Preliminary Druggability Evaluation Of Prodrug Of K-5a2 And Discovery Of Novel DAPYs As HIV-1 NNRTIsTargeting "NNRTI Adjacent" Binding Site

Reverse transcriptase is a pivotal enzyme in life cycle of HIV-1 and is the key research target of antiretroviral chemotherapy.Reverse transcriptase inhibitors are divided into two categories:nucleoside/nucleotide reverse transcriptase inhibitors(N(t)RTIs)and non-nucleoside reverse transcriptase inhibitors(NNRTIs).Based on its unique antiviral activity,high specificity,and low toxicity,HIV-1 NNRTIs occupy an increasingly important position in contemporary HIV-1 therapy(ie,highly effective antiretroviral therapy).However,the efficacy of NNRTIs is gradually hampered by the rapid emergence of drug-resistant viruses,poor pharmacokinetic properties,and severe toxic side effects for long-term treatment,which necessitates the development of new NNRTIs with broader antiviral spectrum and better pharmacokinetic properties.Structural diversity and pharmacophore similarity of reported HIV-1 NNRTIs provide new insights for the discovery and optimization of novel lead compounds.K-5a2,an anti-AIDS drug candidate,is a thienopyrimidine HIV,1 NNRTI discovered by our group with diarylpyrimidines as the lead.K-5a2 has a prominent anti-HIV-1 activity,which has a better or comparable inhibitory activity against WT and mutant strains of HIV-1 than that of ETR.However,K-5a2 has a low oral bioavailability and short half-life.Therefore,it is imperative to improve its pharmacokinetic and druggability properties.HIV-1 RT undergoes a series of conformational changes during viral replication,and its intrinsic flexibility also provides many novel binding sites for the discovery of novel HIV-1 NNRTIs.When maintaining the classical binding mode,targeting other new binding sites will be beneficial to increase the specific binding and affinity between molecules and targets,which will improve anti-resistance profiles of drugs.Consequently,this thesis focused on improving pharmacokinetic and druggability properties of lead compound K-5a2 and optimizing anti-resistance profiles of HIV-1 NNRTIs is divided into the following two parts:Part ?:Design,synthesis and preliminary druggability evaluation of prodrug of K-5a2K-5a2,a promising anti-HIV-1 drug candidate,was discovered by employing a multidisciplinary medicinal chemistry endeavor of structure-based drug design,antiviral activity and preliminary druggability evaluation in our group recently.It is gratifying that the activity of K-5a2 against HIV-1 wild-type strain was 2.8 times better than that of ETR.Antiviral activity against single mutants L100I,K103N,and Y181C was comparable to that of ETR;for single mutants Y188L,E138K and double mutant F227L+V106A,the activity of K-5a2 was about 5-fold more potent than that of ETR.In addition,K-5a2 showed the low toxicity with a high selectivity index(>159101).Meanwhile,K-5a2 presenteded a low acute toxicity in mice(LD50>2 g·kg-1),which merits further development as a potential clinical candidate for the treatment of HIV-1 infection.However,K-5a2 has poor water solubility.As we known,the water solubility of drug molecule is a main factor affecting its pharmacokinetic and druggability properties.At present,different strategies are reported to increase aqueous solubility,especially,prodrug has been widely accepted by pharmaceutical chemists.To improve pharmacokinetics of K-5a2,a novel sulfonamide propyl prodrug of K-5a2(HM-1)was designed,then its water solubility,the preliminary druggability evaluations,including anti-HIV-1 activity,acute toxicity,and pharmacokinetic were evaluated.The results showed that the water solubility of K-5a2 was much lower than 1?g/mL and the water solubility of HM-1(69.66 ?g/mL)with a logP value of 1.22 was at least 70-fold than that of K-5a2.HM-1 inhibited WT HIV-1 strain with an EC50 of 7.99 nM,which was about 7-fold lower than K-5a2(EC50 = 1.17 nM).The results of acute toxicity test(gastric standard:2 g · kg-1)showed that mice taking prodrug of K-5a2 had normal physiological activity and increased 1.7 g averagely in body weight during one week.The data of pharmacokinetic experiment showed that HM-1 can be quickly converted into K-5a2 in rats and HM-1 had an oral bioavailability of 80.71%,which was 3.5 times higher than K-5a2(22.89%),and the T1/2 extended from 3.21 h to 3.53 h.Part II:Design,synthesis and biological evaluation of DAPY NNRTIs targeting HIV-1 RT NNIBP and "NNTRI Adjacent" binding siteAccording to the 3D-architectural characteristics of "NNTRI Adjacen" binding site,a novel series of dual binding site-typed DAPY derivatives was designed to target the NNIBP and "NNRTI Adjacen' binding site simultaneously,which is characterized by a S atom-bearing linker connected to the 5-position of pyrimidine ring with different length and different terminal substituents.These compounds were expected to increase binding affinity with RT and to improve the drug resistance profiles.The synthetic protocol for 5-substituted diarylpyrimidine derivatives totally 21 compounds is started from 2,4-dichloropyrimidine and obtained through nucleophilic substitution,buchwald-hartwig coupling,electrophilic reaction,palladium-catalyzed coupling reaction and oxidation reaction or hydrolysis as well as amide condensation reaction.Both spectral and analytical data of the newly synthesized compounds were consistent with the proposed structures.The results demonstrated that six compounds(H7,H14 and H18?H21)showed excellent activities against WT HIV-1 strain(EC50 = 2.4?3.8 nM),which were more potent than that of ETR(EC50 = 4.0 nM).Furthermore,H7,H14,H20 and H21 showed more potent or equipotent activity against single mutant HIV-1 strains compared to that of ETR.Especially,H7 showed marked antiviral activity,which was 1.5-fold greater against WT and 1.5?3-fold greater against L100I,K103N,Y181C,Y188L,and E138K when compared with ETR with CC50 value of 27.2 ?M and SI value of 10045.In addition,all compounds showed lower toxicity(CC50=5.1?149.2?M)than that of ETR(CC50 = 2.2 ?M).The HIV-1 RT inhibitory assay was further conducted to confirm their binding target.Preliminary structure-activity relationships(SARs),molecular modeling as well as calculated physicochemical properties of selected compounds were also discussed comprehensively in the paper.To sum up,the sulfonamide propyl prodrug of K-5a2 was designed and synthesized to improve its poor pharmacokinetics and low bioavailability,and the preliminary druggability properties were also identified in the second chapter of this thesis,which had laid a solid foundation for the preclinical standardized study of K-5a2.In addition,in the third chapter,novel dual-site DAPYs NNRTIs targeting HIV-1 RT NNIBP and "NNRTI Adjacent" binding site simultaneously were designed and synthesized based on advances in structural biology of HIV-1 RT in order to improve the anti-drug resistance of HIV-1 NNRTIs.Novel lead compound as well as the new binding site that was suitable for further modification were discovered,which provided a new direction for structural optimization of HIV-1 NNRTIs.