Design, Synthesis And Anti-HIV Evaluation Of Novel Non-Nucleoside Reverse Transcriptase Inhibitors

In the life cycle of human immunodeficiency virus type1(HIV-1), reverse transcriptase (RT) is responsible for the conversion of single-stranded viral RNA into double-stranded proviral DNA, a prerequisite for integration into host DNA. Due to its important role in the HIV-1life-cycle, RT has been identified as a prime target for anti-HIV drug discovery. Among currently available RT inhibitors, non-nucleoside reverse transcriptase inhibitors (NNRTIs) represent an important component of drug combination therapy for the treatment of HIV-infected patients. Up to date, five NNRTIs have been approved for clinical application by FDA:nevirapine (NVP), delavirdine (DLV), efavirenz (EFV), etravirine (ETV) and rilpivirine (RPV). First generation of NNRTIs, NVP and EFV, are suffered from severe side-effects over long periods of therapy, as well as inevitable emergence of HIV-1resistant strains. Though the second generation of NNRTIs, DLV, ETV, and RPV perform well against a variety of variants bearing clinically prevalent mutations, the development of novel NNRTIs families are still demanded to address issues of side effects, poor solubility, cross-resistance, and virologic failureThe NNRTI is a class of specific HIV-1RT inhibitors with diverse structures but share some inherent similarities in their pharmacophoric elements and binding mode with the NNRTI binding pocket (NNIBP), which provides valuable information for the lead discovery and drug optimization. More importantly, the extensive crystallgraphic investigations on the NNRTI/RT complexes help to interpret the ligand-enzyme interactions and reveal some important structure features to maintain the antiviral activity against mutant HIV-1strains. In this thesis, we focus our research interest on developing of novel NNRTIs using the structure-based approaches using a combination of traditional medicinal chemistry, structural biology, and computational chemistry. My research work could be divided into three sections, which are summarized as follows:Section One:The Design, Synthesis and Biological Evaluation of DAPY-IOPY Hybrid DerivativesBased on the pharmacophoric similarity and crystallographic overlaps of TMC125and R221239, we designed a novel series of DAPY-IOPY hybrid derivatives through combining their privileged structural features using molecular hybridization strategy:i)2-pyridone ring of IOPY was kept as the central scaffold to develop hydrogen-bondings with K103and K101. Meanwhile, halogen atoms were introduced to the3-position of the2-pyridone ring, respectively, to evaluate their impacts on antiviral activity; ii) Fragment switching of the2-aniline group of DAPY into the6-position of2-pyridione template and modified the substituents at this phenyl ring; iii) Introduction of the favorable substitutions of4-phenoxy moiety of DAPY and IOPY into the4-position of novel2-pyridone template, respectively, to probe the substitution SAR patterns of these novel DAPY-IOPY hybrid derivatives.In total, we synthesized31target compounds which could be classified into two series (Series IA and Series IB). The biological testing results showed that most compounds possessed submicromolar to micromolar level of inhibition activities against HIV-1Ⅲb strains in MT-4cell cultures. The most potent compound IA-7d showed anti-HIV-1IIIB activity with an EC50value of0.15μM, comparable to that of nevirapine, but superior to those of delavidine and zalcitabine. The structure-activity relationship (SAR) analysis indicated that2-pyridone scaffold of these inhibitors was indispensable for their anti-HIV-1activity, and substitution of halogen at the3-position of the2-pyridone ring would decrease the anti-HIV activity. Further biological evaluation demonstrated that IA-7d, IA-7c, IB-7a and IB-7b inhibited the replication of HIV-1primary isolated BK132(X4) or92TH001(R5) strains in cord blood mononuclear cells (CBMCs) at submicromolar level. ELISA based RT enzyme inhibition assay revealed that IA-7d possessed anti-RT activity with an IC50value of5.2μM. Quantitative PCR analysis of viral DNA experience showed that IA-7d, IA-7c, IB-7a and I7-8b could highly effectively reduce the late RT DNA product to1.1%、11.7%、2.1%and2.8%, respectively, compared with the DMSO control group (100%). These results proved these2-pyridone typed DAPY-IOPY hybrid derivatives to be a highly promising NNRTI scaffold, which may serve as a new platform for further modification in search for more potent candidates for anti-HIV chemotherapySection Two:The Structural Optimization of DAPY Derivatives by Introducing Piperidyl, Morpholinyl or Piperazinyl Group into the NNIBP Tolerant RegionBased on the crystallographic studies and molecular modeling investigations of DAPYs, we focus our attention on exploiting the tolerant region I and II of NNIBP by incorporating hydrophilic piperidyl, morpholinyl or piperazinyl group into the pyridine-and pyrimidine-typed DAPY derivatives, with an aim to develop additional interactions between the inhibitors and NNIBP, as wells as to improve the drug solubility.The antiviral activity evaluation results showed that, the most potent compounds of Series ⅡA, viz. ⅡA-8c2, IIA-8c3, IIA-8c4and IIA-8b3could effectively inhibit HIV-1NL4-3strains in MT-4cells with EC50values of0.1nM,0.05nM,0.8nM and0.3nM, respectively, accompanied with excellent SI values ranging from20349to242800. The anti-HIV-1ⅢB activities of IIA-8c2, IIA-8c3, IIA-8c4and IIA-8b3were low to9.1nM,7.4nM,9.3nM and7.8nM, respectively (SI=168-1283), much superior to FDA-aproved drug of NVP,3TC and DLV, and comparable to ETV, AZT and EFV. Additionally, compounds IIA-8b2and IIA-8b3showed good performance against the serious RES056(K103N+Y181C) mutant strains with the EC50values of6.2μM and6.8μM, respectively. The SAR analysis of Series IIA demonstrated that replacement of the2,4,6-trimethyl group on left-wing phenyl ring with4-CN-2,6-dimethy group led to improved potency, and incorporation of OCH3group into the6-position of pyridine instead of the Cl group yielded less active compounds. The substitutions at the Nj position of the right-wing piperidyl contributed to the anti-HIV-1activity in the following order:4-SO2NH2-phenyl>4-SO2Me-phenyl> pyridn-4-yl>4-CONH2-phenyl> phenyl> cyclopropyl or cyclobutyl.The anti-HIV-1NL4-3assay results of Series IIB indicated that the extra morpholinyl or piperazinyl group which was introduced into the lead DAPY derivatives could be well adopted by the NNIBP tolerant site, without significantly reducing the compounds’anti-HIV activities. The most compound IIB-8b possessed an EC50value of9.1nM, with SI value of2596. These compounds of series IIB were expected to possess improved solubility and pharmacokinetic profile, due to the favorable hydrophilic properties of morpholinyl and piperazinyl group.Section Three:Design, Synthesis and Biological Evaluation of N2, N4-Disubstituted-1,1,3-trioxo-2H,4H-pyrrolo[1,2-b][1,2,4,6]thiatriazine Derivatives as Novel HIV-1NNRTIsIn an effort to explore derivatives of N2, N4-2,4-disubstitued-1,1,3-trioxo-2H,4H-thieno[3,4-e][1,2,4]thiadiazines (TTDs) as potent HIV-1NNRTIs, we had designed and synthesized a classes of N2, N4-disubstitued-1,1,3-trioxo-2H,4H-pyrrolo[1,2-6][1,2,4,6]thiatriazine derivatives (PTTDs), which displayed good activities against HIV-1virus. As a continuation of our research, we incorporated diverse groups into the N2position to further investigate the S AR patterns of PTTDs. An alternative synthetic strategy was attempted to firstly prepare PTTD ring, which subsequently underwent regioselectivce N-alkylation at its N2and N4positions, respectively, to give the target PTTDs. However, extensive effort to conduct this strategy eventually lead to the production of another new heterocycle, viz.1,1,3-trioxo-2H-pyrrolo[1,2-6][1,2,5]thiadiazole, rather than the expected PTTD ring. We proposed a plausible reaction mechanism for formation of1,1,3-trioxo-2H-pyrrolo[1,2-6][1,2,5]thiadiazole. Finally, we utilized the improved synthesis route of PTTDs to synthesize the PTTD target compounds.Unfortunately, the biological evaluation results showed that only compound ⅢB-10a10had inhibition activity against wide type HIV-1ⅢB strains. ⅢB-10a10shares a ubiquitous privileged fragment of3,5-dimethyl-allyl group at its N2substitutions with many reported potent NNRTIs, including tetrahydromidazo (TIBO), thiocarboxanilid, diarylamines (Het-NH-Ph-U) and benzothiadiazepines. The molecular modeling study revealed that the3,5-dimethyl-allyl group of ⅢB-10a10generally overlapped the same group of8-TIBO, and occupied the same hydrophobic sub pocket defined by the residues of Y188and Y188. In some extent, this result demonstrated that the intrinsic relationships between the structural similarity of the ubiquitous privileged fragments and their pharmacophoric similarities, which encourages further exploring of bioactive substitutions of PTTDs based on the database of privileged fragments ubiquitous in known NNRTIs with high potency against WT and drug-resistant variants.In summary, taking the DAPYs, IOPYs and PTTDs as leads,75compounds which belong to three categories of novel NNRTIs were designed and synthesized. The new, simple, and convenient synthetic approaches to the title compounds were developed, or improved and optimized. Lastly, through biological evaluation, we find several high potent antiviral compounds which had anti-HIV-1activity ranging from picomolar to nanonolar levels, and some of them also exhibit good performance against the serious resistant strains bearing variants of Y181C and K103N. Additionally, these compounds were expected to have improved pharmacokinetic profiles due to the incorporation of favorable fragments which benefit the drug solubility, which are worth further investigation and development.