Organometallic small molecule kinase inhibitors – direct incorporation of Re and 99mTc into Opaganib®

[(η5-Cp)ReI(CO)3] was incorporated into the kinase inhibitor Opaganib®. The resulting bioorganometallic complex showed a similar anti-cancer activity to Opaganib® against PC-3 cancer cells. The IC50 value for the kinase SK2 is 30x higher than that of Opaganib®. The 99mTc homologue was synthesized, completing a matched-pair for molecular theranostics.

(solvent B)The flow rate was 40 mL min −1 . Detection was performed at between 230 and 270 nm.
Elemental Analysis: Elemental Analysis (EA) measurements were performed on a LecoCHNS-932 elemental analyzer.
Crystal structure determination: Single-crystal X-ray diffraction data for compounds 3, 4 and 6 were collected at 160(1) K on Rigaku OD diffractometers: a XtaLAB Synergy, Dualflex, Pilatus 200K diffractometer for 6 and a SuperNova/Atlas area-detector diffractometer for 3 and 4, both diffractometers using the Cu Kα radiation (λ = 1.54184 Å) from a micro-focus X-ray source and an Oxford Instruments cryojet cooler. The selected suitable single crystals wer mounted using polybutene oil on a flexible loop fixed on a goniometer head and immediately transferred to the diffractometer. Pre-experiment, data collection, data reduction and analytical absorption correction 1 were performed with the program suite CrysAlisPro. 2 Using Olex2, 3 the structures were solved with the SHELXT 4 small molecule structure solution program and refined with the SHELXL2018/3 program package 5 by full-matrix least-squares minimization on F 2 . PLATON 6 was used to check the result of the X-ray analyses. CCDC numbers 2094782 for 4, 2094783 for 3, and 2094784 for 6 contain the supplementary crystallographic data for these compounds, and can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.

Synthesis of 3
A round bottom flask (500 mL) equipped with a rubber septa was charged with 2 (4.69 g, 10 mmol, 1 eq) and CH2Cl2 (100 mL). The solution was overlaid with sat. aq. NaHCO3 (100 mL) and stirred vigorously resulting in a colour change from colourless to bright yellow. 2-bromo-4-chloroacetophenone (2.34 g, 10 mmol, 1 eq) was added and the biphasic solution was stirred for 24 h at r.t. during which the colour turned dark red. The phases were separated, and the aqueous phase was extracted with CH2Cl2 (2×30 mL). The combined organic phases were washed with aq. HCl (1 M, 150 mL), dried over MgSO4 and concentrated in vacuo. The resulting material was dry loaded onto silica gel and purified by column chromatography (silica gel, hexane/EtOAc 5:1). The title compound 3 (589.7 mg, 2.24 mmol, 22%) was obtained as an off-white solid.

Cell culture
Human Caucasian prostate adenocarcinoma (PC3) cells were cultured using F12K media and Retinal pigment epithelium (RPE1) cells using DMEM/F-12 medium supplemented with 10% FBS and 1% Penstrep. The cells were cultivated and maintained in a cell culture incubator at 37 °C with 5% CO2 atmosphere. Before an experiment, the cells were passaged three times.

Cytotoxicity
The cytotoxicity of the compounds was assessed by measuring the cell viability using a fluorometric resazurin assay. The cultivated cells were seeded in sextuplicate in 96 well plates with a density of 4000 cells per well in 100 μL of media. After 24 h, the medium was removed and the cells were treated with increasing concentrations of the compound diluted in cell media achieving a total volume of 100 μL. Dilutions for Opaganib®, 6, and Cisplatin (as a comparison) were prepared as follows: 10 mM stock in DMSO for Opaganib®, 6 and 1.5 mM stock in media for Cisplatin. These solutions were further diluted to 100 µM. After 48 h incubation, the media was replaced with fresh media containing resazurin with a final concentration of 0.2 mg/mL. After 4 h of incubation at 37 °C, the fluorescence signal of resorufin product was measured (λex: 540 nm and λem: 590 nm) in a Tecan microplate Reader. EC50 values were then calculated using GraphPad Prism software. Each experiment was performed in triplicate and an average IC50 value (in μM) was reported with a standard deviation.

SK2 Inhibition
The natural substrate sphingosine is phosphorylated by the kinase SK2. For this process, ATP is converted to ADP. The rate of conversion is competitively inhibited in a concentration dependent manner by e.g. Opaganib or compound 6. The ADP formation is quantified spectroscopically with the ADP-Glo™ Kinase Assay from Promega after fixed time points. 7 In short, SK2 phosphorylates sphingosine in the presence of the inhibitor. After 40 min, the reaction is stopped by the ADP-Glo reagent and excess ATP is removed. The kinase detection reagent is then added which converts the remaining ADP into ATP. The luciferase reaction turns ATP into light emission, which is recorded as the signal.
The SK2 inhibition experiments were performed by the International Centre for Kinase Profiling, MRC PPU Reagents and Services, School of Life Sciences, University of Dundee, Dow Street, Dundee, DD1 5EH, United Kingdom, using the luminescent ADP detection assay ADP-Glo TM of Promega, which measures inhibition as %age (ATP-to-ADP conversion). Inhibition was recorded in triplicates for Opaganib, obtained from Selleck Chemicals and for compound 6. Each data point was measured in duplicates and the IC50 values are the average of three determinations (Tables S1 and S2) Experimental conditions: Sphingosine Kinase 2 (diluted in 100 mM Tris pH = 7.5, 400 mM KCl, 10 mM MgCl2, 2 mM EDTA) is assayed in a total volume of 20 ul containing 0.02 mM Sphingosine and Opaganib or compound 6 in the respective concentrations (Tables S1 and S2) and at apparent ATP Km values. The enzyme is assayed for 60 min after which 20 ul of ADP-Glo TM reagent is added to stop the kinase reaction. 10 µL of Kinase Detection Reagent was added and luminescence output was read following 45-60 min incubation at room temperature with a PerkinElmer Envision instrument for 1 sec/well. This assay follows the guidelines given by the manufacturer as described in ref 7.   Figure S1. Graphical representation of SK2 inhibition by compound 6 (left) and Opaganib ® (right)

Synthesis of [ 99m Tc]4
A microwave vial was charged with 0.5 mL of a 5 mM solution of 3 in CH2Cl2. in 99% RCP as shown in Figure 3 of the manuscript.   Figure S23. ORTEP representation of 3. The displacement ellipsoids are represented at the 30% probability level.    Figure S25. ORTEP representation of 6. The displacement ellipsoids are represented at the 30% probability level.