Design, Synthesis, and Biological Evaluation of 3-(Imidazo[1,2- a]pyrazin-3-ylethynyl)-4-isopropyl- N-(3-((4-methylpiperazin-1-yl)methyl)-5-(trifluoromethyl)phenyl)benzamide as a Dual Inhibitor of Discoidin Domain Receptors 1 and 2

Article abstract of DOI:10.1021/acs.jmedchem.8b01045

Discoidin-domain receptors 1 and 2 (DDR1 and DDR2) are new potential targets for anti-inflammatory-drug discovery. A series of heterocycloalkynylbenzimides were designed and optimized to coinhibit DDR1 and DDR2. One of the most promising compounds, 5n, ti

Full text of DOI:10.1021/acs.jmedchem.8b01045

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Article
Design, Synthesis and Biological Evaluation of 3-(Imidazo[1,2-
a]pyrazin-3-ylethynyl)-4-isopropyl-N-(3-((4-methylpiperazin-1-
yl)methyl)-5-(trifluoromethyl)phenyl)benzamide as Dual
Inhibitors of Discoidin Domain Receptor 1 and 2 (DDR1/2)
Zhen Wang, Yali Zhang, Daniel Pinkas, Alice Fox, Jinfeng Luo, Huocong Huang,
Shengyang Cui, Qiuping Xiang, Tingting Xu, Qiuju Xun, Dongsheng zhu, Zheng-Chao Tu,
Xiaomei Ren, Rolf A Brekken, Alex N. Bullock, Guang Liang, Ke Ding, and Xiaoyun Lu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01045 • Publication Date (Web): 03 Aug 2018
Downloaded from http://pubs.acs.org on August 4, 2018
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of their duties.

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Design, Synthesis and Biological Evaluation of 3ꢀ
(Imidazo[1,2ꢀa]pyrazinꢀ3ꢀylethynyl)ꢀ4ꢀisopropylꢀNꢀ
(3ꢀ((4ꢀmethylpiperazinꢀ1ꢀyl)methyl)ꢀ5ꢀ
(trifluoromethyl)phenyl)benzamide as Dual
Inhibitors of Discoidin Domain Receptor 1 and 2
(DDR1/2)
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Zhen Wang,†,Ƹ,# Yali Zhang,§,# Daniel M. Pinkas,Φ,# Alice E. FoxΦ, JinfengLuo,Ƹ HuocongHuang,&
Shengyang Cui,Ƹ Qiuping Xiang,Ƹ TingtingXu,Ψ QiujuXun,Ƹ Dongsheng Zhu,Ƹ ZhengchaoTu,Ƹ
XiaomeiRen,† Rolf A. Brekken,& Alex N. Bullock,Φ Guang Liang,§, * Ke Ding,†, * Xiaoyun Lu†, *
†International Cooperative Laboratory of Traditional Chinese Medicine Modernization and
Innovative Drug Development of Chinese Ministry of Education (MOE), Guangzhou City Key
Laboratory of Precision Chemical Drug Development, School of Pharmacy, Jinan University,
601 Huangpu Avenue West, Guangzhou 510632, China
Ƹ Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 190 Kaiyuan
Avenue, Guangzhou 510530, China
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§Chemical Biology Research Centre, School of Pharmaceutical Sciences, Wenzhou Medical
University, Wenzhou325035, China
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ΨDepartment of Pulmonary Medicine, the 2nd affiliated hospital, Wenzhou Medical University,
Wenzhou325035, China
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&Nancy B. and Jake L. Hamon Centre for Therapeutic Oncology Research, Departments of
Surgery and Pharmacology, UT Southwestern Medical Centre at Dallas, 5323 Harry Hines
Blvd.Dallas, TX75390, USA
ΦStructural Genomics Consortium, University of Oxford, Old Road Campus Research Building,
Roosevelt Drive, Oxford OX3 7DQ, UK
Key Words: DDR1, DDR2, dual inhibitor, antiꢀinflammation, acute lung injury (ALI)
ABSTRACT: Discoidin domain receptor 1 and 2 (DDR1 and DDR2) are new potential targets
for antiꢀinflammatory drug discovery. A series of heterocycloalkynylbenzimides were designed
and optimized to coꢀinhibit DDR1 and DDR2. One of the most promising compounds, 5n,
tightly bound to DDR1 and DDR2 protein with Kd values of 7.9 and 8.0 nM, and potently
inhibited the kinases with IC50 values of 9.4 and 20.4 nM, respectively, while was significantly
less potent for a panel of 403 wildꢀtype kinases at 1.0 ꢁM. DDR1 and DDR2 kinase inhibition by
5n was validated by Western blotting analysis in primary human lung fibroblasts. The compound
also doseꢀdependently inhibited lipopolysaccharide (LPS)ꢀinduced interleukinꢀ6 (ILꢀ6) release in
vitro, and exhibited promising in vivo antiꢀinflammatory effect in a LPSꢀinduced acute lung
injury (ALI) mouse model. Compound 5n may serve as a lead compound for new antiꢀ
inflammatory drug discovery.
INTRODUCTION
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Discoidin domain receptors (DDRs), including DDR1 and DDR2, are nonꢀintegrin collagen
receptor kinases with a unique extracellular domain homologous to discoidin I protein of
Dictyostelium discoideum.1ꢀ7 DDRs are involved in the regulation of cellular morphogenesis,
differentiation, proliferation, adhesion, migration, and invasion.1ꢀ7 Collective evidence
demonstrates that DDR1 and DDR2 are critical mediators of inflammatory cytokine
secretion.2,4,7 Dysregulation of the receptors has been implicated in a variety of inflammatory
diseases, such as atherosclerosis, osteoarthritis, and organ fibrosis.1ꢀ7 For instance, collagen
induced activation of DDR1b markedly amplifies the production of interleukinꢀ8 (ILꢀ8),
macrophage inflammatory proteinꢀ1α (MIPꢀ1α), and monocyte chemoattractant proteinꢀ1 (MCPꢀ
1) by macrophages during inflammatory responses.8 Renal cortical slices of DDR1 null mice
showed a blunted response of chemokine secretion to lipopolysaccharide (LPS) that was
accompanied by protection against LPSꢀinduced mortality.9 A similar situation was also found in
bleomycinꢀinduced lung injury.10 Pharmacological inhibition of DDR1 by small molecules has
been shown to reduce inflammatory cytokines and demonstrate promising therapeutic effects in
mouse inflammation models.11,12 Activation of DDR2 was also reported to increase the
production of cytokines such as ILꢀ12, tumor necrosis factorꢀα (TNFꢀα), and interferonꢀγ (INFꢀγ)
by human dendritic cells,13,14 and contribute significantly to inflammatory disorders. Collagen I
mediated activation of DDR2 is critical for fibrogenesis and promotes resolution of lung
inflammation.15 Silencing DDR2 expression was reported to decrease alcoholꢀinduced liver
injury and fibrosis in a model for early stage alcoholic liver disease.16 Additionally, DDR2 can
mediate hepatic stellate cell activation, proliferation and migration during acute liver injury,
highlighting the profibrotic activity of DDR2.17 Other studies also reported that activation of
DDR2 by collagen I could induce the expression of DDR1 in primary human lung fibroblasts,18
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indicating potential crosstalk between these two receptors. Therefore, dual inhibition of DDR1
and DDR2 might be a promising strategy for antiꢀinflammatory drug discovery.
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A number of DDR1 and DDR2 inhibitors have been reported to date (Figure 1). However,
most of these molecules suffer from relatively low target specificity.19ꢀ21 For example, In
addition to DDR1/2, inhibitors 1 and 2 also show strong inhibition against Abl, cꢀKit, and cSrc.
4ꢀ[(4ꢀMethylpiperazinꢀ1ꢀyl)methyl]ꢀNꢀ(4ꢀmethylꢀ3ꢀ{[4ꢀ(pyridinꢀ3ꢀyl)pyrimidinꢀ2ꢀ
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yl]amino}phenyl)benzamide (imatinib), 4ꢀmethylꢀNꢀ[3ꢀ(4ꢀmethylꢀ1Hꢀimidazolꢀ1ꢀyl)ꢀ5ꢀ(trifluoro
ꢀmethyl)phenyl]ꢀ3ꢀ[(4ꢀpyridinꢀ3ꢀylpyrimidinꢀ2ꢀyl)amino]benzamide (nilotinib), Nꢀ(2ꢀchloroꢀ6ꢀ
methylphenyl)ꢀ2ꢀ[[6ꢀ[4ꢀ(2ꢀhydroxyethyl)ꢀ1ꢀpiperazinyl]ꢀ2ꢀmethylꢀ4ꢀpyrimidinyl]amino]ꢀ5ꢀ
thiazolecarboxamide (dasatinib) exhibit strong DDR1/2 inhibitory activities, but neither DDR1
nor DDR2 is their primary target.22 It is highly desirable to identify new selective DDR1/2 dual
inhibitors for biological investigation and therapeutic development. Herein, we report the design
and synthesis of heterocycloalkynylbenzimides as new selective DDR1/2 dual inhibitors with
promising in vivo therapeutic effect in a LPSꢀinduced acute lung injury (ALI) mouse model.
Figure 1. Chemical structures of the representative reported DDR1/2 inhibitors.
CHEMISTRY
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The designed DDR1/2 inhibitors were readily prepared using palladiumꢀcatalyzed
Sonogashira coupling23 as the key steps (Scheme 1). Briefly, commercially available methyl 3ꢀ
aminobenzoates (6) went through diazotization and iodization to yield intermediates 7, which
were treated with ethynyltrimethylsilane under palladium catalysis to afford the Sonogashira
coupling products, deprotection of which produced the terminal alkynes (8). Intermediates 8
were reacted with 3ꢀ((4ꢀmethylpiperazinꢀ1ꢀyl)methyl)ꢀ5ꢀ(trifluoromethyl)aniline to produce
intermediate 9 under basic conditions. Compounds 9 coupled with aromatic bromides under
Sonogashira conditions to give compounds 5aꢀ5l. Alternatively, intermediates 8 could also
couple with aromatic bromides and then react with 3ꢀ((4ꢀmethylpiperazinꢀ1ꢀyl)methyl)ꢀ5ꢀ
(trifluoromethyl)aniline under basic conditions to produce final products 5mꢀ5r.
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Scheme 1.Synthesis of Compounds 5aꢀ5r.a
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a
Reagents and conditions: (a) i) conc. H2SO4, sodium nitrite (NaNO2), H2O; ii) potassium
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8
iodide (KI), H2O, 40~70% (two steps); (b) i) trimethylsilyl acetylene, CuI,
bis(triphenylphosphine)palladium (II) chloride (PdCl2(PPh3)2), triethylamine (Et3N), acetonitrile
(MeCN), 60oC; ii) K2CO3, MeOH, room temperature (rt), 88~92% (two steps); (c) tꢀBuOK,
tetrahydrofuran (THF), ꢀ20 oC to rt, 92%; (d) HetꢀBr, CuI, PdCl2(PPh3)2, ethyl diisopropylamine
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o
(DIPEA), N, Nꢀdimethylformamide (DMF), 80 C, 40~85%; (e) CuI, PdCl2(PPh3)2, DIPEA,
o
DMF, 80 C, 64~91%; (f) 3ꢀ((4ꢀmethylpiperazinꢀ1ꢀyl)methyl)ꢀ5ꢀ(trifluoromethyl)aniline, tꢀ
BuOK, THF, ꢀ20 oC to rt, 74~87%.
MOLECULE DESIGN
Compound 4 (7rh) is a newly discovered selective DDR1 inhibitor from our group (Figure
2A),24 which potently inhibited the kinase activity of DDR1 with IC50 value of 9.7 nM. It also
exhibited modest suppressive effect on DDR2 with an IC50 value of 175 nM, but was
significantly less potent against the majority of a panel of 395 nonꢀmutated kinases. In view of
its promising target selectivity and outstanding pharmacokinetic (PK) properties,24 compound 4
was chosen as a starting lead compound for further structural modification to achieve selective
dual inhibition of DDR1 and DDR2.
DDR2 shares an approximate 57% sequence identity with DDR1 in its kinase domain (Figure
S1).7 Thus, a homologous model of DDR2 was first generated based on the DDR1 crystal
structure (PDB code 3ZOS) to provide an initial structural basis for inhibitor optimization. It was
shown that compound 4 could bind to the inactive configurations of DDR1 and DDR2 with
similar type II binding modes (Figure 2A and 2B). The inhibitor was predicted to form four
hydrogen bonds with the Met704, Glu672 and Asp784 residues of DDR1. Favorable van der
Waals contacts could also be formed in the allosteric pocket. However, compound 4 failed to
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form a hydrogen bond interaction with the corresponding Met95 of DDR2 due to the
inappropriate orientation. Further investigation also suggested that the potential stereo hindrance
between pyrazolo[1,5ꢀa]pyrimidin head of 4 and the Tyr94 and Met95 hinge residues might
contribute to significant less potency with DDR2 (Figure 2B). These preliminary computational
analyses indicated that replacement of the pyrazolo[1,5ꢀa]pyrimidine moiety with alternative
hingeꢀbinding heterocycles could be a feasible strategy to achieve dual inhibition against DDR1
and DDR2.
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A
B
C
D
Figure 2. Potential binding modes of new inhibitors with DDR1 and DDR2 proteins. A)
Molecular docking of 4 into DDR1 (PDB: 3ZOX); B) Molecular docking of 4 into the DDR2
homology model; C) Molecular docking of 5j into the DDR1 structure (PDB: 3ZOX); D)
Molecular docking of 5j into DDR2 homology model. Regular hydrogen bonds are indicated by
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black dashed lines. The distances between two atoms are indicated by red dashed line. The key
residues are shown by yellow sticks.
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RESULTS AND DISCUSSION
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Compound 5a with a pyrimidine moiety was first designed and synthesized to exhibit similar
inhibitory potencies against DDR1 and DDR2 to that of inhibitor 4. It was predicted that
introduction of a hydrogen bond donating group at the 2ꢀposition of 5a could potentially capture
an additional hydrogen bond interaction with Met95 residue of DDR2 protein to improve its
kinase inhibitory potency. Indeed, both the 2ꢀaminopyrimidine (5b) and 2ꢀ
(methylamino)pyrimidine (5c) derivatives displayed improved DDR2 inhibitory potencies with
IC50 values of 45.3 and 83.1 nM, respectively (Table 1). The inhibitory activities against DDR1
were also improved by approximately 2 fold with IC50 values of 3.9 and 5.0 nM, respectively.
Not surprisingly, when a dimethylamino group was introduced at the 2ꢀposition, the resulting
compound (5d) almost totally abolished its inhibition of DDR1 and DDR2 kinases. Although
compounds 5b and 5c exhibited good inhibitory potencies against DDR1 and DDR2, they were
almost equally potent against Abl1. The lack of target specificity makes these compounds less
attractive for further investigation. Several 5ꢀmember heterocycles were also utilized as the
potential hinge binding moieties (Table 1). Although the introduction of 3ꢀfuran (5e), 3ꢀ
thiophene (5g) or 2ꢀfuran (5f) significantly decreased the kinase inhibitory potencies against
DDR1 and DDR2, the 1ꢀmethylꢀ1Hꢀimidazole (5h) and 1, 2ꢀdimethylꢀ1Hꢀimidazole(5i)
substituted derivatives exhibited strong inhibition against DDR1 and DDR2 kinases with IC50
values of 16.0, 7.9 and 94.2, 34.1 nM, respectively. Encouraged by the results of compounds 5h
and 5i, biꢀcyclic derivatives (5j, 5k,and 5l) were further designed and synthesized by utilizing a
conformational constraint strategy.25 It was shown the cyclization significantly improved their
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potencies against both DDR1 and DDR2. Compounds 5j, 5k, and 5l suppressed the kinase
activity of DDR2 with IC50 values of 7.0, 13.1, and 10.4 nM, respectively. The compounds also
displayed strong inhibition against DDR1 with IC50 values of 3.2, 3.9, and 6.2 nM, respectively.
Thus, compound 5j represented one of the most potent dual inhibitors against DDR1/DDR2 in
these derivatives. Further computational study suggested that compound 5j bound to DDR1 and
DDR2 with a DFGꢀout conformation (Figure 2C and 2D). The imidazo[1,2ꢀa]pyrazinegroup
could fit nicely into the hinge region of DDR1 or DDR2 with a hydrogen bond interaction with
Met704 or Met95, respectively (Figure 2D). Unfortunately, compound 5j also strongly inhibited
the kinase activity of Abl1 with an IC50 value of 9.4 nM. Thus, further structural optimization
was conducted with the aim to improve the inhibitor’s target selectivity.
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It has been previously demonstrated that the “flag methyl” group was critical for Abl1
inhibitors to achieve potency against the kinase.26 Replacement of an original methyl with an
ethyl group helped us to successfully identify a highly selective DDR1 inhibitor 4.24 It was
hypothesized that the Abl1 inhibition might be diminished by optimizing the “flag ethyl” group
in compound 5j. Compounds 5m, 5n, 5o, and 5p were consequently designed and synthesized
based on this hypothesis. It was shown that the “flag alkyl” moiety indeed had a significant
impact on the inhibitory potency against Abl1. When the ethyl group in 5j was replaced with a
methyl substituent, the resulting compound 5m demonstrated 24 fold improved potency against
Abl1 with an IC50 value of 0.4 nM, but the modification barely affected the DDR1 and DDR2
inhibition. Encouragingly, the isopropyl derivative (5n) exhibited significantly decreased Abl1
inhibitory potency with an IC50 value of 494 nM, while it retained the strong inhibition against
DDR1 and DDR2 with IC50 values of 9.4 and 20.4 nM, respectively. Compound 5n represented
one of the most selective DDR1/DDR2 dual inhibitors over Abl1. The cyclic propyl compound
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5o was less selective, whereas the tertꢀbutyl derivative 5p almost totally abolished the DDR2
inhibition. The isopropyl group substituted compounds 5q and 5r also demonstrated obviously
decreased Abl1 inhibition, but their potencies against DDR2 were also apparently lost. The
relatively weak target inhibitory activities made compounds 5q and 5r less attractive for further
biological investigation.
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Table 1. In Vitro Inhibitory Activities of Compounds 5aꢀ5r against DDR1, DDR2, and Abl1a,b
Kinase inhibition (IC50, nM)
compd
Het
R
DDR1
DDR2
Abl1
Et
Et
Et
Et
Et
9.7±1.7
175±13
308±20
4
8.4± 0.4
3.9 ± 1.1
5.0± 1.4
614± 125
221± 18
45.3 ± 2.7
83.1 ± 4.2
4800± 325
281 ± 12
21.0± 0.1
30.2 ± 2.0
9700± 250
5a
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Et
Et
Et
151±17
1200± 230
695± 110
1540 ± 345
7700± 2300
>10000
5e
5f
2100± 302
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3800± 1000
>10000
5g
Et
Et
Et
16.0± 1.5
7.9± 1.1
3.2± 0.3
94.2± 4.2
34.1± 2.9
7.0± 1.6
346± 35
97.4± 9.5
9.4± 0.1
5h
5i
5j
Et
Et
3.9± 0.4
6.2± 1.0
13.1± 0.6
10.4± 0.9
11.9± 2.3
21.1± 2.6
5k
5l
Me
2.4± 0.5
3.1± 0.5
0.4± 0.1
5m
iꢀPr
9.4± 0.9
20.4± 1.7
40.2± 0.2
494± 64
202± 11
5n
5o
cꢀPr
12.6± 1.3
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tꢀBu
380 ±89
11.4± 0.3
12.5± 1.3
2200± 100
>10000
564± 33
932± 55
5p
5q
5r
iꢀPr
iꢀPr
47.4± 3.8
61.1± 3.3
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aDDR1 and DDR2 inhibition experiments were performed using the LANCE ULTRA kinase
b
assay according to the manufacturer’s instructions. Abl1 activity experiments were performed
using the FRETꢀbased Z′ꢀLyte assay according to the manufacturer’s instructions. The data are
mean values from at least four independent experiments.
To elucidate the details of the interaction of 5n with DDR1, we determined the Xꢀray coꢀ
crystal structure of their complex refined at 2.1Å resolution (Figure 3, Table S3). It was
confirmed that 5n fits nicely into the ATP binding site of DDR1 with a similar binding mode to
that predicted by the docking model of 5j (Figure 2C). The imidazo[1,2ꢀa]pyrazine moiety of 5n
was observed to form an essential hydrogen bond with Met704 in the hinge region. Two
additional hydrogen bonds were also formed between the amide and Glu672 and Asp784,
respectively, while the flag isopropyl fitted nicely into a hydrophobic pocket formed by the
residues Val624, Ala653, Lys655 and Met699.
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Figure 3. Coꢀcrystal structure of 5n with DDR1 (PDB ID: 6GWR). Hydrogen bonds are
indicated by dashed black lines. The key residues are shown as gray sticks. Compound 5n is
shown as magenta sticks.
The DDR1/DDR2 dual inhibition by 5n was validated by determining its binding affinities
with the receptors (conducted by DiscoveRx, San Diego, CA).27 It was shown that 5n bound
tightly to DDR1 and DDR2 with binding constant (Kd) values of 7.9 and 8.0 nM, respectively.
The target selectivity of 5n was further evaluated by conducting a kinase selectivity profiling
study against a panel of 468 kinases (including 403 nonꢀmutated kinases) at 1.0 ꢁM, which is
approximately 125 fold above its Kd values against the DDR1/2 targets. The results indicated that
5n exhibited good target selectivity with S(10) and S(1) scores of 0.032 and 0.017, respectively
(Supporting information).27 For instance, 5n showed 100% competition rate (100% inhibition,
ctrl% = 0) with DDR1 and DDR2 at 1.0 ꢁM, while it only showed obvious binding with a minor
portion of the kinases investigated. The major “off target” (inhibition > 90%, ctrl% < 10)
included Abl1, Ephrin typeꢀA receptor 2 (EPHA2), EPHA7, EPHA8, Ephrin typeꢀB receptor 2
(EPHB2), lymphocyteꢀspecific protein tyrosine kinase (LCK), serine/threonine kinase 10 (LOK),
angiopoietinꢀ1 receptor (TIE2), nerve growth factor receptor A (TrkA), TrkB, and TrkC. The
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binding affinities (Kd) or kinase inhibitory activities (IC50) of compound 5n against these “off
targets” were further determined by using DiscoveRx’s platform or our inꢀhouse kinase assays
(Table 2). It was shown that compound 5n was approximately 10ꢀ46 fold less potent against the
majority of the “off target” kinases. However, this compound seemed to be equally potent to
human disease related LOK, TrkB, and TrkC.28, 29 The offꢀtarget inhibition of 5n on TrkB and
TrkC was further proven by a LANCE ULTRA kinase assay, which exhibited IC50 values of 40
and 18 nM, respectively.
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The inhibitory effect of compound 5n on the activation of DDR1 and DDR2 was also
investigated in primary human lung fibroblasts (Figure 4). The results clearly revealed that 5n
doseꢀdependently inhibited the phosphorylation of DDR1 and DDR2, whereas it did not exhibit
obvious impact on the activation of cꢀAbl at concentrations of 50 and 100 nM.
Table 2. Binding affinities of compound 5n against a panel of “off target” kinases.
Kinase
ABL1
Kd value or IC50 (nM)
Kinase Kd value or IC50(nM)
494b
260a
200a
79a
LOK
TIE2
TRKA
TRKB
TRKC
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10a
370a
EPHA2
EPHA7
EPHA8
EPHB2
LCK
100 a
11a (40) b
9.3a (18) b
ꢀ
260a
180a
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b
aThe binding affinities (Kd) were determined by using DiscoveRx’s platform. The kinase
inhibitory activities (IC50) were evaluated by using “inꢀhouse” kinase assays. Reported data are
means from two independent experiments.
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Figure 4. Effects of DDR1 and DDR2 inhibition on signaling by 5n in primary human lung
fibroblasts. Lysates were probed for the indicated targets by Western blot analysis. Primary
human lung fibroblasts were treated with col I (50 µg/mL) and DMSO or different concentration
of 5n for 8 hours. Cell lysates were harvested and subjected to immunoprecipitation or Western
blot. Activation of DDR2 was detected by immunoprecipitation. Protein lysates were also probed
for pꢀDDR1, DDR1, pꢀcꢀABL, cꢀABL and tubulin.
Given the critical function of DDR1 and DDR2 in the inflammatory process, we determined
the potential antiꢀinflammatory effect of 5n by measuring its capability to suppress the LPSꢀ
induced release of a representative cytokine, ILꢀ6. It was shown that compound 5n doseꢀ
dependently inhibited LPSꢀinduced production of ILꢀ6 in mouse primary peritoneal macrophages
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(MPMs) as evaluated by using an enzyme linked immunosorbent assay (ELISA), supporting its
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promising in vitro antiꢀinflammatory activity (Figure 5).
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Figure 5. 5n inhibited LPSꢀinduced ILꢀ6 release in a doseꢀdependent manner in MPMs. Each bar
represents mean ± SE of 3ꢀ5 independent experiments. Statistical significance relative to LPS is
indicated, ** p <0.01.
The potential therapeutic potential of 5n was further investigated in a LPSꢀinduced mouse
acute lung injury (ALI) model.30 Compound 5n was orally administrated at 20 or 40 mg/kg twice
daily (BID) for seven days prior to the administration of LPS (20 ꢁL, 5 mg/kg) based on its
pharmacokinetics parameters (Table S4). Pretreatment with compound 5n reduced LPSꢀinduced
pulmonary edema as determined by lung Wet/Dry (W/D) ratio (Figure 6A). The total protein
concentrations in bronchial alveolar lavage fluid (BALF) were increased markedly after LPS
administration compared to the control group (Figures 6B), which was doseꢀdependently
inhibited by 5n (Figure 6B). LPS instillation also resulted in significant pulmonary congestion,
thickening of alveolar wall, and interstitial edema (Figure 6C). These pathological changes
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induced by LPS were significantly reduced by treatment with 5n (Figure 6C). Moreover, the
compound was well tolerated and there was no animal death or obvious body weight change
after the mice received 200 mg/kg or 400 mg/kg administration of compound 5n (Supplemented
Figure S3).
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Figure 6. Compound 5n attenuated lung pathophysiologic changes in LPSꢀchallenged mice. A)
The lung W/D ratio. B) Protein concentration in BALF. C) H&E staining. Statistical significance
relative to LPS group was indicated, * p <0.05; ** p <0.01.
Proꢀinflammatory cytokines, which are secreted in the early phase of an inflammatory
response, are critical in ALI. Thus, the levels of proꢀinflammatory cytokines in BALF and serum
were also determined. 5n effectively decreased the levels of TNFꢀα and ILꢀ6 both in BALF and
serum (Figure 7AꢀD). Additionally, LPSꢀinduced elevation of neutrophils and total cell number
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in BALF were also significantly reduced by treatment with 5n (Figure 7E and 7F). We further
examined the effect of 5n on macrophage infiltration in lung tissue through CD68
immunohistochemical staining. As shown in Figure 7G, LPS induced a significant accumulation
of macrophages in the lung, whereas there was no significant difference in the number of CD68
positive macrophages between 5nꢀtreated and control groups. Thus, we concluded that
administration of 5n resulted in significant therapeutic protection from LPSꢀinduced pulmonary
inflammation in vivo.
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Figure 7. 5n attenuated lung inflammation in LPSꢀtreated mice. A) The amount of cytokine
TNFꢀα in BALF. B) The amount of cytokine TNFꢀα in serum. C) The amount of cytokine ILꢀ6
in BALF. D) The amount of cytokine ILꢀ6 in serum. E) The amount of neutrophils in BALF. F)
The amount of total cells in BALF. G) Immunohistochemical of CD68 staining. Statistical
significance relative to LPS group was indicated, *p < 0.05, **p < 0.01.
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To confirm the antiꢀinflammatory effects of 5n, we further evaluated the potency of the
compound on inhibition of inflammatory gene expression. As shown in Figure 8, LPS increased
mRNA levels of proꢀinflammatory cytokines TNFꢀα (A), ILꢀ6 (B), ILꢀ1β(C), ILꢀ12 (D), and
adhesion molecules intercellular cell adhesion moleculeꢀ1 (ICAMꢀ1) (E), vascular cell adhesion
molecule 1 (VCAMꢀ1) (F), while compound 5n treatment significantly abrogated LPSꢀinduction
of these inflammatory markers. The data collectively support that 5n exhibits potent therapeutic
effect on ALI by downꢀregulation of proꢀinflammatory cytokine expression.
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Figure 8. Effects of 5n on the expression of inflammatory genes in lung tissue. The levels of
TNFꢀα (A), ILꢀ6 (B), ILꢀ1β (C), ILꢀ12 (D), ICAMꢀ1 (E), and VCAMꢀ1 (F) were analyzed by
RTꢀqPCR assay. Statistical significance relative to LPS group was indicated,*P < 0.05, **P <
0.01.
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CONCLUSIONS
In summary, a series of heterocycloalkynylbenzimides were optimized to coꢀinhibit both
DDR1 and DDR2. One of the most promising candidates 5n tightly bound to the DDR1 and
DDR2 proteins with Kd values of 7.9 and 8.0 nM, and potently inhibited DDR1 and DDR2
kinase function with IC50 values of 9.4 and 20.4 nM, respectively, while was obviously less
potent against the majority of the 403 wildꢀtype kinases at 1.0 ꢁM. The compound exhibited
promising antiꢀinflammatory effects in vitro and in vivo. To the best of our knowledge, this is the
first in vivo investigation of selective DDR1/2 dual inhibitors as novel antiꢀinflammation agents.
EXPERIMENTAL SECTION
General Methods for Chemistry. All reagents and solvents were used as purchased from
commercial sources without further purification. Flash chromatography was performed using
300 mesh silica gel. All reactions were monitored by TLC using silica gel plates with
1
fluorescence F254 and UV light visualization. H NMR spectra were recorded on a Bruker AVꢀ
400 spectrometer at 400 MHz or a Bruker AVꢀ500 spectrometer at 500 MHz.13C NMR spectra
were recorded on a Bruker AVꢀ500 spectrometer at 125 MHz. Coupling constants (J) are
expressed in hertz (Hz). Chemical shifts (δ) of NMR are reported in parts permillion (ppm) units
relative to an internal standard (TMS). The low or high resolution of ESIꢀMS was recorded on an
Agilent 1200 HPLCꢀMSD mass spectrometer or Applied Biosystems QꢀSTAR Elite ESIꢀLCꢀ
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MS/MS mass spectrometer, respectively. Purity of the final compounds 5a–5r was determined
by utilizing reverseꢀphase high performance liquid chromatography [HPLC, Dionex Summit
HPLC (Column: Diamonsil C18, 5.0 ꢁm, 4.6 × 250 mm (Dikma Technologies); detector: PDAꢀ
100 photodiode array; injector: ASIꢀ100 autoinjector; pump: pꢀ680A)] to be >95%. A flow rate
of 1.0 mL/min was used with mobile phase of 90% MeOH in H2O with 0.1% modifier
(ammonia, v/v).
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4ꢀEthylꢀNꢀ(3ꢀ((4ꢀmethylpiperazinꢀ1ꢀyl)methyl)ꢀ5ꢀ(trifluoromethyl)phenyl)ꢀ3ꢀ(pyrimidinꢀ5ꢀ
ylethynyl)benzamide (5a). To a solution of 4ꢀethylꢀ3ꢀethynylꢀNꢀ(3ꢀ((4ꢀmethylpiperazinꢀ1ꢀ
yl)methyl)ꢀ5ꢀ(trifluoromethyl)phenyl)benzamide (487 mg, 1.13 mmol) in DMF (10 mL) was
added 5ꢀbromopyrimidine (200 mg, 1.26 mmol), DIPEA (0.4 ml, 2.26 mmol), PdCl2(PPh3)2 (80
mg, 0.11 mmol), and CuI (21 mg, 0.11 mmol). The mixture was filled with argon and stirred at
o
80 C overnight. The mixture was poured into iceꢀwater and the precipitate was collected and
further purified by flash chromatography on silica gel to give the final compound 5a (464 mg, 81%
1
yield). H NMR(400 MHz, DMSOꢀd6) δ 10.59 (s, 1 H), 9.23 (s, 1 H), 9.07 (s, 2 H), 8.22 (d, J =
1.6 Hz, 1 H), 8.19 (s, 1 H), 8.02 (s, 1 H), 8.01 (dd, J = 8.0, 1.6 Hz, 1 H), 8.00 (d, J = 1.6 Hz, 1 H),
7.56 (d, J = 8 Hz, 1 H), 7.35 (s, 1 H), 3.54 (s, 2 H), 2.94 (q, J = 7.6 Hz, 2 H), 2.39 (br s, 4 H),
2.34 (br s, 4 H), 2.15 (s, 3 H), 1.28 (t, J = 7.6 Hz, 3 H). 13C NMR (125 MHz, DMSOꢀd6) δ165.1,
159.0, 157.4, 150.3, 141.2, 140.3, 132.7, 131.7, 129.7, 129.6 (q, J = 31.1 Hz), 129.0, 124.6 (q, J
= 270.8 Hz), 124.2, 120.9, 120.4, 119.2, 115.5(d, J = 3.6 Hz), 93.8, 87.2, 61.8, 55.1, 52.9, 46.1,
27.5, 14.9. HRMS (ESI) for C28H28F3N5O [M + H]+, calcd: 508.2319, Found: 508.2316. Purity
99.5% (tR = 12.79 min).
3ꢀ((2ꢀAminopyrimidinꢀ5ꢀyl)ethynyl)ꢀ4ꢀethylꢀNꢀ(3ꢀ((4ꢀmethylpiperazinꢀ1ꢀyl)methyl)ꢀ5ꢀ
(trifluoromethyl)phenyl)benzamide (5b). Compound 5b was prepared by following a similar
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procedure as that of 5a. Yield, 70%. H NMR(400 MHz, DMSOꢀd6) δ 10.53 (s, 1 H), 8.47 (s, 2
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H), 8.19(s, 1 H), 8.12 (s, 1H), 8.01 (s, 1 H), 7.92 (d, J = 8.0 Hz, 1 H), 7.49 (d, J = 8.0 Hz, 1 H),
7.35 (s, 1 H), 7.19 (s, 2 H), 3.54(s, 2 H), 2.89 (q, J = 7.2 Hz, 2 H), 2.40 (br s, 4 H), 2.33 (br s, 4
H), 2.15 (s, 3 H), 1.26 (t, J = 7.2 Hz, 3 H). 13C NMR (125 MHz, DMSOꢀd6) δ 165.3, 162.5,
160.6, 149.5, 141.2, 140.4, 132.6, 131.0, 129.7 (q, J = 30.6 Hz), 128.8, 128.5, 124.6 (q, J = 271.5
Hz), 124.2, 122.3, 120.4, 115.5, 106.3, 90.1, 89.5, 61.9, 55.1, 53.0, 46.1, 27.5, 14.9. HRMS (ESI)
for C28H29F3N6O [M + H]+, calcd: 523.2428, Found: 523.2414. Purity 99.6% (tR = 12.05 min).
4ꢀEthylꢀ3ꢀ((2ꢀ(methylamino)pyrimidinꢀ5ꢀyl)ethynyl)ꢀNꢀ(3ꢀ((4ꢀmethylpiperazinꢀ1ꢀ
yl)methyl)ꢀ5ꢀ(trifluoromethyl)phenyl)benzamide (5c). Compound 5c was prepared by
following a similar procedure as that of 5a. Yield, 76%.1H NMR(400 MHz, DMSOꢀd6) δ 10.53
(s, 1 H), 8.53ꢀ8.48 (m, 2 H), 8.19 (s, 1 H), 8.12 (s, 1 H), 8.02 (s, 1 H), 7.93ꢀ7.91 (m, 1 H), 7.65ꢀ
7.64 (m, 1 H), 7.50 (d, J = 8.0 Hz, 1 H), 7.35 (s, 1 H), 3.54 (s, 2 H), 2.90 (q, J = 7.6 Hz, 2 H),
2.85 (d, J = 4.8 Hz, 3 H), 2.40 (br s, 4 H), 2.34 (br s, 4 H), 2.15 (s, 3 H), 1.26 (t, J = 7.6 Hz, 3 H).
13C NMR (125 MHz, DMSOꢀd6) δ 165.3, 161.5, 160.4, 149.5, 141.1, 140.4, 132.6, 131.0, 129.7
(q, J = 31.4 Hz), 128.8, 128.5, 124.6 (q, J = 270.6 Hz), 124.2, 122.3, 120.4, 115.5, 105.8, 90.1,
89.6, 61.9, 55.1, 52.9, 46.1, 28.2, 27.5, 14.8. HRMS (ESI) for C29H31F3N6O [M + H]+, calcd:
537.2584, Found: 537.2584. Purity 98.7% (tR = 14.44 min).
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3ꢀ((2ꢀ(Dimethylamino)pyrimidinꢀ5ꢀyl)ethynyl)ꢀ4ꢀethylꢀNꢀ(3ꢀ((4ꢀmethylpiperazinꢀ1ꢀyl)
methyl)ꢀ5ꢀ(trifluoromethyl)phenyl)benzamide (5d). Compound 5d was prepared by following
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a similar procedure as that of 5a. Yield, 77%. H NMR (400 MHz, DMSOꢀd6) δ 10.53 (s, 1 H),
8.56 (s, 2 H), 8.19 (s, 1 H), 8.13 (s, 1 H), 8.01 (s, 1 H), 7.92 (d, J = 8.0 Hz, 1 H), 7.49 (d, J = 8.0
Hz, 1 H), 7.35 (s, 1 H), 3.54 (s, 2 H), 3.17(s, 6 H), 2.89 (q, J = 7.2 Hz, 2 H), 2.40 (br s, 4 H), 2.33
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(br s, 4 H), 2.15 (s, 3 H), 1.26 (t, J = 7.2 Hz, 3 H). C NMR (125 MHz, DMSOꢀd6) δ 165.2,
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160.3, 160.1, 149.5, 141.1, 140.4, 132.6, 130.9, 129.6 (q, J = 31.1 Hz), 128.8, 128.5, 124.6 (q, J
= 270.8 Hz), 124.2, 122.2, 120.4, 115.5, 105.2, 90.3, 89.5, 61.9, 55.1, 52.9, 46.1, 37.1, 27.5, 14.8.
HRMS (ESI) for C30H33F3N6O [M + H]+, calcd: 551.2741, Found: 551.2733. Purity 98.5% (tR =
21.11 min).
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4ꢀEthylꢀ3ꢀ(furanꢀ3ꢀylethynyl)ꢀNꢀ(3ꢀ((4ꢀmethylpiperazinꢀ1ꢀyl)methyl)ꢀ5ꢀ(trifluoromethyl)
phenyl)benzamide (5e). Compound 5e was prepared by following a similar procedure as that of
5a. Yield, 50%. 1H NMR(400 MHz, DMSOꢀd6) δ 10.53 (s, 1 H), 8.19ꢀ8.18 (m, 2 H), 8.13 (d, J =
1.6 Hz, 1 H), 8.01 (s, 1 H), 7.95ꢀ7.92 (m, 1 H), 7.81 (t, J = 1.6 Hz, 1 H), 7.50 (d, J = 8.0 Hz, 1 H),
7.35 (s, 1 H), 6.73 (d, J = 1.2 Hz, 1 H), 3.54(s, 2 H), 2.87 (q, J = 7.6 Hz, 2 H), 2.40 (br s, 4 H),
2.34 (br s, 4 H), 2.15 (s, 3 H), 1.26 (t, J = 7.6 Hz, 3 H). 13C NMR (125 MHz, DMSOꢀd6) δ 165.2,
149.6, 146.9, 144.5, 141.1, 140.4, 132.6, 131.1, 129.7 (q, J = 31.3 Hz), 128.8, 128.7, 124.6 (q, J
= 270.7 Hz), 124.1, 122.1, 120.3, 115.5, 112.7, 89.1, 85.4, 61.9, 55.0, 52.9, 46.0, 27.5, 14.8.
HRMS (ESI) for C28H28F3N3O2 [M + H]+, calcd: 496.2206, Found: 496.2204. Purity 98.6% (tR =
14.21 min).
4ꢀEthylꢀ3ꢀ(furanꢀ2ꢀylethynyl)ꢀNꢀ(3ꢀ((4ꢀmethylpiperazinꢀ1ꢀyl)methyl)ꢀ5ꢀ
(trifluoromethyl)phenyl)benzamide (5f). Compound 5f was prepared by following a similar
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procedure as that of 5a. Yield, 46%. H NMR(400 MHz, DMSOꢀd6) δ 10.54 (s, 1 H), 8.18ꢀ8.17
(m, 2 H), 8.01 (s, 1 H), 7.98ꢀ7.96 (m, 1 H), 7.82 (d, J = 1.6 Hz, 1 H), 7.53 (d, J = 8.4 Hz, 1 H),
7.35 (s, 1 H), 6.96 (d, J = 3.6 Hz, 1 H), 6.65ꢀ6.64 (m, 1 H), 3.54(s, 2 H), 2.87 (q, J = 7.6 Hz, 2 H),
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2.40 (br s, 4 H), 2.33 (br s, 4 H), 2.15 (s, 3 H), 1.26 (t, J = 7.6 Hz, 3 H). C NMR (125 MHz,
DMSOꢀd6) δ 165.0, 149.7, 145.6, 141.1, 140.3, 136.1, 132.7, 131.1, 129.7 (q, J = 31.3 Hz), 129.3,
129.0, 124.6 (q, J = 270.8 Hz), 124.2, 121.0, 120.4, 116.7, 115.5, 112.1, 91.5, 83.8, 61.9, 55.0,
52.9, 46.1, 27.5, 14.8. HRMS (ESI) for C28H28F3N3O2 [M + H]+, calcd: 496.2206, Found:
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496.2198. Purity 99.6% (tR = 14.91 min).
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4ꢀEthylꢀNꢀ(3ꢀ((4ꢀmethylpiperazinꢀ1ꢀyl)methyl)ꢀ5ꢀ(trifluoromethyl)phenyl)ꢀ3ꢀ(thiophenꢀ3ꢀ
ylethynyl)benzamide (5g). Compound 5g was prepared by following a similar procedure as that
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of 5a. Yield, 40%. H NMR(400 MHz, DMSOꢀd6) δ 10.54 (s, 1 H), 8.19 (s, 1 H), 8.15 (s, 1 H),
8.02 (s, 1 H), 7.94ꢀ7.93 (m, 2 H), 7.69ꢀ7.67 (m, 1 H), 7.50 (d, J = 8.0 Hz, 1 H), 7.35 (s, 1 H),
7.29 (d, J = 4.8 Hz, 1 H), 3.54(s, 2 H), 2.90 (q, J = 7.6 Hz, 2 H), 2.40 (br s, 4 H), 2.34 (br s, 4 H),
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2.15 (s, 3 H), 1.27 (t, J = 7.2 Hz, 3 H). C NMR (125 MHz, DMSOꢀd6) δ 165.2, 149.8, 141.1,
140.4, 132.6, 131.2, 130.4, 129.9, 129.7 (q, J = 31.3 Hz), 128.7, 127.5, 124.6 (q, J = 270.6 Hz),
124.2, 122.1, 121.5, 120.3, 115.5, 89.4, 86.9, 61.9, 55.1, 52.9, 46.1, 27.6, 14.8. HRMS (ESI) for
C28H28F3N3OS [M + H]+, calcd: 512.1978, Found: 512.1973. Purity 98.3% (tR = 16.21 min).
Nꢀ(4ꢀEthylꢀ3ꢀ((1ꢀmethylꢀ1Hꢀimidazolꢀ5ꢀyl)ethynyl)phenyl)ꢀ3ꢀ((4ꢀmethylpiperazinꢀ1ꢀyl)
methyl)ꢀ5ꢀ(trifluoromethyl)benzamide (5h). Compound 5h was prepared by following a
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similar procedure as that of 5a. Yield, 46%. H NMR(400 MHz, DMSOꢀd6) δ 10.54 (s, 1 H),
8.19 (s, 1 H), 8.16 (s, 1 H) 8.01 (s, 1 H), 7.95 (d, J = 8.0 Hz, 1 H), 7.82 (s, 1 H), 7.52 (d, J = 8.0
Hz, 1 H), 7.37 (s, 1 H), 7.35 (s, 1 H), 3.75 (s, 3 H), 3.54 (s, 2 H), 2.90 (q, J = 7.2 Hz, 2 H), 2.40
(br s, 4 H), 2.33 (br s, 4 H), 2.15 (s, 3 H), 1.27 (t, J = 7.6 Hz, 3 H). 13C NMR (125 MHz, DMSOꢀ
d6) δ 165.2, 149.4, 141.2, 140.3, 140.2, 134.6, 132.7, 130.9, 129.6 (q, J = 31.3 Hz), 128.9, 128.8,
124.6 (q, J = 270.7 Hz), 124.2, 121.7, 120.4, 115.5, 115.4, 94.2, 82.3, 61.8, 55.1, 52.9, 46.1, 32.2,
27.6, 14.9. HRMS (ESI) for C28H30F3N5O [M + H]+, calcd: 510.2475, Found: 510.2471. Purity
99.4% (tR = 12.93 min).
Nꢀ(3ꢀ((1,2ꢀdimethylꢀ1Hꢀimidazolꢀ5ꢀyl)ethynyl)ꢀ4ꢀethylphenyl)ꢀ3ꢀ((4ꢀmethylpiperazinꢀ1ꢀyl)
methyl)ꢀ5ꢀ(trifluoromethyl)benzamide (5i). Compound 5i was prepared by following a similar
procedure as that of 5a. Yield, 48%. 1H NMR(400 MHz, DMSOꢀd6) δ 10.5 (s, 1 H), 8.19 (s, 1 H),
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8.15 (d, J = 1.2 Hz, 1 H), 8.01 (s, 1 H), 7.95ꢀ7.92 (m, 1 H), 7.51 (d, J = 8.4 Hz, 1 H), 7.35 (s, 1
H), 7.24 (s, 1 H), 3.64 (s, 3 H), 3.54 (s, 2 H), 2.89 (q, J = 7.6 Hz, 2 H), 2.40ꢀ2.36 (m, 11 H), 2.15
(s, 3 H), 1.27 (t, J = 7.6 Hz, 3 H). 13C NMR (125 MHz, DMSOꢀd6) δ 165.2, 149.1, 147.1, 141.1,
140.4, 133.0, 132.7, 130.8, 129.6 (q, J = 31.1Hz), 128.9, 128.7, 124.6 (q, J = 270.7 Hz), 124.2,
121.8, 120.3, 115.5, 115.3, 94.0, 83.1, 61.9, 55.1, 52.9, 46.1, 31.2, 27.6, 14.9, 13.6. HRMS (ESI)
for C29H32F3N5O [M + H]+, calcd: 524.2632, Found: 524.2628. Purity 99.6% (tR = 13.87 min).
4ꢀEthylꢀ3ꢀ(imidazo[1,2ꢀa]pyrazinꢀ3ꢀylethynyl)ꢀNꢀ(3ꢀ((4ꢀmethylpiperazinꢀ1ꢀyl)methyl)ꢀ5ꢀ
(trifluoromethyl)phenyl)benzamide (5j). Compound 5j was prepared by following a similar
procedure as that of 5a. Yield, 78%.1H NMR(400 MHz, DMSOꢀd6) δ 10.58 (s, 1 H), 9.21 (d, J =
1.6 Hz, 1 H), 8.64 (dd, J =4.8, 1.6 Hz, 1 H), 8.31 (d, J = 2.0 Hz, 1 H), 8.26 (s, 1 H), 8.19 (s, 1 H),
8.14 (d, J = 4.8 Hz, 1 H), 8.01ꢀ7.98 (m, 2 H), 7.56 (d, J = 8.4 Hz, 1 H), 7.35(s, 1 H), 3.54 (s, 2 H),
2.98 (q, J = 7.6 Hz, 2 H), 2.39 (br s, 4 H), 2.33 (br s, 4 H), 2.14 (s, 3 H), 1.31 (t, J = 7.6 Hz, 3 H).
13C NMR (125 MHz, DMSOꢀd6) δ 165.7, 150.1, 144.2, 141.7, 141.2, 140.8, 140.5, 133.3, 132.1,
131.8, 130.1 (q, J = 31.1 Hz), 129.9, 129.4, 125.1 (q, J = 270.6 Hz), 124.7, 121.6, 120.9, 119.7,
116.0, 109.9, 98.6, 80.2, 62.3, 55.6, 53.4, 46.6, 28.1, 15.4. HRMS (ESI) for C30H29F3N6O [M +
H]+, calcd: 547.2428, Found: 547.2425. Purity 99.8% (tR = 11.47 min).
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4ꢀEthylꢀ3ꢀ(imidazo[1,2ꢀb]pyridazinꢀ3ꢀylethynyl)ꢀNꢀ(3ꢀ((4ꢀmethylpiperazinꢀ1ꢀyl)methyl)ꢀ5ꢀ
(trifluoromethyl)phenyl)benzamide (5k). Compound 5k was prepared by following a similar
procedure as that of 5a. Yield, 76%. 1H NMR(400 MHz, DMSOꢀd6) δ 10.58 (s, 1 H), 8.72 (dd, J
= 4.4, 1.2 Hz, 1 H), 8.26 (dd, J =9.2 , 1.6 Hz, 1 H), 8.23 (m, 2 H), 8.20 (s, 1 H), 8.03 (s, 1 H),
7.99 (dd, J =8.0, 2.0 Hz, 1 H), 7.56 (d, J = 8.0 Hz, 1 H), 7.39 (q, J = 3.6 Hz, 1 H), 7.36 (s, 1 H),
3.55 (s, 2 H), 2.99 (q, J = 7.2 Hz, 2 H), 2.40 (br s, 4 H), 2.34 (br s, 4 H), 2.16 (s, 3 H), 1.31 (t, J =
7.2 Hz, 3 H). 13C NMR (125 MHz, DMSOꢀd6) δ 165.1, 149.8, 145.4, 141.1, 140.3, 140.1, 138.5,
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132.7, 131.0, 129.6 (q, J = 31.2 Hz), 129.2, 129.0, 126.5, 124.6 (q, J = 270.7 Hz), 124.2, 121.5,
120.4, 119.4, 115.5, 112.2, 96.6, 81.1, 61.9, 55.1, 52.9, 46.1, 27.6, 15.0. HRMS (ESI) for
C30H29F3N6O [M + H]+, calcd: 547.2428, Found: 547.2424. Purity 99.5% (tR = 23.41 min).
Nꢀ(4ꢀethylꢀ3ꢀ(imidazo[1,2ꢀa]pyridinꢀ3ꢀylethynyl)phenyl)ꢀ3ꢀ((4ꢀmethylpiperazinꢀ1ꢀyl)
methyl)ꢀ5ꢀ(trifluoromethyl)benzamide (5l). Compound 5l was prepared by following a similar
procedure as that of 5a. Yield, 83%. 1H NMR(400 MHz, DMSOꢀd6) δ 10.57 (s, 1 H), 8.59 (d, J =
6.4 Hz, 1 H), 8.28 (s, 1 H), 8.20 (s, 1 H), 8.07 (s,1 H), 8.03 (s, 1 H), 7.97 (d, J = 8.0 Hz, 1 H),
7.75 (d, J = 8.8 Hz, 1 H), 7.55 (d, J = 8.0 Hz, 1 H), 7.46 (t, J = 7.2 Hz, 1 H), 7.36 (s, 1 H), 7.20 (t,
J = 6.8 Hz, 1 H), 3.54 (s, 2 H), 2.98 (q, J = 7.6 Hz, 2 H), 2.40 (br s, 4 H), 2.34 (br s, 4 H), 2.15 (s,
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3 H), 1.32 (t, J = 7.6 Hz, 3 H). C NMR (125 MHz, DMSOꢀd6) δ 165.3, 149.2, 145.8, 141.2,
140.3, 138.9, 132.8, 131.2, 129.6 (q, J = 31.0 Hz), 128.9, 127.0, 125.9, 124.6 (q, J = 270.6 Hz),
124.2, 121.7, 120.4, 1128.0, 115.5, 114.5, 108.0, 97.4, 81.2, 61.8, 55.1, 52.9, 46.1, 27.1, 14.9.
HRMS (ESI) for C31H30F3N5O [M + H]+, calcd: 546.2475, Found: 547.2465. Purity 99.6% (tR =
16.56 min).
3ꢀ(Imidazo[1,2ꢀa]pyrazinꢀ3ꢀylethynyl)ꢀ4ꢀmethylꢀNꢀ(3ꢀ((4ꢀmethylpiperazinꢀ1ꢀyl)methyl)ꢀ5ꢀ
(trifluoromethyl)phenyl)benzamide (5m). Compound 5m was prepared by following a similar
procedure as that of 9. Yield, 82%.1H NMR(400 MHz, DMSOꢀd6) δ 10.59 (s, 1 H), 9.22 (d, J =
1.2 Hz, 1 H), 8.68 (dd, J = 4.8, 1.6 Hz, 1 H), 8.32 (d, J = 1.6 Hz, 1 H), 8.28 (s, 1 H), 8.20 (s, 1 H),
8.14 (d, J = 4.4 Hz, 1 H), 8.03 (s, 1 H), 7.97 (dd, J = 8.0, 1.6 Hz, 1 H), 7.57 (d, J = 8.0 Hz, 1 H),
7.36 (s, 1 H), 3.55 (s, 2 H), 2.63 (s, 3 H), 2.40 (br s, 4 H), 2.35 (br s, 4 H), 2.16 (s, 3 H). 13C
NMR (125 MHz, DMSOꢀd6) δ 165.6, 144.3, 144.1, 141.6, 141.2, 140.8, 140.4, 133.2, 131.7,
131.6, 130.9, 130.1(d, J = 31.4 Hz), 129.6, 125.1 (d, J = 270.4 Hz), 124.7, 122.3, 120.9, 119.8,
116.0, 109.9, 98.9, 80.6, 62.3, 55.5, 53.4, 46.5, 21.4. HRMS (ESI) for C29H25f3N6O [M + H]+,
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calcd: 533.2271, Found: 533.2275. Purity 95.4% (tR = 7.78 min).
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3ꢀ(Imidazo[1,2ꢀa]pyrazinꢀ3ꢀylethynyl)ꢀ4ꢀisopropylꢀNꢀ(3ꢀ((4ꢀmethylpiperazinꢀ1ꢀyl)methyl)ꢀ
5ꢀ(trifluoromethyl)phenyl)benzamide (5n). Compound 5n was prepared by following a similar
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procedure as that of 9. Yield, 80%. H NMR(400 MHz, DMSOꢀd6) δ 10.60 (s, 1 H), 9.22 (s, 1
H), 8.64 (d, J = 4.0 Hz, 1 H), 8.32 (s, 1 H), 8.28 (s, 1 H), 8.20 (s, 1 H), 8.15 (d, J = 4.0 Hz, 1 H),
8.02 (s, 2 H), 7.62 (d, J = 8.0 Hz, 1 H), 7.36 (s, 1 H), 3.61ꢀ3.56 (m, 1 H), 3.54 (s, 2 H), 2.39 (br s,
4 H), 2.33 (br s, 4 H), 2.15 (s, 3 H), 1.34 (d, J = 6.8 Hz, 6 H). 13C NMR (125 MHz, DMSOꢀd6) δ
165.2, 153.8, 143.7, 141.2, 140.7, 140.3, 140.0, 132.8, 131.8, 131.3, 129.6 (q, J = 31.0 Hz),
129.5, 126.1, 124.6 (q, J = 270.9 Hz), 124.2, 120.7, 120.4, 119.3, 115.5, 109.4, 98.1, 79.9, 61.8,
55.1, 52.9, 46.1, 32.0, 23.2. HRMS (ESI) for C31H31F3N6O [M + H]+, calcd: 561.2584, Found:
561.2580. Purity 99.4% (tR = 14.87 min).
4ꢀCyclopropylꢀ3ꢀ(imidazo[1,2ꢀa]pyrazinꢀ3ꢀylethynyl)ꢀNꢀ(3ꢀ((4ꢀmethylpiperazinꢀ1ꢀyl)methyl)
ꢀ5ꢀ(trifluoromethyl)phenyl)benzamide (5o). Compound 5o was prepared by following a
similar procedure as that of 9. Yield, 85%. 1H NMR(400 MHz, DMSOꢀd6) δ 10.56 (s, 1 H), 9.21
(d, J = 1.2 Hz, 1 H), 8.68ꢀ8.66 (m, 1 H), 8.31 (d, J = 1.6 Hz, 1 H), 8.28 (s, 1 H), 8.19 (s, 1 H),
8.14 (d, J = 4.8 Hz, 1 H), 8.02 (s, 1 H), 7.96ꢀ7.94 (m, 1 H), 7.35 (s, 1 H), 7.15 (d, J = 8.4 Hz, 1
H), 3.54 (s, 2 H), 2.56ꢀ2.53 (m, 1 H), 2.40 (br s, 4 H), 2.34 (br s, 4 H), 2.15 (s, 3 H), 1.23ꢀ1.17
(m, 2 H), 0.93ꢀ0.89 (m, 2 H). 13C NMR (125 MHz, DMSOꢀd6) δ 165.6, 150.2, 144.1, 141.6,
141.2, 140.8, 140.5, 132.5, 131.9, 131.7, 130.1 (q, J = 31.1 Hz), 129.9, 125.1 (q, J = 270.8 Hz),
124.7, 124.5, 122.2, 120.9, 119.9, 116.0, 110.0, 99.1, 80.3, 62.3, 55.6, 53.4, 46.6, 15.0, 11.1.
HRMS (ESI) for C31H29F3N6O [M + H]+, calcd: 559.2428, Found: 559.2427. Purity 98.2% (tR =
14.51 min).
4ꢀ(tertꢀButyl)ꢀ3ꢀ(imidazo[1,2ꢀa]pyrazinꢀ3ꢀylethynyl)ꢀNꢀ(3ꢀ((4ꢀmethylpiperazinꢀ1ꢀyl)methyl)ꢀ
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5ꢀ(trifluoromethyl)phenyl)benzamide (5p). Compound 5p was prepared by following a similar
procedure as that of 9. Yield, 85%.1H NMR(400 MHz, DMSOꢀd6) δ 10.62 (s, 1 H), 9.23 (d, J =
1.2 Hz, 1 H), 8.66ꢀ8.65 (m, 1 H),8.36 (d, J = 2.0 Hz, 1 H), 8.27 (s, 1 H), 8.21 (s, 1 H), 8.16 (d, J
= 4.4 Hz, 1 H), 8.03 (s, 1 H), 8.01ꢀ7.98 (s, 1 H),7.66 (d, J = 8.8 Hz, 1 H), 7.37 (s, 1 H), 3.56 (s, 2
H), 2.42 (br s, 4 H), 2.34 (br s, 4 H), 2.16 (s, 3 H), 1.61 (s, 9 H). 13C NMR (125 MHz, DMSOꢀd6)
δ 165.6, 155.4, 144.3, 141.7, 141.2, 140.8, 140.3, 135.0, 133.3, 131.8, 130.2 (q, J = 31.4 Hz),
129.7, 127.1, 125.1 (q, J = 270.9 Hz), 124.7, 121.0, 120.7, 119.8, 116.0, 110.1, 101.3, 82.5, 62.3,
55.6, 53.4, 46.6, 36.8, 30.7. HRMS (ESI) for C32H33F3N6O [M + H]+, calcd: 575.2741, Found:
575.2735. Purity 99.1% (tR = 15.84 min).
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3ꢀ(Imidazo[1,2ꢀb]pyridazinꢀ3ꢀylethynyl)ꢀ4ꢀisopropylꢀNꢀ(3ꢀ((4ꢀmethylpiperazinꢀ1ꢀ
yl)methyl)ꢀ5ꢀ(trifluoromethyl)phenyl)benzamide (5q). Compound 5q was prepared by
following a similar procedure as that of 9. Yield, 81%. 1H NMR(400 MHz, DMSOꢀd6) δ 10.59 (s,
1 H), 8.73ꢀ8.71 (m, 1 H), 8.27ꢀ8.19 (m, 4 H), 8.02ꢀ8.00 (m, 2 H), 7.61 (d, J = 8.4 Hz, 1 H), 7.40ꢀ
7.35 (m, 2 H),3.66ꢀ3.59 (m, 1 H), 3.56 (s, 2 H), 2.40 (br s, 4 H), 2.33 (br s, 4 H), 2.15 (s, 3 H),
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1.33 (d, J = 6.8 Hz, 6 H). C NMR (125 MHz, DMSOꢀd6) δ 165.6, 154.5, 146.0, 141.7, 140.8,
140.5, 139.1, 133.2, 131.7, 130.1 (d, J = 31.4 Hz), 129.9, 127.0, 126.7, 125.1 (q, J = 270.9 Hz),
124.7, 121.6, 120.9, 120.0, 116.0, 112.7, 97.1, 81.8, 62.3, 55.6, 53.4, 46.6, 32.3, 23.6. HRMS
(ESI) for C31H31F3N6O [M + H]+, calcd: 561.2584, Found: 561.2579. Purity 99.1% (tR = 28.98
min).
3ꢀ(Imidazo[1,2ꢀa]pyridinꢀ3ꢀylethynyl)ꢀ4ꢀisopropylꢀNꢀ(3ꢀ((4ꢀmethylpiperazinꢀ1ꢀyl)methyl)ꢀ5ꢀ
(trifluoromethyl)phenyl)benzamide(5r). Compound 5r was prepared by following a similar
procedure as that of 9. Yield, 80%. 1H NMR(400 MHz, DMSOꢀd6) δ 10.58 (s, 1 H), 8.58 (d, J =
6.8 Hz, 1 H), 8.28 (d, J = 2.0 Hz, 1 H), 8.20 (s, 1 H), 8.07 (s, 1 H), 8.02 (s, 1 H), 8.00ꢀ7.98 (m, 1
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H), 7.75 (d, J = 9.2 Hz, 1 H), 7.60 (d, J = 8.4 Hz, 1 H), 7.48ꢀ7.44 (m, 1 H), 7.36 (s, 1 H), 7.22ꢀ
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139.4, 133.3, 131.9, 130.1 (q, J = 31.4 Hz), 129.6, 127.5, 126.5, 126.4, 125.1 (q, J = 270.7 Hz),
124.7, 121.8, 120.9, 118.5, 116.0, 115.0, 108.5, 97.9, 81.9, 62.3, 55.6, 53.4, 46.6, 32.5, 23.6.
HRMS (ESI) for C32H32F3N5O [M + H]+, calcd: 560.2632, Found: 560.2628. Purity 99.6% (tR =
17.89 min).
Methyl 4ꢀethylꢀ3ꢀiodobenzoate(7a).24 To a suspension of methyl 3ꢀaminoꢀ4ꢀethylbenzoate (10 g,
o
55.8 mmol) in water (100 mL) was added conc. H2SO4 (10 mL) at 0 C and then a solution of
NaNO2 (4.6 g, 67 mmol) in water (50 ml) was added dropwise. After the mixture has been stirred
o
at 0 C for 2 hours, a solution of KI (10.2 g, 61.4 mmol) in water (50 ml) was added dropwise
and the mixture was warmed to rt slowly. The reaction was quenched with saturated Na2S2O3
solution and was extracted with ethyl acetate. The organic layer was washed with brine, dried
over Na2SO4, concentrated in vacuo and further purified by flash chromatography on silica gel to
give the title compound 7a (11.3 g, 70% yield).1H NMR (400 MHz, CDCl3) δ 8.44 (d, J = 1.6 Hz,
1 H), 7.92ꢀ7.90 (m, 1 H), 7.24 (d, J = 2.8 Hz, 1 H), 3.87 (s, 3 H), 2.75 (q, J = 7.6 Hz, 2 H), 1.19
(t, J = 7.6 Hz, 3 H).
Methyl 3ꢀiodoꢀ4ꢀmethylbenzoate (7m). 24 Compound 7m was prepared by following a similar
procedure as that of 7a. Yield, 68%. 1H NMR (400 MHz, DMSOꢀd6) δ 8.46 (s, 1 H), 7.89 (dd, J
= 8.0, 1.2 Hz, 1 H), 7.29 (d, J = 8.0 Hz, 1 H), 3.90 (s, 3 H), 2.47 (s, 3 H).
Methyl 3ꢀiodoꢀ4ꢀisopropylbenzoate (7n). Compound 7n was prepared by following a similar
procedure as that of 7a. Yield, 50%. 1H NMR (400 MHz, DMSOꢀd6) δ 8.33 (d, J = 2.0 Hz, 1 H),
7.93 (dd, J = 8.0, 2.0 Hz, 1 H), 7.47 (d, J = 8.0 Hz, 1 H), 3.84 (s, 3 H), 3.20ꢀ3.10 (m, 1 H), 1.20
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