Journal of Medicinal Chemistry ARTICLE Scheme 1. Autotaxin (ATX) is Responsible for Hydrolyzing the Lipid Lysophosphatidylcholine (LPC) into Lysophosphatidic Acid (LPA) and Choline Figure 1. ATX structure liganded with inhibitor 1 (PDB ID 2XRG). (A) Surface representation of ATX with inhibitor 1 (magenta). (B) Binding of inhibitor 1 to the threonine oxygen nucleophile and two zinc ions. (C) Visualizing the ether linker of inhibitor 1 bound to ATX. (D) Visualizing the degree of freedom for the thiazolidine-2,4-dione core of inhibitor 1 in the ATX binding site. methylene and arylboronic acid moiety are well accessible to solvent (Figure 1C). Binding of inhibitor 1 to the ATX active site is predominately driven by hydrophobic interactions (the inter- 2 action interface is approximately 500 Å ) and by the boronic acid binding to the threonine oxygen nucleophile of ATX.9 The boron oxygen distance observed is ∼1.6 Å, which is consistent with a covalent bond. As expected, this binding is reversible evidenced by the fact that ATX activity can be fully restored upon washing out the inhibitor.13 In addition, one of the boronic acid hydroxyl moieties is tethered by the two zinc ions in the ATX active site. Thus, the boronic acid moiety targets not only the threonine oxygen nucleophile, but also the two zinc ions that are essential for catalytic activity of ATX (Figure 1B). Remarkably, there are no hydrogen bonds or salt bridges that participate in binding of inhibitor 1 to ATX. Inhibitor 1 is locked in a pose with reduced molecular flexibility, forming an ideal starting point for a structure-based approach to further modifications. Previously, we determined that the 4-ﬂuorobenzyl moiety is preferred from over 40 benzylic substituents tested.13 For this reason, we left the 4-ﬂuorobenzyl moiety untouched in this study. We investigated new design options, starting by modifying the ether linker in inhibitor 1. We decided to replace the ether linkage (OCH2) with various amides, an amine, and an E-conﬁgured double bond (CONH (17), CONCH2 (18), NHCO (19), NHCH2 (36), and (E)-CHdCH (20); see Table 1). The thiazolidine-2,4-dione core was investigated next. We substituted Table 1. IC50 Values of the Inhibitors Resulting from the Linker Modiﬁcation a IC50 values have been determined in the choline release assay using 40 μM LPC and 10 nM ATX. The dose response curve of inhibitor 17 shows biphasic curve (see Supporting Information Figure S2). the sulfur atom present with (substituted) amino and methylene moieties (NH (26), NCH3 (28), or CH2 (32)) (Table 2). The carbon double bond conjugated to the thiazolidine-2,4-dione carbonyl moiety forms a possible Michael acceptor. Although this Michael acceptor is resistant to nucleophilic additions in vitro (Supporting Information Figure S1) it may be a biologically active Michael acceptor in vivo, and therefore, we investigated its removal. Chemical Synthesis of Modified Inhibitors. We first ex- plored synthetic routes to replace the ether linkage in 1. The synthesis of target molecules 17 20 (Scheme 2) starts with palladium catalyzed borylation of appropriate aldehydes via a 4620 dx.doi.org/10.1021/jm200310q |J. Med. Chem. 2011, 54, 4619–4626