Platinum(iv) dihydroxido diazido N-(heterocyclic)imine complexes are potently photocytotoxic when irradiated with visible light

Trans,trans,trans-[Pt(N3)2(OH)2(4-picoline)2] is potently photocytotoxic (λirr = 420 nm) towards cancer cell lines whilst being minimally toxic in the absence of irradiation.

Methods NMR spectroscopy. Due to the potential photosensitivity of the compounds, amberised NMR spectroscopy tubes (Goss Scientific) were used. 1 H, 13 C and 195 Pt NMR spectra were acquired at 298 K on Bruker AV-400 (1H: 399.10 MHz), Bruker DPX-400 (1H: 400.03 MHz), Bruker AVIII-600 (1H: 600.13 MHz) or Bruker Avance 700 spectrometers. Spectra were processed using Topspin 3.2. J values are quoted in Hz. 13 C NMR and 1 H NMR were referenced internally to residual solvent or added 1,4dioxane in the case of D 2 O. 2 All chemical shift (δ) values are given in parts per million (ppm). 195 Pt NMR: chemical shifts were externally referenced to K 2 PtCl 6 in 1.5 mM HCl in D 2 O (δ 0 ppm): for spectra of Pt IV species directly bonded to quadrupolar 14 N, typical parameters used were d1 = 0 s, TD 2k, DE 10 μs, 256k scans. Data were processed with a LB of 50 Hz. 14 N NMR: chemical shifts were externally referenced to [ 14 N]NH 4 Cl (1.5 M) in 1 M HCl with a D 2 O coaxial insert and processed with a qfil baseline correction.
Mass Spectrometry: Spectra were acquired on a Bruker Esquire 2000 Trap Spectrometer or Agilent 6130 single Quad, using an automatic sample delivery system. Data were processed using Data Analysis version 3.3 (Bruker Daltonics). MS/MS experiments: positive mode electrospray mass spectra were recorded using a MAXIS UHRQq-TOF (Bruker). ICP-MS was carried out on an Agilent 7500 Series spectrometer. Data acquisition was carried out on ICP-MS Top (version B.03.05) and analysis on Offline Data Analysis (version B.03.05). The standards were prepared from a stock of 1000 ppm Pt solutions obtained from Sigma Aldrich, in 5% HNO 3 with miliQ water at the following concentrations: 200, 50, 10, 5, 1, 0.5, 0.2, 0.05, 0.01 ppb.
UV-visible absorption spectra: UV-vis spectra were recorded on a Perkin-Elmer Lambda 20 UV-visible spectrophotometer or a Varian Cary 300 UV-Vis spectrophotometer, in 1 cm path-length quartz cuvettes purchased from Starna Scientific. Extinction coefficient determination: Extinction coefficients were determined using the Beer-Lambert law (A = εcl, where ε is the extinction coefficient (M -1 cm -1 ), c the molar concentration and l the path length in cm). Pt concentrations for determination of extinction coefficients were measured by ICP-MS. For the shorter wavelengths, solutions of ca. 70 and 50 μM were used whereas for the longer wavelengths the concentrations employed were ca 2 mM and 1 mM, except in the case of complex 29, where a solution of 0.6 mM was used.
HPLC (LC-MS) for monitoring the photochemistry of complex 20 was carried out on a Dionex 3000RS UHPLC coupled with a Bruker MaXis Q-TOF mass spectrometer using a ZORBAX Eclipse Plus C18 (5 μm particle size, 150 × 4.6 mm) column, with 1 mL/min flow rate and 45 μl injection volume. The mobile phase was H 2 O/MeOH with 0.1% TFA. The wavelength of detection was 254 nm. The mass spectrometer was operated in electrospray positive mode with a scan range 50 -2,000 m/z. Source conditions are, end plate offset at -500 V; capillary at -4500 V; nebulizer gas (N 2 ) at 1.6 bar; dry gas (N 2 ) at 8 L/min; dry temperature 180 °C. Ion tranfer conditions were: ion funnel RF at 200 Vp/p; multiple RF at 200 Vp/p; quadruple low mass set at 55 m/z; collision energy at 5.0 ev; collision RF at 600 Vp/p; ion cooler RF at 50-350, Vp/p; transfer time set at 121 µs; pre-pulse storage time 1 µs. Calibration was carried out with sodium formate (10 mM) through a loop injection of 20 μL of standard solution at the beginning of each run. The spectra were processed with Bruker Daltonics Data Analysis.
DFT and TDDFT calculations: All calculations were performed with the Gaussian 03 (G03) program 3 employing the DFT method, with PBE1PBE functionals. 4 The LanL2DZ basis set 5 and effective core potential were used for the Pt atom and the 6-31G**+ basis set 6 was used for all other atoms. Geometry optimizations for complex 18 in the ground state (S 0 ) and lowest-lying triplet state (T 1 ) were performed in the gas phase and the nature of all stationary points was confirmed by normal mode analysis. For the T 1 geometries the UKS method with the unrestricted PBE1PBE functional was employed. Thirty-two singlet excited states with the corresponding oscillator strengths were determined with a Time-dependent Density Functional Theory (TD-DFT) 7,8 calculation.
X-ray crystallography experimental details: In all cases, a suitable crystal was selected and mounted on a glass fibre with Fomblin oil and placed on an Xcalibur Gemini diffractometer with a Ruby CCD area detector. Diffraction data were collected with Mo-Kα radiation (λ = 0.71073 Å). The crystal was held at 100(2) K: (28, (nf2); 20 (es2), 26 (es4)); or 150(2) K (23 (es10), 32 (es9)). Using Olex2 9 the structure was solved with the ShelXT 10 structure solution program using Intrinsic Phasing and refined with the ShelXL 11 refinement package using Least Squares minimisation. The

Cell Studies
Maintenance. Human ovarian carcinoma (A2780), cisplatin-resistant ovarian carcinoma (A2780CIS) and oesophageal adenocarcinoma (OE19) cells were maintained in RPMI medium containing 10% Foetal Calf Serum (FCS). Cell lines were obtained from the European Collection of Animal Cell Cultures (ECACC). Cells were maintained in antibiotic-free medium in a humidified atmosphere of 95% air: 5% CO 2 and subcultured every 7-10 days. Mycoplasma checks were done using the Hoechst staining method. Cells were seeded into dishes the night before experiments at a density of 6 -7×10 4 cells.cm -2 . Experiments were carried out in an adapted laboratory with ambient light levels kept below 1 lux (Solatell, UK).
Irradiation. Cells were irradiated by a bank of TL03 light sources (λ max 420 nm), filtered to attenuate wavelengths below 400 nm. Irradiances, dosimetry and calibrations were traceable to the National Physical Laboratory. 12 Monochromatic light irradiation of the sample was performed using equipment consisting of a 1600W xenon arc light source and a grating monochromator. The apparatus was set to irradiate monochromatic light at 365, 400, 430, 500 and 530 nm. Filters were used to eliminate second order diffraction of shorter wavelengths from the specified longer wavelength monochromatic light. 13 Cells were irradiated in stirred suspension. Sham irradiated cells were treated identically to the test cells but were covered during irradiation.
Treatment. Test compounds were prepared in Earle's Balanced Salt Solution (EBSS) and filtered immediately before use. Photofrin was used as the positive control for visible light irradiation. The positive and negative controls were included on each plate in parallel with the complexes. Cell viability of untreated cells +/-visible light was >90%. Cells were treated for 1 h at 37ºC/5% CO 2 with EBSS containing the test compound, and then irradiated with 5 J.cm -2 visible radiation.
Phototoxic Index and Wavelength-Dependence. The phototoxic Index (PI) was determined 24 h after irradiation using the neutral red uptake assay; 14 following cell uptake the neutral red dye accumulates in lysosomes, the low pH in the lysosomal matrix of live cells causes the dye to becomes charged, allowing it to be retained in this organelle. 15 The dye was then extracted from the cells upon solubilisation and quantified. Protocol: a stock solution of neutral red was prepared in water (4 mg. mL -1 ) and diluted 80-fold in the cell culture medium (RPMI). Prior to addition to the cells, the solution was heated to 37 o C and filtered (no. 1 Whatman paper). The growth medium was removed from the cells, the neutral red dye (100 μL) was added and the cells are incubated for 3 hours in the dark. Upon removal of the dye, the cells were rinsed with PBS (100 μL) and then the plates were shaken for 15 min with PBS (200 μL) for solubilisation. The dye concentration was quantified by measuring the absorbance at 450 nm. An IC 50 value was determined and is defined as the concentration required to inhibit dye uptake by 50%. This was done using non-linear regression (Graphpad Prism). Goodness of fit was determined from the R 2 values of the curves and 95% confidence intervals. All cell experiments were performed in triplicate and repeated independently a minimum of two times.

Syntheses
Caution! No problems were encountered during this work, however heavy metal azides are known to be shock sensitive detonators, therefore it is essential that platinum azides compound are handled with care. The Pt-diazido complexes were synthesised and handled under dim lighting conditions.

Synthesis of mixed trans-N-heterocyclic starting materials
Trans-[Pt(Cl) 2 (pyridine)(n-picoline)], where n = 2, 3 or 4 Cis-[Pt(Cl) 2 (py) 2 ] (0.200 g, 0.47 mmol) was suspended in H 2 O (60 mL) and then n-picoline (4 eq, 0.176 g, 1.89 mmol in the case of 2-picoline; 2 eq, 0.044 g, 0.473 mmol for 3-and 4-picolines) was added and the solution was stirred at 75 o C overnight. When the solution turned colourless (~12 h for 2picoline and 2 h for 3-and 4-picoline), the water was removed by rotary evaporation. The residual white solid was washed with diethyl ether to remove the excess ligand. Then, HCl (2 M, 1.3 mL) and H 2 O (8 mL) were added and the reaction stirred for 4 days at 70 o C. The pale yellow solid was isolated by filtration and washed with water, ethanol and diethyl ether.

General procedure for synthesis of trans-[Pt(N 3 ) 2 (py)(n-pic)] complexes
Trans-[Pt(Cl) 2 (py)(n-pic)] (0.155 g, 0.35 mmol; 0.245 g, 0.56 mmol; 0.24 g, 0.55 mmol for 2-, 3-and 4picoline, respectively) were suspended in H 2 O (204 mL per 1 g). AgNO 3 (2 mol eq) was added and the reaction was carried out at 60 o C for 24 h. The AgCl was removed by filtration on celite on frit and also through an IM filter. NaN 3 (10 mol eq) was added and the mixture was allowed to stir for 24 h at 40 o C. The yellow solid was isolated and washed with cold water, ethanol and diethyl ether. Recrystallisation of these complexes was carried out by dissolving the solid in hot MeOH (50 o C, ~0.5 g/L) and then allowing the solution to stand at -20 o C.

General procedure for synthesis of trans, trans, trans-[Pt(N 3 ) 2 (OH) 2 (py)(n-pic)] complexes
Trans-[Pt(N 3 ) 2 (py)(n-pic)] (0.084 g, 0.18 mmol; 0.138 g, 0.30 mmol; 0.108 g, 0.18 mmol for 2-, 3-and 4-picoline, respectively) was suspended in H 2 O 2 (30% v/v, 70 mL per 1 g) and stirred at 45 o C for 3 hours. A bright yellow solution formed, which was filtered (IM) to remove any unreacted starting material or insoluble side-products. Then, the H 2 O 2 was removed by lyophilization. To isolate the final product, the residual yellow precipitate was suspended in the minimum amount of warm ethanol (45 o C) and then it was allowed to precipitate upon addition of diethyl ether (3-fold) at -20 o C.

Trans-[Pt(Cl) 2 (3-pic) 2 ] (21)
K 2 PtCl 4 (0.700 g, 1.69 mmol) was dissolved in H 2 O (55 mL) and 3-picoline (50 eq, 7.51 mL) was added. The solution was allowed to react under reflux at 100 o C for 24 h. The water and excess ligand were evaporated and the white residue was washed with diethyl ether. Then HCl (2 M, 24 mL) was added and the reaction proceeded for 48 h at 85 o C. The yellow precipitate was collected by filtration (Yield = 0.706 g, 93%).

Trans-[Pt(N 3 ) 2 (4-pic) 2 ] (25)
Trans-[Pt(Cl) 2 (4-pic) 2 ] (0.690 g, 1.53 mmol) was dissolved in DMF (35 mL), then NaN 3 (20 eq, 1.282 g) was added and the mixture was allowed to react for 4 d at room temperature in the dark. The solution was then placed at -20 o C, for 2 d after which the yellow product was isolated by filtration. A second crop of precipitate was collected by the addition an equal volume of diethyl ether to the filtrate, followed by storage at -20 o C for a further 2d. The precipitates were combined, washed with cold solvents (H 2 O, ethanol, ether) to give the product as a yellow solid (Yield = 0.605 g, 85%).

Trans-[Pt(N 3 ) 2 (OH) 2 (4-pic) 2 ] (26)
Trans-[Pt(N 3 ) 2 (4-pic) 2 ] (0.600 g, 1.29 mmol) was suspended in H 2 O 2 (30% v/v, 150 ml) and stirred for 20 h at 50 o C, to give a yellow solution which was lyophilized. Crystallisation was carried out by dissolving the solid in warm methanol (2 mL, 55 o C) and then adding diethyl ether (20 mL). The mixture was placed at -20 o C to give the title compound as a yellow solid. (Yield= 0.193 g, 30%). Crystals suitable for X-ray diffraction were obtained by the slow evaporation at ambient temperature of a methanolic solution of 26.

Trans-[Pt(Cl) 2 (tz) 2 ] (27)
K 2 PtCl 4 (0.800 g, 1.93 mmol) was completely dissolved in H 2 O (16 mL) and thiazole (14 eq, 2.29 mL) was added. The solution was allowed to react, under reflux, at 100 o C for three hours until the mixture turned into a transparent yellow solution (an orange precipitate is formed as an intermediate in the reaction). The solvent was rotary evaporated to dryness and the residual yellow solid was suspended in HCl (2 M, 24 mL) and allowed to react overnight (14 hours) at 85 o C. The yellow precipitate was filtered, washed with water, ethanol and diethyl ether (Yield = 0.750 g, 89%). The product was recrystallised from hot 0.1 M HCl in ethanol, affording yellow crystals suitable for X-ray diffraction.

Trans-[Pt(N 3 ) 2 (tz) 2 ] (28)
Trans-[Pt(Cl) 2 (tz) 2 ] (0.700 g, 1.61 mmol) was completely dissolved in DMF (30 mL) and NaN 3 (20 eq, 2.087 g) was added. The reaction was allowed to proceed for 6 d at room temperature in the dark and then placed in the freezer (-20 o C) overnight. Diethyl ether (30ml) was added and the solution returned to the freezer. After 6 h yellow crystals and a white precipitate formed, the crystals were isolated by filtration and rinsed with minimal cold H 2 O to remove the white precipitate by dissolution. (Yield= 0.411 g, 57%). Crystals suitable for x-ray crystallography were grown by diffusion of diethyl ether into an acetone solution of the product. 1 H-NMR (acetone-d 6 , 400 MHz) δ: 9.57 (dd, 3 J 1H1H = 2.05 Hz, 3

Trans, trans, trans-[Pt(N 3 ) 2 (OH) 2 (tz) 2 ] (29)
Trans-[Pt(N 3 ) 2 (tz) 2 ] (0.400 g, 0.89 mmol) was suspended in H 2 O 2 (30% v/v, 45 mL) and allowed to react at 50 o C for 6 hours. The yellow solution was IM filtered and then the H 2 O 2 removed by freeze drying. Cold acetone was used to rinse the residual yellow solid from the glass and the product was filtered under suction and washed with cold water, ethanol and ether (Yield= 0.215 g, 50%). 1

Trans-[Pt(OH) 2 (N 3 ) 2 (1-methylimidazole) 2 ] (32)
Trans-[Pt(N 3 ) 2 (mim) 2 ] (0.106 g, 0.24 mmol) was suspended in H 2 O 2 (30% v/v, 9.2 mL) and the reaction was heated to 50 o C, until the yellow solution became transparent (1.5 h). Removal of the solvent was carried out via lyophilisation after the solution was IM-filtered. Recrystallisation in a methanol/ DCM solution allowed the growth of crystals suitable for their study by X-ray diffraction (Yield= 0.050 g, 44%).    . The complexes are composed of two hydroxide, two azides and two 4-methyl pyridines. Four complete complexes and eight methanols in the unit cell. One of the methanols (O1-C2) bridges between the OHs of the different complexes. The other methanol (O3 C4) bridges between this methanol and other symmetry related complexes. It was not possible to differentiate the C from the O from examination of the thermal ellipsoids or from the hydrogen bonding pattern so the oxygen and the carbon of that methanol were modelled as 50% occupancy at either position. The hydrogens (O100 and O200) were located on the OH ligands on each crystallographically independent Pt complex but refined with restraints. Any attempt to place hydrogens on the bridging solvent modelled at O3-O4 leads to alerts for short contacts. A model was constructed where the atoms of methanol (O3-O4) were modelled at 50% occupancy of a C and O so the methanol sits 50% one way and 50% the other way round. This makes it difficult to put hydrogens on the bridging methanol O1-C2 as half the time, O1 would be donating a hydrogen bond to C4. This means hydrogens cannot be placed on methanol O1-C2, leading to several crystallographic "B" alerts for a singly bonded carbons with no hydrogens.         Table S11. Decomposition half-lives of complexes (60 μM) when irradiated with blue (463 nm) light, as measured by UV-Vis spectroscopy.

5'
Table S12: 5'-Guanosine monophosphate (5´-GMP) binding of Pt IV -diazido complexes upon blue light irradiation (420 nm, 1 h, 14 mW.cm -2 ) with numbering scheme. The error in % binding is approximated as 5 -10 % due to the uncertainty in 1 H NMR spectral integration of the sugar C 1 ' proton. . The formation of the mono-5'-GMP adduct is shown, with the assignment of the peaks corresponding to the pyridine ligands on the platinum. Assignment was aided by 2D 1H correlation spectroscopy (COSY). (C) Two weeks after the irradiation, where the evolution of a second (bis) 5'-GMP adduct is observed, as shown by the arrows. The spectrum on the right hand side is expanded for the C1 proton region of the sugar ring to illustrate the evolution of the second product 3 days after irradiation. Figure S4: 195 Pt NMR spectra of the Pt(IV) and Pt(II) regions after irradiation (420 nm, 45 min, 7 mW.cm -2 ) of complex 18 (9 mM) in the presence of 5'-GMP. Spectra A and B were obtained immediately after irradiation whereas spectrum C was recorded 2 weeks after the sample was allowed to stand in the dark at ambient temperature. Products X and Y arw assigned to the mono-5'-GMP and bis-5'-GMP adducts, respectively. Z is tentatively assigned to the intermediate trans-[Pt(2-pic)(py)(OH)(5'-GMP).