Biological activity of Sn(IV) and Au(I) thiolates

The activity of organotins is determined by: (1) the nature of the substituents; (2) the coordination availability of the Sn atom to form a bond with the target; (3) a relatively stable L–Sn bond (for example, S–Sn); (4) slow hydrolytic destruction [39]. Synthesis and complex study of the biological activity of Sn and Au complexes containing antioxidant 2,6-di-tert-butylphenol fragment was carried out recently. The structures of compounds (Scheme 2) were confirmed by ¹H, ¹³C NMR, X-ray diffraction and elemental analysis. The mechanisms of their biological action and “structure – activity” correlations has been intensively studied. A comparative study of precursors Ph₃SnCl (Sn-I), AuPPh₃Cl (Au-I) and 2,6-di-tert-butylphenol (RSH) and their complexes Ph₃SnSR (Sn-II) and Ph₃PAuSR (Au-II) in the model processes of interaction with ROS and electron transport was carried out. Cytotoxicity in vitro on human cancer cell lines, cell cycle and influence of various structural fragments on the activity of compounds were studied [31], [32], [40].

Scheme 2:

Structures of tin and gold complexes based on 2,6-di-tert-butyl-4-mercaptophenol. Molecular structure of compounds Sn-II and Au-II (hydrogen atoms are omitted for clarity).

The study of redox activity using the CUPRAC test

The redox activity was studied in CUPRAC (Cupric Ion Reducing Antioxidant Capacity) test [41]. This method is based on the reduction of Cu(II) ions by the studied compound forming Cu(I) complex with neocuproin (2,9-dimethyl-1,10-phenanthroline), which has a maximum absorption at 450 nm. Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid) was used as standard, the results for complexes and starting compounds are presented in Trolox equivalents antioxidant capacity (TEAC) (Table 1) [32], [42].

The initial chlorides Au-I and Sn-I are inactive in the Cu(II) reduction. The most active reducing agent was Sn-II for which the Trolox equivalent was 1.7 times higher than Trolox and 1.4 times higher than organic precursor RSH. Introduction of a phenolic fragment in gold compound Au-II resulted in less antioxidant activity than the Sn-II. It can be assumed that SnPh₃ fragment increases the stability of the phenoxyl radical is responsible for the antioxidant activity.

DPPH-test

The radical scavenging activity of compounds has been studied in the process of hydrogen atom transfer from the phenol moiety to the stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) to give diphenylpicrylhydrazine and phenoxyl radicals (Scheme 3) which can undergo further reactions such as coupling, fragmentation and addition [43].

Scheme 3:

Mechanism of radical scavenging activity of 2,6-di-tert-butylphenols with DPPH.

Antiradical activity was evaluated as the amount of antioxidant necessary to decrease the initial concentration of DPPH by 50% (Efficient Concentration=EC₅₀). The EC₅₀ values for Sn-II and Au-II were 15±4 and 31±2 μM, respectively, whereas the activity of RSH was several times lower (55±10 μM). Compounds Au-I and Sn-I were not active in this test since they do not possess active OH group. The antioxidant activity depends on metal atom nature in the phenol containing complexes. Thus, the antioxidant activity of gold and tin compounds bearing hindered phenol group is mainly determined by the ability to hydrogen atom abstraction from the –OH group and such compounds possess radical scavenging activity.

Evaluation of the ability of compounds to react with NADPH

NADPH affects the pool of adenine nucleotides which plays an important role in antioxidant defense system. The ability of compounds to oxidize NADPH was monitored spectrophotometrically in the presence of NADPH at λ_max 340 nm for 30 min. Since the absorbance and structure of the spectra did not change during incubation, the absence of a reaction between the substances and NADPH can be postulated.

Reaction with ROS

Interaction with superoxide radical anion O₂˙⁻

The activity of the complexes with respect to the superoxide radical anion O₂˙⁻ generated in the xanthine/xanthine oxidase enzymatic system was determined by the change in the reduction rate of tetrazolium blue to formazan (Fig. 1) [44]. All compounds were practically inactive, the initial Au-I gold complex showed little activity (Fig. 1b).

Fig. 1:

Kinetic curves of formazane accumulation in the xanthine/xanthine oxidase system in the presence of different compounds (a) and different concentrations of the Au-I (b).

Assessment of the ability of compounds to react with H₂O₂

Spectrophotometric monitoring of the reactivity of the studied compounds with respect to H₂O₂ in the 200–350 nm range [46] showed no changes in the structure of the spectra during incubation (10 min) indicating the absence of interaction between substances.

Fe³⁺-induced lipid peroxidation

Mitochondria are the main source of intracellular ROS generation. Abnormal level of ROS causes the oxidative damage of intracellular structures as proteins, nucleic acids, lipids, membranes and other biomolecules with consequent injury to cells. ROS are proposed to be involved in all the steps of carcinogenesis [38]. Lipid peroxidation of mitochondrial membranes leads to disturbances of oxidative phosphorylation, a major source of energy. The monitoring of lipid peroxidation in mitochondrial suspension can be followed by the accumulation of substances that reacted with thiobarbituric acid (TBARs) [47]. Oxidative damage (lipid peroxidation) was induced by Fe³⁺. RSH was found to be effective antioxidant with (IC₅₀ 0.2 μM), whereas Sn-II demonstrated moderate antioxidant activity (IC₅₀ 3.1 μM) unlike Sn-I with prooxidant properties. This effect is observed most likely due to the presence of a metal atom (Sn) in their structure [48].

The influence of gold complex Au-II and their precursors RSH and Au-I on lipid peroxidation of isolated rat brain mitochondria was studied. In contrast to Au-I, RSH and the Au-II similarly decreased the TBARs concentration upon Fe³⁺-induced lipid peroxidation of mitochondria (Fig. 2). The tendency to decrease the TBARs level in the presence of phenolcontaining complexes and RSH leads one to suggest the main role of the phenol moiety in the antioxidant mechanism.

Fig. 2:

Influence of gold complex Au-II and precursors (RSH and Au-I) on Fe³⁺-induced lipid peroxidation of isolated rat brain mitochondria.

Toxicity of Sn and Au compounds

Study of apoptosis and cell cycle

The effect of compounds on cell death of cultured cells during incubation for 48 h with compounds at 2·IC₅₀ concentration was studied. After incubation, the cells were removed from the substrate, washed and the percentage of apoptotic and necrotic cells was detected using a cytofluorimetry. This analysis is base on the detection of phosphatidylserine (PS) on the surface of apoptotic cells using fluorescently labeled Annexin V in combination with the dead cell marker 7-AAD [49]. Annexin V is a Ca²⁺-dependent phospholipid binding protein that has a high affinity for PS, a membrane component usually localized to the inner surface of the cell membrane [50]. At the beginning of apoptosis, PS molecules move to the outer surface of the cell membrane, where Annexin V can easily bind them. At the late stage apoptotic cells show a loss of membrane integrity. Membrane-impervious dye 7-AAD is used to distinguish dead cells from early apoptotic cells. Analysis of apoptotic profile can distinguish four cell populations:

(1) Viable cells that do not undergo apoptosis: Annexin V (−) and 7-AAD (−); (2) Early apoptotic cells: Annexin V (+) and 7-AAD (−); (3) Late apoptotic cells: Annexin V (+) and 7-AAD (+); (4) Cells that have died by non-apoptotic pathway: Annexin V (−) and 7-AAD (+). The obtained results of the distribution of 7-AAD-positive and negative and AnV-positive and negative cells in % are presented in Fig. 3 and Table 5 for MCF-7 and HCT-116 cells.

Fig. 3:

Apoptotic profile of cancer cells MCF-7 after treatment with different compounds at μM after 48 h. Concentration of compounds 2·IC₅₀.

For the MCF-7 cells Au-I and Au-II complexes after 48 h of incubation the percentage of late apoptotic cells is maximal. However, there are no significant differences in the percentage of cells in the pool of early and late apoptotic cells for the Au-I compound and Au-II with phenol phragment. The RSH does not induce tumor cell apoptosis. It was shown previously that the Sn-II induces not only depolarization of mitochondria but also mitochondrial swelling and opens the mitochondrial permeability transition pores (MPTp) leading to the cell death [51].

Incubation with compound Sn-II increases in the pool of cells of early apoptosis by 1.5 times compared to the original compound Sn-I, but the percentage of late apoptotic cells was 3 times less. After 48 h of incubation HCT-116 cells were more sensitive to the action of the studied compounds. The percentage of late apoptotic cells for tin and gold complexes was higher than 50%. For Sn-II and Au-II complexes containing a phenolic group the number of late apoptotic cells was lower than early apoptotic cells compared to the precursors Au-I and Sn-I. Thus, for HCT-116 cells the effect of the antioxidant fragment R in Au-II and Sn-II retards the development of apoptosis. For both cell lines, the percentage of late apoptotic cells in the starting substances was higher than in the complexes, furthermore, the number of cells in the early apoptosis stage was higher for compounds with an antioxidant fragment (Fig. 4).

Fig. 4:

Percentage of MCF-7 and HCT-116 cells in early and late apoptosis after 48 h of incubation.

The effect of compounds in 2·IC₅₀ concentration on the cell cycle was studied by mixture of reagents including the nuclear DNA intercalating dye propidium iodide (PI) and RNases in a proprietary formulation after incubation for 24 and 72 h [50].

The technique is based on the determination of DNA content by PI staining to recognize and measure the percentage of cells in each phase of the cell cycle (G0/G1, S and G2/M). PI distinguishes cells at different stages of the cell cycle based on different DNA content in the presence of RNase which increases the specificity of DNA staining. Cells in the G0/G1 phase contain two copies of each chromosome. As cells progress through the cell cycle, they synthesize chromosomal DNA (S-phase). The fluorescence intensity from PI increases until all chromosomal DNA doubles (G2/M phase). At this stage, G2/M cells fluoresce at twice the intensity of the G0/G1 population. G2/M cells divide into two daughter cells at the end of the cycle. The effect of the studied compounds on the cell cycle for MCF-7 and HCT-116 cells is presented in Table 6. For the MCF-7 cell line the effect of the compounds is insignificant. It can be noted that Au-II complex presumably blocks G0/G1 phase. For Au-II and Sn-II complexes decreased the part of cells in G0/G1 phase that seems to be associated with the appearance of dead cells whose DNA is destroyed. The influence of the studied compounds on the cell cycle for HCT-116 cells is presented in Figs. 5 and 6.

Fig. 5:

Cell cycle analysis by flow cytometry in HCT-116 cancer cells were treated by RSH, Sn-I and Sn-II for 24 and 72h, concentration of compounds 2·IC₅₀.

Fig. 6:

Cell cycle analysis by flow cytometry in HCT-116 cancer cells were treated by Au-I and Au-II for 24 and 72h, concentration of compounds 2·IC₅₀.

Au-I and Sn-I do not change essentially the cell cycle of HCT-116 cells. The Au-II causes a block at the G0/G1 stage. RSH results in a minor G1 phase block after 24 h of incubation. Sn-II causes a marked block of the cell cycle in the G2/M phase after 24 and 72 h and initiate cell death. The results demonstrate that the effect on cell cycle depends on compounds and cells type. The pronounced influence was found for HCT-116 cells. High cytotoxicity of Sn-II towards MCF7 and HCT-116 cancer cells indicates the perspectives of this compound for preclinical studies.

Mechanisms of Sn and Au cytotoxicity

The possible mechanisms of Sn-II cytotoxicity including tubulin binding and influence on tubulin assembly in microtubules was investigated previously [51]. Sn-II revealed the significant inhibition of tubulin polymerization without disturbances of microtubules structure (Fig. 7).

Fig. 7:

The influence of Sn-II on tubulin polymerization (a) kinetic curve of tubulin polymerization as the change in fluorescence intensity; (b) the concentration dependence of tubulin polymerization rate (Vp) in the presence of compound Sn-II.

Since tubulin is a sulphhydryl-rich protein with 20 cysteine residues distributed across both subunits, it can react with sulphhydryl-directed reagents, the interaction of Sn-II with tubulin did not provide distinct evidences for the putative binding modes and modes of action of the compounds. For insight of mode of interaction between Sn-II and tubulin, docking simulation for this compound towards well-studied colchicine, paclitaxel and vinblastine binding pockets in the tubulin molecule were carried out. The main contribution into the calculated binding energy score of compoundы was provided by van-der-Waals interactions. A large size of the molecule hampers binding with colchicine site. Predicted binding modes of the compound in paclitaxel and vinblastine binding sites are much more reasonable (Fig. 8).

Fig. 8:

The best scored orientations of compound Sn-II (a, b) in the ligand binding sites of 4EB6 (a) and 1JFF (b). Cognate ligands [vinblastine (a) and paclitaxel (b)] are shown as blue translucent surfaces, organotin is shown as balls-and sticks and red coloured surface [51].

Since organotins form significantly fewer hydrogen bonds than natural compounds, their binding is ruled mostly by shape complementarity. Sn-II can overlap with paclitaxel and vinblastine sites, but their totally different shape requires specific binding modes and this compound cannot penetrate into the deepest parts of the pockets.

Thioredoxin reductase (TrxR) – redox enzyme which maintains the balance between pro- and anti-oxidative states in living cells. TrxR as a major target of auranofin been inhibited by auranofin leads to an oxygen stress, cytotoxicity and induced damage of tissues in which auranofin accumulates that complicate the practical use of auranofin. Many attempts have been made to minimize the pro-oxidant effect of auranofin. Modulation of mammalian TrxR was investigated as a tentative drug target. The Au-II inhibited TrxR with an IC₅₀ value of 0.57 ±0.15 mM. Auranofin showed a higher activity (IC₅₀ 0.016±0.003 mM) whereas RSH had no effect at up to 10 mM [32]. These results justified our attempts of chemical modification of Au-I leading to derivatives with an attenuated prooxidant potency.

Toxicity of Sn and Au compounds in vivo

The experiments were performed on white Balb/c mice weighing 18–20 g, age 8–10 weeks. Compounds Sn-II and Au-II were injected into the abdominal cavity in physiological buffer solution. The volume of injected solutions is 0.5 ml, the volume of solvent (DMSO) does not exceed 125 μl. In the initial experiments, the upper limit of tolerability of administered doses was estimated. Two doses were chosen: 62.5 mg/kg and 125 mg/kg (1.25 mg/mouse and 2.5 mg/mouse, respectively). Each group had 4 animals. After 7 days all mice in both groups (62.5 mg/kg Sn-II or Au-II) were alive. In Sn-II group (125 mg/kg) two mice died on third day. In Au-II (125 mg/kg) group all mice died after first day of experiment. The results of the initial evaluation showed that for compounds Sn-II and Au-II, a dose of 125 mg/kg was lethal. In the following experiments, animals received doses of compounds Sn-II and Au-II from 70 mg/kg to 110 mg/kg once intraperitoneal injection in each group, 6 mice. Observations were continued for 21 days. Table 7 shows the results of the study of acute general resorptive toxicity of compounds Sn-II and Au-II. The introduction was accompanied by toxic manifestations (behavioral-stooping, numbness, sometimes inactivity). When administered doses of 70–80 mg/kg, these manifestations lasted from several hours to 1 day, after which the condition of the animals was restored and, in the future, did not differ from that of intact mice. For compounds Sn-II and Au-II, administration of doses >80 mg/kg was fatal for some animals. Thus, 80 mg/kg is the maximum tolerated dose.