Design, Synthesis, Antimycobacterial Evaluation, and In Silico Studies of 3-(Phenylcarbamoyl)-pyrazine-2-carboxylic Acids

Pyrazinamide, the first-line antitubercular drug, has been regarded the basic component of tuberculosis treatment for over sixty years. Researchers have investigated its effect on Mycobacterium tuberculosis for this long time, and as a result, new potential targets of pyrazinamide or its active form, pyrazinoic acid, have been found. We have designed and prepared 3-(phenyl-carbamoyl)pyrazine-2-carboxylic acids as more lipophilic derivatives of pyrazinoic acid. We also prepared methyl and propyl derivatives as prodrugs with further increased lipophilicity. Antimycobacterial, antibacterial and antifungal growth inhibiting activity was investigated in all prepared compounds. 3-[(4-Nitrophenyl)carbamoyl]pyrazine-2-carboxylic acid (16) exerted high antimycobacterial activity against Mycobacterium tuberculosis H37Rv with MIC = 1.56 μg·mL−1 (5 μM). Propyl 3-{[4-(trifluoromethyl)phenyl]carbamoyl}pyrazine-2-carboxylate (18a) showed also high antimycobacterial activity against Mycobacterium tuberculosis H37Rv with MIC = 3.13 μg·mL−1. In vitro cytotoxicity of the active compounds was investigated and no significant cytotoxic effect was observed. Based to structural similarity to known inhibitors of decaprenylphosphoryl-β-d-ribose oxidase, DprE1, we performed molecular docking of the prepared acids to DprE1. These in silico experiments indicate that modification of the linker connecting aromatic parts of molecule does not have any negative influence on the binding.


In Silico Docking Study
We performed molecular docking studies on DrpE1 of M. tuberculosis H37Rv. DprE1 was chosen as a potential new target involved in mycobacterial cell wall synthesis. We studied only acids 1-18, methyl and propyl esters are considered as prodrugs, which are hydrolyzed in mycobacterium. Molecular Operating Environment (MOE) 2016.08 was used to conduct the in silico study. To verify the docking procedure, the originally co-crystalized ligand was removed and redocked again with RMSD = 0.24 Å.  PDB structure 4P8N (chain A) was chosen for in silico study of DprE1. The predicted poses of individual ligands 1-18 were evaluated with regard to the ligand-receptor interactions and position of original ligand. Eight ligands (3, 4, 6, 9, 10, 11, 16, 18) combining the best docking score, similarity in interactions to the original ligand, and overlapping with the original ligand were considered as the best candidates for DprE1 inhibition. These compounds form interactions between the carbonyl of the carboxylic group (COOH) and side-chain of Lys418 and Tyr60; the 'hydroxyl' oxygen of COOH with side-chain of Arg325; and N-1 of the pyrazine core creates an interaction with Lys418. Pyrazine ring is stabilized by H-π interactions with Val365 and with Gly117 in a similar way to the original ligand. The original ligand had docking score -8.73 kcal/mol, the best studied compound 4 (R = 2,4-diOCH3) had docking score -7.28 kcal/mol. When we calculated ligand efficiency (LE = docking score/number of heavy atoms), compound 4 had better LE = -0.33, compared to original ligand´s LE = -0.31. The best compound according to LE was 11 (R = 4-Br) with LE = -0.36. This compound also exhibited π-π interaction with FAD cofactor as the original ligand, see Figure S1. Compound 16, the most active compound in the whole cell assay, had the docking score -6.96 kcal/mol and LE = -0.33 ( Figure S2). In comparison to the original structure of 2-carboxyquinoxalines, the replacement of -NH-CH2-linker by -CONHgroup does not radically change the character of binding mode. On the other hand, the loss of the condensed ring along with large CF3 substituent seems to decrease antimycobacterial activity. Probably the large lipophilic substituent is needed for the filling of the hydrophobic sub-pocket. enriched with the 0.4% of glycerol (Sigma-Aldrich) and 10% of OADC supplement (oleic acid, albumin, dextrose, catalase; Himedia, Mumbai, India) of declared pH = 6.6 was used for cultivation. Tested compounds were dissolved and diluted in DMSO and mixed with broth (25 μL of DMSO solution in 4.475 mL of broth) and placed (100 μL) into microplate wells. Mycobacterial inocula were suspended in isotonic saline solution and the density was adjusted to 0.5-1.0 McFarland. These suspensions were diluted by 10 −1 and used to inoculate the testing wells, adding 100 μL of suspension to 100 μL of the DMSO/broth solution of tested compound. Final concentrations of tested compounds in wells were 100, 50, 25, 12.5, 6.25, 3.13 and 1.56 μg·mL −1 . Isoniazid (INH) was used as positive control (inhibition of growth). Negative control consisted of broth plus DMSO. A total of 30 μL of Alamar Blue working solution (1:1 mixture of 0.1% resazurin sodium salt (aq. sol.) and 10% Tween 80) was added after five days of incubation. Results were then determined after 24 h of incubation. The minimum inhibitory concentration (MIC; μg·mL −1 ) was determined as the lowest concentration that prevented the blue to pink color change. MIC values of INH were 6.25-12.5 μg·mL −1 against M. avium, 3.13-12.5 μg·mL −1 against M. kansasii, and 0.1-0.2 μg·mL −1 against M. tbc.

Antimycobacterial In Vitro Activity Screening Against Mycobacterium smegmatis
The antimycobacterial assay was performed with fast growing Mycobacterium smegmatis CCM 4622 (ATCC 607) from the Czech Collection of Microorganisms (Brno, Czech Republic). The technique used for activity determination was microdilution broth panel method using 96-well microtitration plates. The culturing medium was Middlebrook 7H9 (MB) broth (Sigma-Aldrich), enriched with 0.4% of glycerol (Sigma-Aldrich, Steinheim, Germany) and 10% of Middlebrook OADC growth supplement (Himedia). Tested compounds were dissolved in DMSO (Sigma-Aldrich), and the MB broth was then added to achieve a concentration of 2000 μg·mL −1 . Standards used for activity determination were INH, rifampicin (RIF), and ciprofloxacin (CPX) (Sigma-Aldrich). Final concentrations were reached by binary dilution followed by the addition of mycobacterial suspension, and were set as 500, 250, 125, 62.5, 31.25, 15.625, 7.81, and 3.91 μg·mL −1 , except for the standards of ciprofloxacin and rifampicin, where the final concentrations were 12.5, 6.25, 3.125, 1.56, 0.78, 0.39, 0.195, and 0.098 μg·mL −1 . The final concentration of DMSO did not exceeded 2.5% (v/v) and did not affect the growth of M. smegmatis. Plates were also sealed with polyester adhesive film and incubated in the dark at 37 °C, without agitation. The addition of 0.01% solution of resazurin sodium salt followed after 48 h. This stain was prepared by dissolving resazurin sodium salt (Sigma-Aldrich) in deionised water, producing a 0.02% solution. Then, a 10% aqueous solution of Tween 80 (Sigma-Aldrich) was prepared. Both liquids were mixed up making use of the same volumes and filtered through a syringe membrane filter. Microtitration panels were then further incubated for 4 h. Antimycobacterial activity was expressed as the minimal inhibition concentration (MIC) and the value was read on the basis of stain color change (blue color-active compound; pink color-not active compound). The MIC values for the standards were in the range of 7.81-15.625 μg·mL −1 for INH, 0.78-1.56 μg·mL −1 for RIF, and 0.098-0.195 μg·mL −1 for CPX. All experiments were conducted in duplicate.

Evaluation of In Vitro Antifungal Activity
Antifungal evaluation was performed using a microdilution broth method against eight fungal strains (Candida albicans ATCC 44859, C. tropicalis 156, C. krusei E28, C. glabrata 20/I, Trichosporon asahii 1188, Aspergillus fumigatus 231, Lichtheimia corymbifera 272 and Trichophyton mentagrophytes 445). Compounds were dissolved in DMSO and diluted in a twofold manner with RPMI 1640 medium, with glutamine buffered to pH 7.0 (3morpholinopropane-1-sulfonic acid). The final concentration of DMSO in the tested medium did not exceed 2.5% (v/v) of the total solution composition. Static incubation was performed in the dark and humidity, at 35 °C, for 24 and 48 h (72 and 120 h for Trichophyton mentagrophytes). Drug-free controls were included. The standards were amphotericin B, voriconazole, nystatin, and fluconazole.
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.
The HepG2 cells were seeded in a density of 1 × 10 4 cells per well on a 96-well plate. The following day (24 h after seeding), they were treated with tested substances dissolved in DMSO (maximal incubation concentration of DMSO was 1%). The tested substances were prepared according to their solubility in DMSO, at incubation concentrations of 1-750 μM. The treatment was carried out in a humidified atmosphere containing 5% CO2 at 37 °C, in triplicate, for 24 h. The controls representing 100% cell viability, 0% cell viability (the cells treated with 10% DMSO), no-cell controls, and vehiculum controls were incubated in triplicate, simultaneously. After 24 h exposure, the reagent from the kit CellTiter 96 ® Aqueous One Solution Cell Proliferation Assay (Promega, Madison, Wisconsin, USA) was added, according to the recommendation by the manufacturer. After 2 h incubation at 37 °C in humidified, 5% CO2 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 a nonlinear regression analysis of the inhibitory curves using GraphPad Prism software (version 7, GraphPad Software, Inc., La Jolla, CA, USA).