Journal of Medicinal Chemistry Figure 2. (A) Focus from inside the protein on the thiazolidine-2,4- dione core of inhibitor 1 bound to ATX. (B) Inhibitor 1 docked into the active site of ATX to validate our docking approach. (C) The three best docking poses for the Z-isomer of 28. (D) The three best docking poses for the E-isomer of 28. Docking poses were generated using the docking program Glide. chiral inhibitor 35, which is still very potent although some activity is lost compared to 1. No signiﬁcant diﬀerence is observed in potency for either the S or R enantiomers of inhibitor 35, with IC50 values of 55 and 59 nM, respectively. In summary, we explored structure activity relations building on boronic acid-based ATX inhibitor 1, which resulted in a number of potent inhibitors. We used the crystal structure of ATX liganded with inhibitor 19 to explain the structure activity relationships observed for a rational inhibitor modiﬁcation approach. Our results suggest that this approach allows rapid structure guided modiﬁca- tion. Finally, molecular docking eﬀorts proved useful to explain unexpected high potency of E-isomer 28 and suggested that the lipophilic pocket near the ATX active site may be better exploited in the future for the design of new inhibitors. ’ EXPERIMENTAL SECTION Chemistry. The R and S enantiomers of building block 33 were purchased from CSPS Pharmaceuticals, San Diego, USA. All other chemi- cals were obtained from Sigma-Aldrich and used without further purification unless otherwise noted. Analytical thin layer chromatography was per- formed on aluminum sheets precoated with silica gel 60 F254. Column chromatography was carried out on silica gel (0.035 0.070, 90 Å, Acros). ARTICLE For isolation by centrifugation a Heraeus Multifuge 3 S-R centrifuge was used. Products were spun at 4400g, at 298 K for 5 min. Nuclear magnetic resonance spectra (1H and 13C NMR) were determined in deuterated dimethyl sulfoxide (d6-DMSO) using a Bruker Avance 300 (1H, 300 MHz; 13C, 75 MHz) at 298 K, unless indicated otherwise. Peak shapes are indicated with the symbols ‘d’ (doublet), ‘dd’ (double doublet), ‘s’ (singlet), ‘bs’ (broad singlet), and ‘m’ (multiplet). Chemical shifts (δ) are given in ppm and coupling constants J in Hz. Dimethyl sulfoxide (δH = 2.50 ppm; δC = 39.51 ppm) was used as internal reference. The purity of all tested compounds was determined by high-perfor- mance liquid chromatography coupled to mass spectrometry (HPLC- MS) and was greater than 95%. HPLC-MS measurements were per- formed on a system equipped with a Waters 2795 Separation Module (Alliance HT), Waters 2996 Photodiode Array Detector (190 750 nm), Atlantis T3 C18 column (2.1 100 mm, 3 μm), and an LCT Orthogonal Acceleration Time of Flight Mass Spectrometer. Samples were run at a ﬂow rate of 0.40 mL min 1 at 313 K, using gradient elution (water/ acetonitrile/formic acid) from 950/50/1 (v/v/v) to 50/950/1 (v/v/v). The preparative HPLC system was equipped with a Waters 1525 Binary HPLC Pump, a Waters 2487 Dual λ Absorbance Detector, and an Atlantis C18 column (19 250 mm, 10 μm). Samples were run at a ﬂow rate of 18 mL min 1 using gradient elution (water/acetonitrile) from 60/40 (v/v) to 10/90 (v/v). General Procedure for Borylation of Aldehydes and Pinacol Deprotection (12 16). In a dry flask, bis(pinacolato)diboron (1.34 g, 5.28 mmol), the appropriate aldehyde (1.80 mmol) and potassium acetate (0.542 g, 5.52 mmol) were added to a solution of Pd(dppf)Cl2 (46.6 mg, 0.0637 mmol) in dimethylformamide (15 mL). The reaction mixture was stirred under an atmosphere of argon for 18 h at 353 K. The reaction mixture was filtered over Hyflo Super Cel medium and diluted with ethyl acetate (100 mL). The solution was washed with brine (50 and 25 mL), dried over magnesium sulfate, and concentrated. The crude product was dissolved in tetrahydrofuran (11 mL) and sodium periodate (2.22 g, 10.4 mmol) and water (2.8 mL) were added. After stirring for 30 min, 1 M hydrochloric acid (1.1 mL) was added, and after 2 h, additional sodium periodate (1.17 g, 5.47 mmol) was added and the solution was stirred for another 2 h. The reaction mixture was diluted with ethyl acetate (20 mL) and washed with water (10 mL). The water layer was extracted with ethyl acetate (15 mL). The combined organic layers were washed with brine (15 mL), dried over magnesium sulfate, and the solution was concentrated under vacuum resulting in a light yellow solid. The resulting product was used without further puriﬁcation. (4-((4-Formylphenoxy)methyl)phenyl)boronic Acid (12). Yield: 70%. 1H NMR: δ = 9.86 (s, 1H), 8.09 (s, 1H), 7.87 (d, J = 8.8, 1H), 7.82 (d, J = 8.1, 1H), 7.42 (d, J = 8.1, 1H), 7.20 (d, J = 8.7, 1H), 5.24 (s, 1H). 13C NMR: δ = 191.75, 163.73, 138.49, 134.74, 132.26, 130.22, 127.15, 115.76, 70.09, 39.95 (C B(OH)2 not visible). MS: m/z [MþH]þ calc. 257.10, obs. 257.10. For experimental details of compounds 13 16, see Supporting Information. General Method for Knoevenagel Condensation (1, 17 20, 26, and E-28). To a solution of 3-(4-fluorobenzyl)thiazolidine-2,4-dione (0.293 mmol) in ethanol (2.5 mL), piperidine (20 μL, 0.207 mmol), and the appropriate aldehyde (0.352 mmol) were added and the solution was refluxed for 22 h. (Z)-4-[(4-{[3-(4-Fluorobenzyl)-2,4-dioxo-1,3-thiazolan-5- yliden]methyl}phenoxy) methyl]benzene Boronic Acid (1). Upon cooling the reaction mixture to room temperature, the product precipitated out of solution. Dissolving the product in dimethyl sulfoxide and precipitating it with 0.5 M hydrochloric acid resulted in pure compound. Yield: 81%. 1H NMR: δ = 8.03 (s, 2H), 7.92 (s, 1H), 7.80 (d, J = 8.1, 2H), 7.60 (d, J = 8.9, 2H), 7.41 (d, J = 8.0, 2H), 7.39 7.31 (m, J = 5.5, 8.8, 2H), 7.26 7.09 (m, J = 4.5, 8.9, 4H), 5.21 (s, 2H), 4.82 (s, 2H). 13C NMR: δ = 167.38, 165.59, 161.66 (d, 1JCF = 244), 160.33, 4623 dx.doi.org/10.1021/jm200310q |J. Med. Chem. 2011, 54, 4619–4626