Design,synthesis And Biological Activities Of 2-Arylheterocyclic Quinazolinone Derivatives As HIV Latency-Reversing Agents

Acquired immunodeficiency syndrome(AIDS)is a highly harmful infectious disease caused by human immunodeficiency virus(HIV).Although the application of highly active antiretroviral therapy(HAART)has successfully transformed acquired immune deficiency syndrome from a deadly disease into a chronic and controllable disease.However,the therapy cessation results in viral rebound because of the presence of latent HIV reservoirs.One approach referred to as ’shock and kill’,is characterized by the use of pharmacological agents to reverse HIV-1 latency while the patient continues HAART.The latency-reversing agents(LRAs)induces viral gene expression and anti-retroviral drugs prevent new productive infections of the released viruses.The virus-producing cell will die due to virus-induced cytopathic effects or via immune-mediated mechanisms.The development of efficient and low toxicity of HIV LRAs for AIDS curative research is particularly important.Recently,Huanzhang Zhu of Fudan university and Lin Li in our group et al reported that the BET inhibitor Apabetalone,can reactivate HIV-1 from latency and without influence on global immune cell activation.Therefore Apabetalone is safer than Prostratin.In addition Apabetalone has no obvious cytotoxicity to PBMCs.But Apabetalone’s activation activity is not strong enough,compared with the classical LRAs Prostratin and JQ1.In this paper,apabetalone was used as a lead to be optimized,we retained its active pharmacophore quinazolinone based on literature,designed and synthesized 65 novel Q1～Q5 series compounds.To determine the effect of these compounds on latent HIV-1 reactivation,we used J-Lat 10.6 cell line as a model of latency,and detected the cells’ green fluorescent protein(GFP)expression level by flow cytometry.We found Q204,Q205,Q207,Q209,Q212,Q214 of Q2 serie,Q313,Q509,Q511 of Q3 and Q5 series,these 9 compounds had good reactivation effect with HIV.Among the 9 compounds,Q205 had the highest reactivation effect,which was better than the apabetalone.And we verified the reactivation effect of Q205 through another two cell lines J-Lat A2 cells and ACH2 cells as HIV-1 latency models by flow cytometry,fluorescence quantitative PCR,and Western blotting.The results showed that Q205 can efficiently induce the expression of GFP in J-Lat A2 cell line.Q205 can significantly promote the expression of HIV-1 related genes Gag,Tat,Vpr,Vif,LTR and capsid protein p24 in ACH2 cells,and the effect was stronger than Apabetalone.By analyzing the structure-activity relationship of Q1-Q5 series compounds,we supposed that the 5,7-dimethoxy group of quinazolinone ring might be critical for maintaining the pharmacological activity.XTT assay was used to investigate the cytotoxicity of Q205 in J-Lat 10.6,J-Lat A2 cells and human PBMCs.The results showed that the cytotoxicity of Q205 was very low,the CC50 of it on PBMCs was more than 200 μM.Based on the experimental results of Q1-Q5 series compounds,we have designed and synthesized 15 XQ series compounds,in which Q205 was used as a lead and modified by bioisostere principle and skeleton transition.And we also detected their reactivation effects,which showed that the activity of XQ14 was stronger than apabetalone and XQ14 had very good reactivation effect at low concentration,maybe XQ14 is a better LRA than Q205,which need to be verified in further tests.And the activities of XQ2 and XQ15 were comparable to those of Apabetalone.The structure-activity relationship indicated that the substitution of the methoxymethyl group of Q205 furan ring side chain with n-propyl group or the methylation of its quinazolinone mother nucleus lactam group leading to loss of detectable potency.We also observed extend Q205 furan ring side chain with fat chain or replace the methoxymethyl with benzyloxymethyl will reduce its activity,replaced the furan ring for the thiophene ring maybe good for potency.And we adopt a new method to synthesis apabetalone,2-bromo-4,6-dimethoxybenzaldehyde was used as a key intermediate to afford apabetalone.The new synthetic route had some advantages,such as mild reaction conditions,simple operation,high yield.In summary,we successfully got four good LRAs Q205,XQ2,XQ14,XQ15,by modifing Apabetalone,Q205 had a better reactivation activity than Apabetalone and its cytotoxicity was low.Our study suggested that Q205 is an attractive potential candidate for HIV-1 cure studies.The mechanism of Q205 is worthy of further study.And we found XQ14,an analog of Q205,which had very good reactivation effect at very low concentration in preliminary experiment,maybe XQ14 is a better LRA than Q205,and it need to be verified in further tests.