Synthesis, Characterization, and In Vitro Cytotoxic Activities of Benzaldehyde Thiosemicarbazone Derivatives and Their Palladium (II) and Platinum (II) Complexes against Various Human Tumor Cell Lines

The palladium (II) bis-chelate Pd (L1−3)2 and platinum (II) tetranuclear Pt4(L4)4 complexes of benzaldehyde thiosemicarbazone derivatives have been synthesized, and characterized by elemental analysis and IR, FAB(+)-mass and NMR (1H, 13C) spectroscopy. The complex Pd(L2)2 [HL2 = m-CN-benzaldehyde thiosemicarbazone] shows a square-planar geometry with two deprotonated ligands (L) coordinated to PdII through the nitrogen and sulphur atoms in a transarrangement, while the complex Pt4(L4)4 [HL4 = 4-phenyl-1-benzaldehyde thiosemicarbazone] has a tetranuclear geometry with four tridentate ligands coordinated to four PtII ions through the carbon (aromatic ring), nitrogen, and sulphur atoms where the ligands are deprotonated at the NH group. The in vitro antitumor activity of the ligands and their complexes was determined against different human tumor cell lines, which revealed that the palladium (II) and platinum (II) complexes are more cytotoxic than their ligands with IC50 values at the range of 0.07–3.67 μM. The tetranuclear complex Pt4(L4)4, with the phenyl group in the terminal amine of the ligand, showed higher antiproliferative activity (CI50 = 0.07–0.12 μM) than the other tested palladium (II) complexes.

The deprotonated thiosemicarbazone ligands usually coordinate to platinum, palladium, copper, ruthenium, and osmium through oxygen, nitrogen, and sulphur donor atoms in their (N, S) bidentate form or (N, N, S or O, N, S) tridentate form, to form metallic complexes of different molecular geometry [9][10][11].
The square planar platinum (II) and palladium (II) complexes of M(HL)Cl 2 and M(L)Cl type with thiosemicarbazone ligands derived from phenylacetaldehyde and 2formylpyridine showed high cytotoxicity in vitro against HL60 leukemia and P388 mouse leukemia cell lines [12], while platinum(II) and palladium(II) binuclear complexes with p-isopropylbenzaldehyde thiosemicarbazone ligands exhibit strong cytotoxic activities on mouse tumor cell growth inhibition [4,13].

Synthesis of the ligands
The thiosemicarbazone derivatives (HL) were prepared according to the literature [20] as shown in Scheme 1.

General method
To a hot solution of thiosemicarbazide (1.82 g, 20 mmol) or 4-phenylthiosemicarbazide (3.3 g, 20 mmol ) in 160 mL, methanol was added dropwise a solution of the corresponding benzaldehyde (20 mmol) in 70 mL methanol during 30 minutes. The mixture was stirred and refluxed for 4 hours, it was filtered and the filtrate was concentrated to half the volume under reduced pressure. After a slow evaporation of the concentrate at room temperature, crystals were collected by filtration, washed with cold ethanol, and dried in vacuo. For ligand HL 4 , the filtrate was kept in the refrigerator and after several hours small rectangular crystals were obtained. These crystals were suitable for structure analysis by X-ray diffraction.

Synthesis of the palladium (II) and platinum (II) complexes (see Scheme 2)
A solution of Pd(acac) 2 (0.30 g, 1.0 mmol) in CH 2 Cl 2 / CH 3 OH (30 mL, 2:1 v/v) or a solution of (NH 4 ) 2 PtCl 4 (0.1865 g, 0.5 mmol) in water/ethanol (2:1, 15 mL) was added dropwise to a stirred solution of the corresponding thiosemicarbazone (2.0 mmol) in 60 mL of methanol. Sodium acetate (0.16 g, 2 mmol) in 3 mL of water was then added. The solution was refluxed for 2 hours and stirred for 24 hours at room temperature. The precipitate was collected by filtration and dried in vacuo.

Palladium (II) complex of m-cyanobenzaldehyde thiosemicarbazone, Pd(L 2 ) 2
Crystals suitable for X-ray structure determination were obtained by slowly evaporating a methanol/dichloromethane

Crystal structure determinations
The data for the crystal structure determinations were collected on a Siemens CCD smart diffractometer (HL 4 , Pt 4 (L 4 ) 4 ) and a Stoe IPDS2 diffractometer (Pd(L 2 ) 2 ) (MoKα radiation, λ = 0.71073Å, graphite monochromator). The intensities were corrected for Lorentz and polarization effects and for absorption using SADABS (Pt 4 (L 4 ) 4 ) and X-RED/X-SHAPE (Pd(L 2 ) 2 ). The structures were solved by direct methods, which revealed the positions of all nonhydrogen atoms and refined on F 2 by a full matrix least-squares procedure using anisotropic displacement parameters with the exception of the solvent molecules in Pt 4 (L 4 ) 4 which were refined isotropically. The hydrogen atoms for HL 4 and Pd(L 2 ) 2 were located from different Fourier syntheses and refined isotropically. For Pt 4 (L 4 ) 4 , the hydrogen atoms were included in calculated positions and refined in riding mode. All calculations were carried out using the SHELXS-97 and SHELXL-97 programs [21,22]. Crystal data collection and refinement details for the ligand HL 4 , the palladium (II) complex Pd(L 2 ) 2 , and the platinum (II) tetranuclear complex Pt 4 (L 4 ) 4 are summarized in Table 1.

Assessment of cytotoxicity
Cells were inoculated into 96-well tissue culture plates at a density of 3000-5000 cells per well and incubated at 37 • C with their corresponding growth medium for 24 hours to allow cells to attach. A plate containing each of these cells was fixed in situ with trichloroacetic acid (TCA) in order to obtain the cell values at zero time before adding the test compounds. The rest of the plates containing the different cell lines received serial dilutions of the ligands and palladium (II) complexes in DMSO to be incubated at 37 • C for 48 hours. The assay was terminated by the addition of cold TCA. The cell numbers in each well was determined using the sulforhodamine B (SRB) assay [23]. TCA-treated plates were incubated at 4 • C for 1 hour and then the cells were washed five times with tap water and dried completely at room temperature. The cells were stained for 20 minutes with a solution of 0.4% sulforhodamine B in 1% acetic acid. At the end of the staining period, unbound dye was removed by washing four times with 1% acetic acid until the washing solution became colorless. After complete drying, bound dye was solubilized with 10 mM Tris buffer (pH 10.5) and the absorbance reads on an automated plate reader at a wavelength of 550 nm. The IC 50 value was defined as the concentration of test sample resulting in a 50% reduction of absorbance as compared with untreated controls that received a serial dilution of the solvent in which the test samples were dissolved, and was determined by linear regression analysis.
For K562 cells, which grow in suspension, instead of fixing and staining with SRB, cells were counted using a Coulter counter.

IR spectra of the ligands and their complexes
The infrared absorption bands become very useful for determining the mode of coordination of the ligands to metal. In the IR spectra, the broad bands of the -NH group observed at 3236-3250 cm −1 for the ligands disappear in Wilfredo Hernándeza et al.  [24]. On complexation, these bands were observed to be shifted to lower frequencies (1570-1590 cm −1 ), which are in agreement with the wave numbers for other bischelate complexes [6,25,26]. These results indicate that the imine nitrogen is coordinated to the metal ion. All ligands showed medium bands in the 880-915 cm −1 range ascribed to ν(C=S) vibrations. These absorption bands shift 65-80 cm −1 to lower frequencies on the coordination of the thiocarbonyl sulfur to palladium (II) or platinum (II) ion. These results are in agreement with other thiosemicarbazone complexes [24,27]. In addition, the vibrational frequencies of the -NH 2 groups remain unchanged for both the ligands and the complexes. This evidence indicates the noncoordination of the -NH 2 group to the Pd(II) center.

NMR spectra of the ligands and their complexes
In the 1 H-NMR spectra of the ligands, the signals of the =N-NH protons were observed as singlets at δ 11.42-11.83. These signals disappeared in the 1 H-NMR spectra of the palladium (II) and platinum (II) complexes indicating the deprotonation of the =N-NH group. The signals of the HC=N protons which appear as singlets at δ 8.03-8.17 in the ligands show a shift to downfield in δ 0.03-0.80 after complexation. This shift indicates the coordination of the imine nitrogen to the metal center [28]. The signals of the aromatic protons of the ligands appeared at δ 7.21-7.91, and the resonance lines found correspond to the calculated multiplicity. These signals do not suffer relevant changes in the chemical shifts for the palladium (II) and platinum (II) complexes. The NH 2 signal in the ligands HL 1 , HL 2 , and HL 3 appears as doublets at δ 7.98-8.45 due to the nonequivalence of the amine protons. This evidence is attributed to the restricted rotation around C-N bond (thiocarbonyl carbon and terminal amine nitrogen) due to its partial double bond character [14,29]. The presence of the phenyl group on the terminal amine (NHPh) of the ligand HL 4 produces a downfield chemical shift at δ 2.1 with respect to the NH 2 group of the ligand HL 1 . This reveals that HL 4 is slightly less basic than HL 1 . The resonance signals of the -NH 2 or NHPh groups in the palladium (II) and platinum (II) complexes do not change, and this evidence indicates that the amine groups are not coordinated to the metal ion [14].
In the 13 C-NMR spectra, the carbon resonance signals of the HC=N group appeared at δ 139.66-148.3. The results are similar to the chemical shifts found for the ligands benzophenone thiosemicarbazide and phenylpropenal thiosemicarbazone (both found at δ 141) [30,31]. The C=S signals observed at δ 178.5-176.0 are characteristic for this group, while the aromatic carbons were observed at δ 139.1-124.5.

Structural data
The molecular structures of HL 4 , Pd(L 2 ) 2 , and Pt 4 (L 4 ) 4 are shown in Figures 1, 2, and 3, respectively, whereas their selected bond lengths and bond angles are presented in Tables 2 and 3.

Bis(3-cyanophenyl-1-benzaldehyde thiosemicarbazonato)palladium (II) Pd(L 2 ) 2
3-cyanophenyl-1-benzaldehyde thiosemicarbazone reacts with palladium (II) acetylacetonate to form a bis-chelate with C i molecular symmetry (see Figure 2). The deprotonated ligand coordinates bidentately through S and N. The coordination of the Pd atom is square planar with a transarrangement of the coordinating atoms. It leads to a lengthening of the S1-C1 bond and a shortening of the N1-C1 bond. The Pd1S1N1N2C1 chelate ring is nearly planar and forms an angle of 11.5(1) • with the C3-C8 phenyl ring.
In the crystal structure, one molecule of water per formula unit of the chelate is included.

Tetrakis (4-phenyl-1-benzaldehyde thiosemicarbazonato)tetraplatinum (II) Pt 4 (L 4 ) 4
4-phenyl-1-benzaldehyde thiosemicarbazone reacts with ammonium tetrachloroplatinate (II) to form a tetranuclear complex with slightly distorted square planar geometry (see Figure 3). The tridentate ligands are deprotonated at the NH group and coordinated through S, N, and C (aromatic ring). The fourth coordination site at each Pt atom is occupied by a sulfur atom of a neighboring ligand. In this way, a puckered eight-membered ring of alternating Pt and S atoms is formed as the core of the molecule. Each of the four Pt atoms belongs to two fused five-membered chelate rings: the C, N metallocycle and the N, S chelate moiety. The Pt-Pt distances range from 3.43Å to 3.84Å. The Pt-S bonds form two distinct groups with significantly differing lengths: Pt-S chelating 2.351(2)Å and Pt-S bridging 2.298(2)Å (mean values). The coordination of the ligand to the Pt atoms leads to a lengthening of the S-C1 bond (increased single-bond character) and a shortening of the neighboring N2-C1 bond (increased double-bond character) compared with the free ligand.
In the crystal structure, two molecules of ethanol per formula unit of the tetranuclear complex are included, which stabilize the crystal structure by hydrogen bonds. The O1 and O2 atoms of the ethanol molecules are bonded by hydrogen bonds to N3 atoms: 2.07Å, N3 · · · O1 2.898Å, N3-H · · · O1 162 • ] and N3- The formation of tetranuclear compounds was previously observed for Pd complexes with similar thiosemicarbazone ligands [14].

Antitumor evaluation
All ligands had a 50% inhibitory concentration (IC 50 ) > 40 μM against the used human tumor cell lines. As shown in Table 4, the palladium (II) and platinum (II) complexes were more cytotoxic (IC 50 = 0.08-12.46 μM) than their respective ligands. These results reveal that the cytotoxic activity increases dramatically when ligands are coordinated to the metal ion [18,19,32].
The Pd(L 2 ) 2 complex with a cyano group in the metaposition of the aromatic ring and the Pt 4 (L 4 ) 4 tetranuclear complex with the phenyl group in the terminal amine of the ligand showed to be more cytotoxic (IC 50 = 0.45-3.67 and 0.07-0.12 μM, resp.) than the other Pd(L 1 ) 2 and Pd(L 3 ) 2 complexes against all tested human tumor cell lines. These results indicate that the cytotoxic activity is enhanced when four ligands are coordinated to four platinum atoms.
Probably, the high cytotoxicity of the Pd(L 2 ) 2 and Pt 4 (L 4 ) 4 complexes may be related to the intercalation of each metal complex between nitrogen bases of the DNA tumor cells, causing greater conformational changes in the double helix of DNA and then producing cell death [33,34].
In summary, we have prepared the palladium (II) bis-chelate complexes and the platinum (II) tetranuclear complex, Pd(L 1-3 ) 2 , and Pt 4 (L 4 ) 4 , which were more cytotoxic on all the human tumor cell lines at low micromolar concentrations with respect to the free ligands. The crystal structure of Pd(L 2 ) 2 shows that the palladium atom has a square-planar geometry with two bidentate ligands having sulfur and nitrogen as donor atoms in transpositions. The complex Pt 4 (L 4 ) 4 has a tetranuclear structure with four tridentate(C,N,S) ligands coordinated to four platinum atoms.

Supplementary material
Further details of the crystal structure determination are available on request from the Cambridge Crystallographic Data Center (CCDC, 12 Union Road, Cambidge CB2 1EZ, UK; fax: +44 1223 336033; email: deposit@ccdc.cam.ac.uk), on quoting the depositing numbers CCDC 694972 for HL 4 , 694973 for Pd(L 2 ) 2 , and 694974 for Pt 4 (L 4 ) 4 , the names of the authors, and the journal citation.