Design, synthesis and antitumor evaluation of novel pyrazolo[3,4-]pyrimidines incorporating different amino acid conjugates as potential DHFR inhibitors.

The present study aimed to investigate the antitumor effect of simultaneous inhibition of dihydrofolate reductase (DHFR) enzyme. We designed some novel pyrazolo[3,4-]pyrimidines bearing different amino acid conjugates as efficient antifolate agents attributable to their structural similarity with methotrexate (MTX) and MTX-related antifolates. All compounds were tested to screen their enzymatic inhibition against DHFR compared with the reference drug MTX and for their antitumor cytotoxicity against six MTX-resistant cancer cell lines. The flow cytometry indicated that the most potent compound arrested MCF-7 cells in the S-phase and induced apoptosis. Western blot for visualisation proved the ability of compound to induce the expression of proapoptotic caspases and Bax proteins in MCF-7 breast cancer cell line beside its ability to diminish the expression of antiapoptotic Bcl-2 protein. Molecular modelling studies concluded that compound displayed better binding energy than that of the normal ligand MTX. HIGHLIGHTSNew pyrazolo[3,4-]pyrimidine derivatives which are structurally similar to the classical methotrexate (MTX) and MTX-related antifolates were synthesised as antitumor agents.Novel -acyl amino acid compound exhibited marked DHFR inhibition activity that are parralel to both the molecular docking results and cytotoxic activity.Compound could induce the expression of proapoptotic caspases and Bax proteins in MCF-7 breast cancer cell line beside its ability to diminish the expression of antiapoptotic Bcl-2 protein.All prepared compounds obey Lipinski rule of five except compound .