3-Substituted N-Benzylpyrazine-2-carboxamide Derivatives: Synthesis, Antimycobacterial and Antibacterial Evaluation

A series of substituted N-benzyl-3-chloropyrazine-2-carboxamides were prepared as positional isomers of 5-chloro and 6-chloro derivatives, prepared previously. During the aminolysis of the acyl chloride, the simultaneous substitution of chlorine with benzylamino moiety gave rise to N-benzyl-3-(benzylamino)pyrazine-2-carboxamides as side products, in some cases. Although not initially planned, the reaction conditions were modified to populate this double substituted series. The final compounds were tested against four mycobacterial strains. N-(2-methylbenzyl)-3-((2-methylbenzyl)amino)pyrazine-2-carboxamide (1a) and N-(3,4-dichlorobenzyl)-3-((3,4-dichlorobenzyl)amino)pyrazine-2-carboxamide (9a) proved to be the most effective against Mycobacterium tuberculosis H37Rv, with MIC = 12.5 μg·mL−1. Compounds were screened for antibacterial activity. The most active compound was 3-chloro-N-(2-chlorobenzyl)pyrazine-2-carboxamide (5) against Staphylococcus aureus with MIC = 7.81 μM, and Staphylococcus epidermidis with MIC = 15.62 μM. HepG2 in vitro cytotoxicity was evaluated for the most active compounds; however, no significant toxicity was detected. Compound 9a was docked to several conformations of the enoyl-ACP-reductase of Mycobacterium tuberculosis. In some cases, it was capable of H-bond interactions, typical for most of the known inhibitors.


Results and Discussion
Most of the compounds presented in this paper with in vitro activity against M. tuberculosis were disubstituted derivatives with two large benzyl substituents on two adjacent positions of the pyrazine core. We were interested to find out whether such sterically demanding derivatives would be able to fit in the active site of InhA in a manner similar to smaller PZA derivatives with single aryl substituent. Therefore, we performed molecular docking of the most active dibenzyl derivative 9a into various conformations of InhA, differing in the size of the active site cavity, which is formed by the highly flexible substrate-binding loop (Fig. S1). Not surprisingly, 9a was not able to fit into closed conformations of InhA (pdb: 2X23; 3FNF) and did not show the expected ligand-receptor interactions. On the other hand, when opened conformation of InhA receptor was used (pdb: 4R9S, 4TZK, or 5G0S), we were able to identify two different binding modes for 9a with scores similar to the score of the cocrystalized ligands and, more importantly, with ligand-receptor interactions known to be typical for InhA inhibitors. See Table S1.
The first binding mode ( Fig. S2) was similar to the one suggested for simpler N-benzylaminopyrazine-2-carboxamides, where the H-bond accepting moiety was the carbonyl oxygen of the carboxamide moiety. In our case, the best pose of 9a was further stabilized by H-bond interaction of carboxamide hydrogen (donor) to sulphur of Met199 (acceptor). The docking score for 9a was -9.0 kcal/mol compared to redocked ligand with -10.72 kcal/mol. However, when we use the docking score weighted by the number of heavy atoms of the ligand ('ligand efficiency'), ligand 9a can be considered more efficient fragment than the original co-crystalized ligand (see Table S1).
In the second binding mode (Fig. S3), the H-bond accepting role of 9a was played by the N-1 nitrogen of the pyrazine core. Consequently, the carbonyl oxygen remained available for the intramolecular H-bond with NH of the benzylamino substituent. This intramolecular H-bond was present in all low energy conformations of 9a as determined by low mode molecular dynamics conformational search. Therefore, the availability of carbonyl oxygen for intramolecular H-bond increases the stability of this binding mode. However, the N-1 nitrogen of the pyrazine core can accept only one H-bond. Interestingly, the interaction between ligand and phenolic hydroxyl of Tyr158 has the form of arene-H interaction (see Fig. S3).
In both binding modes, the ligand 9a fits tightly to hydrophobic areas of the binding pocket as visible from the contour lines on the ligand-interaction diagrams (Fig. S4).

Conclusions
Although the results of molecular docking are not enough to confirm the inhibition of InhA as the mechanism of action of derivative 9a, we have shown that even such sterically demanding derivatives are able to mimic poses and interactions of known InhA inhibitors. Selection of suitable structure of InhA with open conformation was crucial in this docking study.

Note
The primary objective of this molecular docking study was to determine whether sterically demanding derivatives with two large benzyl substituents at the adjacent positions of the pyrazine core would be theoretically compatible with the active site cavity of mycobacterial enoyl-ACP-reductase. This was confirmed as documented above. However, as suggested by a reviewer of the manuscript, we probed the differences in binding between corresponding 3-chloro derivatives (one benzyl substituent) and 3-benzylamino derivatives (two benzyl substituents). Using the same methodology as described below, we performed docking of the most active 3-chloro derivative 3 along 3-benzylamino derivatives (1a, 2a, 3a, 9a) into various forms of InhA. The results can be summarized as follows:  The smaller molecule of 3 had more binding modes then sterically demanding 3a, especially in open InhA conformations.  The docking score (and ranking) of 3 was worse than the score of 3a, as well as other dibenzyl derivatives (1a, 2a, 9a).  With one exception, no predicted poses of 3 formed critical H-bond interactions to neither Tyr158 nor 2'-OH of the ribose of NAD + .  In the best scored pose of 3 in InhA pdb: 2X23, the oxygen of the carboxamide moiety accepted the hydrogen of 2'-OH of the ribose of NAD + . However, in this pose the plane of the carboxamide moiety was significantly rotated out of the plane of the pyrazine core. Many scoring functions would penalize this non-planarity of the conjugated carboxamide moiety.
Judging only from the docking score and ligand-receptor interaction patterns, 3-benzylamino derivatives should be better inhibitors of InhA than 3-chloro derivatives. However, this molecular docking study alone is not enough to suggest the inhibition of InhA as a mechanism of action of neither of the structural classes.