New superior bioactive metal complexes of ligand with N, O donor atoms bearing sulfadiazine moiety: Physicochemical study and thermal behavior for chemotherapeutic application

2.1 Physical measurements

A standard gravimetric method was applied to reveal the metal ion (Vogel, 1966). All metal-comprising complexes were dried out under vacuum over P₄O₁₀. The infrared (IR) spectra were analyzed as KBr and CeBr pellets utilizing Perkin-Elmer 683 spectrophotometer (4000–200 cm⁻¹). The electronic spectral data were qualitatively obtained by Perkin-Elmer 550 spectrophotometer. The conductance of metal complexes (1 × 10⁻³ M solution in DMF) was assessed, at room temperature, using Bibby conductimeter. The ¹H NMR spectra of both the ligand and the Cu(II) and Zn(II) complexes were measured by Perkin-Elmer R32-90-MHz spectrophotometer with TMS being utilized as an internal standard. The mass spectrum of the ligand was determined using JEULJMS-AX-500 mass spectrometer connected to data software. Thermal analyses (DTA and TGA) were done with a temperature ranging from 27 to 800 °C, at a heating rate of 10 °C/min, utilizing Shimadzu DT-30 thermal analyzer. Gouy’s method was applied to measure the magnetic susceptibilities using mercury tetrathiocyanate cobalt(II) as a standard while Pascal constant (Lewis et al., 1967) was used to evaluate the diamagnetic corrections. The magnetic moments were calculated according to equation. The ESR spectra of the metal complexes were identified by Varian E-109 spectrophotometer, at room temperature, with DPPH used as a standard. The TLC verified the purity of all compounds.

2.2 Synthesis of the ligand and its metal complexes

2.3 Cytotoxic effect of ligand and complexes on human cells

Fifteen milliliters of blood was withdrawn from a participant after signing informed consent. The blood (15 ml) was carefully layered over Bicoll in a 15 ml conical tube at a ratio of 1.5 to 1. Centrifugation was carried out at 2000 rpm for 25 min at 20 °C. The mononuclear cell layer was collected and washed twice with phosphate buffer saline (PBS). The supernatant was discarded and the pellet was dislodged. Red blood cells (RBCs) were lysed by lysing buffer followed by centrifugation then the pellet was washed with PBS (Soliman et al., 2017). The cell pellet was resuspended in complete Dulbecco’s Modified Eagle’s Medium (DMEM) which consisted of low-glucose DMEM (Gibco, USA), 30% fetal bovine serum (Biochrom, Germany), 1% penicillin/streptomycin (100 U/mL), and 1% l-glutamine. Cell count and viability were calculated by hemocytometer. The mononuclear cells were cultured in 96-well plates containing complete DMEM, at a seeding density of 1 × 104 cells/well, and the ligand and complexes were added to the cells (1000 µg/mL). Serial dilution of the complexes was done 5 times until reaching a concentration of 31.2 µg/mL. Finally, the cells were incubated in 5% CO₂ and 90% humidified atmosphere at 37 °C for 24 h, and then, cells were counted visually under an inverted microscope (Abd El-RazekZ et al., 2020). Based on the United States National Cancer Institute categorization, the activity of cytotoxicity was classified as high activity when IC₅₀ was below 20 μg/mL, moderate activity when IC₅₀ was 20–100 μg/mL, weak activity when IC₅₀ was 201–500 μg/mL, and no activity when IC₅₀ exceeded 500 μg/mL (Huda et al., 2017).

2.4 Chemotherapeutic effect

The chemotherapeutic effect was evaluated based on the cytotoxic activity of the complexes on human hepatoma cells (HepG2). HepG2 cell line passage number 80≈85 was separately cultured in 25 cm² tissue culture flasks using RPMI medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin. Afterwards, the flasks were incubated in CO₂ incubator at 37 °C until 90% confluence was reached. Then, the media were carefully removed and the flasks were washed with PBS once. Trypsin (0.25%) was added for 7 min and the flasks were checked constantly under an inverted microscope until all the cells were detached. Subsequently, the collected cells were washed with PBS, resuspended in culture media, and inoculated, at a density of 10⁴ cells/well, in 96-well plates which were incubated until sheets of cells were observed. Serial dilutions of drug complexes, in growth media, were added to cells at dilutions of 1000, 500, 250, 125, 62.5, and 31.2 µg/mL, followed by, incubation of the plates for 24 h. The malignant cell viability was assessed by crystal violet staining where crystal violet stain was utilized to stain DNA and to determine the density of well-adherent viable cells. Initially, the media were decanted, the plates were washed with PBS, the cells were stained with 20 µl of 0.5% crystal violet (Sigma-Aldrich Corp., USA), in 30% ethanol, and were left at room temperature for 10 min. Then, the plate wells were cautiously washed 3 times with distilled water. Lastly, the color absorbance values were obtained at an optical density (OD) of 490 nm (Śliwka et al., 2016).

2.5 Antimicrobial activity

The cup-plate agar diffusion approach was done to assess the antimicrobial effect of the different chemical complexes dissolved in DMSO (200 µg/100 µl) (Valgas et al., 2007). Two bacterial microbes; Staphylococcus aureus ATCC6538-P (Gram +ve) and Pseudomonas aeruginosa ATCC 27853 (Gram −ve); one yeast test microbe Candida albicans ATCC 10231, and one fungal test microbe Aspergillus niger NRRL A-326 were picked to determine the antimicrobial activities of the ligand and its complexes. The bacterial and the yeast microbes were grown on nutrient agar medium (g/L): agar (20), beef extract (3), and peptone (10). Whereas, the fungal test microbe was grown on Szapek-Dox agar medium (g/l): agar (20), Sucrose (30), NaNO₃ (3), MgSO₄·7H₂O (0.5), KCl (0.5), FeSO₄·7H₂O (0.055), and K₂HPO₄ (1). The culture media of each test microbe was diluted with pre-sterilized distilled water to 10⁷–10⁸ colony forming units (CFU)/mL. One mL of each was inoculated into 1 L-Erlenmeyer flask having 250 ml of solidified agar media (Youssef et al., 2014). 25 ml of these media were poured in sterilized Petri dishes (10 cm diameter). In each plate, 3–4 cups, measuring 10 mm in diameter were cut. The cups were then filled with 100 uL of samples which were allowed to diffuse, for 2 h, at 4 °C. Afterwards, the inoculated plates were incubated, for 24 h, at 37 °C (for bacteria) and for 48 h, at 30 °C (for fungus). Finally, the diameters of the microbial growth inhibition zones, resulting from the ligand and metal complexes, were measured.

3.1 Mass spectrum of the ligand, (1) and its complexes (2, 4)

The mass spectrum of the ligand revealed a molecular ion peak (m/z) at 404.43, which was coincident with its calculated molecular weight. Proposed mass fragmentation of ligand 1 was illustrated in Fig. 2.

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Fig. 2

Proposed mass fragmentation of ligand (1).

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The mass spectrum of Cu(II)-containing complex (2) indicated a molecular ion peak at 621.5 m/z, coincident with its molecular weight, and the fragmentation pattern split a parent ion peak at (m/z) = 79, 94, 158, 234, 276, 387.5, 523.5, 576.5, and 621.5 a.m.u. corresponding to C₄H₃N_2, C₄H₄N_3, C₄H₄ N₃SO_2, C₁₀H₈N₃SO_2, C₁₁H₁₀N₅SO_2, C₁₁H₁₂N₆SO₄Cu, C₁₅H₂₀N₆ SO₉Cu, C₁₉H₂₅N₆SO₉Cu, and C₂₁H₃₀N₆SO₁₀Cu moieties, respectively. The mass spectrum of Cu(II)-comprising complex (4) demonstrated a molecular ion peak at 592.5 a.m.u. coincident with its molecular weight and the fragmentation pattern split a parent ion peak (m/z) at 18, 97, 112, 176, 252, 294, 460.5, 494.5, 547.5, and 592.5 a.m.u. corresponding to H₂O, C₄H₅N₂O, C₄H₆N₃O, C₄H₆N₃SO_3, C₁₀H₁₀N₃SO_3, C₁₁H₁₂N₅SO_3, C₁₃H₁₄N₆SO₄Cl₂Cu, C₁₁H₁₆N₆SO₆Cl₂Cu, C₁₅H₂₁N₆SO₆Cl₂Cu, and C₁₇H₂₆N₆SO₇Cl₂Cu moieties, respectively (supporting information table 2).

3.2 ¹H – NMR spectra

The ¹H – NMR spectra of the ligand and its Zn(II) metal complex (11), in deuterated DMSO, identified signals consistent with the structure. The NMR showed the differences in the magnetic properties of the various nuclei present and deduced in a large measure where these atoms are within the molecule. The NMR studies also detected the change in the chemical shift of the complexes and the ligand as a result of complexation. The signals observed, at 4.15 and at 3.25 ppm, were owing to the protons of two NH₂ groups (Jeffrey et al., 2015). The proton of azomethine group appeared at 10.4 ppm. The peaks were identified as multiple ones at 6.1–7.5 ppm may be assigned to aromatic protons (Mayadevi et al., 2003). The protons in the ligand were shifted downfield because of the metal ions (Balgavý et al., 1980).

The NMR spectral data of Zn(II) complex (11) revealed changes in the chemical shift and intensity of the bands in relation to those of the free ligand which indicated the addition of the metal to the free ligand proving that the azomethine nitrogen atom coordinated to the metal ion (N - Zn) (Kavitha and Anantha Lakshmi, 2017). Upon complexation, the sulfa-H imino proton signal disappeared suggesting the covalent binding of sulfonamide nitrogen to the metal atom. The aromatic protons appeared in the 6.3–7.9 ppm range. A new signal was noticed at 1.87 ppm produced by the coordinated acetate group protons (Kavitha and Anantha Lakshmi, 2017).

3.3 Conductivity

The molar conductance measurements of the prepared compounds in DMF (1 × 10⁻³ M) presented in (Table 1) indicating that the complexes (2–7), (10), and (11) were not electrolytes. This confirmed that the anion was incorporated into the metal ion. On the other side, complexes (8) and (9) showed a different property as their conductivity values were 165 and 177 Ω mol⁻¹cm², respectively, which might be caused by the expanding ionization potential of the central metal ions that enhanced the lattice energy. The molar conductivity findings showed that the complexes were electrolytes in the solvent DMF and established the stability of theses complexes.

3.4 IR spectra

Careful investigation of the IR spectra of complexes and comparison with that of the ligand was carried out giving information to illustrate the manner of bonding of the ligand towards different metal ions. Thus a detailed characterization of the IR spectrum of the ligand and the effect of metal ion binding on the vibration frequencies was performed. The most fundamental IR spectral bands of all systems and their assignments are collected in (Table 2). The data elicit the following observations:

1. The enolization of sulfonamide moiety in sulfadiazine Schiff base ligand (1) predicated to S⚌O and NH groups, where one NH is responsible for enol – keto form and the other has the same nature as free ligand, as indicative of different mode of vibrations of NH group (ν, δ, γ) (Kavitha and Anantha Lakshmi, 2017).

2. All complexes showed the same modes of vibrations corresponding to NH and S⚌O groups, indicating that the ligand chelates with metal ions in neutral keto–enol–form. The (ν, δ, γ) of NH group showed that sulfonamide moiety doesn’t participate in metal complexation with different metal ions (Kavitha and Anantha Lakshmi, 2017).

3. All complexes display ν(C⚌N) and ν(C⚌O) bands that are shifted to higher wavenumbers than those of free ligand by 2–18 and 3–20 cm⁻¹, respectively, suggesting the participation of nitrogen atom of azomethene group in bonding and oxygen of carbonyl group. (Fouda et al., 2008; Youssef et al., 2010)

4. The ν(SO₂-NH) group and ν(NH₂) ligand bands showed weak effect for all complexes indicating that sulfonamide moiety atoms of sulfadiazine and nitrogen of amino group are not involved in bonding. (Kavitha and Anantha Lakshmi, 2017; Youssef et al., 2010)

5. The spectra of the complexes gave new bands, not observed in that of the ligand, at 560–520 and 470–430 cm⁻¹ assignable to ν(M—N) and ν(M—O). (Youssef et al., 2010; El-Tabl et al., 2015) The ν(C⚌O) predicated to C⚌O in all complexes are coordinated with different metal ions.

6. Additional to the above observation, the ν(NH₂) group band shows more or less shift around 3450 cm⁻¹ and a change in its shape. This is may be due to the formation of inter‐ or intramolecular hydrogen bonding. According to the above observations, the ligand behaves as a neutral bidentate ligand and binds with different metal ions through nitrogen and oxygen atoms of azomethene and carbonyl groups complexes, ν(C⚌N) and ν(C⚌O) bands are shifted to higher and lower wavenumber by 2–18 and 3–20 cm⁻¹ associated to emphasize the coordination of azo‐nitrogen atom and oxygen of carbonyl group with the metal ions.

7. The ligand behaves as a bidentate moiety and chelates metal ions through both carbonyl oxygen atoms of ligand and with azo nitrogen atom.

8. In complexes (2), (6), (7), and (11) the bands were produced by νa(COO) noticed in the 1530–1510 cm⁻¹ and the νs(COO) observed in the 1410–1360 cm⁻¹ ranges. The disparity between these two bands was in the 20–50 cm⁻¹ range signifying that the acetate group coordinated in a unidentate manner with the metal ions (Fouda et al., 2008). Complex (5) showed bands at (1385, 1285, 815, 765) cm⁻¹ which were assigned to coordinated nitrate group (Fouda et al., 2008).

9. Whereas, sulphate complexes (3) and (8–10) exhibited new bands at (1105, 1030, 805, 690, 485), (1108, 1030, 820, 640, 400), (1115, 1020, 805, 625, 420), and (1108, 1010, 802, 620, 410) cm⁻¹ which implied that the sulphate ion coordinated to the metal ion in a unidentate chelating way (El-Tabl et al., 2008; El-Tabl et al., 2013).

10. Complex (4) showed a band at 425 cm⁻¹ which was generated by the coordinated monodentate chloride anion ν(M—Cl) (El-Tabl et al., 2008).

3.5 Electronic transition and magnetic moments

The DMF electronic absorption spectra and the effective magnetic moment values of the ligand and of its metal complexes are reported in Table 4. The ligand displayed three transition bands in the high energy region. The first band which presented at 255 nm was appointed to π → π* transition within the aromatic rings and this band was nearly the same upon complexation. The second and third bands detected at 270 and 330 nm were due to n → π* of the azomethine groups and ν(SO₂ - N) moiety and CT transitions (Mohamed et al., 2005; Aslan et al., 2011). These bands were shifted upon complexation providing the involvement of these transitions in the coordination with the new metal ions. The electronic spectra of Cu(II) complexes (2–5) exhibited bands in the 452–630, 455–645, 460–655, and 445–648 nm ranges which were designated to ²B₁g→ ²A₁g (d_(x2−y2) → d_z2) and ²B₁g→²Eg (d_(x2−y2) → d_xy, d_yz) transitions, respectively which denoted that the Cu(II) ion had a tetragonally distorted octahedral geometry. This could be attributed to the Jahn-Teller effect operated on the d⁹ electronic ground state of six coordinate system, elongating one trans pair of coordinate bonds and shortening the remaining four ones (Mohamed et al., 2005; Aslan et al., 2011). The magnetic moments of all Cu(II) complexes (2–5) were in the 1.68–1.74B.M. range indicated that the complexes had square planar and octahedral geometry (Kumar et al., 2014). The spectra of Ni(II) complexes (7) and (10) showed three bands at (495, 620, 735), and at (496, 618, 736) nm, these bands were corresponding to ³A₂g (F) → ³T₂g (F)(1), ³A₂g (F) → ³T₁g(F)(2), and ³A₂g (F) → ³T₁g(P)(3) transitions, respectively, entailing octahedral Ni(II) complexes formation (Akbar et al., 2011). The lowest value of 2/1 ratio for the complexes was within 1.27–1.34 range which was below the usual range of 1.5–1.75 which was indicative of distorted octahedral Ni(II) complex (Akbar et al., 2011). The magnetic moment values of Ni(II) complexes (7) and (10) were 2.77 and 2.9B.M., respectively, in harmony with two unpaired electrons state and proving octahedral geometry of the Ni(II) ion (Akbar et al., 2011). The electronic spectra of the Co(II) complex (6) showed three d–d transition bands at (555, 620, 715 nm). These three bands were assigned to ⁴T₁g(F)→⁴T₂g(F)(1), ⁴T₁g(F) →⁴T₁g(p)(2), and ⁴T₁g(F) →⁴A₁(F)(3) transitions, respectively, consistent with high spin Co(II) octahedral complexes (Mohamed et al., 2005). The magnetic moment of complex (6) was 4.7B.M. which was within the described range of high spin octahedral Co(II) complex. Iron(III) complex (8) showed six bands at 265, 325, 385, 466, 592, and 640 nm, respectively. The initial three bands were within the ligand while the other bands were resulting from charge transfer and ⁶A₁→⁴T₁ transitions indicating distorted octahedral geometry around the iron(III) metal ion (Mangamamba et al., 2014). Chromium(III) complex (9) showed six bands at 285, 305, 380, 465, 562, and 634 nm, respectively. The first three bands were appointed to ⁴A₂g→⁴T₁g(F), ⁴A₂g→⁴T₂g, and ⁴A₂g→²T₂g transitions, respectively, demonstrating octahedral structure around the Cr(III) ion. Iron(III) complex (8) and chromium(III) complex (9) had magnetic moment values of 5.2 and 3.8B.M. suggestive of a high spin octahedral structure (Mangamamba et al., 2014). Zn(II)-based complex (11) gave bands of octahedral structures and diamagnetic which gave merely intraligand transitions (supporting information table 4).

3.6 Thermal studies [DTA and TGA]

As the IR spectral data indicated the existence of water molecules, thermal analyses (DTA and TGA) were done to assure their character which was listed in supporting information table 5. The thermal curves in 27–800 °C temperature range for complexes (2–11) revealed that these complexes were thermally stable up to 35 °C. Dehydration was illustrated by endothermic peaks within 70–90 °C temperature range, corresponding to loss of water molecules (Surati, 2011; El-Tabl et al., 2016). The decomposition mechanism of ligand 1 was illustrated in Fig. 3.

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Fig. 3

Thermal decomposition mechanism of the ligand 1.

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The decomposition step for complex (2) started with the break of H-bonding associated with an endothermic peak at 45 °C, followed by the loss of two coordinated water molecules with an endothermic peak at 120 °C and with 8.96% weight loss (Calc. 9.01%). A peak was observed at 170 °C with 20.84% weight loss (Calc. 20.78%). It was owing to two acetate groups. The endothermic peak found at 355 °C might be induced by the melting point. Oxidative thermal decomposition occurred in 465–650 °C temperature range with exothermic peaks leaving CuO 17.33% weight loss (Calc. 17.77%) (Kumar et al., 2014; El-Tabl et al., 2016). The decomposition step for complex (3) began with the disruption of H-bonding with endothermic peak at 45 °C, followed by the elimination of two water molecules at 83 °C with weight loss of about 8.65% (Calc. 5.57%), and then by the removal of two coordinated water molecules with a peak at 135 °C and 9.03% weight loss (Calc. 9.04%). Another thermal decomposition peaked at 215 °C with weight loss of approximately 17.55% (Calc. 17.66%) as a result of a coordinated sulphate group loss. An additional endothermic peak was noticed at 320 °C with no weight loss which could be caused by the melting point. Finally, the complex showed exothermic peaks in the 440–660 °C temperature range corresponding to oxidative thermal decomposition which proceeded gradually with a final residue at 660 °C which was allocated to CuO (Kumar et al., 2014; El-Tabl et al., 2016) (see Figs. 4 and 5).

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Fig. 4

Inverted microscopic examination of the human mononuclear cells from peripheral blood treated with a concentration of 62.5 µg/mL of the ligand and the complex compounds.

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Fig. 5

Crystal violet stain colorimetric assay at OD = 590 nm for the nine complexes in compare to control human hepatoma (HepG2) cell line.

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The decomposition step for complex (4) was initiated with H-bonding break associated with an endothermic peak identified at 48 °C, followed by the loss of a hydrated water molecule (H₂O) at 85 °C associated with 3.08% weight loss (Calc. 3.04%) and then by the deletion of two coordinated water molecules with a peak at 145 °C and 6.46% weight loss (Calc. 6.27%). Yet, the endothermic peak observed at 177 °C accompanied by 13.27% weight loss (Calc. 13.18%) was due to two chloride groups. A peak was detected at 345 °C resulting from the melting temperature point. Oxidative thermal decomposition occurred in 455–670 °C temperature range with exothermic peaks, leaving CuO with about 17.02% weight loss (Calc. 17.00%) (Surati, 2011; El-Tabl et al., 2016). The decomposition step for complex (5) began with the break of H-bonding with a peak at 45 °C, followed by the loss of 2 H₂O at 87 °C with weight loss of about 5.75% (Calc. 5.69%). Then, two coordinated water molecules were removed with an endothermic peak at 135 °C and 6.06% weight loss (Calc. 6.04%). A peak spotted at 165 °C, with weight loss of about 22.18% (Calc. 22.14%), was designated to two nitrate groups. The peak at 310 °C might be generated by the melting point. Oxidative thermal decomposition occurred in 440–680 °C temperature range with exothermic peaks, leaving CuO with about 18.42% weight loss (Calc. 18.32%) (Kumar et al., 2014; El-Tabl et al., 2016).

The decomposition step for complex (6) started with H-bonding break which was associated with an endothermic peak at 46 °C. This was followed by the removal of 2H₂O molecules at 87 °C with weight loss of about 5.57% (Calc. 5.51%). Then, two coordinated water molecules were eliminated with a peak at 137 °C and 5.86% weight loss (Calc. 5.84%). The peak which was observed at 186 °C with 20.35% weight loss (Calc. 20.30%) was attributed to two acetate groups. An endothermic peak was detected at 326 °C because of the melting point. Oxidative thermal decomposition occurred in the 460–680 °C temperature range with exothermic peaks, leaving CoO weight loss of about 16.13% (Calc. 16.18%) (Kumar et al., 2014; El-Tabl et al., 2016).

The decomposition step for complex (7) was initiated with H-bonding disruption with a peak at 46 °C, followed by the rejection of three H₂O at 89 °C with about 8.86% weight loss (Calc. 8.73%). Then, two coordinated water molecules were lost through a peak at 125 °C with weight loss of about 6.34% (Calc. 3.38%). Nevertheless, the peak observed at 180 °C associated with 22.03% weight loss (Calc. 22.32%) was caused by two acetate groups. The peak at 350 °C was possibly a melting point. Oxidative thermal decomposition occurred in the 465–660 °C temperature range with exothermic peaks, leaving NiO with weight loss of about 18.23% (Calc. 18.17%) (Kumar et al., 2014; El-Tabl et al., 2016).

The decomposition step for complex (8) was triggered with the break of H-bonding with a peak at 47 °C which was followed by the removal of 2H₂O at 86 °C with 5.77% weight loss (Calc. 5.57%). Then, three coordinated water molecules were eradicated with a peak at 120 °C and 8.55% weight loss (Calc. 8.86%). A peak at 185 °C associated with 17.03% weight loss (Calc. 17.27%) was owing to sulphate group. The endothermic peak at 359 °C was a melting temperature point. Oxidative thermal decomposition occurred in the 460–650 °C temperature range associated with exothermic peaks, leaving Fe₂O₃.

The decomposition step for complex (9) began with H-bonding split accompanied by an endothermic peak at 45 °C with the elimination of 3H₂O at 84 °C and with 8.06% weight loss (Calc. 8.18%). Three coordinated water molecules were eliminated through a peak at 130 °C with weight loss of about 8.76% (Calc. 8.91%). The peak noticed at 180 °C with 17.22% weight loss (Calc. 17.39%) was attributed to one sulphate group. The peak at 350 °C was caused by the melting point. Oxidative thermal decomposition occurred in the 450–670 °C temperature range concomitant with exothermic peaks leaving Cr₂O₃.

The decomposition step for complex (10) was initiated with H-bonding disruption with a peak at 44 °C. Then, 2H₂O were lost at 87 °C with weight loss of about5.34% (Calc. 5.55%). Afterwards, three coordinated water molecules were removed through a peak at 129 °C with 8.45% weight loss (Calc. 8.81%). On the other hand, the peak spotted at 184 °C with 17.23% weight loss (Calc. 17.24%) was resulting from one sulphate group. The endothermic peak appeared at 352 °C was a melting temperature point. Oxidative thermal decomposition occurred in the 440–660 °C temperature range accompanied by exothermic peaks leaving NiO.

The decomposition step for complex (11) started with H-bonding collapse with an endothermic peak at 48 °C before the loss one H₂O at 80 °C with 2.87% weight loss (Calc. 2.73%). Later, two coordinated water molecules were removed at 137 °C with 5.46% weight loss (Calc. 5.61%). A peak detected at 175 °C with weight loss of about 19.23% (Calc. 19.49%) was caused by two acetate groups. The melting point triggered a peak at 335 °C. Oxidative thermal decomposition occurred in the 440–660 °C temperature range with exothermic peaks leaving NiO 16.56% weight loss (Calc. 16.69%).

3.7 Electron spin resonance (ESR)

The ESR spectral data for metal complexes (2), (4), and (9) are stated in (supporting information table 6). The spectra of Cu(II) metal complexes (2) and (4) were representative of species d9 conformation having an axial type of a d_(x2-y2) ground state which is the most frequent type for Cu(II) complexes (Akbar et al., 2011). The metal-based compounds had g|| > g_⊥ > 2.0023 demonstrating an octahedral geometry around the Cu(II) metal ion (El-Tabl et al., 2011). G-values = (g||-2)/(g_⊥-2). If G is>4.0, local tetrahedral axes are misaligned parallel or only slightly misaligned and if G is below 4.0, this means that there is a significant exchange coupling (Thomas and Mohanan, 2017). The g||/A|| values of the new complexes lied within the range expected for the square planar or octahedral metal complexes. The orbital reduction factors (K||, K_⊥, K), which are measures of covalency, were also calculated. Concerning the Cu(II) complexes (2) and (4), the K values signified a covalent bond character (El-Razek et al., 2020). Also, the g-values confirmed the covalent bond character. The in-plane σ- covalency parameter, α2 (Cu) was suggestive of a covalent bonding. The complexes (2) and (4) displayed β₁ values i.e. a covalence character in the in-plane π- bonding. While β² for the complexes denoted an ionic bonding character in the out of plane π- bonding. However, complex (4) had an ionic bond character out of the plane bonding (Thomas and Mohanan, 2017; El-Razek et al., 2020). The Cu(II) complexes calculated orbital populations (a²d) implied a d(x2-y2) ground state. Cr(III) complex (9) showed isotropic spectra with 2.09 value.

3.8 Chemotherapeutic effect

To assess the cytotoxicity and the anticancer effectiveness of the new complexes, the IC50 values (the concentration that decreases the cell viability to 50% of the cell control viability) were determined. These values were calculated utilizing human mononuclear cells incubated with the concentrations of 1000, 500, 250, 125, 62.5, and 31.2 µg/mL of the ligand and compounds for 36 h and visually, the viable cells were counted under the inverted microscope (Fig. 2).

The complexes 1, 3, and 5 showed rare cytotoxic activities while complexes 2, 4, 6, 7, 8, and 9 were weakly cytotoxic (Table 7) (see Tables 8 and 9).

In vitro growth inhibition assays were done on human cancer cell line (HepG2) at various concentrations in comparison to the non-treated HepG2 control. A highly significant anticancer effect, against HepG2 cells, after 24 h exposure, was specified by the crystal violet stain colorimetric assay. The anticancer effects of the ligand and the new metal-containing compounds were directly proportional to their concentration. The Cr(III)-, Fe(III)-, and Cu(II)-based complexes, in order, had the highest anticancer activities while Ni(II) had the lowest activity in comparison to the ligand (Fig. 3). These results were in agreement with Alturiqi et al. (2018) who found that Cr(III) complex exhibited higher in vitro cytotoxicity against both the selected cancer and normal cell lines when compared to the ligand while the Ni(II) and Cu(II) complexes displayed lower anticancer activities than that of the ligand.

3.9 Antimicrobial activity

The antimicrobial activities of the ligand (1) and of its metal complexes were evaluated in vitro by investigating their antibacterial and antifungal effects. The results were compared with Tetracycline which is the standard medication. S. aureus, P. aeruginosa, C. albicans, and A. niger were utilized as bacterial, yeast, and fungal test organisms. The ligand and its metal complexes (2–9) displayed antibacterial and antifungal effects against these life forms (supporting information Table 7). It was detected that the metal-based complexes had a more noteworthy effect than the ligand on the test organisms. Ligand (1) showed direct antibacterial and antifungal activities in comparison to the standard antibacterial (Neomycin) and antifungal (cycloheximide) drugs with clear zones values of 14, 14, 16, and 16 mm for S. aureus, p. aeruginosa, C. albicans and A. niger, respectively. Complexes (2), (5), (4), (3), and (7) were more hostile to G +ve bacteria than ligand (1) with clear zone values of 22, 18, 19, and 19 mm, respectively. Besides, complexes (2), (3), (6), (8) and (9) had higher antibacterial effects on G-ve bacteria than the ligand with clear zone values of 18, 15, 25, 30, 20, and 23 mm, respectively. Moreover, complexes (2), (4), (3), (6), (7), (8), and (9) revealed antifungal activities against the yeast C. albicans which were more elevated than the ligand with values 21, 24, 21, 19, 25, 24, and 26 mm, respectively. Concerning the antifungal properties of both ligand and complexes against A. niger, complexes (6) and (7) exceeded the ligand antifungal activity with clear zone values of 29 and 30 mm, respectively.