Design, Synthesis And Biological Evaluation Of Pyrazolyl-nitroimidazole Derivatives As Potential EGFR/HER-2 Kinase Inhibitors

The occurrence and recurrence and metastasis of Malignancies is a very sophisticated and complex process. With people’s further study in the relationship between development of cancer and cell signaling transduction, people gradually found that the abnormal activation of cell signal transduction pathway might be an important reason of tumorigenesis. As a member of protein tyrosine kinase receptor family, epidermal growth factor receptor (EGFR/HER-2), when combined with epidermal growth factor (EGF/TGF-a), it can activate the intracellular region of EGFR/HER-2, which initiate complex signaling pathways transferred by the The medium material. Genes encoding the epidermal growth factor receptor protein locate on chromosome 7p12, these genes stably express in a variety of epithelial and mesenchymal tissue as well as neuronal tissue. When over expression or mutation of EGFR/HER-2 occurs, it often leads to sustained activation of downstream signaling pathways, Which ultimately leading to cell transformation, proliferation and resistance to apoptosis. In fact, all these above is closely related to the development of tumors. Therefore EGFR/HER-2 as a target for small molecule tyrosine kinase inhibitors has been clinically proved to be a efficient anticancer drugs.Dihydropyrazole, a extremely important nitrogen-containing five-member heterocyclic compound which has strong biological activity, such as anti-bacterial, anti-viral, anti-fungal, anti-tuberculosis, insecticidal activity, attracted wide attention. Chiral properties resulting from the structure, lead to the variability of substituent conformation on dihydropyrazole ring, which shows promising potential for biological activity. In recent years, related research have proved that the dihydropyrazole structure has the potential effect against cancer cells. In addition, a five-member heterocyclic ring containing two nitrogen atoms, imidazole ring, often interact with receptor by forming non-covalent bond such as π-π bond, H bond, coordination bond between nitrogen and acceptor atoms. According to these mechanism, nitroimidazole often appear as tumor vascular disrupting agents, tumor resistance reversal agents, radiation sensitizers for clinical treatment.In summary, in order to get a compound with better small molecule tyrosine kinase inhibitory activity, we design and screen a series of small molecule inhibitors targeting EGFR protein and having pyrazole, nitro-imidazole as basic skeleton based on known active skeleton structure, computer-aided drug design, comparative moleular field analysis so on. Meanwhile, the build-related 3D-QSAR model analysed a quantitative structure-activity relationship group of homologues skeleton with a good prediction and correlation.20 target compounds were designed in this research, which were all first synthesized and made correct structural characterization by utilizing 1H NMR, ESI-MS,13C NMR. As for biological activity, The inhibition activity of all small molecule drugs on EGFR/HER-2 protein was evaluated by the selective experiment of kinase. Wherein the most potent compound 5c exhibited excellent inhibitory activities with IC50 value 0.26±0.03 μM/0.51±0.06/M respectively, while the positive control drug Erlotinib had the IC50 value of 0.20±0.02/0.41±0.02/M as well as the IC50 of Lapatinib stays 0.28±0.03/0.54±0.02 μM. Besides, it is potent in the cell antiproliferative activity in four tested cancer cell lines (MCF-7, Hela, HepG2, B16-F10), with most effective antiproliferation in the Hela. The best behaved active compound was also compound 5c with IC50 value 0.13±0.05μM, while the positive control drug, Erlotinib/Lapatinib having the IC50 value remained 0.20±0.06/0.12±0.02 μM. In addition, compound 5c was further proved to induce Hela dose dependent early apoptosis by flow cytometry with annexin V-FITC/PI apoptosis. In order to display the binding mode between compound 5c and EGFR tyrosine kinase receptor, we performed molecular simulation. The result showed that compound 5c was nicely bound to the EGFR via one hydrogen bond, one van der waals interaction, one π -cationic interaction and three charge interactions. In conclusion, all compounds exhibit promising biological activity against human cervical carcinoma proliferation, metastasis and invasion and provide a guideline to design and optimize more effective EGFR inhibitors, of which compound 5c was expected to be a novel blocker impacting receptor tyrosine protein kinase EGFR signal transduction.