Introduction

The increasing threat of multidrug resistance worldwide has posed significant challenges in the development of new, effective antimicrobial drugs 1. Microbial infections often require prolonged treatment with a combination of powerful antibiotics, contributing to the appearance of dangerous drug-resistant strains 2and increasing the risk of overlooked and potentially deadly infections acquired during hospitalization 3. A persistent need for novel antimicrobial agents that provide broad-spectrum activity while ensuring high safety and efficacy subsequently exists. Among various heterocyclic compounds, tetrazoles have emerged as promising agents because of their diverse pharmacological properties, including antibacterial 4, antifungal 5, antimalarial 6,7, antitubercular 8,9, antihypertensive 10, antiallergic 11, and antiviral effects 12, and their role as cholinesterase inhibitors 13,14,15,16. Several marketed drugs contain the tetrazole moiety, such as cefazolin, azosemide, irbesartan, and valesartan 14 (Fig. 1).

Fig. 1
figure 1

Marketed drugs containing the tetrazole ring.

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Tetrazole is a peculiar five-membered heterocyclic compound consisting of four nitrogen atoms and one carbon atom, theoretically standing in three tautomeric arrangements: 1H, 2H, and 5H (Fig. 2). The 1H and 2H tautomers exhibit aromaticity with 6 π-electrons, whereas the 5H tautomer is nonaromatic and has been proposed theoretically without any experimental evidence 17. In its solid-state form, tetrazole primarily exists as the 1H tautomer, and this form also predominates in polar solvents such as dimethylformamide and dimethyl sulfoxide. In contrast, the 2H tautomer is favoured in the gas phase 17. Tetrazole is often used as a bioisosteric substitute for carboxyl and amide groups, enhancing metabolic stability and improving the ADMET (absorption, distribution, metabolism, excretion, and toxicity) of molecules containing these functional groups.

Fig. 2
figure 2

Tautomers of tetrazole.

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Five-membered nitrogen-containing heterocyclic organic compounds where at least one atom within the ring structure is an element other than carbon, such as nitrogen, oxygen, or sulfur 18,19. Particularly, 1,2,3-triazoles and their examples are of great interest due to their wide variety of bioactivities in medicinal applications 20,21. These compounds have various pharmacological properties, including antibacterial 22,23,24,25,26,27, antifungal 28,29,30, antidiabetic 31,32, antioxidant 33,34,35,36, anticancer 37,38,39, antitubercular 40,41,42, anti-inflammatory 43, and antimalarial activities 44,45,46. The usefulness of 1,2,3-triazoles in drug design and synthesis can be attributed to their resistance to metabolic degradation, as they are stable under both acidic and basic conditions and in various oxidoreductase environments 47. Their polar nature is crucial for effective interactions with biological molecules through dipole‒dipole interactions and hydrogen bonding. Several clinically used medicines feature a 1,2,3-triazole skeleton, as illustrated in (Fig. 3). The most common synthetic approach for 1,2,3-triazoles involves the interaction of terminal alkynes and azides to develop a 1,3-dipolar cycloaddition reaction, first reported by Huisgen 48, which results in the formation of 1,4- and 1,5-disubstituted 1,2,3-triazoles. In 2002, Sharpless and Meldal developed a modified version of Huisgen’s reaction 49,50. This Cu(I)-catalyzed cycloaddition, commonly called a “click reaction,” operates under mild conditions and provides improved yields, producing a single isomeric product. The click reaction is valued for its versatility, selectivity, compatibility with various functional groups, and ability to convert reactants into a single isomer under gentle conditions efficiently.

Fig. 3
figure 3

Listed drugs having a 1,2,3-triazole moiety.

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Molecular hybridization is a strategic approach in rational drug design that involves merging distinct pharmacophoric elements from the molecular frameworks of two or more known bioactive compounds to generate new ligands or prototype molecules. This method plays a crucial role in drug discovery, as it allows for the adjustment of undesirable side effects and the development of dual-acting drugs that integrate the therapeutic effects of different agents 51. This prompted us to explore whether combining these two groups could produce compounds with enhanced antibacterial potential 52 (Fig. 4).

Given the limited availability of triazole‒tetrazole hybrid molecules and our goal to develop new, effective antimicrobial agents, we were inspired to synthesize previously unreported triazole‒linked tetrazole conjugates for antibacterial assessment. In the present work, we designed, synthesized, characterized, and evaluated the antibacterial activity of a novel series of triazole-tethered tetrazole derivatives.

Fig. 4
figure 4

Diagrammatic representation of molecular hybridization for bacterial inhibition.

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Results and discussion

Chemistry

The synthesis of triazole-tethered tetrazole derivatives (6a–6o) is outlined in the experimental procedure (Fig. 5). The synthesis began with the preparation of 5-phenyl-2-(prop-2-yn-1-yl)-2 H-1,2,3,4-tetrazole (4a) from 5-phenyl-1 H-1,2,3,4-tetrazole (2) via propargyl bromide (3) in the presence of triethylamine, which yielded 5-phenyl-2-(prop-2-yn-1-yl)-2 H-tetrazole (4a) in excellent yield while also producing 5-phenyl-1-(prop-2-yn-1-yl)-1 H-tetrazole (4b) in lower yield. Additionally, compound 4a reacted with various substituted aromatic azides (5a–5o) via a copper-catalyzed [3 + 2] azide‒alkyne cycloaddition reaction, resulting in a series of triazole‒tethered tetrazole derivatives (6a–6o) with excellent yields (45–85%). The chemical structures of the synthesized compounds (6a–6o) were confirmed via  1H NMR, 13C NMR, and mass spectrometry. The 1H NMR and 13C NMR spectra of compounds (6a–6o) were recorded in CDCl3, with peak assignments in the characterization section. The 1 H NMR spectra indicate that the proton signals of the triazole rings appear in the range of 8.1 ppm, whereas the methylene protons adjacent to the triazole ring are observed at approximately 6.1 ppm for all the compounds. In the 13C NMR spectra, the methylene carbon (–CH2) attached to the nitrogen of the tetrazole ring is located near 48 ppm for all the compounds, and the (–C=CH) of the triazole appears approx. at 117.52–126.32 ppm. (6a–6o). All the compounds presented the M + 1 peak in mass spectra corresponding to their molecular formula. These spectral data collectively confirmed the successful synthesis of the final compounds.

Fig. 5
figure 5

Experimental procedure for the synthesis of triazole-tethered tetrazole analogues.

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Reaction mechanism for the transformation of compounds 4a into 6(a–o)

The stepwise catalytic cycle begins with the formation of a Cu –alkyne π complex (1), followed by deprotonation of the alkyne proton to form a copper acetylide (2), as shown in (Fig. 6). The coordination of copper increases the acidity of the acetylenic proton and facilitates deprotonation in the aqueous medium. One of the copper ions from the species (2) coordinates with the azide nitrogen (3) and activates it toward attack of the terminal nitrogen of the azide group on the alkyne carbon, leading to the synthesis of the metallacycle (4). This metallacycle undergoes ring contraction via a transannular interaction between the lone pair of electrons present on the azide nitrogen and the carbon-copper double bond. Subsequently, Cu triazolide species (5) is formed, which undergoes protonation to generate 1,4-disubstituted triazole (6) and Cu(I) catalyst 53.

Fig. 6
figure 6

Plausible mechanism for the synthesis of triazole tethered tetrazole hybrids 6(a–o)

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Biological activity

Antibacterial activity

The antibacterial activities of the synthesized compounds (6a–6o) were tested by determining their minimum inhibitory concentrations (MICs, µg/mL) against different bacterial strains. Their effectiveness was specifically assessed against various gram-positive bacteria, such as Staphylococcus aureus (ATCC 25923) and Staphylococcus epidermidis (ATCC 35984), and gram-negative bacteria, such as Escherichia coli (ATCC 25922), Arthemonas hydrophila (ATCC 7966), Pseudomonas aeruginosa (ATCC 27853), Salmonella typhi (clinical isolate), and Salmonella typhimurium (clinical isolate) strains. Intriguingly, the antibacterial activity of these compounds seemed to correlate with the nature and position of the substituents on the phenyl ring (Fig. 7). The MIC activity results are summarized in Table 1. Table 1 clearly shows that the synthesized compounds 6a, 6b, 6c, 6d, 6e, 6g, 6h, 6i, 6j, 6k, 6l, 6m, and 6o exhibited greater potency against the gram-positive bacterial strain S. epidermidis (ATCC 35984). Compound 6a, having a 4-chloro-substituted benzene ring, showed potent activity with an MIC of 3.12 µg/mL against S. epidermidis, which is similar to the standard drug MIC (3.12 µg/mL), and in S. aureus, A. hydrophila, P. aeruginosa, S. typhi, S. typhimurium, and E. coli,it showed moderate to weak activity. Compared with the standard drug, compound 6b, which contains a 2,4-difluoro-substituted benzene ring, had a similar MIC of 3.12 µg/mL against S. epidermidis. In contrast, in S. aureus, A. hydrophila, P. aeruginosa, S. typhi, E. coli, and S. typhimurium showed moderate to weak activity. Compound 6c, bearing a 3-chloro-4-fluoro-substituted benzene ring, exhibited potent activity with an MIC of 1.56 µg/mL against S. epidermidis, which is half of the MIC of the standard drug ciprofloxacin, whereas other bacterial strains displayed moderate to weak activity, ranging from 12.5 to 25 µg/mL. Compound 6d, having a 4-bromosubstituted benzene ring, exhibited potent activity with an MIC of 1.56 µg/mL against S. epidermidis, which is also half of the MIC of ciprofloxacin, whereas in E. coli and S. typhi, the MIC of 6.25 µg/mL was similar to that of the standard drug, and in other tested strains, it showed weak activity. Compound 6e, bearing a 2-chloro-4-fluoro-substituted benzene ring, is potent, with an MIC of 1.56 µg/mL against S. epidermidis, which is half of the MIC of the standard drug, 3.12 µg/mL, whereas in other bacterial strains, it displayed moderate to weak activity, with an MIC of 25-100 µg/mL. Compound 6f, having a 2-chloro- and a 4-methyl-substituted benzene ring, exhibited moderate to weak activity, with an MIC of 12.50-100 µg/mL in all the tested strains. Compound 6g, with a 2,4-dichloro-substituted benzene ring, showed potent activity, with an MIC of 3.12 µg/mL against S. epidermidis and an MIC of 6.25 µg/mL against S. aureus, which is equal to the MIC of the standard drug; however, in the other tested strains, it exhibited weak activity, with MICs ranging from 12.50 to 25 µg/mL. Compound 6h, which has a 4-phenoxyphenyl-substituted benzene ring, showed potent activity, with an MIC of 3.12 µg/mL against S. epidermidis and 6.25 µg/mL against P. aeruginosa, which is equal to the MIC of the standard drug; however, in the other tested strains, it exhibited weak activity, with MICs ranging from 12.50 to 50 µg/mL. Compared with the MIC of the standard drug, compound 6i, with a 4-nitro-substituted benzene ring, had an equal MIC of 6.25 µg/mL against P. aeruginosa and S. typhi. The same compound exhibited moderate activity in other strains, with MICs ranging from 12.50 to 25 µg/mL. Compound 6j, which has a 2-chloro-substituted benzene ring, showed potent activity with an MIC of 3.12 µg/mL against S. epidermidis, which is equal to the MIC of ciprofloxacin, and in other strains, it exhibited weak activity. Compared with the MIC of the standard drug, compound 6k, with 3-chloro-substituted benzene, exhibited an equal MIC of 6.25 µg/mL against S. typhi, whereas other strains presented moderate activity, with an MIC of 12.50-25 µg/mL.Compound6l, containing a 3-fluoro-substituted benzene ring, showed activity at an MIC of 3.12 µg/mL against S. epidermidis, equal to the MIC of the standard drug. In contrast, other strains presented moderate activity, with MICs ranging from 12.50 to 50 µg/mL. The compound 6m 4-trifluoromethoxy-substituted benzene ring exhibited activity with an MIC of 1.56 µg/mL against S. epidermidis, which is just half the MIC of the standard drug, and had a similar MIC of 6.25 µg/mL against P. aeruginosa,whereas other strains presented moderate activity. The compounds 6n and 6o, having 2,3-dichloro- and 3,5-dimethoxy-substituted benzene rings, respectively, exhibited activity against S. epidermidis,with an MIC of 6.25 µg/mL. Both compounds had MICs of 6.25 µg/mL, which were similar to those of ciprofloxacin; however, in the other strains, they presented moderate to weak activity, with MICs ranging from 12.50 to 100 µg/mL. The in vitro antibacterial activity data facilitated preliminary structure‒activity relationship (SAR) analysis of the synthesized compounds (6a–6o). Notably, triazole derivatives containing halogen and phenoxy groups, particularly compounds 6d and 6h with para-substituted, 4-bromo, and 4-phenoxy benzene rings, exhibited superior antibacterial activity against all the tested microorganisms compared with those with ortho- and meta-halo substituents. Additionally, compounds 6a, 6b, 6c, 6e, 6g, 6i, 6j, 6k, 6l, and 6m, featuring electron-withdrawing lipophilic groups such as –Cl, –F, trifluoro, and –NO2 on the benzene ring, demonstrated stronger antibacterial efficacy than those with electron-donating groups. Triazole derivatives linked to tetrazole rings, which are electron-rich and contain heteroatoms, can interact with enzymes or receptors through weak forces such as hydrogen bonds, ion‒dipole interactions, π‒π stacking, hydrophobic interactions, and van der Waals forces, thereby displaying diverse biological activities. Structural modifications in these triazole derivatives, including the addition of bromo, phenoxy, nitro, or chloro groups, as well as the isomerization of the tetrazole moiety, may significantly influence their physicochemical properties, such as lipophilicity, cell membrane permeability, and binding affinity to microbial enzymes or receptors.

Fig. 7
figure 7

Structure‒activity relationships of synthesized potent triazole tethered tetrazole hybrids.

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Table 1 Antibacterial activity (MIC µg/mL) of the test compounds.
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Molecular docking studies

To corroborate the biological findings, we initially performed molecular docking studies on the biologically identified hits (6m, 6d, 6g, 6h, and 6i). We also included the biologically least potent compound 6e for analysis, and ciprofloxacin served as a positive control. All the synthesized ligands were virtually drawn using ChemBioDraw software (PerkinElmer ChemDraw, professional (64-bit), 22.2.0.3300) (https://revvitysignals.com/products/research/chemdraw) and saved in the “sdf” format. The prepared ligands were docked into the active domain of the gyrase protein gyrase B (PDB ID: 4ZVI) (https://www.rcsb.org/), which has 4,5-dibromopyrrolamide as a co-crystallized ligand. The docking protocol was validated by redocking the co-crystallized ligand, which yielded an RMSD of 1.8Å (Fig. 8). The co-crystallized ligand was found to have essential interactions with the residual amino acids ASP49, ARG76, TYR109, and ARG136.The co-crystallized ligand formed hydrogen bonding interactions with ASP49 and hydrophobic interactions with amino acid residues TYR109, GLU50, VAL43, and VAL120.

Fig. 8
figure 8

Overlapping of co-crystalized ligands to validate the docking protocol.

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Following validation, we docked the chosen ligands via the XP mode of the Glide module of the Schrodinger software (Schrödinger: Maestro version 13.9, Schrödinger 2024-1 for virtual screening and visualization) (https://www.schrodinger.com/). The analysis revealed (Table 2) that the docked compounds 6g and 6e were the most potent ligands, with docking scores of − 5.9 and − 5.4 kcal/mol, respectively, in comparison to ciprofloxacin (− 4.8 kcal/mol).

Table 2 Docking scores of the selected ligands with the docking score and amino acids involved in the interaction.
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Furthermore, to understand the binding patterns of the top hits (6g and 6e), we analysed the molecular interaction diagrams of both ligands. The analysis of 6g revealed (Fig. 9A,B) that the ligand stabilizes itself via hydrogen bonding within the active site. The significant H-bond interactions (nitrogen-rich regions of the ligand) include bonding with ARG76, and hydrophobic interactions with TYR109, GLU50, VAL43, VAL120, and ASP73. In addition, halogen bond interactions were also found with the residual amino acid ASN107, which further stabilized the binding. The –Cl was found to engage in nonpolar interactions, chiefly stabilizing the molecule through van der Waals forces.

Fig. 9
figure 9

Binding pattern and molecular interactions [(A) 3D and (B) 2D interactions] of compound 6g within the binding domain of the protein (PDB ID: 4ZVI).

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Next, the analysis of 6e within the active domain revealed (Fig. 10A,B) a similar interaction pattern to that of 6g. 6e was found to be stabilized via hydrogen bonding mediated chiefly via GLU50, ASP73, VAL43, and VAL120, amino acid residues. The interaction with ASP73 also stabilized the ligands’ binding and conformation within the active site. In addition, interactions with ARG76 through water bridge formed hydrogen bonding, which impacted the overall binding energy. The halogen substituents on compound 6g engaged in nonpolar interactions with ASN107, which contributed to the stabilization of 6g via van der Waals forces. In contrast, compound 6e lacks this mode of interaction, resulting in different stabilization characteristics. Other chosen ligands had docking scores lower than those of ciprofloxacin, and their 2d interaction diagrams are presented in the supplementary file (Fig. S1).

Fig. 10
figure 10

Binding pattern and molecular interactions [(A) 3D and (B) 2D interactions] of compound 6e within the binding domain of the protein (PDB ID: 4ZVI).

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Molecular dynamics simulation

Molecular docking is associated with creating a snapshot of binding, i.e., it only predicts the ligand fitting into the active site. However, docking does not consider whether the ligand remains stabilized within the active site over time. To elucidate the best lead 6g stability over a time frame of 100 ns, we performed molecular dynamics (MD). The Desmond program of Schrodinger was used for molecular dynamics. Initially, the RMSD analysis revealed (Fig. 11A) the stability of the protein‒ligand complex over the simulation period. The protein stabilized at approximately 4.5–5.0 Å after 40 ns and presented minimal conformational changes beyond a stabilized system. The ligand conformation remained stable between 2.0 and 3.0 Å, indicating its stability within the binding pocket. The RMSF analysis of the protein (Fig. 11B), which provides information on the residual amino acid fluctuations, suggested that most of the fluctuations were under 2.0 Å, suggesting a stabilized conformation of the protein. The high peak around the residue index of ~ 350 indicates perturbations owing to the flexibility of the active site for ligand binding. The ligand RMSF (Fig. 11C) values exceeded 1.5 Å, indicating the flexibility of the ligand structure (free rotation around bonds). Next, the protein‒ligand (PL) interaction plot (Fig. 11D) was used to identify the key amino acid residues involved in stabilizing ligands within the active domain. TYR109 and ILE94 were found to connect and hold the ligand for approximately 80% of the total simulation period. VAL43 was also critical in maintaining the ligand’s stability and orientation within the binding pocket. The 2D interaction diagram (Fig. 11E) further corroborated this finding. Finally, dihedral angle analysis (Fig. 11F), which reflects conformational stability, revealed stable dihedral angles and restricted conformational changes throughout the simulation time, thus validating the stable P-L interaction.

Fig. 11
figure 11

The illustration reveals the MD simulation analysis of 6g within the protein (PDB ID: 4ZVI) active cavity over the simulation period of 100 ns. (A) RMSD plot highlighting the stability of the protein (shown in blue) and ligand (shown in red) within the simulation period; (B) Protein RMSF indicates the flexibility of amino acid residues; (C) Ligand RMSF reveals the atomic-level fluctuations of the ligand; (D) P-L interaction diagram reveals the key interactions of the ligand with amino acid residues of the protein active site. (E) Interaction map of key contacts between the ligand and residual amino acid residues. (F) Dihedral angle distribution plots, which indicate the conformational stability and flexibility.

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Evaluation of the physicochemical characteristics of potent compounds

The potent compounds from the in-silico study database are analyzed for the physicochemical characteristics of the compounds through the SwissADME (http://www.swissadme.ch/index.php) in Table 3 54.

Table 3 The physicochemical properties of potent compounds with the SwissADME database.
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Experimental section

Chemicals and solvents used in this study were purchased from E. Merck (India) and Sigma–Aldrich chemicals. The reactions were monitored using thin-layer chromatography (TLC) on a pre-coated silica gel 60 F254 (mesh). The results were observed using UV light or an iodine chamber. Merck silica gel (230–400 mesh) was employed for column chromatography. The compounds’ melting points were ascertained using an open capillary method, the results of which were uncorrected. The 1H and 13C NMR spectral data were recorded on the JEOL GS-400 model FT-NMR spectrometer at 400 MHz using TMS as an internal standard. The chemical shifts are reported on a ppm scale for CDCl3 (7.269 ppm) for 1H, (77.00 ppm) for 13C NMR, and DMSO-d6 (2.5 ppm) for 1 H, (3.5 ppm) for moisture, and (40.39 ppm) for 13C NMR as an internal standard. The abbreviations are: s = singlet, d = doublet, t = triplet, q = quartet, dd = double doublet, m = multiplet. The chemical shifts were gauged in parts per million (ppm) on the delta (δ) scale with tetramethylsilane (TMS) acting as the internal reference. Mass spectra were recorded on an Agilent G6530AA (LC-HRMS-Q-TOF) and Sciex X500R QTOF mass spectrometer. Elemental analysis was done on an Elementar GmbH Vario El analyzer.

Synthesis of 5-phenyl-1 H-1,2,3,4-tetrazole (2)

The synthesis of tetrazole was carried out according to the literature procedure 14. Sodium azide (1.5 mmol) was added to a magnetically stirred solution of benzonitrile (1 mmol) in anhydrous DMF and the Cerium ammonium nitrate (10 mmol %). The reaction mixture was constantly stirred for another 10 h at 110 °C under a nitrogen atmosphere. The completion of the reaction was monitored by TLC. The reaction mixture was brought to room temperature, and the solvent was evaporated under vacuum. The crude was obtained and dissolved in ethyl acetate (20 mL), and the solution was washed with acidified water (4 M HCl, 15 mL) twice. The separated organic layer was washed with brine solution, dried over anhydrous Na2SO4, and the solvent was removed under a high vacuum to obtain tetrazole as a white crystalline solid.

White solid, Yield: 90%, mp: 212–215 °C; 1H NMR (400 MHz, DMSO-d6)δ: 8.08–8.05 (m, 2 H, Ar-H), 7.63–7.61 (m, 3 H, Ar-H); 13C NMR (400 MHz, DMSO-d6)δ: 155.30, 131.22, 129.40, 126.94, 124.14; Elemental analysis: molecular formula: C7H6N4. Calculated: C-57.53; H-4.14; N-38.34; Found: C-57.21; H-4.12; N-38.31.

Synthesis of 5-phenyl-2-(prop-2-yn-1-yl)-2 H-1,2,3,4-tetrazole/5-phenyl-1-(prop-2-yn-1-yl)-1 H-1,2,3,4-tetrazole (4a and 4b)

The alkyne synthesis was carried out according to the procedure described in the literature 52. Briefly, a solution of tetrazole (2) (1 mmol) in 15 mL dry acetone was added 1.5 mmol of triethylamine, and the reaction mixture was refluxed for 15–30 min. Subsequently, (1 mmol) of propargyl bromide was added dropwise, and the reaction mixture was refluxed for 36 h. After the completion of the reaction, as seen by TLC, the reaction mixture was brought to room temperature, and precipitated triethylamine hydrochloride was filtered off. Filtrate was concentrated to dryness and purified by column chromatography using hexane and ethyl acetate (70:30) as eluent to give two compounds, major 4a (68%) and minor 4b (20%). The major compound 4a was used to synthesize the next step compound (6a–6o) by click chemistry.

5-phenyl-2-(prop-2-yn-1-yl)-2 H-1,2,3,4-tetrazole (4a)

Oil, Major (2,5 isomer), Yield: 68%,1H NMR (400 MHz, CDCl3)δ: 7.86–7.83 (m, 2 H, Ar-H), 7.61 ( s, 3 H, Ar-H), 5.23–5.22 (d, 2 H, J = 2.4 Hz, –N–CH2–), 2.59–2.58 (t, 1H, J = 2.4 Hz, -C ≡ CH); 13C NMR (400 MHz, CDCl3)δ: 154.22, 131.59, 129.33, 128.75, 123.18, 75.99, 74.97, 37.90; Elemental analysis: Molecular formula: C10H8N4. Calculated: C-65.21; H-4.38; N-30.42; Found: C-65.23; H-4.35; N-30.44.

5-phenyl-1-(prop-2-yn-1-yl)-1 H-1,2,3,4-tetrazole(4b)

Oil, Minor (1,5 isomer), Yield: 20%, 1H NMR (400 MHz, CDCl3)δ: 8.08–8.06 (m, 2 H, Ar–H), 7.40–7.38 (m, 3 H, Ar–H), 5.36–5.35 (d, 2 H, J = 2.7 Hz, –N–CH2–), 2.52–2.51 (t, 1 H, J = 2.1 Hz, –C≡CH); 13C NMR (400 MHz, CDCl3) δ: 165.58, 130.44, 128.82, 126.96, 126.87, 75.94, 73.95, 42.59; Elemental analysis: molecular formula: C10H8N4. Calculated: C-65.21; H-4.38; N-30.42; Found: C-65.23; H-4.35; N-30.44.

General procedure for the synthesis of azides (5a–5o)

The azides were synthesized using the reported procedure 55. Initially, aniline was taken in stoichiometric amounts (1 eq., 5 mmol) and dissolved in a 6 N HCl solution (10 mL/mmol of aniline). This solution was chilled to 0 °C. Subsequently, sodium nitrite (1 eq., 5 mmol) solution was added. This mixture was kept under stirring for 10 min at a temperature between 0 and 5 °C. After that, sodium azide was added to the mixture (1.2 eq., 6 mmol), and the mixture was stirred at ambient temperature for 2–3 h. After completion of the reaction, as monitored by TLC, the mixture was extracted with ethyl acetate (10 mL × 3). The resultant organic layer was treated with brine solution (10 mL) and dried over anhydrous sodium sulfate. The solvent was removed, and the azide was sufficiently pure for further reactions. All the synthesized azides were stored at a temperature of -20 °C to ensure their stability.

General procedure for the synthesis of (2-{substituted-[1-(substituted-phenyl)-1 H-1,2,3-triazol-4-ylmethyl]-5-phenyl-2 H-1,2,3,4-tetrazole derivatives (6a-6o)

The syntheses of compounds (6a–6o) were carried out according to the literature procedure 55. Briefly, 5-phenyl-2-(prop-2-yn-1-yl)-2 H-1,2,3,4-tetrazole (4a) (1 mmol) and various aromatic azides (2 mmol) were suspended in N,N′-dimethylformamide (DMF) (5 mL). Sodium ascorbate (0.3 mmol, in water) was added, followed by copper (II) sulfate pentahydrate (0.03 mmol, in water). The heterogeneous mixture was stirred vigorously overnight, and TLC was used to monitor the completion of the reaction. After completion of the reaction, the reaction mixture was diluted with water, cooled in ice, and the precipitate was collected by filtration.

2-{[1-(4-chlorophenyl)-1 H-1,2,3-triazol-4-yl]methyl}-5-phenyl-2 H-1,2,3,4-tetrazole (6a)

Light pink solid, Yield: 81%, mp: 127 °C, 1H NMR (400 MHz, CDCl3)δ: 8.16–8.14 (m, 2 H, Ar-H), 8.09 (s, 1 H, =CH-of triazole), 7.69–7.67 (d, 2 H, J = 9.2 Hz, Ar–H), 7.52–7.47 (m, 5 H, Ar–H), 6.08 (s, 2 H, –N–CH2–); 13C NMR (400 MHz, CDCl3)δ: 135.08, 130.61, 130.04, 128.91, 126.90, 121.83, 117.23(−C=CH of triazole), 48.39; HRMS m/z (M + H)+: 340.11.Elemental analysis: Molecular formula: C16H12ClN7: Calculated: C-56.89, H-3.58, N-29.03; Found: C-56.87, H-3.55, N-29.00.

2-{[1-(2,4-difluorophenyl)-1 H-1,2,3-triazol-4-yl]methyl}-5-phenyl-2 H-1,2,3,4-tetrazole (6b)

Creamy solid, Yield: 54%, mp: 83 °C; 1H NMR (400 MHz, CDCl3)δ: 8.19–8.18 (d,1 H, J = 4 Hz, =CH-of triazole ), 8.16–8.14 (m, 2 H, Ar-H), 7.93–7.91 (m, 1 H, Ar-H), 7.49–7.47 (m, 3 H, Ar-H), 7.10–7.06 (m, 2 H, Ar-H), 6.09 (s, 2 H, -N-CH2-); 13C NMR (400 MHz, CDCl3)δ: 165.59, 140.91, 130.48, 128.87, 127.03, 126.88, 126.27, 126.18, 124.64, 124.57(− C = CH of triazole), 112.88, 112.84, 112.65, 112.61, 105.71, 105.48, 105.44, 105.16, 48.24;HRMS m/z (M + H)+: 340.08. Elemental analysis: Molecular formula: C16H11F2N7: Calculated: C-56.64, H-3.27, N-28.90; Found: C-56.61, H-3.23, N-28.83.

2-{[1-(3-chloro-4-fluorophenyl)-1 H-1,2,3-triazol-4-yl]methyl}-5-phenyl-2 H-1,2,3,4-tetrazole (6c)

Light green solid, Yield: 83%, mp: 123 °C; 1H NMR (400 MHz, CDCl3)δ: 8.15–8.13 (m, 2 H, Ar–H), 8.08 (s, 1 H, =CH-of triazole), 7.85–7.83 (m, 1 H, Ar–H), 7.64–7.60 (m, 1 H, Ar–H), 7.49–7.47 (m, 3 H, J = 5.2 Hz, Ar–H), 7.31–7.26 (m, 1 H, Ar–H), 6.08 (s, 2 H, –N–CH2–); 13C NMR (400 MHz, CDCl3)δ: 165.64, 159.43, 154.30, 141.57, 130.54, 128.90, 126.96, 126.88, 125.23, 123.23, 121.67, 120.47(−C=CH of triazole), 120.39, 117.86, 117.64, 48.30; Elemental analysis: Molecular formula: C16H11ClFN7: Calculated: C-54.02, H-3.12, N-27.56; Found: C-54.00, H-3.08, N-27.53.

2-{[1-(4-bromophenyl)-1 H-1,2,3-triazol-4-yl]methyl}-5-phenyl-2 H-1,2,3,4-tetrazole (6d)

Creamy solid, Yield: 48%, mp: 151 °C; 1H NMR (400 MHz, CDCl3)δ: 8.15–8.13 (m, 2 H, Ar–H), 8.09 (s, 1 H, =CH-of triazole), 7.67–7.60 (m, 4 H, Ar–H), 7.49–7.48 (m, 3 H, Ar–H), 6.08 (s, 2 H, –N–CH2-); 13C NMR (400 MHz, CDCl3)δ:165.61, 141.43, 135.57, 133.00, 130.52, 128.90, 126.98, 126.88, 122.93, 122.02(− C=CH of triazole), 121.44, 48.36;HRMS m/z (M + H)+: 382.04. Elemental analysis: Molecular formula: C16H12BrN7: Calculated: C-50.28, H-3.16, N-25.65; Found: C-50.23, H-3.12, N-25.61.

2-{[1-(2-chloro-4-fluorophenyl)-1 H-1,2,3-triazol-4-yl]methyl}-5-phenyl-2 H-1,2,3,4-tetrazole (6e)

Dark brown solid, Yield: 47%, mp: 79 °C;1H NMR (400 MHz, CDCl3)δ: 8.15 (s, 3 H, Ar–H and =CH– of triazole), 7.60–7.56 (m, 2 H, Ar-H), 7.49–7.43 (m, 4 H, Ar–H), 6.10 (s, 2 H, –N–CH2–); 13C NMR (400 MHz, CDCl3)δ: 165.58, 142.50, 140.45, 136.64, 133.08, 130.64, 130.49, 129.30, 128.88, 128.49, 128.38, 127.03, 126.87, 125.44 (− C=CH of triazole), 48.28;HRMS m/z (M + H)+: 356.08.Elemental analysis: Molecular formula: C16H11ClFN7: Calculated: C-54.02, H-3.12, N-27.56; Found: C-54.00, H-3.06, N-27.52.

2-{[1-(2-chloro-4-methylphenyl)-1 H-1,2,3-triazol-4-yl]methyl}-5-phenyl-2 H-1,2,3,4-tetrazole (6f)

Light pink solid, Yield: 63%, mp: 97 °C; 1H NMR (400 MHz, CDCl3)δ: 8.09 (s, 3 H, Ar–H and =CH– of triazole), 7.41–7.33(m, 4 H, Ar–H), 7.20–7.15(m,2 H, Ar–H) 6.02 (s, 2 H, –N–CH2–), 2.36 (s, 3 H, CH3); 13C NMR (400 MHz, CDCl3)δ: 165.72, 141.88, 131.09, 130.44, 128.88, 128.66, 127.34, 127.11, 126.93, 126.88 (−C=CH of triazole), 53.42, 21.04;HRMS m/z (M + H)+: 352.10. Elemental analysis: Molecular formula: C17H14ClN7: Calculated: C-58.04, H-4.01, N-27.87; Found: C-58.00, H-4.00, N-27.83.

2-{[1-(2,4-dichlorophenyl)-1 H-1,2,3-triazol-4-yl]methyl}-5-phenyl-2 H-1,2,3,4-tetrazole (6g)

Dark brown solid, Yield: 74%, mp: 104 °C; 1H NMR (400 MHz, CDCl3)δ: 8.06–8.03 (m, 3 H, Ar–H and = CH– of triazole), 7.51–7.50 (m, 1 H, Ar–H), 7.39–7.38 (m, 3 H, Ar–H), 7.25–7.22 (m, 1 H, Ar–H), 7.10–7.05 (m, 1 H, Ar–H), 6.01 (s, 2 H, –N–CH2–); 13C NMR (400 MHz, CDCl3)δ: 165.50, 163.95, 161.41, 140.32, 130.92, 130.88, 130.44, 130.13, 130.01, 129.21, 129.11, 128.92, 128.84, 126.99, 126.82, 125.62, 118.24 (−C=CH of triazole), 117.98, 115.46, 115.23, 48.24; HRMS m/z (M + H)+: 372.05. Elemental analysis: Molecular formula: C16H11Cl2N7: Calculated: C-51.63, H-2.98, N-26.34; Found: C-51.58, H-2.91, N-26.29.

2-{[1-(4-phenoxyphenyl)-1 H-1,2,3-triazol-4-yl]methyl}-5-phenyl-2 H-1,2,3,4-tetrazole (6h)

Brown solid, Yield: 42%, mp: 145 °C; 1H NMR (400 MHz, CDCl3)δ: 8.18–8.17 (m, 3 H, Ar–H and =CH– of triazole), 7.50–7.49 (m, 8 H, Ar–H), 7.12–6.93 (m, 4 H, Ar–H), 5.46 (s, 2 H, –N–CH2–); 13C NMR (CDCl3)δ: 166.50, 130.54, 129.12, 128.99, 128.91, 128.65, 127.04, 127.00, 126.94, 126.85, 126.82, 118.77 (–C=CH of triazole), 111.51, 50.65;HRMS m/z (M + H)+: 396.15.Elemental analysis: Molecular formula: C22H17N7O: Calculated: C-66.82, H-4.33, N-24.80; Found: C-66.78, H-4.30, N-24.78.

2-{[1-(4-nitrophenyl)-1 H-1,2,3-triazol-4-yl]methyl}-5-phenyl-2 H-1,2,3,4-tetrazole (6i)

Light pink solid, Yield: 79%, mp: 178 °C; 1H NMR (400 MHz, CDCl3)δ: 8.43–8.41 (m, 2 H, Ar–H), 8.25–8.23 (m, 2 H, Ar–H), 8.14–8.13 (s, 1 H, =CH– of triazole), 7.99–7.96 (d, 2 H, J = 8.4 Hz, Ar–H), 7.48 (s, 3 H, Ar–H), 6.11 (s, 2 H, –N–CH2–); 13C NMR (CDCl3) δ: 165.70, 147.47, 146.84, 142.06, 140.71, 130.59, 128.92, 126.87, 125.59, 121.54, 120.70, 119.36 (−C=CH of triazole), 48.22; Elemental analysis: Molecular formula: C16H12N8O2: Calculated: C-55.17, H-3.47, N-32.17; Found: C-55.12, H-3.41, N-32.13.

2-{[1-(2-chlorophenyl)-1 H-1,2,3-triazol-4-yl]methyl}-5-phenyl-2 H-1,2,3,4-tetrazole (6j)

Creamy solid, Yield: 87%, mp: 88 °C; 1H NMR (400 MHz, CDCl3)δ: 8.15 (s, 3 H, Ar–H and = CH– of triazole), 7.60–7.57 (m, 3 H, Ar–H), 7.47 (s, 4 H, Ar–H), 6.10 (s, 2 H, –N–CH2–); 13C NMR (CDCl3)δ:165.55, 134.47, 131.08, 130.80, 130.44, 128.87, 128.53, 127.99, 127.76, 127.08, 126.88, 125.54 (−C=CH of triazole), 48.36;HRMS m/z (M + H)+: 338.09.Elemental analysis: Molecular formula: C16H12ClN7: Calculated: C-56.89, H-3.58, N-29.03; Found: C-56.87, H-3.55, N-29.00.

2-{[1-(3-chlorophenyl)-1 H-1,2,3-triazol-4-yl]methyl}-5-phenyl-2 H-1,2,3,4-tetrazole (6k)

Light brown solid, Yield: 60%, mp: 122 °C; 1H NMR (400 MHz, CDCl3)δ: 8.15–8.12 (m, 3 H, Ar–H and = CH– of triazole), 7.77 (s, 1 H, Ar–H), 7.63–7.62 (d, 1 H, J = 6.8 Hz, Ar–H), 7.48–7.42 (m, 5 H, Ar–H), 6.08 (s, 2 H, –N–CH2–); 13C NMR (CDCl3)δ: 165.62, 137.36, 135.68, 130.91, 130.51, 129.26, 128.89, 127.00, 126.88, 121.56, 120.90 (−C=CH of triazole), 118.60, 48.34; HRMS m/z (M + H)+: 338.09.Elemental analysis: Molecular formula: C16H12ClN7: Calculated: C-56.89, H-3.58, N-29.03; Found: C-56.87, H-3.55, N-29.00.

2-{[1-(3-fluorophenyl)-1 H-1,2,3-triazol-4-yl]methyl}-5-phenyl-2 H-1,2,3,4-tetrazole (6l)

Light pink solid, Yield: 57%, mp: 156 °C; 1H NMR (400 MHz, CDCl3)δ: 8.16–8.11 (m, 3 H, Ar-H and = CH-of triazole), 7.54–7.47 (m, 6 H, Ar-H), 7.19–7.13 (m, 1 H, Ar-H), 6.08 (s, 2 H, -N-CH2-); 13C NMR (CDCl3)δ: 165.64, 164.28, 141.42, 131.36, 131.27, 130.52, 128.90, 127.01, 126.90, 121.55(−C=CH of triazole), 116.25, 116.04, 115.96, 115.93, 108.67, 108.35, 48.35;HRMS m/z (M + H)+: 322.12 Elemental analysis: Molecular formula: C16H12FN7: calculated: C-59.81, H-3.76, N-30.51; Found: C-59.78, H-3.70, N-30.46.

5-phenyl-2-({1-[4-(trifluoromethoxy)phenyl]-1 H-1,2,3-triazol-4-yl}methyl)-2 H-1,2,3,4-tetrazole (6m)

Dark brown solid, Yield: 47%, mp: 108 °C; 1H NMR (400 MHz, CDCl3)δ: 8.15–8.10 (m, 3 H, Ar–H and =CH– of triazole), 7.79–7.77 (m, 2 H, Ar–H), 7.49–7.48 (m, 3 H, Ar–H), 7.40–7.38 (d, 2 H, J = 8.4 Hz, Ar–H), 6.09 (s, 2 H, –N–CH2–); 13C NMR (400 MHz, CDCl3)δ: 163.37, 134.94, 130.53, 128.90, 127.00, 126.89, 122.34, 122.16 (−C=CH of triazole), 121.62, 48.35;HRMS m/z (M + H)+: 388.11.Elemental analysis: Molecular formula: C17H12F3N7O: Calculated: C-52.72, H-3.12, N-25.31; Found: C-52.67, H-3.09, N-25.28.

2-{[1-(2,3-dichlorophenyl)-1 H-1,2,3-triazol-4-yl]methyl}-5-phenyl-2 H-1,2,3,4-tetrazole (6n)

Creamsolid, Yield: 44%, mp: 91 °C; 1H NMR (400 MHz, CDCl3)δ: 8.07–8.06 (m, 3 H, Ar–H and =CH– of triazole), 7.58–7.56 (d, 1 H, J = 8.4 Hz, Ar–H), 7.45–7.39 (m, 4 H, Ar–H), 7.33–7.29 (m, 1 H, Ar–H), 6.03 (s, 2 H, –N–CH2–); 13C NMR (CDCl3)δ: 165.58,, 135.98, 134.71, 131.91, 130.47, 128.95, 128.87, 128.03, 127.96, 127.67, 127.03, 126.86, 126.30, 126.13, 125.54, 117.52 (−C=CH of triazole), 117.31, 48.28; HRMS m/z (M + H)+: 372.23.Elemental analysis: Molecular formula: C16H11Cl2N7: Calculated: C-51.63, H-2.98, N-26.34; Found: C-51.58, H-2.91, N-26.29.

2-{[1-(3,5-dimethoxyphenyl)-1 H-1,2,3-triazol-4-yl]methyl}-5-phenyl-2 H-1,2,3,4-tetrazole (6o)

Creamy white solid, Yield: 40%, mp: 198 °C; 1H NMR (400 MHz, CDCl3)δ: 8.17–8.15 (m, 3 H, Ar–H and =CH– of triazole), 7.50–7.48 (m, 5 H, Ar–H), 7.41–7.38 (m, 1 H, Ar–H), 6.07 (s, 2 H, –N–CH2–), 3.73 (s, 6 H, 2×OCH3); 13C NMR (CDCl3)δ: 163.14, 155.68, 141.54, 141.51, 141.46, 137.38, 137.35, 136.90, 135.62, 130.54, 128.95, 128.96, 128.85, 126.87, 124.37 (−C=CH of triazole), 116.15, 30.93; Elemental analysis: Molecular formula: C18H17N7O2: calculated: C-65.24, H-5.17, N-29.59; Found: C-65.19, H-5.12, N-29.53.

Biological assays

Determination of antibacterial activity

Various bacterial strains were utilized to assess in vitro antibacterial potency. These included Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 35984), and gram-negative bacteria such as Escherichia coli (ATCC 25922), Arthemonas hydrophila (ATCC 7966), Pseudomonas aeruginosa (ATCC 27853), Salmonella typhi (clinical isolate), and Salmonella typhimurium (Clinical isolate). These microbial cultures are housed at the Department of Microbiology at the Institute of Medical Sciences, Banaras Hindu University in Varanasi, India. All cultures were obtained from the American Type Culture Collection (ATCC) or were clinical strains. Before experimentation, the microbial cultures were refreshed using normal saline. For antibacterial benchmarking, Ciprofloxacin acted as the standard drug. To determine the Minimum Inhibitory Concentration (MIC), we utilized the micro-dilution approach with each compound’s progressive (10-fold) dilutions. Systematically, these diluted compounds were introduced into a microtiter plate. To be precise, a 10 mL aliquot of standardized inoculum (concentration:12 × 107CFU/mL) was dispensed into every well of this plate. Following this, an aerobic incubation at 37 °C was carried out for a period ranging from 18 to 24 h. The concentration at which the compounds stymied visible bacterial proliferation, evidenced by a clear, non-turbid solution compared to control wells, was deemed the MIC.

Molecular dynamics docking and simulation

Computational protein-ligand docking has become increasingly important in drug discovery, as it helps predict the bound configurations and binding free energies of small-molecule ligands with macromolecular targets 56. Docking techniques utilize a wide range of computational platforms, enhancing our understanding of the intricate interactions between a drug or chemical and its receptor(s), often described by the “key-lock” analogy.

In the present in silico-based analysis, compounds were sketched using ChemBioDraw software to perform the in silico-based studies and saved into .sdf files. The ligands were further refined, and their tautomeric states (if any) were generated using the Ligprep module of the Schrodinger software. The protein (PDB ID: 4ZVI) was downloaded from the protein data bank and optimized for missing loops (Prime) and side chains using the software’s Protein Prep wizard. The docking was performed using the XP module of the Glide, and the scores were represented in kcal/mol units. The Desmond module of the Schrodinger software was used to perform the molecular dynamics 18,19.

Conclusion

A series of novel triazole-tethered tetrazole derivatives (6a–6o) were efficiently synthesized using click chemistry. All synthesized compounds were assessed for their in vitro antibacterial activity against seven bacterial strains. Notably, compounds 6c, 6d, 6e, and 6m exhibited greater potency than the standard drug ciprofloxacin, with an MIC of 1.56 µg/mL against S. epidermidis (ATCC 35984). Compounds 6a, 6b, 6g, 6h, 6j, and 6l also exhibited MIC 3.12 µg/mL against a similar strain. Electron-withdrawing groups at positions 2 and 4 of the benzene ring enhanced antibacterial activity, whereas electron-donating groups reduced activity. Compounds 6h, 6i, 6m, and 6o showed excellent activity against P. aeruginosa (MIC 6.25 µg/mL), similar to standard ciprofloxacin’s efficacy. Molecular modelling and dynamics analysis of the synthetic binding affinity with the DNA gyrase revealed that 6g and 6e possess a higher affinity within the gyrase than ciprofloxacin. The molecular dynamics analysis for 6g revealed a stable conformation within the protein domain during the simulation period. The present work thus opens up the possibility of further exploring the utility of 6g and 6e in delineating their DNA gyrase binding biologically and deducing their mechanistic interventions. The work may further be expanded to recruit more pathogenic-resistant strains, and the inhibitory potential of the compounds may further be analysed.