Design, Synthesis And Antitumor Activity Study Of Lead Compounds Based On HDAC And Nitric Oxide Dual Target

Ⅰ. Research backgroundHistone deacetylases (HDACs) are a family of enzymes, which catalyze acetyl-group removal from the lysine residue in the histone tails and lead to a transcriptionally repressed chromatin state. Abnormal HDAC activity has been found to be associated with the aberrant gene expression and the development of several kinds of cancer and other human ailments. Accordingly, HDAC inhibition restores normal gene expression profile resulting in cancer cell cycle arrest, cell differentiation, and apoptosis. Thus, HDAC inhibitors (HDACIs), which block abnormal HDAC deacetylation have been recently developed and validated as potential anticancer agents, such as hydroxamic acids, short chain fatty acids, cyclic tetrapeptide, and benzamides. Among these HDACIs, hydroxamic acids have been the most well-known ones with SAHA, PXD-101 and LBH-589 approved by the US Food and Drug Administration (FDA) in October 2006, July 2014 and February 2015 for the cancers respectively in the clinic.Nitric Oxide (NO) participates in the vascular regulation, nerve transmission delivery, inflammation, and immune response process as an important massager molecule in organism. NO can also inhibit tumor cells proliferation, angiogenesis, metastasis, and accelerate tumor cell apoptosis. In addition to the inducible nitric oxide synthase (iNOS), which can produce a heavy dose of cellular NO, chemical NO donor is also an effective way to generate a high concentration of cellular NO. Oxadiazole was found in 2008 through a high throughput screen to be an important and potential NO donor, which could produce high levels of NO in vitro and inhibit the growth of tumor in vivo.Over the past decades, an increasing body of research reported that covalent modifications, such as S-nitrosylaion or tyrosine-nitration of proteins by NO can dramatically influenced their cellular functions. Interestingly, many HDAC family members have also been found to be direct or indirect targets of NO, and several reports have illustrated that NO-dependent regulation of HDAC functions. In fact, some studies have shown that HDACI and NO are synergistic in cardiac hypertrophy and wound healing."Multifunctional drugs" through the hybridization strategy is a well-developed approach in drug design process. Based on the above mentioned analysis and the "multifunctional drugs" theory, the goal of this study is to develop novel HDACI containing the NO donor motif that exhibits addictive antitumor activity via HDAC inhibition and NO release.Ⅱ. Design, synthesis, and antitumor evaluation of histone deacetylase inhibitors equipped with phenylsulfonylfuroxan module as a nitric oxide donorPhenylsulfonylfuroxan, a classical type of oxadiazole, can release NO to produce activity in variety of tissues and organs through a non-enzymatic pathway. It was connected with several drugs as NO donor into the hybrids compounds as its stability in acidic and basic conditions, and the activity of hybrids were always higher than original drugs. Therefore, phenylsulfonylfuroxan was designed into the novel NO-HDACI in this paper as NO donor motif and 15 novel target compounds were synthesized and characterized.All synthesized compounds were evaluated for their HDAC inhibitory potency against HeLa cell nuclear extracts as well as for their antiproliferative effects against several tumor cell lines. Several representative compounds were evaluated in the HEL cell line and displayed potent NO releasing activity, with compound 5c being the most potent of those evaluated. Pretreatment of HEL cells with the NO scavenger hemoglobin moderately reduced the antiproliferative activity of 5c, demonstrating the additive effect between NO release and HDAC inhibition. Further mechanistic studies revealed that 5c induced a much stronger apoptotic effect and G1 phase arrest in HEL cells than that of SAHA, which was in line with its ability to inhibit HDACs and to generate NO. Because of its very promising in vitro activity, compound 5c was progressed to a HEL xenograft model and exhibited greater oral antitumor potency than SAHA in a dose-dependent manner. It is worth noting that compound 5c was a remarkably HDAC 6 selective inhibitor at the molecular level, which could be used as a design template for HDAC 6-selective inhibitors. Taken together, compound 5c, a very potent HDAC inhibitor with NO releasing activity, was discovered and deserves further research and development as a promising therapeutic agent for hematologic malignancies.Ⅲ. Design, synthesis, and antitumor evaluation of histone deacetylase inhibitors equipped with benzofuroxan module as a nitric oxide donorBenzofuroxan (benzofurzan oxide) is another important NO donor motif, it has the potential for the treatment of cardiovascular diabetic complications due to its prominent NO-releasing activity. Therefore, benzofuroxan was designed into the structure of NO-HDACI as the other NO donor and 10 novel target compounds were synthesized.Most target compounds displayed potential HDACs inhibition against the Hela cell nuclear extracts, and the primary SAR study focused on the length of linker and the position of furoxan ring on the phenyl group that played crucial roles in HDACs inhibition. By MTT assay, 10d and 15d shown potent inhibitory activities to the proliferation of tumor cells. And high levels of NO could be generated from 10d and 15d in HCT-116 cells. Besides, the inhibitory activities of 10d and 15d against HCT-116 cells could be partially diminished by the NO scavenger hemoglobin, which confirmed our designed compounds were "multifunctional drugs" both on the inhibitory activities of HDACs and NO release. Further structural optimization and in vivo activity evaluation of these promising antitumor agents are underway.Ⅳ. Conclusion and perspectiveIn conclusion, under the guide of discovery of multifunctional drugs and with the strategy of hybrid in pharmaceutical development, NO donors were rationally designed into the research of HDACIs and 25 novel NO-HDACIs were synthesized in this paper. 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 the anti-tumor activities of NO-HDACI hybrids were dramatically higher, especially the compound 5c, which was the more potent against tumor cells proliferatation both in vitro and in vivo than the marked drug SAHA. Interestingly,5c also displayed better selectivity to HDAC 6. According to these results, the further research and development of 5c was underway. In a word, this paper established a great foundation for the research of HDAC selectivity inhibitors and the development of multifunctional drugs.