Journal of Medicinal Chemistry (3:1, 4 mL) and 30 wt % of sodium hydroxide solution was used to adjust the pH to 14. The reaction mixture was heated to 313 K and 4-fluoro- benzyl isocyanate (100 μL, 0.785 mmol) was added. After 2 h of stirring, the mixture was cooled to room temperature and the resulting solid was removed using centrifugation. From the resulting solution, dioxane was evaporated and concentrated hydrochloric acid was used to adjust the pH to 1. After refluxing the reaction mixture for 2 h 30 min, it was cooled to 278 K affording a white precipitate. Washing the precipitate with ice-cold water (3 1 mL) afforded pure compound S-34. Yield: 30 mg, 18%. 1H NMR: δ = 9.37 (s, 1H), 7.35 7.30 (m, 2H), 7.18 7.12 (m, 2H), 7.06 7.03 (m, 1H), 6.65 6.63 (m, 2H), 4.77 (d, J = 16.8, 1H), 4.58 (s, 2H), 4.32 4.27 (m, 2H), 3.05 (m, 1H), 2.82 2.64 (m, 1H). 13C NMR: δ = 172.76, 161.44 (d, 1JCF = 244), 156.07, 154.60, 132.82 (d, 4JCF = 3), 132.35, 130.13, 129.51 (d, 3JCF = 8), 121.45, 115.26 (d, 2JCF = 21), 114.30, 112.67, 54.54, 41.23, 40.56, 29.02. MS: m/z [MþH]þ calc. 327.11, obs. 327.09. (R)-2-(4-Fluorobenzyl)-7-hydroxy-10,10a-dihydroimidazo- [1,5-b]isoquinoline-1,3(2H,5H)-dione (R-34). For reaction de- tails, see compound S-34. Yield: 29%. 1H NMR, 13C NMR, and MS, see Supporting Information. (S)-(4-(((2-(4-Fluorobenzyl)-1,3-dioxo-1,2,3,5,10,10a-hexa- hydroimidazo[1,5-b]isoquinolin-7-yl)oxy)methyl)phenyl)boronic Acid (S-35). To a heated solution (323 K) of compound S-34 (15.3 mg, 0.0469 mmol) in acetone (0.3 mL), potassium carbonate (10.2 mg, 0.0738 mmol) and 4-(bromomethyl)phenylboronic acid (12.4 mg, 0.0577 mmol) were added. After 4 h of stirring and heating, additional potassium carbonate (10.1 mg, 0.0730 mmol) was added and the suspension was stirred overnight. Finally, the reaction mixture was diluted with ethyl acetate and the organic layer was washed with 1 M hydrochloric acid (2 0.5 mL) and brine (0.5 mL), dried over sodium sulfate, and concentrated in vacuo. The resulting solid was further purified using preparative HPLC affording pure compound S-35. Yield: 11 mg, 52%. 1H NMR: δ = 8.06 (s, 2H), 7.78 (d, J = 8.1, 2H), 7.50 7.27 (m, 4H), 7.27 7.05 (m, 3H), 7.02 6.76 (m, 2H), 5.09 (s, 2H), 4.82 (d, J = 17.0, 1H), 4.58 (s, 2H), 4.42 4.20 (m, 2H), 3.19 3.02 (m, 1H), 2.80 2.71 (m, 1H). 13C NMR: δ = 172.72, 161.46 (d, 1JCF = 244), 157.09, 154.60, 138.74, 134.17, 132.81 (d, 4JCF = 3), 132.60, 130.23, 129.55 (d, 3JCF = 8), 126.49, 123.60, 115.28 (d, 2JCF = 21), 113.87, 112.47, 69.20, 54.40, 41.30, 40.60, 29.00 (C B(OH)2 not visible). MS: m/z [MþH]þ calc. 461.17, obs. 461.14. (R)-(4-(((2-(4-Fluorobenzyl)-1,3-dioxo-1,2,3,5,10,10a-hexa- hydroimidazo[1,5-b]isoquinolin-7-yl)oxy)methyl)phenyl)- boronic acid (R-35). For reaction details, see compound S-35. Yield: 56%. 1H NMR, 13C NMR, and MS, see Supporting Information. Choline Release Assay.13 Measuring ATX activity using LPC (18:1) as substrate was determined as follows. In an opaque flat-bottom 96-wells plate (Greiner) was added 0.5 μL dimethyl sulfoxide containing inhibitor, to 25 μL recombinant ATX (∼20 nM) in Tris-HCl buffer (0.01% Triton X-100 and 50 mM Tris-HCl, pH 7.4). Finally, 25 μL of 80 μM LPC (18:1) in Tris-HCl buffer (10 mM MgCl2, 10 mM CaCl2, 0.01% Triton X-100 and 50 mM Tris-HCl, pH 7.4) was added to each well and the plate was incubated at 310 K. The above-described mixture with dimethyl sulfoxide alone was used as a control. LPC without ATX was taken as control for autohydrolysis of LPC. For each inhibitor, ten concentrations were measured covering a range of 0.01 to 30 μM to determine IC50 values. After 4 h of incubation, 50 μL ABTS (2 mM) and horseradish peroxidase (5 U mL 1) was added to the reaction mixture and absorbance was measured and used to correct for absorbance of the molecules. Finally, 50 μL choline oxidase (5 U mL 1) in Tris-HCl (0.01% Triton X-100 and 50 mM Tris-HCl, pH 7.4) was added for colorimetric reaction. Absorbance was measured in a Perkin-Elmer Envision plate reader (λ = 405 nm). Data were analyzed using Graphpad Prism software. In addition, the eﬀect of the inhibitors on the enzymatic coloring reaction was investigated using 40 μM choline at 30 μM inhibitor using ARTICLE the above-described coloring reagents. The enzymatic coloring reaction was not eﬀected by the inhibitors. Docking Experiments, Protein, and Ligand Preparation. The X-ray structure of ATX in complex with inhibitor 1 (PDB ID: 2XRG) was used for the docking studies. The protein structure was prepared using the Schro€dinger Suite 2010 Protein Preparation Wizard (with Epik 2.1,24 Impact 5.6, and Prime 2.2). The initial 3D structures of the ligands were generated using LigPrep 2.4 and the ligand partial charges were ascribed using the OPLS2005 force-field as performed by Glide 5.6.20 22 We defined the binding region by a 20 Å 20 Å 20 Å box centered on the central position of inhibitor 1 in the crystal ATX complex. We used positional constraints for the two oxygen atoms of the boronic acid and the arylic carbon direct next to the boron atom in inhibitor 1. The Glide Emodel score was used to rank the docking poses. Pictures were made using PyMOL 1.3. ’ ASSOCIATED CONTENT bS Supporting Information. Contains Michael acceptor study of inhibitor 1, dose response curves of inhibitors 17, 19, and 36, image visualizing π-stacking of carbonyl inhibitor 1 with ATX residue F274, image of docked and X-ray pose of inhibitor 1 superimposed, experimental details of compounds 13 20, 26, and E-28, details on the syntheses of aldehydes 2 6 and amine linker-based inhibitor 36, and spectral data of all inter- mediates and target molecules. This material is available free of charge via the Internet at http://pubs.acs.org. ’ AUTHOR INFORMATION Corresponding Author *Huib Ovaa, Division of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. Phone: þ31-20-5121979. E-mail: h.ovaa@nki.nl. Notes †Deceased. ’ ACKNOWLEDGMENT In memory of Loes J. D. Hendrickx 06-01-1986/28-05-2010. We want to thank Irene Farre Gutierrez for helpful discussions. This work was supported by grants from The Netherlands Organization for Scientiﬁc Research (NWO), the Dutch Cancer Society (KWF), and The Netherlands Proteomics Centre sup- ported by The Netherlands Genomics Initiative. ’ NON-STANDARD ABBREVIATIONS ABTS, 2,20-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid); ATX, autotaxin; HPLC-MS, high-performance liquid chromatography mass spectrometry; LPA, lysophosphatidic acid; LPC, lysopho- sphatidylcholine; eNPP, ectonucleotide pyrophosphatase and phosphodiesterase ’ REFERENCES (1) Tokumura, A.; Majima, E.; Kariya, Y.; Tominaga, K.; Kogure, K.; Yasuda, K.; Fukuzawa, K. Identiﬁcation of human plasma lysopho- spholipase D, a lysophosphatidic acid-producing enzyme, as autotaxin, a multifunctional phosphodiesterase. J. Biol. Chem. 2002, 277, 39436– 39442. (2) Umezu-Goto, M.; Kishi, Y.; Taira, A.; Hama, K.; Dohmae, N.; Takio, K.; Yamori, T.; Mills, G. B.; Inoue, K.; Aoki, J.; Arai, H. 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