Identification of a Multitargeted Tyrosine Kinase Inhibitor for the Treatment of Gastrointestinal Stromal Tumors and Acute Myeloid Leukemia

Article abstract of DOI:10.1021/acs.jmedchem.9b01229

Gastrointestinal stromal tumors (GISTs) are prototypes of stem cell factor receptor (c-KIT)-driven cancer. Two receptor tyrosine kinases, c-KIT and fms-tyrosine kinase (FLT3), are frequently mutated in acute myeloid leukemia (AML) patients, and these mutations are associated with poor prognosis. In this study, we discovered a multitargeted tyrosine kinase inhibitor, compound 15a, with potent inhibition against single or double mutations of c-KIT developed in GISTs. Moreover, crystal structure analysis revealed the unique binding mode of 15a with c-KIT and may elucidate its high potency in inhibiting c-KIT kinase activity. Compound 15a inhibited cell proliferation and induced apoptosis by targeting c-KIT in c-KIT-mutant GIST cell lines. The antitumor effects of 15a were also demonstrated in GIST430 and GIST patient-derived xenograft models. Further studies demonstrated that 15a inhibited the proliferation of c-KIT- and FLT3-driven AML cells in vitro and in vivo. The results of this study suggest that 15a may be a potential anticancer drug for the treatment of GISTs and AML.

Full text of DOI:10.1021/acs.jmedchem.9b01229

Subscriber access provided by Hong Kong University of Science and Technology Library
Article
Identification of a Multitargeted Tyrosine Kinase Inhibitor for the Treatment of
Gastrointestinal Stromal Tumors (GISTs) and Acute Myeloid Leukemia (AML)
Wen-Hsing Lin, Suying Wu, Teng-Kuang Yeh, Chiung-Tong Chen, Jen-Shin Song, Hui-Yi
Shiao, Ching-Chuan Kuo, Tsu Hsu, Cheng-Tai Lu, Pei-Chen Wang, Tsung-Sheng Wu, Yi-
Hui Peng, Hui-You Lin, Ching-Ping Chen, Ya-Ling Weng, Fang-Chun Kung, Mine-Hsine Wu,
Yu-Chieh Su, Kuo-Wei Huang, Ling-Hui Chou, Ching-Cheng Hsueh, Kuei-Jung Yen, Po-Chu
Kuo, Chen-Lung Huang, Li-Tzong Chen, Chuan Shih, Hui-Jen Tsai, and Weir-Torn Jiaang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01229 • Publication Date (Web): 13 Nov 2019
Downloaded from pubs.acs.org on November 15, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 62
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 62
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Identification of a Multitargeted Tyrosine Kinase Inhibitor for the
Treatment of Gastrointestinal Stromal Tumors (GISTs) and Acute Myeloid
Leukemia (AML)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Wen-Hsing Lin,1,# Su-Ying Wu, Teng-Kuang Yeh, Chiung-Tong Chen, Jen-Shin Song,
1,#
1,#
1
1
1
1
1
Hui-Yi Shiao, Ching-Chuan Kuo, Tsu Hsu, Cheng-Tai Lu, Pei-Chen Wang, Tsung-Sheng
1
1
2
1
1
Wu, Yi-Hui Peng, Hui-You Lin, Ching-Ping Chen, Ya-Ling Weng, Fang-Chun Kung,
1
1
2
1
1
Mine-Hsine Wu, Yu-Chieh Su, Kuo-Wei Huang, Ling-Hui Chou, Ching-Cheng Hsueh,
1
1
1
2,3,5,6
Chuan Shih,1
Kuei-Jung Yen, Po-Chu Kuo, Chen-Lung Huang, Li-Tzong Chen,
Hui-Jen Tsai,2,3,4* and Weir-Torn Jiaang1,*
1Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes,
No. 35, Keyan Rd., Zhunan Town, Miaoli Country 350, Taiwan R.O.C.
2National Institute of Cancer Research, National Health Research Institutes, Tainan City 704,
Taiwan R.O.C.
3Division of Hematology/Oncology, Department of Internal Medicine, National Cheng Kung
University Hospital, College of Medicine, National Cheng Kung University, Tainan City 704,
Taiwan R.O.C
4Division of Hematology/Oncology, Department of Internal Medicine, Kaohsiung Medical
University Hospital, Kaohsiung Medical University, Kaohsiung 807, Taiwan R.O.C.
5Institute of Molecular Medicine, National Cheng Kung University, Tainan City 704, Taiwan
R.O.C.
6Division of Gastroenterology, Department of Internal Medicine, Kaohsiung Medical
University Hospital, Kaohsiung Medical University, Kaohsiung 807, Taiwan R.O.C.
1
ACS Paragon Plus Environment

Page 3 of 62
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
#
Wen-Hsing Lin, Su-Ying Wu, and Teng-Kuang Yeh contributed equally.
Corresponding authors
*
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Abstract
Gastrointestinal stromal tumors (GISTs) are prototypes of stem cell factor receptor
c-KIT)-driven cancer. Two receptor tyrosine kinases, c-KIT and fms-tyrosine kinase (FLT3),
(
are frequently mutated in AML patients, and these mutations are associated with poor
prognosis. In this study, we discovered a multitargeted tyrosine kinase inhibitor, compound
1
5a, with potent inhibition against single or double mutations of c-KIT developed in GISTs.
Moreover, crystal structure analysis revealed the unique binding mode of 15a with c-KIT and
may elucidate its high potency in inhibiting c-KIT kinase activity. Compound 15a inhibited
cell proliferation and induced apoptosis by targeting c-KIT in c-KIT-mutant GIST cell lines.
The antitumor effects of 15a were also demonstrated in GIST430 and GIST patient-derived
xenograft (PDX) models. Further studies demonstrated that 15a inhibited the proliferation of
c-KIT- and FLT3-driven AML cells in vitro and in vivo. The results of this study suggest that
1
5a may be a potential anticancer drug for the treatment of GISTs and AML.
Keywords: Multitargeted tyrosine kinase inhibitor, Gastrointestinal stromal tumors, Acute
myeloid leukemia, GIST430, Patient-derived xenograft model
2
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 62
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Introduction
Gastrointestinal stromal tumors (GISTs) are mesenchymal neoplasms that develop
in the gastrointestinal tract. GISTs are thought to derive from the interstitial cells of Cajal.1
Worldwide, epidemiological data show that the annual incidence of GISTs is approximately
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
10−20 cases per million people. These tumors can occur at any site but are predominantly
1
found in the stomach and small intestine. Approximately 60% of patients with operable
GISTs can be cured by surgery. However, about 40% of localized GISTs develop metastasis.1
Unresectable and/or metastatic GISTs have poor responses to conventional chemotherapy and
radiation therapy. c-KIT mutations were found in most GISTs and act as a driver gene for
3
GISTs. Understanding the underlying cause of GISTs has led to the development and
investigation of new agents targeting c-KIT. These drugs effectively inhibit the activity of
mutant forms of c-KIT in tumors and significantly improve the clinical outcomes for patients
with advanced GISTs.4,5 Currently, there are three c-KIT tyrosine kinase inhibitors (TKIs)
5
approved for treatment of advanced GISTs (Figure 1). The standard first-line treatment is
imatinib (1). Treatment with 1 in advanced GISTs successfully achieved a disease control rate
of 81.6% and significantly improved progression-free survival (PFS) and overall survival (OS)
6
in a phase II clinical trial. The results from a phase II and a phase III trial suggest compound
1 achieves a long-term median OS of 51−57 months in patients with advanced or metastatic
GISTs.7,8
Although compound 1 has good response to GISTs, disease progression or
3
ACS Paragon Plus Environment

Page 5 of 62
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
recurrence still may develop by either primary or secondary resistance to 1.9,10 c-KIT
mutations have been found in approximately 80% of GISTs. Mutations at exon 9 and exon 11
are the most common primary mutations identified at initial diagnosis of GISTs.
Approximately 7% of GISTs have PDGFR gene mutations.11,12 GIST patients who are not
responsive to first-line 1 treatment within 6 months are classified as primary resistance to 1.
Patients with exon 11 mutations have better response and survival to 1, whereas those with
exon 9 mutations have worse treatment outcomes.12−14 GIST patients with wild type c-KIT or
PDGFR gene mutations are usually resistant to 1. Moreover, patients who develop resistance
to 1 after 6 months of treatment are classified as secondary resistance and about 50% of these
patients develop a secondary mutation in either exon 13, 14, 17 or 18 of c-KIT. The majority
of these secondary mutations developed in patents with primary mutations in exon 11 of
c-KIT.15,16 For 1-resistant GIST patient, sunitinib (2), a multitargeted tyrosine kinase inhibitor,
was approved as a second-line treatment for advanced GIST patients after failure of
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
7
compound 1 therapy. In addition, regorafenib (3) is another multitargeted tyrosine kinase
inhibitor approved in 2013 as the third-line treatment for GIST patients who were resistant or
1
8
intolerant to 1 and 2. However, the median PFS achieved by compounds 2 and 3 are only
5
6.8 and 4.8 months, respectively. Therefore, the development and identification of novel
agents for refractory GISTs is urgently required.5,18
Acute myeloid leukemia (AML) is a hematologic malignancy with uncontrolled
proliferation and impaired maturation of hematopoietic cells. A high percentage of AML
4
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 62
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
patients relapse despite the apparent success of initial induction chemotherapy. The
occurrence of oncogenic mutations that leads to aberrant activation and proliferation of
cellular signal transduction pathways results in primary treatment failure in AML and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
9
provides insight into the refractory nature of AML. FMS-like tyrosine kinase-3 (FLT3) is a
member of the class III membrane-bound receptor tyrosine kinase (RTK) family. In addition
to FLT3, RTK class III includes c-KIT, CSF1R (FMS), PDGFR and PDGFR.20 FLT3
mutations have been identified in approximately 30% of all AML patients and these
mutations are indicative of poor prognosis.21,22 The mutations include internal tandem
duplications (ITDs) in the juxtamembrane domain of FLT3 and missense point or short-length
mutations in the activation loop (AL) of the tyrosine kinase domain (TKD).23,24 Notably,
c-KIT are mutated in a subset of AML (core-binding factor (CBF)-AML) with a range of
17-46% mutation rate.25-29 Even though CBF-AML patients usually have longer survival than
other poor-risk patients, CBF-AML patients with c-KIT mutation have higher risk of relapse
and usually have poor survival, particularly with D816V mutation.28-30
Mutations in both FLT3 and c-KIT are significantly associated with poor prognosis
in patients with AML, the development of agents that target these mutations has emerged as
promising therapeutic strategies for the treatment of AML.31 Currently, midostaurin
(PKC-412, 4, Figure 1) is the only approved TKI in combination with standard intensive
chemotherapy for adult patients with newly diagnosed FLT3-mutated AML. The international
phase III trial results indicated that randomized patients to receive compound 4 (Figure 1) had
5
ACS Paragon Plus Environment

Page 7 of 62
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
3
2
a higher median OS, with a hazard ratio of 0.78 compared with those receiving placebo. By
the end of 2018, the Food and Drug Administration approved gilteritinib for the treatment of
3
3
adult patients who have relapsed or refractory AML with a FLT3 mutation. The mutation
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
sites of c-KIT in CBF-AML mainly located on exon 8 (D419) and exon 17 (D816 and
3
4
N822). Several agents targeting c-KIT, such as compound 1 and dasatinib, have been shown
to suppress the proliferation of c-KIT mutant leukemia cells in vitro.35,36 Compound 1 failed
to respond to relapsed AML patients with c-KIT mutations in earlier study.37 Recently,
dasatinib in combination of intensive chemotherapy was shown to induce a higher response
3
8
rate for CBF-AML and the phase III trial is still ongoing.
Cl
HN
HN
CF3
H
N
O
O
F
N
N
O
N
H
N
N
O
N
O
N
N
N
N
N
H
N
F
H
O
HN
N
O
N
H
HN
O
O
O
Imatinib (1)
Sunitinib (2)
Regorafenib (3)
Midostaurin (PKC412, 4)
Figure 1. Structures of c-KIT and FLT3 inhibitors in the clinic.
Design of Multitargeted Tyrosine Kinase Inhibitors
Using a structure-based design, we previously developed a novel class of
5
-phenylthiazol-2-ylamine-based derivatives (Structure O, Figure 2) as a versatile template
for the development of multitargeted kinase inhibitors. This previous study led to the
6
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 62
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
discovery of urea 5, which exhibited promising in vitro and in vivo antitumor activities
against
FLT3-ITD–expressing
MOLM-13
and
MV4;11
cell
lines
and
FLT3-ITD/D835Y–expressing 32D cell lines.39 Kinome-wide selectivity profile of 5 with
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
0
DiscoveRx’s KinomeScan technology demonstrated that 5 bore a low selectivity (S score
(10) = 0.091 at a low concentration of 0.1 μM) among 395 wild-type kinases tested. Both
multikinase inhibitors and single-kinase inhibitors have advantages and disadvantages, which
are related to potential resistance mechanisms, pharmacokinetics, selectivity, tumor
4
1
environment and toxicity. However, we speculate that the off-target profile of 5 might
explain its intrinsic toxicity in vivo. To trim the off-target profile, optimize target inhibition
and increase the therapeutic window with acceptable toxicity, we rationally designed a second
class of heteroaryl-substituted 2-aminothiazole derivatives as multikinase inhibitors (Structure
P, Figure 2). The approach removes an important urea pharmacophore at the phenyl ring of 5
and replaces phenyl group (5) with pyridine or pyrimidine group (Structure P). Based on the
chemical design, a 5-pyridin-4-yl-thiazol-2-ylamine series of compounds (15) was found to
maintain a similar anti-cancer activity to that of 5. In this study, the structure-activity
relationships (SAR) of a series of Structure P derivatives are reported. This study has led to
the discovery of compound 15a, which is a multitargeted kinase inhibitor with excellent
pharmaceutical properties, good pharmacokinetics (PK) profile, and superior efficacy and
tolerability in GIST and AML tumor xenograft models. These promising results demonstrate
the potential of 15a suitable for further preclinical and clinical development.
7
ACS Paragon Plus Environment

Page 9 of 62
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
R2
NH
N
N
S
HN
N
R1
O
SAR Optimization
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
N
NH
N
X
W
NH
Y
N
N
S
HN
N
Z
Rational design
S
HN
N
N
N
R1
R2
N
5
P
Figure 2. Identification of 5- pyridine-thiazol-2-ylamine derivatives as TKIs.
Chemistry
The general synthetic route to 5-aromatic substituted thiazol-2-ylamine pyrimidines
12-16 (Table 1) is shown in Scheme 1. The synthesis began with the preparation of
5-aromatic substituted thiazol-2-ylamines 9 according to a previously published protocol with
4
2
some modifications. 2-Aminothiazole 6 was protected with trimethylacetyl chloride using
Et N to give N-thiazol-2-yl-propionamide 7. The Suzuki coupling of 7 with chloro-substituted
3
pyridine or pyrimidine catalyzed by Pd(0) gave 5-aromatic substituted thiazol-2-ylamides 8.
Amides 8 were hydrolyzed in the presence of a concentrated acid followed by neutralization
to yield free 5-aromatic substituted thiazol-2-ylamines 9. Treatment of 5-aromatic substituted
thiazol-2-ylamines 9 or the commercially available 10 with 4,6-dichloropyrimidine or
4,6-dichloro-2-methylpyrimidine in the presence of sodium hydride (NaH) and
1-methyl-2-pyrrolidinone (NMP) at 0 °C yielded 4-monosubstituted pyrimidine derivatives 11.
8
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 62
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The treatment of these derivatives with 2° amines in DMSO at 100 °C or with
2-morpholin-4-yl-ethanol in the presence of KOH and diglyme at 160 °C yielded
4,6-disubstituted pyrimidines 12−16. The synthetic routes to 4-pyridin-3-yl-thiazol-2-ylamine
pyrimidine 19 and N-(5-pyridin-3-yl-thiazol-2-yl)benzamide 21 (Table 1) are shown in
Scheme 1. Displacement of 6-chloro and 4-chloro substituents of 4,6-dichloropyrimidine with
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
3
sodium salt of 4-pyridin-3-yl-thiazol-2-ylamine 17 and 1-ethylpiperazine, respectively, gave
4,6-disubstituted pyrimidines 19. Benzoyl chloride 20 with a water-solubilizing
1-methylpiperazine group was employed to acylate amine 9b in pyridine to yield amide 21.
The freebase forms of compounds 12−16, 19 and 21 were reacted with 6N HCl/CH OH
3
aqueous to yield their corresponding hydrochloride salts.
O
O
Py
S
N
Py
S
N
S
S
N
a
b
c, d
NH2
NH2
N
H
N
H
N
7
8
9
6
e
X
W
W
X
Z
Y
N
N
Y
1
1
1
2 W, X, Y, Z = C
3 W = N; X, Y, Z = C
4 X = N; W, Y, Z = C
Z
S
"R
S
R
"R
f, g
HN
S
N
HN
e
NH2
15 Y = N; W, X, Z = C
1
N
N
6 X, Z = N; W, Y = C
HCl
1
0
Cl
N
R
R'
N
11
N
N
S
N
S
N
N
N
HN
N
e
HN
f, g
N
S
NH2
N
N
Cl
N
1
7
HCl
N
1
8
1
9
N
N
O
O
S
N
h, g
S
N
+
NH
N
Cl
N
NH2
HCl
N
N
9
b
20
21
2 2
Scheme 1. Reagents and conditions: (a) TEA, CH Cl , 0 °C to rt; (b)3-Chloropyridine, 4-Chloropyridine or
9
ACS Paragon Plus Environment

Page 11 of 62
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
5
1
4
4
-Chloropyrimidine, KOAc, Pd(PPh
2N HCl,
,6-dichloro-2-methylpyrimidine, NaH, NMP,
-(2-hydroxyethyl)morpholine, KOH, diglyme, 160 °C for 15f; (g) 6N HCl/CH
3
)
4
3 4
, DMAc, 150 °C or 2-Chloropyridine, CsF, Pd(PPh ) , DMSO, 160 °C; (c)
H
2
O, 110 °C; (d) NaHCO
3
,
H
2
O, rt to 50 °C; (e) 4,6-Dichloropyrimidine or
°C; (f) 2° amines, DMSO, 100 °C or
OH, 0 °C; (h) pyridine, 0 °C to
0
3
rt.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Results and Discussion
In Vitro Pharmacology. As shown in Table 1, the target compounds were tested
against c-KIT and FLT3 (in-house kinase assays). In addition, these compounds were further
evaluated against the GIST cell line GIST-T1 (expressing a heterozygous deletion in the
c-KIT exon 11) and AML cell line MOLM-13 (expressing wild-type FLT3 and FLT3-ITD
4
4
mutation). The first compound, 12 , which lacks a urea pharmacophore of 5, increased the
inhibitory activity against c-KIT (IC50 = 24 nM) but significantly lost antiproliferative
potency against MOLM-13 cells (GI = 35 nM) compared to urea 5. Next, the effects on
5
0
replacing the phenyl moiety present in 12 with pyridine groups were evaluated. The second
position of pyridine attached to the fifth position of thiazole (13) exhibited similar inhibitory
activities against wild type c-KIT and FLT3 kinases but did result in reduced cellular
potencies (GIST-T1 and MOLM-13 GI = 42 and 36 nM, respectively) relative to 5. When
5
0
the nitrogen atom was moved to the third position, 14a (R = CH ) and 14b (R = H) were as
2
3
2
potent as 5 in the enzymatic assays but were significantly less active in the cellular assays
with GI values of 26−140 nM. Compound 15a (R = CH ), with the fourth position of
5
0
2
3
pyridine attached to thiazole, exhibited similar inhibitory activities against c-KIT and FLT3
kinases compared to 5 and displayed single digit nanomolar activity against cell lines
1
0
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 62
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
GIST-T1 (GI = 7.1 nM) and MOLM-13 (GI = 9.4 nM). When compared with 15a, 15b (R
2
5
0
50
= H) showed potent inhibitory activities against c-KIT and FLT3 (IC < 30 nM) but no
5
0
improvement in cellular potency (GI > 10 nM).
5
0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Next, we examined the effects of water-solubilizing substituents on the fourth
position of the pyrimidine ring and compared the potencies of compounds 15c-f with
N-ethylpiperazine
analog
15a.
(15d),
N-(2-Fluoroethyl)piperazine
(15c),
N-(2-hydroxyethyl)piperazine
N,N-dimethylpiperidin-4-amine
(15e)
and
4-(2-hydroxyethyl)morpholine (15f) did not affect the inhibitory potencies against c-KIT and
FLT3 kinases. Nevertheless, 15e bearing a N,N-dimethylpiperidin-4-amine group slightly
increased the cellular activities (GIST-T1 GI = 1.5, MOLM-13 GI = 3.5 nM), but 15f
5
0
50
bearing morpholine group tethered by a two-carbon ether apparently lost cellular potencies
(GIST-T1 GI = 27, MOLM-13 GI = 42 nM). In this study, limited water-solubilizing
5
0
50
groups were discussed because the effects of some water-solubilizing groups on biological
activities, pharmacokinetics and in vivo toxicities were fully clarified based on the previous
results from the development of 5-phenylthiazol-2-ylamine-based inhibitors (Structure O,
3
9
Figure 2). Extensive SAR studies were carried out by adding a single methyl substituent on
the pyridine ring of 15a and replacing the pyridine moiety of 15a with pyrimidine to yield
analogs 15g and 16, respectively. In comparison with 15a, 2-methylpyridine 15g was 2-fold
to 4-fold less potent against the c-KIT, FLT3 and MOLM-13 cells but maintained potency
against GIST-T1 cells (GI = 7.7 nM). Pyrimidine 16 exhibited only moderate inhibition of
5
0
1
1
ACS Paragon Plus Environment

Page 13 of 62
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
c-KIT (IC = 123 nM) and FLT3 (IC = 163 nM) with a dramatic decrease in cellular
5
0
50
potency (GI > 100 nM).
5
0
After optimizing the 5-pyridin-4-yl-thiazol-2-yl series of pyrimidines (compounds
15, Structure P), we investigated the effects of substitutions on 2-aminothiazole and compared
the potencies of compounds 19 and 21 with 14a, respectively. A significant decrease in both
enzymatic activities (IC > 1,000 nM) and cellular potencies (GI > 1,000 nM) was observed
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
50
when the pyridine ring of 14a was moved from the fifth position to the fourth position (19) of
the thiazole ring. According to prior reports, the 2-aminothiazole moiety is conformationally
well suited to form hydrogen bond interactions with the kinase hinge region of the ATP
4
5
46
pocket, and benzamide is a known scaffold for FLT3 kinase inhibitors. Accordingly, we
replaced the pyrimidine group with a benzamide group bearing a solubilizing substituent at
the para position of a benzene ring, yielding benzamide 21. This approach led to benzamide
21 as only a moderate c-KIT/FLT3 inhibitor with submicromolar activities against GIST-T1
(GI = 647 nM) and MOLM-13 cells (GI = 544 nM). Therefore, further modification of
5
0
50
lead 21 was not carried out.
As for FDA-approved TKIs for GISTs or AML, compound 1 inhibits tyrosine
kinases specifically BCR-ABL, c-KIT and PDGFR and compounds 2−4 are multitargeted
TKIs possessing c-KIT/FLT3 dual inhibition (Figure 1). As shown in Table 1, 1 potently
inhibited c-KIT and GIST-T1 cells with IC and GI values of 53 and 40 nM, respectively.
5
0
50
Among these multitargeted TKIs (2−4), compound 2 exhibited the best enzymatic activities
1
2
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 62
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
against c-KIT and FLT3 (IC50 < 50 nM) and cellular potencies against GIST-T1 and
MOLM-13 cells (GI < 60 nM). Compounds 3 and 4 showed less potent inhibition toward
5
0
MOLM-13 (GI = 887 nM) and GIST-T1 (GI = 235 nM) cells, respectively. Among the
5
0
50
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
pyridine-substituted 2-aminothiazole analogues listed in Table 1, 15a and 15c-e are potent
dual inhibitors of c-KIT and FLT3 with less than 10 nM antiproliferative activities in cellular
assays. In preliminary studies, the pharmacokinetics, toxicities and/or tumor‑inhibiting
activities between 15a and 15c-e were compared in normal mice or SCID mouse xenografts.
The results showed that 15a caused more significant anti-tumor effects in GIST430
xenografts compared to 15c and 15d. Moreover, 15a exhibited lower toxicity in mice
compared to 15c and 15e, and the pharmacokinetic profile of 15a appeared to be more
favorable than that of 15d. Therefore, compound 15a was selected for further biological
activity and in vivo efficacy studies.
Table 1. Inhibition of enzymes and cell proliferation with thiazole analogues.
N
X
N
W
N
N
Y
N
N
N
N
Z
S
S
N
O
HN
N
HN
N
S
N
HN
N
S
HN
N
N
N
R1
R2
N
N
N
1
2 W, X, Y, Z = C
15g
19
21
13 W = N; X, Y, Z = C
1
1
1
4 X = N; W, Y, Z = C
5 Y = N; W, X, Z = C
6 X, Z = N; W, Y = C
IC50 (nM)a
GI50 (nM)a
R1
R2
Compd
AML
c-KIT
wt-FLT3
GIST-T1
MOLM-13
5
97
24
38
38
3.2
8.0
2.0
N
N
1
2
-CH3
35
1
3
ACS Paragon Plus Environment

Page 15 of 62
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
N
N
N
N
N
N
1
3
-CH
-CH
-H
3
3
100
69
24
63
60
42
26
80
36
140
81
1
4a
1
4b
91
N
N
N
N
1
5a
-CH
-H
3
56
29
30
20
7.1
15
9.4
12
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
15b
N
N
1
5c
-CH
-CH
-CH
-CH
3
3
3
3
47
49
53
37
43
35
3.8
2.8
1.5
7.7
6.5
3.5
F
N
N
1
5d
OH
1
5e
N
N
O
1
5f
49
35
64
27
42
39
O
N
N
1
5g
100
7.7
N
1
6
-CH
3
123
>1,000
107
53
163
> 1,000
103
131
31
120
> 1,000
647
280
> 1,000
544
19
2
1
1
2
(imatinib)
(sunitinib)
40
>1,000
54
48
38
3 (regorafenib)
(midostaurin)
116
109
82
119
887
4
40
235
68
aValues are expressed as the mean of three independent determinations and are within ±25%.
Kinase Inhibition Profiles of 15a. To better understand the enzyme inhibition by
the potent compound, 15a was first examined its kinome-wide selectivity profile with
DiscoveRx’s KinomeScan technology in a panel of 468 kinases and mutants at 100 nM
4
0
concentration (competition binding assay). The results demonstrated that 15a was a typical
multitargeted tyrosine kinase inhibitor (S score (10) = 0.074 at 0.1 μM, Supporting
Information, Figure S1). Because the competition binding assay (KinomeScan) may not fully
1
4
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 62
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
reflect inhibitory activity, a continuous kinase enzymatic assay (HotSpot Kinase profiling,
Reaction Biology Corporation) was performed to measure 15a’s inhibition activity against
selected 38 protein kinases at a concentration of 100 nM. These 38 kinases covered the major
oncogenic kinases of the human protein kinome and showed higher affinity interactions with
15a (%Ctrl <20, KinomeScan binding assay). The enzyme activity assay results showed that
15a inhibited 22 kinases by more than 70% at 100 nM out of a panel of 38 tested kinases
(Supporting Information, Table S1). Most importantly, the kinase profiling revealed 15a with
potent activity against clinically important wild-type and mutant kinases, including FLT3
(98%), FLT3-D835Y (99%), FLT3-ITD (99%), PDGFR-T674I (98%), ABL1 (97%), c-Src
(94%, IC = 3 nM), TRKB (93%), TRKA (90%, IC = 4.9 nM), PDGFR (88%), c-KIT
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
50
(85%), PDGFR (75%) and PDGFR-V561D (75%).
Compound 15a Inhibits Enzymatic Activities of a Broad Spectrum of c-KIT
Mutants. There are multiple sites of c-KIT mutation in cancers, with some “hot spots”
corresponding to the intracellular and extracellular juxtamembrane domains (exons 8, 9 and
11) and the activation loop of the kinase domain (exon 17), which lead to disruption of
3
4
autoinhibitory mechanisms. The ability of 15a to potently inhibit activated c-KIT suggested
that the inhibitory activity of 15a among a variety of c-KIT mutants should be examined.45
Accordingly, various single- or double-mutant isoforms of c-KIT were evaluated for their
susceptibility to inhibition by 15a. As shown in Table 3, compound 15a more effectively
inhibited a broad spectrum of c-KIT mutants bearing single mutations in the JM domain,
1
5
ACS Paragon Plus Environment

Page 17 of 62
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
ATP-binding pocket and the A-loop. In addition to single c-KIT mutants, 15a also potently
inhibited c-KIT double mutants containing primary V560G and secondary mutations at the
A-loop (D816V and N822K), although reduced potency was observed in the V559D/V654A
double mutant (54% inhibition at 100 nM concentration, Table 2). Except for ATP binding
site mutants and the V559D/V654A double mutant, 15a was considerably more potent than
compound 2 on the inhibition of the other mutants shown in Table 3. Apparently, the
inhibition of these mutant forms of c-KIT by compound 3 was not comparable to 15a,
especially toward single mutants V654A, D816H and D816V and double mutants
V559D/V654A and V560G/D816V (< 15% inhibition at a concentration of 100 nM).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 2. Inhibition of enzymatic activities of c-KIT mutants by 15a, 2 or 3 (by HotSpot Kinase profiling,
Reaction Biology Corporation)
%
inhibition of enzymatic activities
2 (sunitinib) 3 (regorafenib)
Mutant forms of c-KIT
15a
100 nM
93
10 nM
56
100 nM
10 nM
8
100 nM
67
14
52
50
9
10 nM
JM domain Exon 11
V560G
41
73
80
93
56
47
65
81
85
43
82
52
51
24
3
ATP
Exon 13
Exon 13
Exon 14
Exon 17
Exon 17
Exon 17
Exon 17
Exon 17
Exon 18
V654A
K642E
T670I
74
49
48
60
43
12
5
binding
pocket
94
77
16
9
89
42
D816H
D816V
D820E
D820Y
Y823D
A829P
90
47
0
92
49
10
65
80
88
44
1
5
98
53
16
36
28
6
13
25
27
4
A-loop
99
90
99
93
95
61
Exon 11/13 V559D/V654A
Exon 11/17 V560G/D816V
Exon 11/17 V560G/N822K
54
12
32
9
0
Double
mutant
92
54
0
0
95
61
7
50
7
1
6
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 62
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Crystal Structure of c-KIT Kinase in Complex with 15a. The structure of c-KIT
kinase in complex with 15a was obtained, and the statistics for data collection and refinement
are summarized in Supporting Information, Table S2. The overall structure of 15a bound
c-KIT adopts an autoinhibited conformation (Figure 3A). The JM region of 15a is ordered,
and the compact hairpin loop conformation resembles that of the autoinhibited c-KIT. The
N-terminal half of the hairpin loop (Figure 3A) inserts directly into the interspace between the
C-helix in the N-lobe and the A-loop in the C-lobe. The insertion, together with the other
solvent-exposed half (Figure 3A) of the hairpin loop, stabilizes the autoinhibited state by
replacing and reorienting the A-loop from an activated to an inactive conformation.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Compound 15a binds to c-KIT kinase in the ATP-binding site located between the
N-lobe and C-lobe (Figure 3B). The thiazolylamine scaffold of 15a forms two hydrogen
bonds with the backbone of Cys673 in the hinge region. In addition, the thiazolylamine group
of 15a also forms hydrophobic interactions with Leu595, Tyr672, Cys673 and Leu799. In
particular, the sulfur atom of 15a forms the sulfur- interactions with Phe811 in the DFG
motif of c-KIT kinase. The pyridine group of 15a occupies the back pocket of the
ATP-binding site by forming a hydrogen bond with the side chain of Lys623. Moreover, 15a
induces a conformational rearrangement of Phe811 to form perpendicular “edge-to-face”
aromatic interactions with its pyridine group (Figure 3B). In addition to Phe811, the pyridine
group of 15a also forms hydrophobic interactions with Ala621 and Leu799. The
ethylpiperazine group of 15a is solvent-exposed in the crystal structure of c-KIT, and the
1
7
ACS Paragon Plus Environment

Page 19 of 62
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
design of this moiety is usually for improvement of the physiochemical properties of
compounds. Unlike some type II RTK inhibitors that insert deeply beyond the back pocket of
the ATP-binding site,47 15a does not interact with the C-helix or create any additional
hydrophobic pocket that pushes the JM domain away from its autoinhibited conformation.
Retaining the JM domain in an autoinhibited conformation may therefore contribute to the
high potency of 15a to inhibit c-KIT kinase activity.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. (A) Overall structures of c-KIT in complex with 15a (PDB code: 6KLA). (B) The
interactions of c-KIT with 15a. The juxtamembrane (JM) domain and activation loop are
shown in magenta and yellow, respectively.
Antiproliferative Effects of 15a on c-KIT Mutant GIST Cell Lines. GIST cell
lines, including c-KIT mutated GIST882, GIST48, and GIST430, were treated with 15a and
screened for the inhibitory effect of 15a on cell proliferation via methylene blue assay.
Among the three c-KIT-mutated GIST cell lines, GIST882 carries the exon13 K642E
mutation. GIST430 has a primary c-KIT exon11 in-frame deletion and a heterozygous
secondary exon13 missense mutation (V654A). GIST48 has a primary, homozygous exon 11
1
8
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 62
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
missense mutation (V560D) and a heterozygous secondary exon 17 mutation (D820A). The
GI50 values for GIST cell lines treated with 15a were 5.0, 17 and 21 nM for GIST882,
GIST430, and GIST48, respectively (Table 3). Compared with compounds 1−3, the IC50
values of 15a to suppress the cell proliferation of three c-KIT mutant GIST cell lines are
much lower than the three available tyrosine kinase inhibitors used in clinical practice, as
shown in Table 3.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 3. Inhibitory activity of 1, 2, 3 and 15a against GIST cell lines
GI50 (nM)*
cell line
Primary/secondary mutation
1
(imatinib)
195
2 (sunitinib) 3 (regorafenib)
15a
5.0
17
GIST882
GIST430
GIST48
K642E/none
V560-L576del/V654A
V560D/D820A
64
47
285
785
929
625
2,000
> 2,000
21
*Values of GI50 are expressed as the mean of three independent experiments and are within ±25%.
As c-KIT is the driving oncogene of c-KIT-mutated GIST cells, we evaluated c-KIT
and phosphorylated c-KIT for GIST882, GIST430, and GIST48 cells and found that
phosphorylated c-KIT was significantly suppressed by 15a time-dependently, as shown in
Figure 4. GIST cells were treated with 100 nM of 15a for 24, 48, and 72 h to evaluate the
inhibitory effects of 15a on c-KIT and its downstream signals. Compound 15a exerted
marked suppressive effects on c-KIT phosphorylation in all three GIST cell lines. The
downstream signals, the phosphorylation of AKT, and MEK, were suppressed well in
GIST882 and GIST430 cells by 100 nM of 15a. In GIST48 cells, phosphorylated AKT and
MEK were suppressed by 100 nM of 15a at 24 h but recovered at 48 and 72 h. This effect can
1
9
ACS Paragon Plus Environment

Page 21 of 62
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
be overcome by the use of 15a at a concentration of 1,000 nM, as shown in Figure 4. Since
GIST-T1 cells are sensitive to compounds 1 and 2, western blot analysis to assess the
inhibitory effect of 15a on phosphorylation of mutant c-KIT is not further tested.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Compound 15a effectively suppressed c-KIT phosphorylation and its downstream signaling pathways
in GIST cell lines.
Antiproliferative Effects of 15a on Leukemia Cell Lines. The antiproliferative
activity of 15a was assessed in a panel of leukemic and transfected BaF3 cell lines (Table 4).
Compound 15a potently inhibited the proliferation of FLT3-ITD-positive AML cell lines
2
0
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 62
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
MOLM-13 and MV4;11 with GI50 values of 6.4 and 4.9 nM, respectively. In addition,
Kasumi-1 cells were sensitive to 15a with a low GI value of 12 nM. Kasumi-1 is an AML
5
0
cell line carrying a c-KIT mutation on codon 822 (N822K). In contrast, 15a inhibited B-cell
acute lymphoblastic leukemia cell line RS4;11 (expressing native FLT3), leukemic monocyte
lymphoma cell line U937 (not expressing FLT3) and human chronic myeloid leukemia cell
line K562 (expressing wt-BCR/ABL) at significantly higher concentrations (517−2,905 nM),
strongly suggesting that 15a is a dual FLT3/c-KIT inhibitor. The growth inhibitory effects of
15a on FLT3-driven MOLM-13 and MV4;11 cells were more potent than that of compound 4
(Table 4). Additionally, c-KIT inhibitors 1−4 (Figure 1) showed moderate inhibition against
Kasumi-1 cells with GI values of 1,002, 225, 261 and 284 nM, respectively (Table 4).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
FLT3-ITD secondary TKD mutants, especially at residues D835 and F691, confer
3
9
clinical resistance to the FLT3 inhibitors quizartinib and sorafenib. Due to 15a’s promising
potency toward the FLT3-driven AML cell lines, the ability of 15a to inhibit both D835Y and
F691L secondary mutations of FLT3-ITD should be evaluated. In this study, we examined the
cellular GI values of 4 and 15a against BaF3 cells expressing FLT3-D835Y (activation loop
5
0
mutation), FLT3-ITD-D835Y (ITD and activation loop mutation) and FLT3-ITD-F691L (ITD
and gatekeeper mutation). As shown in Table 4, the cell proliferation–based assays indicated
that the sensitivity toward 15a differed significantly for inhibiting FLT3-D835Y (GI = 17
5
0
nM), FLT3-ITD-D835V (GI 50 = 92 nM) and FLT3-ITD-F691L (GI 50 = 152 nM). Compound
2
1
ACS Paragon Plus Environment

Page 23 of 62
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
15a showed an equal inhibitory effect on the proliferation of BaF3 cells expressing
FLT3-D835Y compared to compound 4 (GI = 16 nM). Nevertheless, the growth inhibitory
5
0
effects of 15a on FLT3-ITD-D835Y and -F691L mutants were slightly less potent than 4,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
with GI values of 43 and 47 nM, respectively, as shown in Table 4.
5
0
In addition, the antiproliferative activities against other cancer cell lines were also
evaluated (Supporting Information, Table S3). The GI values of 15a against eleven cancer
5
0
cell lines ranged from 376 nM (Colo205) to > 20,000 nM (A549 and KYSE-510). Moreover,
15a had no inhibitory effect on Detroit 551 (normal human skin fibroblast cell), with GI >
5
0
20,000 nM.
Table 4. Potency comparison of 4 and 15a in cellular assays.
GI50 (nM)b
4 (midostaurin)
Cell line
Leukemia
15a
6.3
MOLM-13
55
38
MV4;11
Kasumi-1a
RS4;11
U937
4.9
284
12
400
650
2,905
517
1,400
> 20,000
K562
BaF3
GI50 (nM)b
Parent/IL3
540
16
1,100
17
FLT3-D835Y
FLT3-ITD-D835Y
FLT3-ITD-F691L
43
92
47
152
aKasumi-1 GI50: 1 = 1,002 nM; 2 = 225 nM; 3 = 261 nM.
bValues are expressed as the mean of three independent determinations and are within ±25%.
Because inhibitor of 15 exhibits cytotoxicity in BaF3 cells expressing FLT3 TKD
2
2
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 62
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
mutant and FLT3-ITD TKD mutants (Table 4), it was important to verify that the mechanism
of cell death was directly linked to the inhibition of FLT3. Next, a western blot analysis was
performed using HEK293T cells engineered to express FLT3-WT, FLT3-ITD, FLT3-D835Y
or FLT3-ITD-D835Y. As shown in Figure 5, Compound 15a potently inhibited the
phosphorylation of FLT3-WT, FLT3-ITD and FLT3-D835Y with IC values from 0.1 nM to
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
1.0 nM. Consistently, 15a showed moderate potency against autophosphorylation of the
FLT3-ITD-D835Y mutant (IC value of approximately 100 nM), whose inhibition level was
5
0
closely correlated with antiproliferative effect on BaF3 cells expressing FLT3-ITD-D835Y
(GI = 92 nM, Table 4).
5
0
Figure 5. HEK293T cells expressing FLT3-WT, FLT3-ITD, FLT3-D835Y or FLT3-ITD/D835Y were analyzed.
FLT3-transfected HEK293T cells were treated with compound 15a at various concentrations for 1 h. Compound
1
5a potently inhibits the FLT3-WT, FLT3-ITD and FLT3-D835Y signaling pathways.
Pharmacokinetics and In Vivo Efficacy Studies of 15a. Table 5 shows the
pharmacokinetic properties of 15a evaluated in male Sprague–Dawley rats and ICR mice. In
both species, 15a exhibited high volumes of distribution (V = 10.1 L/kg in rats and 14.7
ss
2
3
ACS Paragon Plus Environment

Page 25 of 62
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
L/kg in mice) and plasma clearances (Cl = 39.6 mL/min/kg in rats and 70.7 mL/min/kg in
mice) after intravenous (IV, 2 mg/kg) administration. A single 10 mg/kg oral dose of 15a was
administered as a solution containing 22% hydroxypropyl--cyclodextrin in water and
absorbed with a moderate half-life in rats (t1/2 = 3.9 h) and in mice (t1/2 = 4.1 h). The Cmax
(325 ng/mL) and AUC (1955 ng/mL*h) in mice were slightly higher than those in rats, and
the oral bioavailability ranged from 36% in rats to 68% in mice. After demonstrating
favorable pharmacokinetic properties and excellent cellular potency against GIST and AML
cell lines, 15a was appropriate for continued in vivo investigation to determine the antitumor
activity of 15a in a broad panel of GIST and AML xenograft models (Figure 4).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 5. Pharmacokinetic profile of compound 15a.
IV (dose: 2 mg/kg)
CL
Species (h) (mL/min/kg) (L/kg)
PO (dose: 10 mg/kg)
T
1/2
V
ss
AUC(0-inf)
T
1/2
C
max
AUC(0-inf)
F
(ng/mL*h)
(h)
3.9
4.1
(ng/mL)
153
(ng/mL*h)
(%)
Rats
5.7
1.6
39.6
70.7
10.1
14.7
804
542
1470
36
68
Mice
325
1,955
First, the in vivo pharmacodynamic (PD) effect of 15a was performed in
NOD/SCID mice bearing GIST430 tumors. Three doses of 15a (10, 20 and 40 mg/kg) were
administered orally to subcutaneous GIST430 xenografts and harvested 16 h after 15a
administration. Pharmacodynamic analysis of tumors showed sustained inhibition of p-c-KIT
and p-Akt in a dose-dependent response (Figure 6A). These data demonstrated the
long-lasting effect of 15a on c-KIT inhibition in vivo and that once-a-day oral dosing was
expected to be sufficient for in vivo efficacy in the mouse model. To examine its antitumor
2
4
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 62
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
efficacy in the GIST430 xenograft model, 15a was administered orally at a dose of 30 mg/kg
once daily (qd) on days 1−2, 5−9 and 12−16. As shown in Figure 6B, rapid tumor regression
was observed during the dosing period, with 3 of 5 treated animals exhibiting complete
regression (CR) by day 14. There was no tumor regrowth up to day 30, while the 2-treated
group (80 mg/kg) began to recur after dosing was halted. In an independent GIST430
xenograft model, treatment of 15a with a low dose of 15 mg/kg also led to rapid tumor
regression, but no CR was found in the low-dose group (Supporting Information, Figure S2).
This model was resistant to 1 (approved for GIST first-line treatment) and sensitive to 2
(approved for GIST second-line treatment).48
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Next, the antitumor efficacy of 15a was assessed in two GIST patient-derived
xenograft (PDX) models. The first PDX model performed in NOD/SCID mice bearing the
exon 11 (delW557K558)/exon 17 (Y823D) double mutant was resistant to 1 and 2. Oral
administration of 15a showed significant antitumor effects at 15 mg/kg and 30 mg/kg qd for 5
days a week for 2 weeks in the PDX model. Treatment with 15 mg/kg dosing caused tumor
stasis, whereas tumor regression was observed in the 30 mg/kg dosing group during the
second week of treatment followed by slow tumor regrowth starting approximately 7 days
after cessation of dosing. At the end of the experiment (day 29), the mean percentage of tumor
growth inhibition (TGI) was 76% and 92% in the 15- and 30-mg/kg 15a administration
groups, respectively (Figure 6C). As expected, this PDX model was sensitive to 3 (30 mg/kg),
2
5
ACS Paragon Plus Environment

Page 27 of 62
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
which is approved for GIST third-line therapy and known to show potency against secondary
4
8
c-KIT mutants in exon 17 except for D816H/V. The second PDX model was performed in
NOD/SCID mice bearing the exon 13 (K642E)/exon 17 (N822K) double mutant. As shown in
Figure 6D, 15a at doses of 15 and 30 mg/kg resulted in tumor stasis by the second week of
treatment. Tumors in both groups began to recur after cessation of dosing, with mean values
of 80% and 88% TGI being observed in the 15 and 30 mg/kg dosing groups, respectively, on
day 29. Apparently, 3 (30 mg/kg) was not sensitive to this PDX model with a 39% TGI on
day 29. In the two PDX models, 15a is well-tolerated at both effective doses.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
6
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 62
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 6. Compound 15a inhibits the growth of GIST and AML xenografts. (A) A pharmacodynamics study
was conducted in the GIST430 xenograft model separately from the efficacy study. Levels of phosphorylated
c-KIT (c-KIT-Tyr703) and AKT (AKT-Ser473) were determined by immunoblot. GIST430 xenografts were
treated with 15a (10, 20 and 40 mg/kg) or vehicle for 16 h. Tumors were then harvested, and protein lysates were
subjected to immunoblotting for p-c-KIT, total c-KIT, p-AKT, total AKT and -actin. (B to F) Antitumor
activity of 15a was evaluated against GIST430 xenografts, n = 5/group (B), c-KIT exon 11
2
7
ACS Paragon Plus Environment

Page 29 of 62
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
(
delW557K558)/exon 17 (Y823D) GIST PDX, n = 6/group (C), c-KIT exon 13 (K642E)/exon 17 (N822K) GIST
PDX, n = 6/group (D), c-KIT mutated Kasumi-1 AML xenografts, n = 8/group (E), FLT3-ITD mutated
MOLM-13 AML xenografts, n = 4/group (F). Mice were treated with 15a and reference compounds using the
dosing levels indicated. The gray bars indicate the dosing schedule for 15a and reference compounds. The tumor
size is expressed as the mean ± SEM.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In AML xenograft models, Kasumi-1 AML xenografts (c-KIT exon 17 N822K) in
SCID mice treated with 15a dosed orally at 10, 20 and 30 mg/kg qd on days 1−5 and 8−12.
Treatment with the low dose of 10 mg/kg led to stasis of tumor growth during the
administration period, and apparent tumor regression was observed in the 20 and 30 mg/kg
dosing groups followed by slow tumor regrowth starting within one week after cessation of
dosing (Figure 6E). Four animals in the 30 mg/kg group (n = 8) became and remained
tumor-free (CR) through the end of the experiment. In this model, 15a was well-tolerated at
all doses. Finally, a dose-escalation study was conducted to evaluate the in vivo efficacy and
toxicity of 15a in nude mice bearing MOLM-13 tumors (FLT3-ITD+). Compound 15a was
administered orally at doses of 50 and 100 mg/kg qd on days 1−5 and 8−12. Treatment started
3
after randomization when tumors had reached an average volume of 370 mm . As shown in
Figure 6F, apparent tumor regression was observed for the first 5 days in the 50 and 100
mg/kg dosing groups. At 50 mg/kg, near-complete tumor regression was observed in all
animals by day 15 (85% tumor regression); no tumor growth resumed after dosing was halted.
At 100 mg/kg, complete disappearance of the tumor mass was observed after dosing was
halted. Treatment with 100 mg/kg resulted in 4 of 4 treated animals with CR during the 13
2
8
ACS Paragon Plus Environment