Design,Synthesis And Biological Evaluation Of Novel HIV-1 Integrase/Ribonuclease H Dual Inhibitors And NNRTIs

Acquired immune deficiency syndrome(AIDS)mainly caused by human immunodeficiency virus type-1(HIV-1),continues to be a major leading pandemic disease worldwide.The emergence of "Highly Active Anti-Retroviral Therapy"(HAART),which combines various antiviral drugs for different replication periods of the virus,greatly reduces the morbidity and mortality of AIDS.However,the long term efficacy of HARRT are severely hampered by the rapid occurrence of drug resistance and serious side effcts,forcing people to develop novel antiviral drugs and expand new anti-AIDS drug design ideas.The reverse transcriptase(RT)of HIV-1 is one of the crucial enzymes in the life-cycle of the virus,responsible for the conversion of the viral genomic RNA into proviral DNA.Due to its special and indispensable role in the replication,RT has been identified as a prime target for the development of anti-AIDS drugs.Based on the mechanism of action and structures,the reverse transcriptase inhibitors currently approved by the US FDA can be divided into nucleos(t)ide RT inhibitors(N(t)RTIs)and non-nucleoside RT inhibitors(NNRTIs).Compared with NRTIs,NNRTIs have gained an essential impact in HAART due to their strong antiviral potency,relatively low toxicity and high selectivity.However,its use in the first line treatment is usually limited by potential drug interactions,high rate of adverse effects as well as ineluctable emergence of HIV-1 resistant strains.Therefore,there is an urgent need to develop HIV-1 NNRTIs tageting new binding sites or bearing new structural types with improved antiviral efficacy,good pharmacokinetic and reduced drug-resistance profiles.As another domain of RT,RNase H selectively degrades the RNA strand in the RNA/DNA hybrid strand,plays a key role in the HIV-1 reverse transcription process,and is a very promising new target for drug design.Although RNase H inhibitors with different mechanisms and different structures have been reported in the literature,most of them suffer from the disadvantages of low target activity,poor antiviral activity at the cellular level and high toxicity.HIV-1 integrase and HIV RNase H belong to the nucleotide transferase superfamily and share a very similar active center structure.The metalloenzyme activity is dependent on metal ions located in the catalytic site.Consequrntly,the limitations of molecular simulation researchhave brought great challenges to structure-based rational drug design Even so,four catalytically active site inhibitors of HIV integrase have approved by the US FDA for the treatment of HIV-1.Thus,the metalloenzyme superfamily has attracted great attention as potential drug targets.The shared enzymatic mechanism enployed by many dication-dependent polynucleotide metabolizing enzymes provides a huge opportunity for a multipronged drug discovery effort.Design,synthesis and biological evaluation of a novel hydroxyl-containing N-heterocyclic HIV-1 integrase/RNase H dual-target inhibitor.At present,the most commonly used HAART characterized by the combination of multiple drugs in the clinic can significantly reduce the HIV-1 viral load in AIDS patients and slow the development of the disease course,but also suffers a lot from serious toxicity,side effects,complex drug interactions,and poor patient compliance.Therefore,seeking multi-target HIV inhibitors with a single chemical entity has become a new strategy and hotspot in the development of anti-AIDS drugs.The 3-hydroxyquinazoline-2,4(1H,3H-diones derivatives were designed based on molecular hybridization and all evaluated as effective inhibitors of HIV-1 RNase H with IC50 values ranging from sub to low micromolar concentrations.Among them,ⅡA-6B-4 was the most potent one in enzymatic assays,with an IC50 value of 0.41μM,almost five times lower than the IC50 obtained with β-thujaplicinol.In addition,ⅡA-6B-4 was also effective in inhibiting HIV-1 IN strand transfer activity(IC50=0.85 μM)but less potent than raltegravir(IC50=71 nM).Despite its relatively low cytotoxicity,the efficiency of ⅡA-6B-4 in cell culture was limited by its poor membrane permeability.Nevertheless,structure-activity relationships and molecular modeling studies confirm the importance of tested 3-hydroxyquinazoline-2,4(1H,3H)-diones as useful leads for further optimization.Among the pyrido[2,3-d]pyrimidin-2(1H)-one derivatives,ⅡB-9-4 is the most potent one with an IC50 value of 0.50 μM which is 4 times that of the positive controlβ-thujaplicinol(IC50=1.98 μM).What’s more,ⅡB-9-4 also showed good inhibitory activity in cell-based assay(EC50-HIV-1=14.69 μM,ECso-HIV-2=21.00 μM).The structure-activity relationship lays the foundation for in-depth exploration and structural optimization research.Design,synthesis and biological evaluation of novel indolylarylsulfones as potent HIV-1 NNRTIs.The drug resistance of currently available non-nucleoside reverse transcriptase inhibitors for the treatment of HIV-1 cannot be ignored.In the first part of this chapter,based on the SAR analysis of previously reported indolyl aryl sulfone(IAS)derivatives,we maintained the core of indolyl sulfone and introduced an acrylamide or vinyl sulfonyl chloride group into alkyl rings near the 181C residue in Y181C RT NNIBP to explore whether it could specifically form an interaction with the sulfydryl group of 181C residue through an Michael addition reaction to enhance the activity against the Y181C mutant HIV-1 strain Among the obtained compounds,ⅢA-7(IC50=18.2 μM)showed significant improvement towards Y181C RT over the positive control without the introduction of a covalent group(IC50=75.2 μM.MALDI-TOF mass spectrometry analysis was carried out to confirm its mode of action as a covalent inhibitor.In the second part,a series of non-covalent compounds targeting the conservative amino acid W229 was designed and synthesized based on the structure of IAS.The compounds with introduced π-system substituents greatly increased the activity of the compound against Y181C mutant HIV-1,which confirms our design concept.The most active compound is ⅢB-7R-10,whose activity against wild-type HIV-1 is 2.6 times that of the positive control lamivudine.It is worth noting that the compound displayed a Y181C mutation selectivity,whose inhibitory activity towards Y181C mutant is about twice than the inhibitory activity of its wild strain,3.7 times that of positive control lamivudine,and 11 times that of positive control nevirapine.The above results indicate that both strategies of covalently targeting mutant cysteine or enhancing the force with conserved W229 can improve the activity of the compound against Y181C resistant strains.However,we should also pay attention to the maintenance of the original force to find lead compounds with good activity in both wild strains and drug-resistant strains.Design synthesis and biological evaluation of novel diarylpyrimidines as HIV-1 NNRTIs.This chapter focuses on the severe drug resistance problems that have occurred in the latest generation of NNRTIs currently in clinical use.Under the guidance of DAPYs classic "four-point pharmacophore" model,the following explorations have been carried out:1)Conformational restriction strategy was used to lock the privileged conformation of DAPY and direct the introduced side chain to the tolerant region Ⅱ in order to improve the activity and resistance profile by forming a wide range of forces with the nearby amino acid and solvent interface.Most of the compounds in IVA series show excellent antiviral activity,and some compounds also show outstanding inhibitory activity against mutant strains,with EC 50 of E138K single mutant strains ranging from submicromolar to nanomolar,far exceeding lamivudine and nevirapine and equivalent to zidovudine and etravirine.The inhibitory activity of IVA-7D(EC50ⅢB=0.07μM,EC50-E138K＝0.01 μM)against E138K reached 7 times that of wild strains,and it also showed micromolar inhibitory activity towards L1001,K103N,Y181C and Y188L.2)Inspired by the fused central ring inhibitors,the scaffold hopping strategy is used to split the fused ring into bicyclic compounds connected by a rotatable single bond.Series IVB compounds are all excellent wild-type HIV-1 inhibitors,the inhibitory activity against wild-type HIV-1 ranges from nanomolar to submicromolar,of which the best compounds IVB-5-4 and IVB-5-8 possess an EC50 value of 2.5 nM,which is better than the positive controls NVP,DLV and ETV IVB-5-4 and IVB-5-8 simultaneously maintain nanomolar activity for multiple HIV-1 mutants(L1001,K103N,Y181C,Y188L,E138K),whose inhibitory activity is significantly better than that of the marketed drug NVP and is basically at the same level as EFV and ETR,which were regarded as lead compounds for further optimization.Design,synthesis and biological evaluation of HTV-1 NNRTIs via rapid assembly and screening of focused combinatorial fragment librariesThe fifth chapter report the rapid identification of highly potent HIV-1 NNRTIs based on the rapid assembly and in situ screening of focused combinatorial fragment libraries using CuAAC click chemistry.On the basis of the solvent-exposed regions in the HIV-1 RT and the structural characteristics of IASs,2 alkynes and 40 azide building blocks were applied to construct a combinatorial libraryt by CuAAC click chemistry.Finally,13 compounds selected by enzyme inhibition assay were synthesized and evaluated for their anti-viral activity,which all showed strong inhibitory activity against HIV-1 wild strains,with ECso values ranging from 0.024 to 0.23 μM,most of them are far superior to the first-generation drug nevirapine(EC50=0.16 μM).C1N4(EC50=0.024 μD showed the best anti-HIV-1 ⅢB activity with extremely low cytotoxicity,high selectivity and safety(CC50>215.88 μM).Some compounds also showed high inhibitory activity against HIV-1 single mutant strains such as Y188L,E138K,etc.,which has the value of further investigation.In conclusion,more than 130 compounds which belong to 7 categories of novel anti-HIV agents were designed and synthesized via multiple medicinal chemistry methodologies.The following biological evaluation results demonstrate that several compounds show high antiviral activity with EC50 ranging from micromole to nanonolar levels.Some diarylpyrimidines,such as IVA-6D,IVA-7D,IVA-8V,IVB-5-4,IVB-5-8,etc,exhibit good anti-resistance profiles such as E138K,significantly higher than that of the first-generation drug nevirapine and was in the same order of magnitude as the second-generation drug etravirine,which have great potential for further investigation.Our study also serves as a proof-of-concept for the development and optimization of covalent compounds as potent inhibitors of HIV-1 replication.Our findings demonstrate that there is a tremendous opportunity to apply rapid assembly and screening of focused combinatorial fragment libraries as a promising method to accelerate drug discovery.