Structure-Based Design,Synthesis And Biological Evaluation Of Novel HIV-1 Capsid Inhibitors

Human immunodeficiency virus(HIV)is the pathogen of acquired immunodeficiency syndrome(AIDS),of which HIV-1 is the main pathogenic subtype.Although combination antiretroviral therapy(cART)can reduce the viral load in AIDS patients to below the detection limit,since HIV cannot be completely removed from the body,people living with HIV need to take drugs for life,which inevitably leads to problems such as drug resistance and side effects.Therefore,it is urgent to develop anti-HIV drugs with novel mechanisms and chemotypes to enrich the existing therapies.HIV-1 capsid(CA)is an indispensable structural protein that constitutes a mature virus with infectious ability,which envelops the HIV-1 genome,the key enzymes,and regulatory factors,and plays multiple roles in the replication cycle.CA can participate in and regulate the transportation,nuclear entry,uncoating,reverse transcription,integration in the early stage and the assembly of the virus as well as maturity in the late stage of replication.The CA monomers are assembled into the complete capsid by a variety of protein-protein interaction.Furthermore,destroying these key protein-protein interactions will affect the stability of the capsid,thereby inhibiting the replication of HIV-1.PF74 is a small molecular binding to the NTD-CTD interface.Although the mechanism is unique,poor antiviral activity and unstable metabolic stability have hindered further studies on it.Lenacapavir(LEN)was obtained through multi-round modification based on PF74 with picomole-level anti-HIV activity and strong metabolic stability,which is the first HIV-1 capsid inhibitor approved by FDA.However,a variety of drug-resistant strains that are not sensitive to LEN were found in clinical trials and in vitro screening,and the drug was complicated to synthesis and therefore expensive for medication,which limited its application.Therefore,the development of high-efficiency,anti-drug-resistance and low-cost HIV-1 capsid inhibitors has become the current hotspot of research.Based on the issues associated with LEN,we identified two types of HIV-1 capsid inhibitors with novel chemotypes and mechanisms,employing the molecular hybridization strategy,structure-based drug design strategy,and multi-site binding strategy.1.Design,Synthesis and Biological Evaluation of 4-Quinazolinone-bearing Phenylalanine Derivatives as HIV-1 Capsid InhibitorsIn this chapter,the molecular hybridization strategy was employed to transform the advantages of LEN,GSK878,111 and PF74 for the basic skeleton of the novel compounds.In order to improve the anti-drug-resistance of the novel compounds,based on the conserved NTD-NTD interface,we introduced a variety of substitutions in the R4 ring system extending to the protein-protein interface to enhance the interaction between small molecules and Ser41.Through a "building block" synthesis strategy,I series compounds were synthesized efficiently.In the I series compounds,IC-2i showed the strongest antiviral activity(EC50=0.65 ± 0.27 nM)in vitro and excellent selectivity(SI>1570.0).In the SPR assay,IC-2i exhibited a strong affinity(KD=2.7±0.5 nM)and long residence time(180 min)with CA hexamer.The results of a single-round infection assay illustrated that IC-2i had a strong early-stage inhibiting activity.This effect has nothing to do with the activity of the reversal transcription enzyme,but may be that IC-2i can inhibit the combination of CA and CPSF6,thereby inhibiting CPSF6 regulating functions the in early stage.In the late stage,IC-2i had no promotion of assembly of multiple subtypes of HIV-1 CA and a variety of drug-resistant strains in vitro,but may induce it to form abnormal capsid without virus genomes.Subsequently,we predicted the IC-2i binding model through docking,and calculated the impact of the modification of the privileged structures on the binding of the compounds through FEP,thereby explaining the reasons why the I series compound activity decreased compared with LEN.In the preliminary drug-likeness evaluation,IC-2i had better metabolic stability than PF74 in human liver microsome and human plasma.In the acute and subacute toxicity assays in vivo,IC-2i showed the security that was consistent with the experiment in vitro.IC-2i showed excellent pharmaceutical dynamic characters by s.c.(T1/2=19.9±14.2 h,F=61.73±16.1%).In summary,IC-2i is a HIV-1 capsid inhibitor with strong antiviral activity and novel mechanism different from LEN,which has the potential for further modification to develop long-acting HIV-1 inhibitor.2.Design,Synthesis,and Biological Evaluation of Dimerized Phenylalanine Derivatives as HIV-1 Capsid InhibitorsIn this chapter,to design high-efficiency and low-cost HIV-1 capsid inhibitors,based on multi-site binding strategy,we designed a series of dimerized phenylalanine derivatives with 2-piperazineone or 2,5-piperazinedione as liners.The structure-activity relationship(SAR)indicated that the dimers had stronger antiviral activity than the monomers of the same chemotype.In addition,the inclusion of fluorine substituted phenylalanine and methoxyl substituted aniline was found to be beneficial for antiviral activity.In Ⅱ series of compounds,IIC-1d was the most potent compound with higher EC50(0.57 ± 0.13 μM)than PF74(EC50=0.75 ±0.33 μM).The SPR assays showed that IIC-1d had higher affinity with CA monomer than that of hexamer,which was consistent with its strong inhibiting activity(99.4%under 10time EC50).Competitive SPR experiments with CPSF6 peptides and NUP153 peptides illustrated that IIC-1d binds to the NTD-CTD interface and strongly interferes with the combination of NUP153 and CA hexamer.Single-round infection assay showed that IIC-1d interferes with the life cycle of HIV-1 in a dual-stage manner,affecting both pre-and postintegration.The stability analysis performed in human liver microsome and human plasma demonstrated that although IIC-1d exhibited greater stability than PF74,it still falls significantly short compared to existing drugs and requires further modifications.In summary,the dimerized phenylalanine derivatives provide great potential for modification and represent promising lead compounds.In summary,this thesis extensively employed various medicinal chemistry strategies to address the limitations of LEN and PF74,resulting in the discovery of IC-2i,which exhibits potent antiviral activity and novel mechanism of action,and IIC-1d,which shows promising modification potential for the development of HIV-1 capsid inhibitors.