Design,Synthesis And Biological Evaluation Of HIV-1 Protease Inhibitors

ObjectiveAcquired immune deficiency syndrome（AIDS）,caused by human immunodeficiency virus（HIV）,is an infectious disease.As a HIV-1 protease inhibitor,darunavir（DRV）has potent therapeutic effect.But serious drug resistance and side effects have emerged in the process of medication.Therefore,it is of great significance to modify its structure to develop effective,harmfulless and resistant anti-AIDS drugs.MethodsA series of novel HIV-1 protease inhibitors was designed with DRV as the lead compound.The synthetic routes of the target compounds were determined:using tert-butyl（（S）-1-（（S）-oxiran-2-yl）-2-phenylethyl）carbamate as the starting material,the key intermediates 10-15 were synthesized by ring opening of1,2-epoxides,sulfonylation and de-protection reaction.And then they reacted with 2-morpholinoethy（4-nitrophenyl）carbonate,derivatives of 4-morpholine acetic acid,4-morpholine carbonyl chloride,4-（（2R）-epoxyethyl）morpholine,4-（2-chloroacetyl）morpholine and 4-（2-bromoethyl）morpholine by condensation or acylation or ring-opening of 1,2-epoxide or nucleophilic substitution to produce the target compounds,respectively.The inhibitory activity of the target compounds against HIV-1 protease was determined in vitro by the fluorescence method,using DRV as the positive control.The antiviral activity of the target compounds 27a and 43a against DRV-resistant HIV-1 variants and their inhibition stage of HIV-1 life cycle were evaluated by cell-based assays.The binding modes of compounds 39a、43a and 27b with HIV-1 protease were predicted by molecular modeling software MOE.ResultsIn this paper,sixty-four unreported derivatives of DRV were designed and synthesized.The chemical structures of the intermediates were confirmed by¹³C NMR and¹H NMR,and the chemical structures of the target compounds were confirmed by¹³C NMR,¹H NMR and HRMS.The results of HIV-1protease inhibitory activity test and preliminary structure-activity relationship showed that:（1）The target compound 27a displayed potent inhibitory effect against HIV-1 protease with IC₅₀value of 0.41 n M,which were almost equivalent to DRV(IC₅₀=0.37 n M).（2）When R¹was isobutyl,the inhibitory activity of the compounds against HIV-1 protease was better than that of the correspnding cyclopropyl compounds.（3）Inhibitors with amide chains between P1 and P2 ligands showed enhancement of HIV-1 protease inhibition over that with aliphatic.Carbamate inhibitors exhibited the most potent activity,followed by ureas and acetamides.Further,The activity of compounds with aliphatic chains decreased with the increase of linker length in a certain range.（4）When 2-substituted methyl groups were introduced into R³fragment of the morpholine ring,the inhibitory activity was improved.The inhibitory activity of（S）-2-methylmorpholine compounds was better than that of（R）-2-methylmorpholine compounds.（5）In most cases,when the substituent R²of P2’ligand was methoxy and amino,the inhibitory activity of the compounds were superior to that of the corresponding nitro and trifluoromethyl substituent compounds.The antiviral activity of the target compounds 27a and 43a against DRV-sensitive or resistant pseudotyped HIV-1 showed that compounds 27a and 43a against DRV-resistant mutations differed slightly from that against wild type HIV-1 with 8 and 2 fold enhancement in EC₅₀respectively,comparing with DRV which exhibited 60-fold increase in EC₅₀against DRV-resistant mutations than that of wild type virus.The results of cell-based assays on the inhibition stage of HIV-1 life cycle showed that the target compounds 27a and 43a had significant inhibitory effect on the later stage of HIV-1 life cycle（viral maturation）with inhibition rate of 94%and 91%,respectively.However,the two compounds hardly had effects on the early stage,albeit to a lesser extent with inhibition rate of 5%and 12%,respectively.Compounds 43a and 27b fitted into the HIV-1 protease binding site perfectly,while compound 39a did not fit into the active site very well.All the tested compounds could form strong hydrogen bonds,van der Waals and hydrophobic interactions with amino acid residues.ConclusionsThe structure-modification of the P2,P1’and P2’ligands in darunavir not only enriched the structural diversity of HIV-1 protease inhibitors,but also contributed to broaden the design ideas.The structure-activity relationships could provide valuable clues for the future study of HIV-1 protease inhibitors.