Synthesis, spectroscopic characterization and in vitro antimicrobial studies of Schiff base ligand, H₂L derived from glyoxalic acid and 1,8-diaminonaphthalene and its Co(II), Ni(II), Cu(II) and Zn(II) complexes

2.1 Material and methods

All the reagents used were of A.R. grade. The metal salts M(NO₃)₂·6H₂O [M = Co(II), Ni(II), Zn(II); n = 6, Cu(II); n = 3] and the chemical 1, 8-diaminonaphthalene and glyoxalic acid (All E. Merck) were commerically pure samples used as received.

2.2 Synthesis of Schiff base ligand, H₂L

To a stirring methanolic solution (25 ml) of 1,8-diaminonaphthalene (1 mmol; 0.158 g), methanolic solution (25 ml) of glyoxalic acid (2 mmol, 0.22 ml) was added gradually. The reaction mixture was magnetically stirred for 24 h at room temperature, leading to the isolation of yellow solid product which was filtered, washed with methanol and dried in vacuum over anhydrous calcium chloride.

2.3 Synthesis of complexes, [ML] [M = Co(II), Ni(II), Cu(II), Zn(II)]

To a solution of metal nitrate (1 mmol) dissolved in 10 ml methanol, methanolic solutions of both 1,8-diaminonaphthalene (1 mmol, 0.158 g) and glyoxalic acid (2 mmol, 0.22 ml) were added simultaneously under stirring. The stirring of reaction mixture was continued for 24 h at room temperature, leading to the isolation of coloured solid product which was filtered, washed with methanol and finally dried in vacuum over anhydrous calcium chloride.

2.4 Physical measurements

The elemental analysis was made using Perkin–Elmer 2400 CHN elemental analyser. The FT-IR spectra (4000–200 cm⁻¹) were recorded as KBr pellets on Perkin Elmer-2400 spectrometer. ¹H and ¹³C NMR spectra at room temperature were recorded in DMSO-d₆ using Bruker Avance II 400 NMR spectrophotometer. EPR spectrum was recorded on E112 ESR spectrometer at room temperature. The FAB-Mass spectra were recorded on Joel SX-102 mass spectrometer. The electronic spectra in DMSO were recorded on Pye-unicam-8800 spectrophotometer at room temperature. Magnetic susceptibility measurements were carried out on a Faraday balance at 25 °C. The electrical conductivities of 10⁻³ solution in DMSO were obtained on a systronic type 302 Conductivity bridge equilibrated at 25 °C ± 0.01. Schiff base and its complexes were tested for antimicrobial activity by disk diffusion assay with slight modifications against some Gram-positive, Gram-negative strains of bacteria and some fungi. After that minimum inhibitory concentration (MIC) of the complexes was determined.

2.5 Organism culture and in- vitro screening

Antibacterial activity of ligand H₂L and its Co(II), Ni(II), Cu(II) and Zn(II) complexes were completed by the disc diffusion method with slight alterations. Inoculums of Streptococcus mutans, Pseudomonas aeruginosa, Methicillin resistant Staphylococcus aureus (MRSA + Ve), Staphylococcus pyogenes, Salmonella typhimurium, and Escherichia coli were prepared in BHI medium and incubated for 18 h at 37 °C. Subsequently a suspension of about 10⁵ cfu mL⁻¹ was prepared in autoclaved saline solution according to the McFarland protocol. Then 10 μL of this saline suspension was mixed with10 mL of sterile antibiotic agar at 40 °C and then 9 cm diameter plates were poured in a laminar flow cabinet. Five paper discs of 6.0 mm diameter were placed on these nutrient agar plates. One milligrams of each test compound was dissolved in 100 μl DMSO to get ready stock solution and from this stock solution varied concentrations 10, 20, 25, 50, and 100 μg/μl of the trial compound were set. Subsequently the compound of varied concentrations was poured over disc plate onto it. The disc of chloramphenicol (30 μg) was used as a positive control and DMSO poured disc as a negative control. The susceptibility was assessed on the basis of diameter of zone of inhibition against Gram-positive and Gram-negative strains of bacteria and fungi. The Zone of inhibition (mm) of the ligand, H₂L and its Co(II), Ni(II), Cu(II) and Zn(II) complexes against Gram-positive and Gram-negative strains of bacteria are shown in Table 1. The macro dilution test with standard inoculums of 10⁵ cfu mL⁻¹ was used to assess the minimum inhibitory concentration (MIC) of test ligand and its complexes. First of all, the ligand and its complexes were dissolved in dimethyl sulfoxide (DMSO) then serial dilutions of the test compounds were prepared to final concentrations of 512, 256, 128, 64, 32, 16, 8, 4, 2 and 1 mg/mL to all tube was added 100 mL of a 24 h old inoculums. The MIC is the lowest concentration of the trial compound, which can control the visible growth after 18 h incubation at 37 °C. The MIC was determined visually after incubation for 18 h, at 37 °C. After 18 h of incubation at 37 °C MIC results for bacterial strains are shown in table 2. For assaying antifungal activity, Candida albicans, Candida krusie, Candida parapsilosis and Cryptococcus neoformans were inoculated in Sabouraud Dextrose broth medium (Hi-Media Mumbai) and incubated for 24 h at 35 °C, and subsequently a suspension of about 10⁶ cfu mL⁻¹ was prepared in sterile saline solution according to the McFarland protocol. Autoclaved Sabouraud Dextrose Agar (SDA) was poured onto 9 cm diameter plates in laminar flow cabinet and then with the help of sterilized cotton swabs the suspension of each fungal cell was streaked onto Sabouraud Dextrose Agar (SDA) plates. Five paper discs of 6.0 mm diameter were put onto Sabouraud Dextrose agar plate. 1 mg of test compounds was dissolved in 100 μl DMSO to get ready stock solution and from this stock solution varied concentrations 10, 20, 25, 50, and 100 μg/μl of the trial compound were set. Subsequently the compound of varied concentrations was poured over disc plate onto it. The disc of fluconazole (30 μg) was use as a positive control and DMSO poured disk as a negative control. The susceptibility of different fungal strains against test compounds was assessed on the basis of diameter of zone of inhibition after 48 h of incubation at 35 °C Table 3. MIC results for fungal strains are shown in Table 4. The in vitro antifungal activity assay showed that [CuL] complex was more active antifungal than H₂L and [ML] [M = Co(II), Ni(II) and Zn(II)].

3.1 Infrared spectra

The prominent bands observed in the IR spectra of Schiff base ligand, H₂L and its complexes are listed in Table 6. The spectrum of free ligand shows a strong band at 1635 cm⁻¹ assigned to ν(C⚌N), indicating the formation of desired Schiff base ligand. This band undergoes negative shift of ∼30 cm⁻¹ on complexation indicating the involvement of azomethine nitrogen in chelation with metal ions in the complexes (Singh and Srivastava, 1988). This was further confirmed by the presence of a new band observed in 440–460 cm⁻¹ region assigned to ν(M–N) (Ferraro, 1971). The intense band at 1664 cm⁻¹ present in the IR spectrum of free ligand may be assigned to ν(C⚌O) of the carboxylic acid. However, this band was found to be absent in all the complexes. Instead, IR spectra of all complexes exhibit two bands at 1615–1635 cm⁻¹ and 1370–1400 cm⁻¹ regions attributed to ν_asym (CO₂⁻) and ν_sym (CO₂⁻) vibrations, respectively, indicating the involvement of the carboxylate oxygen atom in coordination to the metal ion. It is to be noted that Δ value [Δ = ν_asym (COO⁻) − ν_sym(COO⁻)] is larger than 200 cm⁻¹ in all complexes indicating the monodentate coordination of the COO⁻ group to the metal ion (Deacon and Phillips, 1980) which is further confirmed by the presence of new band at 430–480 cm⁻¹ assignable to ν(M–O) (Ferraro, 1971).

3.2 ¹H NMR and ¹³C NMR spectra

The formation of Schiff base ligand, H₂L was further supported by the ¹H NMR spectral study. The ¹H NMR spectrum of free ligand, H₂L (Fig. 1) showed a resonance signals at 9.92 and 8.13 ppm assigned to carboxylic and azomethine protons of proposed Schiff base moiety, respectively. The spectrum of free ligand, exhibited a multiplet in 6.30–7.50 ppm region which may be assigned to naphthalene protons (Jeong et al., 2007). However the resonance signal assigned to carboxylic protons in free ligand disappeared in the ¹H NMR spectrum of Zn(II) complex (Fig. 2) indicating the deprotonation of carboxylic proton and the involvement of oxygen of carboxylate ion in chelation (Garg et al., 2002). However, the position of resonance signals for azomethine proton and the aromatic protons undergo a slight downfield shift and appeared at 8.50 and 6.52–7.62 ppm, respectively on complexation with Zn(II) ion (Hage et al., 1988; Krishnapriya and Kandaswamy, 2005).

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Figure 1

The ¹H NMR spectrum of ligand.

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Figure 2

The ¹H NMR spectrum of Zn(II) complex.

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The ¹³C NMR spectrum of Zn(II) complex revealed the presence of expected number of signals corresponding to the different types of carbon atoms. A strong NMR signal appearing at 161 ppm (Shakir et al., 2009) may reasonably be to azomethine carbon and other at 194 ppm (Geeta et al., 2010) assigned to carboxylate carbon COO⁻. The chemical shifts of naphthalene carbons appear at 138, 129, 127, 125, 123 and 120 ppm. These values were found to be downfield shifted by about 1.0–2.0 ppm as compared with free ligand (Shakir et al., 2009).

3.3 Electronic spectra and magnetic moment

The electronic spectrum of Co(II) complex showed bands at 17,064 cm⁻¹ and 22,779 cm⁻¹. The band at 22,779 cm⁻¹ was assigned to π → π^∗ transitions, suggesting a square planar environment around Co(II) ion which was further supported by its magnetic moment value of 1.76 B.M. (Manhas et al., 1995; Loginova et al., 2008). The electronic spectrum of Ni(II) complex showed band at 28,260 cm⁻¹ which may be assigned to charge transfer and other band at 22,280 cm⁻¹ single d-d band is assigned to ¹A_2g ← ¹A₁g transition (Lever, 1984). The electronic spectrum of Cu(II) complex displays two bands at 16,000 cm⁻¹ and 18,000 cm⁻¹ which may reasonably be assigned to ²A_1g ← ²B_1g and ²E_g ← ²B_1g transitions, respectively, consistent with a square planar geometry around Cu(II) ion which is further confirmed by its magnetic moment value of 1.82 B.M (Figgis, 1966).

3.4 EPR spectrum

EPR spectrum of polycrystalline Cu(II) complex (Fig. 3) has been recorded at room temperature. The absence of hyperfine signal may be attributed to the strong dipolar and exchange interaction between Cu(II) ions in the unit cell (Ahuja and Tripathi, 1991). The calculated g_|| = 2.21 and g_⊥ = 2.05 values reasonably support that the ²B₁ is the ground state having an unpaired electron in the d_x2–y2 orbital (Balhahausen, 1962). The g_|| > g_⊥ (2.32 > 2.05) suggests a square planar environment around Cu(II) ion (Ottaviani et al., 1994; Kivelson and Neiman, 1961). The G parameter G = (g_|| − 2)/(g_⊥ − 2) which measures the exchange interaction between the metal centres in polycrystalline solid has been calculated. According to Hathway if G > 4, the exchange interaction is negligible and if G < 4 considerable exchange interaction occurs in the solid complex in the complex (Hathaway et al., 1971). The G value is greater than 4 indicating the exchange interaction is negligible.

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Figure 3

The EPR spectrum of Cu(II) complex.

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3.5 FAB mass spectroscopy

The FAB-mass spectrum of Schiff base ligand, [H₂L] (Fig. 4) showed a molecular ion peak [M]⁺ at m/z 270 which corresponds to its proposed molecular formula. The molecular ion peaks for the schiff base complexes, [ML] [M = Co(II), Ni(II), Cu(II) and Zn(II)] appear at m/z 326, 326, 331 and 333, respectively, consistent with the proposed molecular formulae of the complexes. A reference spectrum of [NiL] has been shown in Fig. 5.

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Figure 4

The FAB mass spectrum of ligand.

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Figure 5

The FAB mass spectrum of Ni(II) complex.

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3.6 In vitro antimicrobial activity

The in vitro antibacterial and antifungal activity of ligand [H₂L] and its complexes type, [ML], [M = Co(II), Ni(II), Cu(II) and Zn(II)], were tested using the bacterial cultures of S. mutans, P. aeruginosa, Methicillin resistant Staphylococcus aureus (MRSA + Ve), S. pyogenes, S. typhimurium, E. coli and fungal cultures of C. albicans, Candida krusei, C. parapsilosis and C. neoformans, by the disc diffusion method (Khan et al., 2007) and then the minimum inhibitory concentration (MIC) of the ligand and complexes was determined. Chloramphenicol (30 μg) was used as positive control in case of bacterial strains and in case of fungi; fluconazole was used as a positive control. While the disk poured in DMSO was used as negative control. The minimum inhibitory concentration (MIC) was assessed by the macro dilution test using standard inoculums of 10⁵ cfu mL⁻¹_. Initially the compounds were dissolved in dimethyl sulfoxide (DMSO) after that serial dilution of the test compound was set to final concentrations of 512, 256, 128, 64, 32, 16, 8, 4, 2 and 1 mg/mL to each tube was added 100 mL of 24 h old inoculums. The MIC is the lowest concentration of the test compound, which can restrain the apparent growth after 18 h incubation at 37 °C. The MIC was determined visually after incubation for 18 h, at 37 °C. The in vitro study results verified that the Cu(II) complex was found to be more active antimicrobial agent than ligand H₂L and Co(II), Ni(II) Zn(II) complexes. Zone of inhibition was visualized after 18 h of incubation at 37 °C. The susceptibility was assessed on the basis of diameter of zone of inhibition against Gram-positive and Gram-negative strains of bacteria and fungi. The zones of inhibition (mm) of ligand and its complexes against Gram-positive and Gram-negative strains of bacteria are shown in Table 3. Zones of inhibition for fungal strains are shown in Table 3. MIC results for bacterial strains are shown in Table 2 and for fungal strains in Table 4.

The higher antimicrobial activity of the metal complexes as compared to Schiff base ligand may be explained in terms of chelation which makes metal complexes to act as more powerful and potent antimicrobial agents, thus inhibiting the growth of the microorganisms (Berejo et al., 1999; Chohan et al., 2003). Moreover, coordination reduces the polarity of the metal ion mainly because of the partial sharing of its positive charge with the donor groups within the chelate ring system. This process, in turn, increases the lipophilic nature of the central metal atom, which favours its permeation more efficiently through the lipid layer of the microorganism, thus destroying them more aggressively.

The Cu(II) complex showed enhanced activity. The distinct difference in the antimicrobial property of compound further justifies the purpose of this study. The importance of such work lies in the possibility that the new compound might be more effective against bacteria for which a thorough investigation regarding the structure activity relationship, toxicity and in their biological effects which would be helpful in designing more potent antibacterial agents for therapeutic use if required.