Design, Synthesis And Biological Evaluation Of Novel HIV-1 NNRTIs And SARS-CoV-2 Main Protease Inhibitors

Viral infections pose a serious threat to human health and public health safety.This paper focuses on HIV-1 reverse transcriptase and SARS-CoV-2 main protease,using a rational drug design strategy based on target structure to design and synthesize a series of compounds.Several lead compounds with good antiviral activity were identified through antiviral activity testing.The main work is divided into the following two parts:(1)Design,Synthesis and Biological Evaluation of Novel Indolylarylsulfones as Potent HIV-1 NNRTIsAcquired immunodeficiency syndrome(AIDS),caused by human immunodeficiency virus type 1(HIV-1)infection,is a chronic infectious disease that poses a serious threat to human health and the global economy.Reverse transcriptase(RT)plays a key role in the life cycle of the HIV-1 virus,and its mechanism of action is clear,with a well-defined protein structure.Non-nucleoside reverse transcriptase inhibitors(NNRTIs)targeting this target have the advantages of high efficiency and low toxicity.However,due to the amino acids in the NNRTI binding pocket(NNIBP)that are prone to mutation,existing NNRTIs can develop drug resistance,which reduces clinical efficacy,there is a need to explore NNRTIs with novel skeleton.Indolylarylsulfones(IASs)are a class of HIV-1 non-nucleoside reverse transcriptase inhibitors(NNRTIs)that possess excellent antiviral activity.However,their clinical use is limited by high cellular toxicity and drug resistance.The crystal structure of the indolylarylsulfone inhibitor and reverse transcriptase complex indicates that the orientation of indole 2 towards the accommodating region II,composed of L100,E138,and V179,has not been occupied,making it a favorable site for structural modification.In addition,the structure of sulfonamide contains multiple hydrogen bond donor receptors,which can produce additional interaction with residues around the binding site,and is widely used in the design of various drugs.In order to explore this binding pocket and establish additional interaction with surrounding amino acid residues while reducing the toxicity of the compound,this paper uses a target structure-based drug design strategy and introduces different lengths of aliphatic chains and various sulfonamides into the indole formamide side chain to design and synthesize 48 novel sulfonamide indolylarylsulfone derivatives in Chapter 2.Cell-based antiviral activity tests against HIV-1 showed that most of the compounds had good inhibitory activity against the wild-type virus(EC50(WT)=0.0041-0.21μM).The most active compounds,R10L4(EC50=0.0070 μM)and R13L2(EC50=0.0041 μM),showed 18-fold and 32-fold increases in antiviral activity against the wild-type HIV-1 compared to the positive control NVP,respectively,and were comparable to the activities of EFV(EC50=0.0038 μM),ETV(EC50=0.0051 μM),and lead compound 10(EC50=0.0041 μM).Besides,R10L4 exhibited significant inhibitory activity and selectivity over nevirapine and etravirine for a variety of clinically common mutant strains L100I(EC50=0.017 μM,SI=13055),E138K(EC50=0.017μM,SI=13123)and Y181C(EC50=0.045 μM,SI=4753).Importantly,compared to the lead compound 10(CC50=5.52 μM),the cell toxicity of R10L4(CC50=216.51 μM,SI=30930)and R13L2(CC50=172.00 μM,SI=41951)was significantly reduced.Acute and subacute toxicity experiments further confirmed that R10L4 is highly safe.Target activity experiments showed that the series of compounds exhibited inhibitory activity against HIV-1 RT.Preliminary pharmacological evaluation showed that R10L4 had a moderate oral half-life(T1/2=1.78 h),but low oral bioavailability(8.5%)that needs further optimization.(2)Design,synthesis and activity evaluation of non-peptide SARS-CoV-2 main protease inhibitors based on hydroxyprolineCoronavirus Disease 2019(COVID-19)is an acute respiratory infectious disease caused by the Severe Acute Respiratory Syndrome Coronavirus 2(SARS-CoV-2).The emergence of SARS-CoV-2 variants with immune escape ability may lead to a second wave of infection in the future.The development of broad-spectrum,safe,effective,low-priced,orally available small molecule drugs with anti-drug resistance is still of great significance.The main protease(Mpro)of SARS-CoV-2 specifically recognizes and cleaves the viral polyprotein into various functional proteins,playing a critical role in key stages of the virus replication cycle.Mpro is highly conserved in coronaviruses,and is highly similar among coronaviruses,making it an ideal target for developing broad-spectrum antiviral small molecule drugs against SARS-CoV-2.GC-14(IC50=0.40 μM,EC50=1.1 μM)is a non-covalent,non-peptide Mpro inhibitor discovered by the research group.Compared with marketed drugs,GC-14 still has a large significant in enzyme and cell activity,and there is a problem of low oral bioavailability.Analysis of the co-crystal structure of GC-14 and Mpro revealed that the overall structure of the molecule occupies three cavities of the active site,we found water bridges and hydrophilic amino acids near the S2c region,and the S1 pocket was unoccupied,providing modifiable space.Hydroxyproline has an advantage skeleton with three branches,which can occupy three cavities simultaneously and is cheaper and easier to obtain than pyrazine carboxylic acid.In summary,in order to further enhance the activity of GC-14 and improve its pharmacokinetics,we uses a drug design strategy of skeleton transition and multi-site binding,The six-membered pyrazine ring was replaced with a five-membered tetrahydro-pyrrole ring.Various advantageous fragments were incorporated into the S2 or S4 regions,and hydrogen-bonding receptor groups were introduced into the S2c region to maximize occupancy of the active pocket of Mpro and formed extensive hydrogen bonds.The results of target activity screening showed that the inhibition rate of most compounds on Mpro was less than 40%at the concentration of 10 μM,and the best active compounds PY-10(IC50=17.61μM)and PH-8(IC50=15.41μM)were still significantly less than the lead compound GC-14(IC50=0.41 μM).The results showed that the two series of compounds showed no anti-SARS-CoV-2 activity at the cellular level.It is speculated that the replacement of piperazine ring by rigid tetrahydropyrrole ring is not conducive to the conformational change of the compound,which reduces the flexibility of the structure and loses the interaction with the surrounding amino acid residues.In conclusion,this paper focuses on HIV-1RT and SARS-CoV-2 Mpro,and uses target-based rational drug design strategies to design and synthesize 48 indole sulfonyl sulfones and 30 hydroxyproline compounds,respectively,in response to the significant clinical needs of anti-AIDS and anti-COVID-19 drugs in the current society.Among indole aromatic sulfones,R10L4 was found to have excellent inhibitory effects on both wild and mutant HIV-1 strains,and significantly reduced the cytotoxicity of the lead compound.Molecular simulations predicted the mechanism of resistance.Among hydroxyproline compounds,the antiviral activity of piperazine ring was significantly reduced after the replacement of piperazine ring by tetrahydropyrrole ring with strong rigidity.Although this work failed to obtain a compound with good activity,it provided guidance for subsequent optimization.