New Metronidazole Derivative and Some of Its Complexes with Antibiofilm study

Synthesis of a new ligand, namely [bis(2-(2-methyl-5-nitro-1 H-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 (NiCl 2 .6H 2 O and CuCl 2 .2H 2 O) 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( 1 H 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.


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 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.

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.

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 0 C.

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 37 o C, 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

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.

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 proposed structures of the synthesized compounds.The thermogram of the ligand (BIB) and nickel complex C1 is shown in Figure 7.8.
Table 6.Electronic transitions of the BIB and its complexes, proposed geometry, molar conductivity, and magnetic susceptibility.

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][26][27][28].

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][30][31][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).

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.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].

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.The proposed structure of the Ni(II) complex was the tetrahedral geometry and the octahedral geometry of the Cu(II) complex, and the results showed that the complexes have electrolytic behavior.Against the Gram-negative bacterium Pseudomonas auroginosa (G-), all synthesized compounds were evaluated as anti-biofilm agents.The results showed that in comparison to other compounds, copper complexes were more active.

Figure 2 .
Figure 2. The suggested structures of synthesized complexes.

Table 2 .
The BIB ligand and its metal ion complexes' name and molecular formula.

Table 3 .
Main band of FTIR for the ligand (BIB) and it complexes.

Table 7 .
Biofilm intensity based on estimated cutoff value*of P. aeruginosa isolates.

Table 8 .
The MIC and sub-MIC of MTN, BIB and all complexes against P. aerougenosa isolates.
Figure 12.The MIC of MTN, BIB and All complexes against P. aerougenosa.