Palladium Complexes with 3-Substituted Derivatives of 5-Methyl-5-( 4-pyridyl ) hydantoins . Synthesis , Study and in vitro Cytotoxicity

Six palladium(II) and palladium(IV) complexes with 3-ethyl-5-methyl-5-(4-pyridyl)hydantoin, 3-propyl-5-methyl-5-(4-pyridyl)hydantoin and 3-benzyl-5-methyl-5-(4-pyridyl)hydantoin were synthesized. The complexes were identified and characterized by elemental analysis, IR, H, C NMR spectra etc. On the data obtained the molecular formulae of the new palladium complexes were proposed. The cytotoxicity of the complexes was evaluated in vitro using a panel of human tumour cell lines. The results demonstrate that the Pd(II) complex with 3-benzyl-5-methyl-5-(4-pyridyl)hydantoin exerts cytotoxicity as compared to the other studied Pd(II) complexes in all tested cell lines.


INTRODUCTION
In recent years much research efforts have been focused upon the design and development of innovative antineoplastic transition metal complexes with superior antiproliferative activity than cisplatin.Cisplatin is a widely used drug in cancer chemotherapy, 1−4 but a wide range of side effects 5 have limited its clinical usefulness.For this reason research has been extended to other metal complexes.On the basis of the structural analogy between platinum(II) and palladium(II) complexes, a variety of studies on palladium(II) complexes as potential anticancer drugs have been carried out. 6mong the first to be used for clinical trials against tumors, were the analogous to cisplatin, complexes of Pd(II), cis-[Pd(En)Cl 2 ] and cis-[Pd(DACH) 2 Cl 2 ] because Pd(II) has a very similar coordination chemistry to Pt(II), capable of forming square planar complexes.But these complexes have lower antitumor activity when compared to the analogous Pt(II) complexes, as well as their high toxicity. 7In general the use of Pd(II) complexes in medicine is limited.However, Pd(II) N, S chelates with inert ligands (e.g.sulfur or nitrogen) were suggested by Das and Livingstone 8 to be more effective antitumor agents than those of other metals, they possess the proper lability to bring the metal to the target (DNA) and allow it to interact with it.Palladium(II) complexes are much more labile than corresponding platinum(II) complexes, the lability of central palladium(II) may be much lower because of the shielding effect. 9Palladium(II) compounds might materialize the concept of tumor targeting, which would result in drugs with other spectrum of activity and a lack of cross-resistance as compared with platinum drugs. 10he present study represents the synthesis, physicochemical evaluation and pharmacological investigation of Pd(II) and Pd(IV) complexes with 3ethyl-5-methyl-5-(4-pyridyl)hydantoin, 3-propyl-5methyl-5-(4-pyridyl)hydantoin and 3-benzyl-5-methyl-5-(4-pyridyl)hydantoin.As a part of our drug discovery program to develop more effective palladium based anticancer drugs, here we report the antiproliferative activity of some Pd(II) and Pd(IV) complexes.

Materials and Physical Measurements
Potassium tetrachloropalladate(II) and potassium hexachloropalladate(IV) were purchased from Aldrich -USA.All other chemicals were of analytical grade.
The IR spectra were recorded on Thermo Scientific Nicolet iS10 spectrophotometer in the range of 4000−400 as pellets KBr and on IFS 113 v Bruker FTIR spectrophotometer in the range of 400−150 cm −1 in polyethylene.
The 1 H and 13 C NMR spectra were registered on a Bruker WM 250 (250 MHz) spectrometer in DMSO-d 6 .

Pharmacology
The present study describes a comparative evaluation of the cytotoxic effects of six newly synthesized Pd(II) and Pd(IV) complexes with some 3,5-disubstituted hydantoins vs. the referent antineoplastic agent cisplatin on a panel of human tumor cell lines, using the standard MTT-dye reduction assay for cell viability.

Cell Culture Conditions
The following cell lines were used for the experiments: (i) SKW-3 or a KE-37 derivative (human T-cell leukemia, established from the peripheral blood of a 61-yearold man with T-cell lymphocytic leukemia); (ii) HL-60 (acute myeloid leukemia, established from the peripheral blood of a patient with acute promyelocyte leukemia); (iii) EJ (human urinary bladder carcinoma), (iv) LAMA-84 (human chronic myeloid leukemia, established from the peripheral blood of a 29-year-old woman with chronic myeloid leukemia).The cell lines were maintained as suspension-type (SKW-3, LAMA-84, HL-60) or adherentmonolayer-type cultures (EJ) in cell-culture flasks using RPMI-1640, supplemented with 10 % heatinactivated fetal calf serum and L-glutamine as a growth medium.The cells were incubated in a controlled environment (37 °C, 5 % carbon dioxide humidified atmosphere).

Cytotoxicity Assessment
Cytotoxicity of the compounds was assessed using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] dye reduction assay as described by Mossman 14 with some modifications. 15Exponentially growing cells were seeded in 96-well microplates (100 L / well at a density of 3.510 5 cells / mL for the adherent and 110 5 cells / mL for the suspension cell lines) and allowed to grow for 24 h prior the exposure to the studied compounds.Stock solutions of the palladium complexes were freshly prepared in DMSO and then diluted with corresponding growth medium.At the final dilutions the solvent concentration never exceeded 0.5 %.Cells were exposed to the tested agents for 72 h, whereby for each concentration a set of 8 separate wells was used.Every test was run in triplicate, i.e. in three separate microplates.After incubation with the tested compounds MTT solution (10 mg / mL in PBS) aliquots were added to each well.The plates were further incubated for 4 h at 37 ºC and the formazan crystals formed were dissolved by adding 110 L of 5 % HCOOH in 2-propanol.Absorption of the samples was measured by an ELISA reader (Uniscan Titertec) at 580 nm.Survival fraction was calculated as percentage of the untreated control.The experimental data were processed using GraphPad Prizm software and were fitted to sigmoidal concentration/response curves, using non-linear regression analysis.

Chemical Studies
Complexes (1), ( 2) and (3) were prepared by adding of water-ethanol solutions of ligands L1, L2 and L3 to the water solution of K 2 [PdCl 4 ] in molar ratio 1 : 2. Complexes (1a), (2a) and (3a) were prepared by adding of water-ethanol solutions of ligands L1, L2 and L3 to the water solution of K 2 [PdCl 6 ] in the same ratio.The mixtures were stirred for 1−2 hours then the precipitates were filtered off, washed with distilled water and dried under vacuum.The substances are soluble in solvents with pronounced donor properties, such as N,N-dimethyl-formamide, dimethylsulfoxide and weakly soluble in water and ethanol.

IR Spectroscopy
Infrared spectroscopy data confirm the coordination of ligands to the palladium(II) and palladium(IV) ions via nitrogen atom from pyridine ring of the ligands.In the spectra of the ligands L1, L2 and L3 the typical stretching vibrational mode of substituted pyridines, ν(C=N) for the uncoordinated ligands is appeared at 1600 сm −1 and were blue shifted to higher wavenumbers -1658, 1618, 1616, 1617, 1618 and 1617 cm −1 in the spectra of the complexes -respectively.The other characteristic bands from the pyridine ring of the metal-free ligands are again blue-shifted upon complexation.These shifts were in agreement with the N-coordination, involving the pyridine N atom of the ligands.New bands in the range of 377-318 cm −1 were assigned to the ν(Pt-Cl) stretching vibrations.In the IR spectra of the all complexes two bands for ν(Pt-Cl) stretching vibrations were observed, implying cis-location of chloride ligands. 16he bands related to the stretching vibrations of the two carbonyl groups in the metal-free ligands did not shift upon coordination of L1, L2 and L3 to palladium(II) and palladium(IV) ions, indicating that the C=O groups were not involved in binding to the metals.

H NMR
The 1 H NMR spectra of the freshly prepared DMSO-d 6 solutions of the ligands and complexes were recorded.In the spectra of complexes 1, 2 and 3 the signals of the protons for Н-2 and Н-6 from the pyridine ring were shifted from 8.61 ppm in the ligands L1−L3 to 8.77, 8.76, 8.77, 8.78, 8.78 and 8.78 ppm in the complexes 1−3 and 1a−3a.The differences between the chemical shifts of the protons of the ligands L1−L3 and those of the corresponding complexes are in the range of 0.15-0.17ppm for all studied complexes.The signals of the Н-3 and Н-5 protons from the pyridine ring were shifted from 7.48 ppm in the ligands L1-L3 to 7.66, 7.66, 7.66, 7.67, 7.67 and 7.68 ppm in the complexes 1-3 and 1a−3a respectively.Here the differences are in the range of 0.18-0.20 ppm.All signals for the protons in the pyridine ring were shifted.This shows that the most probable bounding of the ligands with the palladium ions in all complexes is realized through the nitrogen atom from the pyridine ring. 13C NMR Spectra In the 13  The resonances of the two С=О groups of the hydantoin ring in metal-free-ligands L1, L2 and L3 and in their complexes 1-3 and 1a−3a were the practically same.This shows that the hydantoin ring is not involved in the coordination with the metal ion.
The elemental analysis and spectroscopic data confirm the following structures of the complexes shown in Figure 1.

In vitro Cytotoxicity
The tested palladium(II) and palladium(IV) complexes exerted cytotoxic effects after 72 h continuous exposure, whereby the individual chemosensitivity varied among the different cell lines, as evidenced by the IC 50 values, summarized in Table 2.
The Pd(II) complex with 3-benzyl-5-methyl-5-(4pyridyl)hydantoin (3) proved to be the most active analogue within this series, whose antiproliferative potency in SKW-3 was relative to that of the reference antineoplastic drug cisplatin.

CONCLUSION
Six Pd(II) and Pd(IV) complexes with 3-ethyl, 3-propyland 3-benzyl-5-methyl-5-(4-pyridyl)hydantoins were synthesized according to procedures described in the literature.All complexes were characterized by elemental analysis, IR, 1 H, 13 C NMR spectra.The mode of the coordination of the ligands with palladium ions is realized via the nitrogen atom of the pyridine ring.The coordination mode is identical to those of Pt(II) and Pt(IV) complexes with the same ligands, described in our preceding reports.The cytotoxicity of the prersented palladium complexes was assessed in vitro on a panel of four human tumour cell lines.The results show that the Pd(II) and Pd(IV) complexes with 3-benzyl-5-methyl-5-(4-pyridyl)hydantoin possess higher cytotoxicity in all cell lines than the other Pd(II) and Pd(IV) complexes with 3-ethyl-and 3-propyl-5-methyl-5-(4-pyridyl)hydantoins.This is in keeping with the Pt(II) (where for Pt(II) complexes cytotoxicity was strongly dependent on their calculated logP values) and Pt(IV) complexes with the same ligands.
C NMR spectra of the compounds 1-3 and 1a−3a the signals for the carbon atoms C-2 and С-6 from the pyridine ring were at 153.1, 153.1, 153.2, 153.1, 153.1 and 153.2 ppm, respectively, compared to 150.0 ppm for the metal-free ligands L1−L3.The downfield chemical shifts in the range 3.1 to 3.2 ppm indicate that the pyridine nitrogen atom takes part in the coordination to the metal ions.The shifts of the С-3 and С-5 were smaller in according to their remote positions from the binding nitrogen atom.

Table 1 .
Physical properties and elemental analysis of new palladium complexes