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Platinum(IV) Complexes with Some Derivatives of 5-Methyl5-(4-pyridyl)Hydantoin. Synthesis, Study and Comparative Pharmacological Investigation DOI 10.1055/s-0033-1345109 Drug Res 2013; 63: 420–423 For personal use only. No commercial use, no depositing in repositories.

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420 Original Article

Platinum(IV) Complexes with Some Derivatives of 5-Methyl-5-(4-pyridyl)Hydantoin. Synthesis, Study and Comparative Pharmacological Investigation

Authors Affiliations

Key words ▶ Pt(IV) complexes ● ▶ Cytotoxic activity ● ▶ 3,5-disubstituted hydantoins ●

A. Bakalova1, R. Buyukliev1, Z. Ivanova1, G. Momekov2, D. Ivanov1 1 2

Department of Chemistry, Faculty of Pharmacy, Medical University of Sofia, Sofia, Bulgaria Department of Pharmacology, Faculty of Pharmacy, Pharmacotherapy and Toxicology, Sofia, Medical University of Sofia, Bulgaria

Abstract

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3 Pt(IV) complexes with 3-ethyl-5-methyl-5-(4-pyridyl)hydantoin (4), 3-propyl-5-methyl-5-(4-pyridyl) hydantoin (5) and 3-benzyl-5-methyl-5-(4-pyridyl) hydantoin (6) with general formulae cis-[Pt(L)2Cl4] were synthesized. The novel compounds were characterized by elemental analysis, IR, 1H, 13C, NMR spectra in solid state and in solution. The studies showed that the ligands coordinate to the platinum ions in a monodentate manner through the nitrogen atom from the pyridine ring. The cytotoxic activity in vitro of newly synthesized complexes as well

Introduction

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received 21.02.2013 accepted 29.03.2013 Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1345109 Published online: May 15, 2013 Drug Res 2013; 63: 420–423 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence Assoc. Prof. A. Bakalova Department of Chemistry Faculty of Pharmacy Medical University of Sofia 2 Dunav Strasse 1000 Sofia Bulgaria Tel.: + 359/2/923 6578 Fax: + 359/2/987 9874 [email protected]

Cisplatin is one of the most potent chemotherapy drug used for cancer treatment. Its discovery at 1969 was a corner stone which triggered the interest in platinum(II) and other metal-containing compounds as potential anticancer drugs [1]. Clinical success of cisplatin and its derivatives carboplatin, oxaliplatin, nedaplatin lobaplatin, heptaplatin determined considerable effort to develop other effective metal-based anticancer compounds [2–4]. However its use is limited due to the side toxic effects in normal tissues. One of the approaches to overcome the acquired resistance to cisplatin is to use Pt(IV) complexes. 4 Pt(IV) complexes were in clinical trials – tetraplatin, iproplatin, satraplatin and its adamantylamine analogue LA-12 [5]. It is believed, that Pt(IV) complexes acted as prodrugs via activation by reduction to their Pt(II) species [6]. Pt(IV) based drugs would have better activity and lower side effects when they are reduced in the cell [7]. The bonding of hydantoin molecules with transition metals are of great interest in the chemistry of coordination compounds [8–11]. Biological activity of metal complexes with hydantoin

Bakalova A et al. Pt(IV) Complexes of 5-Meth-5-(4-pyr)hyd … Drug Res 2013; 63: 420–423

as their previously prepared analogous of Pt(IV) with other derivatives like 3-amino-5-methyl-5(4-pyridyl)hydantoin (1), 5-methyl-5-(4-pyridyl) hydantoin (2), 3,5-dimethyl-5-(4-pyridyl)hydantoin (3) was screened against a panel of human tumor cell lines. The tested compounds displayed cytotoxic activity which was invariably superior with the Pt(IV) complex with 3-benzyl-5methyl-5-(4-pyridyl)hydantoin (6) causing 50 % inhibition of cellular viability at micromolar concentration, though the activity of the other studied Pt(IV) complexes proved to greatly decrease in the order 5-4-3-2-1.

derivatives has been widely studied as antitumoral and antibacterial activities [12]. Our aim was taking into account all previously mentioned properties of anticancer drugs to synthesize novel platinum(IV) complexes with 3,5-disubstituted hydantoin ligands that could be potent antitumor agents. The present study represents the synthesis, physicochemical evaluation and pharmacological investigation of Pt(IV) complexes with 3-ethyl-5-methyl-5-(4-pyridyl) hydantoin (4), 3-propyl-5-methyl-5-(4-pyridyl) hydantoin (5) and 3-benzyl-5-methyl-5-(4pyridyl)hydantoin (6) in comparison with previously synthesized and published Pt(IV) complexes with 3-amino-5-methyl-5-(4-pyridyl)hydantoin (1), 5-methyl-5-(4-pyridyl)hydantoin (2), 3,5dimethyl-5-(4-pyridyl)hydantoin (3) and clinically applied drug cisplatin [13–15]. General scheme of the investigated compounds is shown ▶ Fig. 1 in ●

Experimental

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Platinum(IV) chloride utilized for the synthetic procedures was purchased from Heraeus GmbH. All other chemicals were of analytical grade.

Original Article 421

agents for 72 h, whereby for each concentration a set of 8 separate wells was used. Every test was run in triplicate. 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 °C and the formazan crystals formed were dissolved by adding 110 μl of 5 % HCOOH in 2-propanol. The MTT-formazan absorption was measured using a multimode microplate reader (Beckman Coulter DTX880) and the results were normalized as percentage of the untreated control (set as 100 % viable). The data were fitted to sigmoidal dose-response curves and the IC50 values were calculated using non-linear regression analysis (Curve-fir; GraphPad Prizm software for PC).

Synthesis of the Pt(IV) complexes Preparation of cis-tetrachlorido-bis(3-ethyl-5-methyl-5(4-pyridyl)hydantoin)platinum(IV) – cis-[Pt(L4)2Cl4]

Fig. 1 Scheme of the complexes, objects of the recent study.

The newly synthesized Pt(IV) complexes (4), (5) and (6) were characterized by elemental analysis, IR, 1H, 13C NMR spectra. The carbon, nitrogen and hydrogen contents of the compounds were determined by elemental analysis. The elemental analysis was carried out on a EuroEA 3000 apparatus. The IR spectra were recorded on Thermo Scientific Nicolet iS10 spectrophotometer in the range of 4 000–400 as pellets KBr and IFS 113 v Bruker FTIR spectrophotometer in the range of 400– 150 cm-1 in polythene. The 1H and 13C 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. The following cell lines were used for the experiments: (i) SKW-3 or a KE-37 derivative (human T-cell leukemia, established from peripheral blood of a 61-year-old 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), (iiii) LAMA-84 (human chronic myeloid leukemia, established from peripheral blood of a 29-year-old woman with chronic myeloid leukemia). The cell culture flasks and the 96-well microplates were obtained from NUNCLON (Denmark). MTT, FCS and cisplatin were purchased from Sigma Co. The stock solutions of tested compounds (10 mM) were freshly prepared in DMSO. The serial dilutions of the tested compounds were prepared immediately before use. At the final dilutions obtained the concentrations of DMSO never exceeded 1 %. 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 [16] with some modifications [17]. Exponentially growing cells were seeded in 96-well microplates (100 μl/well at a density of 3.5 × 105 cells/ml for the adherent and 1 × 105 cells/ml for the suspension cell lines) and allowed to grow for 24 h prior the exposure to the studied compounds. Cells were exposed to the tested

The complex cis-[Pt(L4)2Cl4] (4) was prepared by a method given in literature [18]. Two water-ethanol solutions of the PtCl4 and of the 3-ethyl-5-methyl-5-(4-pyridyl)hydantoin were prepared. A water solution 6 ml of L4 (0.13 g, 0.6 mmol) was added dropwise to an ethanol solution 3 ml of PtCl4 (0.10 g, 0.3 mmol). The solution was stirred magnetically for 5–6 h at 50 °C, concentrated and cooled to 0 °C. A bright-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 was checked up by thin layer chromatography with the eluent CH3COOC2H5/C2H5OH – 2:1 and elemental analysis. Yield: ca. 39 %, m.p.: > 275 °C (dec.). IR (KBr disc. and polythene): 3 369, 3056, 1 789, 1 717, 1 621, 351, 322; 1 H NMR (DMSO-d6, δ, ppm): 9.05 (s, NH-1); 8.80 (d, 2H, H-2 + H-6); 7.85 (d, 2H, H-3 + H-5); 3.40 (q, 2H, J = 7 Hz, N-CH2); 1.67 (s, 3H, CH3-C-5); 1.06 (t, 3H, J = 7 Hz, N-CH2-CH3); 13C NMR (DMSO-d6, δ, ppm): 174.1 (C = O – 4’); 156.0 (C-2 + C-6); 153.6 (C = O – 2’); 150.0 (C- 4’); 123.0 (C-3 + C-5); 62.2 (C-5); 33.3 (N-CH2); 24.5 (CH3-C-5); 13.1 (N-CH2-CH3). Anal. Calcd. ( %) for [Pt(C11H13N3O2)2Cl4] C, 34.07; H, 3.36; N, 10.84. Found ( %): C, 33.40; H, 3.94; N, 10.70.

Preparation of cis-tetrachlorido-bis(3-propyl-5-methyl-5(4-pyridyl)hydantoin)platinum(IV)–cis-[Pt(L5)2Cl4].H2O The complex cis-[Pt(L5)2Cl4].H2O (5) was synthesized analogously to the procedure for complex (4). Yield: ca. 60 %, m.p.: > 213 °C (dec.). IR (KBr disc. and polythene): 3 358, 3 106, 1 783, 1 714, 1 618, 352, 323; 1H NMR (DMSO-d6, δ, ppm): 9.14 (s, NH-1); 8.83 (d, 2H, J = 7 Hz, H-2 + H-6); 7.87 (d, 2H, J = 7 Hz, H-3 + H-5); 3.35 (q, 2H, J = 7 Hz, N-CH2); 1.74 (s, 3H, CH3 – C-5); 1.53 (m), 2H, J = 7 Hz, N-CH2-CH2); 0.82 (t, 3H, J = 7 Hz, N-CH2CH2-CH3); 13C NMR (DMSO-d6, δ, ppm): 173.2 (C = O – 4’); 155.5 (C-2 + C-6), 154.7 (C = O – 2’); 151.6 (C-4’); 124.1 (C-3 + C-5); 62.4 (C-5); 38.8 (N-CH2); 25.3 (CH3 – C-5); 20.8 (N-CH2-CH2) 11.0 (N-CH2-CH2-CH3). Anal. Calcd. ( %) for [Pt(C12H15N3O2)2Cl4]: C, 35.09; H, 3.90; N, 10.23. Found ( %): C, 34.87; H, 4.19; N, 10.28:

Preparation of cis-tetrachlorido-bis(3-benzyl-5-methyl5-(4-pyridyl)hydantoin)platinum(IV) – cis-[Pt(L6)2Cl4]. 2H2O The complex cis-[Pt(L6)2Cl4].2H2O (6) was synthesized analogously to the procedure for complex (4). Yield 73.5 %, m.p.: 194– 196 °C(dec.). IR (KBr disc. and polythene): 3 352, 1 784, 1 718, 1 619, 350, 320; 1H NMR (DMSO-d6, δ, ppm): 9.30 (s, NH-1); 8.80 (d, 2H, H-2 + H-6); 7.86 (d, 2H, H-3 + H-5); 7.34–7.20 (m, 5H, C6H5); 4.55 (s, 2H, CH2); 1.75 (s, 3H, CH3); 13C NMR (DMSO-d6,


422 Original Article

δ, ppm): 173.1 (C = O – 4’); 155.3 (C-2 + C-6); 151.7 (C = O – 2’); 149.7–123.6 (N-CH2 – C6H5); 136.2 (C-4’); 128.7 (C-3 + C-5); 62.7 (C-5); 41.7 (N-CH2); 25.5 (CH3). Anal. Calcd. ( %) for [Pt(C16H15N3O2)2Cl4].2H2O: C, 41.08; H, 3.64; N, 8.99. Found ( %): C, 41.17; H, 3.54; N, 8.40.

Results and discussion

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Chemistry The ligands 3-ethyl-5-methyl-5-(4-pyridyl)-2,4-imidazolidenedione (L4), 3-propyl-5-methyl-5-(4-pyridyl)-2,4-imidazolidenedione (L5) and 3-benzyl-5-methyl-5-(4-pyridyl)-2,4-imidazolidenedione (L6) were prepared by previously published method [19]. Their Pt(IV) complexes were synthesized using reported procedure with minor revisions [20]. On the basis of the data from the elemental analysis for the new complexes the following formulae can be derived: [Pt(C11H13N3O 2)2Cl4] (4), [Pt(C12H15N3O2)2Cl4].H2O (5), [Pt(C16H15N3O2)2Cl4].H2O (6). In order to evaluate the mode of coordination of the ligand to the metal ion, IR, 1H and 13C NMR spectra of the free ligands as well as of theirs Pt(IV) complexes were recorded.

IR spectra The comparative analysis of the infrared spectra of the complexes 4–6 and of the metal-free ligands L4, L5, and L6 revealed that the absorption bands characteristic for the stretching vibrations of pyridine C = N group were blue shifted from 1 600 сm − 1 for the ligands L4–L6 to 1621, 1618, 1619 cm − 1 for the compounds 4–6 respectively. This indicates that the pyridine nitrogen atom participates in the coordination with platinum ion in all novel platinum(IV) complexes. The other characteristic bands of the pyridine ring of the metalfree ligand are blue-shifted upon complexation as well, providing further evidence for the coordination through the pyridine N atom. In the IR spectra of the complexes 4–6 new 2 bands at 352–322 cm − 1 for the ν(Pt–Cl) stretching vibrations were observed, implying cis-location of chloride ligands [21]. The bands related to the stretching vibrations of the 2 carbonyl groups in the metal-free ligands did not shift upon coordination of L4, L5 and L6 to platinum(IV) ions, indicating that the C = O groups were not involved in binding to the metals.

NMR spectra In the 1H NMR spectra of compounds 4–6 the signals of the protons for Н-2 and Н-6 from the pyridine ring were shifted from 8.61 ppm in the spectra of the ligands L4–L6 to 8.80, 8.83 and 8.80 ppm in the spectra of the complexes 4–6. The differences between the chemical shifts of the protons of the ligands L4–L6 and those of the corresponding complexes are noticeable at 0.19, 0.22 and 0.19 ppm for complexes 4–6. The signals of the Н-3 and Н-5 protons from the pyridine ring were shifted from 7.48 ppm in the ligands L4–L6 to 7.85, 7.87 and 7.86 ppm in the complexes 1–6 respectively. The differences between the chemical shifts of the protons of the ligands L4–L6 and those of the corresponding complexes are noticeable at 0.37, 0.39 and 0.38 ppm for complexes 4–6. All signals for the protons in the pyridine ring were shifted, as H-3 and H-5 were shifted more than H-2 and H-6 due to the conjugation in the pyridine ring. This shows that the most probable bounding of the ligands with the platinum ions is realized through the nitrogen atom from the pyridine ring.

In the 13C NMR spectra of compounds 4–6 the signals for the carbon atoms C-2 and С-6 from the pyridine ring were found at 151.5, 151.6 and 151.7 ppm, respectively, compared to 150.0 ppm for the metal-free ligands L4–L6. The downfield chemical shifts in the range 1.5 to 1.7 ppm indicate that the pyridine nitrogen atom partakes in the coordination to the metal ions. The shifts of the С-3 and С-5 were smaller in accord with their remote positions from the binding nitrogen atom. The resonances of the 2 С = О groups of the hydantoin ring in metal-free-ligands L4, L5 and L6 and in the complexes 4–6 were found at the same chemical shifts. This finding is an indication that the hydantoin ring is not involved in the coordination with the metal ion.

Pharmacology In vitro cytotoxicity The present study describes a comparative evaluation of the cytotoxic effects of three newly synthesized Pt(IV) complexes and three previously synthesized and studied Pt(IV) complexes with other 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. The tested compounds displayed cytotoxic effects in a concentration dependent manner. The nature of the ligands greatly influenced the relative potency of the complexes, whereby superior activity within the series was established with the Pt(IV) complex with 3-benzyl-5-methyl-5-(4-pyridyl)hydantoin, causing 50 % inhibition of cellular viability at micromolar concentration, whereas the activity of the other complexes proved to greatly decrease in the order 5-4-3, while complexes (2) and (1) ▶ Table 1). were far less active (●

Cytotoxicity of cis-[Pt(L5)2Cl4] after co-administration of a biological reducing agent Based on the well appreciated notion that the Pt(IV) complexes are inactive prodrugs whose cytotoxicity is due to conversion to the corresponding Pt(II) species we thought to determine the cytotoxicity of complex (5) in the presence of the ubiquitous biological reducing agent ascorbic acid. To meet this objective SKW-3, HL-60, EJ and LAMA-84 cells were treated for 72 h with complex (5) at 25, 50, 100 or 200 μM alone or in combination with ascorbic acid. The biological reducing agent was applied at non-cytotoxic concentrations – 12.5 or 50 μM. As prove of the principle the established IC50 values indicate that, when coadministered to cells with sub-cytotoxic concentrations of Table 1 Cytotoxicity of all studied Pt(IV) complexes 1–6 cisplatin in four human tumour cell lines.

in comparison to

IC50 values(μM) Cell line

SKW-3a

HL-60b

EJc

Complex 1 2 3 4 5 5 + Vit. C 6 Cisplatin

> 200 > 200 > 200 183.1 44.2 4.2 33.4 11.4

> 200 > 200 131.4 87.2 64.2 31.7 22.5 8.7

> 200 > 200 187.2 152.0 95.8 38.2 42.1 10.2q

a

LAMA-84d > 200 > 200 > 200 140.8 74.2 55.4 44.5 16.9

T-cell leukemia; b acute myeloid leukemia, c urinary bladder carcinoma, d human

chronic myeloid leukemia;


Original Article 423

ascorbic acid the cytotoxicity of the tested compound was greatly augmented, compared to its sole administration.

Conclusion

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3 new Pt(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 as carrier ligands and chloride anions as leaving groups were synthesized. The molecular formulae of the complexes were confirmed by elemental and spectral analysis as IR, 1H-, 13C spectra in solid state and in solution. The different substituted ligands significantly influenced the cytotoxicity of the complexes against the tested cell lines, which was invariably superior with the Pt(IV) complex, with 3-benzyl-5-methyl-5-(4-pyridyl)hydantoin, which caused 50 % inhibition of cellular viability at low micromolar concentrations.

Acknowledgements

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This work was supported by a grant from the Medicinal Science Council at the Medical University of Sofia, Bulgaria.

Conflict of Interest

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The author states no conflict of interest.

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