Design,Synthesis And Preliminary Bio-evaluation Of Novel Histone Deacetylase Inhibitors

As critical epigenetic modulators,histone deacetylases(HDACs)together with histone acetyltransferases(HATs)regulate the dynamic equilibrium of the deacetylation/acetylation on histones and non-histone proteins along with maintenance of homeostasis.However,specific external stimuli or gene mutations can lead to HDACs overexpression or abnormal HDACs activity,which cause compact nucleosome structure as well as downregulation of tumor suppressor genes.This phenomenon can lead to tumor development and progression.The therapeutic intervention of the abnormal function of HDACs in cancer cells using HDACs inhibitors can effectively suppress the growth as well as proliferation of cancer cells and eventually achieve good anti-tumor therapeutic outcome.Decades of efforts have led to the approval of five HDACs inhibitors including Vorinostat,Romidepsin,Belinostat,Panobinostat and Chidamide for the treatment of peripheral T-cell lymphoma,cutaneous T-cell lymphoma and multiple myeloma.Although HDACs inhibitors have achieved a great success in tumor therapy,clinical development conventional pan-HDACs inhibitors originally was hampered by the severe side effects and the poor therapeutic effect on solid tumor.Therefore,the development of novel HDACs inhibitors is still an attractive filed in medicinal chemistry.To overcome the severe side effects of pan-HDACs inhibitors,we developed HDAC6 isoform selective inhibitors.Pan-HDACs inhibitors indiscriminately inhibit all HDAC isoforms causing various toxicities and side effects,whereas HDAC inhibitor with good inhibitory activity and isoform selectivity can solve the problem well.Several studies revealed that HDAC6 knockout does not lead to lethal or teratogenic effect.While HDAC6 inhibition can effectively suppress the growth and proliferation of cancer cells.We firstly investigated the structural features of the HDAC6 protein and explored the rim of HDAC6 catalytic channel using AlphaSpace,and discovered non-polar P1 pocket as well as potential semi-polar P2 pocket.The hit compound LT-0(HDAC6,IC50=21 μM)showed mild HDAC6 inhibitory activity.The binding mode between LT-0 and HDAC6 indicated that the cap group of LT-0 does not accommodate within the P1 and P2 pocket effectively.This may explain its mild HDAC6 inhibitory activity.Therefore,we introduced rigid as well as bulky hydrophobic aromatic rings to construct a hydrophobic cap group for P1 and P2 pocket occupation,and obtained series I HDAC6 selective inhibitors.Unfortunately,series I HDAC6 selective inhibitors exhibit mild HDAC6 inhibitory activities at a concertation of 40 nM.Further studies indicated that the linker of series I HDAC6 inhibitors was too short to allow the hydroxamic acid group to chelate Zn2+,followed by the mild HDAC6 inhibitory activities.Then we optimized the linker of the series Ⅰ HDAC6 inhibitors,and designed as well as synthesized series Ⅱ,Ⅲ HDAC6 inhibitors.The preliminary biological evaluations revealed that series Ⅲ target compounds exhibited nanomolar HDAC6 inhibitory activities and potent compound HSI-30 inhibited HDAC6 with an IC50 value of 4.4 nM.Moreover,HSI-30 also showed good HDAC6 isoform selectivity and its HDAC6 inhibitory activity is 218 times of HDAC1,63 times of HDAC2,53 times of HDAC3,37 times of HDAC10,more than 20000 times of HDAC4,7,8,9 as well as 11.In subsequent biological evaluations,HSI-30 also possessed good in vitro profiles by means of induction of apoptosis induction,caspase-3 activation,cell cycle arrest,and anti-tumor cell proliferation.The structural biology information revealed that HDAC6 possesses the shallower and wider catalytic channel compared with HDAC1 and HDAC2,and HDAC6 inhibitor preferred bulkier and shorter linker.That is consistent with the structure-activity relationship of our series Ⅱ,Ⅲ HDAC6 inhibitors.On the other hand,HDAC6 has more binding pockets in the rim of the catalytic channel compared with HD AC1 and HDAC2.The introduction of different substituents into the cap region to occupy these pockets can further increase HDAC6 protein affinity thereby achieving better HDAC6 inhibitory activity and HDAC6 isoform selectivity.Based on the aforementioned findings,we introduced hydrophobic group and polar group into the cap region of newly designed compound for the occupation of P1 non-polar pocket and the P2 semi-polar pocket.Then the large,short,para-substituted N-hydroxybenzamide fragment was used as linker and ZBG for newly designed series Ⅳ-ⅦHDAC6 inhibitors.Biological evaluations revealed that four-rounds of structural optimization greatly improves the HDAC6 inhibitory activities and HDAC6 isoform selectivity of compounds.The most potent HDAC6 selective inhibitor HSI-70(HDAC6,IC50=2.1 nM)exhibited good HDAC6 isoform selectivity and its HDAC6 inhibitory activity is 5545-fold increase of HDAC1,5864-fold increase of HDAC2 as well as 1638-fold increase of HDAC8.Further biological evaluations suggested that HSI-70 possesses good profiles in terms of HDAC6 inhibitory activity,HDAC6 isoform selectivity,mitochondrial depolarization,caspase-3 activation,apoptosis induction,carcinoma cells growth suppression,in vitro druggability evaluation(normal-tissue cytotoxicity,plasma stability and liver microsomes stability)and in vivo pharmacokinetic properties evaluation(oral bioavailability is 52.5%).To explore the difference in HDAC6 inhibitory activity of the enantiomers of HSI-70,we tried a variety of preparation methods and finally obtained(R)-HSI-70 and(S)-HSI-70.The biological evaluations suggested that the HDAC6 inhibitory activity of(R)-HSI-70 is comparable to that of(S)-HSI-70.In our HDAC6 isoform selective inhibitors study,four-round of structural optimization gave birth to 76 target compounds(series I-VII)and the most potent compound HSI-70 exhibits low nanomolar HDAC6 inhibitory activity,more than 1600-fold HDAC6 isoform selectivity,sub-micromolar antiproliferative activity and good druggability.It is also worth mentioning that the oral bioavailability of the HSI-70 is better than that of the approved drug SAHA(oral bioavailability is 42.5%)in the pharmacokinetic evaluation.Given the good profiles of HSI-70 in biological evaluations,HSI-70 can be used as a candidate compound to develop more effective and safer HDAC6 inhibitor antitumor agent in the future.The poor therapeutic effect of approved HDACs inhibitors on solid tumors is another prominent problem.Despite HDACs inhibitors have been proven to be effective toward a defined subset of hematological tumors,their limited efficacy as a single agent on solid tumors has a great effect on the application of HDACs inhibitors in anti-tumor therapy.And novel HDACs inhibitor combination strategy or chemical hybrid molecule containing HDACs inhibitor fragment is the good approach to overcome the poor therapeutic effect of HDACs inhibitors on solid tumors.Several studies indicated that HDACs inhibition can up-regulate pro-apoptotic proteins(Bax protein).Therefore,we speculated that combining HDACs inhibitors and Bax activator is supposed to enhance Bax-dependent apoptosis,thereby achieving better anti-proliferative activity.To confirm our hypothesis,we explored the combination of HDACs inhibitor SAHA and Bax activator BTSA1(1:1)on HeLa cells.The results indicated that SAHA and BTSA1 can achieve a synergistic effect(CI=0.73)on the anti-proliferative activity against HeLa cells,and that provides a new approach to solve the poor therapeutic effect of HDAC inhibitors on solid tumors.However,there are many problems in drug-combination,such as the complex clinical trial design,poor patient compliance,unpredictable pharmacokinetics(PK)and pharmacodynamic(PD).Therefore,we developed HDAC-Bax multiple ligands that can simultaneously achieve HDACs inhibition and Bax conformation activation.To reduce the redundant structure and improve the rationality of the HDAC-Bax multiple ligands,pharmacophore fusion strategy was used.In brief,we incorporated the cap group of HDACs inhibitors into BTSA1 functional fragment,and then introduced linker and ZBG in the solvent-exposed region of BTSA1.We designed and synthesized three series of HDAC-Bax multiple ligands.The preliminary bio-evaluation results show that our potent HDAC-Bax multiple ligands HIBA-6 possesses HDACs inhibitory activity comparable to SAHA and similar Bax affinity relative to BTSA1,but its anti-proliferative activity is 15-fold better than BTSA1 and 3-fold better than SAHA.The better antiproliferative activity and lower cytotoxicity of HIBA-6 indicated that our HDAC-Bax multiple ligands design strategy achieves a success.Bax protein knockdown assay and subsequent other biological experiments revealed that our HDAC-Bax multiple ligands HIBA-6 could upregulate Bax protein expression and then induce the conformation activation of more Bax protein,thereby enhancing Bax-dependent apoptosis,followed by the better anti-proliferative activity.In our HDAC-Bax multiple ligands study,we firstly explored the combination of HDAC inhibitor and Bax activator,and then designed as well as synthesized a series of HDAC-Bax multiple ligands.Our research exhibits a new paradigm in the treatment of solid tumors by enhancing Bax-dependent apoptosis.In summary,we developed HDAC6 isoform selective inhibitors to overcome the severe side effects of pan-HD ACs inhibitors and developed HDAC-Bax multiple ligands to solve the poor therapeutic effect of HDAC inhibitors on solid tumors.In this thesis,we focus on the practical problems of conventional HDACs inhibitors and designed as well as synthesized 90 novel HDACs inhibitors.Our findings provide potential recipes for future tumor treatment with focus on HDACs inhibitors.