Design, Synthesis And Anti-Cancer Activity Evaluation Of Small Molecular Inhibitors Based On Histone Deacetylases

I. Research BackgroundHistone deacetylases (HDACs) are a class of enzymes which can catalyze the removal of the acetyl group from the lysine residues of histones. This type of enzymes promote chromatin condensation, and then lead to the genes transcriptional repression. So far,18 human HDACs have been identified and grouped into 4 classes. HDACs regulate gene expression and other cellular properties by the deacetylation of histone and non-histone proteins.Aberrant expression and mutation of HDACs could cause a lot of human diseases, especially cancer, which makes HDACs important anti-cancer targets. Therefore, discovery of HDAC inhibitors (HDACIs) have gained popularity in the treatment of cancer. The diverse anti-cancer mechanisms of HDACIs including induction of tumor cell apoptosis and cell cycle arrest, anti-angiogenesis, reduction of tumor cell invasion and metastasis, reduction of DNA repair, induction of autophagy, etc. To date,5 HDACIs, Vorinostat (SAHA), Romidepsin (FK228), Belinostat (PXD-101), Panobinostat (LBH-589) and Chidamide have been approved by for the treatment of cutaneous T-cell lymphoma, peripheral T-cell lymphoma or multiple myeloma. Besides, over 20 HDACIs are in clinical trials as anti-cancer agent.The pharmacophore model of HDACIs consists of three portions, a cap group which interacts with the enzyme’s surface (cap), the active site zinc binding group (ZBG) and a linker occupies the enzyme’s narrow channel and connects the cap and ZBG (linker). We modify these three portions based on the privileged structures of clinical HDACIs and the structure of catalytic active center of HDACs, and finally we get the selective HDACIs with excellent anti-cancer activity.II. Design, synthesis and anti-cancer activity evaluation of 4-cinnamamide-based hydroxamic acid derivatives as HDACIs.N-hydroxycinnamamide is a privileged fragment for the design of HDACIs, and HDACIs PXD101. LBH589 and SB939 containing this fragment are undergoing clinical studies. Therefore, we used N-hydroxycinnamamide as ZBG and linker and introduced indole in cap group in the design of our HDACIs. Finally, we synthesized 32 compounds with new skeleton in this part and the structure of them were confirmed by 1HNMR and HRMS. Part of excellent compounds are> 95% pure by HPLC analysis.We firstly used hela cell extract (mainly contains HDAC1 and HDAC 2) as enzyme resource to screen our compounds. Most of our compounds displayed higher HDACs inhibitory activity than the positive control SAHA. Compound llr exihibited low nanomolar IC50 value against HDAC1 (11.8 nM) and HDAC3 (3.9 nM) while displayed micromolar or sub-micromolar IC50 values against HDAC 2,4,6,8 and 11, which showed llr was a HDAC 1/3 dual selective HDACIs. To date, most of the class I HDACs selective inhibitors are benzamides HDACIs, llr constitutes the first report of HDAC 1/3 selective hydroxamic acid derivatives. Besides, compounds 11, 11p, 11r, llw and lly exihibited potent in vitro antiproliferative activity against several hematological or solid tumor cells. Compounds 11e, 11r, 11w and lly obviously increased acetylation level of histone H3 and H4, in the meanwhile, these compounds decreased the expression of pro-caspase3, which was consistent with the result of apoptotic assay. In vivo anti-cancer assay in U937 xenograft model identified that compound 11r was a potent, orally active HDACI.Ⅲ. Design, synthesis and anti-cancer activity evaluation of 3-cinnamamide-based hydroxamic acid derivatives as HDACIs.The 4-cinnamamide-based HDACIs displayed promising antitumor activity and isoform selectivity, therefore, we next made further modification on this series. We can see from the structure of N-Hydroxycinnamamide-based HDACIs PXD101, LBH589 and SB939 that the substituents of cinnamamide are at different position, in detail, LBH589 is 4-substituted-N-hydroxycinnamamide derivative while PXD101 is 3-substituted-N-hydroxycinnamamide-based HDACI. Inspired by this observation, we designed a novel series of 3-cinnamamide-based hydroxamic acid derivatives as HDACIs. A total of 18 compounds were synthesized in this part, and their structures were confirmed by 1HNMR,13CNMR and HRMS.All of the target compounds were evaluated their HDACs inhibitory activity against hela cell extract, result showed compounds 29m,29q and 29r exhibited much higher activity than positive control SAHA. The HDACs isoform selectivity assay demonstrated representative compound 29k was a pan-HDACI. In addition, further in vitro anti-tumor assay focused on 29k revealed its ability in anti-proliferation and inducting apoptosis in tumor cells U937. Finally, in vivo assay conducted in U937 xenograft model identified 29k as an orally active HDACI. It is worth mentioning that the 3-cinnamamide-based HDACIs were less potent than the 4-cinnamamide-based ones.IV. Design, synthesis and anti-cancer activity evaluation of benzamide-based hydroxamic acid derivatives as HDACIs.In our previous study, HDACIs with N-hydroxybenzamide as ZBG and linker showed good performance in HDACs inhibition, which indicated N-hydroxybenzamide fragment can be used as an active group of HDACIs. Therefore, we introduced N-hydroxybenzamide fragment into the linker and ZBG of our HDACIs.27 compounds were newly synthesized in this part and their structures were confirmed by 1HNMR,13C NMR and HRMS.Among all of the compounds,36q-36v owned low nanomolar ICso values against HDACs, which were much more potent than positive control SAHA, however, the antiproliferative activity of 36r was just slightly higher than SAHA. Poor transcellular permeability of 36r confirmed by HDACs class Ⅰ cellular assay could definitely explain the reason. In addition, HDAC Ⅱa cellular inhibitory activities of 36r and 361 were similar to their class I cellular activities, which indicated 36r and 361 might be pan-HDACIs.V. Design, synthesis and anti-cancer activity evaluation of o-phenylenediamine-based and thiol-based HDACIs with indole-containing cap group.In our previous study, we discovered N-Hydroxycinnamamide-based HDAC1/3 dual selective inhibitors. However, because of the strong chelating ability of hydroxamic acid, our HDAC1/3 selective inhibitor also has some inhibitory activity against HDAC2, HDAC8, HDAC6 and other isoforms. Therefore, we tried to replace hydroxamic acid by other ZBG in order to increase HDACs isoform selectivity of our HDACIs. To the best of our knowledge, most of the class Ⅰ selective HDACIs in clinical are o-phenylenediamine derivatives, such as MS-275, MGCD0103, CI994 and RG2833. Therefore, we kept the cap group of HDAC1/3 selective inhibitor 11y (in our previous study), and introduced ZBG and linker of clinical class Ⅰ selective HDACIs, and finally obtained the novel o-phenylenediamin-based HDACIs. Besides, HDACIs with thiol as ZBG in literatures also displayed HDACs class Ⅰ selectivity, based on this investigation, we designed thiol-based HDACIs.Previous report demonstrated modification in the ZBG o-phenylenediamine by introducing a phenyl or heterocyclic aromatic group on the opposite site of amino could decreases HDAC3 inhibitory activity of HDACIs, while introducting fluorine in the para position of acid amide could get a HDAC3 selective inhibitor. Therefore, we modified the ZBG group of the o-phenylenediamine-based HDACIs in order to obtain more selective profiles.In this section, we synthesized 24 new compounds and confirmed their structures by 1HNMR and HRMS. Most of the o-phenylenediamine-based HDACIs showed potent HDACs inhibitory activity, while the thiol-based HACIs were inactive at the concentration of 10μM. Among all of the o-phenylenediamine derivatives, compound 43b displayed best HDACs inhibitory activity, HDAC1 selectivity and in vitro antiproliferative activity. In vivo study demonstrated 43 b was oral active in leukemia cell U937 and colon cancer cell HCT116 xenograft model. The newly designed phenyl and thienyl compounds exhibited dramatic HDAC1/2 dual selectivity, however, their antiproliferative activity was really unsatisfactory. We confirmed part of the reason was their poor transcellular permeability by conducting cellular uptake assay. Therefore, improvement of transcellular permeability of our HDACIs was our future research emphasize.Ⅵ. Conclusion and perspectiveIn conclusion, in the guidance of medicinal chemistry, biochemistry, computational chemistry and the privileged structure of HDACIs in clinical, a total of 100 novel compounds were designed and synthesized. Then we conducted HDACs inhibitory assay (including isoform selectivity), in vitro and in vivo anti-cancer assays. After structural modification and optimization based on the SAR information, we got target selective HDACIs with high anti-tumor activity, in particular the HDAC1/3 dual selective inhibitors, which provided a new insight into discovery of selective hydroxamic acid derivatives as HDACIs.