249 © 2025 The Author(s). Published by College of Education for Pure Science (Ibn Al-Haitham), University of Baghdad. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License Preparation and Characterization of Mixed Ligands of Flucloxacillin and Proline and Theoretical Treatment to Form Coordination Complexes with Some Metal Ions Shayma A. Hndi 1* and Sahar S. Hassan 2 1,2Department of Chemistry, College of Science for Women, University of Baghdad, Baghdad, Iraq. 1Ministry of Education, Baghdad, Iraq. *Corresponding Author. Received: 26 June 2023 Accepted: 10 September 2023 Published: 20 April 2025 doi.org/10.30526/38.2.3629 Abstract Flucloxacillin (Flx) derivative metal complexes(2S,5R,6R)-6-({[3-(2-chloro-5- fluorophenyl)-5-methylisoxazol-4-yl] carbonyl}amino)-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo [3.2.0] heptane-2-methyl aster&proline with CoII, NiII, CuII, ZnII and CdII. They were synthesized and identified using physiochemical techniques, including (elemental analysis CHNS, metal analysis AA, Uv-Vis, FTIR, TGA, magnetic susceptibility, and conductivity measurement). This study aims to investigate the coordination behavior of flucloxacillin and proline, analyze their structural and electronic properties, and evaluate their potential biological activities against bacterial and fungal strains. Additionally, theoretical calculations using HyperChem software are conducted to assess the stability and electronic configurations of the synthesized complexes. Flucloxacillin reacted with metal ions as a bidentate ligand using the nitrogen of the β-lactone thiazolidine ring and the carboxylate ion, forming a five-membered ring, according to the results and from the oxygen and amine group of proline as mixed ligand complexes, with a six-coordination octahedral geometry proposed for all complexes. The Hyper Chem-8.0.7 software was employed to compute the Heat of Formation (Hf), Binding Energy (∆Eb), Total energy (∆ET), Vibration Spectra, and Electronic Spectra for the mixed ligand and its corresponding metal complexes. The antifungal activity of mixed ligands and their metal complexes was evaluated against Candida albicans, while their antibacterial activity against Staphylococcus aureus and Escherichia coli was also assessed. These tests were conducted at a concentration of 5 mM in a nutritional agar medium. Keywords: Flucloxacillin derivatives, Metal ions complexes, Mixed ligand, Proline, Transition metal ions. 1. Introduction In biological systems, it is common for metal ions to form ternary complexes, wherein the metal ion interacts with two or more different ligands (1). The recent increase in the popularity https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ https://orcid.org/0009-0009-6079-4985 mailto:shaymaalmehmdy@gmail.com https://orcid.org/0000-0002-1985-7878 mailto:saharsabeeh21@yahoo.com IHJPAS. 2025,38(2) 250 of mixed chelation can be attributed to the presence of numerous possible ligands in biological fluids, which compete for metal ions in vivo (2). Ternary coordination complexes are widely recognized for their significant role in various biological processes, as evidenced by the fact that metal ions are known to activate enzymes in several instances. Ternary complexes that incorporate an amino acid as a secondary ligand hold considerable significance due to their potential as models for enzyme-metal ion-substrate complexes. The involvement of ternary complexes in the storage and transportation of active chemicals across membranes has been further impacted by the formation of these species and the electronic configuration of the metal ions involved in the process mentioned above (3). Flucloxacillin, alternatively referred to as floxacillin (Flx), is a widely utilized antibiotic in treating infections. Its mechanism of action involves inhibiting bacterial cell wall synthesis (4). Flucloxacillin, a β-lactam antibiotic, is commonly associated with a significant incidence of drug-induced cholestatic liver damage. While the precise pathogenic mechanisms responsible for developing the clinical phenotype remain unknown, it is plausible that both immune-mediated and nonimmune systems play a role. Forflucloxacillin-induced cholestasis, a nonimmune-mediated process characterized by the activation of HSP27 and subsequent sequential molecular events, has been documented (5). It is especially effective against benzylpenicillin-resistant Staphylococci. This category includes infections of the bones and joints, endocarditis, and soft tissues. In the event of a known penicillin hypersensitivity, it should be avoided. Patients having a history of hepatic impairment brought on by flucloxacillin should be avoided. In porphyria (associated with acute attacks), flucloxacillin is considered unsafe (6). Proline is a non-essential amino acid that the body synthesizes from glutamic acid. It is a key component of proteins, especially collagen, supporting skin, tendons, bones, and connective tissues. Vitamin C enhances its production and is found in citrus fruits, bell peppers, and parsley. Proline-rich foods include meat, fish, cabbage, soybeans, and peanuts. Also known as Pyrrolidine-2-carboxylic acid, it is a white solid with a molecular weight of 115.132 g/mole, a melting point of 205-228°C, and a molecular formula of C5H9NO2. Proline was first isolated in 1900 by Richard Willstätter (7,9). This study aims to synthesize and characterize mixed ligand complexes of flucloxacillin and proline with transition metals (CoII, NiII, CuII, ZnII, CdII). It examines their coordination behavior, structural and electronic properties, and biological activities, especially antibacterial and antifungal effects. HyperChem software is used for theoretical calculations on stability and electronic configuration. 2. Materials and Methods 2.1. Instrumentation Electrical conductivity measurements of the complexes were made with 10–3 M of DMF solvent and with the temperature of the laboratory in the laboratories of the Department of Chemistry, College of Science for Women, University of Baghdad. The melting point of the prepared ligands and complexes was measured using the Gallen Kamp M.F.B-60 thermoelectric melting device in the Department of Chemistry/ College of Science for Women/ University of Baghdad laboratory. The electronic spectra of the ligands and their ion metal complexes were recorded on an 1800-UV Shimadzu spectrophotometer in the range of 200– 1100 nm using concentration 10–3 M by and DMF as a solvent at the laboratories of the Department of Chemistry, College of Science, University of Baghdad. Infrared spectra of IHJPAS. 2025,38(2) 251 ligand in and their complexes were recorded using a device of the type (IR Prestige-21, single beam path) Fourier Transform Infrared spectrophotometer (Shimdizu) using a CsI disk within the range (200-4000) cm-1 at Central Environmental Laboratories, Department of Chemistry/ College of Science/ University of Baghdad. The prepared ligands' 1H-NMR nuclear magnetic resonance spectra were recorded using a Bruker Avance 500 MHz NMR Spectrometer. The measurements were carried out at the University of Tehran/ Iran using DMSO as a solvent and TMS as an internal source. A study of the biological activity of each of the metal salts, ligands, and complexes prepared on special types of positive and negative bacteria and fungi. The tests were conducted in the laboratory of BPC Analysis Center/ Iraq-Baghdad. 2.2. Materials All materials used in the research are supplied by Fluka, with a purity of 99% (methanol, ethanol, CoCl2.6H2O, NiCl2.6H2O, CuCl2.6H2O, ZnCl2.6H2O, CdCl2.6H2O, NaOH, H2SO4, proline, DMF, DMSO). 2.3. Preparation of mixed ligand Flucloxacillin derivative was prepared by dissolving the drug (0.4 g) in (35 mL) methanol with drops of concentrated sulfuric acid to convert the COOH group to OCH3. About 0.11 g of Proline was reacted with (0.04 g) sodium hydroxide (NaOH) to prepare (Flx) salt. 2.4. Preparation of mixed ligand complexes One mmol of Flucloxacillin (L) dissolved with (1 mmol) proline, formed (L-pro), which reacted with the salts of the elements (0.2 g) CdCl2.6H2O, (0.23 g) CoCl2.6H2O, (0.23 g) NiCl2.6H2O, (0.17 g) CuCl2.6H2O, (0.24 g) ZnCl2.6H2O) for the preparation of the ligand complexes (L-pro) (preparation ratio L:proline: M 1:1:1). 3. Results and Discussion The complexes are characterized by their stability at room temperature and distinct melting points and colors. The chloride content of the prepared complexes was measured using the Moore-Titro processor method in Ibn Sina laboratories in Baghdad. The physical properties are illustrated in Table 1. 3.1. Infrared spectra of the complexes prepared from the first mixed ligand The band seen at a wave number of 1735 cm-1, corresponding to the β-lactam carbonyl group (C=O) in the ligand, remains unaltered in all complexes. It is important to note that the oxygen atom of the carbonyl group does not participate in any coordination interactions. In contrast, the participation of the amide nitrogen atom in the β-lactam compound occurs due to its favorable IHJPAS. 2025,38(2) 252 positioning for forming a 5-membered ring. This nitrogen atom is also bent out of a plane about the other three C atoms in the β-lactam ring (10-12), as shown in Table 2. Table 1. Analytical and some physical data for the ligand and its metal complexes. Compounds Elemental analysis cal. (found) % M.W M.P ⸰C Color % yield µscm- 1 C H N S M Cl O C22H27Cl FN3O5S L 52.85 5.44 8.40 6.41 --- 7.09 16.00 499.98 210 Light yello w 92 --- (52.66) (5.21) (8.30) (6.22) --- (7.00) (15.98) C5H8NO2 Pro 52.62 7.07 12.27 ----- --- --- 28.04 114.12 208 White 85 --- (52.40) (7.00) (12.10 ) ----- --- --- (27.96) [C27H37Cl2CoFN4O8S ] Cl2.2H2O L (pro) Co 44.64 5.13 7.71 4.41 8.11 9.76 17.62 726.51 225 Pink 78 18.4 (44.32) (5.01) (7.31) (4.22) (7.97) (9.14) (17.02) [C27H37Cl2NiFN4O8] Cl2.2H2O L (pro) Ni 44.65 5.13 7.71 4.42 8.08 9.76 17.62 726.27 230 Light green 88 12.0 (44.05) (4.93) (7.06) (4.09) (8.01) (9.03) (17.33) [C27H37Cl2CuFN4O8S ] Cl2.2H2O L (pro) Cu 44.36 5.10 7.66 4.39 8.69 9.70 17.51 731.12 235 Dark green 67 14.7 (44.18) (4.82) (7.28) (4.02) (8.05) (9.56) (17.20) [C27H39ClZnFN4O9] Cl2.2H2O L (pro) Zn 45.32 5.49 7.83 4.48 9.14 4.95 20.12 715.55 243 Light yello w 85 76.2 (44.97) (5.03) (7.29) (4.09) (9.00) (4.10) (19.95) [C27H39ClCdFN4O9] Cl2.2H2O L (pro) Cd 42.53 5.16 7.35 4.20 14.74 4.65 18.88 762.55 252 Light yello w 87 89.5 (42.06) (4.78) (7.03) (4.10) (14.56) (4.20) (18.33) The coordination between the oxygen atom of the carbonyl group (C=O) and the nitrogen atom of the β-lactam (C-N) exhibited a decrease in frequency, falling within the range of 5-35 cm-1 for both the ligand (L) and all complexes. Additionally, the C-N band in proline was observed within the range of 2-21 cm-1. This coordination was further substantiated by identifying M-O and M-N bands in the complex spectra, which were observed within the range of 400-550 cm-1 as reported in the literature (13). Table 3 and Figures 1-3 show the FT-IR spectrum's additional significant characteristic peaks. Table 2. Fourier transform infrared spectroscopy bands of L and proline. ѵM-Cl ѵM-N ѵM-O C=O Ester β-Lactam L1 ѵ C-N ѵ(COO)asy ѵ(COO)sym ѵNH Compound --- ---- 1743 1735 1335 ---- 3380 L --- --- ----- 1342 1573 1492 3083 Pro IHJPAS. 2025,38(2) 253 Table 3. Fourier transform infrared spectroscopy bands of complexes. M-Cl M-O (pro) (L) M-N (pro) (L) C=O Ester β-Lactam L C-N (L) (pro) COO (asy) COO (sym) (pro) N-H (L) (pro) Compound --- 474 418 521 547 1793 1735 1300 1340 1575 1458 3033 3411 L (pro) Co --- 476 416 512 543 1745 1735 1330 1355 1573 1460 3101 3390 L (pro) Ni --- 462 406 505 512 1780 1735 1320 1342 1577 1456 3078 3421 L (pro) Cu 235 472 451 501 510 1781 1733 1315 1321 1510 1458 3058 3390 L (pro) Zn 248 480 420 509 530 1770 1735 1320 1353 1508 1438 3122 3417 L (pro) Cd Figure 1. Spectrum of L FTIR. Figure 2. The FTIR spectrum of Pro. IHJPAS. 2025,38(2) 254 Figure 3. The FTIR spectrum of L (pro) Co. 3.2. The UV-Vis spectra of mix ligand and its metal complexes The n→π* and π→ π* transitions to the C-O, C-N, and C=C transitions, respectively, were assigned to the peaks between 220 and 350 nm in the electronic spectrum of the mix of ligands in Figure 4. The utilization of spectra facilitated the prediction of the planned geometry by analyzing the characteristics of the observed peaks in shape and quantity (14). In Table 4, L1(pro)Co complex shows 4 bands in the wave number (14755,16447,23255,34722) cm-1. L1(pro) Ni complex has 4 bands (11173,15873,20576,34843) cm-1. L (pro)Cu complex shows 3 bands in the wave number (12987,2270,41322) cm-1. L (pro)Zn complex shown 2 bands in the wave number (39216) cm-1. L (pro) Cd complex shows 2 bands in the wave number (34722,29411) cm-1 (15,16). The Uv-visb spectra of L (pro) Cu is shown in Figure 5. Table 4. The Uv-visible bands of prepared compounds. Compound Λ nm Absorption Bands cm-1 Assignment L 328 227 221 30487 44528 45248 π-π* (C=C) n-π*(CO, CN) pro 331 281 30211 35871 π-π* (C=C) n-π*(CO, CN) L (pro)Co 686 608 430 288 14755 16447 23255 34722 4T1g → 4T2g 4T1g → 4T1g 4T1g → 4A2g I.L L (pro) Ni 895 630 486 287 11173 15873 20576 34843 3A2g → 3T2g 3A2g → 3T1g(F) 3A2g → 3T1g(P) I.L L (pro) Cu 770 361 242 12987 2770 41322 2Eg → 2T2g Charge Transfer ILCT L (pro)Zn 255 327 39216 ILCT ILCT L (pro)Cd 288 340 34722 29411 ILCT ILCT IHJPAS. 2025,38(2) 255 (a) (b) Figure 4. The Uv-visb spectra of FLU and Pro. Figure 5. The Uv-visb spectra of L (pro) Cu. 3.3. The 1H NMR spectrum of L(pro) The 1HNMR spectrum of the mixed ligand L (pro) was measured using DMSO as a solvent, as illustrated in Figure 6, where the mono signal at σ (2.53) ppm refers to the DMSO solvent and the mono signal at (3.35) that belongs to 3H, CH3-O and the treble signal at σ (3.67) ppm which belongs to 9H, CH3, (7.30) ppm due to CH2-N for β-lactam cycle, and the mixed ligand spectrum showed a signal at σ (8.52) ppm, which is attributed to 3H, NH, amine. It also showed a mono signal at σ (9.20) ppm due to 1H-NH amide. Table 5 shows the apparent signals and the chemical shifted values measured in ppm (17,18). Table 5. The 1H NMR spectrum of L(pro) C25H27ClFN4O7S. σ(ppm) No of proton Compound 3.35 O)-3S(3H, CH )pro(2L 3.67 t(9H ,CH3) 4.82 S(2H, SCH) 5.83 -6.73 2d(7H, cyclo) 7.30 lactam-N) β-2t (2H, CH 8.52 d (3H, NH, amine) 9.20 s (1H, NH, proline) IHJPAS. 2025,38(2) 256 Figure 6. The 1HMNR spectrum of L. 3.4. Theoretical study HyperChemprogramme is an advanced molecular modeler, editor, and potent computational suite renowned for its quality, adaptability, and user-friendliness (19). To optimize geometry, it can calculate electrostatic potential, total charge density, and thermodynamic functions. The semi-empirical method is one of the most essential methods employed. This information is valuable for assessing reactivity and establishing a correlation between computational outcomes and experimental data. 3.5. Electrostatic potential The PM3 method was used to measure the electrostatic potential (E.P) of a molecular system with a positively charged point, with the objective of identifying the most active interaction sites within the positively charged particle species. These entities tend to target a molecule with a highly negative and electrophilic electron pair (20,21). The E.P of the ligands (Flx, Proline) were determined and visualized through 2D and 3D contour plots. These plots were utilized to analyse the reactive sites within the molecules, as illustrated in Figure 7. 3.6. Energies and optimized geometries of metal complexes for the ligand Theoretical treatment was used to find the most stable structures for the metal complexes with ligands (22,23). Depending on the method (PM3), the geometric configurations depicted in Figure 8 correspond to the most favorable computation geometries for both the ligand and its complexes. Semi-empirical techniques are used to calculate the thermodynamic functions (∆Hf, ∆Eb, and ∆ET) of compounds in the gas phase at 0 °K, as tabulated in Table 6. Table 6. Energies of shapes ( KJ.mol-1) for the (L1,L2 and metal complexes). Compounds PM3 Flx ΔHf -475.05 ΔEb -19770.44 ΔET -507738.67 Proline -418.29 - 6930.79 - 143078.96 L (Pro) Co -2656.6 - 29610.09 - 785939.97 L (Pro) Cu - 1645.13 - 28507.77 - 823673.68 L (Pro) Ni - 2379.65 - 29334.82 - 810222.17 L (Pro) Zn - 794.31 - 27449.58 - 711345.43 L (Pro) Cd - 893.47 - 27530.1 - 710949.28 IHJPAS. 2025,38(2) 257 3.7. Optimized vibrational spectra Theoretically, calculated molecular frequencies of vibration deviate from experimentally determined values (24). These deviations are generally accepted in theoretical calculations and are outlined in Table 7. Figures 7-10 show some theoretical vibrational modes of L and proline (19,20). The Suggested structure of the prepared complexes illustrated in Figure 11. Flx Proline Figure 7. The E.P of the (Flx) and (proline) using PM3 methods. IHJPAS. 2025,38(2) 258 Flx Proline L (pro)Cd L (Pro) Co IHJPAS. 2025,38(2) 259 L (Pro) Cu L (Pro) Ni L (Pro) Zn Figure 8. Geometric shapes of (L(Pro)) and its complexes using PM3 method. IHJPAS. 2025,38(2) 260 Table 7. Data of theoretical wave number of (L, proline) compounds (cm-1) and their comparison with experimental vibrations. Compound ʋ NH ʋC=O ʋCOO ʋC-N L 3380* 3339** 0.012*** 1743* 1952** -0.107*** 1335* 1442** 0.074*** Proline 3083* 3393 1573* 1441** -0.09*** 1342* 1326** -0.012*** *experimental, **theoretical, ***Error. ʋ NH ʋCN ʋC=O ʋC=O Figure 9. Some theoretical vibrational modes of L. IHJPAS. 2025,38(2) 261 ʋ NH ʋCOO ʋCN Figure 11. The suggested structure of the prepared complexes. 3.8. Evaluation of antibacterial activity Mix ligand and metal complexes were examined for their in vitro biological screening effects against microorganisms. This was accomplished using the disc diffusion method with nutrient agar as the medium (25,26). The test solution was introduced into the well using a micropipette, and subsequently, 24 hours were spent incubating the dishes at 37°C. Using a ruler in millimeters (mm), the inhibition zone of each pore was measured. To record the results, the growth inhibition surrounding the disc was estimated. Chelation theory and the concept of overtones can explain why the inhibition zone of metal complexes is more significant than that of ligands. The chelation process reduces the polarity of the metal ion due to the overlapping of the ligand orbital and the partial sharing of the positive charge of the metal by the donor groups. Furthermore, it facilitates the dispersion of π-electrons throughout the entirety of the chelating ring and promotes the infiltration of complexes into lipid membranes. Additionally, it obstructs the metal-binding sites of microorganism enzymes. The results obtained for biological activity for the preparation ligand and its metal complexes are shown in Table 8. The free Figure 10. Some theoretical vibrational modes of proline. IHJPAS. 2025,38(2) 262 ligand showed a slightly antimicrobial and antifungal activity against both bacteria. The nickel (II) complex shows higher inhibition against both organisms and antifungal effect than free ligands in both concentrations. The copper (II) and cobalt (II) complexes showed moderate and non-activity, respectively, against all organisms used in this study, which is clarified in Figure 12. The higher inhibition zone of the metal complexes than the ligand can be explained based on chelation theory and overtone concept (27-30). Table 8. The diameter of the inhibition circle in (mm) after being injected (24 hrs.) at 37 0C for some mixed ligand complexes prepared from ligand (L2) and proline. Figure 12. The antibacterial activity of prepared compounds against Staphylococcus aureus, Escherichia coli, and Candida albicans. in 5 mM. 4. Conclusion A new mixed ligand was prepared from a derivative of Floclxaciline and the amino acid (proline). The form and type of bonding between the ligand and the prepared complexes were revealed using spectroscopic methods and nuclear magnetic resonance. The shapes of the Candida albicans Staphylococcus aureus Escherichia coli Compound 19 28 10 Pro 14 27 10 L 8 34 31 L (pro)Ni 16 25 17 L (pro)Cu 9 9 15 L (pro)Zn 10 8 14 L (pro)Co 27 14 13 L (pro)Cd IHJPAS. 2025,38(2) 263 formed complexes were suggested to be octahedral. The Hyperchem-8.07 software was used to forecast the structural engineering of all compounds in the gas phase, with physical properties calculated using the PM3 program. Acknowledgment The authors thank the Department of Chemistry/ College of Science for Women/ University of Baghdad. Conflict of Interest The authors declare that they have no conflicts of interest. Ethical Clearance The study is included in the 2022–2023 academic year research plan form at the University of Baghdad. References 1. Nesa SH, Nasira SU, Ashrafuzzaman M, Habib A, Rashid AM, Haque M. 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