Discovery of OATD-01, a First-in-Class Chitinase Inhibitor as Potential New Therapeutics for Idiopathic Pulmonary Fibrosis

Article abstract of DOI:10.1021/acs.jmedchem.0c01179

Chitotriosidase (CHIT1) and acidic mammalian chitinase (AMCase) are the enzymatically active chitinases that have been implicated in the pathology of chronic lung diseases such as asthma and interstitial lung diseases (ILDs), including idiopathic pulmonary fibrosis (IPF) and sarcoidosis. The clinical and preclinical data suggest that pharmacological inhibition of CHIT1 might represent a novel therapeutic approach in IPF. Structural modification of an advanced lead molecule 3 led to the identification of compound 9 (OATD-01), a highly active CHIT1 inhibitor with both an excellent PK profile in multiple species and selectivity against a panel of other off-targets. OATD-01 given orally once daily in a range of doses between 30 and 100 mg/kg showed significant antifibrotic efficacy in an animal model of bleomycin-induced pulmonary fibrosis. OATD-01 is the first-in-class CHIT1 inhibitor, currently completed phase 1b of clinical trials, to be a potential treatment for IPF.

Full text of DOI:10.1021/acs.jmedchem.0c01179

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Drug Annotation
Discovery of OATD-01, a First-in-Class Chitinase Inhibitor as
Potential New Therapeutics for Idiopathic Pulmonary Fibrosis
Robert Koralewski, Barbara Dymek, Marzena Mazur, Piotr Sklepkiewicz, Sylwia Olejniczak,
Wojciech Czestkowski, Krzysztof Matyszewski, Gleb Andryianau, Piotr Niedziejko, Michal Kowalski,
̇
Mariusz Gruza, Bartłomiej Borek, Karol Jedrzejczak, Agnieszka Bartoszewicz, Elzbieta Pluta,
Aleksandra Rymaszewska, Magdalena Kania, Tomasz Rejczak, Sylwia Piasecka, Michal Mlacki,
Marcin Mazurkiewicz, Michał Piotrowicz, Magdalena Salamon, Agnieszka Zagozdzon,
Agnieszka Napiorkowska-Gromadzka, Aneta Bartlomiejczak, Witold Mozga, Paweł Dobrzanski,
Karolina Dzwonek, Jakub Golab, Marcin Nowotny, Jacek Olczak, and Adam Golebiowski*
́
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ABSTRACT: Chitotriosidase (CHIT1) and acidic mammalian chitinase (AMCase) are the enzymatically active chitinases that have
been implicated in the pathology of chronic lung diseases such as asthma and interstitial lung diseases (ILDs), including idiopathic
pulmonary fibrosis (IPF) and sarcoidosis. The clinical and preclinical data suggest that pharmacological inhibition of CHIT1 might
represent a novel therapeutic approach in IPF. Structural modification of an advanced lead molecule 3 led to the identification of
compound 9 (OATD-01), a highly active CHIT1 inhibitor with both an excellent PK profile in multiple species and selectivity
against a panel of other off-targets. OATD-01 given orally once daily in a range of doses between 30 and 100 mg/kg showed
significant antifibrotic efficacy in an animal model of bleomycin-induced pulmonary fibrosis. OATD-01 is the first-in-class CHIT1
inhibitor, currently completed phase 1b of clinical trials, to be a potential treatment for IPF.
main chitinase produced by tissue activated macrophages3,9−11
INTRODUCTION
■
and expressed in human lung12 and liver.13−15 Moreover,
Chitinases are chitin-degrading enzymes that belong to the
CHIT1 was shown to modulate TGFβ and IL-13 signaling
pathways to promote fibrotic response in animal models of
pulmonary fibrosis.16
Currently, most antifibrotic treatment strategies are limited
to specific diseases or organs. More evidence demonstrates
great similarities among inflammatory and fibroproliferative
diseases, and new drugs are being shown to be effective
antifibrotic therapies across different diseases and organs.17
glycosyl hydrolase family 18, which includes enzymatically
active chitinases and chitinase-like proteins without enzymatic
activity.1−3 Mammals have two distinct genes encoding for
chitinase enzymes, chitotriosidase (CHIT1) and acidic
mammalian chitinase (CHIA, hereafter referred to as AM-
Case).4 Both enzymes are composed of a catalytic domain
linked by a flexible hinge to chitin binding domain belonging
to the carbohydrate-binding module family 14. The catalytic
domain shows a conserved cluster of three acidic residues in its
active-site cleft that are involved in the reaction of chitin
hydrolysis. The highly conserved chitin binding domain
facilitates recognition of crystal chitin.5 Both enzymes were
shown to modulate inflammatory response in chitin-dependent
and -independent manner. While AMCase is mostly produced
at sites of Th2 inflammation and modulates allergic response in
animal models of asthma,6−8 CHIT1 was reported to be the
Received: July 7, 2020
https://dx.doi.org/10.1021/acs.jmedchem.0c01179
© XXXX American Chemical Society
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CHIT1 is having a wide spectrum of scientific and clinical
interest mostly due to its specific localization in chronically
activated tissue macrophages that infiltrate the lungs of patients
suffering from chronic lung diseases, such as severe
asthma,9,18−20 progressive fibrotic interstitial lung diseases
(ILDs), such as idiopathic pulmonary fibrosis (IPF)16,21 or
sarcoidosis,21−23 and chronic obstructive pulmonary disease
(COPD).9,18,24 However, pathological tissue macrophages that
highly express CHIT1 are found also in liver disorders such as
nonalcoholic steatohepatitis (NASH) or hepatic fibrosis and
cirrhosis in general suggesting their role in liver diseases that
progress to fibrosis.14
IPF is a devastating and rapidly progressing fibroproliferative
lung disease, with an estimated 5-year survival of approximately
20%, characterized by epithelial damage, fibroproliferative
matrix deposition, and parenchymal remodeling.25−28 Despite
pirfenidone and nintedanib being approved for the treatment
of IPF, these compounds show moderate efficacy in delaying
the lung functional decline and disease progression. IPF is still
a greatly unmet medical need; therefore the search for new
therapeutic targets to support existing treatments is highly
demanded.
Our early research on chitinases inhibitors started from
natural product, allosamidin, cyclic peptides,35 and the
literature reported molecule 1 (also called Wyeth 1),36 which
was optimized into an advanced lead compound 2.20 The
initial SAR development led to improvement of in vitro activity
by optimizing interactions with Asp213 (mostly by shifting
scaffold design from the central piperazine ring into 4-
aminopiperidine) and by further exploring hydrophobic
interactions to gain selectivity toward murine (m) mAMCase
(e.g., OAT-177),20 human (h) hAMCase (e.g., OAT-1441),37
and mCHIT1 (e.g., OAT-2068) (Table 1).38 For the future
clinical candidate, we decided to focus our work on hCHIT1/
hAMCase inhibitors and progress lead compound 3.39 The
independent, very recent publication on m- and hCHIT1
selective inhibitor and its antifibrotic activity in bleomycin
induced lung fibrosis model in mice was reported by Jiang et
al.40
Molecule 3, compared to many others made in the program
(>3K chitinase inhibitors have been synthesized and tested),
has a unique in vitro profile, being a very strong inhibitor of all
four isoforms of key chitinase targets, i.e., both mouse and
human CHIT1 and AMCase. It also has a very good PK profile
in both mice and rats, but its main liability is relatively high
activity against dopamine transporter (DAT IC50 = 370 nM).
This off-target effect was not surprising considering that the
phenethylamine substructure (also present in all monoamines)
can be easily found in the compound 3. We have seen this off-
target activity in some other of our early inhibitors, e.g., OAT-
177 (DAT 95% inhibition at 10 μM).19
In the first step, we have studied chitinase inhibition activity
of other ring analogs of molecule 3, e.g., compounds 4−8.
Piperidine series (represented by compound 4) were as active
as morpholines but with higher activity against hERG and
monoamine transporters (likely because of higher basicity of
the nitrogen atom in piperidines vs morpholines). Other
replacements of the oxygen atoms (compounds 5−8) proved
to be inferior, with piperazine 6 being the most promising lead
molecule. Further development of the SAR of compound 6 led
to a higher mCHIT1 selectivity at the cost of activity against
human enzymes, and although it gave an interesting tool
molecule, i.e., mCHIT1 selective inhibitor OAT-2068,38 it was
later discontinued.
Alkyl substitution at position 2 of the morpholine ring, i.e.,
molecules 9−17, turned out to be a suitable modification
instantly abrogating activity toward dopamine transporter. This
result represents the classic case of SAR-cliff or “magic methyl”
discovery.43−45 The cis-diasteroisomer 9 turned out to be a
synthetically preferred isomer over the trans compound 10,46
with coincident higher activity against chitinases. Increasing
the size of the 2-substituent (e.g., compounds 11−17) led in
general to equipotent inhibitors but with much inferior PK
(e.g., compounds 11, 15, 16, 17; see Supporting Information).
Activated profibrotic tissue macrophages29 are thought to be
the drivers of fibrosis in IPF lungs. Intriguingly, CHIT1 is the
only chitinase induced in activated macrophages in vitro3,9,12,17
and found to be one of the markers of profibrotic macrophages
in IPF lungs. Initial studies evaluating the role of CHIT1 in the
pathogenesis of pulmonary fibrosis in mice showed that
CHIT1 deficiency resulted in decreased pulmonary fibrosis by
significant modulation of TGF-β- and IL-13-driven fibrotic
responses.16 This suggests a critical role of chitinases in the
development of airway and interstitial fibrotic remodeling and
implicates that pharmacological inhibition of CHIT1 is an
attractive approach for patients suffering from fibrotic lung
diseases.30,31
In addition, CHIT1 was suggested to facilitate tissue
remodeling processes in fibroblastic liver tissue (similar to
the lung) being produced by liver-specific macrophages,
Kupffer cells, which by activating hepatic stellate cells induce
hepatic fibrosis and cirrhosis.14,15 Higher activity of CHIT1 has
been also detected in the sera of patients with chronic hepatitis
C virus (HCV) infection as compared to control individuals
but most importantly also in the sera of patients with more
pronounced fibrosis or liver cirrhosis as compared to patients
with no or minimal liver fibrosis.14,32,33 This significant
correlation between serum CHIT1 and histological fibrosis
stages or liver stiffness further underlines the important role of
CHIT1 along with senescence for liver fibrogenesis.32,34
In the current studies we demonstrate discovery and
development of a selective chitinase inhibitor OATD-01 with
low nanomolar activity toward CHIT1, exhibiting exceptional
PK profile in multiple animal species. Furthermore, we
confirmed antifibrotic properties of OATD-01 in pulmonary
interstitial fibrosis model induced by bleomycin.
RESULTS AND DISCUSSION
■
The main goal of our studies was to develop a chitinase
inhibitor for the treatment of inflammation-driven fibrotic
diseases. We searched for a molecule with single-digit
nanomolar activity toward CHIT1, excellent PK/PD profile,
and therapeutic activity in preclinical animal models of chronic
pulmonary inflammation and fibrosis.
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correlation with earlier established IC50 data (hCHIT1 Ki =
17.3 11.3 nM; mCHIT1 Ki = 26.05 7.7 nM; hAMCase Ki
= 4.8 1.3 nM; mAMCase Ki = 5.7 4.1 nM; n = 3). Off-
target in vitro effects of compound OATD-01 have been
evaluated at 10 μM in the Eurofins Panlabs panel of 98 in vitro
binding and enzymatic assays, involving diverse molecular
classes of proteins. These studies demonstrated no significant
interactions as no inhibition or stimulation higher than 50%
was observed. Similarly, no significant inhibition was observed
in the panel of seven CYPs and 13 transporters (see
Supporting Information for details).
Table 1. Structure−Activity Relationship of Selected
a
Analogs of Compound 3
An X-ray crystallography approach was used to gain insights
into the structure of the hCHIT1−OATD-01 complex and to
understand the increased activity of the compound toward
recombinant hCHIT1 in comparison with the previously
reported OAT-177.20 OATD-01 was cocrystallized with the
catalytic domain of hCHIT1. The obtained crystals belong to
space group P212121 and diffracted X-rays to 1.5 Å resolution
at a synchrotron source. The hCHIT1−OATD-01 complex
structure was solved by molecular replacement, using the
hCHIT1 structure (PDB code 1WB0) as the search model.
The OATD-01 inhibitor occupies the chitin-binding cleft of
hCHIT1 (Figure 1). The binding mode is largely the same as
previously observed for the OAT-177 compound. Two of the
hCHIT1 catalytic triad residues (Asp136, Asp138, Glu140)
bind the OATD-01 aminotriazole ring by direct (Asp138, 2.8
Å) and water-mediated (Glu140) hydrogen bonds. In the apo
structure (PDB code 4WJX) the side chain of Asp138 adopts
two conformations, but in the presence of OATD-01 is
stabilized through a hydrogen bond with Asp136. The position
of the carbonyl group of one of the alternative conformations
of Asp138 in apo protein, in the inhibitor complex structure, is
occupied by a water molecule mediating the Glu140−inhibitor
interaction. The aminotriazole moiety is also directly H
bonded to Tyr212 (2.7 Å) and Tyr27 (3.1 Å). The position
of the ring is stabilized by van der Waals interactions with
Met210, Met356, Trp358, Tyr27, and Ala183. Strong
interaction between carboxylic group of the conserved catalytic
Asp213 and nitrogen of the central ring of the inhibitor is
crucial for the potency of inhibitors toward hCHIT1 and
AMCase. As for compound OAT-177, 4-aminopiperidine core
of the OATD-01 forms stable hydrogen bond of 2.8 Å with
Asp213.
The key difference between OAT-177 and OATD-01 is the
presence of the 2-methylmorpholine ring. The analysis of the
X-ray structure reveals why the inhibitor’s activity is modified
by the addition of the ring. First, the interactions between the
inhibitor and Arg269 residue are altered (compare super-
imposition of the active site residues in hCHIT1 complexed to
OATD-01 and OAT-177; Supporting Information Figure 1).
In the OAT-177 complex the side chain of this residue adopts
two alternative conformations, while in complex with OATD-
01 Arg269 has one conformation and forms van der Waals
interactions with the 2-methylmorpholine ring (3.3−4.0 Å).
Second, O03 oxygen and C04 carbon of the ring form van der
Waals interactions with the side chain of Met300.
Similar to OAT-177, the 4-chlorobenzyl part of OATD-01
occupies a hydrophobic pocket formed by Leu362, Ala302,
Leu301, Tyr267, and Met300 residues. All of these residues
form van der Waals interactions with of OATD-01. Chloride
atom, located deep in the hydrophobic cavity, interacts with
Thr295 within 3.6 Å distance. It was shown20 that this
interaction is key for inhibition of AMCase and CHIT1
a
IC50 data of each compound were determined in 2−3 independent
b
assays, and mean IC50 SD values are shown in the table. Data from
Predictor hERG fluorescence polarization assay41 % inhibition at 10
c
μM. Data from DAT human dopamine transporter binding
(antagonist radioligand) assay, Cerep.42 NT = not tested; IA =
d
inactive.
Thus, compound 9 (OATD-01) was selected for further
studies. To thoroughly determine its affinity toward h/
mCHIT1 and h/mAMCase, the inhibitory constants (Ki) for
all four enzymes have been measured and revealed good
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Figure 1. Structure of OATD-01−hCHIT1 complex (PDB code 6ZE8). The structure was solved by molecular replacement, using the hCHIT1
structure (PDB code 1WB0) as the search model. (A) Stereoview of key residues (shown as green sticks) of hCHIT1 active site. OATD-01
compound is shown as blue sticks, water molecule as red sphere. Protein−ligand hydrogen bonds are represented by orange dashed lines, and
distances are given in Å. (B) Stereoview of active site pocket of hCHIT1 represented as surface and colored according to electrostatic potential. Key
residues of hCHIT1 active site are shown as green sticks. OATD-01 compound is shown as blue sticks and water molecule as red sphere.
a
Scheme 1. Synthesis of OATD-01
a
Reagents and conditions: (a) (R)-2-bromopropionic acid, TBTU; CH2Cl2, rt, 60−80%; (b) NaH; THF, rt, 60−80%; (c) BH3·DMS; THF, reflux,
60−80%; (d) N-Boc-piperid-4-one, NaBH(OAc)3; DCE, AcOH, rt, 50−60%; (e) HCl(g); AcOEt, rt, 90%; (f) S,S′-dimethyl N-
cyanodithioiminocarbonate, K2CO3; MeCN, reflux; (g) hydrazine; MeCN, reflux; ∼80% over two steps.
chitinases activity. Chitin-binding cleft is closed by Trp99
residue. The orientation of Trp99 is the same as that observed
for OAT-177 compound. Trp99 side chain forms van der
Waals interactions with the 2-methyl group of morpholine, and
4-aminopiperidine thus plays a key role in stabilizing the
overall conformation and strengthening the inhibitor binding.
OATD-01 was synthesized in several different ways, and its
early preparation was carried out in the seven steps synthetic
D
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sequence (Scheme 1). Amino alcohol 1839 was acylated using
optically active (R)-2-bromopropionic acid, and the inter-
mediate 19 was subsequently cyclized to form morpholin-3-
one 20. Reduction of the amide bond led to optically pure
morpholine 21, which was used as an amino substrate in the
reductive amination, Boc-deprotection sequence to form
piperidine 22. Previously described20 aminotriazole formation
was used to form the final product OATD-01 with 15% overall
yield.
The pharmacokinetic (PK) properties of OATD-01 were
evaluated based on mouse, rat, and dog single dose
administrations of the compound (iv and po) under fasting
conditions in the following doses: 2 mg/kg (iv), 3 mg/kg (iv),
6 mg/kg (po), 10 mg/kg (po). As formulations, aqua solutions
of solutol, glucose, and/or ethanol were examined. The
concentrations of OATD-01 in animal plasma samples were
monitored by LC/MS−MS analysis, until 24 h after dose
(Figure 2). Mean PK parameters of individual species
calculated by the noncompartmental model (NCA) are
shown in Table 2.
OATD-01 showed a favorable pharmacokinetic profile in
the studied species in all formulations. Following iv dosing,
OATD-01 was characterized by low clearance (0.1−0.35 (L/
h)/kg) and moderate volume of distribution (1−2 L/kg).
Following po administration, systematic exposure expressed by
AUC0−24 ranged between 5.5 and 31 mg·h/L, depending on
species and formulation, while Cmax (in the range of 2.2−7.6
mg/L) was reached in 30 min (dogs) to 2 h (rodents) after
dosing. Following fast absorption, OATD-01 was eliminated
with an elimination half-life of 1.9−4.5 h in rodents and 23 h in
dogs, which is comparable to intravenous administration of the
compound. OATD-01 showed high bioavailability (from 77 to
107%) in tested species and formulations.
Pharmacodynamics in Lungs. In order to evaluate the
PK/PD relationship of OATD-01 in mice, the chitinolytic
activity (the pharmacodynamic marker) in plasma and lungs
upon oral administration of OATD-01 was monitored. Mice
were administered with one or two doses of OATD-01 (30
mg/kg) under fasting conditions. Plasma and perfused lungs,
collected before and at defined time points up to 24 h after the
last dose, were used for the determination of the levels of
OATD-01 and chitinolytic activity. As shown in Figure 3, a
decrease in chitinolytic activity following OATD-01 admin-
istration was concomitant with the increasing concentration of
OATD-01 in plasma and lung tissue, thus demonstrating target
engagement in vivo in both plasma and lungs. Single
administration of OATD-01 resulted in at least 70% reduction
of chitinolytic activity in plasma for up to 12 h. Sequential two
doses of OATD-01, separated by 8 h led to more sustained
inhibition of chitinolytic activity levels over time. Among the
tested time points, the peak of compound activity was detected
at 0.5 h after dosing in either scheme of administration.
In Vivo Therapeutic Efficacy of OATD-01. CHIT1 was
suggested to be the main chitinase active in the human lungs12
that facilitates pulmonary fibrotic response.16 Further studies
were carried out to evaluate direct antifibrotic effects of
OATD-01 due to additional inhibition of CHIT1 in a robust
model of pulmonary interstitial fibrosis induced by admin-
istration of bleomycin. Our recent studies (data not shown)
showed that CHIT1 is induced in the lungs of bleomycin
Figure 2. Plasma concentration time−course for compound OATD-
01 after single intravenous 3 mg/kg (iv) bolus and after 10 mg/kg oral
administration (po) into female BALB/c mice. Free concentrations
were obtained based on in vitro plasma protein binding (PPB);
unbound fraction (fu) in mice is 0.253.
Table 2. Mean Pharmacokinetic Parameters of OATD-01 in Mice, Rats, and Dogs Determined by the Noncompartmental
a
Model (NCA)
species
rat
iv
mouse
dog
iv
rat
po
mouse
dog
po
route
iv
po
sex
male
2
female
3
male
3
male
6
female
10
male
10
dose (mg/kg)
AUC0−24 (mg·h/L)
AUC0−24/D (kg·mg·h/(L·mg))
C0 or Cmax (mg/L)
5.56
2.78
1.00
n/a
n/a
4.49
0.34
0.34
1.82
n/a
A
8.60
2.87
3.97
n/a
n/a
2.09
0.35
0.35
1.01
n/a
B
31.01
10.34
2.47
0.82
n/a
21.94
x
0.10
0.96
n/a
C
20.28
3.38
2.21
0.37
2.00
4.47
n/a
n/a
n/a
107.46
A
22.18
2.22
3.42
0.34
2.00
1.88
n/a
n/a
n/a
77.38
B
94.13
9.41
7.64
0.76
0.5*
22.87
n/a
n/a
n/a
91.42
C
C
max/D (kg·mg/(L·mL))
Tmax (h)
T1/2 (h)
CL ((L/h)/kg) (0−inf)
CL ((L/h)/kg) (0−24)
Vss (L/kg) (0−24)
bioavailability (%)
formulation
a
A, mix of ethanol and solutol (aq solution); B, mix of solutol and glucose (aq solution); C, glucose (aq solution); n/a, not applicable; x, not
calculated as the % AUC extrapolated (% exp) was greater than 20%; Tmax, calculated from analytical data; ∗, median.
E
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Figure 3. Relationship between total OATD-01 concentration in plasma (A, B) and lungs (C, D) following single (A, C) or double (8 h interval
between doses; B, D) administration of OATD-01 at 30 mg/kg.
administered mice; its expression is mostly restricted to
macrophage population and correlated with fibrotic gene
signature in the lungs. Therefore, antifibrotic efficacies of both
OATD-01 (30, 100 mg/kg, po; q.d.) and designated clinically
approved therapeutic, nintedanib (100 mg/kg; po; q.d.), were
evaluated using semiquantitative Ashcroft scoring scale47,48 of
the lungs of bleomycin-induced model in a therapeutic
regimen (d7−d21), (Figure 4A,B). This analysis revealed
that OATD-01 significantly reduced the degree of lung fibrosis
(Figure 4B) in a dose-dependent manner, ultimately achieving
comparable therapeutic efficacy to reference treatment with
nintedanib in this animal model. Moreover, these results
correlated with a significant reduction of bleomycin-induced
chitinolytic activity in plasma (Figure 4C), as opposed to
nintedanib treatment confirming that CHIT1 inhibition is one
of potential mechanisms to reduce fibrotic lesions in
bleomycin-induced pulmonary fibrosis, and potential new
strategy for IPF treatment.
OATD-01 is a highly potent CHIT1 inhibitor, also inhibiting
AMCase, with excellent PK properties across multiple animal
species. Given once daily in a range of doses between 30 and
100 mg/kg orally in vivo, it showed significant antifibrotic
efficacy in preclinical animal model of bleomycin-induced
pulmonary fibrosis in mice. Provided data package for OATD-
01 and its antifibrotic efficacy in preclinical models in vivo will
hopefully lay the grounds for novel therapeutic approach in
IPF and possibly other fibrosis-related clinical indications such
as NASH. The preclinical studies have shown that the OATD-
01 is well tolerated, has low toxicity, and has a favorable
pharmacokinetic profile. OATD-01 is currently being clinically
studied (completed phase 1b).
EXPERIMENTAL SECTION
■
Chemical Methods. All solvents, substrates, and reagents that
were commercially available were used without further purification.
TLC analysis was performed using precoated glass plates (0.2 0.03
mm thickness, GF-254, particle size 0.01−0.04 mm) from
Fluorochem Ltd., U.K. Column chromatography was performed
using high-purity grade silica gel (pore size 60 Å, 220−440 mesh
particle size, 35−75 μm particle size) from Fluka. Preparative HPLC
was performed on LC-20AP Shimadzu with ELSD-LTII detector
equipped with Hypersil GOLD 21.2/250 mm, 5 μm C18 column. 1H
NMR spectra were recorded on Agilent Mercury 400 MHz
CONCLUSIONS
■
An advanced lead molecule 3 was modified and its overall
activity optimized. The strategic placement of the “magic
methyl” group significantly changed its in vitro activity profile
and led to the identification of the molecule OATD-01.
F
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Figure 4. OATD-01 treatment ameliorates bleomycin-induced lung fibrosis in a dose-dependent manner. (A) Representative Masson’s trichrome
staining of pulmonary fibrosis 21 days after bleomycin injury, with or without treatment with OATD-01 or nintedanib. (B) Semiquantitative
analysis of pulmonary fibrosis by Ashcroft score (0−6) in the lung sections (n = 3 per animal) with or without OATD-01 (30 and 100 mg/kg; q.d.)
and nintedanib (100 mg/kg; q.d.) as compared to vehicle treated controls. (C) Chitinolytic activity in plasma collected 24 h after the last dose of
OATD-01 (30 and 100 mg/kg; q.d.) or nintedanib (100 mg/kg; q.d.) as compared to vehicle treated controls. Data are presented as the mean
SEM. P-values of <0.05 were considered as statistically significant and presented as ∗ for p < 0.05, ∗∗ for p < 0.01, ∗∗∗ for p < 0.001, and ∗∗∗∗ for
p < 0.0001.
spectrometer and on Bruker AVANCE DRX500, AVANCE DRX600,
or Bruker AVANCE II PLUS (respectively at 500, 600, or 700 MHz)
NMR spectrometers. All spectra were recorded in appropriate
deuterated solvents (CDCl3, DMSO-d6, D2O, methanol-d4, etc.)
that were commercially available. Resonances are given in parts per
million relative to tetramethylsilane. Data are reported as follows:
chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, m =
multiplet, br = broad), coupling constants (J in Hz), and integration.
LC−MS spectra were recorded on a Shimadzu LC-20AD LPG
separation module with a SPD-M20A UV detector and LCMS-2020
mass detector equipped with Kinetex 2.1/50 mm, 2.6 μm C18 column
eluted with 0.5 mL/min flow of 10−90% gradient (over 6 min) of
acetonitrile in water. Purities of all final reported compounds were
greater than 95% based on HPLC chromatograms. Purification of the
final compounds by preparative HPLC was accomplished on C-18
250 mm × 21 mm column in 0.05% TFA in water/acetonitrile 95:5
→ 45:55 gradient over 30 min followed by freeze-drying of the pooled
fractions containing pure products. In some cases, the so obtained
trifluoroacetate salts of the final compounds were judged to be of
insufficient quality for biological testing due to their physical
appearance (oils with a distinguished scent of trifluoroacetic acid).
Therefore, they were redissolved in a small amount of 0.1 M HCl and
subjected to the second lyophilization providing well-behaving
hydrochloride salts.
Synthesis of compounds 5, 7, 8, and 15−17 are described in
Supporting Information.
(S)-4-(1-(5-Amino-4H-1,2,4-triazol-3-yl)piperidin-4-yl)-3-(4-
chlorobenzyl)thiomorpholine 1,1-Dioxide (5). 1H NMR (500
MHz, DMSO-d6) δ 7.31 (m, JAA′BB′ = 8.5 Hz, 2H), 7.24 (JAA′BB′ = 8.5
Hz, 2H), 5.48 (bs, 2H), 3.71−3.64 (m, 2H), 3.52−3.43 (m, 1H),
3.11−2.96 (m, 3H), 2.94−2.79 (m, 4H), 2.78−2.70 (m, 1H), 2.64−
1
2.52 (m, 2H), 1.65−1.55 (m, 2H), 1.37−1.18 (m, 3H); H NMR
G
https://dx.doi.org/10.1021/acs.jmedchem.0c01179
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Journal of Medicinal Chemistry
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Drug Annotation
(700 MHz, DMSO-d6 + D2O, 348 K) δ 7.32−7.29 (m, 2H), 7.29−
7.20 (m, 2H), 3.79−3.63 (m, 2H), 3.63−3.52 (m, 1H), 3.40−3.31
(m, 1H), 3.19−3.12 (m, 1H), 3.06−2.99 (m, 2H), 2.99−2.76 (m,
5H), 2.72−2.60 (m, 2H), 1.69−1.61 (m, 2H), 1.45−1.25 (m, 2H);
13C NMR (176 MHz, DMSO-d6) δ 138.3, 131.7 (2×), 131.3, 128.7
(2×), 60.4, 57.5, 55.8, 52.2, 50.2, 46.0, 45.9, 41.5, 35.2, 30.2, 28.0,
21.2; HRMS (ESI) m/z calcd for C18H26ClN6O2S [M + H]+
425.1521 found 425.1533.
Step 2. Synthesis of (R)-2-Bromo-N-((S)-1-(4-chlorophenyl)-3-
hydroxypropan-2-yl)propanamide (19). To the solution of alcohol
18 (1 g; 4.5 mmol) and (R)-2-bromopropionic acid (0.4 mL; 4.5
mmol) in DCM (30 mL) at 0 °C, diisopropylethylamine (DIPEA;
2.35 mL; 13.5 mmol) and TBTU (1.44 g; 4.5 mmol) were added
sequentially, and the reaction mixture was stirred at this temperature
for 3 h. After this time TLC control showed complete consumption of
the starting materials, so the reaction mixture was transferred to the
separatory funnel and washed sequentially with 1 M HCl, 1 M
K2CO3. The organic phase was dried over anhydrous MgSO4, filtered,
and concentrated in vacuo, and the crude product was purified by
flash column chromatography (DCM/MeOH 100:1 v/v), and
product 19 was obtained as a white solid in 64% yield (0.6 g; 2.88
mmol). ESI-MS m/z for C12H16BrClNO2 found 320.7/322.7 [M +
(S)-5-(4-(2-(4-Chlorobenzyl)-4-methylpiperazin-1-yl)-
piperidin-1-yl)-4H-1,2,4-triazol-3-amine (6).38 1H NMR (500
MHz, DMSO-d6) δ 10.86 (bs, 1H), 7.32−7.26 (m, 2H), 7.17 (d, J =
8.0 Hz, 2H), 5.59 (s, 2H), 3.77−3.67 (m, 2H) 2.91−2.71 (m, 3H),
2.70−2.51 (m, 7H), 2.00 (s, 3H), 1.98−1.90 (m, 2H),1.75−1.55 (m,
1
2H), 1.49−1.22 (m, 2H); H NMR (700 MHz, DMSO-d6) δ 7.42−
1
H]+; H NMR (500 MHz, CDCl3) δ 7.25 (d, J = 8.2 Hz, 2H), 7.14
7.28 (m, 2H), 7.28−7.15 (m, 2H), 3.88−3.65 (m, 2H), 2.97−2.88
(m, 1H), 2.88−2.79 (m, 2H), 2.76−2.62 (m, 3H), 2.62−2.53 (m,
2H), 2.38−2.23 (m, 2H), 2.11−1.99 (m, 5H), 1.78−1.73 (m, 1H),
1.69−1.64 (m, 1H), 1.53−1.46 (m, 1H), 1.41−1.30 (m, 1H); 13C
NMR (176 MHz, DMSO-d6) δ 139.7, 131.5 (2×), 130.8, 128.6 (2×),
58.8, 56.7, 55.74, 55.66, 46.5, 46.3, 46.1, 43.7, 40.5, 32.7, 30.1, 25.9
(one signal is missing due to overlap); HRMS (ESI) m/z calcd for
C19H29ClN7 [M + H]+ 390.2167 found 390.2173.
(d, J = 8.2 Hz, 2H), 6.58 (d, J = 6.7 Hz, 1H), 4.33 (dd, J = 14.1, 7.1
Hz, 1H), 4.13−4.06 (m, 1H), 3.69−3.64 (m, 1H), 3.61−3.57 (m,
1H), 2.90−2.81 (m, 2H), 2.17 (brs, 1H), 1.82 (d, J = 6.9 Hz, 3H).
Step 3. Synthesis of (2S,5S)-5-(4-Chlorobenzyl)-2-methylmor-
pholin-3-one (20). To the solution of compound 19 (1.46 g; 4.52
mmol) in THF (45 mL) sodium hydride (NaH) (60% in mineral oil;
0.65 g; 13.56 mmol) was added in one portion and stirred at room
temperature for 30 min. TLC showed consumption of the starting
material. The reaction was quenched by addition of water and then
extracted with diethyl ether. Combined organic extracts were then
washed with brine, dried over anhydrous MgSO4, filtered, and
concentrated in vacuo to provide product 20 as a white solid after
washing with hexane in 77% yield (0.83 g; 3.46 mmol). ESI-MS m/z
for C12H15ClNO2 found 240.1/242.1 [M + H]+; 1H NMR (500 MHz,
CDCl3) δ 7.27 (d, J = 8.2 Hz, 2H), 7.10 (d, J = 8.2 Hz, 2H), 6.13
(brs, 1H), 4.18 (m, 1H), 3.76 (d, J = 3.0 Hz, 2H), 3.55−3.50 (m,
1H), 2.91−2.83 (m, 2H), 1.46 (d, J = 6.9 Hz, 3H).
(S)-4-(1-(5-Amino-4H-1,2,4-triazol-3-yl)piperidin-4-yl)-3-(4-
chlorobenzyl)piperazin-2-one 2,2,2-Trifluoroacetate (7). 1H
NMR (500 MHz, DMSO-d6) δ 7.88 (s, 1H), 7.32−7.22 (m, 4H),
3.85−3.59 (m, 4H), 3.15−2.75 (m, 8H), 1.73 (d, J = 12.2 Hz, 1H),
1.66−1.53 (m, 1H), 1.51−1.28 (m, 2H); 1H NMR (700 MHz,
DMSO-d6 + D2O, 348 K) δ 7.34−7.20 (m, 4H), 3.73−3.55 (m, 3H),
3.24−3.17 (m, 1H), 3.11−3.01 (m, 3H), 2.99−2.76 (m, 5H), 1.75−
1.62 (m, 2H), 1.51−1.33 (m, 2H); 13C NMR (176 MHz, DMSO-d6)
δ 159.4, 137.4, 131.9 (2×), 131.5, 128.3 (2×), 62.0, 57.2, 45.7, 45.6,
41.3, 40.0, 38.5, 35.1, 28.3, 27.0, 25.2; HRMS (ESI) m/z calcd for
C18H25ClN7O [M + H]+ 390.1804 found 390.1809.
(S)-4-(1-(5-Amino-4H-1,2,4-triazol-3-yl)piperidin-4-yl)-5-(4-
chlorobenzyl)piperazin-2-one 2,2,2-Trifluoroacetate (8). 1H
NMR (500 MHz, D2O) δ 7.28 (JAA′BB′ = 7.3 Hz, 2H), 7.14 (JAA′BB′
= 7.7 Hz, 2H), 4.07−4.12 (m, 1H), 4.01 (d, J = 16.7 Hz, 1H), 3.86 (d,
J = 16.9 Hz, 1H), 3.73−3.77 (m, 2H), 3.32−3.36 (m, 1H), 3.17−3.22
(m, 2H), 2.81−3.04 (m, 4H), 2.09−2.15 (m, 2H), 1.68−1.75 (m,
2H); 1H NMR (700 MHz, DMSO-d6 + D2O, 348 K) δ 7.41−7.24 (m,
4H), 3.87−3.77 (m, 2H), 3.74−3.69 (m, 1H), 3.67−3.62 (m, 1H),
3.60−3.52 (m, 1H), 3.37−3.23 (m, 3H), 3.12−3.07 (m, 1H), 3.06−
2.99 (m, 1H), 2.99−2.86 (m, 2H), 2.82−2.72 (m, 1H), 2.02−1.91
(m, 2H), 1.71−1.56 (m, 2H); 13C NMR (176 MHz, DMSO-d6) δ
159.2, 135.0, 132.4, 131.7 (2×), 129.2 (2×), 59.2, 54.3, 48.1, 47.4,
45.3, 45.1, 40.3, 40.0, 26.4, 25.4, 24.5; HRMS (ESI) m/z calcd for
C18H25ClN7O [M + H]+ 390.1804 found 390.1821.
Step 4. Synthesis of (2S,5S)-5-(4-Chlorobenzyl)-2-methylmor-
pholine (21). To the solution of lactam 20 (1.03 g; 4.29 mmol) in
tetrahydrofuran (21.25 mL) borane dimethylsulfide complex (1.2 mL;
12.89 mmol) was added dropwise at 0 °C (foaming!). The cooling
bath was removed, and the reaction mixture was refluxed for 2 h. LC−
MS analysis showed consumption of the starting material. The
mixture was cooled to room temperature, and 6 M HCl aq (10 mL)
was carefully added (foaming!), then refluxed for another 1 h. The
reaction mixture was cooled to room temperature and 4 M NaOH aq
was added to reach pH 10 and then extracted with ethyl acetate (2 ×
50 mL). The combined organic phases were dried over MgSO4,
filtered, and concentrated in vacuum to give product 21 as a colorless
oil in 90% yield (871 mg; 3.86 mmol). ESI-MS m/z for C12H17ClNO
1
found 226.4/228.4 [M + H]+; H NMR (500 MHz, DMSO-d6 +
D2O) δ 7.33 (d, J = 8.3 Hz, 2H), 7.23 (d, J = 8.3 Hz, 2H), 3.50−3.48
(m, 1H), 3.49−3.47 (m, 2H), 2.87−2.81 (m, 1H), 2.80−2.76 (m,
2H), 2.65 (dd, J = 12.4 Hz, 8.3 Hz, 1H), 2.58 (dd, J = 12.4 Hz, 3.0
Hz, 1H), 1.09 (d, J = 6.2 Hz, 3H).
5-(4-((2S,5S)-5-(4-Chlorobenzyl)-2-methylmorpholino)-
piperidin-1-yl)-4H-1,2,4-triazol-3-amine Hydrochloride (9).
Step 1. Synthesis of (S)-2-Amino-3-(4-chlorophenyl)propan-1-ol
(18). To a solution of 4-chloro-L-phenylalanine (20.0 g; 100 mmol) in
tetrahydrofuran (200 mL) borane dimethylsulfide complex (28.16
mL; 300 mmol) was added dropwise at 0 °C (foaming!). After
addition was complete, the cooling bath was removed, and the
reaction mixture was refluxed for 5 h. The reaction progress was
monitored by LC−MS. The reaction mixture was cooled to room
temperature and 6 N HCl aq (100 mL) was carefully added
(foaming!) and then refluxed for an additional 1 h. The reaction
mixture was cooled to room temperature, 4 N NaOH aq was added to
reach pH 12 and subsequently extracted with ethyl acetate (2 × 500
mL). The combined organic layers were dried over anhydrous
MgSO4, filtered, and concentrated in vacuo. The oily residue was
triturated with diethyl ether and the solids were filtered off to give
(S)-2-amino-3-(4-chlorophenyl)propan-1-ol (18) as a white solid in
89% yield (16.5 g; 88.88 mmol). ESI-MS m/z for C9H12ClNO found
Step 5. Synthesis of (2S,5S)-5-(4-Chlorobenzyl)-2-methyl-4-
(piperidin-4-yl)morpholine Hydrochloride (22). To a solution of
compound 21 (1.36 g; 6.02 mmol) in 1,2-dichloroethane (4 mL)
Boc-piperidone (1.8 g; 9.03 mmol) was added followed by acetic acid
(0.69 mL; 12.4 mmol), and the reaction mixture was stirred for 4 h at
ambient temperature. Sodium triacetoxyborohydride [NaBH(OAc)3]
(2.61 g; 12.4 mmol) was then added in one portion, and the reaction
mixture was stirred overnight at ambient temperature. To this mixture
5% aq NaHCO3 (10 mL) was added, and after stirring for 5 min the
mixture was extracted with dichloromethane (2 × 20 mL). The
organic phases were combined, dried over anhydrous MgSO4, filtered,
and concentrated in vacuum. The residue was purified by flash
chromatography (DCM/MeOH 100:0 to 200:1 v/v) white foam
(1.10 g; 2.68 mmol; 45% yield) was treated with 3M HCl(g)/AcOEt
(26.8 mL), and the reaction mixture was stirred for 40 min at ambient
temperature. The solvent was removed in vacuo and residue was
triturated with diethyl ether and filtered to give product 22 as a
hydrochloride salt as a white solid in 34% yield (640 mg; 1.85 mmol).
1
185.7/187.7 [M + H]+; H NMR (500 MHz, CDCl3) δ 7.28 (d, J =
8.3 Hz, 2H), 7.11 (d, J = 8.3 Hz, 2H), 3.62 (dd, J = 10.6, 3.8 Hz, 1H),
3.37 (dd, J = 10.5, 6.9 Hz, 1H), 3.11−3.07 (brs, 1H), 2.76 (dd, J =
13.6, 5.4 Hz, 1H), 2.50 (dd, J = 13.6, 8.6 Hz, 1H).
1
ESI-MS m/z for C17H26ClN2O found 309.9/311.9 [M + H]+; H
NMR (500 MHz, DMSO-d6) δ 7.35 (d, J = 8.4 Hz, 2H), 7.33 (d, J =
H
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Journal of Medicinal Chemistry
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Drug Annotation
8.4 Hz, 2H), 4.04 (brs, 2H), 3.59 (brs, 2H), 3.45 (brs, 1H), 3.40 (m,
1H), 3.39−3.36 (m, 2H), 3.10 (brs, 2H), 2.98 (brs, 1H), 2.87 (brs,
2H), 2.31 (brs, 2H), 2.17 (brs, 2H), 1.21 (d, J = 6.2 Hz, 3H).
Step 6. Synthesis of 5-(4-((2S,5S)-5-(4-Chlorobenzyl)-2-
methylmorpholino)piperidin-1-yl)-4H-1,2,4-triazol-3-amine Hydro-
chloride (9). To a solution of piperidine 22 (640 mg; 1.85 mmol) in
2H), 7.36−7.29 (m, 2H), 3.94−3.85 (m, 2H), 3.80−3.77 (m, 1H),
3.74−3.68 (m, 2H), 3.67−3.46 (m, 3H), 3.16−3.10 (m, 2H), 3.10−
3.01 (m, 1H), 3.01−2.90 (m, 2H), 2.28−2.12 (m, 2H), 1.83−1.72
(m, 2H), 1.64−1.48 (m, 2H), 0.95 (t, J = 7.5 Hz, 3H); 13C NMR
(176 MHz, DMSO-d6) δ 135.0, 132.3, 132.0 (2×), 129.2 (2×), 75.1,
64.9, 58.7, 55.5, 51.9, 47.3, 45.3, 45.1, 40.0, 38.9, 27.3, 25.8, 25.7, 9.5;
HRMS (ESI) m/z calcd for C20H30ClN6O [M + H]+ 405.2164 found
405.2166.
̀
acetonitrile (10.0 mL) was added S,S-dimethyl N-cyanodithio-
iminocarbonate (298 mg; 2.03 mmol) followed by potassium
carbonate (563 mg; 4.07 mmol), and the reaction mixture was
refluxed for 1.5 h. LC−MS analysis showed consumption of the
starting material. To this mixture hydrazine monohydrate (0.27 mL;
5.55 mmol) was added, and reflux was maintained for an additional 2
h. The reaction mixture was cooled down to ambient temperature,
filtered, and the filtrate was concentrated in vacuo. The residue was
purified by flash chromatography (DCM/MeOH 250:1 to 50:1 v/v).
The material obtained after evaporation of solvents was dissolved in a
small amount of AcOEt, and then 3 M HCl/AcOEt (20 mL) was
added. The mixture was stirred for 5 min and concentrated in vacuo.
The residue was dissolved in methanol (2 mL) and as a solution was
added dropwise to vigorously stirred diethyl ether (250 mL). Stirring
was continued for an additional 20 min at room temperature and then
the precipitate was filtered off to give product 9 as a hydrochloride salt
5-(4-((2S,5S)-5-(4-Chlorobenzyl)-2-isopropylmorpholino)-
piperidin-1-yl)-4H-1,2,4-triazol-3-amine 2,2,2-Trifluoroacetate
(12). The title compound 12 was obtained as a TFA salt (2% overall
yield) in the same manner as example 9 with the exception that (2R)-
2-bromo-3-methylbutanoic acid instead of (2R)-2-bromopropionic
1
acid was used in the second synthetic step. H NMR (700 MHz,
DMSO-d6) δ 10.15 (s, 1H), 7.45−7.41 (m, 2H), 7.36−7.31 (m, 2H),
7.05 (bs, 2H), 3.88 (t, J = 16.8 Hz, 2H), 3.83−3.75 (m, 1H), 3.63−
3.53 (m, 3H), 3.49−3.37 (m, 2H), 3.17−3.07 (m, 1H), 3.03 (t, J =
12.5 Hz, 1H), 2.94 (t, J = 12.8 Hz, 1H), 2.84 (t, J = 12.5 Hz, 1H),
2.28 (d, J = 12.0 Hz, 1H), 2.22 (d, J = 12.2 Hz, 1H), 1.82 (ddd, J =
13.7, 9.4, 5.9 Hz, 1H), 1.69−1.59 (m, 2H), 0.97 (d, J = 6.9 Hz, 3H),
0.96 (d, J = 6.9 Hz, 3H); 13C NMR (176 MHz, DMSO-d6) δ 134.9,
132.4, 131.9 (2×), 129.2 (2×), 78.2, 64.9, 61.3, 58.8, 55.5, 45.8, 45.3,
45.1, 39.9, 30.7, 27.3, 25.8, 25.6, 18.6, 17.3; HRMS (ESI) m/z calcd
for C21H32ClN6O [M + H]+ 419.2321 found 419.2325.
1
as a white solid in 72% yield (568 mg; 1.33 mmol). H NMR (500
MHz, DMSO-d6) δ 7.39 (d, J = 8.3 Hz, 2H), 7.30 (d, J = 8.3 Hz, 2H),
3.87−3.79 (m, 3H), 3.66 (brs, 2H), 3.58 (brs, 1H), 3.52−3.45 (m,
2H), 3.09 (brs, 2H), 3.00−2.93 (m, 2H), 2.92−2.88 (m, 1H), 2.20
5-(4-((2S,5S)-5-(4-Chlorobenzyl)-2-isobutylmorpholino)-
piperidin-1-yl)-4H-1,2,4-triazol-3-amine 2,2,2-Trifluoroacetate
(13). The title compound 13 was obtained as a TFA salt (3% overall
yield) in the same manner as example 9 with the exception that (2R)-
2-bromo-4-methylpentanoic acid instead of (2R)-2-bromopropionic
1
(brs, 2H), 1.67 (brs, 2H), 1.20 (d, J = 6.2 Hz, 3H); H NMR (700
MHz, C6D6/methanol-d4) δ 7.13 (d, J = 8.2 Hz, 2H), 6.89 (d, J = 8.2
Hz, 2H), 3.99 (br s, 2H), 3.66 (br s, 3H), 3.56 (d, J = 11.4 Hz, 1H),
3.54−3.46 (m, 1H), 3.37 (d, J = 11.4 Hz, 1H), 2.90−2.80 (m, 3H),
2.68 (d, J = 10.4 Hz, 1H), 2.40 (d, J = 11.9 Hz, 2H), 2.32−2.25 (m,
1H), 2.05 (t, J = 11.2 Hz, 1H), 1.81 (d, J = 12.5 Hz, 1H), 1.65 (d, J =
12.5 Hz, 1H), 1.54−1.46 (m, 1H), 1.46−1.37 (m, 1H) and 1.11 (d, J
1
acid was used in the second synthetic step. H NMR (700 MHz,
DMSO-d6) δ 7.43 (d, J = 8.4 Hz, 2H), 7.34 (d, J = 8.4 Hz, 2H), 6.99
(br s, 1H), 3.89−3.85 (m, 3H), 3.79−3.47 (m, 7H), 3.12 (d, J = 7 Hz,
2H), 3.00 (s, 1H), 2.95−2.91 (m, 1H), 2.86−2.83 (m, 1H), 2.23−
2.18 (m, 2H), 1.82−1.76 (m, 1H), 1.64−1.59 (m, 2H), 1.53−1.49
(m, 1H), 1.31−1.27 (m, 1H), 0.91−0.87 (m, 6H); 13C NMR (176
MHz, DMSO-d6) δ 135.0, 132.3, 131.9 (2×), 129.2 (2×), 72.6, 65.1,
60.0, 58.7, 55.6, 47.8, 45.3, 45.1, 41.3, 40.0, 27.3, 25.9, 25.6, 23.9,
23.5, 22.1; HRMS (ESI) m/z calcd for C22H34ClN6O [M + H]+
433.2477 found 433.2489.
((2R,5S)-4-(1-(5-Amino-4H-1,2,4-triazol-3-yl)piperidin-4-yl)-
5-(4-chlorobenzyl)morpholin-2-yl)methanol Hydrochloride
(14). The title compound 14 was obtained as a hydrochloride salt
(8% overall yield) in the same manner as example 9 with the
exception that (2R)-2-bromo-3-tert-butoxypropanoic acid instead of
(2R)-2-bromopropionic acid was used in the second synthetic step.
1H NMR (700 MHz, DMSO-d6 + D2O) δ 7.43−7.37 (m, 2H), 7.37−
7.29 (m, 2H), 3.96−3.80 (m, 4H), 3.77−3.65 (m, 2H), 3.65−3.52
(m, 4H), 3.21−3.07 (m, 3H), 3.04−2.95 (m, 2H), 2.29−2.18 (m,
2H), 1.87−1.74 (m, 2H); 13C NMR (176 MHz, DMSO-d6) δ 135.0,
132.3, 132.0 (2×), 129.2 (2×), 74.7, 64.6, 61.7, 58.7, 55.5, 45.2, 45.1,
44.9, 39.9, 27.1, 25.8, 25.6 (one signal is missing due to overlap);
HRMS (ESI) m/z calcd for C19H28ClN6O2 [M + H]+ 407.1957 found
407.1969.
2-((2R,5S)-4-(1-(5-Amino-4H-1,2,4-triazol-3-yl)piperidin-4-
yl)-5-(4-chlorobenzyl)morpholin-2-yl)propan-2-ol 2,2,2-Tri-
fluoroacetate (15). 1H NMR (600 MHz, DMSO-d6 + D2O) δ
7.39 (JAA′BB′ = 8.5 Hz, 2H), 7.31 (JAA′BB′ = 8.5 Hz, 2H), 3.89−3.77
(m, 2H), 3.75−3.69 (m, 1H), 3.67−3.53 (m, 3H), 3.46−3.37 (m,
2H), 3.21−2.97 (m, 3H), 2.97−2.86 (m, 2H), 2.25−2.16 (m, 2H),
1.64−1.55 (m, 2H), 1.15 (s, 3H), 1.14 (s, 3H); 1H NMR (700 MHz,
DMSO-d6 + D2O, 348 K) δ 7.45−7.35 (m, 2H), 7.35−7.26 (m, 2H),
3.96−3.84 (m, 2H), 3.76−3.62 (m, 4H), 3.54−3.50 (m, 2H), 3.22−
3.16 (m, 1H), 3.12−3.06 (m, 2H), 2.97−2.87 (m, 2H), 2.30−2.13
(m, 2H), 1.73−1.61 (m, 2H), 1.19 (s, 6H); 13C NMR (176 MHz,
DMSO-d6) δ 134.9, 132.4, 132.0 (2×), 129.2 (2×), 70.4, 65.0, 59.4,
59.01, 57.7, 55.4, 52.1, 45.3, 45.2, 44.0, 40.0, 27.2, 27.1, 25.6, 24.6;
HRMS (ESI) m/z calcd for C21H32ClN6O2 [M + H]+ 435.2270 found
435.2282.
1
= 6.2 Hz, 3H); H NMR for free base (400 MHz, methanol-d4) δ
7.32−7.23 (m, 2H), 7.21−7.12 (m, 2H), 3.88−3.77 (m, 2H), 3.66−
3.52 (m, 2H), 3.51−3.42 (m, 1H), 3.02−2.92 (m, 2H), 2.91−2.76
(m, 4H), 2.72−2.63 (m, 1H), 2.43−2.30 (m, 1H), 2.09−1.98 (m,
2H), 1.56−1.37 (m, 2H), 1.18 (d, J = 6.2 Hz, 3H); 13C NMR (175
MHz, C6D6) δ 139.0, 132.1, 131.2, 129.0, 128.1, 72.6, 67.8, 56.5, 55.7,
50.1, 48.9, 45.8, 45.7, 29.7, 29.2, 27.7, 19.3; 13C NMR for free base
(101 MHz, methanol-d4) δ 138.7, 131.5, 130.6 (2×), 128.2 (2×),
72.3, 67.1, 56.2, 55.3, 49.5, 45.26, 45.25, 29.0, 28.5, 26.7, 18.0 (two
signals are missing due to overlap); HRMS (ESI) m/z calcd for
C19H28ClN6O [M + H]+ 391.2008 found 391.2004.
5-(4-((2R,5S)-5-(4-Chlorobenzyl)-2-methylmorpholino)-
piperidin-1-yl)-4H-1,2,4-triazol-3-amine Hydrochloride (10).
The title compound 10 was obtained as a hydrochloride salt (3%
overall yield) in the same manner as molecule 9 with the exception
that (2S)-2-bromopropionic acid instead of (2R)-2-bromopropionic
1
acid was used in the second synthetic step. H NMR (700 MHz,
DMSO-d6) δ 7.41 (d, J = 8.4 Hz, 2H), 7.35 (d, J = 8.4 Hz, 2H), 4.08
(br s, 1H), 3.91−3.84 (m, 2H), 3.83−3.80 (m, 2H), 3.70−3.67 (m,
2H), 3.48 (d, J = 12.7 Hz, 2H), 2.87 (t, J = 11.6 Hz, 1H), 2.75 (t, J =
11.5 Hz, 1H), 2.70 (dd, J = 14.5, 7.7 Hz, 1H), 2.65 (t, J = 12.0 Hz,
1H), 1.80−1.90 (m, 2H), 1.63 (qd, J = 12.3, 4.2 Hz, 1H), 1.11 (d, J =
6.2 Hz, 3H); 13C NMR (176 MHz, DMSO-d6) δ 135.7, 132.1, 131.4
(2×), 129.2 (2×), 69.5, 67.3, 58.7, 57.8, 50.3, 45.6, 45.3, 40.5, 31.7,
26.2, 22.5, 18.6 (one signal is missed due to overlap); HRMS (ESI)
m/z calcd for C19H28ClN6O [M + H]+ 391.2008 found 391.2015.
5-(4-((2S,5S)-5-(4-Chlorobenzyl)-2-ethylmorpholino)-
piperidin-1-yl)-4H-1,2,4-triazol-3-amine Hydrochloride (11).
The title compound 11 was obtained as a hydrochloride salt (7%
overall yield) in the same manner as molecule 9 with the exception
that (2R)-2-bromobutanoic acid instead of (2R)-2-bromopropionic
1
acid was used in the second synthetic step. H NMR (500 MHz,
DMSO-d6 + D2O) δ 7.38 (d, JAA′BB′ = 8.5 Hz, 2H), 7.27 (d, JAA′BB′
=
8.3 Hz, 2H), 3.81 (brd, J = 12.8 Hz, 2H), 3.67−3.69 (m, 1H), 3.52−
3.56 (m, 4H), 3.38−3.41 (m, 1H), 2.99−3.06 (m, 3H), 2.85−2.93
(m, 2H), 2.15−2.21 (m, 2H), 1.48−1.60 (m, 4H), 0.89 (t, J = 7.5 Hz,
3H); 1H NMR (700 MHz, DMSO-d6 + D2O, 348 K) δ 7.41−7.36 (m,
5-(4-((2R,5S)-5-(4-Chlorobenzyl)-2-(methoxymethyl)-
morpholino)piperidin-1-yl)-4H-1,2,4-triazol-3-amine 2,2,2-Tri-
fluoroacetate (16). 1H NMR (500 MHz, DMSO-d6) δ 7.42 (JAA′BB′
I
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Drug Annotation
= 8.3 Hz, 2H), 7.33 (JAA′BB′ = 8.2 Hz, 2H), 3.91−3.80 (m, 4H), 3.64−
3.53 (m, 3H), 3.53−3.44 (m, 3H), 3.30 (s, 3H), 3.15−3.02 (m, 3H),
2.94−2.86 (m, 1H), 2.85−2.79 (m, 1H), 2.21−2.12 (m, 2H), 1.66−
was refined with 98.39% residues in the favored region of the
Ramachandran plot. X-ray data collection and refinement statistic are
summarized in Table 3. Structural analyses and figures were prepared
using PyMol (The PyMOL Molecular Graphics System, version 2.2.1,
1
1.55 (m, 2H); H NMR (700 MHz, DMSO-d6 + D2O, 348 K) δ
7.40−7.35 (m, 2H), 7.35−7.26 (m, 2H), 3.92−3.83 (m, 3H), 3.74−
3.66 (m, 2H), 3.66−3.60 (m, 2H), 3.55−3.50 (m, 2H), 3.44−3.38
(m, 1H), 3.36−3.28 (m, 3H), 3.20−3.07 (m, 3H), 2.96−2.85 (m,
2H), 2.21−2.09 (m, 2H), 1.69−1.57 (m, 2H); 13C NMR (176 MHz,
DMSO-d6) δ 134.9, 132.4, 131.9 (2×), 129.2 (2×), 72.2, 64.9, 59.2,
58.84, 58.76, 55.6, 45.2, 45.1, 39.9, 27.4, 25.91, 25.89, 25.6 (one signal
is missing due to overlap); HRMS (ESI) m/z calcd for C20H30ClN6O2
[M + H]+ 421.2113 found 421.2131.
̈
Schrodinger, LLC).
Table 3. Data Collection and Refinement Statistics of
hCHIT1−OATD-01 Complex Crystal
a
Data Collection
space group
cell dimensions
a, b, c (Å)
α, β, γ (deg)
resolution (Å)
Rmeas (%)
I/σ(I)
CC1/2
completeness (%)
Refinement
unique reflections
work/Rfree (%)
number of atoms
protein
ligand ion
water
P 21 21 21
(2R,5S)-4-(1-(5-Amino-4H-1,2,4-triazol-3-yl)piperidin-4-yl)-
87.34, 93.76, 281.68
90.00, 90.00, 90.00
47.34−1.50 (1.59−1.50)
7.3 (104.6)
17.04 (1.70)
5-(4-chlorobenzyl)-N-methylmorpholine-2-carboxamide
1
2,2,2-Trifluoroacetate (17). H NMR (500 MHz, DMSO-d6, 348
b
K) δ 7.62 (bs, 1H), 7.35 (JAA′BB′ = 8.5 Hz, 2H), 7.30 (JAA′BB′ = 8.5 Hz,
2H), 4.05−3.97 (m, 1H), 3.82−3.74 (m, 2H), 3.63−3.54 (m, 2H),
3.30−3.11 (m, 2H), 3.02−2.82 (m, 6H), 2.66 (d, J = 4.7 Hz, 3H),
1.98−1.92 (m, 2H), 1.57−1.47 (m, 2H); 1H NMR (700 MHz,
DMSO-d6 + D2O, 348 K) δ 7.42−7.32 (m, 4H), 4.37−4.29 (m, 1H),
3.91−3.79 (m, 3H), 3.76−3.63 (m, 3H), 3.33−3.23 (m, 1H), 3.15−
3.08 (m, 2H), 3.04−2.93 (m, 2H), 2.54−2.45 (m, 3H), 2.23−2.11
(m, 2H), 1.84−1.66 (m, 2H); 13C NMR (176 MHz, DMSO-d6) δ
167.8, 134.9, 132.4, 132.0 (2×), 129.1 (2×), 73.2, 64.6, 58.7, 55.9,
45.2, 45.1, 44.7, 39.8, 27.9, 26.0, 25.9, 25.5 (one signal is missing due
to overlap); HRMS (ESI) m/z calcd for C20H29ClN7O2 [M + H]+
434.2066 found 434.2071.
b
b
c
b
99.9 (61.0)
99.6 (97.7)
b
368652
12.3/16.8
20052
17508
162 6
2370
R
Protein Expression and Purification. Gene coding human
chitotriosidase hCHIT1 catalytic domain (residues 1−386) was
cloned into the pCMV2 expression vector. Protein expression was
performed in suspension-cultivated mammalian FreeStyle 293-F HEK
cells using FectoPro transfection reagent (Polyplus transfection). The
secreted protein was purified using three-step protocol. Clarified
culture medium was precipitated with ammonium sulfate added to a
final saturation of 45%. The precipitate was collected by
centrifugation and resuspended in buffer A (20 mM Tris, pH 8.5,
10 mM NaCl, 1 mM DTT). The suspension was concentrated, and
the buffer was exchanged to buffer A to remove excess of ammonium
sulfate. The material was loaded on a 1 mL Resource Q column (GE
Healthcare) equilibrated with buffer A. After loading, the column was
washed with buffer A and bounded proteins were eluted with 1 M
sodium chloride in buffer A. The flow-through fraction containing
hCHIT1 protein was purified by size-exclusion chromatography on a
Superdex 200 Increase 10/30 GL (GE Healthcare) column in buffer B
(20 mM Tris, pH 8.5, 100 mM NaCl, 1 mM DTT). The protein
purity was estimated by SDS−PAGE followed by Coomassie Brilliant
Blue staining. The enzyme concentration was determined from the
absorption at 280 nm using an UV NanoDrop 1000 spectropho-
tometer (Thermo Scientific).
B factor
protein
23.6
21.3
18.1
41.0
ligand/ion
water
rms deviation
bond length (Å)
bond angle (deg)
0.0133
1.14
a
b
The data collection statistics are based on a single crystal. Values in
c
parentheses are for highest-resolution shell. CC1/2, correlation
coefficient between the average intensities in two parts of the
unmerged data, each with a random half of the measurements of each
unique reflection.
Enzymatic Assays. Determination of IC50 toward human and
mouse AMCase and human and mouse CHIT1 was done as described
previously.39
In Vivo Studies. The pharmacokinetics studies were performed in
accordance with protocols by the Institute for Animal Care and Use
Committee and were approved by Warsaw Local Ethics Committee
for Animal Experimentation (Approvals 79/2015, 82/2015, 452/
2017), Poland. The animals used in the bleomycin-induced
pulmonary fibrosis model were cared for following guidelines Act
on Welfare and Management of Animals, Standards Relating to the
Care and Management of Laboratory Animals and Relief of Pain, and
Guidelines for Proper Conduct of Animal Experiments, Japan.
Pharmacokinetics Measurements in Mice. The PK properties
of OATD-01 were measured in female BALB/c mice following single
intravenous bolus (3 mg/kg) or oral (10 mg/kg) administration.
Compound 9 (OATD-01) was prepared in a in 20% solutol/4%
glucose in water vehicle for intravenous bolus and oral administrations
at 5 mL/kg, respectively, and administered to 2 mice/group per time
point with samples collected up to 24 h after dose. Blood collection
was performed by cardiac puncture under anesthesia with sampling of
blood into K2EDTA anticoagulant tubes, followed by centrifugation
to obtain plasma. Samples were stored frozen at −80 °C or lower
prior to compound extraction and LC−MS/MS analysis. Pharmaco-
kinetic parameters of compound OATD-01 in mice were calculated
by standard noncompartmental modeling from the systemic plasma
concentration−time profile.
Crystallization. For crystallization, the hCHIT1 protein was
concentrated to 7.3 mg/mL in buffer B and mixed with OATD-01
ligand in 1:5 molar ratio. Single crystals were obtained for hCHIT1−
OATD-01 complex mixed with an equal volume of reservoir buffer
that contained 0.2 M potassium sodium tartrate (pH 7.2) and 31%
(w/v) PEG 3350 using the hanging drop vapor diffusion method at
18 °C. Crystals were cryoprotected in 30% glycerol and flash frozen in
liquid nitrogen.
Diffraction Data Collection, Structure Solution, and Refine-
ment. X-ray diffraction data for hCHIT1−OATD-01 crystals were
collected at beamline 14.1 at Berliner Elektronenspeicherring-
Gesellschaft fu
̈
r Synchrotronstrahlung (BESSY).34 The diffraction
data were collected to 1.5 Å resolution. Data were processed and
scaled with XDS.49 Phases were determined by molecular replacement
using Phaser-MR module in Phenix.50 The structure of one molecule
of hCHIT1 (PDB code 1WBO) was used as the starting model for
molecular replacement. Six independent copies of hCHIT1 were
found as a MR solution.
The model was improved by manual model building using Coot51
and refined in Phenix.refine with 5% of unique reflections flagged for
Rfree calculation. The structure of the hCHIT1−OATD-01 complex
Pharmacokinetics Measurements in Rats. The PK properties
of OATD-01 were evaluated in male Sprague-Dawley (SD) rats
J
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Drug Annotation
following single intravenous bolus (2 mg/kg) or oral (6 mg/kg)
administration. OATD-01 was prepared in a 10% ethanol/10%
solutol in water vehicle for intravenous bolus and oral administrations
at 1.5 mL/kg or 5 mL/kg, respectively. Blood collection was
performed at 5 min (iv only), 15 min (iv only), 30 min, 2 h, 4 h, 6
h, 12 h, and 24 h after OATD-01 administration (n = 2) serially from
same animal by tail vein puncture without anesthesia. Blood was
collected into K2EDTA anticoagulant tubes followed by centrifugation
to obtain plasma. Plasma samples were snap frozen and stored at −80
°C or lower prior to compound extraction and LC−MS/MS analysis.
Pharmacokinetic parameters of OATD-01 in rats were calculated by
standard noncompartmental modeling from the systemic plasma
concentration−time profile.
Pharmacokinetics Measurements in Dogs. The PK properties
of OATD-01 were evaluated in male Beagle dogs following single
intravenous bolus (3 mg/kg) or oral (10 mg/kg) administration.
OATD-01 was prepared in a 5% aqueous glucose vehicle for
intravenous bolus and oral administrations at 0.75 mL/kg or 2.5 mL/
kg, respectively. Blood collection was performed at 5 min, 15 min, 30
min, 1 h, 2 h, 4 h, 8 h, and 24 h after dose of OATD-01
administration, serially from same animal by jugular vein puncture
without anesthesia. Blood was collected into sodium heparin
anticoagulant tubes followed by centrifugation to obtain plasma.
The concentrations of OATD-01 in dog plasma after extraction were
determined by LC−MS/MS. Pharmacokinetic parameters were
calculated by standard noncompartmental modeling from the
systemic plasma concentration−time profile.
PK/PD Study of OATD-01 (9) in Mice. The PK/PD properties
of compound OATD-01 were evaluated in female C57BL/6N mice
(Charles River, Germany) following single (q.d.) and double (b.i.d.,
interval 8 h) dose of OATD-01 in 0.5% carboxymethyl cellulose
(CMC) administered by oral gavage at 30 mg/kg/dose. A control
group received vehicle only. At the defined time-points up to 24 h
after last compound administration, mice were preanesthetized with
isoflurane, euthanized by overdosing of pentobarbital and blood was
collected to EDTA-coated tubes, mixed, and centrifuged 2000g for 10
min in 4 °C. The collected plasma was stored in −80 °C until analysis.
The perfused lungs were homogenized using bead tissue homogenizer
(Bertin Instruments) in 10% ethanol, centrifuged, and the super-
natants were collected and stored in −80 °C until analysis. OATD-01
concentrations in plasma and tissue homogenate were assayed by
LC−MS/MS. Chitinolytic activity (PD parameter) in plasma and
tissue homogenates was measured as described previously.39
evaluated using the Ashcroft score47,48 for the semiquantitative
histological analysis.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01179.
Molecular formula strings and some data (XLS)
Permeability and physicochemical properties of OATD-
01; CYPs and transporters inhibition data; super-
imposition of the active site residues in hCHIT1
complexed to OATD-01 and OAT-177; PK profiles of
compounds 11 and 15−17; experimental procedures for
compounds 5, 7, 8, 15−17; 1H NMR spectra of
compounds 5−17; HPLC profile of compounds 3−17
(PDF)
Accession Codes
Atomic coordinates and structure factors for the reported
crystal structure have been deposited in the Protein Data Bank
(PDB code 6ZE8) and will be released upon publication.
AUTHOR INFORMATION
■
Corresponding Author
Adam Golebiowski − OncoArendi Therapeutics SA, 02-089
Warsaw, Poland; orcid.org/0000-0001-5960-2995;
Phone: +48 (22) 552 67 24; Email: a.golebiowski@
oncoarendi.com
Authors
Robert Koralewski − OncoArendi Therapeutics SA, 02-089
Warsaw, Poland
Barbara Dymek − OncoArendi Therapeutics SA, 02-089
Warsaw, Poland
Marzena Mazur − OncoArendi Therapeutics SA, 02-089
Warsaw, Poland
Piotr Sklepkiewicz − OncoArendi Therapeutics SA, 02-089
Warsaw, Poland
Bleomycin-Induced Model of Pulmonary Fibrosis. The study
applying bleomycin-induced pulmonary fibrosis model in mice was
performed at SMC Laboratories, Inc., Tokyo, Japan. On day 0, 60
mice were anesthetized with pentobarbital sodium (Kyoritsu Seiyaku,
Japan) and intratracheally administered with bleomycin (lot no.
261810, Nippon Kayaku, Japan) in saline at a dose of 3 mg/kg, in a
volume of 50 μL per animal using a Microsprayer (Penn-Century,
USA). The mice were transferred to a clean cage (resting cage) and
kept until recovery from anesthesia. The bleomycin administration
took place on two separate days, with equal numbers of mice assigned
to each day. Control mice were intratracheally administered with
saline, instead of the bleomycin, and served as the sham-control
group. In each slot, bleomycin-induced pulmonary fibrosis model
mice were divided into 5 groups of 6 mice based on the body weight
changes on the day before the start of treatment at day 7. OATD-01
and nintedanib were prepared in 0.5% carboxymethyl cellulose
solution and administered orally in a volume of 10 mL/kg. OATD-01
was administered at two doses 30 and 100 mg/kg q.d., while
nintedanib was administered at a reference dose of 100 mg/kg q.d.
Chitinolytic Activity in Plasma. The plasma samples were stored
at −80 °C prior to measurement of the chitinolytic activity in these
samples as described previously.39
Sylwia Olejniczak − OncoArendi Therapeutics SA, 02-089
Warsaw, Poland
Wojciech Czestkowski − OncoArendi Therapeutics SA, 02-089
Warsaw, Poland
Krzysztof Matyszewski − OncoArendi Therapeutics SA, 02-089
Warsaw, Poland
Gleb Andryianau − OncoArendi Therapeutics SA, 02-089
Warsaw, Poland
Piotr Niedziejko − OncoArendi Therapeutics SA, 02-089
Warsaw, Poland
Michal Kowalski − OncoArendi Therapeutics SA, 02-089
Warsaw, Poland
Mariusz Gruza − OncoArendi Therapeutics SA, 02-089
Warsaw, Poland
Bartłomiej Borek − OncoArendi Therapeutics SA, 02-089
Warsaw, Poland
Karol Jedrzejczak − OncoArendi Therapeutics SA, 02-089
Warsaw, Poland
Agnieszka Bartoszewicz − OncoArendi Therapeutics SA, 02-
089 Warsaw, Poland
̇
Elzbieta Pluta − OncoArendi Therapeutics SA, 02-089 Warsaw,
Poland
Aleksandra Rymaszewska − OncoArendi Therapeutics SA, 02-
089 Warsaw, Poland
Histopathological Analysis of Pulmonary Fibrosis. Right lung
tissues prefixed in 10% neutral buffered formalin were embedded in
paraffin and sectioned at 4 μm. For fibrosis analysis, the sections were
stained with Masson’s trichrome kit (Sigma, USA) according to the
manufacturer’s instructions. The degree of pulmonary fibrosis was
K
https://dx.doi.org/10.1021/acs.jmedchem.0c01179
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Drug Annotation
Magdalena Kania − OncoArendi Therapeutics SA, 02-089
Warsaw, Poland
Tomasz Rejczak − OncoArendi Therapeutics SA, 02-089
Warsaw, Poland
Sylwia Piasecka − OncoArendi Therapeutics SA, 02-089
Warsaw, Poland
Michal Mlacki − OncoArendi Therapeutics SA, 02-089 Warsaw,
Poland
Marcin Mazurkiewicz − OncoArendi Therapeutics SA, 02-089
Warsaw, Poland
Michał Piotrowicz − OncoArendi Therapeutics SA, 02-089
Warsaw, Poland; orcid.org/0000-0001-7214-858X
Magdalena Salamon − OncoArendi Therapeutics SA, 02-089
Warsaw, Poland
ABBREVIATIONS USED
■
AMCase, acidic mammalian chitinase; hAMCase, human acidic
mammalian chitinase; mAMCase, mouse acidic mammalian
chitinase; CHIT1, chitotriosidase; hCHIT1, human chitotrio-
sidase; mCHIT1, mouse chitotriosidase; IL-13, interleukin 13;
Th2, type 2 helper T cell; TBTU, N,N,N′,N′-tetramethyl-O-
(benzotriazol-1-yl)uronium tetrafluoroborate; DIPEA, N,N-
diisopropylethylamine; Vss, steady-state volume of distribution;
CL, clearance; AUC0−inf, area under the curve; Cmax, peak
plasma concentration of a drug after administration; Tmax, time
to reach Cmax; BAL, bronchoalveolar lavage
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Jakub Golab − OncoArendi Therapeutics SA, 02-089 Warsaw,
Poland; Department of Immunology, Medical University of
Warsaw, 02-097 Warsaw, Poland
Marcin Nowotny − Structural Biology Center, International
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Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01179
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare the following competing financial
interest(s): Some of the authors are current employees of
OncoArendi Therapeutics SA and own company stocks.
ACKNOWLEDGMENTS
■
Studies were supported by two projects: (1) “Development of
a first-in-class small molecule drug candidate for treatment of
idiopathic pulmonary fibrosis through chitotriosidase inhib-
ition” (Grant POIR.01.01.01-00-0551/15), acronym IPF, and
(2) “Preclinical research and clinical trials of a first-in class
development candidate in therapy of asthma and inflammatory
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