Pharmacological and spectral studies of synthetic biomimetic copper complexes derived from 3-hydroxyflavone derivatives as anti-inflammatory agents

2.1 Preparation of Ligand (L¹–L³)

Equimolar amount of 3-hydroxyflavone and o-aminophenol (L¹)/o-aminobenzoic acid (L²)/o-aminothiazole (L³) was dissolved in ethanol (40 mL). Acetic acid (1.0 mL) was added to this solution. The solution was stirred for 3 h and the precipitate was formed. The precipitate was filtered and washed with water and ethanol.

• L¹: Yield: 60%. Anal. Calcd for C₂₁H₁₅NO₃: C, 76.58; H, 4.59; N, 4.25. Found: C, 76.64; H, 4.62; N, 4.28. FAB mass spectrometry (FAB-MS), m/z 330 [M+1]. ¹H-NMR (400 MHz, CDCl₃) δ: 6.6–7.8 (13H, m, Ar-H) and 11.2 and 10.8 (2H, s, O–H, D₂O exchangeable, 3-hydroxyflavone and o-aminophenol moities).¹³C-NMR (400 MHz, CDCl₃, ppm): 150.8 (C-2), 140.6 (C-3), 153.8 (C-4), 112.8(C-5), 145.6 (C-6), 124.4 (C-7), 126.9 (C-8), 156.6 (C-9), 118.8 (C-10), 133.6 (C-1′), 124.3 (C-2′, 6′), 126.5 (C-3′, 5′), 126.4 (C-4′), 130.6 (C-1″), 115.2 (C-2″), 120.6 (C-3″), 119.2 (C-4″), 126.2 (C-5″) and 140.8 (C-6″).

• L²: Yield: 65%. Anal. Calcd for C₂₂H₁₅NO₄: C, 73.94; H, 4.23; N, 3.91. Found: C, 73.98; H, 4.26; N, 3.98. FAB mass spectrometry (FAB-MS), m/z 358 [M+1]. ¹H-NMR (400 MHz, CDCl₃) δ: 6.7–7.9 (12H, m, Ar-H), 11.6 and 10.4 (2H, ¹³C-NMR (400 MHz, CDCl₃, ppm): 149.4 (C-2), 110.2 (C-3), 154.5 (C⚌N), 115.6 (C-5), 146.4 (C-6), 122.8 (C-7), 126.4 (C-8), 152.6 (C-9), 119.9 (C-10), 132.5 (C-1′), 125.4 (C-2′, 6′), 127.6 (C-3′, 5′), 126.5 (C-4′), 148.6 (C-1″), 113.4 (C-2″), 132.6 (C-3″), 116.5 (C-4″), 128.9 (C-5″), 140.8 (C-6″) and 168.2 (COOH).

• L³: Yield: 62%. Anal. Calcd for C₁₈H₁₂N₂O₂S: C, 67.49; H, 3.78; N, 8.75. Found: C, 67.42; H, 3.72; N, 8.69. Fast atom bombardment mass spectrometry (FAB-MS), m/z 322 [M+1]. ¹H-NMR (400 MHz, CDCl₃) δ: 5.9–7.9 (13H, m,Ar-H), 12.9 and 10.8 (2H, s, S–H & O-H, D₂O exchangeable). ¹³C-NMR (400 MHz, CDCl₃): 149.4 (C-2), 110.2 (C-3), 154.5 (C⚌N), 115.6 (C-5), 147.4 (C-6), 122.6 (C-7), 126.9 (C-8), 154.6 (C-9), 121.9 (C-10), 132.5 (C-1′), 125.4 (C-2′, 6′), 127.6 (C-3′, 5′), 126.5 (C-4′), 158.6 (C-11), 103.4 (C-12) and 148.9 (C-13).

2.2 Biological studies

3.1 Molar conductance

The molar conductance data for the copper complexes measured in DMSO solution for the 0.001 M solutions are given in Section 2. The values of the complexes fall in the range of 8–29 Ω⁻¹ cm² mol⁻¹, which is the expected range of 1–35 Ω⁻¹ cm² mol⁻¹ for the complexes to behave as non-electrolytes (Geary, 1971). Thus, the complexes have non-electrolytic nature as evidenced by the involvement of the acetate group in coordination. This result was confirmed from the chemical analysis of CH₃COO⁻ ion not precipitated by addition of FeCl₃. The complexes did not show electrolytic properties.

3.2 IR spectra

In order to study the binding mode of the Schiff base to the metal in the complexes, the IR spectrum of the free ligand was compared with the spectra of the complexes. The IR spectrum of the ligands shows a ν(C⚌N) peak in the region 1645–1632 cm⁻¹. The IR spectra of all complexes show ν(C⚌N) bands at 1629–1590 cm⁻¹ (Bansod et al., 2007) and it is found that the ν(C⚌N) bands in the complexes are shifted to lower energy regions compared to the free ligands. The shift of this band towards energy side is probably caused by an increase in the C⚌N bond order due to the coordination of the nitrogen with the copper atom.

However, the spectra of complexes show two characteristic bands at 1630–1602 and 1404–1344 cm⁻¹ attributed to υ_asy(COO⁻) and υ_sy.(COO⁻), respectively, indicating the participation of the carboxylate oxygen atom in the complexes. The mode of coordination of the carboxylate group has often been deduced from the magnitude of the observed separation between the υ_asy(COO⁻) and υ_sy.(COO⁻), the separation value (A) between υ_asy(COO⁻) and υ_sy.(COO⁻) in metal complexes was more than 200 cm⁻¹ (260–216 cm⁻¹) which suggests the coordination of the carboxylate group in copper complexes of the ligands in a monodentate fashion (Nakamoto, 1988). The Schiff base ligands display a band around 844 cm⁻¹ ascribed to υ C–S (Sandipan Sarkar et al., 2009) which shifts to lower frequencies in their spectra of copper complexes in the region 838–832 cm⁻¹, respectively, suggesting the coordination of copper ions through the sulfur atom of the thioazole moiety. The band at 3466 cm⁻¹ for υ(OH) in the free ligand disappeared on complexation, indicating coordination through a deprotonated oxygen.

The band observed in the region 1534–1526 cm^–1 is due to the ν_C=C stretching of the aromatic ring system. In all the copper-Schiff base complexes, most of the band shifts observed in the wave number region 1142–980 cm⁻¹ are in agreement with the structural changes observed in the molecular carbon skeleton after complexation, which cause some changes in the (C–C) bond lengths. Conclusive evidence of the bonding is also shown by the observation that new bands in the spectra of all copper complexes appear in the low frequency regions at 550–516, 504–498 and 486–448 cm¹ characteristic to υ(M–O), υ(M–N) and υ(M–S) stretching vibrations, respectively, that are not observed in the spectra of free ligands (Anacona et al., 2009). The IR bands at 810–854 and 784–799 cm⁻¹, υ(H₂O) of coordinated water, is an indication of the binding of the water molecules to the metal ions.

3.3 Electronic spectra

The electronic spectra of the Schiff base ligands and their copper complexes in DMSO were recorded at room temperature and the band positions of the absorption maxima; band assignments and the proposed geometry are shown in Table 1. The electronic spectra of the ligands and their complexes were recorded in DMSO as a solvent. The absorption spectrum for L¹ shows band at 225 and 312 nm. These bands can be attributed to n–π^∗and π–π^∗ transitions within the Schiff base molecule. The electronic spectrum of the corresponding [CuL¹(H₂O)] complex in DMSO reveals a broad band at 558 nm assignable to ²B_1g→²A_1g transition (Chandra and Gupta, 2005) which is characteristic of square planar environment around the copper(II) ion. Similar spectral features were assigned for other complexes.

The electronic spectra of all the complexes exhibit bands in the regions 200–225, 272–332 and 362–390 nm, which may be due to the π–π^∗ transition of the benzenoid/or n–π^∗ (COO), π–π^∗ transition of the >C⚌N– chromophore and n–π^∗ transition of the >C⚌N– chromophore, coupled with the secondary band of the benzene ring, respectively. Further, there were a few sharp bands observed in the region 233–257 nm in the spectra of the complexes, which could be assigned as charge transfer bands.

3.4 NMR spectra

The ¹H and ¹³C-NMR spectrum of ligands were recorded in CDCl₃ and are given in Section 2. All the protons were found as to be in their expected region (Li et al., 2008). The conclusions drawn from these studies lend further support to the mode of bonding discussed in their IR spectra. The number of protons calculated from the integration curves and those obtained from the values of the expected CHN analyses agree with each other. The ¹H and ¹³C-NMR spectra of the copper complexes could not be obtained due to their paramagnetic nature.

3.5 FAB mass spectra

Mass spectra provide a vital clue for elucidating the structure of the compounds. The mass spectra of the ligand (L¹) and its copper complex [CuL¹(H₂O)] were recorded and their stoichiometric compositions were compared. The intensity of these peaks reflects the stability and abundance of the ions (Hamming and Foster, 1972). The molecular ion peak for the ligand (L¹) is observed at 424m/z (Fig. 2) whereas its copper complex shows the molecular ion peak at 515m/z (Fig. 3), which confirms the stoichiometry of the metal complexes to be [CuL¹(H₂O)] type. Elemental analysis values are in close agreement with the values calculated from molecular formula assigned to these complexes, which is further supported by the FAB-mass studies of respective complexes. Similar mass spectral features were assigned for other ligands and their copper complexes.

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Figure 2

FAB mass spectrum of Ligand (L¹).

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Figure 3

FAB mass spectrum of [CuL¹(H₂O)] complex.

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3.6 ESR spectra

The ESR spectrum of the [CuL¹(H₂O)] complex was recorded in DMSO at 300 and 77 K. The spectrum at 300 K shows one intense absorption band at high field, which is isotropic due to the tumbling motion of the molecules. However, this complex in the frozen state (77 K) shows four well resolved peaks with low intensities in the low field region and one intense peak in the high field region. The magnetic susceptibility value reveals that the copper complex has a magnetic moment of 1.92 B.M corresponding to one unpaired electron, indicating that the complex is mononuclear in nature. This fact was also evident from the absence of half field signal, observed in the spectrum at 1600 G due to the Δm_s = ±2 transitions, ruling out any Cu–Cu interaction (Gaballa et al., 2007). The ESR spectral data are given in Table 2.

The f value for cu, Zn SOD is 160 cm indicating a tetrahedral distortion from square planar geometry and is one of the features that enhances the catalytic activity of the enzyme. From the above EPR data, the f values for copper complexes were determined to be in the range of 142–158 cm. Therefore, our synthesized copper complexes exhibiting appreciable square planar distortion are expected to show high SOD like activity. Molecular orbital coefficients α² (in-plane σ-bonding), β² (in-plane π-bonding) and γ² (out-plane π-bonding) were calculated using Eqs. 1–3.

3.7 Cyclic voltammetry

Cyclic voltammetry is the most versatile electroanalytical technique for the study of the electroactive species. The important parameters of a cyclic voltammogram are the magnitudes of the anodic peak current (ipa), cathodic peak current (ipc), anodic peak potential (Epa) and cathodic peak potential (Epc). The copper(II) complexes in the DMSO solution undergo one electron reduction and also one electron oxidation to form monovalent and trivalent metal species. The electrochemical behaviours of the Schiff base Cu(II) complexes in DMSO (0.1 M of TBAP as supporting electrolyte (scan rate 100 mVs⁻¹) at 300 K were examined. Table 3 summarizes the potentials and their assignments, which mainly depend on the geometry and environment around the copper ion (i.e., ligand core).

All copper complexes show well-defined redox couple corresponding to copper(II/I). The cathodic peak appearing in the negative potential of 658–768 mV range corresponds to the reduction of copper(II) and the corresponding anodic peak also appears in the negative potential of 658–526 mV which corresponds to the oxidation of copper(I). The measured ΔEp values (116–158 mV) clearly indicate that these redox couples are quasi-reversible in nature. The i_pa/i_pc falls at ca. 0.9–1.1, clearly confirming one electron transfer in this redox process.

The cyclic voltammogram of the [CuL¹(H₂O)] complex in the DMSO solution at 300 K in the potential range +0.4 to −0.8 V at scan rate 0.1 V s⁻¹ was recorded (Fig. 4). It shows a well defined redox process corresponding to the formation of the quasi-reversible Cu(II)/Cu(I) couple. The anodic peak at Epa = 0.065 mV versus Ag/AgCl and the associated cathodic peak at Epc = 0.076 mV correspond to the Cu(II)/Cu(I) couple. The [CuL¹(OAc)] complex exhibits a quasi-reversible behaviour.

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Figure 4

Cyclic voltammogram of [CuL²(H₂O)] complex.

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The cyclic voltammogram of the [CuL²(H₂O)] and [CuL³(H₂O)] complexes in the DMSO solution at 300 K are shown in Figs. 4 and 5. On comparing the cyclic voltammograms, we observed that the variation in oxidation and reduction potential may be due to distortion in the geometry of the complexes which arises due to different donor atoms coordinated to the copper ion. It is concluded that the present ligand systems stabilize the unusual oxidation states of copper ion during electrolysis. Similar electrochemical behaviour was observed and assigned for other complexes.

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Figure 5

Cyclic voltammogram of [CuL³(H₂O)] complex.

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3.8 Thermogravimetric analyses

The thermal decomposition studies of the complexes were carried in the temperature range 30–600 °C with a sample heating rate 5 °C/min in a static nitrogen atmosphere. All the complexes undergo first step decomposition with weight loss between 220 and 250 °C due to loss of the one coordinated water molecule. The complexes show the second step decomposition between 390 and 420 °C due to removal of half part of the ligand. Finally the complexes undergo decomposition between 500 and 530 °C due to loss of the remaining part of ligand. The final residue was analysed by IR spectra and identified as CuO which corresponds to the calculated value. These features support the formulae [CuL¹(H₂O)], [CuL²(H₂O)] and [CuL³(H₂O)], assigned to the complexes.

3.9 XRD

The crystallite size of the complex was calculated from the Scherre‘s formula: $$d_{\text{XRD}}=0.9\lambda /\beta \cos \theta \text{,}$$ where λ is the wavelength, β is the full-width half maximum of the characteristic peak and θ is the diffraction angle for the hkl plane.

From the observed d_XRD patterns, the average crystallite sizes for the copolymer [CuL¹(H₂O)], [CuL²(H₂O)] and [CuL³(H₂O)] complexes are found to be 75, 84 and 62 nm, respectively.

3.10 Antimicrobial activity

To contribute in the field of bioinorganic chemistry, consequently, the compounds synthesized have been evaluated for their antibacterial, antifungal, DNA binding studies. The antibacterial and antifungal tests were carried out using the serial dilution method.

The in vitro antimicrobial activities of the investigated compounds were tested against the bacterial species S. aureus, E. coli, K. pneumaniae, P. vulgaris and P. aeruginosa and fungal species A. niger, R. stolonifer, A. flavus, R. bataicola and C. albicans. The minimum inhibitory concentration (MIC) values of the compounds are summarized in Tables 4 and 5. A comparative study of the ligands and their complexes (MIC values) indicates that complexes exhibit higher antimicrobial activity than the free ligands. In this study, the antimicrobial activity of the ligands may be due to the heteroaromatic residues. Compounds containing >C⚌N group have enhanced antimicrobial activity than >C⚌C< group. The growth of certain microorganisms takes place even in the absence of O₂.

The enhanced activity of the complexes can be explained on the basis of the Overtone’s concept (Anjaneyula and Rao, 1986) and Tweedy’s Chelation theory (Dharamaraj et al., 2001). Chelation can considerably reduce the polarity of the metal ion, which in turn increases the lipophilic character of the chelate. Thus, the interaction between metal ion and the lipid is favoured. This may lead to the breakdown of the permeability barrier of the cell, resulting in interference with the normal cell processes. If the geometry and charge distribution around the molecule are incompatible with the geometry and charge distribution around the pores of the bacterial cell wall, penetration through the wall by the toxic agent cannot take place and this will prevent the toxic reaction with in the pores.

Chelation is not the only criterion for antibacterial activity. Some important factors that contribute to the activity are nature of the metal ion, nature of the ligand, coordinating sites, geometry of the complex, concentration, hydrophilicity, and lipophilicity. Certainly, steric, and pharmokinetic factors also play a decisive role in deciding the potency of an antimicrobial agent. The higher toxicity of the metal complex can be attributed to the effect of metal ion on the normal cell processes. The widespread interaction of metal ions with cellular compounds is due to the fact that all these structures contain a variety of functional groups that can act as metal binding ligands. The problem is how to obtain those interactions in cells and organisms where the non-polar membrane exists to hinder the movement of charged metal ions into the cell, where myriad of metal binding sites exists to compete for the metal ion, and where specificity of cellular interaction must occur in order to obtain therapeutic value.

Apart from this, the mode of action of these compounds may also invoke the hydrogen bond though the imine C⚌N group with the active centres and thus interfere with normal cell processes. Presence of lyphophilic and polar substituents is expected to enhance antimicrobial activities. Heterocyclic ligands with multifunctionality have greater chance of interaction either with nucleoside bases (even after complexation with metal ion) or with biologically essential metal ions present in the biosystem which can be promising candidates as bactericides since they always look to enact especially with some enzymatic functional groups, to achieve higher coordination number. Thus, the antibacterial property of metal complexes cannot be ascribed to chelation alone but it is an intricate blend of all the above contributions.

3.11 SOD activity

A great deal of interest has been shown in the development of therapeutic SOD mimetics for the scavenging of superoxide ( ${\text{O}}_2^{\text{•}-}$ ) which is a precursor to reactive oxygen and nitrogen species (RONS) known to contribute to oxidative stress. The SOD mimetic activities of the copper(II) complexes were determined and compiled in Table 6. In the present study, [CuL³(H₂O)] has higher SOD activity as compared to other complexes. A greater interaction between superoxide ion and Cu(II) complex is induced due to the stronger axial bond, which results in an increased catalytic activity. In addition azomethine ligands containing electron withdrawing substituent stabilizes the Cu(I) complex formed during superoxide dismutation reaction which further reacts with superoxide ion to give hydrogen peroxide. The distorted geometry of these complexes may favour the geometrical change, which is essential for the catalysis as the geometry of copper in the SOD enzyme also changes from distorted square planar geometry. The difference in reactivities of the synthesized complexes may be attributed to the coordination environment and the redox potential of the couple Cu⁺/Cu⁺⁺ in copper(II) complexes during the catalytic cycle. It has been reported that the redox potential of copper(II) ions is affected by the coordination structure of copper(II) complexes (Díaz et al., 2009) (see Table 7).

3.12 Anti-inflammatory activity

The reference drug, indomethacin, is a non-steroidal anti-inflammatory drug, which is used for the treatment of a broad spectrum of inflammation related diseases suggested to be due to inhibition of prostaglandin syntheses. The initial phase is attributed to the release of histamine and serotonin. The oedema maintained between the first and the second phase is due to kinin like substances (Lazzaroni and Bianchi Porro, 2004). The second phase is said to be promoted by prostaglandin like substances. It has been reported that the second phase of oedema is sensitive to drugs like hydrocortisone, phenylbutazone and indomethacin. The results show that the copper complex which has higher activity than other complexes is due to the high diffusion of the copper complex.