342 This work is licensed under a Creative Commons Attribution 4.0 International License IHJPAS. 37 (1) 2024 Ibn Al-Haitham Journal for Pure and Applied Sciences Journal homepage: jih.uobaghdad.edu.iq PISSN: 1609-4042, EISSN: 2521-3407 1Simaa Safaa Mahmoud* 2Asmaa Mohammed Noori 1,2 Department of Chemistry, College of Sciences, University of Baghdad, Baghdad, Iraq. *Corresponding Author: saimaa.safaa1205m@sc.uobaghdad.edu.iq Abstract Synthesis of new ligand, namely [bis(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl) hydrogen borate] (BIB), utilizing the reaction of metronidazole with boric acid in mole ratio (2:1), as well as the metal complexes with [Ni(II) and Cu(II)], were synthesized. All synthesized compounds were characterized by utilizing spectroscopic techniques such as FTIR, 1H-NMR, thermal analysis (T.G., UV-Vis), and atomic absorption (A.A.S.), as well as micro elemental analysis (C.H.N.), melting point (m.p), magnetic susceptibility, molar conductivity, and chloride content measurements. All complexes were paramagnetic, and the electrolyte and the suggested geometries were tetrahedral for nickel and octahedral for copper. In addition, all the transition metal complexes produced were shown to be antibacterial and antifungal against the bacteria Staphylococcus aureus, Escherichia coli, and the fungus Candida. Also, metronidazole and the ligand were evaluated as anticancer agents against human breast cancer (MCF-7). The results showed that ligand was more active as an anticancer than metronidazole. Keywords: Metronidazole, Boric acid, Spectroscopic techniques, Antibiotic, Anticancer. 1. Introduction Nitroimidazole compounds have an imidazole ring with two or five active nitro groups. The pharmacokinetic properties of 5-nitroimidazoles are anti-parasitic, while the effects of 2- nitroimidazoles are anti-ischemic and anti-inflammatory [1]. The 5-nitroimidazole compound, namely [metronidazole (MTN)], is the prototype and most commonly utilized drug in this class. It's one of the most versatile antibiotics in clinical use, effective against a wide range of anaerobic microorganisms ranging from protozoa to bacteria. The World Health Organization has classified it as an essential medication [2]. Metronidazole (MTN) is a crystalline powder and is slightly soluble in water [3]; it exhibits a wide range of pharmacological actions, with a particular emphasis on its antiviral, antibacterial, anti-proliferative, and antifungal activities [4]. Metronidazole's initial clinical trials revealed that it could treat amoebic liver abscesses and invasive amoebic dysentery [5]. Received 2 February 2023, Received 18 February 2023, Accepted 22 February 2023, Published 20 January 2024 Synthesis, Biological and Medicinal Evaluation of New Boric Acid Ester Derived from Antibiotic Drug with Some of its Metal Complexes doi.org/10.30526/37.1.3261 https://creativecommons.org/licenses/by/4.0/ https://jih.uobaghdad.edu.iq/index.php/j/index#1609-4042 https://jih.uobaghdad.edu.iq/index.php/j/index#2521-3407 mailto:saimaa.safaa1205m@sc.uobaghdad.edu.iq https://orcid.org/0009-0000-1731-4434 mailto:saimaa.safaa1205m@sc.uobaghdad.edu.iq https://orcid.org/0000-0002-2877-5663 mailto:asmaa.m@sc.uobaghdad.edu.iq IHJPAS. 37 (1) 2024 343 Boric acid B(OH)3 is a weak inorganic acid that is odorless and soluble in water. It is an efficient acid catalyst in organic synthesis for various selective transformations of simple and complex molecules [6]. It's an antiseptic in mouthwashes, talcum powder, protective ointments, and eye-washes. Also, boric acid is used in industrial applications such as optical and sealing glasses, textile fiberglass, heat-resistant borosilicate glass, porcelain enamels, and ceramic glazes [7]. This study has synthesized a new metronidazole derivative using boric acid as a BIB ligand Figure 1. Additionally, we are synthesizing metal complexes of this (BIB) ligand with [Ni (II) and Cu (II)] metal ions. Figure 2. All synthesized compounds are characterized using physicochemical and spectral studies to prove the proposed structure. The medicinal uses and biological activities of the synthesized compounds were evaluated. 2. Materials and Methods The elemental analyzer EuroEA 3000/Italy recorded elemental microanalyses (CHN) for carbon, hydrogen, and nitrogen. The melting points were determined using the Gallen Kamp melting point apparatus. The FT-IR (Fourier transform infrared) spectrophotometry for ligands in the 400–4000 cm-1 (KBr) range and complexes in the(200 - 4000 cm-1) range UV-Vis spectrum was measured using a Shimadzu 1800-UV spectrophotometer and distilled water as a solvent. The 1H -NMR spectra in DMSO-d6 were measured using an NMR Bruker 500MHz in Germany. Thermal analyses (TG) were recorded (TG sta. 300, Germany). The Auto Magnetic Susceptibility Balance Model by Sherwood Scientific was utilized to record magnetic susceptibility data at room temperature. Metal content was evaluated using atomic absorption spectroscopy on a Nova 350 spectrophotometer. The Mohr technique was utilized to determine the chloride content of complexes. 2.1 Synthesis of bis(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl) hydrogen borate (BIB) The mixture of metronidazole (0.1 g, 0.5842 mmol) in 8 mL of distilled water and boric acid (0.0180 g, 0.2921 mmol) was heated under reflux for 8 hours with stirring. The TLC technique tested the solution, and the eluent was Toluene, chloroform, and methanol (3:2:0.6, v/v/v). A part of the solution was evaporated, and the white product was obtained by cooling in an ice bath, scratching, washing with cold distilled water, and drying in an oven at 80 0C. Figure 1. Structure of the ligand (BIB) IHJPAS. 37 (1) 2024 344 2.2 Synthesis of BIB complexes with (Nii(II) and Cu(II)) metal ions (C1 and C2) The warm solution of the BIB (0.1 gm, 0.2718 mmol) in 5 mL distilled water was added to a solution of metal salt (0.0323 gm, 0.0231 gm (0.1355 mmol)) of NiCl2.6H2O and CuCl2.2H2O, respectively, in 2 mL distilled water. The mixtures were heated under reflux for 5 hours with stirring. The solvent was evaporated, and the products were collected in an ice bath and crashing, washed with cold distilled water, and dried in an oven at 800C. 2.3 Anti-microbial activity Using the diffusion technique with 2×10-2 M in H2O solutions, all synthesized compounds were tested for anti-bacterial and anti-fungal activity against (gram-positive Staphylococcus aurous, gram-negative Escherichia coli and Candida). The diameters of inhibition were examined to assess the anti-bacterial activity [8]. 2.4 Anticancer activity A 96-well plate was used for the MTT cell viability assay to evaluate the cytotoxic effect. The cell lines were seeded at (1×104 cells/ well). Cells were treated with the studied compounds after 24 hours. The effectiveness of the anticancer treatment was studied utilizing literature [9], and absorbance at 575 nm was evaluated. 3. Results and Discussion The physical and analytical data are consistent with the proposed structures of the compounds in the study (Tablee1). Table 1. Data from the analysis as well as the physical properties of the (BIB) ligand and its metal complexes Comp The molecular formula Color Yield % m.pp (°C) M.wtt (g/mol) (Found) Calc. Metall content % Chloride content % C% H% N% BIB C12H17BN6O7 White 97% (148-150) 367.8 39.15 (39.41) 4.65 (5.37) 22.83 (23.22) ___ _____ C1 Ni(II) [C24H36B2N12O15 NiCl]Cl.H2O light green 76% (158-160) 901.29 31.95 (32.71) 4.21 (4.74) 18.63 (19.39) 6.51 (7.33) 7.87 (8.76) C2 Cu(II) [C24H40B2N12O17 CuCl].Cl green 82% (156-158) 924.14 31.16 (32.08) 4.32 (5.18) 18.17 (17.21) 6.87 (7.41) 7.68 (6.75) Table 2. The name of BIB and proposed formula for its metal ion complexes Comp Formal Name BIB C12H17BN6O7 bis(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl) hydrogen borate. C1 Ni(II) [(L1)2Ni(H2O)Cl].Cl.H2O [aqua chloro bis{bis(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl) hydrogen borate} Nickel (II)] hydrate. Chloride. C2 Cu(II) [(L1)2Cu(H2O)3Cl].Cl [Tri aqua chloro bis{ bis(2-(2-methyl-5-nitro-1H- 344midazole-1-yl)ethyl) hydrogen borate} Copper(II)] Chloride. 3.1 The FT-IR spectra The infrared spectra of (Ni and Cu) complexes showed a shifting in the frequency and a change in the profile of (υ OH) Table 3 as a result of coordination with metal ions[10]. IHJPAS. 37 (1) 2024 345 At (1483–1487)cm-1, a new band appears in the spectra of the ligand and its complexes; this band is attributed to the (υ B-O) group [11]. The stretching of (C=N) the imidazole ring did not show any change in frequency or profile, and this was because of no coordination with metal ions through (C=N) [12]. The spectrum of the Ni (II) complex showed the lattice water at (3437) cm-1 and coordinated water at (3365) cm-1, as well as the lower frequency bands [(991) and (678)] cm- 1. The coordinate H2O of Cu (II) complex appeared at (3388) cm-1, and in the lower frequency band [(767) and (680)] cm-1, low-frequency bands appeared in complex spectra due to υ M-O, υ M-Cl [13, 14] as shown in Figure 3 and 4. Table 3. Infrared absorption bands that are specific to the (BIB) ligand and its metal ion complexes Compound υ OH H2O lattice (coordinate) υ C=N υ B-O υ M-O υ M-Cl MTZ 3222 _____ 1535 _____ _____ _____ BIB 3415 _____ 1535 1487 _____ ____ BIB (Ni) C1 3384 3437 (3365) (991) (678) 1535 1487 457 347 BIB (Cu) C2 3406 (3388) (767) (680) 1535 1483 424 333 Figure 2. The suggested structures of synthesized complexes IHJPAS. 37 (1) 2024 346 Figure 3. The FT-IR spectrum of the (BIB) ligand Figure 4. The FT-IR spectrum of the Ni(II) complex C1 3.2 The 1H-NMR spectroscopy Table 4 shows the 1HNMR data for the BIB ligand, which is backed up by Figure 5, Figure 6 shows the NMR spectrum in DMSO-d6. The spectrum of the ligand observed a chemical shift at δ(3.99), which is due to the B-OH proton [15, 16]. The multiple peaks observed in the ranges δ (4.35–4.40) and δ (3.66) referred to NCH2 and O-CH2, respectively [17, 18]. Chemical shifts of the methyl group CH3 and residual DMSO-d6 at δ(2.44) [17] The peak observed at δ(8.04) is attributed to imidazole protons [19]. IHJPAS. 37 (1) 2024 347 Figure 5. Proton positions in structure of the ligand BIB Table 4. Chemical shifts for 1HNMR of BIB Assignments in DMSO-d6 Mark Chemical shifts δ (ppm) CH3 1, 9 (2.44),6H, s CH(imidazole) 2, 8 (8.04),2H, s N-CH2 3, 7 (4.35-4.40),4H, m O-CH2 4, 6 (3.66),4H, m B-OH proton 5 (3.99)1H, m Figure 6. The 1H-NMR Spectrum of BIB 3.3 Thermogravimetric analysis (TGA) The thermogravimetric analysis was conducted under argon gas at a heating rate of (10 ºC/min) and a temperature range of (25 – 800) ºC. This technique characterized the suggested structures and the studied thermal st and synthesized compounds. In the following order, the BIB ligand and IHJPAS. 37 (1) 2024 348 its complex's thermal stability were increased: (C1 > BIB > C2) Table 5 [3]. The thermal decomposition was utilized to confirm the structures where the degradation results exhibit high agreement of found mass loss and calculation, confirming the proposed construction of the synthesized compounds. The ligand (BIB) and nickel complex C1 thermogram are shown in Figures 7 and 8. Figure 7. The thermogram of the (BIB) ligand Figure 8. The thermogram of nickel complex C1 IHJPAS. 37 (1) 2024 349 Table 5. The TGA of ligand (MBIB) and their complexes Comp Compounds (M.wt) (gm/mol) Step Temp. rang of thee Decomposition ºC Suggested Assignment Mass loss% Cal. Found BIB C12H17BN6O7 367.8 1 25-135 CH3 4.078 3.223 2 135-275 N3C7O2H9 45.40 44.54 3 275-420 N3C2O3H5 32.35 32.84 4 420-800 2C+O 10.87 11.85 Residue <800 B+O 7.28 7.55 C1 [C24H36B2N12O15NiCl]Cl.H2O 901.29 1 25-278 2H2O+2Cl+N6O7C13H 20+B 54.34 54.77 2 278-800 N6O4C8H7 27.84 26.93 Residue < 800 O3H7C3B Ni 17.80 18.30 C2 [C24H40B2N12O17CuCl].Cl 924.14 1 25-262 3H2O+2Cl+2(N3O2C6 H8)+O 48.58 48.94 2 262-800 N6O9C12H18B+Cu 50.24 50.26 Residue <800 B 1.16 0.81 3.4 The UV-Vis spectral studies: The UV-Vis spectra of (BIB) and its metal complexes in distilled water are listed in Table 6. The spectrum of the ligand Figure 9 showed an intense band at [313 nm (31948 cm-1)] due to the (π→ π*) transition [20]. The Ni(II) complex Figure 10 exhibited a shift of the ligand band (π→ π*). Two bands showed at [962 nm (10395 cm-1) and 785 nm (12738 cm-1)] which were assigned to [3T1(F) →3A2 and 3T1(F) →3T1(P)] transitions of Tetrahedral Ni(II) complexes [21].The Meff of Ni(II) was 2.78 B.M. This value is in agreement with Tetrahedral geometry [20, 21]. Table 6 shows a list of the data. Two absorption bands were observed in the spectrum of copper complex (C2) Figure 11 at [960 nm (10416 cm-1) and 739 nm (13531 cm-1)] which were attributed to[2B1g→ 2A1g and 2B1g → 2B2g] transitions, respectively [20]. The magnetic moment of the copper complex was 2.23 B.M, and these values of Meff agree with Octahedral geometry [22–24]. In distilled water, the molar conductance for each synthesized complex was measured using (10-3 M). The complexes (C1 and C2) have electrolyte behavior [25–27]. Table 6. Electronic transitions of the BIB and its complexes, proposed geometry, molar conductivity, and magnetic susceptibility Comp ℷ nmm (cm-1) Assignment Molar conductivity (S.cm2 .mol-1) in H2O Meff (B.M) Geometry BIB 313(31948) (π -π*) ______ _____ _______ C1(Ni) 315(31746) 785(12738) 962(10395) (π -π*) 3T1(F) →3T1(P) (υ2) 3T1(F) →3A2(F) (υ1) 131 2.78 Tetrahedral C2(Cu) 357(28011) 739(13531) 960(10416) (π -π*) 2B1g→2B2g (υ2) 2B1g→2A1g (υ1) 139 2.23 Distorted octahedral IHJPAS. 37 (1) 2024 350 Figure 9. The UV-Vis spectrum of BIB Figure 10. The UV-Vis spectrum of C1 complex Figure 11: The UV-Vis spectrum of C2 complex IHJPAS. 37 (1) 2024 351 3.5 Anti-cancer Activity Metronidazole and (BIB) ligands were evaluated for their ability to inhibit human breast cancer (MCF-7) cells by the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assay technique Figures 12 and 13. The results showed that (BIB) ligand was more active as an anticancer than the original material (metronidazole), and the killing capability of cancer cells for the BIB ligand was higher than that of metronidazole [28]. This is due to the presence of a boron atom in the ligand. Table 7. Cytotoxicity effects of metronidazole on (MCF-7) anddWRI-68 cells afterr24 hours incubation at 37 °C Cell line Conc. μg/mL IC50 μg/mL P-value 200.00 100.00 50.00 25.00 12.50 6.25 MCF-7 55.67± 3.64 61.00±1.48 76.08± 1.77 89.82±2.34 92.86±4.65 95.02±1.45 53.29 <0.0001 WRL 77.28±1.62 87.96±1.51 92.13± 1.56 95.41±0.37 95.79±0.71 94.91±2.20 297.4 Figure 12: Cytotoxicity effect of Metronidazole on MCF-7 cells afterr24 hours incubation period at 37 ºC Table 8. Cytotoxicity effects of (BIB) ligand on (MCF-7) and WRI-68 cells afterr24 hours incubation at 37 °C Cell line Conc. μg/mL IC50 μg/mL P-value 200.00 100.00 50.00 25.00 12.50 6.25 MCF-7 43.21±2.20 49.19±4.36 66.09±2.71 72.57± 2.91 86.19±3.92 94.41±0.55 37.42 <0.0001 WRL 74.38±4.21 85.57±2.34 92.94±0.81 95.72± 0.53 94.91±1.51 95.18±0.41 120.7 IHJPAS. 37 (1) 2024 352 Figure 13. Cytotoxicity effect of (BIB) Ligand on MCF-7 cells after 24 hours incubation period at 37 ºC 3.6 Antimicrobial activity: The synthesized compounds' antibacterial and antifungal behavior was evaluated against gram- positive bacteria Staphylococcus aureus, gram-negative bacteria Escherichia coli, and Candida [29-31]. The results indicated that ligand (BIB) has more activity than metronidazole(MTZ) in Staphylococcus aureus, according to the following activity order (BIB > Boric acid > MTZ) depending on inhibition zone (23 > 22 > 20) mm. respectively. In Escherichia coli, according to the following order, boric acid has higher activity (boric acid > BIB > MTZ) depending on the inhibition zone (12 > 11 > 9) mm, respectively. Comparison of the biological activities in Staphylococcuss aureus of BIB and its metal complexes was in the following order (C1 > BIB > C2 = boric acid > MTZ) at the inhibition zone (24 > 23 > 22 > 20) mm. In Escherichia coli, the complexes were more energetic than BIB, and the order was as follows (Cu (C2) > Ni (C1) = boric acid > BIB > MTZ) depending on the inhibition zone (13 > 12 > 11 > 9) mm, respectively [32-34]. Table 9 shows the antibacterial and antifungal date results, while Figures 14, 15, and 16 show the inhibition zones. Table 9. The biological activity of the substances understudy in (2×10-2 M) Compound Staphylococcuss aureus(G+) inhibition zoneediameter (mm) Escherichia coli(G-) Inhibition zonee diameter (mm) Candida (nm) H2O -- -- -- MTZ 20 9 10 Boric acid 22 12 16 BIB 23 11 15 Ni(C1) 24 12 14 Cu(C2) 22 13 12 IHJPAS. 37 (1) 2024 353 Figure 14. The zone of inhibition towards Staphylococcussaureus (G+) in the case of the ligand (BIB) and its metal complexes, boric acid, and metronidazole Figure 15. The zone of inhibition towards Escherichia coli (G-) in the case of the ligand (BIB) and its metal complexes, boric acid, and metronidazole Figure 16. The zone of inhibition towards Candida in the case of the ligand (BIB) and its metal complexes, boric acid, and metronidazole IHJPAS. 37 (1) 2024 354 4. Conclusion A new ligand (BIB) was synthesized from the reaction of metronidazole with boric acid, and its metal complexes with Ni(II) and Cu(II) were synthesized in a 2:1 (BIB: M) mole ratio—spectral and physicochemical methods characterized all synthesized compounds. The proposed structure of the Ni(II) complex was tetrahedral geometry and the octahedral geometry of the Cu(II) complex, and the results showed that the complexes have electrolytic behavior. All synthesized compounds were tested as anti-biofilm agents against (Pseudomonas autogiros (Gram-negative) bacteria (G-). The results showed that, compared to other compounds, copper complexes were more active. The medicinal application (anticancer) in human breast cancer cells (MCF-7) was studied of the ligand (BIB) and gave a good result in testing. Acknowledgment The authors thank the Department of Chemistry/ College of Science/ University of Baghdad staff for their assistance in performing this research. 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