Design, Synthesis, Optimization And Biological Evaluation Of Anti-cancer Agents Targeting BRAF

In recent years, a number of key signaling pathways, membrane receptors, kinases and other biological macromolecules, which play an important role in tumorigenesis and cancer development, have been identified along with the further understanding of the pathogenesis of human cancer. Development of small molecular drugs specially targeting these specific macromolecules provides opportunities to overcome human cancers. Mitogen activated protein kinase (MAPK) signal transduction pathway is one of the most important signaling pathways inside the cell. BRAF, which is one of the most important pro-oncogenes in MAPK pathway, is mutated in approximately8%of human tumors. The most common BRAF mutation is a valine-to-glutamate transition (V600E) that constitutively activates MEK/ERK pathway in cancer cells, leading to tumor development, invasion, and metastasis. There is an accumulating body of experimental evidences validating oncogenic BRAFV600E as a therapeutic target and offering opportunities for anti-cancer drug development.In this thesis, we focus on the identification and modification of novel molecular probes that interfere with the ATP binding region of BRAFV600E as a demonstration of using highly specific and selective small molecule agents to inhibit the protein-kinase activity of BRAFV600E. Virtual screening program and in vitro protein-kinase inhibitory activities evaluation were introduced for initial screening. Then candidate compounds3,5-diarylpyrazoline salicylamide derivative Hit1(IC50=7.22μM), urea derivative Hit4(IC50=3.08μM) and niacinamide derivative Hit5(IC50=6.9μM) were obtained and selected for further optimization and pharmacophore studies. Based on the structure of lead-compound and SAR (Structure-activity Relationship) of synthesized analogs,141pyrazoline derivatives have been designed, synthesized and evaluated in vitro for their pharmaceutical activities as anti-cancer agents.(1) On the basis of the structural skeleton of Hit1and Hit4, various substituents were introduced into C-3, C-5and N-1position of pyrazoline ring, including the thiourea motif in N-1position. Then twenty3,5-diarylpyrazoline salicylamide derivatives and sixteen3,5-diarylpyrazoline urea derivatives were designed and synthesized, and thirty compounds were first reported. Compound S25exhibited the most potent inhibitory activity with an IC50value of0.16μM for BRAFV600E and GI50value of0.24μM for mutant BRAF-dependent WM266.4melanoma cells. The BRAFWT (BRAF wild-type) cellular assay and BRAFV600E cancer cell based pERK assay of compound S25suggested it could selectively inhibit proliferation of mutant BRAF-dependent melanoma cell line through inhibiting oncogenic BRAF. The preliminary SAR analysis and subsequent3D-QSAR study of inhibitor analogues, provide important pharmacophore clues for further structural optimization.(2) According to the skeleton of3,5-diarylpyrazoline niacinamide derivative Hit5(IC5o=6.9μM), further structural modification was performed, all the81synthesized analogs were new compounds. Firstly, various substituents were introduced in C-3and C-5position of pyrazoline ring. Then structural modification was proceeded on the pyridine ring of Hit5, altogether513,5-diarylpyrazoline niacinamide derivatives were obtained in this part. Among them, compound20n exhibited the most potent inhibitory activity with an IC50value of0.17μM for BRAFV600E and GI50value of0.89μM for BRAFV600E-dependent WM266.4melanoma cells. The3D-QSAR models with reliable predictability were established to analyze the structure-activity relationship and provide pharmacophore clues for further structural optimization. The3D-QSAR contour map suggested an increasing binding affinity might be obtained by replacing the niacinamide moiety with a slightly larger template which also can cause a lower electron density. Thus,1,4-benzodioxan motif was introduced in N-1position and thirty2,3-dihydrobenzo[6][1,4]dioxin-containing4,5-dihydro-1H-pyrazole derivatives were designed as potential B-Rafv600E inhibitors and predicted to have a positive progress with a sound cancer therapeutic benefit. Finally, the most potent BRAFv600E inhibitor owing3,5-diarylpyrazoline core, C14(IC50BRAFV600E=0.11μM, GI50WM266.4=0.58μM), was identified. The introduction of2,3-dihydrobenzo[b][1,4]dioxin structure reinforced the combination of our compounds and the receptor, resulting in progress of bioactivity.(3) With the in-depth study of BRAFV600E inhibitors as anti-cancer agents, discovery of MEK inhibitor was considered as possible solution of the treatment for tumor with dyregulated ERK signaling pathway. Two series of novel N-benzyl-N-(4-hydroxy-3-methoxy-benzyl)-N’-phenylurea and thiourea derivatives (mlu-mllu, mlt-mllt) designing for MEK kinase inhibitors have been discovered. Their anti-proliferative activities against the melanoma cell line B16-F10and MEK inhibitory activities were evaluated. Some compounds displayed good inhibitory activities and the SAR have also been studied. Among the compounds tested, we found compound m6t had demonstrated significant MEK inhibitory activity (IC50=1.27μM) and anti-proliferative activity in melanoma cells growth inhibition (GI50=0.33μM). Through DAPI staining and Annexin V-FITC and PI staining for apoptosis assay, it was found compound m6t mainly induced cell death in apoptotic mode. Therefore, compound m6t with potent MEK inhibitory activity would be a potential anticancer agent deserving further study.