Revisiting the anticancer properties of phosphane(9-ribosylpurine-6-thiolato)gold(I) complexes and their 9H-purine precursors

Abstract

New mono- and di-nuclear thio-purine and thio-purine nucleoside gold(I) complexes were synthesized, characterized, and evaluated in vitro for biological activities in comparison to related known purine complexes. By combining known anti-tumoral thio-purines with R₃PAu moieties as present in auranofin, complexes with enhanced effects and selectivities were obtained, which not only act as cytostatics, but also disrupt tumor-specific processes. Their IC₅₀ values in cytotoxicity test with tumor cell lines ranged from three-digit nanomolar to single-digit micromolar, revealing a tentative structure-activity relationship (SAR). Both the residues R² of the phosphane ligand and R¹ at C2 of the pyrimidine ring had a significant impact on the cytotoxicity. In most cases, the introduction of a ribo-furanosyl group at N9 of the purine led to a distinctly more cytotoxic complex. Most complexes were more active against multi-drug-resistant tumor cells or such lacking functional p53 when compared to the respective untreated wild type cell lines. Some nucleoside complexes displayed an interesting dose-dependent dual mode of action regarding cell cycle arrest and DNA repair mechanism. Some phosphane(purine-6-thiolato)gold (I) complexes had a stronger inhibitory effect on the thioredoxin reductase (TrxR) and on the reactive oxygen species (ROS) generation in cancer cells than is typical of other gold complexes. They also led to DNA fragmentation and showed anti-angiogenic effects. Their stability under test conditions was demonstrated by ⁷⁷Se NMR monitoring of an exemplary selenopurine complex.

Keywords: Anticancer compounds; Auranofin; Gold(I) complexes; Nucleosides; Thioredoxin reductase (TrxR) inhibitors; Triorganophosphanes.

Fig. 1

Effect on the cell cycle of 518A2 melanoma cells after 24 h incubation with different concentrations of 6-MP, 6-TG, and auranofin, as well as complexes 5 and 6. Percentage of 518A2 melanoma cells in G₁, S and G₂/M cell cycle phases and the proportion of potentially apoptotic cells (sub-G1) as determined by flow cytometry. Values are means ± SD of at least three independent experiments

Scheme 1

Synthesis of complexes 1–10

Fig. 2

Relative increase of the intracellular concentration of reactive oxygen species in 518A2 melanoma cells after treatment with solvent (0 µM) or 6-MP, 6-TG, auranofin or complexes 5–7 (1 µM and 5 µM) for 24 h. All experiments were performed in sextuplicate. ROS levels were calculated as mean ± SD with respect to untreated control set to 100%

Fig. 3

Concentration-dependent inhibition of TrxR activity in lysates of 518A2 melanoma cells by 6-MP, 6-TG, auranofin and complexes 5 and 6. TrxR-independent substrate reduction was accounted for by experiments in the presence and absence of the specific TrxR inhibitor aurothiomalate. All values are means ± SD of at least four independent experiments with negative controls (0 µM) set to 100%

Fig. 4

Effects on cell migration of DMSO (control), 6-MP, 6-TG, complexes 5–6 and CA-4 as positive control. 518A2 melanoma cells were treated with the respective IC₅₀ concentrations and their measured after 0, 6, 12 and 24 h. All experiments were performed as triplicates. Values are displayed as means ± SD. Migration progress was set to 0% at 0 h

Fig. 5

DNA damage upon treatment with complex 5 (3.3 µM and 33 µM) and CDDP (33 µM) as positive control, documented with comet assays. Treatment with DMSO as a negative control. Photos are representative of the respective compound and concentration. The images were taken at 100-fold magnification and were processed for better recognizability in terms of brightness and contrast. DNA damage is visualized by migration of DNA fragments out of the nucleus, which is reminiscent of a comet tail

Fig. 6

Tube formation assays: Inhibition of tube formation of Ea.hy926 cells through 6-MP, 6-TG, auranofin, 6-MMR and complex 5, as well as DMSO (neg. control) and CA-4 (pos. control). The formation of tubes, some of which are indicated by yellow arrows, mimics vessel formation during angiogenesis. The lack of it is an indication for anti-angiogenic effects of test compounds. Final concentrations were 5 µM, except for CA-4 (0.01 µM). Photos are representative of the respective compound. The images were taken at tenfold magnification and were processed for better recognizability in terms of brightness and contrast