Design,Synthesis And Biological Evaluation Of Novel Piperidine Substituted DAPY Analogues As HIV-1NNRTIs

Acquired immune deficiency syndrome (AIDS) which is mainly caused by human immunodeficiency virus type-1(HIV-1), remains to be one of the leading pandemic diseases worldwide. Currently, the highly active antiretroviral therapy (HAART) plays a crucial role in the treatment of AIDS which contains at least two kinds of anti-HIV-1drugs. Among the FDA approved anti-HIV-1drugs, the reverse transcriptase (RT) inhibitors are the most widely used in clinical, which can be mainly divided into two types:nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs).Among the structurally diverse NNRTIs, diarylpyrimidine (DAPY) derivatives with unique antiviral potency, high specificity and low cytotoxicity have attracted considerable attention over the past few years. As the representatives of DAPYs, Etravirine (TMC125) and Rilpivirine (TMC278) were approved by US FDA in2008and2011respectively due to their superior activity against wild-type and NNRTI-resistant mutant strains of HIV-1.X-ray crystallography and molecular modeling studies showed that the binding conformation of DAPYs resembled a horseshoe or "U" shape in the NNIBP, which can generate numerous conformational variants through the torsional flexibility of the-O-and-NH-linker to keep high binding affinity to the mutant RT. However, Etravirine has low water solubility and poor pharmacokinetic properties, and there will be serious rash after long-term use. Though Rilpivirine has higher antiviral activity, the failure rate of clinical treatment was significantly higher than that of Etravirine. Therefore, it is an urgent need to design and develop novel NNRTIs with low toxicity, good pharmacokinetic properties and high efficiency against drug resistant strains.Based on the above analysis, triazine-amino-piperidine series (Series I),3-nitro/amino pyridine-amino-piperidine series (Series II) and2-amino pyrimidine-amino-piperidine series (Series III) were designed via bioisosterism principle. The newly designed compounds, which retained the left2,4,6-tri-substituted phenyl and the right substituted piperidine group, also made the following structural transformation:1) The para-substituent (R1) on the left phenyl, the substituent (R2) on the central ring and the linker (X) were designed structural diversely to generate many congeners;2) Not only the N-substituted benzyl/phenyl groups, and also the substituted benzoyl were introduced to the NH of piperidine;3) Diverse substituents were introduced to the ortho-, meta-and para-positions of the terminal phenyl;4) The terminal benzene ring (Ph) was replaced by the hydrophilic aromatic heterocycles (Het). Molecular simulation studies of the newly designed compounds using the software SYBYL-X (Surflex-Dock) indicated that the new compounds could bind in to NNIBP in similar mode as lead compounds, and could form hydrophobic interaction and hydrogen bond interaction.Subsequently, a series of novel piperidinylamino-diarylpyrimidine (pDAPY) derivatives (Series IV) was designed through molecular hybridization strategy by introducing the N-benzyl substituted piperidine group to the6position of central pyrimidine ring. Docking studies showed that the novel hybrid compounds could bind into the NNIBP in two modes:1)2,4,6-tri-substituted phenyl formed hydrophobic interaction with aromatic amino acid residues; The4-cyanophenyl group occupied the groove lined by Pro236, Vall06and Leu234, and the newly introduced N-benzyl substituted piperidine group stretched out of the NNIBP through the entrance channel (Mode1).2) N-benzyl substituted piperidine group extended to the solvent/protein interface; And the4-cyanophenyl group was oriented into the entrance channel (Mode2). In both of the two modes, the entrance channel was occupied by N-benzyl substituted piperidine group or4-cyanophenyl group. Further optimization of pDAPY derivatives (shorten the N-benzyl substituted piperidine group) led to Series V, which were kept one binding mode as Etravirine and introduced more flexible and hydrophilic groups in order to occupy the entrance channel effectively.The synthetic routes of target compounds were designed according to the retrosynthetic analysis and related literature. Firstly the starting materials reacted with2,4,6-trisubstituted aniline/phenol and4-amino-l-Boc piperidine respectively to construct the key intermediates S-3, S-7, B-3a, B-3b, B-7, M-4a and M-4b. After transformation of relevant functional groups and removal of Boc groups, the intermediates could connect with different substituents to get the target compounds, of which the structures were confirmed by1H-NMR,13C-NMR, IRand ESI-MS.All the title compounds were tested in a cell-based antiviral assay against HIV (wild-type, ⅢB and a resistant mutant HIV-1strain, containing K103N/Y181C in the RT) and HIV-2(ROD) with nevirapine (NVP), delavirdine mesylate (DLV), efavirenz (EFV) and zidovudine (AZT) as reference drugs. Most of the new compounds were active against wild-type HIV-1with EC50values in the nanomolar concentration range, and some compounds were active against K103N/Y181C resistant mutant strains. However, none of the newly synthesized compounds were active against HIV-2(ROD) at a subtoxic concentration in MT-4cells.Among the triazine-amino-piperidine derivatives (Series Ⅰ), compounds with the N-benzyl substituted piperidine group showed high activity against wild-type HIV-1with EC50values below20nM, and against K103N/Y181C resistant mutant strains with EC50values below5μM. Compound TA-a5showed the highest activity against wild-type HIV-1(EC50=2.2nM, SI=45285), which was89times higher than the reference drug NVP,15times higher than DLV,2.9times higher than EFV and2.7times higher than AZT. Compound TN-a5showed the highest activity against K103N/Y181C resistant mutant strains (EC50=0.12μM, SI=1500), which was4.6times higher than the reference drug EFV (NVP and DLV were not active in the same assay).Among3-nitro/amino pyridine-amino-piperidine analogues (Series Ⅱ), compound BD-e2was the most potent compound with EC50value of5.1nM, which was22times higher than NVP,21times higher than DLV, and comparable to EFV and AZT. Compound BD-cl (EC50=10nM) showed exciting low cytotoxicity with a CC50value greater than146μM and high selectivity (SI>14126), which is comparable to AZT.Most2-amino pyrimidine-amino-piperidine analogues (Series Ⅲ), showed high activity against wild-type HIV-1with EC50values below10nM, which were15-20times higher than NVP and DLV. Whereas, these compounds were less potent against K103N/Y181C resistant mutant strains with EC50values in the micromolar concentration range.The hybrid compound MD-c5(Series Ⅳ) displayed high activity against wild-type and K103N/Y181C resistant mutant strains with EC50values0.038μM and0.95jiM respectively. Further optimization resulted in compound DSC-a4(Series Ⅴ) with higher activity against wild-type and K103N/Y181C resistant mutant strains (EC50=7.8nM and0.65μM) than compound MD-c5.The most active compounds of each series were performed HIV-1RT inhibitory assays using commercial kit. The results indicated that the representative compounds showed high affinity to HIV-1RT, and inhibited the activity of RT in vitro with EC50values comparable to reference drug NVP.In summary, based on the analysis of binding mode and SAR studies of the lead compounds,5series of compounds were designed using bioisosterism principle and molecular hybridization strategy. The newly designed compounds were docked into NNIBP and overlaped with lead compounds to elucidate the binding mode. All the target compounds were synthesized according to the retrosynthetic analysis and related literature, and screened for anti-HIV activity. Some compounds were confirmed to be highly active against wild-type and K103N/Y181C resistant mutant strains of HIV-1with lower EC50values than the reference drugs which have potential for further research. In addition, important structure activity relationship (SAR) information of each series in the thesis were discussed for further research.