Design, synthesis, biological evaluation and molecular docking study of new pyrazolo[1,5-a]pyrimidines as PIM kinase inhibitors and apoptosis inducers

Abstract

This study involved designing and synthesizing new derivatives of pyrazolo[1,5-a]pyrimidine 5a-l, which were prepared via reaction of 4-Arylazo-1H-pyrazole-3,5-diamines 3a,b with arylidenemalononitriles (4a-f) in refluxing methanol. The structure of the synthesized derivatives were confirmed using different spectroscopic techniques. Virtual screening was achieved for the newly designed derivatives using docking simulation between the target pyrazolo[1,5-a]pyrimidine 5j and the active binding site of the PIM-1 enzyme. The cytotoxicity of novel derivatives of pyrazolo[1,5-a]pyrimidine framework was screened on the cancer cell lines: colon (HCT-116), hepatocellular (Hep-G2), and breast (MCF-7). Comparing with doxorubicin, compounds 2,5-diamino-7-(4-ethoxyphenyl)-3-(p-tolyldiazenyl)pyrazolo[1,5-a]pyrimidine-6-carbonitrile; 5h, 2,5-diamino-7-(4-hydroxy-3-methoxyphenyl)-3-(p-tolyldiazenyl)pyrazolo[1,5-a]pyrimidine-6-carbonitrile; 5j, and 2,5-diamino-7-(furan-2-yl)-3-(p-tolyldiazenyl)pyrazolo[1,5-a]pyrimidine-6-carbonitrile; 5l exhibited noteworthy anticancer properties, with half maximal inhibitory concentration (IC50 range) = 1.26–3.22 μM. In addition, the most potent compound, 5j demonstrated a lower level of toxicity towards normal cells (WI-38 cell line), suggesting an increased level of safety. Compounds 5h, 5j, and 5l were evaluated for their activity against three different PIM kinases (PIM-1, PIM-2, and PIM-3 enzymes) in an effort to elucidate their inhibitory potential on PIM kinases. Additionally, the apoptotic potency of 5j was assessed on MCF-7 cells. Results revealed that compound 5j was potent against both PIM-1 and PIM-2 having IC50 0.158 and 0.297 μM, respectively, comparing to staurosporine's IC50 0.294 and 0.477 μM against both enzymes, respectively, while its inhibitory activity against PIM-3 enzyme was moderate. In addition, compound 5j promoted apoptosis via elevating the levels of Bax, p53, and caspase-3 and dropping down Bcl-2 level. G1 phase arrest of cell cycle was also observed. Furthermore, docking analyses of 5j in the PIM-1 binding site was conducted.