Development of autotaxin inhibitors: A series of zinc binding triazoles

Article abstract of DOI:10.1016/j.bmcl.2018.05.030

A series of inhibitors of Autotaxin (ATX) has been developed using the binding mode of known inhibitor, PF-8380, as a template. Replacement of the benzoxazolone with a triazole zinc-binding motif reduced crystallinity and improved solubility relative to PF-8380. Modification of the linker region removed hERG activity and led to compound 12 – a selective, high affinity, orally-bioavailable inhibitor of ATX. Compound 12 concentration-dependently inhibits autotaxin and formation of LPA in vivo, as shown in pharmacokinetic-pharmacodynamic experiments.

Full text of DOI:10.1016/j.bmcl.2018.05.030

Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of autotaxin inhibitors: A series of zinc binding triazoles
Christopher G. Thomsona,⁎, Darren Le Granda, Mark Dowlingb, Cara E. Brocklehurstc,
Colin Chinnd, Lucy Elphickb, Michael Fallere, Mark Freemanb, Vikki Furmingera,
Cornelia Gasserc, Ahmed Hamadia, Elizabeth Hardakerb, Victoria Headf, Johan C. Hillb,
Diana I. Janusa, David Pearcea, Anne-Sophie Poulauda, Emily Stanleya, Lilya Sviridenkoa
a
Global Discovery Chemistry, Novartis Institutes for BioMedical Research, Horsham RH12 5AB, UK
Respiratory Disease Area, Novartis Institutes for BioMedical Research, Horsham RH12 5AB, UK
Global Discovery Chemistry, Novartis Institutes for BioMedical Research, Fabrikstrasse, CH-4056 Basel, Switzerland
Chemical and Pharmaceutical Profiling, Novartis Institutes for BioMedical Research, Horsham RH12 5AB, UK
Department of Chemical Biology and Therapeutics, Novartis Institutes for BioMedical Research, Fabrikstrasse, CH-4056 Basel, Switzerland
Metabolism and Pharmacokinetics, Novartis Institutes for BioMedical Research, Horsham RH12 5AB, UK
b
c
d
e
f
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of inhibitors of Autotaxin (ATX) has been developed using the binding mode of known inhibitor, PF-
8380, as a template. Replacement of the benzoxazolone with a triazole zinc-binding motif reduced crystallinity
and improved solubility relative to PF-8380. Modification of the linker region removed hERG activity and led to
compound 12 – a selective, high affinity, orally-bioavailable inhibitor of ATX. Compound 12 concentration-
dependently inhibits autotaxin and formation of LPA in vivo, as shown in pharmacokinetic-pharmacodynamic
experiments.
Autotaxin inhibition
Idiopathic pulmonary fibrosis
PK/PD
Solubility
hERG inhibition
Introduction
describing our discovery of autotaxin inhibitors for potential treatment
of IPF.
Lysophosphatidic acid (LPA) is a key, serum-borne phospholipid,
regulating a number of cellular processes such as proliferation, migra-
tion and differentiation through its interaction with G-protein coupled
receptors.1 LPA receptor signaling has been implicated in several dis-
ease states including fibrosis,2 cholestatic pruritus3 and tumour me-
tastasis.4 There are a number of forms of LPA, varying in length and
unsaturation levels of the lipid sidechain, as well as at least six known
receptors, whose roles are not all clearly understood.2 Autotaxin (ATX),
also known as ectonucleotide pyrophosphatase/phosphodiesterase 2
(eNPP2), is thought to be the predominant enzyme responsible for
production of the various LPAs.5 Autotaxin exhibits lysophospholipase
D activity, which cleaves lysophosphatidylcholines (LPC) to the re-
spective LPAs (Fig. 1).
Receptor mediated LPA signaling has been shown to be an im-
portant mechanism in lung fibrosis6 and Bristol-Myers Squibb have
been developing the selective LPA1 antagonist, BMS-986020,7 for
treatment of idiopathic pulmonary fibrosis (IPF). Another approach for
blockade of this signaling pathway is inhibition of ATX, preventing
formation of LPA. Galapagos are currently testing this approach with
the autotaxin inhibitor GLPG-1690.8 This paper is the first of two
PF-8380 (Fig. 2), characterised by Pfizer,10 is a high affinity ATX
inhibitor that has been shown by crystallization studies to occupy a
similar binding pocket to LPA (Fig. 3). The benzyl carbamate occupies a
hydrophobic pocket, similarly to the lipophilic chain of LPC, whilst the
benzoxazolone is bound by the catalytic zinc.
Whilst PF-8380 is a potent autotaxin inhibitor and useful chemical
probe for exploring ATX biology, its clinical utility is limited by a lack
of solubility, which can lead to erratic oral exposure, and by activity at
the hERG channel. In an attempt to design a safer and more soluble
molecule, with consistent oral exposure, we began to explore the che-
mical space around PF-8380.
Initially, library work was carried out using the fragment 110
(Scheme 1). In an attempt to improve solubility, a number of alternative
alcohols (ROH) were used to reduce the number of aromatic rings via
saturation.15 Whilst 2b and 2c maintained the activity (Table 1) when
compared with unsubstituted compound 2a, none of these changes
resulted in an improvement in thermodynamic solubility, which re-
mained unmeasurable at physiological pH. Replacement of the di-
chlorophenyl moiety of PF-8380 with heterocycles such as pyridine led
to a significant loss of activity (data not shown).
⁎
Corresponding author.
E-mail address: chris.thomson@pharmaron-uk.com (C.G. Thomson).
https://doi.org/10.1016/j.bmcl.2018.05.030
Received 28 March 2018; Received in revised form 11 May 2018; Accepted 14 May 2018
0960-894X/©2018ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:Thomson,C.G.,Bioorganic&MedicinalChemistryLetters(2018),https://doi.org/10.1016/j.bmcl.2018.05.030

C.G. Thomson et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Fig. 1. Cleavage of 18:1 lysophosphatidylcholine (LPC) to 18:1 lysopho-
sphatidic acid (LPA) by Autotaxin.
Scheme 1. Reagents and conditions: (i) ROH, CDI, DMF, overnight at RT,
30–70% yield.
Table 1
Fig. 2. Autotaxin inhibitor, PF-8380: structure and properties. ATX CRψ IC50
1.1 nM, Rat p.o. pharmacokinetics10 F 43-83%, HT-Eq Solubility11 (pH6.8
)
0.011 mg/ml, Selectivity: hERG dofetilide12 IC50 2700 nM. ψCholine release
assay.9 Assay values represent geometric means of 3–6 determinations.
Compound
R
ATX CRψ IC50/nM
1.1
PF-8380
The linker region of PF-8380 was then modified, in an attempt to
gain solubility via change in geometry or incorporation of heteroatoms,
as shown in Scheme 2, to give a series of amide carbamates (6). Several
potent compounds were identified (Table 2), but again, thermodynamic
solubility remained unmeasurable at physiological pH.
It became apparent at this time, that all compounds explored con-
tained the benzoxazolone, and that all showed high crystallinity. We
hypothesized that the crystallinity was due to the benzoxazolone, and
that high crystal lattice energy was leading to poor dissolution, and
hence poor solubility. In an attempt to reduce the crystal lattice energy,
the benzoxazolone was truncated to the oxazolinone (8). Intermediates
(7) were prepared analogously to 5, then coupled to 2-oxo-2,3-dihy-
drooxazole-5-carboxylic acid (Scheme 3). We were pleased to find that
this led to an improvement in solubility, despite a reduction in activity.
Some activity could be recovered by replacing the piperazine with a
2a
2b
2c
130
240
170
ψ
Choline release assay.9 Assay values represent geometric means of 3–6
determinations.
piperidine to give compound 8b.
Within the series, it had been noted that hydrogen bond donor
(HBD) count greatly affected permeability, as measured in artificial
membrane experiments (PAMPA16). This was reflected in the perme-
ability data (Table 3) for compounds 8a and 8b containing two HBDs.
Fig. 3. 22:6 LPA and PF-8380 bound to Autotaxin. 1. 22:6 Lysophosphatidic acid (LPA) bound to mouse Autotaxin (ATX) – the phosphate coordinated to Zn2+ (blue
spheres) in the catalytic domain, and the lipophilic tail bound in the hydrophobic pocket. The tunnel is thought to deliver LPA to the receptor.13 [PDB ID 3NKR], 2.
Crystal structure of PF-8380 bound to rat ATX. The benzoxazolone is in close proximity to the catalytic site Zn2+ and the benzyl carbamate is bound in the
hydrophobic pocket.14 [PDB ID 5LOK].
2

C.G. Thomson et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Table 3
Cpd.
A
R
HT-Eq Solubilityδ
(pH6.8)/mgml−1
ATX CRψ PAMPAΦfraction
IC50/nM
absorbed/%
Scheme 2. Reagents and conditions: (i) 3,5-Dichlorobenzyl carbonochloridate,
aq. NaHCO3 soln., DCM, 1 h at RT, 90%-quantitative yield. (ii) 4 N HCl in 1,4-
dioxane, 0 °C-RT, 1 h, quantitative yield. (iii) Benzoxazolone-6-carboxylic acid,
T3P®, DIPEA, DMF, overnight at RT, 50–80% yield.
PF-8380 See Fig. 1.
0.01
0.06
0.08
0.04
1.7
370
86
81
35
68
94
8a
8b
8c
N
CH
CH
H
H
Me
25
ψ
Table 2
Choline release assay.9 Assay values represent geometric means of 3–6
determinations.
δ
High-throughput equilibrium solubility. Values are the mean of 2 de-
terminations.11
Φ
Values are the mean of 2 determinations.16
Compound
Linker
ATX CRψIC50/nM
10
6a¥
8c
6b
3.9
Fig. 4. Autotaxin inhibitor, 8c and its properties. ATX CRψ IC50 25 nM HT Eq
Solubility11 (pH6.8) 0.04 mg/ml, Rat microsomal Clint 235 μl/min/mg, Rat
PPB18 fu 0.5%, hERG (dof.12) IC50 > 30000 nM, Q-patch19 IC50 7300 nM,
6c
8
Intravenous PK parameters (1 mg/kg, rat), t 4.8
min/kg, Vss 2.2
25
0.8 h, CL 12
0.9 L/kg, Oral PK parameters (10 mg/kg, rat), AUC(0-inf)
5.9 µM/hour, Cmax 3400 910 nM, tmax 2.5 h, F 80
19%. ψCholine
2.9 mL/
½
6d¥
18
release assay.9 Assay values represent geometric means of 3–6 determinations.
¥
Tested as a racemate.
Choline release assay.9 Assay values represent geometric means of 3–6
ψ
acid degradation was attributed to the oxazolinone head group.
Having taken a step back from affinity to gain better exposure and
solubility, we started work to replace the unstable oxazolinone, and
address the selectivity over the hERG channel. Keeping in mind the
binding mode of the series from crystal structures, triazole was identi-
fied as another mildly acidic zinc-binder. Compound 9 (Table 4) was
prepared analogously to Scheme 3, coupling with 1H-1,2,3-triazole-4-
carboxylic acid. Compound 11 was prepared using ‘Click’ chemistry20
(Scheme 4) which became a robust and efficient method to prepare the
relevant triazole carboxylic acids for all subsequent compounds.
Although 8c showed good oral exposure, this was dependent on
high plasma protein binding, which varied across the series. Compound
9 lost affinity to ATX, but showed good permeability and improved
microsomal stability. Both of these compounds exhibited similar in-
hibition of hERG in the automated patch clamp assay. In an attempt to
disrupt this hERG activity, the amide bond was moved closer to the
piperidine21 (Table 4, 11) to redistribute areas of polarity across the
molecule. For 11, this approach had no significant effect on hERG ac-
tivity, but showed a clear improvement in ATX inhibition.
Attempts were then made to improve the poor permeability of 11 by
lengthening the carbon chain to increase lipophilicity, or by removing a
hydrogen bond donor via methylation, as had been shown with 8c.
Compounds 12, 13 and 15 were synthesized analogously to 11, using
the relevant acetylene acid, and for 13, the relevant 4-amino-
methylpiperidine starting material. Compound 14 was formed as shown
in Scheme 5.
Whilst methylation of the amide nitrogen did indeed improve per-
meability (13, Table 5), ATX inhibition suffered. Similarly, methylation
of the triazole in 14 reduced ATX inhibition considerably, reinforcing
that the interaction of the acidic triazole with the catalytic zinc in ATX
is key. Extension of the carbon chain by one (12) and two methylenes
determinations.
Scheme 3. R = Me or H. Reagents and conditions: (i) 2-Oxo-2,3-dihydroox-
azole-5-carboxylic acid, HATU, DIPEA, DMF, 1 h at RT, 30–50% yield.
Methylation of 8b to give 8c improved this permeability, as well as
leading to another small improvement in activity.
Compound 8c showed high intrinsic clearance in rat microsomes17
,
but was highly bound to plasma protein (Fig. 4).
When compound 8c was dosed in vivo, it showed reasonable ex-
posure, due to lower clearance and a moderate volume of distribution.
This suggested that the compound distributed into tissues, as well as
being highly bound to plasma proteins. However, further profiling of 8c
showed that despite being inactive in a dofetilide radioligand binding
assay, it had significant activity in a hERG automated patch clamp
assay.19 Finally, when tested for chemical stability, compound 8c was
unstable in acid. As all compounds synthesized at this point had con-
tained the carbamate moiety and had shown no signs of instability, the
3

C.G. Thomson et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Table 4
Compound
X
ATX CRψIC50/nM
25
PAMPAΦ fraction absorbed/%
94
Rat microsomal ClintΩ/μl/min/mg
hERG Q-patchΣ IC50/nM
7300
8c
235
9
150
4.2
97
43
51
8600
8800
11
75
ψ
Choline release assay.9 Assay values represent geometric means of 3–6 determinations.
Values are the mean of 2 determinations.16
Values are the mean of 2 determinations.17
Φ
Ω
Σ
Automated patch clamp assay19 using CHO-K1 cells stably expressing the hERG potassium channel. Assay values represent geometric means of 3–6 determi-
nations.
Further exploration of the aryl substitution (Table 6) confirmed
what had been found early on in the development of the series – that
3,5 substitution was optimal. No further improvement was seen in
permeability, but hERG activity could be reduced to > 30 μM by re-
placement of one of the chlorines with a cyano group (16d). However,
this compound demonstrated even further reduced permeability and
was subsequently shown to have poor oral exposure.
At this point, despite lower permeability in the PAMPA assay,
compound 12 was studied further and shown to have good oral ex-
posure in the Sprague Dawley rat. Clearance and volume of distribution
were comparable to 8c, leading to good bioavailability (Fig. 5).
When tested in an in vitro serum assay,22 measuring 18:1 LPA
levels by LCMS, 12 showed ATX inhibition of IC50 9 nM. The oral
exposure levels and activity in the presence of serum led to 12 being
studied in a PK/PD model,23 where inhibition of ATX was measured
by LCMS detection of four isoforms of LPA – 16:0, 18:0, 18:1 and
20:4 (Fig. 6).
Scheme 4. n = 1–3. Reagents and conditions: (i) 3,5-Dichlorobenzyl carbono-
chloridate, aq. NaHCO3 soln., DCM, 1 h at RT, 98% yield. (ii) HCl in 1,4-di-
oxane, 0 °C-RT, 1 h, quantitative yield. (iii) Benzyl azide, Cu(OAc)2, sodium
ascorbate, tBuOH, water, overnight at RT, 90% yield. (iv) 0.35 bar hydrogen,
10% Pd/C, EtOH, 3 h at RT, 97% yield. (v) Acid 10, T3P®, DIPEA, DMF, 1 h at
RT, 67% yield.
When dosed at 0.3 mg/kg to Sprague Dawley rats, a robust drop
in all four LPA levels was seen, the peak reduction of around
70–80% coinciding with the Cmax of 12. This inhibition of LPA
production then decayed in line with the clearance of 12, showing
the observed PK/PD relationship to be direct. This was simulated,
using a turnover model23 to capture inhibition of the formation of
each of the LPA isoforms described above: an IC50 of around 20 nM
was determined for 12.
In conclusion, a series of potent autotaxin inhibitors has been de-
veloped, following a similar binding motif to PF-8380. The benzox-
azolone in this molecule was hypothesised to be causing poor solubility
through high crystal lattice energy, and was replaced with smaller,
monocyclic zinc-binding heterocycles, leading to lower affinity, but
more soluble and better orally absorbed compounds. Subsequent opti-
misation led to compound 12, which shows good oral exposure, and a
concentration dependent inhibition of formation of LPA in vivo. This
compound has superior solubility and hERG selectivity to PF-8380, and
was chosen for further development.
Scheme 5. Reagents and conditions: (i) K2CO3, MeI, DMF, overnight at RT. A
mixture of the three regioisomers was obtained; 14 was isolated in 9% yield.
(15) maintained potency. Improvement of permeability for 15 was
marginal, but a similar level of hERG inhibition was seen. For com-
pound 12, however, the selectivity over hERG inhibition improved.
4

C.G. Thomson et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Table 5
Compound
X
ATX CRψIC50 / nM
4.2
PAMPAΦ fraction absorbed/%
hERG Q-patchΣIC50/nM
8800
11
43
12
13
3.1
93
43
89
20900
–
14
15
1370
3.5
–
–
60
9300
ψ
Φ
Σ
Choline release assay.9 Assay values represent geometric means of 3–6 determinations.
Values are the mean of 2 determinations.16
Automated patch clamp assay19 using CHO-K1 cells stably expressing the hERG potassium channel. Assay values represent geometric means
of 3–6 determinations.
Table 6
16
Cpd.
Ar
ATX CRψIC50/nM
3.1
PAMPAΦ fraction absorbed/%
43
12
16a
39
35
16b
16c
206
55
43
35
16d
7.7
21
Fig. 6. PK/PD relationship of 12 with LPA levels.
ψ
Acknowledgments
Choline release assay.9 Assay values represent geometric means of 3–6
determinations.
Φ
The authors would like to thank Drs. Duncan Shaw, David Sandham,
Laszlo Urban and Dallas Bednarczyk for help in preparation of the
manuscript.
Values are the mean of 2 determinations.16
A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.bmcl.2018.05.030.
12
Fig. 5. Compound 12 in vivo properties and activity. 12, ATX CRψ IC50 3.1 nM, ATX
LCMS21 (100% human serum, 18:1 LPA) IC50 9 nM, Crystalline material24, M.pt.
129 °C, FeSSIF thermodynamic solubility 0.126 mg/ml, Rat microsomal Clint 104 μl/
min/mg, Human microsomal Clint 59 μl/min/mg, Rat PPB18 fu 4%, Human PPB18 fu
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