Benzimidazole-Triazole Hybrids as Antimicrobial and Antiviral Agents

1. Introduction

Heterocyclic compounds have a central place in medicinal chemistry, being used as therapeutic agents to treat most diseases [1–3]. Among these heterocycles, benzimidazole stands out, as a purine-analog pharmacophore, with a wide biological activity, such as antimicrobial [4–8], antiviral [9,10], antihistamine [11,12], anticonvulsant [3,13], antitumor [14–16], proton pump inhibitors [17], antiparasitic [16,18,19], anti-inflammatory [20–22], or antihypertensive [23,24]. Some benzimidazoles are efficient agents in Diabetes mellitus [25–27], while astemizole compounds possess anti-prion activity to treat Creutzfeldt-Jakob disease [5,28]. The literature also reports anti-Alzheimer [29,30], psychoactive, anxiolytic, analgesic  [31,32],  and anticoagulant properties [33,34] of benzimidazole derivatives. Also, for triazole compounds, the literature mentions a series of therapeutic activities, such as antimicrobial [35–38], antitubercular [39,40], potential inhibitors of SARS CoV-2 [41–43], antiviral [43,44] anti-inflammatory [45,46] antitumor [47–50], antihypertensive [50], antioxidant [47,51,52] and antiepileptic [53,54]. Pharmacological applications of triazoles refer to their activity as α-glucosidase inhibitors [55,56], analgesic [50,57], anticonvulsant [53,58], and antimalarial agents [57,59]. Triazole derivatives are efficient in the treatment of Alzheimer's disease [60,61] and are very effective neuroprotective agents [62,63].

The successive events happened from the spring of 2020 up to and including the present, regarding the emergence and development of the COVID-19 pandemic, have led the scientific world to investigate more closely the possibility of treating this infectious disease with various antiviral [64–66], antimicrobial [67], immunomodulatory [68] or anti-inflammatory drugs [69], therefore, the discovery of new molecules with simple or hybrid structures, with biological properties that satisfy the requirements of the treatment of this condition it is absolutely necessary and constitutes the engine of the development of new effective therapeutic agents.

Classical drugs containing benzimidazole and triazole rings recommend these heterocycles as essential in building new target compounds with antimicrobial, antiviral, antiparasitic, etc. properties (Figure 1). In addition, the literature mentions a series of benzimidazole-triazole hybrids with remarkable antimicrobial properties, antiviral activities, including new anti-SARS-COV-2 agents [70–74], with particular importance in the context of the recent pandemic, which led to the study of synthesis methods, antimicrobial properties, structure-property relationships and their biological activities. As expected, the study refers to both 1,2,3-triazole-benzimidazole hybrids and 1,2,4-triazole-benzimidazole hybrids, even if it seems that the literature is richer in the second category, in terms of antimicrobial activity.

In order to highlight the structures of the heterocycles in the discussed compounds, we colored benzimidazole nucleus with red, 1,2,3-triazole with blue and 1,2,4-triazole with green.

Figure 1. Figure 1. Chemical structures of some benzimidazole, 1,2,3-triazole and 1,2,4-triazole-based marketed drugs

Figure 1. Figure 1. Chemical structures of some benzimidazole, 1,2,3-triazole and 1,2,4-triazole-based marketed drugs

The recent literature marks several strategies for the synthesis of 1,2,3-triazoles, like click reaction [75], Bouiton-Katritzky rearrangement [76], oxidative cyclization of hydrazones [77], post-cycloaddition functionalization [78], alkylation or arylation of triazoles [79]. Also, for benzimidazoles, the literature mentions several methods of synthesis, such as reaction of o-phenenediamine with aldehydes or ketones (Phillips-Ladenburg reaction) [3,80,81,82], with acids or their derivatives (Weidenhagen reaction) [81], or green methods of classic syntheses [80, 83–86].

In the following, we will present syntheses of benzimidazole-triazole hybrids with antimicrobial and antiviral properties.

2. Synthesis and antimicrobial activities of benzimidazole-1,2,3-triazoles

2.1. 2-Benzimidazole-R(Ar)-1,4-disubstituted-1,2,3-triazole hybrids

Two series of new hybrids, 2-[4-((1H-benzimidazol-2-ylthio)methyl)-1H-1,2,3-triazol -1-yl]N`-(arylmethylidene)acetohydrazides (2a-2l) and 2-[4-((1H-benzimidazol-2-ylthio) methyl)-1H-1,2,3-triazol-1-yl]N-(α-arylethylidene)acetohydrazides (3a-3f) were prepared by Youssif et al. in two steps starting from 2-[4-((1H-benzimidazol-2-ylthio) methyl)-1H-1,2,3-triazol-1-yl] acetohydrazide 1 (Scheme 1). Compounds 2a-2l exhibited pronounced antibacterial activity which ranged from 35–75% that of standard drug against Staphylococcus aureus and 50–80% that of Ciprofloxacin against E. Coli (MIC values of 3.125–12.5 μmol mL^-1). Compound 2k showed the highest activity against S. aureus (75% activity, MIC = 12.5 μmol mL^-1), while compound 2d was the most active derivative against E. Coli (80% activity, MIC = 3.125 μmol mL^-1). All the synthesized compounds were tested as potential antifungal agents against Candida albicans using Fluconazole as a reference drug. Compound 1 showed the activity of 48% of that of Fluconazole (MIC = 12.5 μmol mL^-1). Compounds 2e and 2k displayed the higher antifungal activity among the other derivatives as they showed 75% activity of that of Fluconazole (MIC = 3.125 μmol mL^-1). Compounds 3a-3f exhibited moderate to good activity against E. Coli and their activity was 50–70% of that of Ciprofloxacin (MIC values of 6.25–12.5 μmol mL^-1), and that compounds 3a and 3f were the most active compounds against E. coli as they showed 70% of that of Fluconazole (MIC = 6.25 μmol mL^-1) while compound 3b showed the highest activity against Staphylococcus aureus (65% of that of Ciprofloxacin, MIC = 18 μmol mL^-1) [87].

Al-blewi et al. used an azide–alkyne Huisgen cycloaddition reaction carried out by simultaneously mixing thiopropargylated benzimidazole 4 with the appropriate sulfa drug azides 5a–5f, copper sulfate and sodium ascorbate in DMSO/ H₂O to regioselectively furnish target mono-1,4-disubstituted-1,2,3-triazole tethered benzimidazole-sulfonamide conjugates 6a–6f with 85–90% yields after 6–8 h of heating at 80 °C (Scheme 2). All compounds were evaluated for their antimicrobial activity (Table 1) against four pathogenic bacterial strains (Gram-positive: Bacillus cereus ATTC 10876, Staphylococcus aureus ATTC 25923 and Gram-negative: Escherichia coli ATTC 25922, Pseudomonas aeruginosa ATTC 27853 and two fungal strains, Candida albicans ATTC 50193, Aspergillus brasiliensis ATTC 16404). As can be seen in Table 1, compound 6a shown the best antibacterial activity against Bacillus cereus and Staphylococcus aureus (64 μgmL^-1) and compounds 6c, 6d and 6e the best antibacterial activity against against Escherichia coli (64 μgmL^-1) [88].

Rashdan et al. synthesized hybrids 10 starting from 2-azido-1H-benzo[d]imidazole derivatives 7a–7b which reacted with acetylacetone in the presence of sodium ethoxide to obtain hybrids molecules 8a–8b. The latter acted as a key molecules for the synthesis of new carbazone derivatives 9a–9b that were submitted to react with 2-oxo-N-phenyl-2 (phenylamino)acetohydrazonoyl chloride to obtain the target hybrid derivatives 10a–10b (Scheme 3). All compounds were screened for their in vitro antimicrobial activity against pathogenic microorganisms Staphylococcus aureus, E. coli, Pseudomonas aeruginosa, Aspergillus niger, and Candida albicans. The results showed that compounds 10a and 10b had strong activity against all the tested pathogenic microbes. Compounds 8a and 9a only showed effects against the Gram-negative and Gram-positive bacteria and had no effect on the tested fungi. In addition, in silico and in vitro findings showed that compounds 10a and 10b were the most active against bacterial strains, and could serve as potential antimicrobial agents (Table 2). The hybrids 8–10 were subjected to molecular docking studies with DNA gyrase B and exhibited binding energy that extended from -9.8 to -6.4 kcal/mol, which confirmed their excellent potency. The compounds 10a and 10b were found to be with the minimum binding energy (-9.8 and -9.7 kcal/mol) as compared to the standard drug Ciprofloxacin (-7.4 kcal/mol) against the target enzyme DNA gyrase B [89].

Hybrids	Inhibition zone diameters using the agar diffusion method (mm)
	S. aureus	E. coli	P. aeruginosa	A. niger	C. albicans
8a	15 ± 0.14	12 ± 1.08	22 ± 1.01	-	-
8b	-	5 ± 0.2	-	30 ± 1.16	27 ± 1.1
9a	23 ± 0.8	-	13 ± 0.65	-	-
9b	-	-	12 ± 0.8	14 ± 0.15	19 ± 1.04
10a	24 ± 0.6	25 ± 0.9	17 ± 0.75	20 ± 0.9	16 ± 0.89
10b	29 ± 1.2	21 ± 1.14	19 ± 0.79	18 ± 0.12	14 ± 0.58
Ciprofloxacin	20 ± 0.9	23 ± 1.02	21 ± 0.9	-	-
Nystatin	-	-	-	22 ± 0.18	23 ± 1.15

Hybrids	Inhibition zone diameters using the agar diffusion method (mm)
	S. aureus	E. coli	P. aeruginosa	A. niger	C. albicans
8a	15 ± 0.14	12 ± 1.08	22 ± 1.01	-	-
8b	-	5 ± 0.2	-	30 ± 1.16	27 ± 1.1
9a	23 ± 0.8	-	13 ± 0.65	-	-
9b	-	-	12 ± 0.8	14 ± 0.15	19 ± 1.04
10a	24 ± 0.6	25 ± 0.9	17 ± 0.75	20 ± 0.9	16 ± 0.89
10b	29 ± 1.2	21 ± 1.14	19 ± 0.79	18 ± 0.12	14 ± 0.58
Ciprofloxacin	20 ± 0.9	23 ± 1.02	21 ± 0.9	-	-
Nystatin	-	-	-	22 ± 0.18	23 ± 1.15

Compounds 11a–11g with terminal acetylene and 2-(azidomethoxy)ethyl acetate were condensed using CuI as catalyst and triethylamine (TEA) under microwave irradiation, to achieve hybrids 1,2,3-triazole connected via benzene to the benzimidazole nucleus 12a–12g with excellent yields (70–90%)(Scheme 2). The cleavage of the acetyl group using potassium carbonate (K₂CO₃) in methanol liberated the hydroxy group of the corresponding hybrid triazoles 13a–13g in almost quantitative yields. Compounds 6a–6g were screened for in vitro antifungal activities against two phytopathogenic fungi Verticillium dahliae Kleb and Fusarium oxysporum f. sp. albedinis. The result of the mycelia linear growth rate indicates that some of the compounds show a weak inhibition against the two fungi, the only compound that shows a significantly increased rate is compound 6e with rate of 29.76% against Verticillium dahliae [90].

Bistrović et al. synthesized in two steps hybrids 19a–19e, 20a–20e and 21a–21e starting from 4-(prop-2-ynyloxy)benzaldehyde 14 (Scheme 5). All compounds were evaluated for their in vitro antibacterial activity against Gram-positive bacteria: S. aureus ATCC 25923, methicillin-sensitive S. aureus, E. faecalis, vancomycin-resistant E. faecium, and Gram-negative bacteria: E. coli ATCC 25925, P. aeruginosa ATCC 27853, A. baumannii ATCC 19606 and ESBL-producing K. pneumoniae ATCC 27736. Generally, compounds showed better activities against Gram-positive than Gram-negative bacteria. Compounds 20a–20e with better binding affinity relative to other amidines, were the most active against S. aureus (MIC = 8–32 µgmL^-1). Compound 19a was the most promising candidate because of its higher potency (MIC = 4 µgmL^-1) against ESBL-producing E. coli [91].

Rao et al. synthesized hybrids 22a–22b (Fig. 1), using click chemistry approach. Compounds had weak activity against Mycobacterium bovis strain (BCG values % inhibition = 27.3 and 26.2 respectively) [92]. Ashok et al. synthesized in three steps hybrids 26a–26j, starting from 1H-indole-3-carbaldehyde 7 (Scheme 6). The compounds were evaluated for their antimicrobial activity against gram-positive Staphylococcus aureus ATCC 6538, Bacillus subtilis ATCC 6633 and gram-negative Proteus vulgaris ATCC 29213, Escherichia coli ATCC 11229 bacteria using Gentamicin as standard. Antifungal activity was tested against Candida albicans ATCC 10231 and Aspergillus niger ATCC 9029 strains with standard drug Fluconazole. Compounds 26b, 26c and 26h with with MIC of

3.125–6.25 μgmL^-1 were found to be the most promising potential antimicrobial molecules [93]. Mallikanti et al. synthesized novel benzimidazole-conjugated 1,2,3-triazole analogues 29a–29l in two steps: 1. formation of benzimidazole intermediate by reaction between 3',5'-difluorobiphenyl-3,4-diamine 27 and 2-hydroxy-4-(prop-2-ynyloxy) benzaldehyde 28, and 2. microwave-assisted copper-catalyzed click reaction (Scheme 7). Compounds 29a-29l have shown minimal inhibition zones against all gram positive (S. aureus, B. subtilis) and gram-negative (E. coli, P. aeruginosa) strains using Ampicillin as standard drug. Among all tested compounds, the 29i and 29k have showed greater activity against P. aeruginosa, S. aureus and B. subtilis than standard reference. Compounds 29a, 29b, 29c, 29d, 29e, 29f, 29g, 29h, 29j and 29l demonstrated moderate antibacterial activity against the same. Also, compounds 29i, 29j and 29k established potent activity against both fungal strains, C. albicans MTCC 183 and A. niger MTCC 9652 stains compared to standard drug Griseofulvin [70]. Chandrika et al. reported hybrids 30–32 with broad spectrum antifungal activity (0.975–3.9 µgmL^-1 against C. albicans; 0.12–0.48 µgmL^-1 against C. parapsilosis) (Figure 2). These compounds also displayed good activity against C. albicans biofilms [94].

2.2. 1-Benzimidazole-R(Ar)-1,4-disubstituted-1,2,3-triazole hybrids

Deswal et al. synthesized a new series of benzimidazole-1,2,3-triazole-indoline derivatives 35 by employing click reaction between substituted N-propargylated benzimidazole derivatives 33 and in situ formed substituted 2-azido-1-(indolin-1-yl) ethanone derivatives 34 in moderate to good yields (Scheme 8). The obtained results indicate stronger inhibitory effect of compound 35d against E. coli, while compound 35g showed good inhibition against all the tested strains except B. subtilis (Table 3). The good antimicrobial activity of the compounds was correlated with the presence of the pyridine ring in position ''2'' of the benzimidazole and the NO₂ group on the indole ring [6].

Saber et al. synthesized new 1,4-disubstituted-1,2,3-triazole containing benzimidazolone derivatives 37a–37d exclusively using click chemistry (Scheme 9). All derivatives exhibited antibacterial activity against tested strains, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa, but compounds 37b and 37d are more effective against Gram-positive bacterium S. aureus (MIC = 3.125 µgmL^-1) and 37b has better activity against Gram-negative bacterium E. coli (MIC = 3.125 µgmL^-1) with Chloramphenicol as standard drug [95]. Mohsen et al. synthesized hybrids 41a–41e in three steps starting from benzimidazole 38, namely two alkylation reactions and a click reaction (Scheme 10). New derivatives exhibited good zone inhibition of 6.8, 5.4, 5.2, 4.5, 5.3 mm for S. aureus and 5.4, 3.8, 4.2, 3.3, 4.9 mm for E. coli strain, indicating that the 1,2,3-triazole core contributed significantly to bacterial growth suppression (Ciprofloxacin showed 10.2 mm for S. aureus and 10.4 mm for E. coli) [96].

2.3. 1,2-. bis-substitutedBenzimidazoles-R(Ar)-1,4-disubstituted-1,2,3-triazole

Rezki reported the intramolecular cyclization of thiosemicarbazides 42a–42d in refluxing aqueous sodium hydroxide (2N) for 6 h with the formation of hybrids 43a–43d with yields of 82–86% (Scheme 11). Among all the 1,2,4-triazole derivatives, N4-phenyl and N4-(4-fluorophenyl) derivatives 43a and 43b were found the most potent with MIC values of 4–8 µg mL^-1. Also, triazoles 43a and 43b exerted the best inhibition against both tested fungal strains, A. brasiliensis and Candida albicans, with MIC values ranging from 0.5–4 µg mL^-1, more potent than the reference drug Fluconazole. Condensation of compound 44 with several benzaldehydes in refluxing ethanol for 4–6 h with a catalytic amount of HCl produced a new class of hybrid Schiff bases 45a–45g with yields of 84–86% (Scheme 12). The antimicrobial bioassay results for the synthesized Schiff bases 45a–45g revealed that all of the tested compounds were more effective towards all of the organisms, with MIC values of 1–16 µg mL^-1. Among them, Schiff bases 45c, 45d and 145e with a fluorine atom at position "2" exhibited the highest antibacterial inhibition potency at MIC 1–8 µg mL^-1. The Schiff base 45e containing a CF₃ group exerted the highest antifungal inhibition activity with MIC of 1 µg mL^-1 [97].

Al-blewi et al. synthesized triazoles 47a–47f in two steps: i. regioselective alkylation of 4 with two equivalents of propargyl bromide in the presence of two equivalents of potassium carbonate as a base catalyst to afford benzimidazole 46 with 91% yield after stirring at room temperature overnight; ii. Copper-mediated Huisgen 1,3-dipolar cycloaddition reaction on compound 46 in good yields (82–88%) (Scheme 13). In general, bis-1,2,3-triazoles 47a–47f exhibited more potent antimicrobial activities than their mono-1,2,3-triazole derivatives 6a-6f. This was attributed to the synergistic effect of the sulfonamoyl and tethered heterocyclic components in addition to the improved

lipophilicity of the bis-substituted derivatives. Among the synthesized compounds, compound 47a was the most potent antimicrobial agent with MIC values ranging between 32 and 64 μgmL^-1 against all tested strains B. cereus, S. aureus, E. coli P. aeruginosa, C. albicans, and A. brasiliensis [88].

Aparna et al. used a similar strategy for obtaining nine new bis-1,2,3-triazol-1H- 4-yl-substituted arylbenzimidazole-2-thiol derivatives 48a-48l (Figure 3). Antibacterial activity of triazole derivatives 48 demonstrates moderate to good activity against gram negative (E. coli, S. typhy, P. aeruginosa) and gram positive (S. aureus) bacterial strains. The products 48i, 48k, and 48l characterized by a broad spectrum of antibacterial activity at concentration of 10 μgmL^-1. The derivative 48l displays the highest dock score of –7.69 kcal/mol and the least dock score of –0.942 kcal/mol is obtained for 48h [98].

2.4. Benzimidazole-R(Ar)-1,2,3-triazole hybrids as antitubecular agents

Ashok reported compound 26h as best antitubercular drug candidate by inhibiting the growth of the MTB (Mycobacterium tuberculosis) strain with MIC = 3.125μ mL^-1 (7.1 μM) (control Rifampicin MIC = 0.04μgmL^-1 and isoniazid MIC = 0.38μgmL^-1). The best antitubercular activity of 26h may be attributed to the presence of nitro group on phenyl ring at ortho position. Compound 26b (MIC = 6.25 μg mL^-1 (14.7μM)) with chlorine substituent, compound 26i (MIC = 6.25 μgmL^-1(14.2 μM)) with trifluoromethyl substituent and compound 26j (MIC = 12.5 μgmL^-1 (28.4μM)) with benzyl substituent exhibited moderate antitubercular activity. Therefore, incorporation of the electron-withdrawing nitro group, electronegative chlorine and trifluoromethyl groups on the phenyl ring was highly favored for antitubercular activity [93]. Gill et al. reported synthesis of hybrids 51a–51d by reaction between 2-(3-fluorophenyl)-1H-benzo[d]imidazole 50 and phenyl-substituted 4-(bromomethyl)-1-phenyl-1H-1,2,3-triazole 49 in DMF at room temperature (Scheme 14). Trifluorosubstituted-compound 51a possessed enhanced anti-mycobacterial activity, > 96% of inhibition at 6.25 µg concentration. Also, compounds 51b and 51c, which had antimicrobial activities superior to the other compounds, were reported as the best choice for the preparation of new derivatives in order to improve effectiveness on intracellular mycobacteria (macrophage) or in infected animal [99]. Anand et al. reported one pot reaction between 2-propargylthiobenzimidazole 4, 4-bromomethyl coumarins/1-aza-coumarins 52/53 and sodium azide under click chemistry conditions to give exclusively 1,4-disubstituted triazoles 54a–54n. Anti-tubercular assays against M. tuberculosis (H37Rv) coupled with in silico molecular docking studies indicated that dimethyl substituents 54c and 54d showed promising activity

(MIC = 3.8 µMol L^-1) with higher C-score values [100]. Khanapurmath et al. synthesized triazoles 55 by click reaction (Figure 4A). Benzimidazolone bis-triazoles 55a–55n showed better activity with MIC in the range 2.33–18.34 μM and most active compounds were 55h and 55m. All compounds exhibited moderate to low levels of cytotoxicity with IC₅₀ values of the human embryonic kidney cells in the range of 943–12294 μM, and none of 14 compounds exhibited any significant cytotoxic effects, suggesting huge potential for their in vivo use as antitubercular agents. Docking studies revealed an additional interaction of benzimidazolone oxygen in these compounds (Figure 4B) [101]. Also, Sharma et al. summarizes 1,2,3-triazoles as antitubercular compounds, and various hybrids with benzimidazole, coumarin, isoniazid, quinolines, etc [39].

3. Synthesis and antimicrobial activities of benzimidazole-1,2,4-triazoles

3.1. 2-Benzimidazole-R(Ar)-1-(1,2,4-triazole)

Pandey et al. synthesized hybrids 59a–59e in three steps: reaction of 7-hydroxy-4-methyl coumarin with thiosemicarbazide to form triazole intermediate571, which underwent Mannich reaction with formaldehyde, and an amino acid to form intermediates 58a–58e, which gave benzimidazolo-1,2,4-triazole hybrids in poor yields by reaction with o-phenylenediamine in pyridine (Scheme 16). Compound 59a displayed promising antifungal activity against Candida albicans and Cryptococcus himalayensis, since the MIC value in each case was found to be 3.5 μg mL^–1. Compound 59b showed low to moderate antifungal activity against all the five fungi, Candida albicans, Cryptococcus himalayensis, Sporotrichum schenkii, Trichophyton rubrum and Aspergillus fumigatus [102].

Jadhav et al. synthesed a series of hybrids 1,2,4-triazolyl-fluorobenzimidazoles in two steps: i. synthesis of 2-(4-(1H-1,2,4-triazol-1-yl)phenyl)-4,6-difluoro-1H-benzo [d]imidazole 62 by reaction between 3,5-difluorobenzene-1,2-diamine 60 and 4-(1H-1,2,4- triazol-1-yl)benzaldehyde 61 in toluene at 110°C, and ii. alkylation of compound 62 in DMF at room temperature, with the formation of the final hybrids 63a-63o (Scheme 17). All compounds were screened for the antimicrobial activity against different gram positive organisms S. aureus, P. aeruginosa and gram negative organisms E. coli and S. typhosa using Gentamycin as a reference standard. The data generated from preliminary screening showed that compounds displayed moderate to better antimicrobial activity. Compounds 63a, 63e, 63f, 63h, 63i, and 63l displayed maximum activity (Table 4)[103].

Barot et al. synthesized hybrid 64 and determined its antimicrobial activity against Bacillus cereus MTCC-430, Enterococcus faecalis MTCC-493, S. aureus MTCC-737, Escherichia coli MTCC-1687, Pseudomonas aeruginosa MTCC-2642, Klebsiella pneumonia MTCC-109, Candida albicans MTCC-3017, Aspergillus niger MTCC-1344 and Fusarium oxyspora MTCC-1755, of MIC = 13–18 µg ml^-1, with Ofloxacine and Fluconazole as standard drugs [104]. Also, Jiang et al. reported antifungal activity for hybrid 65 against Candida albicans, Candida tropicalis, Cryptococcus neoformans, Trichophyton rubrum, and Aspergillus fumigatus, of MIC₈₀ = 1–64 µg mL^-1 considering Fluconazole as standard drug (Figure 5) [105]. Luo et al. reported a series of naphthalimide benzimidazole-1,2,4-triazole hybrids 68a–68h and the corresponding triazoliums salts 69a–69d prepared by convenient and efficient procedures starting from naphthalimide triazole 66 (Scheme 18). 2-Chlorobenzyl triazolium 68g and compound 69b with octyl group exhibited the best antibacterial activities among all the tested compounds, especially against S. aureus with inhibitory concentration of 2 μgmL^-1 which was equipotent potency to Norfloxacin (MIC=2 μgmL^-1) and more active than Chloromycin (MIC= 7 μgmL^-1). Triazoliums 68g and 68f bearing 3-fluorobenzyl moiety displayed the best antifungal activities (MIC=2–19 μgmL^-1) against all the tested fungal strains, C. albicans ATCC 76615, A. fumigatus ATCC 96918, C. utilis, S. cerevisia and A. flavus, without being toxic to PC12 cell line within concentration of 128 μg mL^-1. Further investigations showed that compound 68g could intercalate into calf thymus DNA to

form the 68g-DNA complex which could block DNA replication, exerting powerful antimicrobial activities. [106]. Benzimidazole-1,2,4-triazole Mannich base 70 was active against Bacillus subtilis and Bacillus pumilus (inhibition zone diameters being 19 and 17 mm, respectively, compared to Ciprofloxacin with 28 and 30 mm, respectively) [107]. Kankate et al. reported synthesis of the hybrids 73a–73l (Scheme 19). Antifungal activity of compounds 73a was tested against Candida albicans spores in vitro (turbidimetric

method) and in vivo (kidney burden test). Compound 73i had a good antifungal activity as compared with the other twelve compounds at 0.0075 µmole ml^-1 which is equivalent to Fluconazole activity both in vitro and in vivo [108]. Ahuja et al. reported antifungal activity of compounds 74a–74c against F. verticillioides, D. oryzae, C. lunata and F. fujikuroi (Figure 6). All compounds had increased potency than the standard commercial benzimidazole fungicide, carbendazim (Table 5). Compound 74c exhibited ED₅₀ values lower than triazole fungicide, propiconazole. The results reinforced the synergistic effects of benzimidazole and 1,2,4-triazole combination supported by computational approach [109]. Evren et al. reported synthesis of the compounds 79a–79c in two steps: i. reaction of 1,2,4-triazole 75 with 4-fluorobenzaldehyde 76 in DMF with the formation of 4-(1H-1,2,4-triazol-1-yl)benzaldehyde 77; ii. reaction of aldehyde 77 with 1,2-phenylene diamines 78 (Scheme 20). Although the antibacterial activities of compounds 79a–79c against Escherichia coli ATCC 35218, E. coli ATCC 25922, Klebsiella pneumoniae NCTC 9633, Pseudomonas aeruginosa ATCC 27853, Salmonella typhimurium ATCC 13311, and Staphylococcus aureus ATCC 25923, were weak, the antifungal activities against C. albicans were found promising, with MIC values of 3.9, 7.8, and 3.9 μg mL^-1 respectively, using as reference drug Ketokonazole (MIC = 7.8 μgmL ^-1). Theoretical ADME calculations of the 79a, 79b, and 79c were made, and the compounds were found to have good lipophilicity, moderate water solubility, and within the limiting rules of Lipinski, Ghose, Veber, Egan, and Muegge [110]. Ghobadi et al. reported synthesis of compounds 85a-85e, in two different ways, from 3,4-diaminobenzophenone 80, i. formation of 2-mercapto benzimidazole derivatives 82, 83, and ii. nucleophilic ring opening of various oxiranes 84a-84e with benzimidazoles 82 and 83 using NaHCO₃ in ethanol at room temperature (Scheme 21). Compounds 85a-85e, containing 5-benzoylbenzimidazole scaffold showed better antifungal activity against Candida spp. and Cryptococcus neoformans than related benzimidazole and benzothiazole derivatives. The better results were obtained with the 4-chloro-derivative 85b displaying MICs < 0.063–1 μgmL^-1.. Also, compound 86c, synthesized analogously, is as potent as compound 85b. In vitro and in silico ADMET evaluations of the most promising compounds 85b indicated that the selected compounds have desirable ADMET properties in comparison to standard drug Fluconazole. Docking simulation study demonstrated that the benzimidazol-2-yl-thio moiety is responsible for the potent antifungal activity of these compounds [72].

3.2. 1-Benzimidazole-R(Ar)-2-1,2,3-triazole

Ansari et al. synthesized hybrids 88a–88c in two steps from 2-(2-methyl-1H-benzo [d]imidazol-1-yl)acetohydrazide 87 (Scheme 22). Generally, all benzimidazole-triazole hybrids showed low antimicrobial activity (Table 6) [111]. Tien et al. synthesized hybrids 89a–89d in three steps from 2-(2-methyl-1H-benzo[d]imidazol-1-yl)acetohydrazide 87b (Scheme 23). All compounds exhibited antifugal activity against A. niger (MIC = 50 µg mL^-1). Only compound 89b exhibited activity against F. oxysporum (Table 7) [112]. Kantar et al. reported antimicrobial activity of hybrid 90 (Figure 7) against four Gram-positive, Bacillus cereus 702 Roma (62.5 µg mL^-1), B. megaterium DSM-32 (125 µg mL^-1), B. subtilis ATCC 6633 (62.5 µg mL^-1), Staphylococcus aureus ATCC 25923 (250 µg mL^-1), and four

Gram-negative bacteria, Escherichia coli ATCC 25922 (250 µg mL^-1), Enterobacter cloaceae ATCC13047 (125 µg mL^-1), Pseudomonas aeruginosa ATCC 27853 (250 µg mL^-1), and Yersinia pseudotuberculosis ATCC 911 (125 µg mL^-1) bacteria [113]. Nandwana et al. reported compound 91 synthesized in good yield (70%) with promising antibacterial activity, with minimum inhibitory concentration (MIC) values of 4−8 μg mL^-1 for all bacterial tested strains (Escherichia coli, Pseudomonas putida, Salmonella typhi, Bacillus subtilis, Staphylococcus aureus), as compared to the positive control Ciprofloxacin, and also with pronounced antifungal activity against both tested strains, Aspergillus niger and Candida albicans (MIC = 8−16 μg mL^-1) as compared with Amphotericin B [114]. Al-Majidi et al. synthesized 2-mercaptobenzimidazole derivatives 95, 96 and 97 by cyclization of intermediate precursors 93, 94 and 95 under reflux with 2N NaOH (Scheme 24). The compounds generally showed a moderate antimicrobial activity against all tested strains, as can be seen in Table 8 [115]. El-masry et al. synthesized compounds 98 and 99 and found that they did not exhibit antimicrobial activity (Figure 8) [116]. Menteşe et al. synthesized compounds 100a–100d, for which they found no antimicrobial activity on the ten strains tested [117]. Karale et al. synthesized bis-benzimidazole-1,2,4-triazole hybrids 102a-102e in four steps, from 7-methyl-2-propyl-3H-benzo[d]imidazole-5-carboxylic acid. All compounds 102 did not show antimicrobial activity against the strains tested, C. albicans, A. fumigatus, S. aureus and E. coli [118,119].

3.3. 2-Benzimidazole-R(Ar)-2-1,2,4-triazole

Eisa et al. synthesized compounds 105a and 105b by the reaction between 2-(chloromethyl)-1H-benzo[d]imidazole 103 and 4-phenyl-5-(pyridin-3-yl)-4H-1,2,4-triazole-3-thiol 103 and 4-phenyl-5-(pyridin-3-yl)-4H-1,2,4-triazole-3-thiol 104a or 4-phenyl -5-(thiophen-2-yl)-4H- 1,2,4-triazole-3-thiol 104b, at reflux in absolute ethanol, for 12 hours. Also, they reported synthesis of the compounds 107a and 107b from 2-(2-(phenylthiomethyl)-1H-benzo[d]imidazol-1-yl) acetohydrazide in two steps (Scheme 27). All compounds showed antimicrobial activity against Escherichia coli superior to that of standard Gentamicin. Compound 107a exhibited only a moderate activity against Staphylococcus aureus [120].

Scheme 26. Synthesis of benzimidazole-1,2,4-triazoles 105a-105b

Scheme 26. Synthesis of benzimidazole-1,2,4-triazoles 105a-105b

Nevade et al. synthesized compounds 109a–109h in five steps from 1H-benzo[d]imidazole-2-thiol 108 (Scheme 28). The antimicrobial screening results presented in Table 10 reveal that compounds 109a, 109c, 109e exhibited satisfactory effect against S.aureus and E.coli, while the compounds 109b, 109f, 109g have shown the moderate activity against the same microbes. Also antifungal activity of these compounds was screened against Candida albicans. Compounds 109a and 109d showed highest degree of inhibition against C.albicans when compared with the Standard drug Ketoconazole [121]. Can et al. synthesized hybrids 111a–111h in four steps from methyl 4-(5-methyl-1H-benzo[d]imidazol-2-yl)benzoate 110 (Scheme 29). All compounds were screened for antifungal activity against Candida albicans ATCC 24433, Candida glabrata ATCC 90030, Candida krusei ATCC 6258 and Candida parapsilosis ATCC 22019 (Table 11). Compounds 111i and 111s exhibited significant inhibitory activity against Candida strains with MIC₅₀ values ranging from 0.78 to 1.56 𝜇g mL^-1 [122]. Gencer et al. synthesized compounds 112 in good yields (77–88%) using a similar strategy (Figure 9). Microbiological studies revealed that compounds 112a, 112b, 112c, 112e, 112f, 112g and 112h possess a good antifungal profile against all tested strains, C. albicans, C. glabrata, C. krusei, C. parapsilopsis, with MIC₅₀ = 0.78–1.56 µg mL^-1. Compound 112i was the most active derivative and showed comparable antifungal activity to those of reference drugs Ketoconazole and Fluconazole [123]. The SAR (Structure–activity relationship) on the synthesized benzimidazole-triazole compounds are summarized in Figure 10. It is observed that the presence of chlorine or fluorine in the "5" position of benzimidazole, as well as the presence of fluorine in the "4" position of phenyl increase the antibacterial activity, while the presence of fluorine in the "2" position of phenyl does not change the activity, and the presence of groups CH₃ or C₂H₅ in position "4" in the triazole nucleus does not bring any change in the antibacterial activity of the compounds. Güzel et al. synthesized a new series of benzimidazole-1,2,4-triazole derivatives 113a–113l using the same procedure described in Scheme 29 as potential antifungal agents. All the compounds were screened for their in vitro antifungal activity against four fungal strains, namely, C. albicans, C. glabrata, C. krusei, and C. parapsilopsis and were found to exhibit excellent activity against C. glabrata. Especially, compounds 113b, 113i, and 113j were found to be the most effective

compounds in the series with an MIC value of 0.97 μg mL^-1 [71]. Aryal et al. reported synthesis of new 2-substituted benzimidazole containing 1,2,4-triazoles 114a and 114b (Figure 12). The compounds did not show antimicrobial activity against the tested strains Staphylococcus aureus ATCC 6538P and Staphylococcus epidermidis ATCC 1228 [124]. Kazeminejad et al. did a study on 1,2,4-triazoles as well as structure-activity relationships (SAR) [125].

3.4. 6-substituted-Benzimidazole-R(Ar)-1-1,2,4-triazole

Nandha et al. reported synthesis of 6-substituted-benzimidazoles with 1-(1,2,4-triazole) 115a--115d in three steps from 5-chloro-4-fluoro-2-nitrobenzenamine (Scheme 30). All compounds were screened against M. tuberculosis and four fungal strains, C. albicans, C. glabrata, C. krusei and C. tropicalis. Compound 115c was the most active against M. tuberculosis and all tested fungal strains (MIC = 25 μg mL^-1) [126].

4. Synthesis and antiviral activities of benzimidazole-triazoles

Youssif et al. reported synyhesis of benzimidazole-1,2,3-triazole hybrids 2-{4-[(1-benzoylbenzimidazol-2-ylthio)methyl]-1H-1,2,3-triazol-1-yl}-N-(p-nitro--phenyl)-acetamide 116 and 2-(4-{[1-(p-chlorobenzoyl)-benzimidazol-2-ylthio)methyl]-1H-1,2,3-triazol -1-yl}-N-(p-nitrophenyl)-acetamide 117 which showed significant activity against hepatitis C virus (HCV) (Figure 13). Thus, fifty percent effective concentrations (EC₅₀) of HCV inhibition for compounds 116 and 117 were 7.8 and 7.6 μmol L^–1, respectively, and the 50 % cytotoxic concentrations (CC₅₀) were 16.9 and 21.1 μmol L^–1.The results gave an insight into the importance of the substituent at position 2 of benzimidazole for the inhibition of HCV [73].

Antiviral activity of compounds 59a–59e was tested against two viruses, viz., Japanese encephalitis virus (JEV) (P20778), a RNA virus of higher pathogenicity, and Herpes simplex virus type-I (HSV-I) (753166), the most common virus present in the environment. The antiviral activity of the compounds data are given in Table 12. All but one of the five compounds were found active against JEV. Compound 59b displayed 90% CPE (cytopathic effect) in vitro with an effective concentration of 8 µg mL^–1 while its in vivo activity was less significant (16% protection with a MST of 4 days). The authors sugested that that these compounds are better anti-JEV agents than anti-HSV agents, since two such compounds, namely 59b and 59e, also displayed a measurable degree of anti-JEV activity in vivo. Compound 59c was found antivirally inactive against both viruses. The anti HSV-I activity was found to be in the order of 33, 46, 53 and 64% for compounds 59a, 59b, 59d and 59e, respectively. Since among compounds 59a to 59e only compound 59e contains a methyl group instead of H as R₁, it follows that R₁ does not seem to be responsible for the biological activity [87].

Tonelli et al. synthesized a series of 1-substituted 2-[(benzotriazol-1/2-yl)methyl] benzimidazoles 118–137 and tested for antiviral activity against a large panel of RNA and DNA viruses (Figure 14). Twelve compounds exhibited high activity against RSV (Respiratory Syncytial Virus), with EC₅₀ values in most cases below 1 µM, comparing favorably with the reference drug 6-azauridine, which, moreover, exhibited a high toxicity against both the MT-4 and Vero-76 cell lines (S.I. =16.7). The observed activity against BVDV, YFV, and CVB2 is moderate, with EC50 values in the range of 6 – 55 µM for the best compounds (Table 13) [127].

SARS-CoV-2 and its variants, especially the Omicron variant, remain a great threat to human health. Al-Humaidi et al. reported synthesis a series of benzimidazole-1,2,3-triazoles 138–140 (Figure 15). Molecular docking studies and in vitro enzyme activity revealed that most of the investigated compounds demonstrated promising binding scores against the SARS-CoV-2 and Omicron spike proteins, in comparison to the reference drugs (Table 14).

Three-dimensional binding mode of compound 140 is shown in Figure 16 [74]. Benzimidazole-1,2,3-triazole hybrids can be potent anti-HSV (Herpes simplex virus) agents. These compounds were screened against flaviviruses and pestiviruses. Compound 141 showed excellent activity against respiratory syncytial virus (RSV) with an EC₅₀ value of 0.02 mM (Figure 17) [128].

5. Conclusions

This review summarizes the syntheses of benzimidazole–triazole compounds with antimicrobial an antiviral properties mentioned in the literature. The presence of certain groups grafted on the benzimidazole and trizole nuclei, such as -F, -Cl, -Br, -CF₃, -NO₂, -CN, -NHCO, -CHO, -OH, OCH₃, -N(CH₃)₂, COOCH₃, as well as other heterocycles in the molecule (pyridine, pyrimidine, thiazole, indole, isoxazole, thiadiazole, coumarine), increases the antimicrobial activity of the compounds [4,5,83,84,129,130,131]. From the presented literature data, we can highlight some aspects related to the correlation structure - antimicrobial properties.

- The presence of substituents in the "4" or "5" positions of the benzimidazole nucleus can increase the antimicrobial activity of the benzimidazole-triazole hybrids (compounds 12, 13, 19, 20, 35).

- The presence of the ortho or para substituted phenyl substituent in the "1" position of 1,2,3-triazoles in benzimidazole-triazole hybrids can increase their antimicrobial activity.

- In the case of benzimidazoles substituted in the "1" position with triazoles, the presence of an aliphatic or aromatic radical substituent increases the antimicrobial activity of the hybrids.

- The presence of the oxygen atom in the bridge that connects the benzimidazole and triazole rings is favorable to the antimicrobial activity of the hybrids (compounds 19, 20, 21, 29, 30)

- The presence of the sulfur atom in the bridge that connects the benzimidazole and triazole rings is favorable to the antimicrobial activity of the hybrids, and even to the antitubercular activity (95–97, 105, 107).

- The presence of a supplementary triazole ring in benzimidazole-triazole hybrids improves their antimicrobial activity (compounds 43, 45, 47).

- The presence of the benzoyl substituent in the "5" position of the benzimidazole in the benzimidazole-1,2,4-triazole hybrids clearly improves their antimicrobial activity (compounds 85a-85e).

- The phenyl nucleus as a spacer between the "1" position of 1,2,4-triazole and the "2" position of benzimidazole favors the formation of antimicrobial compounds, and the substituents in the "5" position of the benzimidazole nucleus increase the antimicrobial activity (compounds 79, 111, 112, 113).

- Only benzimidazole-1,2,3-triazole hybrids are mentioned in the literature as having antiviral properties.

- 2-Substituted or 1,2-disubstituted benzimidazoles with 1,2,3-triazoles are mentioned as antiviral compounds and the presence of an additional triazole ring improves the antiviral activity (compound 140).

We hope that this review will be useful for the design and synthesis of new benzimidazole-triazole hybrids with antimicrobial and antiviral properties in the context of exacerbation of microbial and viral infections and of resistance to treatments with drugs known on the market.