Design, Synthesis And Biological Evaluation Of Novel Pyrrolothiadiazines Derivatives As HIV-1 NNRTIs

In the treatment of HIV patients, the non-nucleoside reverse transcriptase inhibitors (NNRTIs) have been widely use as important component of highly active antiretroviral therapy (HAART) for their high selectivity, relatively low toxicity, and potent activity. However, NNRTI therapy is suffered from an inevitable emergence of HIV-1 resistant strains due to rapid mutants of the residues in RT. Therefore, further development of novel NNRTIs with improved pharmacological and resistance profile is critical for a more successful application of NNRTIs in combination therapy.Recently, a series of 2,4-disubstituted-1,1,3-trioxo-2H,4H-thieno[3,4-e][1,2,4] thiadiazines (TTDs) was reported as potent NNRTIs which can effectively inhibit the replication of wide type HIV-1 as well as a variety of HIV-1 mutant strains, including strains that are resistant to AZT. The prototype compounds QM96521 endowed with an EC50 value of 1μM. In an effort to improve its activity, extensive modification work was focused on the substitutions at N4 and N2 positions, which further led to the discovery of QM96539, QM96639 and QM96625. In recent years, our team has been engaged in structural modifications of TTDs scaffold, including replacement of thiophene with polar heterocycles or lager benzene ring, which generally reduced, even diminished the antiviral activity. Hence, the structural modification of five-membered heterocycle moiety of TTDs plays a key role in the generating novel TTDs-type NNRTIs with high potency. Inspired by this foundation, we further designed a series of novel disubstituted pyrrolothiadiazine (PTD) derivatives based on the general principle of bioisosteric replacement. The lipophilic heterocyclic system of pyrrolothiadiazine is considered as an ideal surrogate for thienothiadiazines to maintain its hydrophobic interaction with the highly hydrophobic NNRTI-binding pocket. Additionally, substitutions at N2 and N4 position were obtained from the favoriteπ-system groups elucidated by the previous structure and activity relationship (SAR) of TTDs.For the preparation of pyrrolothiadiazine ring, methyl-pyrrole-2-carboxylate (La-2) was converted to 1-(benzylsulfonyl)-pyrrole-2-carboxylic acid (La-3) through one-pot reaction including chlorosulfonation and hydrolysis. Subsequently, La-3 reacted with non-explosive DPPA agent to give rise to acyl azides, which thermally underwent curtius rearrangement to produce isocynate. The isocynate then underwent intramocular nucleophilic attack reaction to form the pyrrolothiadiazine ring. Totally,23 novel compounds had been synthesized and their structures were identified by IR, MS and 'H-NMR,13C-NMR spectral analysis respectively.The target compounds were evaluated for their activity against HIV-1ⅢB and cytotoxicity in MT-4 cells. Bioactivity assay indicated that most of the target compounds showed good activities against HIV-1. Among them, five compounds displayed inhibitory activity at the lower micromolar concentration ranges (EC50= 5.05～8.90μM), comparable to that of lead QM96521 (EC50=1μM) and NSC287474 (EC50=4μM). Besides, preliminary SAR analysis indicated that the substitutions at N2-benzly group contributed to antiviral activity in the following order:2-CN>3-CN> 2-Cl> 2-Br> H> 2,4-CI2. In order to predict the binding mode of our newly pyrrolothiadiazines derivatives and rationalize the SAR conclusion, molecular modeling of the most potent compounds by docking into the NNRTIs binding pocket (NNIBP) of HIV-1 RT was performed. The results suggested that this type of compounds shares a unique binding mode with classic NNRTIs. Notably, the N2 substituted benzyl group in the pyrrolothiadiazine core fits into the aromatic-rich binding pocket, surrounded by the aromatic side chains of Y188 and W229. Detailed analysis of the binding mode shows that the phenyl ring is parallel to Y188 side chain, giving rise to a positiveπ-stacking interaction. Meanwhile, N4 substituted benzyl group forms hydrophobic interaction with Y181, V179 and E138. The fused pyrrole moiety is close to the flexile P236'hairpin loop of the protein/solvent interface. It is worth mentioned that the inhibitors cyano group and sulfonyl group form extensive network of hydrogen bonds with the adjacent residues, which is assumed as critical factors to contribute to the affinity of inhibitors.In summary, based on SAR study of TTD derivatives, a series of novel of pyrrolothiadiazine derivatives was designed based on the principle of bioisosteric replacement. The new, simple, and convenient "one-pot" synthetic approach to the important intermediate La-3 was developed and optimized. Totally,23 target compounds were synthesized and were structurally identified. Biological evaluation for their anti-HIV-1 activity assay revealed that most compounds were effective NNRTIs with low nanomolar level, which are worth further investigation and development. The preliminary SAR analysis among the newly synthesized compounds are discussed briefly and rationalized by docking studies. We hope that the knowledge and insight on the NNRTI research learnt from this work will provide valuable information for the development of novel potent NNRTIs.