Arene Variation of Highly Cytotoxic Tridentate Naphthoquinone-Based Ruthenium(II) Complexes and In-Depth In Vitro Studies

Abstract

The main purpose of this study was to synthesize a new set of naphthoquinone-based ruthenium(II) arene complexes and to develop an understanding of their mode of action. This study systematically reviews the steps of synthesis, aiming to provide a simplified approach using microwave irradiation. The chemical structures and the physicochemical properties of this novel group of compounds were examined by ¹H-NMR and ¹³C-NMR spectroscopy, X-ray diffractometry, HPLC-MS and supporting DFT calculations. Several aspects of the biological activity were investigated in vitro, including short- and long-term cytotoxicity tests, cellular accumulation studies, detection of reactive oxygen species generation, apoptosis induction and NAD(P)H:quinone oxidoreductase 1 (NQO1) activity as well as cell cycle analysis in A549, CH1/PA-1, and SW480 cancer cells. Furthermore, the DNA interaction ability was studied in a cell-free assay. A positive correlation was found between cytotoxicity, lipophilicity and cellular accumulation of the tested complexes, and the results offer some important insights into the effects of the arene. The most obvious finding to emerge from this study is that the usually very chemosensitive CH1/PA-1 teratocarcinoma cells showed resistance to these phthiocol-based organometallics in comparison to the usually less chemosensitive SW480 colon carcinoma cells, which pilot experiments suggest as being related to NQO1 activity.

Keywords: anticancer; metal-based drugs; piano-stool complexes; ruthenium.

Figure 1

Chemical structures of clinically studied ruthenium compounds. Ru(II) coordination compound TLD–1433 (A) and Ru(III) drug BOLD–100 (B).

Figure 2

Tridentate ruthenium arene complexes with anticancer properties [17,18,19,20].

Scheme 1

Synthetic pathway of phthiocol (L) (A) and complex syntheses (1–6) (B). (i) = H₂O₂, Na₂CO₃, MeOH; 0 °C to room temperature, 1.5 h; (ii) = SiO₂, H₂SO₄, THF, 70 °C; (iii) = MeOH, microwave, 50–60 °C, 20–30 min.

Figure 3

Arene influence towards the chemical shifts of pyrazolate in MeOD-d₄. R = p-cymene = 1, R = benzene = 2, R = toluene = 3, R = indane = 4, R = biphenyl = 5, R = hexamethylbenzene = 6.

Figure 4

The percentage amount of initial complex vs. time in PBS (pH = 7.4) at 20 °C.

Figure 5

DFT calculations regarding the hydrolysis of complexes (1–6). Relative free energies of complexes (1–6) are set to zero (left) in order to compare the energetic order of the corresponding transition states (middle) and aqua complexes (right).

Figure 6

Inhibition of colony formation in SW480 cells exposed to L and 1–6 at two or three different concentrations for 11 days. The results are given in percentage (%) relative to the untreated control. Columns represent the mean ± standard deviation of three independent experiments.

Figure 7

Semi-logarithmic plot of cellular accumulation after 2 h exposure to compounds 1–6 vs. their IC₅₀ values (96 h exposure) in SW480 cells (A). Correlation between drug accumulation and a theoretical lipophilicity coefficient (96 h exposure) (B). The symbols indicate the compound concentrations applied in cellular accumulation experiments, with filled symbols for 2 µM and open symbols for 5 µM.

Figure 8

Impact of the most cytotoxic complexes 1 and 5 on cell cycle distribution in CH1/PA-1 (A) and SW480 cells (B) upon exposure for 24 h. Complex 1 was applied at 50 µM concentration in CH1/PA-1 cells and 0.078 µM in SW480 cells. Complex 5 was applied at 25 µM in CH1/PA-1 cells and 0.078 µM in SW480 cells. Histograms represent the effects of the IC₅₀ (according to MTT assays) relative to untreated control in a representative experiment. The different colors indicate the cell fractions in G1/G0 phase (purple), S phase (yellow) and G2/M phase (green), calculated by integrating the histograms of fluorescence signals by using Watson algorithm (indicated by magenta lines). In the column diagrams, the data are given as means ± SDs from at least three independent experiments. Unpaired t-test with Welch’s correction was performed for statistical evaluation (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns: not significant).

Figure 9

To distinguish the viable (Q4), early apoptotic (Q3), late apoptotic (Q2) and necrotic (Q1) cell counts, SW480 cells treated with complexes 1 and 5 at three different concentrations were subject to annexin V-FITC (x-axis) and PI (y-axis) staining, detected by flow cytometry. As a positive control, 1-P was used [42]. The dot plots represent a representative experiment out of at least three.

Figure 10

Time-dependent impact on NQO1 activity in SW480 (left panel) and CH1/PA-1 (right panel) cell extracts after treatment with the most active complexes, 1 and 5, or dicoumarol (positive control) compared to negative controls with and without DMSO (concentrations corresponding to the content in solutions of the test compounds). Data are expressed as OD (450 nm) per 100 µg/mL of total cellular protein.