الفهرس 
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Abstract
Cancer is a fierce fighter, even after a long ongoing war; it is still fighting back through the emergence of drug resistances. EGFR (Epidermal Growth Factor Receptor) is a known key player in the development of many cancers, namely Non-small-cell Lung Cancer (NSCLC), Colorectal and breast cancer. Targeting EGFR with small molecules is a well-established therapeutic approach since the approval of Gefitinib in 2002. Among the various resistance mechanisms to EGFR inhibitors, our current study focuses on HER2 (Human Epidermal growth factor Receptor 2) amplification. Dual EGFR/HER2 targeting is expected to overcome EGFR inhibitors resistance by being able to simultaneously block the two counterparts of the HER family, thus blocking their downstream signaling pathway. Building on the classical kinase inhibitors design, and guided by molecular modeling, we designed a series of novel thieno[2,3-d]pyrimidine-based compounds with the aim of achieving dual EGFR/HER2 inhibition. The designed compounds were synthesized, purified and structurally confirmed by different analytical and spectral techniques.
The study involved the synthesis of the following unavailable reported intermediates:
(1) 2-chloro-1-((3-fluorobenzyl)oxy)-4-nitrobenzene (Ia)
(2) 2-((2-chloro-4-nitrophenoxy)methyl)pyridine (Id)
(3) 3-chloro-4-((3-fluorobenzyl)oxy)aniline (IIa)
(4) 3-chloro-4-((3-chlorobenzyl)oxy)aniline (IIb)
(5) 3-chloro-4-(pyridin-2-ylmethoxy)aniline (IId)
(6) 2-chloro-4-nitro-1-(3-(trifluoromethyl)phenoxy)benzene (IIIa)
(7) 3-chloro-4-(3-(trifluoromethyl)phenoxy)aniline (IVa)
(8) 3-chloro-4-(m-tolyloxy)aniline (IVb)
(9) 1-benzyl-5-nitro-1H-indazole (V)
(10) 1-benzyl-1H-indazol-5-amine (VI)
(11) (E)-N,N-dimethyl-2-(4-nitrophenyl)ethenamine (IX)
(12) Ethyl 2-amino-5-(4-nitrophenyl)thiophene-3-carboxylate (X)
In addition to the following new intermediates:
(1) 2-chloro-1-((3-chlorobenzyl)oxy)-4-nitrobenzene (Ib)
(2) 1,2-dichloro-4-((2-chloro-4-nitrophenoxy)methyl)benzene (Ic)
(3) 3-chloro-4-((3,4-dichlorobenzyl)oxy)aniline (IIc)
(4) 2-chloro-4-nitro-1-(m-tolyloxy)benzene (IIIb)
(5) 2-((2-chloro-4-nitrophenyl)thio)benzo[d]thiazole (VII)
(6) 4-(benzo[d]thiazol-2-ylthio)-3-chloroaniline (VIII)
(7) 6-(4-nitrophenyl)thieno[2,3-d]pyrimidin-4(3H)-one (XI)
(8) 4-chloro-6-(4-nitrophenyl)thieno[2,3-d]pyrimidine (XII)
Finally, the study comprised the synthesis and the characterization of the following new targeted compounds:
(1) N-(3-bromophenyl)-6-(4-nitrophenyl)thieno[2,3-d]pyrimidin-4-amine (XIIIa)
(2) N-(4-chloro-3-(trifluoromethyl)phenyl)-6-(4-nitrophenyl)thieno[2,3-d]pyrimidin-4-amine (XIIIb)
(3) N-(3-ethynylphenyl)-6-(4-nitrophenyl)thieno[2,3-d]pyrimidin-4-amine (XIIIc)
(4) N-(3-chloro-4-fluorophenyl)-6-(4-nitrophenyl)thieno[2,3-d]pyrimidin-4-amine (XIIId)
(5) N-(3-chloro-4-(3-(trifluoromethyl)phenoxy)phenyl)-6-(4-nitrophenyl)thieno[2,3-d]pyrimidin-4-amine (XIVa)
(6) N-(3-chloro-4-(m-tolyloxy)phenyl)-6-(4-nitrophenyl)thieno[2,3-d]pyrimidin-4-amine (XIVb)
(7) N-(1-benzyl-1H-indazol-5-yl)-6-(4-nitrophenyl)thieno[2,3-d]pyrimidin-4-amine (XV)
(8) N-(4-(benzo[d]thiazol-2-ylthio)-3-chlorophenyl)-6-(4-nitrophenyl)thieno[2,3-d]pyrimidin-4-amine (XVI)
(9) N-benzyl-6-(4-nitrophenyl)thieno[2,3-d]pyrimidin-4-amine (XVIIa)
(10) 6-(4-nitrophenyl)-N-phenethylthieno[2,3-d]pyrimidin-4-amine (XVIIb)
(11) N-(3-chloro-4-((3-fluorobenzyl)oxy)phenyl)-6-(4-nitrophenyl)thieno[2,3-d]pyrimidin-4-amine (XVIIIa)
(12) N-(3-chloro-4-((3-chlorobenzyl)oxy)phenyl)-6-(4-nitrophenyl)thieno[2,3-d]pyrimidin-4-amine (XVIIIb)
(13) N-(3-chloro-4-((3,4-dichlorobenzyl)oxy)phenyl)-6-(4-nitrophenyl)thieno[2,3-d]pyrimidin-4-amine (XVIIIc)
(14) N-(3-chloro-4-(pyridin-2-ylmethoxy)phenyl)-6-(4-nitrophenyl)thieno[2,3-d]pyrimidin-4-amine (XVIIId)
(15) 6-(4-aminophenyl)-N-benzylthieno[2,3-d]pyrimidin-4-amine (XIX)
(16) 6-(4-aminophenyl)-N-(3-chloro-4-((3-fluorobenzyl)oxy)phenyl)thieno[2,3-d]pyrimidin-4-amine (XX)
(17) N-(4-(4-((3-chloro-4-((3-fluorobenzyl)oxy)phenyl)amino)thieno[2,3-d]pyrimidin-6-yl)phenyl)acetamide (XXI)
(18) 6-(4-aminophenyl)-N-(3-chloro-4-(3-(trifluoromethyl)phenoxy)phenyl)thieno[2,3-d]pyrimidin-4-amine (XXII)
(19) N-(4-(4-((3-chloro-4-(3-(trifluoromethyl)phenoxy)phenyl)amino)thieno[2,3-d]pyrimidin-6-yl)phenyl)acetamide (XXIII)
(20) N-(4-(4-((3-chloro-4-(3-(trifluoromethyl)phenoxy)phenyl)amino)thieno[2,3-d]pyrimidin-6-yl)phenyl)methanesulfonamide (XXIV)
(21) Ethyl (4-(4-((3-chloro-4-(3-(trifluoromethyl)phenoxy)phenyl)amino)thieno[2,3-d]pyrimidin-6-yl)phenyl)carbamate (XXV)
(22) (E)-N-(4-(4-((3-chloro-4-(3-(trifluoromethyl)phenoxy)phenyl)amino)thieno[2,3-d]pyrimidin-6-yl)phenyl)-4-methylpent-2-enamide (XXVI)
(23) (Z)-4-((4-(4-((3-chloro-4-(3-(trifluoromethyl)phenoxy)phenyl)amino)thieno[2,3-d]pyrimidin-6-yl)phenyl)amino)-4-oxobut-2-enoic acid (XXVII)
(24) 1-(4-(4-((3-chloro-4-(3-(trifluoromethyl)phenoxy)phenyl)amino)thieno[2,3-d]pyrimidin-6-yl)phenyl)-3-cyclopropylurea (XXVIIIa)
(25) 1-(4-(4-((3-chloro-4-(3-(trifluoromethyl)phenoxy)phenyl)amino)thieno[2,3-d]pyrimidin-6-yl)phenyl)-3-(2-morpholinoethyl)urea (XXVIIIb)
Screening of the compounds for dual inhibitory activity involved cycles of enzymatic and cell-based assays. Firstly, we screened for the optimum 4-anilino derivative through determining the dual EGFR/HER2 inhibitory activity against the isolated enzymes at 10 μM, and the cytotoxicity assay on human epidermoid carcinoma (A431) cell line and human breast adenocarcinoma (MDA-MB-361) cell line. We identified the 3-chloro-4-(3-(trifluoromethyl)phenoxy)aniline (XIVa) as the aniline derivative that can afford satisfactory dual inhibitory activity. Secondly, we tried to optimize the activity of (XIVa) by screening for the optimum solubilizing group. Applying the same assays followed by IC50 determination on the isolated enzymes, we identified three compounds (XXII) (XXIII) (XXVIIIb) with adequate enzymatic and cytotoxic activities, showing IC50 values of 21.4 nM/1.5 μM, 47.7 nM/0.879 μM and 91.7 nM/1.2 μM against EGFR/HER2, respectively, and IC50 values of 3.33 μM/5 μM, 8.13 μM/13.3 μM and 1.33 μM/3.42 μM against A431 / MDA-MB-361 cell lines, respectively.
Finally, molecular modeling studies were performed and were in accordance with the demonstrated patterns of activity, inasmuch as the molecular docking study conformed to the observed pattern of enzymatic activity of the compounds. Moreover, a correlation study of the molecular properties identified the descriptors affecting the cellular activity.