# Study of Synthesis, Characterization, DFT, and In Vitro Biological Activity of Cu(II), Co(II), and Fe(II) Metal Complexes Based on Heterocyclic Azo Pyrazole Dye Ligand

Authors: Albandari Alkurdi; Mutlaq AlJahdali; Abdulmohsen Alshehri
Journal: Journal of Chemistry
 (2022)

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## Abstract

A new azo dye ligand was synthesized by coupling 5-amino-3-methyl-1-phenylpyrazole with a diazonium ion of 2-amino-1-naphthalene sulfonic acid. Its Co(II), Fe(II), and Cu(II) complexes were also synthesized. This ligand and its complexes were characterized based on elemental analysis, IR,1H NMR, UV-vis spectra, and thermogravimetric analysis. The data showed that the proposed complexes have an octahedral geometry around the central metal ion. The nonelectrolytic nature of the complexes was confirmed by molar conductance measurement. X-ray powder diffraction indicates that Co(II) and Fe(II) complexes are amorphous in nature, whereas the Cu(II) complex has crystalline and amorphous phases. The bond strength and molecular stability between the interaction of metal ions and the ligand were investigated by density functional theory (DFT). The in vitro antibacterial activity of the ligand and complexes was tested against two Gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus) and two Gram-negative bacteria (Escherichia coli and Neisseria gonorrhoeae) and a fungus (Candida albicans). The Cu(II) complex exhibited a high antimicrobial effect against all tested microorganisms. The DNA fragmentation percentage and cytotoxic activity against human liver cancer cells (HepG2) were evaluated. All tested compounds showed potential anticancer activity, and the Co(II) complex showed better activity results than the other tested compounds, with IC50 = 19.55 μg/mL.


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## Body

## 1. Introduction

The synthesis of a new ligand with distinctive characteristics and novel reactivity is possibly the most significant step in the evolution of metal complexes. The heterocyclic ligand that contains the pyrazole ring system plays an important role in various pharmacological and medicinal compound syntheses [1]. Furthermore, the survey of the literature revealed that organic acids are a major class of organic molecules with a general structure R-COOH (including fatty acids and amino acids), such as carboxylic acids, amino acids, and naphthoic acids. These organic acids play an important role due to their biological activities and therapeutic applications [2, 3].Azo dyes are the largest and most famous coordination compounds, exhibiting biological activities against bacteria and fungi [4]. Azo dyes are organic compounds bearing the functional group R-N = N-R’, in which R and R’ are usually aryls [5]. An azo coupling reaction is an organic interaction in which a diazonium salt and a coupling agent are combined using a metal catalyst to produce aromatic azo compounds [6]. This reaction is commonly used in the industrial production of dyes and lake pigments. In the coupling reaction, diazonium ions act as electrophiles with activated aromatics, such as aniline or phenols. The substitution reaction usually occurs at the para position; however, if the para position is occupied, the ortho position is preferred. Moreover, the coupling reaction must occur under slightly acidic or neutral conditions, since the reaction does not occur at a considerably low pH [7].In the present study, a new bi-heterocyclic azo dye (AMePhPNA) was synthesized using 5- amino-3-methyl-1-phenylpyrazole as the diazo component and 2-naphthoic acid as the coupling component, due to their low cost, brilliant color, good stability, easy preparation, and pharmacological and industrial importance [8]. The coordination behavior of the azo dye-based ligand toward Cu(II), Co(II), and Fe(II) ions was investigated, and the results were obtained by physical properties, such as thermal analyses, infrared (FT-IR), UV-visible spectroscopy, X-ray powder diffraction (XRD), and DFT calculations. Moreover, the compounds were tested for their in vitro antimicrobial and antioxidant activities, and DNA fragmentation was identified in liver cancer cells (HepG2).


## 2. Experiment

### 2.1. Materials and Methods

All chemicals, reagents, solvents, and metal salts used were of analytical grade.The percentage compositions of C, H, N, and M were determined using a Leco CHNS-932 analyzer. FT-IR spectra (KBr) were recorded at 4000–400 cm−1 on a Bruker Alpha FT-IR spectrophotometer. 1H NMR spectra were recorded on a JEOL instrument using 500 MHz, DMSO-d6 at 25°C. UV-vis spectra were scanned at 200–800 nm on a Shimadzu 1600 spectrometer using DEMSO as the solvent. Melting points of the compounds were determined on an electrothermal apparatus using open capillary tubes and reported without correction. Magnetic moments were recorded by Gouy’s method at room temperature using a Johnson Matty magnetic susceptibility. Differential thermal analysis (DTA)/thermogravimetric analysis (TGA) of the metal complexes was performed in a nitrogen atmosphere from room temperature (25°C) to 800°C using a Shimadzu TG-50 H thermal analyzer. The morphology of the metal complexes was determined by scanning electron microscopy (SEM) images obtained using a JEOL JSM-5410 microscope. X-ray powder diffraction patterns were recorded using a PW3050/60 X-ray diffractometer with CuKα radiation (λ = 1.541 nm) in the 2θ range of 10°–80°.


### 2.2. Synthesis of the Ligand

The 1-((5-amino-3-methyl-1-phenyl-1H-pyrazol-4-yl) diazenyl)-2-naphthoic acid azo dye ligand (HL) was synthesized by applying the diazo coupling reaction [9]. A diazonium solution was prepared by dissolving 5-amino-3-methyl-1-phenylpyrazole (1.73 g, 0.01 mol) in a 50 mL mixture solution (4 mL HCl distilled water/4 mL HCl). The resultant mixture was cooled down to 0–5°C. A solution of NaNO2 (0.69 g, 0.01 mol) in 30 mL of H2O was added to this mixture dropwise at 0–5°C and was continuously stirred for 25 min to form the diazo compound (solution A), as shown in (Scheme 1). The aqueous solution B of 2-amino-1-naphthalenesulfonic acid (2.23 g, 0.01 mol) was dissolved in 50 mL of ethanol and strong alkaline sodium hydroxide (0.84 g, 0.01 mol) at 0–5°C. Solution B was added dropwise with contentious stirring of solution A for 30 min at 0–5°C in an ice bath. It was acidified with dilute HCl at pH 6.0. The brown precipitate of the HL ligand was filtered off and washed several times with distilled water and ethanol. The crystals were dried for 24 h at 25°C.Scheme 1
Synthesis of heterocyclic azo dye ligand (AMePhPNA).


### 2.3. Synthesis of Metal Complexes

The transition metal complexes were synthesized by dissolving 0.001 mol of metal chloride (CuCl2.2H2O [0.170 g], CoCl2.6H2O [0.129 g], and FeCl2·4H2O [0.198 g]) in 45 mL of hot phosphate buffer solution at pH 6.0. The solution was then added dropwise to a hot ethanolic solution (45 mL) containing 1 mM of the prepared ligand HL. The resulting solution was heated to 70°C for 30 min. The precipitate was filtered off and washed with distilled water and warm ethanol. The obtained products were desiccated in a vacuum over CaCl2 (anhydrous) [10].


## 2.1. Materials and Methods

All chemicals, reagents, solvents, and metal salts used were of analytical grade.The percentage compositions of C, H, N, and M were determined using a Leco CHNS-932 analyzer. FT-IR spectra (KBr) were recorded at 4000–400 cm−1 on a Bruker Alpha FT-IR spectrophotometer. 1H NMR spectra were recorded on a JEOL instrument using 500 MHz, DMSO-d6 at 25°C. UV-vis spectra were scanned at 200–800 nm on a Shimadzu 1600 spectrometer using DEMSO as the solvent. Melting points of the compounds were determined on an electrothermal apparatus using open capillary tubes and reported without correction. Magnetic moments were recorded by Gouy’s method at room temperature using a Johnson Matty magnetic susceptibility. Differential thermal analysis (DTA)/thermogravimetric analysis (TGA) of the metal complexes was performed in a nitrogen atmosphere from room temperature (25°C) to 800°C using a Shimadzu TG-50 H thermal analyzer. The morphology of the metal complexes was determined by scanning electron microscopy (SEM) images obtained using a JEOL JSM-5410 microscope. X-ray powder diffraction patterns were recorded using a PW3050/60 X-ray diffractometer with CuKα radiation (λ = 1.541 nm) in the 2θ range of 10°–80°.


## 2.2. Synthesis of the Ligand

The 1-((5-amino-3-methyl-1-phenyl-1H-pyrazol-4-yl) diazenyl)-2-naphthoic acid azo dye ligand (HL) was synthesized by applying the diazo coupling reaction [9]. A diazonium solution was prepared by dissolving 5-amino-3-methyl-1-phenylpyrazole (1.73 g, 0.01 mol) in a 50 mL mixture solution (4 mL HCl distilled water/4 mL HCl). The resultant mixture was cooled down to 0–5°C. A solution of NaNO2 (0.69 g, 0.01 mol) in 30 mL of H2O was added to this mixture dropwise at 0–5°C and was continuously stirred for 25 min to form the diazo compound (solution A), as shown in (Scheme 1). The aqueous solution B of 2-amino-1-naphthalenesulfonic acid (2.23 g, 0.01 mol) was dissolved in 50 mL of ethanol and strong alkaline sodium hydroxide (0.84 g, 0.01 mol) at 0–5°C. Solution B was added dropwise with contentious stirring of solution A for 30 min at 0–5°C in an ice bath. It was acidified with dilute HCl at pH 6.0. The brown precipitate of the HL ligand was filtered off and washed several times with distilled water and ethanol. The crystals were dried for 24 h at 25°C.Scheme 1
Synthesis of heterocyclic azo dye ligand (AMePhPNA).


## 2.3. Synthesis of Metal Complexes

The transition metal complexes were synthesized by dissolving 0.001 mol of metal chloride (CuCl2.2H2O [0.170 g], CoCl2.6H2O [0.129 g], and FeCl2·4H2O [0.198 g]) in 45 mL of hot phosphate buffer solution at pH 6.0. The solution was then added dropwise to a hot ethanolic solution (45 mL) containing 1 mM of the prepared ligand HL. The resulting solution was heated to 70°C for 30 min. The precipitate was filtered off and washed with distilled water and warm ethanol. The obtained products were desiccated in a vacuum over CaCl2 (anhydrous) [10].


## 3. Biological Studies

### 3.1. Antimicrobial Test

The compounds were screened in vitro for antimicrobial properties. The agar plates with bacteria were incubated at 35–37°C for 24–48 h, whereas the agar plates with fungi were incubated at 25°C for 48 h [11]. Standard disks of ampicillin (antibacterial agent) and amphotericin (antifungal agent) served as positive controls for antimicrobial activity; however, filter disks impregnated with 10 μL of DMS as negative controls. In brief, 100 μL of the test bacteria/fungi were grown in 10 mL of fresh media until they reached a count of approximately 108 cells/mL for bacteria and 105 cells/mL for fungi. Approximately 100 μL of microbial suspension was spread on agar plates corresponding to the broth in which they were maintained [12, 13].


### 3.2. Cell Viability by MTT Assay

The cytotoxic evaluation of the azo ligand and its complexes was conducted using the MTT assay [14]. Approximately 1 × 105 cells/mL HepG2 (100 μL/well) were plated in a 96-well plate and incubated at 37°C for 24 h to develop a complete monolayer sheet. The media were aspirated, and the cells were washed with phosphate buffer saline (5 mg/mL). The plates were then kept in 5% CO2 at 37°C for 1–5 h. After incubation, the culture medium was removed, and 20 μL of MTT solution was added to each well. DMSO (200 μL) was then added, and the plates were incubated for 5 min. Cell viability was measured using spectrophotometry at a wavelength of 560 nm and a subtracted background of 620 nm. HepG2 cells were treated with different concentrations of compounds (3.125, 6.25, 12.5, 25, 50, and 100 μg/mL) for 24 h [15].


### 3.3. DNA Fragmentation Assay

The percentage of DNA fragmentation was estimated using the diphenylamine (DPA) assay [16]. HepG2 cells (5 × 105 cells/mL) were cultured and inserted in labeled tubes in a complete RPMI medium, which was centrifuged at 20×g at 4°C for 10 min. After centrifugation at 200×g, the pellet was resuspended in 1 mL of TTE solution (TE buffer at pH 7.4 with 0.2% Triton X-100 stored at 4°C), and 1 mL of 25% trichloroacetic acid (TCA) was transferred in new conical tubes, followed by incubation overnight at 4°C. The DNA was then hydrolyzed by adding 160 mL of 5% TCA to each tube and heating at 90°C for 15 min. Approximately 320 mL of fresh DPA solution was added to each tube, which was then vortexed. The color was allowed to develop by heating at 37°C for 4 h or overnight at room temperature. Two 200 mL aliquots of the colored solution were transferred from each tube to a well of a 96-well microtiter plate. The optical density was determined at 600 nm; however, wavelengths from 560 nm to 620 nm were also considered. The percentage of fragmented DNA was calculated as follows:(1)%fragmentedDNA=S+TS+T+B×100,where S, T, and B are the optical densities (OD 600) of fragmented DNA in the S, T, and B fractions, respectively [17].


## 3.1. Antimicrobial Test

The compounds were screened in vitro for antimicrobial properties. The agar plates with bacteria were incubated at 35–37°C for 24–48 h, whereas the agar plates with fungi were incubated at 25°C for 48 h [11]. Standard disks of ampicillin (antibacterial agent) and amphotericin (antifungal agent) served as positive controls for antimicrobial activity; however, filter disks impregnated with 10 μL of DMS as negative controls. In brief, 100 μL of the test bacteria/fungi were grown in 10 mL of fresh media until they reached a count of approximately 108 cells/mL for bacteria and 105 cells/mL for fungi. Approximately 100 μL of microbial suspension was spread on agar plates corresponding to the broth in which they were maintained [12, 13].


## 3.2. Cell Viability by MTT Assay

The cytotoxic evaluation of the azo ligand and its complexes was conducted using the MTT assay [14]. Approximately 1 × 105 cells/mL HepG2 (100 μL/well) were plated in a 96-well plate and incubated at 37°C for 24 h to develop a complete monolayer sheet. The media were aspirated, and the cells were washed with phosphate buffer saline (5 mg/mL). The plates were then kept in 5% CO2 at 37°C for 1–5 h. After incubation, the culture medium was removed, and 20 μL of MTT solution was added to each well. DMSO (200 μL) was then added, and the plates were incubated for 5 min. Cell viability was measured using spectrophotometry at a wavelength of 560 nm and a subtracted background of 620 nm. HepG2 cells were treated with different concentrations of compounds (3.125, 6.25, 12.5, 25, 50, and 100 μg/mL) for 24 h [15].


## 3.3. DNA Fragmentation Assay

The percentage of DNA fragmentation was estimated using the diphenylamine (DPA) assay [16]. HepG2 cells (5 × 105 cells/mL) were cultured and inserted in labeled tubes in a complete RPMI medium, which was centrifuged at 20×g at 4°C for 10 min. After centrifugation at 200×g, the pellet was resuspended in 1 mL of TTE solution (TE buffer at pH 7.4 with 0.2% Triton X-100 stored at 4°C), and 1 mL of 25% trichloroacetic acid (TCA) was transferred in new conical tubes, followed by incubation overnight at 4°C. The DNA was then hydrolyzed by adding 160 mL of 5% TCA to each tube and heating at 90°C for 15 min. Approximately 320 mL of fresh DPA solution was added to each tube, which was then vortexed. The color was allowed to develop by heating at 37°C for 4 h or overnight at room temperature. Two 200 mL aliquots of the colored solution were transferred from each tube to a well of a 96-well microtiter plate. The optical density was determined at 600 nm; however, wavelengths from 560 nm to 620 nm were also considered. The percentage of fragmented DNA was calculated as follows:(1)%fragmentedDNA=S+TS+T+B×100,where S, T, and B are the optical densities (OD 600) of fragmented DNA in the S, T, and B fractions, respectively [17].


## 4. Results and Discussion

### 4.1. Elemental Analysis and Molar Conductivity Studies

The ligand (AMePhPNA) was prepared by the diazotization coupling reaction of 5-amino-3-methyl-1-phenylpyrazole and 2-amino-1-naphthalenesulfonic acid in a 1 : 1 molar ratio. The results of the elemental analysis (C, H, N, and M) are in a good agreement with those required by the proposed formula, which are apparent in Table1. The prepared NNO doner azo dye ligand is stable at room temperature, brown in color, and soluble in dimethyl formamide and DMSO. The molar conductance values of the complexes in DEMSO are in the range values (ΛM = 24–41 ohm−1cm2mol−1), indicating that the complexes are nonelectrolytic in nature [18]. The proposed structure of the Cu(II), Co(II), and Fe(II) complexes is shown in Figure 1 [19].Table 1
Physical properties and elemental analytical data of the azo dye ligand and its metal complexes.
Elemental analysis (%) (found) calcdComp.Molecular formulaColor M.wt. (g/mol)m.p. °CC%H%N%M%Λm (Ω−1cm2mol−1)μeff (B.M)(HL)[C21H17N5O2]Brown (371)25667.91 (67.87)4.61 (4.60)18.86 (18.82)—24—Cu(II)L[C21H19Cl2CuN5O3].H2OBrown (523)>30048.15 (48.13)3.66 (3.71)13.37 (13.34)12.13 (11.84)301.76Co(II)L[C21H19Cl2CoN5O3].H2OBlue (523)>30048.58 (49.05)3.69 (3.43)13.49 (13.23)11.35 (10.68)414.68Fe(II)L[C21H19Cl2FeN5O3].H2OPale- yellow (516)>30048.87 (48.84)3.71 (3.23)13.57 (13.14)10.82 (10.49)285.15Figure 1
The proposed structure of the metal complexes (M = Cu(II), Co(II), and Fe(II)).


### 4.2. Electronic Spectra and Magnetic Properties

The electronic absorption spectra of the prepared azo dye and its complexes were conducted in DMSO, and the data are represented in Figure2. The diffuse reflectance spectrum of the free ligand displays two main peaks at 42.194 cm−1, and 40.816 cm−1 which are attributed to the (π–π∗) electronic transition of the conjugated (C=C) in the aromatic rings and (C=O) of carboxylic acid group. Another band was observed at 29.240 cm−1, which is attributed to the (π–π∗) electronic transition of the (N=N) azo group. These bands are shifted to high or low wavelengths due to the coordination of the ligand with the metal ions.Figure 2
UV-vis absorption spectra of the azo dye ligand (HL) and its Cu (II), Co(II), and Fe(II) metal complexes in DMSO.The Cu(II) complex presented an absorption band in the UV range 43478–41494 cm−1, which was assigned to ligand to metal charge transfer transition (LMCT). The octahedral geometry of the Cu(II) ion is supported by the observed value of the magnetic moment (1.76 BM) [20]. The magnetic moment value of Co(II) was 4.68 BM, suggesting an octahedral environment of the Co(II) ion in the complex. The electronic spectrum of the complex showed one broad band at 19.569 cm−1 which can be assigned to d-d transitions [21]. The electronic spectrum of the Fe(II) complex shows an absorption band at 32.787 cm−1 which is caused by π⟶π∗ intraligand transition. Although the spectrum displays another two weak bands at 31.847 and 20.161 cm−1 attributed to the electronic transition 5Eg ⟶ 5T2g. The magnetic moment value of 5.15 BM was an additional evidence for an octahedral structure around the metal center [22].


### 4.3. IR Spectral Studies and the Mode of Bonding

The infrared spectra of the free azo dye ligand were compared with the metal complexes to identify the atoms that participate in the complexation (Table2; Figure 3). The IR spectrum of the free ligand shows two stretching vibration bands at 3,430–3,305 cm−1 due to γasym (NH2) and γsym (NH2), and the γ (CH3) stretching vibrations in the region of 2,848–3,118 cm−1 both appear as broad peaks of the O-H stretching band that overlap this region [23]. In addition, the strong vibration bands between 1691 and 1656 cm−1 are attributed to the stretching vibrations γ (C=O) and γ (C=C), respectively [24]. The vibration of the –N=N– azo group is found at 1,473 cm−1 [25, 26]. The IR spectra of all metal complexes display medium broad bands approximately at the 2,476–3,655 cm−1 regions, confirming the presence of a coordinated water molecule in the structure of the metal complex. After coordination between the ligand and its Co(II), Co(II), and Fe(II) metal ions, the γ (C=O) was shifted to a low frequency and was observed at 1,622, 1,627, and 1,615 cm−1, respectively, indicating that the coordination occurred through the oxygen of carbonyl [27]. Moreover, the γ (N=N) and γ (NH2) stretching bands are shifted toward a lower frequency compared with the free ligand, supporting the involvement of one of the azo nitrogen atoms and amino groups in coordination with metal ions. New weak bands were found; these bands have never been observed in the free HL spectrum, which corresponds to γ (M-O), γ (M-N), and γ (M-Cl) stretching [28]. In addition, the IR spectral studies concluded that the HL ligand acts as an NNO-tridentate ligand with three binding sites via the nitrogen atom of the amino group γ (N-H2), the oxygen atom of the carboxylic group γ (OH), and the nitrogen atom of the azo group γ (N=N).Table 2
Important FT-IR spectral bands (cm−1) of the HL ligand and its complexes.
Compoundγ (N-H)γ (C=O)γ (C=C)γ (C=N)γ (N=N)γ (C-OH)γ (M-N)γ (M-O)γ (M-Cl)[HL]3305–343416911656155114731235———[Cu(II)L]3232–334016221591150313861286551619502[Co(II)L)]3244–339216271595150113821289561598439[Fe(II)L]3094–367516151563147414091241543595452Figure 3
IR spectra at the range 400–4000 cm−1 for the (HL) ligand and its metal complexes. (a) HL. (b) Fe(II)L. (c) Cu(II)L. (d) Co(II)L.


### 4.4.1H NMR Spectra

The proton NMR spectra of the synthesized azo dye ligand and its Cu(II) complex were recorded in DMSO-d6 solution as a solvent at room temperature (Figure 4). The three protons signal of the (CH3) group in the (HL) free ligand appears at 2.0 ppm. Also, the spectrum showed multiple peaks in the range of 7.4–7.9 ppm due to an aromatic ring proton of the ligand. The signal at δ 8.5 ppm may be assigned to the O-H proton in the carboxylic group.Figure 4
1H-NMR chemical shifts of compounds (ppm, 500 MHz, DMSO-d6 solutions, room temperature) for (a) HL ligand and (b) Cu(II)L.
(a)(b)In the1H NMR spectrum of the Cu(II) complex, the aromatic protons resonated moved to upfield (∼0.2 ppm) due to coordination. While the signal of OH groups shifted downfield (∼2.0 ppm), which indicates the bonding of OH group to metal ion. However, the signals of NH2 are not observed due to the interaction with the solvent DMSO-d6 [29].


### 4.5. Thermal Studies

The TGA and DTA studies of the complexes were performed at 0–800°C. The thermal decomposition data of the metal complexes are displayed in Table3 and Figure 5. The results are in good agreement with the theoretical formula suggested by the elemental analyses.Table 3
Thermogravimetric analysis (TGA/DTA) data of the Cu(II), Co(II), and Fe(II) complexes.
CompoundStepTemp. range ˚CDTAMax°CTotal weight loss estimated (calcd.), %Decomposition assignmentResidualCu(II)L1st64–16880.412.1 (12.0)Loss of 3½ H2O (lattice)CuO2nd173–62452087.0 (87.8)Loss of 2Cl + 1H2O (coord.water) + C21H17N5O2 ligandCo(II)L1st40–66940092.6 (92.1)Loss of 1H2O (lattice) + 1H2O (coord.water) + 2Cl + C21H17N5O2 ligand¼ Co2O2Fe(II)L1st51–19399.910.3 (10.4)Loss of 2H2O (lattice) + 1H2O (coord.water)FeO2nd194–649524.485.7 (85.6)Loss of 2Cl + C21H17N5O2 ligandFigure 5
Thermal analysis (TGA/DTA) patterns for the (a) Cu(II), (b) Co(II), and (c) Fe(II) complexes.
(a)(b)(c)The TG/DTG curves of the Cu(II) complex were decomposed in two steps. The first step occurred within the temperature range of 64–168°C, which is attributed to the 3½ H2O lattice water loss. The second step occurred at the temperature range of 173–624°C, with the maximum temperature at 520°C due to 87.0% (calcd. 87.8%) weight loss of two moles of chlorine atoms, one molecule of coordinated water, and the C21H17N5O2 ligand, leaving CuO as the residue. The thermal decomposition of Co(II)L showed the complete decomposition of the organic ligand in one step, two chlorine atoms, 1 H2O (lattice), and coordinated H2O at the temperature range of 40–669°C, with a total 92.6% weight loss (calcd. 92.1%). The thermogram of the Fe(II) complex, the initial weight loss of 10.3% (calcd. 10.4%) occurred at the temperature range of 51–193°C, with DTAmax at 99.9°C, due to the loss of 2H2O lattice water molecules and a mole of coordinated water. The second step occurred within a temperature range of 194–649°C, which signifies 85.7% (calcd. 86.0%) loss of 2Cl atoms and ligand molecules, leaving iron oxide as a residue.


### 4.6. Morphological Behavior

The morphological behaviors of the metal complexes of the azo dye were characterized by XRD measurements and SEM. The X-ray patterns for all samples were recorded between 2θ, ranging from 10° to 80° using the CuKα radiation (λ = 1.540 Å). Interplanar spacing or d-spacing was calculated using Bragg’s law nλ = 2dsinѲ, where Θ is the diffraction angle [30]. The lattice parameters were performed using the Bruker EVA software (Bruker package software). The powder diffraction data 2Θ values are shown in Figure 6. The crystallite size (ξ) of the Cu(II) complex was estimated using the Scherer method, as shown in the following equation [31]:(2)ξ=Kλβcosθ.Figure 6
X-ray diffraction patterns of (a) Cu(II), (b) Co(II), and (c) Fe(II) complexes.The equation uses the referenceK, which is the Scherer constant (0.95), β is the full-width peak at half-maximum, and θ is the Bragg diffraction angle. The XRD reflections of the Cu(II) complex show a mixture of crystalline and amorphous phases, as it lacks sharp peaks at maxima 2Ѳ = 29.88°. The CuO phase was a monoclinic system at a room temperature, with lattice parameters a = 4.67 Å, b = 3.45 Å, c = 5.12 Å, α = 90°, β = 98°, γ = 90°, and volume = 81.94 Å3. The value of δ was calculated and was 76 nm. By contrast, crystalline peaks were not observed in the Co(II) and Fe(II) complexes, indicating the amorphous nature of the tested complexes, which could reflect the immeasurably minute sizes of the aggregated particles within the nanosize range.The surface morphology of the Co(II) complex was investigated using a scanning electron microscope. The SEM images were recorded at different magnifications at an acceleration voltage of 20 kV. The images show that these particles have diameters of 3–20μm. However, the structural morphology consists of a foam-like shape, as shown in Figure 7.Figure 7
Scanning electron micrograph images of the (a) Cu(II)L and (b) Co(II)L complexes.
(a)(b)


## 4.1. Elemental Analysis and Molar Conductivity Studies

The ligand (AMePhPNA) was prepared by the diazotization coupling reaction of 5-amino-3-methyl-1-phenylpyrazole and 2-amino-1-naphthalenesulfonic acid in a 1 : 1 molar ratio. The results of the elemental analysis (C, H, N, and M) are in a good agreement with those required by the proposed formula, which are apparent in Table1. The prepared NNO doner azo dye ligand is stable at room temperature, brown in color, and soluble in dimethyl formamide and DMSO. The molar conductance values of the complexes in DEMSO are in the range values (ΛM = 24–41 ohm−1cm2mol−1), indicating that the complexes are nonelectrolytic in nature [18]. The proposed structure of the Cu(II), Co(II), and Fe(II) complexes is shown in Figure 1 [19].Table 1
Physical properties and elemental analytical data of the azo dye ligand and its metal complexes.
Elemental analysis (%) (found) calcdComp.Molecular formulaColor M.wt. (g/mol)m.p. °CC%H%N%M%Λm (Ω−1cm2mol−1)μeff (B.M)(HL)[C21H17N5O2]Brown (371)25667.91 (67.87)4.61 (4.60)18.86 (18.82)—24—Cu(II)L[C21H19Cl2CuN5O3].H2OBrown (523)>30048.15 (48.13)3.66 (3.71)13.37 (13.34)12.13 (11.84)301.76Co(II)L[C21H19Cl2CoN5O3].H2OBlue (523)>30048.58 (49.05)3.69 (3.43)13.49 (13.23)11.35 (10.68)414.68Fe(II)L[C21H19Cl2FeN5O3].H2OPale- yellow (516)>30048.87 (48.84)3.71 (3.23)13.57 (13.14)10.82 (10.49)285.15Figure 1
The proposed structure of the metal complexes (M = Cu(II), Co(II), and Fe(II)).


## 4.2. Electronic Spectra and Magnetic Properties

The electronic absorption spectra of the prepared azo dye and its complexes were conducted in DMSO, and the data are represented in Figure2. The diffuse reflectance spectrum of the free ligand displays two main peaks at 42.194 cm−1, and 40.816 cm−1 which are attributed to the (π–π∗) electronic transition of the conjugated (C=C) in the aromatic rings and (C=O) of carboxylic acid group. Another band was observed at 29.240 cm−1, which is attributed to the (π–π∗) electronic transition of the (N=N) azo group. These bands are shifted to high or low wavelengths due to the coordination of the ligand with the metal ions.Figure 2
UV-vis absorption spectra of the azo dye ligand (HL) and its Cu (II), Co(II), and Fe(II) metal complexes in DMSO.The Cu(II) complex presented an absorption band in the UV range 43478–41494 cm−1, which was assigned to ligand to metal charge transfer transition (LMCT). The octahedral geometry of the Cu(II) ion is supported by the observed value of the magnetic moment (1.76 BM) [20]. The magnetic moment value of Co(II) was 4.68 BM, suggesting an octahedral environment of the Co(II) ion in the complex. The electronic spectrum of the complex showed one broad band at 19.569 cm−1 which can be assigned to d-d transitions [21]. The electronic spectrum of the Fe(II) complex shows an absorption band at 32.787 cm−1 which is caused by π⟶π∗ intraligand transition. Although the spectrum displays another two weak bands at 31.847 and 20.161 cm−1 attributed to the electronic transition 5Eg ⟶ 5T2g. The magnetic moment value of 5.15 BM was an additional evidence for an octahedral structure around the metal center [22].


## 4.3. IR Spectral Studies and the Mode of Bonding

The infrared spectra of the free azo dye ligand were compared with the metal complexes to identify the atoms that participate in the complexation (Table2; Figure 3). The IR spectrum of the free ligand shows two stretching vibration bands at 3,430–3,305 cm−1 due to γasym (NH2) and γsym (NH2), and the γ (CH3) stretching vibrations in the region of 2,848–3,118 cm−1 both appear as broad peaks of the O-H stretching band that overlap this region [23]. In addition, the strong vibration bands between 1691 and 1656 cm−1 are attributed to the stretching vibrations γ (C=O) and γ (C=C), respectively [24]. The vibration of the –N=N– azo group is found at 1,473 cm−1 [25, 26]. The IR spectra of all metal complexes display medium broad bands approximately at the 2,476–3,655 cm−1 regions, confirming the presence of a coordinated water molecule in the structure of the metal complex. After coordination between the ligand and its Co(II), Co(II), and Fe(II) metal ions, the γ (C=O) was shifted to a low frequency and was observed at 1,622, 1,627, and 1,615 cm−1, respectively, indicating that the coordination occurred through the oxygen of carbonyl [27]. Moreover, the γ (N=N) and γ (NH2) stretching bands are shifted toward a lower frequency compared with the free ligand, supporting the involvement of one of the azo nitrogen atoms and amino groups in coordination with metal ions. New weak bands were found; these bands have never been observed in the free HL spectrum, which corresponds to γ (M-O), γ (M-N), and γ (M-Cl) stretching [28]. In addition, the IR spectral studies concluded that the HL ligand acts as an NNO-tridentate ligand with three binding sites via the nitrogen atom of the amino group γ (N-H2), the oxygen atom of the carboxylic group γ (OH), and the nitrogen atom of the azo group γ (N=N).Table 2
Important FT-IR spectral bands (cm−1) of the HL ligand and its complexes.
Compoundγ (N-H)γ (C=O)γ (C=C)γ (C=N)γ (N=N)γ (C-OH)γ (M-N)γ (M-O)γ (M-Cl)[HL]3305–343416911656155114731235———[Cu(II)L]3232–334016221591150313861286551619502[Co(II)L)]3244–339216271595150113821289561598439[Fe(II)L]3094–367516151563147414091241543595452Figure 3
IR spectra at the range 400–4000 cm−1 for the (HL) ligand and its metal complexes. (a) HL. (b) Fe(II)L. (c) Cu(II)L. (d) Co(II)L.


## 4.4.1H NMR Spectra

The proton NMR spectra of the synthesized azo dye ligand and its Cu(II) complex were recorded in DMSO-d6 solution as a solvent at room temperature (Figure 4). The three protons signal of the (CH3) group in the (HL) free ligand appears at 2.0 ppm. Also, the spectrum showed multiple peaks in the range of 7.4–7.9 ppm due to an aromatic ring proton of the ligand. The signal at δ 8.5 ppm may be assigned to the O-H proton in the carboxylic group.Figure 4
1H-NMR chemical shifts of compounds (ppm, 500 MHz, DMSO-d6 solutions, room temperature) for (a) HL ligand and (b) Cu(II)L.
(a)(b)In the1H NMR spectrum of the Cu(II) complex, the aromatic protons resonated moved to upfield (∼0.2 ppm) due to coordination. While the signal of OH groups shifted downfield (∼2.0 ppm), which indicates the bonding of OH group to metal ion. However, the signals of NH2 are not observed due to the interaction with the solvent DMSO-d6 [29].


## 4.5. Thermal Studies

The TGA and DTA studies of the complexes were performed at 0–800°C. The thermal decomposition data of the metal complexes are displayed in Table3 and Figure 5. The results are in good agreement with the theoretical formula suggested by the elemental analyses.Table 3
Thermogravimetric analysis (TGA/DTA) data of the Cu(II), Co(II), and Fe(II) complexes.
CompoundStepTemp. range ˚CDTAMax°CTotal weight loss estimated (calcd.), %Decomposition assignmentResidualCu(II)L1st64–16880.412.1 (12.0)Loss of 3½ H2O (lattice)CuO2nd173–62452087.0 (87.8)Loss of 2Cl + 1H2O (coord.water) + C21H17N5O2 ligandCo(II)L1st40–66940092.6 (92.1)Loss of 1H2O (lattice) + 1H2O (coord.water) + 2Cl + C21H17N5O2 ligand¼ Co2O2Fe(II)L1st51–19399.910.3 (10.4)Loss of 2H2O (lattice) + 1H2O (coord.water)FeO2nd194–649524.485.7 (85.6)Loss of 2Cl + C21H17N5O2 ligandFigure 5
Thermal analysis (TGA/DTA) patterns for the (a) Cu(II), (b) Co(II), and (c) Fe(II) complexes.
(a)(b)(c)The TG/DTG curves of the Cu(II) complex were decomposed in two steps. The first step occurred within the temperature range of 64–168°C, which is attributed to the 3½ H2O lattice water loss. The second step occurred at the temperature range of 173–624°C, with the maximum temperature at 520°C due to 87.0% (calcd. 87.8%) weight loss of two moles of chlorine atoms, one molecule of coordinated water, and the C21H17N5O2 ligand, leaving CuO as the residue. The thermal decomposition of Co(II)L showed the complete decomposition of the organic ligand in one step, two chlorine atoms, 1 H2O (lattice), and coordinated H2O at the temperature range of 40–669°C, with a total 92.6% weight loss (calcd. 92.1%). The thermogram of the Fe(II) complex, the initial weight loss of 10.3% (calcd. 10.4%) occurred at the temperature range of 51–193°C, with DTAmax at 99.9°C, due to the loss of 2H2O lattice water molecules and a mole of coordinated water. The second step occurred within a temperature range of 194–649°C, which signifies 85.7% (calcd. 86.0%) loss of 2Cl atoms and ligand molecules, leaving iron oxide as a residue.


## 4.6. Morphological Behavior

The morphological behaviors of the metal complexes of the azo dye were characterized by XRD measurements and SEM. The X-ray patterns for all samples were recorded between 2θ, ranging from 10° to 80° using the CuKα radiation (λ = 1.540 Å). Interplanar spacing or d-spacing was calculated using Bragg’s law nλ = 2dsinѲ, where Θ is the diffraction angle [30]. The lattice parameters were performed using the Bruker EVA software (Bruker package software). The powder diffraction data 2Θ values are shown in Figure 6. The crystallite size (ξ) of the Cu(II) complex was estimated using the Scherer method, as shown in the following equation [31]:(2)ξ=Kλβcosθ.Figure 6
X-ray diffraction patterns of (a) Cu(II), (b) Co(II), and (c) Fe(II) complexes.The equation uses the referenceK, which is the Scherer constant (0.95), β is the full-width peak at half-maximum, and θ is the Bragg diffraction angle. The XRD reflections of the Cu(II) complex show a mixture of crystalline and amorphous phases, as it lacks sharp peaks at maxima 2Ѳ = 29.88°. The CuO phase was a monoclinic system at a room temperature, with lattice parameters a = 4.67 Å, b = 3.45 Å, c = 5.12 Å, α = 90°, β = 98°, γ = 90°, and volume = 81.94 Å3. The value of δ was calculated and was 76 nm. By contrast, crystalline peaks were not observed in the Co(II) and Fe(II) complexes, indicating the amorphous nature of the tested complexes, which could reflect the immeasurably minute sizes of the aggregated particles within the nanosize range.The surface morphology of the Co(II) complex was investigated using a scanning electron microscope. The SEM images were recorded at different magnifications at an acceleration voltage of 20 kV. The images show that these particles have diameters of 3–20μm. However, the structural morphology consists of a foam-like shape, as shown in Figure 7.Figure 7
Scanning electron micrograph images of the (a) Cu(II)L and (b) Co(II)L complexes.
(a)(b)


## 5. Computational Studies

### 5.1. Energy and Geometry Optimization

The HyperChem 8.0 program was used for the optimization calculations of the free ligand and its metal complexes. The optimized geometries round the coordination sphere of the free ligand HL and its corresponding complexes are shown in Figure8. All bond lengths and bond angles of the prepared compounds were calculated, and the values suggest the octahedral geometry around the metal ions as shown in Table 4. A slightly longer bond is between C9-C40 and C17-C18 in the metal complexes compared to the free ligand by 0.01–0.39 Å during the complexation process. The bond length of C17-N27 in case of free ligand was decreased by 0.01–0.04 Å, while C17-N27 is shortest in the case of metal complexes of ≈0.01–0.05 Å. In the case of Fe(II) complex, the bond length M-O is the longest and shortest in the case of Cu(II) complex with a difference of 0.12 Å (6%). Moreover, the bond angles C36-O37-H45 and O37-C36-O38 were significantly changed in the metal case compared to the free ligand by 0.99–4.77° and 1.31–8.24° upon coordination. The value of the bond angle C14-C16-N19 was decreased by 2.82, 2.52, and 4.7°. The calculated bond angles around the central metal ion suggested the regular octahedral geometry for copper (II), cobalt (II), and iron (II) complexes [32].Figure 8
(a) Optimized geometry of the HL ligand atom numbering. (b) Optimized geometry of the complex where M is the metal atom Cu(II), Co(II), and Fe(II).
(a)(b)Table 4
Structural parameters calculated for azo ligand and its metal complexes.
Bond angles (°)HLSelected bondCu(II)LCo(II)LFe(II)LC9-C7-N28123.62C9-C7-N29120.75120.92120.05C7-N28-N27132.34C7-N29-N28114.75115.04117.85C17-N27-N28131.13C18-N28-N29123.56122.89122.35N19-H34-H35118.14N20-H35-H51107.54106.38109.47C36-O37-H45109.47C39-O40-H50114.24113.71110.46O37-C36-O38109.80O41-C39-O40113.19111.11118.04C17-C16-N19123.39C17-C18-N20126.88125.37126.95C14-C16-N19129.88C15-C17-N20127.06127.36125.18C13-C9-C36116.37N20-M-Cl4382.6280.3778.36C39-O40-M117.94116.28111.39O40-M-N20163.72164.02158.63N29-M-N2099.5396.7999.53Cl-M-N29176.39175.64174.39Bond lengths (Å)C6-C71.46C6-C71.431.451.42C17-N271.40C18-N281.391.351.36C7-N281.40C7-N291.461.431.41N19-H341.01N20-H351.001.011.01N19-H351.01N20-H391.001.011.01N27-N281.25N28-N291.331.351.48C16-N191.23C17-N201.461.451.44C16-N191.32C17-N201.461.451.44C9-C361.46C9-N401.471.481.48C36-O371.36C36-O411.391.421.37C16-C171.34C17C181.421.431.41O38-C361.22N20-M1.921.911.90O37-H450.96O40-M1.961.972.08N29-M1.901.921.83The quantum chemical parameters such as electrophilicity (ω), global hardness (η), global-softness (s), electronegativity (x), and chemical potential (μ) have been estimated depending on the energy gaps between the two molecular orbitals of the highest occupied molecular orbital energy (EHOMO) and the lowest unoccupied molecular orbital energy (ELUMO), which are presented in Figure 9 and Table 5. The energy gap and the parameters were calculated by known equations:Figure 9
The charge density maps of HOMO and LUMO maps of the studied ligand and its complexes.Table 5
EHOMO, ELUMO, Egap, and quantum parameters for the HL ligand and its complexes.
CompoundEHOMO (eV)ELUMO (eV)Egap (eV)ƞ (eV)χ (eV)S (eV)−1μ (eV)ω (eV)HL ligand−11.85−5.176.683.348.510.14−8.5110.84[Cu(II)L]−8.64−5.292.351.1711.280.42−6.9620.61[Co(II)L]−12.51−9.363.152.5710.930.31−10.9337.92[Fe(II)L]−7.89−4.952.941.476.420.34−6.4214.01∆E=ELUMO–EHOMO, μ=EHOMO+ELUMO/2, η=IP−EA/2, σ=1/2η, ω=μ2/2η, χ=IP+EA/2, where IP = –EHOMO, and EA = –ELUMO.The negative values of the energy gap ΔE, showing that all investigated compounds are stabilized [33, 34]. Moreover, the results showed that all metal complexes exhibited smaller values of the energy gap than the free ligand, which indicate the greater biological reactivity than the ligand. The global electrophilicity index (ω) of the free ligand has a small value (10.84 eV) compared to the values in metal complexes, indicating the weak electrophile. Furthermore, the electronegativity (χ) of a molecule measures the atom’s ability to attract electrons from a neighboring atom to which it is bonded. The calculation of absolute hardness values (η) is useful to measure the stabilization in energy and reactivity for molecular compounds, it could be arranged as follows: HL > Co(II) > Cu(II) > Fe(II) [35].


### 5.2. Antibacterial Study

The agar diffusion method was conducted to determine the in vitro antimicrobial and antifungal properties of HL and its metal complexes against two gram-positive bacteria,Bacillus subtilis and Staphylococcus aureus, two gram-negative bacteria, Neisseria gonorrhoeae and Escherichia coli, and a fungus, Candida albicans [36]. The results show that the zone of inhibition (mm) has a larger value for all complexes compared to the free ligand, indicating high effectivity against bacterial and fungal strains, as shown in Figure 10. The higher activity of complexes can be explained by Overtone’s concept and Tweedy’s chelation theory. The polarity of the metal ion is reduced because of the partial sharing of its positive charge with the donor groups and possible π-electron delocalization over the entire chelate ring [37]. Moreover, other factors affect the activities of the complexes, such as coordination sites, size, concentration, geometry, structure, and hydrophobicity. The Cu(II)L complex shows higher antimicrobial activity than the other compounds, which may be attributed to their particle size and metal ion size [38]. However, the antimicrobial activity of the complexes is lower than that of standard drugs. The zone inhibition values (area in mm) are presented in Table 6.Figure 10
In vitro. (a) Antifungal and (b) antibacterial properties of the azo ligand (HL) and its metal complexes at a concentration of 100μL.
(a)(b)Table 6
Antibacterial activity data (zone of inhibition in mm) of the ligand and its metal complexes at a concentration of 100μL.
Inhibition zone diameter (mm)ComplexGram positiveGram negativeFungusBacillus subtilisStaphylococcus aureusEscherichia coliNeisseria gonorrhoeaeCandida albicansControl—————Azo dye ligand32638[Cu(II)L]141721814[Co(II)L]101212416[Fe(II)L]8714613Ampicillin26212510—Amphotericin————25


### 5.3. Anticancer Activity

The in vitro cytotoxic effects of the AMePhPNA ligand and its Cu(II), Co(II), and Fe(II) complexes were evaluated on a human tumor cell line (HepG2) using the MTT assay. The IC50 (concentration inhibiting 50% of cell proliferation) was determined. The IC50 values showed the higher activity of metal complexes at 37°C for 24 h, this may be attributed to the high kinetic stability of the complexes in the growth medium [39]. A microscopy study exposed to different concentrations was recorded and is shown in Figure 11. The results show that the cytotoxic effect of all investigated compounds is higher than that of the cisplatin reference drug. The IC50 value against HepG2 of the free ligand is greater than that of all metal complexes, which exhibit low IC50 values and greater anticancer agent properties. This high cytotoxicity may be attributed to the increase in conjugation of the ligand moiety on complexation [40, 41]. Moreover, the type of metal ion may affect different cytotoxic properties, which follows the sequence of trends: Co(II)L > Fe(II)L > Cu(II)L > HL > cisplatin on HepG2. According to the toxicity level, a proton coupled electron transfer reaction results in the formation of a resonance stabilized carboxyl radical, which is how compounds based on naphthoic acid exhibit antioxidant activity. By enhancing the resonance stabilized structure’s stability and increasing the electron donating effect, substituents can increase the activity of these compounds [42]. The cell viability value of HepG2 was evaluated by the MTT assay, as presented in Figure 12 and Table 7.Figure 11
Morphological changes in liver cancer cells (HepG2) treated with different concentrations of (a) ligand and (b) Cu(II), (c) Co(II) and (d) Fe(II) complexes for 24 h.
(a)(b)(c)(d)Figure 12
The percentage of the cell viability calculated using the MTT assay in liver cancer cells (HepG2) for the azo dye ligand (HL) and its Cu(II), Co(II), and Fe(II) complexes after 24 h of incubation at various concentrations compared with the positive and negative controls.Table 7
DNA fragmentation of (a) azo ligand and its (b) Cu(II), (c) Co(II), and (d) Fe(II) complexes at (0.1, 1μg/mL) on HepG2 cells.
(a)IDConc (μg/ml)Mean O.DST.EViability%Toxicity%IC50HL1 : 020.383990.0085103100022.171000.021980.00044624.37112.1860941500.065030.00687687.3298.92331289250.168820.014726623.3266.6789366112.50.289970.008557244.8429.080777096.250.373500.005597181.043.0419222913.1250.380910.004663294.990.851738241(b)IDConc (μg/ml)Mean O.DST.EViability%Toxicity%IC50Cu(II)L1 : 020.554680.0087534100022.091000.02320.00118184114.0311500.065140.006205511.24105.0198250.226970.011273439.1670.1535712.50.4631930.006758979.9219.538576.250.552950.014060695.410.421093.1250.562320.008967297.030.084218(c)IDConc (μg/ml)Mean O.DST.EViability%Toxicity%IC50Co(II)L1 : 020.465060.008486100019.551000.021180.0076324.55113.5772500.035480.0043088.9109.962250.112960.00785131.1290.0784812.50.21720.00548980.6863.366246.250.392550.00667199.9418.578063.1250.460080.00913599.070.92827(d)IDConc (μg/ml)Mean O.DST.EViability%Toxicity%IC50Fe(II)L1 : 020.575010.0054149100020.161000.021180.00044625.72113.293239500.035480.002717516.93108.4284591250.112960.009032943.9681.8592767312.50.21720.010673175.5123.10770446.250.392550.006042097.260


### 5.4. DNA Fragmentation Study

The DPA assay is a highly useful tool in quantitatively measuring apoptosis by determining the percentage. The free ligand HL and its Cu(II), Co(II), and Fe(II) complexes exhibited good antioxidant activity; hence, we performed DNA fragmentation analysis. The data on the percentage of fragmentation are displayed in Figure13 and Table 8. Compared with the large dose, the damage level dramatically decreased when utilizing the 0.1 value, although it was still much higher than in the control group for 24 h. HepG2 was treated with the HL ligand and its Cu(II), Co(II), and Fe(II) complexes, with a percentage of fragmentation of 41.33% (control, 7.00) at IC50 = 22.17 μg/mL, 42.34% (control, 7.17) at IC50 = 22.09, 45.36% (control, 7.68) at IC50 = 19.55 μg/mL, and 43.68% (control, 7.40) at IC50 = 20.16 μg/mL, respectively. This finding may suggest that the free ligand and its complexes may be considered as a cytotoxic drug by inducing cancer cell death due to DNA damage [43].Figure 13
Quantitative estimation of DNA fragmentation % by the diphenylamine assay at various concentrations.Table 8
DNA fragmentation of (a) azo ligand and its (b) Cu(II), (c) Co(II), and (d) Fe(II) complexes at (0.1, 1μg/mL) on HepG2 cells.
(a)GroupsDNA fragmentation%SDP1 Student’st-testP2 Student’st-testTest of significanceHepG2 treated with 0.1μg/mL14.1002281.2557070.001∗<0.001∗ANOVAF = 560.109∗P<0.001HepG2 treated with 1μg/mL41.3324281.830609<0.001∗Control group7.0047270.574902(b)GroupsDNA fragmentation%SDP1 Student’st-testP2 Student’st-testTest of significanceHepG2 treated with 0.1μg/mL14.4441361.2863340.001∗<0.001∗ANOVAF = 560.109∗P<0.001HepG2 treated with 1μg/mL42.3405361.875258<0.0010∗Control group7.1755740.588924(c)GroupsDNA fragmentation%SDP1 Student’st-testP2 Student’st-testTest of significanceHepG2 treated with 0.1μg/mL15.475861.3782150.001∗<0.001∗ANOVAF = 560.109∗P<0.001HepG2 treated with 1μg/mL45.364862.009205<0.0010∗Control group7.6881150.63099(d)GroupsDNA fragmentation%SDP1 Student’st-testP2 Student’st-testTest of significanceHepG2 treated with 0.1μg/mL14.902681.327170.001∗<0.001∗ANOVAF = 560.109∗P<0.001HepG2 treated with 1μg/mL43.684681.93479<0.0010∗Control group7.403370.60762P1: comparison of both study groups versus the control group. P2: comparison between both the study groups;∗P is significant at <0.05.


## 5.1. Energy and Geometry Optimization

The HyperChem 8.0 program was used for the optimization calculations of the free ligand and its metal complexes. The optimized geometries round the coordination sphere of the free ligand HL and its corresponding complexes are shown in Figure8. All bond lengths and bond angles of the prepared compounds were calculated, and the values suggest the octahedral geometry around the metal ions as shown in Table 4. A slightly longer bond is between C9-C40 and C17-C18 in the metal complexes compared to the free ligand by 0.01–0.39 Å during the complexation process. The bond length of C17-N27 in case of free ligand was decreased by 0.01–0.04 Å, while C17-N27 is shortest in the case of metal complexes of ≈0.01–0.05 Å. In the case of Fe(II) complex, the bond length M-O is the longest and shortest in the case of Cu(II) complex with a difference of 0.12 Å (6%). Moreover, the bond angles C36-O37-H45 and O37-C36-O38 were significantly changed in the metal case compared to the free ligand by 0.99–4.77° and 1.31–8.24° upon coordination. The value of the bond angle C14-C16-N19 was decreased by 2.82, 2.52, and 4.7°. The calculated bond angles around the central metal ion suggested the regular octahedral geometry for copper (II), cobalt (II), and iron (II) complexes [32].Figure 8
(a) Optimized geometry of the HL ligand atom numbering. (b) Optimized geometry of the complex where M is the metal atom Cu(II), Co(II), and Fe(II).
(a)(b)Table 4
Structural parameters calculated for azo ligand and its metal complexes.
Bond angles (°)HLSelected bondCu(II)LCo(II)LFe(II)LC9-C7-N28123.62C9-C7-N29120.75120.92120.05C7-N28-N27132.34C7-N29-N28114.75115.04117.85C17-N27-N28131.13C18-N28-N29123.56122.89122.35N19-H34-H35118.14N20-H35-H51107.54106.38109.47C36-O37-H45109.47C39-O40-H50114.24113.71110.46O37-C36-O38109.80O41-C39-O40113.19111.11118.04C17-C16-N19123.39C17-C18-N20126.88125.37126.95C14-C16-N19129.88C15-C17-N20127.06127.36125.18C13-C9-C36116.37N20-M-Cl4382.6280.3778.36C39-O40-M117.94116.28111.39O40-M-N20163.72164.02158.63N29-M-N2099.5396.7999.53Cl-M-N29176.39175.64174.39Bond lengths (Å)C6-C71.46C6-C71.431.451.42C17-N271.40C18-N281.391.351.36C7-N281.40C7-N291.461.431.41N19-H341.01N20-H351.001.011.01N19-H351.01N20-H391.001.011.01N27-N281.25N28-N291.331.351.48C16-N191.23C17-N201.461.451.44C16-N191.32C17-N201.461.451.44C9-C361.46C9-N401.471.481.48C36-O371.36C36-O411.391.421.37C16-C171.34C17C181.421.431.41O38-C361.22N20-M1.921.911.90O37-H450.96O40-M1.961.972.08N29-M1.901.921.83The quantum chemical parameters such as electrophilicity (ω), global hardness (η), global-softness (s), electronegativity (x), and chemical potential (μ) have been estimated depending on the energy gaps between the two molecular orbitals of the highest occupied molecular orbital energy (EHOMO) and the lowest unoccupied molecular orbital energy (ELUMO), which are presented in Figure 9 and Table 5. The energy gap and the parameters were calculated by known equations:Figure 9
The charge density maps of HOMO and LUMO maps of the studied ligand and its complexes.Table 5
EHOMO, ELUMO, Egap, and quantum parameters for the HL ligand and its complexes.
CompoundEHOMO (eV)ELUMO (eV)Egap (eV)ƞ (eV)χ (eV)S (eV)−1μ (eV)ω (eV)HL ligand−11.85−5.176.683.348.510.14−8.5110.84[Cu(II)L]−8.64−5.292.351.1711.280.42−6.9620.61[Co(II)L]−12.51−9.363.152.5710.930.31−10.9337.92[Fe(II)L]−7.89−4.952.941.476.420.34−6.4214.01∆E=ELUMO–EHOMO, μ=EHOMO+ELUMO/2, η=IP−EA/2, σ=1/2η, ω=μ2/2η, χ=IP+EA/2, where IP = –EHOMO, and EA = –ELUMO.The negative values of the energy gap ΔE, showing that all investigated compounds are stabilized [33, 34]. Moreover, the results showed that all metal complexes exhibited smaller values of the energy gap than the free ligand, which indicate the greater biological reactivity than the ligand. The global electrophilicity index (ω) of the free ligand has a small value (10.84 eV) compared to the values in metal complexes, indicating the weak electrophile. Furthermore, the electronegativity (χ) of a molecule measures the atom’s ability to attract electrons from a neighboring atom to which it is bonded. The calculation of absolute hardness values (η) is useful to measure the stabilization in energy and reactivity for molecular compounds, it could be arranged as follows: HL > Co(II) > Cu(II) > Fe(II) [35].


## 5.2. Antibacterial Study

The agar diffusion method was conducted to determine the in vitro antimicrobial and antifungal properties of HL and its metal complexes against two gram-positive bacteria,Bacillus subtilis and Staphylococcus aureus, two gram-negative bacteria, Neisseria gonorrhoeae and Escherichia coli, and a fungus, Candida albicans [36]. The results show that the zone of inhibition (mm) has a larger value for all complexes compared to the free ligand, indicating high effectivity against bacterial and fungal strains, as shown in Figure 10. The higher activity of complexes can be explained by Overtone’s concept and Tweedy’s chelation theory. The polarity of the metal ion is reduced because of the partial sharing of its positive charge with the donor groups and possible π-electron delocalization over the entire chelate ring [37]. Moreover, other factors affect the activities of the complexes, such as coordination sites, size, concentration, geometry, structure, and hydrophobicity. The Cu(II)L complex shows higher antimicrobial activity than the other compounds, which may be attributed to their particle size and metal ion size [38]. However, the antimicrobial activity of the complexes is lower than that of standard drugs. The zone inhibition values (area in mm) are presented in Table 6.Figure 10
In vitro. (a) Antifungal and (b) antibacterial properties of the azo ligand (HL) and its metal complexes at a concentration of 100μL.
(a)(b)Table 6
Antibacterial activity data (zone of inhibition in mm) of the ligand and its metal complexes at a concentration of 100μL.
Inhibition zone diameter (mm)ComplexGram positiveGram negativeFungusBacillus subtilisStaphylococcus aureusEscherichia coliNeisseria gonorrhoeaeCandida albicansControl—————Azo dye ligand32638[Cu(II)L]141721814[Co(II)L]101212416[Fe(II)L]8714613Ampicillin26212510—Amphotericin————25


## 5.3. Anticancer Activity

The in vitro cytotoxic effects of the AMePhPNA ligand and its Cu(II), Co(II), and Fe(II) complexes were evaluated on a human tumor cell line (HepG2) using the MTT assay. The IC50 (concentration inhibiting 50% of cell proliferation) was determined. The IC50 values showed the higher activity of metal complexes at 37°C for 24 h, this may be attributed to the high kinetic stability of the complexes in the growth medium [39]. A microscopy study exposed to different concentrations was recorded and is shown in Figure 11. The results show that the cytotoxic effect of all investigated compounds is higher than that of the cisplatin reference drug. The IC50 value against HepG2 of the free ligand is greater than that of all metal complexes, which exhibit low IC50 values and greater anticancer agent properties. This high cytotoxicity may be attributed to the increase in conjugation of the ligand moiety on complexation [40, 41]. Moreover, the type of metal ion may affect different cytotoxic properties, which follows the sequence of trends: Co(II)L > Fe(II)L > Cu(II)L > HL > cisplatin on HepG2. According to the toxicity level, a proton coupled electron transfer reaction results in the formation of a resonance stabilized carboxyl radical, which is how compounds based on naphthoic acid exhibit antioxidant activity. By enhancing the resonance stabilized structure’s stability and increasing the electron donating effect, substituents can increase the activity of these compounds [42]. The cell viability value of HepG2 was evaluated by the MTT assay, as presented in Figure 12 and Table 7.Figure 11
Morphological changes in liver cancer cells (HepG2) treated with different concentrations of (a) ligand and (b) Cu(II), (c) Co(II) and (d) Fe(II) complexes for 24 h.
(a)(b)(c)(d)Figure 12
The percentage of the cell viability calculated using the MTT assay in liver cancer cells (HepG2) for the azo dye ligand (HL) and its Cu(II), Co(II), and Fe(II) complexes after 24 h of incubation at various concentrations compared with the positive and negative controls.Table 7
DNA fragmentation of (a) azo ligand and its (b) Cu(II), (c) Co(II), and (d) Fe(II) complexes at (0.1, 1μg/mL) on HepG2 cells.
(a)IDConc (μg/ml)Mean O.DST.EViability%Toxicity%IC50HL1 : 020.383990.0085103100022.171000.021980.00044624.37112.1860941500.065030.00687687.3298.92331289250.168820.014726623.3266.6789366112.50.289970.008557244.8429.080777096.250.373500.005597181.043.0419222913.1250.380910.004663294.990.851738241(b)IDConc (μg/ml)Mean O.DST.EViability%Toxicity%IC50Cu(II)L1 : 020.554680.0087534100022.091000.02320.00118184114.0311500.065140.006205511.24105.0198250.226970.011273439.1670.1535712.50.4631930.006758979.9219.538576.250.552950.014060695.410.421093.1250.562320.008967297.030.084218(c)IDConc (μg/ml)Mean O.DST.EViability%Toxicity%IC50Co(II)L1 : 020.465060.008486100019.551000.021180.0076324.55113.5772500.035480.0043088.9109.962250.112960.00785131.1290.0784812.50.21720.00548980.6863.366246.250.392550.00667199.9418.578063.1250.460080.00913599.070.92827(d)IDConc (μg/ml)Mean O.DST.EViability%Toxicity%IC50Fe(II)L1 : 020.575010.0054149100020.161000.021180.00044625.72113.293239500.035480.002717516.93108.4284591250.112960.009032943.9681.8592767312.50.21720.010673175.5123.10770446.250.392550.006042097.260


## 5.4. DNA Fragmentation Study

The DPA assay is a highly useful tool in quantitatively measuring apoptosis by determining the percentage. The free ligand HL and its Cu(II), Co(II), and Fe(II) complexes exhibited good antioxidant activity; hence, we performed DNA fragmentation analysis. The data on the percentage of fragmentation are displayed in Figure13 and Table 8. Compared with the large dose, the damage level dramatically decreased when utilizing the 0.1 value, although it was still much higher than in the control group for 24 h. HepG2 was treated with the HL ligand and its Cu(II), Co(II), and Fe(II) complexes, with a percentage of fragmentation of 41.33% (control, 7.00) at IC50 = 22.17 μg/mL, 42.34% (control, 7.17) at IC50 = 22.09, 45.36% (control, 7.68) at IC50 = 19.55 μg/mL, and 43.68% (control, 7.40) at IC50 = 20.16 μg/mL, respectively. This finding may suggest that the free ligand and its complexes may be considered as a cytotoxic drug by inducing cancer cell death due to DNA damage [43].Figure 13
Quantitative estimation of DNA fragmentation % by the diphenylamine assay at various concentrations.Table 8
DNA fragmentation of (a) azo ligand and its (b) Cu(II), (c) Co(II), and (d) Fe(II) complexes at (0.1, 1μg/mL) on HepG2 cells.
(a)GroupsDNA fragmentation%SDP1 Student’st-testP2 Student’st-testTest of significanceHepG2 treated with 0.1μg/mL14.1002281.2557070.001∗<0.001∗ANOVAF = 560.109∗P<0.001HepG2 treated with 1μg/mL41.3324281.830609<0.001∗Control group7.0047270.574902(b)GroupsDNA fragmentation%SDP1 Student’st-testP2 Student’st-testTest of significanceHepG2 treated with 0.1μg/mL14.4441361.2863340.001∗<0.001∗ANOVAF = 560.109∗P<0.001HepG2 treated with 1μg/mL42.3405361.875258<0.0010∗Control group7.1755740.588924(c)GroupsDNA fragmentation%SDP1 Student’st-testP2 Student’st-testTest of significanceHepG2 treated with 0.1μg/mL15.475861.3782150.001∗<0.001∗ANOVAF = 560.109∗P<0.001HepG2 treated with 1μg/mL45.364862.009205<0.0010∗Control group7.6881150.63099(d)GroupsDNA fragmentation%SDP1 Student’st-testP2 Student’st-testTest of significanceHepG2 treated with 0.1μg/mL14.902681.327170.001∗<0.001∗ANOVAF = 560.109∗P<0.001HepG2 treated with 1μg/mL43.684681.93479<0.0010∗Control group7.403370.60762P1: comparison of both study groups versus the control group. P2: comparison between both the study groups;∗P is significant at <0.05.


## 6. Conclusion

A new synthesized HL (1-((5-amino-3-methyl-1-phenyl-1H-pyrazol-4-yl) diazenyl)-2-naphthoic acid) and its Cu(II), Co(II), and Fe(II) complexes were prepared and characterized by elemental analysis (H, C, N, and M), spectroscopic analysis (FT-IR, 1H NMR, and UV-vis), and TGA-DTA, which provide good results in thermal stability. The molar conductance values confirmed that all complexes are nonelectrolytes and have octahedral geometry. XRD and electron microscopy (SEM) approved that the metal complexes have different morphologies and free ligand. DFT calculations indicate that the high stability of the synthesis compounds depends on Eg values. The in vitro antibacterial and antifungal activities were evaluated, and the results showed poor antimicrobial activity for the free azo ligand against the tested strains, whereas its metal complexes exhibited good to moderate antibacterial and antifungal activities. The new ligand and its complexes were evaluated as cytotoxic candidates against HepG2 through the MTT assay. The Co(II) complex is more biologically effective than the other compounds against the tested cell line, with IC50 = 19.55 μg/mL. DNA fragmentation and cell death in human liver cancer cells were determined by DPA assay, and the data revealed that the fragmentation percent of each compound increased ca. by 6.5 fold compared with the control cells for 24 h. This result shows the possible application of the investigated compounds as antitumor drugs against human liver cancer cells with further detailed studies and additional verification.



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*Source: 3476954-2022-12-16.xml*