Discovery of N-(3-((1-Isonicotinoylpiperidin-4-yl)oxy)-4-methylphenyl)-3-(trifluoromethyl)benzamide (CHMFL-KIT-110) as a Selective, Potent, and Orally Available Type II c-KIT Kinase Inhibitor for Gastrointestinal Stromal Tumors (GISTs)

Article abstract of DOI:10.1021/acs.jmedchem.6b00200

c-KIT kinase is a validated drug discovery target for gastrointestinal stromal tumors (GISTs). Clinically used c-KIT kinase inhibitors, i.e., Imatinib and Sunitinib, bear other important targets such as ABL or FLT3 kinases. Here we report our discovery of

Full text of DOI:10.1021/acs.jmedchem.6b00200

Article
pubs.acs.org/jmc
Discovery of N‑(3-((1-Isonicotinoylpiperidin-4-yl)oxy)-4-
methylphenyl)-3-(trifluoromethyl)benzamide (CHMFL-KIT-110)
as a Selective, Potent, and Orally Available Type II c‑KIT Kinase
Inhibitor for Gastrointestinal Stromal Tumors (GISTs)
Qiang Wang,†,‡,§ Feiyang Liu,†,∥ Beilei Wang,†,‡,§ Fengming Zou,†,‡,§ Cheng Chen,†,‡,§
Xiaochuan Liu,†,⊥ Aoli Wang,†,∥ Shuang Qi,†,‡,§ Wenchao Wang,†,‡,§ Ziping Qi,†,‡,§
Zheng Zhao,†,‡,§ Zhenquan Hu,†,‡,§ Wei Wang,†,‡,§ Li Wang,†,‡,§ Shanchun Zhang,‡,#
Yuexiang Wang,○,∇,◆ Jing Liu,*,†,‡,§ and Qingsong Liu*,†,‡,§,∥
†High Magnetic Field Laboratory, Chinese Academy of Sciences, Mailbox 1110, 350 Shushanhu Road, Hefei, Anhui 230031,
P. R. China
‡CHMFL-HCMTC Target Therapy Joint Laboratory, 350 Shushanhu Road, Hefei, Anhui 230031, P. R. China
§Center for Precision Medicine, CAS (Hefei) Institute of Technology Innovation, Hefei Institute of Physical Science,
Chinese Academy of Sciences, Hefei, Anhui 230088, P. R. China
∥University of Science and Technology of China, Hefei, Anhui 230036, P. R. China
⊥Department of Chemistry, University of Science and Technology of China, Hefei, Anhui 230036, P. R. China
#Hefei Cosource Medicine Technology Co., LTD, 358 Ganquan Road, Hefei, Anhui 230031, P. R. China
○SIBS (Institute of Health Sciences)-Changzheng Hospital Joint Center for Translational Medicine, Institute of Health Sciences,
Shanghai Changzheng Hospital, Institutes for Translational Medicine (CAS-SMMU), Shanghai 200031, China
∇Key Laboratory of Stem Cell Biology, Institute of Health Sciences, SIBS, Chinese Academy of Sciences/Shanghai Jiao Tong
University School of Medicine, Shanghai 200031, China
◆Collaborative Innovation Center of Systems Biomedicine, Shanghai 200025, China
S
* Supporting Information
ABSTRACT: c-KIT kinase is a validated drug discovery target for gastrointestinal stromal tumors (GISTs). Clinically used c-KIT
kinase inhibitors, i.e., Imatinib and Sunitinib, bear other important targets such as ABL or FLT3 kinases. Here we report our discovery
of a more selective c-KIT inhibitor, compound 13 (CHMFL-KIT-110), which completely abolished ABL and FLT3 kinase activity.
KinomeScan selectivity profiling (468 kinases) of 13 exhibited a high selectivity (S score (1) = 0.01). 13 displayed great antiproliferative
efficacy against GISTs cell lines GIST-T1 and GIST-882 (GI50: 0.021 and 0.043 μM, respectively). In the cellular context, it effectively
affected c-KIT-mediated signaling pathways and induced apoptosis as well as cell cycle arrest. In addition, 13 possessed acceptable
bioavailability (36%) and effectively suppressed the tumor growth in GIST-T1 cell inoculated xenograft model without apparent
toxicity. 13 currently is undergoing extensive preclinical evaluation and might be a potential drug candidate for GISTs.
activation is an essential driving force in most of the early
development stages of GISTs.2 c-KIT kinase belongs to the type
INTRODUCTION
■
Gastrointestinal stromal tumors (GISTs) are the most common
mesenchymal tumors in the digestive tract, and they are generally
resistant to chemotherapy and radiation therapy.1 Most GISTs
express constitutively activated forms of the c-KIT kinase whose
Received: February 6, 2016
© XXXX American Chemical Society
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Figure 1. Chemical structures of the representative c-KIT kinase inhibitors: (A) FDA approved c-KIT inhibitors for GISTs and (B) multitarget
inhibitors possess c-KIT inhibitory activity.
III receptor tyrosine kinase (RTK) family, which also includes
platelet-derived growth factor receptor alpha/beta (PDGFRα/β),
FMS-related tyrosine kinase 3 (FLT3), and colony stimulating
factor-1 receptor (CSF1R).3 Structurally, the type III RTKs are
characterized by five extracellular immunoglobulin-like repeats
and a kinase insert separating the ATP-binding and phospho-
transferase regions of the kinase domain. The physiologic
activation of wild-type KIT is accomplished by binding to its
ligand, stem cell factor (SCF), which promotes KIT dimerization,
kinase activation, and tyrosine cross phosphorylation. However,
these normal mechanisms of KIT activation can be subverted by
oncogenic mutations, which contribute to constitutive activation.4
All c-KIT mutations in GISTs appear to be associated with
constitutive c-KIT tyrosine phosphorylation,5 and this makes
c-KIT a validated target for the anti-GIST therapy.
medicinal chemistry effort starting with the compound 1 core
scaffold which led to the discovery of a selective, potent, and
orally available type II c-KIT inhibitor compound 13 (CHMFL-
KIT-110) that abolished both the ABL and FLT3 kinase activities
(Figure 2).
Figure 2. Schematic illustration of discovery of compound 13.
Currently two of the kinase inhibitors that bear c-KIT kinase
inhibitory activity have been approved for the clinical use of
GISTs. Compound 1 (Imatinib, Figure 1A),2 as the first clinically
approved type II c-KIT kinase inhibitor for GIST treatment,
binds to the inactive conformation (DFG-out) of c-KIT and
achieved excellent overall success. However, the potent ABL
kinase inhibitory activity of compound 1 did not contribute to
the GISTs treatment and has been arguably reported to link to
potential cardiotoxicity.6 Compound 2 (Sunitinib, Figure 1A),7
the second FDA-approved (type I inhibitor, which binds to the
active conformation (DFG-In) of kinases) second line drug for
GISTs, was reported to possess FLT3/c-KIT dual inhibition
which might be related to potential myelosuppression toxicity.8
Several other multitarget inhibitors, such as 3 (Amuvatinib),9
4 (Masitinib),10 5 (Nilotinib),11 6 (Pexidartinib),12 7 (OSI930),13
8 (Dovitinib),14 etc. were also reported to bear c-KIT kinase
inhibitory activity (Figure 1B). However, for most of them, c-KIT
is not their primary target. In order to trim the off-target profile
and increase the therapeutic safety window, herein we report our
RESULTS AND DISCUSSION
■
Structure−Activity Relation (SAR) Exploration. We
started with examination of the c-KIT/compound 1 X-ray
crystal complex (PDB ID: 1T46) and envisioned that this is
a typical type II binding mode (Figure 3A). The key binding
elements include the hinge binding moiety, linker moiety,
DFG-out hydrophobic pocket binding moiety, and the well-
known “Flag” methyl fragment.15 The two key hydrogen bonds
formed between Glu640 located in the α-helix and Asp810
located in the DFG motif with the amide group in the linker
moiety stabilize the DFG-out conformation of the c-KIT kinase.
The hydrogen bond formed between Cys673 and the terminal
pyridine in the hinge binding area and the hydrogen bond
formed between the gatekeeper residue Thr670 with the −NH
of the aminopyrimidine help to improve the binding affinity.
Comparison of the three available type II c-KIT kinase inhibitors,
compounds 1, 4, and 5, revealed that they all share the same
linker moiety and only slightly differ from the hinge binding
B
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Figure 3. Schematic illustration of SAR exploration rationale. (A) Analysis of c-KIT/compound 1 binding mode (PDB ID: 1T46). (B) Comparison of
compounds 1, 4, and 5 type II key binding moieties.
moiety and the DFG-out hydrophobic pocket binding moiety
(Figure 3B). In addition, further analysis revealed that in the
hinge binding area, all of these three drugs bear a terminal
pyridine (R1), which presumably can provide the hinge binding
hydrogen bond. All of the R2 parts share similar aromatic rings
that connect the R1 and the linker moiety. Since the hinge
binding and the typical type II binding hydrogen bonds are
required for the type II binding inhibitor, we envisioned that
variation of the R2 and R3 fragments might provide more
selectivity space to develop the c-KIT specific inhibitor.
For the SAR exploration of the R1 and R3 moieties, we choose
to start with fixing the R2 as the ether linked piperidine ring
(Table 2). Introduction of the methyl group at the 4-position of
the benzene ring (22) to compound 13 gained 6-fold c-KIT
activity improvement (GI50: 0.031 versus 0.19 μM). However, its
selectivity window against parental BaF3 (GI50: 6.39 μM) and
K562 (GI50: 2.32 μM) has remarkably narrowed down. Further
variation of the R1 fragment (23, 24, and 25) all led to gaining
back of BCR-ABL inhibitory activity, although the c-KIT
inhibitory activities were better than 13. Introducing the −Cl
(26, 27) or −F (28) atom at the 5-position of the benzene ring in
the R3 fragment started to gain back the BCR-ABL activity.
Interestingly, changing the benzene ring to pyridine ring in R3
(29 and 30) led to complete loss of activity to both the c-KIT and
BCR-ABL. Replacement of −CF3 group with a larger −OMe
group in R3 (31) brought about 15-fold activity loss against
c-KIT compared to 12 (GI50: 1.48 versus 0.11 μM). A tert-butyl
isoxazole moiety at R3 position (32) only moderately decreased
the c-KIT activity (GI50: 0.34 versus 0.11 μM) compared to 12.
Benzodioxole (33) and quinolone (34) moieties at the R3
position both led to around 50-fold activity loss against c-KIT
kinase. Changing the amide linkage between the linker moiety
and R3 fragment to the urea linkage (35) increased the BCR-ABL
activity meanwhile lowering the c-KIT activity.
Biochemical and Cellular Property Evaluation. Among
all of the compounds tested, 13 displayed the best potency
against c-KIT kinase and selectivity over ABL kinase, therefore
we chose it for further characterization. We first examined its
kinome wide selectivity profile with DiscoveRx’s KinomeScan
technology.17 The results demonstrated that 13 bore a high
selectivity (S score (1) = 0.01 at 1 μM) among 468 kinases and
mutants tested. Besides high binding affinity to c-KIT, it also
displayed strong binding against CSF1R, DDR1, and PDGFRβ
kinases (percent activity remaining <1% at 1 μM) (Figure 4A and
Supporting Information Table 1. Given the fact that KinomeScan
is a binding assay and may not fully reflect the inhibitory activity,
we then used Invitrogen’s Z’lyte and Promega’s ADP-Glo based
biochemical activity assay with the purified kinase proteins to
confirm 13’s inhibition activity against CSF1R, DDR1, c-KIT,
and PDGFRβ kinases. The ADP-Glo assay results showed that
13 inhibited c-KIT kinase with an IC50 of 99 nM and PDGFRβ
with an IC50 of 120 nM. The results of Z’lyte assay demonstrated
that 13 inhibited DDR1 kinase with an IC50 of 126 nM and
CSF1R with an IC50 of 133 nM (Figure 4B).
As the starting point for the SAR exploration, we chose the
meta-CF3-substituted benzene as R3 fragment, which is slightly
smaller than the corresponding part of compounds 1, 4, and 5
in order to leave space for further exploration (Table 1). We
used TEL-c-KIT-BaF3 and parental BaF3 cells to monitor the
c-KIT kinase inhibitory activity and K562 cells to monitor
the BCR-ABL inhibitory activity.16 Replacing the R2 fragment
from amine linked aromatic ring to a more flexible ether linked
aliphatic chain (9 and 10) led to significant activity loss com-
pared to compound 1 in the TEL-c-KIT-BaF3 growth inhibitory
assay. However, when we fixed the conformation of aliphatic
chain with the aliphatic piperidine ring (11), it started to gain the
c-KIT activity and became even more potent than compound 1
(GI50: 0.11 versus 0.40 μM). Although 11 bore a good selectivity
window between the TEL-c-KIT-BaF3 and the parental BaF3
cells (GI50: >10 μM), it also exhibited moderate activity against
K562 cell (GI50: 2.94 μM), indicating the moderate BCR-ABL
inhibitory activity. Shifting the nitrogen atom position in the
R1 fragment, i.e., the terminal pyridine (12 and 13), not only
retained the c-KIT activity (GI50: 0.33 and 0.19 μM, respectively)
but also abolished the BCR-ABL activity completely (K562 cell,
GI50: >10 μM), meanwhile retaining the selectivity over the
parental BaF3 cells (GI50: >10 μM). However, when the pyridine
group was replaced by the isoxazole group (14) in R1, it started
to gain back the BCR-ABL activity (GI50: 0.16 μM). Changing
the R1 fragment from the aromatic rings to the aliphatic chains
such as ethyl (15) and ethylene (16) both led to the c-KIT
activity loss. Switching the ether linkage from the 4-position
of the piperidine to the 3-position in R2 (17 and 18) replaced
the “flag” methyl to hydrogen atom completely lost the c-KIT
activity. In addition, replacing the “flag” methyl group with
hydrogen atom (19) led to the slight c-KIT activity loss com-
pared to the most active lead compound 11 (GI50: 0.49 versus
0.11 μM). Substitution of the “flag” methyl with −Cl (20) further
decreased the c-KIT activity (GI50: 0.96 μM) and −OMe group
(21) led to the complete c-KIT activity loss (GI50: > 10 μM).
These results implied that the piperidine ring in the R2 fraction
is well tolerated and that the “flag” methyl in the R4 fraction is
required for the high inhibitory activity.
Compound 13’s inhibitory activity among a variety of c-KIT
mutants was examined in the TEL-c-KIT-BaF3 cells. The results
showed that 13 was more sensitive to c-KIT/V559D mutant
(GI50: 0.04 μM) than others and that it exhibited a similar trend
to 1 (Table 3). Compound 2 was more potent against these
C
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a
Table 1. SAR Exploration Focused on the R1/R2/R4 Positions
a
All GI50 values were obtained by triplet testing.
mutants, but it also affected the parental BaF3 cells growth
(GI50: 2.78 μM) which indicated the multiple target features.
Compound 13 was next tested against a variety of intact
cancer cell lines. Not surprisingly, it did potently inhibit the
growth of c-KIT-dependent GIST cancer cells such as GIST-T1
(GI50: 0.021 μM) and GIST-882 (GI50: 0.043 μM) but not the
c-KIT-independent GIST cell line GIST48B (GI50: > 10 μM)
(Table 4). In addition, unlike 1, 13 did not exhibit potent
antiproliferative activity against BCR-ABL driven CML cell lines
such as K562 (GI50: > 10 μM), MEG-01 (GI50: 7.43 μM), and
D
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a
Table 2. SAR Exploration Focused on the R1/R3 Positions
a
All GI50 values were obtained by triplet testing.
E
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Figure 4. Kinome wide selectivity profiling of 13. (A) KinomeScan profiling of 13 at a concentration of 1 μM against 468 kinases. (B) Biochemical assay
characterization of 13’s inhibitory activity against c-KIT, PDGFRβ (Promega ADP-Glo), DDR1, and CSF1R (Invitrogen Z’lyte).
Table 3. Antiproliferative Effects of 1, 2, and 13 against a
Variety of TEL-c-KIT-BaF3 Isogenic Cells
Table 4. Antiproliferation Effects of 1, 2, and 13 against a
Variety of Intact Cancer Cell Lines
a
a
1 GI50
(μM)
2 GI50
(μM)
13 GI50
(μM)
cell line
1 GI50 (μM)
2 GI50 (μM)
13 GI50 (μM)
cell line
parental BaF3
GIST-T1
GIST-882
GIST48B
K562
0.008
0.014
>10
0.041
0.11
2.01
1.00
1.20
1.10
1.70
0.001
0.005
0.001
0.87
2.48
2.03
0.021
0.043
>10
>10
7.43
6.71
>10
>10
>10
>10
>10
>10
>10
>10
0.40
0.039
3.0
2.78
>10
0.19
0.04
1.05
1.06
8.24
1.03
0.15
5.31
>10
Tel-c-KIT-BaF3
0.078
0.009
0.006
0.51
Tel-c-KIT/V559D-BaF3
Tel-c-KIT/V559D/V654A-BaF3
Tel-c-KIT/N822K-BaF3
Tel-c-KITt/T670I-BaF3
Tel-c-KIT/V654A-BaF3
Tel-c-KITt/L567P-BaF3
Tel-c-KIT/T670I/V559D-BaF3
Tel-c-KIT/D816V-BaF3
0.12
0.074
0.16
>10
MEG-01
KU812
U937
1.29
>10
2.49
0.10
6.67
>10
0.002
0.02
MV-4−11
MOLM-14
HL-60
>10
0.023
0.053
1.38
>10
>10
REC-1
>10
a
All GI50 values were obtained by triplet testing.
CHL
>10
CHO
>10
a
All GI50 values were obtained by triplet testing.
KU812 (GI50: 6.71 μM). Furthermore, 13 did not show any
apparent inhibitory activity against MV4-11 and MOLM13 cells
that are FLT3-ITD growth dependent, which further confirmed
that it had no FLT3 kinase activity. 13 did not inhibit the growth
of other leukemic cell lines such as U937, HL-60, REC-1, and the
normal Chinese hamster ovary cells CHO and CHL cells either,
indicating a good safety profile. Comparably, 2 exhibited weaker
antiproliferative effect against c-KIT-dependent cell lines and
moderate inhibitory activities against BCR-ABL-dependent
CML cell lines. However, it did show potent efficacies against
several AML cell lines such as MV4-11, MOLM14, and HL-60,
which reflected its multiple target features such as FLT3-ITD and
others (Table 4).
(PDB ID: 1T46) with a typical type II binding mode (Figure 5A).
Surprisingly, instead of using the terminal pyridine as the hinge
binding like 1 does, 13 forms an O−H−N hydrogen bond by the
amide carbonyl connecting the terminal pyridine moiety and
piperidine moiety with the Cys673 in the hinge binding area at a
distance about 2.9 Å. There are also two typical hydrogen bonds
formed between the Glu640 and Asp810 with the amide moiety in
the tail part of 13. In the ABL kinase (PDB ID: 2HYY), although
the docking results showed that 13 could also adopt the similar
type II binding mode, the hydrogen bond formed in the hinge
binding area is a little longer than in the c-KIT kinase (3.6 versus
2.9 Å) (Figure 5B). In addition, the spatially adjacent Tyr253
(3.1 Å to the pyridine moiety) in the P-loop introduces a possible
steric hindrance which prevents 13 from stably binding to the
ABL kinase. This could be more clearly illustrated when super-
imposing 13 and 1 in the 1-ABL kinase X-ray crystal structure
In order to understand the structural binding mechanism of
13 to c-KIT kinase and the selectivity mechanism between the
c-KIT and ABL kinase, 13 was docked into c-KIT kinase and
ABL kinase, respectively. The docking results demonstrated
that 13 could adopt the DFG-out conformation of c-KIT kinase
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Figure 5. Structural basis of the binding and selectivity mechanism for 13 against c-KIT and ABL kinase. Hydrogen bonds are indicated by red-hatched
lines to key amino acid residues (highlighted). (A) 13 (in color by atoms) was docked into c-KIT kinase (PDB ID: 1T46) (in white). (B) 13 (in color by
atoms) was docked into ABL kinase (PDB ID: 2HYY) (in white). (C) Superimposition of 1-ABL kinase X-ray crystal structure (in purple) and docking
model of 13 (in color by atoms) with ABL kinase (PDB ID: 2HYY) (in white). (D) Superimposition of docked model of 13 with ABL kinase (PDB ID:
2HYY) (in purple) and 13 with c-KIT kinase (PDB ID: 1T46) (in white).
(PDB ID: 2HYY) (Figure 5C). The pyridine moiety in 1 forms a
hydrogen bond in the hinge binding area with a distance of 2.9 Å,
and this aromatic moiety moves slightly further from the Tyr253
compared to the pyridine moiety in 13, which presumably
could avoid the potential steric hindrance. While in the c-KIT
kinase, the Tyr253 was replaced by the Gly596, which would
provide enough space for this terminal pyridine moiety to be
accommodated (Figure 5D).
Compound 13’s effect on the signaling pathways was evaluated
in the intact GIST cancer cell lines (Figure 6). As expected,
it significantly affected c-KIT pY719, pY703, and pY823 auto-
phosphorylation sites and downstream signaling mediators
such as pAKT(S473), pERK(T202/204), pS6K(T389), and
pSTAT5(Y694) in the c-KIT growth-dependent GIST cancer
cell lines GIST-T1 and GIST-882. However, in the c-KIT-
independent GIST cancer cell line GIST-48B, despite the c-KIT
(Y719, Y703) autophosphorylation site inhibition, it did not
or only weakly affected pc-KIT(Y823) and downstream
mediators pAKT, pERK, and pS6K, though it still strongly
affected pSTAT5(Y694). In addition, in all of these three cell
lines, 13 greatly inhibited pPDGFR which matched the in vitro
biochemical characterizations. It is noteworthy that in all of
the signaling pathways tested, 13 exhibited similar trends to the
well-established c-KIT inhibitor 1, which further proved its
c-KIT kinase inhibitory activity. In accordance with 13’s effect on
the c-KIT-mediated signaling pathways, it could also induce cell
cycle arrest and apoptosis in GIST-T1 and GIST-882 cells but
not GIST-48B cells (Figure 7).
Compound 13’s antitumor efficacy was tested in GIST-T1 cell
inoculated xenograft mouse model. With oral gavage, none of the
25, 50, and 100 mg/kg/day administrations affected the mice
body weight (Figure 8A). However, a 50 and 100 mg/kg dosage
significantly suppressed tumor progression (Figure 8B−D).
Immunohistochemistry stain showed that 13 could strongly sup-
press the antiproliferation (Ki67 stain) and induce the apoptosis
effect (TUNEL stain) in the tumor tissues (Figure 8E). It is
worth mentioning that the PK profile obtained from rats may not
be directly transformed into mice due to the species difference.
CHEMISTRY
■
Starting from commercially available 2-substituted nitrophenol
derivatives 36a−d, etherification with various Boc-protected R2
groups followed by hydrogenation afforded aniline compound
38. Amide formation with carboxylic acid format of R3 group
followed by Boc-deprotection furnished intermediate 40.
Finally installment of R1 group with amide formation conditions
generated compounds 9−30 (Scheme 1). Compounds 31−34
were obtained with a slightly modified procedure (Scheme 2).
Starting from compound 37a, Boc-deprotection was followed by
amide formation to install the R1 group first. Then hydro-
genation reduction of nitro group and subsequent introduction
of various R3 groups were by amide formation conditions
affording compounds 31−34. The synthesis of urea compound
35 started from 38a, which upon urea formation was first to
install the R3 group, and then R1 group was introduced via 42a
and 42b intermediates (Scheme 3).
In Vivo PK/PD Evaluation. Compound 13’s PK properties
were tested in rats following intravenous and oral administration
(Table 5). The results demonstrated that 13 bore an acceptable
bioavailability (F = 36%) and a suitable half-life (T1/2 = 4.11 h)
for the oral administration. It is noteworthy that in the iv
injection, 13 bore a short half-life (T1/2, 0.45h) and quick
clearance (CLz, 4.988 L/h/kg), which indicated that it might not
be very metabolically stable, which required further detailed full
spectrum of PK characterization.
CONCLUSIONS
■
Starting with the well-known type II BCR-ABL/c-KIT dual
inhibitor Imatinib’s core scaffold, with the hybrid type II inhibitor
design approach, we have discovered a highly selective c-KIT
inhibitor 13 which almost completely abolished the ABL
kinase activity. In addition, 13 has no FLT3 kinase activity
which might avoid the potential myeloid suppression problem of
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Figure 6. Compound 13’s effects on the signaling transduction pathways in the c-KIT-dependent and -independent intact GIST cancer cell lines.
spectra were recorded with a Bruker 400 NMR spectrometer and
referenced to deuterium dimethyl sulfoxide (DMSO-d6) or deuterium
chloroform (CDCl3). Chemical shifts are expressed in ppm. In the
NMR tabulation, s indicates singlet; d, doublet; t, triplet; q, quartet;
m, multiplet; and br, broad peak. LC/MS were performed on an Agilent
6224 TOF using an ESI source coupled to an Agilent 1260 Infinity
HPLC system operating in reverse mode with an Agilent Eclipse Plus
C18 1.8 μm 3.0 × 50 mm column. Flash column chromatography
was conducted using silica gel (Silicycle 40−64 μm). The purities of all
compounds were determined to be above 95% by HPLC.
c-KIT/FLT3 dual kinase inhibitors. The extensive biological
activity study also revealed that 13 potently inhibited PDGFRβ,
DDR1, and CSF1-R kinase, which might strengthen its antitumor
activity since PDGFR plays critical roles in the angiogenesis,18
DDR1 kinase plays a role in the tumor proliferation, migration,
and invasion,19 and CSF1-R is essential for the cell survival,
proliferation, and differentiation.20 13 strongly inhibited the cell
proliferation and also induced the cell cycle arrest and apoptosis
in the c-KIT-dependent GISTs cancer cells (GIST-T1 and
GIST-882) but not in the c-KIT-independent GIST-48B cells. It
is worth noting that 13 is not sensitive to compound 1-resistant
c-KIT mutants such as V654A, D816V, etc., which are important
mutants observed in the clinic. Further detailed SAR study based
on this new pharmacophore or combinatorial with other related
signaling mediator inhibitors was required for achievement of
sensitivity against those drug-resistant mutants. The acceptable
selectivity profile, PK profile, and antitumor efficacy suggest that
13 might be a useful pharmacological tool to further study the
pathology of GISTs, and currently it is under extensive preclinical
PK/PD evaluation to determine whether it might serve as a good
potential drug candidate for further clinical development.
Compounds 9−30 and 35 were prepared following the synthetic
procedure of 13.
N-(2-(2-Methyl-5-(3-(trifluoromethyl)benzamido)phenoxy)ethyl)-
nicotinamide (9). Yield 68%. 1H NMR (400 MHz, DMSO-d6) δ 10.40
(s, 1H), 9.05 (s, 1H), 8.95 (s, 1H), 8.72 (s, 1H), 8.26 (m, 3H), 7.98 (d,
J = 7.0 Hz, 1H), 7.80 (t, J = 6.8 Hz, 1H), 7.51 (m, 2H), 7.32 (d, J =
7.5 Hz, 1H), 7.13 (d, J = 7.1 Hz, 1H), 4.16 (s, 2H), 3.74 (s, 2H), 2.15 (s,
3H). LC/MS (ESI, m/z) = 444.1466 [M + H+].
N-(2-(2-Methyl-5-(3-(trifluoromethyl)benzamido)phenoxy)ethyl)-
isoxazole-5-carboxamide (10). Yield 71%. 1H NMR (400 MHz,
DMSO-d6) δ 10.40 (s, 1H), 9.19 (s, 1H), 8.76 (s, 1H), 8.30 (s, 2H), 7.98
(s, 1H), 7.81 (s, 1H), 7.47 (s, 1H), 7.31 (s, 1H), 7.11 (s, 2H), 4.13
(s, 2H), 3.71 (s, 2H), 2.13 (s, 3H). LC/MS (ESI, m/z) = 434.1933
[M + H+].
N-(4-Methyl-3-((1-nicotinoylpiperidin-4-yl)oxy)phenyl)-3-
EXPERIMENTAL SECTION
1
(trifluoromethyl)benzamide (11). Yield 58%. H NMR (400 MHz,
■
Chemistry. All reagents and solvents were purchased from
DMSO-d6) δ 10.39 (s, 1H), 8.65 (s, 2H), 8.27 (s, 2H), 7.87−7.78 (m,
3H), 7.48 (s, 2H), 7.30 (s, 1H), 7.14 (s, 1H), 4.61 (s, 1H), 3.86−3.36
commercial sources and used as obtained. H NMR and 13C NMR
1
H
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Figure 7. Compound 13’s induction of cell cycle arrest and apoptosis in GIST-T1 and GIST-882 cell lines but not in GIST-48B cell line.
Table 5. Pharmacokinetic Characterization of 13
13
AUC(0‑t) (ng/mL·h) AUC(0‑∞) (ng/mL·h) MRT(0‑t) (h) T1/2 (h) Tmax (h) Vz (L/kg) CLz (L/h/kg) Cmax (ng/mL) F (%)
iv (1 mg/kg) mean
198.069
22.942
202.038
21.202
779.793
65.913
0.484
0.103
4.799
0.941
0.451
0.056
4.114
0.252
0.017
0.000
0.5
3.219
0.142
4.988
0.545
510.610
82.438
125.167
35.483
NA
NA
SD
po (10 mg/kg) mean
SD
714.588
153.466
76.407
6.419
12.888
1.145
36.08
NA
0
(m, 3H), 2.16 (s, 3H), 2.02 (s, 2H), 1.79 (s, 2H). LC/MS (ESI, m/z) =
484.1775 [M + H+].
N-(4-Methyl-3-((1-propionylpiperidin-4-yl)oxy)phenyl)-3-
1
(trifluoromethyl)benzamide (15). Yield 72%. H NMR (400 MHz,
DMSO-d6) δ 10.39 (s, 1H), 8.29 (s, 2H), 7.99 (d, J = 7.4 Hz, 1H), 7.81
(t, J = 7.0 Hz, 1H), 7.50 (s, 1H), 7.31 (d, J = 8.0 Hz, 1H), 7.15 (d, J =
7.9 Hz, 1H), 4.57 (s, 1H), 3.68 (s, 2H), 3.44 (s, 2H), 2.37 (m, 2H), 2.16
(s, 3H), 1.95 (s, 2H), 1.68 (s, 2H), 1.02 (m, 3H). LC/MS (ESI, m/z) =
435.1816 [M + H+].
N-(4-Methyl-3-((1-picolinoylpiperidin-4-yl)oxy)phenyl)-3-
1
(trifluoromethyl)benzamide (12). Yield 62%. H NMR (400 MHz,
DMSO-d6) δ 10.39 (s, 1H), 8.61 (s, 1H), 8.28 (s, 2H), 7.96 (m, 2H),
7.80 (t, J = 7.9 Hz, 1H), 7.61 (d, J = 7.2 Hz, 1H), 7.50 (s, 2H), 7.31 (d, J =
7.7 Hz, 1H), 7.16 (d, J = 8.4 Hz, 1H), 3.96−3.33 (m, 4H), 2.18 (s, 3H),
2.03 (s, 2H), 1.76 (s, 2H). LC/MS (ESI, m/z) = 484.1773 [M + H+].
N-(3-(1-Isonicotinoylpiperidin-4-yloxy)-4-methylphenyl)-3-
(trifluoromethyl)benzamide (13). HATU (0.06 mmol, 23 mg), 40a
(0.05 mmol, 19 mg), and DIPEA (0.075 mmol, 10 mg) were dissolved
in 0.5 mL of DMF and cooled to 0 °C. Isonicotinic acid (0.06 mmol,
7.4 mg) was added to the system, and the mixture was stirred at room
temperature for 2 h. The system was extracted with EtOAc and dried
with anhydrous Na2SO4. The solvent was removed under vacuum, and
the residue was purified by silica gel flash chromatography (DCM/
MeOH = 10/1) to offer the product 13 (16.2 mg, 67%) as a white solid.
1H NMR (400 MHz, DMSO-d6) δ 10.37 (s, 1H), 8.67 (s, 2H), 8.27 (s,
2H), 7.96 (d, J = 6.8 Hz, 1H), 7.79−7.76 (m, 1H), 7.48 (s, 1H), 7.43 (s,
2H), 7.29 (d, J = 8.0 Hz, 1H), 7.14 (d, J = 7.6 Hz, 1H), 4.60 (s, 1H),
3.83−3.28 (m, 4H), 2.16 (s, 3H), 1.95 (brs, 2H), 1.77 (brs, 2H).
13C NMR (101 MHz, DMSO-d6) δ 165.02, 164.34, 155.06, 152.22,
143.67, 138.37, 136.22, 132.38, 130.92, 130.14, 128.52, 124.94, 122.94,
113.44, 106.97, 71.90, 44.21, 39.38, 38.94, 30.94, 30.19, 16.15. LC/MS
(ESI, m/z) = 484.1781 [M + H+].
N-(3-((1-Acryloylpiperidin-4-yl)oxy)-4-methylphenyl)-3-
(trifluoromethyl)benzamid (16). Yield 76%. 1H NMR (400 MHz,
DMSO-d6) δ 10.39 (s, 1H), 8.27 (s, 2H), 7.97 (s, 1H), 7.79 (d, J = 7.9
Hz, 1H), 7.48 (s, 1H), 7.29 (d, J = 8.1 Hz, 1H), 7.13 (d, J = 8.2 Hz, 1H),
6.84 (dd, J = 16.5, 10.2 Hz, 1H), 6.11 (d, J = 16.1 Hz, 1H), 5.68 (d,
J = 10.2 Hz, 1H), 4.58 (s, 1H), 3.77 (s, 2H), 3.56 (s, 2H), 2.14 (s, 3H),
1.97 (s, 2H), 1.70 (s, 2H). LC/MS (ESI, m/z) = 433.1668 [M + H+].
N-(3-((1-Nicotinoylpiperidin-3-yl)oxy)phenyl)-3-(trifluoromethyl)-
benzamide (17). Yield 63%. 1H NMR (400 MHz, DMSO-d6) δ 10.39
(s, 1H), 8.66−8.56(m, 3H), 8.27 (s, 2H), 7.97 (s, 1H), 7.83−7.19 (m,
5H), 6.84 (s, 0.4H), 6.64 (s, 0.6H), 4.51(s, 1H), 4.05−3.30 (m, 4H),
2.07−1.58 (m, 4H). LC/MS (ESI, m/z) = 470.1620 [M + H+].
N-(3-((1-(Isoxazole-5-carbonyl)piperidin-3-yl)oxy)phenyl)-3-
1
(trifluoromethyl)benzamide (18). Yield 56%. H NMR (400 MHz,
DMSO-d6) δ 10.46 (s, 0.43H), 10.40 (s, 0.57H), 8.78 (s, 0.43H), 8.63 (s,
0.57H), 8.27 (s, 2H), 7.99 (d, J = 7.4 Hz, 1H), 7.80 (t, J = 7.7 Hz, 1H),
7.35 (m, 3H), 6.96 (s, 0.43H), 6.78 (s, 1H), 6.68 (d, J = 7.9 Hz, 0.57H),
4.55 (s, 1H), 3.99−3.30 (m,4H), 2.06−1.85 (m, 3H), 1.61 (s, 1H).
LC/MS (ESI, m/z) = 460.1412 [M + H+].
N-(3-((1-Nicotinoylpiperidin-4-yl)oxy)phenyl)-3-(trifluoromethyl)-
benzamide (19). Yield 53%. 1H NMR (400 MHz, DMSO-d6) δ 10.45
(s, 1H), 8.64 (s, 2H), 8.27 (s, 2H), 8.04−7.69 (m, 3H), 7.50 (s, 1H),
7.48(s, 1H), 7.34(s, 1H), 7.27 (s, 1H), 6.78 (s, 1H), 4.64 (s, 1H), 3.95 (s,
1H), 3.52 (s, 2H), 1.98 (s, 2H), 1.70 (s, 2H). LC/MS (ESI, m/z) =
470.1619 [M + H+].
N-(3-((1-(Isoxazole-5-carbonyl)piperidin-4-yl)oxy)-4-methylphen-
yl)-3-(trifluoromethyl)benzamide (14). Yield 57%, 1H NMR
(400 MHz, DMSO-d6) δ 10.39 (s, 1H), 8.76 (s, 1H), 8.27 (s, 2H),
7.96 (s, 1H), 7.79 (s, 1H), 7.49 (s, 1H), 7.29 (d, J = 7.2 Hz, 1H), 7.14 (d,
J = 8.0 Hz, 1H), 6.96 (s, 1H), 4.64 (s, 1H), 3.79−3.50 (m, 4H), 2.17
(s, 3H), 2.03 (s, 2H), 1.81 (s, 2H). LC/MS (ESI, m/z) = 474.1568
[M + H+].
I
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Figure 8. Compound 13’s antitumor efficacy in GIST-T1 xenograft mouse model. Female nu/nu mice bearing established GIST-T1 tumor xenografts
were treated with 13 at 25.0, 50.0, and 100 mg/kg/d or vehicle. Daily oral administration was initiated when GIST-T1 tumors had reached a size of
100 to 200 mm3. Each group contained six animals. Data, mean SEM (A) body weight and (B) tumor size measurements from GIST-T1 xenograft
mice after 13 administration. Initial body weight and tumor size were set as 100%. (C) Representative photographs of tumors in each group after 25.0,
50.0, or 100 mg/kg/d 13 or vehicle treatment. (D) Comparison of the final tumor weight in each group after 21-day treatment period of 13; ns, p > 0.05,
*p < 0.05, **p < 0.01.
N-(4-Chloro-3-((1-nicotinoylpiperidin-4-yl)oxy)phenyl)-3-
4.62 (s, 1H), 3.79 (s, 2H), 3.66 (s, 2H), 2.16 (s, 3H), 2.02 (s, 2H), 1.79
(s, 2H). LC/MS (ESI, m/z) = 504.1496 [M + H+].
1
(trifluoromethyl)benzamide (20). Yield 55%. H NMR (400 MHz,
DMSO-d6) δ 10.57 (s, 1H), 8.67 (s, 2H), 8.27 (s, 2H), 8.00 (d, J =
7.4 Hz, 1H), 7.90 (d, J = 7.1 Hz, 1H), 7.82 (t, J = 7.5 Hz, 1H), 7.72 (s,
1H), 7.58−7.38 (m, 3H), 4.71 (s, 1H), 3.91−3.34 (m, 4H), 2.07 (s, 2H),
1.81 (s, 2H). LC/MS (ESI, m/z) = 504.1229 [M + H+].
N-(3-((1-(Isoxazole-5-carbonyl)piperidin-4-yl)oxy)-4-methylphen-
yl)-4-methyl-3-(trifluoromethyl)benzamide (25). Yield 75%. 1H NMR
(400 MHz, DMSO-d6) δ 10.33 (s, 1H), 8.76 (s, 1H), 8.22 (s, 1H), 8.15
(d, J = 7.7 Hz, 1H), 7.62 (d, J = 7.6 Hz, 1H), 7.49 (s, 1H), 7.28 (d, J =
7.7 Hz, 1H), 7.13 (d, J = 7.9 Hz, 1H), 6.96 (s, 1H), 4.63 (s, 1H), 3.67
(t, J = 47.8 Hz, 4H), 2.16 (s, 3H), 2.02 (s, 2H), 1.79 (s, 2H). LC/MS
(ESI, m/z) = 488.1722 [M + H+].
N-(4-Methoxy-3-((1-nicotinoylpiperidin-4-yl)oxy)phenyl)-3-
1
(trifluoromethyl)benzamide (21). Yield 66%. H NMR (400 MHz,
DMSO-d6) δ 10.34 (s, 1H), 8.65 (s, 2H), 8.27 (s, 2H), 8.04−7.72 (m,
3H), 7.59−7.44 (m, 2H), 7.38 (d, J = 8.0 Hz, 1H), 7.02 (d, J = 8.6 Hz,
1H), 4.52 (s, 1H), 3.97 (s, 1H), 3.78 (s, 3H), 3.5−3.32 (m, 3H), 2.02
(s, 2H), 1.74 (s, 2H). LC/MS (ESI, m/z) = 500.1726 [M + H+].
N-(3-((1-Isonicotinoylpiperidin-4-yl)oxy)-4-methylphenyl)-4-
methyl-3-(trifluoromethyl)benzamide (22). Yield 66%. 1H NMR
(400 MHz, DMSO-d6) δ 10.33 (s, 1H), 8.68 (d, J = 4.1 Hz, 2H), 8.23
(s, 1H), 8.15 (d, J = 7.2 Hz, 1H), 7.64 (d, J = 8.0 Hz, 1H), 7.55−7.36 (m,
3H), 7.29 (d, J = 8.1 Hz, 1H), 7.14 (d, J = 8.0 Hz, 1H), 4.61 (s, 1H), 3.84
(s, 1H), 3.68 (s, 1H), 3.48 (s, 1H), 3.30 (s, 1H), 2.17 (s, 3H), 2.00
(s, 2H), 1.78 (s, 2H). LC/MS (ESI, m/z) = 498.1933 [M + H+].
4-Methyl-N-(4-methyl-3-((1-nicotinoylpiperidin-4-yl)oxy)phenyl)-
3-Chloro-N-(4-methyl-3-((1-nicotinoylpiperidin-4-yl)oxy)phenyl)-
1
5-(trifluoromethyl)benzamide (26). Yield 72%. H NMR (400 MHz,
DMSO-d6) δ 10.44 (s, 1H), 8.65 (s, 2H), 8.31 (s, 1H), 8.23 (s, 1H), 8.13
(s, 1H), 7.87 (s, 1H), 7.46 (s, 2H), 7.27 (s, 1H), 7.15 (d, J = 6.8 Hz, 1H),
4.60 (s, 1H), 3.92−3.34 (m, 4H), 2.17 (s, 3H), 2.01 (s, 2H), 1.77 (s,
2H). LC/MS (ESI, m/z) = 518.1385 [M + H+].
3-Chloro-N-(3-((1-(isoxazole-5-carbonyl)piperidin-4-yl)oxy)-4-
methylphenyl)-5-(trifluoromethyl)benzamide (27). Yield 56%.
1H NMR (400 MHz, DMSO-d6) δ 10.45 (s, 1H), 8.76 (s, 1H),
8.32 (s, 1H), 8.24 (s, 1H), 8.13 (s, 1H), 7.47 (s, 1H), 7.28 (s, 1H), 7.15
(d, J = 7.4 Hz, 1H), 6.96 (s, 1H), 4.63 (s, 1H), 3.79−3.54 (m, 4H),
2.17 (s, 3H), 2.03 (s, 3H), 1.81 (s, 2H). LC/MS (ESI, m/z) = 508.1169
[M + H+].
1
3-(trifluoromethyl)benzamide (23). Yield 63%. H NMR (400 MHz,
DMSO-d6) δ 10.31 (s, 1H), 8.65 (s, 2H), 8.22 (s, 2H), 7.88 (s, 1H), 7.62
(s, 1H), 7.49 (s, 2H), 7.28 (s, 1H), 7.12 (s, 1H), 4.60 (s, 1H), 3.98−3.43
(m, 4H), 2.16 (s, 3H), 2.00 (s, 2H), 1.77 (s, 2H). LC/MS (ESI, m/z) =
498.1923 [M + H+].
4-Methyl-N-(4-methyl-3-((1-(thiazole-5-carbonyl)piperidin-4-yl)-
oxy)phenyl)-3-(trifluoromethyl)benzamide (24). Yield 65%. 1H NMR
(400 MHz, DMSO-d6) δ 10.32 (s, 1H), 9.24 (s, 1H), 8.22 (s, 2H),
8.14 (s, 1H), 7.62 (s, 1H), 7.49 (s, 1H), 7.27 (s, 1H), 7.12 (s, 1H),
3-Fluoro-N-(4-methyl-3-((1-nicotinoylpiperidin-4-yl)oxy)phenyl)-
1
5-(trifluoromethyl)benzamide (28). Yield 66%. H NMR (400 MHz,
DMSO-d6) δ 10.42 (s, 1H), 8.65 (s, 2H), 8.15−8.09 (m, 2H), 7.96 (s,
1H), 7.87 (s, 1H), 7.47 (s, 2H), 7.27 (s, 1H), 7.15 (d, J = 7.4 Hz, 1H),
4.60 (s, 1H), 3.82−3.35 (m, 3H), 2.17 (s, 3H), 1.99 (s, 2H), 1.77 (s,
2H). LC/MS (ESI, m/z) = 502.1669 [M + H+].
N-(4-Methyl-3-((1-nicotinoylpiperidin-4-yl)oxy)phenyl)-6-
1
(trifluoromethyl)picolinamide (29). Yield 58%. H NMR (400 MHz,
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a
Scheme 1. Synthesis of Compounds 9−30
a
Reagents and conditions: (a) for 37a−d: tert-butyl 4-((methylsulfonyl)oxy)piperidine-1-carboxylate; for 37e: tert-butyl 3-hydroxypiperidine-1-
carboxylate, DIAD, PPh3, THF, 0 °C to rt; for 37f: tert-butyl (2-bromoethyl)carbamate, K2CO3, DMF, 90 °C, overnight; (b) Pd/C, H2, EtOAc, rt,
6 h; (c) R3CO2H, HATU, DIPEA, DMF, rt, 2 h; (d) 4 N HCl in EtOAc, rt, overnight; (e) R1CO2H, HATU, DIPEA, DMF, rt, 2 h.
a
Scheme 2. Synthesis of Compounds 31−34
a
Reagents and conditions: (a) 4 N HCl in EtOAc, rt, overnight; (b) nicotinic acid, HATU, DIPEA, DMF, rt, 2 h; (c) Pd/C, H2, EtOAc, rt, 6 h;
(d) R3CO2H, HATU, DIPEA, DMF, rt, 2 h.
DMSO-d6) δ 10.28 (s, 1H), 8.65 (s, 2H), 8.38 (s, 2H), 8.17 (s, 1H),
7.87 (s, 1H), 7.68−7.27 (m, 3H), 7.15 (s, 1H), 4.66 (s, 1H), 3.98−3.34
(m, 4H), 2.17 (s, 3H), 1.97 (s, 2H), 1.77 (s, 2H). HRMS (ESI, m/z) =
485.1729 [M + H+].
(m, 4H), 2.17 (s, 3H), 2.03 (s, 2H), 1.80 (s, 2H). LC/MS (ESI, m/z) =
475.1522 [M + H+].
Compounds 31−34 were prepared following the synthetic procedure
of 33.
3-Methoxy-N-(4-methyl-3-((1-nicotinoylpiperidin-4-yl)oxy)-
phenyl)benzamide (31). Yield 63%. 1H NMR (400 MHz, DMSO-d6)
δ 10.11 (s, 1H), 8.64 (s, 2H), 7.87 (s, 1H), 7.50−7.46(m, 5H), 7.27 (s,
1H), 7.12 (s, 2H), 4.59 (s, 1H), 3.83−3.55 (m, 7H), 2.15 (s, 3H), 2.00
(s, 2H), 1.76 (s, 2H). LC/MS (ESI, m/z) = 446.2010 [M + H+].
N-(3-((1-(Isoxazole-5-carbonyl)piperidin-4-yl)oxy)-4-methylphen-
yl)-6-(trifluoromethyl)picolinamide (30). Yield 61%. 1H NMR
(400 MHz, DMSO-d6) δ 10.29 (s, 1H), 8.75 (s, 1H), 8.37 (d, J = 8.4
Hz, 2H), 8.18 (d, J = 7.6 Hz, 1H), 7.56 (s, 1H), 7.41 (d, J = 7.3 Hz, 1H),
7.16 (d, J = 7.2 Hz, 1H), 7.02−6.86 (m, 1H), 4.69 (s, 1H), 3.79−3.54
K
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a
Scheme 3. Synthesis of Compound 35
a
Reagents and conditions: (a) 1-chloro-4-isocyanato-2-(trifluoromethyl)benzene, CH2Cl2, rt, overnight; (b) 4 N HCl in EtOAc, rt; (c) nicotinic
acid, HATU, DIPEA, DMF, rt, 2 h.
5-(tert-Butyl)-N-(4-methyl-3-((1-nicotinoylpiperidin-4-yl)oxy)-
phenyl)isoxazole-3-carboxamide (32). Yield 59%. 1H NMR
(400 MHz, DMSO-d6) δ 10.51 (s, 1H), 8.67 (s, 2H), 7.90 (d, J = 7.8
Hz, 1H), 7.49 (m, 2H), 7.36 (d, J = 7.5 Hz, 1H), 7.14 (d, J = 7.8 Hz, 1H),
6.68 (s, 1H), 4.58 (s, 1H), 3.96−3.32 (m, 4H), 2.17 (s, 3H), 2.01
(s, 2H), 1.78 (s, 2H), 1.37 (s, 9H). LC/MS (ESI, m/z) = 463.2273
[M + H+].
N-(4-Methyl-3-((1-nicotinoylpiperidin-4-yl)oxy)phenyl)benzo[d]-
[1,3]dioxole-5-carboxamide (33). To a solution of benzo[d][1,3]-
dioxole-5-carboxylic acid (0.05 mmol, 8.3 mg) and 41c (0.05 mmol,
16 mg) in 0.5 mL DMF were added HATU (0.06 mmol, 22.8 mg) and
DIPEA (0.1 mmol, 13 mg). The mixture was stirred at room tempera-
ture for 2 h, and the system was quenched with water, extracted with
EtOAc, and dried with anhydrous Na2SO4. The solvents were removed
under vacuum, and the crude product was purified by silica gel flash
chromatography (DCM: MeOH = 10:1) to give the product 33 (15.3 mg,
67%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.97 (s, 1H),
8.67 (s, 2H), 7.89 (s, 1H), 7.61−7.40 (m, 4H), 7.28 (s, 1H), 7.09 (m, 2H),
6.15 (s, 2H), 4.60 (s, 1H), 3.95−3.37 (m, 4H), 2.17 (s, 3H), 2.04 (s, 2H),
1.78 (s, 2H). LC/MS (ESI, m/z) = 460.1803 [M + H+].
(d, J = 7.6 Hz, 1H), 6.29−6.23 (m, 2H), 4.32−4.28 (m, 1H), 3.64−3.62
(m, 2H), 3.46−3.42 (m, 2H), 1.82 (s, 4H), 1.45 (s, 9H). LC/MS (ESI,
m/z) = 349.1403 [M + Na+].
tert-Butyl 4-(5-Amino-2-methoxyphenoxy)piperidine-1-carboxy-
1
late (38c). Yield (two-step) 69%. H NMR(400 MHz, DMSO-d6) δ
6.67 (d, J = 7.6 Hz, 1H), 6.30 (s, 1H), 6.13 (d, J = 7.6 Hz, 1H), 4.61 (s,
2H), 4.32−4.28 (m, 1H), 3.63−3.60 (m, 5H), 3.16 (s, 2H), 1.97 (s, 3H),
1.81 (s, 2H), 1.51 (s, 2H), 1.40 (s, 9H). LC/MS (ESI, m/z) = 345.1898
[M + Na+].
tert-Butyl 4-(3-Aminophenoxy)piperidine-1-carboxylate (38d).
Yield (two-step) 58%. 1H NMR (400 MHz, DMSO-d6) δ 6.87 (d, J =
4.4 Hz, 1H), 6.15−6.08 (m, 3H), 4.98 (s, 2H), 4.38−4.36 (m, 1H), 3.61
(s, 2H), 3.16 (s, 2H), 1.97 (s, 3H), 1.84 (s, 2H), 1.50 (s, 2H), 1.40 (s,
9H). LC/MS (ESI, m/z) = 315.1783 [M + Na+].
tert-Butyl 3-(3-Aminophenoxy)piperidine-1-carboxylate (38e).
Diisopropyl azodicarboxylate (DIAD) (6 mmol, 1.21g) and triphenyl-
phosphine (6 mmol, 1.57g) in 20 mL dry THF were stirred at 0 °C for
0.5 h. tert-Butyl 3-hydroxypiperidine-1-carboxylate (6 mmol, 1g) and
3-nitrophenol (5 mmol, 595 mg) were added to the system, which was
warmed to room temperature and stirred overnight. The solvent was
removed under vacuum, and the crude product was purified by silica
gel flash chromatography (petroleum ether:EtOAc = 6:1) to give the
intermediate 37e as a yellow solid. The obtained 37e was dissolved in
20 mL EtOAc, and Pd/C (5%) was added. The mixture was stirred
under hydrogen balloon at room temperature for 6 h. Then the system
was filtered through diatomaceous earth, and the filtrate was con-
centrated under vacuum to give 38e (1.17g) as yellow oil with a two-step
yield 63%. 1H NMR (400 MHz, DMSO-d6) δ 6.90 (t, J = 7.7 Hz, 1H),
6.17−6.10 (m, 3H), 5.04 (s, 2H), 4.20 (s, 1H), 3.79−3.11 (m, 4H),
1.90−1.71 (m, 3H), 1.40−1.31 (m, 10H). LC/MS (ESI, m/z) =
315.1793 [M + Na+].
tert-Butyl (2-(5-amino-2-methylphenoxy)ethyl)carbamate (38f).
2-Methyl-5-nitrophenol (5 mmol, 0.77g) and tert-butyl (2-bromoethyl)-
carbamate (8 mmol, 1.79g) were dissolved in 15 mL DMF. K2CO3
(10 mmol, 1.38g) was added to the system and heated at 90 °C overnight.
The system was extracted with EtOAc and dried with anhydrous Na2SO4.
The solvent was removed under vacuum, and the residue was purified by
silica gel flash chromatography (petroleum ether:EtOAc = 6:1) to give
the intermediate 37f as a yellow solid. 37f (4 mmol, 1.13 g) was dissolved
in 20 mL EtOAc, and Pd/C (5%) was added. The mixture was stirred
under hydrogen balloon at room temperature for 6 h. The system was
filtered through diatomaceous earth, and the filtrate was concentrated
under vacuum to give compound 38f (780 mg) as a yellow oil in two-step
yield 59%. 1H NMR (400 MHz, DMSO-d6) δ 6.95 (s, 1H), 6.74 (d, J =
7.7 Hz, 1H), 6.17 (s, 1H), 6.07 (d, J = 7.6 Hz, 1H), 4.82 (s, 2H), 3.82
(s, 2H), 3.30 (d, J = 5.5 Hz, 2H), 1.98 (s, 3H), 1.40 (s, 9H). LC/MS
(ESI, m/z) = 267.1626 [M + H+].
N-(4-Methyl-3-((1-nicotinoylpiperidin-4-yl)oxy)phenyl)quinoline-
1
3-carboxamide (34). Yield 57%. H NMR (400 MHz, DMSO-d6) δ
10.55 (s, 1H), 9.38 (s, 1H), 8.97 (s, 1H), 8.68 (s, 2H), 8.18−8.13 (m,
2H), 7.90 (s, 2H), 7.76 (s, 1H), 7.57 (s, 1H), 7.51(s, 1H), 7.35 (s, 1H),
7.18 (d, J = 7.1 Hz, 1H), 4.64 (s, 1H), 3.87−3.55 (m, 4H), 2.20 (s, 3H),
2.03 (s, 2H), 1.81 (s, 2H). LC/MS (ESI, m/z) = 467.2001 [M + H+].
1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(4-methyl-3-((1-nicoti-
noylpiperidin-4-yl)oxy)phenyl)urea (35). Yield 53%. 1H NMR
(400 MHz, DMSO-d6) δ 9.22 (s, 1H), 8.82 (s, 1H), 8.65 (s, 2H),
8.08 (s, 1H), 7.88 (d, J = 7.9 Hz, 1H), 7.70−7.56 (m, 2H), 7.50 (d, J =
4.7 Hz, 1H), 7.25 (s, 1H), 7.06 (d, J = 8.2 Hz, 1H), 6.87 (d, J = 6.9 Hz,
1H), 4.62 (s, 1H), 3.83−3.52 (m, 4H), 2.14 (s, 3H), 2.00 (s, 2H),
1.78 (s, 2H). LC/MS (ESI, m/z) = 533.1486 [M + H+].
tert-Butyl 4-(5-Amino-2-methylphenoxy) piperidine-1-carboxy-
late (38a). 2-Methyl-5-nitrophenol (36a) (5 mmol, 0.77g) and tert-
butyl 4-((methylsulfonyl)oxy)piperidine-1-carboxylate (10 mmol,
2.79g) were dissolved in 15 mL DMF. Then K2CO3 (10 mmol, 1.38g)
was added to the system and heated at 90 °C overnight. The system was
extracted with EtOAc and dried with anhydrous Na2SO4. The solvent was
removed under vacuum, and the residue was purified by silica gel flash
column chromatography (petroleum ether:EtOAc = 6:1) to give 37a as
a yellow solid. LC/MS (ESI, m/z) [M + Na+] = 359.1693. The obtained
37a was directly dissolved in 20 mL EtOAc, and Pd/C (5%) was added.
The mixture was stirred under hydrogen balloon at room temperature for
6 h. The system was filtered by diatomaceous earth, and the filtered
organic system was concentrated under vacuum. The residue was purified
by silica gel flash chromatography (petroleum ether:EtOAc = 8:1) to
give 38a (1.12g) as a yellow oil with a two-step yield of 73%. 1H NMR
(400 MHz, DMSO-d6) δ 6.74(d, J = 6.0 Hz, 1H), 6.23 (s, 1H), 6.07 (d,
J = 6.0 Hz, 1H), 4.78 (s, 2H), 4.37−4.35 (m, 1H), 3.55 (m, 2H), 3.26
(m, 2H), 1.97 (s, 3H), 1.83 (s, 2H), 1.55 (s, 2H), 1.40 (s, 9H). LC/MS
(ESI, m/z) = 329.1949 [M + Na+].
N-(4-Methyl-3-(piperidin-4-yloxy)phenyl)-3-(trifluoromethyl)-
benzamide (40a). To a solution of 3-(trifluoromethyl)benzoic acid
(3 mmol 570 mg) and 38a (3 mmol, 918 mg) in 15 mL DMF were
added HATU (3.6 mmol, 1.37g) and DIPEA (4.5 mmol, 585 mg). The
mixture was stirred at room temperature for 2 h, and the system was
quenched with water, extracted with EtOAc, and dried with anhydrous
Na2SO4. The solvents were removed under vacuum and provided the
crude product 39a. LC/MS (ESI, m/z) [M + H+] = 479.2069. Then 39a
Compounds 38b−d were prepared following the synthetic procedure
of 38a.
tert-Butyl 4-(5-Amino-2-chlorophenoxy)piperidine-1-carboxylate
1
(38b). Yield (two-step) 71%. H NMR (400 MHz, CDCl3) δ 7.09
L
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Journal of Medicinal Chemistry
Article
was dissolved in 10 mL 4 N HCl in EtOAc. The mixture was stirred
at room temperature overnight and neutralized by NaHCO3 (aq),
then extracted by EtOAc and dried with anhydrous Na2SO4. The
concentrated residue was purified by silica gel flash chromatography
(DCM:MeOH = 10:1) to give the product 40a (782 mg) as a white
solid with two-step yield 69%. 1H NMR (400 MHz, DMSO-d6) δ 10.42
(s, 1H), 8.27 (s, 2H), 7.94 (s, 1H), 7.77 (s, 1H), 7.48 (s, 1H), 7.28 (s,
1H), 7.11 (s, 1H), 4.44−4.42 (m, 1H), 3.07 (s, 2H), 2.82 (s, 2H), 2.13
(s, 1H), 1.97 (s, 3H), 2.01 (s 2H), 1.70 (s, 2H). LC/MS (ESI, m/z) =
379.1564 [M + H+].
7.91−7.65 (m, 2H), 7.47 (d, J = 8.1 Hz, 1H), 4.92 (s, 1H), 3.16 (s, 4H),
2.29 (s, 3H), 2.17 (s, 2H), 1.95 (s, 2H). LC/MS (ESI, m/z) = 237.1159
[M + H+].
(4-(5-Amino-2-methylphenoxy)piperidin-1-yl) (pyridin-3-yl)-
methanone (41c). To a solution of nicotinic acid (3 mmol, 369 mg)
and 41a (3 mmol, 816 mg) in DMF (15 mL) were added HATU
(3.6 mmol, 1.37g) and DIPEA (4.5 mmol, 585 mg). The resulting
mixture was stirred at room temperature for 2 h, and the system was
quenched with water, extracted with EtOAc, and dried with anhydrous
Na2SO4. The solvents were removed under vacuum to yield the crude
product 41b, which was dissolved in EtOAc (20 mL), and Pd/C (5%)
was added. The mixture was stirred under hydrogen balloon at room
temperature for 6 h. The system was filtered through diatomaceous
earth, and the filtrate was concentrated under vacuum to give the
Compounds 40b−i were prepared following the synthetic procedure
of 40a.
4-Methyl-N-(4-methyl-3-(piperidin-4-yloxy)phenyl)-3-
(trifluoromethyl)benzamide Hydrochloride (40b). Yield (two-step)
63%. 1H NMR (400 MHz, DMSO-d6) δ 10.48 (s, 1H), 9.31 (s, 2H), 8.24
(s, 2H), 7.59−7.49 (m, 2H), 7.31 (s, 1H), 7.11 (s, 1H), 4.57−4.55 (m,
1H), 3.17 (brs, 2H), 3.08 (s, 2H), 2.5 (s, 1H), 2.14 (s, 5H), 1.96 (s, 3H).
LC/MS (ESI, m/z) = 393.1719 [M + H+].
1
product 41c (727 mg) as yellow oil. Yield (two-step) 78%. H NMR
(400 MHz, DMSO-d6) δ 8.65 (s, 2H), 7.88 (d, J = 7.5 Hz, 1H), 7.49 (dd,
J = 7.5, 4.7 Hz, 1H), 6.77 (d, J = 7.8 Hz, 1H), 6.28 (s, 1H), 6.09 (d, J =
7.6 Hz, 1H), 4.87 (s, 2H), 4.49 (s, 1H), 3.96−3.38 (m, 4H), 2.02 (s, 5H),
1.72 (s, 3H). LC/MS (ESI, m/z) = 312.1640 [M + H+].
3-Fluoro-N-(4-methyl-3-(piperidin-4-yloxy)phenyl)-5-
1
(trifluoromethyl)benzamide (40c). Yield (two-step) 58%. H NMR
1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(4-methyl-3-(piperidin-
4-yloxy)phenyl)urea Hydrochloride (42b). Compound 38a (5 mmol,
1.53 g) in CH2Cl2 (20 mL) was added to 1-chloro-4-isocyanato-2-
(trifluoromethyl)benzene (5 mmol, 1.1 g), and the mixture was stirred
overnight at room temperature. The system was extracted with EtOAc
and dried with anhydrous Na2SO4. The solvents were removed under
vacuum to give the crude product 42a, which was added directly into 4 N
HCl in EtOAc (30 mL). The mixture was stirred overnight at room
temperature. The solid was collected and washed with EtOAc to give the
(400 MHz, DMSO-d6) δ 10.43 (s, 1H), 8.27−8.05 (m, 2H), 7.97 (d, J =
7.1 Hz, 1H), 7.43 (s, 1H), 7.28 (d, J = 7.6 Hz, 1H), 7.14 (d, J = 8.0 Hz,
1H), 4.40 (s, 1H), 3.03 (s, 2H), 2.72 (s, 2H), 2.13 (d, s, 3H), 1.93 (s,
2H), 1.64 (s, 2H). LC/MS (ESI, m/z) = 397.1466 [M + H+].
3-Chloro-N-(4-methyl-3-(piperidin-4-yloxy)phenyl)-5-
1
(trifluoromethyl)benzamide (40d). Yield (two-step) 61%. H NMR
(400 MHz, DMSO-d6) δ 10.49 (s, 1H), 8.34 (s, 1H), 8.26 (s, 1H), 8.13
(s, 1H), 7.47 (s, 1H), 7.29 (d, J = 7.9 Hz, 1H), 7.15 (d, J = 7.8 Hz, 1H),
4.43 (s, 1H), 3.07 (s, 2H), 2.77 (s, 2H), 2.16 (s, 3H), 1.95 (s, 2H), 1.70
(s, 2H). LC/MS (ESI, m/z) = 413.1172 [M + H+].
1
product 42b (1.6 g) as a white solid. Yield (two step) 69%. H NMR
(400 MHz, DMSO-d6) δ 9.92 (s, 1H), 9.33 (s, 1H), 8.90 (s, 2H), 8.10 (s,
1H), 7.72−7.52 (m, 2H), 7.30 (s, 1H), 7.06 (d, J = 7.9 Hz, 1H), 6.85 (d,
J = 7.8 Hz, 1H), 4.59 (s, 1H), 3.20 (s, 2H), 3.14 (s, 2H), 2.13 (s, 5H),
1.92 (s, 2H). LC/MS (ESI, m/z) = 428.1281 [M + H+].
N-(4-Methyl-3-(piperidin-4-yloxy)phenyl)-6-(trifluoromethyl)-
1
picolinamide Hydrochloride (40e). Yield (two-step) 53%. H NMR
(400 MHz, DMSO-d6) δ 10.29 (s, 1H), 9.26 (s, 2H), 8.96 (s, 1H), 8.45−
8.27 (m, 2H), 8.19 (d, J = 6.8 Hz, 1H), 7.53 (s, 1H), 7.43 (d, J = 8.1 Hz,
1H), 7.18 (d, J = 7.7 Hz, 1H), 4.65 (s, 1H), 3.21 (s, 2H), 3.12 (s, 2H),
2.18 (s, 5H), 1.97 (s, 2H). LC/MS (ESI, m/z) = 416.1280 [M + H+].
N-(4-Chloro-3-(piperidin-4-yloxy)phenyl)-3-(trifluoromethyl)-
benzamide (40f). Yield (two-step) 56%. 1H NMR (400 MHz, DMSO-
d6) δ 10.69 (s, 1H), 8.32 (s, 2H), 8.00 (d, J = 6.6 Hz, 1H), 7.81 (d, J =
9.3 Hz, 2H), 7.46 (s, 2H), 4.64 (s, 1H), 3.19 (s, 2H), 3.04 (s, 2H), 2.13
(s, 2H), 1.91 (s, 2H). LC/MS (ESI, m/z) = 399.1006 [M + H+].
N-(4-Methoxy-3-(piperidin-4-yloxy)phenyl)-3-(trifluoromethyl)-
benzamide (40g). Yield (two-step) 59%. 1H NMR (400 MHz, DMSO-
d6) δ 10.46 (s, 1H), 9.05 (s, 2H), 8.32 (s, 2H), 7.98 (d, J = 7.6 Hz, 1H),
7.80 (s, 1H), 7.58 (s, 1H), 7.40 (d, J = 9.1 Hz, 1H), 7.04 (d, J = 8.7 Hz,
1H), 4.50 (s, 1H), 3.80 (s, 3H), 3.25 (s, 2H), 3.08 (s, 2H), 2.11 (s, 2H),
1.92 (s, 2H). LC/MS (ESI, m/z) [M + H+] = 395.1513.
Cell Lines and Cell Culture. The human GIST-T1 cell line was
purchased from Cosmo Bio Co., Ltd. Tokyo, Japan. GIST-882 and
GIST-48B cell lines were kindly provided by the Group of Professor
Jonathan A. Fletcher, Brigham and Women’s Hospital in Boston,
USA. K562 (CML), KU812 (CML), MEG-01 (CML), MV4-11 (AML),
MOLM14 (AML), U937 (AML), REC-1 (human B-cell lymphoma
cell), HL-60 (human promyelocytic leukemia cells), MEC-1(CLL),
Kasumi-1 (AML), CHL (hamster lung cell), and CHO (hamster ovary
cell) were obtained from American Type Culture Collection (Manassas,
VA). All the cells were grown in a humidified incubator (Thermo, USA)
at 37 °C under 5% CO2. GIST-T1, CHO cells were maintained in
DMEM supplemented with 10% FBS and 1% penicillin/streptomycin.
MV4-11 (AML), GIST-882, and GIST-48B were grown in IMDM
supplemented with 10% FBS and 1% penicillin/streptomycin. All other
cell lines and all the isogenic BaF3 cells were grown in RPMI 1640
medium supported with 10% FBS and 1% penicillin/streptomycin.
Kasumi-1 (AML) was grown in RPMI 1640 medium supported with
20% FBS and 1% penicillin/streptomycin. Adherent cells were grown in
tissue culture flasks until they were 85−95% confluent prior to use. For
suspension cells, cells were collected by spinning down at 700 rpm/min
for 4 min before use.
General Procedure for Antiproliferation Assays. A density of 1
to 3 × 104 cells/mL cells were mixed with various concentrations of
compounds, then 100 μL suspension was added to each well and
incubated for 72 h. Cell viability was determined using the CellTiter-Glo
(Promega, USA) or CCK-8 (Beboy, China). Both assays were per-
formed according to the manufacturer instructions. For CellTiter-Glo
assay, luminescence was determined in a multilabel reader (Envision,
PerkinElmer, USA). For CCK-8 assay, absorbance was measured in
a microplate reader (iMARK, Bio-Rad, USA) at 450 and 655 nm. Data
were normalized to control group (DMSO). GI50 values were calculated
using Prism 5.0 (GraphPad Software, San Diego, CA).
N-(3-(Piperidin-4-yloxy)phenyl)-3-(trifluoromethyl)benzamide
1
Hydrochloride (40h). Yield (two-step) 47%. H NMR (400 MHz,
DMSO-d6) δ 10.58 (s, 1H), 9.18 (s, 2H), 8.28 (s, 2H), 7.98 (d, J =
7.2 Hz, 1H), 7.80 (s, 1H), 7.57 (s, 1H), 7.40 (d, J = 7.7 Hz, 1H), 7.30
(t, J = 8.0 Hz, 1H), 6.81 (d, J = 7.7 Hz, 1H), 4.65 (s, 1H), 3.20 (s, 3H),
3.10 (s, 2H), 2.15 (s, 2H), 1.91 (s, 2H). LC/MS (ESI, m/z) = 365.1396
[M + H+].
N-(3-(piperidin-3-yloxy)phenyl)-3-(trifluoromethyl)benzamide
(40i). Yield (two-step) 52%. 1H NMR (400 MHz, DMSO-d6) δ 10.47 (s,
1H), 8.29 (s, 2H), 7.98 (d, J = 6.6 Hz, 1H), 7.81 (d, J = 7.2 Hz, 1H),
7.59−7.12 (m, 3H), 6.74 (d, J = 7.4 Hz, 1H), 4.31 (s, 1H), 3.19−2.58
(m, 4H), 2.04 (s, 1H), 1.73 (s, 1H), 1.57−1.50 (m, 2H), 1.23−1.20 (m,
1H). LC/MS (ESI, m/z) = 365.1407 [M + H+].
N-(3-(2-aminoethoxy)-4-methylphenyl)-3-(trifluoromethyl)-
benzamide Hydrochloride (40j). Yield (two-step) 56%. 1H NMR (400
MHz, DMSO-d6) δ 10.59 (s, 1H), 8.46 (s, 3H), 8.35 (d, J = 7.4 Hz, 2H),
7.97 (d, J = 7.4 Hz, 1H), 7.79 (t, J = 7.4 Hz, 1H), 7.54 (s, 1H), 7.39 (d, J =
7.8 Hz, 1H), 7.14 (d, J = 8.0 Hz, 1H), 4.20 (s, 2H), 3.26 (s, 2H), 2.19
(s, 3H). LC/MS (ESI, m/z) = 339.1249 [M + H+].
TEL-Isogenic Cell Generation. Retroviral constructs for BaF3-KIT
mutants were made based on the pMSCVpuro (Clontech) backbone.
For TEL-KIT vector, the first 1 kb of human TEL gene with an artificial
myristoylation sequence (MGCGCSSHPEDD) was cloned into the
pMSCVpuro retroviral vector, followed by a 3xFLAG tag sequence and
4-(2-Methyl-5-nitrophenoxy)piperidine Hydrochloride (41a).
Compound 37a (5 mmol, 1.68 g) was added into 20 mL EtOAc (4 N
HCl), and the system was stirred at room temperature for 6 h. The solid
was collected and dried to give the product 41a HCl salt as a yellow
1
solid (1.22g, 90%). H NMR (400 MHz, DMSO-d6) δ 9.35 (s, 2H),
M
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Journal of Medicinal Chemistry
Article
a stop codon. Then, the kinase domain coding sequence of KIT was
inserted in-frame between TEL and 3xFLAG sequences. For full-length
expression vectors, the coding sequences of KIT variants were directly
cloned in pMSCVpuro vector with a 3xFLAG tag at the C-terminal end.
All mutagenesis was performed using the QuikChange Site-Directed
Mutagenesis Kit (Stratagene) following the manufacturer’s instruc-
tions. Retrovirus was packaged in HEK293T cells by transfecting
KIT-containing MSCV vectors together with two helper plasmids.
Virus supernatants were harvested 48 h after transfection and filtered
before infection. Then BaF3 cells were infected with harvested virus
supernatants using spinoculation protocol, and stable cell lines were
obtained by puromycin selection for 48 h. The IL-3 concentrations in
the culture medium were gradually withdrawn until cells were able to
grow in the absence of IL-3.
Kinase Biochemical Assay. The fluorescence resonance energy-
transfer-based Z′-LYTE kinase assay kit−Tyr 06 peptide (Invitrogen,
Carlsbad, CA) was used to evaluate the IC50 value of compound 13 for
inhibition of c-KIT kinase. The reaction was performed on a 384-well
plate with a 10 μL reaction volume per well containing 2 μM Tyr 06
peptide substrate in reaction buffer and 2.5 μL c-KIT kinase with a serial
3-fold dilution of compound 13 (2.5 μL, 10 μM to 1.5 nM). The final
reaction concentration of ATP was 300 μM. After 1 h incubation, a
reaction was developed and terminated, and the fluorescence measured
with an automated plate reader (SpectraMax I3, MD, USA). A dose−
response curve was fitted using Prism 5.0 (GraphPad Software Inc.,
San Diego, CA). The biochemical tests of CSF1R and DDR1 were
provided by Invitrogen (Carlsbad, CA, USA).
The ADP-Glo kinase assay (Promega, Madison, WI) was used to
screen compound 13 for its PDGFRβ inhibition effects. The kinase
reaction system contains 9 μL PDGFRβ (10 ng/μL), 1 μL of serially
diluted compound 13, and 10 μL substrate Poly(4:1 Glu, Tyr) peptide
(0.4 μg/μL) (Promega, Madison, WI) with 100 μM ATP (Promega,
Madison, WI). The reaction in each tube was started immediately
by adding ATP and kept going for 1 h under 37 °C. After the tube was
cooled for 5 min at room temperature, 5 μL solvent reactions were
carried out in a 384-well plate. Then 5 μL of ADP-Glo reagent was added
into each well to stop the reaction and consume the remaining ATP
within 40 min. At the end, 10 μL of kinase detection reagent was added
into the well and incubated for 30 min to produce a luminescence signal.
The luminescence signal was measured with an automated plate reader
(Envision, PE, USA), and the dose−response curve was fitted using
Prism 5.0 (GraphPad Software Inc., San Diego, CA).
Cell Cycle Analysis. GIST-T1, GIST-882, and GIST-48B cells were
treated with DMSO, serially diluted compound 13 (0.3, 1, and 3 μM),
and 3 μM Imatinib for the indicated periods. The cells were fixed in
70% cold ethanol and incubated at −20 °C overnight then stained
with PI/RNase staining buffer (BD Pharmingen). Flow cytometry was
performed using a FACS Calibur (BD), and results were analyzed by
ModFit software.
In Vivo Pharmacokinetics Study. Compound 13 was dissolved in
55% saline containing 5% DMSO and 40% PEG400 by vortex. The final
concentration of the stock solution was 1 mg/mL for administration.
Six 8 week old male Sprague−Dawely rats were fasted overnight
before starting drug treatment via intravenous and oral administration.
Animal blood collection time points were as follows: for groups 1, 3, 5
(intravenous): 1 min, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h before
and after administration was selected; for groups 2, 4, 6 (oral): 5 min,
15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h before and after dosing.
Each time about 0.2 mL of blood was collected through the jugular vein
adding heparin for anticoagulation and kept on ice. Then plasma
was separated by centrifugation at 8000 rpm for 6 min at 2−8 °C. The
obtained plasma was stored at −80 °C before analysis. After finishing
the test, all surviving animals were transferred to the repository or
euthanasia (CO2 asphyxiation).
GIST-T1 Xenograft Model. Six week old female nu/nu mice were
purchased from the Shanghai Experimental Center, Chinese Academy
of Sciences (Shanghai, China). All animals were maintained in a specific
pathogen-free facility and used according to the animal care regulations
of Hefei Institutes of Physical Science, Chinese Academy of Sciences
(Hefei, China), and all efforts were made to minimize animal suffering.
To obtain orthotopic xenograft of human mammary tumor in the mice,
cells were harvested during exponential growth. Five million GIST-T1
cells in PBS were suspended in a 1:1 mixture with Matrigel (BD
Biosciences) and injected into the subcutaneous space on the right
flank of nu/nu mice. Daily oral administration was initiated when
GIST-T1 tumors had reached a size of 100−200 mm3. Animals were
then randomized into treatment groups of six mice each for efficacy
studies. Compound 13 was delivered daily in a HKI solution (0.5%
methocellulose/0.4% Tween80 in ddH2O) by orally gavages. A range of
doses of 13 or its vehicle was administered, as indicated in Figure 8
legends. Body weight and tumor growth were measured daily after 13
treatment. Tumor volumes were calculated as follows: tumor volume
(mm3) = [(W2 × L)/2], where width (W) is defined as the smaller of the
two measurements and length (L) is defined as the larger of the two
measurements.
Signaling Pathway Study. GIST-T1, GIST-882, and GIST-48B
cells were treated with DMSO, serially diluted compound 13, and 1 μM
Imatinib for 2 h. Cells were then washed with cold PBS and lysed in
RIPA buffer (Beyotime, China). Western blotting was performed by
standard methods, as previously described.21 The following antibodies
were used at a range of antibody concentrations as indicated by
the manufacturers to probe for specific proteins: rabbit polyclonal
antibodies to total c-KIT (cat. no. 3308) and phospho-KIT Y719
(cat. no. 3308) and Y703 (cat. no. 3308) were from Cell Signaling
Technology. Rabbit polyclonal antibodies to phospho-KIT Y823 was
from Invitrogen (cat. no. 44−498G). Polyclonal rabbit antibodies to
total p42/44 mitogen activated protein kinase (MAPK) (cat. no. 4695),
phospho-p44/42 MAPK T202/Y204 (cat. no. 4370), phospho-AKT
S473 (cat. no. 4060), total AKT (cat. no. 4691), phospho-Stat5 (cat. no.
9314), total Stat5 (cat. no. 9363), phospho-Stat3 (cat. no. 9145), total
Stat3 (cat. no. 12640), phospho-S6K T389 (cat. no. 9206), total S6K
(cat. no. 9202), phospho-S6 S235/236 (cat. no. 2211), total S6 (cat. no.
2217), phospho-Src Y416 (cat. no. 6943), total Src (cat. no. 2123),
phospho-PDGFRα Y849/PDGFRβ Y857 (cat. no. 3308), and total
PDGFRα (cat. no. 3308) were from Cell Signaling Technology. Beta
actin antibodies were purchased from Sigma (cat. no. A5316).
HE Staining. HE staining was carried out according to the previous
report.20 First the sections were hydrated, and then the slide was dipped
into a Coplin jar containing Mayer’s hematoxylin and agitated for 30 s.
After rinsing the slide in H2O for 1 min, it was stained with 1% eosin
Y solution for 10−30 s with agitation. Subsequently, the sections were
dehydrated with two changes of 95% alcohol and two changes of 100%
alcohol for 30 s each. The alcohol was extracted with two changes of
xylene. Finally, one or two drops of mounting medium were added and
covered with a coverslip.
Ki-67 Staining. For IHC demonstration of Ki-67, tissue sections
were quenched for endogenous peroxides and placed in an antigen
retrieval solution (0.01 M citrate buffer, pH 6.0) for 15 min in a micro-
wave oven at 100 °C at 600 W. After incubation in the casein block,
mouse mAb anti-Ki-67 (ZSGB-BIO, China) was applied to the sections
at dilutions of 1:50. Incubations with primary antibodies lasted
overnight at 4 °C. The secondary detection system was used to visualize
antibody binding. Staining was developed with 3,3′-diaminobenzidine
(DAB), and the slides were counterstained with hematoxylin, dehydrated,
and mounted.
TUNEL Staining. TUNEL staining was performed using the POD in
Situ Cell Death Detection kit (Roche, USA). Briefly, sections were
deparaffinized in xylene, rehydrated in decreasing concentrations of
ethanol, and then treated by nuclease-free Proteinase K for 15 min at
room temperature before the endogenous peroxidase was blocked in 3%
H2O2 in methanol. Terminal deoxynucleotidyl transferase in reaction
buffer was applied to sections for 1 h at 37 °C. Following washes, the
slides were covered by converter-POD solution for 30 min at 37 °C.
Apoptosis Effect Examination. GIST-T1, GIST-882, and GIST-
48B cells were treated with DMSO, serially diluted compound 13, 1 μM
Imatinib for the indicated periods. Cells were collected and analyzed by
Western blotting using the following antibodies: PARP (cat. no. 9532)
and Caspase-3 (cat. no. 9665) from Cell Signaling Technology. Beta
actin antibodies were purchased from Sigma (cat. no. A5316).
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Article
Apoptotic cells were detected after incubation in DAB chromogen
(Beyotime Biotechnology, China) for approximately 8 min, and the
slides were counterstained with hematoxylin.
REFERENCES
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Molecular Modeling. Molecular docking of compound 13 to the
ABL1 kinase and the c-KIT kinase was performed with software YetiX
8.3.22 The kinase domain of chain A in the PDB was used for docking
(PDB ID: 2HYY and 1T46 for ABL1 and c-KIT, respectively).
Alternative conformations of the side chains were manually confirmed,
missing side chains were automatically added using AmberTools,23 the
protonation and tautomeric state at physiological pH were confirmed
by software Reduce,24 and the receptor side-chain structure was further
optimized using YetiX 8.3. Compound 13 was constructed using BioX
4.6,25 and the atomic partial charges were generated by AmberTools.
Template-based induced-fit docking of small molecules to the two
kinases: ABL1 and c-Kit were performed using YetiX 8.3. The docked
models were optimized by the directional Yeti force field.26
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/acs.jmedchem.6b00200.
Table S1 listing the DiscoveRx’s KinomeScan selectivity
profiling data of 13 (PDF)
Compound data (CSV)
AUTHOR INFORMATION
■
Corresponding Authors
*Phone: 86-551-65593186. E-mail: jingliu@hmfl.ac.cn.
*Phone: 86-551-65595161. E-mail: qsliu97@hmfl.ac.cn.
Author Contributions
Q.W., F.L., B.W., F.Z., and C.C. contributed equally. The
manuscript was written through contributions of all authors. All
authors have given approval to the final version of the
manuscript.
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Sobin, L. H.; O’Leary, T. J. Kit mutation portends poor prognosis in
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1633−1636. (b) Lasota, J.; Jasinski, M.; Sarlomo-Rikala, M.; Miettinen,
M. Mutations in exon 11 of c-kit occur preferentially in malignant versus
benign gastrointestinal stromal tumors and do not occur in leiomyomas
or leiomyosarcomas. Am. J. Pathol. 1999, 154, 53−60.
Notes
The authors declare the following competing financial interest(s):
Dr. Shanchun Zhang is a shareholder of Hefei Cosource Medicine
Technology Co., LTD.
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ACKNOWLEDGMENTS
■
Q.L. is supported by the Joint Funds of Research on Major
Science Facilities, Key Program of the National NSFC (no.
U1432250). W.W., J.L., and Q.L. are supported by the grant of
“Cross-disciplinary Collaborative Teams Program for Science,
Technology, and Innovation (2014-2016)” from Chinese
Academy of Sciences. We are grateful for the China “Thousand
Talents Program” support for Q.L. and “Hundred Talents
Program” of the Chinese Academy of Sciences support for J.L.
and W.W. J.L. is also supported by the National Program for
Support of Top-notch Young Professionals. Q.L. is also
supported by the CAS/SAFEA international partnership
program for creative research teams.
ABBREVIATIONS USED
■
CML, chronic myeloid leukemia; GISTs, gastrointestinal stromal
tumors; ABL kinase, Abelson kinase; KIT kinase, v-kit Hardy−
Zuckerman 4 feline sarcoma viral oncogene homologue;
FLT3, FMS-related tyrosine kinase 3; RTK, receptor tyrosine
kinase; PDGFR, platelet derived growth factor receptor;
CSF1R, stimulating factor-1 receptor; SAR, structure−activity
relationship
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20080226747, September 18, 2008.
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