Design,Synthesis And Antitumor Activity Of HDAC/Topo And DHHC20 Inhibitors

Histone deacetylase（HDAC）inhibitors can induce apoptosis,differentiation,and growth arrest in cancer cells,which tightly binds to negatively charged DNA,densely curls chromatin,and inhibits gene transcription.Topoisomerase（Topo）inhibitors show anticancer activities primarily due to the stabilization of the DNA-enzyme cleavable complex by intercalation between DNA base pairs.In view of the fact that HDAC inhibitors and topoisomerase inhibitors exert a synergistic effect on cellular processes in cancer cells,the combined inhibition of two targets is regarded as a rational strategy to improve the effectiveness of these single-target drugs for cancer treatment.In the first part,28 pyrazolo[3,4-d]pyrimidine derivatives as HDAC and Topo dual inhibitors were designed,synthesized and evaluated for their anticancer activity.MTT results showed that the compounds had good antitumor activity.Among them,compounds 8e,8f,8g,10,and 14d had effects on cancer cells He La(IC₅₀=4.58μM),MGC-803(IC₅₀=11.52μM),MCF-7(IC₅₀=7.88μM),and RBL-2H3(IC₅₀=4.58μM)with IC₅₀ values of less than 15μM.Compounds showed powerful antiproliferative activity containing a five carbons chain.Compound 8e（n=5）showed the best cytotoxicity across all cell lines（He La,MGC-803,MCF-7,and RBL-2H3）with IC₅₀below 15μM.Compounds 8c,8e,8f,10,14d,14e,20e,20f and 20g showed stronger inhibitory activities than Vorinostat（a standard HDAC inhibitor）,among which compound 14d displayed the best activity.Almost of the target compounds showed obvious anti-Topo II inhibition activity in vitro topoisomerase II relaxation assay.Among them,compounds 8f,10,14b,20e,20f and 20g showed good inhibitory activities against the dual targets of HDAC and TOPO II.Further studies revealed that compounds 12 and 24 could promote apoptosis in MCF-7 cells.Molecular docking studies were carried out to understand the interactions of compounds 10 and 22,and the results revealed their significant interation with active sites of HDAC2 and Topo II.In addition,in silico prediction of physicochemical properties showed that these compouds conformed well to the the lipinski’s rule of five,suggesting its potential for use a drug like molecule.Human DHHC20 is a subtype of the palmitoyltransferase protein family.Studies indicate that expression levels of DHHC20 are upregulated in ovarian,breast,colon,renal,and prostate cancer compared with normal cells,a potential protein palmitoyltransferase that may play a key protective role in cancer.In the second part,15 isatin derivatives as DHHC20 inhibitors were designed,synthesized and evaluated for their antiproliferative activity against tumour cell lines（He La,MGC-803,MCF-7,RBL-2H3）and normal cell line（L-929）by using the MTT assay.A number of compounds showed low micromolar antiproliferative activities against MCF-7 cell line.Compound 5b exhibited the strongest inhibitory activity with an IC₅₀ value of 12.04?μM.AO/EB staining confirmed that 5b exposure induced apoptosis in MCF-7?cells.Moreover,molecular docking studies were performed to gain insights for the plausible binding interactions and affinities for the target DHHC20-based isatin derivatives within DHHC20 active sites.