SYNTESIS AND CYTOTOXIC ACTIVITY OF NEW PLATINUM …dl.uctm.edu/journal/node/j2013-6/15-A_Bakalova...

A. Bakalova, R. Buyukliev, G. Momekov, D. Ivanov

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Journal of Chemical Technology and Metallurgy, 48, 6, 2013, 631-636

SYNTESIS AND CYTOTOXIC ACTIVITY OF NEW PLATINUM AND PALLADIUM COMPLEXES WITH 3-AMINO-α-TETRALONESPIRO-5’-HYDANTOIN

A. Bakalova1, R. Buyukliev1, G. Momekov2, D. Ivanov1

1Department of Chemistry, Faculty of Pharmacy, Medical University - Sofia, 2 Dunav Str., 1000 Sofia, Bulgaria2 Department of Pharmacology, Pharmacotherapy and Toxicology, Faculty of Pharmacy, Medical University-Sofia, 2 Dunav Str., 1000 Sofia, Bulgaria E-mail: [email protected]

ABSTRACT

Four Pt(II), Pt(IV), Pd(II) and Pd(IV) complexes with 3-amino-α-tetralonespiro-5’-hydantoin as carrier ligand were synthesized. The molecular formulae of the new synthesized complexes were determined by elemental analysis, IR and 1H NMR spectra. On the base of all obtained results from chemical and spectroscopic investigation the coordination mode of the ligand with metal ions was proved. The spectral analyses indicate a cis -square planar structure of all platinum and palladium complexes with ligands coordinated via the NH2 group. Complexes with general formula cis-[ML2Cln], where M is Pt(II), Pt(IV), Pd(II), Pd(IV), n = 2 or 4 were obtained. The cytotoxic activity of the new platinum and palladium complexes were determined in vitro by MTT method against SKW-3 human tumour cell line. All studied complexes evoked concentration-dependent cytotoxic effects, which were much more manifested at micromolar concentrations. The IC50

values of the tested metal complexes showed that Pt(II) complex with 3-amino-α-tetralonespiro-5’-hydantoin has higher cytotoxic activity than the palladium complexes with the same ligand, which confirmed the rule that platinum complexes showed higher activity than the corresponding palladium complexes.

Keywords: Pt and Pd complexes, spirohydantoins, cytoxicity.

Received 11 July 2013Accepted 05 November 2013

INTRODUCTION

Cisplatin is one of the most widely used anticancer drugs for the treatment of diverse solid tumours such as head and neck, squamous cell carcinoma, ovarian cancer, cervical cancer, urinary bladder cancer etc [1-3]. Despite its wide application, cisplatin is associated with many serious side effects like nephrotoxicity, othotox-icity, allergy, myelotoxicity, peripheral neuropathy and haematological toxicity [4-6]. Because of side effects and limited number of cancer types sensitive to cisplatin, other platinum, ruthenium, rhodium, gold, titanium or palladium complexes were synthesized and investigated for cytotoxic activity [7-9]. On the base of the structural

analogy between Pt(II) and Pd(II) complexes, some studies of palladium compounds as suitable drugs have been carried out [10]. Geometry and complex forming processes of palladium(II) are very similar to those of platinum(II) therefore it was speculated that palladium complexes may also have antitumour activities and serve as good models for the understanding of more inert platinum(II) anticancer drugs [11-14]. In order to obtain compounds with superior chemotherapeutic index in terms of increased bioavailability, higher cytotoxic-ity, and lower side effects than cisplatin, a new class of platinum(II) [15-16] and palladium(II) complexes [17] was synthesized. Although palladium(II) complexes are much more labile than corresponding platinum(II)

Journal of Chemical Technology and Metallurgy, 48, 6, 2013

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complexes, the lability of central palladium(II) may be much lower because of the shielding effect [18]. Hydan-toins form a large group of derivatives widely applied in medicine and pharmacy, especially as anticonvulsants, antiarrhythmics, antibacterial drugs, cytotoxic agents etc. [19]

The present study represents the synthesis and phys-icochemical evaluation of four new Pt(II), Pt(IV), Pd(II) and Pd(IV) complexes with 3-amino-α-tetralonespiro-5’-hydantoin(L). Their in vitro cytotoxic activity was assessed and compared to the clinically applied drug cisplatin.

EXPERIMENTAL

Starting Materials Potassium tetrachloroplatinate(II) utilized for the

synthetic procedures was purchased from Merck – Ger-many, platinum(IV) chloride - from Heraeus GmbH, potassium tetrachloropalladate(II) from Fluka and potas-sium hexachloropalladate(IV) from Sigma-Aldrich. All other chemicals were of analytical grade.

InstrumentationThe newly synthesized Pt(II), Pt(IV), Pd(II) and

Pd(IV) complexes with 3-amino-α-tetralonespiro-5’-hydantoin were characterized by elemental analysis, IR and 1H spectra. The carbon, nitrogen and hydrogen contents of the compounds were determined by elemen-tal analysis. The elemental analysis was carried out on a “EuroEA EuroVector apparatus. The IR spectra were recorded on Thermo Scientific Nicolet iS10 spectropho-tometer in the range of 4000-400 as pellets KBr and IFS 113 v Bruker FTIR spectrophotometer in the range of 400-150 cm-1 as polyethylene. The 1H NMR spectra were registered on a Bruker WM 250 (250 MHz) spectrometer in DMSO-d6. Corrected melting points were determined, using a Bushi 535 apparatus.

Synthetic procedureSynthesis of 3-amino-α-tetralonespiro-5’-hydantoin

To 2.16 g (0.01 mole) of α-tetralonespiro-5’-hydantoin was added 10 ml hydrazine hydrate (98%)[20]. The mixture was boiled with refluxing condenser for 3 hours. The resulting solution was placed in 20 ml cold water and the resulting precipitate was filtered off. The obtained compound was recrystallized from 50 %

ethanol, giving white crystals; m.p. 187-188ºC. Yield is 1.2 g (51 %).

Platinum complexesCis-[Pt(L)2Cl2]. Two solutions of K2[PtCl4] and of

the ligand (L) were prepared for the synthesis of the complex cis-[Pt(L)2Cl2] (1). The water-ethanol solution of L (0.1130 g, 0.4818 mmol) was added dropwise to the water solution of K2[PtCl4] (0.1000 g, 0.24096 mmol) at constant stirring at room temperature. After the addition of the ligand, the homogenous solution was stirred for 5-6 h. The solution was concentrated and cooled to 0°C. A light yellow product was obtained, which was filtered off, washed several times with ethyl ether and dried in a vacuum desiccator. The substance is soluble in DMSO. The purity is checked up by thin layer chromatography with the eluent CH3COOC2H5/C2H5OH - 2:1 and el-emental analysis. Yield: ca. 56%, m.p.: >238°C (dec.).

IR (KBr disc. and polythene): 3164, 1780, 1719, 1621, 557, 501, 434, 379;

1H NMR (DMSO-d6, δ, ppm): 9.11 (s, NH-1); 7.45-7.17 (m, 5H, bz); 8.82 (s, N-NH2); 2.85 (t, CH2-4, J=6Hz); 2.35-2.13 (m, CH2-2), 2.06-1.94 (m, CH2-3);

Anal. Calcd. (%) for [Pt(C12H13N3O2)2Cl2] C, 39.57; H, 3.57; N, 11.54. Found (%): C, 39.49; H, 4.22; N, 11.77.

Cis-[Pt(L)2Cl4]. The complex cis-[Pt(L)2Cl4](2) was prepared by a method given in literature [21]. Two solu-tions of the PtCl4 and of the 3-amino-α-tetralonespiro-5’-hydantoin were prepared. The water-ethanol solution of the ligand (0.1372 g, 0.5939 mmol) was added dropwise to the ethanol solution of PtCl4 (0.1000 g, 0.2969 mmol) with stirring magnetically for 5-6 h at 50°C. The solution was concentrated and cooled to 0°C.

A yellow product was obtained and filtered off, washed several times with ethyl ether and dried in a vacuum desiccator. The compound is soluble in DMSO, water and ethanol. The purity is checked up by thin layer chromatography with the eluent CH3COOC2H5/C2H5OH - 2:1 and elemental analysis. Yield: ca. 26%, m.p.: >209°C (dec.).

IR (KBr disc. and polythene): 3220, 1779, 1713, 1618, 562, 551, 351, 322;


Anal. Calcd. (%) for [Pt(C12H13N3O2)2Cl4] C, 36.05;


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H, 3.25; N, 10.52. Found (%): C, 36.39; H, 3.22; N, 10.77.

Palladium complexesCis-[Pd(L)2Cl2] Two solutions of K2[PdCl4] and of

3-amino-α-tetralonespiro-5’-hydantoin were prepared for the synthesis of the complex cis-[Pd(L)2Cl2] (3). The water-ethanol solution of the ligand (0.1415 g, 0.6126 mmol) was added dropwise to the water solution of K2[PdCl4] (0.1000 g, 0.3079 mmol) at constant stirring and after the addition of the ligand the homogenous so-lution was stirred for 1-2 hours. A light-yellow product was obtained, which was filtered off, washed several times with water and dried in a vacuum desiccator. The substance is soluble in DMSO and weakly soluble in water and ethanol. The purity is checked up by thin layer chromatography. Yield: ca. 66%, m.p.: > 222ºC (dec.).

IR (KBr disc. and polythene): 3226, 1782, 1727, 1627, 496, 471, 393, 348


Anal. Calcd. (%) for [Pd(C12H13N3O2)2Cl2] C, 42.65; H, 3.85; N, 12.44. Found (%): C, 42.69; H, 4.39; N, 12.43.

Cis-[Pd(L)2Cl4]. The complex cis-[Pd(L)2Cl4](4) was prepared by a method given in literature [21]. Two solutions of the K2[PdCl6] and of the 3-amino-α-tetralonespiro-5’-hydantoin were prepared. The water-ethanol solution of the ligand (0.113 g, 0.5035 mmol) was added dropwise to the water solution of K2[PdCl6] (0.1000 g, 0.2517 mmol) with stirring magnetically for 5-6 h at 50°C. The solution was concentrated and cooled to 0°C. A yellow product was obtained and filtered off, washed several times with ethyl ether and dried in a vacuum desiccator. The compound is soluble in DMSO, water and ethanol. The purity is checked up by thin layer chromatography with the eluent CH3COOC2H5/C2H5OH - 2:1 and elemental analysis. Yield: ca. 69%, m.p.: >210°C (dec.).

IR (KBr disc. and polythene): 3177, 1783, 1724, 1618, 510, 480, 342, 318

1H NMR (DMSO-d6, δ, ppm): 9.10 (s, NH-1); 7.32-7.10 (m, 5H, bz); 8.71 (s, N-NH2); 2.78(t, CH2-4, J=6Hz); 2.12-2.05 (m, CH2-2), 1.98-1.82 (m, CH2-3);

Anal. Calcd. (%) for [Pd(C12H13N3O2)2Cl4] C, 40.55; H, 3.66; N, 11.83. Found (%): C, 40.96; H, 4.04; N, 12.26.

Biological experimentsCell lines and culture conditions. The cytotoxic

activity of the tested platinum and palladium compounds was assessed against one human tumour cell line: SKW-3 (a KE-37 derivative) (human T-cell leukemia, established from peripheral blood of a 61-year-old man with T-cell lymphocytic leukemia).

Cytotoxicity assessment (MTT-dye reduction as-say). The cell viability was assessed using the standard MTT-dye reduction assay as described by Mosmann [22] with minor modifications. [23]. Method is based on the reduction of the yellow tetrazolium salt MTT to a violet formazan product via the mitochondrial succinate dehy-drogenase in viable cells. Aliquots of 100 μl/well cellular suspension (at a density of 1x105 exponentially growing cells/ml) were seeded in 96-well flat-bottomed micro-plates and after 24h incubation at 37°C were exposed to various concentrations of the tested compounds for 72 h. For each concentration at least 8 wells were used. After the incubation with the test compounds 10 μl MTT solution (10mg/ml in PBS) were added to each well and the microplates were further incubated for 4h at 37°C. Thereafter the formazan crystals formed were dissolved through addition of 100 μl/well 5 % formic acid solu-tion in 2-propanol. The MTT-formazan absorption was measured using an microprocessor-controlled ELISA reader (Labexim LMR-1) at 580 nm. Cell survival fractions were calculated as percentage of the untreated control. In addition IC50 values were calculated from the concentration-response curves. The experimental data were processed by means of GraphPad Prism software and were fitted to sigmoidal concentration-response curves via non-linear regression.

RESULTS AND DISCUSSION

Synthesis of the complex compoundsPt(II) (1), Pt(IV) (2), Pd(II) (3) and Pd(IV) (4)

complexes with 3-amino-α-teralonespiro-5’-hydantoin were prepared by using reported procedures with minor revisions[20, 21]. The obtained metal complexes were characterized by elemental analysis, IR and 1H NMR spectra.

IR spectraIn order to evaluate the mode of coordination of the

ligand to the metal ions, the IR and 1H NMR spectra of


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the pure ligand as well as of its metal complexes were recorded. The comparative analysis of the infrared spec-tra of the complexes 1–4 and of the metal-free ligand L revealed that the absorption bands characteristic for the stretching vibrations of NH2 group were blue shifted from 3237 сm-1 for the L to 3164, 3220, 3226 and 3177 cm–1 for the complexes 1–4, respectively. Absorption band characteristic for the δ(NH2) was blue shifted from 1600 cm–1 for the ligand L to 1621, 1618, 1627 and 1618 cm–1 for the complexes 1-4. This indicates that the amino group from hydantoins ring participates in the coordina-tion to the metal ions in all four complexes.

In the IR spectra of the complexes 1-4 the new bands at 562–471 cm-1 were assigned to the ν(M–N) stretch-ing vibrations which implied cis-location of the amino ligands according to Nakamoto[23]. In the IR spectra of the complexes two new bands in the low-energy region at 434–318 cm-1 were assigned to the ν(M–X) stretching vibrations. This implied cis-location of chloride ligands in the complexes according to Nakamoto[24].

The bands related to the stretching vibrations of the two carbonyl groups at 1768 cm-1 and 1706 cm–1 in the metal-free ligand did not shift upon coordination of L to M(II) and M(IV) indicating that the C=O groups were not involved in bounding to the metal.

1H NMR spectraThe comparative analysis of the 1H NMR spectra

of the complexes 1-4 and the free-metal ligand revealed that there was a big shifting of the signal of N-NH2 group(from 5.00 in the ligand to 8.7-8.8 ppm in the complexes). The signals of the other protons were not shifted extraordinary. This shows that the coordination of the ligand with metal ions was realized by nitrogen atom of the amino group.

On the base of the results from the physicochemical investigation, the following most probable molecular structures of the Pt(II) and Pd(II) complexes with 3-amino-α-teralonespiro-5’-hydantoin could be pro-posed (Fig. 1).

PharmacologyIn vitro cytotoxicity. The present study describes

evaluation of the cytotoxic effects of four newly synthesized platinum and palladium complexes with 3-amino-α-teralonespiro-5’-hydantoin vs. the referent antineoplastic agent cisplatin on SKW-3 human tumor cell line, using the standard MTT-dye reduction assay for cell viability. The concentration-response curves for SKW-3 summarized in Fig. 2. The IC50 values of the synthesized complexes and of the referent cisplatin

IC50 values (µM) Complex (1) (3) (4) Cisplatin Cell line SKW-3a 174.8 208.4 220.2 10.07 aT-cell leukemia

Table 1. Cytotoxicity of the Pt(II), Pd(II) and Pd(V) complexes with 3-amino-α-tetralonespiro-5’-hydantoin 1-4 in comparison to cisplatin in four human tumour cell lines

Fig. 1. Molecular structures of the investigated Pt(II), Pt(IV), Pd(II) and Pd(IV) complexes.

Fig. 2. Cytotoxic effects of the newly synthesized complexes (1), (3) and (4) and cisplatin against the human T-cell leukemia SKW-3.


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are summarized in Table 1. All tested compounds dis-played concentration dependent cytotoxic effects, which were much more manifested at micromolar concentra-tions. The IC50 values obtained were 174.8 mM for the complex (1), 208.4 mM for the complex (3) and 220.2 mM for the complex (4). The referent antineoplastic drug cisplatin induced strong inhibitory activity with IC50 value of 10.07mM. The results show that Pt(II) complex has higher cytotoxic activity than Pd(II) and Pd(IV) complexes. On the other side Pd(II) complex has higher antitumor activity than Pd(IV) complexes, which confirm the rule that Pt(II) and Pd(II) complexes have greater activity than corresponding Pt(IV) and Pd(IV) complexes.

CONCLUSIONS

Four Pt(II), Pt(IV), Pd(II) and Pd(IV) complexes with 3-amino-α-tetralonespiro-5’-hydantoin as carrier ligand were synthesized. The molecular formulae of the new Pt(II), Pt(IV), Pd(II) and Pd(IV) complexes were determined by elemental analysis, IR and 1H NMR spec-tra. In all compounds the mode of coordination of the ligand to the metal ion is one and the same – through the NH2 group. The complexes were studied for antitumor activity in vitro on SKW-3 human tumor cell line. The tested compounds exerted concentration-dependent cy-totoxic effects against the used tumor cell line. The Pt(II) complex with 3-amino-α-tetralonespiro-5’-hydantoin showed higher antitumor activity than the correspond-ing palladium complexes but all metal complexes have lower activity than the referent drug cisplatin.

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