An albumin-based tumor-targeted oxaliplatin prodrug with distinctly improved anticancer activity in vivo † †Electronic supplementary information (ESI) available: Experimental materials and methods, characterization details of all compounds, X-ray crystal structures. CCDC 1499561 and 1499562. For ESI

An oxaliplatin-based platinum(iv) drug which specifically binds to albumin after i.v. application led to several complete responses in tumor-bearing mice.


Materials and methods
Potassium tetrachloridoplatinate (K 2 PtCl 4 ) was purchased from Johnson Matthey (Switzerland). Water for synthesis was taken from a reverse osmosis system and distilled twice before use. For HPLC measurements Milli-Q water (18.2 MΩ•cm, Merck Milli-Q Advantage, Darmstadt, Germany) was used. Other chemicals and solvents were purchased from commercial suppliers (Sigma Aldrich, Merck, Acros, Fluka and Fisher Scientific).
Thereafter, the complexes were oxidized with hydrogen peroxide (50%) using either methanol 3 or acetic acid 4 as a solvent to yield the unsymmetrically oxidized platinum(IV) precursors 3-6. The maleimide-and succinimide-functionalized ligands were prepared as recently published. 5 Electrospray ionization (ESI) mass spectra were recorded on a Bruker amaZon SL ion trap mass spectrometer in positive and/or negative mode by direct infusion.
High resolution mass spectra were measured on a Bruker maXis™ UHR ESI time of flight mass spectrometer. All mass spectra were recorded at the Mass Spectrometry Centre of the University of Vienna. One-and two-dimensional 1 H-, 13 C-, 15 N-and 195 Pt-NMR spectra were recorded on a Bruker Avance III 500 MHz spectrometer at 500. 10 ( 1 H), 127.75 ( 13 C), 50.68 ( 15 N), and 107.51 ( 195 Pt) MHz at 298 K. For 1 H-and 13 C-NMR spectra the solvent residual peak was taken as internal reference, whereas 195 Pt-shifts were referenced relative to external K 2 PtCl 4 and 15 N-shifts relative to external NH 4 Cl. Elemental analysis measurements were performed on a Perkin Elmer 2400 CHN Elemental Analyzer at the Microanalytical Laboratory of the University of Vienna.

Synthesis
General procedure for the synthesis of compounds 7-14: The unsymmetrically, oxidized platinum(IV) compound (3)(4)(5)(6) and the corresponding isocyanate were transferred into a Schlenk flask and set under argon atmosphere. After the addition of dry DMF, the suspension was stirred overnight. A remaining precipitate was filtered, before the DMF was removed under reduced pressure. The residue were taken up as a suspension in methanol and fully precipitated by addition of diethylether. After a few hours in the fridge the crude product was filtered off, washed with diethylether and dried under reduced pressure. The solid was taken up in water and filtered before it was purified by preparative RP-HPLC. Collected product fractions were concentrated, to remove methanol, thereafter lyophilized and dried under reduced pressure.

Purification of compounds 7-14 by preparative RP-HPLC:
All compounds were purified by preparative RP-HPLC using a Waters XBridge C18 column on an Agilent 1200 Series system. Milli-Q water, containing 0.1% formic acid, and methanol were used as eluents.    Single crystal X-ray crystallography The X-ray intensity data were measured on a Bruker D8-Venture equipped with a multilayer monochromator, a Cu-K α INCOATEC micro focus sealed tube and Kryoflex II cooling device.
The results were uploaded to the CCDC (codes see Table S1). The structures were solved by direct methods and refined by full-matrix least-squares techniques. Non-hydrogen atoms were refined with anisotropic displacement parameters. Hydrogen atoms were inserted in calculated positions and refined with a riding model respectively as rotating systems. The following software was used: Frame integration, Bruker SAINT software package 7 using a narrow-frame algorithm, Absorption correction, SADABS, 8 structure solution, SHELXS-97, 9 refinement, SHELXL-2013, 9 SHELXLE, 10 molecular diagrams and OLEX2. 11 Experimental data can be found in Table S1. Crystal data, data collection parameters, and structure refinement details are given in Table S2-S3 and Table S5-S6. Molecular Structures in "Ortep View" are depicted in Figure S1 and Figure S2.

RP-HPLC stability and binding studies
To determine the stability, especially of the maleimide-function, 0.5 mM solutions of the complexes were prepared in triple distilled water and measured repeatedly over several hours. For determination of the binding rate towards human serum albumin (HSA), freshly prepared 1 mM aqueous solutions of a compound and HSA were mixed 1:1 and also measured repeatedly over several hours. All studies were carried out using a reversed-phase Waters XBridge C18 column on a Dionex Summit System. Milli-Q water, containing 0.1% formic acid, and methanol were used as eluents and a standard gradient from 5-95% methanol in 20 minutes was chosen. The autosampler, where the samples were incubated, was temperature controlled at 20°C and the column at 25°C. The loss of the complexes was monitored via UV-VIS detector at 225 nm and evaluated by the decrease of the compound peak area.

SEC-ICP-MS studies
A reagent I grade water (>10 MΩ cm -1 resistance according to ISO 3696 water specifications)  was used as reaction gas. The ICP-MS operation parameters are given in Table S8.  The analysis was carried out in isocratic mode and the flow was split 1:1 after the column since the concentration (100 µM) for the incubation carried out in the autosampler was too high and not suitable for the ICP-MS detector. The chromatographic conditions are shown in Table S9.

SEC-ICP-MS stability and FCS binding studies
A flow injection analysis (FI) was carried out to determine the real concentration of the platinum drugs in FCS and the column recovery. The column recovery was determined at time 0 h of incubation. The results are given in Table S10.

SEC-ICP-MS measurements of in vivo serum samples
The ICP-QMS iCAP Thermo Scientific (Bremen, Germany) was used. Oxygen (purity 4.5, Linde Gas GmbH, Vienna, Austria) was used as reaction gas. The ICP-MS operation parameters are given in Table S12.  Table S13. A flow injection analysis (FI) was carried out to determine the real concentration of the platinum drugs in FCS and the column recovery. The results are given in Table S14.

DLS measurements
The samples were prepared by incubating 100 µM HSA with either 5 eq. of the oxaliplatin-  Table S15. The instrument was tuned on daily basis to achieve maximum sensitivity.     Tables and figures   Table S19: Albumin-binding half-lives of compounds 7-8 and 11-12 evaluated by RP-HPLC.  At day 12 after first treatment, animals were sacrificed and tumor tissues collected, formalin-fixed, paraffin-embedded and H/E-stained. The fractions of apoptotic and mitotic cells were evaluated by counting (4 pictures per sample, at least 300 cells per picture were counted). Each experimental group contained four animals (the respective tumor growth is shown in Figure 4A). a n=3 because of one complete remission. The statistical significance was calculated by One-Way Anova and is indicated with asterisks (* P < 0.05; ** P < 0.01; *** P < 0.001).