Structure-based And Pharmacophore-guided Design, Synthesis And Biological Evaluation Of Novel DAPYs As HIV-1 NNRTIs

HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) played vital roles in preventing and treating AIDS, which are essential components of the highly active antiretroviral therapy (HAART). However, the long term use of NNRTIs were compromised by the severe side effects and multiple drug resistances. Therefore, the development of novel NNRTIs with higher activity, lower toxicity and better anti-drug resistance profiles is still urgently required.Diary lpyrimidines (DAPYs) are a series of NNRTIs with high efficiency and low toxicity, and two representative compounds, Etravirine (2008) and Rilpivirine (2011), have been approved by US FDA for clinical use. By analyzing the crystal structures of DAPYs bonding with RT, it was found that DAPYs can adjust their conformations by wiggling and jiggling when the surrounding amino acid residues in NNRTI binding pocket (NNIBP) mutate, thus keep binding affinity with RT and maintaining their activities against mutant HIV-1 strains. However, DAPYs possess poor oral bioavailability owing to their poor solubility. In addition, the HAART accelerated the development of drug-resistance. Therefore, more and more attentions were paid to developing novel NNRTIs with better anti-resistance profiles and pharmacokinetic profiles.With the aim of developing novel anti-resistance drugs with improved solubility, binding modes of DAPYs were analysed and it was found that the right ring of DAPYs occupied in NNIBP was relatively tolerant (tolerant region I), which can be exploited to improve anti-HIV-1 activities and physicochemical properties. Recently, catechol diethers were reported with picomole activities against wide-type (WT) HIV-1 and good solubility. The main chemical structure difference was the ethyluracil moiety when comparing DAPYs and catechol diethers. Moreover, the ethyluracil moiety and aminobenzonitrile moiety both located in tolerant region I. Therefore, using molecular hybridization based on crystallographic overlays, a new series of compounds were designed by introducing the ethyluracil group into DAPYs. Subsequently, with the help of computer aided drug design (CADD), molecular docking was used to evaluate the designed compounds and 24 compounds were finally synthesized.The starting material uracil was first protected at N-3 using benzoyl chloride, and then reacted with Boc protected 2-bromoethanamine. The protecting groups were deprotected to give intermediate 1-(2-aminoethyl) uracil (A-6). Reaction of halo-pyrimidine with substituted phenols afforded A-15, and then reacted with A-6 to provide the target compounds. Futhermore, the 6-C1 of target compounds could be aminated to give subsequent target compounds.The anti-HIV activities and cytotoxicity of synthesized compounds were tested in MT-4 cells. The antiviral activity results demonstrated that most compounds showed low micromole or sub micromole activity against WT HIV-1 with low toxicity and good selectivity. Among them, the two most potent compounds ZH-7 (EC50=7.8 nM) and ZH-20 (ECso=5.6 nM) showed comparable activities with reference drug Efavirenz (ECso=5.2 nM), and SI of ZH-20 was more than 50000. Meanwhile, the activities against common HIV-1 clinical mutants of ZH-20 and ZH-23 were tested, indicating that both of them were active against L100I, K103N, Y181C and E138K mutant strains, but lost activities against Y188L and double mutant strains (F227L/V106A and Y181C/K103N).Recent advances of DAPYs lead to the construction of a new "Four-point Pharmacophore Model" which conclude the configuration and electrical requirement, and tolerant region Ⅱ in NNIBP was found for DAPYs. Modifications of DAPYs in tolerant region Ⅰ and Ⅱ can not only improve the activities against mutant strains but also reduce side effects.According to the new model, the 5,6-positions of central pyrimidine point to tolerant region Ⅱ, and this region can de occupied by an additional fused ring without loss of activity, like compounds RDEA-427 and RDEA-640. Additionally, introducing a pyrrole ring fused with pyrimidine can also reduce the affinity of DAPYs with CYP enzymes, and thus decrease side effects. In our previous work, a new compound K-5a2 with a fused thienopyrimidine ring displayed comparable activities with Rilpivirine against WT and mutant HIV-1 viruses. All these results indicated that the pyrimidine ring can be replaced by a fused ring without loss of activities. A non-aromatic tetrahydro-thiopyran fused pyrimidine ring was introduced by scaffold hopping to investigate whether an aliphatic ring showed equal effect. In addition, modifications in tolerant region I in previous work can improve the activities against mutant strains by forming additional hydrogen bonds. Thus, the substituted piperidine were also introduced into tetrahydro-thiopyran fused pyrimidine DAPYs and finally 26 compounds were synthesized.Reaction started with methyl 4-chlorobutanoate and methyl thioglycolate, followed by substitution reaction, Dieckmann condensation reaction, cyclization reaction and hydrolysis reaction to provide intermediate B-8. B-8 can react with substituted ethylamine to give target compounds or with Boc protected aminopiperidine to afford B-9. Reaction of B-9 with substituted benzyl bromide (chloride) afforded target compounds.Anti-HIV activities of these title compounds suggested that most compounds were potent against WT and drug-resistant HIV-1 viruses, with ECso values less than 10 nM against WT strain and less than 1 μM against common clinical resistant strains. HZ-3 (1.44 nM) and HZ-18 (2.37 nM) were the most potent inhibitors, which were more active than reference drug Etravirine. In addition, activities of HZ-10 were less than 25 nM against prevalent clinical mutant strains, and was more than two folds potent than Etravirine against L100I, Y181C, F227L/V106A and Y181C/K103N strains.In conclusion, in order to improve the solubilities and activities against mutant HIV-1 virus of DAPYs, molecular hybridization based on crystallographic overlays and newly built "Four-point Pharmacophore Model" were used to guide the design of the new HIV-1 NNRTIs. Two series of 50 compounds were rationally designed, synthesized and evaluated against HIV in cell-based assays. These compounds had novel scaffolds and showed good antiviral activities:the uracil bearing compounds showed nanomole activities against WT HIV-1; the tetrahydro-thiopyran fused pyrimidine compounds showed potent activities against WT and mutant HIV-1 strains and provided two potent leads for next-step modifications. Furthermore, research work in this thesis provide valuable clues for further optimizations of DAPYs.