SYNTHESIS, CHARACTERIZATION AND ANTIMICROBIAL ACTIVITIES OF SOME 5-BROMOURACILMETAL ION COMPLEXES

Six new complexes, [Mn(BrU)2(H2O)2]4H2O (1), [Cd(BrU)2]2H2O (2), [Cu(BrU)2(H2O)2]2H2O (3), [Co(BrU)2(H2O)2]4H2O (4), [Ni(BrU)2(H2O)2]4H2O (5) and [Ag(BrU)(BrUH)]2(H2O) (6) were prepared by the reaction of 5-bromoouracil with MnCl2·4H2O, CdCl2·2.5H2O, CuSO4·5H2O, (CH3COO)2Co·4H2O, (CH3COO)2Ni·4H2O and AgNO3 respectively. The complexes were characterized by melting point, elemental microanalyses, IR and H NMR spectroscopy. The obtained data indicated that the ligand interacted with the metal ions in its mononegatively charged enol form in a bidentate fashion. Thermogravimetric analyses (TGA and DTG) were also carried out. The data obtained agreed well the proposed structures and showed that the complexes were finally decomposed to the corresponding metal or metal oxide. The ligand and its metal-ion complexes were tested for their antimicrobial activities against four bacterial strains (B. subtillis, S. aureus, E. coli and P. aeruginosa) by the agar-well diffusion technique using DMSO as a solvent. The obtained data showed that the complexes were more potent antimicrobial agents than the parent ligand.


INTRODUCTION
Uracil is a naturally occurring nucleic acid [1] and is the most important pyrimidine base that represents remarkable roles in the structures and functions of enzymes and drugs [2]. In recent years, uracil and its derivatives and complexes have been used in the synthesis of antibacterial, antiviral and anti-tumour agents [3][4][5]. Intensive investigations have shed light on the natures of the interactions between metal ions and nucleic acid bases, including their abilities to form complexes [2] which have wide-ranging biological activities, such as antimalarial, antitumoural, antibacterial, and antiviral activities [6,7]. 5-Bromouracil (BrUH), (5-bromopyrimidine-2,4(1H,3H)-dione) (Scheme 1), is a halogenated derivative of uracil which has the ability to terminate DNA replication in viruses and other cell culture systems [8][9][10][11]. This termination of DNA replication occurs via the replacement of a thymine base in the genetic code with 5-bromouracil, resulting in an unusual code that stops the replication process [12,13]. On the other hand, the thymine replacement by 5-bromouracil in the DNA genetic sequence has a significant influence on cancer therapy, leading to higher sensitivity to ionizing radiation [14,15] without influencing the un-irradiated cells. Moreover, 5-bromouracil has a great influence on the growth of viruses, bacteria and other microorganisms [8][9][10][11]. Heterocyclic molecules have tautomeric forms at equilibrium in solutions, where hydrogen atoms can move to various locations within the molecules. In this respect, 5-bromouracil isomers exist in enol and keto tautomeric forms [13], Scheme 2. where X = H (uracil) X = Br (5-bromouracil) Scheme 1. Molecular structure of uracil and 5-bromouracil. At alkaline pH values, the hydrogen atom bonded to the N(3) atom in the keto form of 5bromouracil is removed, indicating that the N(3) hydrogen atom is acidic (pK a = 8); however, the N(1) atom is basic [13,16]. 5-Bromouracil is a nucleotide base that can bind to metals or bind to tissues via metals [17][18][19]. Additionally, its complexes or compounds have been identified as biologically active materials acting as antibacterial and anti-tumour agents [18,20].
In this article, the preparation, characterization and biological activities of Mn(II), Cd(II), Co(II), Ni(II), Cu(II) and Ag(I) complexes with 5-bromouracil has been described. The determination of the binding sites of 5-bromouracil with these metal ions can give additional value by correlating the coordination modes of 5-bromouracil with its biological activity. The obtained complexes were characterized by elemental analysis, infrared (IR) spectroscopy, 1 H nuclear magnetic resonance (NMR) spectroscopy, melting point, conductivity measurements as well as thermal analysis (thermogravimetric analysis, TGA, and differential thermogravimetric analysis, DTG). Analytical Department, Al-Azhar University, Cairo, Egypt). 1 H NMR measurements were performed using DMSO as a solvent on a Bruker spectrometer (Zagazig University, NMR Department, Zagazig, Egypt). TGA was carried out at a heating rate of 10 o C using a Universal TGA Q500 instrument (Science & Technology Center of Excellence, Cairo, Egypt). Molar conductivities of the complex solutions (110 -3 M) in dimethylformamide (DMF) were measured at room temperature using a Jenway 4510 conductivity meter. The heavy metals which include manganese (Mn), cadmium (Cd), copper (Cu), cobalt (Co), nickel (Ni), and silver (Ag) are detected by ICP Spectrophotometer Thermo Jarrel Ash model POEMS 3, using 1000 mg/L (Merck) stock solution for standard preparations. [Mn(BrU) 2

Antimicrobial activity
The in vitro antibacterial effects of the compounds were tested against four bacterial strains, i.e. E. coli and P. aeruginosa (Gram-negative bacteria) and B. subtillis and S. aureus (Grampositive bacteria), by an agar well diffusion method using nutrient agar medium [21][22][23]. All bacteria were inoculated into nutrient broth and incubated for 24 h (1.0 mL of inoculant was added to 50 mL of agar media (50 ºC) and mixed). The agar was poured into 120 mm petri dishes and allowed to cool to room temperature. In the agar well diffusion method, the dilution plate method was used to enumerate microorganisms for 24 h [24,25]. Using a sterilized cork borer (7 mm diameter), wells were dug into the culture plates. The compounds dissolved in DMSO (0.1 mL, 250 μmol/mL) were added to these wells. The petri dishes were left at 5 ºC for 2 h, and then, the plates were incubated at 35 ºC for bacterial growth (24 h). At the end of the period, the inhibition zones formed on the medium were evaluated in millimetres (mm). DMSO (0.1 mL) was used as a control under similar conditions. The inhibition zones based on the zone size around the discs were measured and calculated as means of triplicates and are reported as average values of three experiments.

Free ligand and complexes
All the prepared complexes are insoluble in many organic solvents (methanol, ethanol, acetonitrile, chloroform, carbon tetrachloride) and water. Complexes 1, 4 and 6 are soluble in DMF, while the other complexes 2, 3 and 5 are slightly or sparingly soluble in DMF. These complexes were characterized by IR, 1 H NMR spectroscopy, melting point measurements, elemental analyses and TGA. The complexes have a 1:2 metal-to-ligand stoichiometry.

Conductance
The conductivity values measured at 25  C in DMF for 10 −3 M solutions of the free ligand and its complexes are very small, and their values lie in the range of 3.17-8.2 µs·cm −1 , indicating the non-electrolytic nature of the complexes [26].

IR spectra
The infrared spectrum of the free ligand (Table 1) shows a strong broad band at 1677 cm −1 characteristic of (C(4)=O) and a shoulder at 1617 cm −1 corresponding to (C(2)=O), which are practically overlapped. Two weak/medium bands are observed at 3360 and 3168 cm −1 , which may be assigned to (N(3)−H) and (N(1)−H) stretching vibrations, respectively.
According to the forgoing discussion and based on the proposed molecular formulas, the probable structures of the Mn(II), Co(II), Ni(II), and Co(II) complexes are in octahedral geometries. On other hand, the structures for the Cd(II) and Ag(I) complexes are more likely in tetrahedral geometries, as shown in Scheme 3 [32][33][34][35][36].  Table 1. Selected IR frequencies (cm −1 ) and tentative assignments for 5-bromouracil (as a free ligand) and its metal complexes (1-6).

H NMR spectra
The 1 H NMR spectral data of free 5-bromouracil and its Cd(II) and Ag(I) complexes in DMSO are summarized in Table 2. The spectra reveal a characteristic signal for the aromatic proton in its expected region of 7.00−8.00 ppm. The N(1)H signal is located at 11.13 ppm in the spectrum of the free ligand, while the corresponding signals in the spectra of the Cd(II) and Ag(I) complexes are observed at 10.90 and 10.85 ppm, respectively. The N(3)−H signal in the free ligand spectrum (11.51 ppm) is absent in the spectra of the complexes, which is consistent with the forgoing suggestion that 5-bromouracil reacted with the metal ions through its enol form. The Ag(I) complex exhibits a signal at 10.51 ppm, and such a signal is neither present in the spectrum of the free ligand nor in the spectra of the other complexes. This signal may be attributed to the (OH) proton associated with one of the two ligands coordinated with the Ag(І) ion. Accordingly, one of the two ligands bonded to the Ag(І) ion is a neutral molecule in the enol form, while the other is negatively charged, as shown in Scheme ІІІ. The 1 H NMR spectrum of the free ligand confirms the results obtained from the IR spectrum. The obtained data suggests that 5-bromouracil is in the keto form. Dissolving the base with additional KOH converts the keto form to the enol form, forming a soluble salt with the deprotonation of N(3)−H as the pH is raised to 10 [37,38].

Thermal analyses
To confirm the proposed structures of the complexes, thermogravimetric analyses (TGA) were performed. The thermal data for all complexes are summarized in Table 3. The free ligand completely decomposes in one step at approximately 320 °C, as shown in Figure 1, indicating its pure organic structure.  Figure 1. In these complexes, the first decomposition step proceeds at a temperature between 120 °C and 210 °C with a weight loss ranging from of 6.30−13.50%, associated with the loss of the outer-sphere (uncoordinated) water content. The calculated ratio of the outer-sphere water content in the suggested forms is between 6.82% and 13.27%, in good agreement with the observed values ( Table 3).
The second thermal decomposition step for complexes 1, 3, 4, and 5 displayed weight loss in the range of 6.71−7.50% at a temperature range of 240−275 °C, which may be attributed to the loss of coordinated water. This result agrees with the calculated coordinated water values of 6.59-6.99% in the suggested formulas of these complexes. The TGA thermograms of complexes 2 and 6 shows no weight loss in this temperature range, indicating the absence of any coordinated water, which is consistent with the suggested formulas of both complexes.
The loss of organic content associated with the ligands occurs in the next two to six decomposition steps at maximum temperatures between 283 °C and 925 °C. The weight loss associated with these decomposition steps (65.75−78.49%) is in agreement with the calculated values (66.54−72.59%). The total weight loss throughout the decomposition process lies in the

Antimicrobial activity
The in vitro antibacterial activities of the compounds under investigation were tested against four bacterial strains, i.e. two Gram negative bacteria (E. coli and P. aeru Complexes 1, 3 and 4 showed good activities against the Gram subtillis and S. aureus and moderate activity against Gram P. aeruginosa. Complex 5 Complexes 2 and 6 showed excellent activities compared to the positive control against Gram positive B. subtillis and S. aureus aeruginosa. The remarkable activities of the complexes may geometries of the molecules and the type of metal ion [40] (Table 4). showed good activities against the Gram-positive bacterial strains and moderate activity against Gram-negative bacterial strains E. coli 5 showed good activities against the selected bacterial strains. showed excellent activities compared to the positive control against Gram S. aureus and moderate activity against Gram-negative E. coli and . The remarkable activities of the complexes may be due to structural changes geometries of the molecules and the type of metal ion [40].

Inhibition zone diameter in mm
Gram-positive  3 , respectively. The complexes were structurally characterized by melting point, elemental analyses measurements, electrical conductivity, IR and 1 H NMR spectroscopy. The obtained data indicated that the ligand interacted with the metal ions in its mononegatively charged enol form in a bidentate fashion and the complexes have a 1:2 metal-to-ligand stoichiometry. Thermogravimetric analyses (TGA and DTG) were also achieved. The data obtained agreed with the proposed structures and showed that the complexes were finally decomposed to the corresponding metal oxide or metal. The prepared 5-bromouracilM 2+ complexes were screened for their antimicrobial activities by an agar-well diffusion technique using DMSO as a solvent and showed that the complexes were potent antimicrobial.