Design,Synthesis And Biological Evaluation Of Novel HIV-1 Capsid Inhibitors And RNase H Inhibitors

The human immunodeficiency virus 1(HIV-1)is the causative agent of acquired immunodeficiency syndrome(AIDS)."Highly active antiretroviral therapy"(HAART)significantly increases longevity and life quality of patients.However,the long-term use of antivirals will inevitably bring about complicated drug resistance.Therefore,the discovery of novel targets with draggable potential,especially proteins or enzymes that function at key steps of the HIV-1 replication,such as the capsid protein(CA)and RNase H,and developing highly effective antiretrovirals with novel structures and mechanisms to enrich clinical treatment regimens is an important approach to overcome existing drug resistance.HIV-1 CA is a structural protein enclosed within viral gene and key enzymes.Also,CA plays key regulatory roles in the early and late stages of viral replication.Whereas the NTD-CTD interface is critical for CA assembly,perturbation of this interface can affect CA intrinsic flexibility,and subsequently disrupt viral particle integrity.PF74 is the first compound to be resolved in a co-crystal structure with the CA NTD-CTD interface.Still,it failed to enter clinical development due to its poor antiviral activity and metabolic instability.Finally,Gilead Sciences discovered GS-6207 based on multi-round derivatization of PF74 and is now in the clinical phase.However,it has many drawbacks such as complicated synthesis,huge molecular weight,poor solubility,and drug resistance.It is essential to discover new structural types of HIV-1 CA inhibitors.The HIV-1 RNase H can specifically hydrolyze the RNA strand of the newly generated RNA/DNA hybrid strand during reverse transcription.Once RNase H activity is inhibited,the reverse transcription is difficult to continue.However,most of RNase H inhibitors suffer from weak antiviral activities,low specificities,and high cytotoxicities,and no one has reached the clinical phase.RNase H itself acts as a metalloenzyme,and it has limitations for performing molecular simulation studies,thereby causing difficulties for rational drug design.However,it is a feasible way to find lead compounds with RNase H inhibitory activities through cell-based anti-HIV phenotypic screening of compound library.1.Design,synthesis,and biological evaluation of triazole-containing phenylalanine HIV-1 CA inhibitorsIn this chapter,based on the structural characteristics of 13m previously reported by our group,we comprehensively used the target-based drug design,scaffold hopping and bioisosterism strategies to optimize 13m,as a result,a series of 39 novel triazole-containing phenylalanine HIV-1 CA inhibitors were designed and synthesized.In vitro anti-HIV-1 results showed that most of the IA derivatives displayed moderate antiviral activities(EC50=3.13 ?M?14.81 ?M).Significantly,compounds IA-6a-9,IA-6a-10,IA-6a-11,and IA-5b were found to be more prominent with EC50 values ranging from 3.13 ?M to 3.46 ?M.SPR results indicated that IA-6a-9,IA-6a-10,and IA-5b bound to CA hexamer with KD values close to each other,which were consistent with the trend of their antiviral activities and validated the drug target.The action stage determination assay indicated that IA-6a-9 possessed the characteristics of dual-stage inhibition of HIV-1 replication,and it was more inclined to inhibit the late-stage.The molecular dynamics(MD)simulation of IA-6a-9 found that the formation frequency of H-bonds between IA-6a-9 and CA is low,which may be an important reason why its activity was weak.Moreover,preliminary druggability evaluation revealed that the metabolic stability of IA-6a-9 was slightly better than PF74.Nevertheless,the SARs analysis and MD simulation of the IA series provide helpful guidance for our next round of rational structural optimization.2.Design,synthesis,and biological evaluation of benzenesulfonamide-containing phenylalanine HIV-1 CA inhibitorsThe SARs and MD simulation of IA series confirmed the importance of the H-bond in enhancing the antiviral activities.Therefore,in this chapter,we initially replaced the indole ring of PF74 with benzenesulfonamide moiety to achieve IIA series,and the ring-closure strategy was used to obtain ?B series,and finally,piperazinone-containing derivatives(?C series)were obtained based on ?B series using the scaffold hopping and bioisosterism strategies.Totally,3 series of 37 novel benzenesulfonamide-containing phenylalanine HIV-1 CA inhibitors were designed and synthesized.The in vitro anti-HIV-1 assay indicated that most compounds showed moderate to excellent antiviral activities(EC50=90 nM-10.81 pM).With the iterative structural optimization from ?A(EC50=5.61 ?M-10.81 ?M)to ?B(EC50=2.11 ?M?5.96 ?M)and then to the ?C series(EC50=90 nM?1.06 pM),the activities have also been steadily improved.It was noticed that piperazinone moiety in ?C series plays a key role in achieving a potent activity.?C-31(EC50=90 nM)was found to be the most active CA inhibitor in this thesis,surpassing PF74 nearly 6.times.SPR assay showed that the binding affinities of ?A-3a,?A-3k,?B-2a,?B-2d to C A hexamer are highly consistent with the trend of their antiviral activities,verifying the drug target.?C-31 exhibited the best inhibitory activities on dual-stage of virus replication,and its early-stage inhibitory activity(IC50=8.96 nM)was 6.25 times superior to that of PF74(IC50?56 nM).In the presence of ?A-3k,?B-2a and ?C-31,the p24 production only slightly increased.In the CA assembly assay,?A-3k,?B-2a,and ?C-31 significantly inhibit the process,while PF74 can substantially accelerate the assembly.Also,the co-crystal structure showed that ?C-31 can accurately bind to the six NTD-CTD interface of the CA hexamer at the same time.Simultaneously,the newly introduced piperazinone moiety in ?C-31 has a higher probability of forming additional H-bonds with Arg173,Lys70 and Gln63,which provides a reasonable explanation for the excellent antiviral activities of ?C series.The preliminary druggability evaluation of ?C-31:Initially,the metabolic stabilities of ?C-31 in the HLM and human plasma indicated that it was slightly better than PF74.Later,?C-31 showed acceptable in vivo PK properties and good oral bioavailability(t1/2=1.2 h,F=23.0%).Finally,?C-31 did not show apparent acute toxicity at a dose of 1000 mg/kgOverall,the feasibility of utilizing scaffold hopping and bioisosterism strategies based on the PF74 backbone to improve activities and drug-like properties was verified.And ?C-31 can serve as a lead compound for further structural optimization.3.Discovery of coumarin amide-based HIV-1 RNase H inhibitorsPharmacologically active compounds are the basis for drug research,and screening of compound libraries is the primary way for lead discovery.Especially,our group has accumulated many compounds with diverse structures and good druggabilities,providing an important research basis for discovering novel antiviral compounds through phenotypic screening.It was reported that the hydroxycoumarin skeleton has RNase H inhibition profile,and coumarin derivatives can exert pharmacological activities such as antiviral.To increase the hit rate of RNase H inhibitors and reduce the workload of screening,in this study,we selected coumarin amide-based compounds(DW series)in the in-house library and first conducted in vitro anti-HIV-1 assay on these derivatives.Results showed that DW-3,DW-4,DW-11,DW-25 and DW-31 exhibited comparable inhibitory activities against the double mutant strain RES056 and wild-type ?B.The above results indicated that these compounds have outstanding anti-drug resistance profile.The structural analysis of the five active compounds found that it was consistent with the classic pharmacophore model of HIV-1 RNase H inhibitors.Then the RNase H inhibition assay showed that DW-3,DW-4,DW-25,and DW-31 demonstrated RNase H inhibitory activities(9.9 ?M-68.5 ?M).The above experimental results indicate that such compounds can exert antiviral activities by acting on RNase H.To further explore the antiviral potential of coumarin derivatives,guided by their pharmacophore model and RNase H structure,we designed and synthesized 36 coumarin compounds with novel structures using the scaffold hopping and bioisosterism strategies.The anti-HIV-1 results revealed that 8 compounds demonstrated different levels of antiviral activities(EC50=3.94 ?M?237.34 ?M),and all have very low cytotoxicities(CC50>220 ?M).?B-2a exhibited the most prominent activity(EC50=3.94 ?M)and significantly surpasses lead DW-4(EC50?=101.15?M)nearly 26 times.In addition,?B-2a demonstrated favorable potency against five single mutant strains,mostly maintaining single-digit resistance folds(RF=1.43?11.27),which is close to or equivalent to ETV(RF=0.85?4.09).Subsequent RNase H inhibition assay indicated that most of the compounds showed moderate inhibition rate(57.4?87.2%)at the initial screening concentration of 100 ?M.Besides,?B-2a displayed the best RNase H inhibitory activity(IC50=12.3?M)among the tested compounds,which echoes its best anti-HIV-1 activity,preliminarily verified its drug target.In short,?B-2a can be used as a lead compound for further modification.In summary,although a large number of approved drugs are used in HAART,the emergence of the drug-resistance severely limits their clinical effectiveness.Therefore,the development of new classes of drugs to combat HIV-1 infection is urgently needed.This thesis starts with developing novel HIV-1 CA and RNase H inhibitors under the guidance of drug design and discovery methods such as compound library-based phenotype screening,scaffold hopping,bioisosterism and other medicinal chemistry strategies.Using the strategies mentioned above,we designed and synthesized a total of 6 series of 112 novel small molecule compounds consisting of triazole and benzenesulfonamide-containing phenylalanine as HIV-1 CA inhibitors and coumarin amides as HIV-1 RNase H inhibitors.Through cell and enzyme-based activity assays and HLM and plasma stability assays,PK experiments,etc.,several lead compounds with high potency and druglike properties have been discovered.This thesis also successfully resolved the co-crystal structure of ?C-31 and CA hexamer and clarified molecular mechanism of the piperazinone derivatives with high antiviral activities,laying the foundation of structural biology for the rational design of more potent CA inhibitors.