3. Experimental Section
3.1. General Information
Melting points were measured in open capillary tubes using a Griffin apparatus and are uncorrected. Structures of compounds were confirmed by routine spectrometric analysis. Elemental analyses were carried results were within ±0.4% of the theoretical values. Infrared spectra were recorded on a 435 IR spectrophotometer (Shimadzu Bruker, Tokyo, Japan) using KBr discs. 1H-NMR and 13C-NMR spectra were obtained on a Gemini 500 MHz spectrophotometer (Varian, Polo Alto, Ca, USA) or on a Bruker 500 MHz spectrophotometer, and measured in δ scale using TMS as an internal standard. Mass Spectra were recorded on a 5988 spectrometer (Hewlett Packard, California, USA). Analytical thin layer chromatography (TLC) was performed using silica gel aluminum sheets, 60 F254 (E. Merck, Darmstadt, Germany) for the progress of reactions and visualization with ultraviolet light (UV) at 365 and 254 nm.
3.2. 3-(1-(3-Chlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazol-4-yl)-1-(thiophen-2-yl)prop-2-en-1-one (2)
A mixture of carbaldehyde derivative 1 (0.01 mol) and 2-acetylthiophene (0.01 mol) in of 30% ethanolic solution of NaOH (40 mL) was stirred for 12 h at room temperature. The progress of reaction was monitored by TLC. After completion, the reaction mixture was poured into acidified ice cold water of pH~2. The precipitated solid was filtered, washed with water and recrystallized to afford compound 2 in 87% yield; m.p. 144–146 °C (EtOH); IR (KBr) ν: 3079 (CH-Ar), 1641 (C=O), 1600 (C=C) cm−1; 1H-NMR (DMSO-d6): δ 3.81 (s, 3H, OCH3); 7.00–7.98 (m, 12H, ArH + CH=CH), 8.73 (s,1H, CH of pyrazole) ppm; 13C-NMR (DMSO-d6): δ 55.60, 114.34, 116.62, 117.75, 125.11, 125.95, 127.67, 129.08, 129.81, 131.74, 133.85, 134.47, 135.44, 141.08, 144.32, 151.54, 159.58, 191.96 ppm; MS (EI, 70 eV): m/z (%): 420 (11) [M]+; Anal. Calcd for C23H17ClN2O2S (420.91): C, 65.63; H, 4.07; N, 6.66; Found: C, 65.59; H, 4.16; N, 6.72.
3.3. 1-(3-Chlorophenyl)-4-(4,5-dihydro-3-(thiophen-2-yl)-1H-pyrazol-5-yl)-3-(4-methoxyphenyl)-1H-pyrazole (3)
To a solution of compound 2 (0.01 mol) in ethanol (30 mL) containing a catalytic amount of glacial acetic acid, a solution of hydrazine hydrate (98%, 0.5 mL) was added and the mixture was refluxed for 6 h. The reaction mixture was cooled to room temperature and the precipitated solid was filtered, dried and recrystallization provided compound 3 in 57% yield; m.p. 175–178 °C (EtOH); IR (KBr) ν: 3177 (NH), 1590 (C=C) cm−1; 1H-NMR (DMSO-d6): δ 2.99 (dd, 1H, CH), 3.70 (s, 3H, OCH3), 3.84 (dd, 1H, CH), 5.42 (dd, 1H, CH), 6.39–7.77 (m, 11H, Ar-H), 8.91 (s, 1H, CH of pyrazole), 11.52 (s, 1H, NH D2O exchangeable) ppm; 13C-NMR (DMSO-d6): δ 42.10, 55.61, 60.74, 114.22, 116.78, 117.76, 125.18, 125.90, 126.17, 127.38, 128.67, 129.65, 130.87, 131.26, 134.53, 137.55, 140.98, 151.24, 159.73, 158.76, 161.48 ppm; MS (EI, 70 eV): m/z (%): 434 (18) [M]+; Anal. Calcd for C23H19ClN4OS (434.94): C, 63.51; H, 4.40; N, 12.88; Found: C, 63.59; H, 4.53; N, 12.92.
3.4. 1-(5-(1-(3-Chlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazol-4-yl)-4,5-dihydro-3-(thiophen-2yl)pyrazol-1-yl)-ethanone (4)
To a solution of compound 2 (0.01 mol) in glacial acetic acid (20 mL), hydrazine hydrate (0.01 mol) was added and the mixture was refluxed for 3 h. The reaction mixture was cooled to room temperature and the solid formed was filtered off, dried and recrystallized to get compound 4 in 65% yield; m.p. 150–154 °C (EtOH); IR (KBr) ν: 3080 (CH-Ar), 1677 (C=O), 1619 (C=N), 1588 (C=C) cm−1; 1H-NMR (DMSO-d6): 2.43 (s, 3H, COCH3), 3.30 (dd, 1H, CH), 3.81 (s, 3H, OCH3), 3.88 (dd, 1H, CH), 5.57 (dd, 1H, CH), 6.70–7.65 (m, 11H, Ar-H), 9.18 (s, 1H, CH of pyrazole) ppm; 13C-NMR (DMSO-d6): δ 24.01, 43.81, 55.60, 63.87, 114.26, 116.84, 117.62, 125.35, 125.86, 126.10, 127.56, 129.14, 129.78, 130.69, 131.08, 134.41, 137.82, 140.84, 150.39, 155.19, 159.12, 160.92, 168.19 ppm; MS (EI, 70 eV): m/z (%): 476 (43) [M]+; Anal. Calcd for C25H21ClN4O2S (476.98): C, 62.95; H, 4.44; N, 11.75; Found: C, 63.02; H, 4.35; N, 11.81.
3.5. 5-(1-(3-Chlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazol-4-yl)-4,5-dihydro-3-(thiophen-2-yl)-pyrazole-1-carbothioamide (5)
To a mixture of chalcone 2 (0.01 mol) in absolute ethanol (30 mL), sodium hydroxide (1 g, 0.025 mol) was added. The reaction mixture was heated under reflux for 5 h. The contents were reduced, cooled and poured onto crushed ice. The resulting precipitate was collected by filtration and recrystallized to give in 61% yield; m.p. >300 °C (MeOH); IR (KBr) ν: 3407 (NH2), 1658 (C=N), 1523 (C=C), 1078 (C=S) cm−1; 1H-NMR (DMSO-d6): 3.13 (dd, 1H, CH), 3.84 (s, 3H, OCH3), 4.14 (dd, 1H, CH), 5.51 (dd, 1H, CH), 7.14–8.02 (m, 11H, Ar-H), 9.12 (s, 1H, CH of pyrazole), 9.93 (s, 2H, NH2 D2O exchangeable) ppm; 13C-NMR (DMSO-d6): δ 43.14, 55.64, 62.93, 114.33, 116.72, 117.58, 125.37, 125.80, 126.08, 127.54, 128.79, 129.22, 130.65, 131.16, 138.12, 137.82, 140.85, 151.23, 156.19, 159.30, 161.02, 176.55 ppm; MS (EI, 70 eV): m/z (%): 494 (6) [M]+; Anal. Calcd for C24H20ClN5OS2 (494.03): C, 58.35; H, 4.08; N, 14.18; Found: C, 58.27; H, 4.12; N, 14.23.
3.6. 2-Amino-4-(1-(3-chlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazol-4-yl)-1,6-dihydro-6-oxopyrimidine-5-carbonitrile (6a) and 5-acetyl-2-amino-6-(1-(3-chlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazol-4-yl)-pyrimidin-4(3H)-one (6b)
A mixture of compound 1 (0.01 mol), ethyl cyanoacetate or ethyl acetoacetate (0.01 mol) and (5 mL) of 40% ethanolic sodium hydroxide was stirred for 10 min, followed by addition of guanidine hydrochloride (0.01 mol) and the heating continued under refluxed for 3 h. The reaction mixture was diluted with ice-water and the formed precipitate was collected by filtration, washed several times with water, dried and recrystallized to afford the title compounds 6a,b.
2-Amino-4-(1-(3-chlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazol-4-yl)-1,6-dihydro-6-oxopyrimidine-5-carbonitrile (6a). Yield 64%; m.p. 144–148 °C (EtOH); IR (KBr) ν: 3341, 3145 (NH2, NH), 2219 (C≡N), 1663 (C=O), 1599 (C=C) cm−1; 1H-NMR (DMSO-d6): 2.85 (s, 2H, NH2 D2O exchangeable), 3.88 (s, 3H, OCH3), 7.03–8.75 (m, 9H, Ar-H and NH D2O exchangeable), 9.20 (s, 1H, CH of pyrazole) ppm; 13C-NMR (DMSO-d6): δ 55.63, 113.75, 115.31, 116.79, 117.77, 123.48, 125.21, 126.4, 127.94, 128.30, 129.82, 130.80, 134.57, 140.89, 155.04, 159.88, 160.60, 164.33, 171.88 ppm; MS (EI, 70 eV): m/z (%): 418 (14) [M]+; Anal. Calcd for C21H15ClN6O2 (418.84): C, 60.22; H, 3.61; N, 20.07; Found: C, 60.26; H, 3.58; N, 20.15.
5-Acetyl-2-amino-6-(1-(3-chlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazol-4-yl)pyrimidin-4-(3H)-one (6b). Yield 68%, m.p. 135–137 °C (EtOH); IR (KBr) ν: 3410, 3152 (NH2,NH), 1675 (C=O), 1589 (C=C) cm−1; 1H-NMR (DMSO-d6): 2.30 (s, 2H, NH2 D2O exchangeable), 2.39 (s, 3H, CH3), 3.86 (s, 3H, OCH3), 6.90–7.96 (m, 9H, Ar-H and NH exchangeable with D2O), 9.14 (s, 1H, CH of pyrazole) ppm; 13C-NMR (DMSO-d6): δ 24.38, 55.60, 113.72, 115.94, 116.85, 117.73, 125.10, 126.01, 128.36, 130.76, 132.95, 134.47, 141.05, 151.14, 154.78, 159.60, 160.82, 164.21, 165.38, 182.70 ppm; MS (EI, 70 eV): m/z (%): 435 (7) [M]+; Anal. Calcd for C22H18ClN5O3 (435.86): C, 60.62; H, 4.16; N, 16.07; Found: C, 60.59; H, 4.11; N, 16.12.
3.7. 1-(3-Chlorophenyl)-3-(4-methoxyphenyl)-4-(3-(thiophen-2-yl)isoxazol-5-yl)-1H-pyrazole (7)
A mixture of compounds 2 (0.01 mol) and hydroxylamine hydrochloride (0.01 mol) in ethanol (30 mL) containing sodium hydroxide solution (0.5 g NaOH in 0.5 mL water) was refluxed for 3 h. The reaction mixture was poured onto ice-water, neutralized with drops of conc. Hydrochloric acid and the solid precipitate formed filtered off, washed with water and recrystallized to yield the desired compound 7 in 73% yield; m.p. >300 °C (EtOH); IR (KBr) ν: 3064 (CH-Ar), 1601 (C=C) cm−1; 1H-NMR (DMSO-d6): 3.99 (s, 3H, OCH3), 7.30 (d, 2H, H J = 20), 7.60 (d, 1H, CH); 7.75–8.45 (m, 10H, Ar-H), 9.15 (s, 1H, CH of pyrazole) ppm; 13C-NMR (DMSO-d6): δ 43.19, 55.61, 73.10, 114.38, 116.72, 117.68, 125.68, 125.84, 126.10, 127.43, 129.07, 129.57, 130.81, 131.29, 134.47, 138.21, 140.85, 151.20, 154.63, 160.92, 162.89 ppm; MS (EI, 70 eV): m/z (%): 433 (12) [M]+; Anal. Calcd for C23H16ClN3O2S (433.91): C, 63.66; H, 3.72; N, 9.68; Found: C, 63.74; H, 3.79; N, 9.71.
3.8. 4-(1-(3-Chlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazol-4-yl)-6-(thiophen-2-yl)pyrimidin-2-amine (8)
An aqueous solution 5 mL of 40% sodium hydroxide was added gradually during a period of 3 h to a mixture of chalcone 2 (0.01 mol) and guanidine sulfate (0.01 mol) in ethanol (25 mL). The reaction mixture was refluxed for 5 h and the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was poured onto ice-cold water and the solid product formed was collected by filtration, washed with water then recrystallized to get compound 8 in 67% yield; m.p. >300 °C (MeOH); IR (KBr) ν: 3347 (NH2); 1632 (C=N); 1589 (C=C) cm−1; 1H-NMR (DMSO-d6): 3.80 (s, 3H, OCH3), 6.68-8.08 (t, 12H, Ar-H), 8.93 (s,1H, CH of pyrazole), 10.18 (s, 2H, NH2 D2O exchangeable) ppm; 13C-NMR (DMSO-d6): δ 55.64, 82.10, 114.31, 116.80, 117.59, 125.30, 125.78, 126.09, 127.41, 128.67, 129.48, 130.79, 131.16, 134.49, 139.86, 141.05, 150.88, 152.10, 160.37, 164.21, 166.58, 164.25 ppm; Anal. Calcd for C24H18ClN5OS (459.95): C, 62.67; H, 3.94; N, 15.23; Found: C, 62.63; H, 3.88; N, 15.29.
3.9. 6-(1-(3-Chlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazol-4-yl)-4-(thiophen-2-yl)pyrimidine-2-(1H)-thione (9)
A solution of the chalcone 2 (0.01 mol), thiourea (0.01 mol) and sodium hydroxide (0.1 g) in absolute ethanol (30 mL) was refluxed for 6 h. The reaction mixture was concentrated under vacuum, cooled and neutralized with dilute HCl. The formed product was filtered off, washed with water and recrystallized to get compound 9 in 76% yield; m.p. >300 °C (MeOH); IR (KBr) ν: 3422 (NH), 1595 (C=C), 1176 (C=S) cm−1; 1H-NMR (DMSO-d6): 3.82 (s, 3H, OCH3), 6.90–8.60 (m, 12H, Ar-H), 9.20 (s, 1H, CH of pyrazole), 9.95 (s, 1H, NH D2O exchangeable) ppm; 13C-NMR (DMSO-d6): δ 55.63, 104.56, 113.82, 115.89, 117.66, 125.38, 125.70, 126.03, 127.38, 128.34, 129.45, 130.77, 131.09, 134.42, 138.17, 140.95, 150.76, 157.21, 160.85, 161.80, 162.46, 184.33 ppm; MS (EI, 70 eV): 477 (8) [M]+; Anal. Calcd for C24H17ClN4OS2 (477): C, 60.43; H, 3.59; N, 11.75; Found: C, 60.38; H, 3.66; N, 11.82.
3.10. Measurement of Anticancer Activity
The experimental method used in anticancer screening has been adopted by U.S. National Cancer Institute according to reported standard procedure [
28,
29,
30].