Multi-target rational design and synthesis of novel diphenyl-tethered pyrazolopyrimidines targeting EGFR and topoisomerase II with potential DNA intercalation and apoptosis induction.

[Display omitted] • Novel pyrazolopyrimidines were designed as multitarget-directed drug candidates. • GI % against fifteen human cancer cell lines was recorded. • Cytotoxicity against HNO97, MDA-MB-468, FaDu, and HeLa were measured. • Compound 6l decreased EGFR protein concentration by a 6.10-fold change, compared to imatinib. • Compounds 6a and 6l exhibited IC 50 values of 17.89 and 19.39 μM, respectively, against TOPO II. • DNA fragmentation images described the great potential of 6a and 6l in inducing DNA degradation. • Protein expression analysis for caspases 3, 7, 8, and 9, Bax, p53, MMP2, MMP9, and BCL-2 was conducted. • Cell cycle analysis and molecular docking were performed. Herein, we envisioned the design and synthesis of novel pyrazolopyrimidines (confirmed by elemental analysis, 1H and 13C NMR, and mass spectra) as multitarget-directed drug candidates acting as EGFR/TOPO II inhibitors, DNA intercalators, and apoptosis inducers. The target diphenyl-tethered pyrazolopyrimidines were synthesized starting from the reaction of phenyl hydrazine and ethoxymethylenemalononitrile to give aminopyrazole-carbonitrile 2. The latter hydrolysis with NaOH and subsequent reaction with 4-chlorobenzaldhyde afforded the corresponding pyrazolo[3,4- d ]pyrimidin-4-ol 4. Chlorination of 4 with POCl 3 and sequential reaction with different amines afforded the target compounds in good yields (up to 73 %). The growth inhibition % of the new derivatives (6a-m) was investigated against different cancer and normal cells and the IC 50 values of the most promising candidates were estimated for HNO97, MDA-MB-468, FaDu, and HeLa cancer cells. The frontier derivatives (6a , 6i , 6k , 6l , and 6m) were pursued for their EGFR inhibitory activity. Compound 6l decreased EGFR protein concentration by a 6.10-fold change, compared to imatinib as a reference standard. On the other side, compounds (6a , 6i , 6k , 6l , and 6m) underwent topoisomerase II (TOPO II) inhibitory assay. In particular, compounds 6a and 6l exhibited IC 50 s of 17.89 and 19.39 μM, respectively, surpassing etoposide with IC 50 of 20.82 μM. Besides, the DNA fragmentation images described the great potential of both candidates 6a and 6l in inducing DNA degradation at lower concentrations compared to etoposide and doxorubicin. Moreover, compound 6l , with the most promising EGFR/TOPO II inhibition and DNA intercalation, was selected for further investigation for its apoptosis induction ability by measuring caspases 3, 7, 8, and 9, Bax, p53, MMP2, MMP9, and BCL-2 proteins. Additionally, molecular docking was used to explain the SAR results based on the differences in the molecular features of the investigated congeners and the target receptors' topology. [ABSTRACT FROM AUTHOR]