Design,synthesis And Preclinical Study Of 2-mercaptobenzamide Thioester Prodrug As HIV-1 NCp7 Inhibitor

AIDS is a malignant infectious disease caused mainly by human immunodeficiency virus type 1(HIV-1).At present,combined antiretroviral therapy(cART)is the most effective method for treating AIDS.However,the HIV-1 genome has a high degree of variability,and the emergence of drug-resistant mutants limit the efficacy of cART.Therefore,it is still necessary to develop HIV-1 inhibitors with new structures and mechanisms.HIV-1 nucleocapsid protein 7(NCp7)is a short peptide composed of 55 amino acid residues and has two zinc finger central domains.NCp7 plays a crucial role in the process of HIV-1 reverse transcription and integration.When the structure of NCp7 is changed or cross-linked due to the effect of drugs,HIV-1 replication will be influenced,resulting in the budding and release of incomplete virus with low or no infectivity.Through a mutation test on the nucleotide sequence of NCp7,the results prove that NCp7 is highly conservative and will be inactivated by the change of any amino acids.Therefore,inhibitors for this target are not likely to generate drug resistance due to mutations in the NCp7 gene.Hence,NCp7 has become one of the new attractive targets of anti-AIDS drugs in recent years.Based on the spatial structure characteristics of NCp7 and its mechanism,in the HIV-1 life cycle,a large number of inhibitors for this target are currently in research and show high antiviral activity.There are two main types divided b y the mechanism of action:(1)inhibitors based on the interference of the process of NCp7 binds to nucleic acids;(2)inhibitors based on the mechanism of zinc ion "ejection".Among them,the inhibitor S-acyl-2-mercaptobenzamide thioester(SAMT)based on zinc ion"ejection" mechanism can inactivate NCp7 by changing the structure of the zinc finger of NCp7 through nucleophilic action and eject zinc ions.After that,SAMT is converted to 2-mercaptobenzamide thioester(MT).With the action of intracellular acetyl-CoA,MT converts to SAMT again by acetylation.Through recycling,the half-life of the inhibitor is increased,and the dosage is reduced.Therefore,SAMT and MT are lead compounds with good prospects.However,compared with most drugs used to treat HIV-1 infection,SAMT and MT have relatively weak anti-HIV-1 activity.At the same time,the easily-hydrolyzed thioester bond in SAMT leads to the difficulty to formulate as a therapeutic product.Therefore,the modification of MT is usually modified.Due to the sulfhydryl groups,MT has defects such as high polarity,poor transmembrane properties,easy oxidation,and severe toxicity.Therefore,the use of prodrug modification strategies to improve its defects and antiviral activity is a hot spot in current research.Prodrug strategy has a wide range of applications in the optimization of the ADMET profile of antiviral drug.Especially,designed multiple ligands as an emerging antiviral drug discovery paradigm,using a single entity to inhibit multitargets could yield improved patient compliance,thus reducing the likelihood of drug resistance.The exploration of prodrugs or multifunctional prodrugs has proven valuable for anti-HIV leads discovery.To improve the anti-HIV-1 activity of the NCp7 inhibitor MT and to overcome its poor druggability caused by the existence of sulfhydryl group,this thesis disclosed the design,synthesis and evaluation of 2-mercaptobenzamide thioester(multifunctional)prodrugs as HIV-1 NCp7 inhibitors,which is mainly divided into the following two parts:Part ?:Design,synthesis and evaluation of dual prodrugs based on 2-mercaptobenzamide thioesterHIV-1 reverse transcriptase(RT)plays a vital role in the life cycle of the virus and is unique to the virus with no homologous enzyme in the human body,so it has become one of the important targets for anti-AIDS research.At present,FDA has approved 14 reverse transcriptase inhibitors in total for the treatment of AIDS.According to different mechanisms of action,they are divided into nucleoside/nucleotide reverse transcriptase inhibitors(NRTIs/NtRTIs)and nonnucleoside reverse transcriptase Inhibitors(NNRTIs).Reverse transcriptase inhibitors have excellent anti-HIV-1 activity and take a very important position in cART therapy.However,due to the high variability of HIV-1 genes,under the pressure of reverse transcriptase inhibitor drug selection,it is very easy to produce gene mutations and generate drug resistance(usually cross-resistant).Therefore,it is necessary to develop HIV-1 inhibitors with excellent anti-drug resistance.In contrast,NCp7 is highly conserved and its inhibitors are not likely to generate resistance due to mutations.Since NCp7 often forms protein complexes with reverse transcriptase when it exerts its biological function,this part of the study is based on dual prodrugs strategy,and the highly selective and anti-drug resistant NCp7 inhibitor,2-mercaptobenzene formamide thioester(MT),is selected and connected to a reverse transcriptase inhibitor through an ester bond that can be hydrolyzed spontaneously in the body,and two types of dual-target prodrugs are designed and synthesized.It is expected that the designed compound can have the advantages of two parent drugs,including excellent anti-HIV-1 wild strain activity and excellent anti-drug resistance.According to the different types of reverse transcriptase inhibitors on the market,5 dual-target prodrugs of MT and NRTI and 6 dual-target prodrugs of MT and NNRTI were designed and synthesized.We have conducted cell-level activity tests of this series of compounds on different cell lines and different HIV strains,and selected representative compounds for plasma stability metabolism tests.The results show that the anti-HIV activity of all target compounds of the dual-target prodrugs of MT and NRTI is greatly improved compared to the parent drug MT through the modification of dual prodrugs,and the longer the linker length or the fewer the number of branches shows stronger antiviral activity.In this series of compounds,SSK 1-3 showed the best anti-HIV activity in MT4 cell line and TZM-bl cell line,and their EC50 values for different HIV-1 strains were 0.042 ?M(?B)and 1.329 ?M(RES056)and 0.308 ?M(NL4-3),which is 125 times,6 times and 8 times better than the parent drug MT,respectively.The results of the SSK1-3 plasma stability test show that its metabolism in plasma is relatively sufficient,and the release of the parent drug MT has a good linearity with prodrug properties.However,its metabolic rate in plasma is too fast,and it is almost completely degraded within 10 minutes,which may affect its function to target cells,meaning further modification is required for its plasma stability.MT and NNRTI dual-target prodrug activity testing is in progress.Part ?:Design,synthesis and evaluation of disulfide-based 2-mercaptobenzamide thioester prodrugsDisulfide bond(S-S bond)is an important chemical functional group,which mainly exists in various natural small molecule compounds and biological protein structures.Disulfide bonds can be reduced and broken in body fluids and cytoplasm by glutathione(GSH).GSH is an important part of the intracellular redox system and has a detoxification effect.Besides,the concentration of intracellular GSH is 2000-4000 times greater than that in plasma.Therefore,drugs containing disulfide bonds are usually relatively stable in plasma,and disulfide bonds are prone to undergo reductive cleavage after entering the cell.For the problem of poor plasma stability of MT prodrugs with the ester bond,we designed and synthesized a series of MT prodrugs with disulfide bonds based on the unique redox characteristics of that.We hope that this series of compounds can be degradated as few as possible in plasma,and releases the parent drug to take effectxafter entering the cell.At the same time,the sulfhydryl group of MT was modified to overcome the compound's defect of poor druggability.In this part,we designed and synthesized a total of 7 MT disulfide bond prodrugs and tested the cell-based antiviral potency,and selected representative compounds for plasma stability and GSH metabolic stability tests.The experimental results show that most of the newly synthesized target compounds have higher inhibitory activity against HIV-1 WT(?B)and HIV-2(ROD)strains than the parent drug MT through modification.Among them,SSK 2-3 had the highest activity against HIV-1 WT strain(EC50=2.38?M),which was 2.3 times better than the lead compound MT(EC50=5.52?M)?and SSK 2-2 showed the highest activity against HIV-2 strain(EC50=1.27?M),which is 4.2 times better than the lead compound MT(EC50=5.37?M).However,the cytotoxicity and selectivity index of the disulfide bond prodrug series compounds are worse than those of the parent drug MT,which means further modification is needed.We selected SSK 2-3 as representative compound to carry out plasma stability and GSH stability experiments.The results show that:SSK 2-3 is stable in plasma and hardly decomposes within 120 minutes.At the concentration of GSH in human cells(5mM),SSK 2-3 can rapidly undergo metabolic degradation.The half-life of degradation is 19.2 minutes,and it almost decomposes within 60 minutes to release the original drug,reflecting the advantages of intelligent degradation of prodrugs with the disulfide bond.To sum up,in this thesis,taking 2-mercaptbenzamide thioester(MT)as the lead compound and based on the prodrug strategy,two series of 18 MT prodrugs in total has been designed and synthesized.Biological evaluation results show that many compounds have relatively high HIV inhibitory activity,which is significantly better than MT.The test results are basically consistent with our expectation,which highlights this method with high academic value and broad prospects in this area.