Tetrahydroisoquinoline-7-carboxamide Derivatives as New Selective Discoidin Domain Receptor 1 (DDR1) Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.6b00497

Acute lung injury (ALI) is a deadly symptom for serious lung inflammation. Discoidin Domain Receptor 1 (DDR1) is a new potential target for anti-inflammatory drug discovery. A new selective tetrahydroisoquinoline-7-carboxamide based DDR1 inhibitor 7ae was

Full text of DOI:10.1021/acsmedchemlett.6b00497

Letter
pubs.acs.org/acsmedchemlett
Tetrahydroisoquinoline-7-carboxamide Derivatives as New Selective
Discoidin Domain Receptor 1 (DDR1) Inhibitors
Zhen Wang,†,#,‡ Yali Zhang,§,‡ Sergio G. Bartual,∥ Jinfeng Luo,# Tingting Xu,Ψ Wenting Du,ϕ
Qiuju Xun,# Zhengchao Tu,# Rolf A. Brekken,ϕ Xiaomei Ren,# Alex N. Bullock,*,∥ Guang Liang,*,§
Xiaoyun Lu,*,† and Ke Ding*,†
†School of Pharmacy, Jinan University, 601 Huangpu Avenue West, Guangzhou 510632, China
#State Key Laboratory of Respiratory Diseases, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 190
Kaiyuan Avenue, Guangzhou 510530, China
§Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, People’s Republic
of China
ΨDepartment of Pulmonary Medicine, The Second Affiliated Hospital, Wenzhou Medical University, Wenzhou, People’s Republic of
China
∥Structural Genomics Consortium, University of Oxford, Old Road Campus, Roosevelt Drive, Oxford OX3 7DQ, U.K.
ϕDivision of Surgical Oncology, Department of Surgery and the Hamon Center for Therapeutic Oncology Research, UT
Southwestern Medical Center, Dallas, Texas 75390-8593, United States
S
* Supporting Information
ABSTRACT: Acute lung injury (ALI) is a deadly symptom for serious lung
inflammation. Discoidin Domain Receptor 1 (DDR1) is a new potential
target for anti-inflammatory drug discovery. A new selective tetrahydroiso-
quinoline-7-carboxamide based DDR1 inhibitor 7ae was discovered to
tightly bind the DDR1 protein and potently inhibit its kinase function with
a Kd value of 2.2 nM and an IC50 value of 6.6 nM, respectively. The
compound dose-dependently inhibited lipopolysaccharide (LPS)-induced
interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) release in mouse
primary peritoneal macrophages (MPMs). In addition, 7ae also exhibited
promising in vivo anti-inflammatory effects in a LPS-induced mouse ALI
model. To the best of our knowledge, this is the first “proof of concept”
investigation on the potential application of a small molecule DDR1
inhibitor to treat ALI.
KEYWORDS: DDR1, inhibitor, structure−activity relationship (SAR), inflammation, acute lung injury (ALI)
inflammatory factors in atherosclerosis, organ fibrosis, and
many other inflammatory disorders.15,16 DDR1 not only
induced secretion of inflammatory factors, but more
importantly amplified the effects by other stimuli such as pro-
inflammatory cytokines or bacterial products.17 Direct
activation of DDR1 with agonistic anti-DDR1 Ab augmented
lipopolysaccharide (LPS)-induced interleukin-1β (IL-1β),
interleukin-8 (IL-8), macrophage inflammatory protein-1α
(MIP-1α), and monocyte chemoattractant protein-1 (MCP-1)
release from granulocyte-macrophage colony-stimulating factor
(GM-CSF)-induced human monocyte-derived primary macro-
phages (GM macrophages), although it also induced low-level
release of these proteins without LPS activation.17 Besides, it
was also reported renal cortical slices of DDR1 null mice
cute lung injury (ALI) is an inflammatory condition,
Awhich is characterized by accumulation of neutrophils in
lung tissue.1−3 ALI has clinically proven to be a leading cause of
acute respiratory failure in critically ill patients for which
standard treatment is mainly supportive due to lack of effective
therapies.4 Collective investigations demonstrate that various
inflammatory cytokines, e.g., interleukin-6 (IL-6) and tumor
necrosis factor-α (TNF-α), play crucial roles in mediating,
amplifying, and perpetuating ALI processes.3,5,6 Suppressing the
overproduction of cytokines becomes a new promising strategy
for the clinical management of ALI.
Discoidin domain receptors (DDRs), i.e., DDR1 and DDR2,
are transmembrane receptor tyrosine kinases (RTKs), which
were activated by triple-helical collagens.7−9 DDR1 and DDR2
have been demonstrated to play critical roles in regulation of
cellular morphogenesis, differentiation, proliferation, adhesion,
migration, and invasion.10−14 Increasing evidence has suggested
the potential role of DDRs in regulation of secretion of
Received: December 7, 2016
Accepted: February 9, 2017
Published: February 9, 2017
© XXXX American Chemical Society
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showed a blunted response of chemokines to LPS that was
accompanied by a considerable protection against the LPS-
induced mortality, further suggesting the importance of DDR1
in mediating inflammation and fibrosis.18 Additionally, genetic
inhibition of DDR1 has been reported to alleviate bleomycin-
induced lung fibrosis by blocking p38 mitogen-activated protein
kinase (p38 MAPK) activation.19 These results were further
pharmacologically validated by utilizing our recently disclosed
selective DDR1 inhibitors in a bleomycin-induced mouse
model of lung fibrosis.20 Thus, DDR1 may serve as a novel
potential molecular target for anti-inflammatory drug discovery.
A number of well-characterized kinase inhibitors were
reported to potently suppress the functions of DDR1 and
DDR2.14 However, few of them were developed by using
DDR1 or DDR2 as the primary target. Only recently, several
selective DDR1/DDR2 inhibitors were disclosed with different
selectivity profiles (Figure 1).20−26 In 2016, we reported
Table 1. In Vitro Inhibitory Activities of Compounds 7a−7j
a
against DDR1, DRR2, Bcl-Abl, and c-Kit
Figure 1. Chemical structures of reported selective DDR1/DDR2
inhibitors.
tetrahydroisoquinoline-7-carboxamide derivatives as new selec-
tive DDR1 inhibitors and the arguably first “proof of concept”
investigation on their potential application to treat inflamma-
tion mediated pulmonary fibrosis.20 Herein, we would like to
describe the structural optimization of this class of compounds
and the efforts yielded 7ae as a highly specific DDR1 inhibitor
with promising in vivo therapeutic effect in a LPS-induced
mouse ALI model.
Tetrahydroisoquinoline-7-carboxamides 7a and 7b have been
identified as highly selective DDR1 inhibitors with IC50 values
of 442 and 24.3 nM, respectively, representing promising lead
molecules for optimization (Table 1).20 To minimize our
synthetic burden, R/S racemic molecules were first utilized for
the structure−activity relationship (SAR) exploration. R/S-
Racemic mixture (7c) of 7b displayed an IC50 value of 38.3 nM
under the experimental conditions. The other new molecules
were synthesized by using previously reported protocols
(Schemes S1−S3).20
Our previous investigation suggested that a hydrogen bond
between the pyrimidinyl moiety of 7a or 7b with the NH of
Met704 in the hinge region of DDR1 was critical for the
compounds to exhibit strong DDR1 inhibition.20 Not
surprisingly, replacement of the hinge binding 5-pyrimidinyl
group (7a) by a 4-pyrimidinyl (7d) or a 5-pyrimidinylmethyl
moiety (7e) caused total loss of the DDR1 inhibitory activity,
which likely results from unfavorable orientations of the
heterocyclic heads that prevent the formation of critical
hydrogen bonds. Our previous study also revealed that the
(R)-methyl substituent at R1 position in 7b occupied a small
hydrophobic recess formed by Val624, Ala653, and Met699 of
DDR1 to achieve potency enhancement.20 However, a
dimethyl substitution at R1 position (7g) caused 9-fold potency
loss. When the methyl group was merged to R6 position, the
resulting compound (7f) demonstrated 14-fold less potency
a
DDR1 and DDR2 experiments were performed using LANCE
ULTRA kinase assay according to the manufacturer’s instructions. Bcr-
Abl and c-Kit activity experiments were performed using the forster
resonance energy transfer (FRET)-based Z′-Lyte assay according to
the manufacturer’s instructions. All the data are mean values from at
least three independent experiments.
̈
than the corresponding racemic compound 7c. Nevertheless, all
the compounds exhibited excellent DDR1 selectivity over the
structurally related DDR2, Bcr-Abl, and c-Kit kinases. For
instance, compound 7c exhibited 47-, 55-, and 261-fold less
potency against the mentioned kinases, respectively.
A computational docking study further suggested that the
linker amide in 7b formed two pairs of strong hydrogen bonds
with Glu672 in the C-helix and Asp784 in the DFG motif of the
protein, respectively. In order to validate the contribution of
these hydrogen bonds to DDR1 inhibition, compounds with a
reversed amide (7h) or a urea linker (7j) were designed and
synthesized. It was evident that compound 7h exhibited 3-fold
less potency than that of 7b, while 7j totally abolished the
DDR1 inhibitory activity. Further cocrystal structure determi-
nation confirmed that compound 7h bound into the ATP
binding pocket of DDR1 (PDB ID: 5FDX) with a similar
conformation to that of 7b.20 The methyl group in R1 position
of 7h was also nicely accommodated in the small hydrophobic
recess formed by Val624, Ala653, and Thr701 of DDR1.
However, the reversed amide linker forced the hydrophobic
trifluoromethylphenyl group to moderately rotate away from
the C-helix, and accordingly induced longer distances between
amide moiety and the corresponding Glu672 and Asp784
residues with values of 2.5 and 2.3 Å (Figure 2A). Thus, 7h
B
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Table 2. In Vitro Inhibitory Activities of Compounds 7k−7af
a
against DDR1, DRR2, Bcl-Abl, and c-Kit
Figure 2. ((A) Cocrystal structure of 7h (colored by purple) with
DDR1 (PDB ID: 5FDX). Hydrogen bonds (H-bonds) are indicated
by yellow dashed lines. (B) Superposition of the docking conformation
of 7b (colored by cyan) into the X-ray complex of 7h with DDR1. The
H-bonds are indicated by purple and cyan dashed lines for 7h and 7b,
respectively, and the distances of H-bonds are given by purple and
black, respectively.
might form relatively weaker hydrogen bond networks with the
corresponding amino acids than that of 7b (Figure 2B). This
structural information provides a plausible explanation for the
potency loss of compound 7h. Not surprisingly, the S-isomer 7i
was 8-fold less potent than compound 7h because the methyl
group was out of the small hydrophobic cavity.
Structural investigation also revealed that the trifluorome-
thylphenyl group in 7b bound deeply into a hydrophobic
pocket formed by the DFG-out conformation, while the
hydrophilic 1-(4-methyl)piperazinyl moiety was exposed to a
solvent-accessible pocket. We first examined the contribution of
substituents at R4 position to the DDR1 inhibitory activity by
introducing various hydrophobic groups in this position. It was
found that this position well tolerated a variety of hydrophobic
substituents with different sizes. For instance, racemic
compounds harboring ethyl (7l), isopropyl (7m), tertiary
butyl (7n), and phenyl (7q) groups, exhibited IC50 values of
50.5, 49.9, 66.6, and 44.6 nM respectively, against DDR1
kinase, which were almost equipotent to 7c, whereas the
cyclopropyl (7o) or cyclohexyl (7p) substituted compounds
were obviously less potent with IC50 values of 89.0 and 123 nM,
respectively (Table 2). The results also suggested that a
lipophilic R4-substitution is crucial for the compounds to
maintain their strong inhibition against DDR1. When the CF3
group in 7c was removed, the resulting compound 7k totally
abolished its activity against the kinase.
Further investigation demonstrated that a hydrophilic group
at R2 also contributed greatly to the DDR1 kinase inhibition.
When the 1-(4-methyl) piperazinylmethyl moiety was removed,
the resulting compound 7r exhibited an IC50 value of 191 nM
against DDR1, which was approximately 5-fold less potent than
the original compound 7c. When 1-(4-methyl)-
piperazinylmethyl moiety was moved at R5 position, the
resulting compound 7s totally abolished the kinase activity. A
change of hydrophilic 1-(4-methyl) piperazinylmethyl moiety
from R2 to R3 position (7t) obviously improved the DDR1
inhibitory activity with an IC50 value of 19.9 nM, but the
selectivity over DDR2 and Bcr-Abl was significantly decreased.
It was also found that the 1-(4-methyl)piperazinyl group at R2
position could be replaced by a 1-(4-methyl)piperazinyl (7u),
1-(4-methyl)piperazinylethyl (7v), 1-(4-ethyl)-
piperazinylmethyl (7w), or 1-(4-cyclohexyl)piperazinylmethyl
(7x) to maintain strong DDR1 inhibitory activities with IC50
values ranging from 71.1 to 132 nM. However, when the 1-(4-
methyl)piperazinylmethyl group was replaced by a morpholi-
nomethyl (7y), thiomorpholinomethyl (7z), piperidin-1-
a
DDR1 and DDR2 experiments were performed using LANCE
ULTRA kinase assay according to the manufacturer’s instructions. Bcr-
Abl and c-Kit activity experiments were performed using the forster
resonance energy transfer (FRET)-based Z′-Lyte assay according to
the manufacturer’s instructions. All the data are mean values from at
least three independent experiments.
̈
ylmethyl (7aa), pyrrolidin-1-ylmethyl (7ab), or dimethylami-
nomethyl (7ac) substituent, the resulting compounds were
4.4−6.6-fold less potent than 7c. This might be rationalized by
the fact that all of the new molecules lacked a solvent-exposing
N-atom, which had been shown to form a favorable interaction
with the protein as determined by a 2.3 Å cocrystal structure.20
C
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Encouragingly, 1-methylhomopiperazinemethyl substituted
compound 7ad displayed a similar DDR1 inhibitory potency
to that of 7b. Further evaluation also revealed the R-isomer
(7ae) was 25-fold more potent than the corresponding S-
isomer (7af), with an IC50 value of 6.6 nM. Additionally,
compound 7ae also exhibited target selectivity over DDR2, Bcr-
Abl, and c-Kit with factors of 38-, 1227-, and 1515-fold,
respectively.
anti-inflammatory effect of 7ae by measuring its capability to
suppress the LPS-induced release of cytokines. It was found
that compound 7ae dose-dependently suppressed the LPS-
induced production of interleukin-6 (IL-6) and tumor necrosis
factor-α (TNF-α) in mouse primary peritoneal macrophages
(MPMs) as determined by enzyme-linked immunosorbent
assays (ELISA) (Figure 4), suggesting its promising in vitro
anti-inflammatory activity.
The binding affinity of compound 7ae for the DDR1 kinase
was further determined by using an active-site-dependent
competition binding assay (conducted by DiscoveRx Corpo-
ration, San Diego, USA).27 It was shown that compound 7ae
tightly bound to the ATP-binding site of the kinase with a
binding constant (Kd) value of 2.2 nM, validating its strong
kinase inhibition against DDR1. We further profiled the target
specificity of this compound against a panel of 468 kinases
(including 403 nonmutated kinases) using the DiscoveRx
screening platform at a concentration of 1.0 μM, which was
about 450 times higher than its Kd value with DDR1. The
results revealed that 7ae demonstrated great target specificity
with S(1) and S(10) scores of 0.015 and 0.022, respectively
(Table S1). For instance, 7ae showed almost 100% competition
rate (99.9% inhibition, ctrl% = 0.1) with DDR1 at 1.0 μM, and
it only showed obvious binding to a small portion of the kinases
investigated. The major “off targets” included abelson murine
leukemia viral oncogene (Abl), cyclin-dependent-kinase 11
(CDK 11), DDR2, Ephrin type-A receptor 8 (EPHA8),
hormonally up-regulated Neu-associated kinase (HUNK), and
nerve growth factor receptors A (TrkA), B (TrkB), and C
(TrkC). The binding affinities (Kd) or kinase inhibitory
activities (IC50) of compound 7ae against these “off targets”
were further determined by using DiscoveRx’s platform or our
in-house kinase assays (Table S2). It was shown that compound
7ae was approximately 16−1227-fold less potent against the
majority of these kinases, although it demonstrated similar
binding affinities with TrkB and TrkC to that of DDR1 kinase.
However, the compound exhibited 2.7−8.0-fold less potency
against TrkB and TrkC, respectively, in the biochemical assays
of kinase function.
Figure 4. Compound 7ae inhibited LPS-induced IL-6 and TNF-α
release in a dose-dependent manner in MPMs. Each bar represents
mean SE of 3−5 independent experiments. Statistical significance
relative to LPS is indicated: *p < 0.05, **p < 0.01.
The therapeutic potential of 7ae was further studied in a
LPS-induced ALI model.28 Compound 7ae was orally
administered at 20 or 40 mg/kg twice daily (BID) based on
its pharmacokinetics (PK) parameters (Table S3) for 7 days
prior to the administration of LPS (20 μL, 5 mg/kg). It was
evident that pretreatment with compound 7ae markedly
reduced the LPS-induced pulmonary edema as determined by
lung wet/dry (W/D) ratio (Figure 5A). Meanwhile, the total
The inhibitory effect of compound 7ae on the activation of
DDR1 and downstream p38 signal in primary human lung
fibroblasts was also investigated to confirm its strong DDR1
kinase inhibition (Figure 3). The results revealed that 7ae dose-
dependently inhibited the phosphorylation of DDR1 and the
downstream p38 protein. It was also noteworthy that
compound 7ae displayed obviously stronger signal inhibition
than that of our previously reported DDR1 inhibitor 1.
DDR1 has been implicated as a critical regulator of
inflammation.14,19 We therefore determined the potential
Figure 5. Compound 7ae attenuated LPS induced ALI in mice. (A)
Lung W/D ratio. (B) Total amount of cells in BALF. (C) Protein
concentration in BALF. (D) Hematoxylin and eosin staining.
Statistical significance relative to LPS group was indicated: *p <
0.05, **p < 0.01.
cell number and total protein concentration in bronchial
alveolar lavage fluid (BALF) were increased remarkably after
LPS administration compared to the control group (Figure
5B,C). Administration of 7ae dose-dependently inhibited the
LPS-induced increase in total cell number and total protein
concentration in BALF (Figure 5B,C). LPS treatment also
resulted in significant pulmonary congestion, thickening of
alveolar wall, and interstitial edema (Figure 5D). These
pathological changes were also markedly reduced by the
administration of 7ae (Figure 5D).
In summary, an extensive SAR investigation was conducted
based on our recently disclosed tetrahydroisoquinoline-7-
carboxamide based DDR1 inhibitors. The effort yielded a
highly promising candidate 7ae, which tightly bound the DDR1
Figure 3. Inhibitor 7ae inhibited DDR1-mediated signaling in a
concentration-dependent manner in primary human lung fibroblasts.
Lysates were probed for the indicated targets by Western blot analysis.
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protein with a Kd value of 2.2 nM and potently inhibited its
kinase function with an IC50 value of 6.6 nM. Furthermore, the
compound was notably less potent against most of the 403
nonmutated kinases when tested at 1000 nM (which is
approximately 450 times higher than its Kd value with DDR1),
indicating its great target specificity. In addition, 7ae
demonstrated reasonable pharmacokinetic properties in rats
and exhibited a promising anti-inflammatoty effect in vivo using
a LPS-induced mouse ALI model. Compound 7ae may serve as
a new lead compound for anti-inflammatory drug discovery.
Foundation-FAPESP, Takeda, and Wellcome Trust [106169/
ZZ14/Z].
ABBREVIATIONS
■
DDR, discoidin domain receptor; SAR, structure−activity
relationship; Kd, binding constant; IC50, half maximal (50%)
inhibitory concentration (IC) of a substance; LPS, lip-
opolysaccharide; IL-6, interleukin-6; TNF-α, tumor necrosis
factor-α; MPMs, mouse primary peritoneal macrophages; ALI,
acute lung injure; RTKs, receptor tyrosine kinases; aa, amino
acid; IL-1β, interleukin-1β; IL-8, interleukin-8; MIP-1α,
macrophage inflammatory protein-1α; MCP-1, monocyte
chemoattractant protein-1; GM-CSF, granulocyte-macrophage
colony-stimulating factor; p38 MAPK, P38 mitogen-activated
protein kinase; Val, valine; Ala, alanine; Met, methionine; PDB,
Protein Data Bank; DFG, Asp-Phe-Gly; Abl, abelson murine
leukemia viral oncogene; CDK 11, cyclin-dependent-kinase 11;
EPHA8, Ephrin type-A receptor 8; HUNK, hormonally up-
regulated Neu-associated kinase; TrkA, nerve growth factor
receptor A; ELISA, enzyme linked immunosorbent assays; PK,
pharmacokinetic; BID, twice daily; W/D, wet/dry; BALF,
bronchial alveolar lavage fluid; IL-1β, interleukin-1β; IL-12,
interleukin-12; ICAM-1, intercellular cell adhesion molecule-1;
VCAM-1, vascular cell adhesion molecule- 1
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.6b00497.
Synthetic procedures for compounds 7d−7af, the results
of the kinase selectivity profiling study of compound 7ae,
procedures for Kinomescan screening, in vitro kinase assay,
Western blot analysis, in vitro anti-inflammatory activity,
determination of pharmacokinetic parameters in rats, in
vivo anti-inflammatory experiments protein expression
and purification, crystallization and structure determi-
1
nation, computational study and the H and 13C NMR
spectra of compounds 7d−7af (PDF)
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Accession Codes
Atomic coordinates and experimental data for the cocrystal
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■
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AUTHOR INFORMATION
Corresponding Authors
■
*Tel: +86-20-85228025. Fax: +86-20-85224766. E-mail:
luxy2016@jnu.edu.cn (X.L.).
*E-mail: alex.bullock@sgc.ox.ac.uk (A.N.B.).
*E-mail: wzmcliangguang@163.com (G.L.).
*E-mail: dingke@jnu.edu.cn (K.D.).
ORCID
Wenting Du: 0000-0001-6765-0129
Ke Ding: 0000-0001-9016-812X
Author Contributions
‡These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors appreciate the financial support from National
Natural Science Foundation of China (21572230 and
81425021), Guangdong Province (2013A022100038,
2015A030312014 and 2015A030306042, 2016A050502041),
Guangzhou City (201508030036), and Jinan University. We
also thank Diamond Light Source for beam time (proposal
mx10619) as well as the staff of beamlines I04 and I04-1 for
their assistance with crystal testing and data collection. The
SGC is a registered charity (number 1097737) that receives
funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim,
Canada Foundation for Innovation, Eshelman Institute for
Innovation, Genome Canada, Innovative Medicines Initiative
(EU/EFPIA) [ULTRA-DD grant no. 115766], Janssen, Merck
& Co., Novartis Pharma AG, Ontario Ministry of Economic
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DOI: 10.1021/acsmedchemlett.6b00497
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX