1,2,4-Triazoles as Important Antibacterial Agents

The global spread of drug resistance in bacteria requires new potent and safe antimicrobial agents. Compounds containing the 1,2,4-triazole ring in their structure are characterised by multidirectional biological activity. A large volume of research on triazole and their derivatives has been carried out, proving significant antibacterial activity of this heterocyclic core. This review is useful for further investigations on this scaffold to harness its optimum antibacterial potential. Moreover, rational design and development of the novel antibacterial agents incorporating 1,2,4-triazole can help in dealing with the escalating problems of microbial resistance.


Introduction
Since the discovery of the first antibiotic (Penicillin, 1928), there has been an ongoing "race" between scientists developing new antibacterial agents and pathogenic bacteria harboring various resistance mechanisms. In 2017, the World Health Organization (WHO) published a list of 12 bacteria whose level of resistance to antibiotics is such that they represent a major concern to public health, and grouped them according to their priority as critical, high, and medium. Critical-priority bacteria included Gram-negative bacterial pathogens, namely carbapenem-resistant Acinetobacter baumannii and Pseudomonas aeruginosa, as well as carbapenem-resistant and third-generation cephalosporin-resistant Enterobacteriaceae. The highest ranked Gram-positive bacteria (high priority) were vancomycin-resistant Enterococcus faecium (VRE) and meticillin-resistant Staphylococcus aureus (MRSA). According to another report, entitled "Tackling Drug-Resistant Infections Globally: Final Report and Recommendations", which was a result of an extensive work carried out by an independent commission, drug-resistant infections may be the cause of 10 million deaths annually by 2050, exceeding the number of deaths attributable to road traffic accidents and even cancer [1]. It is not surprising given strong selection pressure exerted on bacteria by antibiotics that are overused and misused in humans and animals as well as in agriculture and food chains, which has also led to considerable environmental pollution. To overcome the development of drug resistance, it is crucial to search for new antibacterial molecules with novel mechanism of action as well as structural modification or optimization of the existing agents by improving both the binding affinity and the spectrum of activity while retaining bioavailability and safety profiles. Searching for new therapeutic options in the treatment of resistant bacterial infections include discovering new synthetic compounds as well as naturally occurring substances (especially from essential oils extracted from plants) [2][3][4].
A wide variety of heterocyclic systems have been explored in order to develop pharmaceutically important molecules. Nitrogen-containing heterocycles are found in many medicines. Derivatives of triazole have particularly interesting therapeutic properties. Triazoles are five-membered rings, which contain two carbon and three nitrogen atoms, with a molecular formula of C 2 H 3 N 3 . According to the position of nitrogen atoms, triazoles exist in two isomeric forms -1,2,3-triazole and 1,2,4-triazole ( Figure 1). The 1,2,4-triazole ring may exist in equilibrium between two forms: 1H-form and 4H-form ( Figure 1). The atoms, triazoles exist in two isomeric forms -1,2,3-triazole and 1,2,4-triazole ( Figure 1). The 1,2,4-triazole ring may exist in equilibrium between two forms: 1H-form and 4H-form ( Figure 1). The calculated energy differences between azole tautomers support preference for the 1H over 4H tautomer [5]. Literature review shows that 1,2,4-triazoles and their fused heterocyclic derivatives show a wide spectrum of biological activities. The 1,2,4-triazole core has been incorporated into a wide variety of therapeutically important agents available in clinical therapy, such as itraconazole, posaconazole, voriconazole (antifungal), ribavirin (antiviral), rizatriptan (antimigraine), alprazolam (anxiolytic), trazodone (antidepressant), letrozole and anastrozole (antitumoral) (Figure 2). In the last few decades, scientists have been paying considerable attention to the synthesis of 1,2,4-triazole derivatives showing such comprehensive biological activities as an antifungal [6,7] antitubercular [8], antioxidant [9], anticancer [10], anti-inflammatory [11], analgesic [12], antidiabetic [13], anticonvulsant [14], and anxiolytic [15] activity. With regard to the antifungal activity, triazole-based pharmacophore has replaced the previously widely used imidazole pharmacophore in systemically active azoles, due to lower toxicity and higher bioavailability of triazole derivatives as well as an increased specificity for fungal cytochrome p450 and a lower impact on human sterol synthesis [16].  Literature review shows that 1,2,4-triazoles and their fused heterocyclic derivatives show a wide spectrum of biological activities. The 1,2,4-triazole core has been incorporated into a wide variety of therapeutically important agents available in clinical therapy, such as itraconazole, posaconazole, voriconazole (antifungal), ribavirin (antiviral), rizatriptan (antimigraine), alprazolam (anxiolytic), trazodone (antidepressant), letrozole and anastrozole (antitumoral) (Figure 2). In the last few decades, scientists have been paying considerable attention to the synthesis of 1,2,4-triazole derivatives showing such comprehensive biological activities as an antifungal [6,7] antitubercular [8], antioxidant [9], anticancer [10], anti-inflammatory [11], analgesic [12], antidiabetic [13], anticonvulsant [14], and anxiolytic [15] activity. With regard to the antifungal activity, triazole-based pharmacophore has replaced the previously widely used imidazole pharmacophore in systemically active azoles, due to lower toxicity and higher bioavailability of triazole derivatives as well as an increased specificity for fungal cytochrome p450 and a lower impact on human sterol synthesis [16]. atoms, triazoles exist in two isomeric forms -1,2,3-triazole and 1,2,4-triazole ( Figure 1). The 1,2,4-triazole ring may exist in equilibrium between two forms: 1H-form and 4H-form ( Figure 1). The calculated energy differences between azole tautomers support preference for the 1H over 4H tautomer [5]. Literature review shows that 1,2,4-triazoles and their fused heterocyclic derivatives show a wide spectrum of biological activities. The 1,2,4-triazole core has been incorporated into a wide variety of therapeutically important agents available in clinical therapy, such as itraconazole, posaconazole, voriconazole (antifungal), ribavirin (antiviral), rizatriptan (antimigraine), alprazolam (anxiolytic), trazodone (antidepressant), letrozole and anastrozole (antitumoral) (Figure 2). In the last few decades, scientists have been paying considerable attention to the synthesis of 1,2,4-triazole derivatives showing such comprehensive biological activities as an antifungal [6,7] antitubercular [8], antioxidant [9], anticancer [10], anti-inflammatory [11], analgesic [12], antidiabetic [13], anticonvulsant [14], and anxiolytic [15] activity. With regard to the antifungal activity, triazole-based pharmacophore has replaced the previously widely used imidazole pharmacophore in systemically active azoles, due to lower toxicity and higher bioavailability of triazole derivatives as well as an increased specificity for fungal cytochrome p450 and a lower impact on human sterol synthesis [16].  Many studies of triazole compounds relates to their antimicrobial properties. This review article highlights recent work (from 2010) carried out on 1,2,4-triazoles with potent antibacterial properties. The work systematizes the compounds in terms of their chemical structure. Antimicrobial properties of triazole hybrids with quinolones, 4-amino-, 3-mercaptotriazoles and fused triazole derivatives are discussed. The method of selecting publications for the review is presented in the diagram below ( Figure 3). Many studies of triazole compounds relates to their antimicrobial properties. This review article highlights recent work (from 2010) carried out on 1,2,4-triazoles with potent antibacterial properties. The work systematizes the compounds in terms of their chemical structure. Antimicrobial properties of triazole hybrids with quinolones, 4-amino-, 3mercaptotriazoles and fused triazole derivatives are discussed. The method of selecting publications for the review is presented in the diagram below ( Figure 3).

Antibacterial Activity of Derivatives of 1,2,4-Triazole
Newly synthesized 1,2,4-triazole compounds were tested for their in vitro growth inhibitory activity against standard Gram-positive and Gram-negative bacterial strains. It is also recommended to test newly obtained substances with potential antibacterial effect on drug-resistant strains (e.g., MRSA, VRE). Additionally, studies on antituberculosis activity were carried out for some compounds. Preliminary screening was performed with the use of the agar disc-diffusion method (cup and plate method), measuring the growth inhibition zones on agar plates. The inhibition zone of compounds was measured using a centimeter scale. In another method, antimicrobial activity was expressed as the lowest concentration that completely inhibits visible growth of a microorganism as detected by

Antibacterial Activity of Derivatives of 1,2,4-Triazole
Newly synthesized 1,2,4-triazole compounds were tested for their in vitro growth inhibitory activity against standard Gram-positive and Gram-negative bacterial strains. It is also recommended to test newly obtained substances with potential antibacterial effect on drug-resistant strains (e.g., MRSA, VRE). Additionally, studies on antituberculosis activity were carried out for some compounds. Preliminary screening was performed with the use of the agar disc-diffusion method (cup and plate method), measuring the growth inhibition zones on agar plates. The inhibition zone of compounds was measured using a centimeter scale. In another method, antimicrobial activity was expressed as the lowest concentration that completely inhibits visible growth of a microorganism as detected by unaided eye (minimum inhibitory concentration-MIC), and values of minimal bactericidal concentration (MBC) were also determined for some substances.
In the group of 1,2,4-triazole-quinolone hybrids, work was also carried out on 6-fluoro analogues (fluoroquinolones), namely norfloxacin, ciprofloxacin, ofloxacin or clinafloxacin. Plech et al. ( , 2016 prepared new hybrids 6 ( Figure 5) using Mannich reaction of 1,2,4-triazole-3-thione derivatives with ciprofloxacin and formaldehyde, and tested them against Gram-positive (MRSA, S. aureus, S. epidermidis, B. subtilis, B. cereus, M. luteus) and Gram-negative bacteria (E. coli, P. mirabilis, P. aeruginosa). The inhibitory effect of selected hybrids on planktonic and biofilm-forming cells of Haemophilus influenzae and Haemophilus parainfluenzae was also investigated. Most ciprofloxacin derivatives were found to possess antibacterial activity higher than the activity of ciprofloxacin, both against Gram-positive and Gram-negative species. Additionally, selected compounds revealed a distinct inhibitory effect against planktonic and biofilm-embedded cells of the Haemophilus spp. Compound 6a exhibited the highest anti-MRSA activity with MIC values about 16and 8-fold lower than in the case of ciprofloxacin and vancomycin, respectively. It was observed that differences in the activity of ciprofloxacin-triazole hybrids resulted from the type of substituent at the C-3 position of the 1,2,4-triazole ring, and the most favorable antibacterial effect was obtained with a hydroxyphenyl fragment [21,22]. Therefore, in a subsequent study,  synthesized compounds 7 ( Figure 5) that differ in terms of the position of the hydroxyl group in the phenyl ring at the C-3 position and the structure of the substituent in the N-4 position of the 1,2,4-triazole core. Microbiological tests revealed that the newly obtained hybrids were, in the vast majority, much more potent than ciprofloxacin itself. Compounds 7a, 7b and 7c showed most potent action against MRSA with MIC of 0.046 µM (MIC for 7c = 0.045 µM) in comparison with vancomycin (MIC = 0.68 µM). Analysis of structure activity relationship (SAR) showed that changes in the position of the hydroxyl group did not significantly affect antimicrobial activity. The position N-4, which has secondary effect on antimicrobial activity, showed a major effect on the toxicity profile of the tested compounds. The presence of methylene linker between triazole and aryl substituent increased toxicity against human cells. Results of enzymatic studies carried out for the selected compounds showed that antibacterial activity of ciprofloxacin-triazole hybrids does not depend solely on the degree of their affinity to bacterial type II topoisomerases (DNA gyrase and topoisomerase IV) [23,24].
1,2,4-Triazole hybrids of 1-[(1R,2S)-2-fluorocyclopropyl]ciprofloxacin 8 ( Figure 5) were synthesized and evaluated for their in vitro antibacterial activity against a broad panel of clinically important drug-sensitive and drug-resistant pathogens (methicillin-sensitive and methicillin-resistant S. epidermidis, methicillin-sensitive and methicillin-resistant S. aureus, E. faecalis, and E. faecium as Gram-positive bacteria, and E. coli and E. coli producting extended spectrum beta-lactamases (ESBLs), K. pneumoniae and K. pneumoniae ESBLs(+), P. aeruginosa, A. coacetious, E. cloacae, E. aerogenes, S. marcescens, M. morganii, P. rettgeri, P. vulgaris, P. mirabilis, S. maltophilia, C. freundii as Gram-negative bacteria) by Gao et al. (2018). Bioassay results revealed that all hybrids had great potency against the tested strains, especially Gram-negative ones, and antibacterial activity was more potent than in the case of the parent 1-[(1R,2S)-2-fluorocyclopropyl]ciprofloxacin, and was comparable to ciprofloxacin and levofloxacin against the majority of tested pathogens. The SAR analysis showed that compounds containing benzyl group in the 4-position of the 1,2,4-triazole inhibited the growth of Gram-positive bacteria more strongly than 4-phenyl derivatives [25].  Gao et al. (2018). Bioassay results revealed that all hybrids had great potency against the tested strains, especially Gram-negative ones, and antibacterial activity was more potent than in the case of the parent 1-[(1R,2S)-2-fluorocyclopropyl]ciprofloxacin, and was comparable to ciprofloxacin and levofloxacin against the majority of tested pathogens. The SAR analysis showed that compounds containing benzyl group in the 4position of the 1,2,4-triazole inhibited the growth of Gram-positive bacteria more strongly than 4-phenyl derivatives [25].
Researchers from Turkey (2017) synthesized phenylpiperazine derivatives of 5-oxoand 5-thioxo-1,2,4-triazole-fluoroquinolone hybrids 9 ( Figure 5). Excellent activity against all tested pathogens (E. coli, Y. pseudotuberculosis, P. aeruginosa, E. faecalis, S. aureus, B. cereus, M. smegmatis) with MIC values ranging from 0.12 to 1.95 µg/mL were observed for all compounds [26]. In another study, Mermer et   showed the highest antimicrobial activity against tested microorganisms. Furthermore, these hybrids displayed good DNA gyrase inhibition with IC 50 values ranging from 0.134 to 1.84 µg/mL. To explain the mode of interaction between compounds and receptors, a molecular docking study was performed. With an average least binding energy of −9.5 kcal/mol, all compounds were found to have remarkable inhibitory potentials against DNA gyrase (E. coli) [27].
Antibacterial activity of new ciprofloxacin derivatives 11 ( Figure 5) was also reported by Mohammed et al. (2019). Coupling of 5-aryl-4H-1,2,4-triazole-3-thione derivatives with acylated ciprofloxacin gave S-bridged hybrids. Biological screening results indicated that the N-allyl derivative with unsubstituted phenyl moiety at the C-5 position of triazole displayed the highest antimycobacterial activity, both against non-pathogenic Mycobacterium smegmatis, compared to the reference isoniazid (MICs: 3.25 µg/mL vs 5 µg/mL), and pathogenic drug-resistant and drug-susceptible strains of Mycobacterium tuberculosis, com-Pharmaceuticals 2021, 14, 224 7 of 28 pared to levofloxacin and moxifloxacin (MICs: 4-32 µg/mL vs 0.03-8 µg/mL). Moreover, N-allyl compound displayed a broad spectrum of antibacterial activity against all bacterial strains tested (S. aureus, K. pneumoniae, P. aeruginosa, E. coli). From the docking studies, it was found that most potent compound showed additional binding interactions with the active site of Mycobacterium gyrase enzyme (extra hydrogen bond interaction between the triazole nitrogen N1 and the amino acid residues Arg D 182), which may explain its enhanced activity against M. smegmatis [28].
In 2015, novel tricyclic fluoroquinolones 12 ( Figure 5) with a functional Mannich base moiety at the C-8 position of the fused system were synthesized and evaluated for their antimicrobial activity against Gram-positive S. aureus and MRSA, Gram-negative E. coli and multidrug-resistant E. coli (MDR E. coli) bacterial strains using ciprofloxacin as a standard. Compounds derived from aliphatic amines were less potent than those derived from heterocyclic amine donors. In particular, 2-methylpiperazine compound 12h was highly active against MDR E. coli bacterial strain with MIC value of 0.25 µg/mL, about 30-fold more potent than ciprofloxacin [29].
Antibacterial activity of new ciprofloxacin derivatives 11 ( Figure 5) was also reported by Mohammed et al. (2019). Coupling of 5-aryl-4H-1,2,4-triazole-3-thione derivatives with acylated ciprofloxacin gave S-bridged hybrids. Biological screening results indicated that the N-allyl derivative with unsubstituted phenyl moiety at the C-5 position of triazole displayed the highest antimycobacterial activity, both against non-pathogenic Mycobacterium smegmatis, compared to the reference isoniazid (MICs: 3.25 µg/mL vs 5 µg/mL), and pathogenic drug-resistant and drug-susceptible strains of Mycobacterium tuberculosis, compared to levofloxacin and moxifloxacin (MICs: 4-32 µg/mL vs 0.03-8 µg/mL). Moreover, N-allyl compound displayed a broad spectrum of antibacterial activity against all bacterial strains tested (S. aureus, K. pneumoniae, P. aeruginosa, E. coli). From the docking studies, it was found that most potent compound showed additional binding interactions with the active site of Mycobacterium gyrase enzyme (extra hydrogen bond interaction between the triazole nitrogen N1 and the amino acid residues Arg D 182), which may explain its enhanced activity against M. smegmatis [28].
In 2015, novel tricyclic fluoroquinolones 12 ( Figure 5) with a functional Mannich base moiety at the C-8 position of the fused system were synthesized and evaluated for their antimicrobial activity against Gram-positive S. aureus and MRSA, Gram-negative E. coli and multidrug-resistant E. coli (MDR E. coli) bacterial strains using ciprofloxacin as a standard. Compounds derived from aliphatic amines were less potent than those derived from heterocyclic amine donors. In particular, 2-methylpiperazine compound 12h was highly active against MDR E. coli bacterial strain with MIC value of 0.25 µg/mL, about 30fold more potent than ciprofloxacin [29].

Antibacterial Activity of 4-Amino-1,2,4-Triazole Derivatives
Indian researchers (2010) reported a synthesis of 4-amino-5-aryl-4H-1,2,4-triazole derivatives 15 ( Figure 8) and screened for in vitro antibacterial properties against E. coli, B. subtilis, P. aeruginosa and P. fluoroscens (recultured). Compound with 4-trichloromethyl group attached to the phenyl ring at the 3-position of triazole was observed to exhibit the highest antibacterial activity (MIC = 5 µg/mL and the zone of inhibition 14-22 mm), equivalent to ceftriaxone, while compounds containing 4-chloro and 4-bromo substituents showed good activity. Acetylation of the NH 2 group at the 4-position and the presence of the free SH group at the 3-position of triazole caused a decrease in antibacterial activity against most bacterial strains [32].

Antibacterial Activity of 4-Amino-1,2,4-Triazole Derivatives
Indian researchers (2010) reported a synthesis of 4-amino-5-aryl-4H-1,2,4-triazole derivatives 15 ( Figure 8) and screened for in vitro antibacterial properties against E. coli, B. subtilis, P. aeruginosa and P. fluoroscens (recultured). Compound with 4-trichloromethyl group attached to the phenyl ring at the 3-position of triazole was observed to exhibit the highest antibacterial activity (MIC = 5 µg/mL and the zone of inhibition 14-22 mm), equivalent to ceftriaxone, while compounds containing 4-chloro and 4-bromo substituents showed good activity. Acetylation of the NH2 group at the 4-position and the presence of the free SH group at the 3-position of triazole caused a decrease in antibacterial activity against most bacterial strains [32].  Figure 8) as antibacterial and anti-inflammatory agents. In vitro assay indicated that compound with 4-hydroxyphenyl moiety inhibited the growth of all bacteria (B. subtilis, S. aureus, P. mirabilis and S. typhi) to the extent comparable to levofloxacin (zones of inhibition of the compound-26-27 mm, compared to 28 mm for levofloxacin). Proteus mirabilis was most sensitive to all tested compounds. Additionally, the selected compounds showed moderate anti-inflammatory activity in a carrageenan-induced rat paw oedema model [33].
A series of methylthio-linked pyrimidinyl-1,2,4-triazoles 20 ( Figure 9) were prepared and screened for their antimicrobial activity by Sekhar et al. (2018). The results of the bioassay indicated that the tested compounds were more active against Gram-negative bacteria than Gram-positive ones. Compound 20f containing 4-nitro substituent showed pronounced activity against P. aeruginosa in comparison with chloramphenicol [37].
A series of methylthio-linked pyrimidinyl-1,2,4-triazoles 20 ( Figure 9) were prepared and screened for their antimicrobial activity by Sekhar et al. (2018). The results of the bioassay indicated that the tested compounds were more active against Gram-negative bacteria than Gram-positive ones. Compound 20f containing 4-nitro substituent showed pronounced activity against P. aeruginosa in comparison with chloramphenicol [37].
Upmanyu et al. (2012) synthesized 4-(substituted acetylamino)-3-mercapto-5-(4substituted phenyl)-1,2,4-triazole derivatives 21 ( Figure 10) and tested them for their in vitro antibacterial activity against four bacterial strains (S. aureus, B. subtilis, P. aeruginosa and E. coli). The SAR analysis of the compounds indicated that 4-methoxy phenyl group is preferable at the 5-position of the triazole ring compared to 4-methyl group. Moreover, antimicrobial activity of the compounds was enhanced with an increase in the number of the carbon atom group (at the C-2 of acetamido group) at position N-4 of the triazole ring, and decreased with branch chain substitution [38].  Figure 10) as antibacterial and antituberculosis agents. Preliminary screening showed that among 4methylphenyl derivatives 22a, compounds containing 6-flouro and 6-methyl substituents at bezothiazole score exhibited activity against Gram-positive bacteria (S. aureus and S. pyogenes), which was equal or even higher than in the case of ampicillin, used as a antituberculosis agents. Preliminary screening showed that among 4-methylphenyl derivatives 22a, compounds containing 6-flouro and 6-methyl substituents at bezothiazole score exhibited activity against Gram-positive bacteria (S. aureus and S. pyogenes), which was equal or even higher than in the case of ampicillin, used as a standard, while compound with 6-nitro substituent showed pronounced activity against Gram-negative bacteria (E. coli and P. aeruginosa), which was 4-to 8-fold higher than in the case of the standard drug. Moreover, 4-methylphenyl 22a and 4-chlorophenyl triazoles 22c with 4-chloro substituent on the benzothiazole ring showed potent antitubercular activity [39,40].
In 2013, results of research on Schiff bases of N-[(4-amino-5-sulfanyl-4H-1,2,4-triazol-3yl)methyl]-4-substituted benzamides 30 ( Figure 11) as antibacterial agents were published by Mange et al. All newly synthesized compounds were evaluated for their antimicrobial activity against Gram-positive (S. aureus and B. subtilis) and Gram-negative bacteria (E. coli and P. aeruginosa). The authors observed that all compounds exhibited the same effect against Staphylococcus aureus as the standard drug, ceftriaxone, and moderate activity against other bacteria [45].
A group of scientists from China (2013) studied Schiff bases of symmetric disulfides connected to the 4-amino-3-(1-benzyl-1H-indol-3-yl]-5-thiomethyl-1,2,4-triazole 31 ( Figure 12) for their antibacterial activity against E. coli, S. aureus and P. aeruginosa. They observed that a compound with 3-bromophenyl substituent showed strong activity against all three bacteria, equal to the reference, sparfloxacin. Compounds bearing an unsubstituted phenyl ring or 2-furyl showed poor activity against all bacterial strains [46]. In another study, the same researchers (2014) synthesized 3-[1-(4-fluorobenzyl)-1H-indol-3-yl]-5-(4-fluorobenzylthio)-4H-1,2,4-triazol-4-amine and its Schiff bases 32 ( Figure 12). Results of antibacterial screening confirmed that the aromatic substituent at the 4-position of triazole played an important role in antibacterial activity and that the presence of halogen and nitro groups significantly enhanced inhibitory activity against all tested bacteria [47].     Figure  13) derived from Schiff and Mannich bases, and evaluated them for antibacterial activity against a panel of bacteria, namely E. coli, Y. pseudotuberculosis, P. aeruginosa, E. faecalis, S. aureus, B. cereus and Mycobacterium smegmatis. Among the new compounds, the Schiff base 33d carrying nitro substituent on the thiophene ring at the 4-position of 1,2,4-triazole showed the highest inhibitory activity against all tested species, 2-or even 35-fold higher than ampicillin. Compounds with morpholine 34 were generally less active [48].   Figure 13) showed the highest activity against Gram-positive bacteria, namely S. aureus and S. pyogenes, with MIC values of 0.264 and 0.132 mM, respectively, which were equal to or higher than the activity of standard drugs, ampicillin and chloramphenicol. Moreover, a dihydrofolate reductase (DHFR) inhibition assay was conducted and the results thereof indicated that all compounds were potent DHFR inhibitors [50].
Antimicrobial activity of Schiff bases of thiazolyl-triazole hybrides 37 ( Figure 13) was tested by Nastasa et al. (2018). The determination of inhibition zone diameters revealed that compounds 37a-b, 37i, and 37j were most potent against Gram-positive L. monocytogenes, with an equal or greater effect than ciprofloxacin. MIC and MBC values were in line with the obtained results. With regard to the activity against Gram-negative strains, most of the compounds inhibited growth of P. aeruginosa, with MIC and MBC values 2-fold more potent than the reference. A molecular docking study, performed on DNA-gyrase A and gyrase B from L. monocytogenes, revealed that all Schiff bases were stronger binders to gyrA than ciprofloxacin (used as the control inhibitor), and formed at least three hydrogen bonds between the azomethine nitrogen and serine (Ser98) and between the triazole nitrogens (N2 and N4) and the valine residues (Val113, Val268), while ciprofloxacin formed two hydrogen bonds between the carboxyl group from position 3 with Gly171 and Ser172 at the active site of enzyme. All compounds were considerably weaker binders to gyrB [51].
In vitro antibacterial activity of 1,2,4-triazole-3-thiones 41 ( Figure 14) with substituted piperazine against S. aureus, B. subtilis, P. aeruginosa and P. mirabilis was studied by a team from India (2011). The results of the study revealed that the presence of phenylpiperazine moiety was crucial for high antibacterial activity against all the microbial strains tested. Additionally, compounds with the phenyl ring at the N-4 position of triazole showed higher activity compared to triazoles substituted with alkyl and alkene groups. However, none of the compounds showed higher activity than norfloxacin, used as a reference drug [55].
5-(2-Aminothiazol-4-yl)-4-substituted-phenyl-4H-1,2,4-triazole-3-thioles 49a and their acetylamine 49b and thioureide derivatives 49c ( Figure 16) were designed, synthesized, and evaluated for their antimicrobial activity against a panel of Gram-positive (S. aureus and B. subtilis) and Gram-negative bacteria (E. coli and P. aeruginosa) by Hassan et al. (2013). Compound with a free 2-amino group and phenoxy moiety at the 4-position of the phenyl ring exhibited potent growth inhibition of all tested bacterial strains, comparable to gentamicin and ciprofloxacin. Diacetylation of the 2-amino-thiazole function (49b) produced moderately active compounds, similar to phenyl-thioureido analogues 49c [62]. synthesized, and evaluated for their antimicrobial activity against a panel of Grampositive (S. aureus and B. subtilis) and Gram-negative bacteria (E. coli and P. aeruginosa) by Hassan et al. (2013). Compound with a free 2-amino group and phenoxy moiety at the 4position of the phenyl ring exhibited potent growth inhibition of all tested bacterial strains, comparable to gentamicin and ciprofloxacin. Diacetylation of the 2-amino-thiazole function (49b) produced moderately active compounds, similar to phenyl-thioureido analogues 49c [62].  mm against all of the tested microorganisms, and were found to be more active than ampicillin and gentamicin [63]. (Figure 16) synthesized by Barot et al. (2017) was observed to exhibit a significant inhibitory effect against Bacillus cereus with MIC of 5 µg/mL, comparable to reference drugs, ofloxacin and metronidazole (MICs: 2-3 µg/mL). Its benzo[d]imidazolyl methyl analogue 51a inhibited bacterial growth to a lesser extent [64].
In vitro evaluation of antibacterial activity was carried out for N-benzothiazole derivatives of 2-[4-(naphthalen-1-yl)-5-(quinolin-6-yl)-4H-1,2,4-triazol-3-ylthio]acetamide 58 ( Figure 18) by researchers from South Korea (2014). The SAR test revealed that the presence of an electron-releasing ethoxy substituent at the C-6 position on the benzothiazole ring was crucial for high activity against P. aeruginosa, providing an MIC value of 25 µg/mL, which was equivalent to that of ampicillin. Analogues with electronwithdrawing substituents, namely fluorine and bromine, exhibited potent action against Gram-positive bacteria, Staphylococcus aureus and Bacillus cereus, respectively, with halffold potency compared to ampicillin [70].  Figure 18) and screened their antibacterial activity against a panel of bacterial strains. Among the tested S-substituted derivatives, optimal activity was shown by the 3,5-trifluormethyl benzyl analogue 59e, being 4-to 8-fold higher against S. aureus, B. subtilis, and P. aeruginosa than in the case of ampicillin and gentamicin, and the activity against M. luteus and E. coli was comparable to the reference drugs [63]. Cui et al. (2016) designed a series of 1,2,4-triazole-pyrimidine derivatives linked by sulfur 60 (Figure 18), and then carried out extensive in vitro and in silico studies of their antimicrobial activity. Preliminary screening against two representative strains (S. aureus and E. coli) revealed two most potent compounds with 2-methyl-or 2-phenylthio moieties at the 2-position of the pyrimidine ring 60a-b. Further studies of antibacterial activity against various strains of bacteria, including methicillin-resistant S. aureus, showed that these compounds significantly inhibited the growth of all bacteria (E. coli, B. subtilis, B. anthracis, and S. aureus isolated) with MIC values ranging from 0.8 to 5.2 µM, and were 10-and even ≥1600-fold more effective against MRSA than most clinically used antibiotics, namely ampicillin, polymyxin B, erythromycin, tetracycline, kanamycin, rifampicin, norfloxacin and even vancomycin. Moreover, compounds 60a-b were highly effective against various MRSA strains with efflux pumps, indicating that they may be helpful in combating multi-drug resistance. Bioassays indicated that the tested 1,2,4-triazolepyrimidine derivatives were potent inhibitors of SecA-dependent protein-conducting  Figure 18) and screened their antibacterial activity against a panel of bacterial strains. Among the tested S-substituted derivatives, optimal activity was shown by the 3,5-trifluormethyl benzyl analogue 59e, being 4-to 8-fold higher against S. aureus, B. subtilis, and P. aeruginosa than in the case of ampicillin and gentamicin, and the activity against M. luteus and E. coli was comparable to the reference drugs [63].
Cui et al. (2016) designed a series of 1,2,4-triazole-pyrimidine derivatives linked by sulfur 60 (Figure 18), and then carried out extensive in vitro and in silico studies of their antimicrobial activity. Preliminary screening against two representative strains (S. aureus and E. coli) revealed two most potent compounds with 2-methyl-or 2-phenylthio moieties at the 2-position of the pyrimidine ring 60a-b. Further studies of antibacterial activity against various strains of bacteria, including methicillin-resistant S. aureus, showed that these compounds significantly inhibited the growth of all bacteria (E. coli, B. subtilis, B. anthracis, and S. aureus isolated) with MIC values ranging from 0.8 to 5.2 µM, and were 10-and even ≥1600-fold more effective against MRSA than most clinically used antibiotics, namely ampicillin, polymyxin B, erythromycin, tetracycline, kanamycin, rifampicin, norfloxacin and even vancomycin. Moreover, compounds 60a-b were highly effective against various MRSA strains with efflux pumps, indicating that they may be helpful in combating multidrug resistance. Bioassays indicated that the tested 1,2,4-triazole-pyrimidine derivatives were potent inhibitors of SecA-dependent protein-conducting channel activity and protein translocation. The SecA protein is a widely conserved membrane protein, responsible for the secretion of virulence factors and directly accessible from the extracellular matrix. Therefore, SecA inhibitors have the potential for being developed as broad-spectrum antimicrobials and can overcome the effect of efflux pumps which are responsible for multidrug resistance [71].    [1,3,4]thiadiazole exhibited the highest activity against all bacterial strains (MICs: 1-2 µg/mL), compared with ceftriaxone (MIC = 1 µg/mL), used as a reference drug [75]. It is interesting, that 3-((quinolin-8-yloxy)methyl)-1,2,4triazolo [3,4-b] [1,3,4]thiadiazol-6(5H)-thione 67b inhibited bacterial growth to a greater extent than its 6-oxo analogue 67a, which showed no antibacterial effect [77]. The results revealed that fused compounds with substituted the phenyl ring attached to the thiadiazine core exhibited high antibacterial activity compared to an unsubstituted derivative. The zone of inhibition of compound 68b at a concentration of 100 µg/mL was greater than that of neomycin, and almost equal to streptomycin, used as standards [78].   [1,3,4]thiadiazol-6(5H)thione 67b inhibited bacterial growth to a greater extent than its 6-oxo analogue 67a, which showed no antibacterial effect [77]. The results revealed that fused compounds with substituted the phenyl ring attached to the thiadiazine core exhibited high antibacterial activity compared to an unsubstituted derivative. The zone of inhibition of compound 68b at a concentration of 100 µg/mL was greater than that of neomycin, and almost equal to streptomycin, used as standards [78].
In 2017, a series of 1,2,4-triazolo [3,4-b] [1,3,4]thiadiazole derivatives 69 ( Figure 21) containing thiouracil moiety were synthesized by structural modifications on a known SecA ATPase inhibitor by Cui et al. All the compounds were evaluated for their antibacterial activity against Bacillus amyloliquefaciens, Staphylococcus aureus, and Bacillus subtilis (expressed as inhibitory rate/% at 25 µg/mL), and the results showed that two compounds containing 2,4-dichlorophenyl group attached to thiouracil moiety exhibited the strongest antibacterial activity against B. subtilis, with inhibitory rate above 90%, higher than that of norfloxacin and the known SecA inhibitor. The SAR analysis suggested that the introduction of additional chlorine atoms on marginal phenyls was beneficial for antibacterial activity [79].
containing 5-methyl-1-phenyl-1H-4-pyrazolyl moiety at the 3-position of triazole, and evaluated their antibacterial activity against four human pathogenic bacteria (E. coli, K. pneumoniae, S. dysentriae, and S. flexnei). The results revealed that fused compounds with substituted the phenyl ring attached to the thiadiazine core exhibited high antibacterial activity compared to an unsubstituted derivative. The zone of inhibition of compound 68b at a concentration of 100 µg/mL was greater than that of neomycin, and almost equal to streptomycin, used as standards [78].  In 2019, an Indian research team synthesized a series of phenylquinoline-1,2,4-triazolo [3,4b] [1,3,4]thiadiazines 70 ( Figure 21) and evaluated their in vitro antimicrobial activity against S. aureus, E. coli, P. aeruginosa. The MIC and zone of inhibition data revealed that compounds with a substitution of the halogen atom at the 6-position of the phenylquinoline ring showed the highest antibacterial activity (MICs: 1-8 µg/mL), comparable to standard ampicillin (MICs: 1-4 µg/mL). Replacement of halogens with electron-donating groups (methyl, nitro, methoxy and tri-methoxy groups) clearly reduced antibacterial potentials [80].
A series of imidazo[2,1-c] [1,2,4]triazoles were synthesized in multicomponent reaction, and evaluated for in vitro antimicrobial potential against B. cereus, S. aureus, E. coli, P. aeruginosa and Salmonella enteritidis (food isolate) by Aouali et al. (2015). Among them, para-chloro substituted compound 71 (Figure 22) emerged as a promising antibacterial agent against B. cereus and S. aureus, respectively, with the diameter of the inhibition zone ranging from 29 to 20 mm, and MIC values of 0.078 µg/mL and 0.312 µg/mL. Gram-negative bacteria were resistant to the tested compounds [81]. In 2017, a series of 1,2,4-triazolo [3,4-b] [1,3,4]thiadiazole derivatives 69 ( Figure 21) containing thiouracil moiety were synthesized by structural modifications on a known SecA ATPase inhibitor by Cui et al. All the compounds were evaluated for their antibacterial activity against Bacillus amyloliquefaciens, Staphylococcus aureus, and Bacillus subtilis (expressed as inhibitory rate/% at 25 µg/mL), and the results showed that two compounds containing 2,4-dichlorophenyl group attached to thiouracil moiety exhibited the strongest antibacterial activity against B. subtilis, with inhibitory rate above 90%, higher than that of norfloxacin and the known SecA inhibitor. The SAR analysis suggested that the introduction of additional chlorine atoms on marginal phenyls was beneficial for antibacterial activity [79].
A series of imidazo[2,1-c] [1,2,4]triazoles were synthesized in multicomponent reaction, and evaluated for in vitro antimicrobial potential against B. cereus, S. aureus, E. coli, P. aeruginosa and Salmonella enteritidis (food isolate) by Aouali et al. (2015). Among them, para-chloro substituted compound 71 (Figure 22) emerged as a promising antibacterial agent against B. cereus and S. aureus, respectively, with the diameter of the inhibition zone ranging from 29 to 20 mm, and MIC values of 0.078 µg/mL and 0.312 µg/mL. Gram-negative bacteria were resistant to the tested compounds [81].   Figure 22) containing diphenylamine moiety. Among them, two compounds with phenyl or 2-thiophenyl rings at the C-4 position of fused triazole exhibited significant antibacterial and antituberculosis activity at a concentration of 12.5 µM (MIC of ampicillin 6.25 µM; MIC of isoniazid >0.2 µM). Triazolo-quinazolinones with a substituted phenyl ring at the 4-position of molecule showed reduced activity when compared to unsubstituted derivative [83].

Miscellaneous 1,2,4-Triazoles with Antibacterial Activity
Antibacterial activity of bis-1,2,4-triazolium derivatives 78 (Figure 24) was reported by Thomas et al. (2019). The MIC values of the compounds were evaluated against four reference strains (S. aureus, E. faecalis, E. coli and P. aeruginosa), but also against four clinical isolates harboring various resistance mechanisms (MRSA, VRE, extended-spectrum blactamase-producing Escherichia, and Pseudomonas aeruginosa resistant, efflux pump). All the prepared bis-1,2,4-triazoliums showed strong activity against the majority of the tested strains, and the most active compound 78a bearing decyl moiety was 2-, 4-and 8fold more potent against the sensitive and the resistant strains of S. aureus, E. faecalis and P. aeruginosa, respectively, than the reference, chlorhexidine. Unfortunately, it also showed high toxicity [86]. The results revealed that the tested compounds showed higher activity against Gram-positive bacteria (Bacillus subtilis, Bacillus thuringiensis) than Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa). Moreover, compounds with electronwithdrawing substituents, such as chloro, bromo, nitro groups exhibited high activity against M. tuberculosis H37Rv with MIC values of 3.125 µg/mL, equal to streptomycin [85].

Miscellaneous 1,2,4-Triazoles with Antibacterial Activity
Antibacterial activity of bis-1,2,4-triazolium derivatives 78 (Figure 24) was reported by Thomas et al. (2019). The MIC values of the compounds were evaluated against four reference strains (S. aureus, E. faecalis, E. coli and P. aeruginosa), but also against four clinical isolates harboring various resistance mechanisms (MRSA, VRE, extended-spectrum b-lactamase-producing Escherichia, and Pseudomonas aeruginosa resistant, efflux pump). All the prepared bis-1,2,4-triazoliums showed strong activity against the majority of the tested strains, and the most active compound 78a bearing decyl moiety was 2-, 4-and 8-fold more potent against the sensitive and the resistant strains of S. aureus, E. faecalis and P. aeruginosa, respectively, than the reference, chlorhexidine. Unfortunately, it also showed high toxicity [86].
Researchers from Jordan (2020) prepared a series of 1,2,4-triazol-3-carbohydrazide derivatives 80 ( Figure 24) and tested them against S. aureus and B. cereus as Gram-positive, and P. aeruginosa and Shigella sp. as Gram-negative bacteria. The results revealed that Bacillus cereus was the most sensitive bacterium, and compounds 80a-c inhibited its growth equally to penicillin (inhibition zone of 10 mm). The calculated MIC values were in line with the obtained results [88].

Structure-Activity Observations
From the biological results, it becomes clear that different substituents on triazole scaffold have a noticeable effect on antibacterial activity. Making a general assessment of the relationship between the molecular structure and biological activity of the described compounds, it might be concluded that: I. In the group of 1,2,4-triazole hybrides of quinolone, isosteric replacement of the COOH group with a 5-membered heterocyclic nucleus (among nalidixic acid and ofloxacine derivatives) or the incorporation of a differently substituted triazole moiety in the side chain at the C-7 position of fluoroquinolones (among nor-, cipro-and clinafloxacin derivatives) provides potent antibacterial properties. In particular, the presence of a hydroxyl group on the phenyl ring at the C-3 position of the triazole has improved antibacterial activity against the screened Gram-positive and Gram-negative bacterial strains. The formation of fused tricyclic fluoroquinolone-7-carboxylic acid derivatives by incorporating a triazole with a functional Mannich-based chain into the 7-and 8-positions of the fluoroquinolone scaffold greatly increased the antibacterial activity against drugresistant bacteria.
II. Among the 4-amino-1,2,4-triazole derivatives, presence of a free -NH2 group and aryl substituents at the C-5 position of triazole provides a broad spectrum of antibacterial activity. The acetylation of an amino group or its replacement with aromatic amines, in particular 1,3-benzothiazol-2-amine, retains potent activity. The presence of an electron withdrawing group on the phenyl ring through -N=CH-linkage in the N-4 position of triazole (Schiff base derivatives), in many cases, is crucial for the high antibacterial activity.
III. The presence of aryl/heteroaryl substituent at C-5 position of 1,2,4-triazolo-3thiones/thioles is crucial for potent antibacterial activity. In the group of 4,5-diphenyl-1,2,4-triazol-3-thione derivatives, the presence of an electron-withdrawing substituents at the phenyl rings enhanced the activity. The substitution of the N-2 position of triazole by various aminomethyl moieties (Mannich base derivatives) retains potent activity, and Researchers from Jordan (2020) prepared a series of 1,2,4-triazol-3-carbohydrazide derivatives 80 ( Figure 24) and tested them against S. aureus and B. cereus as Gram-positive, and P. aeruginosa and Shigella sp. as Gram-negative bacteria. The results revealed that Bacillus cereus was the most sensitive bacterium, and compounds 80a-c inhibited its growth equally to penicillin (inhibition zone of 10 mm). The calculated MIC values were in line with the obtained results [88].

Structure-Activity Observations
From the biological results, it becomes clear that different substituents on triazole scaffold have a noticeable effect on antibacterial activity. Making a general assessment of the relationship between the molecular structure and biological activity of the described compounds, it might be concluded that: I. In the group of 1,2,4-triazole hybrides of quinolone, isosteric replacement of the COOH group with a 5-membered heterocyclic nucleus (among nalidixic acid and ofloxacine derivatives) or the incorporation of a differently substituted triazole moiety in the side chain at the C-7 position of fluoroquinolones (among nor-, cipro-and clinafloxacin derivatives) provides potent antibacterial properties. In particular, the presence of a hydroxyl group on the phenyl ring at the C-3 position of the triazole has improved antibacterial activity against the screened Gram-positive and Gram-negative bacterial strains. The formation of fused tricyclic fluoroquinolone-7-carboxylic acid derivatives by incorporating a triazole with a functional Mannich-based chain into the 7-and 8-positions of the fluoroquinolone scaffold greatly increased the antibacterial activity against drug-resistant bacteria.
II. Among the 4-amino-1,2,4-triazole derivatives, presence of a free -NH2 group and aryl substituents at the C-5 position of triazole provides a broad spectrum of antibacterial activity. The acetylation of an amino group or its replacement with aromatic amines, in particular 1,3-benzothiazol-2-amine, retains potent activity. The presence of an electron withdrawing group on the phenyl ring through -N=CH-linkage in the N-4 position of triazole (Schiff base derivatives), in many cases, is crucial for the high antibacterial activity.
III. The presence of aryl/heteroaryl substituent at C-5 position of 1,2,4-triazolo-3thiones/thioles is crucial for potent antibacterial activity. In the group of 4,5-diphenyl-1,2,4-triazol-3-thione derivatives, the presence of an electron-withdrawing substituents at the phenyl rings enhanced the activity. The substitution of the N-2 position of triazole by various aminomethyl moieties (Mannich base derivatives) retains potent activity, and only after the introduction of large-volume substituents the efficiency of these derivatives decrease.
IV. In the group of triazoles fused with a 5-or 6-membered ring systems, namely 1,2,4triazolo [3,4-b] [1,3,4]thiadiazoles and 1,2,4-triazolo [3,4-b] [1,3,4]thiadiazines, the aryl/heteroaryl substituents in the C-3 and C-6 positions of fused system have an impact on antibacterial activity, and with regard to the effect of the substituent on the phenyl ring among aryl derivatives, the most beneficial for the high antibacterial activity is the presence of halogen atom. The presence of large in volume substituents can decreases activity.

Summary
The conducted review of 1,2,4-triazole and their hybrids with quinolone agents as well as 4-amino-, 3-mercapto-, and fused derivatives of 1,2,4-triazole shows that they have potent antibacterial activity. These compounds inhibit the growth of both Gram-positive and Gram-negative bacteria and the most active compounds are equal or even more potent than the antibacterial drugs commonly used on the market. Moreover, some of 1,2,4triazoles exhibit significant antibacterial activity against drug-resistant bacterial strains (e.g., MRSA, VRE, MDR E. coli) and antimycobacterial activity against Mycobacterium tuberculosis. The most active compounds are listed in Table 1. The study of the mechanism of action of some series of 1,2,4-triazole derivatives reveals that they have inhibitory potential against DNA gyrase, glucosamine-6-phosphate synthase, dihydrofolate reductase (DHFR) and SecA ATPase, which are essential proteins for bacteria. The structure-activity relationship (SAR) analysis provide the knowledge for further research and development of new 1,2,4triazole derivatives with improved potency and maintained safety profile to overcome bacterial resistance.