Metal-Based Biologically Active Compounds: Synthesis, Spectral, and Antimicrobial Studies of Cobalt, Nickel, Copper, and Zinc Complexes of Triazole-Derived Schiff Bases

A series of cobalt, nickel, copper, and zinc complexes of bidentate Schiff bases derived from the condensation reaction of 4-amino-5-mercapto-3-methyl/ethyl-1,2,4-triazole with 2,4-dichlorobenzaldehyde were synthesized and tested as antimicrobial agents. The synthesized Schiff bases and their metal complexes were characterized with the aid of elemental analyses, magnetic moment measurements, spectroscopic and thermogravimetric techniques. The presence of coordinated water in metal complexes was supported by infrared and thermal gravimetric studies. A square planar geometry was suggested for Cu(II) and octahedral geometry proposed for Co(II), Ni(II), and Zn(II) complexes. The Schiff bases and their metal complexes have been screened for antibacterial (Pseudomonas aeruginosa, Bacillus subtilis) and antifungal activities (Aspergillus niger, A. flavus). The metal complexes exhibited significantly enhanced antibacterial and antifungal activity as compared to their simple Schiff bases.


Introduction
Of late due to the constant emergence of antibiotics resistance to clinically used compounds, it is the need of the hour to develop novel antibiotics classes, which eventually would target the lipoid layer of the organisms and other aspects of pathogens' life cycle. Metal complexes may be subjected for the design and synthesis of such possibilities having such biological activities [1][2][3]. The chemistry of Schiff bases and their structural analogues has occupied a place of considerable importance [4] as they easily form stable complexes with most transition metal ions [5,6] and well-established biological properties. 1,2,4-triazoles nucleus and their derivatives emerge rapidly with the advances of modern heterocyclic chemistry, promising a variety of medical applications such as antibacterial, antifungal, anticancer, antitumor, anticonvulsant, anti-inflammatory, and analgesic properties [7][8][9][10][11][12]. Schiff bases of 1,2,4-triazoles find diverse applications and extensive biological activity. The incorporation of the 1,2,4triazole unit into Schiff base macrocycles is of considerable current interest as complexes of 1,2,4-triazoles are being developed for potential use in applications such as magnetic materials and photo chemically driven molecular devices [13]. Schiff bases derived from 3-substituted-4-amino-5mercapto-1,2,4-triazoles show analgesic, antimicrobial, antiinflammatory, and antidepressant activities [14].
The aim of the present study was to modify the bioactivities of 1,2,4-triazole Schiff bases and gain the relative derivatives with better curing effect, optimization of hydrophilic/lipophilic character, and improve the bioavailability by coordinating them with transition metal ions. During the course of this work, a series of cobalt, nickel, copper, and zinc complexes of bidentate Schiff bases derived from the condensation reaction of 4-amino-5-mercapto-3methyl/ethyl-1, 2, 4-triazole with 2, 4-dichlorobenzaldehyde have been synthesized. The synthesized Schiff bases and their metal complexes were characterized with the aid of elemental analyses, magnetic moment measurements, spectroscopic, and thermogravimetric techniques. The Schiff bases and their metal complexes have been screened for antibacterial (Pseudomonas aeruginosa, Bacillus subtilis) and antifungal activities (Aspergillus niger, A. flavus).

Materials and Methods.
All the chemicals used in the present investigation were of Analytical grade and used without further purification. The metal contents were determined using standard gravimetric methods; cobalt was estimated as cobalt pyridine thiocyanate, nickel as nickel dimethylglyoximate, copper as cuprous thiocyanate, and zinc as zinc ammonium phosphate [15].
The Perfit electrical melting point apparatus is used to record melting points of the synthesized complexes and are uncorrected. Carbon, hydrogen, and nitrogen were estimated using Perkin-Elmer 2400 Elemental Analyzer at Punjab University, Chandigarh. Electronic spectra of metal complexes were recorded in DMF on a Systronics 2203 doublebeam spectrophotometer in the region 1100-200 nm. IR spectra were recorded on a MB-3000 ABB spectrometer in KBr/Nujol mulls in the range 4000-250 cm −1 . Proton NMR spectra were recorded in DMSO-d 6 on a Bruker ACF 300 spectrometer at 300 MHz using "tetramethyl silane" as the internal standard. Magnetic moments were measured at Institute Instrumentation Centre, IIT Roorkee on vibrating sample magnetometer (Model 155). The Perkin Elmer (Pyris Diamond) instrument was used to carry out thermal analysis of metal complexes in atmospheric air at the heating rate of 10 • C Min −1 using a reference to alumina powder. The EPR spectrum of the copper(II) complex was recorded at SAIF, IIT, Bombay, using VARIAN E-112 Xband EPR spectrometer with cylindrical quartz sample tube operating at microwave frequency 9.5 GHz. Field calibration was checked using tetracyanoethylene (TCNE) free radical for which g = 2.0027 at room temperature.

Antibacterial Activity.
The antibacterial activity of all the synthesized complexes was evaluated by using agar welldiffusion method. All the microbial cultures were adjusted to 0.5 McFarland standard, which is visually comparable to a microbial suspension of approximately 1.5 × 10 8 cfu/mL. 20 mL of Mueller Hinton agar medium was poured into each Petri plate and plates were swabbed with 100 μL inocula of the test microorganisms and kept for 15 min for adsorption. Using sterile cork borer of 8 mm diameter, wells were bored into the seeded agar plates, and these were loaded with a 100 μL volume with concentration of 4.0 mg/mL of each compound reconstituted in the dimethylsulphoxide (DMSO). All the plates were incubated at 37 • C for 24 hrs. Antibacterial activity of each compound was evaluated by measuring the zone of growth inhibition against the test organisms with zone reader (HiAntibiotic zone scale). DMSO was used as a negative control whereas Ciprofloxacin was used as positive control. This procedure was performed in three replicate plates for each organism [18,19].

Minimum Inhibitory Concentration (MIC) Determination.
MIC of all the compounds against bacterial strains was tested through a modified agar well-diffusion method [20]. In this method, a twofold serial dilution of each compound was prepared by first reconstituting the compound in DMSO followed by dilution in sterile distilled water to achieve a decreasing concentration range of 256 to 0.5 μg/mL. A 100 μL volume of each dilution was introduced into wells (in triplicate) in the agar plates already seeded with 100 μL of standardized inoculums (10 6 cfu/mL) of the test microbial strain. All test plates were incubated aerobically at 37 • C for 24 hrs and observed for the inhibition zones. MIC, taken as the lowest concentration of the chemical compound that completely inhibited the growth of the microbe, showed by a clear zone of inhibition, was recorded for each test organism. Ciprofloxacin was used as positive control.

Antifungal Activity.
The antifungal activity of Schiff bases and their metal complexes was evaluated by poison food technique [21]. The molds were grown on Sabouraud's dextrose agar (SDA) at 25 • C for 7 days and used as inocula. The 15 mL of molten SDA (45 • C) was poisoned by the addition of 100 μL volume of each compound having concentration of 4.0 mg/mL reconstituted in DMSO, poured into a sterile Petri plate, and allowed it to solidify at room temperature. The solidified poisoned agar plates were inoculated at the center with fungal plugs (8 mm diameter) obtained from the colony margins and incubated at 25 • C for 7 days. DMSO was used as the negative control whereas fluconazole was used as the positive control. The experiments were performed in triplicates. Diameter of fungal colonies was measured and expressed as percent mycelial inhibition.
dc: average diameter of fungal colony in negative control sets.
dt: average diameter fungal colony in experimental sets.

Results and Discussion
The Schiff bases ( Figure 1) were soluble in ethanol and methanol. All the metal complexes are colored, nonhygroscopic solids stable in air, insoluble in water and many common organic solvents but soluble in DMF and DMSO, decomposed at higher temperature. The elemental analysis supports the formation of 1 : 1 and 1 : 2 metal complexes of HL 1-2 with Co(II), Ni(II), Cu(II), and Zn(II) metal ions.
The molar conductance values of the complexes (measured in 10 −3 M DMF) are in the range 1.5-3.6 ohm −1 cm 2 mol −1 indicating the nonelectrolytic nature [22]. The purity of ligands and their metal complexes has been checked by TLC.

IR Spectra.
The IR spectral data of the Schiff base ligands and their complexes are listed in Table 1. The IR spectra of the complexes have been compared with those of the free ligand in order to determine the coordination sites that may get involved in chelation. By comparing, it was found that azomethine group frequency ν(N=CH) is present in the free ligands at 1595-1597 cm −1 . This band is shifted to the lower frequency (22-24) by 5-15 cm −1 in the spectra of the complexes, indicating coordination of azomethine nitrogen towards the metal ion (M-N).
A new band at 485-514 cm −1 due to ν(M-N) is further confirmed the coordination of metal to azomethine nitrogen. The Schiff bases exhibit a weak broad band around ∼ 2700 cm −1 due to ν(S-H) vibrations [23,24]. This band disappeared in the spectra of the metal complexes indicating deprotonation and complexation through sulphur. In the spectra of metal complexes, bands appeared at 733-771 cm −1 and 334-352 cm −1 were assigned to ν(C-S) and ν(M-S), respectively [22,25]. In the spectra of metal complexes, a broad band in the region 3200-3490 cm −1 indicated the presence of coordinated water molecules. A strong band in the region 1738-1750 cm −1 has been assigned to ν(OOCCH 3 ) in 1 : 1 metal complexes.

3.2.
1 H NMR Spectra. The 1 H NMR spectra have been recorded for Schiff bases and Zn(II) complexes ( Table 2). The NMR spectra of metal complexes indicated a shift of electron density from the ligand to metal. The ligands [HL 1-2 ] showed characteristic azomethine proton singlet at δ 10.40 and 10.55 ppm, respectively. The characteristic signal, due to azomethine proton deshielded in the spectra of metal complexes, suggests coordination of metal ion to azomethine nitrogen atom [23,25,26]. In addition to this, the signal at δ 11.10-11.09 ppm is ascribed to SH proton. Disappearance of SH protons signal in the spectra of metal complexes supported the deprotonation of thiol group and chelation with metal (M-S). The aromatic protons of Schiff bases appeared as a double of doublet and two doublets at δ 7.35, 7.52 and 8.08 ppm, respectively. These aromatic protons signals show a slight downfield shift upon coordination. In the spectra of 1 : 1 Zn complexes singlet at δ 2.29 and 2.32 ppm are due to methyl group of acetate ion.
The Cu(II) complex has magnetic moment value 1.92-2.11 BM, which fall in the normal range (1.7-2.2 BM). The electronic spectra of Cu(II) complexes showed broad band around 18550 cm −1 , which is assigned to 2 B 1g → 2 A 1g (ν 1 ) transition. It is a characteristic band of square planar geometry around the Cu(II) [31]. Table 1: IR spectral data of the ligands and their metal complexes (cm −1 ).    3.4. Thermal Studies. Thermogravimetric analysis was carried out for Cu(L 2 ) 2 and Zn(L 1 ) 2 ·2H 2 O (Figure 2) from 80 • C to 700 • C in atmospheric air [22,23,25]. The decomposition temperature, pyrolysed products, percentage mass loss of the complexes, and the ash (percent) are given in Table 4. The thermograms for both of these complexes show three decomposition steps. In case of Cu(L 2 ) 2 complex, the first step 50-305 • C results in a mass loss of 45.29% (calcd. 47.90%) corresponding to a loss organic moiety. The 2nd and 3rd steps (305-695 • C) correspond to removal of two triazole molecules with mass loss of 40.32% (calcd. 42.52%) of the ligand leaving metal oxide as residue.
3.5. ESR Spectra. ESR spectra of 1 : 1 and 1 : 2 copper(II) complexes in solid state at 298 K show an intense broad signal having no hyperfine structure. The observed g values for Cu(L 1 )(OAc)·H 2 O at room temperature are g = 2.18, g ⊥ = 2.10, g av = 2.12, G = 1.81 and for Cu(L 1 ) 2 at room temperature are g =2. 19, g ⊥ = 2.12, g av = 2.14, G = 1.59. The trend in g values (g > g ⊥ > 2.00) suggested that the unpaired electron is localized in the dx 2 -y 2 orbital and corresponds to square planar geometry of the complexes. The g < 2.3 value confirms the covalent character of the metal ligand bond. The axial symmetry parameter G is less than 4.0, indicates considerable exchange interaction in the solid complex [32]. inhibitory effect against any of the test organism as shown in Tables 5, 6, and 7. The tested chemical compounds showed zone of inhibition ranging between 15 and 26 mm against the Gram positive bacteria. On the basis of zone of inhibition produced against the test bacterium, Co(L 1 )(OAc)·3H 2 O and Zn(L 1 )(OAc)·3H 2 O were found to be most effective against S. aureus with zone of inhibition ranging between 23.6 mm and 20.6 mm, respectively, and four compounds HL 1 , Co(L 1 )(OAc)·3H 2 O, Zn(L 1 )(OAc)·3H 2 O and Zn(L 2 )(OAc)·3H 2 O were found to be best against B. subtilis, with zone of inhibition ranging between 21.6 mm and 23.6 mm ( Table 5). In the whole series, the MIC of chemical compounds (Figure 3) ranged between 16 and 256 μg/mL against Gram positive bacteria. Compound Co(L 1 )(OAc)·3H 2 O, Zn(L 1 )(OAc)·3H 2 O and Zn(L 2 )(OAc)·3H 2 O were found to be best, as they exhibit the lowest MIC of 32 μg/mL against S. aureus and 16 μg/mL against B. subtilis ( Table 6).
All the synthesized compounds screened for their antifungal activity, among these three compounds HL 1 , Co(L 1 )(OAc)·3H 2 O, and Co(L 1 ) 2 ·2H 2 O showed more than 55% inhibition of mycelial growth against Aspergillus niger, whereas three compounds HL 1 , Co(L 1 )(OAc)·3H 2 O, and    Zn(L 1 )(OAc)·3H 2 O, showed more than 55% inhibition of mycelial growth against A. flavus. Co(L 1 )(OAc)·3H 2 O showed the highest inhibition of fungal mycelium (61%) against A. flavus (Table 7). Among all the tested chemical compounds, Co(L 1 )(OAc)·3H 2 O showed highest antibacterial and antifungal activity. The antimicrobial studies suggested that the Schiff bases were found to be biologically active and their metal complexes showed significantly enhanced antibacterial and antifungal activity against microbial strains in comparison to the free ligands. The overtone's concept [33] and Tweedy's chelation theory [34] can be used to explain the enhanced in antimicrobial activity of the metal complexes. According to the Overtone's concept of cell permeability, the lipid membrane surrounding the cell favors the passage of only lipid-soluble materials; therefore, liposolubility is an important factor which controls the antimicrobial activity. On chelation, polarity of the metal ion is reduced to a greater extent due the overlapping of the ligand orbital and partial sharing of the positive charge of the metal ion with donor groups. Moreover, delocalization of the π-electrons over the whole chelate ring is increased and lipophilicity of the complexes is enhanced. The increased lipophilicity enhances the penetration of the complexes into the lipid membranes and blocks the metal binding sites in the enzymes of microorganisms. These complexes also disturb the respiration process of the cell and thus block the synthesis of proteins, which restricts further growth of the organism. In general, metal complexes are more active than ligands as they may serve as principal cytotoxic species.

Conclusion
The Schiff bases HL 1 and HL 2 coordinate in 1:1 and 1:2 metal-ligand ratios, as confirmed by analytical, IR, PMR, electronic, magnetic measurements, and thermal studies. The presence of coordinated water in metal complexes was confirmed by IR and TG studies. The trend in g values (g > g ⊥ > 2.00) suggested that the unpaired electron is localized in the dx 2 -y 2 orbital and corresponds to square planar geometry of the copper complexes. Based on these, the proposed structures are shown in Figure 4. The antimicrobial studies suggested that the Schiff bases were found to be biologically active, and their metal complexes showed significantly enhanced antibacterial and antifungal activity against microbial strains in comparison to the free ligands. Thus, exhibiting their broad spectrum nature can be further used in pharmaceutical industry for mankind, as an antimicrobial agent, after testing its toxicity to human beings.