Structure-based Discovery Of Novel Fused Pyrimidines HIV-1 NNRTIs As Anti-AIDS Lead Compounds And Drug Candidates

Acquired immune deficiency syndrome(AIDS),which was mainly caused by human immunodeficiency virus type-1(HIV-1),has developed into a worldwide pandemic of disastrous proportions.Non-nucleoside reverse transcriptase inhibitors(NNRTIs)comprise an important class of antiretroviral drugs,and combinations of NNRTIs are widely used in highly active antiretroviral therapy(HAART)regimens to treat HIV infection,owing to their potent antiviral activity,high selectivity,favorable pharmacokinetics.Although the widespread implementation of HAART has greatly reduced the morbidity and mortality of AIDS,the increasing incidence of drug resistant viruses along with the drug toxicity among treated people limited their clinical applications and calls for continuous efforts in developing novel anti-HIV-1 drugs.With the development of structural biology,multivariate co-crystal structures of HIV-1 RT/NNRTIs complexes were well solved,which provide opportunities for understanding inhibitor-protein interactions with greater accuracy,more reliable determination of the structural effects of resistance mutations,and systematic structure based drug design targeting the NNRTI-binding pocket.In accordance with the adaptability requirements of the HIV-1 RT target,a systematic structural modification toward the solvent-exposed regions of NNIBP were carried out from the following four parts in this thesis,with the hope to discovery of novel anti-AIDS drug candidates with significantly improved drug resistance profiles.Structure-based discovery of novel anti-AIDS drug candidates K-5a2 and 25a.Previous research efforts in our lab led to the identification of thiophene[3,2-d]pyrimidine derivative K-5a2 with robust activity profiles against WT HIV-1 and a panel of NNRTI-resistant mutant strains.However,K-5a2(EC50 = 30.6 nM)showed weaker potency for the most challenging double mutant strain RES056 compared to that of ETV(ECso = 17 nM).In the second chapter,to improve the drug resistance profile and to overcome the unpredictability of the precise binding mode between flexible targets and ligands,every region(the left wing,central ring and the substituted piperidine ring of the right wing)of the structure of K-5a2 was further optimized.Finally,a total of 35 novel thienopyrimidine compounds was designed and synthesized via nucleophilic substitution,oxidation,deprotection,and Wittig-Hornor reactions.All the compounds were assayed for their anti-HIV activities and cytotoxicity with MTT method.The results demonstrated that introduce other group and alter N atom position of the 4-aminopiperidine of K-5a2 significantly reduced their antiviral activity.Replacing the center core of K-5a2 with thiophene[2,3-d]pyrimidine,the obtaining compounds showed potent activity against WT and most mutant strains,but still less potent against RES056 compared to ETV.The activity have a considerable improvement when replace the cyano group in the right wing with cyano vinyl.All the compounds displayed potent activity against WT and mutant HIV-1 strains.Among them,IA-19a(25a)proved to be the most potent inhibitor and showed exceptional antiviral activity against the whole viral panel,being about 3—4-fold(WT,EC50 = 1.22 nM;L100I,EC50 = 1.34 nM;K103N,EC50 = 0.958 nM;Y181C,EC50 = 5.00 nM;Y188L,EC50 = 5.45 nM;E138K,EC50 = 4.74 nM;K103N+Y181C,EC50 = 5.50 nM)and 10-fold(F227L+V106A,EC50 = 2.70 nM)more potent than the reference drug ETV in the same cellular assay.Furthermore,K-5a2 and 25a displayed favorable pharmacokinetic properties and low toxicity,all the results indicate that K-5a2 and 25a hold great promise as potential next-generation anti-HIV drug candidates.To investigate the precise binding mode of K-5a2 and 25a,we determined the co-crystal structures of WT and mutant RTs in complex with either K-5a2 or 25a.These structures illustrate the detailed interactions between RT and these two inhibitors,suggesting a possible mechanism for the susceptibilitlies of ETV and RPV to E138K mutation.The results outline the structural features of NNRTIs that can be employed for future drug design to overcome prevalent NNRTI-resistant mutations.Design,synthesis and anti-HIV evaluation of HIV-1 NNRTIs targeting NNIBP Tolerant Region I.In the third chapter,we conducted further activity-oriented structure modification of the right wing targeting NNIBP Tolerant Region I using K-5a2 and DAPY-based NNRTIs RDEA003 as lead compounds according to bioisosterism,molecular hybridization and scaffold hopping strategies.A total of 101 novel compounds were designed and synthesized via nucleophilic substitution,deprotection,acylation and Buchwald-Hartwig cross coupling reaction.The results showed that most compounds of IIA and ?B series exhibited moderate activity against WT HIV-1 strain,?A-5b turned out to be the most potent NNRTIs with EC50 values of 0.0092 ?M.In addition,?A-5b also showed moderate activity against all the single mutant strains and double mutant strain F227L+V106A.However,all compounds lost their activity against double mutant RES056.Seven compounds of IIC series exhibited nanomolar anti-HIV-1 activity(EC50 = 2.20-9.98 nM)against WT HIV-1,which comparable to that of K-5a2(EC50 =1.16 nM).The SAR analysis indicated that the "piperidinamine-methylene-aryl”motif of the right-wing is crucialto its activity.IID series displayed potent activity against WT and mutant HIV-1 srtains.Notably,IID-3e was proved to be the most potent inhibitor,with EC50 values of less than 10nM against all the single mutant strains and F227L+V106A.For RES056,its activity(EC50=21.5 nM)still higher than that of ETV.Furthermore,IID-3e also showed reasonable metabolic stability(T1/2 = 80.1 min)in a human liver microsome(HLM)assay.Design,synthesis and anti-HIV evaluation of HIV-1 NNRTIs targeting NNIBP Tolerant Region II.To increase the affinity with the target RT,multiple lipophilic aromatic rings were introduced to DAPY NNRTIs,which result in their poor solubility for the reason of the strong ?-? interaction.Based on the strategies of bioisosterism,scaffold hopping,crystal packing analysis and addition of polar functional group,the fourth chapter conducted further structure modification of the center core of K-5a2 targeting NNIBP Tolerant Region ?,with the aim to increase drug resistance profiles and solubility simultaneously.A total of 72 novel compounds were designed and synthesized via nucleophilic substitution,deprotection and cyclization.The activity results demonstrated that almost all compounds exhibited nanomolar inhibitory activity against WT HIV-1,and 34 compounds showed more potent activity than ETV.Among them,the activity of compounds with central ring of thiazolo[4,5-d]pyrimidine ?A,thiazolo[5,4-d]pyrimidine ?B,5,7-dihydrofuro[3,4-d]pyrimidine?G and hydrophilic group with hydrogen bond donors and acceptors on the para-position of Ar were superior to that of ETV against RES056.?A and ?B series were the most potent,with EC50 values of 18.1-28.1 nM,which was about 2 times higher than that of ETV(EC50 = 45.4 nM).In addition,?F-5a(EC50 = 28.8 nM)and ?H-5a(EC50 = 25.8 nM)also turned out to be potent inhibitors against RES056.Furthermore,the compounds of ?A,IIIB,?G series and ?F-5a exhibited superior and equivalent activity against other single mutant strains.The resulting potent compounds were further screened by compound ligand efficiency and the results showed ?A-5a? ?A-5g,?B-5a,?B-5c and ?G-5a-d possess good physical and chemical properties,the solubility of ?A-5a,?A-5g,?B-5a and ?B-5c also significantly enhanced.Further pharmacokinetic evaluation of these four lead compounds are in progress.Design,synthesis and anti-HIV evaluation of HIV-1 NNRTIs based on CuAAC click chemistry and in situ screening techniques.The rapid assembly and in situ screening of focused combinatorial fragment libraries using CuAAC click chemistry is a highly robust and efficient strategy for discovering bioactive molecules.The fifth chapter reported the rapid identification of highly potent HIV-1 NNRTIs with this technique for the first time.Based on the three-dimensional space of the HIV-1 RT and the structural characteristics of the solvent-exposed regions,5 alkynes and 39 azide building blocks were designed and then a combinatorial library containing 116 compounds were built by CuAAC click chemistry.Ten compounds with improved inhibitory activity than ETV and K-5a2 were confirmed via in situ screening.The anti-HIV-1 activity results demonstrated that C3A19(EC50 = 3.28 nM,SI>64103)and C3A34(EC50 = 4.38 nM,SI>48544)exhibited potent activity than ETV(EC50 = 5.1 nM,SI>889).Moreover,both of them have much lower cytotoxicity and higher SI values.In addition,C3A19 showed modest activity toward double mutant RES056(EC50 ? 481 nM,SI>437).In conclusion,taking the thiophenepyrimidine NNRTIs K-5a2 and RDEA003 as leads,a total of208 novel compounds were designed with the strategies of bioisosterism,molecular hybridization,scaffold hopping and diversity-oriented structure modification.Moreover,crystal packing analysis was first used to improve the solubility of DAPY NNRTIs.The CuAAC click chemistry and in situ screening technique was also tried for RT target for the first time,yielding a combinatorial library of]16 triazole-containing compounds.Most target compounds exhibited improved drug resistance profiles than ETV.K-5a2 and 25a were confirmed as potential anti-HIV drug candidates for their favorable pharmacokinetic properties and low toxicity in the pharmacokinetic study and safety assessment.IID-3e,?A-5a,?A-5g,?B-5a and?B-5c were identified as promising hit compounds after screening of compound ligand efficiency calculations,solubility measurements,and human liver microsomal stability analysis.Furthermore,we determined the co-crystal structures of WT and mutant RTs in complex with K-5a2 and 25a,including the first co-crystal structure of E138K RT in complex with DAPY NNRTIs,which provide a reliable model to analyze the structural effects of drug-resistant mutations in RT,and will contribute to structure-based design of novel NNRTIs that can effectively target multiple variants of RT.