New pyridopyrimidine derivatives as dual EGFR and CDK4/cyclin D1 inhibitors: synthesis, biological screening and molecular modeling

<b>Aim:</b> A series of pyridopyrimidine derivatives <b>5–20</b> was designed, synthesized and examined for antitumor activity using four types of malignant cells. <b>Materials &amp; methods:</b> Cervical cancer (HeLa), hepatic cancer (HepG-2), breast cancer (MCF-7) and colon cancer (HCT-166) cells, as well as normal human lung fibroblast cells (WI-38) were used to determine the cytotoxicity. <b>Results:</b> Pyrazol-1-<i>yl</i> pyridopyrimidine derivative <b>5</b> was found to be the most active compound against three malignant cells Hela, MCF-7 and HepG-2 with IC₅₀ values of 9.27, 7.69 and 5.91 μM, respectively, related to standard Doxorubicin. Moreover, compounds <b>5</b> and <b>10</b> showed good inhibition against cyclin dependent kinase (CDK4/cyclin D1) and epidermal growth factor (EGFR) enzymes. Pyridopyrimidines derivatives (<b>3–20</b>) have been designed and synthesized through the different reaction conditions. The chemistry of all synthesized compounds were confirmed by the different spectroscopic techniques; IR, ¹H NMR, ¹³C NMR and mass spectroscopy. <i>In vitro</i> cytotoxicity assay has been conducted to evaluate the anti-cancer activities of pyridopyrimidines derivatives toward four types of cancer cell lines. The structure–activity relationship have shown that the cyclization of pyridopyrimidine ring with pyrazole ring and the hybridization with benzylidine resulted in producing derivatives with potent anticancer effects. Enzyme inhibition assay has been performed for compounds <b>5</b> and <b>10</b> which showed strong inhibition of EGFR and CDK4-cyclin D1, utilizing Erlotinib and Palbociclib, respectively as reference drugs. The molecular modeling calculations have been performed which went with the results of enzyme inhibition assay. Cell cycle arrest and apoptosis assay have been done for compounds <b>5</b> and <b>10</b> which showed arrest at G0/G1 phase and induced apoptosis. Real-time Reverse transcription polymerase chain reaction for Bax and Bcl2 has been conducted to compounds <b>5</b> and <b>10</b> to investigate their ability to act as pro-apoptotic or anti-apoptotic inducers. The Lipinski's rule has been calculated using the Molinspiration online program for all new synthesized compounds. Fortunately, compounds <b>5</b> and <b>10</b> have obeyed the Lipinski's rule, so they can be considered as bioavailable drugs.