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Abstract According to statistics from the American Cancer Society (ACS), Cancer is the third most lethal disease after cardiovascular diseases, infectious and parasitic diseases. It may affect humans of all ages, regions and socioeconomic levels. Continuous research worldwide is focusing on developing better therapeutics as well as finding novel druggable targets for better efficacy. Small molecule tyrosine kinase inhibitors provide attractive therapeutic targets, as they are able to block cell signaling associated with many types of cancer. In this study, thienopyrimidine derivatives have been designed and synthesized as targeted EGFR/HER-2 inhibitors. The design focused on exploration of the previous revealed SAR studies, bioisosteric modifications of the lead compounds both in market and in clinical studies, and identification of the key interactions with the binding site in silico. Synthesis of the designed compounds was then accomplished & their structures were confirmed by various spectral and microanalytical data. This study involved the synthesis of the following reported intermediates: 1) 1-(Benzyloxy)-4-nitrobenzene (Ia) 2) 1-Fluoro-3-((4-nitrophenoxy) methyl)benzene (Ib) 3) 1-Fluoro-4-((4-nitrophenoxy) methyl)benzene (Ic) 4) 1-Chloro-4-((4-nitrophenoxy) methyl)benzene (Id) 5) 2-Chloro-1-((3-fluorobenzyl) oxy)-4-nitrobenzene (If) 6) 4-(Benzyloxy) aniline (IIa) 7) 4-((4-Chlorobenzyl) oxy) aniline (IId) 8) 4-(Benzyloxy)-3-chloroaniline (IIe) 9) 3-Chloro-4-((3-fluorobenzyl) oxy) aniline (IIf) 10) 3-Chloro-4-((4-fluorobenzyl) oxy) aniline (IIg) 11)3-Chloro-4-((4-chlorobenzyl) oxy) aniline (IIh) 12)Ethyl -5-amino-4-cyano-3-methylthiophene-2-carboxylate (III) 13) 2-Amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile (VI) 14)Ethyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (IX) 15) 5,6,7,8-Tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4(3H)-one (X) 16)4-Chloro-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine (XI) 17)1-(4-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4- yl)amino)phenyl)ethan-1-one (XII) Also, it comprised the following new intermediates: 1) 1-(Benzyloxy)-2-chloro-4-nitrobenzene (Ie) 2) 2-Chloro-1-((4-fluorobenzyl) oxy)-4-nitrobenzene (Ig) 3) 2-Chloro-1-((4-chlorobenzyl) oxy)-4-nitrobenzene (Ih) 4) 4-((3-Fluorobenzyl) oxy) aniline (IIb) 5) 4-((4-Fluorobenzyl) oxy) aniline (IIc) 6) Ethyl.(E)-4-cyano-5-(((dimethylamino)methylene)amino)-3-methylthiophene- 2-carboxylate (IV) 7) (E)-N’-(3-Cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-N,Ndimethylformimidamide (VII) Also, the study involved the synthesis and the characterization of the following newtargeted compounds: 1) Ethyl4-((4-(benzyloxy) phenyl) amino)-5-methylthieno [2,3-d]pyrimidine-6- carboxylate (Va) 2) Ethyl4-((4-((3-fluorobenzyl)oxy)phenyl)amino)-5-methylthieno[2,3- d]pyrimidine-6-carboxylate (Vb) 3) Ethyl.4-((4-((4-fluorobenzyloxy)phenyl)amino)-5-methylthieno[2,3- d]pyrimidine-6-carboxylate (Vc) 4) Ethyl.4-((4-((4-chlorobenzyloxy)phenyl)amino)-5-methylthieno[2,3- d]pyrimidine-6-carboxylate (Vd) 5) Ethyl4-((4-(benzyloxy)-3-chlorophenyl)amino)-5-methylthieno[2,3- d]pyrimidine-6-carboxylate (Ve) 6) Ethyl.4-((3-fluorobenzyloxy)-3-chlorophenyl)amino)-5-methylthieno[2,3- d]pyrimidine-6-carboxylate (Vf) 7) Ethyl.4-((4-fluorobenzyloxy)-3-chlorophenyl)amino)-5-methylthieno[2,3- d]pyrimidine-6-carboxylate (Vg) 8) Ethyl.4-((4-chlorobenzyloxy)-3-chlorophenyl)amino)-5-methylthieno[2,3- d]pyrimidine-6-carboxylate (Vh) 9) N-(4-(Benzyloxy)phenyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d] pyrimidin-4-amine(VIIIa) 10) N-(4-(3-Fluorobenzyloxy)phenyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3- d]pyrimidin-4-amine (VIIIb) 11) N-(4-(4-Fluorobenzyloxy)phenyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3- d]pyrimidin-4-amine (VIIIc) 12) N-(4-(4-Chlorobenzyloxy)phenyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3- d]pyrimidin-4-amine (VIIId) 13) N-(4-(Benzyloxy)-3-chlorophenyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3- d]pyrimidin-4-amine (VIIIe) 14) N-(3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)-5,6,7,8-tetrahydrobenzo[4,5] thieno[2,3-d]pyrimidin-4-amine (VIIIf) 15) N-(3-Chloro-4-((4-fluorobenzyl)oxy)phenyl)-5,6,7,8-tetrahydrobenzo[4,5] thieno[2,3-d]pyrimidin-4-amine (VIIIg) 16) N-(3-Chloro-4-((4-chlorobenzyl)oxy)phenyl)-5,6,7,8-tetrahydrobenzo[4,5] thieno [2,3-d]pyrimidin-4-amine (VIIIh) 17) (E)-3-(4-Fluorophenyl)-1-(4-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin- 4-yl)amino)phenyl)prop-2-en-1-one (XIIIa) 18) (E)-3-(4-Chlorophenyl)-1-(4-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3- d]pyrimidin-4-yl)amino)phenyl)prop-2-en-1-one (XIIIb) 19) (E)-3-(4-Bromophenyl)-1-(4-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3- d]pyrimidin-4-yl)amino)phenyl)prop-2-en-1-one (XIIIc) 20) (E)-3-(4-Methoxyphenyl)-1-(4-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3- d]pyrimidin-4-yl)amino)phenyl)prop-2-en-1-one (XIIId) 21) (E)-3-(3,4-Dimethoxyphenyl)-1-(4((5,6,7,8−tetrahydrobenzo[4,5]thieno[2,3- d]pyrimidin-4-yl)amino)phenyl)prop-2-en-1-one (XIIIe) 22) 5-(4-fluorophenyl)-3-(4-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin- 4-yl)amino)phenyl)-4,5-dihydro-1H-pyrazole-1-carbothioamide (XIVa) 23) 5-(4-chlorophenyl)-3-(4-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin- 4-yl)amino)phenyl)-4,5-dihydro-1H-pyrazole-1-carbothioamide (XIVb) 24) 5-(4-bromophenyl)-3-(4-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin- 4-yl)amino)phenyl)-4,5-dihydro-1H-pyrazole-1-carbothioamide (XIVc) 25) 5-(4-methoxyphenyl)-3-(4-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3- d]pyrimidin-4-yl)amino)phenyl)-4,5-dihydro-1H-pyrazole-1-carbothioamide (XIVd) 26) 5-(3,4-dimethoxyphenyl)-3-(4-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3- d]pyrimidin-4-yl)amino)phenyl)-4,5-dihydro-1H-pyrazole-1-carbothioamide (XIVe) 27) 4-(4-Fluorophenyl)-2-imino-6-(4-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3- d]pyrimidin-4-yl)amino)phenyl)-1,2-dihydropyridine-3-carbonitrile (XVa) 28) 4-(4-Chlorophenyl)-2-imino-6-(4-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3- d]pyrimidin-4-yl)amino)phenyl)-1,2-dihydropyridine-3-carbonitrile (XVb) 29) 4-(4-Bromophenyl)-2-imino-6-(4-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3- d]pyrimidin-4-yl)amino)phenyl)-1,2-dihydropyridine-3-carbonitrile (XVc) 30) 2-Imino-4-(4-methoxyphenyl)-6-(4((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3- d]pyrimidin-4-yl)amino)phenyl)-1,2-dihydropyridine-3-carbonitrile (XVd) 31) 4-(4-Fluorophenyl)-2-oxo-6-(4-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3- d]pyrimidin-4-yl)amino)phenyl)-1,2-dihydropyridine-3-carbonitrile (XVIa) 32) 4-(4-Chlorophenyl)-2-oxo-6-(4-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3- d]pyrimidin-4-yl)amino)phenyl)-1,2-dihydropyridine-3-carbonitrile (XVIb) 33) 4-(4-Bromophenyl)-2-oxo-6-(4-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3- d]pyrimidin-4-yl)amino)phenyl)-1,2-dihydropyridine-3-carbonitrile (XVIc) 34) 4-(4-Methoxyphenyl)-2-oxo-6-(4-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3- d]pyrimidin-4-yl)amino)phenyl)-1,2-dihydropyridine-3-carbonitrile (XVId) Preliminary molecular docking was performed using C-DOCKER protocol in Discovery Studio 4.0 Software, it was attempted to predict the binding mode of the targeted compounds. The biological evaluation was accomplished through testing both antiproliferative activity and enzyme inhibition activity. The enzymatic assay was performed in BPS Bioscience Corporation; USA. The enzymatic activity of the synthesized compounds was evaluated against EGFR/HER-2 tyrosine kinase at 10 μM concentration. Most of the synthesized compounds showed good to potent EGFR/HER-2 inhibitory activity in particular Va, Vf, VIIIa, VIIIb, VIIIe, VIIIf and VIIIg. The tetrahydrobenzothienopyrimidine derivative bearing a 3-chloro-4-(3- fluorobenzyloxy) aniline (VIIIf) exhibited 100% inhibition on both kinases. Furthermore, five compounds (VIIIa, VIIIb, VIIIe, VIIIf, VIIIg) demonstrated highly potent dose-related EGFR/HER-2 inhibition with IC50 values in submicromolar range which exhibited IC50 values of 1.2, 0.6, 0.3, 0.2, 0.4 µM and 8.2, 3.4, 1.3, 0.5, 2.7 µM on EGFR and HER-2 respectively. Twelve of the final Compounds (Vb, Vc , Vd, Vf, Vh, VIIIa, VIIIb, VIIIc, VIIIe, VIIIf, VIIIg, VIIIh) were selected by the National Cancer Institute “NCI” for single dose screening program at 10 μM in the full NCI 60 cell panel. The thieno[2,3- d]pyrimidine-based derivative (VIIIf) showed remarkably the lowest cell growth promotion, hence good anti-proliferative activity against specific cell lines. Finally, Molecular docking was performed using C-DOCKER protocol in Discovery Studio 4.0 Software, it was attempted to investigate the binding mode of the targeted compounds and interpret their variable inhibitory activity. Computer aided ADMET study was also performed using Discovery Studio 2.5 Software. |