Design,Synthesis And Preliminary Activity Evaluation Of Triazolopyridine HIV Latency Reversing Agents

The widely application of highly active antiretroviral therapy(HAART)has significantly suppressed acquired immunodeficiency syndrome(AIDS)caused by type 1 human immunodeficiency virus(HIV-1)infection,making it a controlled chronic disease from an acute lethal disease.However,HAART can’t completely inhibit virus replication.Once the antiviral treatment is interrupted,virus replication will rebound rapidly within a few weeks.Therefore,patients have to take drugs for life,which will in turn cause side effects,drug resistance and other problems.Currently,AIDS remains an incurable disease mainly due to the existence of validated latent reservoir.After infecting the resting CD4+T cells,HIV forms latent reservoir which can be existed over a long period of time and is difficult to eliminate.This reservoir has been a barrier for effective AIDS therapy.Recently,a "functional cure" concept has been proposed,in which "shock and kill" is a widely accepted and studied strategy,which is a two-step process.First,drugs called latency reversing agents(LRAs)are used to reactivate latent hiding virus in immune cells("shock").The reactivated cells can then be killed by anti-HIV drugs and human immune system("kill").The utilization of effective and safe LRAs is a prerequisite to realize the so-called "shock and kill" strategy.Over the past years,many types of LRAs have been developed,but there was no breakthrough has been achieved since these LRAs,or much or little,have some shortages.Accordingly,the development of novel LRAs with better efficacy and lower toxicity is a hotspot in anti-HIV therapy.Comparing with other types of LRAs,BET protein inhibitors have better efficacy and safety,and can resist virus resistance without affecting normal immune function,making them promising LRAs for anti-HIV-latency therapies.The BET family consists of four bromodomain proteins,BRD2,BRD3,BRD4 and BRDT,among which BRD4 is the most widely studied subtype.The primary structure of BRD4 contains two highly conserved N-terminal bromodomains(BD1 and BD2),an ET domain and a C-terminal domain(CTD).BRD4 can competitively bind the positive transcription elongation factor b(P-TEFb)with Tat protein,the exclusive trans-activation factor of HIV-1,leading to the silence of HIV gene transcription.Thereby,inhibition the binding capacity of BRD4 protein will enhance the binding effect of Tat to P-TEFb complex,and promote the transcriptional extension of the virus,resulting in a latent activation effect.The earliest identified BET inhibitor(+)-JQ1 has displayed obvious reactivation effect in a variety of HIV latent infection cells,but its significant cytotoxicity has limited its clinical application.Consequently,BET inhibitors with improved selectivity,activity and higher safety for the treatment of latent HIV infection are still urgently desired.In this thesis,two series of triazolopyridine derivatives as potent HIV latent activators have been designed and synthesized based on the structural characteristics of the previously reported BRD4 inhibitor(+)-JQl and the clinically studied pan-BET inhibitor ABBV-075.Accordingly,a total of 30 target molecules were obtained,including 26 of A series and 4 of B series,which have been structurally confirmed by 1H NMR,13C NMR and ESI-MS methods.They were then subjected to the in vitro evaluation on HIV latent infection model cell J-Lat A2.Meanwhile,the cytotoxic effects were also tested on J-Lat A2 cells.The results exhibited that most of the A series compounds gave good latent reactivation effects with low cytotoxicity.Among them,8 compounds,YHP-1,YHP-6,YHP-10,YHP-11,YHP-12,YHP-16,YHP-17,YHP-23,showed comparable latent activation effects with the positive control(+)-JQ1,whereas B series compounds did not produce obvious latent reactivation effects.To further confirm the activity,the latent reactivation effects of these compounds were also tested on another model cell line ACH2.It resulted that the latent reactivation effect of most compounds in A series gave exceeded potency than the control(+)-JQ1,and compound YHP-16 stood out with the best reactivation efficacy.In specific,YHP-16 induced 7.85-fold reactivation of latent HIV-1 while(+)-JQ1 only gave a 2.06-fold potency at a concentration of 10 μM.Next,compounds with good reactivation effects were selected for evaluating the enzymatic activity towards BRD4(1)/(2),and all the test compounds demonstrated strong inhibitory activities against BRD4(1)/(2)at a concentration of 1 μM,and the inhibition rate of all compounds is higher than 90%.Finally,the preliminary mechanism of action investigation revealed these compounds exert a latent reactivation effect by inhibiting the binding capacity of BRD4 protein to facilitate the binding of Tat with P-TEFb complex,leading to a promotion of viral transcriptional extension.In summary,the triazolopyridine derivatives developed in our lab are herein expected to be promising LRAs with low toxicity for achieving functional cure of HIV via effective elimination of HIV latent virus reservoirs.