Novel Class of Colony-Stimulating Factor 1 Receptor Kinase Inhibitors Based on an o-Aminopyridyl Alkynyl Scaffold as Potential Treatment for Inflammatory Disorders

Article abstract of DOI:10.1021/acs.jmedchem.9b01912

Colony-stimulating factor 1 receptor (CSF-1R) is involved in inflammatory disorders as well as in many types of cancer. Based on high-throughput screening and docking results, we performed a detailed structure-activity-relationship study, leading to the d

Full text of DOI:10.1021/acs.jmedchem.9b01912

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Article
Novel Class of Colony-Stimulating Factor 1 Receptor Kinase
Inhibitors Based on an o‑Aminopyridyl Alkynyl Scaffold as Potential
Treatment for Inflammatory Disorders
⊥
⊥
⊥
Zhicheng Xie, Bing Wu, Yingqiang Liu, Wenming Ren, Linjiang Tong, Caigui Xiang, Aihuan Wei,
Yuanzhuo Gao, Limin Zeng, Hua Xie,* Wei Tang,* and Youhong Hu*
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ABSTRACT: Colony-stimulating factor 1 receptor (CSF-1R) is involved in inflammatory disorders as well as in many types of
cancer. Based on high-throughput screening and docking results, we performed a detailed structure−activity-relationship study,
leading to the discovery of a new series of compounds with nanomolar IC values against CSF-1R without the inhibition of
5
0
fibroblast growth factor receptors. One of the most promising hits, compound 29, potently inhibited CSF-1R kinase with an IC50
value of 0.7 nM, while it showed no inhibition to the same family member FMS-like tyrosine kinase 3. Compound 29 displayed
excellent anti-inflammatory effects against RAW264.7 macrophages indicated by significant inhibition against the activation of the
CSF-1R pathway with low cytotoxicity. In addition, compound 29 exhibited strong in vivo anti-inflammatory efficacy alongside
favorable drug characteristics. This novel compound 29 may serve as a new drug candidate with promising applications in
inflammatory disorders.
INTRODUCTION
curtail plaque formation in an Alzheimer’s disease in vivo
■
11
model. These experiments and others identify CSF-1R as a
suitable target for macrophage-induced inflammatory disease.
Thus, suppression of the CSF-1/CSF-1R axis through CSF-1R
selective small-molecule inhibitors is of great promise in anti-
inflammation therapy.
A macrophage colony-stimulating factor-1 receptor (CSF-1R,
1
also known as FMS) is a class III receptor tyrosine kinase
family, which also includes FMS-like tyrosine kinase 3 (FLT-
), stem cell factor receptor (KIT), and platelet-derived growth
factor receptor (PDGFR) α and β. CSF-1R is primarily
expressed in macrophage lineages including monocytes, tissue
macrophages, dendritic cells, and osteoclasts. It plays an
important role in signal transduction through CSF-1/CSF-1R
for the differentiation, survival, proliferation, adhesion, and
migration of monocyte/macrophage lineage cells. However,
3
A number of small-molecule CSF-1R inhibitors have been
12
13
2
reported to date. For instance, PLX3397 (1), an oral
tyrosine kinase inhibitor of CSF-1R, KIT, and mutant FLT-3
was approved by the U.S. Food and Drug Administration
(FDA) in 2019 as an orphan drug for the treatment of
pigmented villonodular synovitis (PVNS or dt-GCT). In
2
CSF-1/CSF-1R axis overactivation through downstream path-
1
4
15
addition to PLX3397, BLZ-945 (2), ARRY-382 (3), JNJ-
ways such as PI3K/AKT/NF-κB, PKC/NF-κB, and ROS/
16
17
3
−6
4
0346527 (4), PLX7486 (5, structure undisclosed), and
RAS/RAF/MAPK
inflammatory cytokines including tumor necrosis factor-α
TNF-α) and interleukin-6 (IL-6), among others. The
may lead to aberrant expression of pro-
18
DCC-3014 (6, structure undisclosed) are currently under
clinical development (Figure 1). However, most of the CSF-1R
small-molecule inhibitors in development unfortunately exhibit
poor kinase selectivity. In particular, it has been difficult for the
(
augmentation of these pro-inflammatory cytokines can
promote the pathogenesis of various types of cancer, bone
disorders, and inflammatory diseases. Various groups have
shown that CSF-1R inhibition has the potential to prevent
erosions, reduce symptoms of rheumatoid arthritis (RA) and
Received: November 26, 2019
7
−9
bone osteolysis,
and slow the progression of amyotrophic
1
0
lateral sclerosis. Moreover, recent research has demonstrated
that elimination of microglia through CSF-1R inhibition could
©
XXXX American Chemical Society
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followed by condensation with the different substituted
anilines. Compounds 34−37 (Table 4) were prepared as
illustrated in Scheme 3. The amides 48a−c were obtained by a
condensation reaction with 46 and 47. The designed linear
molecule 34 and reversed amide compound 35 were obtained
through a Sonogashira cross-coupling reaction and a Suzuki
reaction starting from the intermediates 48a and 48b with 39i
and 1-methyl-4-(4,4,5,5-tetramethyl-1,3-dioxolan-2-yl)-1H-
pyrazole, respectively. The amide bond of 48c was further
methylated or reduced to give the intermediates 49 and 50,
which is followed by two-step Pd-catalyzed cross-coupling
reaction to provide the final compounds 36 and 37.
RESULTS AND DISCUSSION
■
Figure 1. Representative chemical structure of CSF-1R kinase
inhibitors.
Design Rationale and SAR. Through screening of our in-
house kinase inhibitor library, we identified a number of
compounds that displayed CSF-1R kinase inhibition including
compounds 7 and 8 (Figure 2), which exhibited moderate
suppression of CSF-1R with an IC50 value of 46.0 ± 17.3 and
23.4 ± 8.7 nM, respectively. Interestingly, differences in
molecular structure revealed that FGFR inhibition activity
(compound 7, FGFR1 IC50 = 1.2 ± 0.4 nM; compound 8,
FGFR1-4 IC50 > 1000 nM) could be eliminated through
removal of a trifluoromethyl group and a N-methylpiperazine
tail. To identify structure-dependent differential binding
modalities, we conducted preliminary computational inves-
tigations (Figure 3). The docking results of 7 and 8 with
FGFR1 (PDB code: 4V04) (Figure 3B,C) indicate potential
binding with the inactive DFG-out conformation (represented
by Phe642) of FGFR1 utilizing a type II binding mode. The
trifluoromethyl group and N-methylpiperazine tail present in
compound 7 likely form interactions crucial for FGFR1 kinase
binding (e.g., multihalogen bonds with Ile546 and Ile639, a
cation−π interaction with His621, electrostatic interactions
with His621 and Cys619, water-bridged hydrogen bonds
between the N-methylpiperazine motif and backbone carbonyl
oxygen atom of Ile620 and His621, among others) that are not
present in compound 8 because of the removal of key
pharmacophores integral to FGFR binding (8, FGFR1-4 IC50
> 1000 nM). Compounds 7 and 8 were then docked into CSF-
1R (Figure 3D−F) to determine the factors contributing to
compound 8’s increased CSF-1R potency poststructural
modification from 7. Although both 7 and 8 fit the DFG-out
conformation of CSF-1R as represented by Phe797 (Figure
3D,F), the large N-methylpiperazine group present in
compound 7 sterically repulsed the JM (juxtamembrane)
domain (Figure 3E) while 8 recruited the JM domain and
formed novel contacts (represented by a π−π stacking with
Trp550) with JM residues upon binding to CSF-1R (Figure
3F). Consistent with prior research, unlike compound 7 which
acts as a conventional type II inhibitor, 8 binds to an
autoinhibited CSF-1R and engages the JM region on a more
physiologically common full-length CSF-1R protein state,
leading to increased potency and reduced secondary kinase
field to distinguish homologous kinases such as FLT-3, KIT,
PDGF-α/β, and so forth. Recent research demonstrates that
the simultaneous inhibition of FLT-3 and KIT could result in
profound side effects including myelosuppression and hair
depigmentation. Therefore, there is large demand to develop
novel CSF-1R inhibitors exhibiting favorable chemical
scaffolding and compound properties for inflammatory disease
therapy. Herein, we report potent and selective CSF-1R
inhibitors with an o-aminopyridylene scaffold that do not
inhibit FLT-3.
1
9
Our previously developed diarylenes-based multitargeted
20
kinase inhibitor 7 exhibited potent activity against fibroblast
growth factor receptors (FGFRs) and moderated inhibitory
activity against CSF-1R. Removal of the N-methylpiperazine
tail produced a novel CSF-1R inhibitor displaying excellent
selectivity to CSF-1R over FGFRs. Following this identi-
fication, structure−activity relationship (SAR) studies adjusted
“head”, “gate”, “linker”, and “tail” moieties, leading to the
discovery of a novel, highly potent, and selective CSF-1R
kinase inhibitor without FGFR and FLT-3 inhibition,
compound 29. In addition, 29 also displayed promising
pharmacokinetic (PK) properties and excellent anti-inflamma-
tory efficacy in both in vitro assays using RAW264.7
macrophages and in vivo investigations utilizing LPS-induced
mouse models of inflammation.
CHEMISTRY
■
Synthesis of the designed compounds 8−22 (Tables 1 and 2)
is outlined in Scheme 1. Commercially available halogenated
heteroaromatic compounds (38) were reacted with ethynyl-
trimethylsilane through Sonogashira cross-coupling reaction to
give the intermediates (39). Compounds 41 were prepared in
high yields by the condensation reaction between 4-
substituted-3-halogenobenzoic acids (40) with the correspond-
ing anilines. The coupling of 41 with 39 gave the final products
(
8−15) and intermediates (42). Compound 42 was
subsequently used in a Suzuki reaction with the boric acid
13
ester to produce the designed molecules (16−22). To explore
cross-reactivity. As a novel-type II inhibitor, 8 exhibited
excellent selectivity to CSF-1R over FGFRs (CSF-1R IC50
the SAR of R moiety (Table 3), an efficient synthetic route
=
2
was designed as shown in Scheme 2. Methyl 3-iodo-4-
methylbenzoate (43) was coupled with 5-bromo-3-
23.4 ± 8.7 nM; FGFR1-4 IC50 > 1000 nM). Consequently,
compound 8 was chosen as a lead compound for further SAR
study.
(
(trimethylsilyl)ethynyl) pyridin-2-amine (39i) at room
temperature to generate the intermediate, which was further
coupled with the related boric acid ester to produce the key
intermediate (44). The designed molecules (23−33) were
readily prepared by hydrolysis of methyl ester (44) and then
Based on the type II kinase inhibitor-binding element hybrid
21
design strategy, we initiated the SAR investigation by varying
the “head”, “gate”, “linker”, and “tail” moieties. First, as a hinge-
binding element, the isoquinoline introduced low atomic
B
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a
Table 1. SAR Exploration Focused on the Het. Moiety
a
All IC50 values (mean ± SD) against CSF-1R kinase were obtained from inhibition ratios at different concentrations (0.1−1000 nM) from two
independent experiments performed in duplicate.
economic efficiency and unfavorable drug characteristics
because of its large rigid planar conjugated system. Therefore,
we first explored the effect of “head” (Het.) moiety and
synthesized a series of compounds with varying aromatic
heterocycles (Het). The biochemical potency of newly
synthesized compounds against CSF-1R was measured and is
displayed in Table 1. The isoquinoline in 8 was replaced with
the dominant fragment imidazo[1,2-b]pyridazine in Ponatinib
and a ring-opening pyridine, leading to compound 9 and
monocyclic compound 10, respectively. However, compound
10 lost more than 9-fold activity against CSF-1R in comparison
with 8. The binding modes of PLX3397 (Figure 3A) and 8
(Figure 3C) suggest that the introduction of an amino group
can serve as a hydrogen bond donor and form an extra
hydrogen bond with a Glu664 backbone carbonyl in the hinge
domain, as in the binding mode of PLX3397. The instillation
of a −NH group in the ortho-position of a pyridine N atom
2
(11, IC = 28.5 ± 9.3 nM) could thus significantly increase
5
0
C
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Table 2. SAR Exploration Focused on the Substitution (R )
Moiety
Table 3. SAR Exploration Focused on the Substitution (R )
Moiety
1
2
a
a
compd.
R2
CSF-1R (IC , nM)
50
compd.
R1
−CH3
−H
−CH(CH3)2
−OCH3
−F
CSF-1R (IC , nM)
50
1
6
3
4
5
6
7
8
9
0
1
2
3
3,5-(OCH3)2
−H
2-OCH3
3-OCH3
4-OCH3
2-Cl
2.6 ± 0.4
6.5 ± 1.7
20.2 ± 4.6
1.4 ± 0.1
1.4 ± 0.2
318.0 ± 54.2
1.3 ± 0.3
0.7 ± 0.1
2.4 ± 0.5
3.0 ± 0.5
5.9 ± 0.1
10.8 ± 0.6
17.3 ± 1.2
1
6
7
8
9
0
1
2
2.6 ± 0.4
16.1 ± 1.9
>1000
263.0 ± 59.3
160.3 ± 22.7
6.5 ± 2.1
>1000
2
2
2
2
2
2
2
3
3
3
3
1
1
1
2
2
2
−Cl
−CF3
3-Cl
4-Cl
PLX3397
14.0 ± 2.9
3-CF3
a
All IC50 values (mean ± SD) against CSF-1R kinase were obtained
4-CF3
2,4-(Cl)2
3,4-(Cl)2
from inhibition ratios at different concentrations (0.1−1000 nM)
from two independent experiments performed in duplicate.
PLX3397
potency to that of compound 8. Structural differences between
compound 8, 11, and PLX3397 revealed an extra binding
pocket in the 5-position outwardly extending from the o-
aminopyridine ring. With this in mind, we hybridized
compound 11 and PLX3397 to create compound 12, which
yielded improvements in activity. Docking studies to CSF-1R
identified plausible binding modes of compound 12 (Figure
a
All IC50 values (mean ± SD) against CSF-1R kinase were obtained
from inhibition ratios at different concentrations (0.1−1000 nM)
from two independent experiments performed in duplicate.
to understand the importance of this substituent in the “head”
(Table 1). As shown in the binding mode of 12 (Figure 4A),
the substituent at the 5-position of o-aminopyridine ring was
bound in a relatively large hydrophilic pocket, and the
introduction of polar groups with heteroatoms or heterocycle
could increase potency because of the electronic fit or the extra
π−π stacking with Tyr665. Interestingly, changing the Cl atom
to a carbonyl group (13−15) remarkably increased the
potency against CSF-1R. Further increasing the size and
polarity of the substituent with a N-methylpyrazolyl group
(16) resulted in the increasing potency of compound 16 for
the inhibition of CSF-1R with an IC50 value of 2.6 ± 0.4 nM.
Based on the results discussed above, the SAR on “head”
(Het.) moiety indicates binding in a suitable hydrophilic
pocket of CSF-1R that is closed to the solvent-exposed areas.
4
A): “head” o-aminopyridine forms double hydrogen bonds
with the hinge regions that are integral to high potency. The
middle benzene ring passes through the “gate” area and allows
the molecule to fit in a hydrophobic pocket. As a “linker”, the
amide motif then forms critical hydrogen bonds with the
conserved DFG-motif (Asp796, Phe797, Gly798) and Glu633
in the c-helix of CSF-1R. Finally, the “tail” of compound 12 (a
benzene ring) binds to the receptor through establishing a π−π
stacking interaction with a Trp550 residue in the JM domain.
Subsequently, we discuss the detailed SAR based on
compound 12 binding modalities.
A series of compounds with different substituent groups to
replace the 5-chloro of Het. in compound 12 were synthesized
a
Scheme 1. Synthesis of Compounds 8−22
a
Reagent and conditions: (a) X = Br: trimethylsilylacetylene, Pd(PPh ) Cl , CuI, MeCN/Et N (4:1, v/v), 80 °C, 4 h; X = I: ethynyltrimethyl-
3
2
2
3
silane, Pd(PPh ) Cl , CuI, Et N, rt, 6 h; (b) HATU, DIPEA, DMF, rt, 4 h; (c) X = Br: Pd(PPh ) Cl , CuI, CsF, Et N, MeCN, 80 °C, 4 h; X = I:
Pd(PPh ) Cl , CuI, CsF, Et N, MeCN, rt, 4 h; (d) 1-methyl-4-(4,4,5,5-tetramethyl-1,3-dioxolan-2-yl)-1H-pyrazole, Pd(PPh ) , K CO , toulene/
3
2
2
3
3
2
2
3
3
2
2
3
3
4
2
3
EtOH/H O (2/1/1, v/v/v), microwave irradiation, 100 °C, 20 min.
2
D
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a
Scheme 2. Synthesis of Compounds 23−33
a
Reagent and conditions: (a) 39i, Pd(PPh ) Cl , CuI, CsF, Et N, MeCN, rt, 5 h; (b) 1-methyl-4-(4,4,5,5-tetramethyl-1,3-dioxolan-2-yl)-1H-
3
2
2
3
pyrazole, Pd(OAc) , X-phos, K CO , THF/H O (4/1, v/v), 90 °C, 6 h; (c) LiOH·H O, THF/MeOH/H O (4/1/1, v/v/v), rt, 12 h; (d) the
2
2
3
2
2
2
substituted phenylamines, HATU, DIPEA, DMF, rt, 4 h.
a
Table 4. SAR Exploration Focused on the Linker Moiety
potencies to compound 16. These results indicate that the R1
substituent binds in a specific hydrophobic binding pocket
with steric and electronic requirements. The importance of this
methyl in maintaining high potency against the CSF-1R kinase
was found by docking compound 16 into CSF-1R, as shown in
Figure 5. The methyl group of 16 was bound in a specifically
sized hydrophobic pocket formed by Val596, Ala614, Lys616
and the gatekeeper Thr663 via extensive van der Waals forces,
and hydrophobic interactions with the receptor. More
importantly, computational analysis hypothesized approximate
orthogonal conformations between the two aromatic systems
of 16 fixed by this methyl group, which aided CSF-1R binding
(Figure 5). Given the outstanding potency of inhibitor 16, the
R1 moiety (methyl group) was further fixed in the follow SAR
investigation.
Computational studies suggest that the substituted benzene
ring “tail” stabilizes the auto-inhibited CSF-1R state by forming
interactions directly with the JM domain (represented by a
π−π stacking interaction with JM-Trp550), which is crucial for
potency and selectivity improvement. To maintain this key
interaction, differential substituent effects were introduced into
the aniline ring (Table 3). 3,5-Dimethoxyl groups were first
removed to yield compound 23, which also exhibited potency
to CSF-1R. Then, we investigated the potential impact of
phenyl ring substituents by introducing electronic effects
compd.
linker
CSF-1R (IC , nM)
50
2
9
4
5
6
7
5-CONH−
4-CONH−
5-NHCO−
0.7 ± 0.1
>1000
7.2 ± 1.8
>1000
3
3
3
3
5-CON(CH )−
3
5-CH NH−
>1000
2
PLX3397
12.0 ± 1.4
a
All IC50 values (mean ± SD) against CSF-1R kinase were obtained
from inhibition ratios at different concentrations (0.1−1000 nM)
from two independent experiments performed in duplicate.
Moreover, the molecular docking results of compound 16
displayed that the N-methylpyrazolyl group increased its
receptor binding affinity because of the extra π−π stacking
interactions forming between its arene π-system with the
benzene ring of Try665 and a π−anion interaction with the
carboxyl negative charge center of Asp670 (Figure 4B).
Because 16 exhibited potent inhibitory efficacy against CSF-
(
MeO, Cl, CF ) at the 2-, 3-, and 4- positions of the benzene
1
R, we maintained the “head” moiety with 5-N-methylpyr-
3
ring (24−31). Ortho-substitution with electron-donating
group MeO yielded lowered activity as compared to 23,
whereas the meta- and para-substitution patterns of methoxyl
groups (25 and 26) improved potency against CSF-1R. A
similar phenomenon was observed with the substitution
patterns of Cl (27−29). These findings suggest that the 3-
or 4- positions might optimally suppress the kinase function of
CSF-1R, with the 4-chloro substitution (29) exhibiting the
azolyl-2-aminopyridine and further explored the “Gate” (R1)
moiety (Table 2). Compound 17 to 22 were initially
synthesized to elucidate the steric and electronic effects
present at this position. It was found that methyl group
replacement with hydrogen (17) and isopropyl (18) decreased
potency, with dramatic loss in activity upon isopropyl
introduction. This finding indicates that optimal R group
1
sizing is crucial for potency. Subsequently, substituent
variations at this position with differing electronic effects
highest potency (IC50 = 0.7 nM). In addition, CF substitution
3
(
MeO in 19, F in 20, and CF in 22) substantially decreased
at the 3- or 4- position maintained CSF-1R potency. Further
3
activity. A Cl group addition (compound 21) yielded similar
investigating di-substitutional effects at favorable meta- and
a
Scheme 3. Synthesis of Compounds 34−37
a
Reagent and conditions: (a) HATU, DIPEA, DMF, rt, 4 h; (b) MeI, NaH, THF, 0 °C to rt, 5 h; (c) BF ·THF (1 M), THF, rt, 12 h; (d) 39i,
3
Pd(PPh ) Cl , CuI, CsF, Et N, MeCN, rt, 4 h; (e) 1-methyl-4-(4,4,5,5-tetramethyl-1,3-dioxolan-2-yl)-1H-pyrazole, Pd(PPh ) , K CO , THF/
3
2
2
3
3
4
2
3
EtOH/H O (2/1/1, v/v/v), microwave irradiation, 100 °C, 20 min.
2
E
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Figure 2. Development schematic of novel o-aminopyridylene scaffold-based CSF-1R selective inhibitor 29 by structural modification of the
previously designed FGFR inhibitor 7.
Figure 3. Binding mode analysis of compounds 7 and 8 to FGFR and CSF-1R kinases. (A) Crystal structure of CSF-1R kinase domain with
PLX3397 (PDB code 4R7H). (B) Molecular docking of compound 7 into FGFR1 kinase (generated from PDB code 4V04). (C) Molecular
docking of compound 8 into FGFR1 kinase (generated from PDB code 4V04). (D) Molecular docking of compound 7 into CSF-1R kinase
(
4
generated from PDB code 4R7I). (E) Structure illustration of compound 7 displaces the JM domain of CSF-1R kinase (generated from PDB code
R7H and 4R7I). (F) Structure illustration of compound 8 recruits the JM domain of CSF-1R kinase (generated from PDB code 4R7H).
para-positions (33) did not display synergistic effects. These
findings indicate that benzene ring electron density plays a
minor role in potency, whereas benzene ring spatial orientation
to JM-Trp550 contributes greatly to activity through π−π
interactions, which requires suitable orientations between the
benzene and indole rings of JM-Trp550. The steric hindrance
of ortho-substituent groups may disrupt the optimal
orientation between the two aromatic rings to lead the
potency loss. Thus, the mono-substitution at meta- or para-
position is more favorable, especially the para-chloro
substitution (29) is optimal.
Finally, we explored the “linker” moiety by fixing the “head”,
“gate”, and “tail” as shown in Table 4. Changing the amide
from the 5-position to the 4-position to yield a linear molecule
F
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Figure 4. Schematic illustration of the SAR exploration rationale. (A) Binding mode study of compound 12 in CSF-1R kinase (generated from
PDB code 4R7H); (B) binding mode study of compound 16 in CSF-1R kinase (generated from PDB code 4R7H).
Figure 5. Molecular modeling analysis of the binding mode of compound 16 in CSF-1R kinase. (A) Binding mode study of 16 in CSF-1R kinase
(
generated from PDB code 4R7H); (B) Approximate orthogonal conformation of the diarylethyne system was controlled by the methyl group in
the middle benzene ring.
Figure 6. Binding mode study of inhibitor 29 in CSF1R kinase (generated from PDB code: 4R7H).
(
34) resulted in total loss of inhibitory potency because of its
Cys666 and 2-amino hydrogen of pyridine with the carbonyl
oxygen atom of Glu664). Beyond this, the aromatic system
formed a π−π stacking interaction with the benzene ring of
Tyr665 and the N-methyl pyrazol, which could enhance the
binding affinity through a π−anion interaction with the
negatively charged carboxyl center of Asp670. Thus, this
“head” motif likely plays a key role in CSF-1R interactions. The
alkynyl linker connects the hinge-binding element with the
back-pocket moiety because of its favorable overall orientation,
while the middle phenyl ring of 29 binds in a hydrophobic
pocket to form a π−cation interaction with the positively
charged center of Lys616. Furthermore, the methyl group of
the phenyl ring extended to another relatively small hydro-
phobic sub-pocket, which may have optimized molecular
conformations, resulting in the observed increased inhibitory
activity against CSF-1R. The amide linker, which can form two
typical hydrogen bonds with Asp796 in the DFG motif and
Glu633 in the c-helix, was critical for the binding. Therefore,
high rigidity and steric resistance upon binding to the target
protein. Furthermore, a reversed amide counterpart of the
linker in 29 (35) resulted in decreasing activity to CSF-1R.
Amide blockage by methylation of −NH (36) and carbonyl
saturation (37) also led to total loss of potency. These results
indicate that the 5-amine linker is crucial for CSF-1R activity
because of the key hydrogen bonding established between the
amide −NH and carbonyl moieties with the DFG-motif and
Glu633 in the c-helix of CSF-1R.
To better understand the interaction between the synthe-
sized compounds and CSF-1R kinase, molecular docking of the
highest potent compound 29 into the ATP binding site of
CSF-1R kinase (PDB code: 4R7H) was performed. The
binding model is depicted in Figure 6. Serving as a desirable
hinge binding element, the 5-(N-methyl)pyrazol-2-amino
pyridine formed double hydrogen bonds with the hinge region
(
the nitrogen of pyridine with amino hydrogen atom of
G
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Table 5. Selectivity Profiling of Compound 29
%
inhibition ratio @ conc.
100 nM
% inhibition ratio @ conc.
100 nM
kinases
1000 nM
10 nM
kinases
1000 nM
10 nM
a
a
a
a
a
a
a
a
a
a
a
a
VEGFR1
VEGFR2
VEGFR3
PDGFR-α
PDGFR-β
RET
c-Kit
Flt-3
EGFR
Src
Abl
89.80
95.50
100.00
92.60
87.90
96.60
98.20
5.90
78.50
83.60
69.40
83.90
70.50
77.60
100.00
0.00
36.20
23.20
27.30
54.60
14.70
42.70
70.40
ErbB2
ErbB4
IGF1R
IR
FGFR1
FGFR2
FGFR3
FGFR4
BTK
9.50
1.60
0.00
0.00
0.00
20.70
2.30
7.40
8.00
10.80
7.40
30.90
8.80
6.60
0.00
2.10
0.00
16.20
0.00
0.00
0.00
14.70
4.20
10.30
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
a
N.T.
N.T.
a
0.00
7.40
72.80
98.30
92.30
61.70
82.10
31.40
1.50
0.00
N.T.
FAK
ITK
ACK1
a
EphA2
a
N.T. = not test.
Figure 7. Cellular effects of compound 29 in the RAW264.7 cell line. (A) Compound 29 inhibited phosphorylation of CSF-1R and its downstream
signaling pathway in RAW264.7 cells as determined by Western blot analysis. (B) Compound 29 exhibited potent activity on LPS-induced TNF-α
and IL-6 release in RAW264.7 cells. Blk: Blank. Data are given as the mean ± standard error of the mean (SEM). Statistical analysis is performed by
one-way analysis of variance (ANOVA). *P < 0.05, **P < 0.01, and ***P < 0.001 compared to LPS. #P < 0.05, ##P < 0.01, and ###P < 0.001
compared to Blk. (C) Cytotoxicity of compound 29 (CC50 = 66.01 μM) and PLX3397 (CC50 = 20.52 μM) over RAW264.7 macrophages was
determined by Cell Counting Kit-8 (CCK-8).
the modification (as shown in Table 4) of this amide linker
could destroy the forming of these crucial hydrogen bonds,
leading to the significant loss of activity. Furthermore, we
identified a π−π stacking interaction between the terminal
phenyl ring with a Trp550 residue in the JM domain of suitable
conformation, which required a suitable spatial conformation
of these two aromatic rings. Because of the steric effect, the
ortho-substituent groups (represented by 24 and 27) are
unfavorable for this spatial requirement, whereas, the para-
chloro substitution (29) was optimal and exhibited the highest
potency.
Selectivity Profiling of Compound 29. The inhibitory
activities of 29 against the other protein kinases, especially the
other members of the class III growth factor receptor family,
that is, FLT-3, KIT, PDGF-α and β, were also evaluated
utilizing our in-house kinase assays (Table 5). The results
H
https://dx.doi.org/10.1021/acs.jmedchem.9b01912
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
a
Table 6. PK Profile of Compound 29 in Rats
t1/2 (h)
Tmax (h)
Cmax (ng/mL)
AUC0−t (h·ng/mL)
CL_obs (mL/min/kg)
MRTINF_obs (h)
Vss_obs (L/kg)
F (%)
p.o.
5
i.v.
2
mg/Kg
2.21
3.33
4.00
165.60
634.02
728.93
5.42
2.30
34.80
mg/Kg
45.98
6.34
a
Determined using LC/MS/MS. AUC0−t: the area under the concentration time curve; MRT: mean residence time; t1/2: elimination half-life; Tmax
the peak time; Cmax: the peak concentration; CL: clearance; V : steady-state distribution volume; F: absolute bioavailability.
:
ss
Figure 8. Inflammation cytokine concentrations in serum of the LPS-induced mice model. The mice were orally administrated with PLX3397 (40
mg/kg), compound 29 (20 and 40 mg/kg) at 4 h before intraperitoneal injection with LPS (n = 6, female). The concentration of cytokines in
serum was detected at 1.5 h for TNF-α and 3 h for IL-6 by ELISA assay. Data are given as mean ± SEM. Statistical analysis is performed by one-
way analysis of variance (ANOVA). *P < 0.05, **P < 0.01, and ***P < 0.001 compared to vehicle. #P < 0.05, ##P < 0.01, and ###P < 0.001
compared to normal.
showed that 29 potently inhibited the activity of KIT
inhibition rate = 70.4% at 10 nM) and PDGFR-α (inhibition
Sprague-Dawley (SD) rats, following intravenous (i.v.) and
oral (p.o.) administration (Table 6). The results demonstrated
that 29 exhibited favorable drug characteristics with acceptable
bioavailability (F = 34.8%), moderate half-life (t1/2 = 2.21 h),
and drug exposure (AUC0−t = 634.02 h·ng/mL) using 5 mg/kg
oral administration. These PK properties indicate that 29 is
suitable for oral administration. In addition, the blockade effect
of 29 on the hERG channel was evaluated using the Qpatch
Automatic Patch-Clamp assays; compound 29 exhibited low
hERG-potassium-ion-channel inhibition with an IC50 value of
>40 μM (Figure S1).
(
rate = 54.6% at 10 nM). This result is not surprising,
considering that KIT and PDGFRα/β kinases belong to the
class III receptor tyrosine kinase family with highly conserved
ATP binding pockets. Interestingly, 29 exhibited no inhibition
against FLT-3, FGFR1-4, EGFR, ErbB2/4, and so forth.
kinases at 1 μM concentration, which could greatly reduce the
potential toxic effects induced by the simultaneous inhibition
of FLT-3 and KIT as previously mentioned.
Cellular Evaluation of Compound 29. In light of its
strong potency and reasonable target selectivity, 29 was
selected as a representative molecule for further cellular
evaluation. We first investigated the CSF-1R-mediated signal-
ing pathway suppression by compound 29 in RAW264.7 cells,
which harbors high kinase levels (Figure 7A). We found dose-
dependent inhibition of CSF-1R and the downstream
mediator, Akt, in the presence of compound 29 with significant
inhibition starting at 10 nM.
CSF-1/CSF-1R signaling-mediated elevated expression of
TNF-α, IL-6, and other proinflammatory cytokines contributes
greatly to the pathogenesis in inflammatory diseases. Signaling
interruptions with CSF-1R inhibitors have shown therapeutic
effects in several inflammatory disorders by reducing the
expression of TNF-α, IL-6, and other proinflammatory
cytokines. Therefore, the potential anti-inflammation effects
of compound 29 were investigated by measuring its capacity to
suppress the release of TNF-α and IL-6 in macrophages
We finally evaluated the in vivo anti-inflammation efficacy of
29 in LPS-induced mouse models of inflammation (Figure 8).
LPS injection in mice resulted in elevation of inflammatory
cytokines, including TNF-α and IL-6, which peak at 1.5 and 3
22
h postinjection, respectively. The compound 29 was orally
administered 4 h prior to LPS injection. Treatment with
compound 29 prominently reduced serum TNF-α and IL-6
concentration as compared with vehicle controls. Therefore,
the results indicate that CSF-1R inhibition by compound 29
can effectively attenuate inflammation and has the potential to
treat inflammatory diseases.
CONCLUSIONS
■
In summary, we started SAR exploration of a novel type II
kinase inhibitor 8, which identified novel o-aminopyridyl
alkynyl scaffold-based CSF-1R selective inhibitors targeting the
autoinhibited-state CSF-1R kinase without FGFR inhibition.
Based on the inhibitor-binding element hybrid design strategy,
we initiated the detailed SAR investigation by varying the
“head”, “gate”, “linker”, and “tail” moieties, leading to the
discovery of a series of potent CSF-1R inhibitors. The
(
Figure 7B). Compound 29 suppressed TNF-α (IC50 = 110
nM) and IL-6 (IC50 = 130 nM) production in RAW264.7
macrophages with low toxicity (CC = 66.01 μM, Figure 7C),
50
which is a stark improvement to that of PLX3397 (CC50
0.52 μM; TNF-α IC50 = 400 nM; IL-6 IC50 = 430 nM),
indicating excellent anti-inflammatory effects in vitro.
=
2
instillation of a −NH group in the ortho-position of a
2
In Vivo PK Evaluation of Compound 29. The
preliminary in vivo PK properties of 29 were investigated in
pyridine (head) of this scaffold forms an extra hydrogen bond
with a Glu664 backbone carbonyl in the hinge domain of CSF-
I
https://dx.doi.org/10.1021/acs.jmedchem.9b01912
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
R and can increase potency against CSF-1R significantly. One
pubs.acs.org/jmc
Article
1H NMR (400 MHz, DMSO-d
(s, 1H), 8.36 (d, J = 8.5 Hz, 1H), 8.28 (s, 1H), 8.25 (s, 1H), 8.00 (t, J
7.6 Hz, 1H), 7.94 (d, J = 8.0 Hz, 1H), 7.83 (t, J = 7.5 Hz, 1H), 7.56
d, J = 8.0 Hz, 1H), 7.11 (d, J = 2.0 Hz, 2H), 6.28 (s, 1H), 3.75 (s,
H), 2.67 (s, 3H). C NMR (126 MHz, DMSO-d ): δ 164.46,
60.37, 152.66, 146.27, 143.51, 140.73, 134.41, 132.78, 132.10,
): δ 10.26 (s, 1H), 9.39 (s, 1H), 8.84
6
1
of the most promising compounds 29 had the highest kinase
inhibitor potency against CSF-1R (IC50 = 0.7 ± 0.1 nM).
Further kinome-screen data revealed that 29 displayed a good
relative selectivity profile, which is the loss of FLT-3 potency.
The effects of 29 on LPS-stimulated RAW-264.7 macrophage
cells were evaluated in vitro, which exhibited low cellular
toxicity and potent inhibition of the CSF-1R-mediated
signaling pathways, resulting a significant suppression of the
release of the inflammatory cytokines. In addition, compound
=
(
6
1
1
1
1
3
6
30.90, 129.97, 128.53, 128.50, 128.44, 127.43, 124.23, 121.84,
14.70, 98.58, 95.82, 94.63, 88.85, 55.11, 20.61. HRMS (ESI, m/z)
+
for C H N O [M + H] : calcd, 423.1703; found, 423.1723. HPLC
2
7
22
2
3
analysis: 22.78 min, 98.92% purity.
N-(3,5-Dimethoxyphenyl)-3-(imidazo[1,2-b]pyridazin-3-ylethyn-
yl)-4-methylbenzamide (9). It is obtained as a brown solid (yield
29 displayed acceptable PK properties in rats without the
1
7
1
8
7
2
6%); mp 95−96 °C. H NMR (400 MHz, DMSO-d ): δ 10.26 (s,
inhibition of hERG. Furthermore, compound 29 potent in vivo
anti-inflammatory effects make it a promising candidate as
potential treatment for inflammatory disorders. Further
preclinical evaluation of compound 29 is currently being
conducting.
6
H), 8.73 (dd, J = 4.4, 1.4 Hz, 1H), 8.27 (dd, J = 9.2, 1.5 Hz, 1H),
.24 (s, 1H), 8.18 (d, J = 1.8 Hz, 1H), 7.92 (dd, J = 8.0, 1.8 Hz, 1H),
.53 (d, J = 8.1 Hz, 1H), 7.40 (dd, J = 9.2, 4.4 Hz, 1H), 7.11 (d, J =
1
3
.2 Hz, 2H), 6.27 (t, J = 2.2 Hz, 1H), 3.74 (s, 6H), 2.60 (s, 3H).
NMR (151 MHz, DMSO-d ): δ 164.39, 160.38, 145.10, 143.25,
C
6
1
1
40.79, 139.67, 138.26, 132.67, 130.12, 130.03, 128.49, 126.14,
EXPERIMENTAL SECTION
General Methods for Chemistry. Unless otherwise noted, all
reagents and solvents employed were purchased commercially and
used as received. All reactions involving air- or moisture-sensitive
21.70, 119.11, 111.74, 98.54, 96.50, 95.83, 81.08, 55.14, 20.40.
■
+
HRMS (ESI, m/z) for C H N O [M + H] : calcd, 413.1608;
found, 413.1604. HPLC analysis: 16.76 min, 98.41% purity.
24 20
4
3
N-(3,5-Dimethoxyphenyl)-4-methyl-3-(pyridin-3-ylethynyl)-
benzamide (10). It is obtained as a yellow solid (yield 81%); mp
reagents were performed under a nitrogen (N ) atmosphere. All
2
1
1
11−113 °C. H NMR (400 MHz, DMSO-d ): δ 10.22 (s, 1H), 8.82
reactions were conducted in microwave vials or flasks containing a
Teflon-coated magnetic stirrer. Microwave irradiation experiments
were performed in a CEM-Discover Lab-Mate mono-mode micro-
wave apparatus equipped with an Intelli-VentTM pressure control
system and a vertically focused IR temperature sensor. Reactions were
monitored by thin-layer chromatography (TLC) on precoated TLC
glass plates (silica gel GF254, 0.2 ± 0.03 mm thickness). TLC glass
plates were developed in a covered chamber and were visualized with
ultraviolet light using a 254 nm fluorescent indicator. Flash column
chromatography was carried out on silica gel (200−300 mesh). All
6
(s, 1H), 8.62 (s, 1H), 8.16 (s, 1H), 8.04 (d, J = 7.5 Hz, 1H), 7.91 (d, J
=
7.5 Hz, 1H), 7.51 (t, J = 8.1 Hz, 2H), 7.10 (s, 2H), 6.27 (s, 1H),
1
3
3
1
1
9
.74 (s, 6H), 2.56 (s, 3H). C NMR (151 MHz, DMSO-d ): δ
6
64.41, 160.38, 151.61, 149.25, 143.67, 140.76, 138.60, 132.66,
30.67, 129.97, 128.55, 123.75, 121.62, 119.32, 98.51, 95.81, 90.64,
+
0.46, 55.13, 20.38. HRMS (ESI, m/z) for C H N O [M + H] :
23 20
2
3
calcd, 373.1547; found, 375.1543. HPLC analysis: 16.86 min, 99.18%
purity.
3-((2-Aminopyridin-3-yl)ethynyl)-N-(3,5-dimethoxyphenyl)-4-
1
13
13
13
methylbenzamide (11). It is obtained as a yellow solid (yield 85%);
NMR spectra were recorded on a Bruker 400 ( H: 400 MHz,
C
C
C
1
19
1
mp 179−180 °C. H NMR (400 MHz, DMSO-d ): δ 10.19 (s, 1H),
NMR: 101 MHz,1 F: 376 MHz), Bruker 500 ( H: 500 MHz,
6
9
1
8.23 (d, J = 1.8 Hz, 1H), 8.01 (s, 1H), 7.85 (dd, J = 8.0, 1.9 Hz, 1H),
7.65 (dd, J = 7.5, 1.4 Hz, 1H), 7.47 (d, J = 8.1 Hz, 1H), 7.10 (t, J =
2.2 Hz, 2H), 6.61 (dd, J = 7.4, 4.9 Hz, 1H), 6.32 (s, 2H), 6.27 (t, J =
NMR: 126 MHz, F: 471 MHz), or Bruker 600 ( H: 500 MHz,
NMR: 151 MHz) spectrometer and referenced to the deuterium
solvent (chloroform-d, MeOH-d or DMSO-d ). Chemical shifts (δ)
4
6
1
3
were expressed in parts per million (ppm) relative to an internal
standard (TMS), and coupling constants were given in Hz. Data for
NMR spectra were reported as follows: chemical shift (δ ppm),
multiplicity (s = singlet, br s = broad singlet, d = doublet, t = triplet, q
quartet, dd = doublet of doublets, m = multiplet), coupling constant
Hz), and integration. Low-resolution mass spectral data were
measured on an Agilent 1200 series LC−MS spectrometer, and
high-resolution mass spectral (HRMS) data were measured on
Micromass Ultra Q-Tof. The purity of all final compounds (>95%)
was determined on an Agilent 1200 series LC system (Agilent
ChemStation Rev.B.03.01; column, ZORBAX Eclipse XD B-C18, 4.6
mm × 150 mm, 5 μm; flow rate, 1.0 mL/min; UV wavelength,
maximal absorbance at 254 nm; temperature, ambient; and injection
2.2 Hz, 1H), 3.74 (s, 6H), 2.54 (s, 3H). C NMR (151 MHz,
DMSO-d ): δ 164.65, 160.38, 159.43, 148.51, 143.10, 140.70, 139.97,
132.59, 130.87, 129.66, 127.78, 122.52, 112.36, 98.49, 98.40, 95.73,
92.70, 90.03, 55.12, 20.51. HRMS (ESI, m/z) for C23 [M +
H] : calcd, 388.1656; found, 388.1653. HPLC analysis: 11.94 min,
98.18% purity.
6
H N O
21 3 3
+
=
(
3-((2-Amino-5-chloropyridin-3-yl)ethynyl)-N-(3,5-dimethoxy-
phenyl)-4-methylbenzamide (12). It is obtained as a brown solid
1
(
yield 82%); mp 195−197 °C. H NMR (400 MHz, DMSO-d ): δ
6
10.20 (s, 1H), 8.25 (s, 1H), 8.03 (s, 1H), 7.86 (d, J = 8.0 Hz, 1H),
7.76 (s, 1H), 7.47 (d, J = 8.0 Hz, 1H), 7.09 (d, J = 2.0 Hz, 2H), 6.61
(s, 2H), 6.26 (s, 1H), 3.73 (s, 6H), 2.53 (s, 3H). C NMR (126
MHz, DMSO-d ): δ 164.67, 160.34, 158.08, 147.66, 146.55, 143.32,
140.73, 139.58, 138.77, 132.63, 131.11, 129.60, 127.98, 122.07, 98.49,
95.71, 93.64, 88.47, 55.08, 20.42. HRMS (ESI, m/z) for
1
3
6
volume, 5 μL; mobile phase, solvent A: H O (with 0.1% HCOOH)
2
and solvent B: MeOH). The gradient elution for compounds 7−11,
+
1
3−16, 18−25, 27−32, and 35−37: 0−5 min, B: 50−50%; 5−10
C
H
23
20ClN
3
O
3
[M + H] : calcd, 422.1266; found, 422.1265. HPLC
min, B: 50−75%; 10−18 min, B: 75−75%; 18−19 min, B: 75−95%;
analysis: 15.27 min, 100.00% purity.
1
5
9−22 min, B: 95−95%; 22−23 min, B: 95−50%; 23−25 min, B:
0−50%; for compounds 12, 17, 26 and 33−34: 0−20 min, MeOH/
6-Amino-5-((5-((3,5-dimethoxyphenyl)carbamoyl)-2-
methylphenyl)ethynyl)nicotinamide (13). It is obtained as a light
yellow solid (yield 72%); mp 253−254 °C. H NMR (400 MHz,
DMSO-d ): δ 10.23 (s, 1H), 8.53 (d, J = 2.3 Hz, 1H), 8.29 (d, J = 1.8
Hz, 1H), 8.12 (d, J = 2.3 Hz, 1H), 7.88−7.82 (m, 2H), 7.48 (d, J =
8.1 Hz, 1H), 7.21 (s, 1H), 7.10 (d, J = 2.2 Hz, 2H), 6.93 (s, 2H), 6.27
(t, J = 2.2 Hz, 1H), 3.74 (s, 6H), 2.56 (s, 3H). C NMR (151 MHz,
1
H O (0.1% HCOOH) (75/25, v/v) (see Table S1 in the Supporting
2
Information).
6
N-(3,5-Dimethoxyphenyl)-3-(isoquinolin-4-ylethynyl)-4-methyl-
benzamide (8). General Procedure for Syntheses of 8−15, 42a−d and
1
3
4
2f. To a solution of 41a (100 mg, 0.25 mmol) in dry MeCN (20
mL) were added 39a (76 mg, 0.34 mmol), CsF (95 mg, 0.63 mmol),
and Et N (105 μL, 0.76 mmol) at rt under a N atmosphere. The
DMSO-d ): δ 166.06, 164.79, 160.75, 160.38, 149.24, 143.20, 140.83,
139.26, 132.67, 131.14, 129.64, 127.88, 122.36, 118.09, 99.97, 98.50,
6
3
2
mixture was stirred at rt for 6 h. The resulting solution was poured
into water and extracted with EtOAc. The organic layer was washed
with brine and dried with Na SO to give the crude product, which
95.73, 92.73, 89.30, 55.13, 20.50. HRMS (ESI, m/z) for C H N O
[M + H] : calcd, 431.1714; found, 431.1718. HPLC analysis: 13.21
min, 96.15% purity.
6-Amino-5-((5-((3,5-dimethoxyphenyl)carbamoyl)-2-
methylphenyl)ethynyl)-N-methylnicotinamide (14). It is obtained as
2
4
22
4
4
+
2
4
was purified by flash column chromatography (SiO , CH Cl /MeOH,
2
2
2
25:1) to give 8 as a white solid (91 mg, yield 85%); mp 177−178 °C.
J
https://dx.doi.org/10.1021/acs.jmedchem.9b01912
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
1
a brown solid (yield 80%); mp 262−265 °C. H NMR (400 MHz,
3-((2-Amino-5-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)ethynyl)-
DMSO-d ): δ 10.20 (s, 1H), 8.49 (d, J = 2.3 Hz, 1H), 8.30−8.26 (m,
H), 8.08 (d, J = 2.2 Hz, 1H), 7.86 (dd, J = 8.1, 1.4 Hz, 1H), 7.48 (d,
J = 8.1 Hz, 1H), 7.09 (d, J = 2.1 Hz, 2H), 6.91 (s, 2H), 6.27 (t, J = 2.0
Hz, 1H), 3.74 (s, 6H), 2.75 (d, J = 4.4 Hz, 3H), 2.56 (s, 3H).
NMR (151 MHz, DMSO-d ): δ 164.75, 164.70, 160.61, 160.37,
48.62, 143.16, 140.78, 138.69, 132.67, 131.10, 129.62, 127.84,
22.32, 118.41, 100.01, 98.49, 95.73, 92.75, 89.25, 55.11, 26.00, 20.48.
HRMS (ESI, m/z) for C H N O [M + H] : calcd, 445.1871;
found, 445.1866. HPLC analysis: 14.05 min, 97.75% purity.
Methyl 6-Amino-5-((5-((3,5-dimethoxyphenyl)carbamoyl)-2-
methylphenyl)ethynyl)nicotinate (15). It is obtained as a yellow
solid (yield 78%); mp 231−232 °C. H NMR (400 MHz, DMSO-d ):
δ 10.19 (s, 1H), 8.56 (s, 1H), 8.29 (s, 1H), 8.05 (d, J = 2.0 Hz, 1H),
.86 (dd, J = 1.6, 8.1 Hz, 1H), 7.48 (d, J = 8.1 Hz, 1H), 7.29 (s, 2H),
N-(3,5-dimethoxyphenyl)-4-methoxybenzamide (19). It is obtained
6
1
2
as a yellow solid (yield 75%); mp 229−230 °C. H NMR (400 MHz,
DMSO-d ): δ 10.13 (s, 1H), 8.27 (d, J = 2.4 Hz, 1H), 8.22 (d, J = 2.3
6
1
3
Hz, 1H), 8.08 (s, 1H), 8.01 (dd, J = 8.8, 2.3 Hz, 1H), 7.83 (d, J = 0.6
Hz, 1H), 7.79 (d, J = 2.4 Hz, 1H), 7.26 (d, J = 8.9 Hz, 1H), 7.10 (s,
C
6
1
3
H), 7.09 (s, 1H), 6.30 (s, 2H), 6.26 (t, J = 2.3 Hz, 1H), 3.98 (s, 3H),
1
1
1
3
.84 (s, 3H), 3.74 (s, 6H). C NMR (101 MHz, DMSO-d ): δ
6
+
164.26, 161.82, 160.37, 157.97, 154.39, 145.16, 140.92, 135.40,
35.28, 131.87, 130.29, 127.06, 126.85, 118.75, 117.60, 111.12,
2
5
24
4
4
1
1
01.24, 98.44, 95.64, 91.14, 90.04, 56.34, 55.13, 38.67. HRMS (ESI,
+
m/z) for C27
H
25
N
5
O
4
[M + H] : calcd, 484.1908; found, 484.0693.
1
HPLC analysis: 22.50 min, 100.00% purity.
6
3
-((2-Amino-5-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)ethynyl)-
N-(3,5-dimethoxyphenyl)-4-fluorobenzamide (20). It is obtained as
7
7
2
1
1
5
1
a yellow solid (yield 88%); mp 236−238 °C. H NMR (400 MHz,
DMSO-d ): δ 10.28 (s, 1H), 8.39 (d, J = 6.9 Hz, 1H), 8.29 (s, 1H),
.10 (s, 1H), 7.09 (s, 1H), 6.27 (s, 1H), 3.80 (s, 3H), 3.74 (s, 6H),
.55 (s, 3H). 13C NMR (151 MHz, DMSO-d ): δ 164.94, 164.75,
6
6
8
9
.09 (s, 1H), 8.03−7.97 (m, 1H), 7.83 (d, J = 2.8 Hz, 2H), 7.51 (t, J =
61.67, 160.37, 150.82, 143.31, 140.78, 140.35, 132.65, 131.25,
29.60, 127.96, 122.18, 113.73, 100.63, 98.49, 95.72, 93.06, 88.52,
.4 Hz, 1H), 7.07 (s, 2H), 6.40 (s, 2H), 6.28 (s, 1H), 3.84 (s, 3H),
1
3
+
3.74 (s, 6H). C NMR (126 MHz, DMSO-d ): δ 163.88, 162.23 (d, J
6
5.11, 51.67, 20.47. HRMS (ESI, m/z) for C H N O [M + H] :
2
5
23
3
5
=
254.5 Hz), 160.38, 157.99, 145.72, 140.61, 136.25, 135.35, 133.22,
31.59 (d, J = 2.9 Hz), 130.42 (d, J = 9.1 Hz), 126.85, 118.59, 117.57,
calcd, 446.1711; found, 446.1713. HPLC analysis: 18.44 min, 99.46%
1
1
9
purity.
3
15.77 (d, J = 21.6 Hz), 110.98 (d, J = 16.2 Hz), 100.27, 98.55, 95.86,
-((2-Amino-5-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)ethynyl)-
1
9
1.15 (d, J = 2.3 Hz), 87.27, 55.12, 38.61. F NMR (376 MHz,
N-(3,5-dimethoxyphenyl)-4-methylbenzamide (16). General Proce-
dure for Syntheses of 16−22. To a solution of 42a (80 mg, 0.17 mmol)
DMSO-d ): δ −106.42 to −106.54 (m). HRMS (ESI, m/z) for
6
+
C H FN O [M + H] : calcd, 472.1780; found, 472.1776. HPLC
in toluene/EtOH/H O (2 mL, 2:1:1, v/v/v) were added 1-methyl-4-
26 22
5
3
2
analysis: 14.02 min, 98.35% purity.
-((2-Amino-5-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)ethynyl)-
-chloro-N-(3,5-dimethoxyphenyl)benzamide (21). It is obtained as
(
4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (71 mg,
3
0
0
.34 mmol), Pd(PPh ) (19 mg, 0.017 mmol), and K CO (59 mg,
3 4 2 3
4
.43 mmol) at rt. After stirring for 5 min under a N atmosphere, the
2
1
a light yellow solid (yield 75%); mp 229−230 °C. H NMR (400
mixture was irradiated at 100 °C for 20 min. The mixture was poured
into water and extracted with EtOAc. The organic layer was separated,
washed with brine, dried with anhydrous Na SO , filtered, and
MHz, DMSO-d ): δ 10.34 (s, 1H), 8.39 (d, J = 2.0 Hz, 1H), 8.31 (d, J
6
=
=
6
2.1 Hz, 1H), 8.10 (s, 1H), 7.94 (dd, J = 8.4, 2.0 Hz, 1H), 7.85 (d, J
2
4
2.2 Hz, 1H), 7.83 (s, 1H), 7.77 (d, J = 8.4 Hz, 1H), 7.08 (s, 2H),
concentrated. The crude product was further purified by flash column
13
.39 (s, 2H), 6.29 (s, 1H), 3.84 (s, 3H), 3.74 (s, 6H). C NMR (101
chromatography (SiO , CH Cl /MeOH/NH OH, 20:1:0.02) to
2
2
2
4
MHz, DMSO-d ): δ 160.41, 158.92, 158.05, 145.98, 140.46, 137.21,
6
afford compound 16 as a light yellow solid (80 mg, yield 85%); mp
1
1
(
36.26, 135.38, 133.94, 132.52, 129.42, 129.19, 126.91, 122.26,
1
2
30−231 °C. H NMR (400 MHz, DMSO-d ): δ 10.20 (s, 1H), 8.27
6
18.59, 117.66, 98.55, 98.46, 95.92, 91.36, 90.90, 55.16, 38.66. HRMS
(
(
d, J = 2.3 Hz, 1H), 8.24 (d, J = 1.9 Hz, 1H), 8.08 (s, 1H), 7.89−7.83
+
ESI, m/z) for C H ClN O [M + H] : calcd, 488.1484; found,
2
6
22
5
3
m, 2H), 7.83 (s, 1H), 7.48 (d, J = 8.1 Hz, 1H), 7.10 (d, J = 2.2 Hz,
4
88.1484. HPLC analysis: 15.57 min, 97.94% purity.
2
2
1
1
9
H), 6.29 (s, 2H), 6.27 (t, J = 2.2 Hz, 1H), 3.84 (s, 3H), 3.74 (s, 6H),
3
-((2-Amino-5-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)ethynyl)-
.57 (s, 3H). 13C NMR (126 MHz, DMSO-d ): δ 164.72, 160.36,
6
N-(3,5-dimethoxyphenyl)-4-(trifluoromethyl)benzamide (22). It is
57.83, 145.25, 143.10, 140.78, 136.30, 135.34, 132.64, 130.93,
29.64, 127.77, 126.82, 122.47, 118.70, 117.61, 101.10, 98.50, 95.73,
2.84, 89.92, 55.11, 38.62, 20.55. HRMS (ESI, m/z) for C H N O
M + H] : calcd, 468.2030; found, 468.2028. HPLC analysis: 14.11
min, 99.65% purity.
-((2-Amino-5-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)ethynyl)-
N-(3,5-dimethoxyphenyl) benzamide (17). It is obtained as a yellow
1
obtained as a bright yellow solid (yield 83%); mp 223−224 °C. H
NMR (400 MHz, DMSO-d ): δ 10.49 (s, 1H), 8.50 (s, 1H), 8.32 (s,
6
2
7
25
5
3
1H), 8.10 (s, 1H), 8.07 (d, J = 8.0 Hz, 1H), 7.99 (d, J = 8.1 Hz, 1H),
+
[
7
3
1
1
.82 (s, 1H), 7.76 (s, 1H), 7.09 (s, 2H), 6.40 (s, 2H), 6.31 (s, 1H),
13
.85 (s, 3H), 3.75 (s, 6H). C NMR (126 MHz, DMSO-d ): δ
6
3
63.83, 160.43, 158.03, 146.29, 140.37, 138.79, 136.34, 135.32,
33.15, 131.22 (q, J = 29.8 Hz), 127.86, 126.92, 126.43 (q, J = 4.5
1
solid (yield 77%); mp 221−222 °C. H NMR (400 MHz, DMSO-d ):
δ 10.30 (s, 1H), 8.26 (d, J = 2.2 Hz, 1H), 8.23 (s, 1H), 8.07 (s, 1H),
6
Hz), 123.40 (q, J = 273.4 Hz), 120.63, 118.47, 117.71, 99.97, 98.62,
19
9
−
5
6.11, 91.74, 89.99, 55.15, 38.59. F NMR (471 MHz, DMSO-d ): δ
6
7
7
1
.93 (d, J = 7.9 Hz, 1H), 7.89−7.83 (m, 2H), 7.81 (s, 1H), 7.58 (t, J =
+
60.95 (s). HRMS (ESI, m/z) for C H F N O [M + H] : calcd,
27 22 3 5 3
.8 Hz, 1H), 7.10 (t, J = 2.1 Hz, 2H), 6.44 (s, 2H), 6.28 (t, J = 2.1 Hz,
22.1748; found, 522.1745. HPLC analysis: 16.56 min, 98.15% purity.
1
3
H), 3.83 (s, 3H), 3.72 (d, J = 10.1 Hz, 6H). C NMR (101 MHz,
3
-((2-Amino-5-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)ethynyl)-
DMSO-d ): δ 164.92, 160.40, 157.94, 145.12, 140.75, 136.48, 135.36,
34.22, 130.53, 128.82, 127.80, 126.80, 122.74, 118.72, 117.47,
00.86, 98.52, 98.43, 95.81, 94.05, 86.26, 55.15, 38.67. HRMS (ESI,
m/z) for C H N O [M + H] : calcd, 454.1874; found, 454.1869.
4-methyl-N-phenylbenzamide (23). General Procedure for Syntheses of
23−33, 38f−g, 41a−g, and 48a−c. To a solution of 45 (100 mg, 0.30
mmol) in DMF (3 mL) were added HATU (148 mg, 0.39 mmol) and
DIPEA (149 μL, 0.90 mmol) at rt, and after stirring for 30 min,
aniline (30 μL, 0.33 mmol) was added, and the mixture was stirred at
rt for 4 h. The resulting solution was poured into water and extracted
with EtOAc. The organic layer was washed with water and brine and
dried with Na SO . The resultant crude material was purified by flash
6
1
1
+
26
23
5
3
HPLC analysis: 2.93 min, 99.16% purity.
3
-((2-Amino-5-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)ethynyl)-
N-(3,5-dimethoxyphenyl)-4-isopropylbenzamide (18). It is obtained
as a bright yellow solid (yield 80%); mp 216−217 °C. H NMR (400
MHz, DMSO-d ): δ 10.22 (s, 1H), 8.28 (s, 1H), 8.25 (s, 1H), 8.09 (s,
1
2
4
6
column chromatography (SiO , CH Cl /MeOH/NH OH, 20:1:0.02)
2 2 2 4
1
1
7
3
H), 7.91 (d, J = 7.9 Hz, 1H), 7.83 (s, 2H), 7.54 (d, J = 8.1 Hz, 1H),
to give 23 as a yellow solid (116 mg, yield 95%); mp 95−96 °C. H
.11 (s, 2H), 6.28 (d, J = 6.4 Hz, 3H), 3.85 (s, 3H), 3.74 (s, 6H),
NMR (400 MHz, DMSO-d ): δ 10.29 (s, 1H), 8.32−8.24 (m, 2H),
6
1
3
.62−3.52 (m, 1H), 1.31 (d, J = 6.7 Hz, 6H). C NMR (151 MHz,
8.08 (s, 1H), 7.90−7.85 (m, 2H), 7.83 (s, 1H), 7.79 (d, J = 7.7 Hz,
DMSO-d ): δ 164.87, 160.41, 157.87, 153.00, 145.29, 140.85, 136.26,
2H), 7.49 (d, J = 8.0 Hz, 1H), 7.36 (t, J = 7.9 Hz, 2H), 7.11 (t, J = 7.4
6
1
3
1
1
35.41, 132.79, 131.54, 128.28, 126.90, 125.30, 121.40, 118.71,
17.66, 101.19, 98.47, 95.73, 92.59, 89.56, 55.14, 38.64, 31.33, 22.81.
HRMS (ESI, m/z) for C H N O [M + H] : calcd, 496.2343;
found, 496.2343. HPLC analysis: 16.51 min, 99.31% purity.
Hz, 1H), 6.31 (s, 2H), 3.84 (s, 3H), 2.57 (s, 3H). C NMR (151
MHz, DMSO-d ): δ 164.70, 157.85, 145.26, 143.04, 139.12, 136.32,
6
+
135.36, 132.71, 131.00, 129.64, 128.62, 127.80, 126.83, 123.69,
122.49, 120.35, 118.72, 117.62, 101.13, 92.88, 89.91, 38.62, 20.55.
2
9
29
5
3
K
https://dx.doi.org/10.1021/acs.jmedchem.9b01912
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
+
HRMS (ESI, m/z) for C H N O [M + H] : calcd, 408.1819; found,
4
Hz, 1H), 7.84−7.80 (m, 3H), 7.49 (d, J = 8.1 Hz, 1H), 7.44−7.39 (m,
13
2
5
21
5
08.1820. HPLC analysis: 13.18 min, 98.93% purity.
2H), 6.29 (s, 2H), 3.84 (s, 3H), 2.57 (s, 3H). C NMR (126 MHz,
3
-((2-Amino-5-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)ethynyl)-
DMSO-d ): δ 164.78, 157.82, 145.26, 143.21, 138.08, 136.30, 135.33,
132.42, 130.98, 129.67, 128.52, 127.79, 127.27, 126.80, 122.52,
6
N-(2-methoxyphenyl)-4-methylbenzamide (24). It is obtained as a
1
brown solid (yield 90%); mp 107−108 °C. H NMR (400 MHz,
121.81, 118.69, 117.61, 101.07, 92.78, 89.96, 38.59, 20.53. HRMS
+
DMSO-d ): δ 9.48 (s, 1H), 8.30−8.23 (m, 2H), 8.08 (s, 1H), 7.87 (t,
(ESI, m/z) for C H ClN O [M + H] : calcd, 442.1429; found,
6
25 20
5
J = 6.5 Hz, 2H), 7.83 (s, 1H), 7.75 (d, J = 7.3 Hz, 1H), 7.48 (d, J =
442.1425. HPLC analysis: 15.48 min, 99.00% purity.
8
(
.0 Hz, 1H), 7.19 (t, J = 7.3 Hz, 1H), 7.10 (d, J = 7.8 Hz, 1H), 6.98
t, J = 7.6 Hz, 1H), 6.31 (s, 2H), 3.84 (br s, 6H), 2.57 (s, 3H).
NMR (126 MHz, DMSO-d ): δ 164.23, 157.84, 151.62, 145.25,
43.06, 136.33, 135.35, 132.28, 130.91, 129.71, 127.56, 126.83,
26.71, 125.84, 124.56, 122.58, 120.19, 118.71, 117.61, 111.44,
3-((2-Amino-5-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)ethynyl)-
1
3
C
4-methyl-N-(3-(trifluoromethyl)phenyl)benzamide (30). It is ob-
1
tained as a deep yellow solid (yield 85%); mp 206−208 °C. H NMR
6
1
1
1
(400 MHz, DMSO-d ): δ 10.63 (s, 1H), 8.30 (d, J = 1.8 Hz, 1H),
6
8.27 (d, J = 2.4 Hz, 1H), 8.26 (s, 1H), 8.09 (d, J = 9.3 Hz, 2H), 7.91
(dd, J = 8.0, 1.9 Hz, 1H), 7.86 (d, J = 2.4 Hz, 1H), 7.82 (d, J = 0.6 Hz,
2H), 7.61 (t, J = 8.0 Hz, 1H), 7.51 (d, J = 8.1 Hz, 1H), 7.46 (d, J = 7.8
01.13, 92.84, 89.90, 55.71, 38.61, 20.53. HRMS (ESI, m/z) for
+
C H N O [M + H] : calcd, 438.1925; found, 438.1923. HPLC
2
6
23
5
2
1
3
analysis: 14.03 min, 98.47% purity.
Hz, 1H), 6.30 (s, 2H), 3.85 (s, 3H), 2.58 (s, 3H). C NMR (126
3
-((2-Amino-5-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)ethynyl)-
MHz, DMSO-d ): δ 165.21, 157.84, 145.28, 143.43, 142.87, 136.32,
6
N-(3-methoxyphenyl)-4-methylbenzamide (25). It is obtained as a
135.34, 132.26, 131.12, 129.75, 129.70, 129.61, 127.93, 126.81,
125.90 (q, J = 3.8 Hz), 124.39 (q, J = 271.3 Hz), 123.57 (q, J = 32.0
Hz), 122.56, 120.14, 118.69, 117.61, 101.06, 92.75, 90.02, 38.60,
1
yellow solid (yield 86%); mp 232−233 °C. H NMR (400 MHz,
DMSO-d ): δ 10.26 (s, 1H), 8.28 (s, 1H), 8.27 (s, 1H), 8.08 (s, 1H),
.86 (t, J = 10.0 Hz, 3H), 7.48 (d, J = 8.5 Hz, 2H), 7.41 (d, J = 8.1 Hz,
H), 7.26 (t, J = 8.1 Hz, 1H), 6.69 (d, J = 6.9 Hz, 1H), 6.32 (s, 2H),
.85 (s, 3H), 3.76 (s, 3H), 2.57 (s, 3H). C NMR (151 MHz,
DMSO-d ): δ 164.74, 159.45, 157.87, 145.28, 143.10, 140.33, 136.34,
35.38, 132.71, 131.00, 129.66, 129.41, 127.82, 126.84, 122.51,
18.75, 117.66, 112.57, 109.18, 106.07, 101.17, 92.91, 89.94, 55.01,
8.62, 20.56. HRMS (ESI, m/z) for C H N O [M + H] : calcd,
6
1
9
7
1
3
20.56. F NMR (471 MHz, DMSO-d ): δ −61.28. HRMS (ESI, m/
6
+
z) for C H F N O [M + H] : calcd, 476.1693; found, 476.1693.
2
6
20
3
5
1
3
HPLC analysis: 16.28 min, 96.39% purity.
6
3-((2-Amino-5-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)ethynyl)-
1
1
3
4
4-methyl-N-(4-(trifluoromethyl)phenyl)benzamide (31). It is ob-
1
tained as a yellow solid (yield 90%); mp 239−240 °C. H NMR (400
+
MHz, DMSO-d ): δ 10.66 (s, 1H), 8.29 (d, J = 1.4 Hz, 1H), 8.27 (d, J
2
6
23
5
2
6
38.1925; found, 438.1925. HPLC analysis: 13.60 min, 98.64% purity.
= 2.2 Hz, 1H), 8.08 (s, 1H), 8.03 (d, J = 8.5 Hz, 2H), 7.90 (dd, J =
7.9, 1.5 Hz, 1H), 7.85 (d, J = 2.2 Hz, 1H), 7.82 (s, 1H), 7.73 (d, J =
3
-((2-Amino-5-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)ethynyl)-
N-(4-methoxyphenyl)-4-methylbenzamide (26). It is obtained as a
8.6 Hz, 2H), 7.50 (d, J = 8.0 Hz, 1H), 6.30 (s, 2H), 3.84 (s, 3H), 2.58
1
13
light yellow solid (yield 91%); mp 180−182 °C. H NMR (400 MHz,
DMSO-d ): δ 10.17 (s, 1H), 8.27 (d, J = 1.9 Hz, 1H), 8.24 (s, 1H),
.09 (s, 1H), 7.86 (d, J = 8.1 Hz, 2H), 7.83 (s, 1H), 7.68 (d, J = 8.8
Hz, 2H), 7.47 (d, J = 8.0 Hz, 1H), 6.93 (d, J = 8.9 Hz, 2H), 6.31 (s,
H), 3.84 (s, 3H), 3.75 (s, 3H), 2.56 (s, 3H). C NMR (126 MHz,
DMSO-d ): δ 164.21, 157.81, 155.54, 145.22, 142.82, 136.28, 135.33,
32.76, 132.14, 130.87, 129.59, 127.67, 126.81, 122.42, 121.92,
18.69, 117.61, 113.73, 101.13, 92.90, 89.81, 55.16, 38.60, 20.50.
(s, 3H). C NMR (126 MHz, DMSO-d ): δ 165.09, 157.84, 145.29,
6
6
143.43, 139.95, 136.31, 135.34, 132.17, 131.05, 129.86, 129.74,
129.33 (q, J = 31.5 Hz), 127.87, 126.82, 124.16 (q, J = 272.2 Hz),
123.76, 122.57, 119.93 (q, J = 3.7 Hz), 118.70, 117.62, 116.36 (q, J =
8
1
3
19
2
4.1 Hz), 101.07, 92.76, 90.00, 38.60, 20.56. F NMR (471 MHz,
6
DMSO-d ): δ −60.31. HRMS (ESI, m/z) for C H F N O [M +
6
26 20
3
5
+
1
1
H] : calcd, 476.1693; found, 476.1694. HPLC analysis: 16.85 min,
98.52% purity.
+
HRMS (ESI, m/z) for C H N O [M + H] : calcd, 438.1925;
found, 438.1925. HPLC analysis: 3.16 min, 99.72% purity.
3-((2-Amino-5-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)ethynyl)-
2
6
23
5
2
N-(2,4-dichlorophenyl)-4-methylbenzamide (32). It is obtained as a
1
3
-((2-Amino-5-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)ethynyl)-
light yellow solid (yield 75%); mp 196−198 °C. H NMR (400 MHz,
N-(2-chlorophenyl)-4-methylbenzamide (27). It is obtained as a
DMSO-d ): δ 10.18 (s, 1H), 8.27 (d, J = 1.9 Hz, 2H), 8.08 (s, 1H),
6
1
yellow solid (yield 75%); mp 175−176 °C. H NMR (400 MHz,
7.90 (dd, J = 8.0, 2.0 Hz, 1H), 7.85 (d, J = 2.4 Hz, 1H), 7.75 (d, J =
2.3 Hz, 1H), 7.61 (d, J = 8.6 Hz, 1H), 7.54−7.45 (m, 2H), 6.30 (s,
DMSO-d ): δ 10.12 (s, 1H), 8.28−8.26 (m, 2H), 8.09 (s, 1H), 7.91
6
1
3
(
dd, J = 1.2, 7.7 Hz, 2H), 7.86 (d, J = 2.0 Hz, 1H), 7.83 (s, 1H),
2H), 3.84 (s, 3H), 2.57 (s, 3H). C NMR (126 MHz, DMSO-d ): δ
6
7
7
.59−7.55 (m, 2H), 7.50 (d, J = 8.1 Hz, 1H), 7.40 (t, J = 7.6 Hz, 1H),
164.66, 157.83, 145.28, 143.53, 136.33, 135.34, 134.25, 131.46,
131.11, 130.82, 130.57, 129.76, 129.60, 129.07, 127.77, 127.65,
126.82, 122.65, 118.69, 117.61, 101.06, 92.71, 89.99, 38.61, 20.58.
1
3
.31 (t, J = 7.6 Hz, 1H), 6.31 (s, 2H), 3.84 (s, 3H), 2.57 (s, 3H).
C
NMR (126 MHz, DMSO-d ): δ 164.58, 157.81, 145.25, 143.34,
6
+
1
1
8
36.31, 135.33, 135.01, 131.71, 131.06, 129.72, 129.56, 128.50,
27.72, 127.51, 127.47, 126.81, 122.61, 118.68, 117.62, 101.08, 92.75,
HRMS (ESI, m/z) for C H Cl N O [M + H] : calcd, 476.1040;
2
5
19
2
5
found, 476.1035. HPLC analysis: 16.58 min, 97.87% purity.
3-((2-Amino-5-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)ethynyl)-
N-(3,4-dichlorophenyl)-4-methylbenzamide (33). It is obtained as a
+
9.92, 38.59, 20.54. HRMS (ESI, m/z) for C H ClN O [M + H] :
2
5
20
5
calcd, 442.1429; found, 442.1426. HPLC analysis: 14.08 min, 98.06%
purity.
1
yellow solid (yield 82%); mp 108−111 °C. H NMR (400 MHz,
3
-((2-Amino-5-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)ethynyl)-
DMSO-d ): δ 10.59 (s, 1H), 8.27 (s, 2H), 8.18 (s, 1H), 8.08 (s, 1H),
6
N-(3-chlorophenyl)-4-methylbenzamide (28). It is obtained as a
yellow solid (yield 85%); mp 204−206 °C. H NMR (400 MHz,
DMSO-d ): δ 10.45 (s, 1H), 8.27 (d, J = 2.3 Hz, 1H), 8.26 (d, J = 1.7
7.92−7.84 (m, 2H), 7.84−7.74 (m, 2H), 7.63 (d, J = 8.8 Hz, 1H),
13
1
7.50 (d, J = 7.5 Hz, 1H), 6.31 (s, 2H), 3.84 (s, 3H), 2.57 (s, 3H). C
NMR (126 MHz, DMSO-d ): δ 165.02, 157.84, 145.30, 143.51,
6
6
Hz, 1H), 8.08 (s, 1H), 7.98 (t, J = 1.9 Hz, 1H), 7.89−7.85 (m, 2H),
139.29, 136.32, 135.34, 132.04, 131.05, 130.84, 130.57, 129.75,
127.86, 126.82, 125.11, 122.59, 121.42, 120.23, 118.69, 117.62,
101.04, 92.72, 90.04, 38.61, 20.56. HRMS (ESI, m/z) for
7
7
3
1
1
1
.83 (s, 1H), 7.73 (dd, J = 7.9, 1.3 Hz, 1H), 7.50 (d, J = 8.0 Hz, 1H),
.39 (t, J = 8.1 Hz, 1H), 7.17 (dd, J = 7.9, 1.8 Hz, 1H), 6.31 (s, 2H),
1
3
+
.84 (s, 3H), 2.57 (s, 3H). C NMR (126 MHz, DMSO-d ): δ
C H Cl N O [M + H] : calcd, 476.1040; found, 476.1042. HPLC
6
25 19
2
5
64.97, 157.85, 145.29, 143.37, 140.61, 136.32, 135.35, 132.94,
32.30, 131.02, 130.35, 129.72, 127.84, 126.83, 123.37, 122.57,
analysis: 9.59 min, 98.70% purity.
4-((2-Amino-5-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)ethynyl)-
N-(4-chlorophenyl)-3-methylbenzamide (34). General Procedure for
Syntheses of 34−37. Step 1: The cross-coupling product was prepared
in 69% yield from 48a and 39i according to the procedure in the
preparation of 8. Step 2: According to the general procedure in the
19.68, 118.70, 118.60, 117.62, 101.07, 92.77, 90.01, 38.62, 20.57.
+
HRMS (ESI, m/z) for C H ClN O [M + H] : calcd, 442.1429;
2
5
20
5
found, 442.1429. HPLC analysis: 15.62 min, 96.20% purity.
3
-((2-Amino-5-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)ethynyl)-
N-(4-chlorophenyl)-4-methylbenzamide (29). It is obtained as a
preparation of 16, the title compound (34) was prepared in 89% yield
1
1
yellow solid (yield 90%); mp 225−226 °C. H NMR (400 MHz,
DMSO-d ): δ 10.40 (s, 1H), 8.27 (d, J = 2.4 Hz, 1H), 8.25 (d, J = 1.8
as a yellow solid; mp 224−225 °C. H NMR (400 MHz, DMSO-d ):
6
δ 10.44 (s, 1H), 8.28 (s, 1H), 8.09 (s, 1H), 7.91 (s, 1H), 7.88−7.78
(m, 6H), 7.42 (d, J = 8.4 Hz, 2H), 6.32 (s, 2H), 3.84 (s, 3H), 2.58 (s,
6
Hz, 1H), 8.08 (s, 1H), 7.87 (dd, J = 8.1, 1.8 Hz, 1H), 7.85 (d, J = 2.4
L
https://dx.doi.org/10.1021/acs.jmedchem.9b01912
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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Article
3
1
1
9
H). 13C NMR (126 MHz, DMSO-d ): δ 164.98, 157.83, 145.46,
8.12 (s, 1H), 7.36 (dd, J = 4.4, 9.2 Hz, 1H), 0.27 (s, 9H). MS (ESI)
m/z: 216.3 [M + H] .
6
+
39.50, 138.07, 136.40, 135.34, 134.11, 131.71, 128.68, 128.52,
27.31, 126.82, 125.64, 125.11, 121.86, 118.66, 117.63, 100.95, 92.90,
3-((Trimethylsilyl)ethynyl)pyridine (39c). General Procedure for
Syntheses of 39c−e and 39i. To a solution of 3-iodopyridine (2.0 g,
+
1.57, 38.60, 20.53. HRMS (ESI, m/z) for C H ClN O [M + H] :
2
5
20
5
calcd, 442.1429; found, 442.1428. HPLC analysis: 5.57 min, 100.00%
9.76 mmol) in dry Et N (50 mL) were added Pd(PPh ) Cl (342 mg,
3 3 2 2
purity.
0.49 mmol), CuI (186 mg, 0.98 mmol), and ethynyltrimethylsilane
(2.1 mL, 14.63 mmol) at rt under a N atmosphere. After stirring for 4
h, the mixture was diluted with EtOAc and filtered through a pad of
Celite. The filtrate was removed by reduced pressure to give the crude
product, which was further purified by column chromatography
N-(3-((2-Amino-5-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)-
ethynyl)-4-methylphenyl)-4-chlorobenzamide (35). It is obtained as
a yellow solid (yield 76%); mp 190−191 °C. H NMR (400 MHz,
DMSO-d ): δ 10.36 (s, 1H), 8.26 (d, J = 2.3 Hz, 1H), 8.09 (s, 1H),
2
1
6
8
1
.05 (d, J = 2.3 Hz, 1H), 8.00 (d, J = 8.5 Hz, 2H), 7.85 (d, J = 2.3 Hz,
H), 7.83 (s, 1H), 7.65−7.61 (m, 3H), 7.31 (d, J = 8.4 Hz, 1H), 6.20
(SiO , petroleum ether/EtOAc, 15:1) to give 39c as a light yellow
2
1
solid (1.4 g, yield 82%). H NMR (400 MHz, Chloroform-d): δ 8.69
(d, J = 1.1 Hz, 1H), 8.52 (dd, J = 4.8, 1.4 Hz, 1H), 7.77−7.69 (m,
1
3
(s, 2H), 3.84 (s, 3H), 2.46 (s, 3H). C NMR (151 MHz, DMSO-d ):
6
δ 164.36, 157.77, 145.03, 136.71, 136.50, 136.20, 135.39, 134.93,
1H), 7.23 (dd, J = 7.8, 4.9 Hz, 1H), 0.27 (s, 9H). MS (ESI) m/z:
+
2
26.3 [M + H] .
1
1
3
33.42, 129.83, 129.62, 128.54, 126.87, 123.51, 122.33, 121.10,
18.73, 117.72, 109.53, 101.43, 93.49, 89.02, 39.94, 39.80, 39.66,
9.52, 39.38, 39.24, 39.10, 38.64, 20.00. HRMS (ESI, m/z) for
3
-((Trimethylsilyl)ethynyl)pyridin-2-amine (39d). It is obtained as
1
a yellow solid (yield 85.0%). H NMR (400 MHz, DMSO-d ): δ 7.95
6
+
(dd, J = 1.9, 4.9 Hz, 1H), 7.50 (dd, J = 1.9, 7.5 Hz, 1H), 6.52 (dd, J =
C H ClN O [M + H] : calcd, 442.1429; found, 442.1429. HPLC
25
20
5
+
4
.9, 7.5 Hz, 1H), 6.10 (s, 2H). MS (ESI) m/z: 191.1 [M + H] .
analysis: 14.72 min, 95.77% purity.
5
-Chloro-3-((trimethylsilyl)ethynyl)pyridin-2-amine (39e). It is
3
-((2-Amino-5-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)ethynyl)-
1
obtained as a yellow solid (yield 93%). H NMR (400 MHz, DMSO-
N-(4-chlorophenyl)-N,4-dimethylbenzamide (36). It is obtained as a
1
d ): δ 7.96 (d, J = 2.5 Hz, 1H), 7.60 (d, J = 2.6 Hz, 1H), 6.37 (s, 2H),
deep yellow solid (yield 80%); mp 109−111 °C. H NMR (600 MHz,
DMSO-d ): δ 8.25 (d, J = 2.4 Hz, 1H), 8.06 (s, 1H), 7.80 (d, J = 0.4
6
+
0
.24 (s, 9H). MS (ESI) m/z: 225.2 [M + H] .
6
6
-Amino-5-((trimethylsilyl)ethynyl)nicotinamide (39f). It is ob-
Hz, 1H), 7.79 (d, J = 2.3 Hz, 1H), 7.67 (d, J = 1.3 Hz, 1H), 7.37−
1
tained as a gray solid (yield 76%). H NMR (400 MHz, DMSO-d ): δ
7.35 (m, 2H), 7.24 (d, J = 8.7 Hz, 2H), 7.16 (d, J = 8.0 Hz, 1H), 7.06
6
8
.48 (d, J = 2.3 Hz, 1H), 7.99 (d, J = 2.3 Hz, 1H), 7.76 (s, 1H), 7.16
(
d, J = 7.8 Hz, 1H), 6.23 (s, 2H), 3.84 (s, 3H), 3.36 (s, 3H), 2.41 (s,
+
H). 13C NMR (126 MHz, DMSO-d ): δ 168.66, 157.76, 145.20,
(s, 1H), 6.70 (s, 2H), 0.24 (s, 9H). MS (ESI) m/z: 234.2 [M + H] .
-Amino-N-methyl-5-((trimethylsilyl)ethynyl)nicotinamide
3
1
1
8
6
6
43.36, 140.91, 136.29, 135.32, 133.81, 131.73, 130.77, 129.09,
28.89, 128.81, 128.05, 126.80, 122.22, 118.68, 117.57, 101.08, 92.64,
9.76, 38.59, 37.83, 20.29. HRMS (ESI, m/z) for C H ClN O [M +
1
(39g). It is obtained as a yellow solid (92%). H NMR (400 MHz,
Chloroform-d): δ 8.43 (d, J = 1.9 Hz, 1H), 7.94 (d, J = 1.8 Hz, 1H),
6.12 (br s, 1H), 5.42 (s, 2H), 2.96 (d, J = 4.7 Hz, 3H), 0.25 (s, 9H).
2
6
22
5
+
H] : calcd, 456.1586; found, 456.1583. HPLC analysis: 14.21 min,
+
MS (ESI) m/z: 248.1 [M + H] .
9
8.57% purity.
Methyl 6-Amino-5-((trimethylsilyl)ethynyl)nicotinate (39h). It is
3
-((5-(((4-Chlorophenyl)amino)methyl)-2-methylphenyl)-
1
obtained as a black solid (yield 78%). H NMR (400 MHz, DMSO-
ethynyl)-5-(1-methyl-1H-pyrazol-4-yl)pyridin-2-amine (37). It is
1
d
3
6
): δ 8.52 (s, 1H), 7.88 (d, J = 2.0 Hz, 1H), 7.06 (s, 2H), 3.77 (s,
H), 0.24 (s, 8H). MS (ESI) m/z: 249.5 [M + H] .
obtained as a brown solid (yield 70%); mp 230−231 °C. H NMR
+
(500 MHz, DMSO-d ): δ 8.24 (s, 1H), 8.07 (s, 1H), 7.82 (s, 2H),
6
5
-Bromo-3-((trimethylsilyl)ethynyl)pyridin-2-amine (39i). It is
7.60 (s, 1H), 7.27 (s, 2H), 7.06 (d, J = 7.4 Hz, 2H), 6.56 (d, J = 7.8
1
obtained as a yellow solid (yield 82.6%). H NMR (400 MHz,
DMSO-d ): δ 8.02 (d, J = 2.4 Hz, 1H), 7.69 (d, J = 2.5 Hz, 1H), 6.37
s, 2H), 0.24 (s, 9H). MS (ESI) m/z: 269.2 [M + H] .
-Iodo-4-isopropylbenzoic acid (40c). Step 1: To a solution of 4-
Hz, 2H), 6.46 (s, 1H), 6.22 (s, 2H), 4.23 (s, 2H), 3.84 (s, 3H), 2.45
s, 3H). 13C NMR (126 MHz, DMSO-d ): δ 157.53, 147.41, 137.83,
6
6
(
+
(
1
1
4
37.50, 136.33, 135.37, 130.39, 129.58, 128.54, 127.63, 126.87,
22.17, 119.07, 118.63, 117.68, 113.66, 101.68, 93.75, 88.91, 45.86,
0.02, 39.85, 39.69, 39.52, 39.35, 39.19, 39.02, 38.61, 20.12. HRMS
3
isopropylbenzoic acid (5.0 g, 30.45 mmol) in DMF (60 mL) were
added K CO (12.62 g, 91.35 mmol) and MeI (482 μL, 36.54 mmol)
+
2
3
(ESI, m/z) for C H ClN [M + H] : calcd, 428.1637; found,
2
5
22
5
at rt, and the mixture was stirred at this temperature for 2 h. After
4
28.1632. HPLC analysis: 17.79 min, 97.09% purity.
being cooled down to 0 °C, saturated aqueous (sat. aq) Na S O
2
2
3
6
-Amino-5-bromonicotinamide (38f). It is obtained as a deep
solution (10 mL) was poured into the resulting solution and stirred at
rt for 20 min. The mixture was poured into water and extracted with
EtOAc. The organic layer was washed with water and brine, dried
with Na SO , filtered, and concentrated under reduced pressure to
1
brown solid (1.6 g, yield 80%). H NMR (400 MHz, DMSO-d ): δ
6
8.47 (d, J = 2.0 Hz, 1H), 8.15 (d, J = 2.0 Hz, 1H), 7.79 (s, 1H), 7.21
+
(
s, 1H), 6.81 (s, 2H). MS (ESI) m/z: 217.1 [M + H] .
2
4
6
-Amino-5-bromo-N-methylnicotinamide (38g). It is obtained as
give methyl 4-isopropylbenzoate as light yellow oil (5.4 g, yield 99%).
The crude product was used directly for the next step without further
purification. Step 2: To a solution of methyl 4-isopropylbenzoate (4.2
g, 23.60 mmol) in AcOH/Ac O (30 mL, 2:1, v/v) were added NaIO
1
a brown solid (yield 91%). H NMR (400 MHz, DMSO-d ): δ 8.44
6
(d, J = 2.1 Hz, 1H), 8.26 (q, J = 4.5 Hz, 1H), 8.13 (d, J = 2.1 Hz, 1H),
+
6
.79 (s, 2H), 2.68 (brs, 3H). MS (ESI) m/z: 231.3 [M + H] .
2
4
4
-((Trimethylsilyl)ethynyl)isoquinoline (39a). General Procedure
(
2.6 g, 12.11 mmol) and I (2.0 g, 8.01 mmol) at 0 °C. Concentrated
2
for Syntheses of 39a−b and 39f−h. To a solution of 4-bromoisoquino-
line (2.0 g, 9.61 mmol) in dry MeCN/Et N (50 mL, 4:1, v/v) were
added Pd(PPh ) Cl (337 mg, 0.48 mmol), CuI (183 mg, 0.96
mmol), and ethynyltrimethylsilane (2.0 mL, 14.42 mmol) at rt under
H SO (10 mL, 184 mmol) was added dropwise into the solution,
2
4
3
and the mixture was stirred at 40 °C for 4 h. Water (40 mL) and sat.
3
2
2
aq Na S O solution (25 mL) was added into the resulting solution
2
2
3
under an ice bath. After stirring at rt for 30 min, the mixture was
extracted with CH Cl . The organic layer was washed with water and
a N atmosphere. After stirring at 80 °C for 4 h under a nitrogen
2
2
2
atmosphere, the mixture was diluted with EtOAc and filtered through
a pad of Celite. The filtrate was removed by reduced pressure to give
the crude material, which was further purified by flash column
chromatography (SiO , petroleum ether/EtOAc, 15:1) to give 39a as
a light brown solid (1.7 g, yield 79%). H NMR (400 MHz, DMSO-
brine and dried with Na SO . The resultant crude material was
2
4
purified by flash column chromatography (SiO , petroleum ether/
2
EtOAc, 50:1) to give methyl 3-iodo-4-isopropylbenzoate as yellow oil
(5.1 g, yield 71.16%). Step 3: To a solution of methyl 3-iodo-4-
2
1
isopropylbenzoate (3.1 g, 10.19 mmol) in THF/MeOH/H O (35
2
d6): δ 9.34 (s, 1H), 8.66 (s, 1H), 8.21 (d, J = 8.2 Hz, 1H), 8.14 (dd, J
mL, 4:1:1, v/v/v) was added LiOH·H O (1.1 g, 26.21 mmol) at rt,
2
=
1.0, 8.4 Hz, 1H), 7.94 (ddd, J = 1.3, 6.9, 8.3 Hz, 1H), 7.78 (ddd, J =
and the mixture was stirred for 5 h. The resulting solution was cooled
down in an ice bath, then the pH was adjusted to 4−5 with diluted
hydrochloric acid (1 M), and the product was precipitated. After
stirring at 0 °C for 30 min, the mixture was filtrated. The filter cake
was rinsed with water (3 × 15 mL) and then dried in a vacuum oven
+
1
.2, 6.9, 8.1 Hz, 1H), 0.32 (s, 9H). MS (ESI) m/z: 226.3 [M + H] .
3
-((Trimethylsilyl)ethynyl)imidazo[1,2-b]pyridazine (39b). It is
1
obtained as a brown solid (yield 82%). H NMR (400 MHz, DMSO-
d6): δ 8.67 (dd, J = 1.3, 4.4 Hz, 1H), 8.21 (dd, J = 1.4, 9.2 Hz, 1H),
M
https://dx.doi.org/10.1021/acs.jmedchem.9b01912
J. Med. Chem. XXXX, XXX, XXX−XXX

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Article
1
to give 40c as a yellow solid (2.8 g, yield 95%). H NMR (400 MHz,
42e−f. To a solution of 41e (200 mg, 0.56 mmol) in dry MeCN (40
mL) were added 39i (205 mg, 0.76 mmol), CsF (214 mg, 1.41
mmol), and Et N (235 μL, 1.69 mmol) at rt under a N atmosphere.
DMSO-d ): δ 12.99 (s, 1H), 8.32 (s, 1H), 7.91 (d, J = 8.1 Hz, 1H),
.45 (d, J = 8.1 Hz, 1H), 3.24 (hept, J = 6.8 Hz, 1H), 1.19 (d, J = 6.7
6
7
3
2
+
Hz, 6H). MS (ESI) m/z: 290.3 [M + H] .
The mixture was heated to 80 °C and stirred for 4 h. The resulting
solution was poured into water and extracted with EtOAc. The
organic layer was washed with brine and dried with Na SO . The
N-(3,5-Dimethoxyphenyl)-3-iodo-4-methylbenzamide (41a). It is
1
obtained as a yellow solid (2.5 g, yield 82%). H NMR (400 MHz,
2
4
DMSO-d ): δ 10.17 (s, 1H), 8.38 (d, J = 1.9 Hz, 1H), 7.88 (dd, J =
.9, 7.9 Hz, 1H), 7.48 (d, J = 8.2 Hz, 1H), 7.07 (d, J = 2.3 Hz, 2H),
.27 (t, J = 2.3 Hz, 1H), 3.73 (s, 6H). MS (ESI) m/z: 398.2 [M +
crude material was purified by flash column chromatography (SiO2,
CH Cl /MeOH, 25:1) to give 42e as a yellow solid (220 mg, yield
6
1
6
H] .
2
2
1
82.8%). H NMR (400 MHz, DMSO-d ): δ 10.29 (s, 1H), 8.39 (d, J
6
+
= 5.8 Hz, 1H), 8.11−8.06 (m, 1H), 8.04−7.97 (m, 1H), 7.83 (dd, J =
9.3, 1.9 Hz, 1H), 7.51 (t, J = 9.0 Hz, 1H), 7.07 (br s, 2H), 6.78 (s,
N-(3,5-Dimethoxyphenyl)-3-iodobenzamide (41b). It is obtained
as a yellow solid (yield 90%). H NMR (400 MHz, methanol-d ): δ
.24 (d, J = 15.2 Hz, 1H), 7.91 (t, J = 8.0 Hz, 2H), 7.28 (t, J = 7.8 Hz,
H), 6.96 (s, 2H), 6.29 (s, 1H), 3.78 (s, 6H). MS (ESI) m/z: 384.1
1
4
1H), 6.70 (s, 2H), 6.28 (s, 1H), 3.74 (s, 6H). MS (ESI) m/z: 470.2
+
8
1
[
M + H] .
3
-((2-Amino-5-bromopyridin-3-yl)ethynyl)-4-chloro-N-(3,5-
+
[
M + H] .
dimethoxyphenyl)benzamide (42f). It is obtained as a yellow solid
1
N-(3,5-Dimethoxyphenyl)-3-iodo-4-isopropylbenzamide (41c). It
is obtained as a yellow solid (yield 85%). H NMR (400 MHz,
DMSO-d ): δ 9.09 (s, 1H), 8.33 (d, J = 2.9 Hz, 1H), 7.88 (dd, J =
5.0, 3.1 Hz, 1H), 7.33 (d, J = 15.0 Hz, 1H), 7.01 (d, J = 3.1 Hz, 2H),
(yield 76%). H NMR (400 MHz, DMSO-d ): δ 10.34 (s, 1H), 8.40
6
1
(d, J = 2.2 Hz, 1H), 8.10 (d, J = 2.5 Hz, 1H), 7.94 (dd, J = 8.5, 2.2 Hz,
1H), 7.83 (d, J = 2.4 Hz, 1H), 7.77 (d, J = 8.4 Hz, 1H), 7.65−7.54
(m, 2H), 7.07 (d, J = 2.2 Hz, 2H), 6.29 (t, J = 2.2 Hz, 1H), 3.74 (s,
6
1
6
1
+
.23 (t, J = 3.0 Hz, 1H), 3.79 (s, 6H), 3.04−2.69 (m, 1H), 1.17 (d, J =
6H). MS (ESI) m/z: 487.1 [M + H] .
+
2.7 Hz, 6H). MS (ESI) m/z: 426.1 [M + H] .
3-((2-Amino-5-bromopyridin-3-yl)ethynyl)-N-(3,5-dimethoxy-
N-(3,5-Dimethoxyphenyl)-3-iodo-4-methoxybenzamide (41d). It
is obtained as a gray solid (yield 84%). H NMR (400 MHz,
methanol-d ): δ 8.37 (s, 1H), 7.97 (d, J = 8.9 Hz, 1H), 7.05 (d, J = 8.7
Hz, 1H), 6.96 (s, 2H), 6.29 (s, 1H), 3.95 (s, 3H), 3.78 (s, 6H). MS
phenyl)-4-(trifluoromethyl)benzamide (42g). It is obtained as a
1
1
yellow solid (yield 60%). H NMR (400 MHz, methanol-d ): δ 8.34
4
4
(s, 1H), 8.06 (s, 2H), 7.92 (d, J = 8.5 Hz, 1H), 7.77 (s, 1H), 7.00 (s,
2H), 6.33 (s, 1H), 5.50 (s, 1H), 3.80 (s, 6H). MS (ESI) m/z: 520.1
+
+
(
ESI) m/z: 414.1 [M + H] .
[M + H] .
3
-Bromo-N-(3,5-dimethoxyphenyl)-4-fluorobenzamide (41e). It
is obtained as a gray solid (yield 76%). H NMR (400 MHz, DMSO-
d6): δ 10.27 (s, 1H), 8.30 (d, J = 6.1 Hz, 1H), 8.02 (m, 1H), 7.54 (m,
H), 7.06 (s, 2H), 6.28 (s, 1H), 3.74 (s, 6H). MS (ESI) m/z: 402.1
M + H] .
-Chloro-N-(3,5-dimethoxyphenyl)-3-iodobenzamide (41f). It is
obtained as a white solid (yield 86%). H NMR (400 MHz, methanol-
d4): δ 8.43 (s, 1H), 7.88 (d, J = 8.2 Hz, 1H), 7.60 (d, J = 8.3 Hz, 1H),
.95 (s, 2H), 6.28 (s, 1H), 3.77 (s, 6H). MS (ESI) m/z: 418.2 [M +
H] .
-Bromo-N-(3,5-dimethoxyphenyl)-4-(trifluoromethyl)-
benzamide (41g). It is obtained as a white solid (yield 77.8%). H
NMR (400 MHz, methanol-d ): δ 8.32 (s, 1H), 8.03 (d, J = 7.9 Hz,
Methyl 3-((2-Amino-5-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)-
ethynyl)-4-methylbenzoate (44). Step 1: To a solution of 43 (5.0
g, 18.11 mmol) in dry MeCN (150 mL) were added 39i (6.3 g, 23.54
mmol), Pd(PPh ) Cl (636 mg, 0.91 mmol), CuI (345 mg, 1.81
1
1
[
3
2
2
+
mmol), CsF (6.9 g, 45.28 mmol), and Et N (7.6 mL, 54.3 mmol) at rt
3
4
under a N atmosphere, and the mixture was stirred for 6 h. The
2
1
resulting solution was filtered through a pad of Celite, and the solvent
was removed by reduced pressure. The resultant crude material was
6
purified by flash column chromatography (SiO , petroleum ether/
2
+
EtOAc, 8:1) to give methyl 3-((2-amino-5-bromopyridin-3-yl)-
ethynyl)-4-methylbenzoate (yellow solid, 6.0 g, yield 96%). The
product obtained above (6.0 g, 17.38 mmol) was dissolved in THF/
3
1
4
H O (150 mL, 4:1, v/v), followed by the addition of 1-methyl-4-
2
1
6
H), 7.91 (d, J = 8.0 Hz, 1H), 6.98 (s, 2H), 6.32 (s, 1H), 3.79 (s,
(
2
4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (5.4 g,
6.07 mmol), Pd(OAc) (0.4 g, 1.74 mmol), X-Phos (1.7 g, 3.48
+
H). MS (ESI) m/z: 452.1 [M + H] .
2
5
-((2-Amino-5-bromopyridin-3-yl)ethynyl)-N-(3,5-dimethoxy-
phenyl)-2-methylbenz-amide (42a). It is obtained as a yellow solid
yield 81%). H NMR (400 MHz, DMSO-d ): δ 10.20 (s, 1H), 8.25
s, 1H), 8.06 (d, J = 2.3 Hz, 1H), 7.86 (d, J = 5.1 Hz, 1H), 7.85 (s,
H), 7.47 (d, J = 8.1 Hz, 1H), 7.09 (d, J = 1.9 Hz, 2H), 6.62 (s, 2H),
.27 (s, 1H), 3.74 (s, 6H), 2.54 (s, 3H). MS (ESI) m/z: 467.3 [M +
mmol), and K CO (6.0 g, 43.45 mmol) under a N atmosphere. The
2
3
2
mixture was refluxed at 90 °C for 6 h. The resulting solution was
filtered through a pad of Celite, and the filtrate was removed by
reduced pressure to give the crude material, which was further
1
(
(
6
1
6
H] .
purified by flash column chromatography (SiO , petroleum ether/
2
1
EtOAc, 2:1) to give 44 as a yellow solid (5.9 g, yield 98%). H NMR
+
(
7
400 MHz, DMSO-d ): δ 8.28−8.23 (m, 2H), 8.08 (s, 1H), 7.88−
6
3
-((2-Amino-5-bromopyridin-3-yl)ethynyl)-N-(3,5-
dimethoxyphenyl)benzamide (42b). It is obtained as a yellow solid
yield 79.6%). H NMR (400 MHz, DMSO-d ): δ 10.29 (s, 1H), 8.22
s, 1H), 8.05 (s, 1H), 7.93 (d, J = 7.2 Hz, 1H), 7.86 (d, J = 8.0 Hz,
H), 7.82 (br s, 1H), 7.59 (t, J = 7.7 Hz, 1H), 7.09 (s, 2H), 6.71 (s,
H), 6.28 (s, 1H), 3.74 (s, 6H). MS (ESI) m/z: 453.1 [M + H] .
-((2-Amino-5-bromopyridin-3-yl)ethynyl)-N-(3,5-dimethoxy-
phenyl)-4-isopropylbenzamide (42c). It is obtained as a yellow solid
.84 (m, 3H), 7.82 (s, 1H), 7.48 (d, J = 8.0 Hz, 1H), 6.35 (s, 2H),
1
3.87 (s, 3H), 3.84 (s, 3H), 2.56 (s, 3H). MS (ESI) m/z: 347.2 [M +
(
(
6
+
H] .
3
-((2-Amino-5-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)ethynyl)-
1
2
+
4-methylbenzoic acid (45). To a solution of 44 (5.9 g, 17.03 mmol)
in THF/MeOH/H O (90 mL, 4/1/1, v/v/v) was added LiOH·H O
2
2
3
(1.8 g, 42.58 mmol) at rt, and the mixture was stirred at this
1
temperature for 12 h. The resulting solution was cooled to 0 °C, and
then the pH was adjusted to 4−5 with diluted hydrochloric acid (1
M), and the product was precipitated. After stirring for 30 min at 0
(
yield 79%). H NMR (400 MHz, DMSO-d ): δ 10.23 (s, 1H), 8.31−
.24 (m, 2H), 8.09 (s, 1H), 7.92 (d, J = 8.2 Hz, 1H), 7.85 (s, 1H),
.55 (m, 1H), 7.12 (d, J = 2.2 Hz, 2H), 6.31 (s, 1H), 6.28 (t, J = 2.1
6
8
7
°
C, the mixture was filtered, and the filter cake was rinsed with water
Hz, 1H), 3.74 (s, 6H), 3.57 (m, J = 13.7, 6.9 Hz, 1H), 1.29 (d, J = 6.8
+
(3 × 15 mL) and then dried in a vacuum oven to give 45 as a yellow-
Hz, 6H). MS (ESI) m/z: 495.1 [M + H] .
1
green solid (5.2 g, yield 92%). H NMR (400 MHz, DMSO-d ): δ
3
-((2-Amino-5-bromopyridin-3-yl)ethynyl)-N-(3,5-dimethoxy-
phenyl)-4-methoxybenzamide (42d). It is obtained as a yellow solid
yield 77%). H NMR (400 MHz, DMSO-d ): δ 10.13 (s, 1H), 8.22
s, 1H), 8.07 (d, J = 2.4 Hz, 1H), 8.01 (d, J = 8.6 Hz, 1H), 7.77 (d, J =
.3 Hz, 1H), 7.62 (s, 1H), 7.26 (d, J = 8.7 Hz, 1H), 7.08 (d, J = 2.1
Hz, 2H), 6.58 (s, 1H), 6.26 (s, 1H), 3.97 (s, 3H), 3.74 (s, 6H). MS
ESI) m/z: 483.3 [M + H] .
-((2-Amino-5-bromopyridin-3-yl)ethynyl)-N-(3,5-dimethoxy-
phenyl)-4-fluorobenzamide (42e). General Procedure for Syntheses of
6
1
3.06 (br s, 1H), 8.26 (s, 1H), 8.22 (s, 1H), 8.08 (s, 1H), 7.88−7.80
1
(m, 3H), 7.45 (d, J = 8.0 Hz, 1H), 6.35 (s, 2H), 3.84 (s, 3H), 2.55 (s,
(
(
6
+
3H). MS (ESI) m/z: 333.2 [M + H] .
N-(4-Chlorophenyl)-4-iodo-3-methylbenzamide (48a). It is ob-
2
1
tained as a light pink solid (yield 90%). H NMR (400 MHz,
chloroform-d): δ 7.92 (d, J = 8.3 Hz, 1H), 7.81 (s, 1H), 7.71 (s, 1H),
7.58 (d, J = 8.6 Hz, 2H), 7.33 (d, J = 8.4 Hz, 2H), 2.50 (s, 3H). MS
+
(
3
+
(ESI) m/z: 371.2 [M + H] .
N
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Article
4
-Chloro-N-(3-iodo-4-methylphenyl)benzamide (48b). It is ob-
PY99 antibody (Santa Cruz Biotechnology, Santa Cruz, CA),
followed by a HRP-conjugated secondary antibody. The wells were
visualized using o-phenylenediamine and read with a multiwell
spectrophotometer (SpectraMax Plus384, Molecular Devices) at an
absorbance of 490 nM.
1
tained as a white solid (yield 88%). H NMR (400 MHz, DMSO-d ):
6
δ 10.32 (s, 1H), 8.31 (d, J = 2.1 Hz, 1H), 8.07−7.89 (m, 2H), 7.70
(
1
dd, J = 8.3, 2.1 Hz, 1H), 7.68−7.56 (m, 2H), 7.30 (d, J = 8.5 Hz,
+
H), 2.34 (s, 3H). MS (ESI) m/z: 371.2 [M + H] .
N-(4-Chlorophenyl)-3-iodo-4-methylbenzamide (48c). It is ob-
Cell Viability Assay. The cytotoxicity of the compounds was
evaluated by the CCK-8. Briefly, the cells (5 × 10 cells/well) were
1
4
tained as a white solid (yield 79%). H NMR (400 MHz, DMSO-d ):
6
δ 10.38 (s, 1H), 8.39 (d, J = 1.8 Hz, 1H), 7.89 (dd, J = 7.9, 1.9 Hz,
seeded into 96-well plates in triplicate with 200 μL of DMEM media
for 24 h in the presence or absence of indicated concentrations of
compounds. Subsequently, a total of 20 μL CCK-8 was added to each
well. After 1 h incubation, the plates were measured at 450 nm (570
nm calibration) using a microplate reader (Molecular Devices,
Sunnyvale, CA, USA), and the cell viability was calculated. The
cytotoxicity of the compounds was expressed as CC , using the log
1
8
H), 7.82 (d, J = 8.9 Hz, 2H), 7.47 (d, J = 7.7 Hz, 1H), 7.41 (d, J =
+
.8 Hz, 2H), 2.44 (s, 3H). MS (ESI) m/z: 371.2 [M + H] .
N-(4-Chlorophenyl)-3-iodo-N,4-dimethylbenzamide (49). A sol-
ution of 48c (1.0 g, 2.69 mmol) in anhydrous THF (25 mL) was
added dropwise to a suspension of NaH (60%, 215 mg, 5.38 mmol) in
anhydrous THF (20 mL) under stirring at 0 °C. After stirring at 0 °C
for 15 min, MeI (335 μL, 5.38 mmol) was added to the reaction
solution. The resulting solution was stirred at rt for 5 h and then
cooled in an ice bath. After addition of water, the aqueous layer was
extracted with CH Cl . The combined organic layers were washed
5
0
(inhibitor) versus normalized response nonlinear fit (GraphPad Prism
8.0).
6
Western Blot Assay. RAW264.7 cells (1 × 10 cells/well) were
seeded into six-well plates and incubated overnight and then treated
with or without different concentrations of compound 29 for 6 h and
then stimulated with or without CSF-1 (100 ng/mL) for 20 min. Cell
samples were then lysed in 1× SDS lysis buffer (Beyotime, Shanghai,
China) and uniformed by the Pierce BCA protein assay kit (Thermo
Fisher Scientific, Pittsburgh, PA, USA). Equal protein amounts were
loaded to 10% sodium dodecyl sulfate polyacrylamide gel electro-
phoresis and transferred to a nitrocellulose membrane (Bio-Rad,
Hercules, CA, USA), which was blocked with SuperBlockTM T20
blocking buffer (Thermo Fisher Scientific, Pittsburgh, PA, USA) and
then incubated overnight at 4 °C with primary antibodies. The bands
were incubated with HRP-conjugated anti-rabbit IgG (Bio-Rad,
Richmond, CA, USA) and further visualized using a SuperSignalTM
West Femto Maximum Sensitivity Substrate kit (Thermo Fisher
Scientific, Pittsburgh, PA, USA) under the ChemiDocTM MP
Imaging System (Bio-Rad, Richmond, CA, USA).
2
2
with sat. aq Na S O solution and brine and dried with Na SO . The
2
2
3
2
4
crude product was purified by flash chromatography (SiO , petroleum
2
ether/EtOAc, 5:1) to give 49 as a light yellow solid (890 mg, 86%).
1
H NMR (400 MHz, DMSO-d ): δ 7.72 (d, J = 1.4 Hz, 1H), 7.34 (d,
6
J = 8.8 Hz, 2H), 7.22 (d, J = 8.7 Hz, 2H), 7.16 (d, J = 7.9 Hz, 1H),
7
.11 (dd, J = 7.9, 1.4 Hz, 1H), 3.32 (s, 3H), 2.27 (s, 3H). MS (ESI)
+
m/z: 385.1 [M + H] .
-Chloro-N-(3-iodo-4-methylbenzyl)aniline (50). To a solution of
8c (1.0 g, 2.69 mmol) in anhydrous THF (20 mL) was added 1.0 M
4
4
BH ·THF (5.4 mL) solution dropwise at rt, and the reaction mixture
3
was stirred at this temperature for 12 h. The resulting solution was
diluted with EtOAc (20 mL) and quenched by the careful addition of
sat. aq NaHCO solution (5 mL). The organic layer was washed with
3
sat. aq sodium potassium tartrate solution (30 mL), 10% aq. Na CO
2
3
solution (30 mL), and brine (30 mL), dried with anhydrous Na SO ,
2
4
filtered, and concentrated under reduced pressure. The resulting
crude product was purified by flash column chromatography (SiO2,
petroleum ether/EtOAc, 10:1) to give 50 as a white solid (427 mg,
In Vitro Anti-Inflammatory Activity Assay. RAW264.7 cells
were incubated with compounds or the media (0.125% DMSO in
DMEM containing 10% FBS), and then the cells were primed with
LPS (1 μg/mL) for 24 h. The supernatants were collected and then
measured using the mouse TNF-α and IL-6 ELISA kit. The IC50
values were estimated using the log (inhibitor) versus normalized
response nonlinear fit (Graph Pad Prism 8.0).
1
yield 44%). H NMR (400 MHz, chloroform-d): δ 7.90 (s, 1H),
7
.35−7.28 (m, 2H), 7.21 (d, J = 8.9 Hz, 2H), 6.63 (d, J = 8.9 Hz,
2
H), 4.33 (s, 2H), 4.15 (br s, 1H), 2.52 (s, 3H). MS (ESI) m/z: 357.3
+
[
M + H] .
Reagents and Antibodies. Compound PLX3397 (batch
number: 160823; purity: 98%) was purchased from Yi shiming
Beijing) biological medicine technology co. LTD. Compounds were
dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich) at a
concentration of 50 mmol/L and then diluted with the Dulbecco’s
modified Eagle’s medium (DMEM) medium (Hyclone, South Logan,
UT, USA) containing 10% fetal bovine serum (FBS, Hyclone). CCK-
was purchased from Dojindo (Kumamoto, Japan). LPS was
purchased from Sigma (St Louis, MO, USA). Primary antibodies
against CSF1R (3152), phosphor-CSF1R (Tyr723, 3151), Akt
Determination of PK Parameters in Rats. Male SD rats (200 ±
20 g) were purchased from B&K Universal Group Co. Ltd. (Shanghai,
China). All the animal studies were performed according to the
protocols and guidelines of the Institutional Care and Use Committee
of the Shanghai Institute of Materia Medica. Rats were housed in a
well-lit air-conditioned animal room at 19−23 °C with 50−60%
relative humidity under standard environmental conditions (12 h
light/dark cycle). Rats were fed with standard rodent chow and water
at libitum. After acclimation to the laboratory for at least 1 week, rats
were randomly divided into two groups (each group 3) and then
fasted for 12 h with free access to water prior to the experiment. One
group was injected with intravenously (iv) formulation at doses of 2
mg/kg compound 29, and the other group was treated by oral gavage
(p.o.) of p.o. formulation at doses of 5 mg/kg compound 29. The p.o.
formulation was prepared with DMSO/HPMC (5/95, v/v). The iv
formulation was made with DMSO/EtOH/PEG300/Saline (5/5/40/
50, v/v/v/v). Blood samples (40 μL) from the femoral vein were
collected into a heparinized tube at 15, 30, 60, 120, 240, 480, and
1440 min after administration. Blood samples were immediately
centrifuged at 4 °C and 12 000g for 5 min to obtain plasma, which
was then transferred to a clean vial and subsequently stored at −20 °C
until analysis. The plasma sample (20 μL) was precipitated with 200
μL of methanol containing the geniposide (IS) (20 ng/mL) and
vigorously vortex-mixed for 1 min. After centrifugation at 12 000g for
5 min at 4 °C, the supernatants from each sample were analyzed using
LC−MS/MS with an Acquity ultra performance liquid chromatog-
raphy system (I-class, Waters, Milford, USA) coupled to a triple
quadrupole mass spectrometer (Xevo TQ-S, Waters, Milford, USA).
The chromatographic separation was performed on a Welch
Xtimate C18 column (50 × 2.1 mm, 1.7 μm) at a low flow rate (0.35
(
8
(
9272), and phosphor-Akt (Ser473, 4060) were purchased were
purchased from Cell Signaling Technology (CST, Boston, MA, USA).
Actin was purchased from Abgent (Abgent, San Diego, CA, USA),
and anti-rabbit IgG horseradish peroxidase (HRP)-linked secondary
antibody was purchased from Bio-Rad (Bio-Rad, Hercules, CA, USA).
Cell Lines. The murine macrophage RAW 264.7 cells (ATCC,
Manassas, VA, USA) were cultured in DMEM containing 10% fetal
bovine serum (FBS), 2 mmol/L of L-glutamine, 100 U/mL penicillin,
and 100 μg/mL streptomycin. The cells were maintained at 37 °C in a
5
% CO incubator.
2
In Vitro Kinase Enzyme Assay. The kinase inhibitory activity of
each compound was determined using an enzyme-linked immuno-
sorbent assay (ELISA). Recombinant proteins, including CSF1R,
VEGFRs, PDGFRs, EGFRs, RET, c-Kit, Flt3, Src, Abl, EphA2,
IGF1R, IR, FGFRs, BTK, FAK, ITK, and ACK1, were purchased from
Millipore (UK, LTD). Briefly, the 96-well ELISA plates were
precoated with substrate poly (Glu, Tyr)4:1 (Sigma, St. Louis, MO,
USA). Active kinases were added into the plate and incubated with
the compound in reaction buffer containing 5 μM ATP. After
incubation at 37 °C for 1 h, the wells were washed and incubated with
O
https://dx.doi.org/10.1021/acs.jmedchem.9b01912
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
mL/min). Plasma amples or calibration standards (5 μL) were
injected into the system, and the temperature of column and
autosampler was maintained at 45 and 10 °C, respectively. Mobile
ASSOCIATED CONTENT
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.9b01912.
■
*
phases: A, H O (5 mM ammonium acetate and 0.1% HCOOH); B,
2
MeCN (0.1% HCOOH). The gradient program was listed as follow:
1
0
.0−0.1 min, 10% (v/v) B; 0.1−0.8 min, 10−95% B; 0.8−1.8 min,
Blockade effect of 29 on the hERG channel, H NMR,
1
3
19
95% B; 1.8−1.81 min, 95−10% B; 1.81−3.0 min, and 10% B. The
C NMR spectra of compounds 7−37, and F NMR
mass spectrometer equipped with an electrospray ionization probe
was operated in a positive ion mode with a capillary voltage 3.0 kV at
a temperature of 500 °C, and the desolvation gas flow 1000 L/h.
spectra of 7, 20, 22, 30, and 31, and the purity data of all
final compounds (PDF)
+
Molecular formula strings and some data (CSV)
Quantitative analysis was performed by monitoring [M + H] for the
compound 29 and IS in multiple reaction monitoring modes. PK
parameters were calculated using a noncompartmental model of
Phoenix 1.3 software (US Pharsight Corporation). The peak
concentration Cmax and the peak time Tmax were measured; the area
under the concentration time curve of AUC0−t value was calculated by
the trapezoidal method; AUC0−∞ = AUC0−t + C /k , where C is the
Accession Codes
PDB code 4R7H was used for modeling docking of compound
, 8, 12, 16, and 29 in CSF-1R; PDB code 4R7I was used for
modeling docking of compound 7 and 8; PDB code 4V04 was
used for modeling docking of compound 7 and 8 in FGFR1.
7
t
e
t
last blood concentration that can be measured, k is the elimination
e
AUTHOR INFORMATION
Corresponding Authors
rate constant; elimination half-life t1/2 = 0.693/k ; mean residence
■
e
time MRT = AUMC/AUC; clearance CL = D/AUC0−∞; steady-state
distribution volume Vss = CL × MRT; absolute bioavailability F =
AUC0−∞(oral)/AUC0−∞(i.v.) × dose(i.v.)/dose(oral) × 100%.
In Vivo Anti-Inflammatory Efficacy Investigation. All the
Hua Xie − Chinese Academy of Sciences, Shanghai, China,
and University of Chinese Academy of Sciences, Beijing,
China; Email: hxie@simm.ac.cn
animal studies were performed according to the protocols and
guidelines of the institutional care and use committee. Inbred 8-week-
old female BALB/c mice were purchased from Shanghai Lingchang
Biotechnology Co., Ltd. (Certificate no. 2013-0018, Shanghai,
China). All mice were housed under specific pathogen-free conditions
and raised in a 12 h light/dark cycle with humidity (60−80%) and
temperature (22 ± 1 °C). All mice were fed standard laboratory chow
and water at libitum and allowed to acclimatize in our facility for 1
week before any experiments started. Mice were randomly divided
into five groups with six mice per group: untreated normal control,
vehicle (0.5% sodium carboxymethylcellulose and 0.25% Tween-80),
PLX3397 (40 mg/kg), and compound 29 (20 and 40 mg/kg).
Vehicle, PLX3397, and compound 29 were orally administrated at 4 h
before intraperitoneal injection with a dose of 5 mg/kg of LPS. Blood
sample (0.1 mL) was collected via orbital sinus puncture at 1.5 and 3
h after LPS application. The blood sample was centrifuged at 4 °C
and 12 000 rpm for 10 min, and then the serum was transferred to a
clean tube and stored at −80 °C prior to analysis. The serum of each
mouse from all groups was determined by using mouse IL-6 and
TNF-α ELISA kits, according to the manufacturer instructions. All
ELISA quantification kits were purchased from BD Pharmingen (San
Diego, CA, USA).
Wei Tang − Chinese Academy of Sciences, Shanghai,
China, and University of Chinese Academy of Sciences,
Beijing, China; Email: tangwei@simm.ac.cn
Youhong Hu − Chinese Academy of Sciences, Shanghai,
China, and University of Chinese Academy of Sciences,
Beijing, China; orcid.org/0000-0003-1770-6272;
Phone: +86-21-50806600-3526; Email: yhhu@
simm.ac.cn
Other Authors
Zhicheng Xie − Chinese Academy of Sciences, Shanghai,
China, and University of Chinese Academy of Sciences,
Beijing, China
Bing Wu − Chinese Academy of Sciences, Shanghai, China,
and University of Chinese Academy of Sciences, Beijing,
China
Yingqiang Liu − Chinese Academy of Sciences, Shanghai,
China, and Fudan University, Shanghai, China
Wenming Ren − Chinese Academy of Sciences, Shanghai,
China
Linjiang Tong − Chinese Academy of Sciences, Shanghai,
China, and University of Chinese Academy of Sciences,
Beijing, China
Molecular Modeling. Ligands were created with ChmBio3D
Ultra 14.0 and minimized according to the MMFF94 force field.
Molecular docking of compounds (7, 8, 12, 16, and 29) into CSF-1R
(
PDB code: 4R7H and 4R7I) and FGFR1 (PDB code: 4V04) kinases
2
3
was performed in AutoDock Vina. For preparation of the
macromolecule and ligands, water was excluded, and Gasteiger
charges were assigned for the protein. Torsional root and the rotatable
bonds of the ligand were identified. Prior to the AutoDock, AutoGrid
was carried out for the preparation of the grid map using a grid box,
which was centered at the original ligand and extended outward the
ligand within the 5 Å domain. A scoring grid was calculated from the
original ligand structure to minimize the computation time. Finally,
AutoDock vina was performed using the Lamarckian Genetic
Algorithm with default settings, and the top ranked poses were
visually inspected. The docking results were shown as the cartoon
model in Pymol.
Caigui Xiang − Chinese Academy of Sciences, Shanghai,
China, and University of Chinese Academy of Sciences,
Beijing, China
Aihuan Wei − Chinese Academy of Sciences, Shanghai,
China
Yuanzhuo Gao − Chinese Academy of Sciences, Shanghai,
China, and University of Chinese Academy of Sciences,
Beijing, China
Limin Zeng − Chinese Academy of Sciences, Shanghai,
China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.9b01912
Statistical Analysis. All experiment data were presented as mean
±
SEM and statistically evaluated by one-way ANOVA followed by
Dunnett’s test between different groups. Statistical analyses were
evaluated using GraphPad Prism 8.0 software. Differences where p <
Author Contributions
Z.X., B.W., and Y.L. contributed equally to this work.
⊥
0.05 were considered statistically significant.
P
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Journal of Medicinal Chemistry
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Article
Notes
Donatelli, R. R.; Chen, Y.; Cheng, D.; Zhou, Z.; Yurkow, E.; Manthey,
C. L.; Player, M. R. Pyrido 2,3-d pyrimidin-5-ones: a novel class of
antiinflammatory macrophage colony-stimulating factor-1 receptor
inhibitors. J. Med. Chem. 2009, 52, 1081−1099.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(8) Illig, C. R.; Manthey, C. L.; Wall, M. J.; Meegalla, S. K.; Chen, J.;
This study was financially supported by the National Science
and Technology Major Project Key New Drug Creation and
Manufacturing of China (2018ZX09711002), Science and
Technology Commission of Shanghai Municipality
Wilson, K. J.; Ballentine, S. K.; DesJarlais, R. L.; Schubert, C.; Crysler,
C. S.; Chen, Y.; Molloy, C. J.; Chaikin, M. A.; Donatelli, R. R.;
Yurkow, E.; Zhou, Z.; Player, M. R.; Tomczuk, B. E. Optimization of a
potent class of arylamide colony-stimulating factor-1 receptor
inhibitors leading to anti-inflammatory clinical candidate 4-cyano-N-
2-(1-cyclohexen-1-yl)-4- 1- (dimethylamino)acetyl -4-piperidi nyl
phenyl -1H-imidazole-2-carboxamide (JNJ-28312141). J. Med.
Chem. 2011, 54, 7860−7883.
(
18431907100), and the State Key Laboratory of Drug
Research (SIMM1803ZZ-01).
ABBREVIATIONS
■
(9) Zhang, C.; Ibrahim, P. N.; Zhang, J.; Burton, E. A.; Habets, G.;
CSF-1R, colony stimulating factor 1 receptor kinase; HTS,
high-throughput screening; FLT-3, FMS-like tyrosine kinase 3;
KIT, stem cell factor receptor; PDGFR, platelet-derived
growth factor receptor; TNF-α, tumor necrosis factor-α; IL-
Zhang, Y.; Powell, B.; West, B. L.; Matusow, B.; Tsang, G.; Shellooe,
R.; Carias, H.; Nguyen, H.; Marimuthu, A.; Zhang, K. Y. J.; Oh, A.;
Bremer, R.; Hurt, C. R.; Artis, D. R.; Wu, G.; Nespi, M.; Spevak, W.;
Lin, P.; Nolop, K.; Hirth, P.; Tesch, G. H.; Bollag, G. Design and
pharmacology of a highly specific dual FMS and KIT kinase inhibitor.
Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 5689−5694.
6
, interleukin-6; RA, rheumatoid arthritis; AD, Alzheimer’s
disease; PVNS/dt-GCT, pigmented villonodular synovitis;
FGFR, fibroblast growth factor receptor; DFG, asparticacid-
phenylalanine-glycine; PDB, Protein Data Bank; Phe, phenyl-
alanine; Ile, isoleucine; HishistidineCys, histidineCyscysteine;
Trp, tryptophan; Glu, glutamicacid; Asp, asparticacid; JM,
juxtamembrane; Gly, glycine; Tyr, tyrosine; Val, valine; Ala,
alanine; Lys, lysine; Thr, threonine; SAR, structure−activity
relationship; IC , half maximal (50%) inhibitory concen-
(10) Martinez-Muriana, A.; Mancuso, R.; Francos-Quijorna, I.;
Olmos-Alonso, A.; Osta, R.; Perry, V. H.; Navarro, X.; Gomez-Nicola,
D.; Lopez-Vales, R. CSF1R blockade slows the progression of
amyotrophic lateral sclerosis by reducing microgliosis and invasion of
macrophages into peripheral Nerves. Sci. Rep. 2016, 6, 25663.
(11) Spangenberg, E.; Severson, P. L.; Hohsfield, L. A.; Crapser, J.;
Zhang, J.; Burton, E. A.; Zhang, Y.; Spevak, W.; Lin, J.; Phan, N. Y.;
Habets, G.; Rymar, A.; Tsang, G.; Walters, J.; Nespi, M.; Singh, P.;
Broome, S.; Ibrahim, P.; Zhang, C.; Bollag, G.; West, B. L.; Green, K.
N. Sustained microglial depletion with CSF1R inhibitor impairs
parenchymal plaque development in an Alzheimer’s disease model.
Nat. Commun. 2019, 10, 3758.
5
0
tration (IC); Het., aromatic heterocycle; HD, hydrogen bond
donor; EGFR, epidermal growth factor receptor; ErbB2/4,
human epidermal growth factor receptor 2/4; VEGFR,
vascular endothelial growth factor receptor; IGF1R, insulin-
like growth factor 1-receptor; IR, insulin receptor; RET, proto-
oncogene tyrosine-protein kinase receptor Ret; BTK, Bruton’s
tyrosine kinase; Src, tyrosine kinases that encoded by the
oncogenic retrovirus, Rous sarcoma virus; FAK, focal adhesion
kinase; Abl, nonreceptor tyrosine kinase Abelson; ITK, IL2-
inducible T-cell kinase; EphA2, ephrin type-A receptor 2;
(12) El-Gamal, M. I.; Al-Ameen, S. K.; Al-Koumi, D. M.; Hamad, M.
G.; Jalal, N. A.; Oh, C.-H. Recent advances of colony-stimulating
factor-1 receptor (CSF-1R) kinase and its inhibitors. J. Med. Chem.
2
(
018, 61, 5450−5466.
13) Tap, W. D.; Wainberg, Z. A.; Anthony, S. P.; Ibrahim, P. N.;
Zhang, C.; Healey, J. H.; Chmielowski, B.; Staddon, A. P.; Cohn, A.
L.; Shapiro, G. I.; Keedy, V. L.; Singh, A. S.; Puzanov, I.; Kwak, E. L.;
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