Journal of Medicinal Chemistry Table 2. IC50 Values of the Inhibitors Resulting from the Core Modiﬁcation a IC50 values have been determined in the choline release assay using 40 μM LPC and 10 nM ATX. E-28 contains 20% of the Z-isomer. Suzuki-Miyaura reaction15 as depicted in Scheme 2 (for synth- eses of aldehydes 2 6 see Supporting Information). This reaction smoothly provided intermediates 7 11. Next, the pinacol protecting group was hydrolyzed under acidic conditions and oxidatively destroyed by NaIO4 giving boronic acid alde- hydes 12 16. In the final step 3-(4-fluorobenzyl)thiazolidine- 2,4-dione is reacted with the boronic acid aldehyde by Knoevenagel condensation to selectively give the Z-isomer of the final products (1 and 17 20). In order to remove the potential Michael acceptor present, we reduced the double bond in inhibitor 1 using hydrogen and ARTICLE palladium on carbon (Scheme 3A), to give compound 21. To reduce the carbonyl moiety also in the thiazolidine-2,4-dione core of 1, we used NaBH4 resulting in hemiaminal 22. After addition of sulfuric acid to the reaction mixture to eliminate the hydroxyl moiety in 22, unsaturated inhibitor 23 was obtained. For the syntheses of imidazolidine-2,4-dione-based inhibitors 26 and 28, imidazolidine-2,4-dione (24) was mono N-alkylated with 4-ﬂuorobenzyl bromide to give intermediate 25 (Scheme 3B). Reaction of 25 by a Knoevenagel condensation with aldehyde 12 selectively resulted in the formation of the Z-isomer of 26. In parallel, intermediate 25 was methylated to give compound 27. Finally, 27 was condensed with aldehyde 12 resulting in target molecule 28. Both the Z- and E-isomers were formed in a 1:4 (Z:E) ratio. To obtain and isolate solely the Z-isomer 28, we N-methylated compound 26. Core-hopping from thiazolidine-2,4-dione to pyrrolidine-2,5- dione is depicted in Scheme 3C. The route starts with the forma- tion of a Wittig reagent starting from 2,5-pyrroledione (29), which is reacted with triphenylphosphine to form ylide 30.16 The Wittig reaction of the carbonyl stabilized ylide 30 with aldehyde 12 selec- tively leads to the E-isomer of intermediate 31 as expected. Finally, compound 31 is N-alkylated with 4-ﬂuorobenzyl bromide resulting in the pyrrolidine-2,5-dione product 32. For the synthesis of a ﬁnal tetrahydroisoquinoline-based core with a more rigid structure, the secondary amine in R- or S- tetrahydroisoquinoline 33 is reacted with 4-ﬂuorobenzyl isocya- nate in the presence of sodium hydroxide to form a urea inter- mediate. The imidazolidine ring is then formed upon acidiﬁca- tion with hydrochloric acid, resulting in compound R- or S-34. In the ﬁnal step, intermediate 34 is O-alkylated with 4-(bromomethyl)- phenylboronic acid resulting in R- or S-35. Structure Activity Relations of Inhibitors and Autotaxin. Activity of the new molecules resulting from the linker and core modifications were determined in an LPC hydrolysis assay described previously,13,17 in which ATX-mediated release of choline from LPC is detected by a two-step enzymatic colori- metric reaction. The IC50 values observed for inhibitors with modified linkers and cores are listed in Tables 1 and 2. When we replaced the ether moiety (OCH2, inhibitor 1, IC50 = 5.7 nM) for an amide linker (CONH, 17), we observed a signi- ﬁcant loss of activity (IC50 = 147 nM). The dose response curve of inhibitor 17 shows a biphasic curve18 that could suggest several binding sites of this inhibitor for ATX (Supporting Information Figure S2). Expanding the CONH linker (17) to a more ﬂexible CONHCH2 linker (18) improved the IC50 value (71 nM) by 2-fold compared to inhibitor 17. Reversing the amide linker in 17 to yield compound 19, results in high potency (IC50 = 10 nM), similar to inhibitor 1. However, inhibitor 19 is not able to achieve full inhibition, and 10% residual ATX activity is observed (Sup- porting Information Figure S3). Apparently, a more rigid amide linker results in suboptimal binding of the inhibitor. Therefore, we synthesized the more ﬂexible amine analogue 36 (for the synthesis of inhibitor 36 see Supporting Information). This resulted indeed in a potent inhibitor (IC50 = 8.3 nM), similar to 1, and with no re- sidual ATX activity (Supporting Information Figure S3). Intro- duction of an (E)-CHdCH linker resulted in compound 20, which was inactive in the nanomolar range (IC50 > 5 μM). This observation can be explained by the fact that the two aromatic rings linked with a ﬂexible OCH2 linker in inhibitor 1 are posi- tioned in an angle of roughly 90 in the ATX structure (see Figure 1B) which cannot be achieved by the (E)-CHdCH linker due to its rigid planar conformation. 4621 dx.doi.org/10.1021/jm200310q |J. Med. Chem. 2011, 54, 4619–4626