Design, Synthesis and Evaluation of N-pyrazinylbenzamides as Potential Antimycobacterial Agents

Three series of N-(pyrazin-2-yl)benzamides were designed as retro-amide analogues of previously published N-phenylpyrazine-2-carboxamides with in vitro antimycobacterial activity. The synthesized retro-amides were evaluated for in vitro growth inhibiting activity against Mycobacterium tuberculosis H37Rv (Mtb), three non-tuberculous mycobacterial strains (M. avium, M. kansasii, M. smegmatis) and selected bacterial and fungal strains of clinical importance. Regarding activity against Mtb, most N-pyrazinylbenzamides (retro-amides) possessed lower or no activity compared to the corresponding N-phenylpyrazine-2-carboxamides with the same substitution pattern. However, the active retro-amides tended to have lower HepG2 cytotoxicity and better selectivity. Derivatives with 5-chloro substitution on the pyrazine ring were generally more active compared to their 6-cloro positional isomers or non-chlorinated analogues. The best antimycobacterial activity against Mtb was found in N-(5-chloropyrazin-2-yl)benzamides with short alkyl (2h: R2 = Me; 2i: R2 = Et) in position 4 of the benzene ring (MIC = 6.25 and 3.13 µg/mL, respectively, with SI > 10). N-(5-Chloropyrazin-2-yl)benzamides with hydroxy substitution (2b: R2 = 2-OH; 2d: R2 = 4-OH) on the benzene ring or their acetylated synthetic precursors possessed the broadest spectrum of activity, being active in all three groups of mycobacterial, bacterial and fungal strains. The substantial differences in in silico calculated properties (hydrogen-bond pattern analysis, molecular electrostatic potential, HOMO and LUMO) can justify the differences in biological activities between N-pyrazinylbenzamides and N-phenylpyrazine-2-carboxamides.

1. Full results of biological assays 1.1

. Full results of antimycobacterial evaluation
Compounds 1h, 1i, 2n, 3a, 3f, 3g, 3k, 3l, and 3n precipitated during the preparation of the basic solution of the compound and therefore the testing was discontinued.

HepG2 cytotoxicity determination
The human liver hepatocellular carcinoma cell line HepG2 (passage 32-34 for compounds 2b-Ac, 2b, 2h, and 2i; and passage 17-18 for 2d-Ac and 3n) purchased from Health Protection Agency Culture Collections (ECACC, Salisbury, UK) was routinely cultured in Minimum Essential Eagle Medium (MEM; Sigma-Aldrich) supplemented with 10% (v/v) fetal bovine serum (PAA, Austria), 1% (v/v) L-glutamine solution (Sigma-Aldrich) and 1% (v/v) non-essential amino acid solution (Sigma-Aldrich) in a humidified atmosphere containing 5% CO2 at 37 °C. For subculturing, the cells were harvested after trypsin/EDTA (Sigma-Aldrich) treatment at 37°C. To evaluate the cytotoxicity, the HepG2 cells treated with the tested substances were used as experimental groups whereas untreated HepG2 cells served as control groups.
HepG2 cells were seeded in a density of 1×10 4 cells per well on a 96-well plate. Next day (24 h after seeding) the cells were treated with tested substances dissolved in DMSO at different concentrations ranging from 0.1 to 1000 µM (depending on the solubility, see Table 2). Maximal incubation concentration of DMSO in a well did not exceed 1% (v/v).
The treatment was carried out in triplicates in a humidified atmosphere containing 5% of CO2 at 37 °C. The controls representing 100% cell viability (untreated cells), 0% cell viability (cells treated with 10% DMSO), no-cell controls and vehiculum controls were incubated in triplicates simultaneously. After 24 h exposure to tested compounds, CellTiter 96® Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA) reagent was added to each well according to the manufacturer`s recommendations. After 2 h incubation at 37 °C in humidified, 5% of CO2 containing atmosphere, the absorbance was recorded at 490 nm. Inhibitory curves were constructed for each compound plotting incubation concentrations vs. percentage of absorbance relative to untreated control. The standard toxicological parameter IC50 was calculated by nonlinear regression analysis of the inhibitory curves using GraphPad Prism software, version 6 (GraphPad Software, Inc., CA, USA).

Confirmatory test of HepG2 cytotoxicity
The HepG2 cells (passage 35-36) were seeded in density 1×10 4 cells per well on a 96-well plate. Next day (24 h after seeding), they were treated with tested substances dissolved in DMSO (maximal incubation concentration of DMSO was 1% v/v). The tested substances were prepared according to their solubility in DMSO at incubation concentrations 0.1-500 µM (see Table 3). The treatment was carried out in a humidified atmosphere containing 5% CO2 at 37 °C in triplicates for 24 h and 48 h. The controls representing 100% cell viability, 0% cell viability (the cells treated with 10% DMSO and the cells treated with Lysis Solution 1:25), no-cell controls and vehiculum controls were incubated in triplicates simultaneously. After 24 h exposure, the reagent from the kit CellTox™ Green Cytotoxicity Assay (Promega, Madison, WI, USA) was prepared and added according to the recommendation of the manufacturer. After 15 min incubation at room