Synthesis of platinum(II) complexes of 2-cycloalkyl-substituted benzimidazoles and their cytotoxic effects

2.1 Materials and instrumentation

All chemicals and solvents used were of reagent grade (Merck, Darmstadt, Germany; Sigma-Aldrich, St. Louis, MO, USA) and were used without further purification. All cell culture reagents were purchased from Gibco Life Technologies (Carlsbad, CA, USA).

Melting points were determined in an Electrothermal 9200 melting point apparatus and are uncorrected. Infrared (IR) spectra were obtained using a Perkin Elmer Spectrum 400 FTIR/FTNIR spectromer equipment with a Universal ATR Sampling Accessory in the range of 4000–400 cm^–1. For the region of 400–200 cm^–1, the samples were analyzed with a FTIR IFS 66/S spectrometer. Elemental analyses were performed with a Leco 932 CHNS analyzer, and proton magnetic resonance (¹H-NMR) spectra were recorded in dimethylsulfoxide (DMSO)-d₆ (Merck) on Varian Mercury 400 MHz FT NMR spectrometer. High resolution mass spectra data (HRMS) were collected in-house using a Waters LCT Premier XE mass spectrometer (high sensitivity orthogonal acceleration time-of-flight instrument) operating in ESI (+) (positive ion mode), coupled to an AQUITY ultra performance liquid chromatography system (Waters Corporation, Milford, MA, USA). Thin-layer chromatography (TLC) was performed on precoated aluminum plates (Silicagel 60 F₂₅₄, Merck). Plates were visualized by ultraviolet light, Dragendorff reagent, or iodine vapor.

2.2 General procedure for the synthesis of the ligands

Five novel Pt(II) complexes of the structure Pt(L₂Cl₂) with some 2-cycloalkyl-benzimidazole carrier ligands (L) were synthesized according to the method of [23]. The ligands had been synthesized previously as indicated below.

Ligands (L¹-L⁵) were synthesized by condensation of the corresponding carboxylic acid with 1,2-phenylenediamine in the presence of 5N hydrochloric acid for L¹, or polyphosphoric acid for L²-L⁵ as shown in Scheme 1.

Scheme 1:

Synthesis of carrier ligand and Pt(II) complexes.

The melting points of L¹ (found m.p.: 236 °C Lit. m.p.: 234–236 °C) [24], L² (found m.p.: 221 °C Lit. m.p.: 229–230 °C) [24], L³ (found m.p.: 259 °C Lit. m.p.: 251–253 °C) [24], L⁴ (found m.p.: 283 °C Lit. m.p.: 282 °C) [25] and L⁵ (found m.p.: 245 °C Lit. m.p.: 239–243 °C) [26] were in accordance with the literature.

2.3 Synthesis of the platinum(II) complexes

2.4 Cell culture

The OVCAR-3 and HeLa cell lines were obtained from the Cell and Virus Bank Department, Foot and Mouth Disease Institute (Ankara, Turkey). The cells were cultured in RPMI 1640 and DMEM medium, respectively, containing 2 mM L-glutamine, streptomycin (100 μg/mL), penicillin (100 U/mL) and 10 % heat-inactivated fetal bovine serum (FBS). The cells were grown in an incubator containing 5 % CO₂ at 37 °C. All cell culture reagents were purchased from Gibco Life Technologies (Grand Island, NY, USA).

2.5 Preparation of test compounds

Stock solutions of the tested complexes were prepared in dimethylformamide (DMF) diluted with culture medium within 1 h, and immediately used in the experiments to avoid the possible ligand exchange between the test compounds and DMF. The final concentration of DMF never exceeded 0.1 % and had no effect on the cells. The culture medium was used as a negative control. Compounds 1–5 in concentrations of 80 and 160 μM, cisplatin at 10, 20 and 40 μM, and carboplatin at 10, 20, 40 and 80 μM were incubated in 96-well plates at 37 °C in humidified air containing 5 % CO₂. The experiments were repeated three times.

2.6 Cell cycle and subG1 peak analyses by flow cytometry

For the analysis of the apoptotic subG1 peak and cell cycle phases, the BD Cycletest Plus DNA Reagent Kit (BD Biosciences, San Jose, CA, USA) was used according to the manufacturer’s recommendations. OVCAR-3 and Hela cells were seeded into 6-well plates (5 × 10⁵ cells/well) treated with the twofold IC₅₀ concentration of the compounds. Cells without additions were used as control. After 72 h treatment, cells were suspended by trypsin-EDTA and washed twice with cold PBS. Cell suspensions were consecutively exposed to 250 μL solution A (trypsin for 10 min, 200 μL solution B (trypsin inhibitor and RNase) for 10 min, and 250 μL solution C (propidium iodine stain) for 10 min at 4 °C in the dark. Cellular DNA content was monitored by a BD FACS CANTO II system with BD Modfit LT 3.2 software (BD Biosciences).

2.7 Annexin V staining apoptosis test

OVCAR-3 or Hela cells (0.5 × 10⁶) were seeded into a 6-well plate and incubated for 24 h. Test compounds were added to the cells at twofold IC₅₀ concentration. The cells were incubated under 5 % CO₂ for 72 h and then collected with trypsin-EDTA. The subsequent procedures were carried out according to the instructions provided by the manufacturer of the FITC AnnexinV Apoptosis Detection Kit (BD Biosciences). Briefly, cells were washed with PBS, suspended in binding buffer, and 5 μL annexinV-FITC and 5 μL propidium iodide (PI) were added and the suspensions incubated for 15 min at RT. The samples were then analyzed in a flow cytometer (BD Biosciences FACS CANTO II system with FACS Diva software).

2.8 Statistical analysis

All statistical analyses were performed using the computer software SPSS for Windows version 11.5 (SPSS Inc., Chiago, IL, USA). The Friedman test was used for multiple statistical comparisons. The Wilcoxon test was used as a post hoc test. A p-value <0.05 was considered statistically significant.

3.1 Chemistry

The carrier ligands L¹–L⁵ were prepared according to the method of [23] as shown in Scheme 1. Their melting points were in accordance with the literature.

The platinum(II) complexes 1–5 were synthesized by the reaction of the corresponding ligands and potassium tetrachloroplatinate in DMF. They were characterized by elemental analysis, HRMS, ¹H-NMR, and IR spectra. Elemental analysis suggested a 1:2 (metal/ligand) stoichiometry for the complexes, which was supported by the HRMS spectra.

The IR spectra of the complexes in the regions of 4000–650 and 700–40 cm^-1 were characteristically different from those of the free ligands. The Pt–N and Pt–Cl vibrations are considered characteristic for dichloro–diamine platinum complexes. But the metal-nitrogen stretching bands could not be distinguished from other ring skeleton vibrations present in the spectra. In the far-IR region of the spectra of compounds 2 and 4, a new broad band centered at ~320 cm^–1 with a half-width of about 30 cm^–1 appeared, due to v (Pt-Cl) and in the spectra of complexes 1, 3 and 5 two bands appeared in the range 336–319 cm^–1, due to ν (Pt-Cl). It is well known that cis-dichloro complexes should show two bands of medium intensity because the vibrations are additive, but in many cases the second band appears only as a shoulder [28]. In some cases, cis-dichloro complexes show only one band due to low resolution independent of cis- or trans-configuration [29].

The ¹H-NMR spectra of the Pt(II) complexes in DMSO-d₆ (Table 1) are indicative of complex formation. These spectra were recorded immediately after dissolving the complexes in DMSO-d₆, in order to avoid a ligand exchange reaction between the respective platinum complex and DMSO-d₆. The spectra of the complexes revealed considerable differences from those of the free ligands. The large downfield shifts in the imidazole N1-H signal of the complexes compared to their ligands are the result of an increase in the N1-H acidic character after platinum binding [30]. The changes in the chemical shift observed upon coordination (Δδ = δ complex – δ free ligand) are mostly positive, indicating a decrease in electronic density on the 2-substituted benzimidazole ligands upon coordination of the platinum. The NH signal appears at around 12 ppm in L¹–L⁵ and is shifted downfield in their complexes (13.2–13.4 ppm).

3.2 Biological evaluation

Cytotoxic activities of compounds 1–5 and and the reference compounds cisplatin and carboplatin were determined for the human OVCAR-3 and HeLa cervix cancer cell lines with the MTT assay are shown in Table 2. Cytotoxic activity of the ligands L¹–L⁵ against the OVCAR-3 and HeLa cell lines was also tested, but not observed.

The relative cytotoxic potencies of the complexes decreased in the order 5 > 3 > 2, 4 > 1 against the HeLa cell line, and 5, 3 > 2 > 4 > 1 against the OVCAR-3 cell line. Compound 4 was an exception from the general rule that the cytotoxic potency increased with the ring size.

3.3 Cell cycle and apoptosis

The effect of the most cytotoxic compound 5, on the cell cycle of the two cell lines was studied. Cisplatin, known to cause DNA damage and apoptosis in various human tumor cells [31] was used for comparision. The two cell lines were exposed to 5 and cisplatin at the twofold concentration of the the respective IC₅₀ value for 72 h. In the OVCAR-3 cell line, 5 caused apoptosis in 72 % of the cells under these conditions, comparable to cisplatin (Figure 1), with a preferential arrest in SubG1, while in the HeLa cells 5 was less active than cisplatin (Figure 2).

Figure 1:

Apoptosis of OVCAR-3 cancer cells treated with 2 × IC₅₀ concentrations of cisplatin and compound 5 was evaluated by flow cytometry after 72 h using annexin-V/propidium iodide (PI) staining. Quadrant 1 (Q1): dead cells; Quadrant 2 (Q2): late apoptotic and necrotic cells; Quadrant 3 (Q3): viable cells; Quadrant 4 (Q4): early apoptotic cells. (A) Images of dot-plot of Annexin-V/PI staining in untreated cells; (B) staining in OVCAR-3 cells treated with cisplatin; (C) cells treated with compound 5. Results are expressed as mean ± SD from three independent experiments. ^*Denotes p < 0.05 compared with control (Friedman test–Wilcoxon signed ranks test).

Figure 2:

Apoptosis of HeLa cancer cells treated with 2 × IC₅₀ concentrations of cisplatin and compound 5 was evaluated by flow cytometry after 72 h using annexin-V/propidium iodide (PI) staining. Quadrant 1 (Q1): dead cells; Quadrant 2 (Q2): late apoptotic and necrotic cells; Quadrant 3 (Q3): viable cells; Quadrant 4 (Q4): early apoptotic cells. (A) The image of dot-plot of annexin-V/PI staining in untreated cells; (B) Staining in HeLa cells treated with cisplatin; (C) Cells treated with compound 5. Results are expressed as mean ± SD from three independent experiments. ^*Denotes p < 0.05 compared with control (Friedman test–Wilcoxon signed ranks test).

Compound 5 induced a significant increase in the SubG1 cell population in both OVCAR-3 and HeLa cells compared to the untreated control and cisplatin. This indicates that compound 5 induces sufficient levels of DNA damage (Tables 3 and 4).

Most of the current antineoplastic drugs presently in use block the cell cycle in the S or G2/M phase [32]. Further studies must be done on compound 5.