Novel Autotaxin Inhibitor for the Treatment of Idiopathic Pulmonary Fibrosis: A Clinical Candidate Discovered Using DNA-Encoded Chemistry

Article abstract of DOI:10.1021/acs.jmedchem.0c00688

The activity of the secreted phosphodiesterase autotaxin produces the inflammatory signaling molecule LPA and has been associated with a number of human diseases including idiopathic pulmonary fibrosis (IPF). We screened a single DNA-encoded chemical library (DECL) of 225 million compounds and identified a series of potent inhibitors. Optimization of this series led to the discovery of compound 1 (X-165), a highly potent, selective, and bioavailable small molecule. Cocrystallization of compound 1 with human autotaxin demonstrated that it has a novel binding mode occupying both the hydrophobic pocket and a channel near the autotaxin active site. Compound 1 inhibited the production of LPA in human and mouse plasma at nanomolar levels and showed efficacy in a mouse model of human lung fibrosis. After successfully completing IND-enabling studies, compound 1 was approved by the FDA for a Phase I clinical trial. These results demonstrate that DECL hits can be readily optimized into clinical candidates.

Full text of DOI:10.1021/acs.jmedchem.0c00688

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Article
A Novel Autotaxin Inhibitor for the Treatment of Idiopathic Pulmonary
Fibrosis: A Clinical Candidate Discovered Using DNA-Encoded Chemistry
John W. Cuozzo, Matthew A. Clark, Anthony D. Keefe, Anna Kohlmann, Mark
J Mulvihill, Haihong Ni, Louis Renzetti, Daniel I Resnicow, Frank Ruebsam,
Eric A Sigel, Heather A Thomson, Ce Wang, Zhifeng Xie, and Ying Zhang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00688 • Publication Date (Web): 25 Jun 2020
Downloaded from pubs.acs.org on June 25, 2020
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A Novel Autotaxin Inhibitor for the Treatment of
Idiopathic Pulmonary Fibrosis: A Clinical Candidate
Discovered Using DNA-Encoded Chemistry
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John W. Cuozzo*, Matthew A. Clark, Anthony D. Keefe, Anna Kohlmann, Mark Mulvihill,
Haihong Ni, Louis M. Renzetti, Daniel I. Resnicow, Frank Ruebsam, Eric A. Sigel, Heather A.
Thomson, Ce Wang, Zhifeng Xie and Ying Zhang*
ABSTRACT
The activity of the secreted phosphodiesterase autotaxin produces the inflammatory
signaling molecule LPA and has been associated with a number of human diseases including
idiopathic pulmonary fibrosis (IPF). We screened a single DNA-encoded chemical library
(DECL) of 225 million compounds and identified a series of potent and selective inhibitors.
Optimization of this series led to the discovery of compound 1 (X-165), a highly potent, selective
and bioavailable small molecule. Co-crystallization of compound 1 with human autotaxin
demonstrated that it has a novel binding mode occupying both the hydrophobic pocket and a
channel near the autotaxin active site. Compound 1 inhibited the production of LPA in human
and mouse plasma at nanomolar levels and showed efficacy in a mouse model of human lung
fibrosis. After successfully completing IND-enabling studies, compound 1 was approved by the
FDA for a Phase I clinical trial. These results demonstrate that DECL hits can be readily
optimized into clinical candidates.
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INTRODUCTION
Autotaxin (ENPP2, Ectonucleotide pyrophosphatase/phosphodiesterase family member
2) is a secreted glycoprotein that hydrolyzes lysophosphatidylcholine (LPC) with various fatty
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acyl chains such as linoleic acid (18:2) to lysophosphatidic acid (LPA) and choline . LPA acts
as a chemoattractant for a wide variety of cell types including fibroblasts via activation of the
2
LPA receptor . Autotaxin is likely the major source of blood-borne LPA since a heterozygous
1
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autotaxin gene deletion in mice reduces LPA blood levels by half . In humans, autotaxin protein
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levels in serum are highly correlated with LPA levels . Genetic studies initially indicated that
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autotaxin may play a role in mouse models of lung fibrosis and a study of idiopathic pulmonary
fibrosis (IPF) patients found increased levels of LPA in bronchoalveolar lavage (BAL) fluid and
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exhaled breath condensate . High levels of autotaxin have also been observed in the lungs of IPF
5
patients . The strongest validation for the link between autotaxin activity and IPF is from a
recently completed Phase II clinical trial of the autotaxin inhibitor GLPG1690 at 600 mg per day
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over a 12-week period in a small cohort of IPF patients . Lung function, as measured by forced
vital capacity (FVC), declined in the placebo group over the course of the study while FVC
values remained stable or improved slightly in GLPG1690 treated patients. Imaging studies
performed on GLPG1690 treated patients also demonstrated significant improvement in airway
volume. An association between reduced disease severity and autotaxin inhibition was
demonstrated by a decrease in plasma LPA 18:2 levels of nearly 90% at week 12 in GLPG1690
8
treated patients. Since approved treatments for IPF only slow the course of the disease , the
observation that an autotaxin inhibitor can stabilize and, in some cases, improve lung function
was an encouraging result for IPF patients and GLPG1690 is currently in two Phase III clinical
9
trials . The Phase II results with GLPG1690 have continued to drive interest in the prospect of
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finding new, more potent, autotaxin inhibitors that could be dosed at lower levels than
GLPG1690 and thus could potentially be combined with approved therapeutics to provide even
more benefit to IPF patients. New autotaxin inhibitors could also potentially be used to treat
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other diseases such as liver fibrosis and some cancers .
DNA-encoded chemical library (DECL) technology utilizes large combinatorial libraries
of small molecules each of which is covalently attached to a DNA sequence. This DNA
sequence encodes the chemical history of each individual library member and may be used to
elucidate its chemical structure. In combination with affinity-mediated selection, PCR
amplification, cloning and deep sequencing, DNA-encoded chemical library technology may be
used to identify rare members within numerically large libraries that bind to targets of interest.
Resynthesis off-DNA and testing in biochemical or biophysical assays can then be used to
determine which enriched library members bind to their cognate targets in a manner that
modulates target activity in the desired fashion. This technology is increasingly being applied to
the identification of novel chemical equity to initiate drug discovery projects and is the subject of
recent reviews12, 13 and references cited therein. Example DECL-initiated drug discovery
projects that have reached the clinic include those directed at Receptor Interacting Protein 1
Kinase (RIP1K)14, 15 and soluble Epoxide Hydrolase (SEH) . We used this approach to identify
a new autotaxin inhibitor that showed nearly twenty-fold superior potency when compared to
GLPG1690 in human plasma along with reduced protein-binding and hERG activity This series
16
of compounds led to the development of compound 1 (X-165) that was recently approved by the
FDA for Phase I human clinical studies17.
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RESULTS AND DISCUSSION
The DNA-encoded library from which the autotaxin inhibitor was identified was
constructed with three cycles of chemistry applying a split-and-pool synthesis strategy with
encoding using DNA oligonucleotides during each cycle (Figure 1). In the first cycle of library
synthesis (cycle A) the DNA headpiece (HP) was split into 300 wells and ligation to a unique
DNA tag occurred within each well. Each of the 300 Fmoc amino acids was subsequently
installed via acylation reaction onto the reaction handle of the HP, also within each well. After
pooling, mixing, deprotection of the N-Fmoc and purification the cycle A material was split into
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00 wells, each was ligated to one of 300 unique DNA tags prior to installing 300 Fmoc amino
acids via acylation. The resulting cycle B material was pooled, mixed and purified prior to being
split into 2,500 wells. The final diversification step (cycle C) was achieved by the installation of
2
,500 carboxylic acids via acylation after each was ligated to one of 2,500 DNA tags. Upon
completion of the final step the wells were pooled, and the resultant completed library was
purified using RP-HPLC resulting in a library of 225 million encoded members.
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Figure 1
Cycle A
Cycle C
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R2
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R1
Cycle B
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Acylation with 300 Fmoc amino acids;
encoding with 300 DNA tags
Deprotection
Acylation with 300 Fmoc amino acids;
encoding with 300 DNA tags
Deprotection
H
NH2
N
NH
Cycle A
O
R1
Cycle B
Acylation with 2500 carboxylic acids;
encoding with 2500 DNA tags
Deprotection
O
R2
H
N
NH
N
O
R1
Cycle C
O
R2
N
H
N
N
O
R1
O
Figure 1. Scheme for synthesis of the DNA-encoded chemical library of 225 million compounds.
Affinity-mediated selection for autotaxin binders was carried out by combining the DNA-
encoded chemical library and FLAG-tagged autotaxin in solution, capturing the DNA-encoded
library and protein complexes on an anti-FLAG matrix, washing, and then heat-eluting prior to
input into a second round of affinity-mediated selection with fresh protein. Encoding
oligonucleotides present in the round two eluate were then amplified, clustered and sequenced.
Sequencing was also performed for PCR-amplified samples of the unselected library and the
output of a no-target selection performed in parallel in the absence of autotaxin. Sequence data
were converted back into encoded chemical information and demographic and statistical
information were calculated for individual building block combinations. We observed a large
number of strongly enriched features in the selection output. The most evident of these
contained a phosphonate-containing amino acid building block that was enriched in a wide range
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of contexts including in the first or second cycles of chemical synthesis. These features were
designated as Families A and B (Figure 2a). The next-most strongly enriched set of highly
structurally related compounds included the chemotype that is the subject of this paper and was
designated as Family C (Figure 2a and 2b).
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Figure 2a
HO
P
HO
O
H
H
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N
DNA
NHR
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O
HO
HO
O
P
NHR
NH
HO
O
O
DNA NH
S
HO OH
P
O
DNA NH
O
HN
N
S
O
O
HN
R
HO OH
P
O
DNA NH
O
O
HN
N
R
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Figure 2b
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NH
O
O
R
Figure 2. 3D scatter plots showing individual enriched features against autotaxin. (a) Library 4
with entire selection output showing phosphonate-containing families in red and green and
family C in blue. (b), as (a), but with phosphonate-containing families removed and the vertical
scale adjusted with the four most enriched disynthons indicated. The x- and y-axes represent
individual building blocks at the indicated cycles of synthesis. The z axis represents a statistical
metric of enrichment significance (ENRv1) for each disynthon (negative log of the asymptotic
1
0
significance value).
Families A and B were both defined by the same amino acid building block, 4-
phosphonomethly-L-phenylalanine. Family A was designated as being comprised of all
compounds for which this phosphonate building block was enriched in the central cycle of
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chemistry with co-enrichment observed for a range of alpha, beta and gamma amino acids at the
initial cycle of chemistry, most of which were aromatic. Building blocks enriched in the
terminal cycle of chemistry in Family A included a wide range of mono and bicyclic aromatic
carboxylates. Family B was designated as being comprised of all compounds for which the
phosphonate building block was enriched in the initial cycle of chemistry with co-enrichment
observed for a range of mostly aromatic alpha, beta and gamma amino acids at the central cycle
of chemistry and a wide range of mono and bicyclic aromatic carboxylates at the terminal cycle.
Resynthesized compounds representative of these families were active in the high nM range and
the similarity of these two families suggests significant opportunities for truncation, but the
presence of the phosphonate and the negative charge it carries led us to decide not to attempt to
progress this series further. Instead of exploring the potential of replacing the phosphonate
group within this series we sought to further examine the selection output data to identify
coenriched building block combinations that did not contain a phosphonate or other negatively
charged groups.
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Family C is distinct from Families A and B in that it does not contain a negatively
charged functional group and is instead defined by an unusual spirocyclic amino acid for which
other closely related building blocks were not present in the library. The co-enriched building
blocks derived from the second chemistry cycle are alpha-amino acids with small hydrophobic
side-chains (Figure 2 and Table 1a), and the third by a wide range of small halogenated aromatic
carboxylates (Table 1b). These data were then used to inform the design of compounds for off-
DNA synthesis.
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Table 1a
Cycle A
Cycle B
Cycle C
O
O
H N
F
2
OH
OH
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OH
N
N
NH
O
O
F
3
4%
H N
2
O
OH
24%
O
H N
2
OH
2
2%
O
H N
2
OH
17%
O
H N
2
OH
2%
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Table 1b
Cycle A
Cycle B
Cycle C
O
O
H N
F
2
OH
OH
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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2
3
4
5
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9
0
OH
N
N
NH
O
O
F
1
.4%
F
O
OH
OH
1
.3%
O
N
1
1
1
.3%
O
OH
O
F
.3%
F
O
OH
F
.2%
Table 1. (a) Feature prevalence table showing structures of the top five building blocks at cycle
B that co-enrich with the indicated cycle A and C building block pair and define tolerated
variability within the active chemotype as indicated in the selection output data at cycle B. The
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3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
relative prevalence of each building block B, in terms of the percent representation in the total
number of co-enriched compounds, is indicated below each building block. (b) As for (a) but
showing all building blocks at cycle C that co-enrich with the indicated cycle A and B building
block pair. No tolerated variation was observed at cycle A presumably because of the relative
uniqueness of this building block.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 1. Synthesis of representative compounds from Family C off-DNAa.
O
O
O
OH
O
N
HN
N
N
(a)
(b)
(c)
N
N
N
Boc
Boc
R1
O
O
R2
HN
N
NHBoc
N
N
O
(d)
(b)
O
NH
N
N
N
N
O
H
O
O
2
3
4
. R = Me, R = F
1 2
. R = Cl, R = H
1
2
. R = R = H
1
2
a
Reagents and conditions: (a) NH Me, EDCI, HOBT, DIEA, DMF, RT; (b) HCl (gas), MeOH, RT; (c) (R)-2-((tert-
2
butoxycarbonyl)amino)-2-cyclohexylacetic acid, EDCI, HOBT, DIEA, DMF, RT; (d) substituted benzoic acid, EDCI,
HOBT, DIEA, DMF, RT.
To determine if the building block combinations enriched by affinity-mediated selection
were able to inhibit the activity of autotaxin we designed and synthesized representative
compounds from Family C without the DNA tag (Scheme 1). The DNA linkage point was
arbitrarily truncated to N-methylamide. A relatively wide range of building blocks were
observed at cycle C but many highly enriched examples contain meta-substituted benzoate
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Page 13 of 60
Journal of Medicinal Chemistry
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moieties and so we synthesized compounds 2 and 3. These two initial compounds were tested as
inhibitors in an autotaxin enzymatic assay using an artificial fluorescent substrate (FS-3) and
they both were able to potently inhibit autotaxin with IC values in the single-digit nM range
5
0
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(Table 2). Compound 4, the corresponding benzoate derivative showed a loss in potency of
greater than 100-fold in the FS-3 assay, indicating the importance of the aromatic substituents at
cycle C. The SAR at the DNA linkage was also explored in the synthesis of compound 5 which
differs from 2 by additional linkage truncation to remove the amide group and showed similar
activity in the FS-3 assay and improved ligand efficiency (LE).
Table 2
L
O
N
A
O
R2
N
N
N
R3
R1
H
O
ATX FS-
IC50
3
ATX LPC
No.
1
L
R1
R2
R3
A
Chirality
(nM)
IC (nM)
50
H
N
2
-F-5-
CF3
N
Methyl
i-Pr
Cy
Cy
Cy
Cy
C=O
CH2
CH2
CH2
CH2
R
R
R
R
R
55
86
O
2
HN
2-F-3-Me
3-Cl
2
O
3
HN
3.5
350
1.6
O
4
HN
H
5
Methyl 2-F-3-Me
56
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Journal of Medicinal Chemistry
Page 14 of 60
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
7
8
9
Methyl
Methyl
Methyl
Methyl
Methyl
3-F
i-Pr
CH2
CH2
CH2
CH2
C=O
CH2
CH2
C=O
C=O
C=O
C=O
C=O
C=O
CH2
R
R
R
S
2.2
2
3-Me
3-Me
3-Me
i-Pr
4-THP
4-THP
i-Pr
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
623
10,000
10,000
819
H
N
2
-F-5-
CF3
N
1
0
1
2
3
4
5
6
7
8
9
S
1
1
1
1
1
1
1
1
1
Methyl 2-F-3-Me
Cy
R
R
R
R
R
R
R
N
Methyl
3-Me
Cy
905
Methyl 2-F-3-Me
i-Pr
683
H
N
N
Methyl
Methyl
Methyl
Methyl
Methyl
Methyl
3-CF3
i-Pr
340
H
N
2
-Me-5-
CF3
N
N
i-Pr
4900
1470
34
H
N
2
-Cl-5-
CF3
i-Pr
H
N
2
-F-5-
CF3
N
N
Cyclopentyl
H
H
N
2
-F-5-
CF3
10,000
13.4
H
N
2
2
-F-5-
CF3
N
i-Pr
R
R
-F-5-
CF3
2
0
Methyl
i-Pr
C=O
100
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Page 15 of 60
Journal of Medicinal Chemistry
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2
2
2
-F-5-
CF3
2
2
2
1
2
3
Methyl
Methyl
Methyl
i-Pr
i-Pr
i-Pr
COOH
C=O
C=O
C=O
R
R
R
31
38
46
-F-5-
CF3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
-F-5-
CF3
Cl
Table 2. SAR table for compounds derived from Family C.
The composition of enriched cycle B building blocks indicates a preference for
hydrophobic side chains at this position (Table 1). The design of compounds with an isopropyl
group was intended to improve the overall logP and LE while maintaining the hydrophobic
nature at the position. Replacing the cyclohexyl group with isopropyl, e.g. compounds 6 and 7,
maintained activity in the FS-3 assay (Table 2). This promising result allowed us to further
optimize other regions of the hit compounds and resulted in potent (nM) binders with improved
physicochemical properties.
As we continued to explore the structure-activity relationships of this series, we
developed an assay using the natural autotaxin substrate LPC rather than the artificial substrate
FS-3. There was a notable shift in the observed IC values of compounds between the FS-3 and
5
0
LPC assays as demonstrated by initial library derived compounds 2 and 5.
It was also observed from the prevalence of enriched cycle B building blocks that the R
configuration of chiral amino acids was preferred when both R and S-isomers were present in the
library. Stereospecific binding was confirmed when LPC IC values were compared for 8 and
5
0
9, and 1 and 10 as matched pairs. In both cases, compounds with R-configuration were
substantially more active than their S-isomers.
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Journal of Medicinal Chemistry
Page 16 of 60
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Family C was defined by a single building block at cycle A for which closely related
variants were not included in the library. The spiro ring structure provides rigidity that locks the
hydrophobic N-phenyl in a conformation that projects it towards the hydrophobic pocket of
autotaxin. We observed over 10-fold loss of potency in the LPC assay relative to 5 when N-
phenyl was replaced by N-cyclopropyl in 11 or N-pyridyl in 12 (Table 2). Optimization at this
position focused on the design of additional spiro moieties (13) which led to the discovery of the
hydantoin series (examples in table 2, 14-18, 20-23), and resulted in compound 1 (X-165) with
potency and ADME properties suitable for testing in animal models.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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Page 17 of 60
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9
Scheme 2. Synthesis of compound 1 and its analogsa
O
F
O
F
CF3
CF3
OH
a, b
OH
N
H
O
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1a
NC H
N
NC H
O
N
c
d
N
N
N
N
N
N
N
Boc
Boc
Boc
H
1b
1c
Cbz
O
O
O
NH
N
N
O
O
O
N
N
N
HN
N
N
Boc
Boc
e, f
h
g
N N
N N
N
N
Cbz
Cbz
Cbz
O
1d
1e
1f
O
N
N
O
O
O
F
O
F
N
N
CF3
N
CF3
N
N
H
j
N
i
H
O
O
N N
HN N
1g
Cbz
1
a
Reagents and conditions: (a) methyl D-valinate.HCl, HATU, DIEA, CH Cl ; (b) LiOH, THF, H O; (c)
2
2
2
1H-indazol-5-amine, TMSCN, AcOH; (d) K CO , CbzCl, THF/H O; (e) NCOSO Cl, CH Cl , HCl/H O;
2
3
2
2
2
2
2
(
f) (Boc) O, K CO , THF; (g) MeI, K CO , DMF; (h) HCl/Dioxane; (i) 1a, HATU, DIEA, CH Cl ; (j)
2 2 3 2 3 2 2
Pd/C, H , MeOH
2
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Journal of Medicinal Chemistry
Page 18 of 60
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6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Scheme 3. Synthesis of N-substituted 1,3,8-triazaspiro[4,5]decan-4-onea
O
O
O
NH
N
N
b
a
N
N
N
N
N
HN
HCl
Boc
O
Boc
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5a
5b
O
O
O
NH2
NH
N
N
NH
N
c
d
a, e
N
N
BocN
BocN
BocN
HN
N
11a
11b
11c
O
O
O
NH
N
NH2
c
N
d
N
H
f
BocN
BocN
BocN
12a
12b
O
O
N
N
N
e
N
BocN
HN
N
N
1
2c
aReagents and conditions: (a) MeI, NaH, THF; (b) HCl (g), MeOH; (c) trimethoxymethane,
AcOH; (d) NaBH4, MeOH; THF; (e) HCl/Dioxane; (f) 2-bromopyridine, Pd (dba) , X-Phos, t-
12d
2
3
BuOK, Toluene
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Page 19 of 60
Journal of Medicinal Chemistry
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7
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9
Scheme 4. Synthesis of N-1,3,8-triazaspiro[4.5]decane-2,4-dionea
NC H
O
O
N
a
b, c
NH
R
N
N
O
Boc
N
N
Boc
Boc
13a R = H
13b R = H
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2
2
1a R = p-CO Me
2a R = p-Me
3a R = m-Cl
R
21b R = p-CO2Me
22b R = p-Me
23b R = m-Cl
2
O
O
N
N
g
d
O
N
O
N
HN
N
Boc
R
R
13d R = H
2
22d R = p-Me
23d R = m-Cl
1e R = p-CO Bn
2
2
1c R = p-CO Me
2
e, f
21d R = p-CO Bn
2
a
Reagents and conditions: (a) substituted aniline, TMSCN, AcOH; (b) NCOSO Cl, CH Cl , HCl/H O;
2
2
2
2
(
c) (Boc) O, K CO , THF; (d) MeI, K CO , DMF; (e) NaOH, MeOH; (f) BnBr, K CO , DMF; (g)
2 2 3 2 3 2 3
HCl/Dioxane
The synthesis of the compounds described above employed two general strategies. The
first resembles the DECL library synthetic route wherein the corresponding amino acids and
carboxylic acids are coupled in a linear fashion (Scheme 1, compounds 2-4). The alternative
approach (Scheme 2) couples the spiropiperidine intermediate to a pre-assembled fragment. The
intermediate 1d was prepared using a hydantoin formation reaction where the cyano intermediate
1c reacted with chlorosulfonyl isocyanate (CSI). Upon methylation of the hydantoin NH,
followed by N-Boc deprotection of the piperidine, the resulting compound 1f was subjected to a
standard amide coupling reaction with the advanced starting material 1a using HATU as
coupling reagent to give 1g. Removal of the N-Cbz protecting group afforded the title
compound 1 in a cumulative yield of 18% (over 4 steps). Compounds 5-23 were prepared
analogously, and their respective intermediates were synthesized via various pathways (Scheme
3
and 4).
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Journal of Medicinal Chemistry
Page 20 of 60
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2
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6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Figure 3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Co-crystal structure of compound 1 in the active site of autotaxin (PDB ID 6W35). The
ligand is shown in green; the binding site residues are depicted in gold; water molecules are
represented as red spheres. Yellow dashes indicate hydrogen bonding networks.
Compound 1 was co-crystallized with human autotaxin (Figure 3). The structure of the
complex was obtained at 1.98Å resolution. The crystals contained one monomer of autotaxin in
the asymmetric unit. The structure revealed the unique binding mode of 1. In contrast to
previously reported structures18, 19 that engage one of the catalytic zinc ions, compound 1 extends
across the binding site to occupy both the hydrophobic pocket and the hydrophobic channel of
autotaxin. The hydrophobic pocket normally binds LPC and the hydrophobic channel is
2
0
proposed to be involved in transport of LPA to target receptors . A similar mode of inhibition
2
1
was previously reported for clinical candidate GLPG1690 but a comparison of the binding
modes revealed different trajectories linking the binding elements in the hydrophobic pocket and
hydrophobic channel.
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Page 21 of 60
Journal of Medicinal Chemistry
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9
The 2-fluoro-5-trifluoromethylphenyl group occupies the hydrophobic pocket and forms
a T-shaped pi-pi interaction with the aromatic side chain of Phe274 and hydrophobic contacts
with surrounding side chains of Leu217, Ile168, Ala305, Leu214 and Phe274. The 2-fluoro is in
a favorable close to co-planar conformation with the amide hydrogen. Removal of the fluoro
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(14) resulted in a 10-fold loss of potency, while substitution with a bulkier non-polar methyl (15)
or chloro (16) pushed the amide out of plane and reduced potency into the micromolar range.
Moreover, replacement of the amide hydrogen with methyl to form the N-Me derivative resulted
in a significant reduction of potency to >10 µM (compound not shown).
The isopropyl group fills the hydrophobic pocket formed by Phe211, Phe275, Leu214
and Trp255. This pocket is large enough to accommodate a substantial hydrophobic substituent,
such as a cyclopentyl (17). Removing the substituent entirely (18) resulted in loss of potency.
Piperidinyl carbonyl forms a water-mediated hydrogen bond to the backbone amine of
Phe274. The hydantoin group is positioned in the hydrophobic channel. The carbonyl at the 4
position is involved in water mediated hydrogen bonds to Trp261 and Tyr215, the carbonyl at the
2
-position hydrogen bonds to Tyr83 and is involved in a water-mediated hydrogen bonding
network with the backbone carbonyl of Phe250. The bifurcated hydrogen bond to Tyr83
potentially weakens the hydrogen bonding potential of the hydroxyl and removal of the oxygen
at the 2-position (compound 19) yielded a more potent compound.
Finally, the indazole group in the hydrophobic channel points toward the solvent, forming
an edge-to-face pi-pi interaction with Phe250 and a hydrogen bond to Ser82. Removal of this
indazole (20) accounts for loss in potency due to loss of the hydrogen bond to Ser82 and the pi-pi
interaction with Phe250. Since the hydroxyl group of Ser82 can function as a hydrogen bond
donor, a carboxylic acid is tolerated at the para position of the phenyl ring without loss of
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Page 22 of 60
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
potency (21). Hydrophobic substitutions at the para and meta positions are also tolerated (22 and
2
3).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 3
Assay
Compound 1 potency (nM)
LPC assay (Ki)
12
7
Mouse Plasma (IC LPA 18:2)
5
0,
Human Plasma (IC LPA 18:2)
14
5
0,
Table 3. Compound 1 potency in the autotaxin LPC enzymatic assay, mouse and human plasma
LC-MS/MS based autotaxin assays.
Table 4
Parameter
Compound 1
hERG (IC , µM)
>50 (MPC, GLP)
5
0
CYP3A4, CYP2D6, CYP1A2 (IC , µM)
>50
10.4
5
0
CYP2C9 (IC , µM)
5
0
CYP2C19 (IC , µM)
46
5
0
ENPP1 (IC , µM)
>10
5
0
LPAR1-3 antagonist and agonist (IC , µM)
>10
5
0
Rat/Human Liver Microsomes T (min)
277 / 533
452
1/2
Thermodynamic solubility at pH 7.4 (µM)
Table 4. Compound 1 selectivity, hERG, CYP activity, solubility and microsomal stability.
MPC: Manual patch clamp assay, GLP: Good laboratory practices
ACS Paragon Plus Environment
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Page 23 of 60
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Compound 1 demonstrated low nanomolar potency in both mouse and human plasma
autotaxin assays, with 60- and nearly 20-fold lower IC values than reported for GLPG1690 in
5
0
2
1
mouse and human plasma respectively (Table 3). Side-by-side plasma LPA inhibition assays
with compound 1 and GLPG1690 in our hands confirmed this potency difference (data not
shown). Compound 1 also demonstrated excellent selectivity against related targets showing no
inhibition of the homologous enzyme ENPP1, which is 45% identical to autotaxin (ENPP2), and
no activity against LPA receptors, which may have been expected if the compound was purely a
substrate mimetic (Table 4). Compound 1 was highly soluble in water and had a low rate of
clearance in rat and human microsomes. Furthermore, compound 1 showed no inhibition of
hERG or cytochromes at concentrations 1000-fold over its inhibitory concentration of LPA
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
21
production in plasma (Table 4) as compared to a 60-fold window for GLPG1690 . Compound 1
2
1
also demonstrated lower protein binding (r/hPPB 72.7/88.7%) than GLPG1690 (>99%) which
may explain the better potency of compound 1 in plasma. Pharmacokinetic experiments in mice
showed that compound 1 demonstrated 80% oral bioavailability and a plasma half-life of 2.5
hours. An oral dose of 30 mg/kg maintained plasma concentrations above the IC value for
9
0
LPA (60 ng/ml, ~100 nM) for 12 hours in mice (Figure 4). Compound 1 was also orally
bioavailable in rats and dogs (Table 5), the designated species for toxicology studies, when dosed
in a 0.5% methylcellulose suspension formulation.
ACS Paragon Plus Environment
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Journal of Medicinal Chemistry
Page 24 of 60
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Figure 4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Compound 1 plasma levels exceed IC for up to 12h when dosed orally in mice. A
9
0
total of 9 mice were dosed orally with compound 1 at 30 mg/kg and plasma concentrations were
determined up to 12h using 3 mice per time point (green). The IC of compound 1 in mouse
9
0
plasma is indicated (blue).
ACS Paragon Plus Environment
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Page 25 of 60
Journal of Medicinal Chemistry
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3
4
5
6
7
8
9
Table 5. Compound 1 Mouse, Rat and Dog PK
Compound 1 PK
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CL (mL/min/kg)
T (h)
54
(iv)
0.57
1.38
744
2587
2.5
1
/2
1
mg/kg
Vss (L/kg)
Mouse
Cmax (ng/mL)
AUClast (h*ng/mL)
T (h)
(
po)
10 mg/kg
1
/2
F%
81
CL (mL/min/kg)
T (h)
36
(iv)
1.5
1
/2
1
mg/kg
Vss (L/kg)
1.98
160
518
1.8
Rat
Cmax (ng/mL)
AUClast (h*ng/mL)
T (h)
(
po)
5 mg/kg
1
/2
F%
24
Dog
(iv)
CL (mL/min/kg)
7.96
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Journal of Medicinal Chemistry
Page 26 of 60
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
mg/kg
T (h)
4.1
1/2
Vss (L/kg)
0.655
374
3081
4.2
Cmax (ng/mL)
AUClast (h*ng/mL)
T (h)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(po)
1
mg/kg
1
/2
F%
100
Figure 5
1
25
100
7
5
2
5
0
5
0
-
9
-8
-7
-6
Compound 1 plasma conc (Log[M]) @ 4h post-dose
Figure 5. Compound 1 inhibits LPA production in vivo. A range of compound 1 doses were
orally administered to rats and both LPA 20:4 and compound 1 levels were measured by LC-
MS/MS in rat blood 4 hours post dosing. Average percent inhibition of LPA levels was then
plotted against plasma levels of compound 1.
ACS Paragon Plus Environment
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Page 27 of 60
Journal of Medicinal Chemistry
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2
3
4
5
6
7
8
9
A study of compound 1 inhibition of plasma LPA production relative to the plasma levels
of the compound was performed in rats (Figure 5). Rats were dosed orally with compound 1 and
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
22
both LPA 20:4 levels, since LPA 20:4 is more abundant in rats than in humans or mice, and
compound 1 levels were measured in plasma at 4 hours post-dose. A dose-dependent
relationship between the plasma compound levels and the percent inhibition of LPA production
was observed with an IC value of 6.5 nM, which is very close to the IC values calculated
5
0
50
from ex vivo experiments using mouse and human plasma (Table 3). The demonstration of a
dose-dependent effect on LPA levels in vivo and the near identity of the in vivo and in vitro
plasma and enzyme assay IC results indicated that the reduction of LPA levels by compound 1
5
0
was consistent with this effect being due to on-target activity.
The development of lung fibrosis in the mouse following bleomycin-induced injury has
been used to demonstrate the anti-fibrotic activity of multiple compounds including those that
23
are currently approved for treating human idiopathic pulmonary fibrosis . The combination of
potency, selectivity and pharmacokinetic properties of compound 1 made it an excellent
candidate for testing its potential anti-fibrotic activity in this model. Compound 1 was tested in
both prophylactic and therapeutic modes at 30, 100 and 300 mg/kg BID (Figure 6). In the
prophylactic mode, compound 1 dosing began one day prior to bleomycin treatment and in the
therapeutic mode compound 1 dosing began seven days after bleomycin treatment. After 21
days post-bleomycin treatment, the mice were sacrificed and the amount of fibrosis in the lung
was quantitated in one group of mice by measuring hydroxyproline levels and in another group
of mice by trichrome staining of lung tissue. GLPG1690, at doses of 30 mg/kg and 100 mg/kg
BID, was used as a control in both models.
ACS Paragon Plus Environment
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Journal of Medicinal Chemistry
Page 28 of 60
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Figure 6a
0
.0625
4
3
2
1
000
000
000
000
0
*
**
*
**
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
e
e
D
I
D
D
I
D
D
I
l
l
I
I
c
c
i
i
B
B
B
B
B
h
h
k
k
k
k
k
e
e
p
p
p
p
p
V
V
m
m
m
m
m
+
0
0
0
0
0
o
3
0
0
3
0
e
1
3
1
l
1
G
B
1
1
+
P
G
+
+
L
P
o
L
e
o
o
G
l
e
l
e
l
G
B
+
B
B
o
+
e
o
l
e
l
B
B
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Page 29 of 60
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Figure 6b
5
4
000
000
*
**
**
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3000
2
1
000
000
0
e
e
D
D
D
I
D
D
I
l
l
I
I
I
c
c
i
i
B
B
B
+
B
B
h
h
k
k
k
k
k
e
e
p
p
p
p
p
V
V
m
m
m
m
m
+
0
0
0
0
0
o
3
0
0
3
0
e
1
3
1
l
1
G
B
1
1
+
P
G
+
+
L
P
L
o
e
o
o
G
l
e
e
l
G
B
l
B
B
o
+
e
o
l
e
l
B
B
Figure 6. Oral dosing of compound 1 demonstrates efficacy in prophylactic (a) and therapeutic
b) models of bleomycin-induced lung fibrosis. Lung hydroxyproline levels are plotted for each
mouse. Median levels and p-values relative to bleomycin + vehicle are indicated for each group
(
(* ≤ 0.05, ** ≤ 0.01). GLPG1690 (GLPG) was used as a control.
The results of the prophylactic study show a clear decrease in hydroxyproline levels at
the lowest dose of compound 1, 30 mg/kg. A statistically significant decrease in hydroxyproline
was observed at both 100 and 300 mg/kg. The GLPG1690 controls reduced fibrosis as expected
and compound 1 was active in the same range of doses as GLPG1690 in this model. Similar
results were also observed in the therapeutic model of fibrosis. Treatment with compound 1
seven days after the initial bleomycin induced injury showed a statistically significant reduction
ACS Paragon Plus Environment
28