Spectroscopic Characterization , In Vitro Cytotoxicity , and Antioxidant Activity of Mixed Ligand Palladium ( II ) Chloride Complexes Bearing Nucleobases

Mixed-ligand palladium(II) chloride complexes bearing the nucleobases, adenine (Ad), cytosine (Cyt), and guanine (Gua), have been synthesized and characterized by UV-vis spectrophotometric methods, magnetic susceptibility, molar conductivity, elemental analysis, FTIR, and H-NMR. The complexes were found to have the composition cis-[PdCl 2 (Gua)(Cyt)], cis-[PdCl 2 (Ad)(Cyt)], and cis-[PdCl 2 (Ad)(Gua)]. A four-coordinated square-planar geometry is proposed for these Pd(II) complexes based on magnetic evidence and electronic spectra. The complexes as well as the free nucleobase ligands were tested for their in vitro cytotoxicity on human promyelocytic leukemia (HL60) and human histiocytic leukemia (U937) cell lines. cis-[PdCl 2 (Ad)(Gua)] showed IC 50 values of 11.29± 2.91 and 8.31± 1.44 μM against HL60 and U937, respectively, which was higher than that of the positive control (curcumin) against U937. The complexes also showed significant antioxidant activity when tested against 2,2-diphenyl1-picrylhydrazylradical (DPPH).


Introduction
The versatility of nucleobases has made them suitable ligands for the synthesis of numerous transition metal complexes [1][2][3][4][5].In particular, palladium complexes of nucleobases and their derivatives have been of current interest since these complexes form faster than their platinum analogues and produce analogous products in solution [6,7].The mode of bonding and structure of these and related ligands have been exhaustively studied, both spectroscopically and crystallographically [8][9][10][11] and have been shown to be monodentate via the N3 of the pyrimidine and N7 of the purine ligand (as shown in Figure 2).In view of the strong electron withdrawing ability of these metal ions, there is a drift of electron density onto the metal centre and in effect ligand hydrogens are easily lost in the presence of a free radical.This makes these metal complexes act as better antioxidants as compared to the free ligand [12,13].
Mixed-ligand complexes play a key role in biological chemistry [14] because mixed chelation occurs commonly in biological fluids as millions of potential ligands are likely to compete for metal ions in vivo [15].Because of this profound role, mixed-ligand complexes have been extensively studied for their thermodynamic [16][17][18] as well as kinetic stability [19][20][21][22][23].These complexes have received tremendous attention in the search for novel drugs against drug resistant diseases [ [24][25][26][27] with cisplatin being a classic example [28].
Metal based antioxidants have received recent attention for their capacity to protect organisms and cells from damage induced by oxidative stress [12].An effective antioxidant, however, should be able to terminate the attack of reactive species like free radicals and prevent them from attacking body cells.The antioxidant activity of a synthetic compound can be measured using the scavenging ability of that compound to trap free radicals [13].Although some palladium(II) complexes of pyrimidine and pyridine derivatives have been synthesized and characterized [1,29], not much has been reported about the antioxidant activity as well as the in vitro cytotoxicity of these complexes.In this study we present the synthesis, spectroscopic characterization, and 2 Journal of Inorganic Chemistry antioxidant as well as in vitro cytotoxicity of palladium(II) complexes with purine and pyrimidine nucleobases as ligands on human promyelocytic leukemia (HL60) and human histiocytic leukemia (U937) cell lines.

Materials and Methods
2.1.Materials.All the ligands were purchased from Molekula (UK) and the palladium(II) chloride was purchased from Merck KGaA (Germany).These were used without further purification.The DPPH was purchased from Sigma-Aldrich (St. Louis, MO, USA).All other chemicals were of analytical grade and obtained from standard suppliers.Palladium content was analysed spectroscopically using dimethylglyoxime and a modification of the method described by Khader and Prasad [30].Free radical scavenging ability of the complexes was used as a measure of the antioxidant activity of the complexes.Thiazoyl blue tetrazolium bromide (MTT) assay was used to determine the cytotoxicity of the complexes against U937 and HL60 cell lines.

Instrumentation and Methods.
Chloride content was determined by the Mohr method. 1 H-NMR was recorded in DMSO-d 6 on a Gemini 2000 instrument (400 MHz) at room temperature and the 1 H chemical shifts referenced to the residual signals of the protons of the NMR solvent quoted in ppm.The IR spectra (KBr disks) were recorded on an Interspec 200-X spectrophotometer.Molar conductivity was recorded on a Wagtech 4510 conductivity meter.Electronic absorbance of the complexes was recorded in DMSO on a T70 UV/VIS spectrometer.Melting points of the complexes were determined with a Thermo Scientific Electrothermal Digital Melting Point Apparatus IA9100.Magnetic susceptibility was determined using a modification of the Gouy method [31,32].

Synthesis of the Palladium(II)
Complexes.The complexes were prepared in a general way as follows: to a 10% HCl ethanolic solution (10 mL) of PdCl 2 (0.354 g, 2 mmol) was added 2 mmol of one nucleobase ligand in 20 mL of boiling ethanol.After stirring the mixture at 100 ∘ C for 30 minutes, 2 mmol of the other ligand dissolved in 20 mL of boiling ethanol was then added.The resulting solution was refluxed at 100 ∘ C with stirring for further 45 minutes.The yellow to orange precipitates were suction-filtered and washed with hot ethanol and dried over anhydrous CaCl 2 .

Procedure for MTT In Vitro Cytotoxicity.
The in vitro cytotoxicity of the compounds was performed on HL60 and U937 cells as described [33].The cells were maintained in RPMI 1640 medium (containing 10% FBS and 0.01% kanamycin).Cells in exponential growth were seeded into 96-well plates at a concentration of 1 × 10 4 cells/100 L/well.The cells were then treated with various concentrations of the test compounds at a concentration range of 0-200 M for HL60 and 0-100 M for U937 using DMSO as the solvent.Negative control (untreated) and positive control (curcumin) experiments were included.
After 72 hours of incubation at 37 ∘ C under 5% CO 2 in humidified atmosphere, 20 L of MTT (3-(4,5dmethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added to each well and the plates were kept in the dark for further 4 hours.Subsequently, 150 L of acidified isopropanol containing Triton-X 100 was added to stop the reaction and also solubilize the formazan crystals formed.Absorbance readings were taken at 570 nm on a Tecan-PC infinite M200 Pro Plate reader after overnight incubation of the plates.Triplicate experiments were performed.Dose response curves were plotted as percentages of cell viability against concentration.Drug sensitivity was expressed in terms of the concentration of drug required for a 50% reduction of cell viability (IC 50 ).The IC 50 values were determined by nonlinear regression analysis.

Procedure for Antioxidant Activity.
The antioxidant activity of the complexes was determined as described [12,34] with slight modifications as follows: on a 96 well plate, the compounds were serially diluted in DMSO to obtain a concentration range of 1.25-20 mM.The reaction mixture consisted of 100 L of 0.5 mM 2,2-diphenyl-1-picrylhydrazylradical (DPPH), and 100 L of each concentration of the test compounds using DMSO as the solvent.For positive control, 2,6-di-tert-butyl-4-methylphenol (BHT) was used at a concentration range of 0.0625-2 mM in methanol.The solvents, DMSO and methanol, were used as blanks.Duplicate experiments were performed.The plates were covered with aluminum foil and kept in the dark for 20 minutes after which the absorbance was read on a Tecan-PC infinite M200 Pro Plate reader at the absorbance wavelength of 517 nm.The percent antioxidant activity was calculated as follows: where  0 is the absorbance of the blank and  1 is the absorbance in the presence of the sample or positive control.Λ  ( 2 ): 15.32 Ω −1 cm −2 mol −1 (0.925). eff = 0.

IR Spectra.
Important vibrational bands were selected by comparing the FTIR spectra of the complexes with those of the respective free nucleobase ligands.The symmetric and antisymmetric N-H stretching frequencies of the complexes were observed in the range of 3424-3170 cm −1 .These peaks were compared with those of the free ligands which appear between 3372 and 3114 cm −1 .The observed shifts can be attributed to hydrogen bonding and other noncovalent interactions in the solid state of these metal complexes [35,36].These observations are supported by the broadening of the spectra observed at 3663-2500 cm −1 [36].Also observed around 1605-1567 cm −1 are important symmetric NH 2 bending vibrations of the ligands which showed slight shifts as compared to those of the free ligands.Furthermore, peaks corresponding to the ring stretching frequencies ]  C=N between 1620-1567 cm −1 in the free ligands were found to have shifted to 1634-1638 cm −1 in the metal complexes, suggesting the involvement of the ring C=N group in coordinating to the metal center [25,36].The far-IR region also revealed bands corresponding to ](Pd-Cl) and ](Pd-N) vibrations which are absent in the spectra of the free ligands.The FTIR spectra of the complexes are summarized in Tables 1 and 2.

1 H-NMR Spectra.
A comparison of the 1 H-NMR spectra of the complexes with those of the free ligands show downfield shifts in the proton signals upon complexation to the palladium(II) ion.As shown in Table 3, the NH 2 cytosine protons of cis-[PdCl 2 (Gua)(Cyt)] which are directly adjacent to the coordinating N3 ring nitrogen show a downfield shift from 7.06 to 7.70 ppm in the complex.Similarly, the NH 2 protons of guanine observed in the free ligand at 5.57 ppm was shifted to 5.95 ppm showing that these protons experienced strong deshielding effects upon complexation [37].Similar chemical shifts were observed for the remaining protons as shown in Table 3. Figure 2 shows the proposed structures of the complexes with labeled atoms.

Electronic Absorption Spectra, Conductivity, and Magnetic
Measurements.The electronic spectra of the complexes were recorded in DMSO (10 −3 M).This was used to study the effect of complexation on the splitting of the d orbitals in the Pd(II) ion.Significant absorption bands were observed in the region of 280-290, 331-333, 403-407, and 531 nm.These transitions may be attributed to intraligand - * as well as the spin allowed LMCT d-d transitions of respectively [37].These transitions confirm the square planar (D 4h ) geometry of the Pd(II) ion [31].Bathochromism was also observed in the spectra of these complexes with increasing concentration as exemplified in Figure 1.This shift in wavelength can be attributed to strong intermolecular hydrogen bonding, a phenomenon which can be likened to the intercalative mode of bonding involving a strong - * stacking interaction between aromatic chromophores and DNA bases [38][39][40].
The molar conductivities of the complexes measured in DMSO (10 −3 M) was between 13.58 and 18.48   indicating the nonelectrolyte nature of these complexes [41][42][43].The effective magnetic moment measurement at room temperature confirmed their diamagnetic characters of these complexes thus square-planar geometry.Based on the above discussion of results and the analytical data obtained from the metal and chloride content analyses we suggest that the structural formulae of the complexes are as given in Figure 2.

In Vitro Cytotoxicity.
The growth inhibition effect of the complexes as well as the free ligands was measured on two leukemia cell lines (human promyelocytic leukemia (HL60) and human histiocytic leukemia (U937)) with curcumin as a positive control.The results as expressed by IC 50 values (M) are presented in Table 4.Of the three nucleobase ligands, guanine was the only cytotoxin on HL60 within the working concentration of 0-200 M.It reported an IC 50 value of 71.38 ± 9.01 M.Guanine was also cytotoxic against U937, recording a value of 53.53 ± 8.55 M.
All the three mixed ligand complexes were significantly cytotoxic with the exception of cis-[PdCl 2 (Cyt)(Ad)] which did not show any activity on HL60 within the working concentration.From Table 4, cis-[PdCl 2 (Ad)(Gua)] was the most cytotoxic against both cell lines with IC 50 values of 11.45 ± 2.91 and 8.31 ± 1.44 M on the respective cell lines.It was more cytotoxic than the positive control (curcumin)  [12,13,46].This assay is based on the measurement of the decrease in molar absorptivity of DPPH at 517 nm after reaction with the test compound.From Figure 3, the antioxidant activity of the free ligands was found to be between 10 and 15% but, upon complexation, it increased significantly (Figure 4).The increased antioxidant activity of these complexes can be attributed to the downfield shift of the 1 H-NMR signals (Table 3).As reported [12,13], this phenomenon is as a result of the electron withdrawing effect of the Pd(II) ion which facilitates the release of hydrogen to reduce the DPPH radical.These proton shifts were very profound in cis-[PdCl 2 (Ad)(Gua)] with the H-1 proton of guanine absorbing at a chemical shift of 14.06 ppm.This could contribute to the higher activity of cis-[PdCl 2 (Ad)(Gua)] with an EC 50 (concentration of complexes needed to scavenge 50% of the initial DPPH concentration) value of 1.00 mM.

Conclusion
In this study, we reported the synthesis and spectroscopic characterization of mixed ligand Pd(II) chloride complexes bearing the nucleobases cytosine, adenine, and guanine.A four-coordinated square-planar geometry was proposed for these complexes based on spectroscopic and magnetic measurements.The ligands and the metal complexes were tested for their antioxidant and in vitro cytotoxic activities.All the complexes were significantly cytotoxic with cis-[PdCl 2 (Ad)(Gua)] showing higher activity than the positive control (curcumin) on U937 cell line.All the complexes also show significant free radical scavenging ability when tested against DPPH.

Figure 2 :
Figure 2: Proposed structures of the palladium(II) chloride complexes.

Figure 3 :
Figure 3: Percent antioxidant activity of the free ligands.

Table 1 :
Major IR/cm −1 absorption bands of the palladium(II) complexes and the nucleobase ligands.

Table 3 :
1H-NMR data of the complexes and the free nucleobase ligands.

Table 4 :
IC 50 values of the complexes tested on the respective cell lines.
3.8.Antioxidant Activity.Various researchers have used scavenging effect of a chemical on DPPH radical as a quick and reliable parameter to measure the in vitro antioxidant activity