Synthesis, Identification and Biological Study of New Pharmaceutical Model Based on Amino Acids with Some of Its Complexes

The synthesis of the MBIB ligand by the reaction of the BIB ligand with methionine in 1:1 ratio, and the metal complexes with Ni(II), Cu(II), and Pt(IV) were described. All synthesized compounds were characterized using spectroscopic methods such as FT-IR, 1 H NMR, UV-VIS, thermal analysis (TG and DSC), atomic absorption (AAS), elemental microanalysis (C.H.N.S), melting point (m.p.), magnetic susceptibility, molar conductivity measurements, and chloride content. All the complexes were electrolytes, and the suggested geometric shapes for the complexes were octahedral. The magnetic properties of the platinum complex were diamagnetic, while those of the nickel and copper complexes were paramagnetic. All synthesized compounds have good anti-biofilm properties against bacteria ( Pseudomonas auroginosa, a gram-negative bacteria), except for C 1 (Ni), which is inactive against the same bacteria. In addition, the ligand was evaluated as an anti-cancer agent against human breast cancer (MCF-7), but its effectiveness has been shown to be less effective compared to metronidazole.


Mahmoud and Khaleel
Iraqi Journal of Science, 2023, Vol. 64, No. 11, pp: 5501-5516 Metronidazole (MTN) is one of several drugs with an imidazole ring [1].MTN is a preferred drug because it is used in both human and veterinary medicine.It is used to treat diseases caused by anaerobic bacteria and protozoa [2].Metronidazole has been studied for its antibacterial activity against gram-negative aerobes and some gram-positive bacteria [3].Boric acid (also called boracic acid or orthoboric acid) is a very weak inorganic acid [4].Boric acid is extensively utilized in the pharmaceutical and chemical industries; it is widely used as an antiseptic, in eye wash preparation, as an insecticide, and as a buffering agent [5].Methionine (Met) is one of the nine amino acids that humans essentially must have for healthy growth and tissue repair.Methionine is utilized as a nutritional element in parenteral nutrition, health foods, infant milk preparations, and sports supplements [6].Methionine is important for the growth of cancer cells and the inhibition of cancer cells.Methionine restriction appears to prevent the growth of cancer cells and may enhance the efficacy of chemotherapeutic medicines, according to a growing body of evidence [7].In this work, the MBIB ligand will be synthesized by the reaction of a metronidazole derivative with methionine (Figure 1).Following this, metal complexes of MBIB with Ni(II), Cu(II), and Pt(IV) metal ions will be formed (Figure 2).All the prepared compounds will be characterized using physicochemical and spectral analyses.The biological and medicinal activities of the prepared compounds will be evaluated.

Methods 2.2.1. Synthesis of 2-amino-4-(methylthio) butanoic(bis(2-(2-methyl -5-nitro-1Himidazol -1yl) ethyl) boric)anhydride (MBIB)
The BIB ligand was synthesized in our previous work [from metronidazole (0.1 g) and boric acid (0.0180 g) in a 2:1 mole ratio and refluxed in H 2 O for 8 hours].The solution of BIB (0.1 gm, 0.276 mmol) in distilled water (8 mL) was added to a solution of methionine (0.0404 g, 0.2718 mmol) in distilled water (4 mL).The mixture was then stirred in a water bath at 42 °C for 8 hours.Part of the solvent was evaporated, and the product was collected using an ice bath and dried in an oven at 80 °C.

Synthesis of MBIB complexes with Ni(II), Cu(II) and Pt(IV))(C 1 , C 2 and C 3 )
A solution of MBIB (0.1 g, 0.2004 mmol) in distilled water (5 mL) and metal salt (0.0238, 0.0170 and 0.0486 gm (0.1000 mmol)) of [(NiCl 2 .6H 2 O), (CuCl 2 .2H 2 O) and (K 2 PtCl 6 )], respectively in distilled water (2 mL) was added.The mixture was then heated to reflux for 5 hours.Part of the solvent was evaporated, and the product was collected using an ice bath and dried in an oven at 80 °C.

Anti-biofilm activity 2.2.3.1. Minimum inhibitory concentrations MIC:
The minimum inhibition concentration of the ligand and complexes was measured in concentrations (32,64,128,256, 512 and 1024 mg/mL) using the microdilution broth method in microtiter plates.

Biofilm formation
The biofilm formation was evaluated in a polystyrene 96-well microplate.Bacterial strains were cultivated overnight for 24 hours at 37 °C in broth culture with glucose (0.2%), 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 methanol (200 µL, 99%).The microplate was washed three times with distilled water, dyed for 15 minutes with crystal violet (200 µL, 0.1%), and dried at room temperature.After the dye that adhered to the biofilm was solubilized in absolute ethanol (200 µL), the absorbance was measured at 630 nm.The experiments were repeated three times, and the data were shown as absorption means [8,9].

Anticancer activity
To evaluate the cytotoxic effect, a 96-well plate was used for the MTT cell viability assay.The cell-lines were seeded (1 × 10 4 cells/ well).Cells were treated with the studied compounds after 24 hours.The effectiveness of the anti-cancer treatment was studied through the literature [10], and absorbance at 575 nm was evaluated.

Results and discussion
The physical and analytical data are consistent with the proposed structures of the compounds in the study (Table 1).

FT-IR spectroscopy
The FT-IR spectrum of the MBIB ligand showed a change in the position of OH band compared with the original BIB ligand because of the insertion of methionine moiety (Table 3) [11].A new band appears in the ligand and complex spectra at 1612-1622 cm -1 , which is assigned to the C=O group [12].In comparison to BIB, the band of C=N for the ligand and its complexes did not change [2,12].The spectra of the ligand and its complexes appeared band at 1473-1487 cm -1 which is due to the B-O band [12,14].Low-frequency bands appeared in complex spectra due to M-O, M-S, M-N, and M-Cl stretching vibrations, as shown in Figures 3-6 [2,12,15].

1 H NMR spectroscopy
The 1 H NMR spectrum of the MBIB ligand is shown in Figure 7, and the data are consistent with the literature [12,14,18, and 19].

Thermogravimetric analysis (TGA)
The TG analysis was carried out under argon gas at a heating rate of 10 C/min.and a temperature range of 25-800 o C. Using this technique, the structures were characterized, as well as the thermal stabilities of the synthesized compounds.In the following order, the MBIB ligand and its complex's thermal stability were increased: (C 1 > C 3 > MBIB > C 2 ) (Table 4) [2].Thermal decomposition was utilized to confirm the structures where the information of degradation exhibits high agreed with found mass loss and calculation, which confirms the proposed structures of synthesized compounds.The thermograms of the MBIB ligand and platinum complex C 3 are shown in Figures 8 and 9.

UV-Vis spectral studies
All the details of the spectra are listed in Table 5.The spectrum of MBIB (Figure 10) exhibited the bands in the region of 323 nm (30959 cm -1 ), which were assigned to the π -π* transition [11].The spectrum of the Ni(II) complex (Figure 11) exhibited a shift in the position of the π -π* transition.Double bands were at 964 nm (10373 cm -1 ), 670 nm (14925 cm -1 ), which noticed 3 A 2 g → 3 T 2 g and 3 A 2 g → 3 T 1 g(P) transition, respectively [21].The magnetic moment of the nickel complex (C 1 ) was 3.12 BM, and this value of M eff agreed with octahedral geometry [22,23].The spectrum of the copper complex (Figure 12) exhibited a shift in the position of the π -π* transition.The paramagnetic C 2 complex showed two-bands at 976 nm (10245 cm -1 ), and 695 nm (14388 cm -1 ).These bands refer to the 2 B 1 g → 2 A 1 g and 2 B 1 g→ 2 B 2 g transitions, respectively.The magnetic moment of the copper complex (C 2 ) was 2.48 BM, and the value of M eff demonstrated distorted octahedral geometry [24].The spectrum of Pt(IV) complexes is shown in Figure 13, which shows a shift in the π -π* transition.The electronic spectrum of diamagnetic C 3 Pt showed a double band at 970 nm (10309 cm -1 ) and 540 nm (18518 cm -1 ), that also refer to the 1 A 1 g→ 1 T 1 g and 1 A 1 g→ 3 T 1 g(H) transitions of an octahedral Pt(IV) complex [25,26].The MC measurements of C 1 , C 2 , and C 3 complexes in distilled water showed that the complexes C 1 and C 2 are 1:1.Complex C 3 has a 1:3 ratio of electrolyte properties [27,28].

Anti-cancer activity
Metronidazole and MBIB ligand 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 14 and 15).Different concentrations of metronidazole and MBIB ligand were used in the MTT assay to determine cell viability and the inhibition rate of the tumor cell line.In comparison with the normal cell line WPL-68, the percentage of viable cells was calculated, as shown in Tables 6 and 7.According to the results, MBIB ligand has been killed the least by metronidazole.This may be because methionine is present, which links to several important metabolic pathways that play key roles in epigenetics, nuclear functions, detoxification, and cellular membranes [29].

Determination of the MIC and sub-mic for(metronidazole, MBIB and all complexes)
MIC is defined as the lowest concentration of a substance tested that prevented the blue to pink change of resazurin [34].The broth microdilution method was used to determine the MIC of MTN, MBIB, and all complexes in a 96-well microtiter plate.The susceptibility of an isolated P. aeruginosa with higher biofilm formation proberity (P.aeruginosa no. 5) against MTN, MBIB, and all complexes was tested by determining the MIC using microtiter plates.The results revealed that the MIC of MTN, C 2 Cu, C 3 Pt complexes which can inhibit bacterial growth, was 1024 μg/mL, and the MIC of MBIB ligand was 512 μg/mL, but C 1 Ni had no inhibitory effect on bacterial growth.The MIC are shown in Table 9 and Figure 16.

Antibiofilm activity of MTN, MBIB, and effective complexes
The MTN, MBIB, and effective complexes were evaluated for their anti-biofilm activity.These compounds exhibited anti-biofilm activity against the tested bacterial isolate (P.aerougenosa), and anti-biofilm activity against them before the biofilm formation showed various degrees of inhibition.It is markedly evident that biofilm was significantly reduced (P< 0.001), evaluated by the microtiter plate assay method as shown in Table 10.The MTN, MBIB, and effective complexes succeeded in preventing them from forming biofilm, and most of them kill live cells inside the biofilm.Metals such as copper and platinum are used as anti-bacterial agents [35].

Conclusion
The new MBIB ligand was synthesized via the reaction of methionine with the BIB ligand.Its metal complexes with Ni(IΙ), Cu (II), and Pt(II) were also synthesized (MBIB:M) in a 2:1 mole ratio.All synthesized compounds were characterized, and the suggested structure utilizes spectral and physicochemical techniques.The results revealed that all complexes have octahedral geometry and an electrolyte character.The biological results revealed that all the prepared compounds had excellent anti-biofilm activity against bacteria (Pseudomonas auroginosa gram-negative) except for C 1 (Ni), which is inactive against the same bacteria.The synthesized MBIB ligand has less activity against human breast cancer (MCF-7) cells than metronidazole.

Figure 1 :
Figure 1: The structure of the MBIB ligand

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

Figure 6 :
Figure 6: FT-IR spectrum of the platinum (IV) complex C 33

Figure 14 :
Figure 14: The cytotoxicity of metronidazole ligand on MCF-7 cells (Log for the original concentration), after 24 hours of incubation at 37 °C

Figure 15 :
Figure 15: The cytotoxicity of MBIB ligand on MCF-7 cells (Log for the original concentration), after 24 hours of incubation at 37 °C

Figure 16 :
Figure 16: The MIC of MTN, MBIB and all complexes against P. aerougenosa.

Table 1 :
Data from analysis as well as the physical characteristics of MBIB ligand and its metal complexes

Table 2 :
The name of MBIB and proposed formula for its metal ion complexes

Table 4 :
TGA of MBIB ligand and their complexes

Table 5 :
Electronic transitions of the MBIB ligand and its complexes, proposed geometry, molar conductivity, and magnetic susceptibility

Table 6 :
Cytotoxicity effects of metronidazole on MCF-7 and WRI-68 cells after 24 hours incubation at 37 °C

Table 7 :
Cytotoxicity effects of MBIB ligand on MCF-7 and WRI-68 cells after 24 hours incubation at 37 °C

Table 8 :
Biofilm intensity based on an estimated cutoff value* of P. aeruginosa isolates

Table 9 :
The MIC and sub-MIC of MTN, MBIB, and all complexes against P. aerougenosa isolates

Table 10 :
Anti-biofilm activity of MTN, MBIB and effective complexes sub-MIC