Diorganotin Complexes of a Thiosemicarbazone, Synthesis: Properties, X-Ray Crystal Structure, and Antiproliferative Activity of Diorganotin Complexes

The synthesis and spectral characterization of novel diorganotin complexes with 3-hydroxypyridine-2-carbaldehyde thiosemicarbazone, H2L(1), [SnMe2(L)] (2), [SnBu2(L)] (3), and [SnPh2(L)] (4) are reported. The single-crystal X-ray structure of complex [SnPh2(L)(DMSO)] (5) shows that the ligand is doubly deprotonated and is coordinated as tridentate ligand. The six coordination number is completed by two carbon atoms of phenyl groups. There are two similar monomers 5a (Sn1) and 5b (Sn51) in the asymmetric unit. The monomers 5a and 5b are linked through intermolecular hydrogen bonds of N–H–O and C–H–S type. C–H → π, intermolecular interactions, intra- and intermolecular hydrogen bonds stabilize this structure and leads to aggregation and a supramolecular assembly. The IR and NMR (1H, 13C and 119Sn) spectroscopic data of the complexes are reported. The in vitro cytotoxic activity has been evaluated against the cells of three human cancer cell lines: MCF-7 (human breast cancer cell line), T-24 (bladder cancer cell line), A-549 (nonsmall cell lung carcinoma) and a mouse L-929 (a fibroblast-like cell line cloned from strain L). Compounds 1, 3, and 4 were found active against all four cell lines. Selectivity was observed for complexes 3 and 4 which were found especially active against MCF-7 and T-24 cancer cell lines.


Introduction
Organotin(IV) compounds find wide applications as catalysts and stabilizers, and certain derivatives are used as biocides, as antifouling agents and for wood preservation. It has been observed that several diorganotin adducts show potential as antineoplastic and antituberculosis agents [1][2][3][4].
Thiosemicarbazone derivatives are of considerable interest due to their antibacterial, antimalarial, antiviral, and antitumor activitiy [5,6]. In our laboratory, the chemistry of thiosemicarbazones has been an extremely active area of research, primarily because of the beneficial biological (namely, antiviral and antitumor) activities of their transition-metal complexes [7][8][9]. 3-Hydroxypyridine-2carbaldehyde thiosemicarbazone (1) is a member of the socalled α-(N)-heterocyclic carbaldehyde thiosemicarbazones (HCTs), which are the most potent known inhibitors of ribonucleoside diphosphate reductase. Compounds (1) and 5-hydroxypyridine-2-carbaldehyde thiosemicarbazone have shown high anticancer activity in animal models but were found to be readily glucuronidated and rapidly excreted [10]. The multiple dissociation constants of the ligand H 2 L (1) and the crystal structure of he complex of Pd(II) with 1, [Pd(HL)Cl] have been studied by us [11]. This work is an extension of previously studied complexes of thiosemicarbazones with palladium(II), platinum(II), zinc(II), and organotin(IV) with potentially interesting biological activities [12][13][14].
The present paper includes the interaction of SnPh 2 O (where R is methyl, butyl, and phenyl groups) with 3-Hydroxypyridine-2-carbaldehyde thiosemicarbazone (H 2 L) and the crystal structure of the complex [SnPh 2 (L)(DMSO)] (5). IR and NMR spectroscopic data are reported. The results of the cytotoxic activity of diorganotin complexes have been evaluated for antiproliferative activity in vitro against human cancer cell lines: MCF-7, T-24, A-549, and a mouse L-929.

Experimental
2.1. General and Instrumental. All reagents were commercially available (Aldrich or Merck) and used as supplied. Solvents were purified and dried according to standard procedures. Melting points (m.p.) were determined in open capillaries and are uncorrected. IR and far-IR spectra were recorded on a Perkin Elmer Spectrum GX Fourier transform spectrophotometer using KBr pellets (4000-400 cm −1 ) and nujol mulls dispersed between polyethylene disks (400-40 cm −1 ). The free ligand was dissolved in (CD 3 ) 2 SO and 1 H, 1 H-1 H COSY and 13 C spectra were acquired on a BRUKER 300 MHz spectrometer. Compounds 2-4 were dissolved in CDCl3 and were spectroscopically characterized by the use of 1 D and 2 D NMR spectroscopy on a Varian 600 MHz spectrometer. Experimental data were processed using VNMR and WinNMR routines. Chemical shifts (δ) are reported in ppm while spectra were referenced by the standard experimental setup. 119 Sn NMR spectra were acquired on the Varian 600 MHz and tin spectra were referenced by the use of external solution of Me 4 Sn in C 6 D 6 . The splitting of proton resonances in the reported 1 H-NMR spectra is defined as s = singlet, d = doublet, t = triplet, and m = multiplet. The chemical shifts are reported in ppm for 1 H and 13 C NMR. Elemental analyses were carried out by the microanalytical service of the University of Ioannina, Greece.

3-Hydroxypyridine-2-Carbaldehyde Thiosemicarbazone (1).
The ligand was synthesized according to a published procedure [11]. The white powder was recrystallized from cold ethanol and was dried in vacuo over silica gel.

X-Ray
Crystallography. Crystals of complex 5, suitable for X-ray analysis, were obtained by slow crystallization of 4 from a mixture of solvents C 6 H 6 /toluene/DMSO/CH 3 CN. Crystal data 5 are given in Table 1, together with refinement details. All measurements of crystals were performed in low temperature using an Oxford Cryosystem device on a Kuma KM4CCD κ-axis diffractometer with graphitemonochromated Mo Ka radiation. The data were corrected for Lorentz and polarization effects. No absorption correction was applied. Data reduction and analysis were carried out with the Oxford Diffraction (Poland) Sp. z o.o (formerly Kuma Diffraction Wroclaw, Poland) programs. Crystal structure was solved by direct methods (program SHELXS97) and refined by the full-matrix least-squares method on all F 2 data using the SHELXL97 [15] programs. Nonhydrogen atoms were refined with anisotropic displacement parameters; hydrogen atoms were included from geometry of molecules and Δρ maps. During the refinement process they treated as riding atoms. Molecular graphics were performed from PLATON2003 [16,17]. Crystallographic data, that is, atomic coordinates, thermal parameters, bond lengths, and bond angles (CCDC number 634270 for 5), have been deposited with the Cambridge Crystallographic Data Centre. Copies of available material can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK, (fax: +44-1223-336033 or e-mail: deposit@ccdc.cam.ac.uk or http://www.ccdc.cam.ac.uk). Compounds.

Antiproliferative Assay In
Test solutions of the compounds tested (1 mg/mL) were prepared by dissolving the substance in 100 μL of DMSO completed with 900 μL of tissue culture medium. Afterwards, the tested compounds were diluted in culture medium to reach the final concentrations of 100, 50, 10, 1 and 0.1 ng/μL. The solvent (DMSO) in the highest concentration used in test did not reveal any cytotoxic activity. Cells. The cell lines are maintained in the Cell Culture Collection of the University of Ioannina. Twenty-four hours before addition of the tested agents, the cells were plated in 96-well plates at a density of 10 4 cells per well. The MCF-7 cells were cultured in the D-MEM (Modified Eagle's Medium) medium supplemented with 1% antibiotic and 10% fetal calf serum. L-929 cells were grown in Hepes-buffered RPMI 1640 medium supplemented with 10% fetal calf serum, penicillin (50 U/mL), and streptomycin (50 mg/mL). A-549 cells were grown in F-12K Ham's medium supplemented with 1% glutamine, 1% antibiotic/antimycotic, 2% NaHCO 3 , and 10% fetal calf serum. The cell cultures were maintained at 37 • C in a humid atmosphere saturated with 5% CO 2 . Cell number was counted by the Trypan blue dye exclusion method. MCF-7, L-929, and A-549 cells were determined by the sulforhodamine B assay [18], while T-24 cells by the MTT assay [19]. The in vitro tests were performed as described previously [20]. In the 1 H NMR spectra of (H 2 L) (1) the broad singlet at δ 11.60 was attributed to OH group in accordance with [22] and the broad signal at δ 9.75 ppm was assigned to NH group. These two groups apparently participate in Hbonding with the nucleophilic solvent molecules (DMSO) or with other ligand molecules. These two signals are abolished upon interaction with the metal, indicating deprotonation of these groups and possible coordination to the tin(IV) atom at 2-4. The absence of peaks corresponding to the imino proton NH and OH proton in the spectrum of 2-4 indicates that the nitrogen and oxygen are present in the deprotonated form and the ligand is dideprotonated. A sharp resonance peak appearing at ca. 5 ppm in all complexes is attributed to the NH 2 group. This is also corfirmed by integration of the 1 H spectral profile while additionally the use of CDCl 3 eliminates the formation of H-bonding or complexation with the participation of solvent molecules as was the case with the ligand alone. In the 1 H-NMR spectra of the complexes 2-4, the formyl H-atom H-C(6) was shifted upon coordination, which indicated variations in the electron density at position 6. This signal was shifted downfield, in accordance with a decreased electron density at site C6 in the complexes.

Results and Discussion
The C=S resonance of the thiosemicarbazone moiety in the free ligand resonated at 178.0 ppm. All complexes showed an upfield shift of C7 peak in the order of ∼10 ppm compared with the free ligand, indicating the complexation of tin(IV) to the sulphur atom which apparently is related with an increased electron density at this site on complexation, due probably to π-back bonding for thiolato sulphur [21]. All the complexes exhibit downfield shifts of the C3, C4, and C6. This deshielding, in accordance with decreased electron density upon complexation, is indicative of Sn-O and Sn-N (azomethine nitrogen) bonds. These data indicate that the complexes are formed by ligand deprotonation followed by metallation, a structural motive that seems to be stable both in the solid state and in CDCl 3 solution. 119 Sn chemical shifts of compounds 2-4 were found between −94.4 and −227.2 in accordance with five-coordinate tin center [23].

Molecular
Structure. Crystals of complex 5, suitable for X-ray analysis, were obtained by slow crystallization of 4 from a mixture of solvents C 6 H 6 /toluene/DMSO/CH 3 CN. The crystal structure is shown in Figure 1. Crystal data are given in Table 1, together with refinement details. Bond lengths and angles are given in Table 2. There are two similar monomers 5a (Sn1) and 5b (Sn51) in the asymmetric unit. The double deprotonated ligand is coordinated as tridentate ligand via the phenolic oxygen O(1), the azomethine nitrogen N(3), and thiolato sulfur S(1) atoms. The molecule of DMSO is coordinated to the tin through oxygen O(2) atom. The six coordination number is completed by two carbon atoms of phenyl groups. The organic molecule acts as anionic tridentate with the ONS donors placed in the same side. The dianionic, tridentate ligand has a ZEZ configuration for the oxygen, nitrogen, and sulfur donor centers. The coordinated ligand consists of three rings, one heterocyclic and two chelates, SnSNNC and SnONCC, I and II, respectively. For monomers 5a and 5b the dihedral angles between the planes of the rings I and II are 14.47(6) and 12.59(6) • , respectively and between the ring II and the pyridyl ring are 7.4(5) and 8.1(2) • , respectively, indicating that the ligand as a whole in the two monomers deviates from planarity.
The C-S bond lengths 1.747(2), 1.749(2)Å for 5a and 5b, respectively, are shorter than a single bond (1.81Å), but longer than a double bond (1.62Å), suggesting partial single bond character. The C(14)-N(3) bond length, 1.301(2)Å, is close to a double bond (1.28Å). The S-C bond distances are consistent with increased single-bond character while both thioamide C-N distances indicate increased double bond character. The negative charge of the deprotonated ligand is delocalized over the thiosemicarbazonato moiety. This is indicative of the coordinated thiosemicarbazone's greater conjugation and more delocalized electron density. The Sn-N(3) bond distance is longer than the sum of the covalent radii (2.15Å), which indicates strong bond, while the bond distance Sn-S, 2.5141(5), 2.519(5)Å, though much shorter than the sum of the van der Waals radii (4.0Å), indicates a weak bond [21]. The C-Sn C bond angle is equal to 105.87 (7) (4) and Cg(5) are referred to the rings C(1)-C(6) and C(7)-C(12); b Cg-Cg is the distance between ring centroids; symmetry transformations, (i) type. Also, the monomers of 5b form hydrogen-bonded dimers and are linked by two hydrogen bonds, the N(51)-H(51A)-N(54) and C(64)-H(64)-N(52) (Figure 2). C-H → π intermolecular interactions intra-and intermolecular hydrogen bonds [24] stabilize this structure and lead to a supramolecular assembly, and Table 3 and   [12]. The diphenylorganotin(IV) complex with pyruvic acid thiosemicarbazone has been tested against MCF-7, T-24, A-549, and L-929 cell lines and was found especially active against MCF-7 and T-24 cancer cells [21]. The antiproliferative activity of compounds is presented in Table 4  The mentioned evident differences in the antiproliferative action of the ligand and its diorganotin(VI) complexes indicate that these complexes really exist under the condition of the biological tests. Interestingly enough, 3 and 4 were found to be more potent cytotoxic agent than the prevalent benchmark metallodrug, cis-platin, under the same experimental conditions measured by us. The superior activity of 3 and 4 assumes significance in light of the fact that cisplatin is undisputedly the most studied and widely used metallopharmaceutical for cancer therapy known to date. It is noteworthy the high selectivity against MCF-7 and T-24 cancer cell lines.