Open AccessArticle Biological Evaluation of Esters of 4-Carboxylate-1,2,3-triazine and Analogs as New Potential Anti- Mycobacterium tuberculosis Agents 1 Laboratorio de Biotecnología Farmacéutica, Centro de Biotecnología Genómica, Instituto Politécnico Nacional, Reynosa 88710, Mexico 2 Department of Chemistry, The University of Texas San Antonio, San Antonio, TX 78249, USA 3 Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago, Chicago, IL 60612, USA 4 Laboratorio de Diseño y Desarrollo de Nuevos Fármacos e Innovación Biotecnológica, Departamento de Posgrado, Escuela Superior de Medicina, Instituto Politécnico Nacional, México City 11340, Mexico 5 Laboratorio de Estudios Epidemiológicos, Clínicos, Diseños Experimentales e Investigación, Facultad de Ciencias Químicas, Universidad Autónoma “Benito Juárez” de Oaxaca, Oaxaca 68120, Mexico 6 Unidad Académica Multidisciplinaria Reynosa-Rodhe, Universidad Autónoma de Tamaulipas, Reynosa 88779, Mexico * Authors to whom correspondence should be addressed. Molecules 2026, 31(12), 1993; https://doi.org/10.3390/molecules31121993 (registering DOI) Submission received: 10 May 2026 / Revised: 2 June 2026 / Accepted: 4 June 2026 / Published: 7 June 2026 Abstract In searching for novel molecules to act as antibacterial agents, particularly against Mycobacterium tuberculosis bacteria, three series of C5- and C6-substituted 1,2,3-triazine compounds were investigated: 1,2,3-triazine-4-carboxylate 1-oxide (series 1), 1,2,3-triazine-4-carboxylate (series 2), and 3,6-dihydro-1,2,3-triazine-4-carboxylate 1-oxide derivatives (series 3). Their structural elucidation was confirmed by 1H-NMR, 13C-NMR, and HRMS. We determined their antibacterial activity (MIC value) using the MABA against the M. tuberculosis H37Rv strain, as well as their physicochemical and pharmacokinetic properties. Finally, to determine their potential mode of action, an inhibition assay against M. tuberculosis DNA gyrase was performed. Compounds 4-ethoxycarbonyl-5-(3-methoxyphenyl)-1,2,3-triazine ( 2l) and 4-ethoxycarbonyl-5 -(n-propyl)-1,2,3-triazine ( 3s) exhibited high activity against M. tuberculosis with MIC values 73% yield) [ 19]. This synthetic route provided a set of 1,2,3-triazine compounds with a carboxylate group at C4 and a variety of substituents (alkyl, aryl, and fused heterocyclic systems) at C5 and/or C6 on the triazine scaffold. Series 1 includes nineteen 1,2,3-triazine 1-oxides, series 2 encompasses fifteen 1,2,3-triazines, and series 3 contains sixteen 3,6-dihydro-1,2,3-triazine 1-oxides. All compounds were characterized by proton and carbon nuclear magnetic resonance ( 1H-NMR and 13C-NMR) and High-Resolution Mass Spectroscopy (HRMS), and some were structurally confirmed by crystallographic studies (See Supplementary Material, Figure S1). The structural characterization obtained was in accordance with previous reports [ 19, 20, 21]. Therefore, these compounds were employed for further biological evaluation studies. 2.2. Anti-Mycobacterium Tuberculosis Activity Since there are no previous reports of anti-mycobacterial activity of 4-carboxylate- 1,2,3-triazine derivatives, series 1, 2, and 3 were screened against M. tuberculosis H37Rv to determine their MIC values [ 22]. Results of antimycobacterial activity are reported as MIC 90 values in µg/mL ( Table 1). The MIC values of triazine carboxylates 1-oxide of series 1 were in the range of 100 to 4.62 µg/mL. In general, the results showed that series 2 and 3 had comparable MIC values, but they performed better than series 1. Compounds 2l, 3c, and 3s had MIC values in the range of 4.62 to 8.72 µg/mL, while 1a, 1e, 1h, 2b, 3b, and 3g, with substitutions at C5 and C6, were less active, with MIC values > 100 µg/mL. The results for these three series of compounds showed that series 2 had MIC values that were reduced by a factor of two to twenty-one times compared with their analogs in series 1. In the same way, compounds of series 2 and 3 had better MIC values than those of series 1, except for 2b, 3b, and 3g. 2.3. Cytotoxic Evaluation Cytotoxic activity against the macrophage J774.2 cell line was measured by calculating the half-maximal cytotoxic concentration (CC 50) for three representative compounds belonging to each triazine series that had MIC values 100 µg/mL). In series 1, compounds 1o– 1q are the most active, with IC 50 values of 25 µg/mL. In series 2, compound 2l had high anti-mycobacterial activity (IC 50 = 4.62 µg/mL). In series 3, compounds 3c and 3s were highly active (IC 50 100 µg/mL); the substitution of the chain with an ethyl group ( 1b) and a propyl group ( 1c) had low activity (MIC = 91.00 and 70.10 µg/mL, respectively). Meanwhile, the introduction of a substitution in the chain ( 1d) improved the activity (MIC = 46.60 µg/mL), but the introduction of N 3 ( 1e) and TBSO ( 1f) terminal groups decreased activity (MIC ≥ 77.26 µg/mL). Furthermore, fused aliphatic rings, such as cyclohexyl ( 1g), and the substitution of oxygen ( 1h) and the NBoc group ( 1i) in the aliphatic ring did not improve activity. These results suggest that the more electronegative the atom in the six-membered ring, the lower the anti-mycobacterial activity. Compounds with aromatic substitutions at the 5-position on the 1,2,3-triazine ring exhibited greater activity compared to those with alkyl substitutions; compound 1j, which has a phenyl ring at the 5-position on the triazine ring, showed increased anti-mycobacterial activity (MIC = 48.51 µg/mL). The para-methyl substituents ( 1k) and the methoxy group at the ortho-position ( 1l) maintained activity (MIC = 47.74 and 48.34 µg/mL, respectively). However, the methoxy group at the meta-position ( 1m) drastically reduced the activity (MIC = 90.84 µg/mL). The incorporation of halogens at the para-position on the aromatic ring, such as -Cl ( 1n), maintained activity (MIC = 47.63 µg/mL), as did compounds ( 1j– 1l), while fluorine ( 1o) or bromine ( 1p) and the CF 3 group ( 1q) at the para-position on the aromatic ring increased biological activity (MIC = 25 µg/mL). A similar activity profile was observed when the phenyl ring at the 5-position incorporated fluoride at the 2- and 6- positions ( 1r) and the 2-naphthyl group ( 1s) at the 5-position (MIC = 48.30 and 47.53 µg/mL, respectively), compared to compounds 1j– 1l and 1n. In summary, aromatic derivatives substituted with halogen atoms (except chlorine) exhibited greater activity; this is potentially due to various factors that may be associated with increased molecular hydrophobicity and, consequently, influence cell wall permeability in mycobacteria; however, further studies are needed that include multiple factors such as logP/logD, polar surface area (PSA), hydrogen bonding capacity, and others, to confirm this association [ 24, 25]. 3.1.2. Series 2: 1,2,3-Triazine In general, the compounds of the 4-carboxylate-1,2,3-triazine series 2 exhibited greater anti-mycobacterial activity (MIC ≤ 100 µg/mL) than their series 1 derivatives. Compounds with aliphatic substitutions with the methyl group ( 2a) exhibited low anti-mycobacterial activity (MIC = 63.7 µg/mL); upon substitution of the chain with an ethyl group ( 2b), activity decreased drastically (MIC > 100 µg/mL); surprisingly, the change to a propyl group ( 2c) and the introduction of a unsaturation in the aliphatic chain ( 2d) and a TBSO terminal group ( 2f) drastically increased the activity (MIC = 16.70, 14.00, and 11.4 µg/mL, respectively). For compounds with the N 3 terminal group ( 2e) and fused aliphatic rings, such as cyclohexyl ( 2g), and the substitution of oxygen ( 2h) or the NBoc group ( 2i) in the aliphatic ring, no activity was determined. Compounds with aromatic substituents at the 5-position on the 1,2,3-triazine ring exhibited behavior similar to that of series 1, where aromatic substitutions exhibit greater activity than aliphatic substitutions. Compounds 2j and 2k, with a phenyl ring and a methyl substitution on the phenyl ring at the 5-position on the 1,2,3-triazine ring, exhibited moderate anti-mycobacterial activity (MIC = 11.35 and 11.01 µg/mL, respectively). Meanwhile, the methoxy group at the meta-position ( 2l) on the phenyl ring exhibited greater anti-mycobacterial activity (MIC = 4.62 µg/mL), and the methoxy group at the ortho-position ( 2m) slightly reduced its activity (MIC = 10.71 µg/mL). Compounds with halogen substitutions at the para-position on the phenyl ring, such as -Cl ( 2n), -F ( 2o), -Br ( 2p), and the CF 3 group ( 2q), exhibited moderate activity (MIC ≥ 12.26 to 46.09 µg/mL). The incorporation of fluoride at the 2- and 6- positions ( 2r) on the phenyl ring and 2-naphthyl ( 2s) at the 5-position on the 1,2,3-triazine ring showed activity similar to that of the compounds with halogens at the para-position on the phenyl ring. Therefore, in series 2, compound 2l was identified as the most active compound in the series, corresponding to the derivative with a methoxy group at the meta-position on the phenyl ring, demonstrating that the position of the substituent on the aromatic ring plays an important role in its biological activity. 3.1.3. Series 3: 3,6-Dihydro-1,2,3-triazine 1-oxide The series 3 of 4-carboxylate-3,6-dihydro-1,2,3-triazine 1-oxide derivatives exhibited a wide range of MIC values against M. tuberculosis. Compound 3a, containing a methyl group, exhibited moderate anti-mycobacterial activity (MIC = 47.01 µg/mL); however, extending the aliphatic chain to an ethyl group ( 3b) resulted in no activity against M. tuberculosis. Surprisingly, extending the aliphatic chain to three carbons (propyl group) in compound 3c dramatically increased activity (MIC = 5.09 µg/mL). Meanwhile, introducing unsaturation into the aliphatic chain ( 3d) slightly decreased activity (MIC = 13.60 µg/mL). However, the incorporation of terminal groups in the aliphatic chain, such as N 3 ( 3e) and TBSO ( 3f) groups, exhibited moderate activity (MIC = 56.08 and 34.90 µg/mL, respectively). For compounds 3g– 3i, the activity was not determined. Compounds with aromatic substituents at the 5-position on the 1,2,3-triazine ring exhibited high and moderate activity. The compound with a phenyl group ( 3j) at the 5-position on the 1,2,3-triazine ring exhibited anti-mycobacterial activity (MIC = 10.47 µg/mL); however, substitutions on the phenyl ring, such as a methyl group at the para-position ( 3k) and a methoxy group at the meta-position ( 3l), reduced the activity (MIC = 19.84 and 22.50 µg/mL, respectively). Meanwhile, the methoxy group at the ortho-position ( 3m) on the phenyl ring exhibited activity similar to compound 3j. Furthermore, halogen substitutions at the para-position on the phenyl ring showed that chlorine ( 3n) and bromine ( 3p) exhibited similar activity (MIC ≥ 19.20 µg/mL), whereas fluorine ( 3o) exhibited greater anti-mycobacterial activity (MIC = 10.50 µg/mL); in contrast, the addition of the CF 3 group ( 3q) at the para-position on the phenyl ring decreased the activity (MIC = 19.20 µg/mL). Finally, the incorporation of fluorine at the 2- and 6- position ( 3r) on the phenyl ring and 2-naphthyl ( 3s) at the 5-position on the 1,2,3-triazine ring showed activity similar to that of compound 3o (MIC ≤ 10.30 µg/mL). In summary, series 3 compounds exhibited high activity, such as compound 3c, which corresponds to aliphatic substitutions, and compound 3s, which corresponds to aromatic substitutions; therefore, this series appears to be the most promising for the development of new antimycobacterial agents. 3.2. Mycobacterium Tuberculosis DNA Gyrase Inhibition Activity Assay Once the anti-mycobacterial activity of 1,2,3-triazine derivatives was determined, we wondered whether 1,2,3-triazine derivatives could inhibit M. tuberculosis DNA gyrase. For this purpose, twelve compounds with MIC 100 63.7 47.01 b CH 3-CH 2- - 91.00 >100 >100 c CH 3CH 2CH 2- - 70.10 16.70 5.90 d cis CH 3-CH=CH 2-CH 2- - 46.60 14.00 13.60 e N 3CH 2CH 2- - >100 ND 56.08 f OTBS-CH 2CH 2- - 77.26 11.4 34.90 g cyclohexyl- CH 284.99 ND ND h cyclohexyl O >100 ND ND i cyclohexyl NBoc 93.92 ND ND j C 6H 5- - 48.51 11.35 10.47 k p-CH 3C 6H 5- - 47.74 11.01 19.84 l m-CH 3OC 6H 4- - 48.34 4.62 22.50 m o-CH 3OC 6H 4- - 90.84 10.71 11.13 n p-ClC 6H 4- - 47.63 19.42 24.42 o p-FC 6H 4- - 25.00 12.26 10.50 p p-BrC 6H 4- - 25.00 36.35 24.10 q p-CF 3C 6H 4- - 25.00 46.09 19.20 r 2,6-F 2C 6H 3- - 48.30 17.30 10.30 s 2-C 10H 7- - 47.53 23.47 8.72 RMP 0.09 INH 0.25 ND = not determined; RMP = rifampicin; INH = isoniazid, MIC = minimum inhibitory concentration. Table 2. Half-maximal cytotoxic activity (CC 50) and selectivity index (SI) of nine 1,2,3-triazine compounds against macrophage J774.2. Table 2. Half-maximal cytotoxic activity (CC 50) and selectivity index (SI) of nine 1,2,3-triazine compounds against macrophage J774.2. Compound CC 50(µg/mL) MIC 90(µg/mL) SI 1c >52.65 70.10 >0.75 1j >49.11 48.51 >1.01 1s 32.85 47.53 0.69 2c 62.23 16.70 3.72 2j 22.44 11.35 1.98 2s 55.86 23.47 2.38 3c 14.64 5.9 2.48 3j 49.45 10.47 4.72 3s 21.69 8.72 2.49 Table 3. Pharmacokinetic profile of selected 1,2,3-triazine compounds determined by SwissADME. Table 3. Pharmacokinetic profile of selected 1,2,3-triazine compounds determined by SwissADME. ID GI Absorption BBB Permeant Pgp Substrate CYP1A2 Inhibitor CYP2C19 Inhibitor CYP2C9 Inhibitor CYP2D6 Inhibitor CYP3A4 Inhibitor Lipinski No. Violations Veber No. Violations 1c High No No Yes No No No No 0 0 1j High No No Yes No No No No 0 0 1s High No No Yes Yes No No No 0 0 2c High Yes No Yes Yes No No No 0 0 2j High Yes No Yes Yes No No No 0 0 2s High Yes No Yes Yes No No No 0 0 3c High No No Yes No No No No 0 0 3j High No No No No No No No 0 0 3s High No No No No No No No 0 0 GI = gastrointestinal absorption; BBB = blood–brain barrier; Pgp = permeability glycoprotein. Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. © 2026 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license. Share and Cite MDPI and ACS Style Rivera, G.; Navarrete-Carriola, D.V.; De Angelis, L.; Paz-González, A.D.; Martínez-Vázquez, A.V.; Ortiz-Pérez, E.; Wan, B.; Franzblau, S.; Martínez-Archundia, M.; Moreno-Rodríguez, A.; et al. Biological Evaluation of Esters of 4-Carboxylate-1,2,3-triazine and Analogs as New Potential Anti- Mycobacterium tuberculosis Agents. Molecules 2026, 31, 1993. https://doi.org/10.3390/molecules31121993 AMA Style Rivera G, Navarrete-Carriola DV, De Angelis L, Paz-González AD, Martínez-Vázquez AV, Ortiz-Pérez E, Wan B, Franzblau S, Martínez-Archundia M, Moreno-Rodríguez A, et al. Biological Evaluation of Esters of 4-Carboxylate-1,2,3-triazine and Analogs as New Potential Anti- Mycobacterium tuberculosis Agents. Molecules. 2026; 31(12):1993. https://doi.org/10.3390/molecules31121993 Chicago/Turabian Style Rivera, Gildardo, Diana V. Navarrete-Carriola, Luca De Angelis, Alma D. Paz-González, Ana Verónica Martínez-Vázquez, Eyra Ortiz-Pérez, Baojie Wan, Scott Franzblau, Marlet Martínez-Archundia, Adriana Moreno-Rodríguez, and et al. 2026. "Biological Evaluation of Esters of 4-Carboxylate-1,2,3-triazine and Analogs as New Potential Anti- Mycobacterium tuberculosis Agents" Molecules 31, no. 12: 1993. https://doi.org/10.3390/molecules31121993 APA Style Rivera, G., Navarrete-Carriola, D. V., De Angelis, L., Paz-González, A. D., Martínez-Vázquez, A. V., Ortiz-Pérez, E., Wan, B., Franzblau, S., Martínez-Archundia, M., Moreno-Rodríguez, A., Palos, I., & Doyle, M. P. (2026). Biological Evaluation of Esters of 4-Carboxylate-1,2,3-triazine and Analogs as New Potential Anti- Mycobacterium tuberculosis Agents. 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