Discovery of potent inhibitors of the lysophospholipase autotaxin

Article abstract of DOI:10.1016/j.bmcl.2016.10.036

The autotoxin–lysophosphatidic acid (ATX–LPA) axis has been implicated in several disease conditions including inflammation, fibrosis and cancer. This makes ATX an attractive drug target and its inhibition may lead to useful therapeutic agents. Through a high throughput screen (HTS) we identified a series of small molecule inhibitors of ATX which have subsequently been optimized for potency, selectivity and developability properties. This has delivered drug-like compounds such as 9v (CRT0273750) which modulate LPA levels in plasma and are suitable for in vivo studies. X-ray crystallography has revealed that these compounds have an unexpected binding mode in that they do not interact with the active site zinc ions but instead occupy the hydrophobic LPC pocket extending from the active of ATX together with the occupying the LPA ‘exit’ channel.

Full text of DOI:10.1016/j.bmcl.2016.10.036

Accepted Manuscript
Discovery of potent inhibitors of the lysophospholipase autotaxin
Pritom Shah, Anne Cheasty, Caroline Foxton, Tony Raynham, Muddasar
Farooq, Irene Farre Gutierrez, Aurore Lejeune, Michelle Pritchard, Andrew
Turnbull, Leon Pang, Paul Owen, Susan Boyd, Alexandra Stowell, Allan Jordan,
Niall M. Hamilton, James R. Hitchin, Martin Stockley, Ellen MacDonald, Mar
Jimenez Quesada, Elisabeth Trivier, Jana Skeete, Huib Ovaa, Wouter H.
Moolenaar, Hamish Ryder
PII:
S0960-894X(16)31064-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.10.036
BMCL 24339
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
31 August 2016
11 October 2016
12 October 2016
Please cite this article as: Shah, P., Cheasty, A., Foxton, C., Raynham, T., Farooq, M., Gutierrez, I.F., Lejeune, A.,
Pritchard, M., Turnbull, A., Pang, L., Owen, P., Boyd, S., Stowell, A., Jordan, A., Hamilton, N.M., Hitchin, J.R.,
Stockley, M., MacDonald, E., Quesada, M.J., Trivier, E., Skeete, J., Ovaa, H., Moolenaar, W.H., Ryder, H.,
Discovery of potent inhibitors of the lysophospholipase autotaxin, Bioorganic & Medicinal Chemistry Letters
(2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.10.036
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Discovery of potent inhibitors of the lysophospholipase autotaxin
Pritom Shaha, *, Anne Cheastya, Caroline Foxtona, Tony Raynhama, Muddasar Farooqa, Irene Farre
Gutierreza, Aurore Lejeunea, Michelle Pritcharda, Andrew Turnbulla, Leon Panga, Paul Owena, Susan
Boyda, Alexandra Stowellc, Allan Jordanc, Niall M. Hamiltonc, James R. Hitchinc, Martin Stockleya,
Ellen MacDonalda, Mar Jimenez Quesadaa, Elisabeth Triviera, Jana Skeetea, Huib Ovaab,d, Wouter H.
Moolenaarb and Hamish Rydera
aCancer Research Technology, Discovery Laboratories, Babraham Research Campus, Cambridge,
CB22 3AT, UK
bNetherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands
cDrug Discovery Unit, Cancer Research UK Manchester Institute, The University of Manchester,
Wilmslow Road, Manchester, M20 4BX, UK
dCurrent address: Department of Chemical Immunology, Leiden University Medical Center,
Einthovenweg 20, 2333 ZC, Leiden, The Netherlands
Keywords:
Autotaxin (ATX)
Lysophosphatidic acid (LPA)
Lysophosphatidylcholine (LPC)
Cancer
Autotaxin (ATX), also known as ectonucleotide pyrophosphatase/phosphodiesterase family member 2
(ENPP2), is a secreted lysophospholipase D (lysoPLD) that cleaves choline from
lysophosphatidylcholine (LPC) forming lysophosphatidic acid (LPA), a mitogen and motility factor
that has been implicated in the pathophysiology of cancer1-3 and many other biological processes such
as vascular development, lymphocyte homing and inflammation.4-6 LPA consists of a single fatty acyl
chain, a glycerol backbone and a free phosphate group. Several structurally diverse forms of LPA
exist with different acyl-chain lengths and saturations. LPA acts through a number of LPA receptors
and the great variety of cellular and biological actions of LPA is explained by the fact that the six
known LPA receptors show broad tissue expression and can couple to at least three distinct G
proteins, which, in turn, feed into multiple effector systems.7 (Figure 1).
ATX
+
LPC
LPA
Choline
Extracellular
Intracellular
G-Protein
Migration Proliferation
Survival

Figure 1. The Autotaxin-LPA axis showing conversion of LPC to LPA mediated by autotaxin and
subsequent activation of LPA receptors
The ATX-LPA axis has been implicated in several disease states including inflammation,8 pulmonary
fibrosis,9 and tumour progression.10 ATX is processed along the classical export pathway and secreted
as a catalytically active glycoprotein. ATX’s major lipid substrate, LPC, is secreted by the liver and is
abundantly present in plasma and interstitial fluids. Potent and selective inhibitors of ATX would
clearly be of value to elucidate the biology of this target and an overview of the patent and primary
literature describing the development of novel ATX inhibitors has recently been published.11 Clinical
trials have recently been initiated with GLPG1690 which is the first autotaxin inhibitor to enter the
clinic and is under evaluation for the treatment of idiopathic pulmonary fibrosis.12 Herein we report
the discovery of a new class of small molecule inhibitors of ATX which have been optimized to
deliver compounds suitable for in vivo studies.
A high throughput biochemical screen using the FS-3 assay13 was run using the CRT compound
collection of 87,865 compounds. The screen was run at 30 M and the confirmed hit rate, defined as
giving >50% inhibition at 30 M, was 1.2%. After IC50 determination in the FS-3 assay, a number of
distinct chemical series were identified. Compounds from each series were then screened in the
enzyme coupled (EC) biochemical assay which uses the more physiologically relevant substrate LPC
C16:0 and measures the release of choline.14 The absolute potencies of the compounds in the enzyme
coupled biochemical assay were generally lower and this helped prioritise between the chemical series
so that chemistry effort could be focused on the most promising scaffolds. Based on the potency,
ligand effiency (LE) and chemical tractability, the imidazo[4,5-b]pyridine 1 (Table 1) was selected as
an attractive starting point for further chemistry.
Table 1. Profile of hit compound 1 selected for further optimisation
LEc
cLogPd
3.1
PSAe
69
MWt
400
FS-3 IC50 (M)a EC IC50 (M)b
0.040 0.473
0.29
aThe FS-3 biochemical assay utilizes a doubly labelled analogue of LPC wherein the fluorophore is
quenched through intramolecular energy transfer. Hydrolysis of the substrate produces an increase in
fluorescence. bEC IC50 - this assay utilises LPC C16:0 as substrate and measures the release of
choline. cLE = Ligand Effiency = (1.36*pIC50)/HAC, HAC = heavy atom count, dcLogP is the
ocatanol/water partition coefficient calculated using StardropTM, ePSA = Polar Surface Area calculated
using StardropTM.
Compounds, prepared to explore the structure activity relationships of the hit 1, were synthesized by
the routes described in Schemes 1-4. The route used to modify the amide group (R2 in compound 9)
and the N-benzyl group on the core (R1 in compound 9) is shown in Scheme 1. Thus, 2-chloro-3-
nitro-pyridine was reacted with Cs2CO3 and the appropriate amine 3 in methanol at reflux to give 4 via
a SNAr reaction. The nitro group in 4 was hydrogenated over 10% Pd/C in ethanol to afford pyridine-
2,3-diamine 5. This diamine could alternatively be obtained through a reduction of the nitro group
with iron in HCl. Compound 5 was reacted with succinic anhydride in dioxane at reflux and then
treated with sulphuric acid in ethanol under reflux to give the ester 6. Reaction of 6 with KOtBu and

the appropriate amine 8 in the microwave at 150°C afforded the final compounds of general formula
9. Alternatively the carboxylic acid 7 obtained from the reaction of 5 with succinic anhydride, was
treated with HBTU, triethylamine and the required amine 8 at room temperature to afford 9.
Alternative coupling agents can also be used to perform this amide formation.
Scheme 1. Synthesis of imidazo[4,5-b]pyridine derivatives 9. Reagents and conditions: (i) Cs2CO3,
R1CH2NH2, MeOH reflux, 61-100%; (ii) H2, 10% Pd/C, EtOH, 1atm or cHCl, Fe powder, EtOH, 88-
100%; (iii) succinic anhydride, dioxane, reflux, 52-81%; (iv) cH2SO4, EtOH, 10-13% combined yield
for steps (iii) and (iv); (v) KOtBu, R2NH2, MW, 4-42%; (vi) HBTU, Et3N, R2NH2, 35-79%
The synthesis of compounds in which the ethyl linker has been modified to the carbamate and urea
derivatives 13, 14 and 15 is shown in Scheme 2. Thus the diamine 5 was reacted with glycolic acid to
give the alcohol 10 followed by Mitsunobu reaction with phthalimide to give 11. Deprotection with
hydrazine gave the amine 12 which was derivatised to the carbamate 13 and ureas 15a-b. The alcohol
10 was converted to the isomeric carbamate 14 by reaction with the appropriate isocyanate. The
oxygen and sulphur linked compounds 21, 24, 25 and 26 were prepared as shown in Scheme 3. The
diamine 5 was converted to the thiol 16 with carbon disulphide and then methylated to give 17.
Oxidation of 17 with potassium permanganate gave the sulphone 18 which was displaced with benzyl
2-hydroxyacetate to give 19. Debenzylation of 19 followed by amide coupling gave the ether linked
compounds 21a-b. For the sulphur linked compounds 24, 25 and 26, thiol 16 was alkylated with tert-
butyl-2-bromoacetate to give 22 which was deprotected with trifluoroacetic acid to give the acid 23.
Amide coupling gave the thioether 24 and oxidation of 24 with potassium permanganate gave the
sulphone 25. Oxidation of 24 with 3-chloroperbenzoic acid gave the sulphoxide diastereomers 26a
and 26b (this reaction gives a pair of diastereomers because R2 is chiral with defined absolute
stereochemistry).

Scheme 2. Synthesis of imidazo[4,5-b]pyridine derivatives, linker modifications. Reagents and
conditions: (i) glycolic acid, 150oC, 78%; (ii) phthalimide, PPh3, DIAD, 60%; (iii) NH2NH2, EtOH,
97%; (iv) R2OH, DMF, CDI, 21%; (v) R2NCO, THF or DCM 63-68%; (vi) R2NCO, THF, 4%.
Scheme 3. Synthesis of imidazo[4,5-b]pyridine derivatives, linker modifications. Reagents and
conditions: (i) CS2, EtOH, 63-65%; (ii) MeI, EtOH, 79-85%; (iii) KMnO4, AcOH, 24-35%; (iv) NaH,
THF, 69-93%; (v) H2, Pd/C, EtOAc, 1atm, 76-100%; (vi) HBTU, DMF, Et3N, R2NH2, 33-40%; (vii)
tert-butyl-2-bromoacetate, acetone, K2CO3, 80%; (viii) TFA, DCM, 94%; (ix) HBTU, DMF, Et3N,
R2NH2, 48%; (x) 3-chloroperbenzoic acid, DCM, 42% combined yield for both diastereomers; (xi)
KMnO4, AcOH, 22%
Compounds bearing the isomeric imidazo[4,5-c]pyridine core were synthesized as shown in Scheme
4. Displacement of the bromide 27 with the appropriate benzylamine15 gave 28 which was reduced to
the diamine 29 with hydrogen and 10% Pd/C. The N-oxide 29 was reduced to the diamine 30 with
iron in acetic acid followed by reaction with succinic anhydride to give the acid 31. Amide coupling
of 31 using HBTU gave the target compounds 32a-c.

Scheme 4. Synthesis of imidazo[4,5-c]pyridine derivatives. Reagents and conditions: (i) R1CH2NH2,
n-propanol, 90oC, 58%; (ii) H2, 10% Pd/C, MeOH, 1atm, 98%; (iii) Fe powder, AcOH, 90oC, 100%;
(iv) succinic anhydride, dioxane, AcOH, 80oC, 31-44%; (v); HBTU, DCM, DIPEA, R2NH2, 16-20%
Table 2. SAR of benzyl group R1 and amide group R2
No R1
R2
acLogP bEC
IC50
cPlasma dCLint
CRA
IC50
(mL/min/g
liver)
(M)
mouse/human
(M)
Ph
Ph
Ph
Ph
Ph
4-Methoxybenzyl
Benzyl
4-Fluorobenzyl
4-Methylbenzyl
3.1
3.2
3.3
3.7
2.3
0.473 8.87
1.242
0.324
0.627
6.774
1
9a
9b
9c
9d
23.9/41.2
15.3/-
Ph
Ph
Ph
Ph
Ph
Ph
Ph
4-Chlorobenzyl
3-Fluorobenzyl
2-Fluorobenzyl
3-Methoxybenzyl
Phenethyl
3.8
3.3
3.3
3.1
3.6
3.8
2.4
0.029 5.8
1.252
1.251
2.974
3.436
9e
9f
9g
9h
9i
Cyclohexylmethyl
0.444 9.7
10.98
30.8/17.4
9j
9k
Ph
Ph
n-Butyl
3.0
3.0
8.811
0.856
9l
9m
Ph
Ph
3.0
3.7
2.43
9n
9o
0.068 0.288
21.4/62.0
35.0/21.4
Ph
3.7
2.631
9p
3.6
3.6
3.8
4.1
4.9
0.017 0.318
1.060 8.5
9q
9r
9s
9t
0.006 0.444
0.003 0.215
0.002 0.016
26.5/-
9u

4.7
4.5
2.8
0.001 0.014
0.002 0.044
0.005 0.026
44.1/9.4
52.8/-
4.4/-
9v
9w
9x
acLogP is the octanol/water partition coefficient calculated using StardropTM; bEC IC50 - this assay
c
utilises LPC C16:0 as substrate and measures the release of choline; CRA, (Plasma choline release
assay). The physiological substrate of ATX in human plasma, LPC, is hydrolyzed by ATX to give
LPA and choline. The release of choline can be detected in a two-step enzymatic reaction; dCLint
intrinsic clearance in mouse and human liver microsomes (<1 mL/min/g liver = Low; 1-5 mL/min/g
liver = Medium, >5 mL/min/g liver = High)
Table 3. Linker Modifications
No
X
Y
Z
R1
R2
acLogP bEC
IC50
cPlasma dCLint
CRA
IC50
(mL/min/g
liver)
(M)
(M)
CH2 NH
CH2
O
4.0
4.0
4.1
3.6
4.4
0.957
13
O
NH
NH
NH
0.366 3.294
0.010 0.414
0.080 1.050
0.005 0.099
21.8
14
CH2 NH
CH2 NH
15a
15b
21a
10.0
10.8
O
CH2 NH
O
CH2 NH
CH2 NH
CH2 NH
CH2 NH
CH2 NH
3.5
4.6
2.9
3.3
3.3
0.039 0.821
0.002 0.019
2.884
15.6
71.5
21b
24
S
SO2
eSO
eSO
25
0.010 0.178
28.6
19.8
26a
0.178 0.207
26b
acLogP is the octanol/water partition coefficient calculated using StardropTM; bEC IC50 - this assay
utilises LPC C16:0 as substrate and measures the release of choline; cPlasma CRA, (Plasma choline
release assay). The physiological substrate of ATX in human plasma, LPC, is hydrolyzed by ATX to
give LPA and choline. The release of choline can be detected in a two-step enzymatic reaction; dCLint
intrinsic clearance in mouse liver microsomes (<1 mL/min/g liver = Low; 1-5 mL/min/g liver =
Medium, >5 mL/min/g liver = High). e Single sulphoxide enantiomer of unknown absolute
stereochemistry

Table 4. Core Modification
No R1
R2
acLogP bEC
IC50
cPlasma dCLint
CRA
IC50
(mL/min/g
liver)
18.1
(M)
(M)
4.7
4.3
3.8
0.003 0.034
0.008 0.032
0.052 0.333
32a
32b
32c
acLogP is the octanol/water partition coefficient calculated using StardropTM; bEC IC50 - this assay
utilises LPC C16:0 as substrate and measures the release of choline; cCRA, (Plasma choline release
assay). The physiological substrate of ATX in human plasma, LPC, is hydrolyzed by ATX to give
LPA and choline. The release of choline can be detected in a two-step enzymatic reaction; dCLint
intrinsic clearance in mouse liver microsomes (<1 mL/min/g liver = Low; 1-5 mL/min/g liver =
Medium, >5 mL/min/g liver = High)
Table 2 summarises the effect of modifications to R1 and R2 in compound 9. Looking at the R2 group
compound 9a shows that removal of the 4-OMe substituent reduced biochemical potency whereas
replacement with 4-fluoro 9b or 4-methyl 9c maintained activity. Introduction of the polar sulphone
substituent 9d significantly reduced potency suggesting that the 4-substituent in R2 is accessing a
lipophilic pocket in the binding site. This is further supported by the 40 fold increase in potency
observed when a chloro group was introduced at the 4-position (9a vs 9e). Despite the improved
biochemical potency for 9e the activity seen in the plasma CRA assay was only modest. Substitution
at the 2- and 3-positions of R2 (9f-h) or chain extension to the phenyethyl group 9i generally reduced
potency. Saturation of the aromatic ring to give 9j was tolerated but replacement with polar aromatic
rings such as the pyridine 9k markedly reduced potency. A major breakthrough in potency was
achieved by introduction of a chiral methyl group on the benzylic carbon of R2 which restricted the
conformation of the aromatic ring (9o and 9p). The (S)-isomer 9o was found to be 38 fold more
potent than the (R)-isomer 9p and the introduction of the (S)-methyl group resulted in an 18 fold
increase in potency compared to the unsubstituted benzyl derivative (9o vs 9a). The separation in
potency between the (S) and (R) isomers is further highlighted in compounds 9q and 9r. The (S)-
isomers 9o and 9q also now showed improved activity in the plasma CRA assay. Combining the two
potency enhancing features from 9e and 9q into a single molecule gave 9t which demonstrated single
digit nanomolar potency in the biochemical assay. Further optimisation of the 4-position of R1
resulted compounds such as 9u and 9v which showed high potency in both the biochemical and
plasma CRA assays. Due to the lipophilic nature of the ATX binding pocket the cLogP of the
molecules also tended to increase as potency was optimised. We were able to address this drawback to
some extent by introduction of the polar hydroxymethyl group shown in 9x. The intrinsic clearance in
mouse microsomes for 9b, 9e, 9j, 9o, 9q and 9u-w was found to be high and the only compound with
moderate intrinsic clearance was the hydroxymethyl derivative 9x which has the lowest cLogD of the
compounds tested.

We then explored the linker portion of the molecule as shown in Table 3. The ether linked compound
21a showed good potency but had high microsomal intrinsic clearance. The urea linked compounds
15a-b showed a drop in potency particularly in the plasma CRA assay and the carbamates 13 and 14
and sulphone 25 were also significantly less active. The thioether analogue 24 was highly potent and,
of the two corresponding sulphoxide diastereomers 26a and 26b, compound 26a was more potent in
the biochemical assay. The difference in potency observed between the thioether 24, the sulphone 25,
and the sulphoxides 26a and 26b suggests that one of the oxygen atoms on the sulphur has an
unfavourable interaction with the protein. Good potency was also achieved when the core scaffold
was modified to the imidazo[4,5-c]pyridine ring system (Table 4). Comparative in vitro potency data
for the published ATX inhibitor PF-838016 is provided in the Supplementary Information.
Having established the structural requirements for potency in both the biochemical and biomarker
(CRA) assays, we evaluated key compounds in the metabolic stability assay. Disappointingly, we
found that all compounds had high intrinsic clearances in mouse microsomes. To ascertain whether
the in vitro clearance correlated to in vivo blood clearance, we evaluated a number of compounds in
intravenous PK studies in CD-1 mice. Table 5 shows that the majority of compounds had high blood
clearances when tested in vivo in CD-1 mice. Compound 9v, however, had a moderate blood
clearance (41 mL/min/kg). Scaling of the in vitro intrinsic clearance of 9v, using the well-stirred
model and incorporating free fraction, predicted an in vivo hepatic clearance of 25 mL/min/kg
indicating that the moderate in vivo clearance of 9v was driven by the high protein binding in the
mouse (Table 6). The same correlation was observed in the rat. Oral bioavailability of 9v was
moderate at 41% in Balb-c nu/nu mice, and the compound showed a proportional increase in exposure
when orally dosed at 10, 30 and 100 mg/kg (Table 7). We conducted PK studies in SD rats and found
that 9v had a reasonable PK profile in this species (Cl = 19 ml/min/kg, half life = 5.4 h, F = 28%). The
compound was well tolerated in Balb-c nu/nu mice following five days of dosing at 100 mg/kg bid.
We demonstrated that 9v reduced LPA levels in vivo in MDA-MB-231-luc tumour bearing Balb-c
nu/nu mice with an IC50 of 0.013 μM for LPA C18:1 (Supplementary Information, Section 10). The
compound was effective in reducing LPA C18:1 levels in vitro in human plasma with an IC50 of 0.038
μM (LPA levels were measured using a UPLC/MS/MS assay, see Supplementary Information,
Section 5). Compound 9v was also shown to inhibit the migration of 4T1 cells with an EC50 of 0.025
μM in a LPC/ATX mediated transwell migration assay with recovery of inhibition in the presence of
LPA (Supplementary Information, Section 6).
Table 5. Mouse Pharmacokinetic Data
a
No
Dose
Route t1/2 bCLb
cVdss
(mg/kg)
(h)
(mL/min/kg (L/kg)
0.5
1
1
1
1
0.5
1
1
1
1
iv
iv
iv
iv
iv
iv
iv
iv
iv
iv
0.17 75
0.33 72
1.0 41
0.37 62
0.16 73
0.2 78
0.15 67
0.2 81
0.22 82
0.14 73
0.19
0.88
1.3
0.4
0.2
0.4
0.3
0.3
0.2
0.2
9q
9u
9v
9w
9x
21a
15b
24
26a
32a
ablood half life; bblood clearance; cvolume of distribution

Table 6. in vitro in vivo correlation unadjusted and adjusted for plasma protein binding
No Species
aCLint
(mL/min/g liver)
bFu
cCLhep pred
(mLmin/kg)
dCLhep pred
(mLmin/kg)
eCLb in vivo
(mL/min/kg)
Unadjusted for Fu Adjusted for Fu
Mouse
Rat
44
15
0.018
0.020
112
78
25
10
41
19
9v
9v
amicrosomal intrinsic clearance; bfraction unbound, cpredicted blood clearance scaled from in vitro
clearance using the well-stirred liver model and unadjusted for Fu; dpredicted blood clearance scaled
from in vitro clearance using the well-stirred liver model and adjusted for Fu. emeasured blood
clearance
Table 7. Mouse dose escalation data
No
Dose
(mg/kg)
10
30
100
Route aCmax
(µM)
bAUC
(µM.h))
3.2
15.2
59.3
ct1/2
(h)
1.4
0.9
1.3
d%F
po
po
po
3.8
10.9
18.1
41
64
75
9v
9v
9v
amaximum concentration; barea under the curve, cblood half life; doral bioavailability
The crystal structure of mouse ATX alone and in complex with LPAs with different acyl-chain
lengths and saturations has recently been reported.17 We solved the x-ray crystal structure of 9v bound
to mouse ATX (PDB accession code 5LIA) and this revealed that the compound interacts with the
protein in an unexpected binding mode (Figure 2). The compound binds to the lipophilic acyl chain
pocket of ATX and also to the LPA exit channel.18 The inhibitor binds approximately 5Å from the
catalytic centre in contrast to some ATX inhibitors reported in the literature which bind to the
catalytic zinc ions and/or threonine 209 residue.19 20 This mode of binding which avoids interaction
with the catalytic zinc ions may offer selectivity advantages over other inhibitors.

Figure 2. Crystal structure of murine ATX (mATX) in complex with 9v determined at 1.92 Å
resolution (PDB 5LIA): (A) Cartoon representation of the domain organisation of mATX
highlighting the compound-binding site. The somatomedin B-like domains 1 and 2 (SMB), catalytic
and nuclease-like domains (NLD) are coloured red, brown and blue, respectively. The active site Zn2+
ions are shown as cyan spheres. 9v, which binds to the catalytic domain, is shown as a stick
representation (C: limon; N: blue; O: red; F: green; Cl: dark green); (B) Zoomed-in view of (A)
showing the 2|Fobs|-|Fcalc| electron density map (blue mesh) for 9v contoured at 1 σ. Key residues
implicated in compound binding and the catalytic threonine (T209) are labelled. 9v binds ~5 Å away
from the active site in the LPA-substrate hydrophobic pocket and LPC exit channel; (C) Equivalent
view to (B) showing the surface of the compound-binding cavity in grey. The hydrogen bond between
the main chain amide of W275 and the compound carboxyamide moiety is shown as a dotted line; (D)
Ligand interaction diagram for 9v produced using Molecular Operating Environment (MOE),
2013.08; Chemical Computing Group Inc., Montreal, Canada. Panels (A)-(C) were generated using
PyMOL (http://www.pymol.org).
In summary we have identified and optimised a novel series of ATX inhibitors based on the
imidazopyridine scaffold.21 This work led to the discovery of a leading compound 9v with excellent
enzyme potency and drug-like properties. The compound potently inhibited the production of LPA in
human plasma and has suitable PK properties for in vivo efficacy studies. We are currently evaluating
the lead compound 9v (CRT0273750) in ATX/LPA-dependent models of cancer.

Supplementary Data
Supplementary information associated with this article can be found in the online version.
Compound 9v (CRT0273750) can be sourced from the XimBio reagent portal
(http://www.ximbio.com).
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