DNA‐Intercalative Platinum Anticancer Complexes Photoactivated by Visible Light

Abstract Photoactivatable agents offer the prospect of highly selective cancer therapy with low side effects and novel mechanisms of action that can combat current drug resistance. 1,8‐Naphthalimides with their extended π system can behave as light‐harvesting groups, fluorescent probes and DNA intercalators. We conjugated N‐(carboxymethyl)‐1,8‐naphthalimide (gly‐R‐Nap) with an R substituent on the naphthyl group to photoactive diazido PtIV complexes to form t,t,t‐[Pt(py)2(N3)2(OH)(gly‐R‐Nap)], R=H (1), 3‐NO2 (2) or 4‐NMe2 (3). They show enhanced photo‐oxidation, cellular accumulation and promising photo‐cytotoxicity in human A2780 ovarian, A549 lung and PC3 prostate cancer cells with visible light activation, and low dark cytotoxicity. Complexes 1 and 2 exhibit pre‐intercalation into DNA, resulting in enhanced photo‐induced DNA crosslinking. Complex 3 has a red‐shifted absorption band at 450 nm, allowing photoactivation and photo‐cytotoxicity with green light.

NMR spectra were recorded on Bruker Avance III 400 MHz (for 1 H) or Bruker Avance III HD 500 MHz (for 1 H) spectrometers with the residual signal of the solvent used as a reference. ESI-MS spectra were recorded on an Agilent 6130B single quadrupole detector instrument and ESI-HR-MS data were collected on a Bruker microTOF instrument for positive ions at 298 K.
Electronic absorption spectra were recorded on a Varian Cary 300 UV-vis spectrophotometer in a 1 cm quartz cuvette and solvent used as reference. Jasco FP-6500 Spectrofluorometer was used to record fluorescence spectra.
LC-MS was carried out on Bruker Amazon X mass spectrometry connected online with an Agilent 1260 HPLC.
Platinum content was analysed on an ICP-MS 7500cx (Agilent) or ICP-OES 5300DV (Perkin Elmer). The emission wavelength detected for Pt in ICP-OES was 265.945 nm and 195 Pt was determined in ICP-MS with 166 Er (50 ppb) as an internal standard.

Synthesis and characterisation.
Caution! Although we encountered no problem during the work reported here, due care and attention with appropriate precautions should be taken in the synthesis and handling of heavy metal azides since they can be shock-sensitive. All work involved metal azides was carried out in the dark. . To the solution of complex FM-190 (50.0 mg, 106 μmol), Fmoc-gly-OH (31.6 mg, 106 μmol), and TBTU (34.2 mg, 106 μmol) in DMF (3 mL), DIPEA (100 μL) was added. The reaction mixture was stirred overnight at 298 K under a nitrogen atmosphere. After evaporation to dryness, the oily residue was collected and purified by column chromatography on silica gel (2% methanol + 98% DCM) to give trans,

Photooxidation of NADH. An aqueous solution with 60 μM Pt(IV) complex and 2 mM NADH
was irradiated with indigo light (420 nm) for 1 h, then analysed by LC-MS immediately.
Photoreaction with 5'-GMP. 30 μM complex was mixed with 2 mol. equiv. of guanosine 5′monophosphate disodium salt hydrate (5′-GMP-Na2) in aqueous solution. The solution was irradiated for 1 h (420/517 nm) and analysed immediately on a Bruker Amazon X mass spectrometer connected online with the HPLC.
LD spectroscopy. LD spectra of calf thymus DNA (3 × 10 -4 M) in the absence or presence of Pt(IV) complexes were recorded with a Jasco J-720 spectropolarimeter using a flow Couette cell consisting of a fixed outer cylinder and a rotating solid quartz inner cylinder, separated by a gap of 0.5 mm, giving a total path length of 1 mm. The spectra represent the mean of two recordings from 600 to 220 nm, at a scan rate of 500 nm/min, using a 0.5 nm step, 2 nm width, and 0.25 s averaging time. All spectra were obtained from samples in 10 mM Tris-Cl, pH 7.4 at room temperature.
Independent duplicate plates were used, one for dark, the other for irradiation experiments. The cells were pre-incubated in drug-free medium with phenol red at 310 K for 24 h. Complexes were dissolved first in DMSO and then diluted in phenol red-free RPMI-1640 to make the stock solution of the drug. These stock solutions were further diluted using phenol-red free cell culture medium until working concentrations were achieved, the maximum DMSO concentration was < 0.5% v/v in these solutions. Cells were exposed to the complexes at different concentrations for 1 h. Then one plate was irradiated for 1 h using blue light (4.8 mW cm -2 per LED at 465 nm) or green light (11.7 mW cm -2 per LED at 520 nm), while the dark plate was kept in the incubator. After irradiation, supernatants of both plates were removed by suction and the cells were washed with phosphate-buffered saline (PBS). Photocytotoxicity was determined after another 24 h recovery at 310 K in drug-free phenol red-containing medium by comparison to untreated controls which were only exposed to vehicle. Untreated controls were also compared between the irradiated and the non-irradiated plates to ensure that the differences in cell survival were not statistically relevant, hence guaranteeing that the differences in cell viability observed were not due to the light source. The SRB assay was used to determine cell viability. S11 Absorbance measurements of the solubilised dye (on a Promega microplate reader) allowed the determination of viable treated cells compared to untreated controls. IC50 values (concentrations which caused 50% of cell death) were determined as the average of triplicates and their standard deviations were calculated. Stock concentrations for all metal complexes used in these biological assays were adjusted/verified after ICP-OES metal quantification.

Platinum accumulation in cancer cells in the dark.
For Pt cellular accumulation studies, ca.
5×10 6 A2780, A549 or PC3 cells were plated in 100 mm Petri dishes and allowed to attach for 24 h, then the plates were exposed to complexes at 10 μM. Additional plates were incubated with medium alone as a negative control. After 1 h of incubation in the dark at 310 K, the cells The excitation light power at 420/450 nm is ca. 1 mW. A 850 nm longpass glass filter from Thorlabs (FGL850S) was used between the light source and detector.

Intracellular ROS generation determined by confocal fluorescence microscopy.
Fluorescence images were recorded on a confocal microscope (LSM 880, AxioObserver).