Cytotoxic Activity of Organotin(IV) Derivatives with Triazolopyrimidine Containing Exocyclic Oxygen Atoms

In this study cytotoxicity of organotin(IV) compounds with 1,2,4-triazolo[1,5-a]pyrimidines, Me3Sn(5tpO) (1), n-Bu3Sn(5tpO) (2), Me3Sn(mtpO) (3), n-Bu3Sn(mtpO) (4), n-Bu3Sn(HtpO2) (5), Ph3Sn(HtpO2) (6) where 5HtpO = 4,5-dihydro-5-oxo-[1,2,4]triazolo-[1,5-a]pyrimidine, HmtpO = 4,7-dihydro-5-methyl-7-oxo-[1,2,4]triazolo-[1,5-a]pyrimidine, and H2tpO2 = 4,5,6,7-tetrahydro-5,7- dioxo-[1,2,4]triazolo-[1,5-a]-pyrimidine, was assessed on three different human tumor cell lines: HCT-116 (colorectal carcinoma), HepG2 (hepatocarcinoma) and MCF-7 (breast cancer). While 1 and 3 were inactive, compounds 2, 4, 5 and 6 inhibited the growth of the three tumor cell lines with IC50 values in the submicromolar range and showed high selectivity indexes towards the tumor cells (SI > 90). The mechanism of cell death triggered by the organotin(IV) derivatives, investigated on HCT-116 cells, was apoptotic, as evident from the externalization of phosphatidylserine to the cell surface, and occurred via the intrinsic pathway with fall of mitochondrial inner membrane potential and production of reactive oxygen species. While compound 6 arrested the cell progression in the G2/M cell cycle phase and increased p53 and p21 levels, compounds 2, 4 and 5 blocked cell duplication in the G1 phase without affecting the expression of either of the two tumor suppressor proteins. Compounds 1 and 2 were also investigated using single crystal X-ray diffraction and found to be, in both cases, coordination polymers forming 1 D chains based on metal-ligand interactions. Interestingly, for n-Bu3Sn(5tpO)(2) H-bonding interactions between 5tpO− ligands belonging to adjacent chains were also detected that resemble the “base-pairing” assembly and could be responsible for the higher biological activity compared to compound 1. In addition, they are the first example of bidentate N(3), O coordination for the 5HtpO ligand on two adjacent metal atoms.


Introduction
Organotin (IV) compounds have been receiving increasing attention over the past years [1][2][3][4][5][6] due to their possible use as biologically active non-platinum chemotherapeutic agents, that might have lower toxicity, better excretion properties and fewer side effects than platinum based drugs [7][8][9]. These compounds, even at low doses, present high anti-proliferative activity which, in some cases, is higher than that of cisplatin. Organotin compounds induce apoptosis by binding to DNA at external phosphate groups and by altering the intracellular metabolism of the phospholipids. Investigations have been carried out to test their antitumor activity and it has been observed that several triorganotin derivatives show very promising in vitro antitumor properties against a wide panel of tumor cell lines of human origin unlike di-and mono-organotin (IV) analogs, due to their ability to bind proteins [10].
1,2,4-triazolo [1,5-a] pyrimidine derivatives have been used as model systems to mimic the reactivity between purines and several metal ions [11] due to their ability to coordinate through different nitrogen atoms with accessible electron pairs in their basic skeleton. Furthermore, their versatility can be increased by changing the number of exocyclic substituents on the triazolopyrimidine rings. Most of the significant investigations with coordination compounds have been directed to the development of anti-tumor compounds of different metals [12] that could show improved therapeutic indexes and wider activity spectra. Such derivatives are interesting for their many biological effects such as antimicrobial [13,14], antitumor [15][16][17][18][19] and antiparasitic [20][21][22] activity.
The ligand 4,5-dihydro-5-oxo- [1,2,4]triazolo-[1,5-a] pyrimidine (5HtpO), the structures of which were described by Abul Haj et al. [23], is a triazolopyrimidine containing exocyclic oxygen atoms and is an isomer of the purine base hypoxanthine. Various studies have shown that the use of additional positions of coordination for oxygen with complexing capacity and the presence of a metal ion improve the antiparasitic [24] and antibacterial [25] activities.
In the polynuclear [Cd 2 (µ-5tpO) 2  Compound 1 exhibits two molecules, A and B, in the asymmetric unit. The two molecules each form a metal coordination polymer, and π-π stacking interactions link the polymers into a three-dimensional network (with contacts ranging around 3.4 Å, Figure 1b predicted for nucleobase analogues such as our oxo-tp ligands but not so commonly shown, seems to dictate the molecular packing of 2. Actually, no aromatic interactions take place. Selected geometric parameters (Å, • ) for 2 are summarized in Table 1.

Cytotoxicity Assay
Cytotoxicity of the organotin(IV) compounds 1-6, characterized by three different ligands 5HtpO, HmtpO and H 2 tpO 2 , was studied on human tumor cell lines HCT-116, HepG2 and MCF-7 using a MTT test. The viability of cellular monolayers after 24 h of treatment with the compounds and their ligands, tested in the 0.01-1 µM range, is reported in Figure 3. While all the ligands and the compounds Me 3 Sn(5tpO) 1 and Me 3 Sn(mtpO) 3 had no influence on cell growth under the conditions used, compounds n-Bu 3 Sn(5tpO) 2, n-Bu 3 Sn(mtpO) 4, n-Bu 3 Sn(HtpO 2 ) 5 and Ph 3 Sn(HtpO 2 ) 6 effectively inhibited cell viability in a dose-dependent manner. Based on the IC 50 values, the cytotoxicity of the compounds on HCT-116 and HepG2 cells followed the order 4 > 6 > 2 > 5, while on MCF-7 cells the compounds appeared less cytotoxic following the order 6 > 4 > 2 > 5 (Table 2). Moreover, complexes 2, 4, 5 showed a different cytotoxic activity related to their ligands being HmtpO > 5HtpO > H 2 tpO 2 . Ph 3 Sn(HtpO 2 ) 6 appeared more effective against the growth of all the cell lines than n-Bu 3 Sn(HtpO 2 ) 5, which has the same ligand. Interestingly, the compounds were more cytotoxic than cisplatin, used for comparison as gold standard (Table 2).
Selective cytotoxicity is a pivotal requirement for anticancer drugs, and activity of the compounds on intestinal normal-like differentiated Caco-2 cells and human bronchial epithelial cells (16-HBE) was assessed in additional experiments. While a reduction of the cell survival was detected after treatment with the 2, 4 and 5 derivatives in the 1-10 µM range on both the cell lines, compound 6 did not affect the cell viability at any assayed concentration ( Figure 4). However, as the calculated selectivity index (SI, Table 3) greatly exceeds the value accepted as threshold for antitumor drugs (SI = 2.0) [35], potential use of all the synthesized complexes as antiproliferative agents could be considered.
Selective cytotoxicity is a pivotal requirement for anticancer drugs, and activity of the compounds on intestinal normal-like differentiated Caco-2 cells and human bronchial epithelial cells (16-HBE) was assessed in additional experiments. While a reduction of the cell survival was detected after treatment with the 2, 4 and 5 derivatives in the 1-10 μM range on both the cell lines, compound 6 did not affect the cell viability at any assayed concentration ( Figure 4). However, as the calculated  selectivity index (SI, Table 3) greatly exceeds the value accepted as threshold for antitumor drugs (SI = 2.0) [35], potential use of all the synthesized complexes as antiproliferative agents could be considered.  Values are the mean ± SD of three separate experiments in triplicate. LC50 concentration, which is lethal to 50% of the normal cells, compared to untreated controls.

Cell Death
In order to analyze the mode of cell death induced by the active organotin compounds, HCT-116 were treated with the individual compounds at 0.1 μM concentration for 24 h before doublestaining with AnnexinV/PI. Cisplatin, one of the most widely used chemotherapy drugs, was used as positive control. The results showed that all tested compounds increased the number of early apoptotic dead HCT-116 cells without causing any necrotic damage, generally accompanied in vivo by inflammatory processes ( Figure 5).

Cell Death
In order to analyze the mode of cell death induced by the active organotin compounds, HCT-116 were treated with the individual compounds at 0.1 µM concentration for 24 h before double-staining with AnnexinV/PI. Cisplatin, one of the most widely used chemotherapy drugs, was used as positive control. The results showed that all tested compounds increased the number of early apoptotic dead HCT-116 cells without causing any necrotic damage, generally accompanied in vivo by inflammatory processes ( Figure 5).

Mitochondrial Dysfunction
In many systems, apoptosis is associated with the loss of mitochondrial inner membrane potential (Δψm), which is responsible for the release of some pro-apoptotic factors from the organelle. We investigated the involvement of mitochondria in apoptosis induced by the organotin compounds using the lipophilic cationic fluorochrome DiOC6, a fluorescent mitochondria-specific and voltagedependent dye. Measurements of Δψm using flow cytometry analysis showed that compounds 2, 4, 5 and 6 at 0.1 μM induced in HCT-116 cells, after 24 h of treatment, a significant decrease in DiOC6 (3) uptake, indicating induction of mitochondrial dysfunction ( Figure 6). Mitochondrial permeability dysfunction appeared comparable to that produced by 20 μM cisplatin ( Figure 6).

Mitochondrial Dysfunction
In many systems, apoptosis is associated with the loss of mitochondrial inner membrane potential (∆ψm), which is responsible for the release of some pro-apoptotic factors from the organelle. We investigated the involvement of mitochondria in apoptosis induced by the organotin compounds using the lipophilic cationic fluorochrome DiOC6, a fluorescent mitochondria-specific and voltage-dependent dye. Measurements of ∆ψm using flow cytometry analysis showed that compounds 2, 4, 5 and 6 at 0.1 µM induced in HCT-116 cells, after 24 h of treatment, a significant decrease in DiOC6(3) uptake, indicating induction of mitochondrial dysfunction ( Figure 6). Mitochondrial permeability dysfunction appeared comparable to that produced by 20 µM cisplatin ( Figure 6). Since the mitochondrial dysfunction may be related to intracellular redox unbalance and induction of apoptosis in various cell types, we explored whether the organotin(IV) complexes could stimulate ROS generation in HCT-116 cells. As shown in Figure 7, compounds 2, 4, 5 and 6 after 24 h of treatment induced, when compared to control, an increase in ROS production, cytofluorimetrically detected by the fluorescent dye DCFH-DA. Cisplatin, at 20 μM, produced a similar effect.

Cell Cycle Analysis
A disturbance of the cellular redox state could induce apoptosis triggering a ROS-dependent signaling pathway leading to block of the cell cycle. The effects of active 2, 4, 5 and 6 complexes on HCT-116 cell cycle distribution were determined using flow cytometric analysis after staining of DNA with propidium iodide (PI). As shown in Figure 8, compounds 2, 4 and 5 after 24 h of treatment induced a block of cell duplication in the G0/G1 phase, whereas compound 6 caused a cell cycle arrest in phase G2/M. Although compounds 5 and 6 share the same ligand (H 2 tpO 2 ), they caused proliferation arrest at different phases of the cell cycle, underlining the relevance of the organotin moieties in the biological mechanism of the complexes. In the end, all compounds caused an increase of the percentage of cells in the sub-G1 phase, which is representative of cells with fragmented DNA, confirming that the cells were carried through apoptosis. 2.2.5. p53-p21 WAF1 Protein Levels p53, a major tumor suppressor protein, orchestrates various biological events including cell cycle arrest, cellular senescence and DNA repair, in addition to apoptosis [36]. Functionally, p53 is a transcription factor forming a homo-tetramer to activate nearly 500 target genes [37]. In mammalian cells, p53 induces a large number of genes involved in various steps of apoptosis signaling and execution [38]. These include BH3 domain-only proapoptotic proteins, death receptors and apoptosis execution factors. In addition, p53 promotes the transcriptional activation of p21 WAF1 that, binding to several cyclins, causes arrest of the cell cycle at different phases [39,40]. To assess the involvement of p53-p21 WAF1 signaling pathway in the apoptosis triggered by the organotin compounds, we investigated the compounds' influence on the expression of both the proteins by immunoblotting. While 24 h treatment of HCT 116 cells with 2, 4, 5 did not vary the level of p53 or p21, incubation with compound 6 resulted in a net increment (about 1, 5fold) of the levels of both the tumor suppressor proteins (Figure 9). 2.2.5. p53-p21 WAF1 Protein Levels p53, a major tumor suppressor protein, orchestrates various biological events including cell cycle arrest, cellular senescence and DNA repair, in addition to apoptosis [36]. Functionally, p53 is a transcription factor forming a homo-tetramer to activate nearly 500 target genes [37]. In mammalian cells, p53 induces a large number of genes involved in various steps of apoptosis signaling and execution [38]. These include BH3 domain-only proapoptotic proteins, death receptors and apoptosis execution factors. In addition, p53 promotes the transcriptional activation of p21 WAF1 that, binding to several cyclins, causes arrest of the cell cycle at different phases [39,40]. To assess the involvement of p53-p21 WAF1 signaling pathway in the apoptosis triggered by the organotin compounds, we investigated the compounds' influence on the expression of both the proteins by immunoblotting. While 24 h treatment of HCT 116 cells with 2, 4, 5 did not vary the level of p53 or p21, incubation with compound 6 resulted in a net increment (about 1, 5fold) of the levels of both the tumor suppressor proteins (Figure 9).

X-Ray Crystallography of Me3Sn(5tpO), 1 and n-Bu3Sn(5tpO), 2
Single crystal data for compounds Me3Sn(5tpO) 1 and n-Bu3Sn(5tpO) 2 were collected at low temperature (200 K) on an Oxford XCalibur S CCD diffractometer equipped with a graphite monochromator (Mo-Kα radiation, λ = 0.71073Å) and with a cryostat CryoStream800. Two twin unit cells were indexed for 1 and 2, and the reflection data were integrated with the default configuration for twinned crystals of CrysAlisPro software. Subsequent structure solution and refinement were performed using the HKLF4 file containing non-overlapped reflections. All non-hydrogen atoms were refined anisotropically. HCH atoms for all compounds were added in calculated positions and refined riding on their respective carbon atoms. SHELX97 [41] was used for structure solution and refinement on F2. The program Mercury [42] was used to calculate intermolecular interactions and for molecular graphics. Data collection and refinement details are listed in Table 4. Crystal data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB21EZ, UK; fax: (+44)1223-336-033; or email: deposit@ccdc.cam.ac.uk). CCDC numbers 1908619-1908620.
3.2. X-ray Crystallography of Me 3 Sn(5tpO), 1 and n-Bu 3 Sn(5tpO), 2 Single crystal data for compounds Me 3 Sn(5tpO) 1 and n-Bu 3 Sn(5tpO) 2 were collected at low temperature (200 K) on an Oxford XCalibur S CCD diffractometer equipped with a graphite monochromator (Mo-Kα radiation, λ = 0.71073Å) and with a cryostat CryoStream800. Two twin unit cells were indexed for 1 and 2, and the reflection data were integrated with the default configuration for twinned crystals of CrysAlisPro software. Subsequent structure solution and refinement were performed using the HKLF4 file containing non-overlapped reflections. All non-hydrogen atoms were refined anisotropically. H CH atoms for all compounds were added in calculated positions and refined riding on their respective carbon atoms. SHELX97 [41] was used for structure solution and refinement on F2. The program Mercury [42] was used to calculate intermolecular interactions and for molecular graphics. Data collection and refinement details are listed in Table 4. Crystal data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB21EZ, UK; fax: (+44)1223-336-033; or e-mail: deposit@ccdc.cam.ac.uk). CCDC numbers 1908619-1908620.

Viability Assay
The synthesized compounds 1-6 with three different ligands 5HtpO, HmtpO and H 2 tpO 2 were dissolved in dimethylsulfoxide (DMSO) and then diluted in culture medium so that the effective DMSO concentration did not exceed 0.1%. HTC116 (human colorectal carcinoma), HepG2 (human hepatocellular carcinoma), MCF-7 (human breast cancer), 16-HBE (human bronchial epithelium) and Caco-2 (human colorectal carcinoma) cell lines were purchased from American Type Culture Collection, Rockville, MD, USA. All cell lines were grown in RPMI medium supplemented with L-glutamine (2 mM), 10% fetal bovine serum (FBS), penicillin (100 U/mL), streptomycin (100 µg/mL) and gentamicin (5 µg/mL). Cells were maintained in log phase by seeding twice a week at a density of 3 × 10 8 cells/L in humidified 5% CO 2 atmosphere, at 37 • C. In all experiments, after plating, cells were allowed to adhere overnight and then treated with the compounds or vehicle alone (control cells), whereas Caco-2 cells were treated 15 days after confluence, at which time the cells are differentiated in normal intestinal-like cells [43]. No differences were found between cells treated with DMSO 0.1% and untreated cells in terms of cell number and viability.
Cytotoxic activity of the organotin compounds against human tumor cell lines (HCT-116, HepG2 and MCF-7) and human normal cells (16-HBE and intestinal-like differentiated Caco2 cells) was determined using the MTT colorimetric assay based on the reduction of 3-(4,5-dimethyl-2-thiazolyl)bromide-2,5-diphenyl-2H-tetrazolium (MTT) to purple formazan using mitochondrial dehydrogenases of living cells, as reported [44]. This method is commonly used to illustrate inhibition of cellular proliferation. Monolayer cultures were treated for 24 h with various concentrations (0.01-1 µM) of the drugs. Cisplatin was used for comparison. Briefly, all cell lines were seeded at 1.0 × 10 4 cells/well in 96-well plates containing 200 µL RPMI. When appropriated, cells were washed with fresh medium and incubated with the compounds in RPMI. After incubation, cells were washed, and 50 µL FBS-free medium containing 5 mg/mL MTT was added. The medium was discarded after a 2 h incubation at 37 • C by centrifugation, and formazan blue formed in the cells was dissolved in DMSO. The absorbance, measured at 570 nm in a microplate reader (Bio-RAD, Hercules, CA, USA) of MTT formazan of control cells was taken as 100% of viability. The growth inhibition activity of compounds was estimated by comparison with the not-treated control. The values for the inhibitory concentration for 50% of the tested compounds (IC 50 ) were calculated using a sigmoidal model using GraphPad Prism 5.02 from GraphPad Software (San Diego, CA, USA). Each experiment was repeated three times in triplicate to obtain the mean values.

Selectivity Index (SI)
Formazan of control cells was taken as 100% viability and the 50% lethal concentrations of the synthesized complexes (LC 50 ) were determined by plotting the graph of cell viability versus the concentrations. The selectivity index (SI) values were calculated for each compound by dividing the LC 50 of normal differentiated Caco2 or 16-HBE cells by the IC 50 of HCT-116 tumor cells. Each experiment was repeated three times in triplicate.

Measurement of Phosphatidylserine Exposure
The externalization of phosphatidylserine to the cell surface was detected using flow cytometry by double staining with annexin V/PI. Tumor cells were seeded in triplicate in 24-well culture plates at a density of 5.0 × 10 4 cells/cm 2 . After an overnight incubation, cells were washed with fresh medium and incubated with the compounds prepared as described above. After 24 h, cells were harvested by trypsinization and adjusted at 1.0 × 10 6 cells/mL with buffer according to the manufacturer's instructions (eBioscience, San Diego, CA, USA). One hundred µL of cell suspended solution was added to a new tube and incubated with 5 µL annexin V and 10 µL of a 20 µg/mL propidium iodide (PI) solution at room temperature in the dark for 15 min. Then samples of at least 1.0 × 10 4 cells were subjected to fluorescence-activated cell sorting (FACS) analysis using Epics XL™ flow cytometer with Expo32 software (Beckman Coulter, Fullerton, CA, USA), using appropriate 2-bidimensional gating method.

Measurement of Mitochondrial Transmembrane Potential
Mitochondrial transmembrane potentials (∆ψm) were assessed using flow cytofluorometry, with the cationic lipophilic dye 3,3 -dihexyloxacarbocyanine iodide(3) (DiOC6) (Molecular Probes, Inc., Life Technologies Italia, Monza, Italy), which accumulates in the mitochondrial matrix. Changes in mitochondrial membrane potential are indicated by a reduction in the DiOC6-induced fluorescence intensity. After 24 h treatment, cells were incubated with DiOC6 at a 40 nmol/L final concentration, for 15 min at 37 • C. After centrifugation, cells were washed with phosphate buffer saline (PBS) and suspended in 500 µL PBS. Then, samples of 1.0 × 10 4 cells were subjected to FACS analysis.

Measurement of Intracellular Reactive Oxygen Species (ROS)
ROS level was monitored by measuring fluorescence changes that resulted from intracellular oxidation of 2 ,7 -dichlorofluorescin diacetate (DCFH-DA). DCFH-DA, at 10 µM final concentration, was added to the cell medium 30 min before the end of the treatment. The cells were collected using centrifugation for 5 min at 2000 rpm at 4 • C, washed, suspended in PBS and immediately subjected to FACS analysis. At least 1 × 10 4 cells were analyzed for each sample.

Cell Cycle Analysis
Cell cycle stage was analyzed using flow cytometry. Aliquots of 1.0 × 10 6 cells were harvested using centrifugation, washed with PBS and incubated in the dark in a PBS solution containing Triton X100 (0.1%, v/v), 20 µg/mL propidium iodide (PI) and 200 µg/mL RNase, for 30 min, at room temperature. Then, samples were immediately subjected to FACS analysis. At least 1 × 10 4 events were analyzed for each sample.

Western Blot Analysis
After treatment with the compound, protein extracts were prepared and equal amounts of protein samples (80 µg/lane), subjected to SDS-PAGE, were transferred to nitrocellulose membrane as previously reported [45]. The immunoblot was incubated overnight at 4 • C with blocking solution (5% skim milk), followed by incubation with anti-p21 (F-5, Cat No SC-6246 Santa Cruz Biotechnology) or anti-p53 (DO-1, Cat No SC-126, Santa Cruz Biotechnology) for 1 h at room temperature. Blots were washed two times with Tween 20/Tris-buffered saline (TTBS) and incubated with a 1:2000 dilution of horseradish peroxidase (HRP)-conjugated anti-IgG antibody (Dako Denmark, Glostrup, Denmark) for 1 h at room temperature. Blots were again washed five times with TTBS and then developed using enhanced chemiluminescence (Amersham Life Science, Arlington Heights, IL, USA.). Immunoreactions were also performed using β-actin antibody as loading controls.

Statistical Analysis
Results are given as means and standard deviations. Unless stated otherwise, three independent observations were performed for each experiment thrice replicated. Calculations and graphs were obtained using the INSTAT-3 statistical software (GraphPad Software, Inc., San Diego, USA) with a test for normality followed by ANOVA, with Tukey's correction for multiple comparisons. In all cases, significance was accepted if the null hypothesis was rejected at the p < 0.05 level.

Conclusions
The spectroscopic properties of compounds 1-6 have been previously reported [25]. In this study we report that compounds 2, 4, 5 and 6 effectively inhibited the cell growth of three different lines of human carcinoma (colon, liver and breast), with calculated submicromolar IC 50 values. Cytotoxic activity of the compounds appeared 2-3 orders of magnitude higher than that of cisplatin used as gold standard, and were highly selective (SI > 90) towards the tumor cells. Among the tri-n-butyltin(IV) derivatives 2, 4 and 5, the complex 4 with HmtpO showed stronger cytotoxicity. Although it has been previously demonstrated that n-Bu 3 Sn(HtpO 2 ) 5 owns a greater antifungal and antibiofilm activity than Ph 3 Sn(HtpO 2 ) 6 [25], the present study carried out on human tumor cells indicates an inverse antiproliferative efficacy.
The cell death mechanism exerted by the organotin(IV) derivatives was apoptosis, as established in HCT-116 cells using flow cytometric analysis of externalization of plasma membrane PS, and occurred via the intrinsic pathway, as ascertained using mitochondrial dysfunction. The signaling pathway of apoptosis induced by the compounds appeared distinct. In fact, while compound 6 arrested the cell progression in the G2/M cell cycle phase and increased p53 and p21 WAF1 levels, compounds 2, 4 and 5 induced a block of cell duplication in the G0/G1 phase without affecting either p53 or p21 WAF1 expression. It is interesting in this regard to mention that a triphenyl derivative with N-tert-butoxycarbonyl-L-ornithine, previously synthesized and characterized in our laboratory, induced apoptosis in human hepatoma HepG2 cells associated with an increase of p53 levels [46], suggesting that the induction pathway of the oncoprotein is influenced by the Ph 3 Sn-moiety. At last, since p53 is defective in >50% of tumors, the ability of the 2, 4 and 5 derivatives to induce apoptosis independently of p53 may offer an advantage in anti-tumor therapy.
A SCXRD investigation was carried out on both compounds 1 and 2, the only ones of the series to yield good enough crystals. As for compound 2, its ability, through metal coordination, to form pairs of hydrogen bonded inter-strand, purine-like ligands may certainly be related to an enhanced propensity for recognition processes towards biological targets, in comparison to 1, which is devoid of it and inactive. These ligand-mediated recognition processes may give a contribution towards organotin binding to DNA and help to potentially deliver new metal-based therapies to the clinic. Therefore, a further investigation on the detailed mechanism of pro-apoptotic action exerted specifically by compounds n-Bu 3 Sn(5tpO) 2 and n-Bu 3 Sn(mtpO) 4 may prove of great interest, and is actually planned in the near future, in order to better elucidate their specific signaling pathways.