Identification of a novel class of autotaxin inhibitors through cross-screening

Article abstract of DOI:10.1039/c5md00081e

Three novel series were generated in order to mimic the pharmacophoric features displayed by lead compound AM095, a lysophosphatidic acid (LPA₁) receptor antagonist. Biological evaluation of this array of putative LPA₁ antagonists le

Full text of DOI:10.1039/c5md00081e

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CONCISE ARTICLE
Identification of a novel class of autotaxin
inhibitors through cross-screening†
Cite this: Med. Chem. Commun.,
2015, 6, 1149
Diana Castagna,a Emma L. Duffy,a Dima Semaan,b Louise C. Young,b
c
c
c
John M. Pritchard, Simon J. F. Macdonald, David C. Budd, Craig Jamieson a and
Allan J. B. Watson
*
a
*
Received 23rd February 2015,
Accepted 7th May 2015
Three novel series were generated in order to mimic the pharmacophoric features displayed by lead
compound AM095, a lysophosphatidic acid (LPA1) receptor antagonist. Biological evaluation of this array of
putative LPA1 antagonists led us to the discovery of three novel series of inhibitors of the ectoenzyme
autotaxin (ATX), responsible for LPA production in blood, with potencies in the range of 1–4 μM together
with good (>100 μg mL−1) solubility.
DOI: 10.1039/c5md00081e
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with both targets. This phenomenon has been reported with
syn- and anti- diastereomers of α-bromophosphonates (BrP-
Introduction
Lysophosphatidic acid (LPA) is a bioactive phospholipid,
accessed via the hydrolysis of lysophosphatidyl choline (LPC)
by the enzyme autotaxin (ATX),1,2 and mediates most of its
LPA), pan-LPA1–3 antagonists and nanomolar inhibitors of
ATX.16 However, these lipid-like compounds are suboptimal
drug development candidates, as their physicochemical pro-
files do not meet conventional medicinal chemistry require-
ments, because they exhibit high molecular weight and lipo-
philicity, two factors which are associated with attrition in
drug development.17 The first non-lipid-like structure acting
as an LPA receptor antagonist was Ki16425.18 This was identi-
fied by the Kirin Brewery through screening of 150 000 low
molecular weight compounds for LPA receptor antagonism. It
was found that the isoxazole derivative Ki16425(1) inhibited
biological effects through a set of six G-protein-coupled recep-
3
tors (GPCRs) known as LPA1–6
.
These receptors induce a
number of downstream signalling pathways and exert a range
of cellular responses, such as calcium mobilisation, transfor-
mation of smooth muscle cells, cell proliferation and migra-
tion, prevention of apoptosis, and chemotaxis, by acting as
agonists at membrane-bound GPCRs.4 Based on this, the
ATX–LPA signalling pathway has been implicated in many dis-
ease states including cardiovascular diseases,5 autoimmune
diseases,6 cancer,7 fibrotic diseases,8 inflammation,9 neuro-
degeneration,10 and pain,11 amongst others. Accordingly, sig-
nificant efforts have been invested in the generation of novel
lead compounds in order to identify new chemotypes for the
treatment of LPA/ATX-related disorders. A recent review has
summarised the current state of lysophospholipid receptors
in drug discovery.12 Among the first LPAR antagonists
synthesised were lipid-like structures,13,14 and similarly the
first patent describing ATX inhibitors was also lipid-like.15 As
both LPA1 and ATX bind LPA, one might expect a degree of
cross-talk between the types of compounds which interact
a WestCHEM, Department of Pure and Applied Chemistry, University of
Strathclyde, Thomas Graham Building, 295 Cathedral Street, Glasgow,
G1 1XL, UK. E-mail: craig.jamieson@strath.ac.uk, allan.watson.100@strath.ac.uk
b Strathclyde Institute of Pharmacy and Biomolecular Science, University of
Strathclyde, John Arbuthnott Building (Hamnett Wing), 161 Cathedral Street,
Glasgow, G4 0RE, UK
c Fibrosis DPU, GlaxoSmithKline, Medicines Research Centre, Gunnels Wood
Road, Stevenage, Hertfordshire, SG1 2NY, UK
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c5md00081e
Fig. 1 Selected LPA1 antagonists 1–4, ATX inhibitor 5, and pan-LPA1–3
antagonist/ATX inhibitor 6.
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LPA-induced actions in a manner highly specific to LPA1 and
LPA3 receptor subtypes.18 Subsequently, AM095(4), a novel
LPA1 receptor antagonist originally developed by Amira Phar-
maceuticals Inc., together with Bristol-Myers Squibb, was
developed. Further classes of LPA1 receptor antagonists have
been disclosed, most recently from Hoffmann-La Roche.3 In
order to identify novel LPA1-selective antagonists, Hoffmann-
La Roche undertook a bioisostere approach based on the
AM095 scaffold and reported the synthesis and biological test-
ing of a novel class of LPA1 antagonists.3 Of the reported
chemotypes, AM095 has progressed to clinical trials and as a
result became the main focus of our current study. Herein,
we report the design and synthesis of a novel palette of com-
pounds, derived from the AM095 chemotype, which display
inhibitory activity in the ATX/LPA pathway (Fig. 1).
biphenyl acetic acid moieties present in AM095, whilst the
aryl indole series challenged this binding hypothesis with the
construction of a series containing a similar structural overlap
but different functionalities. In addition to the representative
examples depicted, the regiochemistry of the biaryl and acetic
acid moieties would be probed. The aim of the three series
was to design a “reduced complexity” (lead-like)19 library using
simple synthetic handles available for chemical synthesis.
In order to qualitatively assess the level of similarity
between AM095 and the three proposed series, a range of
overlays was prepared using energy minimized structures of
AM095 and an exemplar from each series. These were gener-
ated using the Gaussian20 programme and then manually
overlaid using Discovery Visualiser,21 as an efficient overlap
may be indicative of a similar binding mode. These novel
heterocyclic biaryl compounds could potentially provide
selectivity toward LPAR isoforms. Based on the analysis
shown in Scheme 1, all compounds demonstrated a reason-
able degree of overlap with AM095, and accordingly, these
templates were prioritised for synthesis in order to validate
their biological properties against both LPA receptors and
autotaxin.
The synthetic approach used for the construction of the
acyl aniline and the aryl indole series is illustrated in
Schemes 2 and 3, respectively. The acyl aniline series was
divided into three main routes in order to probe (i) the posi-
tion of the acetic acid moiety, (ii) the presence of the acetic
acid moiety, and (iii) the potential for functionalization on
the nitrogen of the aniline in the form of a cyclohexyl (c-Hex)
or a tetrahydropyran (THP) group to mimic the isoxazole
group present in AM095. The synthetic route associated with
each of these three subgroups involved the use of three main
steps as follows: (i) carbamate formation, (ii) Suzuki–Miyaura
cross-coupling, and (iii) hydrolysis. Compounds 7, 16, and 17
were synthesised by reaction of 3-bromoaniline with benzyl
chloroformate to afford carbamate 11, which could then be
taken forward into the Suzuki–Miyaura cross-coupling, using
a range of substituted pinacol boronic esters to install the
desired biaryl functionality.
Compound design and synthesis
The medicinal chemistry strategy implemented was a scaffold
hopping approach in order to identify a new chemotype based
on the Amira (AM095) and Hoffmann-La Roche assets which
belong to the same pharmacophore. To this end, three
unique series were designed by performing simple disconnec-
tions to give the acyl aniline, the aryl indole, and the
hydantoin series, removing the isoxazole as it was assumed to
be a non-essential spacer, as illustrated in Scheme 1. The acyl
aniline and the hydantoin series retained the carbamate and
The final step involved the hydrolysis of the ester moiety
to afford the desired acetic acid compounds. A similar route
Scheme
compounds. a) K2CO3, CbzCl, 2-MeTHF, rt, 5 h; b) 2′-IJdimethylamino)-
2-biphenylyl-palladiumIJII)chloride dinorbornylphosphine complex,
K3PO4, pinacol boronic ester, 1,4-dioxane/water (2 : 1), 130 °C, 30 min,
μW; c) NaOH, THF, 16 h, 40 °C.
2 Route developed for the synthesis of acyl aniline
Scheme 1 Schematic representation of the three proposed series
(7–9) from disconnecting AM095 and their corresponding overlays with
AM095 (grey).
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carboxylic acid. The synthetic route applied to the hydantoin
series is illustrated in Schemes 5 and 6. The construction of
this series required firstly the synthesis of the requisite
hydantoin intermediates using reductive amination of both
2-IJ4-bromophenyl)acetaldehyde and 5-bromopicolinaldehyde
to the secondary amines 34 and 43. Preparation of the target
hydantoins involved the reaction of the appropriate second-
ary amine with the relevant isocyanate system, yielding a urea
derivative. This derivative is not isolated but can be readily
converted to the hydantoin following treatment with a protic
acid, in this case, trifluoroacetic acid. This was applied to the
synthesis of compounds 35–37 (Scheme 5) and 44–45
(Scheme 6). Suzuki–Miyaura cross-coupling was used to pre-
pare the biaryl hydantoins with boronic pinacol esters and
then smoothly hydrolysed to the acetic acid functionality, fur-
nishing compounds 9, 38–41 and 46–47.
Scheme
3 Route developed for the synthesis of the substituted
acyl aniline compounds. a) NaHBIJOAc)3, DCE, AcOH, rt, 24 h,
cyclohexanone or tetrahydropyranone; b) K2CO3, CbzCl, DCE, rt, 24 h;
c) 2′-IJdimethylamino)-2-biphenylyl-palladiumIJII)chloride dinorbornylphosphine
complex, K3PO4, pinacol boronic ester, 1,4-dioxane/water (2 : 1), 130 °C,
30 min, μW; d) NaOH, THF, 16 h, 40 °C.
Pharmacology
was followed for the formation of compound 15, which
involved the reaction of phenyl boronic acid with compound
11 in order to obtain the biaryl species lacking the acetic acid
moiety. Lastly, substitution was introduced on the nitrogen
of the aniline by reductive amination of 10 with cyclohexa-
none and tetrahydropyranone, followed by carbamate forma-
tion using benzyl chloroformate, and finally, hydrolysis to
reveal the acetic acid moiety for compounds 25 and 26.
A similar route was undertaken for the synthesis of the
aryl indole compounds, as illustrated in Scheme 4. However,
hydrolysis of benzylic ester to acetic acid was performed prior
to the introduction of carbamate.
The hydantoin series was accessed via two main routes,
namely (i) benzyl hydantoin and (ii) pyridyl hydantoin. The
phenyl substituted hydantoin possessed a good structural
overlay with AM095; therefore, the isopropyl (i-Pr) and cyclo-
hexyl (c-Hex) groups were designed as isosteres to the phenyl
group with the goal of lowering c log P and reducing the num-
ber of aromatic rings to below three.22 Within the benzyl
hydantoin, the presence/absence of acetic acid would be
probed along with cyclopropyl substitution alpha to the
The synthesised compounds were evaluated for both LPA1
receptor agonism and antagonism using CHO-K1 EDG2
β-arrestin cell line in a single dose format (10 μM), with the
inclusion of 1-oleoyl-LPA, AM095, and Ki16425 as controls.23
The data obtained from the assay are described in terms of
percentage of agonism where 100% refers to complete
agonism and 0% refers to complete antagonism. The data
obtained from this assay unfortunately indicated that none
of the synthesised compounds were acting as LPA agonists,
and in terms of antagonist activity only the controls, 1-oleoyl-
LPA, AM095 (11% activity) and Ki16425 (16% activity),
showed clear LPA1 antagonism. From the design strategy of
our novel series, we speculated that the isoxazole of AM095
was a spacer between essential carbamate and biaryl acetic
acid moieties. From these data, it is now clear that isoxazole
replacement is not tolerated in terms of LPA1 antagonism
within this chemotype. However, it can be derivatized to
triazole and pyrazole analogues, as demonstrated by Qian
et al.3 In parallel, the three series were evaluated for ATX
inhibitory activity with bis-IJp-nitrophenyl)phosphate as the
substrate and PF-8380, a known ATX inhibitor,24 as the stan-
dard for comparison. As well as generating key developability
Scheme
4
Route developed for the synthesis of aryl indole
Scheme 5 Route developed for the synthesis of the hydantoin
compounds. a) 2′-IJDimethylamino)-2-biphenylyl-palladiumIJII)chloride
dinorbornylphosphine complex, K3PO4, pinacol boronic ester, 1,4-
dioxane/water (2 : 1), 130 °C, 30 min, μW; b) NaOH, THF, 40 °C, 16 h;
c) NaH, DMF, N-IJbenzyloxycarbonyloxy)succinimide, 40 °C, 16 h.
compounds. a) Glycine methyl ester hydrochloride, EtOH, NEt3, MeOH,
NaBH4, rt, 2 h; b) CH2Cl2, isocyanate, TFA; c) pinacol boronic ester,
Pd2IJdba)3, PCy3, K3PO4, 1,4-dioxane/H2O (1.3 : 0.7), 100 °C, 16 h;
d) THF/H2O (3 : 1), LiOH, rt, 3 h.
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Table 2 Biological and physicochemical data for the substituted acyl ani-
line series
Cpd.a
R
Ki (μM) log Db
Sol.c (μg mL−1
)
Papp (nm s−1
)
d
25
26
c-Hex
THP
2
>30
4.92
3.13
189
212
150
23
Scheme
6 Route developed for the synthesis of the pyridine
a
b
c
Cpd., compound number. log D, Chrom log D at pH 7.4. Sol.,
chemiluminescent nitrogen detection (CLND) kinetic aqueous
solubility assay. d Papp, permeability pH 7.4 assay.
hydantoin compounds. a) Glycine methyl ester hydrochloride, MeOH,
NEt3, NaBHIJOAc)3; b) CH2Cl2, isocyanate, TFA; c) 2′-IJdimethylamino)-2-
biphenylyl-palladiumIJII)chloride dinorbornylphosphine complex, K3PO4,
4-IJcarboxymethyl)phenylboronic acid pinacol ester, 1,4-dioxane/water
(2 : 1), 100 °C, 16 h; d) THF/H2O (3 : 1), LiOH, rt, 3 h.
Table 3 Biological and physicochemical data for aryl indole compounds
data,28 (log D,25 solubility,26 and permeability27) concentra-
tion–response (1 nM–30 μM) studies were performed to deter-
mine the potency of all compounds against ATX.28 The inhib-
itor constants (Ki) are summarized in Tables 1–4. To enable
comparison with our progenitor, hit compounds AM095 and
Ki16245 were also tested as ATX inhibitors at 30 μM and
showed less than 20% ATX inhibition. Pleasingly, it was
found that several of the compounds tested exhibited Ki
values less than 5 μM (7–9, 20, 27, 34, 35, 37, 38, 43, and 44).
The active compounds were all equipotent; however, they var-
ied significantly in their physicochemical profiles; solubility
was generally high (>100 μg mL−1), as measured by a high
throughput solubility assay. However, permeability varied
between the series, with the acyl aniline and indole com-
pounds exhibiting increased membrane permeability, com-
pared to those in the hydantoin series.
Cpd.a
Pos. Ki (μM) log Db
Sol.c (μg mL−1
)
Papp (nm s−1
)
d
8
32
5
6
1.1
>30
7.25
7.43
70
110
445
510
a
b
c
Cpd., compound number. log D, Chrom log D at pH 7.4. Sol.,
chemiluminescent nitrogen detection (CLND) kinetic aqueous
solubility assay. d Papp, permeability pH 7.4 assay.
The overlay diagrams indicated that the backbone of all four
compounds contained the same conformation. In terms of
the three acetic acid moieties, these all pointed in the same
spatial orientation, indicating the potential beneficial interac-
tion within the catalytic pocket. This would further support
why its removal led to a complete loss of activity.
From the acyl aniline series, it became evident that the
presence of the acetic acid moiety was essential as its
removal in compound 15 caused a complete loss of activity
compared to compounds 7, 16, and 17. The acetic acid moi-
ety is potentially binding to the Zn2+ ions present in the
active site of ATX, an interaction that has been noted with
other acidergic groups.29 An additional set of overlays was
conducted, as illustrated in Scheme 7, with compounds 7, 15,
16 and 17 in order to rationalise the biological data which
implied that the position of the acetic acid (o-, m-, or p-) is
not important; however, its presence is essential for activity.
It was also noted that substitution on the nitrogen of the
aniline was tolerated, as illustrated by compound 25, with
the introduction of a cyclohexyl group, and resulted in a Ki
value of 2 μM. However, the introduction of the tetra-
hydropyran (THP) group, compound 26, in this position was
inactive, perhaps suggesting an unfavourable polar interac-
tion with the oxygen present in the THP group which is
absent in the cyclohexyl group, thus implying that other polar
functionalities may also not be tolerated in this position.
The aryl indole compounds, however, illustrated that the
substitution of the biaryl ring was in fact important as
Table 1 Biological and physicochemical data for the acyl aniline series
Cpd.a
R
Pos R′
R1
Ki (μM)
log Db
Sol.c (μg mL−1
)
Papp (nm s−1
)
d
15
16
17
7
H
H
H
H
—
1
2
H
>30
4
3.5
4.5
7.52
3.06
3.10
3.19
5
287
221
198
450
140
64
CH2CO2H
CH2CO2H
CH2CO2H
3
73
a
b
c
Cpd., compound number. log D, Chrom log D at pH 7.4. Sol., chemiluminescent nitrogen detection (CLND) kinetic aqueous solubility
assay. d Papp, permeability pH 7.4 assay.
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Table 4 Biological and physicochemical data for the hydantoin series
Cpd.a
R
X
Y
Ki (μM)
log Db
Sol.c (μg mL−1
)
Papp (nm s−1
)
d
38
9
Ph
Ph
Ph
i-Pr
c-Hex
i-Pr
c-Hex
C
C
C
C
C
N
N
H
1.3
3.6
>30
1.2
1.8
4
6.24
2.18
1.50
1.10
1.12
−1.38
−0.35
7.5
540
3
16
3
22
CH2CO2H
CIJc-propyl)CO2H
CH2CO2H
CH2CO2H
CH2CO2H
CH2CO2H
174.5
195
190
185.5
108
93
39
40
41
46
47
<10
<30
3.9
a
b
c
Cpd., compound number. log D, Chrom log D at pH 7.4. Sol., chemiluminescent nitrogen detection (CLND) kinetic aqueous solubility
assay. d Papp, permeability pH 7.4 assay.
suggests that the inhibitory effect is due to the compounds
blocking the endogenous production of LPA by ATX. This can
be rationalised by the fact that PC3 cells express significant
levels of endogenous autotaxin30 that drives the generation of
lysophosphatidic acid (LPA) which in turn provides an auto-
crine/paracrine feedback loop to drive basal proliferation of
these cells. Visual inspection of the cells showed no apparent
cell death.
Scheme 7 Overlay diagram of compounds 7 (blue), 15 (purple), 16
(red), and 17 (green) to illustrate the position of the acetic acid moiety.
Compounds 7, 25, and 38 were also tested for cytotoxicity
in an A2780 ovarian cancer cell line at 30 μM using an
alamarBlue® Assay. Gratifyingly, none of the compounds
showed any signs of cytotoxicity.
moving the aryl carbamate from the 5 to the 6 position led to
inactivity with Ki values of 1.1 and >30 μM, respectively.
Strikingly, these data imply that the acid moiety which was
previously believed to be essential for activity is in fact not
important, therefore indicating a potential alternative hydro-
phobic binding mode.
Lastly, within the third series, phenyl substituted
hydantoins in the presence or absence of an acetic acid moi-
ety were active, 38 and 39, 1.3 and 3.6 μM, respectively,
potentially indicating a different binding mode to both the
acyl aniline and the aryl indole series. Surprisingly however,
the addition of a cyclopropyl group adjacent to the acid
group 36 rendered the compound inactive, potentially
suggesting an unfavourable steric clash within the active site
of the constrained and bulky cyclopropyl group. The isopro-
pyl and cyclohexyl substituted hydantoins (40, 41, 46, and 47)
all showed significant inhibitory activity of ATX with poten-
cies in the range of 1–4 μM.
To examine whether the inhibitors had an impact on in-
cell proliferation via ATX modulation, three of the prepared
ATX inhibitors (7, 25, and 38) and AM095 were examined
using a [3H]-thymidine incorporation assay where the quan-
tity of tritiated thymidine measured is proportional to DNA
synthesis, as illustrated in Fig. 2. There was a significant
increase in DNA synthesis within PC3 cells when stimulated
with LPA (p < 0.05). Upon treatment with 7, 25, and 38 as
well as AM095, DNA synthesis was inhibited to a statistically
significant level (p < 0.05) with respect to the control. Com-
pounds 7, 25, and 38 show ATX inhibitory potency and block
baseline [3H]-thymidine incorporation into PC3 cells; this
Fig.
2
3H-thymidine incorporation into LPA (2 μM), AM095 (a),
compound 7 (b), compound 25 (c), or compound 38 (d) (10 μM); in the
stimulated prostate cancer (PC3) cell line, cells were quiesced for 24
hours and treated for 18 h with compounds (AM095, 7, 25, or 38). N =
9, data are presented as mean S.E.M.
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Conclusions
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Using a range of literature templates as a basis to design new
compounds which could interact in the LPA–ATX signalling
pathway, three novel chemotypes have been prepared and
their inhibitor activities at LPA and ATX have been assessed.
Although the compounds prepared did not show any activity
against the LPA1 receptor, cross-screening against ATX
revealed that several exemplars had significant levels of
inhibitory activity against the enzyme. Allied to this, measure-
ments of the key developability parameters indicated that
several of the hits identified had promising physicochemical
profiles, suggesting that the series identified could offer
potential for further optimisation. Efforts to achieve this are
on-going and will be reported in due course.
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Acknowledgements
We thank the EPSRC National Mass Spectrometry Service
Centre, Swansea University for analyses and GlaxoSmithKline
for funding. We thank Alan Hill for physicochemical analysis
and Professor Susan Pyne and Professor Nigel Pyne of the
Strathclyde Institute of Pharmacy and Biomedical Sciences.
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