Organotin(IV) Derivatives of L-Cysteine and their in vitro Anti-Tumor Properties

The synthesis and characterization of the organotin compounds [(n-C4H9)2Sn(cys)] (1), [(C6H5)2Sn(cys)] (2), [(C6H5)3Sn(Hcys).(H2o)] (3), {[(CH3)2Sn(Kcys)2].2(H20)} (4), {[(n-C4H9)2Sn(Kcys)2].2(H20)} (5) and {[(C6H5)2Sn(Kcys)2].2(H20)} (6) (where H2cys = L-cysteine) are reported. The compounds have been characterized by elemental analysis and 1H-NMR, Uv-Vis, FT-IR and MOssbauer spectroscopic techniques. Attempted recrystallization of (2) in DMSO/methanol 2:1 solution yielded after several days unexpectedly the dimeric compound bis(tri-phenyltin)sulphide {[(C6H5)3Sn]2S} (7) which has been characterized by x-ray analysis. The structure of the parent complex (2) as well as the mechanism of the decomposition of cysteine are being further investigated. The in vitro anticancer activity of complexes (I)- (6), against human leukemia (HL60), human liver (Bel7402), human stomach (BGC823) and human cervix epithelial human carcinoma (Hela), nasopharyngeal carcinoma (KB) and lung cancer (PG) tumor cells, were evaluated.


I. INTRODUCTION
The biological activity of organotin(IV) compounds is well known/1-3/. Most organotin(IV) compounds are generally toxic /3/. The binding of organotins by thiol groups, on the other hand, is significant in biological systems /4/. A great deal of work has been reported thus far, on the interaction of organotin compounds with thiol-containing mercapto-amino acids such as cysteine and its derivatives/3, 5-14/. The interpretation of the results however, was rather controversial. Thus, based on M6ssbauer spectroscopy Barbieri et  Another demand for such studies raised from the potential pharmaceutical application of organotin compounds/5, 15/. Today, a number of organotin (IV) derivatives are known to have an efficient anti-turnout activity/15/. A comparison of the structures of the active and inactive compounds suggests that all active compounds should conform to the following requirements: (i) to have available coordination positions around Sn, (ii) to have a relatively stable ligand-Sn bond (e.g. Sn-N and Sn-S) with low hydrolytic decomposition/5/. The aim of the present work is the exploration of new synthetic routes for the synthesis of organotin(IV) complexes with cysteine, the better characterization of the compounds formed and the evaluation of anticancer activity of the complexes derived and characterized.

Materials and Instruments:
All solvents used were reagent grade, while organotin chlorides (Aldrich), cysteine (Merck) were used with no further purification. Elemental analysis for C, H, N, and S were carried out with a Carlo Erba EA Model 1108. Infra-red spectra in the region of 4000-370 cm were obtained in KBr discs while far-infra-red spectra in the region of 400-50 cm were obtained in polyethylene discs, with a Perkin-EImer Spectrum GX FT-IR spectrometer. A Jasco Uv/Vis/NIR V 570 series spectrophotometer was used to obtain the electronic absorption spectra.. The H-NMR spectra were recorded on a Bruker AC250 MHFT-NMR instrument in CDCI. solutions. Chemical shifts i are given in ppm using TMS as an internal reference. The gsn Christos 7". Chasapis. et al.
MTT solution was added to each well. After incubation for 4 h at 37 C, acid-isopropanol was added to all wells. The measurements of absorbance of the solutions related to the number of living ceils were carried out on a Bio-Rad Model 450 Microplate Reader at 570 nm.
Characteristic vibration bands (cm!) in infra-red spectra of complexes or ligands L-Cys  Table  1) indicating coordination of cysteine through the carboxyl group to tin atom/10, 13/. The magnitudes of the [Vas(COO) vs(COO)] (Av) separation in complexes (I)-(3) (Av=239+8) are comparable to those reported for organotin derivatives of L-cysteine and L-cysteine ethyl ester/12, 13, 2 I/. The conclusions drawn above are further supported by the presence in the IR spectra of a sharp band at 560+20 cm -, which was assigned to the Sn-O stretching vibration/2 I/( Table 1). The organotin compounds (4)- (6) (6) indicate coordination of cysteine through the carboxyl group to tin atom as well/12, 13, 21/. The stretching frequencies for NH2 group in the ir spectra of compounds (I)-(6) can help to distinguish coordinated from free amino groups. The v(NH2) of the free amino group is observed at 3166cm in cysteine/13, 22/. The assignment of the-NHz group has been made by deuterization of the samples using D20. The corresponding v(NH2) vibrations of the organotin derivatives (I)-(6) remain at the same frequency indicating that coordination through the amino group of cysteine to tin atoms does not take place. These conclusions are further supported by the absence in the far-IR of a band assignable to v(Sn-N)/21/. The absence of v(S-H) bands in the IR spectra of compounds (I)-(6) shows a coordination of sulfur to tin atom. The stretching frequency of C-S bond in IR spectra of cysteine is observed at 691 cm-I/13, 22/. The corresponding vibration v(C-S) of organotin derivatives (!)- (6) shifts to lower frequencies (Table 1) supporting the same conclusion.
119SFl MOssbauer spectra: Mssbauer parameters of compounds (!)-(6) are listed in Table 2, while the quality of the 19Sn M6ssbauer spectrum of the compounds measured at 85K are shown in Figure 1. Table 2 M0ssbauer parameters of compounds (1)- (6) Compound 6 (mms-) AEq (mms-) The occurrence of two symmetric Lorentzian doublets in the spectra of compounds (1)- (6) show two distinct coordination sites with tin/2, 23/. The values between the quadruple splitting resonances of the two symmetric Lorentzian doublets in the spectrum of compound (3) ( Table 2) differ significantly, allowing the assignment of the environment around the tin atom/7/to be made. In the case of compound (3), a resonance with smaller splitting (tin moiety with contribution of 80% in the sample) is similar to that in [(Ph3Sn)2Cys] /7/ and is assigned to a tetrahedral [Ph3SnS] environment, while that with the higher splitting resonance corresponds to the penta-coordinated Ph3Sn(Cys,S-)(OH2) environment /7/. For compound (5), the inner doublet was assigned to the BuSn-S moiety while the outer doublet was assigned to the BuSn-O moiety/12/.
H-NMR spectrum of free cysteine in DMSO-d6 shows signals at 2.86 and 3.12 ppm for-CH2-and -CHprotons respectively. The signal in H-NMR spectrum of complex (2) in DMSO-d6 at 2.91 ppm is assigned to the -CH2-and -CHprotons of coordinated cysteine.
The corresponding resonance signals of-CH2-and-CH-groups in H-NMR of free cysteine in CD3OH are observed at 3.10 and 4.02 ppm respectively; these are shifted to 2.94-2.98 ppm and 3.7 ppm respectively in the case of (4) and at 2.89-2.98 ppm and 3.6 ppm in the case of complex (5), indicating coordination of cysteine to tin(IV) atom. 5O Christos T. Chasapis et al. H-NMR spectrum of complex (I) in CDCl3 shows signals for the -CH2-and -CHprotons of cysteine at 2.67-3.04 and 3.48 ppm.

Bioinorganic Chem.istry and Applications
The 3C-NMR spectrum of free cysteine in DMSO-d6 shows signals at 169.86 ppm for the -COO and at 56.50 ppm, 26.12 ppm for the Ca and Cb atoms respectively. The 3C-NMR spectrum of the complex (1) shows resonance signals at 174.34 ppm for the -COO and at 57.25, 31.14 ppm for the Ca and Cb atoms respectively. The signals of the carboxylic-COO and Cb atoms have been shifted downfield by 4.5 and 5.0 ppm respectively towards the corresponding signals of free cysteine indicating the cysteinato-k2S,O coordination mode in the case of complex (1). The signal at 57.25 ppm is attributed to the Ca atom of the coordinated cysteine while four new signals also appear in the 3C-NMR spectrum of complex (I) at 28.33, 27.16 (J Sn-Ca '= 51 Hz), 23.25 and 14.54 ppm which are attributed to the Cb', Ca', Cc' and Cd' atoms of the butyl group.
Results show that complexes (1), (2) and (5) exhibit high cell toxicity against Hela while complexes (1), (3) and (6) show high cell toxicity against BGC. Cell toxicity at concentrations of 10 JM and the cells were viable to less than 10 %. A further systematic investigation of the biological properties of these metal compounds is recommended focusing on their mutual significance for tumor induction of neoplastic growth.

ACKNOWLEDG EM ENTS.
This work was carried out in partial fulfillment of the requirements for a M.Sc. thesis of Mr Christos Chasapis within the graduate program EPEAEK in Bioinorganic Chemistry financed by the Ministry of Education of Greece and coordinated by Professor N. Hadjiliadis. Vol. 2, Nos. I-2, 2004 Organotin (IV) Derivatives of L-Cysteine and their in vitro Antitumor Properties Table 4 The in vitro anticancer activity of the complexes (1)- (6), against Human cervix epithelial human carcinoma (Hela), Human stomach (BGC823), Human leukemia (HL60), Lung Cancer (PG), Human liver (Be17402) and Nasopharyngeal Carcinoma (KB) tumor cells Sample () (4) (4)  Vol. 2, Nos. [1][2]2004 Organot# (IV) Derivatives of L-Cysteine and their in vitro .Antitumor Properties