Platinum(II)/palladium(II) complexes with n-propyldithiocarbamate and 2,2^′-bipyridine: synthesis, characterization, biological activity and interaction with calf thymus DNA

Abstract

Two Pd(II) and Pt(II) complexes ([Pt(bpy)(pr-dtc)]Br and [Pd(bpy)(pr-dtc)]Br, where bpy=2, 2^′-bipyridine and pr-dtc = n-propyldithiocarbamate) were synthesized and characterized by elemental analysis (CHN), molar conductivity measurements, Fourier transform infrared, ¹H nuclear magnetic resonance and UV–visible techniques. In these complexes, the dithiocarbamato ligand coordinates to Pt(II) or Pd(II) center as bidentate with two sulfur atoms. The binding of these complexes to calf thymus DNA (CT-DNA) was investigated using various physicochemical methods such as spectrophotometric, spectrofluorometric and gel filtration technique. The experimental results indicate that Pt(II) and Pd(II) complexes interact with CT-DNA in the intercalative mode. Both complexes unexpectedly denatured DNA at low concentration. Gel filtration studies indicated that the binding of complexes with DNA is strong enough and does not break readily. The cytotoxic activity of these metal complexes has been tested against human cell tumor lines (K562) and revealed much lower 50% cytotoxic concentration (Cc₅₀) less than that of cisplatin. Several binding and thermodynamic parameters are also described.

Keywords:

• Pt(II)/Pd(II) complexes
• Cytotoxicity
• DNA binding

1. Introduction

Platinum drugs have played a key role among the metal-based anticancer agents. The initial discovery in 1969 of the antitumor properties of cisplatin by Rosenberg et al. [1] was suddenly followed by clinical trials demonstrating its efficacy toward a variety of solid tumors. However, cisplatin exhibits several side effects, such as nausea and vomiting and high nephrotoxicity in particular. To reduce the toxicity of platinum(II)-based drugs, sulfur-containing compounds (especially thiols and dithiocarbamates) was administered as antidotes [2–4]. This is perhaps due to the strong binding of platinum with dithiocarbamate, which prevents or at least limits the reaction with other sulfur-containing renal proteins [5].

The need for less toxic analogs than cisplatin led to the promotion of various chemical approaches; one of which provided the synthesis of complexes which had heterocyclic N-donor ligands coordinated to the cytotoxic Pt(II) moiety [5,6]. Replacement of the NH₃ groups by planar aromatic ligands generally reduced the toxicity of the Pt(II) complexes [6]. The ligand planarity of these complexes would have a significant effect on their DNA-binding affinity [7].

Following our recent works [8–10] and in order to obtain compounds with superior chemotherapeutic index in terms of increased bioavailability, high cytotoxicity and low side effects, we synthesized two new platinum(II) and palladium(II) complexes (Figure 1). Both of which bear a planar 2,2^′-bipyridine ligand. This planar aromatic ligand along with square planar geometry around Pt(II) or Pd(II) centers may make the complexes susceptible to intercalate in DNA. Moreover, we attached a bidentate dithiocarbamate to Pt(II) and Pd(II) centers which can protect a variety of animal species from renal, gastrointestinal and bone marrow toxicity induced by cisplatin [11]. We used a number of physical methods including UV–visible (UV–VIS) spectroscopy, gel filtration and emission spectroscopy to study the binding of Pt/Pd(II) complexes with calf thymus DNA (CT-DNA), because a detailed knowledge of binding studies between DNA and these complexes is beneficial for medical science and pharmacokinetics. Furthermore, these water-soluble complexes have been tested against human cell tumor lines K562.

Figure 1. Proposed structures and NMR numbering of [M(bpy)(pr-dtc)]NO₃.

2. Experimental

2.1 General and instrumental

All reagents were commercially available (Aldrich or Merck) and used as supplied. Solvents were purified according to standard procedures. Other used chemicals were of analytical reagent or of higher purity grade. [Pt/Pd(bpy)Br₂] was made similar to that of [Pt/Pd(bpy)Cl₂] [12]. The Fourier transform infrared (FT-IR) spectra (KBr pellets) were recorded using a J_ASCO-460 plus FT–IR spectrophotometer in the range of 4000–400 cm⁻¹. ¹H nuclear magnetic resonance (NMR) spectra were recorded on Brucker DRX-500 Avance spectrometer at 500 MHz in DMSO-d₆ using tetramethylsilane as internal reference (chemical shifts are given in ppm (δ scale)). Melting points of the synthesized compounds were recorded on a Unimelt capillary melting point apparatus. Conductivity measurements of the above platinum and palladium complexes were carried out on a Systronics conductivity bridge 305, using a conductivity cell of 1.0 cell constant. UV–VIS spectra were recorded on a J_ASCO UV/VIS-7850 recording spectrophotometer. The fluorescence spectra were recorded on a Varian spectrofluorimeter model Cary Eclips, and elemental analyses were performed using a Herause CHNO–RAPID elemental analyser.

2.2 Synthesis of the ligand and the complexes

2.2.1 Synthesis of sodium n-propyldithiocarbamate

The sodium n-propyldithiocarbamate was prepared according to a reported procedure with some modifications [13]. A cold solution of sodium hydroxide (0.1 mol) in 15 mL doubly distilled water was added to n-propyl amine (0.1 mol) in 20 mL acetone-water (1: 1 v/v) mixture followed by the addition of carbon disulfide (0.15 mol, excess). The mixture was stirred for an hour in an ice-salt bath and at room temperature for 3 h. Most of the solvent evaporated until the volume of the solution reduced to 20 mL and filtered out the small solid residue. Diffusion of acetone into this filtrate led to the creation of the product after three days. Yield: 8.8 g (56%), m.p. 90–94°C. Anal. Calcd for C₄H₈NS₂Na(%): C, 30.57; H, 5.10; N, 8.91. Found: C, 30.61; H, 5.11; N, 8.90. Selected infrared (IR), ν (cm⁻¹): 1507 (N–C₁), 1158 (N–C₂), 1016 (C₁˭ S)_as and 748 (C₁˭ S)_s [14]. ¹H NMR (DMSO-d₆, , , broad,) –0.844 (t, 3H, H_a), 1.38–1.53 (m, 2H, H_b), 3.27–3.35 (m, 2H, H_c) and 8.07 (sb, 1H, H_d).

2.2.2 Preparation of complexes

Sodium n- propyldithiocarbamate (1.5 mmol) in acetone (50 mL) was slowly added to a suspension of [Pt/Pd (bpy)Br₂] (1.5 mmol ) in acetone (80 mL).The mixture was heated with stirring in dark for 4 h at 60°C and then for 5–6 h at room temperature and filtered. In order to complete dryness, the clear yellowish filtrate was evaporated at 35–40°C to complete dryness. A yellow precipitate was obtained and recrystallized in dichloromethane/methanol (1: 1) solvent mixture and then dried in vacuum.

(2,2′ -bipyridine)(n-propyldithiocarbamato)- platinum(II) bromine: [Pt(bpy)(pr-dtc)]Br Complex was obtained as orange solid. Yield: 0.399 g, (47%), decomposes at 231–234°C. Anal. Calcd for C₁₄H₁₆N₃S₂PtBr(%): C, 29.73; H, 2.83; N, 7.43. Found: C, 29.70; H, 2.81; N, 7.44. Molar conductance measurement for the complex is 116.14 Ω⁻¹ mol⁻¹ cm² indicating 1: 1 electrolytes [15]. Selected IR, ν (cm⁻¹): 1563 (N–C₁), 1107 (N–C₂), 833 (C₁˭ S)_as, 760 (C₁˭ S)_s and 3436 (N–H) [14]. ¹H NMR (DMSO-d₆, , , broad, ) –0.931 (t, 3H, H_a), 1.57–1.62 (m, 2H, H_b), 3.41–3.45 (t, 2H, H_c) and 11.65 (sb, 1H, H_d), 7.75 (t, 2H, H_{3, 8}), 8.21 (sb, 2H, H_{4, 7}), 8.36 (t, 2H, H_{5, 6}) and 8.66 (d, 2H, H_{2, 9}). Electronic spectra exhibited four bands. The bands at 220 (log ), 253 (log ), 281 (log ) and 341 (log ) might have been assigned to intraligand and transitions of 2,2^′-bipyridine ligand as well as CSS⁻ group [8].

(2,2^′ -bipyridine)(n-propyldithiocarbamato)-palladium(II) bromine: [Pd(bpy)(pr-dtc)]Br Complex was obtained as yellow solid. Yield: 0.400 g, (56 %), decomposes at 217–221°C. Anal. Calcd for C₁₄H₁₆N₃S₂PdBr(%): C, 30.57; H, 5.10; N, 8.91. Found: C, 30.61; H, 5.11; N, 8.90. Molar conductance measurement for the complex is 103.2 Ω⁻¹ mol⁻¹ cm² indicating 1: 1 electrolytes [15]. Selected IR, ν (cm⁻¹): 1544 (N–C₁), 1105 (N–C₂), 958 (C₁˭ S)_as, 765 (C₁˭ S)_s and 3422 (N–H) [14]. ¹H NMR (DMSO-d₆, , , broad, ) –0.935 (t, 3H, H_a), 1.60–1.65 (m, 2H, H_b), 3.42–3.45 (t, 2H, H_c) and 11.67 (sb, 1H, H_d), 7.72 (t, 2H, H_{3, 8}), 8.19 (sb, 2H, H_{4, 7}), 8.34 (t, 2H, H_{5, 6}) and 8.66 (d, 2H, H_{2, 9}). Electronic spectra exhibited four bands. The bands at 217 (log ), 248 (log ), 307 (log ) and 312 (log ) might have been intraligand and transitions of 2,2^′-bipyridine ligand as well as CSS⁻ group [8].

2.3 Cytotoxic activity

The following procedure was similar to the one reported earlier [16]. Here, also 2×10⁴ cells per mL of K562 chronic myelogenous leukemia were used in Tris–HCl buffer solution of PH 7.0, as well. The cells were then grown in this medium supplemented with l-glutamine (2 mM), streptomycin and penicillin (5 μg.mL⁻¹), and 10% heat-inactivated fetal calf serum, at 37°C under a 5% CO₂/95% air atmosphere. In this study, the harvested cells were seeded into 96-well plate (1×10⁴ cell mL⁻¹) with various concentrations of metal complexes ranging from 0 to 0.25 mM and incubated for 24 h [17]. The 50% cytotoxic concentration (Cc₅₀) of the Pt(II) and Pd(II) complexes were found.

2.4 DNA-binding studies

The difference between UV–VIS absorption and fluorescence emission intensities was used to determine the mode/modes of binding of [M(bpy)(pr-dtc)]Br ((II), Pd(II)) complexes to CT-DNA. The procedures followed have been reported earlier [18].

3. Results and discussion

3.1 Cytotoxicity screening

[Pt(bpy)(pr-dtc)]Br and [Pd(bpy)(pr-dtc)]Br complexes were screened for their anti-tumor activity against K562 leukemia cell lines. In this study, various concentrations of above complexes ranging from 0 to 250 μM of stock solution (2 mM) were used to culture the tumor cell lines for 24 h. The 50% cytotoxic concentration (Cc₅₀) of these complexes was determined to be 64 μM for Pt(II) and 45 μM for Pd(II) complexes (Figure 2). As shown in Figure 2, cell growth was significantly reduced in the presence of various concentrations of the platinum and palladium complexes after 24 h. Furthermore, Cc₅₀ value of cisplatin was determined under the same experimental conditions. This value (154 μM) is much higher as compared with the Cc₅₀ values of the above complexes [18].

Figure 2. The growth suppression activity of [Pt(bpy)(pr-dtc)]Br and the inset, [Pd(bpy)(pr-dtc)]Br on K562 cell line. The tumor cells were incubated with varying concentrations of the complexes for 24 h.

3.2 Spectrophotometric assays

3.2.1 Thermodynamics of DNA denaturation process

The drug–DNA interaction can be detected by UV–VIS absorption spectroscopy by measuring the changes in the absorption properties of the drug or the DNA molecules. UV–VIS absorption spectrum of DNA exhibited a broad band (200–350 nm) in the UV region with its maximum peak at 260 nm. Absorption titration can monitor the denaturation of CT-DNA by Pt(II) and Pd(II) complexes, which was done by looking at the changes in the UV absorption spectrum of DNA solution at 260 nm upon addition of platinum(II) and palladium(II) complexes. Addition of metal complexes to DNA solution continued until no further changes in the absorption readings were observed. These experiments were carried out separately at two temperatures of 300 and 310 K in Tris–HCl buffer medium.

The profiles of denaturation of CT-DNA by [Pt(bpy)(pr-dtc)]Br and [Pd(bpy)(pr-dtc)]Br (inset) complexes are shown in Figure 3. As Figure 3 shows, the concentrations of metal complexes in the midpoint of transition, [L]_1/2, are 0.68 mM and o.64 mM for Pt(II) complex and 0.102 mM and 0.091 mM for Pd(II) complex at 300 and 310 K, respectively. These values indicate that the increase in the temperature lowers the stability of the DNA against denaturation caused by these complexes. The important observation of this work is the low values of [L]_1/2 for these complexes [19–21], that is, both complexes (in particular Pd(II) complex) can denature DNA at low concentrations. Thus, if these complexes are used as anti-tumor agents, low doses will be needed, so this may have fewer side effects. These values are comparable with [L]_1/2 values of binding of analogous complexes [Pt/Pd(bpy)(Et-dtc)]NO₃ [10] and [Pt/Pd(bpy)(Bu-dtc)]NO₃ [18] with CT-DNA.

Figure 3. The changes of absorbance of CT-DNA at λ_max=260 nm due to increasing the total concentration of [Pt(bpy)(pr-dtc)]Br and the inset, [Pd(bpy)(pr-dtc)]Br, [L]_t, at constant temperature of 300 and 310 K.

The basic rationale to carry out thermodynamic studies of drug–DNA denaturation is to determine what factors are responsible for the overall denaturation affinity and strength of the drug. The first step is to experimentally determine the unfolding equilibrium constant, K, and the unfolding free energy change, LnK). Using the CT-DNA denaturation plots (Figure 3) and Pace method [22], the value of K, and Δ G⁰ of CT-DNA at two temperatures of 300 and 310 K in the presence of Pt(II) and Pd(II) complexes have been calculated. A straight line is obtained when Δ G⁰s are plotted versus concentrations of each metal complex in transition regions at 300 and 310 K separately; these plots are shown in Figure 4 for Pt(II) complex and the inset for Pd(II) complex. The, m, slope of these plots (a measure of the metal complex ability to denature DNA) and the intercept on ordinate, Δ G⁰(H₂O) (conformational stability of DNA in the absence of metal complex) are summarized in Table 1. The values of m for Pd(II) complex are higher than that of Pt(II) complex, which indicate the higher ability of Pd(II) to denature DNA. As we know, the higher the values of Δ G⁰(H₂O), the larger the conformational stability of CT-DNA. However, the values of Δ G⁰(H₂O) decreases as the temperature rises. This is as expected, because, in general, the decrease in Δ G⁰(H₂O) value is the main reason for the decrease in DNA stability [20]. Molar enthalpy of CT-DNA denaturation in the absence of Pt(II) and Pd(II) complexes Δ H⁰(H₂O) is an another important thermodynamic parameter. For this, we calculated the molar enthalpy of DNA denaturation in the presence of each metal complex, or , in the range of the two temperatures 300–310 K using Gibbs–Helmholtz equation [23]. On plotting the values of these enthalpies versus the concentrations of each metal complex, straight lines were obtained which are shown in Figure 5 for [Pt(bpy)(pr-dtc)]Br and the inset for [Pd(bpy)(pr-dtc)]Br. Interpolation of these lines (intercept on ordinate, i.e.

Figure 4. The molar Gibbs free energies plots of unfolding (Δ G⁰ vs. [L]_t) of CT-DNA in the presence of [Pt(bpy)(pr-dtc)]Br. Inset: in the presence of [Pd(bpy)(pr-dtc)]Br.

Figure 5. Plots of the molar enthalpies of CT-DNA denaturation ( or ) in the interaction with [Pt(bpy)(pr-dtc)]Br and the inset with [Pd(bpy)(pr-dtc)]Br complexes in the range of 300–310 K.

Table 1.  Thermodynamic parameters and values of L _1/2 of DNA denaturation by Pd(II) and Pt(II) complexes.

	Temperature		(kJ mol
Compound	(K)		(mmol L	(kJ mol K)	(kJ mol	(kJ mol
[Pt(bpy)(pr-dtc)]Br	300	0.68	18.47	14.79	35.33	0.068
	310	0.64	21.61	14.10		0.068
[Pd(bpy)(pr-dtc)]Br	300	0.102	158.91	14.85	35.10	0.067
	310	0.091	170.50	14.17		0.067

^aMeasure of the metal complex ability to denature CT-DNA.

^bConformational stability of CT-DNA in the absence of metal complex.

^cThe heat needed for CT-DNA denaturation in the absence of metal complex.

^dThe entropy of CT-DNA denaturation by metal complex.

absence of metal complex) gives the values of Δ H⁰(H₂O) (Table 1). These plots show that in the range of 300–310 K, the changes in the enthalpies in the presence of Pt(II) and Pd(II) complexes are ascending. These observations indicate that if the concentration of Pt(II) and Pd(II) complexes increases, the stability of CT-DNA raises too. Moreover, the entropy of CT-DNA unfolding by Pt(II) and Pd(II) complexes Δ S⁰(H₂O) has been calculated using equation and the data are given in Table 1. These data show that the metal-DNA complexes are more disordered than those of native CT-DNA, because the entropy changes are positive for Pt(II)- or Pd(II)-DNA complexes in the denaturation processes of CT-DNA. These thermodynamic parameters are compared favorably well with those of palladium (II) complexes as reported earlier [9,24,25].

3.2.2 Measuring binding parameters

For drug–DNA interactions, spectrophotometric methods and sensitive routes for obtaining K_b are convenient. In these experiments, binding-induced changes in the spectral properties of the drug (or DNA) are monitored using an UV–VIS technique. Spectroscopic titrations (carried out by taking a fixed DNA concentration and titrating with drug, or vice versa) can then be used to construct equilibrium-binding isotherms.

Here a fixed amount of each metal complex was titrated with increasing concentration of CT-DNA in total volume of 2 mL at 300 and 310 K. The values of , that is, the change in the absorbance when all binding sites on CT-DNA were occupied by metal complex, are given in Table 2 and Figure 6 for [Pt(bpy)(pr-dtc)]Br and the inset for [Pd(bpy)(pr-dtc)]Br. These values were used to calculate the concentration of metal complex bound to CT-DNA, [L]_b, and the concentration of free metal complex, [L]_f and ν, the ratio of the concentration of bound metal complex to total [DNA] in the next experiment, that is, titration of fixed amount of CT-DNA with varying amounts of each metal complex in total volume of 2 mL at 300 and 310 K, separately. Using these data (ν and [L]_f) the Scatchard plots [26] were constructed for the interaction of each metal complex at two temperatures of 300 and 310 K. These plots are shown in Figure 7 for [Pt(bpy)(pr-dtc)]Br and the inset for [Pd(bpy)(pr-dtc)]Br. These curvilinear concave downward plots suggest co-operative binding [27]. Substituting ν and [L]_f in Hill's equation, , a series of equation with unknown parameters n, K and g were obtained. Using Eureka software [28], the theoretical values of these parameters could be deduced (Table 2). The, K, apparent binding constant in the interaction of [Pd(bpy)(pr-dtc)]Br with DNA is higher than that of [Pt(bpy)(pr-dtc)]Br with DNA (Table 2). This indicates that the interaction affinity of Pd(II) complex to DNA is more than Pt(II) complex. Because palladium complexes are about 10⁵ times more labile than their platinum analogs [29]. Values of n, the Hill coefficient (as a criterion of co-operativity), for palladium complex are higher than that of platinum analog. Similar trends are observed in the results of cytotoxic studies of these two compounds.

Figure 6. The changes in the absorbance of fixed amount of metal complexes in the interaction with varying amount of CT-DNA at 300 and 310 K. The linear plot of the reciprocal of Δ A vs. the reciprocal of [DNA] for [Pt(bpy)(pr-dtc)]Br. Inset: for [Pd(bpy)(pr-dtc)]Br.

Figure 7. Scatchard plots for binding of [Pt(bpy) (pr-dtc)]Br with CT-DNA. The inset is Scatchard plots for binding of [Pd(bpy)(pr-dtc)]Br with CT-DNA.

Table 2.  Values of Δ A_max and binding parameters in the Hill equation for interaction between CT-DNA and Pd(II)/Pt(II) complexes in 20 mmol L⁻¹ Tris–HCl buffer and pH 7.0.

Compound	Temperature (K)			(mol L		^eError
[Pt(bpy)(pr-dtc)]Br	300	0.162	2	14.31	2.58	0.0001
	310	0.214	2	16.72	4.17	0.0002
[Pd(bpy)(pr-dtc)]Br	300	0.339	4	30.94	2.61	0.002
	310	0.335	4	82.52	8.90	0.0007

^aChange in the absorbance when all the binding sites on CT-DNA were occupied by metal complex.

^bThe number of binding sites per 1000 nucleotides.

^cThe apparent binding constant.

^dThe Hill coefficient (as a criterion of co-operativity).

^eMaximum error between theoretical and experimental values of ν.

Figure 7 also shows the experimental values of ν obtained from Scatchard (lines) and theoretical values of ν from Hill (dots) and their superimposability on each other. In addition, these values of ν were plotted versus the values of Ln[L]_f. The results are sigmoidal curves and are shown in Figure 8 for Pt(II) complex and the inset for Pd(II) complex at 300 and 310 K. These plots indicate positive co-operative binding at both temperatures for both complexes. Finding the area under the above plots of binding isotherms and using the Wyman–Jons equation [30], , (where and K_app are concentrations of free metal complex and apparent binding constant for each particular ν, respectively), the values of K_app can be calculated at the two temperatures of 300 and 310 K for each particular ν [31]. Using the values of K_app, we can determine the corresponding values of molar Gibbs free energy of binding ( from equation , molar enthalpy of binding ( from equation and molar entropy of binding ( from equation – [23]. Plots of the values of versus the values of [L]_f are shown in Figure 9 for [Pt(bpy)(pr-dtc)]Br and the inset for [Pd(bpy)(pr-dtc)]Br at 300 K. Deflections are observed in both plots. These deflections indicate that at particular [L]_f, there is a sudden change in enthalpy of binding which may be due to binding of metal complex to macromolecule or macromolecule denaturation. Similar observations can be seen in the literature where Pd(II)/Pt(II) complexes have been interacted with CT-DNA [9,24,25].

Figure 8. Binding isotherm plots for [Pt(bpy) (pr-dtc)]Br in the interaction with CT-DNA. Inset: for [Pd(bpy)(pr-dtc)]Br.

Figure 9. Molar enthalpies of binding in the interaction between CT-DNA and [Pt(bpy)(pr-dtc)]Br (inset: [Pd(bpy)(pr-dtc)]Br) vs. free concentrations of complexes at pH 7.0 and 300 K.

3.3 Spectrofluorometric assays

No luminescence is observed for the [Pt(bpy)(pr-dtc)]Br and [Pd(bpy)(pr-dtc)]Br complexes; it is hard to monitor the intercalation of these complexes with DNA by employing direct fluorescence emission methods. In order to test whether the Pt(II) and Pd(II) complexes could bind to DNA by intercalation, ethidium bromide (EBr) was employed, as EBr interacts with DNA as a typical indicator of intercalation [32]. The experiments of DNA competitive binding with EBr were carried out in the Tris–HCl buffer by keeping [DNA] ( μM, μM) and varying the concentrations of the metal complexes (0–100 μM). The buffer used in the binding studies was 20 mM Tris–HCl, pH 7.0, containing 10 mM NaCl. The sample was incubated 2 h at 300 K before spectral measurements. For all fluorescence measurements, the entrance and exit slits were maintained at 0.5 nm, respectively. The emission range was set between 500 and 750 nm. The fluorescence emission spectra of the intercalated EBr with increasing concentrations of Pt(II) complex and the inset for Pd(II) complex are shown in Figure 10 which shows a significant reduction of the DNA-EBr emission intensity by adding different concentrations of Pd(II) and Pt(II) complexes. It indicates that the fluorescence intensity of DNA intercalated EBr is quenched when ethidium is removed from the duplexes of DNA by the action of palladium or platinum complexes and is released into buffer medium. Thus, it allows us to conclude that our two complexes possibly intercalate in DNA through the planar bpy ligand present in their structures.

Figure 10. Fluorescence emission spectra of interacted EBr-DNA in the absence (1) and presence of different concentrations of [Pt(bpy)(pr-dtc)]Br and the inset for [Pd(bpy)(pr-dtc)]Br: 15 μM (2), 30 μM (3), 45 μM (4), 60 μM (5), 75 μM (6), 90 μM (7), and EBr alone (8).

The fluorescence Scatchard plot was performed to study the binding constant determination by the luminescence titration method. A fixed amount of CT-DNA (60 μM) in the absence and presence of each metal complex was taken, incubated for 2 h, titrated with increasing concentration of EBr (2, 4, 6,  … , 20 μM) and their fluorescence emission intensity at 605 nm was measured. Hence, by carrying different sets of DNA-metal complexes corresponding to different r_f values (r_f is the ratio of the concentration of metal complex to DNA concentration), the number of EBr molecules intercalated to DNA (C_b) was then calculated using Scatchard equation, , where I₀ is the fluorescence emission intensity of EBr alone, I_t is the fluorescence emission intensity of -metal complex, K is the slope of the plot of I₀ versus C₀ (where C₀ is concentration of EBr added) and ν is the ratio of fluorescence emission intensity of the bound and free EBr under the same condition of excitation wavelength, concentration, temperature and solvent. By knowing C_b, r was calculated, which is the ratio of bound EBr to total DNA concentration and C, the concentration of free EBr. On plotting the r/C versus r, the binding isotherms were constructed and were represented as fluorescence Scatchard plots. The fluorescence Scatchard plots obtained for binding of EBr to CT-DNA in the absence (⧫) and presence (▲, •, ⬨) of various concentrations of Pt(II) and Pd(II) (inset) complexes were shown in Figure 11. This figure shows that these complexes inhibit competitively the EBr binding to CT-DNA (type-A behavior) [33], where number of binding sites n (intercept on the abscissa) remain constant and the slope of the graphs, that is K_app, (apparent association constant) decreases with increasing the concentration of Pd(II) and Pt(II) complexes (Table 3). This further proves that both complexes are intercalating in CT-DNA and thereby competing for intercalation sites occupied by EBr. The above metal complexes by themselves do not show any fluorescence. There was no interaction between metal complexes and EBr. In addition, the metal complexes did not quench the fluorescence. Comparing their K_app values with those of other known CT-DNA-intercalative complexes which possess analogical structure; Pt(II) and Pd(II) complexes in our article have similar or stronger affinities with CT-DNA [14,34].

Table 3.  Binding parameters for Pd(II) and Pt(II) complexes on the fluorescence of EBr in the presence of CT-DNA.

Compound		(M)
[Pt(bpy)(pr-dtc)]Br	0.00	0.90	0.0032
	0.42	0.49
	0.84	0.23
	1.26	0.11
[Pd(bpy)(pr-dtc)]Br	0.00	0.63	0.0038
	0.25	0.44
	0.50	0.31
	0.75	0.20

^aFormal ratio of metal complex to nucleotide concentration.

^bAssociation constant.

^cNumber of binding sites (n ) per nucleotide.

Figure 11. Competition between [Pt(bpy)(pr-dtc)]Br and the inset for [Pd(bpy)(pr-dtc)]Br with EBr for the binding sites of CT-DNA (Scatchard plot). In curve no. 1(⧫), Scatchard plot was obtained with CT-DNA alone. Its concentration was 60 μM. In curves nos. 2(▲), 3(•), and 4(⬨), respectively, 25, 50 and 75 μM for Pt(II) complex and15, 30 and 45 μM, for Pd(II) complex were added, corresponding to molar ratio [complex]/[DNA] of 0.42, 0.84, and 1.26 for Pt(II) complex and 0.25, 0.50, and 0.75 for Pd(II) complex. Solutions were in 20 mM NaCl and 20 mM Tris–HCl (pH 7.0). Experiments were done at room temperature.

3.4 Gel filtration

[Pt(bpy)(pr-dtc)]Br and [Pd(bpy)(pr-dtc)]Br complexes were incubated with CT-DNA for 2 h at 300 K in Tris–HCl buffer, pH 7.0. Then, DNA-metal complexes were passed through a Sephadex G-25 column equilibrated with the same buffer. The elusion of the column fraction of 2.0 mL was monitored at 283 and 260 nm for DNA-Pt(II) complex system and at 313 and 260 nm for DNA-Pd(II) complex system. These results are given in Figure 12 for Pt(II) complex and the inset for Pd(II) complex. These plots show that the peak obtained for the two wavelengths (at 260 nm for DNA and at 283 nm for Pt(II) or at 313 nm for Pd(II) complexes) are not resolved and suggests that CT-DNA has not separated from the metal complexes. Consequently, it implies that the binding between CT-DNA and the metal complexes is not reversible under such circumstances. This is due to the fact that if the interaction between CT-DNA and metal complexes was weak, the CT-DNA should have come out of the column separately. Also peaks due to DNA and each of the metal complexes should have appeared in different places of the plots. Thus, the interaction affinities are not only effective, but also strong enough not to break readily [9].

Figure 12. Gel chromatograms of [Pt(bpy)(pr-dtc)]Br and the inset for [Pd(bpy)(pr-dtc)]Br obtained on Sephadex G-25 column, equilibrated with 20 mol L⁻¹ Tris–HCl buffer of pH 7.0 in the presence of 20 mmol L⁻¹ sodium chloride.

4. Conclusion

Two water soluble Pt(II) and Pd(II) complexes of formula [M(bpy)(pr-dtc)]Br (where (II) and Pd(II), -bipyridine and pr--propyldithiocarbamate) have been prepared and characterized by spectroscopic methods. The DNA binding of these complexes have been examined by UV–VIS, fluorescence spectroscopic and gel filtration techniques. They can denature CT-DNA at low concentrations (in particular Pd(II) complex). The results support the notion that complexes can co-operatively bind to CT-DNA by intercalation. They have been found to be better cytotoxic agents than cisplatin against chronic myelogenous leukemia cell line, K562. Several binding and thermodynamic parameters are also presented. Remarkably, most of the experimental results indicate that the tendency of the Pd(II) complex to interact with DNA and its antitumor activity against K562 is more than that of its Pt(II) analog.

Funding

Financial assistance from the Research Council of University of Sistan and Baluchestan and of University of Tehran is gratefully acknowledged.