Structure-based design of novel boronic acid-based inhibitors of autotaxin

Article abstract of DOI:10.1021/jm200310q

Autotaxin (ATX) is a secreted phosphodiesterase that hydrolyzes the abundant phospholipid lysophosphatidylcholine (LPC) to produce lysophosphatidic acid (LPA). The ATX-LPA signaling axis has been implicated in inflammation, fibrosis, and tumor progression

Full text of DOI:10.1021/jm200310q

ARTICLE
pubs.acs.org/jmc
Structure-Based Design of Novel Boronic Acid-Based Inhibitors of
Autotaxin
Harald M. H. G. Albers,‡,§ Loes J. D. Hendrickx,‡,† Rob J. P. van Tol,‡ Jens Hausmann,r Anastassis Perrakis,r
and Huib Ovaa*,‡,§
‡Division of Cell Biology, §Netherlands Proteomics Centre, and rDivision of Biochemistry, The Netherlands Cancer Institute,
Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands
S Supporting Information
b
ABSTRACT: Autotaxin (ATX) is a secreted phosphodiesterase that hydrolyzes
the abundant phospholipid lysophosphatidylcholine (LPC) to produce lysopho-
sphatidic acid (LPA). The ATX-LPA signaling axis has been implicated in
inflammation, fibrosis, and tumor progression, rendering ATX an attractive drug
target. We recently described a boronic acid-based inhibitor of ATX, named
HA155 (1). Here, we report the design of new inhibitors based on the crystal
structure of ATX in complex with inhibitor 1. Furthermore, we describe the
syntheses and activities of these new inhibitors, whose potencies can be
explained by structural data. To understand the difference in activity between
two different isomers with nanomolar potencies, we performed molecular
docking experiments. Intriguingly, molecular docking suggested a remarkable
binding pose for one of the isomers, which differs from the original binding pose of inhibitor 1 for ATX, opening further options for
inhibitor design.
’ INTRODUCTION
nucleophile of ATX with a hard matching Lewis acid. The crystal
structure of ATX in complex with HA155 (1)9 confirmed our
hypothesis that this inhibitor targets the threonine oxygen
nucleophile in the ATX active site via the boronic acid moiety,
while the hydrophobic 4-fluorobenzyl moiety of inhibitor 1
targets the hydrophobic pocket responsible for lipid binding
(Figure 1).
Here, we report a number of synthetic routes, systematically
substituting linkers and the thiazolidine-2,4-dione core in 1, while
keeping the boronic acid moiety untouched. The observed structureꢀ
activity relationscould wellbeexplainedfromtheATXstructure in
complex with inhibitor 1. A remarkable binding pose of a novel
inhibitor, as predicted from molecular docking experiments,
suggests additional avenues for further inhibitor design.
The secreted glycoprotein autotaxin (ATX) is a phosphodies-
terase responsible for the hydrolysis of lysophosphatidylcholine
(LPC) into lysophosphatidic acid (LPA) and choline, as depicted
in Scheme 1.1,2 The bioactive lipid LPA stimulates migration,
proliferation and survival of cells by activating specific G protein-
coupled receptors.3 The ATX-LPA signaling axis is involved in
cancer, inflammation and fibrotic disease.4ꢀ6 Potent and selec-
tive ATX inhibitors are needed to elucidate the contribution of
ATX action to signaling cascades that may result in disease in case
of malfunction.
ATX, also known as eNPP2, is a unique member of the ecto-
nucleotide pyrophosphatase/phosphodiesterase (eNPP) family
of proteins. It is the only family member capable of producing
LPA by hydrolysis of LPC.7 Recently reported crystal structures
of mouse8 and rat9 ATX confirmed that a threonine residue and
two zinc ions are necessary for activity of ATX.10 From these
structures, it could be concluded that ATX hydrolyzes its sub-
strates through a typical alkaline phosphatase/phosphodiesterase
mechanism.11,12 Furthermore, these structures showed that ATX
specifically binds its lipid substrates in a hydrophobic pocket
extending from the active site of ATX. This pocket accommo-
dates the alkyl chain of the lipids in different poses as was also
shown in various crystal structures.8
’ RESULTS AND DISCUSSION
Design of Inhibitors. The structure of inhibitor 1 bound to
the ATX active site (Figure 1) showed that its 4-fluorobenzyl
moiety binds into the hydrophobic lipid binding pocket of ATX
(Figure 1C,D).9 This pocket also accommodates the lipid tail of
LPA, the hydrolysis product of LPC.8 The thiazolidine-2,4-dione
core of 1 and the conjugated aromatic ring are located between
the hydrophobic pocket and the catalytic site (Figure 1D). The
ether linker, bridging the two aromatic rings in 1, and especially a
Recently, we described the discovery of a boronic acid-based
ATX inhibitors that helped to reveal the short half-life (∼5 min)
of LPA in vivo.13,14 We introduced a boronic acid moiety in the
inhibitor structure to rationally target the threonine oxygen
Received: March 18, 2011
Published: May 26, 2011
r
2011 American Chemical Society
4619
dx.doi.org/10.1021/jm200310q J. Med. Chem. 2011, 54, 4619–4626
|

Journal of Medicinal Chemistry
ARTICLE
Scheme 1. Autotaxin (ATX) is Responsible for Hydrolyzing the Lipid Lysophosphatidylcholine (LPC) into Lysophosphatidic
Acid (LPA) and Choline
Table 1. IC50 Values of the Inhibitors Resulting from the
Linker Modification
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-
action interface is approximately 500 Å2) and by the boronic
acid binding to the threonine oxygen nucleophile of ATX.9 The
boronꢀoxygen 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
a IC50 values have been determined in the choline release assay using
active site. Thus, the boronic acid moiety targets not only the
40 μM LPC and 10 nM ATX. The doseꢀresponse curve of inhibitor 17
threonine oxygen nucleophile, but also the two zinc ions that are
shows biphasic curve (see Supporting Information Figure S2).
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.
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 17ꢀ20 (Scheme 2) starts with
palladium catalyzed borylation of appropriate aldehydes via a
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
4620
dx.doi.org/10.1021/jm200310q |J. Med. Chem. 2011, 54, 4619–4626

Journal of Medicinal Chemistry
ARTICLE
Table 2. IC50 Values of the Inhibitors Resulting from the
Core Modification
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.
StructureꢀActivity 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 doseꢀresponse 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.
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 2ꢀ6 see Supporting Information). This
reaction smoothly provided intermediates 7ꢀ11. Next, the
pinacol protecting group was hydrolyzed under acidic conditions
and oxidatively destroyed by NaIO4 giving boronic acid alde-
hydes 12ꢀ16. 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 17ꢀ20).
In order to remove the potential Michael acceptor present,
we reduced the double bond in inhibitor 1 using hydrogen and
4621
dx.doi.org/10.1021/jm200310q |J. Med. Chem. 2011, 54, 4619–4626

Journal of Medicinal Chemistry
ARTICLE
Scheme 2. Synthetic Route Towards Linker Modified Inhibitors
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
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.20ꢀ22 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.
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.
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

Journal of Medicinal Chemistry
ARTICLE
For isolation bycentrifugation 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 (190ꢀ750 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 minꢀ1 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 minꢀ1 using gradient elution (water/acetonitrile) from
60/40 (v/v) to 10/90 (v/v).
General Procedure forBorylation of Aldehydes andPinacol
Deprotection (12ꢀ16). In a dry flask, bis(pinacolato)diboron (1.34 g,
5.28 mmol), the appropriate aldehyde(1.80 mmol) and potassiumacetate
(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
layerswere washed withbrine (15mL), dried overmagnesium 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 (CꢀB(OH)2 not visible). MS: m/z
[MþH]þ calc. 257.10, obs. 257.10.
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 structureꢀactivity 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 structureꢀactivity 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.
For experimental details of compounds 13ꢀ16, see Supporting
Information.
GeneralMethod forKnoevenagel Condensation(1, 17ꢀ20,
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), 7.39ꢀ7.31 (m,
J = 5.5, 8.8, 2H), 7.26ꢀ7.09 (m, J = 4.5, 8.9, 4H), 5.21 (s, 2H), 4.82 (s,
’ 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 (0.035ꢀ0.070, 90 Å, Acros).
1
2H). 13C NMR: δ = 167.38, 165.59, 161.66 (d, JCF = 244), 160.33,
4623
dx.doi.org/10.1021/jm200310q |J. Med. Chem. 2011, 54, 4619–4626

Journal of Medicinal Chemistry
ARTICLE
3
138.19, 134.28, 133.48, 132.33, 131.81 (d, 4JCF = 3), 129.95 (d, 3JCF = 8),
3), 129.66 (d, JCF = 8), 115.21 (d, 2JCF = 21), 46.00, 40.29. MS: m/z
2
126.67, 125.55, 117.89, 115.81, 115.47 (d, JCF = 21), 69.52, 43.90
[MþH]þ calc. 209.07, obs. 208.93.
(CꢀB(OH)2 not visible). MS: m/z [MþH]þ calc. 464.11, obs. 464.19.
For experimental details of compounds 17ꢀ20, 26, and E-28 see
Supporting Information.
3-(4-Fluorobenzyl)-1-methylimidazolidine-2,4-dione (27). To
a cooled solution (273 K) of 3-(4-fluorobenzyl)imidazolidine-2,4-dione
(98.1 mg, 0.471 mmol) in dimethylformamide (0.5 mL), sodium hydride
(60% in oil, 21.1 mg, 0.530 mmol) was added. Subsequently, iodomethane
(33 μL, 0.53 mmol) was added to the reaction mixture. The mixture was
allowed to warm up to room temperature and was stirred for 3 h. Then, the
mixture was poured into ice water (2.5 mL) and hexane (2.5 mL) was added.
After a night at 277 K, the precipitate was filtered and dried to give a white
solid. Yield: 75 mg, 72%. 1H NMR: δ = 7.34ꢀ7.12 (m, 4H), 4.52 (s, 2H),
4.01 (s, 2H), 2.86 (s, 3H). 13C NMR: δ = 170.20, 161.46 (d, 1JCF = 244),
156.24, 132.92 (d, 4JCF = 3), 129.68 (d, 3JCF = 8), 115.21 (d, 2JCF = 21),
51.36, 40.74, 29.21. MS: m/z [MþH]þ calc. 223.09, obs. 223.06.
(Z)-(4-((4-((1-(4-Fluorobenzyl)-3-methyl-2,5-dioxoimidazoli-
din-4-ylidene)methyl) phenoxy)methyl)phenyl)boronic Acid
(Z-28). To a cooled solution (273 K) of hydantoine 26 (10.1 mg,
0.0224 mmol) and sodium hydride (60% in oil, 1.46 mg, 0.0365 mmol) in
DMF (0.15 mL), iodomethane (2.25 μL, 0.0361 mmol) was added. The
mixture was allowedtowarmuptoroomtemperatureandwas stirredfor 4
h. Final product was isolated by using preparative HPLC. Z-configuration
confirmed by the chemical shift of the vinyl and methyl proton reported in
literature.23
(4-((4-((3-(4-Fluorobenzyl)-2,4-dioxothiazolidin-5-yl)methyl)
phenoxy)methyl) phenyl)boronic Acid (21). A mixture of com-
pound 1 (50.1 mg, 0.108 mmol) and 10 wt % Pd/C (24.0 mg) in
degassed methanol (3 mL) was stirred under a hydrogen atmosphere for
2 h. Extra 10 wt % Pd/C was added (12.0 mg) and the reaction was
allowed to continue for one night. The mixture was filtrated and
concentrated to dryness. Preparative HPLC afforded the title com-
pound. Yield: 18.3 mg, 77%. 1H NMR: δ = 8.03 (s, 2H), 7.80 (d, J = 8.1,
2H), 7.39 (d, J = 8.1, 2H), 7.28ꢀ6.99 (m, 6H), 6.88 (d, J = 8.7, 2H), 5.06
(s, 2H), 5.00 (dd, J = 4.4, 8.0, 1H), 4.60 (dd, J = 15.0, 21.5, 2H), 3.14 (dd,
J = 8.0, 14.2, 1H). 13C NMR: δ = 173.71, 171.00, 161.50 (d, 1JCF = 244),
157.44, 138.74, 134.16, 131.58 (d, 4JCF = 3), 130.56, 129.66 (d, 3JCF = 8),
127.98, 126.52, 115.21 (d, 2JCF = 21), 114.57, 69.12, 50.94, 43.56, 35.79
(CꢀB(OH)2 not visible). MS: m/z [MþH]þ calc. 466.13, obs. 466.25,
[M-H2OþH]þ calc. 448.12, obs. 448.23.
(4-((4-((3-(4-Fluorobenzyl)-4-hydroxy-2-oxothiazolidin-5-
yl)methyl)phenoxy) methyl)phenyl)boronic Acid (22). To a
solution of compound 1 (50.0 mg, 0.108 mmol) in dimethyl sulfoxide
(0.5 mL), sodium borohydride (16.3 mg, 0.430 mmol) was slowly
added. After 9 h of stirring, the reaction mixture was diluted with ethyl
acetate (4 mL) and was washed with water (2 ꢁ 2 mL). The organic
layer was dried over calcium chloride and concentrated in vacuo,
Yield: 6.97 mg, 68%. 1H NMR: δ = 8.11 (s, 1H), 7.80 (d, J = 8.1, 2H),
7.50ꢀ7.30 (m, 6H), 7.26ꢀ7.10 (m, 2H), 7.05 (d, J = 8.8, 2H), 6.76 (s,
1H), 5.14 (s, 2H), 4.67 (s, 2H), 2.92 (s, 3H). 13C NMR: δ = 163.01,
1
4
159.92, 156.78 (d, JCF = 249), 138.52, 134.19, 132.55 (d, JCF = 3),
131.28, 129.75 (d, 3JCF = 8), 128.50, 126.58, 124.66, 115.31 (d, 2JCF
=
1
resulting in the title compound. Yield: 30.2 mg, 60%. H NMR: δ =
21), 114.55, 111.56, 69.30, 41.12, 30.35 (CꢀB(OH)2 not visible). MS:
8.06 (s, 2H), 7.80 (d, J = 8.1, 2H), 7.39 (d, J = 8.0, 2H), 7.36 ꢀ 7.11 (m,
5H), 6.94 (d, J = 8.7, 2H), 6.87 (d, J = 8.7, 2H), 6.71 (d, J = 6.2, 1H), 5.06
(s, 2H), 4.72 (dd, J = 7.7, 15.1, 1H), 4.10 (d, J = 15.1, 1H), 3.67 (t, J = 7.9,
1H), 3.35 (s, 1H), 2.81 (dd, J = 7.3, 13.9, 1H), 2.70 (dd, J = 8.4, 13.9,
m/z [MþH]þ calc. 461.17, obs. 461.12.
(E)-(4-((4-((2,5-Dioxopyrrolidin-3-ylidene)methyl)phenoxy)
methyl)phenyl)boronic Acid (31). To a heated (343 K) solution of
compound 30 (159 mg, 0.442 mmol)16 in methanol (5 mL) aldehyde 12
(106 mg, 0.414 mmol) was added. After 1 h of heating, the reaction
mixture was cooled using an ice bath resulting in precipitation of the title
compound. The precipitate was filtered and washed with ice-cold
methanol resulting in compound 31. Yield: 88 mg, 63%. 1H NMR: δ =
11.34 (s, 1H), 8.04 (s, 2H), 7.80 (d, J = 8.0, 2H), 7.57 (d, J = 8.9, 2H), 7.41
(d, J = 8.0, 2H), 7.33 (t, 4J = 2.1, 1H), 7.10 (d, J = 8.8, 2H), 5.19 (s, 2H),
1
1H). 13C NMR: δ = 169.70, 161.55 (d, JCF = 244), 157.13, 138.85,
4
3
134.15, 133.14 (d, JCF = 3.0), 130.01, 130.00 (d, JCF = 8), 129.73,
126.54, 115.38 (d, 2JCF = 21), 114.64, 84.02, 69.11, 51.42, 44.02, 39.65.
(CꢀB(OH)2 not visible). MS: m/z [MþH]þ calc. 468.15, obs. 468.23.
(4-((4-((3-(4-Fluorobenzyl)-2-oxo-2,3-dihydrothiazol-5-yl)
methyl)phenoxy) methyl)phenyl)boronic Acid (23). To a
solution of compound 1 (45.7 mg, 0.0989 mmol) in dimethyl sulfoxide
(0.75 mL), sodium borohydride (29.1 mg, 0.769 mmol) was slowly
added. After 7 h of stirring the reaction mixture, concentrated sulfuric
acid (2 ꢁ 50 μL) was added over a 15 min interval. The reaction mixture
was stirred for an additional 5 h. Ethyl acetate (25 mL) was added and
the mixture was washed with water (4 ꢁ 10 mL). The organic layer was
dried over calcium chloride and concentrated in vacuo, affording pure
compound 23. Yield: 31.8 mg, 72%. 1H NMR: δ = 8.05 (s, 2H), 7.79 (d, J =
8.1, 2H), 7.38 (d, J = 8.1, 2H), 7.36ꢀ7.16 (m, 4H), 7.13 (d, J = 8.7, 2H),
6.95 (d, J = 8.7, 2H), 6.87 (s, 1H), 5.07 (s, 2H), 4.80 (s, 2H), 3.73
(s, 2H). 13C NMR: δ = 170.43, 161.61 (d, 1JCF = 244), 157.13, 138.85,
4
3.60 (d, J = 2.2, 2H). 13C NMR: δ = 175.80, 172.09, 159.47, 138.42,
134.22, 132.00, 131.31, 126.96, 126.54, 124.16, 115.31, 69.31, 34.71
(CꢀB(OH)2 not visible). MS: m/z [MþH]þ calc. 338.12, obs. 338.11.
(E)-(4-((4-((1-(4-Fluorobenzyl)-2,5-dioxopyrrolidin-3-ylidene)
methyl)phenoxy) methyl)phenyl)boronic Acid (32). To a
solution of compound 31 (30 mg, 0.0890 mmol) in dimethylformamide
(0.3 mL) sodium hydride (60% in oil, 3.65 mg, 0.0913 mmol) was
added. After addition of 4-fluorobenzyl bromide (24 μL, 0.19 mmol),
the reaction mixture was stirred for 4 h. In addition, potassium carbonate
(2.04 mg, 0.0148 mmol) was added and the reaction mixture was stirred
overnight. Then, the mixture was poured into ice water (0.9 mL) and
hexane (0.3 mL) was added. After a night at 277 K, the precipitate was
filtered, dried, and purified using preparative HPLC to give a white solid.
E-Configuration confirmed by the chemical shift of the vinyl proton
reported in literature.16 Yield: 13 mg, 32%. 1H NMR: δ = 8.04 (s, 2H),
7.80 (d, J = 8.1, 2H), 7.61 (d, J = 8.9, 2H), 7.45 (t, 4J = 2.1, 1H), 7.41 (d,
J = 8.0, 2H), 7.38ꢀ7.29 (m, 2H), 7.23ꢀ7.04 (m, 4H), 5.19 (s, 2H), 4.66
(s, 2H), 3.74 (d, 4J = 2.1, 2H). 13C NMR: δ = 174.19, 170.59, 161.44 (d,
1JCF = 244), 159.65, 138.39, 134.22, 132.59 (d, 4JCF = 3), 132.31, 132.17,
4
3
134.17, 133.10 (d, JCF = 3), 130.63, 130.63, 129.76 (d, JCF = 8),
129.42, 126.48, 121.56, 117.78, 115.50 (d, 2JCF = 21), 114.86, 69.16,
46.78, 32.91 (CꢀB(OH)2 not visible). MS: m/z [MþH]þ calc. 450.13,
obs. 450.22.
3-(4-Fluorobenzyl)imidazolidine-2,4-dione (25). To a cooled
solution (273 K) of hydantoin (8.01 g, 80.1 mmol) in dimethylformamide
(140 mL) sodium hydride (60% in oil, 1.80 g, 45.0 mmol) was added. A
solution of 1-(bromomethyl)-4-fluorobenzene (5.0 mL, 41 mmol) in
dimethylformamide (5 mL) was added to the reaction mixture. The
mixture was allowed to warm up to room temperature and was stirred for
6 h. Then, the mixture was poured into water (200 mL) and hexane
(200 mL) was added. After a night at 277 K, the precipitate was filtered
and dried to give a white solid. Yield: 4.7 g, 56%. 1H NMR: δ = 8.14 (s,
1H), 7.45ꢀ7.21 (m, 1H), 7.26ꢀ7.09 (m, 1H), 4.51 (s, 2H), 3.97 (s, 2H).
129.73 (d, 3JCF = 8), 126.85, 126.54, 122.27, 115.36, 115.22 (d, 2JCF
=
21), 69.33, 40.71, 33.74 (CꢀB(OH)2 not visible). MS: m/z [MþH]þ
calc. 446.16, obs. 446.12.
(S)-2-(4-Fluorobenzyl)-7-hydroxy-10,10a-dihydroimidazo-
[1,5-b]isoquinoline-1,3(2H,5H)-dione (S-34). Compound S-33
(100 mg, 0.518 mmol) was dissolved in a mixture of dioxane and water
13C NMR: δ = 171.91, 161.45 (d, 1JCF = 244), 157.29, 133.04 (d, 4JCF
=
4624
dx.doi.org/10.1021/jm200310q |J. Med. Chem. 2011, 54, 4619–4626

Journal of Medicinal Chemistry
ARTICLE
(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), 7.35ꢀ7.30(m,2H),7.18ꢀ7.12(m, 2H),7.06ꢀ7.03
(m, 1H), 6.65ꢀ6.63 (m, 2H), 4.77 (d, J = 16.8, 1H), 4.58 (s, 2H), 4.32ꢀ
4.27 (m, 2H), 3.05 (m, 1H), 2.82ꢀ2.64 (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.
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 Schr€odinger 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 5.6.20ꢀ22 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.
(R)-2-(4-Fluorobenzyl)-7-hydroxy-10,10a-dihydroimidazo-
[1,5-b]isoquinoline-1,3(2H,5H)-dione (R-34). For reaction de-
’ ASSOCIATED CONTENT
1
tails, see compound S-34. Yield: 29%. H NMR, 13C NMR, and MS,
S
see Supporting Information.
Supporting Information. ContainsMichaelacceptorstudy
b
of inhibitor 1, doseꢀresponse 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 13ꢀ20,
26, and E-28, details on the syntheses of aldehydes 2ꢀ6 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.
(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), 7.50ꢀ7.27
(m, 4H), 7.27ꢀ7.05 (m, 3H), 7.02ꢀ6.76 (m, 2H), 5.09 (s, 2H), 4.82
(d, J = 17.0, 1H), 4.58 (s, 2H), 4.42ꢀ4.20 (m, 2H), 3.19ꢀ3.02
’ 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.
1
(m, 1H), 2.80ꢀ2.71 (m, 1H). 13C NMR: δ = 172.72, 161.46 (d, JCF
4
= 244), 157.09, 154.60, 138.74, 134.17, 132.81 (d, JCF = 3), 132.60,
3
2
130.23, 129.55 (d, JCF = 8), 126.49, 123.60, 115.28 (d, JCF = 21),
113.87, 112.47, 69.20, 54.40, 41.30, 40.60, 29.00 (CꢀB(OH)2 not
visible). MS: m/z [MþH]þ calc. 461.17, obs. 461.14.
’ ACKNOWLEDGMENT
(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 mLꢀ1) 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 mLꢀ1) 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 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. Autotaxin
has lysophospholipase D activity leading to tumor cell growth and
In addition, the effect of the inhibitors on the enzymatic coloring
reaction was investigated using 40 μM choline at 30 μM inhibitor using
4625
dx.doi.org/10.1021/jm200310q |J. Med. Chem. 2011, 54, 4619–4626

Journal of Medicinal Chemistry
ARTICLE
motility by lysophosphatidic acid production. J. Cell Biol. 2002, 158,
227–233.
Francis, P.; Shenkin, P. Glide: a new approach for rapid, accurate
docking and scoring. 1. method and assessment of docking accuracy.
J. Med. Chem. 2004, 47, 1739–1749.
(21) Friesner, R.; Murphy, R.; Repasky, M.; Frye, L.; Greenwood, J.;
Halgren, T.; Sanschagrin, P.; Mainz, D. Extra precision glide: docking
and scoring incorporating a model of hydrophobic enclosure for protein-
ligand complexes. J. Med. Chem. 2006, 49, 6177–6196.
(22) Halgren, T.; Murphy, R.; Friesner, R.; Beard, H.; Frye, L.;
Pollard, W. T.; Banks, J. Glide: a new approach for rapid, accurate
docking and scoring. 2. enrichment factors in database screening. J. Med.
Chem. 2004, 47, 1750–1759.
(23) Tan, S.; Ang, K.; Fong, Y. (Z)- and (E)-5-Arylmethylenehy-
dantoins: spectroscopic properties and configuration assignment.
J. Chem. Soc., Perkin Trans. 2 1986, 1941–1944.
(3) Moolenaar, W. H.; van Meeteren, L. A.; Giepmans, B. N. The ins
and outs of lysophosphatidic acid signaling. Bioessays 2004, 26, 870–881.
(4) Kanda, H.; Newton, R.; Klein, R.; Morita, Y.; Gunn, M.; Rosen,
S. Autotaxin, an ectoenzyme that produces lysophosphatidic acid,
promotes the entry of lymphocytes into secondary lymphoid organs.
Nat. Immunol. 2008, 9, 415–423.
(5) van Meeteren, L.; Moolenaar, W. Regulation and biological
activities of the autotaxin-LPA axis. Prog. Lipid Res. 2007, 46, 145–160.
(6) Tager, A.; LaCamera, P.; Shea, B.; Campanella, G.; Selman, M.;
Zhao, Z.; Polosukhin, V.; Wain, J.; Karimi-Shah, B.; Kim, N.; Hart, W.;
Pardo, A.; Blackwell, T.; Xu, Y.; Chun, J.; Luster, A. The lysophosphatidic
acid receptor LPA1 links pulmonary fibrosis to lung injury by mediating
fibroblast recruitment and vascular leak. Nat. Med. 2008, 14, 45–54.
(7) Stefan, C.; Jansen, S.; Bollen, M. Modulation of purinergic
signaling by NPP-type ectophosphodiesterases. Purinergic. Signal
2006, 2, 361–370.
(24) Shelley, J.; Cholleti, A.; Frye, L.; Greenwood, J.; Timlin, M.;
Uchimaya, M. Epik: a software program for pKa prediction and
protonation state generation for drug-like molecules. J. Comput.-Aided
Mol. Des. 2007, 21, 681–691.
(8) Nishimasu, H.; Okudaira, S.; Hama, K.; Mihara, E.; Dohmae, N.;
Inoue, A.; Ishitani, R.; Takagi, J.; Aoki, J.; Nureki, O. Crystal structure of
autotaxin and insight into GPCR activation by lipid mediators. Nat.
Struct. Mol. Biol. 2011, 18, 205–212.
(9) Hausmann, J.; Kamtekar, S.; Christodoulou, E.; Day, J.; Wu, T.;
Fulkerson, Z.; Albers, H.; van Meeteren, L.; Houben, A.; van Zeijl,
L.; Jansen, S.; Andries, M.; Hall, T.; Pegg, L.; Benson, T.; Kasiem, M.;
Harlos, K.; Kooi, C.; Smyth, S.; Ovaa, H.; Bollen, M.; Morris, A.;
Moolenaar, W.; Perrakis, A. Structural basis of substrate discrimination and
integrin binding by autotaxin. Nat. Struct. Mol. Biol. 2011, 18, 198–204.
(10) Gijsbers, R.; Aoki, J.; Arai, H.; Bollen, M. The hydrolysis of
lysophospholipids and nucleotides by autotaxin (NPP2) involves a
single catalytic site. FEBS Lett. 2003, 538, 60–64.
(11) Gijsbers, R.; Ceulemans, H.; Stalmans, W.; Bollen, M. Structur-
al and catalytic similarities between nucleotide pyrophosphatases/
phosphodiesterases and alkaline phosphatases. J. Biol. Chem. 2001,
276, 1361–1368.
(12) Zalatan, J.; Fenn, T.; Brunger, A.; Herschlag, D. Structural and
functional comparisons of nucleotide pyrophosphatase/phosphodies-
terase and alkaline phosphatase: implications for mechanism and
evolution. Biochemistry 2006, 45, 9788–9803.
(13) Albers, H.; van Meeteren, L.; Egan, D.; van Tilburg, E.;
Moolenaar, W.; Ovaa, H. Discovery and optimization of boronic acid
based inhibitors of autotaxin. J. Med. Chem. 2010, 53, 4958–4967.
(14) Albers, H. M.; Dong, A.; van Meeteren, L. A.; Egan, D. A.;
Sunkara, M.; van Tilburg, E. W.; Schuurman, K.; van Tellingen, O.;
Morris, A. J.; Smyth, S. S.; Moolenaar, W. H.; Ovaa, H. Boronic acid-
based inhibitor of autotaxin reveals rapid turnover of LPA in the
circulation. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 7257–7262.
(15) Miyaura, N.; Suzuki, A. Palladium-catalyzed cross-coupling
reactions of organoboron compounds. Chem. Rev. 1995, 95, 2457–2483.
(16) Mizufune, H.; Nakamura, M.; Mitsudera, H. Process research
on arylnaphthalene lignan aza-analogues: a new palladium-catalyzed
benzannulation of [alpha],[beta]-bisbenzylidenesuccinic acid deriva-
tives. Tetrahedron 2006, 62, 8539–8549.
(17) Cui, P.; Tomsig, J.; McCalmont, W.; Lee, S.; Becker, C.; Lynch,
K.; Macdonald, T. Synthesis and biological evaluation of phosphonate
derivatives as autotaxin (ATX) inhibitors. Bioorg. Med. Chem. Lett. 2007,
17, 1634–1640.
(18) Fischer, G.; Mutel, V.; Trube, G.; Malherbe, P.; Kew, J. N. C.;
Mohacsi, E.; Heitz, M. P.; Kemp, J. A. Ro 25ꢀ6981, a highly potent and
selective blocker of N-methyl-d-aspartate receptors containing the
NR2B subunit. characterization in vitro. J. Pharmacol. Exp. Therapeutics
1997, 283, 1285–1292.
(19) Jain, A.; Purohit, C.; Verma, S.; Sankararamakrishnan, R. Close
contacts between carbonyl oxygen atoms and aromatic centers in
protein structures: π-π or lone-pair-π interactions? J. Phys. Chem. B
2007, 111, 8680–8683.
(20) Friesner, R.; Banks, J.; Murphy, R.; Halgren, T.; Klicic, J.;
Mainz, D.; Repasky, M.; Knoll, E.; Shelley, M.; Perry, J.; Shaw, D.;
4626
dx.doi.org/10.1021/jm200310q |J. Med. Chem. 2011, 54, 4619–4626