Complexes of palladium ( ii ) with 1-phenyl-1-hydroxymethylene bisphosphoniс aCid and their antitumor aCtivity

1Vernadsky Institute of General and Inorganic chemistry, National academy of Sciences of Ukraine, Kyiv; 2Technical centre of National academy of Sciences of Ukraine, Kyiv; 3Institute of Organic chemistry, National academy of Sciences of Ukraine, Kyiv; 4SI “Institute of Pharmacology and Toxicology, National academy of Medical Sciences of Ukraine”, Kyiv; 5Kavetsky Institute of experimental Pathology, Oncology and radiobiology, National academy of Sciences of Ukraine, Kyiv; e-mail: complex@ionc.kiev.ua


O, O] ring, and two chlorine anions. The formation of palladium(II) equimolar complexes with PhhMBP and bidentate coordination of the ligand to the central metal cation was confirmed by 31 P NMr spectroscopy. cytotoxic activity (Ic 50 based on metal content) of the synthesized Pd(II) complexes with PhhMBP against human MG-63 osteosarcoma and McF-7 mammary tumor cells was compared with cisplatin on in vitro models. It was established that cytotoxic activity of the Pd complexes was lower than that of cisplatin. The acute toxicity (lD 50 based on metal content) of solutions of Pd(II) complexes with PhhMBP was found to be lower compared to cisplatin. It was shown that the use of solutions of palladium(II) complexes with PhhMBP inhibited tumor growth in mice with sarcoma 180. K e y w o r d s: palladium(II) complexes, bisphosphonic acid, cytotoxic activity, toxicity.
C isplatin is still one of the most famous and efficient anticancer drugs used in the clinical practice [1].Nevertheless, searching for new compounds with antineoplastic action continues to eliminate the serious side effects, to improve the clinical efficacy and to broaden the antitumor spectrum.In particular, palladium(II) bisphosphonates are promising species for the treatment of bone cancer and metastases: complexes include a cytotoxic metal (palladium) and a bisphosphonic acid, which possesses affinity for bone tissue and can ensure targeted delivery of the cytotoxic metal to the lesion site [2][3][4][5][6][7][8][9].The P-C-P moiety present in bisphospho nic acids provides their active link to the bone matrix, and two side substituents determine their physicochemical and pharmacologic properties.To study the effect of the structure of complex and chemical nature of the substituents bonded to the carbon atom of bisphosphonate moiety on the biological activity, the formation of palladium(II) complexes with 1-phenyl-1-hydroxymethylene bisphosphoniс acid was investigated, which, besides two phosphonic groups, contain a hydroxy group and a phenyl moiety.
The aim of the work is to determine the composition, structure, stability and biological activity of palladium (II) complexes with PhHMBP.

materials and methods
The following reagents were used in the work: 1-phenyl-1-hydroxymethylene bisphosphonic acid (PhHMBP, H 4 L) synthesized by the procedure presented in [10], concentrated solution of pure carbonate-free potassium hydroxide, palladium black.Potassium tetrachloropalladate was prepared according to the standard scheme [11]: The palladium content of PdCl 2 was determined by the reduction of a weighed amount of the compound in a hydrogen stream to metallic palladium [12].
The standard solution of hydrochloric acid was prepared from a titrimetric standard substance.The potassium hydroxide solution was prepared right away before using from a saturated KOH solution.The exact KOH concentration was determined by titration with the standard solution of hydrochloric acid.The exact concentration of PhHMBP was determined by titration with KOH.
Weighting was performed on a Vibra HT-120 CE balance.The measurements were made a Metrohm 827 pH lab pH meter.The device was calibrated using standard buffer solutions with pH 1.68, 6.86, 9.18 (25 °C).
The electronic absorption spectra of the solutions were recorded on a Specord M-40 spectrophotometer (range 50000-11000 cm -1 ).The recording was performed in quartz cuvettes with l = 1 cm.
To prepare solutions for pH potentiometric and spectrophotometric studies, freshly distilled water was used.
The formation constants of complexes and their equilibrium concentrations were calculated using the PSEQUAD program [13] from the data of EASs and pH of equilibrium solutions from the formulas: The 31 P NMR spectra with proton decoupling ( 31 P-{ 1 H}) were recorded on a Bruker AVANCE 400 spectrometer without deuterium stabilization of the resonance conditions.The value of 31 P NMR chemical shift was determined relative to 85% H 3 PO 4 .
All measurements were carried out a day after the preparation of solutions, the period required for the establishment of chemical equilibrium.
To carry out the studies of biological activi ty, solutions of a Pd(II) complex with PhHMBP (С Pd(II) = 10.0 mg/ml (0.094 mol/l); С H4L = 0.094 mol/l, C KCl = 0.15 mol/l, рН = 4.56) were prepared.To study in vitro cytotoxic activity, we used MG-63 human osteosarcoma and cells of the MCF-7 line (cell line obtained from human breast cancer).The cells were cultured in a DMEM culture medium (Biowest, France) from 10% fetal calf serum (FCS) (HyClone, France) at 37 °C in a humid atmosphere in the presen ce of 5% CO 2 .The obtained cell suspensions were placed on a 96-well plate in a concentration of 1.2-1.5‧10 4 cells/well (in a volu me of 0.1 ml) and incuba ted for 72 h at 37 °C in the presence of 5% CO 2 .The solution of palladium complex under investigation was added four or two times (100 μl each time) with two-fold dilution from a concentration of 200 μg/ml in the first wells to a concentration of 1.56 μg/ml.The wells intact cell cultures were used as a control.After the addition of a solution of palladium complexes, the test and control cultures in the plates were incubated for 72 h.The cytotoxici ty of palladium(II) complexes with PhHMBP was assessed on the basis of the number of alive cells in the test and control wells (MTT test) [15].To this end, after the completion of incubation, the supernatant with dead cells and detritus was completely removed, and 50 μl of a crystal violet solution was put in each well to fix and color the cells on the substrate.The plate was incubated for 10 min at room temperature.Then the dye was removed, the plate wells were washed with water and dried at room temperature.The optical density, which is proportional to the number of alive cells in the wells, was determined with a Labsystem Multiscan Plus spectrometer at an excitation wavelength of 540 nm.
The in vivo toxicity of Pd(II) complexes with PhHMBP was assessed by determining the halflethal dose (LD 50 ) and studying the percentage of sarcoma 180 (S-180) growth inhibition in while mice in the case of introducing solutions of Pd(II) complexes with PhHMBP.The half-lethal dose of Pd(II) complexes with PhHMBP (LD 50 ) was determined according to [16].The solutions were injected intraperitoneally in while mice once before feeling in different doses.The screening studies of the anticancer activity of Pd(II) complexes with PhHMBP were carried out on while nonlinear mice of the same sex with a body weight of 18-20 g on a sarcoma 180 model [17].The number of animals in the test group was 7 and in the control group 22.The substance under investigation was injected 24 h after tumor transplantation in a dose of 1/3 LD 50 daily (7 injections).The effect parameter was the percentage of tumor growth inhibition by mass.The minimum activity criterion is tumor growth inhibition of over 50%.

results and discussion
According to the results of a pH potentiometric titration in the pH range 2.2-11.3,PhHMBP is a tetrabasic acid (H 4 L).
The determined formation constants of PhHMBP forms of different degree of deprotonation are listed in Table 1.They were calculated from the data of two parallel titrations (each titration included more than 50 points) (according to the measurement data in two parallel experiments).The first dissociation constant value for PhHMBP (pK 1 ) is close to 2, which complicates its calculation from the data of pH potentiometric titration.The values of pK 2 , pK 3 and pK 4 , which correspond to the consecutive disso-ciation of OH protons from the phosphonic groups, within the experimental error agree with the published data [18].The geminal hydroxy group remains protonated below the pH of 13.
The When a K 2 [PdCl 4 ] solution is titrated in 0.15 mol/l KCl with a solution of PhHMBP neutralized with three KOH equivalents, the maximum in the electronic absorption spectra (EASs) shifts from 21650 cm -1 to approx.24800 cm -1 (pH 4.91-5.24)due to the formation of a palladium(II) complex with PhHMBP (Fig. 2).
The formation constants of complexes of the compositions [PdНLCl 2 ] 3-(lgβ = 24.51(0.3))and The values of formation constant calculated for complexes of equimolar composition were used to construct a diagram of the equilibrium concentration distribution of palladium complexes as a function of pH (Fig. 4).

T a b l e 1. Formation constants of protonated PhhMBP forms and corresponding dissociation constants
It is seen from the diagram that the anionic complex of the equimolar composition [PdLCl 2 ] 4-is predominant in the solution at the physiological concentration of chlorine anions and pH close to 7.
The composition of the complexes and the mode of the coordination of ligand to the central metal ion, determined from spectrophotometric data, are corroborated by the results of investigation of the system K 2 [PdCl 4 ] : PhHMBP = 1 : 1 and 1 : 2 by the 31 P NMR method.
The 31 P NMR spectra (Fig. 5, a) of a set of solutions with the ratio K 2 [PdCl 4 ] : PhHMBP = 1 : 1 The coordination mode of ligand to the central metal ion was established on the basis of the dependence of the position of the absorption band maximum on the composition of the first coordination sphere (chromophore) of square-planar palladium complexes [19].In particular, in the case of bidentate coordination of ligand by two oxygen atoms of phosphonic groups the absorption band with max- It should be noted that the 31 P NMR spectra of solutions with pH<3 exhibit low-intensity signals in the δ 31 Р range 24.3-23.5 ppm, which probably correspond to a complex with PhHMBP monodentately coordinated to palladium(II) by phosphonic group oxygen atom.This was not established by electron spectroscopy and pH potentiometry (C = 2‧10 -3 mol/l).
The 31 P NMR spectrum of bidentately coordinated PhHMBP consists of three signals with close δ values.As we have previously shown, the observed nonequivalence of the phosphorus nuclei in the coordinated ligand may be both due to the conformation isomerism of the formed complexes and a result of forming an intraligand hydrogen bond [20].
The in vivo anticancer activity of a palladium(II) complex with PhHMBP was determined on an MG-63 and MCF-7 cell line models (IC 50 21  and 41.7 μg/ml (based on palladium content), respectively, Fig. 6).The found values are lower than those found for cisplatin (2.2 and 1 μg/ml (based on palladium content), respectively).
The acute toxicity of solutions of palla dium(II) complexes with PhHMBP (LD 50 = 41 mg/kg (based on palladium content)) is lower than that for cisplatin, for which LD 50 is found to be 8.42 mg/kg (based on palladium content).
When determining the antineoplastic action of solutions of palladium(II) complexes with PhHMBP on sarcoma 180 bearing mice, the found percentage of tumor growth inhibition is over 50% (minimum activity criterion) (Table 2).
Depending on pH, the PhHMBP ligand forms in water solutions with the physiological concentration of chlorine anions (0.15 mol/l) the anionic complexes of the equimolar compositions, [PdНLCl 2 ] 3- and [PdLCl 2 ] 4 , as indicated by pH potentiometry, electron and NMR spectroscopy.The first coordination sphere involves two oxygen atoms of two phosphonic groups of ligand with closure of the six-membered [O, O] ring and two chlorine anions.
The solutions of palladium(II) complexes with PhHMBP demonstrates moderate in vitro cytotoxic activity (IC 50 ) against MG-63 and MCF-7 tumor cells that does not exceed that of cisplatin.At the same time, solutions of palladium(II) complexes with PhHMBP exhibit lower toxicity as compared with cisplatin.They also provide inhibition for sarcoma 180 growth in mice.