Synthesis of Trans-bis-(2-hydroxypyridine)dichloroplatinum(II) and its Activity in Human Ovarian Tumour Models

Characterization

Microanalyses: Platinum was determined by graphite furnace atomic absorption spectroscopy (AAS) using a Varian Spectra-240 Atomic Absorption Spectrophotometer at the University of Sydney. C, H, N, and Cl were determined at the Australian National University. DH3: Calc.% C: 26.3; H: 2.2, N: 6.1; Cl: 15.6; Pt: 42.8. Obs.%: C: 26.3±0.4, H: 2.2±0.4, N: 6.1±0.4, Cl: 15.9±0.4, Pt: 42.8±1.0.

Molar conductivity: The limiting molar conductivity value (in Ω⁻¹ cm² mol⁻¹) at zero concentration (Λ₀) for DH3 and cisplatin are 730 and 270, respectively, indicating DH3 is more fully ionized than cisplatin in solution in the 1:10 (v:v) mixture of DMF and mQ water. Spectral studies: Infrared, mass and ¹H-NMR) spectra were used to aid in the structural characterization of DH3.

Infra-red (IR) Spectral analysis: (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 structure of DH3. In particular it indicates the presence of various functional groups. 3245.4 cm⁻¹: N-H stretch; 1597.4, 1538.4, 1403.8 cm⁻¹: C=N and C=C stretch; 1239.9 cm⁻¹: C–N stretch; 920.0 cm-1: pyridine ring stretch; 983.4 cm⁻¹: C–H in-plane bending; 818.0, 503.3 cm⁻¹: C–H out-of-plane bending; 473.7, 449.0 cm⁻¹: Pt–N stretch, 239.8 cm-1: Pt–Cl stretch.

Mass spectroscopy: 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). EIS-MS (mQ water) (m/z: M=378). The peak at m/z = 383 corresponds to (M–2Cl–2H) and the peak at m/z=415 corresponds to (Pt(2-hydroxypyridine)2Cl–2H) and the peak at m/x=288 corresponds to (Pt(2-hydroxypyridine)–2H).

¹H-NMR: ¹H-NMR spectrum of DH3 dissolved in dimethylsulfoxide (DMSO) was recorded in a Bruker DPX400 spectrometer (Bruker Australia Pty Ltd Sydney NSW Australia) using a 5 mm high-precision Wilmad NMR tube at 300. ¹H NMR DMSO δ ppm: 8.50 (s, due to OH); 7.94 (d, due to ortho CH); 4.50 (d, due to meta CH); 3.50 (q, due to meta CH); 2.69 (q, due to para CH); NH–Pt); 2.55 (d, due to dimethylsulfoxide (DMSO)).

Cytotoxicity assays. Cytotoxicity of DH3 against human ovarian A2780, A2780^cisR, and A2780^ZD0473R cancer cell lines was determined using the 3-(Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-terazolium bromide (MTT) reduction assay (14, 15). Cisplatin was used as a reference. A2780 (parent), A2780^cisR (cisplatin-resistant type) and A2780^ZD0473R (ZD0473-resistant type) ovarian cancer cell lines were obtained from Ms. Zhang from Royal Prince Alfred Hospital, University of Sydney, Australia. Briefly, between 4500 to 5500 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 cells to attach. Platinum complexes dissolved first in a minimum volume of DMF were diluted to the required concentrations with mQ 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. The plates were left to incubate under normal growth conditions for 72 h. The inhibition of cell growth was determined using the MTT assay (15). Four hours after the addition of MTT (50 μl per mg ml⁻¹ MTT), the yellow formazan crystals produced were dissolved in 150 μl of DMSO and the absorbance of the resulting solution was read with Bio-Rad Model 3550 Microplate Reader (BioRad Sydney Australia) set at 570 nm.

Platinum accumulation and platinum–DNA binding. DH3 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=1×10⁶ cells ml⁻¹). 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 3500 rpm for 2 min at 4°C (16). Ice-cold phosphate-buffered saline (PBS) was used to wash the cells twice and the pellets were stored at −20°C until assayed. At least three independent experiments were performed.

For the determination of drug accumulation in the cells, 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 (17) was used. Briefly, high molecular weight DNA was isolated from cell pellets using JETQUICK Blood DNA Spin Kit/50. The cell pellets were resuspended 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 to the mixture followed by incubation for 10 min at 70°C. Absolute ethanol (200 μl) was added and mixed thoroughly to prevent any precipitation of nucleic acids due to high local alcohol concentration. The samples were centrifuged for 1 min at 10600 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 K2, low-salt buffer to change the high-salt conditions on the silica membrane to low-salt, and centrifuged for 1 min at 10600 rpm. The columns were centrifuged again for 2 min at full speed (13000 rpm) to further clear the silica membrane of residual liquid. The receivers were changed and the purified DNA in the column was eluted from the membrane with 200 μl of 10 mM Tris-HCl buffer (pH 8.5). DNA content was determined using UV spectrophotometry set at 260 nm (Varian Cary 1E UV-Visible spectrometer with Varian Cary Temperature Controller). Platinum levels were determined by graphite furnace AAS. A260/A280 ratios were found to be between 1.75 and 1.8 for all samples, reflecting high purity of the DNA.

Interaction with pBR322 plasmid DNA. Interaction between DH3 and cisplatin with pBR322 plasmid DNA, with and without BamH1 digestion, was studied using gel electrophoresis using a method described by Stellwagen (18). Exactly 1.5 μl of supplied pBR322 plasmid DNA in solution was added to solutions of DH3 and cisplatin at different concentrations ranging from 0.55 μM to 70 μM. The DNA blank was prepared by adding 18.5 μl mQ 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. The reaction was stopped by rapid cooling to 0°C for 20 min. The samples were mixed with 1 μl of marker dye ethidium bromide and 16 μl of each sample was loaded onto 1% agarose gel made in (TAE) buffer. The gel was photographed using Eastman Kodak Company, Molecular Imaging Systems (Carestream Health Inc. Rochester New York USA).

BamH1 digestion. BamHI is a type II restriction endonuclease that hydrolyses phosphodiester bonds. It binds at the recognition sequence 5’-GGATCC-3’, and chops these sequences just after the 5’-guanine on each strand (19). 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. The same identical set of drug-DNA mixtures as described previously, was first 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 and 4 μl of ethidium bromide was added to each sample before loading onto the gel. The gel was photographed following the method described previously.