Synthesis, Characterization and Antiproliferative Activity of the Co(II), Ni(II), Cu(II), Pd(II) and Pt(II) Complexes of 2-(4-Thiazolyl)Benzimidazole (Thiabendazole)

Complexes of 2-(4-thiazolyi)benzimidazole (thiabendazole, THBD) with Co(II), Ni(II), Cu(ll) of general formula ML2(NO3)2 H2O and complexes of Pd(II) and Pt(II) of general formula ML2Cl2 H2O have been obtained and characterized by elemental analyses, IR and far IR spectroscopy and magnetic measurements. The X-ray crystal structure of the copper(II) complex has been determined. The in vitro cell proliferation inhibitory activity of these compounds was examined against human cancer cell lines A 549 (lung carcinoma), HCV-29 T (urinary bladder carcinoma), MCF-7 (breast cancer), T47D (breast cancer), MES-SA (uterine carcinoma) and HL-60 (promyelocytic leukemia). Pt-THBD has been found to exhibit an antileukemic activity of the HL-60 line cells matching that of an arbitrary criterion.


INTRODUCTION
Co-ordination compounds of metals have found increasing application for the treatment of many diseases. For instance, they have been used as haematopoietic, antiphlogistic, bactericidal, antiarthiric and antitumor drugs [1][2][3]. Studies of antitumor properties of platinum complexes have been triggered by the detection by Rosenberg [4,5] of selective inhibition of cell division by cis-Pt(NH3)2CI2 and cis-Pt(NH3)zCI4 complexes. Their antitumor activity rests on the inhibition of DNA synthesis in the tumor cell, and the structure of a compound being formed resembles that of cross-linked chains [6,7].
Many research workers have also studied the antitumor properties of complexes with azole ligands [12][13][14][15][16]. In this connection it seemed worthwhile to study the activity of complexes of Co(II), Ni(1I), Cu(II), Pd(II) and Pt(II) with a popular antiparasitic drug, 2-(4-thiazolyl)benzimidazole (thiabendazole, THBD; Fig.l). The distribution of charge densities in the ligands was calculated by PM3 semiempirical methods using a Hyper Chem 5.01 computer program for Windows 95 from Hypercube Inc. The purpose of this contribution was to obtain an information about the composition of the complexes, co-ordination arrangement of the central ion and to identify electron-donating atoms of the ligand as well as to determine the antiproliferative activity of the complexes against cells of selected human tumor lines. Synthesis, Characterization and Antiproliferative Activity of the Co(ll), Cu(II), Pd(lI) and Pt(II) Complexes of 2-(4-Thiazolyl)Benzimidazole (Thiabendazole) MATERIALS AND METHODS Reagents 2-(4-thiazolyl)benzimidazole (thiabendazole, THBD) was purchased from Sigma. The cobalt(II), nickel(II) and copper(II) nitrates (POCh, Gliwice, Poland) were crystallized from redistilled water. The preparation of PdCl_ and K2PdCI4 was described previously [17].

Physical measurements
Magnetic measurements at ambient temperature were run on a MSB-MKI (Sherwood Scientific Ltd.) instrument. Elemental analyses were run on a Model 240 Perkin-Elmer CHN Analyzer.
The IR spectra (4000-250 cm"!) were taken on a Beckmann Model 4240 spectrophotometer in KBr discs. The far-IR spectra (450-80 cm") were recorded on a DiGiLab FTS-60 spectrophotometer by applying suspensions of the compounds in acetone onto the polyethylene window. Melting points were determined on a Boetius apparatus. Magnetic measurements were accomplished on a MSB-MKI Instrument (Sherwood Scientific Ltd) at ambient temperature. X-ray measurements were performed at room temperature using a Kuma KM-4 four-circle diffractometer and the graphite monochromatic CuKo radiation 18].
The structures were solved by heavy-atom methods with SHELXS-86 and refined by the full-matrix leastsquares methods using SHELXL-93 with anisotropic parameters for all non-hydrogen atoms [19,20]. No correction for absorption has been made. The positions of all hydrogen atoms were determined from the differential Fourier synthesis [21,22]. The positions of the hydrogen atoms have not been refined. The atomic scattering factors were those of neutral atoms incorporated in SHELXL-93 [20]. Details of the measurements of the crystal data of the complexes (M-THBD) and the refinement parameters are listed in Table 2. The solutions were left for crystallization, the complexes were filtered off, washed successively with hot ethanol, acetone and diethyl ether, and dried in vacuo at 25C. The yields were 70-80%. The antiproliferative.assay in vitro Test solutions of the compounds (1 mg/ml) were freshly prepared for each test by dissolving them either in 100 or 200 lal of DMSO + 900 or 800 lal of water pro injectione or, if soluble, in water only. Then the compounds were diluted in a culture medium (described below) to reach final concentrations of 100, 10, 1, 0.1 and 0.01 tg/ml. Cells of the following human cancer lines were used: A 549 (non-small cell lung carcinoma), MCF-7 and T47D (breast cancer), HCV-29 Y (bladder cancer), MES-SA (uterine cancer) and HL-60 (promyelocitic leukemia). All lines were obtained from the American Type Culture Collection (Rockville, Maryland, USA) and cultured in Cell Culture Collection of the Department of Tumor Immunology, Institute of Immunology and Experimental Therapy, Wroclaw, Poland. Twenty four hours before application of the tested compounds, the cells were placed in 96-well plates (Costar, USA) at density of 104 cells per well in 100 l.tl. The cells were cultured in an opti-MEM medium supplemented with 2 mM glutamine (Gibco, Warszawa), 50 l.tg/ml streptomycin (Polfa, Jelenia Gora). 50 U/ml penicillin (Polfa, Jelenia Gora) and 5% fetal calf serum (Gibco, Grand Island, USA). The cells were cultured with the agents for 72 hrs at 37C in a humid atmosphere saturated with 5% CO2.
The in vitro proliferation inhibition test, SRB (cell lines A-549, MCF-7, T47D, HCV-29 T and MES-SA) was applied as described in Skehan's paper [23]. Zero adjustment of the photometer was accomplished by the so-called negative control, i.e. by the liquid taken from the well containing the culture medium without cells. In each experiment, samples of particular concentration of the compounds were applied in triplicate. The tests were likewise carried out in triplicate.

RESULTS AND DISCUSSION
In order to determine the composition of the coordination compounds, elemental analyses (C, H, N, metal) were run ( Table 1). The results of magnetic measurements for the Co(II), Ni(II) and Cu(II) complexes of THBD match the theoretical ones, while the Pd(II) and Pt(II) compounds turned out to be diamagnetic.  dia. Fig.1 shows a schematic structure of the THBD molecule together with calculated electron densities on optional electron-donating atoms. As seen, the optional electron-donating atom is the azomethine nitrogen of the imidazole ring (-0.044). However, complexation through the nitrogen atom of the thiazole ring, carrying negligible positive charge (+0.004) cannot be ruled out.

CRYSTALLOGRAPHY
Monocrystals needed for the determination of the crystal structure could only be obtained with the Cu-THBD compound.
Details of the measurements for complex (3) crystal data together with the refinement parameters are shown in Table 2. As seen in Fig. 2, the Cu(II) ion in coordinated by lone electron pairs of the azomethine nitrogen atoms of the imidazole and thiazole rings and, in addition, by one of the oxygen atoms of the nitrate group to form a pentacoordinate structure. Such a coordination mode is in excellent agreement with the calculated electron densities (Fig.l).
Small differences in bond lenghts in the immediate surrounding of the central ion can be explained in terms of hydrogen bonds between one of the hydrogen atoms of water molecule and an oxygen atom of the coordinated nitrato group as well as between the oxygen atom of the non-coordinated nitrate group and the hydrogen atoms of the benzimidazole moiety. A chelating effect cannot be excluded, as well.

Infrared spectroscopy
The IR spectrum (4000-400 cm l range) of the complex resembles that of the non-coordinated ligand. Certain differences are noted over the stretching vibration range of the system of conjugate C=C and C=N double bonds of the compounds. Table 3 lists absorption bands characteristic of thiabendazole and its complexes. As seen, the largest displacements due to complexation relative to thiabendazole are noted for the C=N and C=C bands.
The C-N bands are only little shifted, whereas those due to C-S remain unchanged. Remarkable are bands due to stretching and deformation vibrations of the water molecules as well as of coordinated and noncoordinated NO3 groups [24,25].
These are due to the bending and stretching vibrations of the metal-oxygen bonds (for the Co(II), Ni(II) and Cu(II) complexes) and of the metal-nitrogen bonds (for the Pd(II) and Pt(ll) compounds). Absorption bands at 291,296, 315, 350 and 347 cm are likely to be due to vibrations of the dative metalnitrogen linkages for the complexes of Co(If), Ni(II), Cu(II), Pd(ll) and Pt(ll), whereas those at 437, 437 and Marek Z Wisniewski et al.
Synthesis, Characterization and Antiproliferative Activity of the Co(ll), Cu(ll), Pd(ll) and Pt(lI) Complexes of 2-(4-Thiazolyl)Benzimidazole (Thiabendazole) 438 cm " are due to M-O linkages in the complexes of Co(II), Ni(II) and Cu(II), respectively, with thiabendazole [26,27].   On the basis of these findings, it can be speculated that the copper(II) compound of THBD has a pentacoordinate structure. A similar structure can be assigned to the cobalt(II) and nickel(II) complexes, whilst those of palladium(II) and platinum(II) form planar square structures.  Table 4.
The adopted activity criterion for the compounds in the in vitro screening was an IDs0 level not exceeding 4 lag/ml [28]. No compound satisfied that criterion.
As seen in Table 4, only the Pt(II)-THBD complex exhibited activity matching that set by the criterion in an assay against the HL-60 leukemia cells and those of urinary bladder carcinoma HCV-29 T cells. Of the cancer cells tested, only the leukemia cells have been found to be sensitive to all the coordination compounds tested. Besides, the copper(lI) and platinum(II) complexes of THBD exhibited the ability to inhibit proliferation of cells of all the cancer lines tested. Again, the Co(II)-THBD compound inhibited proliferation of some cell lines only, notably those of T47D, MES-SA and HL-60, whereas the palladium(lI) and nickel(II) complexes exhibited only a low activity against the HL-60 cells.
To sum up, as far as the in vivo antiproliferative activity assay conducted is concerned, it can be concluded that further studied should be focused on the HL-60 leukemia cells.