Journal of Medicinal Chemistry Scheme 2. Synthetic Route Towards Linker Modiﬁed Inhibitors ARTICLE Scheme 3. Synthetic Routes Towards Core Modiﬁed Inhibitors Next, we explored the activity of compounds with a modiﬁed core. Reducing the carbon double bond in 1 resulted in little loss of activity in 21 with an IC50 value of 25 nM. Thus, although rigidity is preferred, the Michael acceptor can easily be removed without signiﬁcant loss in activity. Reducing both the carbon double bond and the neighboring carbonyl in inhibitor 1 to hemiaminal 22, led to a signiﬁcant loss in potency (IC50 = 1.6 μM). Inhibitor 23, where the hydroxyl moiety in compound 22 is removed is not very potent either (IC50 = 683 nM), indicates that the carbonyl moiety is important for binding. It appears from the crystal structure of inhibitor 1 bound to ATX that π-stacking between the phenyl ring of phenylalanine residue 274 (F274) and the carbonyl moiety in 1 is very likely seen their distance (4.1 Å, Supporting Information Figure S4).19 By remo- ving the carbonyl moiety in 1 or by changing it into a hydroxyl moiety, π-stacking will be lost resulting in lower potencies as observed for inhibitor 22 and 23. The sulfur heteroatom in the thiazolidine-2,4-dione core was replaced with other atoms and moieties. We started by replacing the sulfur atom in 1 with a methylene moiety gives compound 32 with an IC50 value of 7.3 nM, comparable to inhibitor 1. Replacement of the sulfur atom for an amino group (26, IC50 = 26 nM) resulted in little loss in activity compared to inhibitor 1. When the amine in compound 26 is methylated, potency for the resulting inhibitor Z-28 (IC50 = 6.7 nM) is slightly increased. Interestingly, the E-isomer of 28 (IC50 = 5.3 nM) is marginally more potent than Z-28 or inhibitor 1, a ﬁnding that we did not anticipate. Intrigued by the characteristics of inhibitors Z-28 and E-28, we decided to calculate likely binding poses. For this purpose, we used the Glide docking software.20 22 To validate our docking approach where we constrain the boronic acid moiety, we ﬁrst docked inhibitor 1 back into the ATX active site, resulting in a pose (Figure 2B) very similar to the original crystal structure (Figure 2A) with a rmsd value of 1.1 Å (for superimposed image see Supporting Information Figure S5). Next, we docked the Z- and E-isomers of inhibitor 28 (the three best docking poses are depicted in Figure 2C and D). The docking poses of Z-28 correspond to the pose of inhibitor 1 in the ATX structure. However, two of the best three docking poses for E-28 suggest that the 4-ﬂuorobenzyl moiety likely binds to a diﬀerent area in the hydrophobic pocket (Figure 2D). The imidazolidine-2, 4-dione core of E-28 is ﬂipped around the double bond axis in the ATX binding site compared to its Z-isomer (compare Figure 2C with D). This observation is in agreement with the conformations of both isomers. In addition, the binding poses of Z-28 and E-28 resulting from our docking study suggest that the current 4-ﬂuorobenzyl moiety could be expanded from the methylene moiety in the 4-ﬂuorobenzyl substituent with other substituents in future ATX inhibitors. Finally, we evaluated inhibitors in which we introduced a rigidiﬁed three-ring system (Table 2) incorporating a tetrahydro- isoquinoline motif that we deemed likely to bind. This modiﬁca- tion avoids the presence of a Michael acceptor, while introducing rigidity and a new core structure. This modiﬁcation resulted in the 4622 dx.doi.org/10.1021/jm200310q |J. Med. Chem. 2011, 54, 4619–4626