Sustainable synthesis of bivalent Ni²⁺, Co²⁺, and Cu²⁺ Schiff base complexes via ball milling as a green approach: A study of spectroscopic characterization, cyclic voltammetry, bioactivity, DFT analysis, SAR, drug likeness, and molecular docking

2.1. Preparation of ligand and its Ni²⁺, Co²⁺, and Cu²⁺ metal chelates

3-(2-hydroxybenzylidene)-N-(benzothiazol-2-yl)hydrazine-3-oxopropanamide were refluxed for 3 hrs in absolute ethanolic solution (95%) to create pure H₂BT, as Scheme 1S illustrates. Solid chelates of the acetate salts of Ni²⁺, Co²⁺, and Cu²⁺ were synthesized using the high-energy ball milling approach, which shortens the reaction time (20 mins) and yields a pure compound with a high yield of about 98% (Scheme 1). This approach adheres to sustainability ideals by minimizing the usage of dangerous chemicals and consuming less energy. Additional purification was done by recrystallization using absolute ethanol.

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Scheme 1.

The outline synthesis of solid complexes. Scheme 1S, the synthesis of the ligand, Scheme 2S, the images of the instruments used.

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2.2. Equipment

Scheme 2S shows every total of apparatus accustomed the suggested geometries of the prepared compounds.

2.3. Theoretical calculation

Utilizing DMOL3 and the Materials Studio package, the theoretical conclusions were made [19,20]. The geometry optimization of the resulting compounds was verified by using DFT, utilizing the Lee-Yang-Parr non-local correlation functional (B3LYP) [21].

2.4. Evaluation of biological activity

2.5. Molecular docking calculations

Using the AD4/MGLTools-1.5.7 software suite on the Windows platform, a computational molecular docking analysis was performed to elucidate and evaluate the binding empathies of the investigated complexes with the energetic site of the BCL2 gene [30,31]. This was accomplished by using the AD 4 program in combination with the Lamarckian Genetic Algorithm (LGA) to evaluate the compounds’ binding affinities for the indicated biomolecules. The protein-ligand rigid-flexible docking process was conducted using the Lamarckian Genetic Algorithm (LGA) with the following parameters: 27,000 generations, a maximum of 250,000 energy estimations, and alteration and border rates of 0.02 and 0.8, correspondingly. This assay comprised multiple procedure such as grid building, ligand and protein manufacturing, and selection. The crystallographic structure of the ligand-attached BCL2 receptor (PDB: 2w3l) was discovered using the RCSB protein database (PDB database) [32]. The original structure was brought into the visualizer, and then water molecules, heteroatoms, chain B, and any remaining atoms were eliminated. Next, the active site linked to the co-crystallized DRO ligand was found [33]. Within the BCL2 exploration domain, a 35 x 35 x 35 Å grid box with 0.375 Å grid spacing was used. We utilized the x,y, and z coordinates of 40.91, 27.92, and -10.02, respectively.

2.6. Cyclic voltammetry

Three primary electrodes were employed to construct the electrochemical cell that we used in our experiments:

• i.

  Using 3M KCl as a reference electrode and Ag/AgCl.

• ii.

  A functional electrode was the glassy carbon.

• iii.

  Platinum wire served as the auxiliary.

• iv.

  As a supporting electrolyte, 30 mL of HCl (0.l M) solution was added to each electrode. The US convention provided CV data once the DY 2100 potentiostat was connected.

3.1. The isolation of complexes

The results detailing the physical properties and elemental analysis of H₂BT, along with the synthesized chelates, have been presented in Table 1. Both the chemical compositions and the proposed formulas were examined through the comparison of practical and calculated percentages derived from the analysis. The melting points of the synthesized complexes exceeded 300°C, indicating their stability. These complexes were formed in a 1:1 molar ratio (metal to ligand). The prepared ligand displayed solubility in hot absolute ethanol, whereas the isolated solid complexes were soluble in dimethyl sulfoxide (DMSO) and dimethylformamide (DMF).

3.2. ¹³C/¹H NMR analysis

Figure 1S displayed the H₂BT ligand ¹H and ¹³C NMR analysis spectra, which were obtained in Dimethyl sulfoxide (DOMS). The ¹H NMR spectrum showed three signals at 11.56, 12.56, and 11.035 ppm, which were identified as originating from the protons (OH), (NH₁), and (NH₂), respectively. A strong signal at 3.62 ppm was identified as coming from the protons (CH₂). Additionally, multiplet signals of aromatic protons were found in the 6.56–8.27 ppm area, with the (N=C—H) signal appearing at 8.45 ppm. Additionally, Table 1S displayed the coupling constant that was determined for each doublet and triplet signal.

The (C=O)₁ as well as (C=O)₂ were identified by two signals at 166.913 and 166.407 ppm in the ¹³C NMR spectra for H₂BT, (C=N)_azo & (C=N)_thiazol were identified by two signals at 157.880, 168.00 ppm, and (C―OH) was identified by a signal at 162.067 ppm [34].

3.3. Mass spectra

The molecular ion peak [M]⁺ at m/z = (20.84%) in the ligand’s mass spectrum corresponded to the ligand moiety [(C₁₇H₁₄N₄O₃S) atomic mass 487.13 U] and was equal to its molecular weight. Additionally, a base peak that matched the target H₂BT primary fragmentation molecular weight was seen at m/z =5 7.09. The different molecular ion peaks that appeared in the H₂BT mass spectrum (abundance variety 2-100%) were associated with ligand fragmentation, which was caused by the breaking of several bonds within the molecule through successive degradation that led to many essential peaks because of the production of many radicals (Scheme 8S).

3.4. Infra-red spectra

Table 1 was provided an explanation of the physical parameters and elemental analysis data for H₂BT in addition to its manufactured chelates. Following their correlation, the chemical compositions and the proposed formula were both examined using experimental and computed percentages.

The vibrational bands seen at 1690, 1657, 3448, 2927, 3214, 3187, 1605, and 1624 cm^-1 in the infrared spectra of H₂BT (Figure 2S) were attributed to (C = O)₁, (C = O)₂, (O – H), (CH₂), (N– H)₁, (N– H)₂, (C = N)_azo, then (C = N)_thiazol, correspondingly, as shown in Table 2S [35].

In IR spectrum of [Cu(BT)(H₂O)₂] complex, H₂BT functioned as a binegative tetradentate ligand via the two deprotonated enolized carbonyl oxygens (=C– O-)₁ & (=C– O-)₂, and two nitrogen’s groups (C=N)_azo & (C=N)_thiazol [36]. This mode of coordination was supported by: (i) The shift of vibrations of both nitrogen’s groups (C=N)_azo & (C=N)_thiazol to lower wave numbers; (ii) The disappearance of both (C=O)₁ & (C=O)₂ with parallel appearance for new vibrational bands at 1202 cm^-1 and 1153 cm^-1, which are attributed to (C – O)_1(enolic) & (C– O)_2(enolic); (iii) The disappearance of (NH)₁ & (NH)₂ with new bands appearing at 1637 cm^-1 and 1632 cm^-1 which are attributed to (C = N*)₁ & (C=N*)₂; and (iv) The appearance of new vibrations at 511 & 485 cm^-1, which may be related to (Cu – O) & (Cu – N), respectively.

Also, in IR spectra of [Co(HBT)(H₂O)(OAc)].2H₂O and [Ni(HBT)(H₂O)(OAc)].3H₂O chelates, the H₂BT acted as a mononegative tridentate via (C = N)_azo, (C = O)₁, and (=C– O-)₂ groups [36]. This mode of complexation was suggested based on the following observations: (i) The (C=N)_azo as well as (C=N)_thiazol bands shift to lower wave number numbers; (ii) The (C=O)₂ and (NH)₂ bands disappearance with analogous appearance of novel vibrations at (1635, 1605) and (1153, 1155) cm^-1 that are assigned to (C=N*)₂ & (=C – O-)₂ [37]; (iii) Novel vibrational bands appeared at (505, 580) & (464, 476) cm^-1 that may be movable to (M – O) & (M – N), correspondingly; (iv) The appearance of new vibrations at (1471, 1469) & (1346, 1377) which related to υ_s(O – C – O) and υ_as(O – C – O), respectively indicated the bidentate manner of acetate groups.

3.5. UV-visible and magnetic moment

As shown in Table 3S and Figure 3S, the results indicate that the prepared complexes of Ni²⁺ and Co²⁺ possessed octahedral geometry, whereas the Cu²⁺ complex exhibited deformed octahedral geometry [37].

The electronic spectrum of an octahedral Ni²⁺complex showed two bands at 17241 and 27027 assignable to ³A₂ → ³T_1g (F) & ³A_2g → ³T_1g (P) transitions, correspondingly. Also, the calculated values of (D_q=1104), (B=736), (β=0.758) & (υ₂/υ₁=1.55) confirmed the proposed octahedral structure. In addition to, the value of the magnetic moment (3.12 B.M.) can be considered additional evidence for an octahedral geometry.

The electronic spectrum of the octahedral Co²⁺ complex displayed two bands at 14815 cm^-1 and 19802 cm^-1 ascribed to ⁴T_1g → ⁴A_2g(F) & ⁴T_1g → ⁴T_1g(P) transitions, correspondingly. Also, the calculated values of (D_q=793), (B=933), (β=0.961) & (υ₂/υ₁=2.15) suggested an octahedral structure. Furthermore, octahedral geometry around the Co(II) ion was confirmed by the value of the magnetic moment (5.12 B.M.). In the electronic spectrum of Cu²⁺, the complex showed a broad band at 16129 cm^-1 with a shoulder at 14388 cm^-1, which are assigned to ²B_1g→²E_g and ²E_g→²A_1g transitions, correspondingly, in a tetragonally distorted octahedral configuration. Also, the octahedral geometry around the Cu(II) ion was confirmed by the magnetic moment value (2.03 B.M.).

3.6. Electron spin resonance

The room-temperature solid state ESR spectrum (Figure 4S) of [Cu(BT)(H₂O)₂] complex exhibited axially symmetric g-tensor parameters with “g_||>g_⊥ >2.0023”, indicating that the copper site had a dx²-y² ground-state characteristic of tetragonally distorted octahedral stereochemistry [38]. Moreover, in the Cu²⁺ complex, there was no band related to the forbidden magnetic dipolar transition at half-field (ca. 1500 G, g = 4.0); this suggested that there was no Cu–Cu interaction, and thus, the Cu²⁺ complex was mononuclear [39]. The exchange interaction between Cu²⁺ centers in the solid complex in the axial symmetry was expressed in Eq. (1) as follows:

As suggested by Hathaway [40], if G > 4, there is an exchange contact between the Cu²⁺ centers; if G < 4, there is a significant exchange interaction in the solid complex. The G value of 4.60 is the calculated value. Also, the f factor (g_||/A_||) was calculated from the ESR spectrum to measure the extent of distortion of the copper (II) complex which equal to 138, which indicated the existence of important dihedral angle distortion in the xy-plane and a typical deformed octahedral structure surrounding the copper site. Additionally, Eqs. (2) & (3) [41] were used to determine the molecular orbital coefficients, such as α² that measures the covalency in-plane β-bonding between a copper 3d orbital and the ligand orbitals and β² that measures the covalency in-plane π-bonding:

Through strong in-plane π-bonding in the Cu²⁺ chelate, the magnitude of α² (0.80) and β² (0.77) showed that both in-plane σ-bonding and in-plane π-bonding are dependable and significantly covalent.

3.7. Thermal investigation

The investigated complex was thermally stable up to 30°C, as indicated by the Cu²⁺ complex’s TG curve, after which the chelate began to dehydrate. The 2H₂O molecules were proposed to be outside the coordination sphere of the copper ion with a 3.57% weight loss over the temperature range of 30-119°C, which is consistent with relatively low temperature [42]. Next, as shown by the Thermogravimetric (TG) curves (Table 4S and Figure 5S), the second temperature range (119-235)°C indicated the existence of 2H₂O inside the coordination sphere with an 8.04% weight loss. The organic moiety’s thermal degradation began at 230°C. The elimination of the ligand’s C₆H₆O, and C₇H₅NS fragments may be the cause of the 48.88% weight loss seen in the TG curve during the temperature range of 235–587°C. Ultimately, the breakdown of the remaining organic moiety (C₄N₃O) accounted for 21.79% of the weight loss in the temperature range of 587-850°C. Copper oxide, which made up 16.92% of the initial mass of the chelate, was the final burned product.

Additionally, the [Co(HBT)(H₂O)(OAc)].2H₂O TG curve. In the temperature range of 30–124°C, the Co(II) complex exhibited a 6.26% weight loss, which corresponded to two water molecules that were not coordinated (Table 4S and Figure 5S). One molecule each of coordinating water and acetic acid were linked to the weight loss in the temperature range of 124–257°C, with a weight loss of 15.37%. The obtained curves showed a 37.48% decrease in the temperature range of 257–447°C, which can be attributed to the presence of organic moieties (C₆H₆O, C₄N₃O). Lastly, the elimination of the remaining moiety of the organic molecule (C₇H₆NS) was responsible for the 26.61 percent weight loss in the temperature range of 447–800°C. Cobalt oxide (CoO) was the end product at 14.28% above 800°C.

3.8. EDX investigation

One analytical technique for determining the chemical makeup of a sample under investigation is EDX analysis. According to the spectra displayed in Figure 6S, the complex formation and the lack of impurities in the sample were confirmed by the complexes involving M= (Co²⁺, Cu²⁺, and Ni²⁺), C, N, O, and S [34,43].

3.9. Scanning electron microscope (SEM) investigation

By using a concentrated electron beam to scan the surface, the SEM technique produces images of the materials under investigation (Co²⁺, Cu²⁺, and Ni²⁺). The target sample’s atoms and electrons then interact to produce a variety of signals that provide precise details about the sample’s composition and surface topography. As shown in Figure 1, SEM was authorized to examine the complexes’ surface geometry. The results showed that Cu²⁺ complexes with spherical grains in terms of relative crystallinity. However, the other pictures displayed a rocky appearance, which could be the result of large particles or amorphous samples [44,45].

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Figure 1(a-c).

SEM of isolated solid complexes.

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3.10. X-ray diffraction invest Vigation

A good non-destructive analytical technique for examining the composition, orientation, and phase crystal structure of solid, liquid, and powder materials is X-ray diffraction. As shown in Figure 7S, the Cu⁺² chelate was examined at room temperature (RT) using (Cu, Kα) radiation. The compound under examination had an amorphous character, as evidenced by the diffraction pattern, which was adjusted within the range of “10° < 2 θ < 90°” [46].

3.11. Molecular chemical parameters

In chemistry and materials research, DFT is a powerful quantum computational mechanical modeling method that can be used to calculate a wide range of parameters of nearly any type of atomic system. Figure 2 displayed the optimal structure of H₂BT and its solid chelates, which were created in the gas phase. Furthermore, the dipole moment, binding energy, exchange-correlation energy, total energy, spin polarization energy, and electrostatic energy frontier atomic orbitals (E_HOMO, E_LUMO) [47] are some significant quantum parameters that could be evaluated and indicate the stability of the compound. These have been included in Table 2.

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Figure 2.

Optimized structure of isolated solid complexes.

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The following conclusions can be drawn from the results:

• i.

  As shown in Table 3 and Figure 3, the significant reliability of H₂BT and its metal chelates is demonstrated by the negative magnitude of the E_HOMO and E_LUMO energies.

• ii.

  The lengthening of the bonds at the active sites of the free ligands that took part in coordination-prepared complexes (Table 5S & Figure 8S).

• iii.

  As shown in Table 6S & Figure 9S, the shift in bond angles of prepared chelates suggested an octahedral geometry surrounding central metal ions with sp3d2 or d2sp3 [48,49].

• iv.

  Solid chelates have larger binding energy values than H₂BT, indicating greater stability for the produced chelates.

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Figure 3.

The HOMO and LOMO for isolated solid compounds. HOMO: Highest occupied molecular orbital, LUMO: Lowest unoccupied molecular orbital.

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3.12. Cyclic voltammetry technique

A versatile electrochemical technique, cyclic voltammetry (CV) is typically employed to examine the redox characteristics of Schiff bases. The following are the primary goals of using CV to Schiff base complexes, especially those containing transition metals like copper (II):

• -

  To comprehend their electrochemical characteristics and possible uses for the target Schiff base, the CV approach was used.

• -

  The CV approach sheds light on the reversibility of the redox processes, the stability of various oxidation states, and the mechanisms underlying electron transport. This can provide information about the ligand effects and coordination environment.

• -

  For applications like fuel cells and sensors, CV aids in assessing their catalytic stability and efficiency.

• -

  Comprehending the electrochemical characteristics of these complexes helps direct the creation of novel materials with specialized qualities for certain uses, such electrical or energy storage devices.

3.12.1. In case of copper (II)

By means of a supporting electrolyte (0.1 M KCl) in 50% DMSO-H₂O (V/V), a GC working electrode within the potential range (1.2 to -1.2 V), and a 0.1 V/s scan rate, the voltammetric work of Cu²⁺ solution was carried out at 291.15 K, as shown in Figure 4. Since the backward scan shows two anodic peaks the (Cu⁰/Cu¹⁺) first potential peak (E_Pa1) at 0.321 V and the (Cu¹⁺/Cu⁺²) other peak (E_Pa2) at - 0.102 V it is evident that the Cu²⁺ solution is electro-active. Two reduction peaks can be seen in the front scan: the first peak (E_Pc1), which is (Cu²⁺/Cu¹⁺), appeared at -0.701V, and the second peak (E_Pc2), which is (Cu¹⁺/Cu⁰), appeared at 0.058 V. Consequently, the following might be used to show the electrochemical mechanism:

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Figure 4.

The cyclic voltammogram of CuCl₂ in (a) Absenteeism and (b) Existence of varying concentrations of ligand at 291.15 K. Electrochemical bands: A,B

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The oxidation process: Cu ⁰ →Cu ⁺¹+e⁻ (0.321 V)

${\text{Cu}}^{+\text{1}}\to {\text{\&nbsp;Cu}}^{+\text{2}}+{\text{e}}^{-}\left(0.\text{1}0\text{2\&nbsp;V}\right)$

The reduction process: Cu ⁺²+e⁻→ Cu ⁺¹ (-0.701 V)

${\text{Cu}}^{+\text{1}}+{\text{e}}^{-}\to {\text{\&nbsp;Cu}}^0\left(0.0\text{58\&nbsp;V}\right)$

Randles-Sevcik was used to determine the peak current (Ip) [51,52]. Additionally, Eqs. (4,5) [53] were used to evaluate the difference in potential (∆E_P):

(4)

$Ip = 0.4463nFAC (nF\nu D / RT) \mathrm{½}$

n: Number of transferred electrons

C: Molar concentration in mol/L

I_p: Current in ampere

ν: Scan rate in volts/s

D: Diffusion coefficient of redox particles in cm²/s

A: Electrode surface area in cm²

(5)

$\Delta Ep = Ep,a - Ep,c$

Numerous significant factors (Q, αna, & ks) can be calculated using the previously discovered values (I_p, E_pc, & E_pa). Eq. (6) can be used to compute the redox ions of surface concentration [54].

(6)

$\Gamma =ip4RT/n2F2A\nu$

F: The Faraday constant that equal 96485.33 C/mol

n: The e ̅ numbers in redox reactions

R: The gas constant that equal 8.314 J/mol.K

T: Temperature in Kelvin

Eq. (7) has been used to estimate the charge transfer coefficient “α” [55]:

(7)

$\alpha na=1.857 RT/(Epc – Epc/2)$

Eq. (8) [56] has been used to evaluate the charge quantity consumption in the electrochemical processes:

(8)

$\text{Q}=\text{NFAΓ}$

Eq. (9) can be used to determine the heterogeneous charge transfer rate constant “ks”:

(9)

$ks = 2.18 \left[Dc \alpha naF \nu / RT\right]1/2 exp \left[\alpha 2 nF \Delta Ep / RT\right]$

As shown in Table 7S, the components that were taken into consideration (Dc, T, R, F, n, ΔE_P, α_na, & v) were computed for both the cathodic and anodic peaks. Additionally, as the CuCl₂ concentration rose, most of the desired components increased, favoring the occurrence of diffusion [57].

Supplementary Table 7

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3.12.3. Effect of scan rate on Cu-H₂BT complex

The voltammograms obtained for Cu²⁺, both in the absence and presence of H₂BT at 291.15 K in 0.1 M KCl, have been presented in Figure 5, showcasing various scan rates (0.01, 0.02, 0.05, and 0.1 V/sec). The data revealed the following observations:

• (i)

  The peak current increased linearly with the scan rate.

• (ii)

  The cathodic and anodic peak positions shifted toward negative and positive potentials, respectively.

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Figure 5.

The cyclic voltammogram of CuCl₂ at various scan rates in (a) absenteeism and (b) presence of H₂BT at 291.15 K. Electrochemical bands: A,B

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These findings were supported by the linear relationship between the peak currents and the scan rates, as illustrated in Figure 6, which suggests a diffusion-controlled redox mechanism. The ratio of the current peaks was greater than 1, indicating the system’s quasi-reversibility. The solvation and kinetic data for Cu²⁺ at various scan rates, both in the presence and absence of H₂BT, have been detailed in Tables 9S and 10S.

Supplementary Table 9

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Supplementary Table 10

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Figure 6.

Relation (i_p vs. ν^1/2) for CuCl₂ at final adding in (a) absenteeism and (b) presences of H₂BT K for wave (A).

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3.13. Biological examines

3.13.1. Antimicrobial investigation

The discussion on the outcomes of the activity index for H₂BT and its metal chelates is crucial for a comprehensive understanding of their antibacterial and antifungal properties, especially in comparison to standard antibiotics such as Ampicillin and antifungal agents like Colitrimazole. Here, we can expand upon several key aspects of the findings, focusing on their implications and contextual importance in the field of medicinal chemistry and microbiology [58].

3.13.1.1. Comparative analysis of activity index values

The moderate activity observed for H₂BT and its metal chelates relative to Ampicillin suggests that while these compounds exhibit some level of antibacterial effectiveness, their efficacy may not yet match that of established antibiotics. This pointed to the potential for further optimization of H₂BT or it chelates to enhance their antibacterial properties.

3.13.1.2. Specific bacterial strains and activity levels

Notably, the lower activity values for the metal chelates against B. subtilis and E. coli could indicate selective sensitivity or resistance patterns that warrant in-depth investigations. This trend raises questions about the mechanisms of action and how these compounds interact with specific bacterial targets. Understanding the biological pathways affected by these chelates could provide insights into their antibacterial mechanism and still pave the way for designing compounds that overcome existing resistance.

3.13.1.3. Highlighting the ni complex

The [Ni(HBT)(H₂O)(OAc)].3H₂O complex generally showed the highest percentage activity among the tested chelates, signifying that nickel coordination could enhance the bioactivity of H₂BT. This observation warrants further exploration of metal ion influence on the pharmacodynamics of the ligand. For instance, research into coordination chemistry might elucidate why nickel enhances activity, possibly due to improved solubility, stability, or affinity for microbial targets.

3.13.1.4. Antifungal activity observations

With respect to antifungal activity, the finding that H₂BT exhibits moderate values against Candida albicans in comparison to Clotrimazole aligns with current trends in exploring novel antifungal agents as resistance to conventional treatments rises. Again, the performance of the metal chelates against fungal targets and the superior activity of the nickel complex could reflect a similar enhancement mechanism. So, further studies should be focused on understanding the interactions between these chelates and fungal cell membranes or metabolic pathways.

3.13.1.5. Future directions for research

The results from Table 13S and Figure 7 provide a solid foundation for potential future research avenues. Investigating the broader spectrum of metal complexes involving other transition metals or varying ligand structures could lead to novel discoveries in the quest for new antibacterial and anti-fungal agents. Moreover, conducting in vivo studies could bridge the gap between empirical laboratory findings and practical therapeutic applications.

Supplementary Table 13

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Figure 7.

(a-c) Antimicrobial results in terms of % activity index of (1) H₂BT, (2) [Cu(BT)(H₂O)₂].2H₂O, (3) [Co(HBT)(H₂O)(OAc)].2H₂O, (4) [Ni(HBT)(H₂O)(OAc)].3H₂O, and (5) standard drugs (Clotrimazole in case of fungi & Ampicillin in case of bacteria).

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3.13.1.6. Conclusion

In summary, while the reported activity indices of H₂BT and its metal chelates demonstrate moderate effectiveness against bacterial and fungal strains, the distinctions observed, particularly the prominence of the [Ni(HBT)(H₂O)(OAc)].3H₂O complex, suggest areas ripe for exploration [59]. A deeper understanding of these mechanisms and their biological implications could lead to more effective applications in combating microbial resistance. The value of % activity index was estimated through Eq. (10):

(10)

$\% Activity Index=\frac{inhibitory zone for test compound,diameter}{inhibitory zone for standard, diameter }\times 100$

3.13.2. Antioxidant investigation

The L-ascorbic acid, a common antioxidant, is used to evaluate the antioxidant rank of the generated Schiff base compounds using the ABTS colorimetric assay, which is recognized as illustrated in Figure 8 [60]. According to the experimental results shown in Table 14S, the ligand (H₂BT) and the standard ascorbic acid showed values that were roughly comparable, while all of the complexes that were tested had lower antioxidant values that were comparable to those of the standard ascorbic acid (88.50%) and the ligand (H₂BT) (85.70%) while [Co(HBT)(H₂O)(OAc)].2H₂O Out of all the substances examined, showed the highest activity index (40.60%).

Supplementary Table 14

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Figure 8.

Antioxidant activity of (1) Ascorbic acid, (2) H₂BT, (3) [Cu(BT)(H₂O)₂].2H₂O, (4) [Co(HBT)(H₂O)(OAc)].2H₂O, (5) [Ni(HBT)(H₂O)(OAc)].3H₂O compounds. (a) The ABTS colorimetric examine using ascorbic acid as standard (b) The Erythrocyte hemolysis using Ascorbic acid as standard.

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DFT outcomes data can provide valuable insights into the antioxidant properties of Schiff base compounds by elucidating the electronic structures and reactivity patterns of the molecules involved. Specifically, DFT calculations can help identify the active sites on the ligand and metal complexes that are responsible for their antioxidant activity.

The relationship between DFT studies and antioxidant activity can be further understood through the following points:

• 1.

  A lower HOMO-LUMO gap generally indicated a higher reactivity, which may correlate with enhanced antioxidant properties.

• 2.

  Also, by computing the energy changes involved in these reactions, we can predict how effectively a compound can donate electrons or hydrogen atoms to neutralize free radicals.

• 3.

  Moreover, correlating DFT-derived properties (like binding energies or reaction pathways) with experimental results from assays like the ABTS colorimetric assay can help clarify why certain complexes exhibit higher or lower antioxidant capacities.

3.14. Molecular docking

The BCL2 family proteins, which are essential for either promoting or preventing apoptosis, have been the subject of much research over the past ten years. Their crucial role in controlling apoptosis, carcinogenesis, and reactions to anticancer therapies is the reason for this increased interest [66]. By blocking its anti-apoptotic action, many tiny compounds known as BCL2 inhibitors increase the vulnerability of cancer cells to apoptosis. These inhibitors work by attaching themselves to the groove of the protein and preventing it from doing its job [67]. To assess the modalities of drugs, the degree of their binding, and their interactions with a particular BCL2 inhibitor, this study used a comparative molecular docking analysis. The best docking positions were examined, along with the interactions of metal and ligand complexes with the 2w3l receptor. The medication was used as a standard reference for comparative analysis. Both the 2D and the hydrogen bond surface representations at the metal complex/drug-receptor interaction site have been shown in Figure 9. Furthermore, further information on the binding interactions has been tabulated in Tables 5 and 15S. The results showed that while the H₂BT ligand and Co²⁺ complex showed lower binding energies, the potential binding energy of the Ni²⁺ and Cu²⁺ metal complexes was higher than that of the original DRO ligand.

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Figure 9.

Interactions between the 2w3l target and the (a) H₂BT ligand, (b) Co²⁺ complex, (c) Ni²⁺ complex, (d) Cu²⁺ complex, and (e) DRO ligand in two and three dimensions (the acceptor area of the hydrogen bond surface is represented by green, and the donor area of the amino acid residues by pink).

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With a docking score of -6.89 kcal/mol, the Cu²⁺ complex showed the highest affinity for binding to the 2w3l target among the four examined isolated compounds. In contrast to the DRO ligand, this implies a stronger binding. As seen in Figure 9, the Cu²⁺ complex displayed interactions in which the residues GLU73, ALA108, MET74, and PHE71 participated in π-Alkyl, π-Sigma, π-Donor Hydrogen bond, and Amide-π stacking interactions. Similarly, a theoretical binding energy of -5.51 kcal/mol was obtained by docking the Ni²⁺ complex with the 2w3l target. The interaction between the Ni²⁺ complex and 2w3l is depicted in Figure 9, emphasizing residues ALA108, MET74, and GLU73. Additionally shown is the H₂BT ligand’s binding mode inside the pocket, which results in a molecular docking score of -4.07 kcal/mol. π-alkyl interactions with the MET74, ARG105, and ALA108 residues as well as a π-π T-shaped interaction with the PHE63 residue are involved in this interaction. With a docking score of -4.11 kcal/mol, the Co²⁺ complex interacts with ASP70 via a traditional hydrogen bond and with the residues VAL92, LEU96, and ALA108 through three π-alkyl interactions.

3.14.1. Docking validation

To evaluate the efficacy of the docking procedure, re-docking was conducted using a similar process, as shown in Figure 10. The 3D molecular interactions of the co-crystallized complex with the BCL2 protein (PDB ID: 2w3l) matched the 3D chemical contacts of the re-docked inhibitor with the same cell line. In comparison to both the original and re-docked complexes, the re-docked inhibitor scored -5.32 kcal/mol and displayed an Root mean square deviation (RMSD) of 0.9364 Å.

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Figure 10.

Co-crystalline ligand (green) and red locked ligand (red) hydrogen bond surfaces with (2w3l) DNA Helix (RMSD= 0.9364 Å).

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3.15. In silico pharmacokinetics analysis

It is crucial to evaluate the pharmacokinetic properties of any molecule being considered as a possible pharmacological agent before conducting in vivo experiments. Absorption, distribution, metabolism, excretion, and toxicity (ADME) variables are all included in this assessment. By analyzing the ADME properties of the examined compounds, the SwissADME server (http://www.swissadme.ch/), which follows Lipinski’s rule of five, may predict the drug-likeness of those compounds. According to Lipinski’s rule, typical drug-like compounds have a molecular weight of 500 Da or fewer, a Log P magnitude of 5 or less, and no more than 10 hydrogen bond donors and 5 hydrogen bond acceptors [68]. Compounds that violate certain requirements may have problems with bioavailability. But according to the ADME analysis, none of the substances went against Lipinski’s rule of five.

The pharmacokinetic properties of synthesized drugs are presented in Table 6. All of the molecules under investigation fell below the 500 threshold, with molecular weights ranging from 354 to 489. This suggests that, in contrast to larger molecules, these chemicals are likely to be more easily absorbed, transported, and disseminated. Furthermore, it was found that the tested compounds’ hydrogen bond acceptor and donor counts fell within the bounds established by Lipinski’s rule. The range of hydrogen bond donors was 3 (<5), and the range of hydrogen bond acceptors was 5 to 8 (<10). There were seven rotatable bonds in the H₂BT ligand, three in the Co²⁺ and Ni²⁺ complexes, and one in the Cu²⁺ complex (<10). By analyzing six physicochemical characteristics lipophilicity, solubility, size, flexibility, polarity, and saturation the bioavailability radar rapidly determines a compound’s drug-likeness. A molecule’s radar plot has to fall completely into a specified physicochemical range on each axis, denoted by a pink area, in order to be classified as drug-like. Lipophilicity (-0.7 – +5.0), size (150 – 500 g/mol), polarity (20-120 Ų), solubility (Log S ≤ 6), saturation (≥0.25), and flexibility (≤9 rotatable bonds) are all within the ideal ranges shown by the pink area. Because of their strong polarity (TPSA: 127-159 Ų), all the compounds were inappropriate for oral delivery, as seen in Figure 11S.

3.16. Structure activity relationship

SAR studies often focus on linking the theoretical insights derived from density functional theory (DFT) to the biological activity observed in the compounds under examination. Specific molecular parameters, including band gap energy (E_gap), chemical potential (μ), and electronegativity (χ), are relevant to both the stability and chemical reactivity of these isolated compounds. Generally, compounds that exhibit a low E_gap alongside high μ and χ are considered effective electron donors. The ABTS assay highlights the reactivity of these compounds, identifying the most effective antioxidants based on their reactivity. Results from the ABTS assay indicated that the Co²⁺ complex exhibited the highest antioxidant activity, likely due to its low E_gap, along with high μ and χ values.

Additionally, the dipole moment provided valuable insights into the biological activity of the synthesized compounds. A larger dipole moment suggests a stronger interaction with and greater affinity for the receptor amino acids in microbial protein envelopes. Antimicrobial tests identified Ni²⁺ as the most potent compound against microbes, attributed to its high dipole moment of 12.581 De. Molecular electrostatic potentials (MEPs) obtained through DFT are instrumental in predicting reactive sites within the optimized structures of the compounds studied. Variations in chemical potential are visually represented by a color gradient, blue (lowest) to red (highest), with green indicating zero potential. In this context, the red zone indicated areas prone to electrophilic attack, while the blue zone signified regions favoring nucleophilic attack. MEP studies are typically linked with cytotoxicity assessments, wherein compounds with significant negative potential areas are often deemed more toxic. Cytotoxicity assays demonstrated that the ligand (H₂BT) was the most toxic towards carcinogenic cells, supporting these findings. The docking results, alongside theoretical analyses, corroborated the cytotoxicity outcomes, suggesting that both the ligand and its Ni²⁺ complex could serve as effective anticancer agents [69].