Design,Synthesis And Biological Evaluation Of The New C-Met Receptor Inhibitors

Tumor is the body’s neoplasm which is resulted from the abnormal proliferation and differentiation of the local tissue cells which were acted by a variety of induce tumor factor and lost the normal regulation of growth at the level of gene.Currently,the number of each year’s new cases of malignant tumor patients is about 3.6 million in our country,and the incidence rate reached to 264.85 / 100,000,which is more than nearly 20% of the world level of 187.83 / 100,000. About 2.2million people died from malignant tumors per year in China,and the mortality rate was about 161.49 / 100,000, which is more than nearly 50% of the world level.Thus,cancer with a higher incidence and mortality has replaced the heart and brain vascular disease and become the number one killer endangering people’s lives and safety.Tumor metastasis is the main reason causing tumor progression, difficult to be effectively treatment and ultimately lead to the death of tumor patients.The discovery and validation of anti-metastatic target and the development of anti-metastatic drugs have become the focus of upday’s anticancer drug research because of their extremely important clinical and economic value.The metastasis and development of tumor cells are closely related to the abnormal expression of many signal pathways in the cell.C-Met protein is a hepatocyte growth factor receptor(Hepatocyte growth factor receptor, HGFR)encoded by the c-Met proto-oncogene with tyrosine kinase activity, which can mediate multiple signaling pathways after bening active,mainly including the MAPK pathway, PI3 K signaling pathway, STAT signaling pathway, nuclear factor termediating,which is variety of cellular functions.It plays a very important role in cellular signal trs and plays an important role in cell proliferation and mitosis aspects.Mutation of met gene and activation will result to overexpression of HGF/SF or MET,and produce autocrine signaling, gene amplification, and then promote the proliferation of tumor cells, metastasis and angiogenesis. The basical and clinical medicine proved that inhibiting the overexpression of c-MET gene or inhibiting the function of c-Met protein can effectively inhibit tumor growth and metastasis ofcancer cells.At present, c-Met protein has become an important target for the anticancer drug design, and the small molecule inhibitor of c-Met protein is becoming a more and more hot spot in anticancer drug discovery.The current developed c-Met inhibitors can be divided into 2-amino-pyridines, anthraquinones, 3,5-disubstituted and3,5,7-trisubstituted quinoline, 2,4-diamino-aryl pyridine classes. Crizotinib, the representative drug, has been approved in August 2011, for non-small cell lung cancer(NSCLC, non-small cell lung cancer) treatment. The compound LY2801653 developed by Lilly company is another promising oral small molecule c-Met inhibitor,which is currently in phase II clinical trials.The published data showed that have good anti-tumor activity in non-small cell lung cancer(NSCLC), pancreatic cancer, breast cancer and renal cancer cell lines, and has potent anti-proliferative and anti-angiogenic.LY-2801653 can inhibit the activity of the ROS1 fusion protein with the similar activity of XL184(Cabozantinib) and XL880(Fretinib),and with better activity than that of crizotinib.In vivo studies, LY2801653 at a dose of 20 mg / kg, the inhibition rate of cancer cells reached 86.5%.Another advantage of LY-2801653 compared with the XL-184 and XL-880 is that it can effectively inhibit the activity of Met and MKNK-1/2 with mutation. In this paper, LY2801653 was taken as the template molecule, and the structure-activity relationship analysis result of this kinds of compounds, bioisosterism principle, using pyridine ring substituted pyrazole ring,using malonic amide substituted pyridone to increase hydrogen bonding interaction and ability to binding with the enzyme. These strategies were used to design the first class of the target compounds.In addition, according to the similar compounds’ structure-activity analysis result,and keeping pyridone side chain, using benzyl to substitute azaindole and using dimethyl ethoxy quinoline to substitute benzo pyrazole, respectively, the second and third classes of target compounds were designed.In this paper, the synthesis route design, chemical synthesis and structure confirmation of the LY-2801653 lead compound and the three kinds of c-Met receptor inhibitors were completed.The main research results are summarized as follows:The method of LY-2801653 synthesis was optimized, and the positive drug synthesis method with easy to get started marterial,safe, high efficient, lower cost,easy to operate and suitable for the industrial amplification was obtained. Thatwas,taken 4-Fluoroaniline as the starting material, via acylation, Mike addition steps to synthetize 1-(4-fluorophenyl)-6-methyl-2-oxo-1,2-dihydropyridine-3 carboxylic acid(2). Then to take 3-hydroxy-benzaldehyde as the starting material, via bromination, imide reaction, nucleophilic cyclization, reduction reaction steps to synthetize intermediates 4-((6-bromo-1-methyl-1H- indazol-5 yl) oxy)-3-fluoroaniline(6). The intermediate(7), N-(4-((6- bromo-1-methyl-1H- indazol-5-yl) oxy)-3-fluorophenyl) 1-(4-fluorophenyl)-6-methyl-2-oxo-1,2-dihydropyridine-3-carboxamide was synthetized by the amide condensation reaction between the intermediate(2) and(6). The intermediate(7) reacted with tert-butyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) l H-pyrazole-1-carboxylic acid tert-butyl ester to obtaine the positive drug LY-2801653 by the Suzuki reaction.The first class of target compounds were designed and synthesized. That is using 3-hydroxybenzaldehyde as the starting material, via bromination reaction to obtain the intermediates of 2,4-dibromo-5-brominated hydroxy benzaldehyde(TDC-1a),and then the intermediate(TDC-1a) reacted with methyl hydrazine sulfate to obtain the intermediate TDC-1b, which continued to conduct cyclization catalyzed by cuprous chloride under reflux temperature over night to give intermediate TDC-1c.Taken tetratriphenylphosphine palladium as catalyst, cesium carbonate as base, in the mixed solution of dioxane and water, the intermediate TDC-1c and 3-pyridyl boronic acid via Suzuki reaction to obtain the intermediate 5-(2-fluoro 4-nitrophenyl)-1-methyl-6-(pyridin-3-yl) l H-indazole(TDC-1d),this intermediate was reduced by iron power,and then reacted with the compounds with various carboxylic acids to give the first kind of target compounds.According to this synthetic route,11 target compounds were synthesized.The second class of target compounds were designed and synthesized.That is using 7-azaindole as a starting material, via oxidizing of nitrogen atom of pyridine by m CPBA to give N-oxo-7-azaindole,after chlorinating with phosphorus oxychloride,then to reacted with benzyl bromide under basic conditions and heating and stirring overnight to give intermediate 1-benzyl-4-chloro--1H- pyrrolo [2,3-b]pyridine(TDC-2b). This intermediate reacted with 4-hydroxyaniline firstly,and then reacted with the compounds containing carboxyl functional group to give the second class target compounds.According to this synthetic route, 3 target compounds were synthesied.The third class of target compounds were designed and synthesized.That is using4-chloro-6,7-dimethoxy quinoline as starting materials to react with a hydroxyl aniline to obtain the compound 4-((6,7-methoxy-quinolin-4-yl) oxy) aniline.This intermediate reacted with the compounds containing carboxyl functional group to give the third class target compounds.According to this synthetic route, 5 target compounds were synthesied.In this paper, the positive compound LY2801653 and the 19 target compounds with new structure were synthesied.The structures of all the new compounds have been confirmed by 1HNMR and MS.The c-Met protease inhibitory activity of all the target compounds was determined. Test results showed : the IC50 value of the compounds TDC-4-1,TDC-4-12, TDC-4-2, TDC-4-4, TDC-4-24, TDC-4-28, TDC-4-30, TDC-4-34,TDC-4-36, TDC-4-40, TDC-4-44, TDC-4-46, TDC-4-48 was under 1n M, has a certain inhibitory activity on c-Met protease.The inhibitory activity(IC50) of the five compounds TDC-4-1, TDC-4-2, TDC-4-4, TDC-4-24, TDC-4-48 are under 100 n M.TDC-4-1、TDC-4-48 with the IC50 of 16.89 n M and 24.43 n M were the best C-Met protease inhibitos,While their inhibitory activity was still less than that of the positive compound.In this paper, the structure activity relationship(QSAR) of the c-Met receptor inhibitors was discussed. Among the three kinds of compounds, the activity of the third kinds of target compounds was the best, which was generally higher than that of the first class. and the activity of the second kinds of target compounds was poor. This indicates that the hydrogen atom on the N atom of pyrrole can not be replaced by other substitute, otherwise the activity will be reduced.Dimethoxy-quinoline displayed high ability to form hydrogen bond with c-Met protein,which can increase the binding ability of inhibitor with the protein and their inhibitory activity. The length of the side chain and the hydrophobicity had effect on the activity, and usually the short side chain or hydrophobic side chain was beneficial to the improvement of the activity.