Discovery of BI-2545: A Novel Autotaxin Inhibitor That Significantly Reduces LPA Levels in Vivo

Article abstract of DOI:10.1021/acsmedchemlett.7b00312

In an effort to find new therapeutic interventions addressing the unmet medical need of patients with idiopathic pulmonary fibrosis, we initiated a program to identify new autotaxin (ATX) inhibitors. Starting from a recently published compound (PF-8380), we identified several highly potent ATX inhibitors with improved pharmacokinetic and safety profiles. Further optimization efforts resulted in the identification of a single-digit nanomolar lead compound (BI-2545) that shows substantial lowering of LPA in vivo and is therefore considered a valuable tool for further studies.

Full text of DOI:10.1021/acsmedchemlett.7b00312

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Letter
Discovery of BI-2545: A Novel Autotaxin Inhibitor
that Significantly Reduces LPA Levels in vivo
Christian Andreas Kuttruff, Marco Ferrara, Tom Bretschneider, Stefan Hoerer,
Sandra Handschuh, Bernd Nosse, Helmut Romig, Paul Nicklin, and Gerald J Roth
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.7b00312 • Publication Date (Web): 08 Nov 2017
Downloaded from http://pubs.acs.org on November 8, 2017
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Discovery of BI-2545: A Novel Autotaxin Inhibitor that Significantly
Reduces LPA Levels in vivo.
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Christian A. Kuttruff*,a, Marco Ferrarab, Tom Bretschneiderc, Stefan Hoerera, Sandra Handschuha,
Bernd Nossea, Helmut Romigc, Paul Nicklind, Gerald J. Rotha.
aBoehringer Ingelheim Pharma GmbH & Co. KG, Medicinal Chemistry, 88397 Biberach an der Riss, Germany
bprevious affiliation: Boehringer Ingelheim Research Italia S.a.s. di BI IT S.r.l., Via G. Lorenzini 8, 20139 Milano, Italy;
current affiliation: Promidis S.r.L., San Raffaele Hospital, Via Olgettina 60, 20132 Milano, Italy
cBoehringer Ingelheim Pharma GmbH & Co. KG, Drug Discovery Sciences, 88397 Biberach an der Riss, Germany
dBoehringer Ingelheim Pharma GmbH & Co. KG, Immunology & Respiratory, 88397 Biberach an der Riss, Germany
KEYWORDS: Autotaxin, IPF, LPA, tool compound
ABSTRACT: In an effort to find new therapeutic interventions addressing the unmet medical need of patients with idiopathic
pulmonary fibrosis (IPF), we initiated a program to identify new Autotaxin (ATX) inhibitors. Starting from a recently published
compound (PFꢀ8380), we identified several highly potent ATX inhibitors with improved pharmacokinetic and safety profiles. Furꢀ
ther optimization efforts resulted in the identification of a singleꢀdigit nanomolar lead compound (BIꢀ2545) that shows substantial
lowering of LPA in vivo and is therefore considered a valuable tool for further studies.
Autotaxin is a therapeutic target for the treatment of fibrotic
conditions such as idiopathic pulmonary disease (IPF). IPF is a
rare and chronic lung disease with unknown cause affecting
approximately three million people worldwide. The disease is
characterized by a progressive scarring of lung tissue which
leads to worsening of dyspnea and lung function and is ultiꢀ
mately fatal within 3ꢀ5 years from the onset of symptoms.1 IPF
is one of the most common and severe forms of idiopathic
interstitial pneumonia (IIP) and shows a characteristic pattern
known as usual interstitial pneumonia (UIP), the latter being a
lung disease characterized by progressive fibrosis of both
lungs. Patients that suffer from IPF are usually over 50 years
old and show symptoms ranging from dry, nonꢀproductive
cough over progressive exertional dyspnea to clubbing of the
finger tips and toes. For many years, there were no treatment
options for IPF until 2014, when the FDA approved nintedanib
(Ofev)2ꢀ5 and pirfenidone (Esbriet)6 for the treatment of IPF.
Both drugs not only help to slow down the decline of the lung
function but also reduce acute IPF exacerbations. In spite of
this advancement in the treatment of IPF, there is still a high
medical need for new and efficient IPF treatment options.
In recent years, it has been shown that patients with IPF
have elevated levels of lysophosphatidic acid (LPA) in their
bronchoalveolar lavage fluid (BALF) and exhaled breath
condensate.7, 8 LPAs are bioactive lysophospholipids that exert
a variety of cellular responses including cell proliferation, cell
motility and cell survival through their interaction with six Gꢀ
drug target for the treatment of IPF. In recent years, several
LPAR1 antagonists have been reported11ꢀ13 and some of these
compounds are currently being evaluated in clinical trials for
the treatment of IPF. Nevertheless, the pathophysiology of
fibrotic disorders is complex and is likely to involve multiple
cell types. Indeed, there is evidence implicating multiple cell
types and LPARs 17, 14, 215, 316 and 617 in fibrosis. Consequentꢀ
ly, there is high interest in therapeutic approaches with the
potential to inhibit the overꢀactivation of multiple LPARs
caused by elevated LPAꢀlevels in disease. This may be
achievable by controlling the rate of formation of LPA speꢀ
cies, thereby inhibiting the LPAR1ꢀn drivers of pathology.
Therefore, inhibition of the enzyme Autotaxin (ATX or
ENPP2), which is responsible for the formation of LPA from
lysophosphatidylchloline (LPC) is an attractive therapeutic
concept.18 Autotaxin is an extracellular enzyme which was
first isolated as a motilityꢀstimulating protein from A2058
melanoma cells19 and that belongs to the family of ectonucleoꢀ
tide pyrophosphatase/phosphodiesterase (ENPP) proteins. The
intense interest in discovering compounds capable of inhibitꢀ
ing ATXꢀLPA signaling is reflected in the substantial increase
in publications and filed patents describing small molecule
ATX inhibitors from numerous pharma companies in recent
20ꢀ27
years.18,
The only ATX inhibitor which is currently in
clinical development for the treatment of IPF is GLPG1690
(1) (Figure 1A).28, 29
N
A)
B)
N
O
N
S
N
H
N
10
proteinꢀcoupled receptors (GPCRs) known as LPAR1ꢀ6.9,
Cl
N
O
N
N
O
N
O
N
LPAR1 has been identified to be the predominant LPA recepꢀ
tor in lung fibroblasts of IPF patients that is responsible for
enhanced fibroblast cell migration and vascular leak.7 It is
therefore envisaged that LPAR1 antagonists will be a potential
O
Cl
F
HO
N
O
GLPG-1690 (1)
PF-8380 (2)
Figure 1. A) Structure of Autotaxin inhibitor GLPGꢀ1690. B)
Structure of Autotaxin inhibitor PFꢀ8380.
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Our ongoing efforts to find new therapeutic treatments for
assay using the natural substrate 18:1 LPC.31 In addition to
1
2
3
4
5
6
7
8
IPF motivated us to identify smallꢀmolecule ATX inhibitors.
In addition to running a traditional highꢀthroughput screen of
our compound collection, we also utilized a tool compound
published by Pfizer, PFꢀ838030 (2) (Figure 1B), as a starting
point for the design of new ATX inhibitors.
that, we determined an IC50 of 307 nM for PFꢀ8380 in rat
whole blood (rWB). However, additional profiling of PFꢀ8380
revealed liabilities such as low metabolic stability in human
liver microsomes (70% of hepatic blood flow) and a rather
prominent inhibition of the hERG channel with an IC50 of 480
nM (for details, see Supporting Information). Based on these
undesirable properties, our goal was to develop a compound
with comparable or improved potency but at the same time a
clean hERG profile and ultimately improved pharmacokinetic
properties.
We tested the hypothesis that removing a basic center in 2
could dial out the offꢀtarget activity. Therefore the basic piperꢀ
azine nitrogen of 2 (marked in magenta in Figure 4) was reꢀ
moved and in addition the core region of the molecule was
rigidified by the incorporation of a [3.1.0]ꢀbicycle. The resultꢀ
ing compound 3 displayed an IC50 of 3 nM in the LPA enꢀ
zyme assay and a fourꢀfold higher potency (IC50: 56 nM) in rat
whole blood. In line with our hypothesis, it had a clean hERG
profile (no inhibitory effect up to a concentration of 10 µM)
and also had increased metabolic stability (41% of hepatic
blood flow in human liver microsomes) compared to PFꢀ8380.
In order to support our optimization activities, we first evalꢀ
uated the binding mode of 2 by solving the crystal structure in
complex with ATX (detailed information about synthesis of 2,
crystallization and structure solution can be found in the Supꢀ
porting information). According to the crystal structure that
we obtained (see Figures 2 and 3), the benzoxazolidinone of 2
(pka = 7.6) occupies the Zn binding pocket and coordinates to
the Zn2+ ion in the proximity to Asp311 and His474. The oxygen
of the carbonylꢀgroup coordinates a water molecule which is
involved in a strong hydrogen bonding network. Through the
linker region, PFꢀ8380 elongates in the direction of the hydroꢀ
9
10
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17
18
19
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phobic pocket (Leu213, Leu216, Ala217, Ile167, Ser169, Trp260
,
Phe273), where finally the dichlorobenzene is situated. There
are few direct interactions of the linker region between the
benzoxazolidinone and the dichlorobenzene. The piperazineꢀ1ꢀ
carboxylate is partially sandwiched between Tyr306 and Phe274
.
The carbonyl group of the carbamate forms a weak hydrogen
bond with the backbone nitrogen of Trp275. There is no ionic
interaction of the positively charged nitrogen. Phe274 flanks the
opening of a hydrophobic channel (bottom Tyr214, Trp260). In
addition, within the crystal ten atoms of polyethyleneꢀglycol
from the crystallization buffer are visible in the hydrophobic
channel.
Linker region
Replacement
of core
O
H
O
O
H
N
Cl
Cl
containing
basic amine
O
N
O
N
N
O
O
H
O
N
N
H
O
H
3
Cl
Hydrophobic pocket residue
Cl
O
Zn-binding residue
PF-8380 (2)
Figure 4. Replacement of the basic piperazine core in PFꢀ8380 as
a strategy to dial out hERG activity and topology of 3 for strucꢀ
tureꢀactivity relationship studies.
With 3 as a potent starting point in hand, we began to exꢀ
plore SAR of the different regions of the inhibitor (see Figure
4). We started off with introducing additional modifications in
the linker region of the molecule to see if the carbamate preꢀ
sent in PFꢀ8380 was the optimal linker between the [3.1.0]ꢀ
bicyclic core and the residue binding to the hydrophobic pockꢀ
et. A variety of linker groups was evaluated at this position
and the results are summarized in Table 1. Replacing the carꢀ
bamate present in 3 with a cinnamic acidꢀderived amide (4)
did not affect the potency in our enzymatic assay but led to a
dramatic drop in rat whole blood potency, accompanied by
increased hERG inhibition. The corresponding saturated anaꢀ
log of 4, compound 5, suffered from very poor stability in
liver microsomes which precluded further measurement in rat
whole blood. In order to further rigidify the backbone of our
compounds, we also introduced a cyclopropylꢀlinker. For the
resulting compound 6, despite having desirable potency in our
primary assay, we observed a dramatic drop in rat whole blood
potency in addition to unfavorable hERG inhibition. Changing
the carbamate to an urea linker resulted in a compound (7) that
not only exhibited decreased potency in the enzymatic assay
but also low permeability and high efflux. Further modificaꢀ
tions of the linker region such as increased steric bulk in the
alphaꢀposition of the carbamate (see methylꢀderivative 8) or
cyclization to a bicyclic linker as present in 9 led to 30ꢀ40 fold
loss in ATX inhibitory potency. Based on these results, we
decided to keep the carbamate linker present in 3.
Figure 2. Cocrystal structure of PFꢀ8380 (2) bound to Autotaxin.
Figure 3. Cocrystal structure of PFꢀ8380 (2) bound to Autotaxin.
Carbon atoms of 2 are shown in dark green, Znꢀions and water
molecules are depicted as brown and red spheres, respectively.
Figures showing structural data have been prepared with PyMOL.
The reported in vitro potency (IC50) of PFꢀ8380 (2) is 3 nM
in an enzyme assay, using recombinant ATX and the artificial
substrate surrogate FSꢀ3.30 We confirmed this potency of PFꢀ
8380 in our recently developed massꢀspectrometryꢀbased ATX
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Table 1. Structure-Activity Relationship for linker region.
1
O
H
O
2
O
N
R
N
O
H
3
N
H
H
4
5
6
7
8
9
Cacoꢀ2
aꢀb 10ꢀ6 [cm/s]/
Efflux
Solubility
[µg/ml] at pH
2.2/4.5/6.8
Metabolic stabilꢀ
ity hLM/rLM
[%Qh]
hATX LPA
IC50 (nM)a
Rat WB IC50
(nM)a
hERG IC50
(µM)
Compound
R
clogP
3.8
Cl
3
O
3.1±2.3
(3)
56±22
(2)
>10
<1/<1/<1
41/64
137/1
Cl
Cl
Cl
Cl
4
5
6
7
8
9
1.7±1.0
(3)
1904±698
(2)
2.7
1.9
1.9
2.3
4.2
3.1
4.2
8.1
4.7
>10
8.5
n.d.
<1/<1/<1
<1/<1/<1
<1/<1/<1
<1/<1/<1
<1/<1/<1
<1/<1/<1
25/<22
94/88
ꢀ/<22
36/35
57/70
ꢀ/53
38/4
18/7
64/2
2/38
118/1
47/3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
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56
57
58
59
60
2.3±0.9
(3)
7±4.4
(2)
Cl
Cl
4.8±2.0
(3)
2399±185
(2)
Cl
Cl
N
13.9±7.0
(3)
77±30
(2)
H
66.9±24.3
(3)
208±79
(2)
Cl
O
Cl
N
82.6±6.4
(3)
n.d.
Cl
aValues are reported as arithmetic means with standard deviations. Number of replicates in parentheses.
Table 2. Structure-Activity Relationship for hydrophobic pocket residues.
O
H
O
R
O
N
N
O
O
H
N
H
H
Solubility
[µg/ml] at pH
2.2/4.5/6.8
Metabolic stabilꢀ
ity hLM/rLM
[%Qh]
Cacoꢀ2
aꢀb 10ꢀ6 [cm/s]/
Efflux
hATX LPA
IC50 (nM)a
Rat WB IC50
(nM)a
hERG IC50
(µM)
Compound
R
clogP
F
F
F
2.3±0.2
(3)
4.5±6.0
(2)
10
11
4.2
3.2
5.2
<1/<1/<1
<1/<1/<1
<23/47
48/56
108/1
44/3
F
F
F
O
F
1.9±1.3
(3)
94±n.d.
(1)
F
F
>10
N
49.0±1.4
6.9±3.2
12
13
14
2.2
1.4
0.7
>10
>10
>10
48/36/25
>92/66/56
>86/>93/63
98/97
25/<22
<23/<22
6/21
1/68
2/30
N
(3)
1530±n.d.
(2)
1607±880
N
(1)
1674±1800
(2)
n.d.
N
O
(3)
aValues are reported as arithmetic means with standard deviations. Number of replicates in parentheses.
Table 3. Structure-Activity Relationship for various Zn-binding residues.
H
O
O
F
F
R
N
O
N
H
F
H
N
Solubility
[µg/ml] at pH
2.2/4.5/6.8
Metabolic stabilꢀ
ity hLM/rLM
[%Qh]
Cacoꢀ2
aꢀb 10ꢀ6 [cm/s]/
hATX LPA
IC50 (nM)a
Rat WB IC50
(nM)a
hERG IC50
(µM)
Compound
R
clogP
Efflux
O
1.5±0.1
18±5.4
N
15
16
17
18
N
N
3.9
2.3
2.2
2.9
>10
>10
>10
>10
<1/<1/<1
32/26/5
29/28
44/43
8/17
7/16
3/12
0/17
N
H
(3)
1.3±0.3
(2)
33±15
O
N
N
N
H
(3)
5.5±1.4
(2)
65±10
N
O
N
75/70/66
28/47
N
H
(3)
10±1.5
(2)
6±n.d.
O
O
N
N
<1/<1/>113
<23/<22
(3)
(1)
HO
aValues are reported as arithmetic means with standard deviations. Number of replicates in parentheses.
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Subsequently, we explored the hydrophobic pocket SAR of reducing LPAs. Furthermore, we achieved a higher in vivo
1
2
3
4
5
6
7
8
ATX (see Table 2). Interestingly, within a clogP range of 2.5ꢀ
4, a fairly broad variety of substituted benzylic residues was
tolerated and led to moderately to highly potent ATX inhibiꢀ
tors (more than 30 analogs were prepared with potencies in
IC50 range between 1ꢀ100 nM in enzymatic assay, not shown).
The most lipophilic representative of the selected compounds
shown in Table 2, ATX inhibitor 10 displayed excellent poꢀ
tency in our rat whole blood assay, favorable metabolic stabilꢀ
ity in human liver microsomes, excellent permeability but also
inhibited the hERG channel with an IC50 of 5.2 µM. Whilst
less potent in our rat whole blood assay, compound 11 was
devoid of hERG inhibition and showed a moderate overall
profile. When more polar residues were introduced, solubility
could be improved but potency could not be maintained, as
illustrated with selected examples 12ꢀ14. Based on the data,
we decided to keep the 3ꢀ(trifluoromethoxy)benzonitrile resiꢀ
due for subsequent SAR studies of the Znꢀbinding motif.
potency compared to PFꢀ8380 (see Table 4 in the Supporting
Information). As the most attractive candidate from this chemꢀ
ical series, 19 was profiled against a broad panel of in vitro
pharmacology assays. At a concentration of 10 µM (~4,500ꢀ
fold the IC50 for ATX), it was significantly, but incompletely,
crossꢀreactive in only four assays: 5ꢀHT2a (55%), Lꢀtype
Calcium channel (80%), Na+ channel site 2 (66%) and Norepiꢀ
nephrine transporter (61%; see Table 5 in the Supporting
Information for the full profile). These data confirm the selecꢀ
tivity of BIꢀ2545 and, together with its in vitro and in vivo
profile, highlights its suitability as an ATX inhibitor tool comꢀ
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
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39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
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57
58
59
60
pound.
Table 4: In vitro and in vivo profile of BI-2545 (19).
F
O
H
H
O
F
F
N
O
N
N
N
H
N
H
F
F
BI-2545 (19)
F
We next investigated a number of potential Znꢀbinding
groups as an alternative for the benzoxazolone (Table 3). We
found that by changing the benzoxazolone present in 11 to a
benzotriazole (compound 15), both potency in rat whole blood
and metabolic stability could be improved. Unfortunately, 15
still displayed very poor solubility which led us to consider
Parameter
In vitro
IC50 potency on human/rat ATX [nM]a
IC50 potency on ATX in human/rat
whole blood [nM]a
Solubility [µg/ml] at pH 2.2/4.5/6.8
hERG IC50 (µM)
2.2±1.0 (3)/3.4±2.1 (2)
29±5.0 (2)/96±32.6 (2)
<1/<1/<1
>10
more polar groups. When using
a 1H,4H,5H,6H,7Hꢀ
[1,2,3]triazolo[4,5ꢀc]pyridine as a Znꢀbinding residue, the
resulting compound 16 displayed improved potency in our
rWB assay and increased solubility at pH 2.2 and 4.5 comꢀ
pared to our starting point 11. We could also increase solubiliꢀ
ty at neutral pH while retaining favorable potencies when the
benzoxazolone was replaced by a triazole that was linked to
our [3.1.0] core via a C2ꢀ (not shown) or C3ꢀlinker (compound
17). However, this also led to reduced Cacoꢀ2 permeability.
ATX inhibitor 18, which displayed the highest solubility at pH
6.8 contained a 4H,5H,6H,7Hꢀ[1,2]oxazolo[5,4ꢀc]pyridinꢀ3ꢀol.
This fairly acidic moiety is an interesting Znꢀbinding moiety
that led to a compound with favorable metabolic stability.
Unfortunately, permeability was low so that we did not further
followꢀup on this residue. Notably, all compounds shown in
Table 3 had a clean hERG profile.
Metabolic stability hLM/rLM [%Qh]
Caco Permeability [·10-6 cm sec-1]
Caco efflux ratio
PPB (h/r)
cLogP
In vivo
Dose [mg·Kgꢀ1]
Dose normalized AUC [nM·h]
Clearance [%Qh]
23/32
9
1.4
>99.8/>99.8
4.9
i.v.
1
2320
10
10
0.9
p.o.
10
675
%QH [%]
Vss [L·Kg-1]
Dose normalized cmax [nM]
Tmax [h]
t1/2 [h]
149
1.7
3.4
30
1.8
F [%]
IC50 potency on ATX [nM]
100
Disappointingly, when ATX inhibitors 15 and 16 were furꢀ
ther evaluated in an in vivo rat PK study, both compounds
showed high clearance (for further details, see Supporting
Information) and consequently, we shifted our attention to the
bisꢀtrifluoromethyl residue, as this residue had resulted in
superior potency in rat whole blood and metabolic stability.
When combining this residue with a benzotriazole as the Znꢀ
binding group, we obtained compound 19 (BIꢀ2545), which
had the best overall profile (structure of BIꢀ2545 see Table 4).
Since BIꢀ2545 (19) not only displayed good potency in the
LPA and rat whole blood assay but also had favorable ADME
properties (see Table 4), it was further evaluated in a rat
PK/PD model.
%QH: Normalized clearance based on liver blood flow; Vss: volume of distribuꢀ
tion at steady state, Cmax: maximal concentration; Tmax: time point at Cmax; t1/2: halfꢀ
life; F: bioavailability; aValues are reported as arithmetic means with standard
deviations. Number of replicates in parentheses.
BIꢀ2545 (19) proved to be stable in human and moderately
stable in rat liver microsomes. In vitro, 19 showed high Cacoꢀ
2 permeability and low efflux. Consequently, low clearance
was observed in vivo in rats and in vivo oral dosing to rats
resulted in good oral exposure. Under these conditions, the
sum of the plasma LPA species was reduced up to 90% (see
Figure 5), demonstrating high and sustained in vivo efficacy in
Figure 5: PK profile of BIꢀ2545 (19) and the plasma LPA level
reduction of the respective LPA species. Error bars represent
standard error of the mean (N = 3).
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A representative synthesis of compound 3-16 and 19 (exꢀ
1
emplified by compound 11) is shown in Scheme 1. Commerꢀ
cially available Bocꢀprotected primary amine 20 was coupled
with 2ꢀoxoꢀ3Hꢀ1,3ꢀbenzoxazoleꢀ6ꢀcarboxylic acid to give 21.
After acidic deprotection of the Bocꢀgroup, intermediate 22
was reacted with 3ꢀ(hydroxymethyl)ꢀ5ꢀ(trifluoromethoxy)ꢀ
benzonitrile to afford the corresponding carbamate 11.
Scheme 1. Synthesis of compound 11.
2
3
4
5
6
7
8
9
O
H
H
O
N
a
H
N
O
N
O
H
N
H2
N
O
O
Figure 6. Cocrystal structure of BIꢀ2545 (19) bound to Autotaxin
and overlay with PFꢀ8380.
H
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
20
21
b
O
N
H
N
In summary, by applying knowledgeꢀbased design on the
recently disclosed ATX inhibitor PFꢀ8380 (2), we identified
BIꢀ2545 (19) which inhibits ATX in the singleꢀdigit nanomoꢀ
lar region and translates into good potency in human whole
blood. Our ATX inhibitor BIꢀ2545 (19) is orally available in
rodents, devoid of hERG channel inhibition and shows an
excellent PK/Target Engagement relationship. It is therefore
considered a valuable tool for further in vivo studies.
H
H
N
N
O
H
O
c
H
NH
N
O
H
O
N
O
H
O
F
O
H
O
22
F
11
F
Reagents and conditions: (a) 2ꢀOxoꢀ2,3ꢀdihydroꢀbenzoxazoleꢀ
6ꢀcarboxylic acid, TBTU, Et3N, DMF, then 20, RT, 12 h, 71%;
(b) 4 N HCl in dioxan, RT, 12 h, 69%; (c) 3ꢀ(hydroxymethyl)ꢀ5ꢀ
(trifluoromethoxy)benzonitrile, CDI, THF, then 22, 50 °C, 12 h,
56%.
ASSOCIATED CONTENT
Supporting Information
Procedures for the preparation of 2ꢀ19 (including intermediates), biologiꢀ
cal and in vitro ADME assays, crystallization and structural determination,
PK data and pharmacology profile of BIꢀ2545.
For the synthesis of compounds 17 and 18, we made use of
the readily available secondary amine 23 as the starting point.
As depicted in Scheme 2, the hydrophobic pocket residue was
attached by treating 23 with 3ꢀ(hydroxymethyl)ꢀ5ꢀ
(trifluoromethoxy)benzonitrile in the presence of CDI. Subseꢀ
quent Bocꢀdeprotection afforded the primary amine 25 which
was now set to be coupled with a wide range of Znꢀbinding
groups through standard amide coupling conditions. This is
exemplified by reaction of 24 with 1Hꢀ1,2,3ꢀbenzotriazoleꢀ5ꢀ
carboxylic acid to afford the ATX inhibitor 18.
AUTHOR INFORMATION
Corresponding Author
*Email: christian.kuttruff@boehringerꢀingelheim.com, Tel.: +49 (7351)
54ꢀ144985. Present Address: Boehringer Ingelheim Pharma GmbH & Co.
KG, Medicinal Chemistry, 88397 Biberach, Germany
Scheme 2. Synthesis of compound 18.
O
H
N
HO
O
N
H
O
NH
N
O
H
a,b
c
N
O
N
H
N
N
N
H
O
H
ACKNOWLEDGMENT
H2
N
O
H
O
F
F
O
H
O
F
F
18
23
F
The authors thank Laura Carrettoni, Paolo Giuseppe Andrea Comito,
Marion Heitmann, Michael Hörmann, Siegfried Kolb, Stefanie Rausenꢀ
berger, AnnaꢀLuisa Reiser, Sarah Scherer, Michaela Walter and Marina
Winter for their dedicated and excellent synthesis support. The authors
also thank Anita Bloching for excellent support in protein crystallization
and the staff of the protein crystallography beamlines at the Swiss Light
Source and Expose GmbH for data collection. We also thank Vanessa
Hiller for her help with the Supporting Information and Udo Fischer for
support with NMR spectroscopy.
24
F
Reagents
and
conditions:
(a)
3ꢀ(hydroxymethyl)ꢀ5ꢀ
(trifluoromethoxy)ꢀbenzonitrile, CDI, DMF, 35 °C, 1 h, then 23,
DIPEA, 45 °C, 12 h, 66%; (b) 4 N HCl in dioxan, RT, 20 h, 87%;
(c) 24, CDT, Et3N, DCM, RT, 1 h then 4,5,6,7ꢀTetrahydroꢀ
isoxazolo[5,4ꢀc]pyridinꢀ3ꢀol, 35 °C, 12 h 38%.
We could confirm the binding mode of BIꢀ2545 (19) into
ATX with a crystal structure (see Figure 6). 19 superimposes
almost perfectly onto 2 (PFꢀ8380) with minor deviations in the
3.1.0ꢀbicycle and the linker between that moiety and the benꢀ
zotriazole. The space filling of the hydrophobic pocket is
optimized by the two trifluoromethylꢀgroups compared to the
dichloroꢀphenyl of 2. The linker between the [3.1.0]ꢀbicycle
and the benzotriazole is shifted by 1.2Å compared to the posiꢀ
tion of the linker between the piperazine and the benzoxazoliꢀ
dinone in 2. Due to the flexibility of the linker this does not
translate into a major rearrangement of the two Znꢀbinding
moieties. The benzotriazoleꢀring is tilted by 14° with respect
to the benzoxazolidinone, but the position of the Znꢀ
coordinating oxygen and nitrogen atoms remains unchanged.
All other hydrogenꢀbonds are also preserved among the two
compounds.
ABBREVIATIONS
ATX, Autotaxin; LPA, lysophosphatidic acid; IPF, idiopathic pulmonary
fibrosis; hERG, human Etherꢀaꢀgoꢀgo Related Gene; SAR, structure
activity relationship; TBTU, Oꢀ(Benzotriazolꢀ1ꢀyl)ꢀN,N,N',N'ꢀ
tetramethyluronium tetrafluoroborate; DMF, dimethylformamide; CDI,
1,1'ꢀCarbonyldiimidazole; CDT, 1,1'ꢀCarbonylꢀdiꢀ(1,2,4ꢀtriazole); THF,
tetrahydrofuran; DIPEA, N,NꢀDiisopropylethylamine; RT, room temperaꢀ
ture; ADME, absorption distribution metabolism excretion; PK, pharmaꢀ
cokinetics; PD, pharmacodynamics
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Insert Table of Contents artwork here
1
2
3
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5
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7
8
9
1.0
4.0
2.0
0.0
F
H
H
O
O
F
F
N
N
O
N
0.5
0.0
H
N
N
H
F
F
ATX inhibitor
F
BI-2545
t [h]
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
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43
44
45
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BI-2545
Sum LPA
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