Mitigating Antimicrobial Resistance through Strategic Design of Multimodal Antibacterial Agents Based on 1,2,3-Triazole with Click Chemistry

Abstract

Drug-resistant bacterial infections impose a major threat to human health, as current antibiotic treatments are becoming increasingly ineffective. Priority has been given to the development of alternative medications to curb the development of resistance or agents that can work on the resistance strains. Among various promising approaches, 1,2,3-triazole-based molecular hybrids have emerged as excellent candidates owing to their ease of synthesis, high structural diversity, functional tunability, and biocompatibility. The rapid advancement of biological understanding of 1,2,3-triazole has been greatly aided by the discovery of the Click reaction. Drugs with a single molecular target often fail to kill the bacteria effectively, and even if they do, the bacteria eventually become resistant by virtue of mutations or other mechanisms. In this context, the 1,2,3-triazole group has been explored to design novel molecular hybrids to combat antimicrobial resistance in an effective manner. Different types of 1,2,3-triazole-based hybrids have been developed that have shown inhibitory effects on critical bacterial enzymes, the ability to produce intracellular reactive oxygen species, and the ability to disrupt the cell membrane. Herein, we discuss the strategic design principles of triazole-based hybrids, their antibacterial potential, especially focusing on the drug resistance issue, and future perspectives to critically assess their potential for multitargeting antibacterial agents. The presented information can lead to the development of novel multifaceted antibacterial agents in the future by means of their unique chemical features to address the growing challenge of drug resistance.

1. Introduction

The rapid emergence of antimicrobial resistance (AMR) over the past few decades has raised questions regarding the efficacy of existing medications. It is estimated that bacterial AMR resulted in 4.95 million deaths across the globe in 2019. The number is expected to rise to 10 million within the next 25 years if no effective measures are implemented. In the coming years, the death rate due to AMR could match that of the SARS-CoV-2 (COVID-19) pandemic, which has also been associated with a large number of secondary bacterial infections. There has been a massive use of antibiotics to treat these bacterial infections or as a precaution during the pandemic period, which may have exacerbated the AMR crisis. Unfortunately, current drug development cannot match the pace of AMR in the very near future. At present, only 1% of the antimicrobial drugs in development are entering clinical trials, with relatively few targeting resistant bacteria. Considering the fact that most of the antibiotics approved in the last 40 years are derived from the already existing chemical structures drives us to explore novel strategies by chemical modifications and derivatization.

Bacteria develop various mechanisms of resistance to antibiotics, of which some are intrinsic, where they use existing genes to survive. The other type is called “acquired”, where they gain new genetic material that provides new capacities to evade the action of antibiotics. In 2017, the World Health Organization (WHO) identified antibiotic resistant pathogens as a significant global health threat, categorizing them into three priority groups: medium, high, and critical. Among these, ESKAPE pathogens fall into the “critical group”, consisting of Enterococcus faecium (E. faecium), Staphylococcus aureus (S. aureus), Klebsiella pneumoniae (K. pneumoniae), Acinetobacter baumannii (A. baumannii), Pseudomonas aeruginosa (P. aeruginosa), and Enterobacter species. , These microbes are widely recognized for their ability to evade traditional antibiotics using various advanced mechanisms such as inactivation of antibiotics by either irreversible modification or degradation by enzymes (β-lactamases, chloramphenicol acetyltransferases, and aminoglycoside modifying enzymes), the use of efflux pumps to expel drugs from the bacterial cell, alterations in cell membrane permeability to prevent antibiotic uptake, and structural modifications of antibiotic target sites, making it difficult for drugs to bind effectively. For example, Staphylococcus aureus (S. aureus), a Gram-positive bacterium, employs the mecA gene to impart methicillin resistance through the alteration of penicillin-binding proteins, whereas K. pneumoniae, a Gram-negative bacterium, acquires carbapenem resistance via carbapenemases and extended-spectrum β-lactamases (ESBLs). P. aeruginosa can resist β-lactams via β-lactamase and metallo β-lactamase. In this manner, bacteria developed resistance to several antibiotics such as aminoglycosides (gentamicin, streptomycin, kanamycin), β-lactams (penicillins, cephalosporins, cephamycins, carbapenems, monobactams), glycopeptides (vancomycin), macrolides (azithromycin, erythromycin), quinolones and fluoroquinolones (ciprofloxacin). Additionally, to prevent the entry of foreign molecules in to the cell an extensive outer lipid layer and a polysaccharide capsule coat were produced by certain bacteria such as Gram-positive mycobacteria. Recently, porin structure modification has also been observed as an important contributor to resistance development. For example, modification of the nonselective porin OmpK36 through a selected insertion of Gly115-Asp116 into loop 3 of the porin has caused significant constriction and increased tolerance to the antibiotic carbapenems in Klebsiella pneumoniae. Similarly, multiple mutations in OmpC affect the permeability of antibiotics such as cefotaxime, gentamicin or imipenem in multidrug resistant E. coli. These resistance mechanisms hamper the treatment of diseases, such as pneumonia and bloodstream infections, highlighting the need for novel therapeutic approaches.

Heterocyclic scaffolds are omnipresent as the common cores in a wide range of active pharmaceuticals and thus naturally have caught the significant attention of medicinal chemists over the decades. Among various heterocyclic scaffolds, nitrogen-containing ones hold particular importance due to their widespread occurrence in nature. These structures serve as integral subunits in various natural products, including vitamins, hormones, antibiotics, etc. Analysis of the U.S. Food and Drug Administration (FDA) databases has shown that approximately 60% of unique small-molecule drugs contain nitrogen-based heterocycles, underscoring their structural importance in drug design and discovery. Of these, triazoles featuring a five-membered ring of two carbon atoms and three nitrogen atoms have been recognized as an important scaffold due to their unique physicochemical properties. , The name triazole was first coined by J. A. Bladin in 1885. Since then, its chemistry and biology have undergone tremendous advancement, facilitated by efficient synthesis methods and applications. Triazoles act as versatile alternatives to a range of aromatic rings and functional groups. These properties render it an attractive heterocycle for drug design, offering a less lipophilic alternative to phenyl rings and serving as a substitute for other heterocycles such as homologous azines. Triazole demonstrates unique physicochemical properties, characterized by its weak basicity, diverse dipole moments, and dual capabilities as both a hydrogen bond donor and acceptor, which are critical in mediating drug-target interactions. The intrinsic polarity of triazole contributes to a reduced logP value, indicating potential enhancements in water solubility. The presence of nitrogen atoms with lone pairs that are capable of metal coordination also makes triazole a valuable scaffold for designing inhibitors. The ease of electrophilic substitutions on the triazole ring leads to the development of novel scaffolds with improved therapeutic effects.

1,2,3-Triazoles are five membered heterocyclic compounds with a 6π electron ring system composed of two carbon and three nitrogen atoms. Several methods for the synthesis of 1,2,3-triazoles have been reported in the literature. However, Click chemistry made it considerably easier to synthesize 1,2,3-triazoles allowing researchers to create hybrids of choice with the desired functionalities. Sharpless established the notion of Click chemistry in 2001 to alleviate the problems and obstacles associated with conventional synthetic techniques. The concept of Click chemistry focuses on efficiency and selectivity in terms of both regio and stereoselectivity. The Click technique paves the way to the discovery of lead compounds and allows for fast exploration of chemical space. It improves optimization through an efficient structure–activity relationship (SAR) by providing various scaffolds. 1,2,3-Triazoles can bind with biomolecular targets like enzymes, receptors, and proteins through various noncovalent interactions such as hydrogen bonding, van der Waals interactions, etc. Due to its similar dipole moments, size, and hydrogen-bond acceptor properties, the 1,2,3-triazole structure has gained attention in drug discovery as a bioisostere for amides. , These physiochemical properties of the 1,2,3-triazole ring attracted researchers for the development of novel scaffolds with various therapeutic applications such as antibacterial, , anticancer and fungicidal activity. Several 1,2,3-triazole-containing drugs including seviteronel, cefatrizine, tazobactam, rufinamide, daridorexant, mubritinib, and suvorexant (Figure ) have already been approved by the FDA for clinical use in anticancer, antibacterial, and antiepileptic therapies. These approvals highlight the growing clinical significance of 1,2,3-triazoles in modern medicine.

1.

Chemical structures of some of the FDA approved 1,2,3-triazole-containing marketed drugs.

Molecular hybrids generated by a combination of two or more active pharmacophores have emerged as a promising alternative to traditional therapeutics, offering increased molecular diversity through the combination of multiple subunits. The concept of designing molecular hybrids for therapeutic purpose was first introduced by Fink et al. in 1996 where they tried to design a single molecular framework to inhibit both acetylcholine esterase and monoamine oxidase for treating Alzheimer’s disease. Since then, there has been a sharp upsurge in research output from both academia and industries on various molecular hybrids to target complex multifaceted diseases such as cancer, diabetes, Alzheimer’s, microbial infections, , etc. Among various molecular hybrids designed so far, 1,2,3-triazole-based ones have attracted significant attention due to their facile synthesis which involves readily available or custom-synthesized alkyne and azide components. Additionally, these compounds demonstrate high biocompatibility and show considerable potential in combating antibiotic-resistant bacteria. The antibacterial activity of 1,2,3-triazoles hybrids highly depends on their molecular composition. The presence of lone pairs on the nitrogen atom of 1,2,3-triazoles confers a coordination effect that is most likely observed with certain metalloproteins as well as free metal ions present in bacteria, resulting in the inhibition of metalloenzyme activities and thus reduces their biological functions, depriving the availability of essential nutrients and inhibiting their growth. The 1,2,3-triazole-based hybrids have been reported to inhibit several critical bacterial enzymes such as DNA gyrase, dihydrofolate reductase, sterol 14-α-demethylase causing bacterial cell death. It is observed that 1,2,3-triazole-based hybrid molecules can target both Gram-positive and Gram-negative strains by generating reactive oxygen species (ROS), thus affecting the metabolic activity and disrupting membrane integrity. It also interacts with the DNA polymerase enzyme responsible for the replication of chromosomal DNA, affecting the survival of bacterial cells by disrupting their cell division. Moreover, the molecular hybridization of 1,2,3-triazole with existing antibiotics resulted in more potent antibacterial drugs that can operate as a framework to improve antibacterial properties more effectively than combination treatment. The incorporation of a 1,2,3-triazole ring improves the overall stability of the molecule and also makes it less susceptible to bacterial resistance due to the multimodal action. Furthermore, triazole hybrids have been shown to inhibit biofilm formation and make the bacteria more susceptible to antibiotics. Thus, 1,2,3-triazole can be an excellent candidate for developing antibacterial agents with multiple targets. In this Perspective, we aim to analyze the structural and functional aspects of recently reported 1,2,3-triazole hybrid molecules and their primary modes of action. Additionally, we examine the role of 1,2,3-triazole as a pharmacophoric unit in enhancing antibacterial activity and explore how 1,2,3-triazole structures may contribute to combating the development of antimicrobial resistance. Finally, we discuss the limitations and challenges associated with 1,2,3-triazole-containing hybrid compounds with the goal of providing a clear understanding of their potential and current constraints. The multifaceted processes by which these chemicals exert their effects, together with their structural diversity, open up new possibilities for the generation of successful treatments.

2. Chemical Properties and Synthesis of 1,2,3-Triazole-Based Molecular Hybrids

1,2,3-Triazoles serve as versatile linkers that connect two bioactive moieties as well as function as a robust pharmacophore capable of binding to various biological targets. This feature has been frequently used in medicinal chemistry in the development of hit compounds, particularly for bidentate inhibitors. In hybrid drug design, the triazole ring is often used to form homodimers or heterodimers, which are made up of two identical or distinct pharmacophores, respectively. In heterodimers, it connects two pharmacophoric units that may simultaneously target multiple sites or pathways. In the case of bidentate inhibitors, the triazole linker is crucial to enable the molecule to interact with two distinct binding sites on a target protein. , This dual engagement often results in a stronger binding affinity and improved selectivity, making these inhibitors more effective in modulating the activity of their target proteins.

In a five-membered ring structure, triazoles can exist in two isomeric forms, 1,2,3- and 1,2,4-triazoles, based on the arrangement of nitrogen atoms in the ring, and there are two tautomeric forms for each of them (Figure a). For 1,2,3-triazoles, it can be 1H-1,2,3-triazole and 2H-1,2,3-triazole whereas the 4H-1,2,3-triazole is generally less stable due to its nonaromatic character. For 1,2,4-triazoles, the tautomeric forms can be 1H-1,2,4-triazole and 4H-1,2,4-triazole. The structural aspects of 1,2,3-triazoles enable them to mimic various functional groups, particularly amide bonds, making 1,2,3-triazoles widely used as bioisosteres in drug design to create novel active compounds. Amide bonds are susceptible to hydrolysis in the presence of endogenous peptidases/proteases, and thus have a poor pharmacokinetic profile. , Therefore, in drug designing, amides have often been replaced by triazoles, which offer better metabolic stability. Despite variation in the dipole moment and substituent spacing, their structural characteristics enable efficient mimicry and overlap with amide-binding groups. Amides and triazoles are similar in several key aspects including substituent distances (3.8–3.9 Å for amides and 5.0–5.1 Å for 1,2,3-triazoles), dipole moments (approximately 4 D for amides and 5 D for 1,2,3-triazoles), and hydrogen bond donor/acceptor capacities (Figure b). Substituted 1,2,3-triazoles can be of two types, 1,4-disubstituted and 1,5-disubstituted. 1,4-Disubstituted 1,2,3-triazoles are excellent Z-trans-amide isosteres: The C-4 atom serves as an electrophilic site; the C–H bond functions as a hydrogen bond donor, and the lone pair of electrons on N-2 and N-3 acts as a hydrogen bond acceptor. On the other hand, 1,5-disubstituted 1,2,3-triazoles effectively mimic E-cis-amides through the optimal spatial overlap of their substituents. However, there are some polarity differences due to the difference between the carbonyl group of amides and electronegative nitrogen atoms of the triazole ring. Trans amides are naturally occurring, whereas cis-amides are sterically unfavorable due to substituent repulsion. This steric barrier is responsible for the higher utilization of 1,4-disubstituted triazoles over their 1,5-disubstituted counterparts, despite the emergence of ruthenium-based catalysts to synthesize the 1,5-regioisomer. This isosteric replacement is not only limited to amides but also to other groups such as phenyl, trans olefins as well as for carboxylic acids and esters upon proper substitutions.

2.

(a) Isomeric forms of the triazole nucleus and their tautomerization, (b) 1,2,3-triazoles as nonclassical bioisosteres for amides, and (c) synthesis of 1,2,3-triazole before and after the advent of Click chemistry.

1,2,3-Triazoles are planar molecules capable of experiencing strong dipole moments up to 5.2–5.6 D depending on the substitution patterns. The 1,2,3-triazole ring often effectively interacts with the target rather than just functioning as a linker because of its capacity to engage through a variety of strong intermolecular interactions such as hydrogen bonding, π–π stacking interactions, etc., by virtue of its high dipole moment. Its structural and electronic features can dramatically increase the compound’s biological activity by directly influencing target interactions. 1,2,3-triazoles typically interact with hydrophobic residues in binding pockets of the proteins, especially with tryptophan (Trp) residues. Their planar, aromatic structure allows for edge-face and face–face stacking interactions. The superior stability exhibited by the 1,2,3-triazole ring against metabolic degradation made it an important scaffold in drug development. They are often resistant toward both acidic and basic hydrolysis, oxidative, and reductive conditions. In a comprehensive metabolic study, Massarotti et al. reported that the 1,4-disubstituted 1,2,3-triazoles exhibited unexpected stability against enzymatic degradation as compared to the 1,5 disubstituted 1,2,3-triazoles highlighting the prolonged availability of the former for therapeutic action with no significant toxicity. The favorable spatial arrangement of 1,5-substituted 1,2,3-triazoles toward enzymatic degradation (e.g., cytochrome P450-mediated oxidation) could be one of the reasons for this distinct metabolic stability of these two isomers. Additionally, it has been reported that 1,5-disubstituted 1,2,3-triazoles can undergo oxidation to form triazole N-oxide when incubated with rat liver microsomes. However, N-oxide formation was not observed for 1,4-disubstituted 1,2,3-triazoles, further establishing their superior metabolic stability. ,

Since the advent of the Click chemistry concept in 2001 by Sharpless, Kolb and Finn, it stands out as the most efficient, simple and eco-friendly method for the synthesis of 1,2,3-triazoles. This approach is based on the copper­(I)-catalyzed azide–alkyne cycloaddition (CuAAC) reaction that offers a highly selective and straightforward route for the synthesis of 1,4 disubstituted 1,2,3-triazole compared to earlier efforts (Figure c). By combining an azide and an alkyne under mild conditions with a copper­(I) catalyst, Click chemistry facilitates the formation of 1,4-disubstituted 1,2,3-triazoles with high yield and purity that has been widely explored for making various library of molecules for drug discovery. Subsequently, Fokin and co-workers developed a ruthenium catalyzed azide alkyne Click reaction (RuAAC) using Cp*RuCl­(PPh₃)₂ complex for the selective synthesis of 1,5 disubstituted 1,2,3-triazoles. In CuAAC, two copper­(I) atoms facilitate the formation of 1,4-substituted 1,2,3-triazoles, where the terminal nitrogen of the azide attacking the substituted carbon of the sigma-bonded alkyne. In RuAAC, a single metal complex is involved, where the first carbon nitrogen bond is formed when the terminal nitrogen attacks the more electronegative and sterically demanding carbon of the pi-coordinated alkyne, selectively producing the 1,5-substituted isomer. Apart from metal catalyzed Click reaction, a metal free strategy has also been reported in 2016 for the synthesis of 1,5-disubstituted triazole where primary amines, enolizable ketones, and 4-nitrophenyl azide are heated together in the presence of acetic acid as a catalyst. Organocatalytic 3 + 2 cycloaddition has also been reported for the synthesis of 1,5 disubstituted 1,2,3-triazoles using organic catalysts such as 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU). Other methods used for the synthesis of 1,2,3-triazoles include ionic liquids as catalysts, microwave and ultrasound-assisted synthesis, etc.

3. Antibacterial Activity of 1,2,3-Triazole-Based Hybrid Compounds

Bacteria continue to develop resistance to antibiotics available on the market, which is a serious public health problem and this demands new antibiotics that would be effective against resistant strains, yet avoid the development of resistance. Designing new alternative medicines requires an innovative and modular approach to address the issue of resistance development. Currently, most existing antibiotics follow a one-drug, one-target model, making them highly susceptible to target modifications, such as mutations. A multitargeting technique would be advantageous, because if one encounters resistance, the other will still work. In this context, we observe that 1,2,3-triazole is a simple and efficient molecule that can be used to combine several distinct mechanisms of action in a single molecule. 1,2,3-triazole hybrids are effective against a broad spectrum of microorganisms, including drug-resistant pathogens. They function through various modes of action (Table ), which are covered in detail in this perspective. These mechanisms include the inhibition of important bacterial enzymes, the production of intracellular ROS, and the rupture of the phospholipid bilayer of the bacterial cell. The strategic combination of multiple modes of action in a single triazole-based compound could result in effective antibacterial agents. In the following section, we present an in-depth overview of various triazole-based hybrids that have demonstrated notable antibacterial activity with a particular focus on their effectiveness against resistant strains and the primary mode of action through which they exert their action.

1. Antibacterial Activity of the 1,2,3-Triazole Hybrids Discussed and Their Major Mode of Action .

Compound	Antibacterial activity	Mode of action	References
1	MIC	Enzyme inhibition (DNA gyrase)
	E. coli, 0.0016 μg/mL
	B. subtilis, 0.0016 μg/mL
	S. epidermidis, 0.0032 μg/mL
	P. aeruginosa, 0.0032 μg/mL
2	MIC	Enzyme inhibition (DNA gyrase)
	E. coli, 25 μg/mL
	K. pneumoniae, 100 μg/mL
	B. subtilis, 125 μg/mL
	S. pyogenes, 100 μg/mL
	S. aureus, 100 μg/mL
3	MIC	Enzyme inhibition (DNA gyrase)
	E. coli, 25 μg/mL
	K. pneumoniae, 100 μg/mL
	P. vulgaris, 100 μg/mL
	S. aureus, 200 μg/mL
4	MIC	Enzyme inhibition (DNA gyrase, sterol 14α-demethylase)
	B. subtilis, 6.2 μg/mL
	S. aureus, 12.5 μg/mL
	E. coli, 6.2 μg/mL
	P. aeruginosa, 12.5 μg/mL
5	MIC	Enzyme inhibition (DNA gyrase)
	S. aureus, 0.195 μg/mL
	K. pneumoniae, 0.195 μg/mL
6	MIC	Enzyme inhibition (DNA gyrase)
	S. aureus: 0.391 μg/mL
7	MIC	Enzyme inhibition (DNA gyrase)
	E. faecalis, 0.018 μg/mL
8	MIC	Enzyme inhibition (DNA polymerase)
	MRSA, 1.2–2.4 μg/mL
9	MIC	Enzyme inhibition (DHFR)
	S. aureus, 10 μg/mL
	E. coli, 36 μg/mL
	K. pneumoniae, 70 μg/mL
	P. putida, 60 μg/mL
10	Disk diameter in mm	Enzyme inhibition (DHFR)
	E. coli, 19 mm
	S. aureus, 16 mm
	Well diameter in mm
	E. coli, 21 mm
	S. aureus, 18 mm
11	MIC	Enzyme inhibition (cysteine synthase)
	E. coli, 1.6 μg/mL
12	MIC	Enzyme inhibition (Inh A, CYP121)
	M. tuberculosis, 1.6 μg/mL
	E. coli, 50 μg/mL
	S. aureus, 50 μg/mL
	P. aeruginosa, 100 μg/mL
13	MIC	Enzyme inhibition (MurB)
	S. aureus C-18:0.65 μg/mL
	S. aureus ″Viotko″ strains: 3.75 μg/mL
14	MIC	Enzyme inhibition (MurB)
	E. coli, 0.15 μg/mL
	B. subtilis, 0.02 μg/mL
15	MIC	Enzyme inhibition (transpeptidases)
	P. aeruginosa, 1.25 μg/mL
16	MIC	Ribosomes (50s subunit)
	MRSA, 0.25 μg/mL
17	MIC	Ribosomes (50s subunit)
	MRSA, 16 μg/mL
18	MIC	Ribosomes (50s subunit)
	VRSA, 16 μg/mL
19	MIC	Membrane disruption
	MRSA, 4 μg/mL
	E. faecalis, 4 μg/mL
	S. pneumoniae, 4 μg/mL
	C. difficile, 4 μg/mL
20	MIC	Membrane disruption
	MRSA: 4 μg/mL
	E. faecalis-8 μg/mL
	S. pneumoniae-8 μg/mL
	C. difficile-16 μg/mL
21	MIC	Membrane disruption
	MRSA, 4 μg/mL
	E. faecalis, 4 μg/mL
	S. pneumoniae, 4 μg/mL
	C. difficile, 8 μg/mL
22	MIC	Membrane disruption
	S. aureus, 4 μg/mL
	E. faecalis, 16 μg/mL
	S. agalactiae, 8 μg/mL
	E. coli, 8 μg/mL
	P. aeruginosa, 8 μg/mL
23	MIC 50	Membrane disruption
	S. pneumoniae, 62.53 μg/mL
	E. faecalis, 36.66 μg/mL
	E. coli, 15.28 μg/mL
	K. pneumoniae, 261 μg/mL
	P. aeruginosa, 134.7 μg/mL
	S. enterica, 402 μg/mL
24	MIC	ROS generation
	E. coli, 134 μg/mL
25	MIC	ROS generation
	S. aureus, 7.8 μg/mL
	B. subtilis, 7.8 μg/mL
	M. luteus, 3.9 μg/mL
	K. planticola, 15.6 μg/mL
	E. coli, >250 μg/mL
	P. aeruginosa, >250 μg/mL
26	MIC	ROS generation
	K. pneumoniae, 2 μg/mL
27	EC 50	ROS generation
	Xac, 2.87 μg/mL

^a M. luteus-Micrococcus luteus, S. pyogenes-Streptococcus pyogenes, P. vulgaris-Proteus vulgaris, M. tuberculosis-Mycobacterium tuberculosis, C. difficile-Clostridioides difficile, S agalactiae-Streptococcus agalactiae, S. enterica-Salmonella enterica, S. pneumoniae-Streptococcus pneumoniae, K. planticola-Klebsiella planticola, Xac-X. axonopodis pv citri

3.1. 1,2,3-Triazole Hybrids That Target Critical Bacterial Enzymes

One of the primary mechanisms of action attributed to 1,2,3-triazole hybrids is the inhibition of the critical enzymes required for bacterial survival. Here, we discuss some of those enzymes that are targeted by 1,2,3-triazole containing hybrids and validated by experimental analysis or computational studies. Enzymes or proteins are essential for both the survival and proliferation of bacteria and the generation of antibiotic resistance. Targeting these enzymes can disrupt essential metabolic processes, leading to inhibition of bacterial growth. The enzyme DNA gyrase is one such target that plays a key role in bacterial cells by introducing negative supercoils to DNA, which is essential for DNA replication, transcription, etc. It is an important and clinically validated target of many antibiotics due to its critical function. A library of novel dehydroacetic acid (DHA) chalcone-1,2,3-triazole hybrids were designed and synthesized by Lal and co-workers in 2018, as potent antibacterial agents against a variety of pathogenic bacteria such as Staphylococcus epidermidis (S. epidermidis), Bacillus subtilis (B. subtilis), Escherichia coli (E. coli) and P. aeruginosa. Chalcone represents an important class of natural product, flavonoids, that has large number of replaceable hydrogens that attracted synthetic chemists to introduce structural diversity. Structurally, chalcones are α,β-unsaturated aromatic ketones and display a wide range of pharmaceutical and biological activities including anticancer, antidiabetic, anti-inflammatory, and antibacterial. The DHA-chalcone alkyne and substituted benzyl azide derivatives were Clicked to obtain a library of compounds. They demonstrated antibacterial activity comparable to standard antibiotic ciprofloxacin, which exhibited a minimum inhibitory concentration (MIC) of 0.0015 μg/mL. In the synthesized library, compound 1 (Figure ) was found to be the most effective against E. coli and B. subtilis with an MIC value of 0.0016 μg/mL. Molecular docking analysis further indicated that compound 1 effectively binds to the active sites of DNA gyrase through hydrogen bonding, hydrophobic interaction, and electrostatic interactions. The SAR studies revealed that the attachment of substituted benzyl groups through the triazole ring increased antibacterial efficacy, highlighting the importance of the 1,2,3-triazole unit. Recently, a novel series of 1,2,3-triazole hybrids was designed and synthesized by Mokariya et al. using an indole scaffold which exhibited excellent antibacterial activity against both Gram-positive and Gram-negative bacterial strains. They synthesized different derivatives by changing the substituents such as Cl, F, NO₂, Me, COOH, etc. on the benzene ring attached to the 1,2,3-triazole group. The most potent compound in their library, compound 2 (Figure ) with a meta-chloro substitution on benzene ring attached to the triazole exhibited an MIC of 25 μg/mL against E. coli. Compound 2 was also found to interact with the active site of DNA gyrase in the molecular docking study. Similarly, Suryapeta et al. synthesized 1,4-disubstituted 1,2,3-triazole derivatives with an indole-triazole-peptide conjugate. In their library, compound 3 (Figure ) exhibited the most potent activity against four different bacterial strains tested, including drug-resistant S. aureus (MIC = 200 μg/mL) and K. pneumonia (MIC = 100 μg/mL). They observed that the activity changes according to the nature and position of the R group on the benzene ring linked to the 1,2,3-triazole core. When docked with DNA gyrase, compound 3 exhibited strong interactions with the target protein. In another study by Yadav and co-workers, the design and synthesis of indole-chalcone linked 1,2,3-triazole hybrids was demonstrated that exhibited strong antibacterial properties against Gram-positive bacteria, Gram-negative bacteria, and fungal strains. From the SAR analysis, they identified the most potent compound, compound 4 (Figure ), with an MIC of 6.2 μg/mL against B. subtilis and E. coli. The bromine substitution on the benzene ring imparted better activity as compared to the chlorine substituent. In-silico studies revealed that the reported compounds interact with the bacterial DNA gyrase enzyme through hydrophobic interactions, leading to antibacterial activity. Furthermore, in-silico studies with sterol 14α-demethylase (CYP51), a cytochrome P450 enzyme, which is an important enzyme in sterol biosynthesis and a primary drug target for antifungal azoles, showed that the triazole-containing compound exhibited π–π stacked interactions and other hydrophobic interactions, resulting in overall inhibition of the enzyme’s functioning and potential antimicrobial effects.

3.

Chemical structures of potent 1,2,3-triazole hybrids that have been reported to inhibit critical bacterial enzymes with their respective MICs against the tested bacterial strains.

In another report, Patel et al. synthesized a library of ciprofloxacin-conjugated 1,2,3-triazoles, where the C-3 carboxylic group of ciprofloxacin was linked to various substituted 1,2,3-triazole rings and other variations were further introduced in the piperazine moiety. The antibacterial efficacy of the synthesized compounds was evaluated against various bacterial strains. Most of the reported compounds demonstrated strong antibacterial activity, particularly against strains such as E. coli, S. aureus, and clinical isolates of A. baumannii. MIC data indicated that the nature, type, and position of the substituents on the benzene ring significantly influenced the antibacterial efficacy. The SAR studies showed that compounds with acetyl and benzoyl groups on the piperazine moiety exhibited enhanced activity, especially against S. aureus, E. coli, and A. baumannii. However, compound 5 (Figure ) with an unsubstituted piperazine moiety showed excellent antibacterial activity against S. aureus with MIC of 0.195 μg/mL, which is 32 times more effective than the standard drug ciprofloxacin, having MIC of 6.25 μg/mL. In-silico studies such as molecular docking and molecular dynamics simulations revealed a strong interaction of the active compounds with the DNA gyrase enzyme. Further, the same has been experimentally validated via an in vitro DNA gyrase inhibition assay. This assay suggested that compound 6 (MIC 0.391 μg/mL against S. aureus) (Figure ) having a bromine substituent on the benzene ring exhibited the highest inhibition of the DNA gyrase enzyme, and the inhibition was better than the unmodified ciprofloxacin used as a positive control.

Further, a series of 2-quinolone-1,2,3-triazole-α-amino phosphonates was synthesized by Gadali et al. leveraging the structural similarity of α-amino phosphonates to α-amino acids, peptides, and natural phosphates. The study detailed the synthesis of novel compounds through a molecular hybridization approach, combining α-aminophosphonate, 1,2,3-triazole, and 4-methyl-2-quinolone moieties in a single scaffold. Four bacterial strains, E. coli, P. aeruginosa, Enterococcus faecalis (E. faecalis), and S. aureus were used to evaluate the antibacterial activity of the synthesized compounds. The chloro-substituted hybrid, compound 7 (Figure ) showed an MIC of 0.018 μg/mL which is 8.7-fold and 10-fold better than the standard antibiotics, ampicillin and rifampicin, respectively against E. faecalis. Derivatives that had 2-bromo and 4-iodo phenyl substitutions on the benzene ring showed MIC values of 0.036 μg/mL against E. faecalis. Molecular docking analysis of compound 7 with bacterial DNA gyrase demonstrated strong interactions such as hydrogen bonding and hydrophobic contacts, which contributed to the inhibition of DNA gyrase activity.

Methicillin resistant Staphylococcus aureus (MRSA) is one of the most dangerous superbugs that cause deleterious effects such as sepsis and endocarditis in humans. Recently, Tang et al. synthesized triazolyl pterostilbene derivatives and tested against MRSA. A Click reaction was performed with propargylated sterostilbenes and selected azides in the presence of sodium ascorbate and copper sulfate to yield a library of hybrids. Among them, significantly low MIC values (1.2–2.4 μg/mL) were exhibited by Compound 8 (Figure ) as compared to pterostilbene (41–161 μg/mL) indicating a great enhancement in anti-MRSA activity. The SAR studies showed that the presence of a carboxylic acid functionality and its spacing from triazole played a key role in the antibacterial activity of the compounds. The compound showed antibacterial properties by inhibiting the replication of bacterial DNA. The mechanism of action was analyzed via experimental as well as molecular docking analysis; the DNA replication test showed the ability of the compounds to inhibit DNA polymerase, and the reduction of PCR products in a dose-dependent manner confirmed the target of the compounds as DNA polymerase. The in-silico studies also indicated that the triazole group and the oxygen that connect to the pterostilbene scaffold formed hydrogen bonds with Lys 450 and Arg 435, respectively. These findings support the experimental validation and suggest a potential interaction between the triazole group and DNA polymerase enzyme, which plays a crucial role in chromosomal DNA replication.

Aarjane et al. have designed and synthesized novel 1,2,3-triazoles from acridone, another nitrogen containing heterocycle with a wide pharmacological application from antimicrobial to anticancer. , The synthesis was started with the alkyne functionalization of acridone using propargyl bromide on the acridone skeleton which were presynthesized using an Ullman condensation reaction 2-bromobenzoic acid and aniline derivatives. The final compound was synthesized by using a Click reaction with aromatic azide derivatives. The antibacterial activity of the synthesized 1,2,3-triazole compounds were evaluated against S. aureus and Pseudomonas putida (P. putida), K. pneumoniae, and E. coli. It was found that, S. aureus was most sensitive to the synthesized compounds. The SAR studies showed that compounds which feature an O-methylphenyl group on the acridone-1,2,3-triazole skeleton, demonstrated the highest antibacterial activity against S. aureus whereas carboxylic acid functionalization decreased the activity. Compound 9 (Figure ) exhibited the most potent activity with an MIC of 10 μg/mL against S. aureus (Table ). The mechanism of action of these compounds was revealed to be through inhibition of the dihydrofolate reductase (DHFR) enzyme, which plays an important role in thymidylate biosynthesis and cell proliferation. Molecular docking studies revealed effective binding of O-methylated derivatives into the active site of DHFR.

In 2022, Malah et al. synthesized a library of ten isatin-1,2,3-triazole hybrids through a Cu­(I)-catalyzed cycloaddition of N-propargylated isatin with various azide derivatives. The N-propargylated isatins were prepared by reacting 1H-indole-2,3-dione with propargyl bromide in an alkaline DMF solution. The antimicrobial activity of the final compounds was evaluated against different pathogenic microorganisms, including E. coli, S. aureus, C. albicans, and A. niger, and it was observed that the activity was highly dependent on the substituents on the N-phenyl group. The compounds showed antibacterial effects through different mechanisms such as reduction of ATP production in bacterial cells, inhibition of DHFR, which is an essential cytoplasmic protease with NADPH as a cofactor involved in the reduction of dihydrofolate to tetrahydrofolate thereby playing a crucial role in DNA synthesis, DNA translation, RNA transcription, protein replication, and controlling cell proliferation. Compound 10 (Figure ) exhibited the most potent activity in the library with zone of inhibition (ZOI) (disk diameter in mm) in E. coli, S. aureus, C. albicans, A. niger as 19 ± 0.08, 16 ± 0.28, 14 ± 0.09, and 12 ± 0.11 respectively. Polar substituents enhanced the activity, whereas nonpolar groups reduced the efficacy. The inhibition of DHFR was found to be the key mechanism of action, using molecular docking studies. Similarly, in 2020, Wallace et al. demonstrated the antimetabolite action of 1,2,3-triazole-thioacetamide hybrids. Their lead compound 11 exhibited a very low MIC of 1.6 μg/mL against E. coli, and the mechanism was found to be inhibition of the cysteine synthase A (CysK). CysK present in bacteria plays an important role in several fundamental pathways such as cysteine biosynthesis, sulfur assimilation, and homocysteine biosynthesis. Inhibition of these critical pathways disrupts sulfur metabolism and redox homeostasis in bacteria, resulting in elevated levels of intracellular oxidative stress and affect their resistance development against antibiotics.

Reddyrajula et al. synthesized 36 novel phenothiazine incorporated 1,2,3-triazole hybrids and evaluated their activity against M. tuberculosis. Compared to the tuberculosis standard drug, pyrazinamide, more than half of the synthesized compounds exhibited superior antibacterial activity. Interestingly, these compounds were inactive against other bacteria such as S. aureus, P. aeruginosa, and E. coli, indicating their specificity toward M. tuberculosis. Compound 12 (Figure ) exhibited the lowest MIC of 1.6 μg/mL against M. tuberculosis. To get an insight into its mechanism of action, they demonstrated molecular docking studies with two target enzymes, namely, enoyl reductase (Inh A) and CYP121. A strong enzyme-ligand binding affinity was observed as a result of hydrogen bonding and pi-pi stacking interactions.

In 2019, Lipeeva et al. reported the antibacterial efficacy of 1,2,3-triazole-coumarin hybrids made from plant-derived coumarin. These hybrids exhibited greater potency against S. aureus compared to standard antibiotics, ceftriaxonum and streptomycin. In particular, hybrid 13 (Figure ) (MIC: 0.65 and 3.75 μg/mL) showed substantial activity against S. aureus C-18 and S. aureus “Viotko” strains, indicating its potency in treating S. aureus infections. The SAR analysis showed that carboxylic acid at the para-position of the phenyl ring was essential for excellent activity against S. aureus. Molecular docking studies further indicated a strong interaction of compound 13 with UDP-N-acetylenolpyruvylglucosamine reductase (MurB) protein, suggesting a potential enzyme inhibition leading to the observed antibacterial activity. MurB is an essential enzyme present in bacteria that plays a major role in the synthesis of cell wall precursor (uridine diphosphate N-acetyl muramic acid (UNAM)). Thus, targeting this enzyme results in disruption of cell wall biosynthesis leading to inhibition of bacterial growth. Adding a furocoumarin moiety in the hybrids significantly improved their efficacy against E. coli and B. subtilis, as observed in the coumarin-1,2,3-triazole-furocoumarin hybrid 14 (Figure ). Compound 14 exhibited MICs of 0.15 μg/mL and 0.02 μg/mL, respectively, against E. coli and B. subtilis which are significantly improved as compared to the standard antibiotic streptomycin with MICs of 1 μg/mL and 2 μg/mL against E. coli and B. subtilis, respectively. Further studies revealed that the linker between the 1,2,3-triazole and coumarin moieties, as well as its position on the coumarin ring, had a significant impact on the activity.

β-Lactam antibiotics are a large class of antibiotics that includes penicillins, cephalosporins, nocardicins, carbapenems and monobactams. The azetidine-2-one ring system is the key to biological activities in β-lactam antibiotics. The primary mode of action of β-lactams to kill bacteria is inhibition of transpeptidases, which form peptidoglycan by cross-linking the peptides. The hybridization of 1,2,3-triazole with the β-lactam motif has gained significant interest among medicinal chemists, as it may have the potential to overcome drug resistance. , For example, in 2020, Kaur et al. reported triazole β-lactam conjugates using Click chemistry. Compound 15 (Figure ) containing 3-nitro or 3-chloro substituted benzene reported in their library exhibited an MIC of 1.25 μg/mL against B. subtilis and P. aeruginosa. In addition to focusing on the different essential proteins and enzymes required for bacterial survival, antibacterial drugs have been created that target the ribosome itself, which is the precise cellular organelle that makes proteins. Since protein biosynthesis is a fundamental process in all living organisms, the ribosome has become a key target for a wide variety of antibiotics. To address the challenges associated with these antibacterial agents, such as low bioavailability, resistance development and toxicity, researchers have explored the hybridization of antibiotics with 1,2,3-triazoles. Pleuromutilin, first isolated in 1951 from Pleurotus mutilus, functions by inhibiting bacterial protein synthesis through interaction with the 23S rRNA of the 50S ribosomal subunit. Several pleuromutilin hybrids have been developed for enhanced antibacterial action, showing a weak tendency to induce cross-resistance. However, these hybrids often suffer from poor bioavailability and CYP450 inhibition. To address these limitations, 1,2,3-triazole-pleuromutilin hybrids have been developed. In 2021, Zhang et al. reported the antibacterial activity of synthesized pleuromutilin analogs containing substituted 1,2,3-triazole moieties against MRSA. Their most potent hybrid, compound 16 featuring a tertiary amine group, demonstrated a 8-fold improvement in the MIC (0.25 μg/mL) against MRSA whereas pleuromutilin alone exhibited an MIC of 2 μg/mL. Similarly, macrolide antibiotics such as clarithromycin, erythromycin, and azithromycin are a key group of drugs used to treat bacterial infections. Several studies have demonstrated that these antibiotics can bind to some specific nucleotide residues, such as A2058 and A2059 on the domain V of 23s rRNA present on the 50S subunit of the bacterial ribosome inhibiting the elongation of peptide chains and thereby selectively blocking bacterial protein synthesis. In 2021, Qin et al. reported 1,2,3-triazole azithromycin hybrids showing potent activity against various drug-resistant strains such as MRSA and penicillin-resistant S. aureus (PRSA). Interestingly, compound 17 (Figure ), upon the introduction of a pentyl group to the 1,2,3-triazole ring exhibited the most potent antibacterial activity against MRSA with an MIC of 16 μg/mL whereas the standard drug azithromycin showed MIC > 256 μg/mL. Molecular docking analyses revealed that hybrid 17 has the potential to interact with the A752 base of the 23S rRNA within the bacterial ribosome, likely through a combination of hydrophobic and electrostatic interactions and contribute to exceptional antibacterial activity. Oxazolidinone class of antibiotics are known to attach 50S ribosomal subunit and inhibit the formation of the 70S initiation complex by blocking the assembly of the N-formyl-methionyl-tRNA, ribosome, and mRNA ternary complex. Resistance to oxazolidinone antibiotics occurs mainly due to mutation in 23S rRNA. In 2014, Phetsang et al. reported azide functionalized analogue of oxazolidinone antibiotic linezolid retaining its antibacterial activity. Further Click-mediated hybridization of azido linezolid led to the creation of a library of compounds showing excellent antibacterial activity against a number of drug-resistant Gram-positive bacteria. The most potent hybrid, compound 18 (Figure ) with a 7-nitrobenzofuran moiety showed promising antibacterial activity against VRSA with an MIC value of 16 μg/mL whereas the standard antibiotic vancomycin showed an MIC > 64 μg/mL. In-silico studies and crystal structure analysis revealed that azide-functionalization promoted fitting of the compound in 50S ribosomal subunit similar to that of oxazolidinones and contributes to the antibacterial efficacy.

4.

Representative compounds containing 1,2,3-triazole hybridized with antibiotics along with their respective MICs against the tested bacterial strains.

3.2. 1,2,3-Triazole Hybrids That Disrupt the Bacterial Cell Membrane

The cell membrane is an essential component of bacteria because it provides protection, resistance, and integrity to the cell by regulating the entry and exit of substances. Cell membrane disruption is one of the primary mechanisms by which antibiotics function. It has advantages over other mechanisms as the possibility of resistance development is comparatively low, as they completely destroy the bacteria other than suppressing their replication or cell division. Cell membrane targeting substituents can be attached to 1,2,3-triazole to develop an efficient antibacterial agent. In this regard, Tague and co-workers developed cationic biaryl 1,2,3-triazolyl peptidomimetic amphiphiles. They have synthesized 43 compounds in three distinct classes using CuAAC. The cationic biaryl amphiphile, 19 (Figure ) was identified as the most effective agent with an MIC of 4 μg/mL against MRSA. The SAR studies suggested that the ratio of cationic residues to hydrophobic residues within the peptidomimetic amphiphiles has a strong influence on their antimicrobial activity, hemolytic activity, and thus membrane selectivity.

5.

Representative 1,2,3-triazole hybrids that function through bacterial cell membrane disruption along with their respective MICs against the tested bacterial strains.

Antibacterial agents derived from basic amino acids, such as lysine and arginine, have shown the ability to disrupt the bacterial membrane. In 2019, Tague and colleagues synthesized a series of 1,2,3-triazole-arginine hybrids. In their synthesized library, compound 20 (Figure ) with ethylbenzene substituent showed excellent activity against a variety of bacterial strains such as MRSA clinical isolates, vancomycin-resistant S. aureus (VRSA) and Vancomycin-resistant E. faecalis (VRE), etc. with MIC values as low as 4 μg/mL. Compound 21 (Figure ) with a cyclohexyl substituent also showed promising antibacterial activity against MRSA with an MIC value of 4 μg/mL. The obtained MIC values were comparable with that of the standard antibiotic vancomycin (MIC of 1–32 μg/mL) against all these strains. Cytoplasmic membrane depolarization and disruption of membrane potential were found to be the key mechanism of bacterial inactivation.

In 2017, Bakka et al. reported a library of 29 1,4-substituted 1,2,3-triazoles containing amphiphilic antimicrobial peptides (AMPs) that can disrupt the bacterial cell membrane. The AMPs’ cationic charge can be drawn to the bacteria’s negatively charged cell membrane, whereupon the hydrophobic portion enters the phospholipid bilayer and disrupts the membrane. The hypothesis behind the incorporation of triazoles into AMPs was to bypass its limitations such as poor bioavailability and metabolic stability. Click chemistry allowed them to easily connect the hydrophilic and lipophilic parts through the triazole ring. Compound 22 (Figure ) with a guanidium substituent exhibited the most potent antibacterial activity with an MIC of 4 μg/mL against S. aureus. Similar to conjugation with AMPs, Aneja et al. designed and synthesized 1,2,3-triazole analogues of natural bioactive precursors for potential antibacterial activity against resistant bacterial strains using Click reaction. Carvacrol, naphthoquinone, and 8-hydroxyquinoline were used as precursors for modification. The most potent compound 23 (Figure ) exhibited significant antibacterial activity against S. pneumoniae (MIC₅₀= 62.53 μg/mL. where MIC₅₀ is the concentration that inhibits 50% bacterial growth), E. faecalis (MIC₅₀= 36.66 μg/mL,), and E. coli (MIC₅₀= 15.28 μg/mL). The growth kinetics on S. pneumoniae and E. coli treated with 23 exhibited an extended lag phase attesting its bactericidal effect. Transmission electron microscopy studies revealed morphological changes and damage to the cell membrane upon treatment with these compounds. They were also found to inhibit the biofilm formed by S. pneumoniae and E. coli. Together with the membrane disruption property, the added biofilm inhibitory function can aid in preventing the development of bacterial resistance to antibacterial drugs, making it a viable option against MDR pathogens.

3.3. 1,2,3-Triazole Containing Hybrid Molecules That Generate Intracellular ROS

The production of ROS has been found to be a significant contributor to the antibacterial activity as well as efficacy against multidrug-resistant organisms. ROS include radical and nonradical oxygen containing species such as hydroxyl radicals (OH^•), superoxide anion (O₂ ^–), hydroxyl anions (OH^–) singlet oxygen (¹O₂), peroxide (O₂ ^2–), and hydrogen peroxide (H₂O₂). Under normal conditions, ROS levels are tightly regulated by the cellular antioxidant defense system. However, during oxidative stress, ROS levels can exceed the cell’s capacity to neutralize them, leading to toxicity. Compared to mammalian cells that are equipped with a better antioxidant defense system, bacteria are prone to severe damage in the presence of ROS. Apart from lipids, ROS also affects DNA by forming cross-links, modification of the bases, and breaking the strands. Similarly, ROS oxidizes proteins, leading to the alteration of its structure and loss of activity.

Although the 1,2,3-triazole ring itself may not directly contribute to ROS production, ROS generating structural elements can be strategically integrated to the 1,2,3-triazole nucleus. So far, there are only a few reports on the antibacterial activity of ROS producing 1,2,3-triazole hybrids. However, significant studies have been conducted on other biological applications of ROS generating 1,2,3-triazole hybrids, such as anticancer, antifungal, etc. This implies that it is important to highlight the possibility of increasing the usage of these compounds in antibacterial applications. In this respect, Hayden et al. designed and synthesized several water-soluble triazole phenazine derivatives. In their library, the most potent molecule, compound 24 (Figure ) exhibited an MIC of 134 μg/mL against E. coli. One putative mode of action for its antibacterial effect is the incorporation of the phenazine portion of the molecule into the cell membrane, affecting membrane-associated cation transporters and compromising the membrane structural integrity. This can interfere with cellular redox balance, leading to the generation of ROS and ultimately to cytotoxicity. Additionally, phenazine may intercalate with DNA, downregulating DNA-directed protein expression and disrupting essential cellular processes. The toxicity of phenazine is primarily attributed to the formation of radicals within the phenazine structure, which then facilitates the reduction of molecular oxygen, producing ROS.

6.

Chemical structures of some of the 1,2,3-triazole hybrids that generate intracellular ROS are represented along with their respective MICs against tested bacterial strains.

Another class of ROS generating antibacterial 1,2,3-triazole hybrids has been reported by T. Vijai Kumar Reddy and co-workers using 1,2,3-triazole analogues of short-chain C12-sphinganine in 2016. These compounds were able to target both Gram-positive and Gram-negative strains with promising antibacterial efficacy. The most potent compound 25 (Figure ), exhibited antimicrobial effects with an MIC of 3.9 μg/mL against M. luteus. It also showed antibiofilm action against the tested microorganisms such as C. albicans, M. luteus, and S. aureus. These hybrids could generate elevated levels of intracellular ROS such as superoxide anions and hydrogen peroxides as observed in the fluorescence assay using DCFH-DA. In another report, Aljohani et al. developed 1,2,3-triazole derivatives complexed with Zn that exhibited antibacterial activity against K. pneumoniae. Among various hybrids reported, 1,2,3-triazole-sulfadiazine-ZnO could target bacteria that were resistant to the known antibiotic, carbapenem. Compound 26 (Figure ) showed antibacterial properties against K. pneumoniae with an MIC of 128 μg/mL, and its hybrid with ZnO showed enhanced antibacterial properties with an MIC of 2 μg/mL. The primary mode of action for its antibacterial activity was reported as the generation of ROS such as free radicals, superoxide anions, and peroxides. The presence of zinc ions improved the antibacterial properties through their penetration and interaction with nucleic acids, resulting in the inactivation of the enzymes involved in the electron transport chains and enhanced cytokine release. The molecular docking studies conducted on metallo-β-lactamase, a diverse group of enzymes involved in the hydrolysis of a wide range of β-lactam antibiotics, demonstrated the potential for interaction with the enzyme through hydrophobic interactions, resulting in potent inhibition. Additionally, the in vitro enzymatic assay of synthesized compounds against metallo-β-lactamase exhibited a similar inhibitory trend.

In 2021, Huang et al. reported ROS generating 1,2,3-triazole tailored carbazoles as efficient antibacterial agents and tested against plant pathogens such as Xanthomonas oryzae pv oryzae (Xoo), Xac, and Pseudomonas syringae pv actinidiae (Psa). Compound 27 (Figure ) showed potent antibacterial activity against Xac with the lowest EC₅₀ value of 2.87 μg/mL. The most potent compound in their library showed antibacterial activity through the ROS generation mechanism, as experimentally studied by the DCFH-DA assay. As previously mentioned, 1,2,3-triazole hybrids have been explored for various other biomedical applications, leveraging their ability to generate ROS. For example, in 2024, Souza et al. reported a library of naphthoquinone based 1,2,3-triazole hybrids that were shown to have ROS mediated anticancer property. Electron transfer from the quinonoid substructure to molecular oxygen generates ROS that results in irreversible DNA damage in the cells. The most potent compound exhibited the highly elevated levels of ROS production in the synthesized library within 2 h of treatment at a concentration of 6 μM. Another such example is reported by Guo et al., where the authors synthesized a series of 28 cabotegravir derivatives incorporating a 1,2,3-triazole moiety. Electron microscopic analysis revealed that their best compound exhibited a large accumulation of intracellular ROS upon treatment with HuH-7 cells at varying concentrations, which resulted in mitochondrial membrane damage. These studies suggest that 1,2,3-triazole hybrids can be engineered to effectively induce substantial ROS generation, disrupt bacterial membranes, and inhibit key enzymes making them potential candidates for combating multidrug resistant bacteria.

3.4. Combination Therapy

Combination therapy is an emerging and promising strategy for treating diseases in which two or more drugs are used in tandem to achieve better therapeutic outcomes. This offers several advantages over conventional monotherapy, including the multitargeting effect, which can improve treatment efficacy. Combination therapy can also make cells sensitive to other treatments, thus, enhancing the overall therapeutic outcome. Furthermore, it reduces the possibility of drug resistance development and minimizes potential toxicity by using lower doses of individual drugs. Metallo-β-lactamases (MBLs) are a diverse set of enzymes that catalyze the hydrolysis of a broad range of β-lactam antibiotics, rendering them ineffective and significantly contributing to antimicrobial resistance. Muhammad and co-workers reported a series of 1,2,3-triazoles in 2020, following a three-step reaction protocol involving a Banert cascade reaction. The synthesized compounds were found to inhibit different types of MBLs such as Verona integron-encoded metallo-β-lactamase (VIM-2) and New Delhi metallo-βlactamase (NDM-1) exhibiting an IC₅₀ value in nanomolar range. The most effective compounds were then tested for synergistic effects with Meropenem against 3 clinical bacterial strains. A 64- and 16-fold improvement in MIC was observed when treated in combination against P. aeruginosa and E. coli, respectively, compared to Meropenem alone. In a separate study, Rogers et al. reported the synergistic effect of 2-aminoimidazole-triazole conjugate and antibiotics toward the biofilm production/dispersion of S. aureus. Bacteria produce biofilms to protect themselves from harsh conditions and antibiotics for their survival. Due to this protective layer, most antibiotics become ineffective, and treating infections becomes challenging. Therefore, creating drugs with both antibacterial and biofilm-inhibiting properties is beneficial. The 2-aminoimidazole-triazole conjugate showed potential to inhibit and disperse the bacterial biofilms of several microorganisms. A 26-fold enhancement in the activity was also observed when used in combination with 0.1 μM novobiocin antibiotic. Similarly, Su and co-workers evaluated 4,5-disubstituted-2-aminoimidazole–triazole conjugates for the resensitization of antibiotics against multidrug resistant bacteria such as MRSA and A. baumannii. They were also found to disperse or inhibit biofilm formation, and desensitization to the antibiotic oxacillin was observed to be of 2–4 fold. In another report, Negi et al. evaluated the synergistic effects of metronidazole-triazole hybrids against MRSA with oxacillin antibiotic displaying a 4-fold improvement in MIC values compared to the monotreatment. These findings clearly imply that 1,2,3-triazole scaffolds can be successfully used to create hybrids that have a synergistic impact on making bacteria more susceptible to antibiotic exposure.

4. Metabolic Stability and Biocompatibility

Triazoles contain a stable heterocyclic structure which is resistant to hydrolysis, oxidation and reduction. The triazole moiety is commonly metabolized via the phase II N-glucuronidation process. It has been reported that aldehyde glucuronidation occurs at the triazole nitrogen in liver microsomes, with the specific site of glucuronidation varying between organisms. Triazoles have been widely explored in medicinal chemistry to enhance metabolic stability and biocompatibility particularly in the case of inhibitor development. For example, Kuttruff et al. developed potent autotaxin (ATX) inhibitors where they introduced a triazole group in their lead compound that exhibited improved pharmacokinetics and safety of the compound. Substitution of benzimidazolone with benzotriazole, not only enhances metabolic stability but also improves its potency. Brequinar, a human dihydroorotate dehydrogenase (hDHODH) inhibitor, was initially abandoned due to its high toxicity. Development of a 1,2,3-triazole based compound by Sainas et al. in 2017 avoided toxicity toward Jurkat T cells, holding potential for further research in autoimmune diseases. Replacing imidazoles with triazoles in antifungal agents has also enhanced metabolic stability and therapeutic effects. Furthermore, five membered heterocyclic compounds including triazole have been found to have better metabolic stability compared to their six membered counterparts.

5. Antibacterial Activity of 1,2,4-Triazole-Based Hybrid Compounds

Similar to 1,2,3-triazoles, researchers have explored 1,2,4-triazole based hybrids for antibacterial activity against antibiotic sensitive as well as resistant organisms. 1,2,4-Triazole also offers structural diversity for designing antibacterial agents with low cytotoxicity and evading resistance development, although the synthesis pathway is completely different from that of 1,2,3-triazoles (Click reaction). The 1,2,4-triazoles have been extensively explored for the development of small molecule inhibitors particularly as antifungal agents and aromatase inhibitors. They majorly function by inhibiting the active site of the enzymes belonging to the cytochrome P450 family. Letrozole and anastrozole (used for the treatment of breast cancer), fluconazole (antifungal agent), rizatriptan (used against migraines) are some examples of 1,2,4-triazole containing compounds approved by the FDA for the clinical treatment. 1,2,4 triazole moiety has been hybridized with other antibacterial pharmacophores to enhance the biological properties. Some of the recently explored 1,2,4-triazole hybrids for antibacterial activity include 1,2,4-triazole–azole, 1,2,4-triazole-coumarin, 1,2,4-triazole-β-lactam, 1,2,4-triazole-pyrimidine, 1,2,4- triazole-quinoline, 1,2,4-triazole-quinolone, and triazole quinazoline hybrids etc. A detailed accounts on the development of 1,2,4-triazole based antibacterial hybrids can be found in several recently published review articles. ,−

6. Conclusion and Future Outlook

The emergence of antimicrobial resistance continues to pose a threat to our healthcare system, necessitating the development of novel classes of antimicrobial drugs that are less likely to develop resistance. In this perspective, we have discussed strategically designed 1,2,3-triazole hybrids that could work via multiple mechanisms of action to treat bacterial infections. The advantage of multifaceted therapy or multitargeting antibacterial agents is its low propensity to develop resistance against them. Compared to a one-drug, one-target approach that becomes ineffective upon drug resistance, a one-drug multitarget approach offers multiple modes of action for bactericidal effect. While small molecules that target critical enzymes required for bacterial survival or replication have been found effective in antibacterial drug development, these enzyme inhibitors are prone to gain resistance by the virtue of mutation or other mechanisms discussed earlier. In this regard, membrane-targeting antibacterial drugs and/or those capable of producing considerable amounts of ROS work through nonspecific interactions, making it difficult for bacteria to build resistance to these treatments. In the past decade, it has been shown how a 1,2,3-triazole ring can bring various pharmacophores together to bring about multiple mechanisms of action to combat bacterial infection. The 1,2,3-triazole ring can act as a linker for two biologically active molecules, providing an opportunity to fine-tune different functionalities in a single molecule and, hence, diversifying their structural aspects. Moreover, 1,2,3-triazole hybrids have been found to bind to different sites of key enzymes critical for bacterial survival. The hydrogen bond acceptor/donor capabilities, biocompatibility, and ease of synthesis make it an attractive class of compounds. 1,2,3-triazole hybrids can be designed to target bacterial cell membranes and for generating intracellular ROS which can cause damage to different biomacromolecules such as lipids, nucleic acids, and proteins. Targeting various bacterial survival mechanisms (Figure ) is essential to lower the likelihood of drug resistance development, as if one is rendered inactive, the others will continue to fight. Therefore, 1,2,3-triazole hybridization offers a modular, simple, and readily synthesizable method for developing multitarget antibacterial drugs. Nonetheless, there are still a few aspects that require further attention, though. Out of the box design of novel hybrids is warranted which would allow engagement with multiple targets to eliminate pathogenic bacteria, including drug-resistant strains. Molecular hybridization can really be a game changer. Novel 1,2,3-triazole hybrids need to be designed considering the diversity and biological activity of potent pharmacophores capable of exhibiting bactericidal effects.

7.

Design of multitargeting antibacterial 1,2,3-triazole-based hybrids using the Click reaction.

For the past few decades, 1,2,3-triazole hybrids have been extensively explored for various biological activities. The major advantage of these heterocycles is their biocompatible nature emerging out of its chemical stability, nonreactivity and relatively simple structure. However, the cytotoxicity of these hybrids can vary, depending on the substituents attached to the triazole nucleus. To minimize cytotoxicity with selectivity, the hybrid design can be made in such a way as to target unique bacterial proteins. Substituents with a positive charge can interact with the negatively charged bacterial cell membrane, providing selectivity over mammalian cells. This selectivity arises because the outer leaflet of the bacterial cell membrane contains a greater proportion of anionic phospholipids as compared to mammalian cells. Furthermore, the introduction of ROS generating functionalities can provide better killing efficiency in bacterial cells compared to normal cells as the former lacks a well-organized antioxidant defense system. Despite all of the aforementioned advantages of 1,2,3-triazoles, there are concerns that warrant further consideration and investigation. The low water solubility of 1,2,3-triazoles is well recognized and can have a substantial effect on their pharmacokinetics and therapeutic effectiveness. To address this challenge, design can be made in such a way as to attach hydrophilic groups or polyethylene glycol linkers, thereby improving the bioavailability of the triazole hybrids. Nevertheless, in a short period of time, 1,2,3-triazole hybrids have witnessed massive growth in their medicinal applications, particularly as anticancer drugs, antibacterial agents, etc. Most of the 1,2,3-triazole hybrids exhibited excellent antibacterial activity against drug-resistant strains. Therefore, rational development of molecular hybrids with the 1,2,3-triazole nucleus with other pharmacophores having antibacterial activity is a promising strategy to design novel antibacterial lead compounds.

The authors declare no competing financial interest.