Novel Benzyloxyphenyl Pyrimidine-5-Carbonitrile Derivatives as Potential Apoptotic Antiproliferative Agents

Abstract

Background: Pyrimidine-5-carbonitrile had a broad spectrum of biological activities such as antiviral, antioxidant and anticancer activity. Among similar compounds, monastrol being the most prominent due to cell-permeant inhibitor of mitosis therefore, we investigated the new Pyrimidine-5-carbonitrile as a cytotoxic agent for p53 pathway. Objective: Several new benzyloxyphenyl pyrimidine-5-carbonitrile derivatives were designed, synthesized, characterized, and their cytotoxicity was evaluated. The most active compounds were tested for their activity against p53 as a mechanistic target for antiproliferative action. Method: The key intermediate tetrahydropyrimidine-5-carbonitrile derivative 4 was prepared by a multicomponent reaction (MCR) of Biginelli type. S-alkylation of the key intermediate with the required alkyl or aralkyl halides or refluxing 4 with POCl3 followed by an amino acid yielded the target compounds. The cytotoxicity of 5c-e, 7a-c, 9, 10a, b and 11 was evaluated using A549 cell line of human lung adenocarcinoma, HepG2 liver cell line, and MDA-MB-231 cell line of breast cancer using the MTT assay. The transcription effects of 7a, 7c, and 11 on the p53 were assessed and compared with the reference doxorubicin. Results: Compounds 7a, 7c, and 11 have the highest cytotoxic effect when applied to most cancer cells. The tested compounds with 5-FU showed a significant increase in the anticancer activity more than 5-FU alone. Compounds 7a, 7c, and 11 increased the level of active caspase 3 by 4-6-fold, compared to untreated control cells in human liver cancer cell line (HepG2). Compounds 7a, 7c, and 11 increase the levels of caspase 8 and 9, indicating activation of both intrinsic and extrinsic pathways and showed potent induction of Bax, down-regulation of Bcl-2 protein levels and over-expression of Cytochrome C levels in HepG2 cell lines. Compound 11 exhibited cell cycle arrest at the Pre-G1 and G2/M phases in the cell cycle analysis of HepG2 cell line. The results revealed an increase of 12.40-19.10 in p53 level compared to the test cells and that p53 protein level of 7a, 7c, and 11 was significantly inductive (636, 861 and 987 pg/mL, respectively) in relation to doxorubicin (1263 pg/mL). Conclusion: Pyrimidine-5-carbonitrile derivatives have potent apoptotic and antiproliferative properties.