Design, Synthesis And Antitumor Activity Study Of Novel Histone Deacetylase Inhibitors

Ⅰ. Research backgroundTumor is the secondary killer of human life next to cardiovascular and cerebrovascular diseases. In current China, the growth rate of malignant tumor incidence and mortality is as high as2.5%and1.3%, respectively.Histone deacetylases (HDACs) are enzymes responsible for deacetylation of lysine residues in various proteinaceous substrates such as nucleosomal histones. The human HDACs family consists of18isoforms, which can be divided into4classes.Many experiments revealed that expression and activity of HDACs are upregulated in many tumor types. The hydrolysis of the acetyl group from the histones results in condensation of chromosomal DNA and transcriptional repression. Besides, aberrant deacetylation of other non-histone proteins can influence protein stability and localization, protein-DNA interaction and protein-protein interaction, many of which can promote tumorigenesis and development. The effects of HDACs in tumor mainly contain:1. promoting tumor cell proliferation and invasion;2. promoting tumor angiogenesis;3. enhancing resistance to chemotherapy and radiotherapy;4. prohibiting tumor cell differentiation and apoptosis, etc.Given the pleiotropic functions of HDACs in tumorigenesis and development, the diverse antitumor mechanisms of HDACs inhibitors are slowly unfolding: inducing tumor cell apoptosis and autophagy, causing tumor cell cycle arrest, anti-angiogenesis, reducing DNA damage repair, enhancing chemotherapy and radiotherapy, reducing tumor cell invation and motility, etc. Currently, over ten HDACs inhibitors are in clinical trials as antitumor agents and two of them, SAHA and FK228, have been approved by the FDA for the treatment of cutaneous T-cell lymphoma (CTCL). Therefore, pursuing potent HDACs inhibitors with low toxicity has become the hot topic in the field of antitumor research.Despite the variety of structural characteristics, most HDACs inhibitors can be broadly described by a common pharmacophore, which mainly contains3parts:a zinc ion binding group (ZBG) and a surface recognition domain, joined by a linker domain with proper length. In this study, according to the analysis of the HDACs active site structure and known HDACs inhibitors pharmacophore, we rationally designed and synthesized series of HDACs inhibitors with novel chemical scaffolds. Through several rounds of evaluation, SAR analysis and structural optimization, we would like to find some lead compounds with promising in vitro and in vivo antitumor activity, which could lay a solid foundation for the research and development of novel antitumor drugs with our own intellectual property.Ⅱ. Design, synthesis and antitumor activity study of tetrahydroisoquinoline-3-carboxylic acid-based hydroxamic acid derivatives as HDACs inhibitorsOne effective strategy in novel HDACs inhibitors design is introducing diverse chemical scaffolds to any part of the common pharmacopore. In this study, we applied classical conformational restriction strategy to design HDACs inhibitors with rigid linker. Increasing selectivity and reducing toxicity of lead compounds is the principal advantage of conformational restriction. Moreover, another intriguing advantage is that rigid compounds tend to have higher bioavailability and stability.Based on our literatures investigation, we found that Tic, short for1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, is an unnatural a-amino acid with distinct geometrical conformation and varied biological activity. The structure of Tic has been contained in many active compounds. In this part, we introduced the rigid Tic fragment to the linker part of HDACs inhibitors and the rationality of our design was confirmed by compouter aided drug design (CADD) system. We totally synthesized66compounds, which were evaluated in enzyme inhibitin assay, MTT assay and in nude mouse models. All target compounds are novel without any report before us and their structures were confirmed by1HNMR, HRMS and IR. Part compounds with good activity are≥95%pure by HPLC analysis.On the basis that all Zn2+dependent HDACs are highly conserved in their active sites and the crystal structure of HDACS active site is now available, we firstly used HDAC8as the enzyme source to screen our target compounds. Most tetrahydrpisoquinoline-based hydroximic acid derivatives exhibited more potent HDAC8inhibition than the positive control SAHA. Compounds with good enzyme inhibition were further evaluated in MTT assay and in nude mouse xenograft models. Among these analogues, compounds13e,22a and22c exhibited comparable even more potent in vivo antitumor activities in a human breast carcinoma (MDA-MB-231) xenograft model, a human colon tumor (HCT116) xenograft model and a mouse hepatoma-22(H22) pulmonary metastasis model. The most potent compound22c has been chosen as the antitumor candidate for further preclinical research and development due to its superior antitumor potency compared with SAHA.Ⅲ. Design, synthesis and antitumor activity study of tyrosine-based hydroxamic acid derivatives as HDACs inhibitorsThe promising antitumor activity of tetrahydroisoquinoline-based HDACs inhibitors inspired us to make some modifications and optimizations on this scaffold. In order to investigate the influence of molecular flexibility on HDACs inhibitory activity, the tetrahydroisoquinoline scaffold was simplified to the more flexible tyrosine scaffold. In this part, total13compounds were designed and synthesized with their structures confirmed by1HNMR and HRMS. All these compounds are novel without any report before us.Enzyme inhibition assay revealed that tyrosine-based derivatives exhibited more potent HDAC8inhibitory activities relative to their corresponding tetrahydroisoquinoline analogues and SAHA. Among these new derivatives, compounds Q1, Q2, Q3and Q4were over10-fold more potent against HDAC8than SAHA. However, their antiproliferative activities against human breast carcinoma (MDA-MB-231), human lung carcinoma (A549) and human colon tumor (HCT116) were disappointing. Further enzyme inhibition assay against HeLa cell nuclear extract (mainly HDAC1and HDAC2) indicated that Q1-Q4were much less potent than SAHA, which might explain why their antiproliferative activities were poor because HDAC1and HDAC2were reported to be closely related to tumor cell proliferation. These results gave us the information that the tyrosine-based hydroxamic acid scaffold was HDAC8isoform selective to some degree, which could be further derivatized to give rise to HDAC8selective inhibitors.Ⅳ. Design, synthesis and antitumor activity study of linear l,4-Dithia-7-azaspiro[4,4]nonane-8-carboxylic acid-based hydroxamic acid derivatives as HDACs inhibitorsIn this part, a novel series of peptidomimetics HDACs inhibitors with linear linker was designed and synthesized based on the common HDACs inhibitors pharmacophore. In order to probe its influence on activity and selectivity, the unnatural amino acid1,4-Dithia-7-azaspiro[4,4]nonane-8-carboxylic acid was introduced into the surface recognition domain of our HDACs inhibitors. Newly synthesized30compounds are all novel without any report before us with their structures confirmed by'HNMR and HRMS. Some representative compounds were analyzed by13CNMR.Enzyme inhibition assay revealed that most of these derivatives were very potent against HDAC8. It was notable that compounds33f,331,341,33k,33m and33n were more potent against HDAC8than SAHA, with the IC50of331and33k being as low as0.021±0.004mmol and0.035±0.007mmol, respectively. However, their antiproliferative acitivities against human breast carcinoma (MCF-7, MDA-MB-231) and prostate carcinoma (PC-3) were not satisfactory. The following enzyme inhibition assay against HDAC1showed that compounds331and33k were more prefer to HDAC8rather than HDAC1. So these two compounds could be used as the lead to design HDACs isoform selective inhibitors.Ⅴ. Conclusion and perspectiveIn conclusion, based on the combination of medicinal chemistry, chemical biology and computational chemistry, we rationally designed and synthesized109novel HDACs inhibitors, which could be divided into3series according to their scaffolds. All the target compounds were tested in HDACs enzyme inhibition assay and compounds with good activities were progressed to in vitro antiproliferative assays and in vivo experiments. Our research revealed that tetrahydroisoquinoline scaffold could be used to obtain pan-HDACs inhibitors with potent in vitro and in vivo antitumor activities. Among these tetrahydroisoquinoline-based hydroxamic acid derivatives, compound22c exhibited more potent in vitro and in vivo antitumor potency than approved drug SAHA, and further research and development are underway. While the tyrosine scaffold and the1,4-Dithia-7-azaspiro[4,4]nonane-8-carboxylic acid scaffold are promising in developing HDACs isoform selective inhibitors, which could be very useful in treating diseases (such as inflammation, neurodegenerative disease, etc) caused by disorder of some specific HDACs isoform.