3.1. Instrumentations
Melting points were measured using a Gallan-Kamp device and are uncorrected. The compounds' purity was confirmed by TLC and spectroscopic examination.
IR spectra were obtained using a Shimadzu 470 IR-spectrophotometer (KBr; vmax in cm-1). The 1HNMR and 13C NMR spectra were obtained on a Varian A5 500 MHz spectrometer with DMSO-d6 as the solvent and tetramethylsilane (TMS) as the internal reference. Coupling constants (J values) are expressed in Hertz. Elemental studies were carried out using a Perkin Elmer 2400 LS Series CHN/O analyzer.
Chemicals used in this work include ortho nitrobenzaldhyde, cyanothioacetamide, piperidine, methyl iodide, ethyl chloroacetate, 2-chloroacetamide, chloroacetonitrile, ethanol, and sodium acetate.3H2O, sodium carbonate) were acquired from Sigma Aldrich CO.
The human liver cancerous cell line (HEPG2), the human colon cancerous cell line(HCT116), and the human normal skin cell line (HSF) were obtained from national cancer institute, Cairo, Egypt.
3.2.1. 7-Acetyl-4-cyano-1,6-dimethyl-6-hydroxy-8-(2-nitrophenyl)-5,6,7,8-tetra-hydroisoquinoline-3(2H)-thione (2).
A mixture of compound 1 (3. 3 g, 10 mmol), 2-cyanothioacetamide (1.0 g, 10 mmol) and piperidine or morpholine (2 mmol) in ethanol (100 mL) was refluxed for 2 h. The yellow crystals that formed on hot were collected, washed with methanol, dried in air to give compound 2. Yield: 95 %; m. p: 280 °C. IR: 3411 (O-H); 3278 (N-H); 2979, 2922 (C-H, sp2); 2219 (C≡N); 1690 (C=O, acetyl). 1H NMR: δ 13.82 (s, 1H, NH); 7.3-7.82 (dd, J = 160.0, 98.3 Hz, 4H, Ar-H); 4.95- 5.03(d, J =38.8 Hz, 2H, OH, C8H), 3.08-3.11 (d, J =16.0 Hz, 2H, C5H, C7H), 2.82-2.85 (d, J =17.5 Hz, 1H, C5H); 1.94 (s, 6H, CH3, COCH3);1.25 (s, 3H, CH3). Anal. calcd for C20H19N3O4S (397.1): C, 60.44; H, 4.82; N, 10.57, Found C, 60.70; H, 4.80; N, 10.78%.
Reaction of compound 2 with ethyl iodide, ethyl chloroacetate, 2-chloroacetamide or its N-aryl-2-chloroacetamides 8a-e; Synthesis of compounds 3, 4, 5 and 9a-e; general method.
A mixture of 2 (3.3 g, 10 mmol) appropirate halocompound (10 mmol) and sodium acetate trihydrate (1.50 g, 11 mmol) in ethanol (100 mL) was refluxed for one hour. The solid that formed after cooling was collected and then recrystallized from ethanol to give white crystals of compounds 3, 4, 5 and 9a-e.5.
3.2.2. 7-Acetyl-4-cyano-1, 6-dimethyl-3-ethylthio-6-hydroxy-8-(2-nitrophenyl)-5,6, 7,8-tetrahydroisoquinoline (3): It was synthesized by reaction of 2 with ethyl iodide. Yield: 87 %;170 m.p.: °C. IR: 3515 (O-H); 3066-2965 (N-H); 2932-2896 (C-H, sp2); 2217 (C≡N); 1705 (C=O, acetyl). 1H NMR: 7.19-7.82 (dd, J = 18 Hz, 17 Hz, 4H, Ar-H); 5.09 (d, J = 30 Hz , 1H,OH) , 4.96- (s, J =38.8 Hz, 1H, C8H), 3.12-3.19 (dt, J =12.6, 6.8 Hz , 4H, CH2, C5H, C7H), 2.82-2.87 (dd, J =17.2, 5.5 Hz , 1H, C5H); 1.95-2.02 (m, 6H, COCH3, CH3);1.23 (s, 6H, 2 CH3). Anal. calcd for C22H23N3O4S ( 425.1): C, 62.10; H, 5.45; N, 9.88. Found C, 61.85; H, 5.87; N, 10.10 %.
3.2.3. Ethyl 2-[(7-acetyl-4-cyano-1,6-dimethyl-6-hydroxy-8-(2-nitrophenyl)-5,6,7,8-tetra-hydroisoquinolin-3-yl)thio]acetate (4): It was synthesized by reaction of 2 with ethyl chloroacetate. Yield: 91%; m.p.: 182°C. IR: 3445 (O-H); 2992(N-H); 2968, 2944 (C-H, sp2); 2214 (C≡N); 1728,1714 (C=O, acetyl). 1H NMR : 7.62 (s, J = 8.0 Hz, 1H, Ar-H); 7.60 (d, J = 7.6 Hz , 1H, Ar-H); 7.52(d, J = 8.0 Hz , 1H, Ar-H); 7.19-(d, J = 7.8 Hz , 1H, Ar-H); 5.13 ( d, J = 9.7 Hz 1H,OH) , 4.95(d, J = 33.2 Hz, 1H, C8H),4.05-4.09( m, 4H, 2CH2 acetate) 3.16(t, J =12.6, 6.8 Hz , 2H, C5H, C7H), 2.90 (d, J = 17.3 Hz, 1H, C5H);1.95 (sd, J = 18, 16 Hz 6H,CH3, COCH3 ), 1.30 (m, 3H, CH3), 1.13(t, J = 7.1 Hz, 3H, CH3).
13C NMR (101 MHz, DMSO) δ 209.16, 168.94, 151.09, 149.34, 137.30, 134.13, 131.62, 129.01128.8, 125.31, 115.31, 104.52, 68.13, 65.05, 61.42, 43.48, 32.53, 31.84, 28.07, 24.03, 14.44.
Anal. calcd for C24H27N3O6S( 485.2) : C, 59.37; H, 5.60; N, 8.65. Found C, 57.95; H, 5.77; N, 8.80%.
3.2.4. 2-[(7-Acetyl-4-cyano-1,6-dimethyl-6-hydroxy-8-(2-nitrophenyl)-5,6,7,8-tetra-hydroisoquinolin-3-yl)thio]acetamide (5): It is synthesized by reaction of 2 with chloroacetamide. Yield: %;95 m.p.:210 °C. IR: IR: 3426 (O-H); 3312 (N-H); 2965 (C-H, sp2); 2222 (C≡N); 1711 (C=O, acetyl). 1H NMR : δ 7.82 (s, J = 8.0 Hz, 1H, 1Ar-H) ; 7.57 -7.48 (d, J = 7.6 Hz , 4H, 2Ar-H, NH2); ; 7.15-(d, J = 10 Hz , 1H, 1Ar-H); 5.09 ( d, J = 9.7 Hz. 2H, OH, C8H), 3.86(m, 2H, C5H, C7H,), 3.15(dd, J =12.6, 2H, CH2 acetate), 2.84 (d, J = 17.3 Hz, 1H, C5H), 1.98 (s, 6H, COCH3, CH3), 1.25(s, 3H, CH3).
Anal. calcd for C22H22N4O5S (454.13): C, 58.14; H, 4.88; N, 12.33%. Found: C, 58.55; H, 4.95; N, 12.49%.
3.2.5. ethyl 7-acetyl-1-amino-8-hydroxy-5,8-dimethyl-6-(2-nitrophenyl)-6,7,8,9-tetrahydrothieno[2,3-c]isoquinoline-2-carboxylate (6): It was synthesized by reaction of 2 with ethyl chloroacetate. Yield: 88%; m.p.: 285°C. IR: 3425 (O-H); 3343(N-H); 2981(C-H, sp2); 1728,1715,1705 (C=O, acetyl). 1H NMR : 10.45( s, IH, NH), 7.83 (s, J = 10.0 Hz, 1H, Ar-H); 7.69 (d, J = 7.6 Hz , 2H, 2Ar-H); 7.60(d, J = 8.0 Hz , 1H, Ar-H); 7.19-(d, J = 7.8 Hz , 2H, NH2); 5.13 ( d, J = 9.7 Hz 1H,OH) , 4.95(d, J = 33.2 Hz, 1H, C6H),4.05-4.09( m, 4H, C9H, C7H , CH2 acetate) 3.16(d, J =12.6, 6.8 Hz , 1H, C9H), 2.90 (d, J = 17.3 Hz, 3H, CH3 at C5H);1.95 (sd, J = 18.5 Hz 3H,CH3, COCH3 ), 1.30 (m, 3H, CH3), 1.13(t, J = 7.1 Hz, 3H, CH3).
Anal. calcd for C24H27N3O6S (485.2): C, 59.37; H, 5.60; N, 8.65. Found C, 58.75; H, 5.69; N, 8.92 %.
3.2.6. 7-acetyl-4-cyano-1,6-dimethyl-6-hydroxy-8-(2-nitrophenyl)-5,6,7,8-tetra-hydroisoquinolin-3-yl)thio]acetoniitrile (7): It was synthesized by reaction of 3a with chloroacetoniitrile. Yield: 93 %; m.p.:178-180 °C. IR: 3463 (O-H); 3332,3229 (N-H); 2961, 2915 (C-H, sp2); 2202 (C≡N); 1709 (C=O, acetyl). 1H NMR: δ, 7.82( t, J = 63.0, 98.3 Hz, 1H,1 Ar-H),7.59 (t, J = 60.0, 58.3 Hz, 1H, 1Ar-H), 7.49(d, J = 60.0, 68.3 Hz, 1H, 1Ar-H), 7.15 (d, J = 61.0, 48.3 Hz, 1H,1 Ar-H); 6.52( s, 2H,CH2), 5.21 (d, J =38.8 Hz, 1H, OH), 4.74(s, 1H, C8H), 3.55 (d, J =16.0 Hz , 1H, C5H), 3.35(d, 1H C7H),3.14(d, J =17.5 Hz , 1H, C5H); 2.07 (d, 6H, COCH3, CH3);1.32 (s, 3H, CH3).
13C NMR (101 MHz, DMSO) δ 209.67 ,158.96, 158.37, 152.52, 149.51, 144.26, 137.83, 133.86, 131.71 ,128.81, 128.38, 125.24, 121.49, 116.22, 73.89, 67.82, 64.80 , 41.95 , 31.83 , 28.40 , 24.15 .
Anal. calcd for C23H20N4O5S(464.1): C, 59.47; H, 4.34; N, 12.06. Found C, 59.71; H, 4.22; N, 12.21 %.
3.2.7. 2-[(7-Acetyl-4-cyano-1,6-dimethyl-6-hydroxy-8-(2-nitrophenyl)-5,6,7,8-tetra-hydroisoquinolin-3-yl)thio]-N-phenylacetamide (9a). It was synthesized by reaction of 2 with N-phenyl-2-chloroacetamide (9a).Yield : 96%; m.p.: 186 °C. IR: 3476 (O-H); 3308, 3139 (N-H); 2970, 2918 (C-H, sp2); 2216 (C≡N); 1687(C=O, acetyl).
1H NMR (400 MHz, DMSO) δ 10.19 (s, 1H, NH), 7.85 (d, J = 7.9 Hz, 1H, Ar-H), 7.55 (m, 4H, Ar-H), 7.25 (m, 3H, Ar-H), 7.11 (d, J = 7.3 Hz, 1H, Ar-H), 5.12 (d, J = 9.6 Hz, 1H,OH), 4.94 (s, 1H, C8H), 4.10 (m, 2H,CH2), 3.17 (dd, J = 29.1, 13.5 Hz, 2H,C5H,C7H), 2.91 (d, J = 17.2 Hz, 1H,C5H), 1.98 (d, J = 29.0 Hz, 6H, COCH3, CH3), 1.30 (s, CH3, 3H).
13C NMR (101 MHz, DMSO) δ208.28, 166.79, 160.51, 160.21, 160.00, 150.79, 149.93, 149.62, 139.09, 137.85, 134.12, 131.95 ,131.62, 129.61, 125.03 , 119.99, 115.71 ,101.17, 72.42, 68.13, 67.94, 66.99, 65.07 , 43.47 , 40.50, 40.30, 40.09, 39.88, 39.67, 35.27 , 31.82 , 28.08 , 24.06.
Anal. Calcd. for C28H26N4O5S (530.16): C, 63.38; H, 4.94; N, 10.56 %. Found: C, 63.55; H, 5.00; N, 10.45%.
3.2.8. 2-[(7-Acetyl-4-cyano-1,6-dimethyl-6-hydroxy-8-(2-nitrophenyl)-5,6,7,8-tetra-hydroisoquinolin-3-yl)thio]-N-(4-tolyl)acetamide (9b). It was synthesized by reaction of 2 with N-(4-tolyl)-2-chloroacetamide (9b).Yield : 94%, m.p.: 210°C. IR: IR: 3457 (O-H); 3305 (N-H); 3005, 2918(C-H, sp2); 2215 (C≡N); 1693 (C=O, acetyl).
1H NMR (400 MHz, ) δ 10.10 (s, 1H, NH ), 7.85 (d, J = 8.2 Hz, 1H,Ar-H), 7.59 (d, J = 7.7 Hz, 1H, Ar-H), 7.50 (t, J = 7.6 Hz, 1H, Ar-H), 7.40 (d, J = 8.4 Hz, 2H, Ar-H), 7.19 (d, J = 7.8 Hz, 1H, Ar-H), 7.08 (d, J = 8.3 Hz, 2H, Ar-H), 5.12 (d, J = 9.6 Hz, 1H,OH), 4.94 (s, 1H, C8H), 4.17 – 4.05 (m, 2H,CH2), 3.17 (dd, J = 28.9, 13.4 Hz, 2H, C5H, C7H), 2.90 (d, J = 17.2 Hz, 1H, C5H), 2.24 (s, 3H, CH3), 2.00 (d, J = 29.1 Hz, 6H COCH3, CH3), 1.30 (s, 3H, CH3 ).
13C NMR (101 MHz, DMSO) δ 209.22, 166.20, 160.50, 158.29, 137.35, 136.86, 134.13, 132.77, 131.60, 129.56, 128.96, 128.59, 125.29, 119.64, 115.41, 104.48, 68.13, 65.04, 56.51, 43.47, 35.22, 31.84, 28.06, 24.07, 20.86.
Anal. Calcd. Anal. calcd for C29H28N4O5S (544.18): C, 63.95; H, 5.18; N, 10.29 %. Found: C, 64.12; H, 5.21; N, 10.19 %.
3.2.9. 2-[(7-Acetyl-4-cyano-1,6-dimethyl-6-hydroxy-8-(2-nitrophenyl)-5,6,7,8-tetra-hydroisoquinolin-3-yl)thio]-N-(4-acetylphenyl)acetamide (9c): It was synthesized by reaction of 2 with N-(4-acetylphenyl)-2-chloroacetamide (9c). Yield: 96 %; m.p.: 210-212°C: IR: 3447 (O-H); 3267 (N-H); 3005, 2973(C-H, sp2); 22156 (C≡N); 1685 (C=O, acetyl). 1H NMR (400 MHz, DMSO) δ 10.55 (s, 1H, NH), 7.87 (dd, J = 22.1, 8.2 Hz, 3H,Ar), 7.67 (d, J = 8.3 Hz, 2H,Ar-H), 7.58 (d, J = 7.5 Hz, 1H, Ar-H), 7.49 (d, J = 7.8 Hz, 1H, Ar-H), 7.18 (d, J = 7.8 Hz, 1H, Ar-H), 5.11 (d, J = 9.7 Hz, 1H, OH), 4.94 (s, 1H, C8H ), 4.21 (m, 2H,CH2), 3.27 (dd, J = 28.3, 13.5 Hz, 2H, C5H, C7H), 2.91 (d, J = 17.3 Hz, 1H, C5H ), 2.51 (s, 4H, C7, CH3), 2.04 (s, 3H,CO CH3), 1.93 (s, 3H, CH3), 1.30 (s, 3H,CH3). 13C NMR (101 MHz, DMSO) δ 209.72 ,167.54, 167.41, 160.52, 158.09, 150.97, 149.25, 143.55, 137.10, 134.14, 132.30, 131.50, 129.93, 129.00, 125.25, 118.90, 115.37, 104.38, 68.16, 64.69, 43.45,35.27, 33.27, 32.11, 27.88, 26.75, 23.93.
Anal. Calcd. for C30H28N4O6S (572.17): C, 62.92; H, 4.93; N, 9.78%. Found: C, 62.80; H, 5.12; N, 9.89 %.
3.2.10. 2-[(7-Acetyl-4-cyano-1,6-dimethyl-6-hydroxy-8-(2-nitrophenyl)-5,6,7,8-tetra-hydroisoquinolin-3-yl)thio]-N-(4-chlorophenyl)acetamide (9d): It was synthesized by reaction of 2 with N-(4-chlorophenyl)-2-chloroacetamide (9d). Yield: 92 %; m.p.: 202-204°C. IR: 3539 (O-H); 3218 (1H, N-H); 3029, 2976 (C-H, sp2); 2209 (C≡N); 1716,1697 (C=O, acetyl) . 1H NMR : δ (400 MHz) δ 10.34 (s, 1H), 7.85 (d, J = 8.0 Hz, 1H, Ar-H), 7.65 – 7.44 (m, 4H, Ar-H), 7.32 (d, J = 8.8 Hz, 2H, Ar-H), 7.18 (d, J = 7.8 Hz, 1H, Ar-H), 5.11 (d, J = 9.8 Hz, 1H, OH), 4.94 (s, 1H, C8H), 4.13 (d, J = 5.2 Hz, 2H, CH2), 3.19 (dd, J = 26.8, 13.5 Hz, 2H, C5H, C7H), 2.90 (d, J = 17.4 Hz, 1H, C5H), 2.04 (s, 3H, CH3), 1.95 (s, 3H,CO CH3), 1.30 (s, 3H, CH3). 13C NMR : δ (101 MHz, DMSO) δ 209.30, 166.74, 160.49, 158.18, 151.04, 149.28, 138.31, 137.30, 134.15, 131.58 , 129.13 ,128.97, 127.36, 125.31 , 121.07 , 115.43 , 104.38, 68.09, 64.98, 56.51, 43.48, 35.22 , 31.89, 27.99 , 24.07.20.01. Anal. Calcd. for C28H25ClN4O5S (564.12): C, 59.52; H, 4.46; N, 9.92%. Found : C, 59.63; H, 4.20; N, 9.99%.
3.2.11. 2-[(7-Acetyl-4-cyano-1,6-dimethyl-6-hydroxy-8-(2-nitrophenyl)-5,6,7,8-tetra-hydroisoquinolin-3-yl)thio]-N-(4-methoy- phenyl)acetamide (9e): It was synthesized by reaction of 2 with N-(4-methoxyphenyl)-2-chloroacetamide (9e). Yield: 89 %; m.p.: 197 °C. IR: IR: 3583 (O-H); 3303 (N-H); 2998-2944 (C-H, sp2); 2213 (C≡N); 1698 (C=O, acetyl). 1H NMR (400 MHz, DMSO) δ 10.05 (s, 1H, NH), 7.86 (d, J = 8.0 Hz, 1H,Ar-H), 7.65 – 7.36 (m, 4H,4 Ar-H), 7.19 (d, J = 7.8 Hz, 1H,Ar-H), 6.85 (d, J = 8.7 Hz, 2H, 2Ar-H), 5.12 (d, J = 9.6 Hz, 1H,OH), 4.94 (s, 1H, C8H ), 4.10 (d, J = 6.8 Hz, 2H,CH2), 3.71 (s, 3H, OCH3), 3.18 (dd, J = 27.9, 13.5 Hz, 2H, C7H, C5H), 2.91 (d, J = 17.3 Hz, 1H, C5H), 2.01 (d, J = 24.9 Hz, 6H, COCH3, CH3), 1.30 (s, 3H,CH3).
13C NMR (101 MHz, DMSO) δ 209.24, 165.96, 160.50, 158.30, 155.80, 150.98, 149.33, 137.34, 134.13, 132.50, 131.60, 128.97, 128.58, 125.28, 121.22, 115.41 , 114.36 , 104.49 , 68.13 , 65.03, 55.63,40.64, 35.13 , 31.86 , 28.06, 24.08.
Anal. Calcd. for C29H27N4O6S (559.2): C, 62.24; H, 4.86; N, 10.01. Found: C, 62.38; H, 4.90; N, 9.89%.
4. Synthesis of 7-Acetyl-1-amino-2-(N-arylcarbamoyl)-5,8-dimethyl-8-hydroxy-6-(2-nitrophenyl, 3-nitrophenyl or 4-nitrophenyl)-6,7,8,9-tetrahydrothieno[2,3-c]isoquinolines 10a,d; general methods.
4.1. Method A)
To a suspension of 9a, d (10 mmol) in abs. ethanol (60 mL), a methanolic sodium methoxide was added (prepared by dissolving 0.25 g of sodium in 30 ml of methanol). The reaction mixture was stirred for one hour at room temperature. The yellow solid that formed after dilution with water (60 ml) was collected, washed with water, dried in air and then recrystallized from dioxane to give 10a, d respectively.
4.1.1. 7-Acetyl-1-amino-5,8-dimethyl-8-hydroxy-6-(2-nitrophenyl)-N-phenyl-6,7, 8,9-tetrahydrothieno[2,3-c]isoquinoline-N-phenyl-2-carboxamide (10a).
It was obtained by cyclization of compound 9a. Yield: 93%; m.p.: 286-290°C. IR: IR: 3585 (O-H); 3432 (N-H); 2925 (C-H, sp2); 1697 (C=O, acetyl). 1H NMR (400 MHz, DMSO) δ 9.40 (s, 1H, NH), 7.80 (d, J = 8.0 Hz, 1H, Ar-H) 7.60 (m, 2H, 2Ar-H), 7.50 (d, J = 7.8 Hz, 1H, Ar-H), 7.33 (d, J = 8.7 Hz, 1H, Ar-H), 7.18 (d, J = 9.6 Hz, 2H, 2Ar-H), 7.1 (m, 4H,NH2,2 Ar-H), 5.21 (d, J = 6.8 Hz, 1H, OH, ), 4.78 (s, 1H, C6H ), 3.56 (dd, J = 27.9, 13.5 Hz, 2H, C9H, C7H ), 3.1 6 (d, J = 17.3 Hz, 1H, C9H ), 2.07 (d, J = 28 Hz, 6H, COCH3, CH3), 1.34 (s, 3H, CH3).
13C NMR (100 MHz, DMSO) δ 166.86, 149.79, 128.85, 128.72, 122.02, 121.84, 108.92, 67.91, 40.79, 40.56, 40.34, 32.52, 32.21, 28.49, 28.35, 28.17, 27.86, 7.05.
Anal. Calcd. for C28H26N4O5S (530.16): C, 63.38; H, 4.94; N, 10.56 %. Found: C, 63.57; H, 5.12; N, 10.22%.
4.1.2. 7-Acetyl-1-amino-N-(4-chlorophenyl)-5,8-dimethyl-8-hydroxy-6-(2-nitro-phenyl)-6,7,8,9-tetrahydrothieno[2,3-c]isoquinoline-2-carboxamide (10d). It was obtained by cyclization of compound 9b. Yield:94 %; m.p.: 300°C. IR: 3450 (O-H); 2924 (C-H, sp2); 1698 (C=O, acetyl). 1H NMR (400 MHz, DMSO) δ 9.52 (s, 1H, NH), 7.83 (d, J = 8.0 Hz, 1H, Ar-H) 7.74 (d, 2H, 2Ar-H), 7.59 (d, J = 7.8 Hz, 1H, Ar-H), 7.50 (d, J = 8.7 Hz, 1H, Ar-H), 7.38 (d, J = 9.6 Hz, 2H, Ar-H), 7.15 (m, 3H, NH2, Ar-H), 5.21 (s, 2H, OH ), 4.78 (s, 1H, C6H ), 3.50 (dd, J = 25, 27 Hz, 2H, C9H, C7H ), 3.2 (s, 1H, C9H), 2.09 (d, J = 28 Hz, 6H, COCH3, CH3), 1.34 (s, 3H,CH3).
Anal. Calcd. for C28H25ClN4O5S (564.12): C, 59.52; H, 4.46; N, 9.92%. Found: C, 60.03; H, 4.32; N, 9.84%.
4.2. Method B).
To mixture of compound 3a-c (10 mmol) and respective N-aryl-2-chloroacetamide 9a-e (10 mmol) in ethanol (60 mL),a methanolic sodium methoxide (1.30 g) was added. The resulting mixture was refluxed for 110 mins hours. The solid that formed while hot was recrystallized from dioxane to give compounds 10a, d; yield: 80-86%.
5. 1 Cytotoxicity against human cancer cell lines
In this work, all produced compounds anticancer activity were assessed for IC50 against two cell lines HEGP2 and HCT116 cells and the human normal skin cell line (HSF) which were obtained from national cancer institute, Cairo, Egypt. were examined using the sulphorhodamine-B (SRB) test. Cells were seeded at a density of 3 *103 cells/well in 96-well microtiterplates [32]. They were allowed to connect for 24 hours before incubating with our compounds. Cells were treated with various doses of our synthesized compounds (0, 12.5, 25, 50, and 100 µg/mL). For each concentration, three wells were employed, and the incubation period lasted 48 hours. DMSO was employed as the control vehicle (1% v/v). At the end of the incubation, the cells were fixed with 20% trichloroacetic acid and stained with 0.4% SRB dye. The optical density (O.D.) of each well was measured spectrophotometrically at 570 nm with an ELISA microplate reader. The proportion of cell survival was determined as follows: Survival fraction = O.D. (treated cells) / O.D. (control cells). Sigmoidal dose-response curve-fitting models (GraphPad Prizm software, version 5) were used to obtain the IC 50 (concentration that inhibits cell growth by 50%) for each chemical.
5.2. Cell cycle analysis:
The cell cycle arrests of compound 3 against HEGP2 at their IC50 concentration was carried out according to Abcam method (code ab139418), (www.abcam.co.jp) . Thus, HEGP2 cells were collected using 75% ice-cold ethanol and kept at -20 °C for 1 hour following treatment with an IC50 dose of our compounds 3. The cells were then centrifuged, washed twice with ice-cold PBS, and incubated at 4°C for 20 minutes. The cell cycle was assessed using a flow cytometry kit (Propidium Iodide [ab13941]). Finally, use the Cell Quest software to undertake statistical analysis of the cell fractions in sub-G0/G1, S, and G2/M phases [33-34].
5.3. Annexin-V FITC apoptosis assay
The Annexin-V FITC apoptosis assay of compounds 3 against HEGP2 at their IC50 values was performed according to (BioVision Research Products (code k101-25). (www.biovision.com).Thus, HEGP2 cell line were treated with (6.4 µM) of the compound 3 for 24 h then collected by trypsin, centrifuged then rinsed with PBS and suspended in binding buffer, then dual-stained with Annexin V-FITC (5 μL) and propidium iodide (5 μL) in the dark for 15 min at romm temp. using flow cytometry to measured the cells with an excitation wavelength of 488 nm and an emission wavelength of 530 nm. The final results were analyzed with the Cell quest software [35-36].