Copper based azo dye catalysts for phenoxazinone synthase mimicking efficiency: Structure characterization and bioactivity evaluation

3.1.1 Mass spectra

Electron impact (EI) mass spectra of the two ligands H₂TNBS (Z) and H₂INBS (G) are given in Figs S1 & S2. As shown in the figures, the molecular ion peaks are apparent at m/z = 410.49 and 422.14 for H₂TNBS and H₂INBS, successively, that were found to be consistence with the theoretical molecular weights of the ligands’ suggested structures (calculated molecular weights are 410.47 and 422.46 for H₂TNBS and H₂INBS, respectively).

3.1.2 ¹H NMR

¹H NMR spectra of H₂TNBS and H₂INBS assigned in deuterated DMSO solvent and in existence of tetramethylsilane (TMS) internal standard are illustrated in Fig. 2 and S3, respectively.

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Fig. 2

¹H NMR spectrum of Structure of H₂TNBS ligand.

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The spectrum of H₂TNBS shows singlet at 15.77 ppm that assigned to the phenolic OH proton. the two singlet signals at, 12.8 and 9.71 ppm are assigned to sulfonamide OH and NH protons, respectively, and hence supports the coexistence of the ligand H₂TNBS in the two tautomeric structures I and II (Scheme 1) (Kanaani et al., 2016). The peaks located within the range 8.41–6.38 ppm were attributed to the aromatic protons whose integration assuring the existence of the two tautomeric structures I and II (Scheme 1).

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Scheme 1

The two tautomeric structures (I and II) of the ligand H₂TNBS.

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For the ligand H₂INBS, the singlet appearing at 9.9 ppm assigned to NH proton. The multiple signals appearing within the range 7.59–6.35 ppm have been integrated for the aromatic protons whereas the two singlet signals assigned to the 2 methyl protons appeared at 2.04 and 1.65 ppm (El-Ghamry et al., 2022).

3.2.1 Stoichiometry and general composition

on the basis of elements content data and molar conductance values, the emperical formulae of the generated chelates and the two synthesized ligands are displayed in Table 1 together with information of formula weights, color, and melting temperatures. The values of elements’ percent, which also completely consistent with the data indicated in Table 1, validated the separated chelate structures. Accordingly, these Cu chelates were assured to be produced in a 2: 1 (metal: ligand) ratio for complexes 1–3 and 1:1 for complexes 4–6. From molar conductivity values (measured using 10^-4 M in DMSO) which found to be inside the range 8.63–35.6 Ω^-1cm²mol⁻¹ range supported non-electrolytic complexes formation (Geary, 1971; Fawzy et al., 2020). It was also shown that the complexes maintained their stability despite prolonged subjection to air. Solubility tests demonstrated the ease solubility of all compounds in DMF and MDSO however, in other polar and non-polar solvents, they provided either poor solubility or total insolubility.

3.2.2 EI- mass spectra

Since mass spectrometry offers appropriate spectral analysis to establish the molecular weights and, in turn, the molecular formulae of the synthetic compounds, it has been used to evaluate the acquired Cu(II) complexes, 1–6. The mass spectra of complexes 1–6 are shown in Figs. 3, 4, and S4–S7. These figures show that the molecular ion peaks for complexes 1–6 are observed at m/z = 657.98, 689.25, 659.19, 538.12, 562.75, and 564.43, respectively. These molecular ion peaks are consistent with the calculated molecular weights of 660.93, 689.66, 659.56, 538.64, 562.05, and 565.02 (excluding lattice H₂O molecules) for complexes 1–6, successively.

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Fig. 3

Mass spectrum of complex 5.

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Fig. 4

Mass spectrum of complex 6.

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3.2.3 Infrared spectra

The coordinated function groups in the ligand with the metal ions can be determined from the IR spectra of both of them upon comparison with each other, making FTIR spectral analysis an important tool for characterizing the structure of metal complexes. Distinctive function groups are shown in Table 2 along with their vibration wavenumbers.

This comparison inferred that the two spectral bands occurring at 1499 and 1208 cm⁻¹ in the spectrum of H₂TNBS (Z) and at 1452 and 1207 cm⁻¹ in the spectrum of H₂INBS (G) which assigned to the vibration wavenumber of N = N and C-O groups have been shifted to higher or lower values in the spectra of all complexes (1–6) by at least 7 cm⁻¹ and hence ensuring their incorporation in connection to the copper ion centers in metal complexes (Khedr et al., 2019; Khedr et al., 2022). The azomethine groups’ stretching wavenumbers of thiazole and isoxazole rings appeared in the ligands’ H₂TNBS (Z) and H₂INBS (G) spectra at 1568 and 1552 cm⁻¹, respectively. The first band has been shifted to lower position in the spectra of complexes 1–3 compared with H₂TNBS (Z) ligand whereas the second band appeared in complexes’ 4–6 spectra in a position very close to its position in the spectrum of ligand H₂INBS (G) assuring that the C = N shared in complex formation in complexes 1–3 but in contrast this band kept free in the spectra of complexes 4–6. Similarly, the great shift in the position of the of asymmetric and symmetrical vibrations of SO₂ group in the spectra of complex 1 confirm the involvement of its oxygen atom in complex formation while in complexes 4–6, such group has been confirmed to remain free due to its appearance almost at the same wavenumber as in the ligand H₂INBS spectra. Vanishing of SO₂ bands in spectra of compounds 2 and 3 is explained by coordination of sulfonamide O to the Cu center in the deprotonated enolic form. The non-ligands bands appearing between 558 and 527 cm⁻¹ and between 481 and 426 cm⁻¹ have been assigned to stretching wavenumbers of Cu–O and Cu–N bonds, successively.

In addition, analysis of the IR spectra allows a precise assignment of the coordinated anion's method of binding to the metal center. The two non-ligand bands that are shown in the spectra of the acetate-containing complexes 2 and 5 have appeared at 1593 and 1341 cm⁻¹ for complex 2 and at 1588 and 1351 cm⁻¹ for complex 5 and attributed to the v(COO⁻)_asy and v(COO⁻)_sym, respectively, of CH₃COO groups,. The difference between v(COO⁻)_asy and v(COO⁻)_sym has been calculated and equals 252 and 237 cm⁻¹, matching with the Δν value reported for monodentate acetate groups (Olar et al., 2013).

For 3 and 6 (containing NO₃ coordinating groups), the two bands occurring at 1427 & 1297 cm⁻¹ in complex 3 spectrum and at 1402 & 1216 cm⁻¹ for complex 6 with Δν differences of 130 and 186 cm⁻¹, successively, assured the bi- and unidentate ligation fashion of nitrato groups in complex 3 and 6, successively (Takroni et al., 2020).

3.2.4 Magnetic moment

Cu(II) complexes 1–3 were discovered to have magnetic moment values that fell within the range of 2.38–2.56B.M. for the two Cu(II) centers (Table 3). Such values are quite close to the calculated one corresponding to bimetallic chelates with two Cu(II) centers that are not in interaction (i.e. 2.45B.M.) (Li and Jin, 2009).

For complexes 4–6, the magnetic moment values were measured to be 1.77, 1.79 and 1.81B.M., respectively, which are almost within the acceptable spin-allowed limit for a single electron (i.e. 1.73B.M.) in mononuclear Cu(II) chelates (Takroni et al., 2020).

3.2.5 Electronic spectra

The most important study for identifying geometry of the ligand atoms surrounding the core metal ion in metal chelates is the electronic absorption spectra (UV–Vis). Accordingly, the nujol-Mull method was used to measure the UV–Vis spectra of compounds 1–6 over the wavelength range of 200–800 nm.

In all complexes’ spectra, Figs. 5, S8 and Table 3, the bands appearing within the ranges 251–282, 209–348 and 360–392 nm with medium to high absorbance are characteristic for π → π* transitions of benzene ring, π → π* and n → π* transitions in C = N & N = N groups, respectively (Yarkandi et al., 2017). The broad bands apparent in 493–503 nm range assigned to LMCT (Cadranel et al., 2017).

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Fig. 5

UV–Vis spectra of compounds 5–8 using nujol mull technique.

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The low and broad intensity bands apparent in the spectra of six complexes in the range 707–738, has been assigned to ²B_1g→²A_1g transition familiar for square planar architectures (Uysal and Kurşunlu, 2011).

3.2.6 Thermogravimetric analysis (TGA)

The thermogravimetric (TGA) analytical tool is one of the useful instruments that is frequently used to gain understanding of the structure and stability of inorganic compounds. The full analysis of the TG thermograms of complexes 1–6 along with the derivative thermogravimetric data (DTG) the is shown in Figs. 6 & S9 and interpreted in Table 4.

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Fig. 6

TG/DTG thermograms of complexes 1–3.

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Compounds 1–6 underwent disintegration in three decomposition ranges as for complexes 5 and 6, four decomposition ranges as in the complexes 1 and 3 or five decomposition ranges as in the complexes 2 and 4.

A closer look at the results given in Table 4 revealed that only lattice or lattice and coordinated water molecules are lost during the first stage of disintegration, which began at ambient temperature and continued up to 150 ^°C. The next step of decomposition occurred between 72 and 403 ^°C, inside which coordinated water or anions get lost in some compounds. The subsequent steps of breakdown are attributed to the progressive breakdown of the organic ligand molecules included in the complexes’ structures. Table 4 also lists and illustrates the DTG temperatures for each stage. The following Scheme (Scheme 2) also indicate the thermal decomposition of complexes 1 and 2, as example:

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Scheme 2

Decomposition stages of complexes 1 and 2.

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3.2.7 X-ray diffraction investigations

In light of current advancements in material science technology and knowledge, X-ray diffraction (XRD) is considered as one of the most significant and frequently employed material characterization technique. It is commonly applied to gather structural information on chemical compounds' microcrystalline structures in the solid state (Ali et al., 2022). As a result, the diffraction patterns of organic ligands H₂INBS & H₂TNBS and inspected metal complexes (1–6) were implemented within 10° <2θ > 80° scattering angle range (Figs. 7 & S10). The ligands' diffraction patterns allocations are drastically distinct from those of the equivalent metal complexes, which supports complex formation and shows that there are no contaminants or beginning reactants that are smeared (Khedr et al., 2022). The acquired x-ray diffraction patterns denoted that ligand H₂TNBS displayed high crystallinity with diffraction patterns totally distinct from those of complexes 1–3 which exhibited good to weak crystallinity. Also, high crystallinity afforded for ligand H₂INBS, good crystallinity of complex 4 and weak crystallinity of complex 5, with completely different diffraction patterns, whereas complex 6 is fully amorphous. Depending upon the Deby-Scherrer equation calculations (Sanad et al., 2022), the average crystallite sizes have been calculated: $$\tau =\frac{K\lambda }{\beta COS\theta }$$

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Fig. 7

XRD spectra of H₂INBS complex 4.

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where, τ is the mean size (crystallite size) of the ordered (crystalline) domains, which may be smaller or adequate to the grain size, λ is the X-ray wavelength, K mean a dimensionless shape factor and its value amounts to unity (0.9), but varies with the actual shape of the crystallite, β refers to line broadening at half the maximum intensity (FWHM), after subtraction of the instrumental line broadening.

Applying this equation to the obtained spectra, the ligand H₂TNBS, complex 1, complex 2, and complex 3 exhibited 20.7, 19.0, 5.5 and 16.3 average crystallite sizes, respectively, and ligand H₂INBS, complex 4, and complex 5 found to be 33.7, 25.3 and 8.0 nm, respectively. The irregular arrangement of the materials’ particles through their precipitation may be indicated by the complexes' amorphous nature. This amorphous state will predominate if the complexation process is rapid and/or the produced chemical is cooled quickly. Additionally, many of the structural elements of the complex cannot be accommodated in crystalline form. The amorphous compounds with probable small sizes are usually investigated by TEM technique to examine if they are having particles sizes in the nano-metric range. These nanometer-scale compounds usually receive a lot of interest due to their improved functional properties and wide range of potential technological applications, including optics, microelectronics, catalysis, bio- and chemical sensors (Gaber et al., 2017).

3.2.8 Investigation of TEM images

Transmittance electron microscopy (TEM) is one of the most common techniques to study a wide range of fascinating nano-sized materials. It's a method that's usually employed to look at the shape and dimensions of solid materials. Therefore, the particle sizes and crystallinities of the produced complexes 1–6 were further investigated by TEM technique. Images captured by the transmittance electron microscope in bright field for complexes 1–6 are shown in Fig. 8 & S11-S14 demonstrating that the samples are made up of teeny, different-sized nanoparticles. These photos make it abundantly notable that the complexes powders are made up of nanoscale particles with spherical-like shape (either regular or not). Additionally, it was determined that the particle size of metal complexes ranged between 15.9 and 45.6 nm for example complex 1 and 27.89–39.37 nm for 2 while complex 3′s was 18.19–34.12 nm, complex 4′s was 9.48–23.25 nm, complex 5′s was 12.13–18.45 nm and complex 6′s was 13.80–20.63 nm.

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Fig. 8

Compounds 2 and 6 TEM images.

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Accordingly, in comparison to their bulk analogues, the biological effectiveness of these innovative synthetic compounds is suspected to be stronger at these nanometric scales. There is a lot of interest that might be sparked by the novel functional properties of the nanosized chelates and the wide range of potential uses (Saad et al., 2019).

As a result, and in light of all the investigations previously deliberated, the structures of compounds 1–6 are depicted in Figs. 9 and 10.

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Fig. 9

Concluded structures for complexes 1–3.

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Fig. 10

Concluded structures for complexes 4–6.

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3.3.1 Antimicrobial investigation

As described in the experimental part, the examined two ligands, H₂TNBS and H₂INBS, and their complexes (1–6) were subjected to antimicrobial screening applying a well diffusion method (Jahangirian et al., 2013) (Table 5). All inspected compounds displayed very strong and promising against S. aureus within inhibition zones ranged from 15 to 26 mm which are higher than the inhibition zone of the used standard Gram-positive ant-bacterial agent Amoxycillin-clavulinic (10 mm) by one and half to two and half. Also, the inhibition zones of examined ligands and complexes towards B. cereus varied from 12 to 28 mm, which exceeds the inhibition zone of standard Gram-negative antibacterial agent (12 mm) by two and half times of value. Complexes 1, and 5 exhibited inhibition zones equal 9 equivalent to that of the standard bactericide (8 mm) against E. coli, whereas complexes 2 showed 19 mm, more than the double value of the common standard towards the same organism. respectively. Complex 4, displayed antibacterial activity (16 mm) double that of the applied standard (8 nm) against S. typhi. At the same time, ligand H₂TNBS and complex 3 showed 12 and 9 mm inhibition zones towards it. Only ligand H₂INBS showed excellent antifungal activity against the multicellular fungus A. flavus, with 26 nm inhibition zone which is higher than double the value of applied standard fungicide (11 mm). Ligands H₂INBS and H₂TNBS exhibited inhibition zones 16 and 12 mm against C. albicans. Upon complexation the inhibition zones of complexes 3 and 5 increased compared with the corresponding ligands and arrived 16and 18 mm. These values are equivalent or greater than the standard Amphotericin-p (13 mm) towards the same unicellular fungus. The selected species demonstrate diversity of microorganisms, including Gram-positive and Gram-negative bacteria, unicellular and multicellular fungus (Khedr et al., 2022). In most instances, the increased antibacterial effect during chelate formation can be ascribed to metal ions existence, with more hyper sense to microbial cells than the chelating ligands (Saad et al., 2017). Moreover, the greater action of the metal chelates may be caused by the increased lipophilicity during complexation. The coordinated metal ions may cause detrimental interactions between cellular components, or they may stop cellular enzyme activity. Obvious variation in the efficacy of the alternative compounds under study contra distinct organisms are brought on either by the impermeability of the cells of microorganisms or variations in the ribosomes in microbs’ cells (Singh et al., 2007). Additionally, Overton's notion and Tweedy's chelation hypothesis offer the greatest explanations for this improved efficiency upon complex development (Saad et al., 2019). According to the available information, the studied ligands and their complexes hold a great deal of promise for becoming broad-spectrum, novel, highly effective, fungicides and bactericides.

3.3.2 Performance as antitumor agents

The vast number of cytotoxic chemicals that are currently available includes a significant number of metal-based complexes. Platinum (II) complexes, such as cisplatin, oxaliplatin, and carboplatin, which are particularly target genomic DNA are frequently utilized in clinical settings to treat a variety of malignancies. In about 50% of chemotherapy-treated cancer patients, either alone or in combination, a platinum medication is administered. Despite playing a key role in cancer chemotherapy, platinum medicines have significant limitations, including a narrow range of antitumor activity, systemic dose-related toxicity, and a propensity to induce drug resistance, which frequently results in treatment failure (Gamberi and Hanif, 2022). The exploration of nonplatinum metal-based medications as an efficient substitute has sparked a great interest in response to these observations (Saad et al., 2018). In this regard, we seek to evaluate the antitumor efficacy of azo ligands H₂TNBS & H₂INBS and their chelates (1–6) against human Lung carcinoma (A-549) and Pancreatic carcinoma (Panc-1) and cells (Table 6 and Fig. 11). Untreated cells used as a control and vinblastine sulphate (VS) as a reference drug which had IC₅₀ values of 4.68 and 24.6 and µg/ml contra pancreatic and lung cancer cell lines, respectively. The inspected compound concentration that caused 50% decrease in cell reproduction are referred IC₅₀. IC₅₀ is defined as the concentration that, under usual conditions, inhibits cell growth by 50%. The average of three separate experimental repetitions is averaged to produce each final value (Khedr et al., 2019). The obtained data made obvious that inspected compounds exhibited an increasing propensity to colonies lung cancer cell lines; H₂TNBS < H₂INBS < 6 < 4 < 5 < 1 < 3 < 2. The examined compounds reduced the viability of cancer cells, which was significantly influenced by their structures, mainly with the type of anions attached to the complex and geometrical arrangement around the core metal ion (Gaber et al., 2018). With IC₅₀ values of 10.41, 4.99, 7.44, 19.62, 12.65 and 29.54 μg ml⁻¹ for complexes 1–5, respectively, demonstrated strong and very encouraging anticancer actions toward A-549 cells, overcoming the standard vinblastine sulphate having IC₅₀ of 24.60 μg ml⁻¹. Complex 6 displayed very good anticancer efficiency with IC₅₀ equals 29.54 μg ml⁻¹, substantially equal to VS against the same tumor cells. Furthermore, an increase in anticancer effectiveness and inhibition of cell viability against Panc-1 were seen for all drugs under consideration in increasing order; H₂TNBS < H₂INBS < 6 < 4 < 5 < 1 < 3 < 2. With IC₅₀ values of 2.57 and 3.98 g μg ml⁻¹, respectively, complexes 2 and 3 demonstrated strong and extremely promising anticancer actions toward Panc-1 cells, exceeding the standard drug vinblastine sulphate with IC₅₀ equal 4.68 μg ml⁻¹. Also, complexes 5 and 1 displayed excellent and encouraging Panc-1 antitumor efficiency within 9.20 and 6.54 μg ml⁻¹, IC₅₀ value whereas complex 1 showed acceptable IC₅₀ (14.23 μg ml⁻¹). Depending on these findings, these compounds may undoubtedly be regarded as efficient and potential anticancer therapies for the examined tumor cells.

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Fig. 11

The in-vitro anticancer evaluation test as IC₅₀ (μg/ml) of H₂TNBS, H₂INBS and their metal chelates (1–6) against A-549 and PANC-1 cancer cells.

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3.3.3 Effectiveness toward phenoxazinone synthase and kinetic studies

Recording of APX absorption peak that produced consequent to the oxidation of OAP by the power of Cu(II) chelates; 1–6, exhibited a raise of the main peak APX with time. This band emerged in Cu(II) complexes’ spectra around 435 nm, and consequently supported their catalytic effect for phenoxazinone synthase like activity. Figs. 12, 13 & S15-S18 show the time dependent spectra of complexes 1–6 with concentration of 3x10^-5 M with addition of 10^-2 M of OAP in DMF medium under aerobic condition. For these catalytic reactions, once OAP was added to the complexes’ solutions, time started, and the compounds’ spectra were recorded each 5 min up to 70 min. When the same experiments were run without catalysts, no observed increase in APX peak was seen (blank test). The observed rate constant (k_obs) and the initial rate of reaction (V_o) have been assessed using the initial rate method (Table 7) (ε_(APX) = 18300 M⁻¹cm⁻¹) (Podder and Mandal, 2020).

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Fig. 12

The increase in APX spectral peak at ≈ 435 nm upon addition of 10^-2 M of OAP to 3x10^-5 M of catalyst 4, the reaction medium is DMF.

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Fig. 13

The increase in APX spectral peak at ≈ 435 nm upon addition of 10^-2 M of OAP to 3x10^-5 M of catalyst 6, the reaction medium is DMF.

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The kinetics of the reactions were studied in order to assign the applied Cu(II) complexes' level of catalytic performance in comparison to one another and to other known comparable compounds. In these reactions, 3 x10^-5 M solutions of each catalyst was treated with series of substrate concentrations (with concentrations in the range 1–8 × 10^-3 M). The substrate concentration 10 times, at least, higher than the concentration of the catalyst to make sure that pseudo-first order kinetics is applied. For each catalytic solution, the absorption peak of APX, which is formed as the product of substrate oxidation, was taken once the reaction started and each 3 min intervals until the time of the reaction reached 21 min. The recorded absorbances were used to assign the initial rate for each catalytic solution. Plotting the substrate concentrations ([S]) versus initial rate (V_o) inferred that the majority of the Cu complexes under investigation provided rate saturation kinetics (Figs. 14, 15, and S19–S22). The outcomes clearly demonstrate the pre-equilibrium production of the complex-substrate intermediate, and the irreversible substrate oxidation is the rate-determining stages for catalytic cycle, which eventually developed APX, as demonstrated by the equation:

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Fig. 14

Michaelis–Menten plot (left) and Lineweaver–Burk plot (right) of complex 5 for catalytic oxidation of OAP.

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Fig. 15

Michaelis–Menten plot (left) and Lineweaver–Burk plot (right) of complex 6 for catalytic oxidation of OAP.

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The applied catalysts afforded saturation kinetics, so Michaelis-Menten equation is the most significant equation for treatment of such reactions. The following equation illustrates the connection between [substrate] (i.e [S]) and the rate of the enzyme reaction (V) using this kinetic-like model: $$V=\frac{V_{\mathrm{m}\mathrm{a}\mathrm{x}}[S]}{K_M+[S]}$$

V_max is the greatest rate which the system can achieve when [substrate] reaches saturation levels. The Michaelis constant, or K_M, refers to the substrate’s concentration at which the reaction rate is half of V_max. By linearizing the Michaelis-Menten equation, a double reciprocal Lineweaver-Burk plot is created, which is applied to examine different kinetic parameters like V_max and K_M. The formula serves as an example of the Lineweaver-Burk Equation. $$\frac{1}{[S]}=\frac{K_M}{V_{\mathrm{m}\mathrm{a}\mathrm{x}}[S]}+\frac{1}{V_{\mathrm{m}\mathrm{a}\mathrm{x}}}$$

Fulfillment of the previous equation to the kinetic process of the examined catalysts (Figs. 14 & 15), result in the calculation of the kinetic parameters V_max, K_M and the turnover number (k_cat, h⁻¹) which are collected in Table 7.

Table 7 shows that H₂INBS chelates (compounds 4–6) had higher activity than H₂TNBS chelates (complexes 1–3), indicating that the ligand structure is one of the factors influencing the activity of the complexes.

On comparing the activity of the synthesized catalysts; 1–6, relatively according to each other, the following remarks can be summarized:

For complexes 1–3, the catalytic activity of these complexes that deduced from TOF values indicated complex 1 to exhibit the uppermost efficacy with k_cat of 60.8 h⁻¹ followed by compound 3 having k_cat value of 32.15 h⁻¹ and finally the least activity afforded by compound 2 with k_cat of 4.34 h⁻¹. On the other hand, for H₂INBS chelates; 4–6, the obtained results showed an extreme activity for compound 6 having 467.01 h⁻¹ k_cat value and the next is compound 4 having k_cat equals 390.48 h⁻¹ and the least activity observed for complex 5 having k_cat equals of 22.82 h⁻¹.

It is observed that APX generation through oxidation of OAP by the catalytic effect of copper chelates can only take place in existence of molecular dioxygen since there is hardly any peak of APX appears without the availability of O₂ in a similar behavior of reported work (Sohtun et al., 2021; Mandal et al., 2020). The suggested mechanism is shown in Scheme S1 (Sarkar et al., 2017).

Comparing the activity of the metal chelates with each other can lead to conclude that the metal center oxidation number and its geometry. The distance between metals in bimetallic center and the structure of organic ligand are also important factors that contributes to catalysts’ activity. Comparing the activity of the metal complexes incorporating the same ligand (i.e. compounds 1–3 with each other and compounds 4–6 with each other) inferred that the key factor controlling the activity is the type of incorporated anion sine all the other factors among each group of compounds are the same. Cloro complexes, 1 & 3, and nitro complexes, 3 & 6, provided the highest activity whereas the other complexes, which are only weakly active, contain CH₃COO^– anions in their structure (i.e., complexes 2 and 5). Depending on the approachability of the labile sites in the first coordination sphere, which is better in case of chloro and nitro groups, it is possible to explain why Cl^- or NO₃^– complexes have greater activity than CH₃COO^– complexes (Panja et al., 2018). The comparable efficacy may also be elucidated based on the steric impact brought on by the CH₃COO^– group's bulkiness when compared to Cl^- or NO₃^–.