Benzimidazole-Triazole Hybrids as Antimicrobial and Antiviral Agents: A Systematic Review

Abstract

Bacterial infections have attracted the attention of researchers in recent decades, especially due to the special problems they have faced, such as their increasing diversity and resistance to antibiotic treatment. The emergence and development of the SARS-CoV-2 infection stimulated even more research to find new structures with antimicrobial and antiviral properties. Among the heterocyclic compounds with remarkable therapeutic properties, benzimidazoles, and triazoles stand out, possessing antimicrobial, antiviral, antitumor, anti-Alzheimer, anti-inflammatory, analgesic, antidiabetic, or anti-ulcer activities. In addition, the literature of the last decade reports benzimidazole-triazole hybrids with improved biological properties compared to the properties of simple mono-heterocyclic compounds. This review aims to provide an update on the synthesis methods of these hybrids, along with their antimicrobial and antiviral activities, as well as the structure–activity relationship reported in the literature. It was found that the presence of certain groups grafted onto the benzimidazole and/or triazole nuclei (-F, -Cl, -Br, -CF₃, -NO₂, -CN, -CHO, -OH, OCH₃, COOCH₃), as well as the presence of some heterocycles (pyridine, pyrimidine, thiazole, indole, isoxazole, thiadiazole, coumarin) increases the antimicrobial activity of benzimidazole-triazole hybrids. Also, the presence of the oxygen or sulfur atom in the bridge connecting the benzimidazole and triazole rings generally increases the antimicrobial activity of the hybrids. The literature mentions only benzimidazole-1,2,3-triazole hybrids with antiviral properties. Both for antimicrobial and antiviral hybrids, the presence of an additional triazole ring increases their biological activity, which is in agreement with the three-dimensional binding mode of compounds. This review summarizes the advances of benzimidazole triazole derivatives as potential antimicrobial and antiviral agents covering articles published from 2000 to 2023.

1. Introduction

Heterocyclic compounds have a central place in medicinal chemistry, being used as therapeutic agents to treat most diseases [1,2,3]. Among these heterocycles, benzimidazole stands out, as a purine-analog pharmacophore, with a very diverse therapeutic activity. The very broad spectrum of biological activities it treats include antimicrobial [4,5,6,7,8], antiviral [9,10], antihistamine [11,12], anticonvulsant [3,13], antitumor [14,15,16], proton pump inhibitors [17], antiparasitic [16,18,19], anti-inflammatory [20,21,22], or antihypertensive [23,24] activities. Some benzimidazoles are efficient agents in Diabetes mellitus [25,26,27], 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.

Additionally, triazole compounds possess a diversity of biological activities as antimicrobial [35,36,37,38], antitubercular [39,40], potential inhibitors of SARS-CoV-2 [41,42,43], antiviral [43,44], anti-inflammatory [45,46],antitumor [47,48,49,50], antihypertensive [50], antioxidant [47,51,52], and antiepileptic [53,54]. Pharmacological applications of triazoles refer to their activity as α-glucosidase inhibitors [55,56], analgesics [50,57], anticonvulsants [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 that occurred from the spring of 2020 until now, 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 [64,65,66], antimicrobial [67], immunomodulatory [68] or anti-inflammatory [69] drugs, therefore, the discovery of new molecules with simple or hybrid structures, which meet the requirements of the treatment of this condition is absolutely necessary and constitutes the engine for the development of new effective therapeutic agents.

Why did I choose the study of benzimidazole-triazole compounds? 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, and antiviral activities, including new anti-SARS-CoV-2 agents [70,71,72,73,74], 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.

Therefore, this review aims to provide an update on the synthesis methods of the benzimidazole-triazole hybrids, along with their antimicrobial and antiviral activities, as well as the structure–activity relationship and DFT studies reported in the literature. The advantages of the study of benzimidazole-triazole hybrid compounds refer to a wider range of antimicrobial activities, compared to simple precursor heterocycles, to their better minimum inhibitory concentrations compared to simple component heterocycles, as well as to the need to hire specialized personnel to carry out this research.

The main disadvantages are material because the synthesis of some hybrid compounds requires high costs compared to simple heterocycles, as well as greater time consumption. Consequently, if the synthesized hybrids have increased biological properties compared to simple precursor heterocycles, the balance clearly tilts towards the advantage of the synthesis of hybrid compounds. However, access to hybrid compounds will not be without both sides, advantages and disadvantages, which requires careful prospecting of all the components involved in the production of hybrids.

As expected, the literature mentions benzimidazole-triazole hybrids with other biological properties than those studied in this review, such as antitumor [15,48,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92], antioxidant [93,94,95], anti-Alzheimer [96,97,98,99], antidiabetic [100,101,102,103,104], and anti-inflammatory [105] properties, which is additional proof of the therapeutic potential of these hybrids and the need to study these hybrids on the topic proposed in the title. 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.

The recent literature marks several strategies for the synthesis of 1,2,3-triazoles, like click reaction [106], Bouiton-Katritzky rearrangement [107], oxidative cyclization of hydrazones [108], post-cycloaddition functionalization [109], alkylation or arylation of triazoles [110]. Also, for benzimidazoles, the literature mentions several methods of synthesis, such as the reaction of o-phenylenediamine with aldehydes or ketones (Phillips-Ladenburg reaction) [3,111,112,113], with acids or their derivatives (Weidenhagen reaction) [81], or green methods of classic syntheses [111,114,115,116,117].

Why this review is necessary and what exactly it proposes I will clarify in what follows. This article summarizes for the first time in the literature: various synthesis methods of benzimidazole-1,2,3-triazole hybrids as well as benzimidazole-1,2,4-triazoles, their antimicrobial and antiviral activities, as well as SAR studies and DFT performed on the mentioned hybrids. Where necessary, for compounds with superior biological activities, several examples from the literature were given, and the various studies performed on them (in vitro, in vivo, in silico, etc.) were mentioned. All of these aim at directing the syntheses of hybrid compounds with specific structures and superior antimicrobial and antiviral properties, taking into account the mentions reported in the literature up to now.

The database search methodology used in this review was the use of keywords, which can be found in the title, such as benzimidazole, 1,2,3-triazole, click reaction, 1,2,3-triazole, benzimidazole-triazole hybrids, antimicrobial, antiviral, or therapeutic properties, in different websites, such as PubMed, MDPI, Science Direct, Springer, The Royal Society Chemistry, ACS Publications, and Taylor & Francis. The selection of scientific articles for the last ten years was made according to the novelty brought in the benzimidazole-triazole hybrids and their antimicrobial and antiviral properties, as well as the therapeutic properties of the reported compounds.

Generally, articles from the last ten years have been selected. For the hybrids found, first, the syntheses and then their biological properties were presented, with special emphasis on those with improved properties (active on a larger range of microbial strains, with better minimum inhibitory concentrations, or where SAR studies were performed, DFT, etc.). In the following, we will present syntheses of benzimidazole-triazole hybrids with antimicrobial and antiviral properties. In order to highlight the structures of the heterocycles in the discussed compounds, we colored the benzimidazole nucleus with red, 1,2,3-triazole with blue, and 1,2,4-triazole with green.

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 to 75% of that of the standard drug against Staphylococcus aureus and 50–80% of 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 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 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) [118]. 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. Antimicrobial screening results of compounds 6a–6f presented as MIC (μg mL⁻¹).

Compound	Gram-Positive Organisms		Gram-Negative Organisms		Fungi Organisms
	B.c.	S.a.	P.a.	E.c.	A.b.	C.a.
6a	64	64	256	128	128	128
6b	128	128	128	128	256	256
6c	256	128	256	64	256	156
6d	256	128	256	64	256	256
6e	256	128	256	64	256	256
6f	512	512	256	256	512	512
Ciprofloxacin	8	4	8	4	-	-

) against four pathogenic bacterial strains (Gram-positive: Bacillus cereus ATTC 10876, Staphylococcus aureus ATTC 25,923, and Gram-negative: Escherichia coli ATTC 25922, Pseudomonas aeruginosa ATTC 27,853 and two fungal strains, Candida albicans ATTC 50193, Aspergillus brasiliensis ATTC 16404). As can be seen in Table 1, compound 6a showed the best antibacterial activity against Bacillus cereus and Staphylococcus aureus (64 μg mL⁻¹), and compounds 6c, 6d, and 6e showed the best antibacterial activity against Escherichia coli (64 μg mL⁻¹) [119]. Evaluation of in silico physicochemical properties or ADMET (adsorption, distribution, metabolism, excretion, and toxicity) as a robust tool to confirm the potential of a drug candidate was applied for these compounds [120]. As per Lipinski’s rule of five, an orally administered drug should have a log p ≤ 5, a molecular weight (MW) < 500 Daltons, and an HBD ≤ 5 [121] to be in the acceptable range. Results have shown that all hybrids have in good agreement in terms of HBD. 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 molecule 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. In vitro antimicrobial screening of hybrids 8, 9, and 10 using the agar diffusion method.

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

). 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, as summarized in Figure 2 [122].

ADMET analysis of compounds 8–10 exhibited that they have good absorption properties (%HIA) ranging from 99.57 to 100% [123]. For distribution, the compounds do not permeate the blood–brain barrier (BBB). Moreover, the molecules were negative in the AMES toxicity and carcinogenicity test, which suggests that they are non-mutagenic. 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 (Scheme 4). in almost quantitative yields. Compounds 13a–13g 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 13e, with a rate of 29.76% against Verticillium dahliae in the sporulation test [124].

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 19,606 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. Results of anti-trypanosomal evaluations showed that p-methoxyphenyl substituent in 19b–21b enhanced activity, with 20b (IC₅₀ = 1.1 mM and IC₉₀ = 3.5 mM) being more potent than Nifurtimox. In contrast to the observed correlation between antimicrobial activity and DNA binding, the antiprotozoal effects of 20b did not correlate with its DNA affinity [125].

Rao et al. synthesized hybrids 22a–22b (Figure 3) using the click chemistry approach. Compounds had weak activity against Mycobacterium bovis strain, with BCG values % inhibition = 27.3 and 26.2, respectively, at 30 µM concentration, using a turbidometric assay. Compounds 22a and 22b also showed moderate antiproliferative activity against human breast cancer cell line MCF-7, with IC₅₀ values of 31.9 and 25.1 μM, respectively [126]. 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 11,229 bacteria using Gentamicin as standard. Antifungal activity was tested against Candida albicans ATCC 10,231 and Aspergillus niger ATCC 9029 strains with the standard drug Fluconazole. Compounds 26b, 26c, and 26h with MIC of 3.125–6.25 μg mL⁻¹ were found to be the most promising potential antimicrobial molecules [127]. The authors calculated various physicochemical parameters such as clogP, drug score, and drug-likeness of 26a–26j using the Osiris Property Explorer software [128]. For all the compounds, the calculated clogP values were found to be below five according to Lipinski’s rule-of-5 and also exhibited positive values for drug score. Mallikanti et al. synthesized novel benzimidazole-conjugated 1,2,3-triazole analogs 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 showed minimal inhibition zones against all Gram-positive (S. aureus, B. subtilis) and Gram-negative (E. coli, P. aeruginosa) strains using Ampicillin as A standard drug. Among all tested compounds, the 29i and 29k showed higher activity against P. aeruginosa, S. aureus, and B. subtilis than the standard reference. Compounds 29a, 29b, 29c, 29d, 29e, 29f, 29g, 29h, 29j, and 29l showed moderate antibacterial activity against tested strains (Inhibition zone: 10–25 mm compared with 18–20 mm for Ampicillin). Compounds 29i, 29j, and 29k also established strong activity against both fungal strains, C. albicans MTCC 183 and A. niger MTCC 9652, compared to the standard drug Griseofulvin [70]. To understand the binding mode of novel compounds, docking simulations were performed against the crystal structures of glucosamine-6-phosphate synthase (GlmS) (PDB ID: 2VF5) of E. coli and secreted aspartic proteinase (Sap) 1 (PDB ID: 2QZW) of C. albicans retrieved from the protein data bank. The best active compound, 29l, scored the highest binding affinity value of about −10.0 kal/mol, which demonstrated two key interactions with the active site amino acid Asp549 of Glms with a bond distance of 2.66 and 2.81 Å. Further, the hydrophobic interactions were taken with Tyr312, Ser316, Asp474, Asn523, Ala572, and Ala551 of Glms, among which one π-π T-shaped interaction with Tyr312, and halogen bond [129] interactions with Tyr312, Asn523, and Asn551 (Figure 4). The binding energies and interactions of all compounds are better than that of Ampicillin, which proves that these molecules could best fit into the cavity of Glms [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 5). These compounds also displayed good activity against C. albicans biofilms (3.9–15.6 µgmL⁻¹ against C. albicans) [130].

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 a 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 a 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. Antimicrobial activity of the compounds 35 in terms of MIC (µmol mL⁻¹).

Compound	S. aureus	E. coli	B. subtilis	S. epidermitis	A. niger	C. albicans
35a	0.028	0.056	0.056	0.056	0.056	0.056
35b	0.031	0.062	0.062	0.062	0.062	0.062
35c	0.029	0.058	0.058	0.058	0.058	0.058
35d	0.060	0.030	0.060	0.030	0.060	0.060
35e	0.029	0.056	0.056	0.056	0.056	0.056
35f	0.026	0.052	0.052	0.052	0.052	0.052
35g	0.031	0.026	0.052	0.026	0.026	0.026
Norfloxacin	0.020	0.039	0.039	0.039	-	-
Fluconazole	-	-	-	-	0.04	0.020

). 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. Furthermore, in vitro α-glucosidase inhibition of all synthesized derivatives identified 35e (IC₅₀ = 0.015 ± 0.0003 μmol mL⁻¹) and 35g (IC₅₀ = 0.018 ± 0.0008 μmol mL⁻¹) as potent inhibitors of α-glucosidase, even better than standard drug Acarbose [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. The expected inhibition efficiency, 37c > 37a > 37b, was attributed to the favorable effect of the side carbon chain of the triazole moiety, according to DFT calculations, in this process [131,132]. 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, and 5.3 mm for the S. aureus strain and 5.4, 3.8, 4.2, 3.3, 4.9 mm for the 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). Compared with Gram-negative bacteria, all compounds showed a strong effect against Gram-positive bacteria [94].

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 to be the most potent with MIC values of 4–8 µg mL⁻¹. Also, triazoles 43c and 43d exerted the best inhibition against both tested fungal strains, A. brasiliensis and Candida albicans, with MIC values ranging from 0.5 to 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 45e 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 [133]. 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). Generally, 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 μg mL⁻¹ against all tested strains B. cereus, S. aureus, E. coli P. aeruginosa, C. albicans, and A. brasiliensis. Pharmacophore elucidation of the compound 47a–47f was performed based on in silico ADMET evaluation of the tested compounds. Screening results of drug-likeness rules showed that all compounds follow the accepted rules, meet the criteria of drug-likeness, and follow Lipinski’s rule of five. In addition, the toxicity results showed that all compounds are non-mutagenic and noncarcinogenic [119].

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 6). 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 are characterized by a broad spectrum of antibacterial activity at a concentration of 10 μg mL⁻¹. The synthesized 1,2,3 triazole derivatives were studied for their molecular docking on the high-resolution X-ray crystal structure of FabI of Staphylococcus aureus (pdb id:4FS3) obtained from the protein data bank [134]. The highest dock score of –7.69 kcal/mol and the lowest dock score of –0.942 kcal/mol were obtained for molecules 48l and 48h, respectively [135].

2.4. Benzimidazole-R(Ar)-1,2,3-Triazole Hybrids as Antitubecular Agents

Ashok reported compound 26h has the best antitubercular drug candidate by inhibiting the growth of the MTB (Mycobacterium tuberculosis) strain with MIC = 3.125 μ mL⁻¹ (7.1 μM) (control Rifampicin MIC = 0.04 μg mL⁻¹ and isoniazid MIC = 0.38 μg mL⁻¹). The best antitubercular activity of 26h may be attributed to the presence of the nitro group on the phenyl ring at the ortho position. Compound 26b (MIC = 6.25 μg mL⁻¹ (14.7 μM)) with chlorine substituent, compound 26i (MIC = 6.25 μg mL⁻¹(14.2 μM)) with trifluoromethyl substituent and compound 26j (MIC = 12.5 μg mL⁻¹ (28.4 μM)) with benzyl substituent exhibited moderate antitubercular activity. Therefore, the incorporation of the electron-withdrawing nitro group, electronegative chlorine, and trifluoromethyl groups on the phenyl ring was highly favored for antitubercular activity. The authors calculated various physicochemical parameters and found from the theoretical data that compounds 26a–26j also exhibited positive values for drug score [127]. Gill et al. reported syntheses 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 animals [136]. Anand et al. reported a one-pot reaction between 2-propargylthiobenzimidazole 4, 4-bromomethylcoumarins/1-aza-coumarins 52/53 and sodium azide under click chemistry conditions to give exclusively 1,4-disubstituted triazoles 54a–54n. (Scheme 15). Antitubercular 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. Surflex-Dock was used to investigate detailed intermolecular interactions between the ligand and the target protein. Three-dimensional structure information on the target protein was taken from the PDB entry 4FDO. Processing of the protein included the removal of the co-crystallized ligand and water molecules, as well as the addition of essential hydrogen atoms. All 14 inhibitors 54a–54n were docked into the active site of ENR, as shown in Figure 7a, and Figure 7b indicates the superimposition of compounds 54a and 54d with ligand [137]. Khanapurmath et al. synthesized triazoles 55 by click reaction (Figure 8a). Benzimidazolone bis-triazoles 55a–55n showed better activity with MIC in the range 2.33–18.34 μM, and the 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 the 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 8b) [138]. Also, Sharma et al. summarize 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 intermediate 57, which underwent Mannich reaction with formaldehyde, and an amino acid to form intermediates 58a–58e. Intermediates 58a–58e reacted with o-phenylenediamine in pyridine to give benzimidazole-1,2,4-triazole hybrids in poor yields (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 five fungi, Candida albicans, Cryptococcus himalayensis, Sporotrichum schenkii, Trichophyton rubrum, and Aspergillus fumigatus [139].

Jadhav et al. synthesized 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 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. Antimicrobial activity of the compounds 63a–63o using the agar diffusion method.

Compound	Inhibition Zone Diameters Using the Agar Diffusion Method (mm)
	S. aureus	P. aeruginosa	E. coli	S. typhosa
63a	28	26	21	19
63b	23	18	16	14
63c	21	23	18	19
63d	20	22	23	23
63e	25	23	21	24
63f	27	26	24	20
63g	19	20	15	13
63h	29	26	22	24
63i	26	22	19	18
63j	14	12	16	16
63k	22	21	20	18
63l	25	23	19	21
63m	21	18	18	16
63n	24	22	22	21
63o	19	21	18	14
Gentamycin	34	35	31	30

) [140].

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 [141]. 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 a standard drug (Figure 9). From the antifungal activity data, preliminary SARs was obtained. In general, the amine linker was important for antifungal activities. Substituted piperazine derivatives were comparable or superior to the corresponding N-methyl derivatives. [142]. Luo et al. reported a series of naphthalimide benzimidazole-1,2,4-triazole hybrids 68a–68h and the corresponding triazolium 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 an inhibitory concentration of 2 μg mL⁻¹ which was equipotent potency to Norfloxacin (MIC = 2 μg mL⁻¹) and more active than Chloromycin (MIC = 7 μg mL⁻¹). Triazoliums 68g and 68f bearing 3-fluorobenzyl moiety displayed the best antifungal activities (MIC = 2–19 μg mL⁻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. [143]. 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) [144].

Kankate et al. reported the synthesis of hybrids 73a–73l (Scheme 19). The antifungal activity of compound 73 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 µM mL⁻¹, which is equivalent to Fluconazole activity both in vitro and in vivo. The antifungal activity decreased with the increasing alkyl length of N1 of benzimidazole (methyl to ethyl). This was proven for compounds 73i and 73l, which showed MICs of 0.0075 and 0.015 µM mL⁻¹, respectively. The ligand fit method was performed to study and predict the binding mode of the hybrids 73 with the target enzyme (homology modeled) cytochrome P450 lanosterol 14-α-demethylase of C. albicans. All compounds showed binding in the active site of the enzyme. The 1,2,4-triazole ring of compounds 73a–73l is positioned almost perpendicular to the porphyrin plane, with a ring nitrogen (N-4) atom coordinated to the heme iron (Figure 10) [145,146]. Ahuja et al. reported antifungal activity of compounds 74a–74c against F. verticillioides, D. oryzae, C. lunata, and F. fujikuroi (Figure 11). All compounds had increased potency than the standard commercial benzimidazole fungicide, carbendazim (

Table 5. ED₅₀ values (µg mL⁻¹) of compounds against test fungi.

Compound	F. verticillioides	D. oryzae	C. lunata	F. fujikuroi
74a	35	50	28	45
74b	30	25	18	30
74c	16	12	10	15
Carbendazim	230	-	-	150
Propiconazole	20	25	22	21

).

Compound 74c exhibited ED₅₀ values lower than triazole fungicide, propiconazole. The results reinforced the synergistic effects of the benzimidazole and 1,2,4-triazole combination supported by a computational approach. Hydrogen bonding interactions were more pronounced in compounds 74a–74c in the binding pockets of both the target enzymes, in comparison to standards. In compound 74c, two H-bonds were formed with Gln11 present in the binding cleft of the active pocket of β-tubulin (Figure 12). In all three compounds, the right position of the N-atoms of both 1,2,4-triazole and benzimidazole, that O-atoms of methoxy and carbonyl groups, contributed well to strong binding into the active site of enzymes via H-bonding [147]. Evren et al. reported the 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 μg mL ⁻¹). Protein-ligand interactions and binding poses of the 79a, 79b, and 79c compounds on the CYP51 active site were examined. As shown in Figure 13, an H bond of 2.11 Å between compound 79a and Met508, π-π stacking interactions with Tyr118, Hie377, Phe233, generated hydrophobic interactions with Pro230, Leu376, Tyr64, Phe228, and Tyr505. 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 (Figure 14) [148]. Ghobadi et al. reported the 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 a 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 4-chloro-derivative 85b displaying MICs < 0.063–1 μg mL⁻¹. Also, compound 86c, synthesized analogously, is as potent as compound 85b. The docking experiments were conducted to further rationalize the obtained antifungal activity data and investigate the type of interactions between compound 85b and the active site of lanosterol 14α-demethylase (CYP51). As shown in Figure 13, the coordinated bond-forming distance between the N4 atom of the triazole nucleus of compound 85b and the iron atom in the heme group of active site were 2.71 and 2.40 Å, respectively. A hydrogen-bonding interaction between Tyr132 and the sulfur group of (S)-85b was observed. 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 the standard drug Fluconazole. A 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. Antimicrobial activity of compounds 88a–88c expressed as MIC in μg mL⁻¹.

Compound	S. aureus	B. subtilis	S. mutans	P. aeruginosa	C. albicans
88a	NT	NT	16	16	32
88b	8	16	16	16	NT
88c	8	16	32	32	32
Ampicillin	2	2	<1	4	NT
Kanamycin	2	<1	4	2	NT

NT = not tested.

) [149]. 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 antifungal activity against A. niger (MIC = 50 µg mL⁻¹). Only compound 89b exhibited activity against F. oxysporum (

Table 7. The minimum inhibitory concentrations (µg mL⁻¹) of the compounds against fungi.

Compound	Concentration (µg mL−1)	Aspergillus niger	Fusarium oxysporum
89a	50	50	-
89b	50	50	50
89c	50	50	-
89d	50	50	-

) [150]. Kantar et al. reported antimicrobial activity of hybrid 90 (Figure 15) 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 25,923 (250 µg mL⁻¹), and four Gram-negative bacteria, Escherichia coli ATCC 25,922 (250 µg mL⁻¹), Enterobacter cloaceae ATCC13047 (125 µg mL⁻¹), Pseudomonas aeruginosa ATCC 27,853 (250 µg mL⁻¹), and Yersinia pseudotuberculosis ATCC 911 (125 µg mL⁻¹) bacteria [151]. 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 [152]. 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 moderate antimicrobial activity against all tested strains, as can be seen in

Table 8. Antimicrobial activity of compounds 89a–89c.

Compound (800 µg mL−1)	S. aureus	P. aerugnosa	B. subtilis	A. baumannii	C. albicans
95	18	14	15	-	10
96	19	11	12	-	11
97	17	15	14	12	-
Amoxicillin	33	32	33	-	-
Fluconazole	-	-	-	-	25

[153]. El-masry et al. synthesized compounds 98 and 99 and found that they did not exhibit antimicrobial activity (Figure 16) [154]. Menteşe et al. synthesized compounds 100a–100d, for which they found no antimicrobial activity on the ten strains tested [155]. Karale et al. synthesized bis-benzimidazole-1,2,4-triazole hybrids 102a–102e (Scheme 25) 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 [156,157].

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

Eisa et al. synthesized compounds 105a and 105b (Scheme 26) (

Table 9. Antimicrobial activity of compounds 105a–105b and 107a–107b.

Compound	Minimum Inhibitory Concentrations (μg mL−1)
	Gram-Positive Bacteria		Gram-Negative Bacteria
	B. subtilis	S. aureus	E. coli	P. aeruginosa
105a	98	-	52	-
105b	-	-	65	-
107a	75	105	62	-
107b	79	-	72	-
Gentamycin *	64	56	72	48

* Concentration of Gentamycin = 30 μg mL⁻¹.

) 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 104a or 4-phenyl -5-(thiophen-2-yl)-4H-1,2,4-triazole-3-thiol 104b, at reflux in absolute ethanol, for 12 h. 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 moderate activity against Staphylococcus aureus [158]. 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. Antibacterial activity of compounds 109a–109h.

No	Compound	Zone of Inhibition (mm)
		E. coli	S. aureus	C. albicans
1	109a	15	13	18
2	109b	13	11	12
3	109c	17	16	14
4	109d	12	13	16
5	109e	13	17	9
6	109f	10	8	11
7	109g	8	11	12
8	109h	12	7	10
9	Ampicilline	24	25	-
10	Ketokonazole	-	-	20

reveal that compounds 109a, 109c, and 109e exhibited satisfactory effects against S.aureus and E.coli, while compounds 109b, 109f, and 109g showed moderate activity against the same microbes. Also, the antifungal activity of these compounds was screened against Candida albicans. Compounds 109a and 109d showed the highest degree of inhibition against C.albicans when compared with the standard drug Ketoconazole [159]. 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 22,019 (

Table 11. MIC₅₀ (μg mL⁻¹) values of compounds 111a–111s.

Compound	C. albicans	G. glabrata	C. krusei	C. parapsilosis
111a	12.5	6.25	6.25	12.5
111b	6.25	3.12	6.25	6.25
111c	12.5	6.25	6.25	12.5
111d	6.25	12.5	6.25	6.25
111e	12.5	6.25	12.5	12.5
111f	6.25	3.12	3.12	6.25
111g	3.12	6.25	6.25	6.25
111h	12.5	6.25	12.5	6.25
111i	0.78	1.56	1.56	0.78
111j	12.5	6.25	12.5	12.5
111k	12.5	6.25	12.5	12.5
111l	6.25	12.5	6.25	12.5
111m	3.12	3.12	3.12	6.25
111n	3.12	3.12	1.56	3.12
111o	3.12	3.12	6.25	6.25
111p	12.5	12.52	6.25	6.25
111r	6.25	3.12	3.12	3.12
111s	0.78	1.56	1.56	0.78
Ketokonazole	0.78	1.56	1.56	1.56
Fluconazole	0.78	1.56	1.56	0.78

). Compounds 111i and 111s exhibited significant inhibitory activity against Candida strains with MIC₅₀ values ranging from 0.78 to 1.56 μg mL⁻¹ [160]. Gencer et al. synthesized compounds 112 in good yields (77–88%) using a similar strategy (Figure 17). 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 [161]. The SAR (Structure–activity relationship) on the synthesized benzimidazole-triazole compounds is summarized in Figure 18. 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. Furthermore, toxicological and ADME studies indicated the relative potency of hybrids 112h and 112i, according to the literature [162,163,164,165,166]. Compound 112i also inhibited ergosterol biosynthesis concentration dependently. Results of ergosterol level quantification assay and fluorescence microscopy studies revealed that the mechanism of action of hybrids is associated with the inhibition of ergosterol biosynthesis, which may subsequently result in altered membrane fluidity, plasma membrane biogenesis, and functions of fungi. 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 (Figure 19). 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]. According to the molecular docking study, compounds 113b, 113i, and 113j fit into the LDM enzyme active pocket. In a previous study [167], the Tyr118 amino acid and HEM601 protein were described as essential residues, and in this study, the synthesized active compounds interacted significantly with Tyr118, His377, and HEM601 residues. The interactions with HEM were seen as π−π stacking and π−cation interactions. Therefore, the antifungal effects of compounds 113b, 113i, and 113j were considered to be caused by the destruction of cell integrity due to the inhibition of the LDM enzyme. The authors identified compound 6i with higher inhibitory activity due to H-bonding with Tyr132, unlike the other two compounds. Aryal et al. reported synthesis of 2-substituted benzimidazole containing 1,2,4-triazoles 114a and 114b (Figure 20). The compounds did not show antimicrobial activity against the tested strains Staphylococcus aureus ATCC 6538P and Staphylococcus epidermidis ATCC 1228 [168]. Kazeminejad et al. did a study on 1,2,4-triazoles as well as structure–activity relationships (SAR) [38].

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) [169].

4. Synthesis and Antiviral Activities of Benzimidazole-Triazoles

Over 200 viruses are known to cause disease in humans, yet currently approved antiviral drugs are available to treat only about 10 of these viral infections [170,171]. The past decade has underscored the global threat posed by emerging viruses. An alternative solution is the development of broad-spectrum antiviral drugs. One advantage of this approach is reduced time and cost associated with the early stages of drug development per approved indication. It can also diminish the clinical risks in more advanced stages of development [172,173]. Youssif et al. reported the synthesis of benzimidazole-1,2,3-triazole hybrids 2-{4-[(1-benzoylbenzimidazol-2-ylthio)methyl]-1H-1,2,3-triazol-1-yl}-N-(4-nitro-phenyl)-acetamide 116 and 2-(4-{[1-(4-chlorobenzoyl)-benzimidazol-2-ylthio)methyl]-1H-1,2,3-triazol -1-yl}-N-(4-nitrophenyl)-acetamide 117 which showed significant activity against hepatitis C virus (HCV) (Figure 21). 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].

The antiviral activity of compounds 59a–59e was tested against two viruses, viz., Japanese encephalitis virus (JEV) (P20778), an 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 is given in

Table 12. Anti-JEV and anti-HSV activity of compounds 59a–59e.

Compd.	In Vitro				In Vivo
	CT50 (µg mL–1)	EC50 (µg mL–1)	TI	CPE Inhibition (%)	Dose (µg per Mouse per Day)	MST (days)	Protection (%)
Anti-JEV
59a	125	4	31	30	200	-	-
59b	125	8	16	90	200	4	16
59c	-	-	-	-	-	-	-
59d	125	4	31	30	200	-	-
59e	250	62.5	4	50	200	2	10
Anti-HSV
59a	125	62.5	2	33	-	-	-
59b	125	62.5	2	46	-	-	-
59c	-	-	-	-	-	-	-
59d	125	31.25	4	53	200	-	-
59e	250	7.8	32	64	200	-	-

CT₅₀—50% cytotoxic concentration, EC₅₀—50% effective concentration, TI—therapeutic index (TI = CT_50/EC₅₀). CPE—cytopathic effect, MST—mean survival time.

. 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 in vivo activity was less significant (16% protection with an MST of 4 days). The authors suggested 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 [139].

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 22). 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 high toxicity against both the MT-4 and Vero-76 cell lines (S.I. = 16.7). The observed activity against BVDV (Bovine Viral Diarrhea Virus), YFV (Yellow Fever Virus), and CVB2 (Coxsackie virus B2) is moderate, with EC₅₀ values in the range of 6–55 µM for the best compounds (

Table 13. RSV, BVDV, YFV, and CVB2 Inhibitory Activity of hybrids 118–137 expressed as EC₅₀ (µM).

Compound	Anti-RSV Activity	Anti-BVDV Activity	Anti-YFV Activity	Anti-CVB2 Activity
118	0.7	-	-	-
119	2.3	-	-	-
120	0.7	>100	80	>100
121	0.7	63	>90	>100
122	0.3	53	>70	>100
123	0.15	51	>60	>100
124	0.03	-	-	-
125	0.7	-	-	-
126	0.06	90	>100	>100
127	0.1	72	>54	>100
128	0.9	15	6	40
129	0.05	19	>21	>88
130	0.02	14	>20	26
131	10.0	-	-	-
132	7.0	-	-	-
133	1.9	67	>36	>100
134	>36	15	>18	>36
135	9	-	-	-
136	11	80	>45	>100
137	23.0	80	27	>83
6-Azaurine	1.2	>100	26	>100

). Though not particularly impressive, the presently uncovered activity against BVDV, YFV, and CVB2 is of some interest because it may lead, through the identification of the target, to the development of broad spectrum antiviral agents. In this respect, the definition of the mode of action of the above compounds is mandatory. Furthermore, since the activity against these viruses was influenced by the presence and nature of the substituents in position “5” of the benzimidazole ring, it will be worthwhile to further explore the effect of diversified substitutions as a possibility to improve activity and/or decrease cytotoxicity [174]. SARS-CoV-2 and its variants, especially the Omicron variant, remain a great threat to human health [10]. More novel variants of SARSCoV-2 are also expected to originate in the future. Therefore, efforts should be made to develop wide-ranging measures to prevent future outbursts of zoonotic origin. Recent articles reported essential and up-to-date information about SARS-CoV-2 variants, antiviral drugs, and vaccines used to fight it [175,176].

Al-Humaidi et al. reported the synthesis of a series of benzimidazole-1,2,3-triazoles 138–140 (Figure 23). 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. Antiviral activity of benzimidazole-1,2,3-triazole hybrids 138–140.

Compound	CC50 (µg mL−1)	EC50 (µg mL−1)	Selectivity Index (SI)
Ceftazidime	1045.53	85.07	12.29
138	1065.51	155.05	6.87
139	1530.5	306.1	5.0
140	1028.28	80.4	12.78

).

Data proved the promising activity of the tested compound 140, with its IC₅₀ reaching 75.98 nM against the Omicron spike protein and 74.51 nM against the SARS-CoV-2 spike protein. The three-dimensional binding mode of compound 140 is shown in Figure 24. 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 25) [74]. Seliem et al. designed and synthesized some quinolone–triazole conjugates against SARS-CoV-2. It was revealed that 4-((1-(2-chlorophenyl)-1H-1,2,3-triazol- 4-yl)methoxy)- 6-fluoro-2-(trifluoromethyl)quinoline and 6-fluoro-4-(2-(1-(4-methoxyphenyl) -1H-1,2,3- triazol-4-yl)ethoxy)-2-(trifluoromethyl)quinoline have high antiviral activity with a high selectivity index (SI) against SARS-CoV-2 in comparison to the reference drugs. They explained that the fluorine atoms in the tested compounds have a major role in the observed antiviral activity [43]. The importance of the 1,2,4-triazole ring in antiviral compounds is reviewed by El-Sebaey, who emphasizes the importance of the substituents in the triazole nucleus, as well as the important role of other heterocycles in the molecule [177].

5. Conclusions

This review summarizes the syntheses of benzimidazole–triazole compounds with antimicrobial and antiviral properties mentioned in the literature. The presence of certain groups grafted on the benzimidazole and triazole 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, coumarin), increases the antimicrobial activity of the compounds [4,5,114,115,165,178,179,180]. 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).

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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.

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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.

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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).

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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).

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

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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).

The presence of both the benzimidazole ring and the triazole ring in a single molecule enhanced the effectiveness of the antimicrobial activities, as seen in the sections above. The recent ADME and SAR studies mentioned in this review are also important for directing new syntheses of benzimidazole-triazole hybrids in close correlation with their properties.

As mentioned in the cited literature, it is extremely useful, both from a therapeutic and economic point of view, that the synthesized compounds, such as the benzimidazole-triazole hybrids analyzed in this review, possess both antimicrobial and antimicrobial biological activity antiviral, to meet the medical requirements demanded especially lately, for better action, especially in the case of SARS-CoV-2.

The ADME studies performed on the benzimidazole-triazole hybrids mentioned in this review recommend the compounds as antimicrobials and antivirals and open new horizons to create new compounds, following the conclusions found here, with improved biological properties.

The articles researched on this topic, although they report the general characteristics of these molecules (lipophilicity/hydrophilicity), in order to have the desired antimicrobial or antiviral properties, refer only to liquid formulations in the form in which the compounds were tested, and so far not no article is reported that formulates in the form of nanosystems, nanoparticles for better availability of the active substance. This remains an open research topic for future studies.

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 resistance to treatments with drugs known on the market.