Synthesis and Characterization of New Palladium(II) Thiosemicarbazone Complexes and Their Cytotoxic Activity against Various Human Tumor Cell Lines

The palladium(II) bis-chelate complexes of the type [Pd(TSC1-5)2] (6–10), with their corresponding ligands 4-phenyl-1-(acetone)-thiosemicarbazone, HTSC1 (1), 4-phenyl-1-(2′-chloro-benzaldehyde)-thiosemicarbazone, HTSC2 (2), 4-phenyl-1-(3′-hydroxy-benzaldehyde)-thiosemicarbazone, HTSC3 (3), 4-phenyl-1-(2′-naphthaldehyde)-thiosemicarbazone, HTSC4 (4), and 4-phenyl-1-(1′-nitro-2′-naphthaldehyde)-thiosemicarbazone, HTSC5 (5), were synthesized and characterized by elemental analysis and spectroscopic techniques (IR and 1H- and 13C-NMR). The molecular structure of HTSC3, HTSC4, and [Pd(TSC1)2] (6) have been determined by single crystal X-ray crystallography. Complex 6 shows a square planar geometry with two deprotonated ligands coordinated to PdII through the azomethine nitrogen and thione sulfur atoms in a cis arrangement. The in vitro cytotoxic activity measurements indicate that the palladium(II) complexes (IC50 = 0.01–9.87 μM) exhibited higher antiproliferative activity than their free ligands (IC50 = 23.48–70.86 and >250 μM) against different types of human tumor cell lines. Among all the studied palladium(II) complexes, the [Pd(TSC3)2] (8) complex exhibited high antitumor activity on the DU145 prostate carcinoma and K562 chronic myelogenous leukemia cells, with low values of the inhibitory concentration (0.01 and 0.02 μM, resp.).

The synthesis of thiosemicarbazones (R-CH=N-NH-CS-NHR 1 ) has been developed due to the facility to replace the R and R 1 substituent groups by alkyl, aryl, or heterocyclic derivative and thus leading to a broad spectrum of new bidentate (N,S or N,N) and tridentate (N,N,N or N,N,S) and also tetra-and pentadentate ligands, capable of coordinating to metal centres [6,[20][21][22].
In previous articles, we have reported the cytotoxic activity of the ligands derived from benzaldehyde and furaldehyde thiosemicarbazone and their palladium(II) bischelate complexes.In vitro antitumor studies against different human tumor cell lines revealed that these metal complexes (IC 50 = 0.21-12.46M) were more cytotoxic than their corresponding ligands (IC 50 > 60 M).On the other hand, the platinum(II) tetranuclear, [Pt 4 L 4 ] (HL = 4-phenyl-1benzaldehyde thiosemicarbazone), exhibits higher antiproliferative activity with IC 50 values in the range of 0.07-0.12M [27].

General Method.
To a hot solution of 4-phenyl thiosemicarbazide (3.34 g, 20 mmol) in methanol (100 mL) was added a solution of acetone (1.47 mL, 20 mmol) in 40 mL of methanol with a few drops of glacial acetic acid.The reaction mixture was refluxed for 2-3 h and stirred for 24 h at room temperature.The solid product was filtered, washed several times with ethanol, and dried in vacuo.A similar procedure was applied using o-chloro-benzaldehyde (2.25 mL, 20 mmol) in 60 mL of methanol, m-hydroxy-benzaldehyde (2.44 g, 20 mmol) in 60 mL of methanol, naphthaldehyde (2.72 mL, 20 mmol) in 40 mL of methanol, or 1-nitro-2naphthaldehyde (4.02 g, 20 mmol) in 70 mL of methanol.Single crystals suitable for X-ray crystallography for both HTSC 3 and HTSC 4 were obtained by slow evaporation of the solvent at room temperature.(0.153 g, 0.5 mmol) in dichloromethane/ethanol (2 : 1, 45 mL) was added dropwise to a stirred hot solution of the corresponding thiosemicarbazone (1.0 mmol) in 70 mL of methanol.Then, sodium acetate (0.082 g, 1 mmol) in 5 mL of water was added.The solution was refluxed for 2-3 h and stirred for 24 h at room temperature.The precipitate was collected by filtration, washed three times with ethanol (30 mL), and dried under vacuum.For the complex [Pd(TSC 1 ) 2 ] (6), single crystals suitable for X-ray diffraction studies were grown by slow evaporation from an acetone solution.2.4.Crystal Structure Determinations.Crystallographic measurements were made using an IPDS1 diffractometer (graphite monochromated Mo-K radiation ( = 0.71073 Å)).Data were collected using Φ scan technique with a scan width of 0.7 ∘ .The structures were solved by direct methods using the program SIR2004 [28] and were refined using anisotropic approximation for the nonhydrogen atoms using SHELXL-97 software [29].

Assessment of Cytotoxicity.
The assay was performed as described previously [30].Briefly, 3000-5000 cells were inoculated in each well of 96-well tissue culture plates and incubated at 37 ∘ C with their corresponding culture medium during 24 h.The ligands HTSC 1-5 (10-250 M), palladium(II) complexes (0.01-10 M), or cisplatin (1-10 M) in DMSO were then added and incubated for 48 h at 37 ∘ C with a highly humidified atmosphere, 5% CO 2 and 95% air.After the incubating period, cell monolayers were fixed with 10% trichloroacetic acid and stained for 20 minutes using the sulforhodamine B dye.Then, the excess dye was removed by washing repeatedly with 1% acetic acid.The protein-bound dye was solubilized with 10 mM Tris buffer (pH 10.5) and the absorbance values were obtained at 510 nm using a microplate reader.The IC 50 value was defined as the concentration of a test sample resulting in a 50% reduction of absorbance as compared with untreated controls and was determined by linear regression analysis.
Analytical and spectroscopy data obtained for the thiosemicarbazone ligands and their palladium(II) complexes are in agreement with the proposed structures.
The ligand HTSC 3 (3) and the complex [Pd(TSC 1 ) 2 ] (6) were recrystallized from acetone, and single crystals suitable for X-ray crystallography were obtained, while single crystals of the ligand HSTC 4 (4) were obtained by slow evaporation of the solvent from the final reaction mixture.

Infrared Spectra.
The broad bands of the -NH group observed at 3140-3182 cm −1 in the spectra of the free ligands disappeared in the spectra of the corresponding complexes, thus indicating the deprotonation of the =N-NHgroup.The strong bands observed in the range of 1598-1626 cm −1 were assigned to (C=N) stretching vibrations of the free thiosemicarbazones.These bands were shifted to lower frequencies (10-22 cm −1 ) after coordination, which is in agreement with the observed behaviour of other bischelate complexes [26,[34][35][36][37][38][39].These results indicate the coordination of the azomethine nitrogen to the metal ion.The ](C=S) vibrations observed at 815-1088 cm −1 in the spectra of the free ligands shift 20-138 cm −1 towards lower frequencies upon complexation, indicating the involvement of the thione sulphur in the bond formation to the metal ion [40,41].

NMR Spectra.
The 1 H NMR and 13 C NMR spectra of the ligands and their metal complexes were recorded in DMSOd 6 .In the 1 H NMR spectra of the ligands HTSC 1-5 , the signal of the =N-NH proton appears as a singlet at  10.07-12.03,while on complexation these signals disappeared, thus indicating the deprotonation of the =N-NH group [25,33,[42][43][44][45][46].In the 1 H NMR spectra of the ligands HTSC 2-5 , the signal of the HC=N proton appeared as a singlet at  = 8.07-9.08.These signals are shifted by 0.26-0.59ppm upfield for [Pd(TSC 2-3 ) 2 ] complexes (7,8).These results are consistent with the IR spectral data and suggest the coordination of palladium to the imine nitrogen [24,25,[43][44][45].For all ligands, the resonance lines found at  = 9.83-10.35were assigned to the proton of the NHPh group.The presence of the phenyl group on the terminal amine induces the shift of these signals by 1.9 ppm downfield, as compared to the resonance lines of the -NH 2 terminal group found for other thiosemicarbazone derivatives [33,44].On the other hand, the aromatic proton signals of the phenyl amine group in all the ligands were observed at  = 7.15-7.61,and these resonance lines show the expected calculated multiplicity.For the ligands HTSC 2 (2) and HTSC 3 (3) the aromatic proton signals of the phenyl fragment bound to the −CH=N group were affected by the presence of the chloro and hydroxy substituents in the C-2  and C-3  positions, respectively, of the phenyl moiety.For the HTSC 2 (2) ligand, these signals are shifted downfield for the protons in the positions C-3  (1 ppm) and C-4  (0.1 ppm), while for HTSC 3 (3) ligand they are shifted upfield for the protons in the positions C-2  (0.55 ppm) and C-4  (0.21 ppm), with respect to the unsubstituted phenyl moiety [33].For the HTSC 5 (5) ligand, the presence of the nitro substituent group in the naphthoyl moiety affected the resonance signals of the aromatic protons.These signals are shifted downfield for the protons in the positions C-3  (0.73 ppm) and C-6  (0.19 ppm), while for the protons in the positions C-4  , C-5  , C-7  , and C-8  these are shifted upfield by 0.16-0.25 ppm, relative to the HTSC 4 (4) ligand with the unsubstituted naphthoyl moiety.Thus, the aromatic protons signals in all the ligands do not suffer relevant changes in their chemical shifts after complexation.
In the 13 C NMR spectra, the carbon resonance signals of the C=N group appear at  = 152.8-176.8.These results are similar to the chemical shifts found for other ligands derived from benzaldehyde thiosemicarbazone [33,40].The C=S signals observed at  = 176.4-193.2are characteristic for the thiocarbonyl group present in all the ligands.For [Pd(TSC 1-5 ) 2 ] complexes (6-10), the C=N and C=S signals are shifted downfield by 1.3-13.3ppm and upfield by 0.5-26.1 ppm, respectively, with respect to their ligands.These results confirm the coordination of the thiocarbonyl sulphur and azomethine nitrogen atoms to the palladium(II) ion [36,47].For all ligands, the aromatic carbons of the NHPh group Bioinorganic Chemistry and Applications   2 and 3.
The complex [Pd(TSC 1 ) 2 ] (6) (Figure 3) crystallizes in the monoclinic space group C2/c with four molecules in the unit cell and with a C 2 molecular symmetry.The sulfur and nitrogen donor atoms are in a cis arrangement.The deprotonated ligand coordinates bidentately to Pd II ion through S and N. It leads to lengthening of the C4-S1 bond (1.773 Å) and shortening of the N2-C4 bond (1.29 Å) and these results are in agreement with those found for other palladium(II) bischelate complexes of the type [PdL 2 ] with thiosemicarbazone ligands [27,42].
The chelate ring with the atoms Pd1, N1, N2, C4, and S1 has an envelope configuration.For the plane formed by the atoms N1, N2, S1, and C4, the average deviation is 0.003 Å, while the deviation of the Pd atom from this plane is 0.664 Å; this distortion indicates a pseudo square planar coordination geometry.

Antitumor Evaluation.
The cytotoxic potential of the ligands derived from thiosemicarbazones and their respective palladium(II) complexes were investigated in the following six human tumor cell lines: H460, DU145, MCF-7, M14, HT-29, and K562.For comparison purposes, the cytotoxicity of cisplatin was evaluated under the same experimental conditions.
The results of the cytotoxic activity of the ligands, palladium(II) complexes, and cisplatin are expressed as IC 50 values (micromolar concentration inhibiting 50% cell growth), and these compounds were evaluated in vitro against the different human tumor cell lines, as shown in Table 4.In general, the palladium(II) complexes (IC 50 = 0.01-9.87M) exhibited higher antiproliferative activity than their free ligands (IC 50 = 23.48-70.86 and >250 M).Figure 6 shows the antiproliferative activity of the ligands HTSC 1-5 and their palladium(II) complexes [Pd(TSC 1-5 ) 2 ] against H460 and K562 human tumor cell lines after 48 h incubation time.These results indicate that the cytotoxicity is enhanced when the ligands are coordinated to the Pd(II) ion.Probably, the palladium(II) bis-chelate complexes of square planar geometry act as intercalating agents between the pyrimidine and guanine bases of the DNA tumor cells, inducing conformational changes on the DNA double helix specific that finally produce tumor cell death [33,44,48].
In summary, we have synthesized palladium(II) bischelate complexes with ligands derived from acetone, benzaldehyde, and naphthaldehyde thiosemicarbazone.The molecular structure of [Pd(TSC 1 ) 2 ] (6) shows a squareplanar geometry with deprotonated ligands coordinated to Pd(II) through the azomethine nitrogen and thione sulfur atoms in a cis arrangement.
Of all the studied complexes, the hydroxy-substituted [Pd(TSC 3 ) 2 ] (8) complex resulted to be more cytotoxic in all tumor cell lines at low micromolar concentrations, compared to the other complexes and the free ligands.

a
IC 50 corresponds to the concentration required to inhibit 50% of the cell growth when the cells are exposed to the compounds during 48 h.Each value is the average of two independent experiments.b Lung large cell carcinoma (H460), prostate carcinoma (DU145), breast adenocarcinoma (MCF-7), amelanotic melanoma (M-14), colon adenocarcinoma (HT-29), and chronic myelogenous leukemia (K562).

Figure 4 :
Figure 4: Double chain structure of HTSC 3 in the crystal.

Figure 5 :
Figure 5: Helix structure of HTSC 4 in the crystal.