Synthesis , Characterization and Antitumor Activity of New Organotin ( IV ) Methoxyethyldithiocarbamate Complexes

Two new organotin(IV) dithiocarbamate complexes of the type R3SnL and R2SnL2 (L = methoxyethyldithiocarbamate and R = C6H5 or C4H9) were synthesized in good yields. The organotin complexes were suitably characterized by elemental analysis, FT-IR, 1H, and 13C NMR spectroscopies. These complexes were prepared in situ. Elemental analysis data (carbon, hydrogen, nitrogen, and sulfur) showed an agreement with the suggested formula structures. The infrared spectra of these complexes showed three important peaks for n(C=N), n(C=S), and n(Sn-S) in the region of 1450–1463, 992–994, and 324–326 cm-1 respectively. Data for 13C NMR spectroscopy showed an important peak in the region of dC197–201 ppm that corresponded to the NCS2 group. These complexes were evaluated for their in vitro anti proliferative activities against HL-60 cell lines. The results showed that both of these complexes had high cytotoxicities toward HL-60 cell lines with the IC50 values below 1 mM.

Dithiocarbamates are sulfur-and nitrogencontaining ligands that display a rich and varied coordination chemistry, thus providing a wide range of transition and main group metal complexes (Hill et al. 1985).These ligands are versatile chelating with diverse applications in industry, agriculture, and medicine (Hulanicki 1967;Coucouvanis 1970).Dithiocarbamates deal with a great interest in inorganic synthesis as they have a number of applications.These ligands have attracted the attention among scientists because of their potential biological activities (Leka et al. 2006).Their metal complexes present striking structural features and have diversified applications such as high-pressure lubricants and accelerators used in vulcanization (Beer et al. 2001).Some dithiocarbamates have also been found to be pharmacologically active, used for the treatment of alcoholism (Jacobsen 1950), and tested in clinical trials for various infections including HIV (Hersh et al. 1991;Kaplan et al. 1989; Lang et al. 1988) and cancers (Dufour et al. 1993;Francis et al. 1993;Verma et al. 1990).The antitumor effects of these dithiocarbamates can in part be attributed to their ability to complex tumor cellular copper, leading to binding to and inhibition of the proteasome and in turn initiating tumor cell-specific apoptosis (Buac et al. 2012).Besides, these dithiocarbamates have also been used in the treatment of bacterial and fungal infections (Menezes et al. 2004).
In view of the wide-range applications of organotin(IV) dithiocarbamate complexes, we repor t in this ar ticle the synthesis and characterization of dibutyltin(IV) and triphenyltin(IV) methoxyethyldithiocarbamate complexes and the cytotoxic study of these complexes to evaluate their potential as anticancer agents.

MATERIALS ANd METHOdS
All chemicals and solvents used in this experiment were purchased from Merck Company and used without purification due to their high purity.The melting points were determined in open capillary tubes using an electrothermal 9300 digital melting point apparatus.The percentage compositions of carbon, hydrogen, nitrogen, and sulfur were determined using an elemental analyzer, CHNS-O Model Fison EA1108.Solid state infrared spectra were recorded as potassium bromide discs using a Perkin-Elmer spectrophotometer GX.The 1 H and 13 C nuclear magnetic resonance spectra were recorded using a BRUKER FT-NMR 600 MHz Cryo-Probe spectrometer with DMSO-d 6 /CDCl 3 as solvent.

Synthesis of Organotin(IV) dithiocarbamate Complexes
Organotin(IV) dithiocarbamate complexes were prepared using a direct reaction between 5m mol of carbon disulfide and 5 mmol of ethanolic solution of methoxyethylamine.The reaction mixture was then stirred for 1 h at 277 K temperature and added dropwise to organotin(IV) (dibutyltin(IV) or triphenyltin(IV)) chloride (suitable amount) in 20 mL of ethanol.The precipitate formed was filtered and washed with cold ethanol and then dried in a desiccator.These complexes were then recrystallized from chloroform.

MTT Cytotoxicity Assay
The cytotoxicity of the synthesized compounds was screened using a 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazholium bromide (MTT) assay as described by Mosmann (1983).Briefly, the HL-60 (1 × 10 6 cells/mL) cells were treated with the synthesized compounds in a series of concentrations in a 96-well plate.Following 24 h of incubation, 20 mL of MTT (Sigma-Aldrich, USA) (5mg/mL in PBS solution) was added into each well while excluding ambient artificial light and further incubated for 4 h.Then, 180 mL of supernatant was carefully removed from each well, and 180 mL DMSO (Fisher Scientific, UK) was added to dissolve the formazan crystals formed.After 15 to 20 min of incubation, the absorbance of each well was measured using an ELISA microplate reader (iMark) (Bio-Rad Laboratories, USA) at 570 nm.The graphs were plotted as a percentage of viable cells vs. compound concentrations.The IC 50 values were determined based on the plotted graphs where by the IC 50 values represented the reduction of 50% of the cell population in the treated cells compared to the untreated cells.Doxorubicin hydrochloride was used as a positive control.

Synthesis
Two new organotin(IV) dithiocarbamate complexes were prepared using an insertion technique (Abdul Muthalib et al. 2011), which is a reaction involving organotin(IV) chloride, methoxy ethylamine, and carbon disulfide at 277 K in an ethanol solvent to give stable complexes.The scheme for the reaction involved in the synthesis is shown in Figure 1.Both complexes exhibited as a white solid and was stable in air and highly soluble in chloroform.Recrystallization process was done to the complexes using chloroform as a solvent.The resulting solution was slowly evaporated, and colorless crystals of the complexes were obtained.The elemental analysis showed that the experimental values were in agreement with the theoretical values based on their chemical formula (see Table 1).The percentage of tin in the complexes was determined using gravimetric analysis.

Infrared Spectroscopy
The important infrared absorption bands of complexes 1 and 2 are presented in Table 2. Based on the spectral data, each complex exhibited a peak assigned as thioureide band, n(C=N) in the region of 1470-1500 cm "1 (Bonati & Ugo 1967; Sharma et al. 1996).The infrared spectra of the dithiocarbamate complexes showed very in tense absorptions in the region of 1487-1470 cm "1 , attributed to thioureide vibration (Honda et al. 1968).Furthermore, another important peak observed in these compounds was n(Sn-C), and the peaks within the range 544-559 cm "1 signified the presence the of Sn-C stretching bands for the compounds with phenyl or butyl moiety.The Sn-Sulfur coordination was supported by the presence of medium absorptions in the region of 386-425 cm -1 , verifying the bonding of the tin metal with sulfur atom of the methoxy ethyldithiocarbamatelig and (Shahzadi et al. 2006;Santacruz-Juarez et al.

2008).
NMR Spectroscopy 1 H NMR spectra for these complexes were recorded in CDCl 3 solution, and tetramethylsilane was used as an internal standard at room temperature.The proton of methoxy group for both complexes exhibited a sharp singlet signal at d H 3.35 ppm (see Table 3).The protons of ethylene groups, N-CH 2 and -CH 2 attached to the nitrogen atom exhibited a triplet signal, respectively, at d H 3.70 and d H 4.13 ppm for dibutyltin(IV) complex and d H 3.72 and d H 4.13 ppm for triphenyltin(IV) complex.
In the case of the dibutyltin(IV) compound, i.e., complex 1, a triplet signal was observed for the methyl protons at d H 0.94 ppm, and three sets of broad signals for the methylene protons were also observed in the ranges of d H 1.41-1.47,1.88-1.93,and 2.04-2.07ppm.The attachment of the butyl group to the electropositive Sn atom via carbon nuclei would cause a shielding effect, experienced through the carbon chain (Gomez-Ortiz et al. 2002).A complex multiplet was found at d H 7.27-7.74ppm in triphenyltin(IV) complex due to the aromatic protons of the phenyl group directly attached to Sn atom (Khan et al. 2013).
The important data of 13 C NMR of these complexes are depicted in Table 4.The 13

In vitro Cytotoxic Activity
The efficiency of the synthesized dibutyland triphenyltin(IV) complexes as potential antitumor agents were preliminarily tested in vitro against HL-60 cell lines.The results of the in vitro cytotoxic activity of complexes 1 and 2(see Table 5 and Figure 2) against HL-60 cell lines were compared using doxorubicin hydrochloride as a positive control.Previous studies carriedout by our group demonstrated that the organotin (IV) dithiocarbamate complexes are potent to be developed as anticancer agents (Awang et al. 2010; Khan et al. 2014a;Khan et al. 2014 b).The values are expressed asI C 50 , i.e., the concentration of compound (in mM) that inhibits a proliferation rate of the tumor cells by 50% as compared to the untreated cells as a control.
The results signified that the tested organotins induced a concentration-dependent anti proliferative effect toward HL-60 cells upon treatment for 24 h.These two complexes exhibited high antiproliferative effects;thus, this finding was in agree ment with the previous study by Awang et al. (2010).Compared to the value of triphenyltin (IV) complex, the dibutyltin (IV) complex displayed a higher potency against the HL-60 cells, and the IC 50 values were on the other hand much lower.Therefore, both of these complexes have the potential to be developed as antitumor agents due to their potent cytotoxiceffect at micromolar concentrations.As these results are preliminary, further mechanistic studies on the antitumor activities of these complexes are highly recommended.

CONCLUSION
The formation of the dibutyltin(IV) and triphenyltin(IV) methoxyethyldithiocarbamate complexes were confirmed via characterization analysis.The micro elemental composition in both complexes were in good percentages and in agreement with the suggested molecular formulas.The presence of the thioureide bands, n(C=N) and the Sn-S stretching frequencies in the complexes indicated the formation of the dithiocarbamate groups and the bonding between the Sn(IV) with the dithiocarbamate ligands, respectively.The formation of dithiocarbamates were further supported by the NCS 2 peaks recorded via 13 C NMR.The cytotoxicity assay of these complexes showed a high cytotoxic activity on the HL-60 cell lines.Further in vitro and in vivo studies are recommended as the next research stages on these potential complexes.

Fig. 2 :
Fig. 2: graphical representation of the cytotoxic activity of complexes 1 and 2 and doxorubic in against HL-60 cell lines.The red line (-----) indicates the 50 % of viable cells population