Design, Synthesis And Anti-HIV Evaluation Of Novel Arylazolyl (Azinyl) Thioacetanilides As Potent NNRTIs

The human immunodeficiency virus type 1 (HIV-1) is the main cause of the acquired immunodeficiency syndrome (AIDS), which was first identified in the Western world in 1981. Since then, AIDS has developed into a worldwide pandemic of disastrous proportions. Considerable progress has been made in treating HIV-infected patients using highly active antiretroviral therapy (HAART) involving multidrug combinations. However, the increasing incidence of drug resistant viruses along with the drug toxicity among treated people calls for continuous efforts of developing anti-HIV-1 drugs.HIV-1 reverse transcriptase (RT) is one of the main targets for the action of anti-AIDS drugs. Drugs targeted at HIV RT can be divided into two categories:(i) nucleoside and nucleotide analogue RT inhibitors (NRTIs/NtRTIs), which, following activation to their triphosphate forms, compete with the RT substrate and also act as terminators of DNA synthesis after incorporation into the primer strand; and (ⅱ) nonnucleoside RT inhibitors (NNRTIs), including the approved drugs nevirapine, delavirdine, efavirenz and etravirine, which, although having wide structural variation, all bind at a similar site distal to the active site within RT. NNRTIs currently in clinical use have a low genetic barrier to resistance and therefore, the need for novel NNRTIs active against drug-resistant mutants selected by current therapies is of paramount importance.In recent year, in spite of the rapid growth of HIV-1 RT 3D-structural information, the difficulty in structure-based de novo design of NNRTIs scaffolds and docking based virtual screening approach lies in the following two aspects:i) The flexibility of NNIBP, formed by conformational changes in the RT on binding of the NNRTI ligand;ⅱ) The NNRTI resistance mutations located in and around the NNIBP. Therefore, structure-based and ligand-based combined drug design methodology was carried out to facilitate both drug lead generation and lead optimization. And considerable cases illustrated the benefits for NNRTIs design of closely coupled traditional medicinal chemistry, structural biology, computational chemistry methodology, and many others.From high-throughput screening (HTS) of compounds library, several interesting sulfanyltriazole and sulfanyltetrazole-typed leads were identified as novel NNRTIs, which have simple, yet distinctively different chemical structure from the HIV inhibitors reported in the literature. Extensive structural modification and bioactivity research demonstrated that most of them showed submicromolar activity in a cell assay and significant in vitro activity on the WT or K103N-Y181C HIV-1 RT. With a suitable combination of substitution patterns on the aryl linked to the tetrazole/triazole core (hydrophobic interaction domain), the anilide aryl (the solvent exposed region), five-membered moiety and thioacetamide linker (scaffold and hydrogen bond interaction domain), it is possible to identify compounds which maintain the same intrinsic activity on the wild-type HIV-1 enzyme and the clinically relevant K103N mutant. Based on the above, we hypothesized that alternate, potentially better scaffolds could be designed.Bioisosteric replacement principle is an excellent tool of Medicinal Chemistry for lead optimization to produce the desired potency and selectivity and the requisite ADME profile for a marketable drug. On the basis of extensive SAR and molecular modeling studies of sulfanyltriazole and sulfanyltetrazole-typed NNRTIs, we replace the triazole or tetrazole five-membered core in the leads by other five-membered heterocycles (1,2,3-thiadiazole,1,2,3-selenadiazole, imidazole, triazole) and six-membered heterocycles (pyridazine, triazine) to obtain the novel scaffolds, i.e. Arylazolyl(azinyl)thioacetanilides based NNRTIs.In chemoinformatics, searching for compounds which are structurally diverse and share a biological activity is called scaffold hopping. On account of the structural similarity of NNRTIs families, scaffold hopping or chemotype switching via dismantlement and simplification of known NNRTIs is important since it can be used to obtain alternative structures with improved efficiency and unexpected side-effects. Based on the "scaffold hopping" concept and the pharmacophore characteristic of lead compounds, sulfamoylaminoacetamide and sulfamoyl pyrrolidine-2-carboxamide scaffold were designed, which exhibited similar binding conformation to tetra(tri)azole thioacetanilides. In addition, the SAR analysis and the similarity of molecular modeling of tetra(tri)azole thioacetanilides based NNRTIs and DAPY-based NNRTIs permitted the scaffold hopping to a novel series of substituted thiazole thioacetanilides NNRTIs.Multiple ligands are emerging in anti-HIV drug discovery strategies, using a single entity to inhibit multitargets could yield improved patient compliance, thus reducing the likelihood of drug resistance. The exploration of such multifunctional ligands has proven valuable for anti-HIV leads discovery. Our design began with the general knowledge that a diketoacid (DKA) group and an directly connected aromatic ring are the two indispensable structural features for the DKA class of HIV-1 integrase (IN) inhibitors. The key to multiple ligands design strategy would be to incorporate these two features into a tolerant region in the RT inhibitors. Molecular modelling has shown that the amide connected phenyl group of azole thioacetanilides-based NNRTIs is situated in an open area controlled by the P236 loop where structural modification could be tolerated. Based on these general knowledge and the multiple ligands design strategy, azolthioacetamidophenyl-2-hydroxy-4-oxobut-2-enoic acid-based RT/IN dual inhibitors were designed via incorporation of an IN pharmacophore to this tolerant region of the azole thioacetanilides NNRTIs. Virtual compounds library was constructed by introducing superior substituent group of lead compounds to the designed core:1,2,3-thiadiazole thioacetanilides, 1,2,3-selenadiazole thioacetanilides, imidazole thioacetanilides, triazole thioacetanilides, pyridazine thioacetanilides, triazine thioacetanilides, sulfamoylaminoacetamide and sulfamoyl pyrrolidine-2-carboxamide scaffold, substituted thiazole thioacetanilides and azolthioacetamidophenyl-2-hydroxy-4-oxobut-2-enoic acid-based scaffold. Virtual compounds were evaluated by docking, program, carried out using FlexX, which is a widely used docking algorithm in drug design whose ability in predicting a binding mode of the ligand very close to its X-ray structure has been widely described in literature.In this dissertation,206 title compounds belong to 9 novel scaffolds were designed and synthesized, starting from the commercially available starting material, and were structurally identified by IR, Mass spectroscopy,'H-NMR and/or 13C-NMR spectral analysis respectively. These compounds and the new synthesis approaches have not been reported in literature.The preliminary activity and cytotoxicity screening of the newly designed and synthesized target compounds 1,2,3-thiadiazole thioacetanilides,1,2,3-selenadiazole thioacetanilides and imidazole thioacetanilides were tested in MT-4 cells for inhibition of HIV-1 (strain IIIB) and HIV-2 (strain ROD). Bioactivity assay indicated that most of the title compounds showed good activities against HIV-1 and none of the compounds exhibited inhibitory activity against HIV-2.①In particular,10 compounds in 1,2,3-thiadiazole thioacetanilide (TTA) series showed anti-HIV-1 activities at sub-micromolar concentrations. Interestingly, the cytotoxicity of TTAs was generally low. Owing to the above reasons, their SI values were in many cases similar to that of the reference drug. In particular, compound I-A7c displayed the most potent anti-HIV-1 activity (EC50=36.4 nM), inhibiting HIV-1 replication in MT-4 cells more effectively than NVP (by 7-fold) and DLV (by 8-fold).②The results showed that some 1,2,3-selenadiazole thioacetanilide derivatives, such as II-7f (EC50=2.45μM), possess similar HIV-1 inhibitory activity compared with that of sulfanyltriazole series, but most of these derivatives exhibited decreased anti-HIV-1 specificity due to a significantly increased cytotoxicity. Although the pharmacological results are not very encouraging, this study provides useful information to further design new anti-HIV agents.③Most of the tested imidazole thioacetanilides derivatives inhibited HIV-1 replication in a lower micromolar concentration range. The most potent HIV-1 inhibitors were III-A4e (EC50=0.18μM, CC50=28.81μM, SI=162), andⅢ-A4b (EC50= 0.20μM, CC50=35.24μM, SI=170). The EC50 values of these two compounds were lower than those of one triazole lead compound (EC50=2.053μM) and of the reference drugs NVP and DLV. Other compounds,Ⅲ-A4c,Ⅲ-A4d,Ⅲ-C4e, andⅢ-A4a, also showed higher anti-HIV-1 potency (EC50=0.64,0.73,1.03, and 1.78μM, respectively) compared with the respect to that of the triazole lead compound, indicating that the imidazole is also an acceptable isosteric replacement for the triazole, tetrazole, or 1,2,3-thiadiazole in the lead compounds.④Several compounds in 1,2,4-series and chloro-substituted thiazole series (Ⅳ-7a,Ⅳ-7c,Ⅳ-7e,Ⅳ-7g,Ⅳ-71,Ⅳ-7m andⅨ-B8a,Ⅸ-B8e,Ⅸ-B8f,Ⅸ-B8g) proved to be active against HIV-Ⅰ(Ⅲb). These compounds also showed an appreciable activity against mutant HIV-1 strains (E138K,K103N,L1001).⑤All of the tested compounds in sulfamoylaminoacetamide series, sulfamoyl pyrrolidine-2-carboxamide series and amino-substituted thiazole series lost their anti-HIV activity.⑥Two azolthioacetamidophenyl-2-hydroxy-4-oxobut-2-enoic acid derivatives (Ⅹ-3A andⅩ-3B) retained moderate activity against wild-type HIV-1 and the mutant strains E138K, K103N and L1001. On the basis of the chemical structure and the fact that the target compounds inhibit HIV-1, but not HIV-2 replication, these molecules can be proposed to act as genuine HIV-1 NNRTIs. The preliminary structure-activity relationships (SAR) of the newly synthesized congeners are discussed. Molecular modeling (docking) and 3D-QSAR studies of some potent compounds in complex with HIV-1 RT are described, allowing rationalization of some SAR conclusions. Moreover, the anti-HIV activity against an NNRTI-resistant strain of the newly synthesized derivatives is in progress, from which we are expecting to get some useful information. Some selected target compounds (54 compounds) were randomly tested for their anti-influenza virus activity. Preliminary result showed that two amino-substituted thiazole derivatives (IX-A8g and IX-A8h) displayed potent and selective inhibitory activities against influenza A H1N1 subtype. And these compounds could be used as "hit" compounds to further design influenza inhibitors.In summary, taking the sulfanyltriazole and sulfanyltetrazole-typed NNRTIs as leads, night series of arylazolyl(azinyl)thioacetanilides-based NNRTIs were designed and synthesized in this dissertation according to "bioisosteric replacement" principle, "scaffold hopping" concept and "multiple ligands design" strategy, respectively. The new, simple, and convenient synthetic approaches to the title compounds were developed, or improved and optimized. Lastly, through biological evaluation, we find many high potent antiviral agents, the 1,2,3-thiadiazole/imidazole thioacetanilide scaffolds were identified as novel NNRTIs, amino-substituted thiazole derivatives were discovered as promising anti-influenza virus agents in random screening, which are worth further investigation and development. We hope that the knowledge and insight on the NNRTI research learnt from the work will help a lot on the battle against the virus and benefit human health and life.