Molecular Design, Synthesis And SAR Research Of 2,5,6-trisubstituted S-DABO And 6-naphthylmethyl Substituted N-DABO Analogues As Nonnucleoside HIV-1 Reverse Transcriptase Inhibitors

Acquired immunodeficiency syndrome(AIDS) is a worldwide pandemic disease caused by the human immunodeficiency virus(HIV).The reverse transcriptase(RT) of HIV,being the primary enzyme responsible for the conversion of the viral single-stranded RNA to the double-stranded DNA chain that subsequently is incorporated into the DNA of the infected host cell,remains one of the most attractive targets for the development of new anti-HIV/AIDS drugs.Among all the currently FDA-approved anti-HIV-1 drugs,14 drugs exert their therapeutic efficacy by inhibiting HIV-1 RT.Dihydroalkoxybenzyloxopyrimidines(DABOs) are one of the most representative classes of non-nucleoside reverse transcriptase inhibitors(NNRTIs) of HIV-1 and have been considered as interesting lead compounds for the design of new anti-retroviral drugs.Our continuing interest in the search for new NNRTIs led us recently to describe the design and synthesis of a new series of 6-naphthylmethyl and 6-naphthylthio substituted S-DABO analogues as highly potent HIV-1 inhibitors.The follow-up structure-activity relationships(SAR) studies on S-DABOs revealed that a major determinant of increased potency in these new congeners was the improved interactions between the residues Tyr188,Tyr181 in the hydrophobic binding pocket of RT and the C-6 aromatic ring of the inhibitors.The SAR studies also indicated that the C-2 substituent of S-DABOs situated in a large and flexible hydrophobic region of the binding site of RT,which could accommodate a large number of different kinds of subsitutent varied from alkyl to arylmethyl and arylcarbonylmethyl groups.As for the C-5 substituent,the factors responsible for the improved inhibitory profiles of S-DABOs remain to be elucidated.In this paper,we studied the putative binding mode of S-DABOs to the active site of RT by Autodock program to achieve further modifications on this series of NNRTIs. In particular,we mainly focused our attention on the structural variations on the C-2, C-5 and C-6 substitutents of S-DABOs.These includes a benzyl moiety at the C-6 position of the pyrimidine ring with different substituents such as Br,Cl,CH₃ and CF₃ groups at the ortho or meta positions of the benzene ring,which was expected to improve the interactions between the C-6 substituent of S-DABOs and Tyr188, Tyr181 of RT;the introduction of H,Me,Et and iso-propyl group at the C-5 position of the pyrimidine ring.In addition,a variety of well-known good moieties such as 4-methoxyphenylcarbonylmethyl,4-methoxybenzyl,sec-butyl,and iso-propyl groups were chosen as the substituents at the C-2 sulfur atom bound to the pyrimidine nucleus with the aim to determine which one was the optimum for S-DABOs concerning the activity against both wild-type HIV-1 and clinically important mutant strains.Based on the above analysis,a new series of 2,5,6-trisubsituted S-DABO analogues was designed and synthesized with the hope to find more potent HIV-1 RT inhibitors and to obtain further insights of the structure-activity relationships of S-DABOs.Due to the structural similarity of N-DABO and S-DABO series,they may share some common chemical requirements for HIV-1 inhibitory potency.Thus,the SAR conclusions of S-DABOs could further guide the design of N-DABOs.Based on the docking analysis of N-DABOs/RT complexes,we postulated that the introduction of a naphthylmethyl moiety at the C-6 position of the pyrimidine ring would be favorable for their anti-HIV-1 activity by increasing the putativeÏ€-stacking interactions between the ligand and the Tyr188 or Tyr181 of RT.In light of these,we designed a new series of 6-naphthylmethyl subsitituted N-DABOs with different substituents at the C-2 and C-5 positions of the pyrimidine ring aimed to delineate the structure-activity relationships of N-DABOs.Different synthetic routes were explored and applied successfully to obtain these two series of target compounds.In particular,two synthetic methods,Clay method and Blaise method were employed to prepareβ-ketoesters,which were the key synthetic intermediates for both S-DABOs and N-DABOs.According to the Clay method,β-ketoesters could be readily prepared starting from substituted malonate with high purity.On the other hand,the rest ofβ-ketoesters could be prepared using Blasie method.In addition,a general procedure for the synthesis of substituted guanidines commenced from different amines was explored successfully.The S-DABO derivatives could be readily prepared starting fromβ-ketoesters. Condensation of thiourea withβ-ketoesters in the presence of NaOEt in refluxing ethanol furnished the corresponding 2-thiouracils,which were thioalkylation with RX (X=Br,Cl or I) in anhydrous DMF in the presence of K₂CO₃ to afford the required S-DABO analogues WYPS001-WYPS069.The synthesis of N-DABO analogues were achieved using two synthetic routes. Compounds WYPN001-WYPN013 were prepared by direct condensation of β-ketoesters with different guanidines,while compounds WYPN014-WYPN018 were obtained from the starting materials 2-thiouracils through a six-step reaction sequence including thioalkylation,chlorination,methoxylation,oxidation, nucleosubstitution and deprotection.More than 150 intermediates and final products were synthesized in this paper, and among them 88 compounds were the target molecules.Except for compound WYPS018 and WYPS068,all the title compounds are new compounds and have not been reported in literature.The structures of all these compounds were confirmed by IR,MS,¹H NMR,¹³C NMR,and elemental analysis or X-ray analysis.The novel S-DABO and N-DABO analogues were evaluated for their cytotoxicity and anti-HIV activity in MT-4 cells.The results revealed that the arylcarbonylmethylthio moiety was the optimal subsitutent at the C-2 position of the pyrimidine ring,since S-DABOs featuring this kind of C-2 substituent were endowed with highly potent HIV-1 inhibitory potency.In particular,compound WYPS015 turned out to be the most potent inhibitor with a 50%effective concentration(EC₅₀) of 0.010μM against HIV-1 replication in cell culture,being about 7-fold compared to nevirapine and delavirdine.More importantly,it also exhibited exceptionally high selectivity index (SI) of＞31800.Some compounds,WYPS005,WYPS007,WYPS010 and WYPS014 show also high anti-HIV-1 activity(EC₅₀=0.054,0.044,0.040,and 0.018μM, respectively) and excellent selectivity indices(SI=5704,6318,4675,and 13278, respectively),surpassing those found for the reference drugs nevirapine and delavirdine.It is worth noting that compound WYPS015 also exhibited activity against K103N+Y181C HIV-1 double mutant strain at micromolar concentration; although less active than efavirenz,its selective index is almost equal to that of efavirenz.Thus,compound WYPS015 was chosen as a new lead compound for further optimization,with the expectation to find more potent antiviral agents, especially against HIV-1 mutant strains.As for the newly synthesized 19 N-DABO congeners,13 compounds showed more potent anti-HIV-1 activity than that of HEPT and DDI.Among them,compound WYPN004 was the most promising compound,being about 25-fold more potent than HEPT and DDI.In addition,all N-DABO analogues were also evaluated for their capability to inhibit the HIV-2 multiplication in MT-4 cells,but none was found effective except for compound WYPN006 with an EC₅₀ against HIV-2 ROD of 3.79μM. Finally,the 3D QSAR(CoMFA and CoMSIA) models of S-DABOs and N-DABOs were established,wherein the active conformations of these ligands were obtained by performing docking analysis.Based on the 3D QSAR models,the further SAR of these newly synthesized compounds were explored in the aspects of steric, electrostatic,hydrophobic and H-bond donor fields.The conclusions summarized from these results will provide useful information for the development of new NNRTIs with more potent anti-HIV-1 activity and selectivity.