Mixed ligand complex: Synthesis, characterization, and investigation of biomedical activity

A new complex, CuCl2.2H2O (M), was synthesized to react with melatonin (L1) and one of the amino acids (L-cysteine, L2) in a mole ratio of (1:1:1) (M: L1:L2) to produce the metal complex. The synthesized complex was then characterized using spectral techniques including infrared (FT-IR), thermal analysis (TG), flame atomic absorption (AAS), micro elemental analysis (CHNS), melting point (m.p.) measurement, and determination of chloride. Octahedral geometry was proposed for the complex. The copper complex was paramagnetic and non-electrolyte. The synthesized complex was evaluated as an antibacterial and antifungal agent against Escherchia coli (G) and Staphylococcus aureus (G+). fungus Candida. The results showed that the copper complex was more active in 10–3 M than the two ligands; the complex's anti-cancer properties were also evaluated and gave a good result in the test.


Introduction
According to recent research, mixed ligand complexes play an important function in biological systems 1 , the main method for adjusting the properties of transition metal ions to achieve the intended applications involves the use of various ligands with different structures and properties 2 .The mixed ligand complexes can be di ligand, tri ligand, tetra ligand, or multi ligand complexes depending on the number of ligands linked to the metal ion.The development of mixed ligand complexes and their characteristics can be critical in terms of the kinetic impact 3 .
Transition ion complexes have a wide range of commercial and technical uses, including antibacterial, antifungal, and anticancer medications, as well as catalysts.The metal atom itself may play a variety of functions in these complexes, depending on its oxidation state, coordination geometry, and magnetic, electronic, and photochemical properties 4 .It has been proved clearly that new chemical ligands show improvement as physiologically active 5 .One type of ligand in the compound increases the chances of variation in the expected properties of the compound 6 .
Many biological processes require organic molecules with pyridine rings.So complexes containing the pyridine ring (cyclic nitrogen) have been shown to have high anticancer efficacy as well as tumor size reduction 1 .
Published Online First: https://doi.org/10.21123/bsj.2024.8582P-ISSN: 2078-8665 -E-ISSN: 2411-7986 Baghdad Science Journal Parkinsonism, Alzheimer's disease, and cancer.Melatonin also plays several physiological roles in humans.Exogenous MLT may also decrease agerelated oxidative processes and act as a skin UV radiation protector in addition to its other possible applications.Although being applied topically in cosmetic goods, it also acts as a skin protectant against UV rays 8,9 .Melatonin is a circadian rhythmregulated and multifunctional molecule that plays a neuroprotective role against the pathogenesis of AD 10 , Parkinson's illness 11 Breast cancer, depression, and glaucoma, breast and prostate cancer 5 .
Cysteine (Cys), the main sulfur-containing amino acid that contains sulfur, is a semi-essential amino acid since it may be taken from food or synthesized through the transsulfuration path during the hydrolysis of methionine.It is believed to act as a biomarker of sulfur-containing amino acids in the mammalian diet 12 , It becomes essential.
Transsulfuration is a metabolic mechanism in the liver that allows cysteine to be supplied by converting a necessary amino acid: methionine 13 .
Cysteine is considered a proteinogenic amino acid since it serves as a building component for about 2% of proteins and plays an essential role in biological processes in our bodies.Several key metabolic processes are catalyzed by it 12 .Cysteine residues provide a variety of roles in proteins.In eukaryotes, the sulfur atom in cysteine, which is oxidatively sensitive and highly ionizable, responds to redox conditions and the pH of subcellular organelles 14 , Cysteine is oxidized at the thiol group (-SH), which can form a covalent bond by reacting with free radicals and other groups 15 Cysteine acts as an antimicrobial to heal damaged skin 16 .It involves lipid biosynthesis 15 , Iron-sulfur biosynthesis14 is an essential component of skeletal muscles and a source of taurine, glutathione, and coenzyme A 17 .
In this research, a mixed ligand copper complex was prepared and its biological and toxic activity was studied.

The chemical and apparatus
All chemicals were used exactly as provided, with no additional purification.CHNS Elemental Analyzer Euro EA 3000/Italy was used to record micro elemental analyses (CHNS).The melting points of all compounds were determined using the Gallenkamp melting point instrument.The FTIR (Fourier Transform Infra-Red) spectra were obtained using a SHIMADZU 8400 s spectrophotometer for ligands in the (4000-400) cm -1 range with KBr and complexes in the range 4000-250 cm -1 with CsI.The electronic spectra (Uv-Vis).Thermal analysis (TG) was recorded by METTLER TA 4000 SYSTEM).The metal content was determined using a Nova350 spectrophotometer and flame atomic absorption spectroscopy.The Mohr technique was used to determine the chloride concentration in the complexes.Bruker 400 MHz NMR spectrometer was used to measure 1 H-NMR spectroscopy in d-DMSO 6 .

Synthesis Cu (II) complex
A mixed ligand complex was prepared from copper salts as chloride (CuCl2.2H2O),melatonin (L1) as a primary ligand, and cysteine (an amino acid) (L2) as a secondary ligand.
To an aqueous solution (10 ml) of (0.134g, 1 mmol) Cu(II), an aqueous solution (10 ml) of melatonin (0.232 g, 1 mmol) containing NaOH (0.04 g, 1 mmol) was added.The reaction mixture was stirred and kept in a boiling water bath for 10 minutes.To this hot solution, an aqueous solution (5 ml) of cysteine (0.121g, 1 mmol) was added with constant stirring.The PH of the resultant mixture was adjusted to 7.5-8.0using NaOH.The mixture was heated again in a water bath (reflux 3.5 h), and during this period, a gray color appeared for the precipitated complex.The precipitate was collected by filtration, washed with deionized water, then with absolute ethanol, and finally dried with ether in the oven.

Biomedical evaluation (antimicrobial and anticancer activity).
The antibacterial and antifungal activity of the synthesized complex was accomplished using the agar diffusion technique with 10 The preparation of metal-based anticancer drugs is considered one of the most important areas in this field.A lot of drugs that are used for cancer treatment are cytotoxic.As a result, the most important challenge these days is to develop novel anticancer drugs with high efficacy and low toxicity.

Results and Discussion
The physical properties, elemental analysis data illustrated in Table 1.
Table 1.Analysis of data and physical properties of the two ligands and their metal complex.

FT-IR
In the characteristics of L-cysteine chelated, a band at 3307 cm -1 was noticed due to stretching vibration of (NH2) in the complex of Cu(II), which differed slightly from the band seen in free cysteine, indicating coordination through the nitrogen atom of the amino group of Cysteine 18 .The strong band assigned to NH (indole) of melatonin at 3473 cm -1 shifted to 3244 cm -1 in complex, which may be linked to the formation of a nitrogen-metal bond.The complex's band of (C=O) carboxylic acid didn't change in comparison to the two ligands (melatonin and cysteine), indicating that the -(C=O) group of cysteine and melatonin was not involved in the coordination with metal 19 .
The Cysteine ligand spectrum indicated a band at 2551 cm -1 that refers to (SH), which shifted to a lower frequency in the complex spectrum due to coordination with metal ions via the SH group, and the corresponding vibration (C-S) in the mixed ligand complex was also shifted to a lower frequency, indicating coordination of L-cysteine via the sulfur atom 19 .New bands formed at 424-474 cm - 1 , indicating the appearance of metal ions coordinated via the N atom of the ligand 20 . ).

H-NMR Spectroscopy
The 1 H-NMR spectra of the two ligands showed characteristic peaks of (SH, CH2, CH, NH2) for cysteine and aromatic protons and cyclic protons (CH3, CH2, CH2, CH3O) for melatonin.The 1 H-NMR spectra of cysteine showed peaks ranging from (δ 0.87) ppm that were attributed to SH proton and peaks ranging from (δ 2.79-2.93)and (3.31-3.33)ppm that refer to CH2 and CH protons, respectively, as well as peaks at (δ 6.61) that were assigned to NH2 proton.Later, the donor atoms SH and OH showed a shift to a lower frequency because of the coordination of metal ions.
All the chemical shifts δ (ppm) of the peaks mentioned above were in agreement with literature 19, 22   .As shown in Figs.4-7 and Tables 3 and 4.

Thermal analysis of the synthesized complex
The TG analysis of the complex was performed under nitrogen gas at temperatures ranging from 25 to 800) °C and a heating rate of (10 ˚C/min) 23 .Fig .8.This technique (heat dissociation) was used to evaluate the thermal stability of the synthesized compound and to describe its proposed structure.The weight losses for the compound obtained from the thermal graph were utilized to calculate the decomposed species.The water molecule outside the coordination sphere (lattice water) was lost in the first step, and at higher temperatures, the water molecule within the coordination sphere was lost as well, followed by other lightweight components of the complex.
Fig. 9. Since the results showed good agreement in the real and theoretical percentages of mass loss, Table 5 Table 5. Thermal decomposition data of the Cu-complex.

Electronic spectra (UV-Visible) of the ligands and their Cu-complex
The electronic absorption data of the ligands (L1, L2) and their metal ion complex in (1*10 -4 M) were recorded in DMSO at room temperature, and they are shown in Table 6 and Figs. 10, 11, 12.The electronic spectrum of the first ligand melatonin (L1) exhibits one band at 218.2 nm (45829) cm -1 due to (π → π*) transition 24 .The electronic spectrum of the second ligand cysteine (L2) exhibits one band at 277 nm (36101) cm -1 due to (π → π*) transition 19 .

Antimicrobial activity
The antibacterial and antifungal activity of the synthesized complex was tested against Escherichia coli (G-) (E.coli), Staphylococcus aureus (S. aureus) (G+), and fungi (Candida) (C.albicaus)] The results showed that the synthesized complex (Cu(ll) complex) and ligands have good activity against gram-negative Escherichia coli and very good activity against Staphylococcus aureus, and it is noted that the complex has more activity than the ligands.As shown in Table 7 and Figs. 13, 14.
The results indicated that L1 and L2 have activities approximately equal to each other in Escherichia coli (G-), while L1 was more active than L2 in Staphylococcus aureus (S. aureus) (G+), according to the following activity order: L1>L2, depending on inhibition zone (8>5) mm.The inhibition diameters were measured for the evaluation of antimicrobial activity.

Anticancer activity
The cytotoxic effect of the Cu-complex on human colon cells (CACO -2 ) was tested using the 3-(4,5dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay procedure in various concentrations 26 , (HdFn: normal cell, CACO -2 : cancer cell), the results exhibited that the Cucomplex had the ability to inhibition (CACO -2 ) cells viability in different concentration at range 400 -6.25 µg/mL and slight inhibition for normal cells at the same concentrations As shown in Table 8 and Fig. 15.From the result above, it concluded that the Cucomplex needs a low concentration to kill the CACO - 2 cell line and at the same time its effect is little on normal cells (HdFn).

Conclusion
Melatonin and cysteine were reacted with CuCl2.2H2O in a mole ratio of 1:1:1 (L1:M:L2), yielding a new metal complex.The produced compound was analyzed, and the suggested structure was supported using spectral and physicochemical approaches.According to the findings, the compound has octahedral geometry and a non-electrolyte feature.The scientific results demonstrated that the synthesized complex has high antibacterial action against Escherchia coli (G-) (E.coli), Staphylococcus aureus (G+), and Candida albicaus.The synthesized complex that was created has anticancer properties.

Figure 9 .
Figure 9.The suggested structural formula of the Cu-complex.

Figure 13 .Figure 14 .
Figure 13.The inhibition zones versus bacterial gram positive and gram negative of two ligand.