الفهرس 
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Abstract
Heterocyclic nucleus is a scaffold of biological interest. Herein, the present study focuses on pyrazolopyrimidines that are fused heterocyclic ring systems structurally resemble purines which prompted different biological investigations to assess their potential therapeutic significance1.
A new series of pyrazolo[3,4-d]pyrimidine derivatives have been synthesized with the aim to explore their biological activities especially the anticancer activity.
Our starting synthetic material ethyl-(ethoxymethylene)cyanoacetate I was cyclized with phenyl hydrazine to obtain ethyl 5-amino-1-phenyl-1H-pyrazole-4-carboxylate II which underwent further cyclization with formamide to afford the pyrazolo[3,4-d]pyrimidinone III.
Compound III was chlorinated using phosphorous oxychloride to obtain 4-chloro-1-phenyl-1H-pyrazolo[3,4-d]pyrimidine (IV). The key intermediates 4-{(1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-amino}-benzoic acid (V) and 3-{(1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)amino}benzoic acid (VIII) were prepared through reaction of compound IV with 4-amino benzoic acid or 3-amino benzoic acid, respectively.
Reaction of our key intermediates V and VIII with 1H-benzotriazole afforded the novel N-acyl benzotriazole compounds VI and IX that were condensed with a series of different amino acids to obtain N-acyl amino acids compounds; 2-{4-[(1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)amino]benzamido}alkanoic acid (VIIa-m) and 2-{3-[(1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)amino]benzamido}alkanoic acid (Xa-m), respectively.
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On the other side, the key intermediate benzoic acid derivative VIII was esterified with ethanol to obtain the ethyl ester derivative XI which underwent hydrazinolysis to afford the novel acid hydrazide XII that represent a new key intermediate for obtaining the novel hydrazone derivatives (XIIIa-g) through its condensation with different aromatic aldehydes.
Moreover, the target thiosemicarbazide derivatives XIVa,b were prepared via reaction of the novel acid hydrazide XII with different aryl isothiocyanate derivatives which were then cyclized through 3 pathways; firstly via condensation with a series of phenacyl bromides to afford a new thiazole derivatives XVa-h, secondly by using sulfuric acid to afford a novel 1,3,4 thiadiazole derivatives XVIa,b and the third procedure via cyclization with sodium hydroxide to give the new 1,2,4-triazole derivatives XVIIa,b.
The dihydrofolate reductase (DHFR) inhibition activity of the novel synthesized compounds VI, VIIa-m, IX and Xa-m was evaluated using the BioVision’s Dihydrofolate Reductase Inhibitor Screening Kit and methotrexate (MTX) as a positive control. The results revealed that compounds Xe, Xg and Xf displayed the highest inhibition activities in submicromolar level against DHFR enzyme with IC50 values of 0.24, 0.25 and 0.30 μM respectively which are approximately 23, 22 and 19 fold more active than MXT reference drug (IC50 5.57 μM). In addition, compounds VIIf and VIId were proved to have superior inhibition activity of DHFR with IC50 values of 0.31 and 0.43 μM, respectively.
The molecular modeling and docking studies calculations for the most stable conformers of newly synthesized pyrazolo[3,4-d]pyrimidine
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derivatives VI, IX, VIIa-m and Xa-m come compatible with the IC50 enzyme assay results. The procedures were carried out using molecular operating environment MOE version (2014.0901) into the binding pocket of DHFR-binding domain (pdb code ID: 1U72) using methotrexate (MTX) reference drug as a normal ligand. The top results revealed that compound VIIf elucidated binding energy (S= - 9.2584 Kcal/mol) that is better than that of the normal ligand MTX (S= - 8.3951 Kcal/mol). In addition, compound VIId (S= - 8.6795 Kcal/mol) exhibited considerable salt bridge between carboxylate group and the crucial amino acid Arg70. Furthermore, the most stable pose of compound Xe (S= - 8.4390 Kcal/mol) was proved by 4 considerable interactions; the substantial salt bridge of the carboxylate group with Arg70, arene-arene interaction between phenyl ring and Phe31 residue and the distinctive (HBA) effect of the carbonyl oxygen with Asn64 (-1.6 Kcal/mol).
It is clear from the present study that some of the newly synthesized compounds showed promising effect regarding DHFR inhibitory activity such as the N-acyl amino acids Xe, VIIf and VIId with prominent binding interaction to the DHFR active site that require a future further investigation.
On another hand, the EGFR inhibitory activity of the novel compounds XI, XII, XIIIa-g, XIVa,b, XVa-h, XVIa,b and XVIIa,b was estimated based on BPS-Bioscience measuring using microplate reader. The results revealed that the hydrazone compounds XIIId and XIIIf beside the thiazoles XVd and XVh demonstrate the highest EGFR inhibition activities with IC50 values of 0.111 and 0.113 nM respectively for hydrazones as well, 0.097 and 0.115 nM for thiazoles respectively comparable to geftinib reference standard (IC50= 0.166 nM), while the 1,2,4 triazole compound XVIIa exhibited anti-
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EGFR activity of (IC50= 0.162 nM) that is approximately equal to that of geftinib reference drug.
Molecular docking study was performed to identify the most preferred binding modes of the novel compounds XI, XII, XIIIa-g, XIVa,b, XVa-h, XVIa,b and XVIIa,b into the EGFR enzyme active site (pdb code ID: 4WKQ) using geftinib normal ligand as reference drug. The docking simulation of compound XVd showed that it fit the EGFR active site with bonding score of (- 8.1691 Kcal/mol) that significantly preferable than geftinib reference drug (S= - 6.3978 Kcal/mol). While compound XVh and XIIIf displayed bonding score of (S= - 7.7912 Kcal/mol) and (- 7.6632 Kcal/mol) respectively. Also, the docking pose of the novel 1,2,4 triazole XVIIa (S= - 7.0697 Kcal/mol) relative to geftinib (S= - 6.5026 Kcal/mol).
It is apparent from the present investigation that some of the newly synthesized compounds showed considerable effect regarding EGFR inhibitory activity such as the hydrazone containing compounds XIIId and XIIIf beside the thiazole derivatives XVd and XVh in addition to the triazole compound XVIIa with significant binding interaction to the EGFR active site that require a further study.