Studies on the mixed ligand complexes of copper(II) involving a sulfa drug and some potentially bi or tridentate ligands under physiological conditions

2.1 Materials and physical measurements

All the ligands used were extra pure Sigma products. Double distilled conductivity water was used for the preparation of all solutions. The pH titrations were carried out at 37 °C and I = 0.15 mol dm⁻³ (NaClO₄) under nitrogen with a digital pH meter with glass and calomel combined electrode assembly as described earlier (Irving et al., 1967; Nair et al., 1999; Nair et al., 1982; Nair and Santappa, 1981). Titrations on the mixed ligand complex systems were done on 50 ml portions of solutions containing low concentrations (0.002 & 0.0025 M) of Cu(ClO₄)₂, stz (A) and ligand (B) in 1:1:1 and 1:1:2 ratios with known volumes of standard CO₂− free NaOH. The auxiliary data in all the binary systems have been taken from the literature (Nair and Regupathy, 2010; Nair et al., 1980, 1997). The pH profiles obtained for the mixed ligand complex systems were processed using SCOGS computer program (Sayce, 1968; Sayce and Sharma, 1972) and the results are given in Tables 1 and 2.

2.2 Preparation of complexes

The CuAB complexes in Cu(II)-stz(A)-gly, 2aba, dapa and orn(B) systems were prepared by mixing 5 mmol of copper acetate in 20 ml of water, 5 mmol of stz in 20 ml aqueous ethanol and 5 mmol each of gly/2aba/dapa/orn in 20 ml water. The pH of the reaction mixture was maintained at 6.0 by adding few drops of 0.002 M NaOH. The contents were kept in a water bath maintained at 60 °C with constant stirring. The precipitated solid complexes were filtered, washed with water and ether. It was further recrystallised using ethanol and the products were dried in vacuum over fused calcium chloride.

The microanalysis was performed using Heraeus micro analyzer. Magnetic susceptibility measurements were carried out using the Gouy balance at 304 K using mercury tetra(thiocyanato)cobaltate(II) as a calibrant. Conductivity measurements were carried out at room temperature on freshly prepared 10⁻³ M DMSO solution using Systronics 305 conductivity meter. The electronic spectra of the complexes were recorded on Perkin–Elmer 402 spectrometer. The IR spectra of the samples were recorded on a Perkin–Elmer 783 spectrometer as KBr discs. The ESR spectrum of the complexes was recorded in DMSO on Varian ESR spectrometer. Powder XRD was performed using Shimadzu XD-3 diffractometer. SEM measurements were carried out using JSM-5610 scanning electron microscope. TGA and DTA were recorded on Perkin–Elmer 7 series thermal analyzer equipped with Pyres software under a dynamic flow of nitrogen (10 l/min) and heating rate of 10 °C/min from ambient temperature to 700 °C. Cyclic voltammetric data of the complexes in MeCN were collected using BAS C.V 50 electrochemical analyzer. The three-electrode cell contains a reference Ag/AgCl electrode, Pt wire auxiliary electrode and glassy carbon working electrode. [Me₄N]ClO₄ was used as supporting electrolyte and all the potentials are referred to Ag/AgCl.

The in vitro growth inhibitory activity of the Cu(II)-stz(A)-gly, 2aba, dapa and orn(B) complexes were tested against bacteria S. typhi, yeast S. cerevisae by agar diffusion method (Pelczar et al., 1998) and fungi L. theobrome and F. oxysporum by potato dextrose agar method (Pelczar et al., 1998) using agar as the nutrient. The standard drug ampicillin dissolved in DMF, which acts as a control was also tested at the same concentration under conditions similar to that of the complexes. The liquid medium containing the bacterial subcultures were auto claved for 20 min at 121 °C at 15 lb pressure before incubation. The bacteria were incubated in Nutrient Broth at 37 °C for 24 h, and the fungi and yeast were incubated in Sabouraud Dextrose Broth (SDB) at 25 °C for 48 h. The bacteria, fungi and yeast were injected into petri dishes (100 × 70 mm) in the amount of 0.01 cm³, 15 ml of potato dextrose agar were homogenously distributed onto the sterilized petri dishes. All the complexes were injected into empty sterilized antibiotic discs having the diameter of 6 mm in the amount of 30 ml. The complexes were dissolved in DMF to a final concentration of 2000 ppm and soaked in filter paper. The petri dishes were kept at 4 °C for 2 h, plates inoculated with fungi and yeast was incubated at 25 °C for 24 h. The width of the growth inhibition zone around the disc was measured after 24 h incubation and the activity of each treatment was made in duplicate.

The cleavage of CT DNA by the Cu(II)-stz(A)-gly, 2aba, dapa and orn(B) complexes were determined by agarose gel electrophoresis experiments by incubation of the samples containing CT DNA and H₂O₂ in Tris–HCl/NaCl buffer at 37 °C for 2 h. After incubation, the samples were electrophoresed for 2 h at 50 V on 1% agarose gel using tris-acetic acid-EDTA buffer. The gel was then stained using ethidium bromide and photographed under ultraviolet light at 360 nm.

3.1 Mixed ligand complex equilibria

The Cu(II)-stz(A)-gly, 2aba, dp and tp(B) systems showed the presence of CuAB and CuAB₂ species, while in the Cu(II)-stz(A)-3aba, dapa and daba(B) systems CuABH and CuAB species were detected. The Cu(II)-stz(A)-orn(B) system showed the presence of CuABH and CuAB₂H₂ species.

3.2 Structure and stability of CuAB, CuABH, CuAB₂ and CuAB₂H₂

The $\text{log}{K}_{\text{CuAB}}^{\text{CuB}}$ values in all the systems (Table 2) compare favorably to each other indicating a similar type of binding of stz (A) in CuAB species i.e. stz(A) is bidendate in the mixed ligand species as also its binding in the binary species (Nair and Regupathy, 2010; Mukherjee and Basu, 1999). Again, the $\text{log}{K}_{\text{CuAB}}^{\text{CuA}}$ values (Table 2) for Cu(II)-stz(A)-gly, 2aba, 3aba, dp and tp (B) systems correspond to the bidendate binding of gly, 2aba, 3aba, dp, and tp in the respective CuAB complexes. The bidendate binding of both stz(A) and gly, 2aba, 3aba, dp and tp(B) ligands in the CuAB species in Cu(II)-stz(A)- gly, 2aba, 3aba, dp and tp (B) system reveals a tetra coordination to the metal ion. The log β_CuAB values (Table 2) in Cu(II)-stz(A)-dapa (B) system is higher than that in Cu(II)-stz(A)-gly, 2aba, and dp(B) systems suggesting tridendate binding of dapa in its CuAB species. Similarly, the log β_CuAB values (Table 2) in Cu(II)-stz(A)-daba (B) system is higher than that in Cu(II)-stz(A)-3aba and tp(B) systems suggesting tridendate binding of daba in its CuAB species. In the CuAB₂ species in gly/2aba/dp/tp(B) systems the metal would be hexa coordinated. The Δlog K_CuAB (Sigel, 1975) values calculated in all the systems are positive, indicating that the ligand B adds to CuA binary species rather than to the aquated metal ion (Sigel, 1975; Sigel, 1971–1997). The formation of CuAB species is accompanied by a color change in the solution i.e., 1:1:1 solutions were found to have a deep violet coloration at pH 6.0 and the respective λ_max values of 660, 658, 652, 654, 664, 596 and 586 nm were observed for gly, 2aba, 3aba, dp, tp, dapa and daba, (B) systems. The absorbance of the solutions increases with rise in pH but the λ_max value remain unaltered.

The CuABH and CuAB₂H₂ species were identified in Cu(II)-stz(A)-dapa, daba and orn(B) systems. No protonated binary species has been detected in the Cu(II)-stz(A) binary system. The ${\mathit{pK}}_{\text{CuABH}}^H$ values (Table 2) computed in these systems are not comparable to each other. Also, $\text{log}{K}_{\text{CuABH}}^{\text{CuA}}$ values obtained in these systems compare favorably with $\text{log}{K}_{\text{CuBH}}^{\text{Cu}}$ values obtained in the dapa and daba (B) systems. This clearly demonstrates that the proton in CuABH is attached with the terminal amino group of dapa and daba (B). In the orn(B) ligand system, the parameter could not be computed. However, the trend in $\text{log}{\beta }_{\mathit{CuABH}}$ values in dapa,daba and orn(B) systems (Table 2) clearly indicates that the extra proton in CuABH species in the orn(B) system also resides with the terminal amino group of orn(B) ligand. As expected the $\text{log}{K}_{\text{CuABH}}^{\text{CuBH}}$ values (Table 2) obtained in these systems bear favorable comparison with each other. The Δlog K_CuABH (Sigel, 1975) values calculated (Table 2) indicate marked stabilization of the protonated mixed ligand complexes compared to the binary analogues.

3.3 Species distribution diagram

The species distribution diagrams for all the systems under investigation have been obtained for different metal to ligand A and B ratio solutions. The CuABH species have been found to be in maximum concentration in the pH range of 4.5–5.5 and accounted for ∼50% of total metal ion. The CuAB₂H₂ species in Cu(II)-stz(A)- dapa, daba and orn (B) were found to be present in the pH region of 4–6 and accounted for upto 15% of the total metal ion. As the pH increases the CuAB species has been found to be generally favored and accounted for ∼70% of the metal ion in 1:1:1 system. The CuAB₂ species in gly/2aba/dp/tp (B) systems has been found to be favored above pH 6.0 and accounted for 20% of the total metal ion in 1:2:2 systems. In order to show the qualitative trends found in the species distribution diagrams, the plot obtained in the Cu (II)-stz(A)-daba(B) system is given in the Fig. 2

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Figure 2

Species distribution diagram of Cu(II)-stz(A)-daba(B) complex system in 1:1:1 solution. (1) Free Cu(II). (2) CuA. (3) CuBH. (4) CuB. (5) CuABH. (6) CuAB. (7) CuAB₂H₂ (curves for CuA₂, CuB₂ and CuB₂H₂ could not be drawn due to their very low concentrations).

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3.4 Solid state studies

All the complexes prepared are insoluble in water and common non polar solvents like benzene and ether. They are soluble in methanol, ethanol, dimethyl sulphoxide and acetonitrile. The color, melting point, elemental analytical data, magnetic susceptibility data and molar conductance of the complexes are given in the Table 3. Elemental analysis data are consistent with the formulations of CuAB in the Cu (II)-stz(A)-gly,2aba,dapa and orn (B) systems. This is reasonable, because the species distribution diagrams in all these systems demonstrate that CuAB species is predominantly formed at around the pH 6.0. The molar conductance of the complexes in DMSO for ≈10⁻³ M solutions at room temperature is low which indicates the non electrolytic nature of the complexes. The μ_eff values for the Cu(II)-stz(A)-gly,2aba,dapa and orn(B) complexes fall in the range 1.78–1.82 BM, which are characteristic of d⁹ Cu(II) complexes (Figgis, 1965). The magnetic moment values observed indicate that they are monomeric and paramagnetic.

3.4.2

3.4.2 Electronic absorption spectra

The electronic absorption spectra of the CuAB complexes in the Cu(II)-stz(A)- gly, 2aba, dapa & orn(B) systems were recorded at 300 K using DMSO as solvent. The absorption region assignment and geometry of mixed ligand complexes are given in Table 4. The complexes display a broad band in 565–590 nm region for the electronic transition ²B_1g → ²A_1g favoring square planar geometry (Dunn, 1960; Lever, 1971). The probable structures for the CuAB species in the Cu(II)-stz(A)-gly/dapa(B) systems based on the magnetic, IR and electronic data in the solid state are given in Fig. 3.

Table 4 Electronic absorption spectral data of CuAB complexes at 300 K.

Systems	${\lambda }_{\text{max}}$ (nm)	Band assignments	Geometry
Cu(II)-stz(A)-gly(B)	586	2B1g → 2A1g	Square planar
Cu(II)-stz(A)-2aba(B)	566	2B1g → 2A1g	Square planar
Cu(II)-stz(A)-dapa(B)	558	2B1g → 2A1g	Square planar
Cu(II)-stz(A)-orn(B)	579	2B1g → 2A1g	Square planar

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Figure 3

Proposed structure of CuAB species in (a) Cu(II)-stz(A)-gly(B) and (b) Cu(II)-stz(A)-dapa(B) systems in solid state.

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3.4.3

3.4.3 ESR spectra

The X band ESR spectra of the Cu(II)-stz(A)-gly & orn(B) complexes were recorded in DMSO at 300 and 77 K. The spin Hamiltonian parameters calculated are given in Table 5. The spectra of the complexes at 300 K show one intense absorption band in the high field and are isotropic due to the tumbling motion of the molecules. However, four well resolved peaks with low intensities in the low field region and one intense peak in the high field region are observed in the frozen state (Fig. 4). No band corresponding to m_s = ±2 transition was observed in the spectra, ruling out any Cu–Cu interaction. The values in Table 5 show that A_|| >  $A_{\perp }$ and g_|| >  $q_{\perp }$ . This trend indicates that the unpaired electron lies in the ${\text{d}}_{x^2-y^2}$ orbital (Anthonisamy et al., 1999). The covalent bonding parameters α² (in-plane σ bonding), β² (in-plane π bonding) and γ² out of plane π bonding) have also been calculated (Table 5). The α² value of 0.5 indicates complete covalent bonding, while the value of α² equals 1.0 suggests complete ionic bonding (Drago et al., 1983). The observed α² values in Table 5 demonstrated that the complexes have more covalent character. The observed β² and γ² values indicate that there is an interaction in the in-plane π bonding between the metal ion and ligand. This is also confirmed by orbital reduction factors (West, 1984) calculated using the relation K_ll = α²β² and $K_{\perp }$  = α²γ². The trend that K_ll <  $K_{\perp }$ implies a considerable in-plane π bonding and K_ll >  $K_{\perp }$ shows out-plane π bonding between the metal ion and ligand. The K_ll and $K_{\perp }$ values (Table 5) suggest an in-plane π bonding in metal ligand interaction. The g values are related with exchange interaction coupling constant (G). If the G value is larger than four, the exchange interaction is negligible because the local tetragonal axes are aligned parallel or slightly misaligned and if the G value is less than four, the exchange interaction is considerable and the local tetragonal axes are misaligned (Hathaway and Tomlinson, 1970). The present G values (Table 5) indicate that the local tetragonal axes are aligned parallel or slightly misaligned and consistent with a ${\text{d}}_{x^2-y^2}$ ground state.

Table 5 The Spin Hamiltonian parameters of CuAB complexes in DMSO at 77 K.

Systems	Hyperfine constant
	A||	A⊥	Aiso	g||	g⊥	giso	α2	β2	γ2	Kll	K⊥	g||/A||	G
	×10−4 cm−1
Cu(II)-stz(A)–gly(B)	134	84	104	2.28	2.07	2.02	0.75	0.93	0.96	0.69	0.72	170	4.1
Cu(II)-stz(A)–orn(B)	135	95	116	2.26	2.06	2.04	0.69	0.86	0.75	0.59	0.52	167	4.5

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Figure 4

ESR spectrum of CuAB species in Cu(II)-stz(A)-gly(B) system at (a) 300 K (b) 77 K.

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3.4.4

3.4.4 Powder XRD analysis and SEM analysis

The representative diffractogram of Cu(II)-stz(A)-gly(B) and Cu(II)-stz(A)-orn(B) complexes are shown in Fig. 5(a) and (b) and the d-values of the complexes and the ASTM data d-values are listed in Table 6. Powder XRD data (d-values) of these complexes match with the ASTM data d – value. The Cu (II)-stz(A)-gly/orn(B) complexes confirm the presence of copper in α-form. The ASTM data of sulfathiazole matches with the d-values of the complexes, which confirm the presence of sulfathiazole moiety in the complex. Similar observation is noted with the ASTM data d-values in the Cu (II)-stz(A)-gly & orn(B) complexes. It showed the incorporation of gly & orn moiety in the complex. From the figure, it is evident that the strong and broad peak at 2θ = 12.5° and 19.5° confirms the complex formation and the appearance of large feeble peaks indicates that the complex is microcrystalline (Abd et al., 2004). The grain size d_XRD of the ternary complexes was calculated using Scherre’s formula (Cullity, 1977). The grain sizes have an average values of 43 and 49 nm, respectively in Cu(II)-stz(A)-gly/orn(B) systems suggesting that the complexes are in nanocrystalline state. The surface morphology of Cu (II)-stz(A)-gly)/orn(B) complexes were studied using SEM and the respective figures are shown in the Fig. 5(a) and (b). The Cu(II)-stz(A)-gly(B) shows a disc-like morphology, while Cu(II)-stz(A)-orn(B) shows monoclinic shaped microcrystalline structure. The particle size of the complexes was in the diameter range of 45 and 50 μm.

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Figure 5

XRD pattern of CuAB in (a) Cu(II)-stz(A)-gly(B) and (b) Cu(II)-stz(A)-orn(B) systems.

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Table 6 X-ray powder diffraction data of CuAB complexes.

Systems Exp. Data [d Ao]		ASTM DATA [d A°]
Cu(II)-stz(A)-gly(B)	Cu(II)-stz(A)-orn(B)	Cu-acetate	α-Cu	stz	gly	orn
12.89	13.01	5.87	12.79	7.77	4.95	7.50
10.33	10.38	4.28	8.63	5.80	4.43	5.60
8.54	8.58	3.45	4.79	5.46	4.05	4.95
7.44	8.15	2.61		4.79	3.73	3.78
5.80	7.47			4.31	3.19	3.24
5.57	5.54			4.01	2.54	3.09
4.89	4.93
4.47	4.72
4.30	4.48
4.05	4.31
3.78	4.07
3.46	3.76
3.18	3.19
3.07
2.55

3.4.5

3.4.5 TGA and DTA studies

The thermal studies of the Cu(II)–stz(A)-gly/orn(B) complexes were carried out and the thermatogram of Cu(II)-stz(A)-gly(B) as a representative example is shown in Fig. 6. The results show that the complexes decompose in two steps. These complexes did not show any change upto 250 °C suggesting hydrated and coordinated water molecules are not present in the complex (Abd et al., 2004). The CuAB (B = gly/orn) complexes within the temperature range 425–475 °C undergo decomposition with a mass loss of 19.5% (20.0) and 16.8% (17.3), respectively corresponding to the elimination of gly/orn ligand attached to the metal. In the final step these complexes undergo complete decomposition in the temperature range 500–580 °C with a mass loss of 51.8 % (52.4) and 53.6% (54.5) corresponding to the removal of remaining ligand with the formation of metal oxide (28%) as residue (El-Baradie et al., 1994). The analysis suggests the formation of metal oxide as the end product from which the metal content can be calculated and compared with that obtained from analytical determination.

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Figure 6

SEM photograph of CuAB in (a) Cu(II)–stz(A)-gly(B) and (b) Cu(II)–stz(A)-orn(B) systems.

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3.4.6

3.4.6 Electrochemical behaviour

The cyclic voltammograms of Cu(II)-stz(A)-gly/orn(B) complexes were recorded in MeCN solution at room temperature in the absence of molecular oxygen in the potential range of 1.2–1.6 V. The scan rate used was 100 mV s⁻¹. The cyclic voltammetric data obtained for these complexes are given in Table 7. The cyclic voltammograms show a well defined quasi reversible redox peak corresponding to the formation of Cu^II → Cu^III at Ep_a = 0.88 V and the associated cathodic peak for Cu^III → Cu^II at Ep_c = 0.62 V. It exhibits two irreversible peaks characteristics for Cu^II → Cu^I (Ep_c = −0.58 V) and Cu^I→ Cu⁰ (Ep_c = −1.06 V). After an initial scan, if the potential is reversed from −1.60 V, a stripping peak was observed and is due to the oxidation of deposited metal Cu to Cu^II (Roositer and Hamilton, 1986; Jeyasubramanian et al., 1996). The cyclic voltammogram of the complexes Cu(II)-stz(A)-gly(B) and Cu(II)-stz(A)-orn(B) are shown in Fig. 7(a) and (b).

Table 7 Cyclic voltammetric data of CuAB complexes in MeCN solution containing 0.1 M [Me₄ N]ClO_4, Scan rate 100 mV s⁻¹ at 300 K.

Systems	Couple	Epc (V)	Epa (V)	Ipc (μA)	Ipa (μA)	Ipc/Ipa
Cu(II)-stz(A)-gly(B)	Cu(II)/Cu(III)	–	0.88	–	11.17	–
	Cu(III)/Cu(II)	–	–	12.08	–	–
	Cu(II)/Cu(I)	0.62	–	11.18	–	1.08
	Cu(I)/Cu(0)	−0.58	–	11.04	–	1.07
	Cu(0)/Cu(II)	−1.06	−0.16	–	10.46	1.06
Cu(II)-stz(A)-orn(B)	Cu(II)/Cu(III)	–	0.78	–	10.90	–
	Cu(III)/Cu(II)	0.52	–	12.24	–	–
	Cu(II)/Cu(I)	−0.62	–	11.56	–	1.12
	Cu(I)/Cu(0)	−1.10	–	11.14	–	1.10
	Cu(0)/Cu(II)	–	0.18	–	10.48	1.06

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Figure 7

Thermal analytical curve of CuAB in Cu(II)-stz(A)-gly(B) system.

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3.4.8

3.4.8 DNA studies

The oxidative CT DNA cleavage activity of Cu(II)-stz(A) and Cu(II)-stz(A)-gly/2aba/orn(B) complexes were studied using gel electrophoresis and the respective photographs are shown in Fig. 8. The cleavage efficiency of the complexes is compared with the control DNA to study the binding ability. The experiment with the control DNA does not show any significant cleavage of CT DNA. From Fig. 8, it is clear that the metal complexes show higher ability to cleave the CT DNA than the control and the mixed ligand complexes show higher ability to cleave the CT DNA than the binary complex. This is due to higher binding ability of mixed ligand complexes with DNA. Further, the presence of smear in the gel diagram indicates the radical cleavage (Zhang and Lippard, 2003) by the abstraction of hydrogen from sugar units of DNA. The metal complexes were able to convert super coiled DNA into open circular DNA (Zhang and Lippard, 2003). The reaction is modulated by the metallocomplexes bound hydroxyl or peroxo radical generated from the oxidant H₂O₂. (See Fig. 9)

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Figure 8

Cyclic voltamogram of CuAB in (a) Cu(II)-stz(A)-gly(B) and (b) Cu(II)-stz(A)-orn(B) systems.

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Figure 9

Changes in the agarose gel electrophoretic pattern of calf-thymus DNA induced by mixed ligand complexes in presence of H₂O_2. Lane 1 Control DNA. Lane 2 DNA + Cu(II)-stz(A) + H₂O₂. Lane 3 DNA + Cu(II)-stz(A)-gly(B) + H₂O_2. Lane 4 DNA + Cu(II)-stz(A)-2aba(B) + H₂O_2. Lane 5 DNA + Cu(II)-stz(A)-dapa(B) + H₂O_2. Lane 6 DNA + Cu(II)-stz(A)-orn(B) + H₂O₂.

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