Pt(II) and Pd(II) derivatives of ter-butylsarcosinedithiocarbamate

Journal of Inorganic Biochemistry 93 (2003) 181–189 www.elsevier.com / locate / jinorgbio

Pt(II) and Pd(II) derivatives of ter-butylsarcosinedithiocarbamate Synthesis, chemical and biological characterization and in vitro nephrotoxicity a, a a b c d e D. Fregona *, L. Giovagnini , L. Ronconi , C. Marzano , A. Trevisan , S. Sitran , B. Biondi , F. Bordin b a

University Department of Inorganic, Metallorganic and Analytical Chemistry, via Loredan 4, 35131 Padova, Italy b University Department of Pharmaceutical Sciences, via Marzolo 5, 35131 Padova, Italy c University Department of Environmental Medicine and Public Health, via Giustiniani 2, 35128 Padova, Italy d Institute of Chemistry and Inorganic Technologies and Advanced Materials, C.N.R., Research Area, Corso Stati Uniti 4, 35127 Padova, Italy e Biopolymers Research Centre, C.N.R., via Marzolo 1, 35131 Padova, Italy Received 6 June 2002; received in revised form 5 September 2002; accepted 23 September 2002

Abstract This work reports on the synthesis, characterization and biological activity of new coordination compounds of the type [M(TSDTM)X 2 ] (M5Pt(II), Pd(II); X5Cl, Br; TSDTM5ter-butylsarcosine(S-methyl)dithiocarbamate) and [Pd(TSDT)X] n (TSDT5terbutylsarcosinedithiocarbamate) in order to study their behavior as potential antitumor agents. All the synthesized compounds were characterized by means of elemental analysis, FT-IR, 1 H and 13 C-NMR spectroscopy and thermogravimetric analysis, suggesting a chelate S,S9 structure of the TSDTM / TSDT ligand in a square-planar geometry. Finally, the synthesized complexes have been tested for in vitro cytotoxic activity against human leukemic HL60 and adenocarcinoma HeLa cells; the most active compound [Pt(TSDTM)Br 2 ], characterized by IC 50 values very similar to those of the reference compound (cisplatin), was also tested for in vitro nephrotoxicity showing a very low renal cytotoxicity as compared to cisplatin itself.  2002 Elsevier Science Inc. All rights reserved. Keywords: Platinum(II) complexes; Palladium(II) complexes; Cytotoxic activity; Nephrotoxicity

1. Introduction Most of the major classes of pharmaceutical agents contain examples of metal compounds which are in current clinical use [1] and new areas of application are rapidly emerging. Platinum(II) complexes are now amongst the most widely used drugs for the treatment of cancer; four injectable Pt(II) diamine compounds (cisplatin, carboplatin, nedaplatin and oxalilplatin) have been approved for clinical use and several others cis-diamine complexes are in clinical trials, including an oral Pt(IV) complex [2]. Today, cisplatin is one of most effective drugs used for the treatment of testicular, ovarian, small cell lung, bladder, cervical and head and neck carcinomas [3]. Despite its *Corresponding author. Tel.: 139-49-827-5159; Fax: 139-2700-500560. E-mail address: [email protected] (D. Fregona).

high effectiveness, there are some clinical problems related to the use of cisplatin in the curative therapy, such as severe normal tissue toxicity and the frequent occurrence of initial and acquired resistance to the treatment [4]. An important side effect of cisplatin is nephrotoxicity, which results from injury to renal tubular epithelial cells and can be manifested as either acute renal failure or a chronic syndrome characterized by renal electrolyte wasting [5]. These renal adverse effects are correlated to platinum binding and inactivation of thiol-containing enzymes [6]. Continuous efforts are still being made to reduce the toxicity of platinum anticancer complexes toward normal cells, circumventing acquired resistance to cisplatin and decreasing its nephrotoxicity [7]. The interest in the chemical and biochemical properties of platinum and palladium complexes with thiocarbonyl donors stems from the use of sulfur ligands as detoxicant agents against metal-containing drugs [8–12]. For a long time, dithiocarbamates have been evaluated for their

0162-0134 / 02 / $ – see front matter  2002 Elsevier Science Inc. All rights reserved. PII: S0162-0134( 02 )00571-8

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efficacy as inhibitors of cisplatin induced nephrotoxicity [13]; they were shown to protect against cisplatin renal injure in several animal models by reversing cellular damage. In particular, dithiocarbamates selectively remove platinum from the enzyme-thiol complexes by nucleophilic attack of the chelating sulfur atoms to the platinum moiety [6]; moreover, they selectively protect normal tissue without inhibiting the antitumor effect [6,14]. In order to obtain compounds with superior chemotherapeutic index in terms of increased bio-availability, higher cytotoxicity and lower side effects than cisplatin, we recently synthesized a new class of platinum(II) and palladium(II) mixed complexes containing dithiocarbamate and amino ligands of the type [M(ESDT)(L)Cl] (M5 Pt(II), Pd(II); ESDT5ethylsarcosinedithiocarbamate; L5 pyridine, n-propylamine, cyclobutylamine, ethylendiamine). These complexes were characterized for their chemical properties and evaluated for in vitro cytotoxicity against human tumor cell lines [15]. In particular the [Pt(ESDT)(Py)Cl] complex has been found to be more effective as anti-proliferative agent than cisplatin towards several cellular lines and especially towards the cisplatinsensitive human ovarian 2008 and the isogenic resistant C13* cell lines. Toxicity tests on the kidney were also performed by means of a renal cortical slices model: this platinum(II) complex showed a very low renal cytotoxicity compared to cisplatin; in particular, lipid peroxidation induced by cisplatin appeared to be about five times higher than that induced by [Pt(ESDT)(Py)Cl] [16]. These encouraging results induced us to extend the study to Pt(II) and Pd(II) complexes with the TSDTM ligand that presents a ter-butyl ester group with a greater steric hindrance compared to ESDT ligand. This was made in order to investigate the chemical properties and the cytotoxic activity against some tumor cell lines. In this paper we report on the chemical characterization of some complexes of the type [M(TSDTM)X 2 ] (M5Pt(II), Pd(II); X5Cl, Br; TSDTM5ter-butylsarcosine(S-methyl)dithiocarbamate), carried out by means of elemental analysis, 1 H-NMR and FT-IR spectroscopy and thermogravimetric analysis. Finally, the synthesized complexes have been tested for in vitro cytotoxic activity against human leukemic HL60 and adenocarcinoma HeLa cells; moreover, the most effective compound, [Pt(TSDTM)Br 2 ], has been tested for in vitro nephrotoxicity by means of a renal cortical slices model.

2. Experimental

2.1. Measurements Melting points were determined on an Electrothermal IA9300 instrument. FT-IR spectra were recorded in nujol between two polyethylene tablets on a Nicolet Vacuum Far FT-IR 20F

spectrophotometer for the range (50–600) cm 21 , and in solid KBr on a Nicolet FT-IR 55XC spectrophotometer for the range (400–4000) cm 21 . 1 H-NMR spectra were recorded in CDCl 3 and DMSOd 6 on a Bruker Avance DRX400 spectrometer, equipped with a Silicon Graphics O2 workstation operating in Fourier transform, using TMS as internal standard. Elemental analysis were performed by Laboratorio di microanalisi, University Department of Inorganic, Metallorganic and Analytical Chemistry, Padova, Italy, with a Carlo Erba 1108 CHNS-O microanalyzer. The thermogravimetric and thermodifferential curves were obtained using a Netzsch STA429 thermoanalyzer. The measurements were carried out in the range (35– 1200) 8C in alumina crucibles under air (flux rate 30 cm 3 min 21 ) and at a heating rate of 5 8C min 21 , using alumina as reference. The UV-Vis spectra were recorded on a double beam Perkin-Elmer Lambda 15 spectrometer.

2.2. Materials Sarcosine ter-butyl ester hydrochloride, carbon disulfide, chloroform-d (99.8% D atoms), DMSO-d 6 (99.9% D atoms), n-pentane, acetonitrile and dichloromethane were purchased by Aldrich (Milano, Italy); platinum(II) chloride and bromide and palladium(II) chloride and bromide are Johnson Matthey products (Milano, Italy); ethanol, benzene, methyliodide and sodium hydroxide were supplied by Carlo Erba (Rodano (MI), Italy). Cisplatin and DMSO were supplied by Fluka (Buchs, Switzerland); L-glutamic acid monosodium salt, nutrient mixture F12 Ham medium and trypan blue were supplied by Sigma Chemical Co. (St. Louis, USA); fetal calf serum was a Biochrom-Seromed GmbH and Co. (Berlin, Germany); RPMI-1640 medium was supplied by Whitiaker Bioproduct (Walkersville, USA); trypsin was supplied by Boehringer (Manheim, Germany).

2.3. Preparation of the TSDTM ligand TSDTM ligand was prepared by dropwise addition of CS 2 (2.7310 22 mol) to an ethanol solution of sarcosine ter-butyl ester hydrochloride (5.5310 22 mol) under stirring. Once the CS 2 was dissolved, an aqueous solution of 22 NaOH (5.5310 mol) was added, the solution turning from clear yellow into darken brown with the precipitation of a white solid (NaCl) that was filtered off. After one h stirring, the addition of CH 3 I (2.7310 22 mol) yielded to a red oil that was re-solubilized in ethanol and still left stirring for another hour; the solution thus obtained was joined of water to incipient precipitation and, after one night at 5 8C, a white solid was obtained. A further fraction of TSDTM was obtained by treating the mother solution with CS 2 (1.4310 22 mol), NaOH (2.7310 22 mol) and

D. Fregona et al. / Journal of Inorganic Biochemistry 93 (2003) 181–189

CH 3 I (1.4310 22 mol) following the same procedure. Re-crystallization from EtOH / H 2 O yielded a white solid (84% final yield). C 9 H 17 NO 2 S 2 : calcd. C 45.93, H 7.28, N 5.95, S 27.24; found C 45.70, H 7.29, N 5.94, S 26.58. M.p. 131–132 8C.

2.4. Preparation of the complexes The [M(TSDTM)X 2 ] complexes (M5Pt(II), Pd(II); X5Br, Cl) were isolated from a benzene solution of the corresponding metal halides and the ligand in 1:1 molar ratio. For example, [Pt(TSDTM)Cl 2 ] was obtained by stirring a suspension of PtCl 2 (3.0310 23 mol) in a TSDTM (3.0310 23 mol) benzene solution, in the dark, for 2 days. The initially formed deep red solution, probably containing the 1:2 (metal:ligand) complex, slowly reacted with the residual PtCl 2 to yield an orange solid. A small amount of acetonitrile was necessary for the reaction proceeding. The orange solid thus obtained was filtered off, washed with n-pentane and dried in vacuo (yield5 90%). The reaction between palladium(II) and platinum(II) dihalides and TSDTM in 1:2 molar ratio in dichloromethane or benzene was not quantitative, the final product being a mixture of the 1:2 adducts (main product) and demethylated species such as [M(TSDT) 2 ] and [M(TSDT)X] n . The presence of these side products was established by means of FT-IR and 1 H-NMR spectroscopy. The pure [Pd(TSDT)X] n was obtained from the mother solution of the [Pd(TSDTM)X 2 ] reaction on standing. Results from the chemical analysis for all the synthesized compounds complexes are summarized below. [Pt(TSDTM)Cl 2 ]: orange yellow solid (93% final yield). C 9 H 17 Cl 2 NO 2 PtS 2 : calcd. C 21.55, H 3.41, N 2.79, S 12.78; found C 21.11, H 3.05, N 2.79, S 12.71. [Pt(TSDTM)Br 2 ]: dark orange solid (69% final yield). C 9 H 17 Br 2 NO 2 PtS 2 : calcd. C 18.21, H 2.89, N 2.36, S 10.84; found C 18.19, H 2.51, N 2.28, S 9.57. [Pd(TSDTM)Cl 2 ]: pink solid (91% final yield). C 9 H 17 Cl 2 NO 2 PdS 2 : calcd. C 26.19, H 4.15, N 3.39, S 15.54; found C 26.02, H 4.40, N 3.56, S 15.51. [Pd(TSDTM)Br 2 ]: orange solid (91% final yield). C 9 H 17 Br 2 NO 2 PdS 2 : calcd. C 22.55, H 3.42, N 2.80, S 12.78; found C 22.25, H 3.20, N 2.84, S 13.10. [Pd(TSDT)Cl] n : orange yellow solid (55% final yield). C 8 H 14 ClNO 2 PdS 2 : calcd. C 26.59, H 3.86, N 3.87, S 17.68; found C 26.14, H 3.67, N 3.91, S 17.83. [Pd(TSDT)Br] n : orange solid (57% final yield). C 8 H 14 BrNO 2 PdS 2 : calcd. C 23.61, H 3.68, N 3.44, S 15.74; found C 23.21, H 3.21, N 3.24, S 15.23.

2.5. Cytotoxicity assays Compounds were dissolved in DMSO (4.5 mM) just before the experiments; a calculated amount of the drug solution was added to the growth medium to a final solvent

183

concentration of 0.5%, which had no discernible effect on cell viability. HeLa cells (kindly provided by Prof. F. Majone, University Department of Biology, Padova, Italy) were grown as monolayers in nutrient mixture F12 Ham medium supplemented with 10% fetal calf serum. HL60 cells were grown in RPMI-1640 medium containing 5% fetal calf serum, supplemented with 25 mM HEPES buffer and L-glutamine. Both HeLa and HL60 media were supplemented with antibiotics penicillin (50 units ml 21 ) and streptomycin (50 mg ml 21 ) and cell growth was accomplished at 37 8C in a 5% carbon dioxide atmosphere. Cytotoxicity against human leukemic HL60 cells was studied using the trypan blue dye exclusion test [39]. Cells at a concentration of 2310 5 ml 21 were incubated for 24 h in the presence of different concentrations of the tested compounds. Cells were then incubated for 4 min with 0.25% trypan blue. Viable cells were identified by their ability to exclude dye, whereas the dye diffuses into non-viable cells. At least 100 cells were counted for each experimental point recorded. Cytotoxicity on the HeLa cells was evaluated by means of MTT (tetrazolium salts reduction) test [40,41]. Briefly, HeLa cells (5310 4 cells ml 21 ) were seeded in 96-well microplates in growth medium (100 ml) and then incubated at 37 8C in a 5% carbon dioxide atmosphere. After 24 h, the medium was removed and replaced with a fresh one containing the compound to be studied at the appropriate concentrations (12.5–25–50 mM). Quadruplicate cultures were established for each treatment. Twenty four h later, each well was treated with 10 ml of a 5 mg ml 21 MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) saline solution, and after 5 h of incubation, 100 ml of a sodium dodecylsulfate (SDS) solution in HCl 0.1 M was added. After an overnight incubation, the inhibition of cell growth induced by the various complexes was detected by measuring the absorbance of each well at 570 nm using a Camberra-Packard microplate reader. For comparison purposes, the cytotoxicity of cisplatin was evaluated under the same experimental conditions.

2.6. In vitro toxicity studies Young (200610 g) naive male Wistar rats (Harlan, Italy) were sacrificed and kidneys were quickly removed and placed in cold saline. Renal cortical slices (100610 mg wet tissue, thickness 250 mm) were prepared with a Brendel-Vitron slicer and placed in a medium composed by 97 mM NaCl, 40 mM KCl and 0.74 mM CaCl 2 in sodium phosphate buffer 7.4 mM (pH57.4), until all slices could be prepared to rinse free of blood and enzymes released from damaged cells during the slicing process. After preparation, the slices were transferred into 25 ml Erlenmeyer flasks containing 4 ml of incubation medium, treated with 10 ml of [Pt(TSDTM)Br 2 ] or cisplatin (0.125–

D. Fregona et al. / Journal of Inorganic Biochemistry 93 (2003) 181–189

184

Fig. 1. Isomeric forms of TSDTM ligand.

5.0310 24 M, final concentration) dissolved in DMSO added with a 10 ml Hamilton microsyringe. Control slices were treated with DMSO only. The flasks were stoppered, gassed for 5 min with 100% oxygen and incubated at 37 8C for 90 min in a Dubnoff metabolic shaker (100 cycles min 21 ). At the end of the incubation period, the incubation medium was processed to measure malondialdehyde (MDA) according to Younes and Siegers [17] as modified by Kornbrust and Bus [18] as expression of lipid peroxidation. The values were expressed as mmol 100 mg 21 of tissue.

3. Results and discussion

3.1. Characterization of the TSDTM ligand The dithiocarbamates are a large family of compounds presenting a partial double bond character between the carbon and the nitrogen atoms, producing an energetic rotation barrier [19–21] and forcing the molecule into a planar configuration (Fig. 1). The presence of substituents to the nitrogen atom introduces, in the two isomeric forms, different chemical environments and, subsequently, different interactions with the other groups [22]. The 1 H-NMR spectrum in CDCl 3 of TSDTM shows a signal for the ter-butyl group (1.47 ppm), a signal for the S–CH 3 (2.65 ppm) and two singlets for the N–CH 3 (3.56 and 3.41 ppm) and the N–CH 2 (4.68 and 4.39 ppm) as reported in Table 1. The greater signals (66%) are attributed to the isomeric form E and the smaller ones (34%) to isomeric form Z. In fact, it is known that the N–CH 3 group in cis with respect

to the C=S has its signal at lower fields than that corresponding in trans position [23]. The singlet of the S–CH 3 demonstrates that in both the isomeric forms, there is no magnetic interaction between these protons and the near groups. The study of the near IR region (4000–400 cm 21 ) (Table 2) allows us to individualize the characteristic absorption bands of the ligand. We can see the asymmetrical stretching of the carboxylic group at 1734 cm 21 and the asymmetrical stretching O–C–C and C–N at 1099 cm 21 and 1480 cm 21 respectively [24,25]. The 1000–800 cm 21 region held up the C–S stretching of the two not-equivalent sulfur atoms because of the presence of the methyl group (995 and 958 cm 21 ) [26]. The signal at 839 cm 21 is assignable to the vibration of the SCS group and the band at 747 cm 21 is assigned to the S–CH 3 stretching. The electronic spectra of the ligand in the 190–700 nm region were performed in different solvents (benzene, ethanol or chloroform) at different concentrations (2.03 10 26 –2.0310 24 M). In 2.0310 24 M benzene solution, the spectrum shows a band of strong intensity at 277.8 nm due to the electronic transition p → p * localized on the NCS group and a weak band at 346 nm due to the p → p * transition localized on the SCS group [27].

3.2. Characterization of the complexes For reaction of the ligand with the MX 2 (M5Pt(II), Pd(II); X5Cl, Br) species in different molar ratio we obtained the S-methylated 1:1 complexes of the type [M(TSDTM)X 2 ] and the demethylated specie [Pd(TSDT)X] n . We were not able to obtain the pure [Pt(TSDT)X] n demethylated species and the complexes in 1:2 metal to TSDTM ratio, the product being always a mixture of the 1:2 adducts and demethylated species. FT-IR spectroscopy has been used mainly to understand the geometry of the molecule by hunting out the bands that belong to the different types of metal-ligand coordination. The CN stretching mode is at 1480 cm 21 in the free

Table 1 1 H-NMR data (CDCl 3 , 298.15 K, ppm)a Compound

(CH 3 ) 3 (s)

CH 2 –N(s)

CH 3 –N(s)

S–CH 3 (s)

TSDTM

1.47 1.50 / 1.51

3.41 (66%) 3.56 (66%) 3.43 / 3.63

2.65

[Pt(TSDTM)Cl 2 ] [Pt(TSDTM)Br 2 ]

1.50 / 1.52

3.29 / 2.58

3.10 7 3.20

[Pd(TSDTM)Cl 2 ]

1.49 / 1.51

3.56 / 3.62

2.98 / 3.11

[Pd(TSDT)Cl] n [Pd(TSDTM)Br 2 ]

1.48 (br) 1.50 / 1.51

3.25 (br) 3.57 / 3.63

– 2.99 / 3.15

[Pd(TSDT)Br] n

1.45

4.39 (34%) 4.68 (34%) 4.74 / 4.71 / 4.17 / 4.13 / 5.11 / 5.07 / 4.37 / 4.33 4.76 / 4.72 / 4.11 / 4.07 / 5.09 / 5.05 / 4.37 / 4.32 4.33 / 4.37 / 5.06 / 5.11 / 4.73 / 4.68 / 4.67 / 4.63 4.17 4.29 / 4.31 / 4.42 / 4.45 / 4.76 / 4.80 / 5.04 / 5.09 4.24

3.20

–

a

s5singlet; br5broad.

3.09 / 3.22

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185

Table 2 Selected IR frequencies (cm 21 )a Compound

n (C=O)

n (C–N)

n (O–C–C)

n (C–S)

n (SCS)

n (SCH 3 )

n (M–S)

n (M–X)

TSDTM

1734 s

1480 s

1099 m

839 m

747 w

–

–

[Pt(TSDTM)Cl 2 ]

1741 s

1574 s

1101 m

836 mw

747 w

377 m

[Pt(TSDTM)Br 2 ]

1740 s

1576 s

1105 m

838 mw

748 w

373 m

[Pd(TSDTM)Cl 2 ]

1742 s

1576 s

1102 m

835 mw

747 w

366 m

[Pd(TSDTM)Br 2 ]

1740 s

1569 s

1099 m

835 mw

747 w

379 m

[Pd(TSDT)Cl] n [Pd(TSDT)Br] n

1742 s 1739 s

1539 s 1532 s

1104 m 1101 m

995 m 958 m 984 br 900 w 969 w 910 w 984 wbr 903 w 987 wvbr 900 w 900 w 898 w

834 mw 833 w

– –

354 w 370 w

325 s (as) 317 s (s) 202 s (as) 194 s (s) 324 s (as) 300 m (s) 202 m (as) 193 m (s) 291 s 200 s

a

s5strong; m5medium; w5weak; br5broad; v5very; (s) /(as)5symmetric / asymmetric.

ligand, while in the complexes showing a chelate S–S metal coordination it shifts to higher energy of about 100 cm 21 (about 1575 and 1570 cm 21 for Pt and Pd complexes respectively) (Table 2) with an increase of the double bond character on coordination. In the case of a singular coordination with the thiocarbonyl sulfur there should be a shift of |20 cm 21 only [28–32]. Further evidence of dithiocarbamate-metal coordination type comes out from the bands in the 1000–800 cm 21 region characteristic of the different CS 2 group vibrations. The presence of three bands identifies a S–S(CH 3 ) chelate coordination [33,34]: the C=S(1) stretching is near 1000 cm 21 ; the second band, corresponding to the C–S(2)–C stretching is at about 900 cm 21 and the third band assignable to the SCS vibration is at about 830 cm 21 . If the two sulfur atoms are symmetrical (the absence of the S–CH 3 group in our case) the first band is not present (Fig. 2). In the 600–50 cm 21 region the complexes show two M–X bands at 325, 317 cm 21 and 324, 300 cm 21 for the platinum and palladium chloro complexes respectively and at 202, 194 cm 21 and 202, 193 cm 21 for the bromo complexes, as expected for a cis structure [35,36]. The 1 H-NMR data are reported in Table 1. The S–CH 3 group makes the N–CH 2 protons non equivalent and splits the signals into two doublets for each isomer. The 1 HNMR spectra in CDCl 3 performed at different time periods, show that only one isomeric form is present in the [Pt(TSDTM)Cl 2 ] species obtained from benzene solution: in fact, in the starting solution only one signal is present for the S–CH 3 (3.22 ppm) and N–CH 3 (3.63 ppm) groups,

Fig. 2. Symmetrical and asymmetrical coordination of the dithiocarbamate moiety.

while two doublets for the N–CH 2 protons (4.17, 4.13, 4.74, 4.71) are present. After 24 h, both isomeric forms are present in the same amount (Fig. 3). To check the solvent role in the isomerization reaction of the [Pt(TSDTM)Cl 2 ], the complex was dissolved in chlorinated solvents (CH 2 Cl 2 , CHCl 3 ) and dried under reduced pressure; the 1 H-NMR spectra immediately performed in CDCl 3 , contain the signals of both isomeric species. Moreover, the FT-IR spectra after dissolution show a broad band for CN group shifted to lower energy with respect to the species obtained from benzene solution, an absorption split into two signals for the n S–CH 3 group (749, 736 cm 21 ) and the broadening of the n CS at 900 and 984 cm 21 . The 1 H-NMR in CDCl 3 of the [Pd(TSDTM)Cl 2 ] complex, ab initio shows the presence of two isomeric forms with two signals for the S–CH 3 groups; for the N–CH 2 there is a double doublet for the two non equivalent protons of one of the isomeric forms and a broad signal (4.68 ppm) originated from an AB system for the other isomer (Fig. 4). On the other hand, the FT-IR spectrum of a crude solid product obtained from benzene solution is very similar to the platinum analogue derived from the same solvent in one isomeric form, while the FT-IR of the sample resolubilized in dichloromethane, shows the broadening n CN band shifted at 1569 cm 21 , the n S–CH 3 split into two bands (746 and 734 cm 21 ) and the n CS at 984 and 903 cm 21 are broader than those of the starting product. These IR and 1 H-NMR data suggest that the isomerization in chlorinated solvents is very fast. The 1 H-NMR of the [Pd(TSDTM)Br 2 ] performed at different time, shows a slow demethylation in solution with the disappearance of the S–CH 3 signal of the methylated species and the appearance of a singlet for N–CH 3 and N–CH 2 in agreement with formation of [Pd(TSDT)Br] n species. Furthermore, a new signal at 2.66 ppm, well ascribed to CH 3 Br volatile species, appears. The proton NMR spectra in DMSO-d 6 , was periodically recorded to check the stability of the complexes and show

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186

Fig. 3. 1 H-NMR spectra of [Pt(TSDTM)Cl 2 ] in CDCl 3 performed in the time: immediately (a); after 30 min (b); after 5 h (c); after 24 h (d).

Fig. 4. 1 H-NMR spectrum of the complex [Pd(TSDTM)Cl 2 ] in CDCl 3 .

that in this solvent, two isomers are always present for all the complexes, except for [Pd(TSDT)Cl] n that is unstable in DMSO. The thermal behavior of the synthesized compounds has been studied to confirm the proposed stoichiometry and to

support the conclusions reached upon application of the spectroscopic techniques [37,38]. The results of such analyses have been summarized in Table 3; a good correlation exists between calculated and found values. The thermogravimetric curves of the [Pt(TSDTM)Cl 2 ]

Table 3 Thermal data a Compound

[Pt(TSDTM)Cl 2 ] [Pt(TSDTM)Br 2 ] [Pd(TSDTM)Cl 2 ] [Pd(TSDTM)Br 2 ] a

Decomposition range (8C) 190–350 127–209 127–470 65–145 65–850 110–161 110–850

exo5exothermic; endo5endothermic.

Weigh loss (%) Exp.

Calcd.

61.1 25.2 65.6 27.5 74.0 24.7 78.2

60.5 (To Pt) 25.9 (–CH 3 Br; –C 6 H 6 ) 66.8 (To Pt) 26.2 (–CH 3 Cl; –C 6 H 6 ) 74.9 (To Pd) 25.9 (–CH 3 Br; –C 6 H 6 ) 78.8 (To Pd)

DTA peak temperature (8C) 319 endo / 354 exo 209 endo / 335 exo 380 exo / 465 exo 97 endo / 141 endo / 368 exo 828 endo 127 endo / 161 endo 564 endo / 826 exo

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187

Fig. 5. Thermogram of [Pd(TSDTM)Cl 2 ].

complex do not show any particular feature, sample degradation being complete below 400 8C. The [Pd(TSDTM)Cl 2 ] degradation (Fig. 5), occurs in two successive steps, the first one being deriving from evolution of a CH 3 Cl molecule and C 6 H 6 coordinated molecule (weight loss of 25.7% against a calculated value of 26.2%) to yield the [Pd(TSDT)Cl] n species. The shape of the thermogram above 400 8C depends on the formation of PdO in air flux which releases oxygen to form palladium at 828 8C. In Fig. 6 the thermogram of the [Pt(TSDTM)Br 2 ] complex is reported: the contemporary loss of CH 3 Br and C 6 H 6 molecule (25.9%) is in agreement with the experimental weight loss (25.2%), the decomposition to Pt being complete at 470 8C.

3.3. Preliminary cytotoxicity studies The new complexes were examined (with the exception of [Pd(TSDTM)Cl] n because of its instability in DMSO) for their cytotoxic properties on human leukemic HL60 cells by means of dye exclusion test. Cells were treated for 24 h with increasing concentrations of tested compounds and the obtained results are summarized in term of IC 50 values in Table 4; cisplatin was used as reference. Both palladium(II) complexes carrying the ter-butylsarcosinedithiocarbamate moiety were completely ineffective; the demethylated specie [Pd(TSDT)Br] n displayed a certain activity with, however, IC 50 values about 10-fold higher than the reference compound. Among Pt(II) com-

Fig. 6. Thermogram of [Pt(TSDTM)Br 2 ].

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Table 4 In vitro cytotoxic activity

MDA into the incubation medium, about four times higher than that of [Pt(TSDTM)Br 2 ].

Compound

Vital dye exclusion HL60 cells IC 50 (mM)6S.D.a

MTT test HeLa cells IC 50 (mM)6S.D.a

TSDTM [Pt(TSDTM)Cl 2 ] [Pt(TSDTM)Br 2 ] [Pd(TSDTM)Cl 2 ] [Pd(TSDTM)Br 2 ] [Pd(TSDT)Br] n cis–DDP

.100 .100 5.2060.99 .100 .100 35.4063.29 4.3060.19

.100 .100 11.0160.43 .100 .100 45.4062.89 8.7460.33

a

S.D.5standard deviation; IC 50 values were calculated by probit analysis (P,0.05; x 2 test).

plexes, only [Pt(TSDTM)Br 2 ] was able to induce an evident cytotoxic effect with a IC 50 value very similar to those of cisplatin. Similar findings were observed measuring the human adenocarcinoma HeLa cells viability by the MTT test after a 24 h treatment with increasing concentrations of tested compounds. Table 4 shows the results obtained; for comparison purpose, the activity of cisplatin was always evaluated under the same experimental conditions. Again, [Pt(TSDTM)Br 2 ] was the most potent derivative with an IC 50 value comparable to that of cisplatin; the drug inhibitory effect on cell viability was clearly concentration dependent (data not showed). It is worth noting that the free ter-butylsarcosine(Smethyl)dithiocarbamate, tested in both cell lines, resulted completely ineffective.

4. Conclusions Although none of the complexes here reported has been obtained in the crystalline state, and thus the structure cannot be undoubtedly proposed. The other results suggest that coordination of the dithiocarbamate derivatives takes place in a near square-planar geometry through the sulfur atoms, the –NCSS moiety coordinating the metal atom in a bidentate asymmetrical / symmetrical mode, for Smethylated ([M(TSDTM)X 2 ], M5Pt(II), Pd(II); X5Cl, Br) and demethylated ([Pd(TSDT)X] n , X5Cl, Br) compounds respectively, and lying in the same plane. It has been also demonstrated that S-methylated derivatives give rise to an isomerization equilibrium in chlorinated solvents due to the presence of the S–CH 3 group, and that the compound [Pd(TSDTM)Br 2 ] even slowly demethylates in chloroform leading to the [Pd(TSDT)Br] n polymeric derivative. In vitro cytotoxic activity tests against human leukemic HL60 and adenocarcinoma HeLa cells showed that the [Pt(TSDTM)Br 2 ] compound was able to induce a noticeable reduction of cell viability with IC 50 values very similar to those of the reference compound (cisplatin) tested under same conditions. Moreover, performing toxicity tests on the kidney by means of a renal cortical slices model [Pt(TSDTM)Br 2 ] showed a very low renal cytotoxicity as compared to cisplatin.

3.4. Toxicity results The [Pt(TSDTM)Br 2 ] complex shows a very low renal cytotoxicity as compared with cisplatin as reported in Fig. 7. The results confirm that cisplatin causes high cytotoxicity in renal cells with high lipid peroxidation. In fact, cisplatin at the highest dose induced a high release of

5. Abbreviations TSDTM TSDT cisplatin ESDT DMSO TMS MDA DTA MTT SDS

ter-butylsarcosine(S-methyl)dithiocarbamate / t-BuOOCCH 2 N(CH 3 )CSSMe ter-butylsarcosinedithiocarbamate /tBuOOCCH 2 N(CH 3 )CSS cis-diaminodichloroplatinum(II) /cis-DDP ethylsarcosinedithiocarbamate / EtOOCCH2 N(CH 3 )CSS dimethyl sulfoxide tetramethylsilane malondialdehyde differential thermal analysis 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide sodium dodecylsulfate

Acknowledgements Fig. 7. MAD determination; the error bars are expressed as standard deviations.

Partial support for this work by Ministero dell’Universita` e della Ricerca Scientifica e Tecnologica (Pharmaco-

D. Fregona et al. / Journal of Inorganic Biochemistry 93 (2003) 181–189

logical and Diagnostic Properties of Metal Complexes) is gratefully acknowledged.

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