Studies on the Activity of Two Trans-Planaramine Platinum(II) Complexes Coded as DH4 and DH5 in Human Ovarian Tumour Models

Materials and Methods

Potassium tetrachloroplatinate (K2PtCl4), N,N-dimethylformamide (DMF), 2-methylimidazole and 2,3-diaminopyridine were obtained from Sigma Chemical Company, St. Louis, Mo, USA; hydrochloric acid was obtained from Asia Pacific Specialty Chemicals Ltd., (NSW, Australia); acetone and silver nitrate from Ajax Chemicals, (Auburn, NSW, Australia); silver acetate from Sigma Chemical Company, Germany and charcoal from Sigma Aldrich Chemical Company (Milwaukee, WI, USA); 200 mM L-glutamine, 5.6% sodium bicarbonate, foetal calf serum (FCS), RPMI-1640 from Trace Biosciences Pty Ltd. Australia; other chemicals were obtained from Sigma-Aldrich, Sydney, Australia. A2780, A2780^CisR and A2780^ZD0473R ovarian cancer cell lines were gifts from Ms. Mei Zhang, Royal Prince Alfred Hospital, Sydney, Australia. Stock solutions of platinum compounds (1 mM) were prepared in 1:1 DMF and milli Q water mixture.

Synthesis. Cisplatin: Cisplatin required for the synthesis of DH4 and DH5, and also used as a reference was prepared according to the modified Dhara method (3). Briefly, to one millimol of tetrachloroplatinate dissolved in 5 ml of milli-Q water was added potassium iodide (4.12 mmol) dissolved in 1 ml of mQ water. The mixture left on a shaking water bath, at 37°C, for 5 min, produced K2PtI4 which then reacted with 2 mmol of aqueous ammonia at 37°C for 1 h. The dark-yellow precipitate of cis-Pt(NH₃)₂I₂ was washed with ice-cold water and ethanol and air dried. Silver nitrate (2 mmol) and cis-Pt(NH3)2I2 (1 mmol) were mixed together followed by the addition of 4 ml of mQ water. The mixture was left on a shaking water-bath at 37°C for 30 min to produce cis-Pt(NH₃)₂(NO₃)₂ and solid silver iodide. The mixture was centrifuged. The supernatant consisting of cis-Pt(NH₃)₂(NO₃)₂ in solution was collected. Potassium chloride (0.11 g, 1.5 mmol) was added to the filtrate. The mixture was left on a water bath at 37°C for 30 min for crystals of cisplatin to form.

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Figure 1.

Structures of DH4 (a) and DH5 (b).

DH4: Although synthesis of DH4 was reported earlier (4), no work on the activity of the compound has been performed. Briefly the method of synthesis was as follows: 1 mmol (0.415 g) of potassium tetrachloroplatinate was dissolved in 4 ml of mQ water to which 0.25 ml of concentrated hydrochloric acid were added. The solution was heated to 50°C. 10 mmol (0.82 g) of 2-methylimidazole, dissolved in 2 ml of DMF, were slowly added to the solution of potassium tetracholoroplatinate. The temperature of the solution was increased to 65°C and maintained at that temperature for 1 h while the solution was stirred. The volume of the solution was reduced to about 5 ml, following which 40 ml of 6 M hydrochloric acid were added and the mixture was heated under reflux for 24 h at 90°C. The mixture was cooled to room temperature, resulting in the formation of dark-purple precipitate of DH4, which was collected by filtration, giving a yield of 35%.

DH5: From the reaction of 1 mmol (0.3 g) of cisplatin with 2 mmol (0.218 g) of 2,3-diaminopyridine, synthesis of 1 mmol of trans-dichloro(ammino)(2,3-diaminopyridine)platinum(II) was attempted, giving a yield of 45%. The method of synthesis was as follows: Cisplatin (0.300 g, 1 mmol) was added to 8 ml of milli-Q water. 2,3-Diaminopyridine (0.218 g, 2 mmol), dissolved in 2 ml of DMF, was added to the cisplatin suspension. The mixture was heated under reflux for 1 h at 70°C to cause 2,3-diaminopyridine ligand to replace chloro-ligands. The mixture was cooled to room temperature to stop the reaction. Concentrated hydrochloric acid (1.22 ml, 12.2 mmol) was added to the mixture, followed by heating to 70°C under reflux, to produce dark-black crystals of DH5. The crystals of DH5 formed were collected by filtration, washed with ice-cold water and ice-cold ethanol, and air dried, giving a yield of 45%.

Characterization. Microanalyses: Pt was determined by graphite furnace atomic absorption spectroscopy (AAS) using a Varian Spectra-20 Atomic Absorption Spectrophotometer. C, H, N, and Cl were determined at the Australian National University.

DH4: Calc.% C: 22.4; H: 2.4, N: 13.1; Cl: 16.6; Pt: 45.6; Obs.%: C: 22.3±0.4, H: 2.8±0.4, N: 13.1±0.4, Cl: 16.4±0.4, Pt: 45.2±1.0. DH5: Calc.% C: 19.3; H: 2.6, N: 14.3; Cl: 18.1; Pt: 49.8; Obs.%: C: 19.6±0.4, H: 2.3±0.4, N: 14.5±0.4, Cl: 18.5±0.4, Pt: 50.2±1.0.

Molar Conductivity: The limiting molar conductivity value (in ohm⁻¹cm²mol⁻¹) at zero concentration (Λ₀) for DH4, DH5 and cisplatin are 254, 470 and 304 respectively, indicating that the degree of ionization is slightly less than that of cisplatin, but that of DH5 is much greater than that of cisplatin.

Spectral studies: Infrared, mass and 1H (NMR) spectra were used to aid in the structural characterization of DH4 and DH5.

IR (KBr). IR spectrum was collected using a Bruker IFS66 spectrometer equipped with a Spectra-Tech Diffuse Reflectance Accessory (DRA) (Bruker Australia Pty Ltd Bruker Sydney NSW Australia), an air-cooled DTGS detector and a KBr beam splitter. Spectrum was recorded at a resolution of 4 cm⁻¹, with the addition of 128 scans and a Blackman-Harris 3-term apodization function was applied. The peaks listed below can be seen to provide support for the suggested structures of DH4 and DH5.

DH4: 3367.4 and 3121.8.4 cm⁻¹: N-H stretch; 1621.5 and 1426.3 cm⁻¹: C=N stretch; 1283.7 cm⁻¹: imidazole ring stretch; 539.4 cm-1: Pt–N stretch.

DH5: 3307 and 3121.8.4 cm⁻¹: N-H stretch; 1623.5 and 1584.1 cm⁻¹: C=N and C=C stretch; 1222.4 cm⁻¹: ring stretch; 449.4 cm-1: Pt–N stretch.

Mass: The mass spectrum of DH3 was obtained using a Finnigan LCQ ion trap mass spectrometer (Thermo Scientific Massachusetts USA) in which fragmentation was produced by electrospray ionization (EIS).

MH4: (EIS-MS) (milli Q water) (m/z: M=428). The peak at m/z=427 corresponds to (M–H)).

MH5: (EIS-MS) (milli Q water) (m/z: M=392). The peak at m/z=316 corresponds to (Pt (C₅H₇N₃)(NH₃)–5H).

¹H-NMR. ¹H-NMR spectra of DH4 and DH5 dissolved in DMSO were recorded in a Bruker DPX400 spectrometer (Bruker Australia Pty Ltd Sydney NSW Australia) using a 5mm high precision Wilmad NMR tube.

DH4: ¹H-NMR DMSO δ ppm: 12.42 (d, due to CH); 11.34 (d, due to CH); 6.66 (s, due to CH₃); 5.70 (s, due to NH); 4.63 (s, due to NH-Pt); 2.50 (due to DMSO).

DH5: 1H-NMR DMSO δ ppm: 7.95 (s, due to CH ortho); 7.10 (t, due to CH meta); 3.33 (d, due to CH para); 2.80 (s, due to NH₂); 2.70 (s, due to NH₂); 2.40 (s, due to NH₃); 2.50 (t, due to DMSO).

Cytotoxicity assays. The cell killing effect of DH4 and DH5 in human ovarian A2780, A2780^cisR, and A2780^ZD0473R cancer cell lines was determined using 3-(Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-terazolium bromide (MTT) reduction assay (5, 6), where cisplatin was used as a reference. Briefly, between 4,500 to 5,000 cells were seeded into the wells of the flat-bottomed 96-well culture plate in 10% FCS/RPMI-1640 culture medium. The plate was incubated for 24 h at 37°C in a humidified atmosphere to allow for cells to attach. Platinum complexes dissolved first in a minimum volume of DMF were diluted to the required concentrations with milli-Q water and filtered to sterilise. Serial dilutions ranging from 0.0064 μM to 0.8 μM in 10% FCS/RPMI 1640 medium were prepared and added to equal volumes of cell culture in quadruplicate wells following which the cells were incubated under normal growth conditions for 72 h, followed by the addition of MTT. Four hours after the addition of MTT, the yellow formazan crystals produced were dissolved in 150 μl of DMSO and the absorbance was measured at 570 nm.

Platinum accumulation and platinum–DNA binding. DH4, DH5 and cisplatin (at 50 μM final concentration) were added to culture plates containing exponentially growing A2780, A2780^cisR and A2780^ZD0473R cells in 10 ml 10% FCS/RPMI 1640 culture medium (cell density of 1×10⁶ cells ml⁻¹) (7). The cells containing the drugs were incubated for periods of 2 h, 4 h and 24 h, at the end of which cell monolayers were trypsinized and cell suspension (5 ml) was transferred to centrifuge tubes and spun at 3,500 rpm for 2 min at 4°C. Cells were washed twice with ice-cold phosphate-buffered saline (PBS) and cell pellets were stored at −20°C until assayed. At least three independent experiments were performed. For the determination of drug accumulation, cell pellets were suspended in 0.5 ml of 1% triton-X, held on ice then sonicated (5 min). Total intracellular platinum contents were determined by graphite furnace AAS.

For the determination of the level of platinum–DNA binding, the modified method of Bowtell (8) was used. Briefly, high molecular weight DNA was isolated from cell pellets using JETQUICK Blood DNA Spin Kit/50. The cell pellets were re-suspended in PBS to a final volume of 200 μl and mixed with 10 μl of RNase A, then incubated for 4 min at 37°C. 25 μl Proteinase K and 200 μl buffer K1 (containing guanidine hydrochloride and a detergent) were added, followed by incubation for 10 min at 70°C. Absolute ethanol (200 μl) was added and mixed thoroughly. The samples were centrifuged for 1 min at 10,600 rpm through the silica membrane using JETQUICK micro-spin columns. The columns containing the samples were washed with 500 μl of buffer KX (containing high-salt buffer to remove residual contamination), then centrifuged for 1 min at 10,600 rpm. The columns were washed again with 500 μl buffer K2, low-salt buffer, and centrifuged for 1 min at 10,600 rpm. The columns were centrifuged again for 2 min at the full speed (13,000 rpm) to further clear the silica membrane of the residual liquid. The purified DNA in the column was eluted from the membrane with 200 μl of 10 mM Tris-HCl buffer (pH 8.5). The DNA content was determined using UV spectrophotometry at 260 nm (Varian Cary 1E UV-Visible spectrometer with Varian Cary Temperature Controll). Platinum contents were determined by graphite furnace AAS. A₂₆₀/A₂₈₀ ratios were found to be between 1.75 and 1.8 ensuring high purity of the DNA.

Interaction with pBR322 plasmid DNA. Interaction between DH4, DH5 and cisplatin with pBR322 plasmid DNA combined with BamH1 digestion, was carried out using gel electrophoresis following a method described by Stellwagen (8). BamH1 is a type II restriction endonuclease that binds at the recognition sequence 5’-GGATCC-3’ and chops these sequences just after the 5’-guanine on each strand (9). pBR322 plasmid DNA contains a single restriction site for BamH1 that converts the supercoiled form I and singly-nicked circular form II to linear form III DNA. Exactly 1.5 μl of supplied pBR322 plasmid DNA, in solution, was added to the solutions of DH4, DH5 and cisplatin at different concentrations ranging from 5 μM to 30 μM.

The DNA blank was prepared by adding 18.5 μl milli Q water to 1.5 μl of pBR322 plasmid DNA. The samples and the DNA blank were incubated for 4 h on a shaking water bath at 37°C and then subjected to BamH1 (10 units μl⁻¹) digestion. To each 20 μl of the incubated drug–DNA mixture was added 2 μL of 10× digestion buffer SB first and then 0.1 μL BamH1 (1 unit). The mixtures were left in a shaking water bath at 37°C for another 1 h, at the end of which the reaction was terminated by rapid cooling. Four microlitres of ethidium bromide were added to each sample before loading onto the gel. The gel was photographed using Eastman Kodak Company, Molecular Imaging Systems.