Synthesis, Characterization, and Antimicrobial Activity of Novel Sulfonated Copper-Triazine Complexes

Metallotriazine complexes possess interesting biological and medicinal properties, and the present study focuses on the synthesis, characterization, and antimicrobial activity of four novel copper-triazine derivatives in search of potent antibacterial and antifungal drug leads. In this study, 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-4,4′-disulfonic acid monosodium salt (L1, ferrozine) and 3-(2-pyridyl)-5,6-di(2-furyl)-1,2,4-triazine-5,5′-disulfonic acid disodium salt (L2, ferene) have been used as ligands to study the complexation towards copper(II). The synthesized complexes, [CuCl2(ferrozine)]·7H2O·MeOH (1), [CuCl2(ferrozine)2]·5H2O·MeOH (2), [CuCl2(ferene)]·H2O·MeOH (3), and [CuCl2(ferene)2]·H2O·MeOH (4), have been characterized spectroscopically, and preliminary bioassays have been carried out. FTIR spectroscopic data have shown that N=N and C=N stretching frequencies of complexes have been shifted towards lower frequencies in comparison with that of the ligands, confirming new bond formation between Cu and N, which in turn lowers the strength of N=N and C=N bonds. In addition, a bathochromic shift has been observed for UV-visible spectra of complexes (1), (2), (3), and (4). Furthermore, elemental analysis data have been useful to obtain empirical formulas of these complexes and to establish the purity of each complex. Complexes (1) and (2) have shown antibacterial activity for both S. aureus (ATCC® 25923) and E. coli (ATCC® 25922) at 1 mg/disc concentration, and ferrozine has shown a larger inhibition zone against the clinical sample of C. albicans at 1 mg/disc concentration in comparison with the positive control, fluconazole.


Instrumentation.
Elemental analysis was carried out on PerkinElmer 2400 Series II CHNS/O Elemental Analyzer at Atlantic Microlabs, USA. IR spectra were recorded using ermo Scientific NICOLET iS10 spectrophotometer in the spectral range 4000-650 cm −1 for both ligands and complexes.
ermo Spectronic Helios alpha UV-Vis double-beam spectrophotometer was used to measure the absorbance in the range of 190-1100 nm, and baseline correction was performed using matched quartz cuvettes. High-resolution mass spectra were recorded on an Agilent 6210 ESI TOF LCMS mass spectrometer.

Preparation of [CuCl 2 (ferene) 2 ]·H 2 O·MeOH (4).
A procedure similar to above was followed using copper chloride dihydrate (0.25 mmol, 0.0435 g) and ferene (0.50 mmol, 0.2472 g). e resulting mixture was stirred for 5 hours. A brown-yellow colour crystalline precipitate was obtained after 1 day and collected by filtration (yield: 0.1912 g, 65%   measured and expressed as a mean of three replicates. Gentamycin and flucanazole were used as positive controls, and methanol was used as the negative control.

Results and Discussion
3.1. Synthesis. Copper chloride and the relevant ligands were used in 1 : 1 and 1 : 2 ratios to synthesize the desired metal complexes (Figure 2). in-layer chromatography (TLC) was initially used to monitor the progress of reaction, and visualization of spots was done using an iodine bath.

FTIR Analysis.
FTIR data were recorded for dried crystals of ligands and complexes (1)-(4), and literature values were utilized where relevant [41]. e stretching frequency of the pyridine ring (] C�N ) and stretching frequency of the triazine ring (] N�N ) are considered mostly, because their values change upon formation of new bonds serving as good indicators of complex formation.
Stretching frequencies of N�N and C�N in complexes (1) and (2) have shifted to lower frequencies as expected, compared to those values of the free ferrozine ligand, due to σ donation of N lone pair which lowers strength of N�N and C�N bonds (Table 1). Furthermore, a broad band around 3400-3300 cm −1 was observed due to OH groups from methanol or water.
Similarly, stretching frequencies of N�N and C�N in complexes (3) and (4) were observed at lower frequencies in comparison with those of the free ferrozine ligand (Table 1), and a broad band was observed around 3400-3300 cm −1 due to OH groups of solvent. (1, 2, 3, and 4) were recorded in methanol at room temperature ( Figure 3, Table S1, Supplementary Materials). e absorption wavelengths of complexes (1)- (4) have shifted towards longer wavelengths (bathochromic shift) compared to the wavelengths of the reactants (copper, ferrozine, and ferene). Both ferrozine and ferene have aromatic ring systems, and π-π * transitions are thus possible [42]. ese results are in agreement with those previously reported for zinc complexes of ferene and ferrozine [39] where a bathochromic shift was observed for both mono and bis complexes in comparison with that of the free ligand.

Elemental Analysis.
Empirical formulas related to experimental values aided in obtaining the exact molecular formulas of all four complexes (Table 2). It can be seen that experimental values are within ±0.4% of expected values indicating purity of the synthesized complexes.
3.5. Antimicrobial Activity. All four complexes and ligands were studied in vitro for their antimicrobial activity against Gram-positive Staphylococcus aureus ATCC ® 25923 and negative bacteria Escherichia coli ATCC ® 25922 as well as the unicellular fungal species, Candida albicans. Inhibition zones were obtained by adding a concentration of 1 mg/disc, and the diameters of the zones are given in Table 3 for  bacteria and Table 4 for fungi.   Analysis of the inhibition zone diameter revealed that only complex (1) and complex (2) show moderate antibacterial activity when compared to the positive control. It is interesting to see that ferrozine ligand demonstrates antifungal activity.
Antimicrobial activity reported here is of moderate value. Further studies are warranted to optimize this system for greater activity.

Conclusions
We have described the synthesis of four novel water-soluble copper complexes bearing sulfonated pyridyl triazine ligands. FTIR spectroscopic data have confirmed the existence of Cu-N bonds in all four complexes because stretching frequencies of N�N and C�N complexes have been shifted towards lower frequencies in comparison with that of the ligands. In UV-Vis spectra, a bathochromic shift has been observed for complexes (1)-(4). Furthermore, elemental analysis data have been useful to obtain empirical formulas of these complexes and to establish the purity of each complex.
Preliminary bioassays in antimicrobial activity showed moderate antibacterial activity with complexes (1) and (2) whereas ferrozine showed antifungal activity against Candida albicans. To the best of our knowledge, we are the first to report on the antifungal activity of ferrozine. ese findings provide a potential lead for antimicrobial drug development.

Data Availability
e data used to support the findings of this study are included within the article and within the Supplentary Information file.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Acknowledgments
Financial assistance from the University of Sri Jayewardenepura (Grant no. ASP/01/RE/SCI/2015/19) is gratefully acknowledged. Clinical sample of Candida albicans was provided by the Department of Microbiology, Faculty of Medicine, University of Colombo. An earlier version of this work with two of the complexes detailed here was presented as an abstract at Chemistry in Sri Lanka 2015 as found in the link http://www.ichemc.edu.lk/wp-content/uploads/2015/10/ vol-31-no.-2.pdf. e authors also acknowledge the Centre for Advanced Material Research of the University of Sri Jayewardenepura. Table S1: comparison of UV-Vis data of ferrozine, ferene, and complexes (1)