Synthesis, Spectroscopic, Structural and Molecular Docking Studies of Some New Nano-Sized Ferrocene-Based Imine Chelates as Antimicrobial and Anticancer Agents

Abstract

The newly synthesized organometallic acetyl ferrocene imine ligand (HL) was obtained by the direct combination of 2-acetyl ferrocene with 2-aminothiophenol. The electronic and molecular structure of acetyl ferrocene imine ligand (HL) was refined theoretically and the chemical quantum factors were computed. Complexes of the acetyl ferrocene imine ligand with metal(II)/(III) ions (Cr(III), Mn(II), Fe(III), Co(II), Ni(II), Cu(II), Zn(II) and Cd(II)) were fabricated. They were inspected by thermal (DTG/TG), spectroscopic techniques (FT-IR, ¹H NMR, mass, UV–Vis), molar conductivity, and CHNClM to explicate their structures. Studies using scanning electron microscope (SEM) were conducted on the free acetyl ferrocene imine ligand and its Cd(II) chelate to confirm their nano-structure. To collect an idea about the effect of metal ions on anti-pathogenic properties upon chelation, the newly synthesized acetyl ferrocene imine ligand and some of its metal chelates were tested against a variety of microorganisms, including Bacillus subtilis, Staphylococcus aureus, Salmonella typhimurium, Escherichia coli, Aspergillus fumigatus, and Candida albicans. The ligand and its metal chelate were tested for cytotoxic activity in human cancer (MCF-7 cell viability) and human melanocyte cell line HBF4. It was discovered that the Cd(II) chelate had the lowest IC₅₀ of the three and thus had the prior activity. Molecular docking was utilized to investigate the interaction of acetyl ferrocene imine ligand (HL) with the receptors of the vascular endothelial growth factor receptor VEGFR (PDB ID: 1Y6a), human Topo IIA-bound G-segment DNA crystal structure (PDB ID: 2RGR), and Escherichia coli crystal structure (PDB ID: 3T88).

1. Introduction

Imine compounds are a vast class of organic compounds made by interacting primary amino compounds with aldehydes or ketones. They are frequently employed in industry and can catalyze the oxidation of organic molecules and polymers [1,2].

They are potentially capable of creating stable complexes with diverse metal ions particularly in the creation of imine metal chelates [3,4,5]. Imine ligands with sulfur and nitrogen donor atoms in their structures are effective metal chelating agents and have a wide range of therapeutic applications [6,7,8]. Imine compounds produced from 2-aminothiophenol, 2-aminophenol, 2-aminobenzoic acid, and 2-amino-3-hydroxy pyridine, in particular, have antipathogenic activity, inhibition of protein tyrosine phosphatases, and nuclease activity [9]. These activities could be owing to the inclusion of both hard and soft donor groups in a single ligand, which boosts the potential to coordinate with both hard and soft acidic metals [10].

The catalytic activity of acetyl ferrocene imine metal chelates has been investigated in a variety of processes, including oxidation, polymerization, ketones reduction, epoxidation, allylic alkylation, and Michael addition [11,12,13,14]. Acetyl ferrocene imine compounds represent an efficient class of compounds in the pharmaceutical and medicinal fields owing to their potential applications. Properties, such as antitumor, anticancer, antifungal, anticonvulsant, antibacterial, antitubercular, antimalarial, antioxidant, anti-HIV, and anti-inflammatory activities [11,15,16,17], demonstrate their importance.

Redox-active molecules with potential uses in fields such as molecular electronics materials [18,19] and physiologically active chemicals [20] have recently attracted a lot of attention. The use of ferrocenyl amines as a chelating agent for some transition metal ions has piqued the researchers’ curiosity in this study. This work aims to prepare a new Acetyl ferrocene imine derived from the condensation of 2-acetylferrocene and 2-aminothiophenol to prepare its complexes in first-row transition metal series, confirming their structure with some spectroscopic tools, elemental analyses, and thermal studies. Some theoretical studies (DFT and docking) were performed. Their biological applications as antimicrobial and anticancer agents were screened.

2. Experimental

2.1. Reagents and Materials

All of the chemical compounds utilized were analytical reagent grade (AR) and of the utmost precision. They included 2-aminothiophenol and 2-acetylferrocene, CdCl₂, FeCl₃.6H₂O, CrCl₃.6H₂O, CoCl₂.6H₂O, and MnCl₂.2H₂O which were provided from Sigma-Aldrich while ZnCl₂, CuCl₂.2H₂O, and NiCl₂.6H₂O were purchased from BDH, Merck, and Strem Chemicals, respectively. EtOH (90%), Et₂O, and (CH₃)₂NCH were utilized as organic solvents. In most cases, de-ionized water was used in all treatments.

2.2. Solutions

All Stock solutions of the prepared compounds employed in the current investigation and solutions for the anticancer study were mentioned in detail in Supporting Information.

2.3. Instruments

All instruments employed in the current investigation were mentioned in detail in Supporting Information.

2.4. Molecular Structure

The Gaussian03 program suite was used to establish the acetyl ferrocene imine ligand’s molecular structure [21,22]. They were completely refined using the LANL2DZ basis set and the DFT-based B3LYP approach. The TDDFT approach (together with the LANL2DZ basic set) was used to predict the electronic transition spectra of the ligand to evaluate the influence of the solvent around the molecule. The molecular orbital contribution to HOMO and LUMO was also computed.

2.5. Molecular Docking

MOE 2008 software, a constrictive molecular docking software, was used to assess the different binding interactions of the most active drugs versus the receptor of Vascular endothelial growth factor receptor VEGFR (PDB ID: 1Y6a), human Topo IIA-bound G-segment DNA crystal structure (PDB ID: 2RGR), and Escherichia coli crystal structure (PDB ID: 3T88) [23].

Gaussian03 software was used to generate the ligand structure in PDB format. The crystal structures of the Vascular endothelial growth factor receptor VEGFR (PDB ID: 1Y6a), human Topo IIA-bound G-segment DNA (PDB ID: 2RGR), and Escherichia coli (PDB ID: 3T88) were obtained from the database of proteins (http://www.rcsb.org./pdb, accessed on 1 April 2022).

2.6. Synthesis of Acetyl Ferrocene Imine Ligand

Refluxing a mixture of 2-acetyl ferrocene (20.86 mmol, 4.76 g) and 2-aminothiophenol (20.86 mmol, 4.76 g) dissolved in EtOH yielded the ferrocene imine ligand (HL). After swirling the mixture under reflux for about 4 h, a dark brown solid chemical was separated. After being filtered, recrystallized, and washed with Et₂O, it was vacuum-dried.

2.7. Synthesis of Acetyl Ferrocene Imine Metal Chelates

The acetyl ferrocene imine Cr(III), Mn(II), Fe(III), Co(II), Ni(II), Cu(II), Zn(II), and Cd(II) chelates were synthesized combinations of 1:1 molar mixture of hot EtOH solution (65 °C) of chloride salts (1.19 mmol) and the acetyl ferrocene imine ligand (HL) (0.4 g, 1.19 mmol). The resulting mixture was agitated for 1 h under reflux, after which the metal chelates separated. They were extracted through filtering and purified by washing with Et₂O multiple times.

2.8. Biological Activity

2.8.1. Anti-Pathogenic Activity

The antimicrobial activity of the compounds under investigation was screened according to the Well diffusion approach [24,25,26]. The detailed method was shown in the Supporting Information file. The biological activity values for the prepared compounds were obtained as means of duplicate experiments.

2.8.2. Optimization of Anticancer Study

The cytotoxicity of the prepared compounds against the MCF-7 cell line was investigated according to employed methods in the literature [25,27]. The detailed method was shown in the Supporting Information.

3. Results and Discussion

3.1. Identification of the Acetyl Ferrocene Imine Ligand

The organometallic acetyl ferrocene imine ligand (cyclopenta-2,4-dien-1-yl(2-(1-((2-mercaptophenyl)imino)ethyl)cyclopenta-2,4-diene-1-yl)iron) (HL) which conducted by the direct combination reaction between 2-acetylferrocene and 2-amino thiophenol in hot ethanol as solvent, is a dark brown solid powder, very stable in air, and soluble in the most common organic solvents, such as methanol, ethanol, DMF, and DMSO. The elemental analyses (C, H, N, S, and M) data with the molecular formula of the free organometallic ligand, HL, are presented in

Table 1. Analytical and physical data of organometallic acetyl ferrocene imine ligand and its metal chelates.

Compound (Molecular Formula)	Colour (%yield)	M.p. (°C)		% Found (Calcd.)					Λm Ω−1mol−1 cm2
			C	H	N	S	Cl	M
HL (C18H17NSFe)	Dark brown (85)	190	64.38 (64.40)	5.07 (4.93)	4.17 (4.20)	9.54 (9.95)	------	16.84 (16.50)	------
[Cr(HL)(H2O)3Cl]Cl2.2H2O (C18H27NO5SCl3CrFe)	Dark brown (86)	>300	36.99 (37.00)	4.62 (5.00)	2.40 (2.78)	5.50 (5.55)	18.54 (18.93)	18.58 (19.00)	138
[Mn(HL)(H2O)4]Cl2.2H2O (C18H29NO6SCl2MnFe)	Brown (84)	258–260	36.70 (36.73)	4.93 (5.03)	2.38 (2.82)	5.44 (5.65)	12.06 (12.56)	18.35 (18.55)	111
[Fe(HL)(H2O)3Cl]Cl2.3H2O (C18H29NSO6Cl3Fe2)	Brown (85)	>300	35.31 (35.45)	4.78 (5.00)	2.31 (2.30)	5.28 (5.68)	17.16 (17.20)	18.63 (18.96)	100
[Co(HL)(H2O)3Cl]Cl.2H2O (C18H27NO5SCl2CoFe)	Brown (70)	>300	38.95 (39.50)	4.87 (5.30)	2.52 (2.70)	5.77 (5.80)	12.40 (12.50)	20.65 (20.80)	78
[Ni(HL)(H2O)3Cl]Cl.2H2O (C18H27NSO5Cl2NiFe)	Dark brown (80)	237–240	38.88 (39.37)	4.86 (5.30)	2.52 (2.60)	5.76 (5.71)	12.38 (12.55)	20.70 (20.78)	82
[Cu(HL)(H2O)3Cl]Cl.H2O (C18H25NSO4Cl2CuFe)	Dark brown (90)	197–200	39.85 (40.00)	4.41 (4.50)	2.58 (2.90)	5.90 (6.00)	13.10 (13.70)	22.14 (22.50)	54
[Zn(HL)Cl2].2H2O (C18H21NSO2Cl2ZnFe)	Dark brown (75)	125–128	42.56 (42.23)	4.24 (4.60)	2.76 (2.42)	6.31 (6.50)	13.99 (14.00)	23.94 (24.28)	18
[Cd(HL)(H2O)2Cl2] (C18H21NSO2Cl2CdFe)	Dark brown (72)	157–160	38.05 (38.18)	3.39 (3.50)	2.52 (2.48)	5.77 (6.00)	12.10 (12.30)	30.39 (30.60)	4

. The experimental results were in good accordance with the suggested molecular formula, C₁₈H₁₇NSFe. The optimized structure of the free organometallic acetyl ferrocene imine (HL) ligand is represented in Figure S1. The FT-IR spectra of the organometallic acetyl ferrocene imine ligand,

Table 2. IR spectra (4000–400 cm⁻¹) of HL ligand and its metal chelates.

HL.	HLCr	HLMn	HLFe	HLCo	HLNi	HLCu	HLZn	HLCd	Assignment
-------	3408 br	3426 br	3401 br	3430 br	3410 br	3430 br	3437 br	3443 br	υ(OH)
2359 s	2348 s	2337 s	2376 s	2357 m	2369 w	2372 w	2367 w	2355 s	υ(SH)
1656 sh	1626 m	1628 m	1641 w	1644 sh	1634 m	1642 m	1616 m	1607 sh	υ(C=N)
-------	878 w, 1060 w	876 w, 970 w	843 s, 920 s	864 m, 990 s	825 w, 980 s	820 m, 883 w	826 m, 893 w	823 w, 881 w	υ(H2O)
748 m	751 w	750 s	748 w	752 w	751 w	752 w	755 m	748 sh	υ(C-S)
-------	608 w	562 w	602 w	613 w	615 s	617 w	624 w	614 sh	M-O stretch of coordinated water
-------	550 s	476 s	520 s	550 s	537 s	570 s	580 s	560 s	M-N
-------	450 s	461 s	489 s	470 m	450 s	468 w	491 w	495 w	M-S

, show a sharp strong absorption band at 1656 cm⁻¹ due to the imine group indicating the formation of the acetyl ferrocene imine ligand, while the absence of primary amine peaks at 3285 and 3267 cm⁻¹ of amino thiophenol and the υ(C=O) peak at about 1700 cm⁻¹ of acetyl ferrocene confirmed the ligand formation [28,29]. In addition, the IR spectrum shows a small band at 2359 cm⁻¹ and a medium band at 748 cm⁻¹ which was assigned to SH and CS vibrations, respectively [30]. The harmonic vibrations of the optimized structure have been analyzed theoretically. The vibrational frequencies found using quantum chemistry approaches such as DFT levels have long been known to have systematic inaccuracies. To rectify the effects of anharmonicity and the disregarded component of electron correlation, a scaling factor of 0.96 was applied for the LanL2DZ level [31,32]. Figure S2a,b represents the theoretical and experimental FT-IR spectra of HL which show characteristic bands at 2498 cm⁻¹ and 1669 cm⁻¹ due to υ(SH) and υ(C=N), respectively, that confirmed the experimental data. The disappearance of the NH₂ protons in the ¹HNMR spectrum indicated the formation of acetyl ferrocene imine ligand as there were no signals at (δ 8.34–8.96 ppm) [33,34].

Figure 1 shows a very clear peak in the mass spectrum of the acetyl ferrocene imine ligand at m/z = 334 g/mol, with a relative intensity of 4% which is very close to the expected molecular weight of 335.24 g/mol.

Geometrical Optimization of the Investigated Acetyl Ferrocene Ligand

At the DFT level of theory, the full configuration of the free acetyl ferrocene imine ligand HL was accomplished [35,36]. The optimized structure’s bond distances and angles are represented in Table S1. Figure S1 shows the fully optimized shape of the free ligand as well as the atom numbering.

Table 3. The different quantum chemical parameters of the free acetyl ferrocene imine ligand.

The Calculated Quantum Chemical Parameters
E (a.u.)	−884.28
Dipole moment (debey)	2.1158
EHOMO (eV)	−5.78
ELUMO (eV)	−1.58
ΔE (eV)	4.20
χ (eV)	3.68
η (eV)	2.1
σ (eV)−1	0.48
Pi (eV)	−3.68
S (eV)−1	0.24
ω (eV)	3.22
ΔNmax	1.75

shows the computed quantum chemical factors. Equations (1)–(8) were used to derive further characteristics, such as E, absolute electro-negativities, chemical potentials, Pi, absolute hardness, absolute softness, global electrophilicity, global softness, S, and additional electronic charge, N_max [37,38,39].

∆E = E_LUMO − E_HOMO

(1)

χ = −(E_HOMO + E_LUMO)/2

(2)

η = (E_LUMO − E_HOMO)/2

(3)

σ = 1/η

(4)

P_i = −χ

(5)

S = 1/2η

(6)

ω = P_i²/2η

(7)

∆N_max = −Pi/η

(8)

The electric and optical characteristics, as well as molecular electronic (UV–Vis) spectra and chemical reactions, are all influenced by the border molecular orbitals [40]. The energy variance, E, between the HOMO and LUMO levels reflects the ligand’s stability, and it has also aided in the development of theoretical models for describing various molecular systems’ structural and conformational barriers [41,42]. The predicted data showed that high softness indexes S and ω indicate that our HL is a soft ligand that is reactive and has flexible donation towards the cations.

The free ligand HL can accept electrons from metal ions, which decreases its energy and increases its stability; so, the electronic chemical potential is negative and the electrophilicity index is positive [43]. The high total energy negativity of the free ligand indicated its high stability.

3.2. Metal Chelates Characterization

3.2.1. Molar Conductance and Elemental Analyses Studies

The reaction of acetyl ferrocene imine ligand with metal ions gave metal chelates which were separated in good yield (more than 70%). The experimental results of elemental analyses of the complexes were compatible with their proposed compositions and confirmed the formation of metal chelates in a ratio of 1:1 (Metal: ligand) (Table 1). Some metal chelates showed high stability as they have a high melting point (>300 °C), other complexes had melting points ranging from 125 °C to 260 °C. The percent yield of the complexes is in the range of 70–90%. All metal chelates are dissolved in most organic solvents such as DMSO and DMF but insoluble in ethanol, methanol, and acetone. Molar conductivity of metal chelates showed that Cr(III), Mn(II) and Fe(III), Co(II), Ni(II), and Cu(II) chelates were electrolytes but Zn(II) and Cd(II) chelates were non-electrolytes (Table 1).

3.2.2. Vibration Spectra for the Prepared Compounds

The IR spectral data of acetyl ferrocene imine ligand HL and its metal chelates and their possible assignments are given in Table 2. The IR spectra of the metal chelates showed a broad peak in the range 3401–3443 cm⁻¹ which demonstrated that υ(OH) of coordinated water. Three distinct bands were seen in the free acetyl ferrocene imine ligand at 2359 cm⁻¹, 1656 cm⁻¹, and 748 cm⁻¹ due to υ(SH) [44], υ(C=N) [45,46], and υ(CS) [44] vibrations, respectively. In the metal chelates, these bands altered to be in ranges of 2337–2376 cm⁻¹ for υ(SH), 1607–1644 cm⁻¹ for υ(C=N), and 748–755 cm⁻¹ for υ(CS) which differ also in intensity. This shift indicated coordination with metal ions in the sites of nitrogen of the imine group and sulfur of thiophenol which resulted in the appearance of new bands M-S, M-N, and M-O (of coordinated water) in ranges of 450–495 cm⁻¹ [47], 476–580 cm⁻¹, and 562–624 cm⁻¹, respectively [48]. Also coordinated water showed bands at 820–878 cm⁻¹ and 881–1060 cm⁻¹ [49]. Based on these findings, the HL acted as a neutral bidentate ligand that coupled to metal chelates via NS sites.

3.2.3. HNMR Spectroscopic Studies

The ¹H NMR data for the free ligand and Zn(II) complex were recorded in DMSO-d₆ and showed four significant signals at (δ 1.05 ppm) [49], (δ 3.32 ppm) [30], (δ 4.23–4.77 ppm) [50], and (δ 5.42–7.06 ppm) [51] due to 3H of the methyl group of azomethine, SH group, 9H of acetyl ferrocene, and 4H of an aromatic moiety, respectively. The spectrum of Zn(II) complexes showed some variations from the ligand which explained the formation of new bonds with metal ions due to the shift of the signals of the azomethine methyl group, SH, 2-acetylferrocene, and aromatic moiety to (δ 1.25 ppm), (δ 3.48 ppm), (δ 4.18–4.96 ppm), and (δ 6.42–7.96 ppm), respectively.

3.2.4. UV–Vis Absorption Studies

Molecular transition spectra of the free acetyl ferrocene imine ligand and its metal chelates were recorded in (ethanol: DMF) solution in equal ratios. The absorption spectra for the free ligand (HL) exhibit two main peaks, the first appears at 236 nm which was ascribed to benzene and ferrocene π-π* transitions and the second peak appears at 306 nm which was assigned to the azomethine n-π* transitions [52]. These bands moved in metal chelates to 233 nm for π-π* sulfur atom transitions and 287–307 nm for n-π* transitions. In addition, there is the appearance of new bands at 537–630 nm which indicated d-d splitting. Theoretically, the two main absorption bands at 236 and 325 nm (in DMF) are in agreement with the experimental spectrum (Figure S3a,b). The transitions of HOMO-1 to LUMO + 4 with excitation energy of 5.86 eV give rise to the higher energy band at 236 nm. The lower energy band, which occurred at 325 nm and showed a transformation from HOMO to LUMO + 2 with excitation energy of 5.14 eV, is depicted in

Table 4. Main UV-calculated optical transitions with composite ion in terms of molecular orbitals of free ligand.

Compound	Transition	Excitation Energy (eV)	λmax Calcd. nm/(eV)	λmax Exp. nm/(eV)
HL	HOMO→LUMO + 2 (48.9%)	5.14	325 (3.81)	306 (4.06)
	HOMO-1→LUMO + 4 (66.1%)	5.86	236 (5.24)	236 (5.26)

, and Figure 2 illustrates the molecular orbital transitions.

3.2.5. Thermal Analysis

Thermal degradations were performed under an air environment at a rate of heating of 10 °C min⁻¹ from ambient temperature to 1000 °C.

Table 5. Thermoanalytical results (DTG/TG) of the organometallic acetyl ferrocene imine ligand and its metal chelates.

Complex	TG Range (°C)	DTGmax (°C)	n *	Mass Disposal Total Mass Disposal Found (Calcd)%	Assignment	Residues
HL	90–1000	272, 317	2	46.59 (47.39) 47.86 (47.39)	- Removal of C8H17NS.	Fe + 10C
HLCr	40–500	71, 250	1	38.05 (38.27)	- Removal of 3HCl, C6H6, and 2H2O. - Removal of C2H16NSO0.5.	½Cr2O3 + FeO + 10C
				16.31 (16.09)
	500–1000	882	1	54.88 (54.02)
HLMn	15–100 100–245 245–1000	56 109, 160 252	1 2 1	7.01 (6.32) 15.31 (15.72) 27.48 (27.48) 50.07 (49.52)	- Removal of 2H2O. - Removal of 2H2O, HCl and NH3 - Removal of C6H17SCl.	MnO + FeO + 12C
HLFe	45–130 130–1000	67 179, 606	1 2	7.74 (8.90) 44.99 (44.11) 52.98 (51.57)	- Removal of 3H2O. - Removal of C7H23Cl3NSO0.5.	½Fe2O3 + FeO + 11C
HLCo	10–100 100–1000	85 125, 269	1 2	4.27 (3.25) 41.41 (42.20) 46.72 (45.47)	- Removal of H2O. - Removal of 2H2O and C5H21NSCl2.	CoO + FeO + 13C
HLNi	45–125 125–1000	81 369, 876	1 2	7.52 (6.48) 41.17 (41.04) 51.12 (52.48)	- Removal of 2H2O. - Removal of H2O and C6H21NSCl2.	NiO + FeO + 12C
HLCu	30–120 120–240 240–1000	92 140 410	1 1 1	4.05 (3.32) 9.38 (10.06) 26.64 (27.58) 40.06 (40.96)	- Removal of H2O. - Removal of H2O and HCl. - Removal of C4H20NSCl.	CuO + FeO + 14C
HLZn	20–170 170–360 360–1000	92 318 558, 931	1 1 2	11.30 (10.54) 12.71 (12.81) 36.29 (36.95) 60.26 (60.30)	- Removal of H2O and NH3. - Removal of C5H5. - Removal of C9H12SCl.	ZnO + FeO + 4C
HLCd	95–530 530–1000	196, 259 569, 677	2 2	30.76 (30.84) 32.65 (33.00) 63.51 (63.84)	- Removal of NH3, CH4 and 2HCl. - Removal of C5H5. - Removal of C12H7S.	CdO + FeO

n * = number of decomposition steps.

and Figure S4 summarize the degradable segments against different temperature ranges. The free acetyl ferrocene imine ligand was decayed in two stages at T_s = 272 and 317 °C with a total loss of mass of 47.86% (predicted = 47.39%) and the remaining contaminated FeO residue. The Cr(III) complex demonstrated two degradation stages. The first occurred in the temperature range 40–500 °C, with a found loss of mass = 38.05% (predicted = 38.27%) due to loss of two water molecules, three HCl molecules, and a benzene molecule. The second degradation stage occurred at T_s = 882 °C with a found loss of mass = 16.31% (predicted = 16.09%) due to loss of C₂H₁₆NSO₀.₅ moiety leaving ten carbon atoms contaminating metals oxides as residues.

The Mn(II) complex decayed through four stages; firstly, removal of water of crystallization at T_s = 56 °C; next, two degradation stages occurred at 100–245 °C in which 2H₂O, HCl, and NH₃ molecules were lost with the found loss of mass of 15.31% (predicted = 15.72%). Finally, the last degradation stage occurred at temperature maxima of 252 °C due to the loss of C₆H₁₇SCl moiety of the ligand leaving MnO, FeO, and 12C as residues with a total found loss of mass of 50.07% (predicted = 49.52%). For [Fe(HL)(H₂O)₃Cl] Cl₂.3H₂O, it showed three degradation stages; the 1st stage occurred at T_s = 67 °C indicating a loss of three molecules of water of crystallization. The 2nd and 3rd degradation stages took place in the range of 130–1000 °C at which C₇H₂₃Cl₃NSO₀.₅ molecules were lost with the found loss of mass of 44.99% (predicted = 44.11%), leaving contaminated iron oxides. The Co(II) complex TG curve showed three degradation stages where the first stage occurred in a temperature range of 10–100 °C with a found loss of mass of 4.27% (predicted = 3.25%) due to loss of hydrated water molecule. The last two stages occurred in the range of 100–1000 °C at which two molecules of coordinated water and the organic moiety of the ligand were lost leaving cobalt and iron oxides residues contaminated with carbon with a total loss of mass (found = 46.72%, predicted = 45.47%). In the temperature range 45–125 °C, the TG curve of the [Ni(HL)(H₂O)₃Cl] Cl.2H₂O complex demonstrated a peak at 81 °C, equivalent to a weight loss of 7.52% (found weight loss = 6.48%) related to the removal of two hydrated water molecules. The loss of the water molecule and C₆H₂₁NSCl₂ fragment may be attributed to the second and third phases of thermal breakdown, which occurred in the range 125–1000 ºC with two maxima at 369 and 876°C (found 41.17%; predicted 41.04%). At the end of the TG curve, NiO and FeO have remained as residues with carbon contamination. The Cu(II) complex produced a three-stage breakdown pattern from 30 °C to 1000 °C. The first stage was a single-stage between 30 and 120 °C, with a high temperature of 92 °C, signifying the loss of hydrated water molecules with a loss of mass of 4.05% (predicted = 3.32%). Within the temperature range of 120–240 °C and with a temperature maximum of 140 °C, the second stage was similar to the first stage, representing the loss of H₂O and HCl molecules with a loss of mass of 9.38% (predicted = 10.06%). The final stage was to reflect the loss of the C₄H₂₀NSCl fragment with a loss of mass of 26.64% (predicted = 27.58%) between 240 and 1000 °C. At the end of the thermogram, contaminated Cu(II) and Fe(II) oxides were residues. On the other hand, The [Zn(HL)Cl₂].2H₂O complex describes four stages of breakdown. The complex loses H₂O (hydrated) and ammonia gas with a found loss of mass of 11.30% (predicted = 10.54%) in the first stage in the temperature range of 20–170 °C, with one maximum at 92 °C. The second stage occurred at 170–360 °C with T_s = 318 °C, in which the complex loses the C₅H₅ molecule with a found loss of mass of 12.71% (predicted = 12.81%). The final two stages at 360–1000 °C correspond to the breakdown of C₉H₁₂SCl with a loss of mass of 36.29% (predicted = 36.95%). Carbon-contaminated ZnO and FeO were left as the degradation residue. The overall weight loss amounted to 60.26% (predicted = 60.30%). From 95 to 1000 °C, the Cd(II) complex disintegrated in four phases as follows. The first two phases were carried out at 95–530°C, resulting in the loss of NH₃, CH₄, C₅H₅, and 2HCl molecules, with a loss of mass of 30.76% (predicted = 30.84%). The final stages described the breakdown of the C₁₂H₇S molecule with a loss of mass of 32.65% (predicted = 33.00%) with T_s = 569 and 677 °C. Finally, FeO and CdO were the residues with a total found loss of mass of 63.51% (predicted = 63.84%). The presence of carbon contamination with the residues of the ligand and all complexes except the Cd(II) complex can be explained by the high stability of these compounds as they cannot be decomposed completely up to 1000 °C.

Carbon residues were found in practically all metal chelates with metal oxides, which might be explained by the high stability of the complexes including ferrocene derivatives. Complexation with metals strengthened their stability, thus, they could not be totally dissolved at high temperatures.

3.2.6. SEM Inspection

Scanning electron microscope (SEM) images of the ligand and Cd(II) complex at 50,000× magnifications indicated the existence of these compounds at nanoscale (Figure 3a,b). The free ligand appeared as a coral reef structure in size 49.45 nm, while upon complexation with Cd(II), it converted to a spongy shape with size 15.69 nm. These data confirmed that the surface morphology of the complex differs from that of the ligand and this can account for the binding of ligand to Cd(II) ion as it causes changes in the skeleton of the ligand. The presence of the ligand and Cd(II) complex in nanosize can be assigned to the improved biological and anticancer activities.

3.2.7. Structural Manipulation

Elemental studies, IR, ¹H NMR, mass and UV–visible spectra, molar conductivity, SEM, and thermal investigations (DTG/TG), were used to confirm the structures of the free organometallic acetyl ferrocene imine ligand (HL) and its metal chelates. As illustrated in Figure 4, the suggested structural formulas of the investigated metal chelates were described as three types of coordination (8), and their general formulas were [M(HL)(H₂O)₃Cl]Cl_x.nH₂O (M = Cr(III); x = 2, n = 2), (M = Fe(III); x = 2, n = 3), (M = Co(II) and Ni(II); x = 1, n = 2) and (M = Cu(II), x = n = 1), [M(HL)(H₂O)_xCl₂].nH₂O (M = Zn(II); x = 0, n = 2), (M = Cd(II); x = 2, n = 0) and [Mn(HL)(H₂O)₄]Cl₂.2H₂O.

3.3. Antimicrobial Efficiency

The newly synthesized acetyl ferrocene imine ligand, Cr(III), Mn(II), Fe(III), Co(II), Ni(II), and Cu(II) chelates were screened for their antifungal and antibacterial activities against (Aspergillusfumigatus and Candidaalbicans) and (Staphylococcusaureus and Bacillussubtilis, gram-positive, Salmonellatyphimurium and Escherichia coli, gram negative), respectively. The results are represented in Table S2 and Figure 5. The free ligand showed the highest antifungal activity (18 and 16 mm/mg), followed by Mn(II) complex which had inhibition zone diameter values of 17 and 13 mm. Co(II) and Cu(II) complexes showed high antibacterial activities, and the values can be arranged as follows:

• For Staphylococcus aureus Co(II) > Cu(II) but for Bacillus subtilis Cu(II) > Co(II).
• For Salmonella typhimurium Co(II) > Cu(II) but for Escherichia coli Cu(II) > Co(II).

It was found that the free ligand had no antibacterial activities against Staphylococcus aureus, Bacillus subtilis, and Salmonella typhimurium, while some complexes had activities. In addition, it is observed that free ligand had activity against E. coli is lower than some complexes, this can be explained.

The enhancement in the antibacterial activity of the investigated acetyl ferrocene metal chelates compared to acetyl ferrocene imine ligand can be supported by Overtone’s concepts [53,54,55]. Because chelation strengthens the ligand’s ability to operate as more effective and potent bactericidal agents by limiting bacterial growth, metal complexes were discovered to have higher zones of inhibition than free ligands. Because of the partial sharing of positive charge on metal with the hetero donor atoms of the ligand and also due to electron delocalization over the entire chelate ring system, chelation diminishes the polarity of the metal ion significantly. Lipids and polysaccharides are essential components of the cell wall and membranes, and they are excellent for metal–ion interaction. Apart from that, the cell walls contain phosphates, carbonyl, and cystenyl ligands that help maintain the membrane’s integrity by acting as a diffusion barrier and also provide adequate support [56].

The differences in metal complex action against different bacteria are due to differences in the ribosomes of the microbial cells or the impermeability of the microbes’ cells. The reduced activity of complexes relative to others could be due to limited lipid solubility, preventing the metal ion from reaching the cell wall’s favorable site of action and interfering with normal cell activity. Although chelation plays an important role in evaluating antibacterial activity of complexes, other variables, such as solubility, size, dipole moment, coordinating sites, redox potential of metal ions, solubility, bond length between metal and ligand, geometry of complexes, steric, pharmacokinetic, concentration, and hydrophobicity also play a role. As a result of the findings, it is clear that increased antibacterial activity of metal complexes may be attributable to a complex combination of various additional mechanisms in addition to chelation [57].

According to the equation, activity index (A) = ((Inhibition zone of compound (mm)/Inhibition zone of the standard drug (mm)) × 100) [57,58], the activity indexes of the tested substances were computed and plotted in Figure S5a,b. The figures show that the free ligand and Mn(II) complex had high activity indexes, while Ni(II) complex had no activity index.

3.4. Anticancer Activities

The efficiency of the free organometallic acetyl ferrocene imine ligand and its metal chelates as a potential antitumor drug has been tested in vitro on the MCF7 cell line (derived from human breast cancer) and HBF4 cell line (derived from normal human melanocyte). The results for MCF7 expressed as the concentration of the complex required to inhibit the tumor cell growth by 50% (IC₅₀) are recorded in Table S3 and these data are represented in Figure 6. Only four complexes were found to have a certain inhibitory effect of >70%, while the free ligand and other complexes could not inhibit >70% of cancerous cells. Mn(II), Cu(II), Zn(II), and Cd(II) complexes have MCF7 inhibitory effect while the most potent with the lowest IC₅₀ value (5.57 µg/mL) was Cd(II) which showed the most potent cytotoxic results versus the cell line which may be revealed to its nano-size and morphology. In addition, Co(II), Cu(II), Zn(II), and Cd(II) chelates demonstrate surviving fractions 52–62% when tested for HBF4 cell line at concentration 100 µg/mL. By comparing IC₅₀ of the Cd(II) complex with that of 3-(butylamino)-2-[(E)-2-(2-methoxyphenyl) ethenyl]-4H-chromen-4-one compound [59], we found that Cd(II) (5.57 µg/mL) was three times lower than the other compound (15.62 µg/mL) which had high cytotoxic effect. This provides motivation to continue studies on our complex to develop a new antibreastic cancer drug.

3.5. Molecular Docking

For the purpose of rational drug development and discovery, molecular docking is a vital tool for predicting the most preferred mode of binding of a small molecule to its targeted protein, between the targeted molecule (receptor) and the desired compounds (such as ligand and its metal chelates) with low binding free energy but high binding affinity. The MOE 2008 program suite was used to dock the free organometallic ligand HL and the Cd(II) chelate with the vascular endothelial growth factor receptor VEGFR (PDB ID: 1Y6a), the crystal structure of human Topo IIA-bound G-segment DNA (PDB ID: 2RGR), and crystal structure of Escherichia coli (3T88).

Antibodies (inhibitors) to the vascular endothelial growth factor receptor (VEGFR) are important in the treatment of a variety of malignancies [60]. As a result, VEGFR-2 has been chosen as the biological target for docking investigations on active molecules.

Table 6. Energy values obtained in docking calculations of HL with crystal structure of (1Y6a), the receptors of (2RGR), crystal structure of Escherichia coli (3T88).

Receptor	Ligand Moiety	Receptor Site	Interaction	Distance (°A)	E (kcal/mol)
1Y6a	C16	O GLU 915 (A)	H-donor	3.33	−0.7
	O4	ND2 ASN 921 (A)	H-acceptor	3.04	−2.6
	5-ring	CD1 LEU 838 (A)	pi-H	4.07	−0.8
	6-ring	CG1 VAL 846 (A)	pi-H	3.83	−0.8
	S35	NZ LYS 866 (A)	H-acceptor	3.61	−1.3
	6-ring	CG2 VAL 846 (A)	pi-H	4.14	−0.8
2RGR	S35	O GLN 527 (A)	H-donor	10.14	−0.8
	Fe9	OD2 ASP 528 (A)	metal	7.11	−1.9
	C15	MG MG 1 (A)	ionic	8.28	−1.6
	6-ring	CG LYS 603 (A)	pi-H	6.87	−0.9
	6-ring	CE LYS 603 (A)	pi-H	8.13	−1.2
3T88	N20	NH1 ARG 230 (A)	H-acceptor	3.06	−1.1
	5-ring	CB ASP 67 (A)	pi-H	4.22	−0.8

shows the binding energies of the free ligand HL and

Table 7. Energy values obtained in docking calculations of Cd(II) chelate with crystal structure of (1Y6a), the receptors of (2RGR), crystal structure of Escherichia coli (3T88).

Receptor	Ligand Moiety	Receptor Site	Interaction	Distance (°A)	E (kcal/mol)
1Y6a	S35	OD1 ASN 1031 (A)	H-donor	3.53	−1.6
	O43	O ARG 1030 (A)	H-donor	2.74	−1.2
	O43	OD1 ASN 1031 (A)	H-donor	2.89	−14.3
	Fe9	O LEU 838 (A)	metal	2.28	−2.8
2RGR	O43	OE2 GLU 808 (A)	H-donor	2.81	−20.7
	O43	OD1 ASP 809 (A)	H-donor	2.79	−3.8
	O43	OD2 ASP 809 (A)	H-donor	3.54	−0.9
	N20	OE2 GLU 808 (A)	ionic	3.22	−3.2
	S35	OD1 ASP 809 (A)	ionic	3.99	−0.5
	O40	OD1 ASP 809 (A)	ionic	3.51	−1.9
	O43	OE2 GLU 808 (A)	ionic	2.81	−5.9
	O43	OD1 ASP 809 (A)	ionic	2.79	−6
	O43	OD2 ASP 809 (A)	ionic	3.54	−1.7
3T88	S35	O ASP 67 (A)	H-donor	3.65	−0.7
	S35	OD2 ASP 67 (A)	H-donor	3.11	−5.2
	S35	OD1 ASN 68 (A)	H-donor	3.14	−2.3
	O40	OD1 ASN 68 (A)	H-donor	2.87	−10.1
	O43	OD1 ASP 67 (A)	H-donor	2.96	−13.4
	O43	OD2 ASP 67 (A)	H-donor	2.61	−7.1
	N20	OD1 ASP 67 (A)	ionic	3.72	−1.2
	S35	OD2 ASP 67 (A)	ionic	3.11	−3.8
	O43	OD1 ASP 67 (A)	ionic	2.96	−4.8
	O43	OD2 ASP 67 (A)	ionic	2.61	−7.6

shows the binding energies of Cd(II) complex. From these data, it was found that the Cd(II) complex with binding energy −14.3 kcal/mol is more favorable than the free ligand (−2.6 kcal/mol). It is also more potent than the 2-(4-Benzothiazol-2-yl-phenylimino)-5-(4-nitro-benzylidine)-thiazolidin-4-one compound which had a binding energy of −13.3 kcal/mol [60].

This research was also expanded to look at the mechanism of action of our drugs with the human DNA topoisomerase II enzyme. For medicinal chemists working on anticancer medicines, DNA topoisomerases were a key molecular target. The free organometallic ligand bonded to the enzyme via hydrogen, ionic, and metallic bonds with higher positive binding energy (−1.9 kcal/mol) than the Cd(II) complex which exhibits a binding energy of −20.7 kcal/mol [61,62].

On studying binding of HL and its Cd(II) chelate with the crystal structure of E.coli, it was found that both compounds were bounded by H-bonds with E.coli receptor and upon complexation, the binding energy became more negative. Figure 7 shows 3D interactions of both the free ligand and Cd(II) chelate.

From the previous data, it was concluded that the Cd(II) complex had the lowest binding energy with 2RGR receptor (−20.7 kcal/mol) which indicated that human DNA topoisomerase IIα enzyme was the most effective binding protein.

4. Conclusions

The metal chelates of Cr(III), Mn(II), Fe(III), Cu(II), Ni(II), Co(II), Zn(II), and Cd(II) were synthesized from the bi-dentate ligand derived from condensation of 2-acetyl ferrocene with 2-aminothiophenol (HL) and characterized using different spectroscopic methods. The ligand acted as neutral bidentate (NS) ligand, and all complexes showed octahedral geometry except Zn(II) was tetrahedral. All complexes are electrolytes, except Zn(II) and Cd(II) complexes, which are non-electrolytes, according to molar conductivity data. The complexes have M(HL)-type compositions with generic formulae, according to elemental analysis data, [M(HL)(H₂O)₃Cl] Cl_z.nH₂O (M = Cr(III); x = 2, n = 2), (M = Fe(III); x = 2, n = 3), (M = Co(II) and Ni(II); x = 1, n = 2) and (M = Cu(II), x = n = 1), [M(HL)(H₂O)_xCl₂].nH₂O (M = Zn(II); x = 0, n = 2), (M = Cd(II); x = 2, n = 0) and [Mn(HL)(H₂O)₄]Cl₂.2H₂O. SEM graphs showed our ligand and the Cd(II) complex in nanosize with different shapes. The antimicrobial test referred that the HL had the highest antifungal activity while Co(II) and Cu(II) complexes had high antibacterial activities. Furthermore, cytotoxicity of the Cd(II) complex revealed that it has better anticancer activity than the others, with an IC₅₀ of 5.57 g/mL, suggesting that it could be used in clinical trials or as a biological agent.