Chemical and instruments
The analytical grade chemicals and reagents were utilized by itself in experiments without any purification. Decibel melting point apparatus was adapted for monitoring the melting point of the synthesized compounds and are expressed as uncorrected. The thin-layer chromatography (TLC) was fascinated for observing the reaction progress. FT-IR (Diffuse Reflectance Method (DRS) -8000A, Shimadzu, Japan) spectrophotometer was utilized for recording infrared spectra and the Bruker Avance III, 400 MHz NMR spectrometer was employed for nuclear magnetic resonance spectra (1H NMR, 13C NMR, Chemical shift δ values- ppm). DPPH (High Media), Nutrient broth and Sabouard dextrose broth (Hi-Media) have been used for in vitro biological studies.
General procedure for synthesis of (E)-2-(3-(substitutedstyryl)-5-(substitutedphenyl)-4,5-dihydropyrazol-1-yl)benzo[d]thiazole derivatives (Z1-Z20)
Synthesis of (1E,4E)-1,5-bis (substitutedphenyl)penta-1,4-dien-3-one (III)
First of all, 40 mmol benzaldehyde (II) was taken in a round bottom flask and 20 ml of ethanol was added. After dissolution, 20 mmol acetone (I) was added in above mixture. The solution was vigorously stirred for 15 min on magnetic stirrer. RBF was placed in an ice bath for maintaining temperature 1–4 ºC and 20 ml of a freshly prepared 20% sodium hydroxide solution was added drop by drop into the solution with continuous stirring. After complete addition of 20% sodium hydroxide solution, the resulting mixture was continuously stirred for 1 h. The resultant product was neutralized by 10% HCl solution (approximately 50–70 ml). After neutralization the separated product was filtered, washed with water and then dried at room temperature [4].
Synthesis of 1-(benzo[d]thiazol-2-yl)hydrazine (V)
1.5 ml of hydrazine hydrate (99%) was taken in a 50 ml round bottom flask and 1.5 ml concentrated HCl was added drop by drop with stirring the flask at 5–10 °C temperature. After complete addition of conc. HCl, 15 ml of ethylene glycol was added slowly, mixed and 0.75 g of benzo[d]thiazol-2-amine was added. Then flask was vigorously shaken and refluxed for 3 h. Mixture was cooled at room temperature and the mixture was poured drop by drop into crushed ice to obtain solid precipitate, which were filtered off and dried [26].
Synthesis of (E)-2-(3-(substitutedstyryl)-5-(substitutedphenyl)-4,5-dihydropyrazol-1-yl)benzo[d]thiazole derivatives (Z1-Z20) (VI)
2 mmol of (1E,4E)-1,5-bis (substitutedphenyl)penta-1,4-dien-3-one (III) was taken in a 50 ml round bottom flask and 15 ml of glacial acetic acid was added and shaken vigorously to dissolve completely. Then 2 mmol of 1-(benzo[d]thiazol-2-yl)hydrazine (V) was added in the solution and refluxed until the completion of reaction monitored by TLC. The reaction was cooled at room temperature and pour the solution into crushed ice, drop by drop, to obtain solid precipitate. The product was filtered and washed it with cold water and dried [4].
Physicochemical and spectral characterization
(E)-2-(3-(4-Chlorostyryl)-5-(4-chlorophenyl)-4,5-dihydropyrazol-1-yl)benzo[d]thiazole (Z1): Yield: 66.6%; m.p.: 129–132 ºC; Rf: 0.7 (Benzene:Chloroform 5:5); FT-IR (KBr), vmax (cm−1): 2923.36, 2857 (C–H stretching aliphatic), 1650.30 (C = N stretching), 1536.28 (C = C stretching aliphatic), 1490.68 (C = C stretching aromatic), 1327.02 (C–N stretching), 753.74 (C–S–C stretching), 710 (C–Cl stretching); 1HNMR (400 MHz, CDCl3, δ ppm): 7.69–7.73 (d, 2H, C4″ of benzothiazole ring), 7.56–7.58 (d, 2H, C7″of benzothiazole ring), 7.40–7.43 (t, 3H, C5″ and C6″ of benzothiazole ring), 7.30–7.31 (d, 2H, C3 and C5 of phenyl ring A), 7.15–7.17 (d, 2H, C3 and C5 of phenyl ring B), 7.12–7.14 (d, 2H, C2 and C6 of phenyl ring B), 7.04–7.08 (d, 2H, C2 and C6 of phenyl ring A), 6.69–6.75 (dd, 2H, J 16 MHz, ethylene group), 5.79–5.84 (dd, Hx, C4ʹ of pyrazole ring), 3.78–3.85 (dd, 1Hb, C3ʹ of pyrazole ring), 3.14–3.19 (dd, 1Ha, C3ʹ of pyrazole ring). 13C NMR (300 MHz, CDCl3, δ, ppm), 188.47 (N = CS-N), 142.08 (C = N), 140.21 (C–S), 138.72 (C–N, pyrazooline), 136.53 (C–N, benzothiazole), 135.11 (C–Cl), 134.0.6, 133.22 (CH = CH), 129.56, 129.30, 129.26, 129.23, 128.28, 127.96, 126.37, 125.72, 122.89, 121.11, 120.51, 119.50, 63.57.
(E)-2-(3-(3-Chlorostyryl)-5-(3-chlorophenyl)-4,5-dihydropyrazol-1-yl)benzo[d]thiazole (Z2): Yield: 54.6%; m.p.: 90–93 ºC; Rf: 0.7 (Benzene:Chloroform 5:5); FT-IR (KBr), vmax (cm−1): 3062.39 (C–H stretching aromatic), 2925.49, 2860.4 (C–H stretching aliphatic), 1655.23 (C = N stretching), 1476.82 (C = C stretching aliphatic), 1429.08 (C = C aromatic stretching), 1320.09 (C–N stretching), 793.59 (C–S–C stretching), 711.90 (C–Cl stretching).
(E)-2-(3-(3-Nitrostyryl)-5-(3-nitrophenyl)-4,5-dihydropyrazol-1-yl)benzo[d]thiazole (Z3): Yield: 43.1%; m.p.: 102-105ºC; Rf: 0.6 (Toluene:Methanol 7:3); FT-IR (KBr), vmax (cm−1): 3084.43(C–H stretching aromatic), 2924.2, 2926.85 (C–H stretching aliphatic), 1630.89 (C = N stretching), 1564.41 (assym. NO2 stretching), 1526.81(C = C stretching aliphatic), 1478.26(C = C aromatic stretching), 1351.48 (sym. NO2 stretching), 1203.43 (C–N stretching), 736.35 (C–S–C stretching); 1HNMR (400 MHz, DMSO,d6 δ, ppm): 8.64 (s, 1H, C2 of phenyl ring A), 8.57 (s, 1H, C2 of phenyl ring B), 8.52–8.53 (t, 1H, C5 of phenyl ring B), 8.41–8.44 (d,1H,C4 of phenyl ring A),8.26–8.30 (t, 1H, C5 of phenyl ring A), 8.20–8.21 (d, 1H, C4 of phenyl ring B), 7.95–7.99 (d, 1H, C6 of phenyl ring A and B), 7.73–7.84 (t, 2H, C5 and C6 of benzothiazole ring), 7.69–7.73 (d, 1H, C4″ of benzothiazole ring), 7.56–7.60 (d, 1H, C7″ of benzothiazole ring), 6.00–6.05 (dd, 2H, J 12 MHz, ethylene group), 5.69–5.76 (dd, Hx, C4ʹ pyrazole ring), 3.40–3.45 (dd, 1Hb, C3ʹ of pyrazole ring), 3.26–3.32 (dd, 1Ha C3ʹof pyrazole ring).
(E)-2-(3-(4-Nitrostyryl)-5-(4-nitrophenyl)-4,5-dihydropyrazol-1-yl)benzo[d]thiazole (Z4): Yield: 27.8%; m.p.: 167–170 ºC; Rf: 0.8 (Toluene:Methanol 7:3); FT-IR (KBr), vmax (cm−1): 2941.7, 2857 (C–H stretching aliphatic), 1630.35 (C = N stretching), 1517.20 (Assym. NO2 stretching), 1444.43 (C = C aliphatic stretching), 1411.63 (C = C aromatic stretching), 1347.79 (Sym. NO2 Stretching), 1191.63 (C–N Stretching), 746.49 (C–S–C stretching).
(E)-2-(3-(2-Nitrostyryl)-5-(2-nitrophenyl)-4,5-dihydropyrazol-1-yl)benzo[d]thiazole (Z5): Yield: 36.8%; m.p.: 138-141ºC; Rf:0.8 (Benzene:Chloroform 5:5); FT-IR (KBr), vmax (cm−1): 2928.1, 2857 (C–H stretching aliphatic), 1625.76 (C = N stretching), 1570.77 (Assym. NO2 stretching), 1521.91(C = C aliphatic stretching), 1443.81(C = C stretching aromatic), 1344.93 (Sym. NO2 stretching), 1201.05 (C–N stretching), 748.79 (C–S–C stretching).
(E)-2-(3-(2-Chlorostyryl)-5-(2-chlorophenyl)-4,5-dihydropyrazol-1-yl)benzo[d]thiazole (Z6): Yield: 60%; m.p.: 111–114 ºC; Rf:0.6 (Benzene:Chloroform 5:5); FT-IR (KBr), vmax (cm−1): 3062.79 (C–H stretching aromatic), 2924.28 (C–H stretching aliphatic), 1660.66 (C = N stretching), 1565.52(C = C aliphatic stretching), 1471.72 (C = C stretching aromatic), 1316.26(C–N stretching), 752.45(C–S–C stretching), 692.04 (C–Cl stretching).
(E)-2-(3-(3-Hydroxystyryl)-5-(3-hydroxyphenyl)-4,5-dihydropyrazol-1-yl)benzo[d]thiazole (Z7): Yield: 75.6%; m.p.: 157–160 ºC; Rf:0.9 (Toluene:Methanol 7:3); FT-IR (KBr), vmax (cm−1): 3436.49 (OH str.), 2965.4, 2826.5 (C–H stretching aliphatic), 1620.83 (C = N stretching), 1539.90 (C = C aliphatic stretching), 1451.79 (C = C aromatic stretching), 1276.55 (C–N stretching),760.55(C–S–C stretching); 1HNMR (400 MHz, DMSO,d6 δ, ppm): 9.66 (s, OH), 8.05 (s, 2H, C2 of phenyl ring A and B), 7.79–7.81 (d, 1H, C4 of phenyl ring A), 7.67–7.71 (d, 1H, C4 of phenyl ring B), 7.05–7.82 (m, 4H, of benzothiazole ring), 6.86–6.88 (d, 2H, C6 of phenyl ring A and B), 6.64–6.68 (t, 2H, C5 of phenyl ring A and B), 6.70–6.76 (dd, 2H, J 16 MHz, ethylene group), 5.69–5.73(dd, Hx, C4ʹof pyrazole ring), 3.82–3.90 (dd, 1Hb, C3ʹ of pyrazole ring), 3.13–3.18 (dd, 1Ha, C3ʹ of pyrazole ring). 13C NMR (300 MHz, DMSO-d6, δ, ppm), 188.95 (N = CS–N), 162.49 (C–OH), 158.23 (C = N), 155.69 (C–S), 152.67 (C–N, pyrazooline), 143.51 (C–N, benzothiazole), 138.39, 137.57 (CH = CH), 136.49, 130.41, 126.02, 122.36, 121.71, 120.07, 118.19, 115.35, 115.10, 114.43, 63.30.
(E)-2-(3-(4-Hydroxystyryl)-5-(4-hydroxyphenyl)-4,5-dihydropyrazol-1-yl)benzo[d]thiazole (Z8): Yield: 53.7%; m.p.: 197–200 ºC; Rf:0.8 (Toluene:Methanol 7:3); FT-IR (KBr), vmax (cm−1): 3454.05 (OH str.), 2928.1,2874 (C–H stretching aliphatic), 1637.60 (C = N stretching), 1573.34 (C = C aliphatic stretching), 1450.39 (C = C aromatic stretching), 1264.79 (C–N stretching), 752.98 (C–S–C stretching).
(E)-2-(3-(4-Bromostyryl)-5-(4-bromophenyl)-4,5-dihydropyrazol-1-yl)benzo[d]-thiazole(Z9): Yield: 58.9%; m.p.: 148–151 ºC; Rf:0.8 (Benzene:Chloroform 5:5); FT-IR (KBr), vmax (cm−1): 2928.1,2850.2 (C–H stretching aliphatic), 1648.04 (C = N stretching), 1564.19 C = C aliphatic stretching), 1486.95 (C = C aromatic stretching), 1325.34 (C–N stretching), 754.57 (C–S–C stretching), 593.40 (C–Br stretching).
(E)-2-(3-(4-Methylstyryl)-5-(4-methylphenyl)-4,5-dihydropyrazol-1-yl)benzo[d]thiazole (Z10): Yield: 35.8%; m.p.: 143–146 ºC; Rf:0.62 (Benzene:Chloroform 5:5); FT-IR (KBr), vmax (cm−1): 2918, 2853.6 (C–H stretching aliphatic), 1621.40 (C = N stretching), 1540.90 (C = C aliphatic stretching), 1442.87 (C = C aromatic stretching), 1228.88 (C–N stretching), 749.58 (C–S–C stretching); 1HNMR (400 MHz, CDCl3, δ, ppm): 7.72–7.76 (d, 2H, C2, C6 of phenyl ring B), 7.64–7.646 (d, 2H, C2, C6 of phenyl ring A), 7.53–7.55 (d, 2H, C3, C5 of phenyl ring B), 7.38–7.40 (d, 2H, C3, C5 of phenyl ring A),7.05–7.28 (m, 4H, of benzothiazole ring), 6.68–6.75 (dd, 2H, J 16 MHz, ethylene group), 5.76–5.79 (dd, Hx,C4ʹ of pyrazole ring), 3.76–3.83 (dd, 1Hb, C3ʹ of pyrazole ring), 3.16–3.21 (dd, 1Ha, C3ʹ of pyrazole ring), 1.99 (s, 3H, CH3).
(E)-2-(3-(4-Methoxystyryl)-5-(4-methoxyphenyl)-4,5-dihydropyrazol-1-yl)benzo[d]-thiazole (Z11): Yield: 26%; m.p.: 96–99 ºC; Rf:0.53 (Benzene:Chloroform 5:5); FT-IR (KBr), vmax (cm−1): 2961.60, 2840 (C–H stretching aliphatic), 1631.46 (C = N stretching), 1511.24 (C = C aliphatic stretching), 1442.26 (C = C aromatic stretching), 1251.28 (C–N stretching), 1030.50 (OCH3 stretching), 757.24 (C–S–C stretching); 1HNMR (400 MHz, CDCl3, δ, ppm): 7.86–7.88 (d, 4H, C3, C5 of phenyl ring A and B), 7.59–7.61 (d, 4H, C2, C6 of phenyl ring A and B),6.95–7.02 (m, 4H, of benzothiazole ring), 6.69–6.76 (dd, 2H, J 16 MHz, ethylene group),3.88 (s, 3H, OCH3), 3.79 (d, Hx, C4ʹ of pyrazole ring), 2.39–2.41 (d, 1Hb, C3ʹ of pyrazole ring), 2.13–2.17 (d, 1Ha, C3ʹ of pyrazole ring).
(E)-2-(5-Phenyl-3-styryl-4,5-dihydropyrazol-1-yl)benzo[d]thiazole (Z12): Yield: 55.2%; m.p.:97–100 ºC; Rf:0.63 (Benzene:Chloroform 5:5); FT-IR (cm−1): 3056.38,3026.3 (C–H stretching aromatic), 2928.1,2857 (C–H stretching aliphatic), 1651.32 (C = N stretching), 1536.43 (C = C aliphatic stretching), 1447.94 (C = C aromatic stretching), 1195.50 (C–N stretching), 762.34 (C–S–C stretching); 1HNMR (400 MHz, CDCl3, δ, ppm): 7.75–7.79 (d, 1H, C4 of benzothiazole ring),7.64–7.66 (t, 2H, C5 and C6 of benzothiazole ring), 7.34–7.54, (m, 10H, phenyl ring A and B), 7.10–7.14 (d, 1H, C7 of benzothiazole ring), 6.73–6.78 (dd, 2H, J 16 MHz, ethylene group), 5.78–5.83 (dd, Hx, C4ʹ of pyrazole ring), 3.80–3.87 (dd, 1Hb, C3ʹ of pyrazole ring), 3.19–3.25 (dd, 1Ha, C3ʹ of pyrazole ring).
(E)-2-(3-(4-Fluorostyryl)-5-(4-fluorophenyl)-4,5-dihydropyrazol-1-yl)benzo[d]thiazole (Z13): Yield: 55.4%; m.p.:113–116 ºC; Rf:0.6 (Benzene:Chloroform 5:5); FT-IR (KBr), vmax (cm−1): 2928.1, 2850.2 (C–H stretching aliphatic), 1653.14 (C = N stretching), 1537.67(C = C aliphatic stretching 1414.98 (C = C aromatic stretching), 1280.22 (C–F stretching), 1193.39 (C–N stretching), 754.90 (C–S–C stretching); 1HNMR (400 MHz, CDCl3, δ, ppm): 7.70–7.75 (d, 2H, C3,C5 of phenyl ring A), 7.56–7.58 (d, 2H, C3 and C5 of phenyl ring B), 7.46–7.50 (d, 1H, C4″ of benzothiazole ring), 7.33–7.37 (d, 1H, C7″ of benzothiazole ring),7.11–7.17 (t, 2H, C5″ and C6″ of benzothiazole ring), 7.06–7.07 (d, 2H, C2 and C6 of phenyl ring B), 6.99–7.04 (d, 2H, C2 and C6 of phenyl ring A), 6.69–6.76 (dd, 2H, J 16 MHz, ethylene group), 5.84–5.88 (dd, Hx, C4ʹ of pyrazole ring),3.78–3.85 (dd, 1Hb, C3ʹ of pyrazole ring), 3.16–3.21 (dd, 1Ha, C3ʹof pyrazole ring).
(E)-2-(3-(3-Methoxystyryl)-5-(3-methoxyphenyl)-4,5-dihydropyrazol-1-yl)benzo[d]- thiazole (Z14): Yield:81.8%; m.p.:100–103 ºC; Rf:0.71 (Benzene:Chloroform 5:5); FT-IR (KBr), vmax (cm−1): 3056.8, 3000.18 (C–H stretching aromatic), 2936.24; 2835 (C–H stretching aliphatic), 1601.66 (C = N stretching), 1541.86 (C = C aliphatic stretching), 1435.70 (C = C aromatic stretching), 1261.77 (C–N stretching), 1046.74 (OCH3 stretching) 755.35 (C–S–C stretching).
(E)-2-(3-(2-Methoxystyryl)-5-(2-methoxyphenyl)-4,5-dihydropyrazol-1-yl)benzo[d]-thiazole (Z15): Yield: 92.5%; m.p.:96–99 ºC; Rf:0.55 (Benzene:Chloroform 5:5); FT-IR (KBr), vmax (cm−1): 3060.2 (C–H stretching aromatic), 2935.31 (C–H stretching aliphatic), 1614.99 (C = N stretching), 1538.13 (C = C aliphatic stretching), 1443.06 (C = C aromatic stretching), 1245.62 (C–N stretching), 1025.38 (OCH3 stretching), 751.56 (C–S–C stretching).
(E)-2-(3-(3-Bromostyryl)-5-(3-bromophenyl)-4,5-dihydropyrazol-1-yl)benzo[d]thiazole (Z16): Yield: 73.9%; m.p.: 201–204 ºC; Rf:0.67 (Benzene:Chloroform 5:5); FT-IR (KBr), vmax (cm−1): 3060.55,3016.2 (C–H stretching aromatic), 2972.2,2926.16 (C–H stretching aliphatic), 1617.02 (C = N stretching), 1561.29 (C = C aliphatic stretching), 1466.39 (C = C aromatic stretching), 1364 0.16 (C–N stretching), 752.39 (C–S–C stretching) 617.41 (C–Br stretching).
(E)-2-(3-(2,3-Dichlorostyryl)-5-(2,3-dichlorophenyl)-4,5-dihydropyrazol-1-yl)benzo-[d]thiazole (Z17): Yield: 82%; m.p.: 100–103 ºC; Rf:0.72 (Benzene:Chloroform 5:5); FT-IR (KBr), vmax (cm−1): 3070.4 (C–H stretching aromatic), 2931.5,2860.4 (C–H stretching aliphatic), 1618.00 (C = N stretching), 1450.93 (C = C stretching aliphatic), 1411.07 (C = C aromatic stretching), 1181.02 (C–N Stretching), 749.52(C–S–C stretching), 722.10 (C–Cl stretching).
(E)-2-(3-(2,6-Dichlorostyryl)-5-(2,6-dichlorophenyl)-4,5-dihydropyrazol-1-yl)benzo- [d]thiazole (Z18): Yield: 94.5%; m.p.: 205–208 ºC; Rf:0.52 (Benzene:Chloroform 5:5); FT-IR (KBr), vmax (cm−1): 3063.6(C–H stretching aromatic), 2931.5,2857(C–H stretching aliphatic), 1617.96(C = N stretching), 1437.90 (C = C stretching aliphatic), 1427.31 (C = C aromatic stretching), 1177.26 (C–N Stretching), 718.76 (C–Cl stretching), 746.32 (C–S–C stretching); 1HNMR (400 MHz, CDCl3, δ, ppm): 7.82–7.87 (d, 2H, C3, C5 of phenyl ring A), 7.69–7.71(d, 2H, C3, C5 of phenyl ring B), 7.36–7.42 (t, 1H, C4 of phenyl ring A),7.34–7.36 (t, 1H, C4 of phenyl ring B), 7.14–7.27 (m, 4H, of benzothiazole ring), 6.85–6.90 (dd, 2H, J 16 MHz, ethylene group), 6.42–6.47 (dd, Hx, C4ʹ of pyrazole ring), 3.77–3.85 (dd, 1Hb, C3ʹ of pyrazole ring), 3.38–3.45 (dd, 1Ha, C3ʹ of pyrazole ring).
(E)-2-(3-(2,5-Dimethoxystyryl)-5-(2,5-dimethoxyphenyl)-4,5-dihydropyrazol-1-yl) benzo[d]thiazole (Z19): Yield: 95.9%; m.p.:137–140 ºC; Rf:0.57 (Benzene:Chloroform 5:5); FT-IR (KBr), vmax (cm−1): 3002.6,3063.6 (C–H stretching aromatic), 2941, 2834.73(C–H stretching aliphatic), 1615.19(C = N stretching), 1446.47 (C = C stretching aliphatic), 1420.51 (C = C aromatic stretching), 1178.12 (C–N stretching), 1021.77 (OCH3 stretching), 747.32 (C–S–C stretching).
(E)-2-(3-(3,4,5-Trimethoxystyryl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihydropyrazol-1-yl) benzo[d]thiazole (Z20): Yield: 97.7%; m.p.: 95–98 ºC; Rf:0.74 (Benzene:Chloroform 5:5); FT-IR (KBr), vmax (cm−1): 2995.9 (C–H stretching aromatic), 2939.94,2838.24 (C–H stretching aliphatic), 1619.76 (C = N stretching), 1538.45, 1455.37 (C = C stretching aliphatic), 1418.06 (C = C aromatic stretching), 1187.75 (C–N Stretching), 1040.88 (OCH3 stretching), 757.07 (C–S–C stretching); 1HNMR (400 MHz, CDCl3, δ, ppm):7.67–7.71 (t, 1H, C6″ of benzothiazole ring), 7.57–7.59 (d, 1H, C4″ of benzothiazole ring), 7.14–7.16 (t, 1H, C5″ of benzothiazole ring), 6.98–7.02 (d, 1H, C7″ of benzothiazole ring), 6.87 (s, 2H, C2 and C6 of phenyl ring B), 6.70–6.76 (dd, 2H, J 16 MHz ethylene group), 6.56 (s, 2H, C2 and C6 of phenyl ring A), 5.70–5.74 (dd, Hx, C4 of pyrazole ring), 3.94 (s, OCH3 group), 3.89–3.90 (dd, 1Hb, C3 of pyrazole ring), 3.83–3.84 (dd, 1Ha, C3 of pyrazole ring).
In vitro biological evaluation
Anti-oxidant activity
The different concentrations (500, 250, 125, 62.5 and 31.25 µg/ml) of synthesized compounds (Z1–Z20) in DMSO were prepared and 1 ml of sample was taken in a test tube, 1 ml of DPPH solution was added in each test tube and a purple color was observed. The test tubes were placed in dark chamber for 30 min, purple color changed into yellow and after 30 min absorbance was determined by UV spectroscopy at 517 nm wavelength. DMSO was used as blank to set zero [39].
Anti-inflammatory activity
The synthesized compounds (Z1–Z20) were used for the preparation of different concentrations (500, 250, 125, 62.5 and 31.25 ug/ml) in DMSO and 1 ml of each resulting solutions was taken in different test tubes. Then 1.4 ml of freshly prepared phosphate buffer (pH 6.4) and 0.1 ml egg albumin from fresh egg was transferred in each test tube containing different solutions for determining anti-inflammatory activity. The resulting mixtures in test tubes were incubated in a BOD for 15 min at 37 ± 2 ºC and then heated for 5 min at 70 ºC temperature. The mixture of test tubes was cooled at room temperature and absorbance was determined by UV spectroscopy at 660 nm wavelength [40].
Antimicrobial activity
1 ml of test sample was taken in a test tube having 1 ml of nutrient medium and serial dilutions of 50, 25, 12.5, 6.25 and 3.125 µg/ml were prepared. Then inoculation of test strains was done by micropipette and incubated at 37 ºC for 24 h for bacterial strains and 48 h for C. albicans and 120 h for R. oryzae. Results were calculated by visual turbidity observed in test tubes. MIC was calculated by using lowest concentration that inhibits microbial growth [41, 42].
Molecular docking
AutoDock Vina, the advanced docking program was employed to estimate the binding charcaterstics of synthesized compounds into the active sites of target protein [38, 43]. The crystal structures of PDB: 6COX Cyclooxygenase-2 (prostaglandin synthase-2) complexed with a selective inhibitor, SC-558 IN I222 space group) [44], PDB: 2CAG (Catalase compound II) [13], PDB:1U4G, Elastase of P. aeruginosa with an inhibitor [45] and PDB:1EA1 (cytochrome P450 14 alpha-sterol demethylase (CYP51) from Mycobacterium tuberculosis in complex with fluconazole [46] were retrieved from the protein data bank (www.rcsb.org). AutoDock tools were utilized for the enlightenment of A chain of the proteins in pdbqt format. Water molecules which did not participate in interactions were removed and polar hydrogen atoms were introduced. The 2D structures of ligands were figured in MarvinSketch and saved in mol2 format, and then AutoDock tools were utilized to convert into pdbqt format. Energy minimization was accomplished using MMFF94 force field. The docking studies were executed according to requisite conditions of grid box by AutoDock tools. The search grid was identified as center_x = 21.72, center_y = 23.606, center_z = 47.846 (PDB:6COX); center_x = 58.613, center_y = 15.29, center_z = 16.972 (PDB:2CAG); center_x = 19.067, center_y = 26.357, center_z = − 4.427 (PDB:1U4G); center_x = − 16.172, center_y = − 5.396, center_z = 62.468 (PDB:1EA1), for target proteins with dimension size_x = 60, size_y = 60, size_z = 60, respectively. The exhaustiveness was set to be 8. The results were visualized using PyMol and Discovery studio visualizer [47].
Pharmacokinetic parameters
ADMET analysis of synthesized compounds was performed by Molinspiration online tool kit, OSIRIS property explorer and Pre ADMET online server [48,49,50].