Hydroxamic Acid Based HDAC3/BRD4 Dual Inhibitors And Selective HDAC6 Inhibitors:Design,Synthesis And Biological Activity Research

HDAC3 plays an important role in gene expression and signal pathway by regulating the acetylation status of histone or non-histone substrate.In recent years,it has become one of the promising targets in cancer treatment.A lot of evidence has shown that HDAC3 and BRD4 are correlated in many signal pathways and the combination of the two inhibitors can solve the problem of drug resistance and side effects.The development of dual-target inhibitors of HDAC3 and BRD4 is called upon to acquire additional safe and effective anti-tumor drugs.In the previous activity screening of our in-house compound library,BHA-0119 and BHB-0351 were identified as hit compounds to discovery HDAC3 inhibitors.Based on the results of molecular docking and fragment growth method,a series of compounds were designed by using indole and benzodihydropyrazole as skeletons,respectively.Taking indole as starting material,after acylation,reduction,N-alkylation and ammonolysis,1,3-disubstituted indole derivatives A01-A09 were achieved concisely.Taking α-tetralone as starting material,after condensation,cyclization,N-alkylation,hydroxylation,amidation and ammonolysis,1(2),3-disubstituted benzodihydropyrazole derivatives A10-A16 were achieved concisely.The chemical structure was confirmed through 1H NMR,13C NMR and HRMS spectra.Compounds A09 and A15 showed potent HDAC3 inhibitory activity(IC50=0.062 μM,0.059 μM)and selectivity(SF=9.4,6.6).Preliminary structure-activity relationship showed that the introduction of large volume hydrophobic substituents at C-3 position of indole skeleton and N-1 or N-2 position of benzodihydropyrazole skeleton could enhance the selectivity of compounds to HDAC3.According to the principle of pharmacophore fusion,the indolyl alkyl hydroxamic acid of BHA-0119 was hybridized with the 4-aryl-3,5-dimethylisoxazole of P-0014,a BRD4 inhibitor,to give a series of 1,2,3-trisubstituted indole derivatives(A17-A32).Taking 5-bromoindole as starting material,after acylation,reduction,Suzuki coupling,N-alkylation and ammonolysis,compounds A17-A32 were synthesized.The chemical structure was confirmed through 1H NMR,13C NMR and HRMS spectra.Compound A24 was identified as the most potent and selective HDAC3 inhibitor with an IC50 value of 0.005 μM and a SF value of 36.2.A24 also showed potent BRD4 inhibitory activity with the inhibition rate of 88%at 10 μM.Priliminary structure-activity relationship suggested that:(i)the activities of compounds with a 6 carbon atoms length linker at N-1 position of indole are better than 5 carbon atoms length linker,(ii)compounds with various functional groups on C-3 side chain terminus followed the trends of para-substitution>meta-substitution>ortho-substitution,electron-withdrawing group>electron-donating group=no substitution.Western-blot analyses indicated that treatment of THP-1 cells with A24 led to obviously upregulation of Ac-H3 and downregulation of c-Myc.Dual inhibitor A24(GI50=8.79 μM)showed more potent anti-proliferative activity against THP-1 cells than HDAC3 inhibitors A20,A22,A25,A30,A33(GI50=12.13-23.34 μM).Based on the docking model of A24 to HDAC3 and BRD4,indole skeleton was replaced by benzodihydroindazole with fine tuning of the position and structure of substituent to give 1(2),3,7-trisubstituted benzodihydroindazole derivatives(A17-A32).Taking 6-amino-3,4-dihydronaphthalen-1(2H)-one as starting material,after brominated,suzuki coupling,condensation,etc.,compounds A33-A35 were obtained.The chemical structure was confirmed through 1H NMR,13C NMR and HRMS spectra.Compound A47 showed potent HDAC3 inhibitory activity and selectivity with the IC50 value of 0.004 μM and SF value of 113.The BRD4 binding activity of A47 was also potent(ΔT=8℃).In addition,a few principles can be observed.Compounds with various substituents at benzodihydropyrazole N-2 position exhibited superior activities than those of N-1 regioisomeric counterparts.Most of this series of compounds were more sensitive to THP-1 and HCT-116 cells than MCF-7,MDA-MB-231 and PC-3 cells.Compounds A44 and A47 showed strong anti-proliferative activity against THP-1 cells(GI50=1.68 μM,1.17 μM).And compounds A40 and A47 showed strong anti-proliferative activity against HCT-116 cells(GI50=1.15 μM,0.97 μM).Western-blot analyses indicated that treatment of THP-1 cells with A47 led to obviously upregulation of Ac-H3 and downregulation of c-Myc with no influence on the level of Ac-tubulin.Moreover,A47 were more stable in mouse serum than liver microsome.The occurrences of persistent neuroinflammation and oxidative stress reaction are important markers of Alzheimer’s disease.Contemporary studies have shown that HDAC 6 is closely related to the mechanisms of the occurrence of neuroinflammation and the release of ROS.Studies have shown that selective HDAC6 inhibitors could play neuroprotective role in neurological diseases.Based on the structural specificity of active pocket of HDAC6,using the hydroxamic acid as ZBG which can bind to the zinc ion,benzene ring as linker is to shorten the length and increase the hydrophobic property,phenyl pyrazole or phenylamino pyrazole fragment as SRM to prolong and enlarge the volume,a series of compounds(B16-B31)were designed and synthesized.Taking different aniline as starting material,after amidation,cyclization and ammonolysis,compounds B01-B14 were obtained.The chemical structure was confirmed through 1H NMR,13C NMR and HRMS spectra.B02 was identified as the most potent compound against HDAC6 with the IC50 value of 0.076 μM.Preliminary structure-activity relationship indicated that the substitution of p-position of the benzene ring is superior to that of ortho or meta,and the activity of electricity-withdrawing substituents is equal to that of non-substituents,while the substitution of electron-donating group decreases the activity.Taking different acetophenone as starting material,after the synthetic method analogous to A10-A16,compounds B15-B31 were synthesized.The chemical structure was confirmed through 1H NMR,13C NMR and HRMS spectra.Among them,B23 showed the most potent inhibitory activity against HDAC6(IC50=0.005 μM)and B24 was the most selective compound(SF=101.1).In addition,a few principles can be observed.(ⅰ)Compounds with linker at pyrazole N-2 position exhibited superior activities than those of N-1 regioisomeric counterparts but the selectivities are contrary.(ⅱ)The activity of the compounds decreased with the increase of the volume of substituents on benzene rings when the linker at pyrazole N-2 position.The anti-proliferative activity of compound B16-B31 against N9 cells and the content of NO in N9 cells were determined by MTT and Griess assay respectively.The results showed that all the compounds could not inhibit the growth of N9 cells at 1 μM.Compound B23 showed potent anti-NO release activity(IC50=0.03 μM).Flow cytometry and MTT analysis showed that,compound B23 could inhibit the release of TNF-α,IL-6,ROS and the protect PC-12 cells from oxidative stress reaction.In conclusion,in order to search for HDAC3/BRD4 dual inhibitor,39 1,2,3-trisubstituted indole derivatives and 1(2),3,7-trisubstituted benzodihydroindazole derivatives were designed and synthesized.Using HDAC6 as the target,31 phenylpyrazole derivatives and phenylpyrazole derivatives were designed and synthesized.The results of preliminary activity screening showed that the dual inhibition of HDAC3 and BRD4 could increase the anti-proliferative activity of the compounds against tumor cells and selective HDAC6 inhibitor B23 showed neuroprotective and anti-neuroinflammatory activities;further in vivo activity studies are under way.