Metal based pharmacologically active agents: Synthesis, structural elucidation, DNA interaction, in vitro antimicrobial and in vitro cytotoxic screening of copper(II) and zinc(II) complexes derived from amino acid based pyrazolone derivatives

2.4.1 Synthesis of binuclear copper complex (1)

An ethanolic solution of Schiff base L (0.325 g, 1 mM) was refluxed for ca. 4 h with an ethanolic solution of copper sulfate (0.52 g, 2 mM). The solid complex was separated, filtered and washed with ethanol and dried in vacuo. Yield: 48%.

2.4.2 Synthesis of copper(II) and zinc(II) complexes with 1:2 netal/ligand ratio

An ethanolic solution of L (0.65 g, 2 mM) was refluxed for ca. 4 h with the ethanolic solution of metal salts such as copper(II) chloride (2)/zinc(II) chloride (3) (0.34 g/0.237 g, 1 mM). The solid complexes separated were filtered and washed with ethanol and dried in vacuo. Yield: 52% for complex (2) and 58% for complex (3).

2.4.3 Synthesis of copper(II) and zinc(II) complexes with 1:1 metal/ligand ratio

An ethanolic solution of L (0.325 g, 1 mM) was refluxed for ca. 4 h with the ethanolic solution of metal salts such as copper(II) chloride (4)/zinc(II) chloride (5) (0.34 g/0.237 g, 1 mM). The solid complexes separated were filtered and washed with ethanol and dried in vacuo. Yield: 56% for complex (4) and 49% for complex (5).

2.5.1 Absorption spectroscopic studies

Absorption titration experiments were performed by maintaining the metal complex concentration as constant at 50 μM while varying the concentration of the CT DNA within 40–400 μM. While measuring the absorption spectrum, equal quantity of CT DNA was added to both the complex solution and the reference solution to eliminate the absorbance of CT DNA itself. From the absorption data, the intrinsic binding constant K_b was determined from the plot of[DNA]/(ɛ_a − ɛ_f) versus[DNA] using the following equation: $$[\text{DNA}]/({\varepsilon }_a-{\varepsilon }_f)=[\text{DNA}]/({\varepsilon }_b-{\varepsilon }_f)+[K_b({\varepsilon }_b-{\varepsilon }_f)]-1$$ where[DNA] is the concentration of CT DNA in base pairs. The apparent absorption coefficients ɛ_a, ɛ_f and ɛ_b correspond to A_obsd/[M], the extinction coefficient for the free metal(II) complex and extinction coefficient for the metal(II) complex in the fully bound form, respectively (Reichmann et al., 1954). K_b is given by the ratio of slope to the intercept.

2.5.2 Electrochemical methods

Cyclic voltammetry and differential pulse voltammogram studies were performed on a CHI 620C electrochemical analyzer with three electrode system of glassy carbon as the working electrode, a platinum wire as auxiliary electrode and Ag/AgCl as the reference electrode. Solutions were deoxygenated by purging with N₂ prior to measurements. The freshly polished glassy electrode was modified by transferring a droplet of 2 μL of 2.5 × 10⁻³ M of CT DNA solution onto the surface, followed by air drying. Then the electrode was rinsed with distilled water. Thus, a CT DNA-modified glassy carbon electrode was obtained.

2.5.3 Viscosity measurements

Viscosity experiments were carried on an Ostwald viscometer, immersed in a thermostated water-bath maintained at a constant temperature at 30.0 ± 0.1 °C. CT DNA samples of approximately 0.5 mM were prepared by sonication in order to minimize complexities arising from CT DNA flexibility (Charies et al., 1982). Flow time was measured with a digital stopwatch three times for each sample and an average flow time was calculated. Data were presented as (η/η⁰)^1/3 versus the concentration of the metal(II) complexes, where η is the viscosity of CT DNA solution in the presence of complex, and η⁰ is the viscosity of CT DNA solution in the absence of complex. Viscosity values were calculated after correcting the flow time of buffer alone (t₀), η = (t − t₀)/t₀ (Satyanarayana et al., 1983).

2.5.4 Gel electrophoresis

DNA cleavage experiment was monitored by agarose gel electrophoresis method. The gel electrophoresis experiment was performed by incubation at 35 °C for 1.5 h as follows: The samples containing SC pUC19 DNA 30 μM, 50 μM copper complex and 500 μM MPA in 50 mM Tris–HCl buffer (pH = 7.2) were electrophoresed for 2 h at 50 V on 1% agarose gel using Tris-boric acid-EDTA buffer (pH = 8.3). After electrophoresis, the gel was stained using 1 μg/cm³ ethidium bromide (EB) and snapped under UV light.

2.6.1 In vitro antimicrobial assay

The synthesized ligand and its complexes were tested for their in vitro antimicrobial activity against the Gram-positive bacteria Staphylococcus aureus, Bacillus subtilis, the Gram-negative bacteria Escherichia coli, Klebsiella pneumoniae by the disk diffusion method, using agar nutrient as the medium and against the fungi Aspergillus niger, Fusarium solani, Curvularia lunata and Rhizoctonia bataicola by disk diffusion method using potato dextrose agar as medium. The stock solution (10⁻² M) was prepared by dissolving the compounds in DMSO, the solutions were then serially diluted in order to find out the minimum inhibitory concentration (MIC) values. Streptomysin and nystatin were used as control drugs.

2.6.2 Effect of Cu(II) and Zn(II) complexes on in vitro cytotoxicity

Short-term cytotoxicity was assessed by incubating 1 × 10⁶ EAC cells in 1 mL phosphate buffer saline with varying concentrations of the complexes at 37 °C for 3 h in CO₂ atmosphere ensured using a McIntosh field jar. The viability of the cells was determined by the trypan blue exclusion method (Sheeja et al., 1997).

2.6.3 Statistical analysis

All values were expressed as mean + SEM. The data were statistically analyzed by one-way ANOVA followed by Dunnett’s test, the data of hematological parameters were analyzed using ANOVA followed by Tukey multiple comparison test and data of solid tumor were analyzed using Student’s ‘t’ test. P values < 0.05 were considered significant.

3.8.1 Electronic absorption titration

Absorption spectrum of the copper complex (1) in the absence and in the presence of CT DNA is shown in Fig. 6. With increasing CT DNA concentration, for copper complex, the hypochromism in the band at 368.4 nm reaches 27% with a blue shift of 8.2 nm. These spectral characteristics obviously suggest that the copper complex interacts with DNA most likely through a mode that involves a stacking interaction between the aromatic chromophore and the base pairs of DNA. After intercalating the base pairs of DNA, the π^* orbital of the intercalated ligand can couple with the π orbital of the base pairs thus decreasing the π–π^* transition energy and resulting in bathochromism. On the other hand, the coupling π orbital is partially filled by electrons thus decreasing the transition probabilities and concomitantly resulting in hypochromism. The intrinsic binding constant K_b can be obtained from the ratio of the slope to the intercept of the plots of[DNA]/(ɛ_a − ɛ_f) versus[DNA]. To compare quantitatively the affinity of the synthesized complexes towards DNA, the intrinsic binding constants K_b of the synthesized complexes to CT DNA are shown in Table 3. From this table, it is clear that complex (1) has higher binding efficacy than other complexes.

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Figure 6

Electronic absorption spectrum of (1) in the absence (—) and the presence (—) of increasing amount of CT DNA; R = [NP]/[complex].

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3.8.2 Redox behavior

Cyclic and differential pulse voltammetric techniques are extremely useful in probing the nature and mode of DNA binding of metal complexes. Typical cyclic voltammogram of copper complex (2) in the absence and in the presence of varying amount of[DNA] is shown in Fig. 7. In the absence of CT DNA, the first redox couple cathodic peak appeared at 0.384 V for Cu(III)/Cu(II) (E_pa = 0.945 V, E_pc = 0.384 V, ΔE_p = 0.561 V, and E_1/2 = 0.664 V) and the second redox couple cathodic peak at 0.138 V for Cu(II)/Cu(I) (E_pa = 0.794 V, E_pc = 0.138 V, ΔE_p = 0.653 V, and E_1/2 = 0.464 V). Incremental addition of CT DNA to the complex caused a negative shift in E_1/2 and an increase in ΔE_p. The i_pc/i_pa values also decreased in the presence of DNA. The decrease of the anodic and cathodic peak currents of the complex in the presence of DNA is due to the decrease in the apparent diffusion coefficient of the Cu(II) complex upon its complexation with the DNA macromolecule. These results show that the Cu(II) complex stabilizes the duplex (GC pairs) by intercalation. Electrochemical parameters of the synthesized copper complexes are shown in Table 4. These results indicate that the synthesized complexes may stabilize the duplex DNA.

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Figure 7

Redox behavior of complex (2) in the absence (—) and the presence (—) of different concentration of DNA using cyclic voltammogram; Supporting electrolyte, 50 mM NaCl, 5 mM Tris–HCl, pH = 7.1. Scan rate 100 mV s⁻¹.

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In the absence of CT DNA, the redox couple anodic peak appeared at −0.294 to −0.342 V. Incremental addition of DNA to the Zn(II) complexes showed a decrease in the current intensity and a negative shift of the oxidation peak potential. The resulting changes in the current and potential demonstrate that there is an interaction between Zn(II) and DNA.

Differential pulse voltammogram of the complex (4) in the absence and in the presence of varying amount of[DNA] is shown in Fig. 8. An increase in the concentration of DNA caused a negative potential shift along with a significant decrease of the current intensity. The shift in the potential is related to the ratio of the binding constants $$E_b^{°\prime }-E_f^{°\prime }=0.059\log (K_{+}/K_{2+})$$ where $E_f^{°\prime }$ and $E_b^{°\prime }$ are the formal potentials of the, Cu(II)/Cu(I) couple in the free and bound forms, respectively. The ratio of the binding constants (K₂₊/K₊) for DNA binding of the synthesized complexes were calculated and found to be less than one. The above electrochemical experimental results indicated the preferential stabilization of Cu(II) forms on binding to DNA over other forms.

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Figure 8

Redox behavior of complex (4) in the absence (—) and the presence (—) of different concentration of DNA using differential pulse voltammogram; Supporting electrolyte, 50 mM NaCl, 5 mM Tris–HCl, pH = 7.1. Scan rate 100 mV s⁻¹.

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Differential pulse voltammogram of the presented Zn(II) complex showed a negative potential shift along with a significant decrease of current intensity during the addition of increasing amounts of DNA. This indicates that the zinc ion stabilizes the duplex (GC pairs) by intercalation. Hence, for the complex of the electroactive species (Zn(II)) with DNA, the electrochemical reduction reaction can be divided into two steps. $$\begin{array}{cc}\hfill & {\text{Zn}}^{2+}-\text{DNA}\rightleftharpoons {\text{Zn}}^{2+}+\text{DNA}\hfill \\ \hfill & {\text{Zn}}^{2+}+{\text{2e}}^{-}\rightleftharpoons {\text{Zn}}^0\hfill \end{array}$$ The dissociation constant (K_d) of the Zn(II)-DNA complex was obtained using the following equation (Blackburn and Gait, 1996; Hathaway and Billing, 1970): $$i_p^2=\frac{K_d}{[\text{DNA}]}\left(i_{p^{°}}^2-i_p^2\right)+i_{p^{°}}^2-[\text{DNA}]$$ where K_d is dissociation constant of the complex Zn(II)-DNA, $i_{p^o}^2$ and $i_p^2$ are reduction current of Zn(II) in the absence and presence of DNA respectively. The low dissociation constant values (10⁻¹⁰ order) of Zn(II) ions were indispensable for structural stability of complexes Zn(II)-DNA which participate in the replication, degradation and translation of genetic material of all species.

3.8.3 Viscosity measurements

In addition to the peak potential shift in cyclic voltammetry, the spectral shift in UV absorption titration and the dependence of the ionic strength on the binding constant, viscosity measurements were carried out to provide further information on the nature of the interaction between the complex and DNA. A classical intercalation model demands that the DNA helix lengthens as base pairs are separated to accommodate the bound ligand leading to an increase of the DNA viscosity. In contrast, a partial non-classical intercalation of the ligand could bend (or kink) the DNA helix, reduce its effective length and concomitantly also its viscosity in order to further elucidate the binding mode of the present complex. Viscosity measurements were carried out by varying the concentration of the added complex. The effect of the complexes on the viscosity of DNA is shown in Fig. 9. Relative viscosity of DNA increased with the increase in the concentration of the metal complexes, which is similar to that of the classical intercalators (Wang et al., 2007). The viscosity results unambiguously show that complexes bind with DNA in the intercalation mode.

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Figure 9

Ratio of the specific viscosity of DNA in the presence of complex to that of free CT DNA versus 1/R (=[Complex]/[NP]) in the presence of complexes (1) (♦), (2) (▴), (3) (○), (4) (○) and (5) (○).

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3.8.4 Chemical nuclease activity

The DNA cleavage of ligand alone is inactive in the presence and absence of any external agents. The results indicate the importance of the metal in the complex for observing the chemical nuclease activity. Copper complexes can cleave DNA both through hydrolytic and oxidative processes. In the latter instance, these complexes have been shown to react with molecular oxygen or hydrogen peroxide to produce a variety of active oxidative intermediates (reactive oxygen species or ROS), including diffusible hydroxyl radicals and non diffusible copper-oxene species. Normally, a reducing agent like 3-mercaptopropionic acid (MPA) is required to initiate and sustain the radical reaction, but particularly with the employment of DNA derived from biological sources, the presence of an adventitious reducing agent cannot be ruled out. In order to obtain information about the active chemical species which effect DNA damage, we have looked for the formation of three activated oxygen intermediates: hydroxyl radical, singlet oxygen and superoxide. Figs. 10a and 10b show the influence of the radical scavengers on the DNA cleavage of the copper complexes. From Figs. 10a and 10b, it is evident that the hydroxyl radical scavenger, DMSO, significantly diminished the nuclease activity of copper complexes, which indicates the involvement of the diffusible hydroxyl radical in the cleavage process. Sodium azide, scavenger of singlet oxygen or singlet oxygen-like species, both reduce the DNA damage by introducing the participation of the singlet oxygen or a singlet oxygen-like entity. In fact, the SOD enzyme actually increases the nuclease process, which suggests that in this case the dismutation of the superoxide produced by the enzyme gives rise to ROS which break the DNA strands to a greater extent than the complexes plus reducing agents.

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Figure 10a

Gel electrophoresis diagram showing the cleavage of SC pUC19 DNA (0.2 μg) by the synthesized complexes (50 μM) in the presence of oxidant (5 mM): lane 1, DNA + (1) + H₂O₂; lane 2, DNA + (1); lane 3, DNA + (1) + distamycin; lane 4, DNA + (1) + DMSO (4 μL); lane 5, DNA + (1) + MPA + SOD (1 U); lane 6, DNA + (2); lane 7, DNA + (2) + H₂O₂ + MPA; lane 8, DNA + (2) + MPA + distamycin (50 μM).

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Figure 10b

Gel electrophoresis diagram showing the cleavage of SC pUC 19 DNA (0.2 μg) by the synthesized complexes (50 μM) in the presence of MPA (5 mM): lane 1, DNA control; lane 2, DNA + L (50 μM); lane 3, DNA + (5) + MPA; lane 4, DNA + (5); lane 5, DNA + (5) + DMSO (4 μL) + MPA; lane 6, DNA + (5) + NaN₃ + MPA; lane 7, DNA + (3) + distamycin (50 μM) + MPA; lane 8, DNA + (5) + MPA + SOD (1 U).

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The presence of distamycin, respective binder of the minor groove of DNA, inhibited the breakage of DNA strands, thus implying that complex (1) interacts with DNA in the minor groove. But, the complexes (2) and (5) did not inhibit the cleavage of DNA in the presence of minor groove binder. It indicates that complexes (2) and (5) interact with DNA in the major groove.

Several possible reaction pathways can account for the cleavage dependent on the presence of hydrogen peroxide. The first step is the interaction of copper(II) complex with DNA through the outer sphere while the second step consists of the reduction of copper(II) complex to copper(I) complex through reaction with the reducing agent. Once copper(I) is formed, a metal-catalyzed Haber–Weis reaction, which makes used of the Fenton chemistry may be considered to be the major mechanism by which the highly reactive hydroxyl radical is generated in biological systems: (Fenton, 1894; Kehrer, 2000) $$\begin{array}{cc}\hfill & \text{Cu}(\text{II})\text{L}+{\text{e}}^{-}\to \text{Cu}(\text{I})\text{L}\hfill \\ \hfill & \text{Cu}(\text{I})\text{L}+{\text{O}}_2\to \text{Cu}(\text{II})\text{L}+{\text{O}}_2^{-}\hfill \\ \hfill & 2{\text{O}}_2^{-}+2{\text{H}}^{+}\to {\text{H}}_2{\text{O}}_2+{\text{O}}_2\hfill \\ \hfill & \text{Cu}(\text{I})\text{L}+{\text{H}}_2{\text{O}}_2\to \text{Cu}(\text{II})\text{L}+{\text{OH}}^{-}+{\text{OH}}^{\text{•}}\hfill \\ \hfill & {\text{O}}_2^{-}+{\text{H}}_2{\text{O}}_2\to {\text{O}}_2+{\text{OH}}^{-}+{\text{OH}}^{\text{•}}\hfill \end{array}$$

Hydrogen peroxide can also react with another equivalent of Cu(I)L to produce hydroxyl radical-species which could be metal bound. This species, which may be considered analogous to a metal-oxo system, is responsible for initiating DNA strand scission chemistry (Sigman, 1986).

Although the cleavage reaction through zinc complexes does not require additional external agents, we carefully investigated the possibility that diffusible OH radical, singlet oxygen (¹O₂) or superoxide anion radical were involved in this reaction. The cleavage efficiency does not change in the presence of DMSO as potential OH radical scavenger, NaN₃ as potential ¹O₂ inhibitor, or SOD as a superoxide anion radical inhibitor (Cheng et al., 1993; Lesko et al., 1980; Khan, 1976). In conclusion, the results presented here rule out the involvement of any oxidation inhibitors in the strand cleavage, and this reaction most probably occurs through a hydrolytic mechanism. Moreover, experiments support this assumption. It is well known that in DNA hydrolytic cleavage 3′-OH and 5′-OPO₃ (5′-OH and 3′-OPO₃) fragments remain intact and that these fragments can be enzymatically ligated and end-labeled. Fig. 11 shows that the linear DNA fragments cleaved by zinc complexes can be relegated by T4 ligase just like the linear DNA mediated by EcoR1. Hence, this result implied that the process of DNA cleavage by the complex occurs via a hydrolytic path.

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Figure 11

Gel electrophoresis diagram for ligation of SC pUC19 DNA linearized by zinc complex: lane 1, DNA markers; lane 2, DNA + (3); lane 3, DNA + (3) + T4 DNA ligase; lane 4, DNA + (3) + EcoR1; lane 5, DNA + (3) + EcoR1 + T4 DNA ligase.

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3.9.1 Antimicrobial assay

The synthesized ligand and their complexes were tested for their in vitro antimicrobial activity against the Gram-positive bacteria S. aureus, B. subtilis, the Gram-negative bacteria E. coli, K. pneumoniae by the disk diffusion method, using agar nutrient as the medium and fungi A. niger, F. solani, C. lunata and R. bataicola by well diffusion method using potato dextrose agar as medium. Tables 5 and 6 summarize the minimum inhibitory concentration (MIC) values of the investigated compounds. It is clear from the tables that the observed MIC values indicate that most of the complexes have higher antimicrobial activity than the free ligand (L). This is the result of the coordinated metal which plays a significant role for the antibacterial activity. The chelation theory explains that a decrease in the polarizability of the metal can change the lipophilicity or hydrophobicity of complexes. These properties are now seen as important parameters related to membrane permeation in biological systems. Many of the processes of drug disposition depend on the ability or inability to cross membranes and hence there is a high correlation with measures of lipophilicity. Moreover, many of the proteins involved in drug disposition have hydrophobic binding sites, further adding to the importance of lipophilicity.

By consideration of the structures of compounds that exhibit antimicrobial activity, it can be concluded that the metal moiety may play a role in determining the antibacterial activity. From the results which indicated that the tested compounds were more active against Gram-positive than Gram-negative bacteria, it may be concluded that the inhibitory activity of the studied compounds is related to the cell wall structure of the bacteria. This is possible because the cell wall is essential to the survival of bacteria and some antibiotics are able to kill bacteria by inhibiting a step in the synthesis of peptidoglycan. Gram-positive bacteria possess a thick cell wall containing many layers of peptidoglycan and teichoic acids, but in contrast, Gram-negative bacteria have a relatively thin cell wall consisting of a few layers of peptidoglycan surrounded by a second lipid membrane containing lipopolysaccharides and lipoproteins. These differences in cell wall structure can produce differences in antibacterial susceptibility and some antibiotics can kill only Gram-positive bacteria and is ineffective against Gram-negative pathogens (Shakir et al., 1995).

3.9.2 Effect of Cu(II) and Zn(II) complexes on cytotoxicity in vitro

The short term in vitro cytotoxicity study of EAC-bearing mice showed the GI₅₀ of complexes to be 100, 200, 300, 400 and 500 μg/mL. The GI₅₀ values for all of the complexes are given in Table 7. All of the complexes tested, (1) has lowest GI₅₀ value of 111.31 compared to the standard 5-FU (110.12 μg/mL). From this result (1) complex has higher cytotoxicity effect on EAC cancer cell line than other complexes. The cytotoxicity order of the complexes is in the following order: (1) > (4) ≈ (5) > (2) ≈ (3).