Synthesis, characterization and biological screening of some Schiff base macrocyclic ligand based transition metal complexes as antifungal agents

2.1.1 Synthesis of ligand: 6, 7, 14, 15-tetrahydroxy-1, 4, 9, 12-tetraazacyclohexadecane-5, 8, 13, 16-tetrone (1)

The hot ethanolic solution (25 ml), of tartaric acid (8.30 g, 0.05 mol) and a hot ethanolic solution (25 ml) of ethylenediamine (3.00 g, 0.05 mol) were mixed slowly with constant stirring. This mixture was refluxed at 60–70 °C for 7 h in presence of few drops of concentrated hydrochloric acid. On keeping it overnight at 0 °C, a cream white precipitate was formed, which was filtered, washed with ethanol and dried in vacuo over P₄O₁₀ and recrystallized from methanol.

Yield: 75%; mp > 300 °C; IR (KBr) cm⁻¹: 3443 (O–H), 3196 (N–H), 3010 (C–H), 1617 (C⚌O), 1411 (C–N), 1066, 885, 771; ¹H NMR (300 MHz, δ ppm from TMS in DMSO-d₆, 300 k): δ 11.67–12.56 (4H, Br N–H), δ 4.20–4.35 (s, 4H, C–H), δ 2.94–3.02 (s, 8H, C–H₂); ¹³C NMR (CDCl₃) (δ, ppm): 173.5 (C⚌O), 38.09 (4CH₂); ESI MS (m/z) 348 [M] ⁺, 349 [M+1]⁺; Elem. Anal. Calcd. C 41.39%, H 5.70%, O 36.76%, N 16.09%; found C 41.40%, H 5.72%, O 36.78%, N 16.10%.

2.1.2 Synthesis of cobalt(II) complex

Solution of CoCl₂·6H₂O (0.711 g, 3 mmol) dissolved in 20 ml methanol was added dropwise to a methanolic solution (20 ml) of the ligand (1.044 g, 3 mmol) with continuous stirring. The resulting solution was stirred for 7 h at 30 °C and the solution was reduced to half of its volume. It was then allowed to stand overnight in a refrigerator. A light pink product separates out, which was isolated by filtration under vacuum. It was washed thoroughly with hexane and dried in vacuo over fused CaCl₂. The compound was recovered in solid state. It was recrystallized from methanol.

Yield: 65%; mp > 300 °C; UV–Vis (DMSO) cm⁻¹: 12,748–13,320, 16,885–17,487, 24,722–25,350; IR (KBr) cm⁻¹: 3443 (O–H), 3194 (N–H), 2977 (C–H), 1606 (C⚌O), 1369 (C–N), 1085, 881, 637, Far IR (CsI, cm⁻¹) 452 (Co–N), 345 (Co–Cl). ¹H NMR (300 MHz, δ ppm from TMS in DMSO-d₆, 300 k): δ 11.70–12.60 (4H, Br, N–H), δ 4.22–4.40 (4H, C–H), δ 2.97–3.08 (8H, C–H₂), ¹³C NMR (CDCl₃) (δ, ppm) 171.2 (C⚌O), 46.00 (4CH₂). ESI MS (m/z) 478 [M]⁺, 480 [M+2]⁺. Molar conductance, Λ_m (Ω⁻¹cm⁻² mol⁻¹, 10⁻³ DMSO, r.t.): 32. μ_eff (r.t., BM): 4.98. Elem. Anal. Calcd. C 30.15%, H 4.10%, O 26.77%, N 11.72%; found C 30.17%, H 4.11%, O 26.78%, N 11.75%.

2.1.3 Synthesis of nickel(II) complex

This compound was also synthesized by the above same procedure except that NiCl₂·6H₂O was used instead of CoCl₂·6H₂O. A light green product was obtained which was recrystallized from methanol.

Yield 62%; mp > 300 °C; UV–Vis (DMSO) cm⁻¹: 11,668, 13,351, 14,662, 19,120, 24,390; IR (KBr) cm⁻¹: 3417(O–H), 3190 (N–H), 2978 (C–H), 1602 (C⚌O), 1386 (C–N), 1063, 811, 614, Far IR (CsI, cm⁻¹) 468 (Ni–N), 340 (Ni–Cl). ¹H NMR (300 MHz, δ ppm from TMS in DMSO-d₆, 300 k): δ 11.71–12.60 (4H, Br, N–H), δ 4.25–4.37 (4H, C–H), δ 2.96–3.10 (8H, C–H₂).¹³C NMR (CDCl₃) (δ, ppm) 176.5 (C⚌O), 49.50 (4CH₂). ESI MS (m/z) 479 [M]⁺, 481 [M+2]⁺. Molar conductance, Λ_m (Ω⁻¹cm⁻¹ mol⁻¹, 10⁻³ DMSO, r.t.): 34. μ_eff (r.t., BM): 2.97. Elem. Anal Calcd. C 30.16%, H 4.10%, O 26.79%, N 11.72%; found C 30.17%, H 4.12%, O 26.80%, N 11.73%.

2.1.4 Synthesis of copper(II) complex

This compound was also synthesized by the above same procedure except that CuCl₂·2H₂O was used instead of CoCl₂·6H₂O. A Sky blue product was obtained, which was recrystallized from methanol.

Yield: 67%; mp > 300 °C; UV–Vis (DMSO) cm⁻¹: 13,824–14,392, 18,892–19,365, 24,870–25,435; IR (KBr) cm⁻¹: 3419 (O–H), 3189 (N–H), 2976 (C–H), 1600 (C⚌O), 1390 (C–N), 1075, 888, 644, Far IR (CsI) cm⁻¹: 431 (Cu–N), 335 (Cu–Cl). ¹H NMR (300 MHz, δ ppm from TMS in DMSO-d₆, 300 k): δ 11.72–12.61 (4H, N–H), δ 4.23–4.39 (4H, C–H), δ 2.97–3.11 (8H, C–H₂); ¹³C NMR (CDCl₃) (δ, ppm): 174.9 (C⚌O), 44.80 (4CH₂). ESI MS (m/z) 483 M⁺, 485 [M+2]⁺. Molar conductance, Λ_m (Ω⁻¹cm⁻¹ mol⁻¹, 10⁻³ DMSO, r.t.): 29. μ_eff (r.t., BM): 1.98; Elem. Anal. Calcd. C 29.86%, H 4.10%, O 26.52%, N 11.60%; found C 29.88%, H 4.12%, O 26.55%, N 11.63%.

2.2.1 In vitro antifungal activity

2.2.2 MTT assay

H9c2 rat cardiac myoblasts were cultured and maintained as monolayer in Dulbecco’s modified Eagle’s medium (DMEM), high glucose, supplemented with 10% fetal bovine serum (heat inactivated), 100 units/ml penicillin, 100 μg/ml streptomycin, and 2.5 μg/ml amphotericin B, at 37 °C in humidified incubator with 5% CO₂ (Gupta et al., 2006). Only viable cells were used in the assay. Exponentially growing cells were plated at 1.2 × 104 cells per well into 96-well plates and incubated for 48 h before the addition of drugs to achieve the maximum confluency of the cells. Stock solutions were prepared by dissolving the compounds in 20% (v/v) DMSO and further diluted with fresh complete medium to achieve 1 M concentration. Cells were incubated with different concentrations of the standard drug SD and compounds KNi, KCu and ligand (K) for 48 h at 37 °C in 5% CO₂ humidified incubator together with untreated control sample. At appropriate time points, cells were washed in PBS, treated with 50 μl MTT solution (5 mg/ml, tetrazolium salt) and incubated for 45 min at 37 °C. After 45 min of incubation at 37 °C, the cell supernatants were discarded, MTT crystals were dissolved with acid isopropanol and the absorbance measured at 570 nm. All assays were performed in triplicate. Percent viability was defined as the relative absorbance of treated versus untreated control cells. Plates were analyzed in an ELISA plate reader (Labsystems Multiskan RC, Helsinki, Finland) at 570 nm with a reference wavelength of 655 nm.

3.1.1 Cobalt(II) complex

The electronic spectrum of the mononuclear cobalt(II) complex exhibits absorption bands in the range 12,748–13,320, 16,885–17,487 and 24,722–25,350 cm⁻¹, which may be assigned to ⁴T_1g(F)→ ⁴T_2g(F)(ν₁), ⁴T_1g→ ⁴A_2g(ν₂) and ⁴T_1g(F)→ ⁴T_1g(P)(ν₃) transitions, respectively, suggesting an octahedral geometry around a Cobalt(II) ion, in the complexes under study. Furthermore, the magnetic moment measurements recorded at room temperature lie at 4.98 BM. This value is indicative of an octahedral geometry (Carlin, 1965; Chandra and Gupta, 2004) of these complexes.

3.1.2 Copper(II) complex

The electronic spectrum of the mononuclear copper(II) complex recorded at room temperature, in DMF solution, shows broad band absorption in the range 13,824–14,392, 18,892–19,365 and 24,890–25,435 cm⁻¹, which may be assigned to ²B_1g→ ²A_1g, ( ${\text{d}}_{x^2}-y^2$ → $d_{z^2}$ )(ν₁), ²B_1g→ ²B_2g, ( ${\text{d}}_{x^2}-y^2$ → d_zy)(ν₂), and ²B_1g→ ²E_g, ( ${\text{d}}_{x^2}-y^2$ → d_zy, d_yz)(ν₃) transition and it is in conformity with octahedral geometry, an indication of the most probable geometric configuration of the synthesized metal complexes is their magnetic moment values. So, it has been further confirmed by the magnetic moment measurements, room temperature values lie at 1.98 BM. corresponding to the presence of one unpaired electron and it supports an octahedral geometry (Chandra, 2009).

3.1.3 Nickel(II) complex

The magnetic moment of the Ni(II) complex at room temperature lies at 2.97 BM. These values are in tune with high spin configuration and show the presence of an octahedral environment around the Ni(II) ion. The electronic spectra of the nickel(II) complex showed transition bands at 11,668, and 13,351, 14,662 cm⁻¹ which are assigned to the transition ³A_2g→³T_2g, 19,120(shoulder), 24,390 cm⁻¹ which are assigned to the transition ³A_2g→³T_1g(F). These bands indicate an octahedral geometry around the nickel(II) ion in its complex.

3.2.1 Macrocyclic ligand

IR spectrum of the ligand does not exhibit any band corresponding to the free primary amine group. The absence of a broad absorption band in the region 3100–2500 cm⁻¹ characteristic for (O–H) group of carboxylic acid and presence of an absorption band in the region 1675–1775 cm⁻¹ characteristic for the carbonyl group (C⚌O) in tartaric acid, indicates that the OH group of tartaric acid was detached from the COOH group to form a bond between carboxyl carbon atom and amino group nitrogen of ethylenediamine, also suggesting complete condensation of the reactants and the elimination of water molecule. This has been confirmed by the appearance of a strong signal at 1411 cm⁻¹ which may be attributed to the C–N bond. A sharp medium intensity band observed at 3196 cm⁻¹ may be assigned to υ(N–H) of the secondary amine group. The ligand also shows a signal for the C⚌O at 1617 cm⁻¹ and C–H at 3010 cm⁻¹ vibrations. The low frequency of C⚌O group as compared to acetone (1715 cm⁻¹) is attributed to resonance with lone pair of the nitrogen.

3.2.2 Complexes

The molar conductance measurements of the complexes in DMF correspond to nonelectrolytic nature. Thus, the complexes may be formulated as [M(K)Cl₂] where M = Ni(II), Co(II), and Cu(II) and K is [(C₁₂H₂₀O₈N₄)]. The shifting in the band of υ(C–N) toward the lower wave number in the metal complexes indicates that the coordination takes place through the nitrogen of the υ(C–N) group, hence implying that the ligand (K) is tetradentate. This indicates the flow of electron density toward the metal atom through the C–N group. This has been finally established through far IR spectra by the appearance of new signals seen at 482, 470, 452 cm⁻¹ in the spectra of metal complexes which gives us clear proof for the presence of metal–nitrogen bond in Co(II), Ni(II), and Cu(II) complexes, respectively. Other vibrating signals seen at 347, 337 and 330 cm⁻¹ in the spectra of metal complexes give us proof of the presence of metal–chlorine bond in Co(II), Ni(II) and Cu(II) complex, respectively.

3.5.1 Macrocyclic ligand

The thermal analysis (TG/DTA) of the ligand and its metal complexes was recorded under nitrogen atmosphere at the heating rate of 10 °C/min. The ligand is stable up to 215 °C and shows a continuous weight loss up to 380 °C, Therefore the whole ligand gets decomposed in a single step (Fig 1). The DTA of the ligand shows two endothermic peaks; one broad endothermic peak at 228 °C with a shoulder at 210 °C, which correspond to the melting and the first inflexion point. The second inflexion on the DTA curve occurs at 351 °C which represents a small weight loss step from 360 °C to 380 °C.

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Figure 1

TG/DTA spectrum of macrocyclic ligand (K).

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3.5.2 Complexes

The thermal gravimetric (TG) analysis was used as a probe to prove the associated water or solvent molecules to be in coordination sphere or in crystalline form (Emara and Omima, 2007). The thermo gram of copper(II), nickel(II) and cobalt(II) complexes are more stable than the macrocyclic ligand and does not decompose up to 255 °C, 253 °C and 250 °C, respectively (Fig. 2). It shows a major step of decomposition from 255 °C to 330 °C which is detected by DTA at 320 °C, this corresponds to the loss of two phthalic acids and two ethylenediamine moieties (observed weight 75.5%, theoretical weight 72.85%). It is very interesting to note that the complexes gain some 7% weight from 335 °C to 410 °C and do not decompose further. This weight gain of complexes may be attributed to the migration of the metal (M_layer) to the new vacant sites produced by the partial reduction of M²⁺ to M¹⁺ in case of Copper and M⁴⁺ to M²⁺ in case of cobalt and nickel, then subsequent oxidation of M¹⁺, M²⁺ in copper, cobalt and nickel complexes, respectively (Gaillot et al., 2005; Cheney et al., 2008).

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Figure 2

TG/DTA spectrum of Co(II) complex.

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3.6.1 Minimum inhibitory concentration (MIC)

Table 1 summarizes the in vitro susceptibilities of three Candida species. The data are reported as MICs required to inhibit the growth of the tested species. All the Candida species were found to be more sensitive to Ni(II) and Cu(II) complexes, compared to Co(II) complex and the ligand.

3.6.2 Sterol assay

Sterol quantitation method is employed to determine ergosterol content of fungal cell membrane. An important mechanism of action by which antifungal drugs inhibit yeast cell growth is through disruption of the normal sterol biosynthetic pathway, leading to a reduction in ergosterol biosynthesis (Kelly et al., 1995). Ergosterol is the major sterol component of the yeast cell membrane and is responsible for maintaining cell integrity and function (Bard et al., 1978; Rodriguez et al., 1985). Therefore, disruption of this pathway by drugs or additional compounds leads to fungistasis. Total sterol content of samples treated with varying concentrations of Ni(II) complex, Co(II) complex, Cu(II) complex and ligand was studied (Table 2). From the results, it is clear that with increase in test compound concentrations, % ergosterol inhibition increased and finally at MIC values, a flat line was observed indicating absence of ergosterol in the sample. Fig. 3(A, B, C, D) shows typical scans of % ergosterol inhibition in C. albicans ATCC 10261 in the presence of different test compound concentrations. It was observed that Ni(II) complex and Co(II) complex drastically reduces ergosterol content of cell membrane followed by the Cu(II) complex and the ligand itself.

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Figure 3

UV spectrophotometric sterol profile of Candida albicans. (A) In presence of Ni(II) complex in concentration range of 20–40 μg/ml. (B) Cu(II) complex in concentration range of 30–90 μg/ml. (C) Co(II) complex in concentration range of 50–140 μg/ml. (D) Ligand in concentration range of 50–190 μg/ml. (Data represents mean of three experiments).

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