Design,Synthesis And Antitumor Activity Of Novel Histone Deacetylase Inhibitors

Aim:Histone deacetylase is one of the important targets in cancer treatment research.In this paper,two kinds of hydroxamic acid compounds with skeletal structure were designed and synthesized by means of pharmacochemistry and organic chemistry.In the first subject,the skeleton structure of piperidazole triazole isohydroxyoxime acid was included and targeted to histone deacetylase?HDAC6?.In the second subject,HDAC structural fragments were used as scaffolders to attach key structural fragments of heat shock protein?Hsp90?inhibitors.In the second research,the skeleton structure can inhibit both HDAC and Hsp90targets.This dual target inhibitor may have a greater therapeutic benefit than a single agent in preventing drug resistance and enhancing synergistic effects.Methods:Through the method of pharmaceutical chemical design,twenty target compounds were synthesized by introducing 20 aromatic acyl chloride substituents from piperidine triazole as a hetero-hydroxamic acid compound with a parent nucleus of WY series structure.VER49009,an Hsp90 inhibitor reported in the literature,was used as the CAP part of the HDAC inhibitor,and the structure of isohydroxamic acid was connected by aliphatic chain.A series of target compounds are synthesized by changing the length of the aliphatic chain.The length of four to eight carbon chains is considered to have the best inhibitory effect on HDAC.The purity of WY series and WBL series compounds was preliminarily identified by thin layer chromatography with ferric chloride and HPLC.The structure and purity of the two skeleton compounds were confirmed by mass-NMR,hydrogen-NMR and carbon-NMR and growth inhibition of U937 cells were screened in vitro,and the double concentration?1 and 10?M?inhibitory activity of WY series 20 final products on HDAC6 was screened.The anti-proliferative activity of compounds WY-12and WY-15 against HL-60?U937?NCI-H929?U266?Sy5y?MCF-7 cells was detected by MTT.The subtype selectivity of WY-15 to HDAC1,2,3,6,8 was detected by using the HDAC fluorescence detection kit.Western blot was used to detect the changes in the expression level of acetylhitin H3?Ac-HH3?in HL-60 cells treated with WY-15 at 8concentrations and verify the anti-hdac activity of WY-15.Using in vitro cell cycle detection tests,we evaluated the cell cycle blocking effect of WY-15 on Sy5y cells.Using LeDock molecular docking software,Danio rerio HDAC6 catalytic domain,HPB complexation crystal structure?PDB code:5WGK?was used for molecular simulation of WY-15 compound.The activity of 5 compounds of WBL series was screened by McF-7and Eca-109 cells of esophageal cancer to observe their anti-proliferation effect.Results:In this study,small-molecule HDAC inhibitors and dual HDAC-Hsp90 target inhibitors were made,and their structures were confirmed by ¹H-NMR?¹C-NMR and MS.All 20 compounds show moderate to strong inhibitory activity against HDAC6 at two concentrations,among which WY-3,WY-7,WY-10,WY-12,WY-15 show strong inhibitory activity against HDAC6.The inhibition rate of these compounds was over 50%at 1?M and over 90%at 10?M.It suggests that they are effective inhibitors of HDAC6.indicating that they were effective inhibitors of HDAC6.Only two compounds WY-12 and WY-15,showed good anti-proliferative activity and had appropriate cLogP values,which facilitated smooth entry into the cell membrane.Compounds WY-12 and WY-15 showed moderate to excellent anti-proliferative activity in 6 tumor cell lines,with GI₅₀ values in the micromolar range.The activity of WY-15 was superior to WY-12.WY-15 showed strong inhibitory activity against NCI-H929 and Sy5y cells,and the GI₅₀ values of the compound were 1.20and 1.88 uM,respectively,and the positive control SAHA were 0.85 and 1.11 uM,respectively.Wy-15 showed weak selectivity to HDAC6 and HDAC8,Similar to the control compound SAHA.WY-15 induced significant expression of Ac-HH3 in a dose-dependent manner from 6?M to 100?M,and the results showed that WY-15 was an HDAC inhibitor with cellular activity.After WY-15 treatment,Sy5y cells were significantly inhibited in the G0/G1 phase in a concentration-dependent manner.Significantly,the observed percentage of Sy5y cells in G0/G1 phase?0?M?increased from 57.3%to 80.04%?10?M?.The molecular simulation results of compound 15 on HDAC6 show that the 2-naphthalacetyl group of WY-15"CAP"is in good agreement with the hydrophobic inlet of HDAC6,the part of Linker is 4-piperidine 4-triazole can be inserted into the pocket,and the isohydroxamic acid group of WY-15 successfully chelates with the Zn²⁺of HDAC6 in a single-tooth manner.Five compounds in the WBL series have anti-proliferative activity against human breast cancer MCF-7 and esophageal cancer Eca-109 cells.Its GI₅₀ value was in the range of micromole,and the less the number of carbon chain,the stronger the activity.Further tests of the pharmacological activity of the WBL series will be carried out.Conclusion:Twenty hydroxamic acid WY compounds based on 4-piperidine triazole and five hydroxamic acid WBL series compounds containing the key pharmacophore of Hsp90inhibitor were designed and synthesized in this paper,which were used as novel HDAC 6selective inhibitors and hsp90-HDAC double-target inhibitors respectively.The presented compounds WY-12 and WY-15 both exhibited satisfying anti-tumor potency.