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Journal of Organometallic Chemistry 696 (2011) 1600e1608 Contents lists available at ScienceDirect Journal of Organometallic Chemistry journal homepage: www.elsevier.com/locate/jorganchem Synthesis and characterization of glycoconjugate tin(IV) complexes: In vitro DNA binding studies, cytotoxicity, and cell death Sartaj Tabassum a, *, Rais Ahmad Khan a, Farukh Arjmand a, Subrata Sen b, Jyoti Kayal b, Aarti S. Juvekar b, Surekha M. Zingde b a b Department of Chemistry, Aligarh Muslim University, Aligarh 202002, Uttar Pradesh, India Advanced Centre for Treatment Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai 410210, Mumbai, India a r t i c l e i n f o a b s t r a c t Article history: Received 21 November 2010 Received in revised form 24 December 2010 Accepted 11 January 2011 New glycosyl derived ligand and its complexes, with SnCl4$5H2O (1) and (CH3)2SnCl2 (2) were synthesized and characterized by spectroscopic (IR, 1H, 13C, and 119Sn NMR, UVevis, ESI-MS) and analytical methods. Interaction studies of 1 and 2 with CT DNA were studied by using various biophysical techniques, which showed high binding affinity of 2 with CT DNA. In vitro cytotoxicity of complexes 1 and 2 were evaluated against different human cancer cell lines of different histological origins by employing SRB Assay. The organotin(IV) complex 2 exhibited remarkable activity against DWD (oral cancer) cell lines with GI50 values <10 mg/ml. Complex 2 induced apoptosis of DWD cell line at a very low concentration of 1e4 mg/mL. Ó 2011 Elsevier B.V. All rights reserved. Keywords: GlycoconjugateeSn(IV) complexes In vitro DNA binding studies Cytotoxicity Nuclear morphology 1. Introduction One of the most rapidly developing areas of pharmaceutical research is the discovery of robust drugs for treating cancers. Cisplatin (cis-diamminedichloroplatinum(II)) [1,2] is an archetypical metal-based drug widely used for treating solid tumors viz; testicular, ovarian, bladder and head/neck cancers, however, it possesses serious limitations because of inherent or acquired resistance in tumor cells and severe side effects [3]. Although, cisplatin and its other second generation cisplatin drugs remain the top selling drugs (exceeding $1.4 billion in the US in 2008), still the toxicity issues pertaining to these drugs remain major impediments in its use. Therefore, there is an exigency to identify effective metal-based therapeutics particularly, those that overcome inherent and acquired resistance to drug therapy and show improved therapeutic properties, stimulating the ongoing investigations of alternative molecular targeted metal-based drugs [4]. Anticancer drugs targeting biomolecules represent a rational advancement in the modern drug discovery. DNA and enzymes involved in replication and transcription represent the most targeted bioreceptors for small molecules and a target for the control * Corresponding author. Tel.: þ91 05712703893. E-mail address: [email protected] (S. Tabassum). 0022-328X/$ e see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jorganchem.2011.01.012 of gene expression [5e7]. Most anticancer drugs bind to DNA and proteins either in a reversible or irreversible manner suggesting a direct relationship between their interactions with macromolecules, hence, leading to their therapeutic effect [8,9]. There is considerable promise in enhancing the targeting and efficiency of metal-chelators through ligand design. Ligands can significantly alter the biological properties by modifying reactivity or substitution related inertness. Carbohydrates are of primary importance as energy sources for living organisms [4,10,11]. Due to the properties inherent to this class of molecules, carbohydrates have been utilized to prepare bioactive materials [12], and bettertargeted drugs [13,14]. Appending a carbohydrate moiety to drug candidates to form new drugs and/or pro-drugs, offers great potential in increasing the solubility of the molecule and minimizing the toxicity, and enhancing the stability [15]. Present investigation focuses on the design and synthesis of new saccharide derivative chelator by the introduction of salicylaldehyde as anchoring group which can provide well defined binding environment as well as increases the stability of the resultant metal complexes. D-glucosamine (an amino monosaccharide) which is widely taken as dietary supplement to relieve discomfort of osteoarthritis related joint pains, has been reported as an inhibitor of cancer cells in vitro and in vivo [16e19]. A potential benefit of utilizing this approach is that the carbohydrate can remain pendant, thereby being freely available to interact with carbohydrate transport and metabolic pathways in the body. Tin(IV) and organotin(IV) S. Tabassum et al. / Journal of Organometallic Chemistry 696 (2011) 1600e1608 compounds exhibit wide spectrum biological activity [20e24] and are notably important in the discovery of new metal-based anticancer chemotherapeutic agents [3,25e27]. Tin(IV) complexes prefer to bind to the oxygen atom of the phosphate of the polyanionic structure of DNA. Tin(IV) ions possess a hard Lewis acid nature, neutralize the negative charge of the phosphate moiety of the DNA backbone and thus brings conformational changes in DNA [28,29]. This class of compounds has gained tremendous impetus because their in vitro antitumor activity is greater than in vivo activity of cisplatin, especially used for treating testicular, ovarian, bladder and head/neck cancers [3]. Besides this, tin compounds are known to act as strong apoptotic directors, activate apoptosis directly via p53 tumor suppressor pathway. In continuation of our previous work [21,22,29e34], herein we report the synthesis and characterization of N-glycoside derived ligand and its complexes with SnCl4$5H2O (1) and (CH3)2SnCl2 (2), respectively. The interaction of complexes 1 and 2 with CT DNA was carried out by employing various biophysical techniques. The in vitro cytotoxicities of 1 and 2 were tested against the 14 human carcinoma cell lines of different histological origins. The changes in nuclear morphology of the DWD (oral cancer cell line) upon addition of 2 at 1e3 mg/mL concentrations were photographed. 2. Experimental section 2.1. Materials Glucosamine hydrochloride, dimethyl tin(IV) dichloride, ethylenediamine (SigmaeAldrich), tin(IV) tetrachloride, tris(hydroxymethyl)aminomethane (E. Merck), imidazole (Fluka), Disodium salt of calf thymus DNA (highly polymerized stored at 4 C), agarose (SigmaeAldrich) and pBR322 supercoiled plasmid DNA (Genei) were used as received. 2.2. Methods and instrumentation Microanalysis (CHN) was carried out with a Carlo Erba Analyzer Model 1108. Molar conductances were measured at room temperature on a Eutech conductivity bridge. Interspec 2020 FTIR spectrometer was used for recording IR spectra of KBr pellets in the range of 4000e400 cm1. Electronic spectra were recorded on UV-1700 PharmaSpec UVevis spectrophotometer (Shimadzu). Emission spectra were determined with a Hitachi F-2500 fluorescence spectrophotometer. 1H, 13C and 119Sn NMR were recorded on Bruker Avance II 400 NMR spectrometer at 25 C. Electrospray mass spectra were recorded on Micromass Quattro II triple quadrupol mass spectrometer. Cleavage experiments were performed with the help of Axygen electrophoresis supported by Genei power supply with a potential range of 50e500 V, visualized and photographed by Vilber-INFINITY Gel documentation system. DNA binding experiments that include absorption spectral studies, fluorescence studies are confirmed to the standard methods [35e38] and practices previously adopted by our laboratory [30e32]. Standard error limits were estimated using all data points. 2.3. Cleavage activity The cleavage experiments of supercoiled pBR322 DNA (300 ng) by complexes 1 and 2 (10e40 mM) in (5 mM TriseHCl, 50 mM NaCl) buffer at pH 7.2 were carried and observed using agarose gel electrophoresis. The samples were incubated for 1 h at 37 C. A loading buffer containing 25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol was added and electrophoresis was carried out 1601 at 60 V for 1 h in TriseHCl buffer using 1% agarose gel containing 1.0 mg/ml ethidium bromide. Similarly in photocleavage studies, reaction mixture was carried out under illuminated conditions at 365 nm (12 W) monochromatic light source. The samples were incubated for 0.5 h at 37 C and analyzed for photocleaved products using gel electrophoresis as discussed above. The standard protocols were followed for these experiments. 2.4. In vitro antitumor activity The human tumor cell lines used for in vitro screening for antitumor activity were Hop62 and A549 (lung), PC3 and DU145 (prostate), A498 (kidney), DWD (oral), Colo205, HT29 HCT15 and SW620 (colon) T24 (bladder) MIA-PA-CA-2 (pancreas), MCF7 and ZR-75-1 (breast), SiHa, ME180 and HeLa (cervix) U373MG (astrocytomaeglyoblastoma), A2780 (ovary), K562 (erythroleukemia). The human malignant cell lines were procured and grown in RPMI-1640 medium supplemented with 10% Fetal Bovine Serum (FBS) and antibiotics to study growth pattern of these cells. The inhibition of the cell proliferation upon treatment with the synthesized compounds was determined using the Sulforhodamine-B (SRB) semi-automated assay. Cells were seeded in 96-well plates at an appropriate cell density to give optical density in the linear range (from 0.5 to 1.8) and were incubated at 37 C in CO2 incubator for 24 h. Stock solutions of the complexes were prepared as 100 mg/ml in DMSO and four dilutions, i.e. 10 mg/ml, 20 mg/ml, 40 mg/ml, 80 mg/ml, in triplicates were tested, each well receiving 90 mL of cell suspension and 10 mL of the drug solution. Appropriate positive control (Adriamycin) and vehicle controls were also run. The plates with cells were incubated in CO2 incubator with 5% CO2 for 24 h followed by drug addition. The plates were incubated further for 48 h. Termination of experiment was done by gently layering the cells with 5 mL of chilled 30% TCA (in case of adherent cells) and 50% TCA (in case of suspension cell lines) for cell fixation and kept at 4 C for 1 h. Plates were washed under running tap water, air-dried and stained with 50 mL of 0.4% SRB for 20 min. The excess dye was removed by washing with 1% acetic acid. The bound SRB was eluted by adding 100 mL 10 mM Tris (pH 10.5) to each of the wells. The absorbance was read at 540 nM with 690 nM as reference wavelength. All experiments were repeated three times. 2.5. Effect of compound 2 on DWD cells A modified procedure for detection of apoptosis in 96-well plates was used. DWD cells (2.0 104/well) were seeded in quadruplicate wells in a 96-well plate for each treatment and kept overnight. Next day the cells were treated with the complex 2 at 1, 2, 3 and 4 mg/ml and the cells were maintained for 24 h at 37 C in 5% CO2 incubator. The plates containing the control and treated cells were centrifuged at 1000 rpm for 5 min using a plate centrifuge (model R-23, Remi, India). The pelleted cells were stained with acridine orangeeethidium bromide (EB/AO) dye mixture (1:1), (100 mg/ml in PBS, 4 ml/well). Cells were viewed under the Zeiss Axiovert 200 M inverted microscope fitted with FITC (408/490) and rhodamine (550/573) filters. The images of DWD cells treated with complex 2 were captured using Axiocam MRM camera. For each treatment at least 500 cells were counted. Percent live, dead and apoptotic (early þ late) cells were calculated by formula: n=total no: of cells counted 100; where ‘n’ represents number of live/dead or apoptotic cells counted per treatment group. 1602 S. Tabassum et al. / Journal of Organometallic Chemistry 696 (2011) 1600e1608 2.6. Synthesis was stirred for 4 h, which yielded pale yellow coloured product. It was filtered, washed with cold MeOH and Et2O, dried under vacuum over P2O5. Yield ¼ 56%. Anal. Calcd for C15H22N3O5SnCl3 (1): C 32.79, H 4.04, N 7.65. Found: C 32.82, H 4.05, N 7.66. LM (1 103 M, DMSO): 17.0 U1 cm2 mol1 (non-electrolyte). IR (KBr) (nmax/cm1): 3287 (eOH sugar, broad), 1642 (HC]N), 1474, 1451 (dOCH, dCH2, dCCH), 643 (SneO), 432 (SneN), 293 (SneCl). 1H NMR (400 MHz, DMSO, d): 10.19 (s, NH, 1H), 8.69 (s, N]CH, aldimine, 1H); 7.52 (m, AreCH, 2H), 6.93 (m, AreCH, 2H), 6.26 (s, br, NH2, GLcN, 2H), 4.20 (t, CH sugar, 2H), 3.31e3.08 (m, OH, CH, sugar, 9H), 2.56 (s, CH2 of en, 4H). 13C NMR (100 MHz, DMSO, d): 165.24 (carbon of characteristic aldimine), 145.22, 136.18, 135.98, 122.46, 118.40, 117.51 (six aromatic carbons), 78.51, 78.18, 77.85, 58.24, 55.84, 51.32 (six carbons of GlcN), 40.12, 39.91 (two carbons of en, CH2). 119Sn NMR (149.19 MHz, DMSO, d): 570.23. ESI-MS (m/z): 549 [C15H22N3O5SnCl3]þ. 2.6.1. Synthesis of ligand (L) To a methanolic solution (20 mL) of N-glycoside synthesized by a reaction between D-glucosamine hydrochloride (0.215 g, 1 mmol) and ethane-1,2-diamine (0.06 ml, 1 mmol) in MeOH at 80 C for 1 h, [39] was added methanolic solution (10 mL) of salicylaldehyde (0.122 ml,1 mmol) dropwise, and allowed to reflux for 1 h. During the course of reflux, the mixture turned yellow. The reaction mixture was then allowed to cool at room temperature. The solvent was removed by rotavapor and dried under vacuum over P2O5. The product was obtained as a yellow liquid. Yield ¼ 48%. IR (KBr) (nmax/cm1): 3436 (AreOH), 3289 (eOH sugar), 1646 (HC]N); 1472, 1450 (dOCH, dCH2, dCCH). 1H NMR (400 MHz, DMSO, d): 13.27 (s, AreOH, 1H), 10.20 (s, NH, 1H), 8.77 (s, N]CH, aldimine, 1H), 7.50 (m, AreH, 2H), 6.94 (m, AreH, 2H), 5.32 (s, br, NH2 sugar, 2H), 4.19 (t, CH sugar, 2H), 3.65e3.05 (m, OH, CH, sugar, 9H), 2.53 (s, CH2 of en, 4H). 13C NMR (100 MHz, DMSO, d): 172.40 (carbon of characteristic aldimine), 146.48, 136.67, 136.53, 122.60, 122.26, 118.85 (six aromatic carbons), 73.84, 71.39, 71.31, 63.58, 51.31, 51.02 (six carbons of GlcN), 40.33, 40.06 (two carbons of en, CH2). ESI-MS (m/z): 326.4 [C15H23N3O5 þ 1H]þ. 2.6.3. Synthesis of C17H28N3O5SnCl (2) This complex was prepared in a manner analogous to that of complex 1, using (CH3)2SnCl2 (0.219 g, 1 mmol) in place of SnCl4$5H2O. On cooling a yellow coloured complex was obtained, which is filtered, washed with cold MeOH and Et2O, dried under vacuum over P2O5. Yield ¼ 52%. Anal. Calcd for C18H34N3O7SnCl (2): C 38.70; H 6.13; N 7.52. Found: C 38.73, H 6.14, N 7.54. LM (1 103 M, DMSO): 14.0 U1 cm2 mol1 (non-electrolyte). IR 2.6.2. Synthesis of C15H24N3O6SnCl3 (1) To a methanolic solution (20 mL) of SnCl4$5H2O (0.352 g, 1 mmol) was added the ligand (L) (0.325 g, 1 mmol). The solution HO O OH OH + OH NH2CH2CH2NH2 HO MeOH 70oC, 1h OH OH NH3+ Cl- Glucosamine hydrochloride HO OH OH NH2 HN O HO + NH3+ HO CHO in situ OH OH - Cl O NH2 HN O NH3+ Cl- N NH HO NH3+ ClLigand (L) L + SnCl4. 5H2O MeOH Reflux HO O OH HN Cl N H2 OH N O Sn Cl Cl Complex 1 L + (CH3)2SnCl2 MeOH Reflux HO O OH OH N HN H3C Sn N H2 Cl Complex 2 Scheme 1. O CH3 S. Tabassum et al. / Journal of Organometallic Chemistry 696 (2011) 1600e1608 (KBr) (nmax/cm1): 3292 (OeH sugar, broad), 1639 (C]N), 1473, 1450 (dOCH, dCH2, dCCH), 1411 (CeN), 635 (SneO), 429 (SneN), 289, 281, 276 (SneCl). 1H NMR (400 MHz, DMSO, d): 10.17 (s, NH, 1H), 8.54 (s, N]CH, aldimine, 1H), 7.37 (d, AreCH, 1H), 7.21 (t, AreCH, 1H), 6.90 (m, CH, aromatic, 2H), 6.49 (s, br, NH2, GLcN, 2H), 3.94 (t, CH sugar, 2H), 3.31e3.05 (m, OH, CH, sugar, 9H), 2.56 (CH2 of en, 4H), 1.26 (s, CH3, bonded to Sn, 6H). 13C NMR (100 MHz, DMSO, d): 166.55 (C]N, characteristic aldimine); 144.42, 131.96, 131.37, 118.32, 118.23, 116.24 (six aromatic carbons), 78.94, 78.61, 78.28, 58.84, 56.44, 51.34 (six carbons of GlcN); 45.54, 40.12 (two carbons of en, CH2), 8.94, 8.51 (two carbons of methyl group). 119Sn NMR (149.19 MHz, DMSO, d): 603.01. ESIMS (m/z): 508.4 [C17H28N3O5SnCl]þ. 3. Results and discussion 3.1. Synthesis and characterization The glucosamine-based ligand was synthesized by the reaction of N-glycoside formed through condensation of 2-amino-2-deoxyD-glucose hydrochloride with ethylenediamine, by salicylaldehyde in methanol. The reaction of Schiff base ligand with SnCl4$5H2O and (CH3)2SnCl2 in 1:1 molar ratio, respectively, led to the formation yellow coloured complexes 1 and 2 (Scheme 1). Both the complexes are soluble in H2O and DMSO. Elemental analyses obtained are in good agreement with the suggested chemical formulae of the compounds. Structural proposals are based on IR, 1 H, 13C, 119Sn NMR, ESI-MS. The results obtained through these techniques are in agreement with the proposed 1:1 stoichiometry between the Sn(IV) moieties and ligand. The molar conductance values of 103 M solutions of the synthesized complexes 1 and 2 are in the range of non-electrolytes. The in vitro binding studies of 1 and 2 with CT DNA were carried out by using absorption, emission spectroscopic titrations, and gel electrophoresis. In vitro cytoxicity of the complexes on human cell lines was evaluated by SRB assay. 1603 eOH and the eNH of the linker amine. However, on complexation, the phenolic eOH peak disappears which suggest the deprotonation of eOH and bonding to central tin(IV) ion. The 1H NMR spectra of the ligand and its complexes 1 and 2, displayed the characteristic aldimine peak at 8.77e8.54 ppm. The aromatic proton signatures were observed in the range of 7.55e6.86 ppm. A broad peak for the NH2 of the glucosamine appeared at around 5.32 ppm in the ligand, which on complexation undergoes a significant chemical shift suggestive of coordination with the central tin(IV) ion. Furthermore, the characteristic signal for the sugar moiety appeared in the range of 4.20e3.05 ppm, due to the typical-saccharide coupled system; however, the individual assignments of the peak were not possible. The protons of CH2 of ethylenediamine appeared at around w2.5 ppm [44,45]. In case of complex 2, the signal of the methyl group bound to tin appeared at 1.26 ppm. The absence of phenolic OH peak in the complexes suggested the deprotonation and confirms the SneO bonding. 13 C NMR spectrum of the ligand exhibited the strong characteristic signal of aldimine carbon at 172 ppm. A large shift to 165e166 ppm was observed in the spectra of the complexes 1 and 2 which ascertained the coordination of tin(IV) to nitrogen atom. The six aromatic carbon peaks appeared in the range 146e116 ppm. The six distinct carbon signals of the D-glucosamine were exhibited in the range of 78.94e51.32 ppm [46,47]. The two carbons of the ethylenediamine moiety appeared in the range 45.54e39.91 ppm. The additional peak in complex 2, for the two methyl groups bonded to tin was observed at 8.94 and 8.51 ppm [23]. The validation of the geometry of tin(IV) metal ion in 1 and 2 was done by 119Sn NMR spectroscopy. It is known that 119Sn chemical shift d (119Sn) is sensitive towards the coordination sphere around the tin atom. 119Sn NMR showed a single peak at 570.23 ppm and 603.01 ppm for complexes 1 and 2, respectively, confirming the octahedral geometry around the tin atoms in both the complexes which was in good agreement with the previous literature reports [22,30]. 3.2. IR spectroscopy 4. Binding studies In the IR spectrum of the free D-glucosamine, the sharp bands of n(OeH) were observed in the range of 3200e3500 cm1. The characteristic envelop at around 3400 cm1 merged and broadened due to the complexation [40]. The IR spectrum of ligand shows a very sharp and strong band at 1646 cm1 assignable to characteristic n(HC]N) [24]. The IR spectra of the complexes 1 and 2 exhibited significant shift in n(HC]N) to lower wave numbers viz; 1642 and 1639 cm1, respectively, indicating the coordination of Sn(IV) moieties to aldimine nitrogen. The bands appearing between 1475 and 1449 cm1 were attributed to d (OCH, CH2, CCH) for the sugar moiety [41]. The above modes of coordination of ligand are further supported by the appearance of two new bands at 446 cm1 and 432 cm1 in the spectra of tin (IV) complexes, which are assigned to n(SneN) [42,43], respectively. Additional, absorption bands at 647e643 cm1 for n(SneO) were observed which correspond to deprotonation of phenolic OH due to coordination with central tin(IV) atom [30]. In the IR spectrum of the complex 2, a medium intensity band at 572 cm1 corresponds to n(SneC). The n(SneCl) was also revealed by the presence of the bands at w290 to 270 cm1, in the far IR region [22]. 3.3. NMR spectroscopy The 1H NMR spectrum of the ligand exhibited signals at 13.27 and 10.20 ppm, which were attributed to the phenolic 4.1. Absorption studies Titration with UV absorption spectroscopy is an effective method to examine the binding mode of DNA with metal complexes [48]. The interaction of complexes with DNA is expected to perturb the ligand based transitions of the complexes. It is known that the metal complexes can bind to DNA via both covalent and/or non-covalent interactions [49,50]. The absorption spectra of 1 and 2 in the absence and presence of CT DNA (at a constant concentration of complexes, 0.16 104 M) are shown in Fig. 1. In the UV region complexes 1 and 2 exhibited bands at 252 nm and 254 nm, respectively, which are assigned to the pep* transitions, due to long living triplet excited state of the aromatic moiety. The other bands at around 372 nm are associated with LMCT bands and in the region of MLCT bands at w435 nm. Upon increasing concentration of CT DNA (0e0.4 104 M), the UV region exhibited an increase in absorption intensity ‘hyperchromic’ effect with a red shift of 2e4 nm in pep* region. However, spectral changes in the LMCT transition and MLCT region also showed hyperchromism but to a lower extent compared with the pep* region. The strong hyperchromic effect with a significant red shift is suggestive of higher binding propensity to CT DNA, possibly by electrostatic mode, and stabilization of the complex-DNA adduct. The Sn(IV) ions (non-transition metal ion) of the complexes exhibit preferential selectivity 1604 S. Tabassum et al. / Journal of Organometallic Chemistry 696 (2011) 1600e1608 absorbance of the pep* bands with increasing concentration of CT DNA: ½DNA=3a 3f ¼ ½DNA=3b 3f þ 1=Kb 3b 3f (1) where [DNA] represents the concentration of DNA, 3a, 3f and 3b are the apparent extinction coefficient Aobs/[M], the extinction coefficient for free metal complex and the extinction coefficient for metal complex in the fully bound form, respectively. In the plots of [DNA]/ 3a 3f versus [DNA], Kb is given by the ratio of the slope to the intercept. The intrinsic binding constants of 1 and 2 were found to be 1.2 104 M1 and 9.4 104 M1, respectively. The Kb values revealed that 2 exhibits greater propensity towards CT DNA, thereby shows stronger binding affinity. 4.2. Fluorescence interaction studies 4.2.1. Luminescence studies The interaction of complexes 1 and 2 were also investigated by monitoring its fluorescence intensities both in the presence and Fig. 1. Variation of UVevis absorption (a) for complex 1 and (b) complex 2, respectively, with increase in the concentration of CT DNA in buffer 5 mM TriseHCl/50 mM NaCl, pH ¼ 7.2 at 25 C. Inset: plots of [DNA]/3a 3f versus [DNA]. towards the phosphate group of the DNA backbone [30]. Sn(IV) complex interacts with the phosphate group of DNA, and cause the contraction and conformational change of DNA helix, due to the fact that phosphate group can provide the suitable anchors for coordination in Sn(IV) complexes and consequently results in breakage of the secondary structure of the DNA. Furthermore, the Schiff base ligand would encourage minor groove binding by engaging in hydrogen-bonding between coordinated eNHe and eOH with the functional groups positioned on the edge of DNA bases [22,30]. However, the presence of carbohydrate in a drug is important in molecular recognition at the specific site of DNA and has profound effect on biological activity. This effect was first studied in family of anthracycline antibiotics [51], which showed that carbohydrate makes the van der Waal’s contacts with the minor groove of the DNA. Aminosugars that interact with the nucleic acid backbone mainly through electrostatic interactions between the positive charged amino groups and the negative charged phosphate of the furanose moiety. In addition to electrostatic interactions, other kinds of forces involving non-polar interactions between carbohydrates cannot be excluded [52]. Therefore, the above results indicate that complex 2 may first bind with the phosphate group of DNA, neutralize the negative charge of DNA phosphate group, and cause the contraction and conformational change of DNA. To evaluate quantitatively, the binding strengths of 1 and 2 with CT DNA, the intrinsic binding constants Kb of the complexes were determined with Eq. (1) by monitoring the changes in Fig. 2. Emission spectra of (a) complex 1, and (b) complex 2, in the absence and presence of CT DNA in buffer 5 mM TriseHCl/50 mM NaCl, pH ¼ 7.2 at 25 C. S. Tabassum et al. / Journal of Organometallic Chemistry 696 (2011) 1600e1608 1605 absence of CT DNA [22,30,53]. The complexes 1 and 2 exhibited emission bands at 450 nm when excited at 594 nm. In the absence of CT DNA, 1 and 2 emit weak luminescence in 5 mM TriseHCl, 50 mM NaCl buffer at ambient temperature. The enhancements in the emission intensity of 1 and 2 with increasing CT DNA concentration are depicted in Fig. 2a and b. Since it is found that complexes with increased hydrophobicity show greater increase in emission intensities upon binding to polyelectrolytes viz; DNA, thus, we conclude that there is some interaction between the CT DNA and complexes that occurs due to the hydrophobicity of both the molecules. The CT DNA binding abilities of the complexes were obtained from Scatchard equation and follow the order, 2 (K ¼ 7.6 104 M1) > 1 (K ¼ 1.4 104 M1). These results are consistent with the findings obtained from UVevis spectral studies. the adjacent DNA base pairs. Addition of second molecule, which binds to DNA more strongly than EthBr, would quench the DNAinduced EthBr. The extent of quenching of the fluorescence of EthBr bound to DNA would reflect propensity of DNA binding of 1 and 2. On addition of complexes 1 and 2, to CT DNA pretreated with EthBr ([DNA]/[EthBr] ¼ 1) the decrease in emission intensity was observed. The emission intensity in the absence and presence of 1 and 2 with EthBreDNA are depicted in Fig. 3. As there is incomplete quenching of the EthBr-induced emission intensity, thus the intercalative mode for 1 and 2 was ruled out. Furthermore quantitatively the quenching extents Ksr were evaluated for 1 and 2, were found to be 2.1 104 M1 and 8.9 104 M1, respectively. The higher value of Ksr for 2 was attributed to increased hydrophobicity of the complex. 4.2.2. Ethidium bromide displacement assay To further investigate the mode of binding of the complexes 1 and 2, the ethidium bromide displacement assay was carried out [22]. The molecular fluorophore EthBr emits intense fluorescence in the presence of CT DNA due to its strong intercalation between 4.2.3. Effect of phosphate group on the binding of complexes with DNA To further validate the interaction of the complexes 1 and 2 with the DNA, the fluorescence titrations were performed in the Fig. 3. Quenching spectra of CT DNA bound ethidium bromide in the presence of (a) complex 1 and (b) complex 2, in buffer 5 mM TriseHCl/50 mM NaCl, pH ¼ 7.2 at 25 C. Fig. 4. Emission spectra of (a) complex 1, and (b) complex 2, with increasing concentration of K2HPO4, in the absence and presence of CT DNA in TriseHCl buffer at pH ¼ 7.2 at 25 C. 1606 S. Tabassum et al. / Journal of Organometallic Chemistry 696 (2011) 1600e1608 presence of K2HPO4 at 25 C. With increasing amount of K2HPO4, fluorescence intensity of complexes 1 and 2 increase appreciably demonstrating a competitive binding behavior between the phosphate group of K2HPO4 and the phosphate component of DNA backbone [54]. This competitive interaction of the phosphate groups weakens the interaction of the complexes with DNA [55] which provide supportive evidence for electrostatic interaction of the Sn(IV) complexes with the phosphate backbone of DNA (Fig. 4 (a) and (b)). However, the enhancement in the intensity of complex 2 is higher in comparison to complex 1, due to hydrophobicity of the complexes. These observation provides supportive evidence for electrostatic interaction of the Sn(IV) complexes which exhibit selective binding to the phosphate group of DNA double helix. 5. Cleavage experiments 5.1. Chemical nuclease activity a Form II Form I 10 20 The DNA cleavage activity of the complex 2 at 10e40 mM concentration was studied using pBR322 DNA (w80% Supercoiled form) in 5 mM TriseHCl/50 mM NaCl buffer at pH 7.2 and upon irradiation with UV light of 365 nm [58,59]. On increasing the concentration, complex 2 produces conversion of SC DNA (Form I) to NC form (Form III) completely. The ligand moiety could generate the photoexcited (nep*) and/ or (pep*) states, causing the cleavage of DNA as observed. The photocleavage experiment results suggested, the possible involvement of a metal assisted photoexcitation process in the visible light-induced DNA cleavage activity of the complex 2. Thus as evident from the photoinduced electrophoretic pattern of 2, the photonuclease activity observed for 2 at micro molar concentration upon irradiation of 365 nm UV light for a shorter time of 10 min exhibited significantly promising activity (Fig. 6). 6. In vitro anticancer activity There has been considerable interest in DNA endonucleolytic cleavage reactions that are activated by metal complexes [56,57]. Thus, DNA mobility shift assays were carried out to investigate the ability of Sn(IV) complexes to interact with plasmid DNA. The initial amount of pBR322 DNA (300 ng) was constant and incubated with increasing concentrations of the complexes 1 and 2. The complex 1 (10e40 mM) cleaves double stranded supercoiled plasmid DNA (SC form: Form I) (300 ng) on incubation in 5 mmol TriseHCl/50 mmol NaCl buffer into nicked circular form (NC form: Form II) at physiological pH 7.2 and temperature 37 C as depicted in Fig. 5(a). Upon gel electrophoresis of the reaction mixture, with varied concentration of complex 2 (10e40 mM) and keeping the DNA concentration (300 ng) constant efficient cleavage was observed (Fig. 5b). The DNA results suggested that the efficient nuclease activity was exhibited by the complex 2 at the concentration of 40 mM. In the concentration-dependent electrophoretic pattern exhibited conversion of SC form (Form I) to NC form (Form II) with increase in the concentration of 2. The distribution of supercoiled and nicked forms of DNA in the agarose gel electrophoresis provides a measure of the extent of hydrolysis of the phosphodiester bond which suggested the more binding propensity of complex 2 as compared to 1. CT 5.2. Photonuclease activity 30 40 µM b In vitro anticancer activity of complexes 1 and 2 was screened against 20 different human carcinoma cell lines of different histological origin. The Sulforhodamine-B (SRB) assay was used to assess cellular proliferation [60]. The result showed good potential of the complexes 1 and 2 as drug candidate. Interestingly, complex 2 showed specificity and exhibited good cytotoxic activity only against DWD (human oral carcinoma cell line). The results in terms of GI50 values are given in Table 1. The reason for its selectivity needs further investigations. 7. Cell death induced by complex 2 To ascertain whether the cell death induced by complex 2 involves changes in nuclear characteristics, EB/AO dual staining was performed (Fig. 7). To identify the possible involvement of apoptosis, DWD cells treated with complex 2 (1e4 mg/ml for 24 h) were stained with EB/AO dual stain. Changes in the nuclear morphology such as extensive chromatin aggregation or nuclear condensation were observed in the treated cells. This can be explained by the fact that, in dual staining with EB/AO, AO permeates the cells and stains the nucleus green. However, EB is taken up only by the cells when cell membrane integrity is lost and stains nucleus orange [61,62]. Thus with dual staining, the live cells show green coloured intact nucleus while dead or necrotic cells show structurally normal nucleus with even orange staining. The early apoptotic cells, where membrane integrity is not lost show green coloured condensed or fragmented nucleus while late apoptotic cells display fragmented and condensed chromatin which is stained orange. The cells treated with complex 2 exhibited fragmented orange coloured chromatin, a hall mark of late apoptotic cells even at a concentration as low as 1 mg/ml A five to nine fold increase in percent apoptotic cells was observed in treated groups (at 1e4 mg/ml concentrations) as compared to control group. Form II Form II Form III Form I Form I CT 10 20 30 40 µM Fig. 5. Agarose gel electrophoresis patterns of pBR322 plasmid DNA (300 ng) cleaved by (a) complex 1 (10e40 mM), and (b) complex 2 (10e40 mM), after 1 h incubation time in buffer (5 mM TriseHCl/ 50 mM NaCl, pH ¼ 7.2 at 25 C) (concentration dependent). CT 10 20 30 40 µM Fig. 6. Photoinduced electrophoretic separations showing the cleavage of pBR322 plasmid DNA (300 ng) by induced 2 after 0.5 h incubation time, in buffer (5 mM TriseHCl/50 mM NaCl, pH ¼ 7.2 at 25 C). S. Tabassum et al. / Journal of Organometallic Chemistry 696 (2011) 1600e1608 1607 Table 1 Summary of the screening data of 1 and 2, for the in vitro anti tumor activity GI50 (in mg/ml).a Compound Hop62 A549 PC3 DU145 A498 DWD Colo205 HT29 HCT15 SW620 2 1 ADRb 16 79 <10 50 >80 <10 36 76 <10 33 65 <10 61 57 <10 <10c <10c <10 26 50 <10 >80 >80 <10 12 76 <10 49 >80 <10 Compound 2 1 ADR T24 29 49 <10 MIA-PA-CA2 23 >80 <10 MCF7 22 44 <10 ZR-75-1 47 71 <10 SiHa 33 60 <10 ME180 53 >80 <10 HeLa 58 >80 <10 U373MG 45 >80 <10 A2780 25.6 47.5 <10 K562 55 >80 <10 a b c GI50 ¼ growth inhibition of 50% (GI50) calculated from [(Ti Tz)/(C Tz)] 100 ¼ 50, drug concentration results in a 50% reduction in the net protein increase. ADR ¼ adriamycin (positive control compound). GI50 value <10 mg/ml is considered to demonstrate activity. phosphate backbone of DNA double helix. Furthermore, 2 exhibited an efficient DNA photocleavage activity. Complex 2 showed significantly good cytotoxic activity against DWD cell lines (human oral carcinoma cell line). Indeed, complex 2 is an illustration of an antitumor drug candidate which could exhibit fewer side effects, low toxicity, specificity (due to glycosylated unit) and therefore, warrants further in vivo investigations. Acknowledgements We express our gratitude to Department of Biotechnology, New Delhi, for generous financial support (Scheme No. BT/PR6345/Med/ 14/784/2005). Thanks to RSIC, Panjab University, Chandigarh for providing NMR facility, RSIC, CDRI Lucknow for providing CHN analysis data, ESI-MS. References Fig. 7. Effect of complex 2 on nuclear morphology of DWD cells: (A) control cells without any treatment. Pink arrows showing normal green coloured nuclei and white arrow showing occasional orange coloured necrotic or dead cells. (B) Cells treated with complex 2, at 1 mg/mL showing fragmented, condensed orange coloured chromatin (yellow arrows) indicating late apoptotic cells while blue arrow indicating early apoptotic cells with green condensed and fragmented chromatin. (C) (LefteRight) showing a magnified view of necrotic, live, early and late apoptotic cells, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). 8. Conclusions We have described herein, the synthesis and characterization of new water soluble glycoconjugate derived Sn(IV) complexes 1 and 2, as antitumor drug candidates. The in vitro DNA binding studies of complexes 1 and 2 revealed that Sn(IV) ions interact through the [1] B. Rosenberg, L. Vancamp, T. Krigas, Nature 205 (1965) 698e699. [2] B. Rosenberg, L. VanCamp, J.E. Trosko, V.H. Mansour, Nature 222 (1969) 385e386. [3] J.J. Bonire, S.P. Fricker, J. Inorg. Biochem. 83 (2001) 217e221. [4] T. Storr, K.H. Thompson, C. Orvig, Chem. Soc. Rev. 35 (2006) 534e544. [5] R. Palchaudhuri, P.J. Hergenrother, Curr. Opin. Biotechnol. 18 (2007) 497e503. [6] J.L. Nitiss, Nat. Rev. Cancer 9 (2009) 338e350. [7] J.L. Nitiss, Nat. Rev. Cancer 9 (2009) 327e337. [8] D.E. Thurston, Br. J. Cancer 80 (1999) 65e85. [9] L.H. Hurley, Nat. Rev. Cancer 2 (2002) 188e200. [10] K.H. Thompson, C. Orvig, Science 300 (2003) 936e939. [11] J. Reedijk, Proc. Natl. Acad. Sci. U S A 7 (2003) 3611e3616. [12] P. Sears, C.H. Wong, Angew. Chem. 111 (1999) 2446e2471. [13] M. Rouquayrol, W. Gaucher, J. Greiner, A.M. Aubertin, P. Vierling, R. Guedj, Carbohydr. Res. 336 (2001) 161e180. [14] M.P. Sathisha, S. Budagumpi, N.V. Kulkarni, G.S. Kurdekar, V.K. Revankar, K.S.R. Pai, Eur. J. Med. Chem. 45 (2010) 106e113. [15] T. Storr, M. Merkel, G.X. Song-Zhao, L.E. Scott, D.E. Green, M.L. Bowen, K.H. Thompson, B.O. Patrick, H.J. Schugar, C. Orvig, J. Am. Chem. Soc. 129 (2007) 7453e7463. [16] J.H. Quastel, A. Cantero, Nature 171 (1970) 2905e2912. [17] J.G. Bekesi, R.J. Winzler, Cancer Res. 30 (1953) 252e254. [18] H.J. Oh, J.S. Lee, D.K. Song, D.H. Shin, B.C. Jang, S.I. Suh, W.K. Baek, Biochem. Biophys. Res. Commun. 360 (2007) 840e845. [19] J.-Y. Park, J.-W. Park, S.-I. Suh, W.-K. Baek, Biochem. Biophys. Res. Commun. 382 (2009) 96e101. [20] S.K. Hadjikakou, N. Hadjiliadis, Coord. Chem. Rev. 253 (2009) 235e249. [21] S. Tabassum, C. Pettinari, J. Organomet. Chem. 691 (2006) 1761e1766. [22] M. Chauhan, F. Arjmand, J. Organomet. Chem. 692 (2007) 5156e5164. [23] X. Shang, J. Cui, J. Wu, A.J.L. Pombeiro, Q. Li, J. Inorg. Biochem. 102 (2008) 901e909. [24] M. Nath, P.K. Saini, A. Kumar, J. Organomet. Chem. 695 (2010) 1353e1362. [25] T.S.B. Baul, C. Masharing, S. Basu, E. Rivarola, M. Holcapek, R. Jirasko, A. Lycka, D. deVos, A. Linden, J. Organomet. Chem. 691 (2006) 952e965. [26] M. Gielen, E.R.T. Tiekink, in: M. Gielen, E.R.T. Tiekink (Eds.), Metallotherapeutic Drug and Metal-based Diagnostic Agents: 50Sn Tin Compounds and Their Therapeutic Potential, Wiley, New York, 2005, pp. 421e439 (chapter 22). [27] A. Chaudhary, A.K. Sing, R.V. Singh, J. Inorg. Biochem. 100 (2006) 1632e1645. [28] F.H. Zelder, A.A. Mokhir, R. Kramer, Inorg. Chem. 42 (2003) 8618e8620. [29] Z. Shi, S. Tabassum, W. Jiang, J. Zhang, S. Mathur, J. Wu, Y. Shi, Chem. Bio. Chem. 8 (2007) 1e9. [30] M. Chauhan, K. Banerjee, F. Arjmand, Inorg. Chem. 30 (2007) 3072e3082. 1608 S. Tabassum et al. / Journal of Organometallic Chemistry 696 (2011) 1600e1608 [31] N. Hoti, D. Zhu, Z. Song, Z. Wu, S. Tabassum, M. Wu, J. Pharmacol. Exp. Ther. 311 (2004) 22e33. [32] N. Hoti, J. Ma, S. Tabassum, Y. Wang, M.J. Wu, Biochemistry 134 (2003) 521e528. [33] G. Lupidi, F. Marchetti, N. Masciocchi, D.L. Reger, S. Tabassum, P. Astolfi, E. Damiani, C. Pettinari, J. Inorg. Biochem. 104 (2010) 820e830. [34] S. Tabassum, R.A. Khan, F. Arjmand, A.S. Juvekar, S.M. Zingde, Eur. J. Med. Chem. 45 (2010) 4797e4806. [35] J. Marmur, J. Mol. Biol. 3 (1961) 208e218. [36] M.E. Reicmann, S.A. Rice, C.A. Thomas, P. Doty, J. Am. Chem. Soc. 76 (1954) 3047e3053. [37] A. Wolfe, G.H. Shimmer, T. Meehan, Biochemistry 26 (1987) 6392e6396. [38] J.R. Lakowicz, G. Webber, Biochemistry 12 (1973) 4161e4170. [39] S. Yano, Y. Sakai, K. Toriumi, T. Ito, H. Ito, S. Yoshikawa, Inorg. Chem. 24 (1985) 498e504. [40] S. Mathur, S. Tabassum, Chem. Biodivers. 3 (2006) 312e325. [41] J. Guo, X. Zhang, Carbohydr. Res. 339 (2004) 1421e1426. [42] M. Nath, X. Sulaxna, G. Song, Eng. J. Organomet. Chem. 691 (2006) 1649e1657. [43] L.S. Zamudio-Rivera, R. George-Tellez, G. Lopez-Mendoza, A. Morales-Pacheco, E. Flores, H. Hopfl, V. Barba, F.J. Fernandez, N. Cabirol, H.I. Beltran, Inorg. Chem. 44 (2005) 5370e5378. [44] J.C. Pessoa, I. Tomaz, R.T. Henriques, Inorg. Chim. Acta 356 (2003) 121e132. [45] J. Costamagna, L.E. Lillo, B. Matsuhiro, M.D. Noseda, M. Villagran, Carbohydr. Res. 338 (2003) 1535e1542. [46] T. Tanase, T. Onaka, M. Nakagoshi, I. Kinoshita, K. Shibata, M. Doe, J. Fujii, S. Yano, Inorg. Chem. 38 (1999) 3150e3159. [47] S. Yano, S. Inoue, Y. Yasuda, T. Tanase, Y. Mikata, T. Kakuchi, T. Tsubomura, M. Yamasaki, I. Kinoshita, M. Doe, Dalton Trans. (1999) 1851e1856. [48] T.M. Kelly, A.B. Tossi, D.J. McConnell, T.C. Strekas, Nucleic Acids Res. 13 (1985) 6017e6034. [49] Q.L. Zhang, J.G. Liu, H. Chao, G.Q. Xue, L.N. Ji, J. Inorg. Biochem. 83 (2001) 49e55. [50] A. Tarushi, G. Psomas, C.P. Raptopoulou, D.P. Kessissoglou, J. Inorg. Biochem. 103 (2009) 898e905. [51] J.W. Lown, Anthracycline and Anthracenedione-based Anticancer Agents. Elsevier, New York, 1988. [52] J. Rojo, J.C. Morales, S. Penadés, Topics in Current Chemistry, vol. 218, Springer-Verlag, Berlin Heidelberg, 2002, 45e92. [53] G.D. Liu, J.P. Liao, S.S. Huang, G.L. Shen, R.Q. Yu, Ana. Sci. 17 (2001) 1031e1036. [54] C. Tong, Z. Hu, W. Liu, J. Agric. Food Chem. 53 (2005) 6207e6212. [55] G. Han, P. Yang, J. Inorg. Biochem. 91 (2002) 230e236. [56] J.A. Cowan, Curr. Opin. Chem. Biol. 5 (2001) 634e642. [57] A. Sreedhara, J.A. Cowan, J. Biol. Inorg. Chem. 6 (2001) 337e347. [58] A.K. Patra, T. Bhowmick, S. Ramakumar, M. Nethaji, A.R. Chakravarty, Dalton Trans. (2008) 6966e6976. [59] D. Lahiri, T. Bhowmick, B. Pathak, O. Shameema, A.K. Patra, S. Ramakumar, A.R. Chakravarty, Inorg. Chem. 48 (2009) 339e349. [60] V. Vichai, K. Kirtikara, Nat. Protocols 1 (2006) 1112e1116. [61] Y.J. Lee, E. Shacter, J. Biol. Chem. 274 (1999) 19792e19798. [62] J.E. Coligan, A.M. Kruisbeck, D.H. Margulies, E.M. Shevach, W. Strober, in: R. Coico (Ed.), Related Isolation Procedures and Functional Assay, Current Protocols in Immunology, John Wiley & Sons, Inc., New York, 1995, p. 3.17.1.