Design,Synthesis And Biological Evaluation Of Novel DAPYs As NNRTIs Targeting Primer Grip Region Of HIV-1 Reverse Transcriptase

Human immunodeficiency virus type 1(HIV-1),as the main pathogen of AIDS,is one of the viruses that pose a serious threat to human health.Reverse transcriptase(RT)which is a key enzyme in the HIV-1 replication cycle,is an ideal target for anti-HIV-1 drugs.Nonnucleoside reverse transcriptase inhibitors(NNRTIs)have become a research hotspot for antiHIV-1 drugs due to their excellent antiviral efficacy and high specificity.However,the problems of drug-resistant strains and side effects in clinical treatment limit their application.Therefore,the development of NNRTIs with high efficiency,low toxicity and improved drug resistance profiles is still an important task in the field of anti-HIV-1 drug research.The rapid development of HIV-1 RT structural biology provides new information for the rational design of potential anti-HIV-1 agents.The primer grip region(β12 and β13),near the hydrophobic channel of NNRTIs’ binding pocket,is responsible for locating end of the primer strand at the appropriate position,which plays an important role in the catalytic activity of RT.This region is a conserved amino acid sequence,which contains three key amino acid residues in hydrophobic channel:F227,W229,L234,of which W229 is highly conserved.Targeting highly conserved amino acid residues is of great significance for the discovery of new compounds with improved drug resistance profiles.This paper focused on the primer grip region,a new ligand binding site of HIV-1 RT,and modify the left wing of diarylpyrimidinetype(DAPYs)NNRTIs.The specific content was divided into the following two parts.Design,synthesis and biological evaluation of novel DAPYs targeting primer grip region.In order to further explore the chemical space of the primer grip region,RPV was used as the lead compound.In view of its high cytotoxicity,poor solubility and clinical drug-resistant mutants,the left wing was modified by structural diversity(introduction of aromatic rings and spiro rings).Two series of new NNRTIs with a total of 40 structures were designed and the antiviral activity and antienzyme activity of the target compounds were tested.Series I Design,synthesis and biological evaluation of DAPYs substituted with aromatic rings in the left wing as HIV-1 NNRTIsTo target highly conserved amino acid residues in the primer grip region,using-CH2Oand-CH2S-as linkers,substituted benzene rings or pyridine rings were introduced into the left wing of DAPYs.A total of 29 compounds with new structures were synthesized.The results of antiviral activity test against wild-type HIV-1 showed that all compounds showed potent inhibitory activity(EC50(WT)=7.6 nM～199 nM).Among them,TF2(EC50=7.6 nM),TF4(EC50=7.8 nM)and TF12(EC50=7.8 nM)showed the most prominent activity,which was better than that of positive drugs AZT(EC50=0.127 μM)and NVP(EC50=0.023 μM),and was about one-seventh of RPV.Notably,compared with RPV(CC50=3.98 μM),TF2(CC50>279.33 μM)showed low cytotoxicity.For the tested HIV-1 mutants,most of the compounds showed tens to hundreds of nanomoles of inhibitory activity against K103N and E138K.Especially for K103N mutant,the activities of TF2(EC50=28 nM),TF12(EC50=35 nM)and TF13(EC50=28 nM)were better than those of positive control NVP(EC50=7.495μM)and EFV(EC50=95 nM).In addition,all the compounds showed strong enzyme inhibition,and the IC50 values ranged from 0.036 μM to 0.483 μM,which was better than that of NVP(IC50=0.568 μM).Molecular simulation studies showed that the high activity may be due to the extensive π-π stacking interaction between the aromatic substituents and the surrounding amino acids.Series II Design,synthesis and biological evaluation of DAPYs substituted with spiral rings in the left wing as HIV-1 NNRTIsThis series abandoned the aromatic structure which was often introduced in left-wing modification of DAPYs and replaced it with lipophilic spiro rings with high Fsp3 values.Increasing Fsp3 value can improve the physicochemical properties and ADME properties of the compounds to some extent.A total of 11 DAPY derivatives were synthesized in this series.The results of antiviral activity test showed that most of the compounds maintained sub-micro to micro-molar activity against wild-type HIV-1.The EC50 value of TT6,the most active compound,was 0.17μM,which was better than that of positive drug 3TC(EC50=8.01 μM),and was about 121 times lower than that of RPV(EC50=0.001 μM).The cytotoxicity of TT6(CC50=9.92 μM)was slightly lower than that of RPV(CC50=5.40μM).For mutants,only some compounds showed inhibitory activity.The activity of TT6 against K103N mutants was 0.69 μM,which was better than that of positive drugs 3TC(EC50=3.68 μM)and NVP(EC50=4.62 μM).The prediction results of physical and chemical properties showed that the introduction of high Fsp3 spiral ring improved its solubility,and the molecular simulation results showed that the introduction of spiral ring led to the loss of π-π stacking between leftwing and surrounding aromatic amino acid residues,which may be an important reason for the decrease or even loss of the activity.To sum up,in order to explore the primer grip region and target the highly conserved amino acid residues,this paper focused on the modification of the left wing of the DAPYs.Taking the marketed drug RPV as lead compound,a total of 40 NNRTIs with new structures were synthesized.,After biological activity test,a number of high-efficiency and low-toxicity anti-HIV-1 lead compounds were found,some of which reached the nanomolar level,and the structure-activity relationship and molecular simulation laid the foundation for the further development of such compounds.