Synthesis, Characterization, Antimicrobial, Density Functional Theory, and Molecular Docking Studies of Novel Mn(II), Fe(III), and Cr(III) Complexes Incorporating 4-(2-Hydroxyphenyl azo)-1-naphthol (Az)

Abstract

This work synthesized three new CrAz₂, MnAz₂, and FeAz₂ complexes and investigated them using IR, mass, UV spectroscopy, elemental analysis, conductivity and magnetic tests, and thermogravimetric analysis. The azo-ligand, 4-(2-hydroxyphenylAzo)-1-naphthol (Az), couples with metal ions via its nitrogen (in −N=N– bonds) and oxygen (in hydroxyl group) atoms, according to the IR spectra of these complexes. Through thermal examination (TG/TGA), the number and location of water in the complexes were also determined. Density functional theory (DFT) theory is applied to ameliorate the structures of the ligand (Az) and metal complexes and analyze the quantum chemical characteristics of these complexes. The antifungal and antibacterial activity of the ligand and its complexes opposed to several hazardous bacteria and fungi was investigated in vitro. Metal complexes were discovered to have a higher inhibitory impact on some organisms than the free ligand. The MnAz₂ complex exhibited the best activity among the studied materials, whereas the CrAz₂ complex had the lowest. The compounds’ binding affinity to the E. coli (PDB ID: 1hnj) structure was predicted using molecular docking. Binding energies were calculated by analyzing protein-substrate interactions. These encouraging findings imply that these chemicals may have physiological effects and may be valuable for a variety of medical uses in the future.

1. Introduction

Over the course of the past four decades, azo dyes that include heterocyclic rings have made substantial contributions to the field of coordination chemistry.¹ Research in the fields of biology, electrochemistry, and analysis may all make use of these substances in a variety of different ways.² Because of their potential cytotoxicity as well as their antibacterial, antifungal, and antioxidant capabilities, they have garnered a lot of studies.^3,4 There has been a significant amount of research done on the production of metal(II) complexes of azo compounds, as well as their biological activity.^5,6 Because their azo group possesses powerful medicinal properties, azo dyes are advantageous chelating agents for a variety of metal ions,^7,8 due to the azo group of these dyes. In addition to this, it has been demonstrated that azo metal complexes can be beneficial as metal anticorrosion agents and as thermally stable optical storage devices,^9,10

There is an urgent need for new metal-based compounds that can prevent the development of hazardous germs that have become worldwide spread. These microorganisms have the potential to pollute food, water, and soil, as well as cause sickness in humans, animals, and plants.^11,12 In recent years, bioinorganic chemistry, which is the study of coordination chemistry with biological ligands, has experienced tremendous expansion. Bio-metals such as manganese, iron, cobalt, nickel, and copper have been major contributors to this rise. The study of metal-bioligand complexes is essential to gaining an understanding of the processes that occur in living organisms.^13,14 Because coordination chemistry has a history of being successful in the field of pharmacotherapy, academics and researchers have been keenly interested in the topic for a significant amount of time.

Metal ions are selected based on their broad biological history. Previously, complexes of Cr(III), Mn(II), and Fe(III) with azo ligands were reported to show anticancer, antioxidant, and antibacterial activity.^7,15−18 Therefore, we speculate that complexes of Cr(III), Mn(II), and Fe(III) with azo ligands will show more antibacterial and antifungal activity. These findings prompted us to start a project on Cr(III), Mn(II), and Fe(III)-azo-ligand complexes, involving an investigation of their structural characterization, antibacterial, and antifungal properties.

As a result, the purpose of this work is to develop novel coordination compounds based on azo dyes that contain CrAz₂, MnAz₂, and FeAz₂ and then analyze the bioactivity of these compounds in vitro and computationally. In addition, density functional theory (DFT) simulations were utilized so that the three-dimensional (3D) chemical structure of the complexes could be determined. Authors anticipate that the current effort may result in the discovery of a new family of chemicals that due to the ease with which they can be produced and the remarkable efficacy they possess, may represent promising candidates for the treatment of bacterial and fungal diseases.

2. Experimental Section

2.1. Synthesis

2.1.1. Materials and Reagents

The purity of the chemicals used was suitable for use as analytical reagents (AR). These included 4-(2-hydroxyphenylAzo)-1-naphthol, chromium(III) chloride, manganese(II) chloride, and iron(III) chloride, in addition to organic solvents, such as EtOH and CH₃CN.

2.1.2. Synthesis of the 1-(2-Hydroxyphenylazo)-2-naphthol Azo-Ligand (Az)

1-(2-Hydroxyphenylazo)-2-naphthol as the azo-ligand (Az) was prepared as previously reported¹⁹ with slight modifications.

2.1.2.1. Step I: Preparation of Diazonium Salt of 2-Aminophenolas Azo Molecules

A cooled solution (0 °C) of NaNO₂ (0.69 g, 0.01 mol) in 25 mL of water was gradually added to a cooled solution of 1.09 g (0.01 mol) of 2-aminophenol and 36 mL of HCl under stirring in an ice bath for 20 min. The diazonium salt solution was stored in the refrigerator at all times (Scheme 1).

Scheme 1. Preparation of Az Ligands and Its Complexes.

2.1.2.2. Step II: Coupling Procedure

The cooled solution of diazonium salt was gradually added to the cooled solution (0 °C) of 1-naphthol (1.44 g, 0.01 mol) in NaOH (10% w/v) in an ice bath. Then, the azo dye will be precipitated. The final azo product was produced by filtration of the crude precipitate, multiple cold water washes, and recrystallization from the suitable solvent; Scheme 1.

2.1.2.3. 4-(2-Hydroxyphenyl azo)-1-naphthol

Brown–red solid; yield (91%); mp. 190–191 °C; IR (cm^–1): 3114–3218 (OH), 3001 (Ar-H); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 11.25 (s, 1H, OH), 8.56 (s, 1H, OH), 8.26–6.98 (m, 10H, Ar-H); ¹³C NMR (100 MHz, DMSO-d₆) δ (ppm): 157.99, 154.55, 139.65, 132.84, 128.50, 125.93,123.20, 123.07, 122.43, 120.31, 118.34, 115.53, 109.05; elemental analysis for C₁₆H₁₂N₂O₂ (calcd/found); C, 72.72/72.62; H, 4.55/4.41; N, 10.60/10.71.

2.1.3. Preparation of the Metal Complexes

During the process of heating the combination, a solution of the metal salt (2.0 mmol) in water (10 mL) was progressively added to ethanol that already contained the ligand (4.0 mmol). The solution was stirred consistently during the ten-hour process of refluxing in a water bath at 90 °C. The product was recrystallized after it had been filtered, dried, and washed with a water–ethanol mixture with a ratio of 1:2. It was established how much of each product could be obtained as well as the melting and decomposition temperatures; Scheme 1.

2.2. Characterization

The various physicochemical techniques used for the characterization of the free Az ligand and its metal complexes are listed in the Supplementary data file.

2.3. DFT Calculations

To optimize the geometry of the AZ ligand and its complexes, the cc-pVQZ,^20,21 the basis set as a full-electron basis set for all elements composing the investigated compounds in conjunction with the hybrid correlation functional (B3LYP),²²⁻²⁴ was used in ethanol as the solvent. The solvent (ethanol) effect was solved using the polarizable constant model (IEFPCM).^25,26 The DFT calculations²⁷ were done using Gaussian 09 W,²⁸ and the optimized structures, highest occupied molecular orbitals (HOMO)–lowest unoccupied molecular orbitals (LUMO) orbitals, and molecular electrostatic potential (MEP) map were visualized using GaussView 5.²⁹ Also, natural bond orbital (NBO) analysis of the current compounds was performed using the NBO 3.1 programme³⁰ as applied in the Gaussian 09 program. To determine quantum chemical parameters such as ionization potential (IP = −E_HOMO), electronegativity (EN = (I.P. + E.A.)/2), the energy gap (ΔE = E_LUMO – E_HOMO), chemical potential (CP = −EN), electron affinity (EA = −E_LUMO), softness (S = 1/CH), chemical hardness (CH = (I.P. – E.A.)/2)), and electrophilicity index (EP = EN²/2CH), the LUMO and HOMO energies of the relevant compounds were utilized.^31,32

2.4. In Vitro Investigation of Bioactivity

The antimicrobials activity of the compounds was examined using the disc diffusion method,^33,34 against a panel of bacteria (Escherichia coli (−ve), Pseudomonas aeruginosa (−ve), Bacillus cereus (+ve), and Staphylococcus aureus (+ve)) and fungi. The results showed that the compounds were effective against all of the bacteria and fungi tested (Trichophyton rubrum, Candida albicans, and Aspergillus flavus). The inhibitory zone that surrounded the disc was measured in millimeters and the compounds’ activity was compared to that of the standard chloramphenicol antibiotic using the percentage activity index (AI% = IZ of the test compound/IZ of the standard) multiplied by 100.^35,36

2.5. Molecular Docking

Molecular docking simulations of the specified compounds were performed against the 1HNJ protein to validate the therapeutic efficacy of these compounds. 1HNJ is the structural representation of the FabH-CoA complex in E. coli. It is believed that the FabH receptor is targeted with a view to learning about the potential antibacterial properties of chemicals that are present in nature.^37,38 The enzyme FabH plays a role in the synthesis of fatty acids.

The three-dimensional structure of the objective protein receptor was attained by consulting the protein database (http://www.rcsb.org). The chemicals that are researched are utilized as a substrate. The molecular docking investigation is carried out with the use of a molecular overeating environment (MOE).³⁹

After creating a new database, optimizing each chemical by reducing the amount of energy required for substrate preparation, and finally storing the results in the MDB format, in order to prepare the receptor, the target receptor underwent processes including the addition of hydrogen atoms, the connection of receptor types, the fixing of potential energy, and, finally, the manufacture of active pockets and dummies.^40,41 Docking patterns and interaction parameters were exported so that interaction features could be analyzed, and inhibitory activity could be ranked according to a scoring function (S, kcal/mol).^42,43

3. Results and Discussion

3.1. Structural Configuration of the Azo-Ligand Ligand (Az)

The chemical structure of the azo-ligand (Az) was assured by its spectral (IR, ¹H NMR, and ¹³C NMR) and elemental analyses. During the Az analysis, a wide OH absorption band was found between 3114 and 3218 cm^–1, and an aromatic C–H band was identified at 3001 cm^–1. At 1542 cm^–1, the characteristic band that can be produced by the N=N group during stretching vibration absorption was also seen.

Singlet broad signals for the −OH group were seen in the ¹H NMR spectra of Az at 11.25 (s, 1H, OH) and 8.56 (s, 1H, OH), as well as aromatic signals at 8.26–6.98 ppm (m, 10H, Ar–H). Signals were seen in the¹³C NMR spectra at the following frequencies: 157.99, 154.55, 139.65, 132.84, 128.50, 125.93, 123.20, 123.07, 122.43, 120.31, 118.34, 115.53, 109.05 which is characteristic of CH of phenyl and naphthyl moiety, Supporting data; Figure S1. Also, the elemental analysis of the Az ligand referred to good agreement between the found and calculated values for C, H, and N, Table 1, which confirms the proposed structure; Scheme 1.

Table 1. Physical Properties, UV–Vis, Conductivity, Magnetic, and FT-IR Results.

		Az	CrAz2	MnAz2	FeAz2
physical properties	color	brown–red	white–green	white yellow	dark-violet
	melting point (°C)	190–191	>300	>300	>300
	yield (%)	97	90	87	89
conductivity μv, Ω–1 cm2 mol–1	Acetonitrile		11.78	12.04	11.44
	DMF		10.15	9.48	10.07
	assignment		Non-electrolyte
UV–vis	λmax, nm	380	615	410	505
magnetic	μeff (B.M)		3.84	1.96	1.84
	assignment		d3 (t2g3)	d5 low spin (t2g5)	d5 (t2g5)
stoichiometry	M:L		1:2	1:2	1:2
IR spectra	υ (−OH)	3318	3478	3488	3460
	υ (−N=N)	1542	1464	1460	1470
	υ (M–O)		551	547	544
	υ (M–N)		485	478	484
EA found (calc.) %	C	72.62 (72.72)	60.47 (60.81)	62.53 (62.24)	60.28 (60.45)
	H	4.41 (4.55)	3.55 (3.83)	4.66 (4.24)	3.66 (3.80)
	N	10.71 (10.60)	8.69 (8.86)	8.87 (9.07)	8.62 (8.81)
	M		8.54 (8.23)	8.72 (8.90)	8.99 (8.78)

3.2. Structural Clarification of the Metal Complexes

3.2.1. Conductivity Measurements and Elemental Analysis

The metal complexes that are formed are stable even when the temperature is kept at room temperature. Although they are insoluble in water, they are dissolvable in DMF and acetonitrile. The molar conductivity (10^–3 M in both DMF and acetonitrile solution) as well as the elemental analysis of the generated compounds is displayed in Table 1. The theoretical and experimental elemental analyses of metal complexes are very well in agreement with one another. The proposed formula of the title complexes, from the correlation of the elemental analyses data, are C₃₂H₂₄ClCrN₄O₅, C₃₂H₂₆MnN₄O₆, and C₃₂H₂₄ClFeN₄O₅, for the Cr(III), Mn(II), and Fe(III) complexes, respectively. The metal complexes do not have an electrolytic character, which may be deduced from the low molar conductivity values.

3.2.2. IR Spectra

The purpose of the infrared spectral collection was to achieve a deeper comprehension of the relationship that exists between the Az ligand and the metal ion. The most significant infrared bands that may be used to determine the ligand–metal combination that works best are outlined in Table 1 which can be found here. These bands are almost certainly the result of interactions between the coordinating sites of the ligand and the metal ions in the molecule.

At a frequency of 1542 cm^–1, the azo (−N=N−) band was identified. This band showed up in the IR spectra of the Az ligand after coordination with the metal ion, but it did so at a lower wave number, 1460–1470, as shown in Table 1. This indicates that coordination occurred with the azo-nitrogen.

In the infrared spectra of the unbound ligand, the phenolic–OH band could be seen anywhere between 3114 and 3218 cm^–1 in wavelength. The vibration phenolic–OH was similarly eliminated in the metal complexes, which lends credence to the theory that the phenolic oxygen of the ligand plays a role in the formation of the C–O–M bond during deprotonation;^44,45Table 1.

The broadband in the complexes was positioned at >3400 cm^–1; it was determined that this band was caused by −OH groups on water molecules.^46,47 In addition, the complexes have two additional spectral bands, which may be found at 478–485 and 544–551, respectively, and are referred to as (M–N)^48,49 and (M–O);^50,51Table 1.

Our research shows that the Azo-ligand, Az, functions as a mononegatively bi-dentate ligand, meaning that it generates complexes by way of both its azo-nitrogen (−N=N−) and phenolic oxygen (−OH) atoms. This is the conclusion that we have drawn from our data.

3.2.3. Magnetic Moment and Electronic Spectra Measurements

The UV–V of the Az ligand and its metal complexes were measured in acetonitrile between 200 and 800 nanometers. Electronic transitions that may be attributed to n → π* states were observed to take place in the unbound Az ligand when it was observed at 380 nm. When a metal ion was present, the band shifted to longer wavelengths than it normally would have been. With a view to determining the internal structure of the transition metal complexes, the effective magnetic moment, denoted by the formula μ_eff = 2.83 (((X_g*Mwt) – (dia magnetic correction*T))^0.5), was utilized as another method.

In the case of the CrAz₂ complex, the electronic spectrum band at 615 nm was connected to the transition of ⁴A_2g(F) → ⁴T_2g(F) in the octahedral geometry complex.⁵² The 3.84 B.M magnetic moment found for the CrAz₂ complex may be described by the occurrence of a d³ (t_2g³) electron configuration in an octahedral geometry around the Cr(III) center. The results from the electronic transitions are supplemented by an effective magnetic moment value (3.84 μ_B), which is within the experimentally acceptable range (3.70–3.90 μ_B) for Cr(III) complexes.⁵³

In the case of the MnAz₂ complex, the band in its electronic spectra that is located at 24390.24 cm^–1 may be ascribed to ⁶A_1g(F) → ⁴A_1g(G) transition, which hints at an octahedral geometry around the Mn(II) center;^54,55Table 1. The value of 1.96 B.M for the μ_eff of the MnAz₂ complex is indicative of the occurrence of a d⁵ low-spin (t_2g⁵) electron configuration in an octahedral geometry around the center of Mn(II);⁵⁶Table 1.

The electronic spectrum of FeAz₂ shows a transition band at 19,801.98 cm^–1. This visible band is assigned to the ligand-to-metal charge transfer (LMCT) band characteristic of octahedral iron(III) complexes. In general, high-spin Fe(III) complexes have magnetic moment values of around 5.90 μ_B with no possibility of orbital contribution, while low-spin complexes (S = 1/2) have values close to 1.70 μ_B.⁵⁷ The μ_eff value for FeAz₂ is found to be 1.84 μ_B, which is compatible with a return to the d⁵ (t_2g⁵) configuration;⁵⁸Table 1. This suggests that the complex has an octahedral geometry with Fe(III) as its core.

3.2.4. Metal Complexes Stoichiometry

The continuous variation method developed by Jobs was applied in the process of determining the chemical formula of metal complexes.⁵⁹ The detailed procedures for the Jobs method of continuous variation are listed in the Supporting Information. The absorbance curve that was generated by using this method demonstrated the highest level of absorption at a mole fraction of the ligand equal to 0.65. This finding hints that the complex was formed with a metal-to-ligand ratio of 1:2 (as seen in Figure 1), as was previously mentioned.

Figure 1.

(a) UV–Vis Spectral and (b) stoichiometry of the CrAz₂, MnAz₂, and FeAz₂ complexes.

3.2.5. Mass Spectra

The patterns of mass spectra are analyzed to determine the proportional amounts of the constituent parts of a substance. During the course of our investigation, we looked at the mass spectra of several metal complexes, as can be shown in Figure 2. The molecular ion peak, denoted by the symbol M+, can be found in the mass spectra of the CrAz₂, MnAz₂, and FeAz₂ complexes, and its values (631.87, 617.74, and 635.58) are excellent matches for the values that were predicted (631.5, 617, and 635.5). It is possible that the particular characteristics of the metal complexes are responsible for the presence of other peaks in the mass spectrum. The results of our analysis of the mass spectrum are in line with the previously established values for carbon, hydrogen, and nitrogen, as well as the hypothesized structure.

Figure 2.

Mass spectra and TG/ DTG curves of the CrAz₂, MnAz₂, and FeAz₂ complexes.

3.2.6. Thermal Analysis of the Prepared Complexes

It is necessary to ascertain the proportion of coordinated to uncoordinated water molecules in order to get an understanding of the structural transformations that take place in metal complexes as a result of heat treatment (Figure 2 and Table 2).^60,37,61

Table 2. Thermal Decomposition of the Prepared Complexes.

	TG (°C)	DTG (°C)	mass loss (%)		assignment	residue
			found	calculated
CrAz2	30–430	215	53.84	53.76	1H2O + C20H14O2Cl	Cr
	430–650	520	38.22	38.01	C12H8O2N4
MnAz2	30–420	295	52.29	52.19	2H2O + C20H14O2	Mn
	420–640	530	39.13	38.90	C12H8O2N4
FeAz2	30–440	255	53.24	53.42	1H2O + C20H14O2Cl	Fe
	440–680	540	37.91	37.77	C12H8O2N4

The first stage of deterioration was observed at temperatures between 30 and 430 °C, 30 and 420 °C, and 30 and 440 °C, with calculated weight loss percentages of (53.84 (53.76), 52.29 (52.19)), and (37.91 (37.77)) corresponding to the removal of 1H₂O + C₂₀H₁₄O₂Cl, 2H₂O + C₂₀H₁₄O₂, and 1H₂O + C₂₀H₁₄O₂Cl for CrAz₂, MnAz₂, and FeAz₂ complexes, respectively (Figure 2 and Table 2).

The 2nd degradation step was seen at temperatures ranging from 430 to 650 °C, 420 to 640 °C, and 440 to 680 °C, with calculated weight loss percentages of (38.22 (38.01)), (39.13 (38.90)), and (37.91 (37.77)), which corresponded to the elimination of C₁₂H₈O₂N₄ for both CrAz₂ and MnAz₂ complexes, respectively; Figure 2 and Table 2.

At the conclusion of the thermal degrading process, the metal remained as a metallic residue, as seen in Figure 2 and Table 2. Following analysis of the TG and DTG curves, it was concluded that none of the CrAz₂, MnAz₂, or FeAz₂ complexes contain any water molecules that contribute to their hydration despite the fact that the coordination spheres of the CrAz₂, MnAz₂, and FeAz₂ complexes each contain one, two, and one coordinated water molecules, respectively.

3.3. DFT Calculations

3.3.1. Geometry Optimization

The Az free ligand and its metal complexes are shown in Figure 3, each with their optimal structural representations. It was found that all of the CrAz₂, MnAz₂, and FeAz₂ complexes have hexa-coordinate geometry as [Cr(Az)₂(Cl)(H₂O)], [Mn(Az)₂(H₂O)₂], and [Fe(HPN)₂(Cl)(H₂O)]. The bond parameters (bond length, bond angles, and dihedral angles) of the free ligand and its metal complexes are found in the supplementary data file (Supporting Information data file).

Figure 3.

3D optimized structure of the subject compounds.

The properties in terms of total energy (E_Total), energy gap (ΔE), hardness (CH), and softness (S) were calculated for free ligand and compared with that in its metal complexes.

To perform a comparative study of the relative stability of the studied complexes, the binding energy (BE) or the complexation energy of the studied complexes was calculated, Table S1, as follows:⁶²

The more negative BE corresponds to the most stable complex. From the calculated values, Table S1, one can notice that all complexes are more stable than their corresponding ligands, and the order of the complex’s stability is [Fe(HPN)₂(Cl)(H₂O)] −692.261 eV) > [Mn(Az)₂(H₂O)₂] (−589.062 eV) > [Cr(Az)₂(Cl)(H₂O)] (−475.781 eV).

3.3.2. Frontier Molecular Orbitals (FMOs) and Reactivity Parameters

The FMOs, especially the HOMOs and the LUMOs, have been widely used to understand the chemical stability, optical, reactivity, and electrical features of various chemical systems.^63,64 The plots of the HOMO and LUMO are provided in Figure 4.

Figure 4.

HOMO–LUMO of the optimized structures.

In this particular piece of research, HOMO–LUMO energies were used to calculate a number of different properties, including the energy gap, ionization potential, electronegativity, electron affinity, chemical hardness, chemical potentials, softness, and electrophilicity index. The stability, biological activity, polarizability, reactivity, and hardness–softness of a molecule are all affected by the HOMO and LUMO orbitals.^65,66

It is also possible to forecast the reactivity of a molecule by making use of the energy gap that exists between these orbitals. As a consequence of this, molecules that have a lower ΔE are more conducive to the docking process (MnAz₂ > FeAz₂ > CrAz₂ > Az).

Hard acids react more frequently with strong bases, whereas soft acids react more frequently with weak bases, according to the hard-soft acid–base rule, which may be used to forecast the chance of one molecule interacting with another. In interactions with biological molecules, soft molecules are preferred over hard molecules, which is to be expected given the prevalence of soft molecules in living things. As a consequence of this, it is possible that soft molecules interact with biological molecules more effectively than hard molecules.^67,68 As a direct consequence of this, the ranking of MnAz₂, FeAz₂, CrAz₂, and Az in Table 3 is as follows: MnAz₂ > FeAz₂ > CrAz₂ > Az.

Table 3. Calculated E_HOMO (eV), E_LUMO (eV), ionization Potential (IP, eV), Electronegativity (EN, eV), the Energy Gap (ΔE, eV), Chemical Potential (CP, eV), electron Affinity (EA, eV), Softness (S, eV), Chemical Hardness (CH, eV), and Electrophilicity Index (EP, eV) of the Subject Compounds.

	EHOMO (eV)	ELUMO (eV)	ΔE (eV)	IP (eV)	EA (eV)	EN (eV)	CP (eV)	CH (eV)	S (eV–1)	EP (eV)
Az	–10.22	–1.17	9.05	10.22	1.17	5.69	–5.69	4.52	0.11	3.58
CrAz2	–14.10	–8.14	5.96	14.10	8.14	11.12	–11.12	2.98	0.34	20.76
MnAz2	–17.83	–12.35	5.48	17.83	12.35	15.09	–15.09	2.74	0.37	41.58
FeAz2	–10.26	–4.61	5.65	10.26	4.61	7.44	–7.44	2.83	0.35	9.78

The chemical potential and electrophilicity index of the compounds in question represent both the substances’ inherent stability as well as their predisposition for engaging in the electrophilic activity.^52,69

The electrophilicity and nucleophilicity indices are also crucial. The electrophilicity index indicates electron-accepting capacity, whereas the nucleophilicity index demonstrates electron-donating ability.^53,70 As an electrophile, the chemical reactivity ranks in the same direction as a growing electrophilicity index. Thus, in terms of chemical reactivity, MnAz₂ is more reactive as an electrophile, followed by CrAz₂, than FeAz₂ and finally the Az ligand.

3.3.3. Molecular Electrostatic Potential (MEP)

Electrostatic potential (MEP) is a measure of the force acting on a positive test charge (a proton) near a molecule due to the electrical charge distribution of the molecule’s electrons and nuclei. MEP can be used to visualize the charge distribution of molecules and how they interact with each other. MEP can also be used as a general and versatile indicator for electronic substituent effects in various chemical reactions,^71,72 A color-coded map is used in MEP representations to highlight the various charge values of the electronic potential that was present at the surface of the molecule. These charge values were observed. The region that is lacking electrons is depicted as a positively charged zone in blue, whilst the region that is abundant in electrons is depicted as a negatively charged region in red. The mapping MEP of the compounds under study were explored theoretically and is provided in Figure 5. In spite of the fact that the majority of the positive sites were found on the hydrogen atoms, while the negative charges were found on hetero oxygen and nitrogen atoms. This indicated that the ligand is an electron donor through the hetero oxygen and nitrogen atoms.

Figure 5.

MEP of the title compounds.

3.3.4. Natural Charge Analysis

Because they represent the physicochemical characteristics of a molecule (the electronic structure, vibrational spectra, dipole moment, polarizability, and other molecular properties), atomic charges play a significant role in molecules,^73,74 The atomic charges of the free ligand and its Cr(III), Mn(II), and Fe(III) complexes in the current investigation were calculated using NBO analysis at the B3LYP/cc-pVQZ level of theory in ethanol as the solvent. The findings are presented in Figure 6 and in the Supporting Information file.

Figure 6.

Plot of natural charge distribution of the free ligand and its Cr(III), Mn(II), and Fe(III) complexes computed using the B3LYP/cc-pVQZ level of theory in ethanol as the solvent.

Understanding electronegativity equalization and charge transfer in the chemical reactivity of the title molecules is made easier with the use of NBO analysis. The NBO analysis of the molecules reveals that carbon atoms in the free ligand and its Cr(III), Mn(II), and Fe(III) complexes contain both positive and negative charges. Positive carbons are seen for carbon atoms coupled to the electron-withdrawing oxygen and nitrogen atoms, as illustrated in Figure 6, and the values are tabulated in the Supporting Information file, including (C1, C15, C6, C13), (C32, C46, C1, C15, C6, C37, C13, C44), (C1, C32, C46, C15, C6, C37, C44, C13), and (C32, C1, C46, C15, C6, C37, C44, C13) atoms in the tiled free ligand and its Cr(III), Mn(II), and Fe(III) complexes, respectively. On the other hand, the other carbon atoms including carbon atoms (C3, C17, C16, C8, C10, C14, C9, C7, C18, C5, C2, C4), (C40, C7, C38, C14, C17, C48, C5, C9, C45, C10, C18, C36, C49, C41, C3, C16, C39), (C14, C40, C17, C38, C7, C18, C48, C10, C5, C3, C36, C45, C49, C8, C9, C41, C34), and (C3, C34, C17, C16, C49, C45, C36, C48, C38, C7, C18, C41, C10, C8, C39) have a negative charge in the tiled free ligand and its Cr(III), Mn(II), and Fe(III) complexes, respectively. More specifically, the C1 (0.36179 e), C32 (0.30866 e), C1 (0.31891 e), and C32 (0.34781 e) atoms are the largest positive carbons, while the carbon atoms C3 (−0.28405 e), C40 (−0.25088 e), C14 (−0.28139 e), C3 (−0.25998 e) atoms are the largest negative carbons, for the free ligand and its Cr(III), Mn(II), and Fe(III) complexes, respectively. All the hydrogen atoms in the free ligand and its Cr(III), Mn(II), and Fe(III) complexes have positive charges. Specifically, the H32 (0.50109 e), H65 (0.51891 e), H64 (0.50612 e), H63 (0.52542 e) atoms are the positive hydrogen atoms in the free ligand and its Cr(III), Mn(II), and Fe(III) complexes, respectively.

The net charges of the Cr(III), Mn(II), and Fe(III) ions in the Cr(III), Mn(II), and Fe(III) complexes are (1.4412 e), (1.51852 e), and (1.74594 e), respectively, whereas the valence of the free Cr(III), Mn(II), and Fe(III) ions is +3, +2, and +3, respectively. The decrease in the positive charge of the Cr(III), Mn(II), and Fe(III) ions in the Cr(III), Mn(II), and Fe(III) complexes was due to accumulation of the electronic charges from the associated coordinated ions.

3.3.5. NBO Analysis

The NBO study gives evidence for interactions between donors and acceptors and predicts the energy required for their stabilization with second-order perturbations.⁷⁵ In order to demonstrate the donor–acceptor interactions that take place between the HOMO and LUMO levels of the molecule, the NBO analysis was carried out using the B3LYP/cc-pVQZ method. Supporting Information data provide a summary of the natural population analysis that was calculated as well as the natural electronic configuration of the investigated compounds.

The NBO method is well recognized as a fast approach to learning about the features of the electronic structure. Donor–acceptor analysis is facilitated by this method, as is analysis of charge transfer, delocalization, and conjugative interactions in molecules.⁷⁶

The NBO analysis tool is a helpful method for investigating charge transfer or hyper-conjugative interactions, as well as for analyzing intra- and intermolecular bonding. The NBO quantitatively examines bonding and anti-bonding interactions caused by second-order disturbance by expressing the energy of these interactions as E⁽²⁾.⁷⁷⁻⁷⁹ To calculate the off-diagonal NBO matrix element E⁽²⁾ = ΔE_ij = qi(F(i,j)2/E_j – E_i), where E_i and E_j are the diagonal elements, qi is donor orbital occupancy and F_i,j is the NBO off-diagonal matrix element.

According to observations of perturbation energy E⁽²⁾ for various transitions between these donors and acceptors, the Supporting Information file, the following transitions for free ligands are extremely likely to occur; for free ligands; C1–C3 → C2–C4 (132.62 kJ/mol, π → π*), C13–C15 → C17–C18 (116.7 kJ/mol, π → π*), C13–C15 → C14–C16 (76.25 kJ/mol, π → π*), O19 → C13–C15 (27.81 kJ/mol, LP → π*), for Cr(II) complex; C7–C8 → C9–C10 (88.93 kJ/mol, π → π*), C35–C37 → C40–C41 (53.76 kJ/mol, π → π*), C1–C2 → C7–C8 (41.96 kJ/mol, π → π*), C4–C6 → C9–C10 (38.89 kJ/mol, π → π*), for Mn(II) complex; C1–C2 → C4–C6 (720.14 kJ/mol, π → π*), C32–C33 → C35–C37 (400.09 kJ/mol, π → π*), C34–C36 → C35–C37 (338.46 kJ/mol, π → π*), C44–C46 → C45–C47 (62.76 kJ/mol, π → π*), for Fe(III) complex; C13–C15 → C17–C18 (82.91 kJ/mol, π → π*), C13–C15 → C14–C16 (62.76 kJ/mol, π → π*), C44–C46 → C45–C47 (44.13 kJ/mol, π → π*), C44–C46 → C48–C49 (38.44 kJ/mol, π → π*), are the most probable transitions.

Using NBO calculations, significant knowledge on the structures and characteristics of the Cr(III), Mn(II), and Fe(III) complexes was collected. The NBO analysis of the compounds indicated substantial inter- and intramolecular interactions between the acceptor NBOs and the donor NBOs. These interactions were between the donor NBOs and the acceptor NBOs.

3.4. In Vitro Antimicrobial Activity

The newly synthesized compounds’ antibacterial and antifungal activity was tested against a diversity of types of bacteria and fungi strains, such as Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Aspergillus flavus, Bacillus cereus, Trichophyton rubrum, and Candida albicans. The purpose of these tests was to gain a better understanding of the newly synthesized compounds. The biological activity of each compound was evaluated by determining how much of an impact it had on the size of the inhibition zone (IZ). This zone serves as a measurement of how effective the chemical is in inhibiting the development of microorganisms. Table 4 demonstrates that the metal complexes had an inferior minimum inhibitory concentration (MIC) and a broader inhibition zone compared to the pristine ligand.

Table 4. Anti-Bacterial and Anti-Fungal Activity of the Titled Compounds in Terms of Inhibition Zone (IZ, mm) and Activity Index (%).

anti-bacterial activity
bacterial strains		Az	CrAz2	MnAz2	FeAz2
Pseudomonas aeruginosa	IZ	9	15	16	16
	%	50.00	83.33	88.89	88.89
	MIC	50	12.5	6.25	6.25
Escherichia coli (−ve)	IZ	9	17	18	17
	%	45.00	85.00	90.00	85.00
	MIC	25	6.25	6.25	6.25
Staphylococcus aureus (+ve)	IZ	8	15	15	15
	%	44.44	83.33	83.33	83.33
	MIC	25	12.5	6.25	6.25
Bacillus cereus (+ve)	IZ	9	14	15	14
	%	50.00	77.78	83.33	77.78
	MIC	50	6.25	6.25	6.25

anti-fungal activity
fungal strains		Az	CrAz2	MnAz2	FeAz2
Aspergillus flavus	IZ	9	16	16	16
	%	47.37	84.21	84.21	84.21
	MIC	25	12.5	6.25	6.25
Trichophyton rubrum	IZ	9	17	18	17
	%	40.91	77.27	81.82	77.27
	MIC	50	12.5	6.25	12.5
Candida albicans	IZ	9	17	17	17
	%	42.86	80.95	80.95	80.95
	MIC	50	12.5	6.25	6.25

The chelation hypothesis⁸⁰⁻⁸² proposes that metal ions have a positive charge that can be neutralized through the process of chelation or the creation of a complex with a ligand. This theory is one of several that have been proposed as potential explanations for this phenomenon. This electron delocalization across the ring might make the molecule more lipophilic, which would make it easier for it to enter the lipid bilayer that makes up the cell membrane. Once inside, the complex may cause the binding sites to become unstable and may change the metabolic pathways, which may ultimately result in the bacterium’s death.

The inhibition zone values of the newly found compounds are compared in Table 4 with those of the chloramphenicol antibiotic, which has a high activity index and is therefore expected to have a high inhibition zone value. Strong antibacterial activity was shown by the MnAz₂ complex against Escherichia coli (−ve), Pseudomonas aeruginosa (−ve), Bacillus cereus (+ve), and Staphylococcus aureus (+ve), respectively, with the activity index (%) of 88.89, 83.33, and 90.00, respectively. Additionally, it demonstrated significant antifungal effectiveness against Trichophyton rubrum, Aspergillus flavus, and Candida albicans, with activity indexes (%) of 81.82, 84.21, and 80.95, respectively.

The MIC also known as the lowermost dose of a drug that may prevent the growth of bacteria or fungus is frequently used as a starting point in larger preclinical trials of potential antibacterial drugs. These trials are often conducted in preparation for clinical testing. The MIC value was found by repeatedly diluting a sample until the desired concentration was reached. The MIC values that were calculated for the drugs being studied are presented in Table 4. This table demonstrates that the metal complexes had a lower MIC (about 6.25 ppm) than the free ligand did (approximately 50 ppm).

To make a comparison between the biological activity of the compounds that are the subject of this research and that of the metal complexes that have been described in the previous scientific literature,⁸³⁻⁸⁹ Supporting Information; Table S2. The findings indicated that the current compounds had significant values of biological activity which suggests that they could be effective antimicrobials.

3.5. Molecular Docking

To provide a deep understanding of the interactions between the synthesized complexes and target protein E. coli (PDB ID: 1hnj), we performed the molecular docking study by using the MOE program. The docking score (S) and hydrogen bonding (H) interactions were visualized, and interaction energies were calculated for the title complexes. The obtained energy results are presented in Table 5. Figure 7 demonstrates the structures of synthesized complexes interacting with amino-acid residues through some hydrogen bonding interactions. The significance of the metal ion and ligand is figured out by the energy values of hydrogen bonding interactions forming between the same amino-acid residues and noncoordinated parts of ligands around metal ions.^90,49

Table 5. Molecular Docking Data.

	ligand	receptor	interaction	distance	E (kcal/mol)	S (kcal/mol)	Ki (μM)
Az	O 19	THR 81	H-donor	2.85	–1.40	–6.55	16.17
CrAz2	C 5	LEU 191	H-donor	3.21	–0.80	–7.26	4.85
	O 65	LEU 191	H-donor	2.65	–5.10
MnAz2	O 20	GLY 06	H-donor	2.90	–0.90	–7.98	1.43
	O 65	ASN 193	H-donor	2.66	–12.60
	6-ring	PRO 192	pi–H	3.62	–0.70
FeAz2	O 52	THR 81	H-donor	2.90	–2.50	–7.88	1.69
	6-ring	ALA 09	pi-H	3.81	–0.60

Figure 7.

3D orientation of the substrate–protein complex.

The docking data showed that the active site of the protein had strong interactions with the substrates, including hydrophobic and hydrogen bonding interactions.⁹¹ After all the compounds were investigated, it was found that MnAz₂ had the greatest capacity to inhibit, followed by FeAz₂, CrAz₂, and Az. As can be seen in Table 5 and Figure 7, the most efficient molecule, MnAz2, interacted with the protein through the formation of hydrogen bonds as well as hydrophobic interactions.

The inhibition constant, often known as the Ki value, of a molecule, can be utilized to evaluate the drug’s potential as a hit, lead, or therapeutic candidate.^92,93 A higher Ki rating denotes a more powerful action, which is advantageous because of its increased potency. Throughout the course of this study, the Ki values for the compounds ranged anywhere from 1.43 for MnAz₂ to 4.85 for CrAz₂. According to these data, MnAz₂ appears to be a potential therapy method due to the fact that it has a low Ki value.

The findings indicate that molecular docking may be used to predict chemical binding affinity to an antimicrobial target protein and provide potential treatment choices. In general, these findings illustrate that molecular docking can be used. These findings will need to be validated via more research and the efficacy of these chemicals as potential medicines will also need to be evaluated.

4. Conclusions

In this work, we synthesized CrAz₂, MnAz₂, and FeAz₂ complexes and thoroughly analyzed them using a variety of spectroscopic and physicochemical methods. The Az ligand acted as a monobasic bi-dentate NO ligand with 1:2 molar ratios when attached to Cr(III), Mn(II), and Fe(III). Several analytical and spectroscopic techniques were employed to ascertain the geometric structures of the CrAz₂, MnAz₂, and FeAz₂ complexes. Our results showed that the CrAz₂, MnAz2, and FeAz₂ complexes all have octahedral geometries. To further our understanding of the properties of the CrAz₂, MnAz₂, and FeAz₂ complexes, we also used quantum chemistry methods to estimate their theoretically perfect molecular structures. As a result, we were able to foresee and verify the electronic structures of these complexes, which shed light on their prospective medicinal chemistry uses. In addition to analyzing the properties of the free ligand and its metal complexes, we looked into their potential antibacterial and antifungal effects. To test the effects of the free ligand and its metal complexes, we used pathogenic bacterial and fungal strains that are frequently found in Egypt’s polluted environments. Based on the results, metal complexes have the potential to be used as efficient antimicrobial agents as they are more active against bacteria than the free ligand. At last, we used molecular docking to examine whether or not the free ligand and its metal complexes were effective in restraining E. coli growth (PDB ID: 1hnj). By testing several compounds, we were able to see if any of them were able to bind to the receptor of interest and perhaps alter its function. One of the most promising strategies for inhibiting E. coli growth is the MnAz₂ complex, which has the highest affinity to the receptor. Our study has elucidated the structure and antimicrobial activity of CrAz₂, MnAz₂, and FeAz₂ complexes in great detail. Having this information can help researchers create and perfect these complexes for use in medicinal chemistry.

Data Availability Statement

The datasets supporting this article have been uploaded as part of the electronic Supplementary Material.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.3c01413.

• ¹H NMR of the AZ free ligand; ¹³C NMR of the AZ free ligand; binding energy of the studied complexes based on DFT/B3LYP with the cc-PVQZ basis set in ethanol as the solvent; comparison between the minimum inhibition concentrations (MIC) of the current compounds with previously reported compounds in the literature survey against E. coli; bond parameters (bond length, bond angles, and dihedral angles) of the free ligand and its metal complexes; summary of natural population analysis; NBO data of the ligand; summary of natural population analysis of the Cr(III) complex; NBO data of the Cr(III) complex; summary of Natural Population Analysis of the Mn(II) complex; NBO data of the Mn(II) complex; summary of natural population analysis of the Fe(III) complex; and NBO data of the Fe(III) complex (PDF)

Author Contributions

H.M.A.E.-L.: Investigation, Funding acquisition, Writing-original draft, Writing-review and editing. M.M.K.: Investigation, Methodology, Resources, Formal analysis, Data curation, Funding acquisition, Writing-original draft, Writing-review and editing. M.K.: Writing-original draft, Writing-review and editing. A.A.: Investigation, Supervision, Methodology, Resources, Formal analysis, Data curation, Funding acquisition, Writing-original draft, Writing-review and editing. A.A.A.: Investigation, Supervision, Methodology, Resources, Formal analysis, Data curation, Funding acquisition, Writing-original draft, Writing-review and editing. A.A.A.: Investigation, Supervision, Methodology, Resources, Formal analysis, Data curation, Funding acquisition, Writing-original draft, Writing-review and editing. All authors have read and agreed to the published version of the manuscript.

This work was funded by the Deanship of Scientific Research, at King Faisal University, Saudi Arabia (GRANT 3191).

The authors declare no competing financial interest.