IHJPAS. 36 (4) 2023 274 This work is licensed under a Creative Commons Attribution 4.0 International License *Corresponding Author: saimaa.safaa1205m@sc.uobaghdad.edu.iq Abstract Synthesis of a 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 a (2:1) mole ratio The metal complexes were synthesized utilizing the reaction of (NiCl2.6H2O and CuCl2 .2H2O) with (BIB) ligand in a 2:1 (L:M) mole ratio. All synthesized compounds were characterized utilizing spectroscopic techniques such as infrared (FTIR), nuclear magnetic resonance of protons(1H NMR), ultra violet and visible radiation (UV-Vis), thermal analysis (TG), atomic absorption (A.A.S.), micro elemental analysis (C.H.N.S.), melting point (m.p.), magnetic susceptibility, molar conductivity, and chloride content measurements. All complexes were paramagnetic and electrolyte, and the suggested geometries were the tetrahedral of nickel and the distorted octahedral of copper complexes. Against the Gram-negative bacterium Pseudomonas auroginosa (G-), all synthesized compounds were evaluated as anti-biofilm agents. Strikingly, the copper (II) complexes tested exhibit significant activity against biofilms and were better at removing biofilms than metronidazole (an antibiotic that is currently used to treat infections), ligand (BIB), and nickel (II) complexes. Keywords: Metronidazole, Boric acid, FT-IR, Anti-biofilm. 1.Introduction Nitroimidazole chemical compounds have active nitro groups on either the 2' or 5' positions of the imidazole ring. While 2'-nitroimidazoles exhibit pharmacological properties that are anti-ischemic and anti-inflammatory, 5-nitroimidazoles exhibit pharmacological characteristics that are anti-parasitic [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 doi.org/10.30526/36.4.3157 Article history: Received 22 December 2022, Accepted 18 January 2023, Published in October 2023. Ibn Al-Haitham Journal for Pure and Applied Sciences Journal homepage: jih.uobaghdad.edu.iq New Metronidazole Derivative and Some of Its Complexes with Antibiofilm study Simaa Safaa Mahmoud * Department of Chemistry, College of Science, University of Baghdad,Baghdad Iraq. Sarah.wathib@gmail.com Asmaa Mohammed Noori Khaleel Department of Chemistry, College of Science, University of Baghdad,Baghdad Iraq. https://creativecommons.org/licenses/by/4.0/ mailto:saimaa.safaa1205m@sc.uobaghdad.edu.iq mailto:Sarah.wathib@gmail.com mailto:saimaa.safaa1205m@sc.uobaghdad.edu.iq mailto:asmaa.m@sc.uobaghdad.edu.iq IHJPAS. 36 (4) 2023 275 microorganisms ranging from protozoa to bacteria. The World Health Organization has classified it as an essential medication [2]. Metronidazole (MTN) is a crystalline powder that is slightly soluble in water [3]. Metronidazole exhibits a wide range of pharmacological effects, with an emphasis on its antiviral, antibacterial, anti-proliferative, and antifungal activities in particular [4]. Metronidazole's initial clinical trials revealed that it was effective in treating amoebic liver abscess and invasive amoebic dysentery [5]. Boric acid B(OH)3 is a weak inorganic acid that is odorless and soluble in water. It is utilized as an efficient acid catalyst in organic synthesis for various selective transformations of simple and complex molecules [6]. B(OH)3 is utilized as an antiseptic in mouthwashes, talcum powder, protective ointments, and eyewashes. Also, boric acid is used in industrial applications such as optical and sealing glasses, textile fiber glass, heat-resistant borosilicate glass, porcelain enamels, and ceramic glazes [7]. In this study, a new metronidazole derivative was synthesized using boric acid as a BIB ligand (Figure 1). In addition, this (BIB) ligand is being mixed with [Ni (II) and Cu (II)] metal ions to create metal complexes (Figure 2). To prove the proposed structure, all synthesized compounds are characterized using physicochemical and spectral studies. The biological activity of synthesized compounds was evaluated. 2.Experimental Design Material and Instruments Without additional purification, all chemicals were used as supplied (utilizing Hayman, Fieser, BDH, HiMedia, Aarti Drugs Ltd., Fluka-Garantie, Merck, Sigma, Ajenta Pharm, Intron Biotech, and PSI). 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 solution was tested by TLC technique, and the eluents were toluene, chloroform, and methanol (3:2:0.6, v/v/v), respectively. A part of the solution was evaporated, and he white product was obtained by cooling in an ice bath, scratching, washing with cold distilled water, and drying in an oven at 80 °C. Figure 1: Structure of the ligand (BIB). IHJPAS. 36 (4) 2023 276 Synthesis of BIB complexes with (Ni (II) and Cu (II)) metal ions (C1 and C2) : A 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) in 2 ml of distilled water for NiCl2.6H2O and CuCl2.2H2O, respectively. The mixtures were heated for 5 hours under reflux with stirring. The products were collected by the partially evaporated solvent in the presence of an ice bath and crashing, and they were then washed with cold distilled water and dried in an oven at 80 0C. Anti-Biofilm: Determination of the minimum inhibitory concentration (MIC) of Ligand (BIB) and its Complexes: Following a microdilution method, different concentrations of this compound (8 - 1024 mg/mL) were utilized to estimate the MIC. Biofilm quantification protocol: The biofilm formation was evaluated on a polystyrene 96-well microplate. Bacterial strains were cultivated overnight for 24 hours at 37 °C in brain heart infusion (BHI) broth culture with 0.2% glucose, both in the presence and absence of MIC concentrations of chemical compounds. After removing the medium, biofilm-containing wells were washed three times with normal saline before being fixed with 200 µl of 99% methanol. The microplate was washed three times with distilled water, dyed for 15 minutes with 200 µl of (0.1%) crystal violet, and dried at room temperature. After the dye that was adhered to the biofilm was solubilized in 200 µl of pure ethanol, The absorbance was measured at 630 nm [8]. The experiments were repeated three times, and the data were shown as absorption means. Effect of Ligand (BIB) and Complexes at Sub- MIC on Biofilm: The same approach was employed for the previously mentioned biofilm formation study (one isolate was chosen). On the other hand, (BHI) broth contains such compounds at sub-MIC concentrations. At 37oC, the plates were incubated for 24 hours. Following that, all wells were rinsed, stained, and read at 630 nm. Positive controls were also performed by adding 200μl of a compound-free fresh bacterial strain (compatible with the 0.5 McFarland standards). 3.Results The hypothesized structures of the investigated compounds were supported by the physical and analytical data (Table 1). 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 Yied % m.p (°C) M.wt (g/mol) Theoretical% (Experimental) % M % Cl % 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) [C24H36B2N12O1 5NiCl]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) [C24H40B2N12O1 7CuCl].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) IHJPAS. 36 (4) 2023 277 Table 2. The BIB ligand and its metal ion complexes' name and molecular formula. Comp The Molecular Formula 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- 277midazole- 1-yl)ethyl) hydrogen borate} Copper(II)] Chloride. FT-IR spectra: The infrared spectra of (Nickel and Copper) complexes showed changes in profile and shifting in the frequency of (υ OH) (Table 3) as a result of coordination with metal ions [9]. 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 [10]. The stretching of (C=N) the imidazole ring has not shown any change in frequency or profile, and these are because of the lack of coordination with metal ions through (C=N)[11]. The spectra of the Ni(II) complex exhibit lattice water at (3437) and coordinated water at (3365) cm-1 as well as the lower frequency bands [(991) and (678)]cm-1.The coordinate H2O of the Cu(II) complex appeared at (3388)cm-1 and the lower frequency band [(767) and (680)]cm-1. Low-frequency bands appeared in complex spectra due to υM-O, υM-Cl [12,13] as shown in Figure 3 and 4. Table 3. Main band of FTIR for the ligand (BIB) and it 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 _____ ____ (Ni) C1 3384 3437 (3365) (991) (678) 1535 1487 457 347 (Cu) C2 3406 (3388) (767) (680) 1535 1483 424 333 Figure 2. The suggested structures of synthesized complexes. IHJPAS. 36 (4) 2023 278 Figure 3.FT-IRsspectrum of the (BIB) ligand. Figure 4. FT-IRsspectrum of the Cu(II) complex C2. 1H-NMR Spectroscopy: The data on 1HNMR for BIB ligand are listed in Table 4, which is supported by Figure 5, and the 1HNMR spectrum in DMSO-d6 is shown in Figure 6. The spectrum of the ligand exhibits a chemical shift at (δ3.99 ppm) because of the (B-OH) proton [14, 15]. The multiple peaks noticed in the range (δ4.35-4.40ppm) and (δ3.66ppm) referred to N-CH2 and O-CH2, respectively [16, 17]. Chemical shifts of the methyl group CH3 and residual DMSO at (δ2.44ppm) [16] The peak observed at (δ8.04ppm) attributed to imidazole protons [18]. IHJPAS. 36 (4) 2023 279 Figure 5.Proton positions in (BIB) structure. Table 4. Chemical shifts for 1HNMR of BIB. Assignments in D6-DMSO 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,s Figure 6.1H-NMR Spectrum of BIB. Thermogravimetric analysis (TGA): The ligand's thermal degradation (BIB) and its complexes were investigated by (TGA) and from (25-800) o C in argon. Using this technique, the suggested structures were characterized, as was the thermal stability of the synthesized compounds studied. In the following order, the BIB ligand and its complexes thermal stability increased: (C1 > BIB > C2) (Table 5) [3].Thermal decomposition was utilized to confirm the structures where the results of degradation exhibit high agreement with the found mass loss (practical 100% and theoretical 99.9%), which confirms the IHJPAS. 36 (4) 2023 280 proposed structures of the synthesized compounds. The thermogram of the ligand (BIB) and nickel complex C1 is shown in Figure 7.8. Figure 7.The thermo-gram of the (BIB) ligand. Figure 8.The thermo-gram of Nickel complex C1. IHJPAS. 36 (4) 2023 281 Table 5. TGA of ligand (MBIB) and their complexes: Comp. Compounds (M.wt)(gm/mol) Step Temp. rang of the Decomposition C° 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 Ni C1 [C24H36B2N12O15NiCl]Cl .H2O 901.29 1 25-278 2H2O+2Cl+N 6O7C13H20+B 54.34 54.77 2 278-800 N6O4C8H7 27.84 26.93 Residue <800 O3H7C3B Ni 17.80 18.30 Cu C2 [C24H40B2N12O17CuCl]. Cl 924.14 1 25-262 3H2O+2Cl+2( N3O2C6H8)+O 48.58 48.94 2 262-800 N6O9C12H18B +Cu 50.24 50.26 Residue <800 B 1.16 0.81 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) exhibits the band at 313nm (31948 cm-1) due to the π→ π* transition [19]. This band shifted to a lower frequency in nickel and copper complexes, and this is a result of the coordination with metal ions. The Ni (II) complex (Figure 10) appears to have two bands at [962 nm (10395 cm-1) and 785 nm (12738 cm-1)] which were attributed to[3T1(F) →3A2(F) and 3T1(F) →3T1(P)] transitions of the tetrahedral Ni (II) complex [20]. The Meff of Ni(II)C1 was 2.78 B.M. This value agrees with the tetrahedral geometry of nickel complexes [20, 21]. In the spectrum of the copper (C2) complex, there were two absorption bands at [960 nm (10416 cm-1) and 739 nm (13531 cm-1)] that were assigned to the 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 the copper complex's (C2) distorted octahedral geometry [21, 22]. The molar conductance in distilled water for each synthesized complex was measured using (10-3 M). The complexes (C1and C2) are (1:1) exhibit electrolyte behavior [23, 24]. IHJPAS. 36 (4) 2023 282 Table 6. Electronic transitions of the BIB and its complexes, proposed geometry, molar conductivity, and magnetic susceptibility. Figure 9. UV-Vis spectrum of BIB . .complex 1Vis spectrum of C-UV .10 Figure Comp ℷ nm (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) 169 2.23 Distorted Octahedral IHJPAS. 36 (4) 2023 283 .complex 2Vis spectrum of C-UV.11 Figure 4.Anti-biofilm Activity Assessing biofilm formation of P. aeruginosa isolates To quantify biofilm intensity, absorbance at 630nm was determined using a microplate reader.As a result, the absorbance values correlate to the degree of biofilm thickness formed utilizing the isolates in question. Based on the limits summarized the obtained results were categorized into four groups (strong, moderate, weak, and non-biofilm producer) (Table 7). The present study indicated that out of 15 (Pseudomonas aeruginosa) isolates, 4 (26.6%) formed a weak biofilm, 5 (33.3%) formed a moderate biofilm, whereas 6 (40.0%) formed a strong biofilm [25-28]. Table 7. Biofilm intensity based on estimated cutoff value*of P. aeruginosa isolates. ID Biofilm intensity OD630 Limits number of isolates Number of isolates percentage % 1 Non-biofilm producer < 0.05 0 0 2 Weak 0.05 - 0.10 4 26.6 % 3 Moderate 0.10 - 0.20 5 33.3 % 4 Strong ≥ 0.20 6 40.0 % *cutoff value = 0.05 (defined as the Mean of control OD630 plus 3* Standard deviation). Determination of the (Metronidazole, BIB and all complexes) MIC MIC is the lowest antibiotic concentration (μg /mL) that inhibits the growth of a given strain of bacteria [29-32]. The susceptibility of the one higher biofilm producer isolate of P.aeruginosa (P.aeruginosa #5) towards metronidazole, BIB, and all complexes was tested. Variations in susceptibilities to metronidazole, BIB, and all complexes for isolate (P.aeruginosa no.5) were observed (Table 8, Figure 12). IHJPAS. 36 (4) 2023 284 Table 8.The MIC and sub-MIC of MTN, BIB and all complexes against P. aerougenosa isolates. Comp compound code P. aerougenosa isolates MIC(μg/ml) Sub MIC (μg/ml ) MTN As P5 1024 512 BIB As1 P5 512 256 C1Ni As3 P5 1024 512 C2Cu As2 P5 512 256 Figure 12.The MIC of MTN, BIB and All complexes against P. aerougenosa. The Effect of Sub MIC of (MTN, BIB and all Complexes) on Bio-film Formation As shown in Table 9, the study's results showed that metronidazole, its ligand (BIB), and all of its complexes at sub-MIC levels were very good at killing the biofilms of bacteria that were being tested. However, the effects of various compounds vary. Obviously, the biofilms were significantly (P< 0.001) reduced in isolates C2Cu>MTZ >BIB > C1Ni. Table 9. Anti-biofilm activity of MTN, BIB, MBIB and all complexes sub-MIC. Comp. code Bacteria OD Before Treatment After Treatment P MTN 5 Mean± SD 0.492±0.045 0.277±0.068 < 0.001 BIB 5 Mean± SD 0.492±0.045 0.281±0.086 < 0.001 C1Ni 5 Mean± SD 0.492±0.045 0.306±0.038 < 0.001 C2Cu 5 Mean± SD 0.492±0.045 0.222±0.032 < 0.001 Metals such as copper are used as antibacterial agents. The coordination of metal ions with aromatic ligands leads to increase antibacterial and cytotoxic activity [33, 34]. 5. Conclusion The reaction of metronidazole with boric acid generated a new ligand (BIB), and its metal complexes with Ni(II) and Cu(II) were generated in a 2:1 (BIB: M) mole ratio. Spectral and physicochemical techniques were used to characterize each compound that was synthesized. 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