Design,Synthesis And Biological Evaluation Of Novel DAPYs As HIV-1 NNRTIs Targeting The Hydrophobic Region Of NNIBP

Acquired immunodeficiency syndrome(AIDS)is a chronic infectious disease caused by the human immunodeficiency virus type-1(HIV-1),which leads to the destruction of the human immune system.AIDS is the second leading cause of death globally,accounted for 33 million deaths and 38 million living cases.In the HIV-1 life cycle,the reverse transcriptase(RT)enzyme plays a vital role in viral reverse transcription and is the critical research target of antiretroviral chemotherapy.Based on their mechanism of action and structures,RT inhibitors(RTIs)are divided into two categories:nucleos(t)ide RT inhibitors(N(t)RTIs)and non-nucleoside RT inhibitors(NNRTIs),Due to the high selectivity,relatively low toxicity,and strong antiviral potency,NNRTIs have an essential impact on highly active antiretroviral therapy(HAART).However,its use in clinical applications has been hampered by the inevitable emergence of HIV-1 resistant strains and a high rate of adverse effects.Therefore,novel HIV-1 NNRTIs with high antiviral potency,good pharmacokinetics,and reduced drug-resistance profiles are urgently needed.With the development of structural biology,numerous crystal structures of NNRTIs-RT complexes were well analyzed,which provide opportunities for understanding the structural features of resistance mutations and interactions with the target protein and systematic structure-based drug design targeting the NNRTI-binding pocket(NNIBP).The high flexibility of NNRTIs and the highly conserved amino acid residues in the NNIBP provide tremendous possibilities for the structural modification of diarylpyrimidine(DAPY)NNRTIs.Based on the structural biology information and computer-aided drug design,this thesis involved the structural modifications towards the hydrophobic region of NNIBP with the hope of discovering novel anti-HIV drug candidates with significantly improved drug resistance profiles.Design,synthesis and biological evaluation of HIV-1 NNRTIs targeting the hydrophobic region of NNIBP.To improve the pharmacological and physicochemical properties(cytotoxicity,poor solubility and drug resistance)of the second-generation NNRTI,the systemic structural modifications of RPV was carried out by considering RPV as a lead compound.Based on the observed crystal structure of RPV and its bonding with RT and bioisosterism strategy,the left-wing of the RPV consisting of cyanovinyl group was extended to the hydrophobic region of NNIBP to generate additional new interactions with the surrounding amino acid residues,which may be the significant factor to enhance the anti-HIV activity and reduce the cytotoxicity of RPV.16 novel compounds were designed,synthesized,and the structure-activity relationship(SAR)studies were also discussed.The synthesized molecules were evaluated for antiviral activity and cytotoxicity in the cell culture with the MTT assay method.Most of the compounds(except FS13)exhibited submicromolar to nanomolar potency from the tested series against wild-type HIV-1 with the EC50 values ranging from 0.016 to 0.722 μM.Among all,FS2 displayed the most potent HIV-1 activity with an EC50 value of 16.0 nM against WT HIV-1,which is superior to the reference drugs 3TC(EC50=5.234 μM),NVP(EC50=0.16 μM);equivalent to AZT(EC50=0.021 μM);inferior to the second-generation NNRTIs ETV(EC50=3.0 nM)and RPV(EC50=1.3 nM).In addition,FS2 exhibited remarkable inhibitory activity against the most prevalent resistance-associated mutation K103N(EC50=39 nM),which is better than 3TC,NVP and EFV(EC50=2.792,6.745,0.103μM,respectively).It also showed good performance against RES056(K103N+Y181C)double mutant strain with the EC50 value of 1.463 μM.In addition,compared with RPV(CC50=4.38 μM),the cytotoxicity of 7 compounds reduced 3.8-51.8 fold,which initially reduced the toxicity of lead compound.The HIV-1 RT inhibitory assay confirmed further their binding to the target.Preliminary SAR,molecular modelling and calculated physicochemical properties of representative compounds were also discussed in this chapter.Discovery of novel HIV-1 NNRTIs:Click-chemistry-based micro-synthesis and in situ screening technique.The rapid assembly and in situ screening of focused combinatorial fragment libraries using click chemistry has become a highly efficient and robust strategy for discovering bioactive leads.Based on the hydrophobic region in the HIV-1 RT and the structural characteristics of DAPYs,one hydroxylamine and 116 aldehydes building blocks were applied to construct an oxime combinatorial library by aldol reaction.Screening of these oxime derivatives against HIV-1 WT RT led to the identification of 5 potent hit compounds(FQ53,FQ54,FQ77,FQ78,FQ79)with IC50 values ranging from 0.34 to 0.60 μM.Most of these compounds are equipotent to the second-generation drug RPV(IC50=0.23μM).Further,to evaluate the anti-HIV activity in MT4 cells,these hit compounds were again prepared in small quantities,and work is being carried out.In conclusion,to improve the solubility and activity of DAPYs against mutant HIV-1 virus,16 novel DAPY derivatives targeting NNIBP were designed and synthesized by utilizing target-based rational drug design,computer-aided drug design and diversity-oriented structure modification of the lead compound RPV.Click chemistry and in situ screening techniques were also tried based on the three-dimensional space of the HIV-1 RT,which yielded a combinatorial library of 116 oxime-containing compounds.The observed anti-HIV evaluation results indicate that several compounds exhibited prominent antiviral activity against wild-type HIV-1 and single mutant strains.Furthermore,this thesis’s research provides valuable insights for further optimizing DAPY and NNRTIs with high efficiency,low toxicity,and improved physicochemical properties.