Rational design and synthesis of Co(II), Ni(II) and Cu(II) complexes bearing 1,2,4-triazole scaffold for biological applications

Abstract

The chemistry of 1,2,4-triazole-incorporating compounds and their metal complexes has attracted widespread attention due to their potential therapeutic activities. Therefore, this paper presents the synthesis, characterization, biological studies, and theoretical molecular docking evaluation of 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole (AHMT) and its Co(II), Ni(II), and Cu(II) complexes. The proposed structures were confirmed by elemental and spectroscopic analyses, yielding trans-N₂S₂ in an equatorial arrangement within an octahedral geometry. Numerous biological studies have been conducted on AHMT, Co(II)-AHMT, Ni(II)-AHMT, and Cu(II)-AHMT. The synthesized scaffolds have been screened for their antibacterial activity against various bacterial and fungal species. Notably, Ni(II)-AHMT is more potent than the other hybrids. Indeed, the cytotoxic activity of the synthesized hybrids was assessed against four human cancer cell lines: HePG2, MCF-7, HeP-2, and HeLa. The findings revealed that Ni(II)-AHMT has the most toxicity with IC₅₀ values of 8.70 ± 0.5 µM and 9.72 ± 0.7 µM against MCF-7 and HeLa cell lines, respectively. The enhanced antimicrobial and anticancer activities of the metal complexes relative to the free AHMT ligand are attributed to Tweedy's chelation theory, which explains that reduced metal polarity increases lipophilicity and membrane penetration. Mechanistic studies revealed that Ni(II)-AHMT induces cell death primarily via apoptosis and causes prominent G0/G1 cell-cycle arrest in MCF-7 cells. DNA-binding assays of the synthesized compounds were performed. Additionally, the activity of superoxide dismutase (SOD) in the compounds has also been evaluated using the NBT assay. The free ligand AHMT and its nickel complex Ni(II)-AHMT demonstrate notable radical-scavenging capacity, achieving 75.8% and 70.2% inhibition, respectively, compared to the reference antioxidant L-ascorbic acid (78.2%). Molecular docking experiments were conducted to understand the binding interactions of AHMT and its metal complexes with DNA, β-lactamase, and human serum albumin. The docking simulations were consistent with the in vitro findings and suggested that Ni(II)-AHMT is a potential DNA groove binder and a potent antimicrobial candidate that merits further in vivo investigations.

---

1. Introduction

Recently, there has been a rise in microbial and multidrug resistance in the clinical area, adversely impacting health by causing various diseases due to the ineffectiveness and cytotoxicity of organic-based medications, prolonged infection durations, and elevated mortality rates. To address these issues, it is essential to develop novel pharmaceuticals with broad-spectrum biological activity to effectively eliminate microorganisms. Coordination chemistry addresses this issue by facilitating the creation of metal-coordinated molecules. In the same context, metal complexes containing triazole ligands exhibit a broad spectrum of biological activity, capable of combating resistant bacteria.^1,2 Metal-triazole complexes are more likely to traverse lipid bilayer cell membranes. On the other hand, humans require trace amounts of metals for optimal physiological function, and new studies indicate that incorporating metals into pharmaceuticals can enhance their biological efficacy through chelation.

Recently, triazole scaffolds have attracted considerable interest as a prominent field of medicinal and coordination chemistry research due to their high reactivity, biological potential, flexibility, good photostability, and large Stokes shift. In addition, triazole entities are present in various marketed pharmaceutical drugs, including fluconazole, rizatriptan, ribavirin, and anastrozole (Fig. 1) for antifungal activity, treating migraines, an anticoronaviral agent, and a non-steroidal aromatase inhibitor, respectively.^3–5 Also, triazoles can be employed as radiopharmaceutical agents in bioimaging techniques owing to their consequent luminescent properties.⁶

Fig. 1 Marketed drugs based on triazole hybrid.^3–5

While the structural and electronic features of triazole constitutions enable them to be rigid linking units and be a critical factor in the electronic characters of a peptide bond, without a hydrolytic cleavage effect. In the same context, triazoles exhibit a much stronger dipole moment and dual functionality as hydrogen donors and acceptors, enhancing their extraordinary efficiency in the synthesis of coordination network structures.^6,7

In particular, 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole (AHMT) (Fig. 2), which is commercially available as Purpald, and its derivatives show various pharmacological activities involving promising antifungal,⁸ antimicrobial,⁹ antitumor agents against hepatocellular carcinoma,¹⁰ pediatric brain tumor,¹¹ antiviral¹² and tyrosinase inhibitors.¹³ On the other hand, AHMT has high sensitivity and selectivity properties so that it is utilized as a ratiometric electrochemical sensor for the detection of formalin in residual food samples,^14,15 pesticides,¹⁶ and toxic cations (Cd(II) and Cr(III)^17,18) in environmental and industrial wastewater. Additionally, many biochemical materials containing picric, uric, and ascorbic acids, as well as lipoic acid, can be easily detected by the AHMT sensor.¹⁹

Fig. 2 The chemical structure of AHMT.

In contrast to Bagihalli et al.,²⁰ who described Co(II), Ni(II), and Cu(II) complexes with Schiff bases from 3-substituted-4-amino-5-mercapto-1,2,4-triazoles and 8-formyl-7-hydroxy-4-methylcoumarin focusing on antimicrobial effects via O,N-donor ligation, and Calu et al.,²¹ investigated thermal, magnetic, and biological features of Co(II), Ni(II), Cu(II), and Zn(II) complexes with a Schiff base from 3-amino-4H-1,2,4-triazole and salicylaldehyde featuring O,N-chelation. The present study pioneers the understudied 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole (AHMT) ligand as unique polyfunctional architecture offering versatile N,N,S coordination modes via tautomeric thiol–thione (–SH/S), hydrazino (–NHNH₂), and amino groups that surpass simple 1,2,4-triazole derivatives, enabling novel distorted octahedral geometries in Co(II), Ni(II), and Cu(II) complexes. Its dual proton donor/acceptor capabilities further foster strong H-bonds (e.g., –NH⋯S, –SH⋯O=C stronger than conventional amide bonds) with DNA base pairs, protein side chains, and biological thiols, mimicking sulfur-modified nucleosides/phosphorothioates for enhanced redox biology, enzyme inhibition, and biomolecular interactions beyond the rigid imines of prior works.^22–25 In earlier research, metal complexes of AHMT with Pt and Pd;²⁶ Sn;²⁷ Ru(II);²⁸ [M(CO)₆ (M = Cr, Mo, W), Mn(CO)₃Cp]²⁸ and oxidovanadium(V)⁹ were produced.

While AHMT coordination is not entirely unprecedented, preliminary spectral studies date back over 35 years (e.g., Garg et al. 1987;²⁹ Varma et al. 1992 ³⁰), limited to basic IR/UV-vis techniques on select metals (Cu(II),^29,30 Co(II),²⁹ Ni(II)²⁹) without biology. Unlike Garg et al. (1987)²⁹ sulfate-based DMSO–water reflux and Varma et al. (1992)³⁰ alkaline aqueous hot-plate Cu(II) acetate/nitrate synthesis, this introduces chloride salts (including novel CuCl₂), neutral methanol reflux (3 : 1 ratio, 8 h at 80 °C), yielding unique octahedral N₂S₂X₂ geometries involving thiol-protonated structures with chloride coordination (see Fig. 3) with comprehensive characterization (FTIR, elemental analysis, TGA, conductance, UV-vis, ESR, EI-MS, magnetic moments) absent previously with detailed d–d bands, g-values, µ_eff, and molecular ions. Most importantly, this is the inaugural report of AHMT-M(II) with targeted biological profiles: cytotoxicity, antibacterial effects, DNA binding, superoxide dismutase activity, antioxidants, apoptosis, cell cycle analysis, and docking, transforming foundational chemistry into therapeutic potential.

Fig. 3 The chemical structures of the prepared complexes.

2. Experimental

2.1. Materials and instruments

The SI contains detailed information on materials, instruments, and biological studies.

2.2. Synthesis of AHMT

The ligand (AHMT) (Fig. 2) has been prepared according to the reported methodology³¹ as shown in Scheme 1. The structure and purity of the ligand have been confirmed by ¹H-NMR in d₆-DMSO and D₂O (Fig. S1 and S2), ¹³C-NMR (Fig. S3), FT-IR spectrometry (Fig. S4), and CHNS analysis.

Scheme 1 The outline synthesis of the AHMT ligand.

AHMT: ¹H NMR (DMSO-d₆, 500 MHz): δ (ppm) = 4.09 (br, 2H, NH₂), 5.26 (s, 2H, NH₂), 7.14 (s, 1H, NH), 12.68 (s, 1H, SH); all protons are exchangeable with D₂O; ¹³C NMR (DMSO-d₆, 125 MHz): δ (ppm) = 164.4 (1C), 154.7 (1C); FT-IR (cm⁻¹): 3263, 3211, 3186, 3075, 3018 (NH₂, NH), 1646 (NH₂), 1591 (C=N), 2920 (S–H), 1456–1281 (C–N + C–C), 1156 (C–S). Color white powder; m.p. 231–233 °C.³¹ CHN analysis calcd (%) for C₂H₆N₆S (146.17): C, 16.43; H, 4.14; N, 57.50; S, 21.93; found: C, 16.41; H, 4.19; N, 57.43; S, 22.02. UV/vis λ_max (DMSO)/nm: 266.

2.3. Synthesis of metal complexes

Three targeted complexes (Fig. 3) were synthesized by adding a methanolic solution of 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole (AHMT) (3 mmol, 0.45 g) to metal chloride (CoCl₂·6H₂O, NiCl₂·6H₂O, CuCl₂·2H₂O, 1 mmol, 0.24 g, 0.24 g, and 0.17 g, respectively) dissolved in a hot methanolic solution. The reactions were refluxed for 8 h at 80 °C.

[Co(AHMT)₂Cl₂]: FT-IR (cm⁻¹): 3292, 3264, 3212, 3181, 3061, 3020 (NH₂, NH), 1648 (NH₂), 1596, 1543 (C=N), 2941 (S–H), 1443–1236 (C–N + C–C), 1158 (C–S). Color brown powder; m.p. > 300 °C. CHN analysis calcd (%) for C₄H₁₂N₁₂S₂CoCl₂ (422.18): C, 11.38; H, 2.87; N, 39.81; S, 15.19; found: C, 11.31; H, 3.09; N, 39.62; S, 15.36. µ_eff (5.35µ_B) and conductivity: 2.1 Ω⁻¹ cm² mol⁻¹. UV/vis λ_max (DMSO)/nm: 268, 288, 416, 672.

[Ni(AHMT)₂Cl₂]·H₂O: FT-IR (cm⁻¹): 3388, 3263, 3210 (NH₂, NH), 1646 (NH₂), 1593 (C=N), 2931 (S–H), 1446–1235 (C–N + C–C), 1196, 1157, 1157 (C–S). Color light green powder; m.p. > 300 °C. CHN analysis calcd (%) for C₄H₁₄N₁₂OS₂NiCl₂ (439.95): C, 10.92; H, 3.21; N, 38.20; S, 14.58; found: C, 11.14; H, 3.66; N, 37.32; S, 13.83. µ_eff (2.14µ_B) and conductivity: 3.5 Ω⁻¹ cm² mol⁻¹. UV/vis λ_max (DMSO)/nm: 272, 384.

[Cu(AHMT)₃]Cl₂: FT-IR (cm⁻¹): 3264, 3212, 3185, 3076, 3019 cm⁻¹ ν(NH₂, NH), 1647 (NH₂), 1594 cm⁻¹ (C=N), 2918 cm⁻¹ (S–H), 1461–1212 (C–N + C–C), 1157 (C–S). Color grey powder; m.p. 192 °C. CHN analysis calcd (%) for C₆H₁₈N₁₈S₃CuCl₂ (572.96): C, 12.58; H, 3.17; N, 44.0; S, 16.79; found: C, 13.59; H, 3.12; N, 44.13; S, 17.67. µ_eff (0.99µ_B) and conductivity: 80.3 Ω⁻¹ cm² mol⁻¹. UV/vis λ_max (DMSO)/nm: 268, 296, 556.

3. Results and discussion

3.1. General aspects

AHMT was reacted with CoCl₂, NiCl₂, or CuCl₂ at a 3 : 1 ligand/metal ratio, leading to the formation of neutral and cationic complexes with chemical formulae: [Co(AHMT)₂Cl₂], [Ni(AHMT)₂Cl₂]·H₂O and [Cu(AHMT)₃]Cl₂ (Fig. 3). The complexes were powdery in appearance and practically insoluble in water as well as in most of the common organic solvents. Conductivity measurements in DMSO indicate the 1 : 2 electrolyte behavior for [Cu(AHMT)₃]Cl₂ (80.3 Ω⁻¹ cm² mol⁻¹),³² whereas [Co(AHMT)₂Cl₂] and [Ni(AHMT)₂Cl₂]·H₂O (2.1, 3.5 Ω⁻¹ cm² mol⁻¹, respectively) are non-electrolytes.³²

3.2. Computational studies

Based on the earlier studies,^33–35 AHMT ligand can exist in both thiol and thione forms due to tautomerism (Fig. 4). As shown in Fig. 5, DFT calculations were performed through the Gaussian package confirms that the thiol tautomeric form is a more bioactive coordinator than the other tautomeric form according to the simulated electronic features involving energy gap (ΔE), hardness (η), and softness (S).

ΔE = (E_HOMO − E_LUMO) (1)

η = 1/2(E_HOMO − E_LUMO) (2)

S = 1/η (3)

Fig. 4 Thiol and thione forms of AHMT ligand.

Fig. 5 FMO levels for two tautomeric forms of AHMT and their chemical parameters.

As the thiol conformer of AHMT (E_gap = −5.686 eV) displays greater biological and chelation potentiality through thiol and amino functions, compared to the thione conformer (E_gap = −5.465 eV). On the other hand, the softness as a bioactivity concept assures the relative convergence in the energies, value of both geometrical tautomers of the thiol form with a value (S = −0.351) in front of S = −0.366 for thione.

3.3. Characterization of the isolated complexes

3.3.1. Vibrational spectra. The important infrared frequencies exhibited by AHMT and the isolated complexes are listed in the Experimental section. The ligand molecule contains a tautomeric thioamide moiety: HN–C=S or N=C–SH and hence gives rise to four characteristic bands in the IR spectrum (Fig. S4). These bands are observed around 1456, 1329, 1156, and 650 cm⁻¹. Because of the redistribution of the electron cloud, as a result of complex formation, there is a systematic shift in these bands in the IR spectra of the complexes as compared to those of the ligand (Fig. S1). These shifts are consistent with the coordinating ability of the sulfur atom to metal.³⁵

AHMT's IR spectra (Fig. S4) showed characteristic absorption bands at 3263, 3211, 3186, 3075, and 3018 cm⁻¹ assigned for NH₂, NH–NH₂ groups,^36,37 1591, 1281 cm⁻¹ corresponded to C=N, C–N groups^36–38 and 2920 cm⁻¹ specified for S–H group.⁹ Whereby, in the IR spectra of the metal complexes, it was observed that the NH₂ group bands were shifted, indicating binding of the NH₂ group with the metal ion. The characteristic bands for ν(S–H) and ν(C–S) were clearly shifted in the spectra of the Co(II)-AHMT and Ni(II)-AHMT complexes (Fig. S4), indicating that the thiolic –SH group was still protonated and was involved in coordination with metal ions.^39,40 Consequently, the coordination environment around the Co and Ni atoms involves NH₂ and SH groups. While ν(C=N) and ν(C–N) bands shifted downward in the CuCl₂-AHMT complex, indicating that the nitrogenous atom is involved in the coordination to the copper atom. Therefore, the coordination sphere consists of NH₂ and C=N groups around the copper atom. Whereas, a broad medium band is allocated in the range 3200–3500 cm⁻¹, attributable to the ν(O–H) vibration of solvent molecules.⁴¹ The spectra (Fig. S4) showed bands in the 416–544 and 542–668 cm⁻¹ regions, which are assigned to ν(M–N) and ν(M–S), respectively.^42,43

3.3.2. Electronic spectra. The UV spectrum of AHMT ligand (Fig. S5) exhibits an absorption peak at 266 nm. This peak arises from the conjugated aromatic triazole ring system. The electronic spectrum of [Co(AHMT)₂Cl₂] (Fig. 6) shows two broad bands in the visible region at 672 nm (14 837 cm⁻¹) and 416 nm (24 038 cm⁻¹) assigned to ⁴T_1g → ⁴A_2g (ν₂) and ⁴T_1g → ⁴T_1g(P) (ν₃) characteristic for an octahedral geometry. Moreover, the value of the room temperature magnetic moment (µ_eff = 5.35µ_B) is taken as evidence for the existence of one high-spin cobalt atom supporting an octahedral geometry.

Fig. 6 Electronic spectra of complexes Co(II)-AHMT and Cu(II)-AHMT.

The electronic absorption spectrum of [Ni(AHMT)₂Cl₂]·H₂O complex (Fig. S5) shows a sole weak shoulder absorption band at 384 nm, which is attributed to the ³A_2g(F) → ³T_1g(P) (ν₃) transition, in favor of octahedral geometry. Also, the magnetic moment value of Ni(II) complex assured the presence of two unpaired electrons (d⁸) in the octahedral geometry (2.14µ_B). However, the observed magnetic moment of 2.14µ_B is lower than the spin-only value (∼2.83µ_B) expected for a high-spin Ni(II) (d⁸) complex. It can be concluded that the low µ_eff (2.14µ_B) indicates that [Ni(AHMT)₂Cl₂]·H₂O adopts a low-spin configuration in a distorted octahedral geometry, with strong ligand field effects and spin–orbit coupling reducing the number of unpaired electrons. These findings align with studies showing similar magnetic behavior in Ni(II) complexes with strong-field ligands.^44–46

The electronic absorption spectrum for [Cu(AHMT)₃]Cl₂ complex (Fig. 6) shows a broad band at 556 nm, which corresponds to a d–d transition typical for Cu(II) complexes in a distorted octahedral environment. The magnetic moment (µ_eff) of 0.999µ_B is significantly lower than the spin-only value (∼1.73µ_B) expected for an isolated Cu(II) ion with one unpaired electron. This reduced magnetic moment indicates strong antiferromagnetic coupling between Cu(II) centers in the complex, likely mediated by bridging ligands or close metal–metal interactions.^44,45

3.3.3. Thermal gravimetric analysis. The thermogravimetric analysis (TGA) data reveal a three-stage decomposition process for the Co(II)-AHMT complex (Fig. S6). In the initial stage (28–60 °C), a 57.30% mass loss aligns closely with the theoretical value of 57.59%, attributed to the release of volatile fragments including Cl₂, 4CN and 2H₂S. This suggests efficient dehydration and elimination of small molecules at low temperatures. The second stage (60–502 °C) shows a 27.71% loss (theoretical: 28.45%), likely from the release of volatile fragments including 4N₂ + 4H₂. Finally, the residue (502–800 °C) constitutes 13.96% (theoretical: 14.99%) as metallic cobalt (Co), confirming its thermal stability and role as an end product. The thermogravimetric analysis (TGA) of Ni(II)-AHMT complex (Fig. 7) demonstrates a multi-stage decomposition process. In the first stage (28–201 °C), a 12.88% mass loss (theoretical: 12.38%) corresponds to the release of H₂O and HCl, likely from dehydration and elimination of coordinated ligands. The second stage (201–285 °C) shows a 21.89% loss (theoretical: 22.40%), attributed to HCl, N₂, and H₂S, indicating decomposition of nitrogen- and sulfur-containing ligand fragments. At higher temperatures (285–434 °C), 24.54% mass loss (theoretical: 24.57%) arises from 4CN and 2H₂, reflecting cleavage of cyanide groups and hydrogen evolution. The fourth stage (434–541 °C) involves 17.59% loss (theoretical: 17.98%) due to H₅N₃S, suggesting further breakdown of nitrogen–sulfur hybrid moieties. The final residue (23.10%, theoretical: 22.89%) comprises NiN₃, confirming the formation of a thermally stable nickel-azide compound.

Fig. 7 The TGA curve of Ni(II)-AHMT and Cu(II)-AHMT complexes.

Finally, the thermogravimetric analysis (TGA) of Cu(II)-AHMT complex (Fig. 7) reveals a two-stage decomposition process followed by residue formation. The initial stage (30–217 °C) exhibits a 24.88% mass loss (theoretical: 24.95%), attributed to the release of Cl₂, 2N₂, and NH₂, suggesting the elimination of chlorine-containing ligands and nitrogen-rich fragments. The second stage (217–572 °C) shows a substantial 58.01% loss (theoretical: 58.36%), corresponding to the evolution of 2H₂S, 6HCN, 3N₂, NH₄⁺, and H₂, indicating the breakdown of sulfur- and nitrogen-based ligands, including hydrogen cyanide and ammonium-related species. The final residue (572–800 °C) constitutes 17.11% (theoretical: 16.68%) as CuS, confirming the formation of a thermally stable copper sulfide compound.

3.3.4. Electron spin resonance. The Cu(II)-AHMT complex exhibits an anisotropic ESR spectrum (Fig. 8) with distinct g_∥ (2.10694) and g_⊥ (2.16285) values, indicative of an axially distorted environment around the Cu(II) ion. This pattern is characteristic of a d_x²−y² ground state, typically observed for copper(II) ions in distorted octahedral or elongated square planar geometries, and confirms the presence of a tetragonal distortion due to the Jahn–Teller effect. Complementary evidence from ESR parameters, optical absorption features, and magnetic moment measurements persuasively supports the conclusion that the [Cu(AHMT)₃]Cl₂ complex adopts a distorted octahedral geometry around the copper(II) ion. This structure is stabilized by the Jahn–Teller effect, which elongates along the z-axis and gives rise to the observed spectral and magnetic behaviors.

Fig. 8 X-band ESR spectrum of Cu(II)-AHMT.

3.3.5. EI-mass spectrometry. The mass spectra for Co(II)-AHMT and Ni(II)-AHMT complexes, shown in Fig. S7 and S8, display molecular ion peaks [M]⁺ at m/z values of 422.59 and 439.06 with relative abundances of 23.53% and 23.83%, respectively. While the EI-mass spectrum of Cu(II)-AHMT exhibits a molecular ion peak [m/z] at 524.68 with relative abundance 42.57%, which corresponds to the fragment pattern [M–NH₂–SH]⁺ shown in Fig. S9. These observed peaks closely match the calculated molecular weights, thereby confirming the proposed chemical structures of the complexes.

3.4. Evaluation of biological activity

3.4.1. Antibacterial activity. The AHMT ligand and its metal complexes exhibit varying antibacterial and antifungal activity, depending on the coordinated metal ion (Fig. 9, S10 and Table S1). The AHMT exhibits moderate antifungal efficacy against C. albicans (16 mm inhibition zone, 59.2% activity index compared to clotrimazole), surpassing its antibacterial effects against E. coli (9 mm, 34.6%) and S. aureus (11 mm, 45.8%). The Ni(II)-AHMT combination exhibits the most potent antibacterial activity among the metal complexes, especially against S. aureus (14 mm, 58.3% activity index), outperforming the Cu(II)-AHMT complex (9 mm, 37.5%) and the Co(II)-AHMT complex (5 mm, 20.8%). This indicates that Ni(II)-AHMT coordination enhances antibacterial efficacy compared with the unbound ligand. Nonetheless, the ligand alone shows enhanced antifungal efficacy relative to its metal complexes, as evidenced by a larger inhibition zone against C. albicans (16 mm vs. 8–13 mm for the metal complexes). The diminished activity of the Co(II)-AHMT complex against all examined pathogens (3–8 mm inhibitory zones) underscores the pivotal importance of metal ion selection in influencing efficacy. These results align with research indicating that 1,2,4-triazole-metal complexes often exhibit superior antibacterial activity compared to their ligands, but the degree of enhancement depends on the metal's electronic and steric properties.^1,47

Fig. 9 Antimicrobial activity of AHMT and its metal complexes.

The antimicrobial screening revealed a clear correlation between inhibition zone diameters (Fig. 9 and Table S1) and Minimum Inhibitory Concentration (MIC) (Fig. 9 and Table S2).^48,49 The free ligand AHMT exhibited moderate antibacterial activity against E. coli (MIC = 8 µg mL⁻¹) and S. aureus (MIC = 16 µg mL⁻¹), while demonstrating notable antifungal potency against C. albicans (MIC = 4 µg mL⁻¹). Upon coordination with metal centers, the biological activity shifted significantly, illustrating the critical role of the metal ion in modulating the antimicrobial profile. The Ni(II)-AHMT complex emerged as the most effective agent in this series, demonstrating superior activity against both Gram-negative and Gram-positive bacteria and fungi. Notably, it achieved an MIC of 2 µg mL⁻¹ against S. aureus, representing an eight-fold increase in potency compared to the free AHMT ligand (16 µg mL⁻¹). It also showed strong inhibition against E. coli (4 µg mL⁻¹) and C. albicans (8 µg mL⁻¹). This broad-spectrum efficacy suggests that the nickel(II) center facilitates optimal membrane permeability and interaction with multiple microbial molecular targets. The Cu(II)-AHMT complex showed selective potency against S. aureus (4 µg mL⁻¹), though it was less effective against E. coli and C. albicans compared to the free ligand. Conversely, coordination with Co(II) resulted in a significant reduction in bioactivity (MICs 16–64 µg mL⁻¹), suggesting that the cobalt(II) center may hinder the ligand's biological availability or pharmacophore orientation. The enhanced antimicrobial activity of the metal complexes compared to the free AHMT ligand is primarily governed by Tweedy's chelation theory.⁵⁰ The partial sharing of the metal ion's positive charge with the ligand's donor groups (N and S atoms) results in a decrease in the polarity of the metal ion and a significant increase in the lipophilicity of the complex. This promotes easier penetration through the lipoid layers of the microbial cell membranes. Furthermore, coordination might enhance the compounds' ability to block the metal-binding sites of microbial enzymes or to interfere with the respiratory chain, ultimately leading to superior inhibitory effects, as evidenced by the lower MIC values.

3.4.2. In vitro anticancer activity. The AHMT ligand and its metal complexes demonstrate varying cytotoxicity profiles across the tested cancer cell lines (Fig. 10, 11 and Table S3), with metal coordination playing a significant role in determining potency. The AHMT exhibits moderate cytotoxicity, especially towards HePG-2 (IC₅₀ = 18.01 ± 1.4 µM) and HeLa (21.46 ± 1.5 µM), surpassing the efficacy of its Cu(II)-AHMT complex (40.62–55.58 µM) and Co(II)-AHMT complex (42.84–64.48 µM). The Ni(II)-AHMT complex exhibits significantly increased activity, particularly against MCF-7 (8.70 ± 0.5 µM) and HeLa (9.72 ± 0.7 µM), exceeding the efficacy of both the unbound ligand and the reference drug sorafenib (SOR) in these cell lines. This highlights the role of nickel in enhancing efficacy, potentially attributed to improved DNA interaction or redox activity.^51–53 Notably, the compounds' potential toxicity was assessed using the MTT assay on MCF-10A normal cells. The findings demonstrated low cytotoxicity towards normal cells at concentrations effective against cancer cells, as presented in Fig. 11 and Table S3. Based on Tweedy's chelation theory,⁵⁰ coordination reduces the polarity of the metal ion and increases the lipophilicity of the complex. This allows preferential penetration through the more fluid, negatively charged membranes of cancer cells. Consequently, the complexes reach lethal intracellular concentrations in malignant cells more effectively than in normal cells, explaining the observed selectivity.

Fig. 10 The plot of series dilutions versus cell viability% in cell lines. Each data point represents the mean ± SD (n = 3), with statistical significance indicated by (*p < 0.05).

Fig. 11 Cytotoxicity IC₅₀ (µM) of AHMT and its metal complexes.

Co(II)-AHMT and Cu(II)-AHMT complexes exhibit reduced cytotoxicity, underscoring the metal-dependent nature of their antitumor effects. The Ni(II)-AHMT complex exhibits potency comparable to that of doxorubicin (DOX) in MCF-7 cells (8.70 vs. 4.17 µM), indicating its potential as a targeted chemotherapeutic agent. The selectivity index of Ni(II)-AHMT toward MCF-7 cells, is approximately 14.3, indicating that the compound is about fourteen times more toxic to cancer cells than to normal cells and therefore exhibits high selectivity and a potentially favorable safety margin, suggesting that Ni(II)-AHMT exerts its anticancer effects through ligand-specific mechanisms rather than nonspecific nickel toxicity, as free Ni²⁺ ions typically exhibit much lower selectivity and higher toxicity toward normal cells. These trends align with research indicating that triazole-metal complexes often enhance cytotoxicity by stabilizing ligand–metal interactions and selectively targeting cells.⁵²

3.4.3. Apoptosis assay and cell cycle analysis. Cycle and apoptosis analyses (Fig. 12 and 13) have been conducted on the Ni(II)-AHMT complex to investigate the mechanism of cytotoxicity. Whereas, flow cytometry results show that the Ni(II)-AHMT complex induces cytotoxicity in MCF-7 breast cancer cells by arresting the cell cycle specifically at the G1 phase. Whereas, Ni(II) complex triggered a substantial accumulation of cells in the G0–G1 phase (81.96%) compared to the untreated control (59.38%). Consequently, there was a significant lessening in the percentage of cells progressing to the S phase (13.39% vs. 28.75% in the control) and to the G2/M phase (4.65% vs. 11.87% in the control). On the other hand, the cell death mechanism induced by Ni(II)-AHMT is primarily apoptosis rather than necrosis, a desirable characteristic for potential pharmacophores. The obtained data show that Ni(II)-AHMT induced a total apoptosis rate of 31.61%, representing a substantial increase over the 2.77% observed in spontaneous apoptosis in control cells.

Fig. 12 Cell cycle phases diagrams, (a) plot of cell cycle phases percentages of treated and untreated cells, (b) treated cells with Ni-AHMT, (c) untreated MCF-7 cells.

Fig. 13 Apoptosis dot plots of MCF-7 cells: (a) quantification of total, early, and late apoptosis, as well as necrosis, in treated and untreated cells, (b) cells treated with Ni-AHMT, (c) untreated cells.

3.4.4. Superoxide (SOD)-like activity. The AHMT ligand and its metal complexes exhibit substantial superoxide dismutase (SOD) activity (Fig. 14 and Table S4), with efficacy varying depending on metal coordination. The unbound ligand exhibits significant radical-scavenging ability, attaining 75.8% inhibition, which is equivalent to the reference antioxidant L-ascorbic acid at 78.2%. Nickel coordination (Ni(II)-AHMT) maintains high activity (70.2%), but Cu(II)-AHMT (42.9%) and Co(II)-AHMT (30.9%) complexes exhibit diminished efficacy.

Fig. 14 Superoxide (SOD)-like activity of AHMT and its isolated complexes.

This metal-dependent trend corresponds to SOD processes, in which nickel's redox versatility facilitates effective superoxide disproportionation via cyclic Ni(II)/Ni(III) transitions, whereas cobalt's diminished activity may arise from reduced metal–ligand cooperativity in electron transfer. The ligand's independent efficacy indicates that its mercapto and hydrazino groups facilitate radical stabilization, possibly via hydrogen bonding or direct electron donation. These findings underscore AHMT's dual function as an active antioxidant and a framework for modulating metal-specific SOD activity, with nickel complexes demonstrating notable potential for alleviating oxidative stress.^54,55

3.4.5. Antioxidant activity. The DPPH assay results demonstrate the antioxidant activity of the AHMT ligand and its metal complexes, assessed by their efficacy in scavenging free radicals at different doses (Fig. 15 and Table S5). The unbound ligand (AHMT) exhibits significant radical scavenging efficacy, achieving an inhibition rate of 98.1% at 100 µM and an IC₅₀ value of 20.90 ± 0.14 µM, signifying a robust antioxidant capability comparable to that of the reference vitamin C (IC₅₀ = 16.81 ± 0.10 µM). The Ni(II)-AHMT complex exhibits notable activity, achieving 92.8% inhibition at 100 µM and an IC₅₀ of 27.81 ± 0.19 µM, surpassing both Cu(II)-AHMT (IC₅₀ = 33.51 ± 0.22 µM) and Co(II)-AHMT (IC₅₀ = 62.05 ± 0.48 µM), which demonstrate increasingly diminished antioxidant properties. The data indicate that nickel coordination marginally reduces but predominantly maintains the ligand's antioxidant activity, whereas cobalt complexation substantially diminishes it. The DPPH assay substantiates the robust free radical scavenging capacity of the AHMT ligand and underscores the role of metal ions in altering its antioxidant characteristics.

Fig. 15 Antioxidant activity of AHMT and its isolated complexes.

3.4.6. Colorimetric assay for DNA binding affinity. The DNA affinity of the AHMT ligand and its metal complexes was assessed using a DNA/methyl green displacement assay, with IC₅₀ values denoting the concentration necessary to displace 50% of the dye (Fig. 16 and Table S6). The findings indicate that the Ni(II)-AHMT complex demonstrates the most potent DNA binding among the evaluated drugs, with an IC₅₀ of 38.32 ± 1.7 µM, closely rivaling the reference medication doxorubicin (DOX), which has an IC₅₀ of 31.54 ± 1.5 µM. The unbound ligand AHMT exhibits a moderate binding affinity of 41.85 ± 1.9 µM, but the Cu(II)-AHMT and Co(II)-AHMT complexes display increasingly diminished interactions with DNA, with IC₅₀ values of 57.60 ± 2.2 µM and 71.13 ± 2.8 µM, respectively. This pattern indicates that nickel coordination amplifies the ligand's capacity to bind with DNA, possibly via enhanced intercalation or groove binding, whereas copper and cobalt complexes diminish this affinity. These findings underscore the importance of metal ion selection in shaping the DNA-binding properties of triazole-based drugs, with nickel complexes demonstrating significant potential for DNA-targeted therapeutic applications.

Fig. 16 DNA/methyl green colorimetric assay of AHMT and isolated complexes.

3.5. Molecular docking studies

3.5.1. DNA binding. Cytotoxic agents are thought to exert their effects by binding to DNA base pairs. Intercalation and groove binding are two main binding modes of metal complexes, including Co(II), Ni(II), and Cu(II), with DNA. Metal complexes can interact with DNA via covalent hydrogen bonds (HB) or non-covalent electrostatic forces.³⁰ Intercalator metal complexes can effectively insert into the base pairs of the DNA double helix, disrupting its function.⁵⁶ Groove binders disrupt DNA replication and transcription and, accordingly, can induce apoptosis and cell cycle arrest, making them effective cytotoxic and antimicrobial agents.^57,58 Metal complexes could also bind within the grooves of the DNA double helix. The positively charged metal and the negatively charged phosphate backbone may also interact via electrostatic forces. The presence of an aromatic ligand, such as 1,2,4-triazole, bound to a metal is expected to enhance DNA binding affinity through intercalation.^59,60 Considering these proven scientific facts, we decided to evaluate the potential DNA-binding activity of 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole (AHMT) and its complexes with Co(II), Ni(II), and Cu(II). This has been conducted in vitro, and the obtained results were validated and rationalized using in silico methods, including molecular docking. The free ligand (AHMT) acted as a DNA groove binder through the formation of three hydrogen bonding interactions within a distance range of 2.89–3.28 Å. The terminal primary amine functionality of the hydrazine group (NH–NH₂) formed one HB with the nucleotide base DA10, whereas the adjacent secondary NH group of the hydrazine side chain built another HB with the amidic C=O group of thymine (DT31). In the last HB interaction, the NH₂ group attached to N-4 of AHMT served as a hydrogen bond donor, while the carbonyl group of cytosine (DC32) served as an acceptor. The docking score for this ligand was −4.21 kcal mol⁻¹. The cobalt complex Co(II)-AHMT displayed −3.90 kcal mol⁻¹ as a free energy of binding and interacted with DNA through a set of four HB interactions. This metal complex interacted with the DNA nucleotide bases DG37, DA5, and DG6. In this set of HBs, the terminal hydrazine moieties of Co(II)-AHMT acted as HB donors in two interactions, whereas in the other two, one of its chlorine atoms served as an acceptor. Fig. 17 displays the extraordinary mixed approach of intercalation and groove-binding pattern of the 1,2,4-triazole-nickel complex Ni(II)-AHMT. This ligand displayed the highest docking score (−4.42 kcal mol⁻¹) and interacted with DNA by five HBs and one π-H stacking contact. A bidirectional HB has been observed between the nucleotide base DG4 and both chlorine atoms of the metal complex. One of the terminal hydrazine moieties formed a HB with the nucleotide base guanine DG6, while the mercapto group acted as a HB acceptor to build one more HB with DG38 within 4.22 Å. A π-H stacking contact has been formed between this nickel complex and adenine nucleotide base (DG3). The relatively high docking score and the extraordinary interaction pattern of this Ni(II)-AHMT complex may help rationalize the better in vitro results of this ligand in DND binding and cytotoxicity assessments.

Fig. 17 3D interaction patterns of AHMT (A), Co(II)-AHMT (B), Ni(II)-AHMT (C), and Cu(II)-AHMT (D) with DNA double helix.

The copper complex Cu(II)-AHMT displayed a relatively lower binding energy (−2.24 kcal mol⁻¹) and revealed five HBs and one π-H stacking interaction. The HB donor groups of this ligand interacted with the nucleotide bases DA10, DT12, DC13, DC32, and DT31, whereas the π-H stacking has been developed between the 1,2,4-triazole core and the pyrimidine ring of DT31. Table S7 summarizes the docking scores, interaction distances, interacting nucleotides, and interaction types for the free ligand AHMT and its metal complexes.

3.5.2. β-Lactamases inhibition. One major mechanism of microbial resistance to commonly prescribed antibiotics is the production of β-lactamases, a group of enzymes that hydrolyze β-lactam antibiotics, rendering them ineffective.⁶¹ A wide range of bacteria, such as E. coli and S. aureus, can develop this type of antibiotic resistance. Thus, we decided to investigate the virtual binding of AHMT and its metal complexes, Co(II)-AHMT, Ni(II)-AHMT, and Cu(II)-AHMT, with β-lactamases. AHMT exhibited a docking score of −4.00 kcal mol⁻¹ and displayed two HBs and two π-H stacking interactions with the biological target studied. N-1 and the terminal amino group of the hydrazine functionality in AHMT worked as HB acceptors to form two interactions with the amino acid residues Gly236 and Arg244. Within 3.47 Å, one hydrophobic interaction developed between the aromatic ring of Tyr105 and the N-4 attached amine in AHMT. One additional hydrophobic interaction formed between the 1,2,4-triazole ring and the aliphatic side chain of Ala237. This virtual binding pattern may support the relatively wider-diameter in vitro inhibition zones of AHMT against E. coli (9.00 mm) and S. aureus (11 mm). Co(II)-AHMT produced the lowest docking score (−3.11 kcal mol⁻¹) and displayed five hydrogen bonds and one ionic (electrostatic) interaction with five amino acid residues of E. coli β-lactamase (Met272, Glu104, Glu240, Asn132, and Tyr105). This result is almost consistent with the in vitro antimicrobial testing. Conversely, the nickel complex Ni(II)-AHMT has shown the best combined results (scoring energy = −5.77 kcal mol⁻¹; RMSD = 1.88; Table S8). Six HBs and one hydrophobic interaction have been built between this antimicrobial candidate and the amino acid backbones of E. coli β-lactamase. One of the terminal hydrazine functionalities of Ni(II)-AHMT acted as HB donor/acceptor to form three covalent interactions with Asn170 and Glu166 residues within 2.81–3.10 Å. Ser130 and Ser235 residues developed two favorable HB interactions with Ni(II)-AHMT within 3.59 and 2.77 Å, respectively. The aliphatic chain of Ala239 formed one π-H stacking contact with the 1,2,4-triazole ring of Ni(II)-AHMT. This unique interaction profile contributes to rationalizing the superior in vitro activity of Ni(II)-AHMT in antimicrobial testing (% activity indices for E. coli and S. aureus = 46.1% and 58.30%, respectively). With −5.53 kcal mol⁻¹ as a free energy of binding, Cu(II)-AHMT interacted with β-lactamase through a set of covalent and non-covalent interactions. Glu104 served as an HB donor and an anionic partner, forming three hydrogen bonds and five ionic interactions. Likewise, Glu240 formed an additional hydrogen bond and one more ionic interaction, whereas Asn170 and Asn132 acted as HB donors and acceptors, forming two covalent bonds. Table S8 and Fig. 18 show the detailed docking results of AHMT, Co(II)-AHMT, Ni(II)-AHMT, and Cu(II)-AHMT into E. coli β-lactamase.

Fig. 18 3D interaction patterns of AHMT (A), Co(II)-AHMT (B), Ni(II)-AHMT (C), and Cu(II)-AHMT (D) with E. coli β-lactamase.

3.5.3. Binding with human serum albumin (HSA). Because of its ability to bind and transport anticancer, antimicrobial, and other medications, human serum albumin (HSA) is considered a key determinant and a major contributor to drug delivery and pharmacokinetics.⁶² Considering this importance, we examined the in silico binding potential of AHMT and its metal complexes, Co(II)-AHMT, Ni(II)-AHMT, and Cu(II)-AHMT, to HSA and compared them with the native HSA binder, warfarin. Results of docking experiments showed that the original warfarin–HSA complex (Fig. 19) shows three classical HBs with Tyr150, Arg222, and Lys199 and one hydrophobic interaction with Ala291.⁶²

Fig. 19 Binding pattern of warfarin with the HSA. HBs are shown as red dotted lines, while hydrophobic interactions are shown as faint green dotted lines.

The binding interaction of AHMT with HSA (Fig. 20) includes a conventional HB with the amino acid Ser192 within 3.27 Å, and one hydrophobic interaction with Lys195 within 4.29 Å. The critical amino acid residues included in the binding interaction of warfarin (Ala291, Lys199, and Arg222) were also found in the binding interactions of Co(II)-AHMT and Cu(II)-AHMT with HSA. The triazole ring in each ligand interacted through hydrophobic stacking with the aliphatic chain of Ala291. Ni(II)-AHMT displayed one of the highest docking scores (−4.06 kcal mol⁻¹; Table S9), indicating its potential to effectively bind with HSA, and consequently better distribution and delivery. Fig. 20 shows the detailed docking results of AHMT, Co(II)-AHMT, Ni(II)-AHMT, and Cu(II)-AHMT into the active pocket of HSA.

Fig. 20 3D interaction patterns of AHMT (A), Co(II)-AHMT (B), Ni(II)-AHMT (C), and Cu(II)-AHMT (D) into the active pocket of HSA.

3.6. Structure–activity relationships (SAR)

The biological activities of AHMT and its Co(II), Ni(II), and Cu(II) complexes are intricately associated with their structural characteristics, which affect stability, solubility, and interactions with targets. The planar triazole core of AHMT facilitates π–π stacking and hydrogen bonding with biomolecular targets such as DNA. Additionally, its amino (–NH₂), hydrazino (–NH–NH₂), and mercapto (–SH) groups offer various coordination sites for metal binding and radical scavenging. [Ni(AHMT)₂Cl₂]·H₂O (Ni(II)-AHMT) and [Co(AHMT)₂Cl₂] (Co(II)-AHMT) exhibit a monomeric octahedral structure, incorporating two bidentate AHMT ligands alongside two chloride ions. This configuration enhances DNA groove binding and β-lactamase inhibition by optimizing the balance of hydrophobicity and charge distribution. On the other hand, [Cu(AHMT)₃]Cl₂ (Cu(II)-AHMT) displays a cationic octahedral geometry coordinated by three AHMT ligands. The charged nature of DNA enhances electrostatic interactions; however, steric hindrance from the three ligands may diminish binding efficiency to enzymatic targets such as SOD. Moreover, the d⁸ configuration of Ni(II) enhances ligand field stabilization and promotes redox inactivity, thereby supporting the formation of stable DNA adducts and prolonged antimicrobial effects. In contrast, the d⁹ configuration of Cu(II) facilitates redox cycling, contributing to oxidative stress-mediated cytotoxicity but with lower selectivity. The hydrazino group in AHMT enhances the stability of metal chelation, whereas the mercapto group contributes to antioxidant activity through radical-scavenging mechanisms. The combination of these groups in Ni(II)-AHMT yields 70–75% SOD-like inhibition, surpassing that of Co(II) and Cu(II) analogs. Consequently, the structural insights elucidate Ni(II)-AHMT's enhanced DNA-binding efficacy through groove interactions, its antimicrobial activity via β-lactamase inhibition, and its cytotoxicity, with an IC₅₀ of approximately 8–9 µM against MCF-7/HeLa cells. The findings highlight the importance of metal–ligand coordination geometry and electronic properties in optimizing triazole-based therapeutics. The selectivity of the synthesized AHMT complexes toward malignant cell lines over normal cells can be rationalized through the synergy between Tweedy's chelation theory⁵⁰ and cancer cell physiology. The increased lipophilicity of the chelates facilitates uptake across the hyperpolarized, fluid membranes of cancer cells. Furthermore, the acidic microenvironment characteristic of tumor tissues likely promotes the selective intracellular release of the metal ions. Once internalized, these complexes induce targeted apoptosis by exploiting the high basal oxidative stress levels of cancer cells, a mechanism that normal cells are better equipped to resist due to their robust homeostatic and antioxidant capacities.

4. Conclusion

In conclusion, the synthesis and evaluation of 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole (AHMT) and its Co(II), Ni(II), and Cu(II) complexes demonstrate their significant potential in biomedical applications. Structural characterization confirmed the proposed configurations of the compounds, while biological assays highlighted Ni(II)-AHMT as the most promising candidate. It exhibited superior antibacterial and fungal activity, and strong DNA-binding affinity, suggesting that DNA is a key biotarget. Notably, both AHMT and Ni(II)-AHMT displayed remarkable antioxidant activity via superoxide dismutase (SOD) mimicry, achieving ∼75% inhibition in radical-scavenging assays. Interestingly, Ni(II)-AHMT exhibited potent cytotoxicity against MCF-7 and HeLa cancer cell lines (IC₅₀ values of 8.70 ± 0.5 µM and 9.72 ± 0.7 µM, respectively), as evidenced by a pronounced G0/G1 cell-cycle arrest (81.96% vs. 59.38% in control) accompanied by a strong induction of apoptosis (total apoptotic cells 49.90% vs. 3.51% in control), highlighting its potential as a promising apoptotic and cell-cycle-modulating agent. Molecular docking studies further validated these findings, revealing Ni(II)-AHMT's potential as a DNA groove binder and antimicrobial agent through stable interactions with biological targets like β-lactamase and human serum albumin. These results position Ni(II)-AHMT as a multifaceted therapeutic candidate, combining antimicrobial, anticancer, and antioxidant properties. Future in vivo studies are warranted to explore its pharmacokinetics, safety profile, and efficacy in complex biological systems, paving the way for novel drug development inspired by 1,2,4-triazole-based metal complexes.

Ethical approval

We have no financial conflicts or personal connections to declare. Also, this research did not involve the use of either humans/or animals. The biological assays were conducted in strict accordance with the guidelines and approval of the Ethics Committee at Mansoura University (Faculty of Science). No material was extracted from live vertebrate animals.

Conflicts of interest

The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper.

Data availability

The data are in the supplementary information (SI). Supplementary information: spectroscopic data of the compounds, materials, and methods. See DOI: https://doi.org/10.1039/d5ra09383j.

Acknowledgements

This research was supported by Royal Society of Chemistry Research Fund grants through project (R23-0040908760) (Azza A. Hassoon) and project (R25-3563728902) (Azza A. Hassoon).

References

1. L. Todorov and I. Kostova, Front. Chem., 2023, 11, 1247805.
2. A. Kumar, S. Devi, S. Kumar and K. Lal, Coord. Chem. Rev., 2025, 536, 216675.
3. S. M. El Rayes, Molecules, 2010, 15, 6759–6772.
4. H. Benaissa, N. N. Adarsh, K. Robeyns, J. J. Zakrzewski, S. Chorazy, J. G. M. Hooper, F. Sagan, M. P. Mitoraj, M. Wolff, S. Radi and Y. Garcia, Cryst. Growth Des., 2021, 21, 3562–3581.
5. C. Amaral, C. F. Almeida, M. J. Valente, C. L. Varela, S. C. Costa, F. M. Roleira and G. Correia-da-Silva, Cancers, 2025, 17, 165.
6. T. Hosseinnejad, F. Ebrahimpour-Malmir and B. Fattahi, RSC Adv., 2018, 8, 12232–12259.
7. E. A. Ghaith, A. B. Abdallah, A. A. El-Sawi and G. G. El-Bana, J. Heterocycl. Chem., 2025, 62, 1863–1875.
8. L. Qi, M.-C. Li, J.-C. Bai, Y.-H. Ren and H.-X. Ma, Bioorg. Med. Chem. Lett., 2021, 40, 127902.
9. D. Domyati, S. A. Zabin, A. A. Elhenawy and M. Abdelbaset, Processes, 2021, 9(6), 1008.
10. R. R. Radwan, N. H. Zaher and M. G. El-Gazzar, Chem.-Biol. Interact., 2017, 274, 68–79.
11. M. Ajmal, U. Yunus, R. M. Graham and R. M. Leblanc, ACS Omega, 2019, 4(27), 22280–22291.
12. P. C. Jilloju, L. Persoons, S. K. Kurapati, D. Schols, S. D. Jonghe, D. Daelemans and R. R. Vedula, Mol. Diversity, 2022, 26, 1357–1371.
13. A. Mermer and S. Demirci, Eur. J. Med. Chem., 2023, 259, 115655.
14. Y. Feng, L. Wu, Y. Luo, Y. Hao, P. Zhang, R. Zeng and S. Chen, Talanta, 2025, 291, 127858.
15. C. Liu, J. Huang, J. Chen, Q. Xue, H. Yan, D. Kong, Z. Ma, W. Shen, H. K. Lee and S. Tang, ACS Sens., 2025, 10(3), 2060–2072.
16. M. Z. Abedeen, M. Sharma, H. S. Kushwaha and R. Gupta, TrAC, Trends Anal. Chem., 2024, 176, 117729.
17. P. Srinivasan, S. P. Sivaraman, D. K. Madhu, P. Sengupta, B. Kattela, S. Nagarajan, A. M. Mohan and P. Deivasigamani, J. Hazard. Mater., 2024, 469, 133960.
18. P. Kumar and H. K. Sharma, Electrochim. Acta, 2013, 87, 925–929.
19. Vinita, M. Tiwari and R. Prakash, Appl. Surf. Sci., 2018, 449, 174–180.
20. G. B. Bagihalli, P. S. Badami and S. A. Patil, J. Enzyme Inhib. Med. Chem., 2009, 24, 381–394.
21. L. Calu, M. Badea, M. C. Chifiriuc, C. Bleotu, G.-I. David, G. Ionita, L. Marutescu, V. Lazar, N. Stanica, I. Soponaru, D. Marinescu and R. Olar, J. Therm. Anal. Calorim., 2015, 120, 375–386.
22. J.-K. Wang, C.-H. Wang, C.-C. Wu, K.-H. Chang, C.-H. Wang, Y.-H. Liu, C.-T. Chen and P.-T. Chou, J. Am. Chem. Soc., 2024, 146, 3125–3135.
23. H. S. Biswal and S. Wategaonkar, J. Phys. Chem. A, 2009, 113, 12763–12773.
24. K. Mazmanian, K. Sargsyan, C. Grauffel, T. Dudev and C. Lim, J. Phys. Chem. B, 2016, 120, 10288–10296.
25. Y. Y. Zheng, Y. Wu, T. J. Begley and J. Sheng, RSC Chem. Biol., 2021, 2, 990–1003.
26. R. N. Pandey, R. N. Sharma, A. N. Sahey, S. Kumar, S. Kumar and S. Singh, Asian J. Chem., 1993, 5, 813–817.
27. A. B. J. Tadros, J. Chem. Technol. Biotechnol., 1989, 45, 213–221.
28. (a) L. Mishra and A. Kyadaw, Proc. – Indian Acad. Sci., Chem. Sci., 2000, 112, 449–458; (b) S. Sener, I. Kul and K. Bhat, Synth. React. Inorg., Met.-Org., Nano-Met. Chem., 2015, 45, 495–501.
29. B. H. Garg and P. K. Sing, Synth. React. Inorg. Met.-Org. Chem., 1987, 17, 923–936.
30. R. C. Varma and K. Varma, Indian Chem. Soc., J., 1992, 69, 577–578.
31. R. G. Dickinson and N. W. Jacobsen, Org. Prep. Proced. Int., 1974, 6, 156–157.
32. W. J. Geary, The use of conductivity measurements in organic solvents for the characterisation of coordination compounds, Coord. Chem. Rev., 1971, 7, 81–122.
33. R. V. Gadag and M. R. Gajendragad, Curr. Sci., 1979, 2, 839.
34. R. V. Gadag and M. R. Gajendragad, Indian J. Chem., Sect. A, 1980, 19A, 705–707.
35. G. R. Burns, Inorg. Chem., 1968, 7, 277.
36. A. A. Hassoon, R. G. Harrison, N. Nawar, S. J. Smith and M. M. Mostafa, J. Mol. Struct., 2020, 1203, 127240.
37. A. A. Hassoon, S. J. Smith and R. G. Harrison, Inorg. Chem. Commun., 2025, 178, 114464.
38. A. A. Hassoon, S. J. Smith and R. G. Harrison, RSC Adv., 2024, 14, 31850–31860.
39. N. S. Al-Radadi, E. M. Zayed, G. G. Mohamed and H. A. Abd El Salam, J. Chem., 2020, 1, 1–12.
40. Y. Deswal, S. Asija, D. Kumar, D. K. Jindal, G. Chandan, V. Panwar, S. Saroya and N. Kumar, Res. Chem. Intermed., 2022, 48, 703–729.
41. K. Nakamoto, in Infrared and Raman Spectra of Inorganic and Coordination Compounds, Part B: Applications in Coordination, Organometallic, and Bioinorganic Chemistry, John Wiley & Sons, Inc., Hoboken, NJ, 6th edn, 2009.
42. J. Selbin, J. Chem. Educ., 1964, 41, 86–92.
43. J. R. Ferraro, Low Frequency Vibrations of Inorganic and Coordination Compounds, Plenum Press, New York, 1966.
44. A. B. P. Lever, Inorganic Electronic Spectroscopy, Elsevier, Amsterdam, 1984.
45. F. A. Cotton, G. Wilkinson, C. A. Murillo and M. Bochmann, Advanced Inorganic Chemistry, Wiley, New York, 6th edn, 1999.
46. W. Ferenc, A. W. Dziewulska, J. Sarzynski and B. Paszkowska, Eclética Quím., 2006, 31, 53–60.
47. M. Strzelecka and P. Świątek, Pharmaceuticals, 2021, 14, 224.
48. A. Adak, V. Castelletto, B. Mendes, G. Barrett, J. Seitsonen and I. W. Hamley, ACS Appl. Bio Mater., 2024, 7, 5553–5565.
49. A. Adak, S. Ghosh, V. Gupta and S. Ghosh, Biomacromolecules, 2019, 20, 1889–1898.
50. B. Tweedy, Phytopathology, 1964, 55, 910–914.
51. A. Adak, G. Das, V. Gupta, J. Khan, N. Mukherjee, P. Mondal, R. Roy, S. Barman, P. K. Gharai and S. Ghosh, J. Med. Chem., 2022, 65, 13866–13878.
52. Z. H. Chohan and M. Hanif, J. Enzyme Inhib. Med. Chem., 2010, 25, 737–749.
53. Y. Draoui, S. Radi, Y. Bahjou, A. Idir, A. El Mahdaoui, A. Zyad, H. N. Miras, M. Ferbinteanu, A. Rotaru and Y. Garcia, RSC Adv., 2023, 13, 36158–36167.
54. V. Pelmenschikov and P. E. M. Siegbahn, J. Am. Chem. Soc., 2006, 128(23), 7466–7475.
55. P. O'Neill, E. M. Fielden, D. Cocco, G. Rotilio and L. Calabrese, Biochem. J., 1982, 205, 181–187.
56. Y. Thakur, R. Agrawal, M. Tripathi, M. K. Siddiqi, E. Mohapatra, R. H. Khan and R. Pande, J. Mol. Struct., 2019, 1197, 691–706.
57. X. Cai, P. J. Gray and D. D. Von Hoff, Cancer Treat. Rev., 2009, 35, 437–450.
58. M. P. Barrett, C. G. Gemmell and C. J. Suckling, Pharmacol. Ther., 2013, 139, 12–23.
59. W. Zafar, S. H. Sumrra and Z. H. Chohan, Eur. J. Med. Chem., 2021, 222, 113602.
60. P. P. Utthra, N. Pravin and N. Raman, J. Photochem. Photobiol., B, 2016, 158, 136–144.
61. K. Bush, Curr. Opin. Microbiol., 2010, 13, 558–564.
62. R. D. Zanetti, G. A. da Cunha, M. B. Moreira, R. L. Farias, R. F. F. de Souza, P. R. D. V. de Godoy, M. S. Brassesco, F. V. Rocha, M. A. Lima, A. E. Mauro and A. V. G. Netto, Polyhedron, 2023, 229, 116185.

---