Development of some 1,2,3-triazole-based complexes incorporating hydroxy and acetamide groups: Synthesis, characterization, DFT calculation, and biological screening supported by molecular docking approach

2.4.1. TTMCo complex (Diacetato Cobalt(II) (1-(4-methylphenyl)-1H-1,2,3-triazol-4-yl)methanol):

Color : Light pink; Yield = 86 %; m.p. > 300°C; IR (KBr, υ): 3215 (OH), 3074 (Ar-CH), 2926 (Al-CH), 1562 (N=N), 1519 (C=C), 1413 (asyCOO^-), 1359 (syCOO^-), 1242 (C-O), 541 (Co-O) and 459 cm^-1 (Co-N); Analysis for [Co(TTM)(CH₃COO)₂].2H₂O; C₂₄H₃₂CoN₆O₈, 591.48: Calculated: C, 48.73; H, 5.45; N, 14.21; Co, 9.96% Found: C, 48.49; H, 5.25; N, 14.52; Co, 9.65 %. Λ _m* in DMF (ohm^-1cm² mol^-1): 12.15; µ_eff (B.M.); 3.38.

2.4.2. TTMNi complex (Diacetato Nickel(II) (1-(4-methylphenyl)-1H-1,2,3-triazol-4-yl)methanol):

Color: Turquoise; Yield = 84 %; m.p. > 300°C; IR (KBr, υ): 3205 (OH), 3089 (Ar-CH), 2926 (Al-CH), 1566 (N=N), 1517 (C=C), 1411 (asyCOO^-), 1359 (syCOO^-), 1251 (C-O), 549 (Ni-O) and 443 cm^-1 (Ni-N); Analysis for [Ni(TTM)(CH₃COO)₂]. H₂O; C₂₄H₃₀NiN₆O₇, 573.22: Calculated: C, 50.29; H, 5.28; N, 14.66; Ni, 10.24 % Found: C, 49.95; H, 5.15; N, 14.82; Ni, 10.55 %. Λ _m* in DMF (ohm^-1cm² mol^-1): 9.45; µ_eff (B.M.); 2.25.

2.4.3. TTMCu complex (Diacetato Copper(II) (1-(4-methylphenyl)-1H-1,2,3-triazol-4-yl)methanol):

Color : Cyan; Yield = 81 %; m.p. > 300 °C; IR (KBr, υ): 3211 (OH), 3091 (Ar-CH), 2924 (Al-CH), 1579 (N=N), 1516 (C=C), 1388 (asyCOO^-), 1334 (syCOO^-), 1257 (C-O), 563 (Cu-O) and 474 cm^-1 (Cu-N); Analysis for [Cu(TTM)(CH₃COO)₂]. 2H₂O; C₂₄H₃₂CuN₆O₈, 596.09: Calculated: C, 48.36; H, 5.41; N, 14.66; Cu, 10.66 % Found: C, 48.55; H, 5.25; N, 14.85; Cu, 10.35 %. Λ _m* in DMF (ohm^-1cm² mol^-1): 13.15; µ_eff (B.M.); 1.75.

2.4.4. TTMZn complex (Diacetato Zinc(II) (1-(4-methylphenyl)-1H-1,2,3-triazol-4-yl)methanol):

Color: light beige; Yield = 80 %; m.p. > 300 °C; IR (KBr, υ): 3205 (OH), 3118 (Ar-CH), 2926 (Al-CH), 1548 (N=N), 1517 (C=C), 1440 (asyCOO^-), 1381 (syCOO^-), 1244 (C-O), 621 (Zn-O) and 462 cm^-1 (Zn-N), Analysis for [Zn(TTM)(CH₃COO)₂]. H₂O; C₂₄H₃₀ZnN₆O₇, 578.92: Calculated: C, 49.71; H, 5.21; N, 14.49; Zn, 11.28 % Found: C, 49.57; H, 5.05; N, 14.67; Zn, 11.43 %. Λ _m* in DMF (ohm^-1cm² mol^-1): 8.45; µ_eff (B.M.); diamagnetic.

2.11.1. Antimicrobial activity

The disc diffusion technique was employed to evaluate the antibacterial and antifungal activities in vitro, as described in references [71-74]. The study included bacterial strains such as M. luteus (ATCC 4698), E. coli (ATCC 25922), and S. marcescens (ATCC 13880), as well as fungal species, including G. candidum (ATCC 204307), A. flavus (ATCC 9643), and F. oxysporum (ATCC 48112). Compounds were dissolved in DMSO to create a 0.001 mol stock solution. Nutrient agar medium was prepared, cooled to 47°C, and inoculated with the test microorganisms. The medium contained 1% peptone, 0.2% beef extract, 0.4% yeast extract, 1% NaCl, and 3% agar-agar. Once solidified, 5 mm diameter holes were punched using a sterile cork borer. The TTM ligand and its metal complexes were dissolved in DMSO at a concentration of 1.0 × 10⁻³ M and placed into the prepared Petri plates. These plates were then incubated at 37°C for 20 hrs to allow bacterial growth. The diameter of the inhibition zones was measured in mm to assess activity. After 24 hrs at 37°C, the plates were examined for inhibition zones, and the diameters were measured again. The final results were averaged from three readings of antimicrobial activity [75]. The mean ± SD was computed to statistically analyse the data collected over these three occasions.

2.11.2. Anti-cancer activity

The MTT assay, as previously described [76,77], was conducted to assess the viability of cancer cells in response to the synthesized compounds. HCT-116 colon cancer cells, Hep-G2 liver cancer cells, and MCF-7 breast cancer cells were plated at a density of 5 × 10³ cells per well in 96-well plates. After a 24-hrs incubation period, the cells were treated with different concentrations of the compounds, ranging from 0 to 10 μg/mL, for 48 hrs. Following this exposure, the medium containing the compounds was removed, and 10 μL of MTT solution (5 mg/mL in phosphate-buffered saline) was added to each well. The plates were incubated for three hrs at 37°C to allow the formation of purple formazan crystals. To dissolve these crystals, 100 μL of DMSO was added to each well, and the OD was measured at 570 nm. Five control wells were included, and each concentration was tested in triplicate, with vinblastine as the standard drug.

The absorbance readings were expressed as a percentage of cell viability, with control wells representing 100% viability, as detailed in Eq. (5). Dose-response curves were generated using Origin software, and IC₅₀ values were calculated to determine the concentration needed to inhibit 50% of cell viability, as detailed in Eq. (6). Each concentration was tested three times, and the mean values ± SD were recorded.

The percentage growth inhibition (IC₅₀) was computed using (Eq. 6):

2.11.3. Antioxidant activity

The radical scavenging ability of the substances against 2,2-diphenyl-1-picrylhydrazyl (DPPH) was evaluated using Blois’ method [78,73]. DPPH is a stable free radical with a distinct absorption band at 517 nm due to its unpaired electron. This absorption diminishes, and decolorization occurs when the radical is neutralized by a scavenger. Different concentrations of the test complexes were prepared and diluted to 100 µL with methanol. Approximately 5 mL of a 0.1 mM DPPH methanolic solution was added to each sample and standard (Drolox), followed by vigorous shaking. A negative control was made by mixing 100 µL of methanol with 5 mL of the 0.1 mM DPPH solution. The samples were left to stand for 20 mins at 27°C, and the absorbance was measured at 517 nm against a methanol blank.

3.2.1. Ft-IR, of TTM ligand and its complexes

The analysis of IR spectra yields valuable information on the binding modes surrounding the metal atom by comparing the spectra of the metal chelates with the TTM ligand. The vibration wavenumbers of the most characteristic function groups of the ligand and their chelates have been presented in Table 1.

The TTM ligand exhibits a distinct band at 1591 cm^-1, which is attributed to the azo bond (-N=N-) [80]. This peak is associated with lower wavenumber findings (1579–1548 cm^-1), suggesting a coordinating role of the azo group [81,82]. Further analysis of the complexes reveals the presence of new peaks in the ranges of 443–474 cm^-1 and 541–621 cm^-1, which confirms the chelation of the metal ions to the N₂ and O₂ atoms of the ligand [83,84]. Additionally, the complexes show the presence of the (CH₃COO) group, whose mono-dentate nature is demonstrated by the additional absorption peaks observed at 1242–1257 cm^-1. The chelated TTM ligand’s υ(O–H) stretch is thought to be in the range of 3442–3419 cm^-1. Moreover, the vibrations of the (OAc)_υas and (OAc)_υs groups have been found at 1388–1440 cm^-1 and 1334–1381 cm^-1, respectively, in all the investigated complexes.

3.2.2. C, H, N percent and conductivity measurement in the compounds under investigation

The expected chemical structure of the metal chelates served as the foundation for the inferences made from the elemental analysis (C, H, and N) of the complexes. The elemental analysis results indicated that the TTM ligand is coordinated to the metal ions in a 2:1 ligand-to-metal ratio. The metal chelates were found to have high melting (decomposition) points and were stable when exposed to air. This suggests the formation of thermally stable metal-ligand complexes. Furthermore, the molar conductance data for the prepared TTM metal chelates revealed that they exhibit a non-electrolyte nature. The specific molar conductance values were as follows: TTMCo (12.15), TTMNi (9.45), TTMCu (13.15), and TTMZn (8.45).

3.2.3. Electronic spectra and magnetic moment measurements for the compounds under inspection

The ligand and its complexes with Zn(II), Ni(II), Cu(II), and Co(II) were analyzed by electronic spectroscopy in DMF at room temperature. The results have been presented in Table S1 and Figure 1. The band at 260 nm in the TTM ligand is attributed to the intraligand π-π* transition. The absorption profile of the TTMCo complex shows three peaks: at 260 nm (π→π* transition), 523 nm (LMCT transition), and 580 nm (d→d transition). The TTMCu complex exhibits bands at 261 nm (π→π* transition) and 705 nm (d→d transition). In the TTMNi complex, three peaks are observed at 260 nm (π→π* transition), 390 nm (LMCT transition), and 640 nm (d→d transition). The TTMZn complex shows two peaks at 260 nm and 330 nm, corresponding to π→π* and LMCT transitions, respectively [85]. The magnetic moment values of the metal complexes were measured at ambient temperature. The magnetic moment of the Co(II) complex was measured to be 3.38 BM, suggesting an octahedral coordination environment for the Co(II) metal center [40]. The Ni(II) metal chelate had a magnetic moment value of 2.25 BM, consistent with two uncoupled electrons. The magnetic moment of the TTMCu complex was found to be 1.75 BM, which is attributed to its electronic configuration (3d⁹). However, higher magnetic moment values have also been observed for Cu(II) complexes due to spin-orbit coupling [86]. The TTMZn complex showed no magnetic moment, indicating the diamagnetic nature of the Zn(II) metal center.

Close

Figure 1.

Molecular electronic transitions of (a) 1x10^-4 M and (b) 1x10^-3 M for the prepared compounds.

Export to PPT

3.2.4. Thermal analysis

Understanding thermal analysis may help one better learn about complexes’ thermal stability as well as the makeup of the solvent or water molecules that make them up. It facilitates the distinction between lattice water and coordinated water molecules and helps to put out a basic plan for the compounds’ thermal breakdown. TGA was conducted on certain metal complexes under a nitrogen atmosphere, with temperatures ranging from 40 to 1000°C and a heating rate of 10°C/min [87]. The thermal behavior of the Zn(II), Ni(II), Cu(II), and Co(II) complexes is influenced by the specific metal ion present. The thermal decomposition of these complexes occurred in multiple stages, and the observed mass losses align with the suggested formulas derived from analytical data (Table 2). For the TTMCo complex, the first stage involved a 6.12% mass loss (calculated 6.09%) between 45-130°C, attributed to the loss of 2 water molecules. The second stage showed a 19.92% mass loss (calculated 19.97%) between 135-285°C, corresponding to the loss of a C₄H₆O₄ moiety. In the third stage, a 33.19% mass loss (calculated 33.17%) was observed, associated with the loss of a C₆H₈N₆O₂ moiety. Finally, a 30.78% mass loss (calculated 30.81%) occurred between 290-370°C, resulting from the decomposition of a C₁₄H₁₄ moiety, leaving a 6.96% Co residue at 520°C. The three other complexes, TTMCu, TTMNi, and TTMZn, exhibited similar thermal decomposition patterns, with the first stage involving the loss of hydrated water molecules between 45-145°C. The second stage represented the loss of the C₄H₆O₄ moiety between 135-290°C. The subsequent stages involved the degradation of the TTM ligand, culminating in the formation of the respective metal residues.

3.2.5. Characterization of the synthesized compounds’ stoichiometry using spectrophotometry:

The stoichiometry of the complexes formed was identified via the spectrophotometric molar ratio and Job’s methods [88]. The molar ratio graph for the prepared complexes (Figure S5) shows a linear component crossing at a molar ratio of approximately 2, suggesting that the metal complexes were formed in a 2:1 ratio (L:M). Additionally, Job’s method indicated maximum absorbance at a ligand mole fraction of 0.62. These findings, shown in Figure 2, confirmed the molar ratio results and indicated the formation of [Zn(II), Ni(II), Cu(II), and Co(II)] complexes in a 2:1 (L:M).

Close

Figure 2.

The stoichiometry of the inspected complexes in solutions.

Export to PPT

3.2.6. The synthetic complexes’ apparent formation constants

The formation constants (K_f) and stability constants (pK) were estimated and have been presented in Table 3, with the values ranked as follows: TTMCo > TTMCu > TTMNi > TTMZn. Additionally, the Gibbs free energy (ΔG*) values calculated for the complexes were negative, indicating the spontaneity of the complexation reaction [89].

Based on the correlation of all collected analytical, chemical, and physical data for the compounds under investigation, it can be concluded that the examined complexes have an octahedral geometry, as seen in Scheme 1.

3.2.7. pH stability compounds under investigation

The anticipated pH stability profiles of the complexes were highly consistent over the pH = 4 - 10 range, as shown in Figure 3, suggesting that the compounds were quite comparable to one another. They are safe for use in several applications because they maintain their stability throughout a large pH range.

Close

Figure 3.

The stability behavior of the inspected complexes in media with different pH levels.

Export to PPT

3.2.8. Structure optimization using DFT calculation

The stability of optimized structure for TTM free ligand and its complexes TTMCu, TTMCo, TTMZn and TTMNi, chelates were investigated and confirmed by executing the ‘‘stabile’’ keyword and validated by the absence of imaginary frequencies, which indicated the stationary point of the computed structure at ground state [90,91]. The optimized structures have been given in Figure 4. The optimized structure of TTM free ligand exhibited a coplanar conformation with respect to the phenyl moiety, while TTMCu, TTMCo, TTMZn, and TTMNi complexes showed octahedral conformations, as depicted in Figure 4.

Close

Figure 4.

3D optimized structures of (a) TTM, (b) TTMZn, (c) TTMCo, (d) TTMCu, (e) TTMNi compounds.

Export to PPT

The stability and reactivity of the compounds are mainly linked to the electron acceptor and electron donor orbitals (i.e., highest occupied and lowest unoccupied), which are energetically known as E_HOMO and E_LUMO or referred as FMOs. Furthermore, the energy gap (E_HOMO-E_LOMO) is the energetic key to explore the kinetic stability and reactivity of the compounds, through shaping the values of hardness ( η), electronegativity ( χ), softness ( σ), electrophilicity ( ω), electron affinity ( A), chemical potential ( CP) and ionization potential ( I) of the investigated compounds [92]. The surface illustration of HOMO and LUMO orbitals of the title compounds are presented in Figure 5, showing +ve and -ve phases as green and red, respectively. The calculated values for FMOs energy and other parameters are listed in Table 4.

Close

Figure 5.

(a-e) Graphical illustration of HOMO-LUMO distributions.

Export to PPT

Based on the DFT values of energy gap and other parameters, especially, hardness which points to the stability and softness which points to more reactivity of the compounds for which the studied compound values are ranging from 0.354 to 3.362 with reactivity order of TTMCu > TTMCo > TTMNi > TTMZn> TTM (see Table 4). In the literature survey, some researchers [93], computed DFT/B3LYP/6-31g softness for the ligand metallophthalocyanine (M) with its complexes, MZn, MMn, MNi, MCu and MCo in the range from 0.5522 to 1.4612. Recently, [38] reported the softness ranging values from 0.24 to 1.16 for novel divalent chelates of Co(II), Cu(II), Zn(II) and Ni(II) with 1-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl)methanol ligand, respectively, under DFT/B3LYP/LANL2DZ/6-311 g(d,p). Likewise, the DFT/B3LYP-6-311G(d,p) work of Cd, Ni, Co, Zn, and Cu complexes with (E)-N’-(3,5-di-Tert-Butyl-2-Hydroxybenzylidene) Isonicotino Hydrazide ligan reported hardness values between 0.53 to 1.69 [39]. Based on the above, the highest softness and reactivity of our complexes, especially TTMCu, TTMCo, and TTMNi, are clear. This justifies the high docking scores and bioactivity behaviour of these complexes in docking results (see docking part). According to the HSAB law (hard-soft/acid–base) regulatory, the chemical reactivity with softness and hardness of the compounds is considered a vital rule in bio-activity of the compounds, like proteins and cells. Because the soft compounds are well-interacting with a biomedical system in comparison to hard ones [94].

The 3D plot of the MEP is usually linked to the electron density plotted onto the surface of iso-electron density, which can be utilized to characterize the structural and topological features of substrates in a 3D system. MEP specifies the greatest effect of the electrons or nuclei on the molecular system through coloring system fluctuating from maximum electrophilic sites (high positively charged) blue >bluish green (medium positively)> green (less positively) > white (zero potential) > yellow (less negatively) > orang (medium negatively) > red (high negatively charged) at maximum nucleophilic sites. The positive and negative potentials indicate the ability of sites to donate and accept electrons, respectively, through the interactions [92].

MEP representation of the studied molecules has been given in Figure 6. Owing to the richness of electrons on the oxygen and nitrogen atoms, the most negative sites are located around them, so they act as an attractive target for electrophilic attack to construct acceptor H-bonds. On the other side, the highest positive areas are mostly covered by the coordinated hydrogen moiety, which tends to form donor hydrogen bonds in substrate-protein interactions (see docking part). While the bluish green is widely used to paint aromatic rings. This result of DFT-MEP computation closely matches the reported data of similar compounds [36,38,39,93].

Close

Figure 6.

(a-e) MEP diagram of the synthesized compounds.

Export to PPT

The TD-DFT computations of the free ligands and its complexes are depicted in Figure S6, in which the wavelength is extending from 200 to 700 nm to include all possible transitions. The values of computed excitation energy (E), wavelength (Λ_max), transitions (major contribution) and oscillator strength (f) of the investigated compounds are listed in Table 5. The free ligand (TTM) possesses Λ_max of 251 nm with 2.466 eV of energy value and f of 0.1855 which ascribed to the electronic transitions HOMO>LUMO (45%) and HOMO>LUMO+1 (32%). These values are found to be harmonized with the experimental data and reported values for similar free energy ligands [79]. As expected, all complexes suffered from red shift, which ascribed to the presence of the metal atom. The Λ_max for TTMZn complex is pointed at 365 nm having E of 2.607 eV, f of 0.1080, for the electronic transitions HOMO>LUMO (87%). The researchers computed in the literature with similar ligand-Zn complex values of Λ_max = 420 E=2.326 eV and f = 0.116, which are in good agreement with the data [38]. In case of TTMCo the value of Λ_max reached 290 nm with E of 2.142 eV and f of 0.1070, which attributed to the electronic transitions HOMO>LUMO (48%) and HOMO2 > LUMO (25%). The calculated Λ_max of TTMCu complex in the current work is located at 507 nm with 2.160 eV and 0.1644 values of E and f, respectively, which majorly ascribed to the electronic transitions HOMO>LUMO (91%). There are some researchers observed Λ_max for similar complexes at 530 and 674 nm [95]. TTMNi complex, has Λ_max of 325 nm with E of 2,057 eV, and f of 0.1140, which is attributed to the electronic transitions HOMO> LUMO (61%), which is in the range of experimental values and reported data of similar compounds [36,38,39].

The computed vibrational frequencies in cm^-1 units for all studied compounds, have been given in Figure 7. Owing to the different environment between DFT simulation (gas phase) and experiment, it is common to get some variations between vibrational frequency values resulting from the former and latter methods. Generally, the DFT values of vibration modes would be higher compared to the experimental ones. The simulation of vibrational frequencies for all compounds is achieved by the same functional (DFT/B3LYP) 6-311G(d,P) and LANL2DZ.

Close

Figure 7.

Computed IR spectra of the synthesized compounds.

Export to PPT

3.2.9. Molecular docking

The prediction of anti-breast cancer, anti-fungal, and anti-microbial activities of the title compounds was performed by docking studies against 3HB5, 6DRS, and 4JIT target proteins, respectively. These proteins were recognized as a potent target for the stated activities. Table 6 summarizes details of the best conformations for protein-compound docking results, including docking score (S) and bonding interactions. The 3D and 2D plots of the best protein-compound conformations have been depicted in Figures 8 and 9, respectively. Additionally, Figures S7 and S8, in the supplementary material, portray 2D&3D plots for the remaining protein-compound conformations.

Close

Figure 8.

3D visualization of TTMCu, TTMNi, TTMZn, and original inhibitors docked with target proteins.

Export to PPT

Close

Figure 9.

(a-i) 2D interaction plots of TTMCu and TTMNi complexes and original inhibitors docked at the active site pocket of the proteins showing π-H and hydrogen bond interactions.

Export to PPT

The outcomes of compound-receptor modelling revealed substantial docking score values with numerous H-bond formations reinforced with pi interactions. Generally, all the synthesized chelates (TTMCo, TTMNi, TTMCu, and TTMZn complexes) exhibited higher results compared to the TTM ligand, more (-ve) S values, indicating the potent inhibitory activity of the chelates against target proteins compared to the TTM ligand.

The docking simulation of synthesized compounds against the vital target of breast cancer 3HB5 exposed the potent inhibitory activity of TTMCu complex with the highest docking score value of (-8.45 kcal/mol) associated with two hydrogen bond donors between C25 and C33 with ASN152 and SER222, respectively, at distances of 2.32 and 2.19Å, in addition, there are four hydrogen bond acceptors formed between O48 with PRO187 at distance 2.22Å and O52 with SER142 with distance of 2.22Å as well as O54 with the both of GLY144 and SER142 residues having distances of 2.62 and 1.89Å, respectively. Furthermore, this conformation is strengthened by two π-H interactions linking ILE14 and PHE226 amino acids with the centroid of phenyl and triazole rings at distances of 3.98 and 3.25Å, respectively, (see Figures 8 and 9).

The docking score of TTMNi complex recorded a value of (-7.92 kcal/mol), as the second potent inhibitor of breast cancer with one hydrogen bond donor linked C25 with ASN152 at 2.57Å length and one hydrogen bond acceptor between O54 and GLY144 at a distance of 2.31Å. Besides, the conformation also exhibited two π-H interactions between C45 with the centroid of the phenyl ring in TYR155 residues at a distance of 3.00Å and ILE14 with the centroid of the TTMNi phenyl ring having an average distance of 2.94Å, as portrayed in Figures 8 and 9.

The complex TTMCo attained a -7.26 kcal/mol value of docking score against the 3HB5 receptor with one hydrogen bond acceptor connected O52 with VAL188 residue at a distance of 2.42Å and two hydrogen bond donor linked C45 and C60 with GLY186 and VAL188 residues at average distances of 2.32 and 2.17Å, respectively. Additionally, the centroid of the TTMCo phenyl ring interacted with ILE14 amino acid through π-H interaction at a distance of 2.86 Å, as depicted in Figures S7 and S8.

On the other side, TTMZn complex and TTM ligand obtained -7.13 and -5.19 kcal/mol value of docking scores, respectively. The TTMZn complex constructed two hydrogen bond acceptors of O54 with GLY144 and TYR155, at distances of 2.24 and 2.37 Å, respectively, as well as one π-H interaction linked PHE226 with the centroid of TTMCo triazole ring at distances of 2.95Å. While the TTM ligand shaped only one hydrogen bond acceptor between N6 and GLY144 with a distance of 2.53 Å, reinforced with one π-H interaction connecting the VAL225 residue with the centroid of the TTM phenyl ring with an average distance of 3.45 Å, as shown in Figures S7 and S8. Therefore, the ranking of the chemicals’ inhibitory effectiveness against breast cancer is as follows: TTMCu > TTMNi > TTMCo > TTMZn > TTM. In the existing literature, researchers docked (E)-N’-(3,5-di-Tert-Butyl-2-Hydroxybenzylidene) Isonicotino Hydrazide ligand and its complexes with Cd, Ni, Co, Zn, and Cu with same 3HB5 protein and found binding energy values in the range of −4.43 to −6.65 kcal/mol, accompanied with H-bond numbers ranging from 1-6 per compound at distances of 3.3 to 3.5 Å, in which the ligand-Cu complex showed the best activity for inhibiting breast cancer [39]. This literature data supports our result for TTMCu and exhibits its potential in comparison.

In the case of antifungal activity (6DRS), the highest docking score belong to TTMZn and TTMNi complexes with the same value of -7,54 kcal/mol. TTMZn complex formed one hydrogen bond acceptor between O54 and ARG80 with 2.63Å bond length and one π-H interaction found between VAL70 residue and centroid of TTMZn phenyl ring at a distance of 3.29Å, as shown in Figures 8 and 9. While the TTMNi complex exhibited one hydrogen bond acceptor at distance of 1.77Å for O55-ARG80 interaction and another one donor between O24 and MET41 residue, with average distance of 3.01 Å, associated with unique π-H interaction linked ARG36 amino acid with centroid of TTMNi phenyl ring at average distance of 3.07Å, as presented in Figures 8 and 9.

Regarding the predicted antifungal activity of TTMCu and TTMCo compounds, the simulation resulted in docking score values of -7.47 and -7.23 kcal/mol for the former and latter, respectively. The O54 atom of TTMCu constructed two hydrogen bond acceptors with ARG80 and ALA45 residues at distances of 2.29 and 2.33 Å, respectively, while the O24 atom exhibited one hydrogen bond donor with MET41 amino acid at an average distance of 2.41 Å. Furthermore, O24 and O55 atoms on TTMCo formed one hydrogen bond donor and one acceptor with MET41 and PHE44 residues with bond distances of 3.70 and 2.38 Å, respectively, as well, one π-H interaction between the LEU77 residue and centroid of the TTMCo triazole ring, having distance of 3.00Å.

The activity of TTM ligand as an antifungal compound came in the last scale with a docking score value of -5.47 kcal/mol. It was associated with a unique hydrogen bond donor that appeared between O24 and the TYR162 residue at a 1.90Å bond length. Therefore, the antifungal activity of the compounds can be arranged as TTMZn = TTMNi > TTMCu > TTMCo > TTM. The literature data for the triazole-thiole ligand and its chelates showed binding energy values in the range of -3.2 to -5.9 kcal/mol [96].

Regarding the anti-bacterial activity of the investigated compounds, the docking simulation exhibited TTMCu as the most potent compound with a docking score value of -7.43 kcal/mol, with one hydrogen bond acceptor belonging to the O54-RG69 interaction, and one donor for the C60-ASP92 interaction, at distances of 2.03 and 2.01Å, respectively. Also, the centroid of one TTMCu phenyl ring shaped two π-H interactions with ASP92 and VAL91 residues at distances of 3.15 and 3.20 Å, respectively. The TTMNi with a docking score value of -7.39 kcal/mol, got the second order as anti-bacterial compound with one hydrogen bond acceptor for the interaction of O54 with SER62 at distance of 1.73Å, reinforced by two π-H interactions connected one TTMCu phenyl ring with SER61 and SER62 residues at average distances of 2.98 and 3.26Å, respectively.

Sequentially, TTMZn and TTMCo, recorded the third and fourth order in the anti-bacterial activity, with -7.14 and -6.73 kcal/mol docking score values, for which each one exhibited one hydrogen bond acceptor and one donor, as listed in Table 6. Finally, the TTM ligand with a docking score value of -5.18 kcal/mol recorded the lowest anti-bacterial activity among all docking results, with one hydrogen bond acceptor and three π-H interactions, as illustrated in Figures S7 and S8. Consequently, the activity of the compounds as anti-bacterial candidates is TTMCu > TTMNi > TTMZn > TTMCo > TTM. There are also some other docking references to literature, evaluated chelates of Co(II), Cu(II), Zn(II) and Ni(II) with 1-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl) methanol as antibacterial and reported the best binding energy values of -7.93 and - 7.74 kcal/mol for ligand-Cu and ligand-Ni, respectively, with 4H-bonds for each compound having distances between from 2.75 Ǻ and 3.56 Ǻ [38].

Moreover, The docking simulations in the current study have been validated through redocking the original inhibitors, cocrystal ligands, 3-{[(9beta,14beta,16alpha,17alpha)-3,17-dihydroxyestra-1,3,5(10)-trien-16-yl]methyl}benzamide (E2B), 3-{[(3R)-7,9-diamino-3-methyl-2,3-dihydrofuro[2,3-f]quinazolin-4-yl]oxy}benzonitrile (H8A) and {2-[(3S)-3-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)pyrrolidin-1-yl]-2-oxoethyl}phosphonic acid (3ZF) with 3HB5, 6DRS and 4JIT, respectively. This validation work revealed a nice fitting of the original inhibitors in the same active site pocket and equivalent H-bonds construction analogous to the contacts that resulted in our TTM ligand and its complexes. Also, the neighboring residues surrounding the binding pocket of both the co-crystal ligand and compounds are found to be parallel, as portrayed in Figures 8 and 9.

Based on the docking simulation, the presence of metals in the scaffold structure enhanced the biological activity, and among all complexes, the TTMCu and TTMNi exhibited promising anti-breast cancer, anti-fungal, and anti-bacterial activities, especially TTMCu, which might be utilized to open new fields as a lead complex for discovery and developing breast cancer drug and therapy.

3.3.1. In-vitro antimicrobial evaluation

The TTM ligand and its complexes were evaluated for antibacterial activity using the well diffusion approach. Antibacterial activities were tested against M. luteus, E. coli, and S. marcescens, while antifungal activities were assessed against G. candidum, A. flavus, and F. oxysporum at various concentrations. Sabouraud Dextrose Agar and Nutrient Agar were used as the media for antifungal and antibacterial screenings, respectively. Fungal growth inhibition was measured in mm. Ofloxacin and Fluconazole were used as reference medications to evaluate their antibacterial and antifungal properties, respectively (Figures 10 and 11). The antimicrobial screening data confirmed the biocidal activities of all the compounds. Comparisons between the ligand and its metal complexes revealed that the complexes had superior inhibition properties. The TTMCu complex exhibited particularly strong bioactivity, with minimum inhibition concentration (MIC) values of 2.50 µg/mL against S. marcescens and 3.00 mg/mL against G. candidum, the lowest among the tested compounds (Tables 7 and 8). The increased antimicrobial activity of the ligand after complexation can be explained by Overton’s concept and chelation theory [97]. The metal complexes’ lipophilicity increases as the metal ion charge decreases, disrupting cell permeability and hindering cellular processes [98,99]. Once inside, the complex may interfere with cellular processes, leading to microbial cell death. Chelation reduces the polarity of the metal ion due to partial sharing of its positive charge with donor atoms of the ligand, increasing its lipophilicity. Moreover, the antimicrobial activity of metal ions is significantly influenced by their complex structure and charge on the metal ions. Microbial membranes are generally negatively charged due to phospholipids and lipopolysaccharides. Positively charged metal ions (e.g., Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺) strongly interact with microbial surfaces, leading to membrane disruption, increased permeability, and leakage of cellular contents [100].

Close

Figure 10.

Inhibition activity of the compounds under investigation against Microccus Luteus (+ve) bacteria.

Export to PPT

Close

Figure 11.

Inhibition activity of the compounds under investigation against Getrichm Candidum fungi.

Export to PPT

Biological screening showed that inhibition increased with concentration (Table S2) [101]. The antimicrobial abilities of the produced complexes were verified by determining the potency index, as demonstrated in Table 7. Tables S2 and Table 8 show a comparison between the antimicrobial activity of the investigated compounds, with other compounds in the literature showing promising enhancement activity [102,103].

3.3.2. In vitro anticancer activity

The promising results from the molecular docking studies of the ligand and its Zn(II), Ni(II), Cu(II), and Co(II) complexes led the researchers to evaluate their cytotoxic potential against various cancer cell lines. Vinblastine was used as a positive control to assess the anticancer properties of the ligands and their complexes. The results showed that all the new complexes exhibited significant cytotoxic activity (Figure 12 and Table S3). The order of effectiveness based on the IC₅₀ values was: TTM < TTMCo < TTMZn < TTMNi < TTMCu, across the MCF-7 (breast cancer), Hep-G2 (hepatocellular carcinoma), and HCT-116 (colon cancer) cell lines. Notably, the TTMCu complex demonstrated the highest cytotoxic potency against MCF-7 cells, with an IC₅₀ of 4.25 μg/mL, which was slightly lower than the positive control, Vinblastine (IC₅₀ = 3.12 μg/mL). This suggests that the TTMCu complex could be a promising therapeutic agent for cancer treatment. The high cytotoxic activity of the TTMCu complex may be attributed to its strong ability to bind DNA through groove and intercalation modes, as well as the potential of the Cu(II) center to generate reactive oxygen species (ROS). These mechanisms could interfere with the transcription of cancer cell DNA and induce apoptosis. The strong correlation between the complexes’ DNA-binding capacity and their cytotoxicity supports this conclusion [104].

Close

Figure 12.

Cytotoxicity of the compounds under investigation against selected cancer cell lines.

Export to PPT

3.3.3. Antioxidant activity

The DPPH radical scavenging assay serves as a prominent and precise technique for assessing the in vitro antioxidant activity of various substances or molecules [105]. This entails examining the reduction in DPPH’s molar absorptivity at a wavelength of 517 nm following the interaction with the experimental substance. The antioxidant effect on DPPH radical scavenging is due to the compounds’ ability to donate hydrogen or act as radical scavengers [106]. The interaction involving the scavenging action of an antioxidant (H-D) with DPPH can be expressed as follows in Eq. (7):

As the DPPH radical produces DPPH-H, the presence of antioxidants decreases the amount of stable free radicals in DPPH, leading to a reduction in absorbance. This decrease in absorbance indicates the hydrogen-donating potential and scavenging activity of the antioxidants or compounds [107]. Figure 13 presents the dose-response curve for the DPPH radical scavenging activity of the TTM ligand and its Zn(II), Ni(II), Cu(II), and Co(II) complexes, as compared to Trolox, a standard antioxidant compound. The results show that the Cu(II) complexes exhibit a greater activation of the TTM ligands compared to the free TTM ligands. The IC₅₀ value, which represents the concentration required to scavenge 50% of the DPPH radicals, was observed to be 50% at the screening level (100 µg/mL) for the unbound TTM ligand. However, upon complexation with the metal ions, the IC₅₀ values considerably decreased, ranging from 20 to 32 μg/mL. This indicates that the metal complexation enhanced the DPPH radical scavenging activity of the TTM ligand, with the Cu(II) complex showing the most potent antioxidant properties among the tested compounds.

Close

Figure 13.

Free radical suppression of the compounds under investigation.

Export to PPT

3.3.4. Structure-activity relationship (SAR)

The Structure activity relationship (SAR) analysis for the biological reactivity of Co(II), Cu(II), Ni(II), and Zn(II) complexes with 1-p-Tolyl-1H-[1,2,3]triazol-4-yl-methanol ligand considers factors such as coordination geometry, electronic configuration, ligand-metal interactions, and their influence on biological activity [12,108].

Cu-based complexes have shown significant attention in medicinal chemistry, especially with coordination with heterocyclic cores due to their versatile biological activities. This biological activity depends mainly on structural features, the nature of the metal-ligand-metal, the oxidation state of Cu and the molecular stability. Based on the obtained results, Cu-based complexes among all tested complexes exhibited potent bioactivity as anti-microbial, antioxidant and anticancer. Cu-based complexes have shown promising antimicrobial activity against bacteria and fungi. These results agreed with the reported literature that Cu-complexes can interact and damage the microorganism’s cell wall, leading to cell death. Additionally, Cu-complexes can bind to and inhibit the enzymes for microbial survival, such as those involved in DNA replication [109]. Cu-based complexes have shown promising anticancer activity against a panel of cancer cells by induction of apoptosis that can trigger both intrinsic and extrinsic pathways, or by activating the antioxidant capacity by ROS scavenging activity, or by interaction with DNA that can interfere with replication of transcription [110]. Cu-based complexes have shown promising antioxidants by scavenging free radicals, or by mimicking the antioxidant activity like catalase (CAT) and superoxide dismutase (SOD), or by chelating activity [111].