz, 2H, C5 of thiazolidinone), 5.92 (d, 1H, CH, C2 of thiazolidinone), 7.3 (s, 1H, C5 of pyrazole), 6.66 (s, 1H, CH, C2 of 4-methoxyphenyl), 6.70 (s, 1H, CH, C4 of 4-methoxyphenyl), 7.24 (s, 1H, CH, C5 of 4-methoxyphenyl), 6.69 (s, 1H, CH, C6 of 4-methoxyphenyl), 7.20 (s, 1H, CH, C5); 13C-NMR (CDCl3, 500 MHz, δppm): 149.8 (4-pyridine C3 & C5), 122.8 ( 4-pyridine C2 & C6), 167.0 (C=O), 151.8 (pyrazole C3), 46.1 (pyrazole C4), 71.1 (pyrazole C6), 126.0 (4-methoxyphenyl C1), 130.0 (4-methoxyphenyl C2 & C6), 114.0 (4-methoxyphenyl C3 & C5), 163.0 (4-methoxyphenyl C4), 55.9 (O–CH3), 47.8 (thiazolidin-4-one C2), 34 (thiazolidin-4-one C5), 170.9 (thiazolidin-4-one C4 (C=O)), 134 (4-methoxyphenyl C1), 122.6 (4-methoxyphenyl C2 & C6), 114 (4-methoxyphenyl C3 & C5), 156.0 (4-methoxyphenyl C4), 55.9 (O-CH3).; LC–MS: m/z 486 (M+); Anal.: Calcd. for C26H22N4O4S C, 64.188; H, 4.568; N, 11.528; S, 6.598; O, 13.158; Found: C, 63.752; H, 4.576; N, 11.273; S, 6.320; O, 13.614..
2-(1-isonicotinoyl-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-4-yl)-3-(2-methoxyphenyl)thiazolidin-4-one (P-14) Yellow solid (84.3%); m.pt. 336–338 °C; IR (KBr, cm−1): 3217 (C–H str. Ar), 2968 (CH–S str.), 1681 (C=O str., pyrazole), 1735 (C=O str., thiazolidinone), 1645 (C=N str.), 1257 (Ar–N str.), 1174 (R–C-O str.), 1338, 1415 (C=N str.), 1310 (C–O–C asymm. str.), 1120 (C–O–C symm. str.); 1H NMR (500 MHz, DMSO) δ 9.06 (d, J = 6.3 Hz, 2H, CH, C2 & C6 of 4-pyridine), 7.96 (d, J = 6.3 Hz, 2H, CH, C3 & C5 of 4-pyridine), 33.43 (s, J = 5.6 Hz, 2H, C5 of thiazolidinone), 5.92 (d, 1H, CH, C2 of thiazolidinone), 7.3 (s, 1H, C5 of pyrazole), 6.82 (s, 1H, CH, C3 of 2-methoxyphenyl), 7.13 (s, 1H, CH, C4 of 2-methoxyphenyl), 7.87 (s, 1H, CH, C5 of 2-methoxyphenyl), 6.99 (s, 1H, CH, C6 of 2-methoxyphenyl), 3.73 (s, 3H, OCH3).
2-(3-(4-bromophenyl)-1-isonicotinoyl-1H-pyrazol-4-yl)-3-phenylthiazolidin-4-one (P-15) Brown solid (84.7%); m.pt. 256–258 °C; IR (KBr, cm−1): 3398 (C–H str. Ar), 2848 (CH–S str.), 1680 (C=O str., pyrazole), 1730 (C=O str., thiazolidinone), 1649 (C=N str.), 1226 (Ar-N str.), 1198 (R–C-O str.), 1420 (C=N str.), 560 (C–Br str.); 1H NMR (500 MHz, DMSO) δ 9.06 (d, J = 6.3 Hz, 2H, CH, C2 & C6 of 4-pyridine), 7.96 (d, J = 6.3 Hz, 2H, CH, C3 & C5 of 4-pyridine), 33.43 (s, J = 5.6 Hz, 2H, C5 of thiazolidinone), 5.92 (d, 1H, CH, C2 of thiazolidinone), 7.3 (s, 1H, C5 of pyrazole),7.10 (d, J = 4.5 Hz, 2H, CH, C2 & C6 of bromo phenyl ring), 7.31 (d, J = 4.7 Hz, 2H, CH, C3 & C5 of bromophenyl ring), 7.24 (m, 1H, C4 of bromophenyl).
2-(3-(4-bromophenyl)-1-isonicotinoyl-1H-pyrazol-4-yl)-3-(4-nitrophenyl)thiazolidin-4-one (P-16) Yellowish brown solid (68.9%); m.pt. 268–270 °C; IR (KBr, cm−1): 3398(C-H str. Ar), 2900 (CH-S str.), 1690 (C=O str., pyrazole),1740 (C=O str., thiazolidinone), 1649 (C=N str.), 1229 (Ar–N str.), 1154 (R–C–O str.), 1418 (C=N str.); 1520 (NO2 asymm. str.), 1310 (NO2 symm. str.), 1150 (Ar–N str.), 570 (C–Br str.).
2-(3-(4-bromophenyl)-1-isonicotinoyl-4,5-dihydro-1H-pyrazol-4-yl)-3-(2-nitrophenyl) thiazolidin-4-one (P-17) Brown solid (72.0%); m.pt. 252–254 °C; IR (KBr, cm−1): 3200 (C–H str. Ar), 2850 (CH–S str.), 1690 (C=O str., pyrazole), 1735 (C=O str., thiazolidinone), 1649 (C=N str.), 1226 (Ar-N str.), 1157 (R–C–O str.), 1418 (C=N str.); 1518 (NO2 asymm. str.), 1350 (NO2 symm. str.), 1198 (Ar-N str.), 580 (C–Br str.).
2-(3-(4-bromophenyl)-1-isonicotinoyl-1H-pyrazol-4-yl)-3-(4-chlorophenyl)thiazolidin-4-one (P-18) Brown solid (79.8%); m.pt. 244–246 °C; IIR (KBr, cm−1): 3215 (C–H str. Ar), 2936 (CH–S str.), 1680 (C=O str., pyrazole), 1735 (C=O str., thiazolidinone), 1560 (C=N str.), 1228 (Ar–N str.), 1157 (R–C–O str.), 1414 (C=N str.); 690 (C–Cl str.), 578 (C–Br str.); 1H NMR (500 MHz, DMSO) δ 9.06 (d, J = 6.3 Hz, 2H, CH, C2 & C6 of 4-pyridine), 7.96 (d, J = 6.3 Hz, 2H, CH, C3 & C5 of 4-pyridine), 33.43 (s, J = 5.6 Hz, 2H, C5 of thiazolidinone), 5.92 (d, 1H, CH, C2 of thiazolidinone), 7.3 (s, 1H, C5 of pyrazole), 7.5 (m, 4H, CH of C2, C3, C5 & C6 of 4-bromophenyl),7.04 (d, J = 4.6 Hz, 2H, CH, C2 & C6 of 4-chlorophenyl), 7.32 (d, J = 4.6 Hz, 2H, CH, C3 & C5 of 4- chlorophenyl).
2-(3-(4-bromophenyl)-1-isonicotinoyl-4,5-dihydro-1H-pyrazol-4-yl)-3-(3-chlorophenyl)thiazolidin-4-one (P-19) Brown solid (72.3%); m.pt. 248–250 °C; IR (KBr, cm−1): 3125 (C–H str. Ar), 2910 (CH–S str.), 1670 (C=O str., pyrazole), 1735 (C=O str., thiazolidinone), 1589 (C=N str.), 1224 (Ar–N str.), 1150 (R–C-O str.), 1410 (C=N str.); 694 (C–Cl str.), 570 (C–Br str.).
2-(3-(4-bromophenyl)-1-isonicotinoyl-1H-pyrazol-4-yl)-3-(4-methoxyphenyl) thiazolidin-4-one (P-20) Brown solid (83.2%); m.pt. 252–254 °C; IR (KBr, cm−1): 3120 (C-H str. Ar), 2924 (CH-S str.), 1669 (C=O str., pyrazole), 1740 (C=O str., thiazolidinone), 1589 (C=N str.), 1226 (Ar–N str.), 1159 (R–C–O str.), 1340, 1415 (C=N str.); 570 (C–Br str.), 1317 (asymm. C–O–C str.), 1090 (symm. C–O–C str.); 1H NMR (500 MHz, DMSO): δ 9.06 (d, J = 6.3 Hz, 2H, CH, C2 & C6 of 4-pyridine), 7.96 (d, J = 6.3 Hz, 2H, CH, C3 & C5 of 4-pyridine), 3.43 (s, J = 5.6 Hz, 2H, C5 of thiazolidinone), 5.92 (d, 1H, CH, C2 of thiazolidinone), 7.3 (s, 1H, C5 of pyrazole), 7.5 (m, 4H, CH of C2, C3, C5 & C6 of 4-bromophenyl), 6.61 (s, 1H, CH, C2 of 4-methoxyphenyl), 6.75 (s, 1H, CH, C4 of 4-methoxyphenyl), 7.20 (s, 1H, CH, C5 of 4-methoxyphenyl), 6.66 (s, 1H, CH, C6 of 3-methoxyphenyl), 3.73 (s, 3H, OCH3); 13C-NMR (CDCl3, 500 MHz, δppm): 149.8 (4-pyridine C3 & C5), 122.8 (4-pyridine C2 & C6), 167.0 (C=O), 151.8 (pyrazole C3), 46.1 (pyrazole C4), 71.1 (pyrazole C6), 133.0.0 (4-bromophenyl C1), 131.0 (4-bromophenyl C2 & C6), 131.0 (4-bromophenyl C3 & C5), 125.4 (4-bromophenyl C4), 47.8 (thiazolidin-4-one C2), 34 (thiazolidin-4-one C5), 170.9 (thiazolidin-4-one C4 (C=O)), 134 (4-methoxyphenyl C1), 122.6 (4-methoxyphenyl C2 & C6), 114 (4-methoxyphenyl C3 & C5), 156.0 (4-methoxyphenyl C4), 55.9 (O-CH3); LC–MS: m/z 535 (M+); Anal.: Calcd. for C25H19BrN4O3S: C, 56.088; H, 3.588; Br, 14.928; N, 10.468; S, 5.998; O, 8.968. Found: C, 56.078; H, 3.548; Br, 14.878; N, 10.438; S, 5.978; O, 8.938.
2-(3-(4-bromophenyl)-1-isonicotinoyl-4,5-dihydro-1H-pyrazol-4-yl)-3-(2-methoxy phenyl) thiazolidin-4-one (P-21) Yellowish brown solid (69.1%); m.pt. 260–262 °C; IR (KBr, cm−1): 3337 (C–H str., Ar), 2924 (CH–S str.), 1670 (C=O str., pyrazole), 1710 (C=O str., thiazolidinone), 1549 (C=N str.), 1225 (Ar–N str.), 1157 (R–C-O str.), 1338, 1415 (C=N str.), 590 (C–Br str.), 1311 (asymm. C–O–C str.), 1095 (symm. C–O–C str.); 1H NMR (500 MHz, DMSO) δ 9.06 (d, J = 6.3 Hz, 2H, CH, C2 & C6 of 4-pyridine), 7.96 (d, J = 6.3 Hz, 2H, CH, C3 & C5 of 4-pyridine), 3.43 (s, J = 5.6 Hz, 2H, C5 of thiazolidinone), 5.92 (d, 1H, CH, C2 of thiazolidinone), 7.3 (s, 1H, C5 of pyrazole), 7.5 (m, 4H, CH of C2, C3, C5 & C6 of 4-bromophenyl), 5.59 (s, 1H, CH, C2 of 2-methoxyphenyl), 6.25 (s, 1H, CH, C4 of 2-methoxyphenyl), 7.18 (s, 1H, CH, C5 of 2-methoxyphenyl), 6.66 (s, 1H, CH, C6 of 2-methoxyphenyl), 3.73 (s, 3H, OCH3); 13C-NMR (CDCl3, 500 MHz, δppm): 149.8 (4-pyridine C3 & C5), 122.8 ( 4-pyridine C2 & C6), 167.0 (C=O), 151.8 (pyrazole C3), 46.1 (pyrazole C4), 71.1 (pyrazole C-6), 133.0.0 (4-bromophenyl C1), 131.0 (4-bromophenyl C2 & C6), 131.0 (4-bromophenyl C3 & C5), 125.4 (4-bromophenyl C4), 47.8 (thiazolidin-4-one C2), 34 (thiazolidin-4-one C5), 170.9 (thiazolidin-4-one C4 (C=O)), 134 (4-methoxyphenyl C1), 120.4 (2-methoxyphenyl C2 & C6), 114 (4-methoxyphenyl C3 & C5), 152.0 (2-methoxyphenyl C4), 55.9 (O-CH3); LC–MS: m/z 535 (M+); Anal.: Calcd. for C25H19BrN4O3S: C, 56.088; H, 3.588; Br, 14.928; N, 10.468; S, 5.998; O, 8.968. Found: C, 56.088; H, 3.548; Br, 14.878; N, 10.438; S, 5.978; O, 8.938.
Biological evaluation
In vitro cytotoxicity assay/cell viability assay
When investigating a new drug, whether natural or synthesized, we should examine its safety for the host cell or the cytotoxic effect on the cancer cell. This is illustrious as the cell viability test. There are several methods to determine cell viability, among which the MTT test is a frequently used method. The MTT test was carried out using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) colorimetric test [33, 34]. MTT is the frequently used method to assess cell viability and cytotoxicity for drug detection. The MTT test based on the reduction of MTT (yellow color) depends on cellular metabolic activities due to H-dependent cellular NAD (P) oxidoreductase enzymes [33]. Healthy fast-growing cells show high rates of reduction of MTT to formazan while dead or inactive cells do not. The final product of MTT reduction is a purple formazan that can be easily dissolved in DMSO. The high intensity of the purple color indicates greater cell viability, while a decrease in the intensity of the purple color means a reduced number of cells and, therefore, the cytotoxicity of the given substance. The systematic conversion of MTT to formazan is illustrated in Fig. 5 [34].
Procedure [33, 34]
In the present study, systematic experimental steps as shown in Fig. 6 to determine the potential cytotoxicity of the drug at different concentrations using the MTT assay. The decrease in absorbance at 540 nm is shown in cells treated with an increasing drug concentration compared to control cells without any treatment. A decrease in absorbance in cells treated with drugs suggests cytotoxicity. The MTT test considerably helps investigators to determine whether one of the compounds to be tested has cell toxicity or antiproliferative activity [33, 34].
To test the anticancer activities of the synthesized compounds (selected based on docking studies), we evaluated the antiproliferative activity of compounds P (1–21) against HCT116 and MCF-7 cell lines. All the experiments were repeated at least thrice independently, the 50% inhibitory concentration (IC50) of new hybrids was determined (as the anticancer drug concentration causing a 50% reduction in cell viability) and calculated using a trendline equation. IC50 values of the new hybrids in μM against various cell lines are given in Table 2.
Aurora A kinase inhibition assay
The assay was carried out by the following method:
At 30 °C, dilute enzyme (aurora A), substrates (NADH, NaCl, MgCl2, PEP, LDH, PK, and histones H3), ATP, and inhibitors (selected compounds as per in silico studies) in kinase buffer (HEPES) and this to 96 well plates and incubate for 60 min. Once the ATP substrate was added, the reaction began. With the reaction, NADH was continuously transformed into NAD+ The activity of Aurora-A can be assayed by measuring the consumption of nicotinamide adenine dinucleotide plus hydrogen (NADH) at 340 nm. Absorbance at 340 nm was continuously recorded for 10 min. Inhibition of recombinant human Aurora-A by inhibitors was initially screened via an enzyme-coupled continuous spectrophotometric assay (Kishore et al., 2008; Aixia et al., 2011; Fancelli D et al., 2006). The aurora kinase inhibitor, VX-680 (Tozasertib) (Duong et al., 2016) was used as the positive control [12, 13, 20, 21, 36].
The pyrazole derivatives P (1–21) were examined for their capability to inhibit the activity of Aurora A kinase (Table 2) to clarify their structure–activity relationships [22, 23]. The IC50 value of compounds was calculated by using the trendline equation as shown in Table 2.
Docking studies
To study the binding mode and explore the molecular interactions between the ligands and protein, docking studies were carried out for the synthesized pyrazole derivatives with the saluted co-crystallized structure of Aurora-A Kinase by using AutoDock Vina and the graphical user interface, AutoDock Tools (ADT) installed on Windows-7 [38].
Following protocol was followed for the docking studies:
Ligand preparation The 2D structures of the designed pyrazole derivatives were drawn by using Chem Sketch (ACDLABS 12.0) and stored in a database in SDF structure format [39]. All the structures were converted to 3D structures with the help of 3D optimization tool i.e., Ligprep, and storing them as a PDBQT file.
Protein preparation The X-ray co-crystal structure of Aurora-A Kinase protein was obtained from the RCSB protein data bank (http://www.rcsb.org/pdb). After evaluating the numbers of entries, the best protein (PDB entry 2bmc) [25, 37] was selected by analyzing all the proteins and choosing one with the highest resolution i.e., 2.00 A The PDB file of Aurora Kinase A was abridged with the help of PyMOL and α chain was removed along with the complexed inhibitor. All water molecules and interacting ions were removed (The PyMOL Molecular Graphics System). The PDBQT file for the protein was generated with the help of Auto Dock Tools by the addition of all polar hydrogen atoms followed by charge assignment to the macromolecule.
Validation of docking protocol in glide The most suitable method of evaluating the accuracy of the docking procedure is to determine how closely the lowest energy pose predicted by the scoring function resembles an experimental binding mode as determined by X-ray crystallography.
Generation of grid and ligand docking Docking studies on designed derivatives prepared through Ligprep were carried out in the active site of the protein. The calculations of grid parameters were accomplished by the Grid tool in ADT. The grid parameter files possessing all the information about the size of grid, protein, ligand, and geometry of search space were prepared and saved as ‘Conf.txt’. The optimized ligand molecules in PDBQT format were docked in the active site of Aurora Kinase A with Auto Dock Vina. Docking runs were launched from the command line, followed by the generation and scoring of best poses, for every ligand using the scoring function. At the end of the docking, ligands with the most favorable free energy of binding were selected. The protein–ligand interactions were further analyzed for the docked ligands by using PyMOL and the best poses in the binding site were drawn [38, 39].