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

: Acute lung injury (ALI) is a deadly symptom for serious lung in ﬂ ammation. Discoidin Domain Receptor 1 (DDR1) is a new potential target for anti-in ﬂ ammatory 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 K d value of 2.2 nM and an IC 50 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-in ﬂ ammatory e ﬀ ects in a LPS-induced mouse ALI model. To the best of our knowledge, this is the ﬁ rst “ proof of concept ” investigation on the potential application of a small molecule DDR1 inhibitor to treat ALI. is 4 Collective investigations demonstrate that various in ﬂ ammatory 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.


* 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 tetrahydroisoquinoline-7-carboxamide based DDR1 inhibitor 7ae was discovered to tightly bind the DDR1 protein and potently inhibit its kinase function with a K d value of 2.2 nM and an IC 50 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) A cute lung injury (ALI) is an inflammatory condition, which 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 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 proinflammatory 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 macrophages (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 showed a blunted response of chemokines to LPS that was accompanied by a considerable protection against the LPSinduced mortality, further suggesting the importance of DDR1 in mediating inflammation and fibrosis. 18 Additionally, genetic inhibition of DDR1 has been reported to alleviate bleomycininduced 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 tetrahydroisoquinoline-7-carboxamide derivatives as new selective DDR1 inhibitors and the arguably first "proof of concept" investigation on their potential application to treat inflammation 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 IC 50 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 IC 50 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 R 1 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 R 1 position (7g) caused 9-fold potency loss. When the methyl group was merged to R 6 position, the resulting compound (7f) demonstrated 14-fold less potency 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 determination 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 R 1 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

ACS Medicinal Chemistry Letters
Letter 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 trifluoromethylphenyl 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 R 4 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 IC 50 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 IC 50 values of 89.0 and 123 nM, respectively ( Table 2). The results also suggested that a lipophilic R 4 -substitution is crucial for the compounds to maintain their strong inhibition against DDR1. When the CF 3 group in 7c was removed, the resulting compound 7k totally abolished its activity against the kinase.
Further investigation demonstrated that a hydrophilic group at R 2 also contributed greatly to the DDR1 kinase inhibition. When the 1-(4-methyl) piperazinylmethyl moiety was removed, the resulting compound 7r exhibited an IC 50 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 R 5 position, the resulting compound 7s totally abolished the kinase activity. A change of hydrophilic 1-(4-methyl) piperazinylmethyl moiety from R 2 to R 3 position (7t) obviously improved the DDR1 inhibitory activity with an IC 50 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 R 2 position could be replaced by a 1-(4-methyl)piperazinyl (7u), 1 -( 4 -m e t h y l ) p i p e r a z i n y l e t h y l ( 7 v ) , 1 -( 4 -e t h y l )piperazinylmethyl (7w), or 1-(4-cyclohexyl)piperazinylmethyl (7x) to maintain strong DDR1 inhibitory activities with IC 50 values ranging from 71.1 to 132 nM. However, when the 1-(4methyl)piperazinylmethyl group was replaced by a morpholinomethyl (7y), thiomorpholinomethyl (7z), piperidin-1-ylmethyl (7aa), pyrrolidin-1-ylmethyl (7ab), or dimethylaminomethyl (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

ACS Medicinal Chemistry Letters
Letter 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 Sisomer (7af), with an IC 50 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.
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 Corporation, San Diego, USA). 27 It was shown that compound 7ae tightly bound to the ATP-binding site of the kinase with a binding constant (K d ) 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 K d 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 (K d ) or kinase inhibitory activities (IC 50 ) 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.
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 dosedependently 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 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 LPSinduced 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 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 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-7carboxamide based DDR1 inhibitors. The effort yielded a highly promising candidate 7ae, which tightly bound the DDR1

ACS Medicinal Chemistry Letters
Letter protein with a K d value of 2.2 nM and potently inhibited its kinase function with an IC 50 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 K d 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.

* S Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmedchemlett.6b00497.
Synthetic procedures for compounds 7d−7af, the results of the kinase selectivity profiling study of compound 7ae, procedures for Kinome scan 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 determination, computational study and the 1 H and 13 C NMR spectra of compounds 7d−7af (PDF)

Accession Codes
Atomic coordinates and experimental data for the cocrystal structure of 7h with DDR1 (PDB ID: 5FDX). Author Contributions ‡