Study On Design?Synthesis And Biological Activity Of Heat Shock Protein 90 (HSP90) Inhibitors

Heat shock protein 90 (HSP90) is an important anti-tumor targets. HSP90 as the molecular chaperone combining with co-chaperone and binding client protein together form a complex that make these proteins to facilitate proper assembly and folding, so as to achieve its active conformation and increase its stability. Mutations and over-expressing of HER2, EGFR, AKT, p53, BCR-ABL and Raf in non-small cell lung cancer, breast cancer, stomach cancer, chronic myelogenous leukemia, prostate cancer and epidermoid carcinoma or other cancer are all HSP90 client proteins. These proteins involved in the occurrence, proliferative, metastasis and tumor angiogenesis process, closely related to tumor invasion. Therefore, the development of new and efficient HSP90 inhibitors for cancer treatment is an important way.HSP90 inhibitors began in the study on natural product inhibitors. Such as: Gedunin (terpenes), the bitter component in bark of Melia toosendan Sieb.et Zucc. or Melia azedarach L.; Celastrol (triterpenoids) in root of Tripterygium wilfordij Hook.f.; Epigallocatechin gallate (flavonoids) in green tea; Natural products: Derrubone (isoflavones); Novobiocin (coumarin); Geldanamycin, and so on. Their further study are limited by weak effect of anti-cancer, poor stability, cytotoxicity and other shortcomings. Subsequently, based on the natural product inhibitors developed the first generation of HSP90 inhibitors, but clinical studies have found that there are some side effects, such as:liver toxicity, cardiac toxicity. The second generation of HSP90 has over 9 synthesis of small molecule inhibitors in clinical studies. Many second-generation HSP90 inhibitors exhibit reversible toxicity characteristics. Despite strong anti-tumor activities are showed in pre-clinical, due to the evolution of drug resistance, ineffective dose of drug, the lack of a sensitive customer protein or lack of sustained suppression cause the single HSP90 inhibitors show failure effectiveness in clinical tests. Up to now, there has not been a HSP90 inhibitor approved by the FDA, research and development of new, high efficiency, low toxicity of HSP90 inhibitors have very important significance. This subject which is supported by Foshan Municipal Science and Technology Innovation Project (2013HK100012), Guangzhou Technology Support Program (2014J4100222) and the National Natural Science Foundation of China (No.21402205) is used for the development of new research HSP90 inhibitors.This thesis was divided into four chapters. In the first chapter, we reviewed recent progress in the development of HSP90. First, the biological nature of human shock protein 90 (HSP90) was introduced. Then the research progress of HSP90 inhibitors was paid attention:1) HSP90 inhibitors of natural products.2) HSP90 inhibitors in clinical.3) HSP90 inhibitors based on isoxazole nucleus. At last, basis of vertical title was clarified.In the second chapter, design ideas for the new HSP90 inhibitors were stated. We use the following means:NVP-AUY922 as a precursor compound combines C6-C3 skeleton of natural product HSP90 inhibitors, structure of Luminespib (NVP-AUY922) in clinical stage ? and cocrystallization in HSP90 active aite to analysis the efficacy of the compound by a group of computer simulation technology and summarize pharmacophore of structural features by the activity data of isoxazole skeleton of small molecule HSP90 inhibitors to design the new skeletal structure:1) 4-substituted 5-aryl-alkynyl isoxazole-3-carboxamide that the alkynyl was less molecular weight (52), greater ?-? effect than the phenyl.2) 4-triazole-5-aryl isoxazole-3-carboxamide that aromatic triazole ring substituted the phenyl. At first, the structural segment of novel design HSP90 inhibitors were cut and inverse synthesis was analyzed. Finally, an economically viable organic synthesis route was obtained.In the third chapter, the process of organic synthesis of the target compounds was described. We selected market readily available, low-cost 2.4-hydroxyacetophenone as a starting raw material in organic synthesis experiment of this subject. Phenolic hydroxyl group protected by the benzyl (yield 79%). Witting reaction (79% yield), Pd/C catalyzed hydrogen olefin reduction (80% yield), Fried el-Crafts reaction (yield 99.4%), phenolic hydroxyl group protected by the benzyl (yield 81%), Aldol condensation (96% yield). cyclization of hydroxylamine (83% yield), aminolysis of ester (yield 96%) and halo (yield 70%) to afford the key intermediate 5-(2.4-benzyloxy-5 isopropyl)-N-ethyl-4-iodo-isoxazole-3-carboxamide. Then the key intermediate was used to substrate:two 4-phenyl-substituted-5-aryl-N-ethyl-3-carbox-ylic acid amine of novel HSP90 inhibitors were obtained, after Suzuki reaction, reductive amination. deprotection of the benzyl organic synthesis reaction; 38 4-substituted-5-aryl-alkynyl-N-ethyl-isoxazol-3-carboxamides of novel HSP90 inhibitors were obtained, After Sonogashira reaction, deprotection of the benzyl organic synthesis reaction; 124-substituted-5-aryl-alkynyl-N-ethyl-isoxazol-3-carbo-xamides novel HSP90 inhibitors were obtained, after Sonogashira reaction, Clink reaction, a amino group substituted reaction, debenzylation reaction. All new chemical entities have been confirmed by 1H NMR (400MHz.500MHZ).13C NMR (100MHz.125MHz). and MS (ESI-APC1).In the fourth chapter, the test of the biological activity was described and the structure-activity relationship of new chemical precursors was analyzed. Fluorescence polarization assay analyzed binding capacity of new chemical entities and HSP90a, HSP90?, two subtypes of HSP90, And we discussed the structure-activity relationships of new chemical entities based on the above results. Anti-proliferative activity of new chemical entities in human lung cancer cell line (A549), human breast cancer cell line (MCF-7), chronic myelogenous leukemia cell line (K562), prostate cancer cell lines (DU145) and skin test cancer cell lines (Hela) were evaluated by MTT assay. The results showed that:In addition to the isoxazole ring-opened product (compound 1) has no biological activity, the rest of the new chemical entities have HSP90 activity and anti-proliferative activity. Especially,4-phenyl-substituted isoxazole derivative (Compound 2) biological activity reached the nanomolar levelThis paper provides the theoretical basis for study on novel HSP90 inhibitors and the experimental basis for technology research and development of new drugs of C-4-substituted isoxazole-class novel HSP90 inhibitors in the futureInnovation point:(1) They were designed by using computer simulation technology and combining the basic skeleton of natural products and synthetic drugs skeleton. Two new kinds of chemical precursors with independent intellectual property rights of HSP90 inhibitors through an economically viable synthesis were obtained: 4-alkynyl substituted -5-aryl-N-ethyl-isoxazole-3-carboxamide,4-triazolesubstituted-5-aryl-N-ethyl-isoxazole-3-carboxamide. This provided a new way for the research of new HSP90 inhibitors for the C-4-substituted isoxazoles HSP90 inhibitors and an experimental basis for industrial production.(2) Fluorescence polarization assay analyzed binding capacity of new chemical entities and HSP90a, HSP90?, two subtypes of HSP90, And we discussed the structure-activity relationships of new chemical entities based on the above results, which provided theoretical and practical value for research and development of good activity of HSP90 inhibitors to HSP90a and HSP90?.(3) MTT assay was used to evaluate anti-proliferative activity of the chemical precursors in human lung cancer cell line A549, human breast cancer cell line MCF-7, chronic myelogenous leukemia cell line K562, prostate cancer cell line DU145 and proliferation of epidermal carcinoma cell line Hela, which provided foundation for broad-spectrum anti-cancer drug research and development.