Discovery of N-((1-(4-(3-(3-((6,7-Dimethoxyquinolin-3-yl)oxy)phenyl)ureido)-2-(trifluoromethyl)phenyl)piperidin-4-yl)methyl)propionamide (CHMFL-KIT-8140) as a Highly Potent Type II Inhibitor Capable of Inhibiting the T670I gatekeeper Mutant of cKIT Kinase

Article abstract of DOI:10.1021/acs.jmedchem.6b00902

cKIT kinase inhibitors, e.g., imatinib, could induce drug-acquired mutations such as cKIT T670I that rendered drug resistance after chronic treatment. Through a type II kinase inhibitor design approach we discovered a highly potent type II cKIT kinase inhibitor compound 35 (CHMFL-KIT-8140), which potently inhibited both cKIT wt (IC₅₀ = 33 nM) and cKIT gatekeeper T670I mutant (IC₅₀ = 99 nM). Compound 35 displayed strong antiproliferative effect against GISTs cancer cell lines GIST-T1 (cKIT wt, GI₅₀ = 4 nM) and GIST-5R (cKIT T670I, GI₅₀ = 26 nM). In the cellular context it strongly inhibited c-KIT mediated signaling pathways and induced apoptosis. In the BaF3-TEL-cKIT-T670I isogenic cell inoculated xenograft mouse model, 35 exhibited dose dependent tumor growth suppression efficacy and 100 mg/kg dosage provided 47.7% tumor growth inhibition (TGI) without obvious toxicity. We believe compound 35 would be a good pharmacological tool for exploration of the cKIT-T670I mutant mediated pathology in GISTs.

Full text of DOI:10.1021/acs.jmedchem.6b00902

Subscriber access provided by CORNELL UNIVERSITY LIBRARY
Article
Discovery of N-((1-(4-(3-(3-((6,7-dimethoxyquinolin-3-yl)oxy)phenyl)ureido)-2-
trifluoromethyl)phenyl)piperidin-4-yl)methyl)propionamide
(
(CHMFL-KIT-8140) as a Highly Potent Type II Inhibitor Capable
of Inhibiting the T670I “Gatekeeper” Mutant of cKIT Kinase
Binhua Li, Aoli Wang, Juan Liu, Ziping Qi, Xiaochuan Liu, Kailin Yu, Hong Wu, Cheng Chen, Chen
Hu, Wenchao Wang, Jiaxin Wu, Zhenquan Hu, Ling Ye, Fengming Zou, Feiyang Liu, Beilei Wang,
Li Wang, Tao Ren, Shaojuan Zhang, Mingfeng Bai, Shanchun Zhang, Jing Liu, and Qingsong Liu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00902 • Publication Date (Web): 22 Aug 2016
Downloaded from http://pubs.acs.org on August 22, 2016
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 free 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 accessible to all
readers and 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.
Journal of Medicinal Chemistry 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 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 59
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 Nꢀ((1ꢀ(4ꢀ(3ꢀ(3ꢀ((6,7ꢀ
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
dimethoxyquinolinꢀ3ꢀyl)oxy)phenyl)ureido)ꢀ2ꢀ
(trifluoromethyl)phenyl)piperidinꢀ4ꢀ
yl)methyl)propionamide (CHMFLꢀKITꢀ8140) as a
Highly Potent Type II Inhibitor Capable of
Inhibiting the T670I “Gatekeeper” Mutant of cKIT
Kinase
1,2,7
1,3,7
1,7
1,2,7
1,7
1,3
Binhua Li , Aoli Wang , Juan Liu , Ziping Qi , Xiaochuan Liu , Kailin Yu , Hong
1
1,2
1,3
1,2
1,3
1,2
4
Wu , Cheng Chen , Chen Hu , Wenchao Wang , Jiaxin Wu , Zhenquan Hu , Ling Ye ,
1
,2
1,3
1,2
1,2
4
5
Fengming Zou , Feiyang Liu , Beilei Wang , Li Wang , Tao Ren , Shaojuan Zhang ,
5
2,6
1,2*
1,2,3,4*
Mingfeng Bai , Shanchun Zhang , Jing Liu , Qingsong Liu
1. High Magnetic Field Laboratory, Chinese Academy of Sciences, Mailbox 1110, 350
Shushanhu Road, Hefei, Anhui 230031, P. R. China
2. CHMFLꢀHCMTC Target Therapy Joint Laboratory, 350 Shushanhu Road, Hefei Anhui
2
30031, P. R. China
3. University of Science and Technology of China, Hefei, Anhui 230036, P. R. China
ACS Paragon Plus Environment

Page 3 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
4
. Precision Targeted Therapy Discovery Center, Institute of Technology Innovation, Hefei
Institutes of Physical Science, Chinese Academy of Sciences, Hefei, Anhui 230088, P. R.
China
5
. Molecular Imaging Laboratory, Department of Radiology, University of Pittsburgh,
Pittsburgh, PA 15219, USA; Department of Bioengineering, University of Pittsburgh,
Pittsburgh, PA 15261, USA; University of Pittsburgh Cancer Institute, Pittsburgh, PA 15232,
USA.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
6
. Hefei Cosource Medicine Technology Co. LTD., 358 Ganquan Road, Hefei, Anhui 230031,
P. R. China
7. These authors contribute equally
ABSTRACT
cKIT kinase inhibitors, e.g. Imatinib could induce drugꢀacquired mutations such as cKIT
T670I that rendered drug resistance after chronic treatment. Through a type II kinase inhibitor
design approach we discovered a highly potent type II cKIT kinase inhibitor compound 35
(CHMFLꢀKITꢀ8140), which potently inhibited both cKIT wt (IC : 33 nM) and cKIT gatekeeper
50
T670I mutant (IC : 99 nM). Compound 35 displayed strong antiꢀproliferative effect against
5
0
GISTs cancer cell lines GISTꢀT1 (cKIT wt, GI : 4 nM) and GISTꢀ5R (cKIT T670I, GI : 26
5
0
50
nM). In the cellular context it strongly inhibited cꢀKIT mediated signaling pathways and induced
apoptosis. In the BaF3ꢀTELꢀcKITꢀT670I isogenic cell inoculated xenograft mouse model, 35
exhibited dose dependent tumor growth suppression efficacy and 100 mg/kg dosage provided
4
7.7% tumor growth inhibition (TGI) without obvious toxicity. We believe compound 35 would
be a good pharmacological tool for exploration of the cKITꢀT670I mutant mediated pathology in
GISTs.
INTRODUCTION
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 59
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
Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the
gastrointestinal tract. There are approximately 1ꢀ2/100,000 newly diagnosed patients with GISTs
1
each year in the US. Almost all GISTs express cKIT kinase and about 80–85% of all GISTs
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
cases are associated with gainꢀofꢀfunction mutations in the cKIT gene. cꢀKIT kinase is a
member of the type III transmembrane receptor tyrosine kinase (RTK) family. Under
physiological conditions, it gets activated upon binding of the extracellular stem cell factor
(SCF). cKIT plays important roles in cellular transformation and differentiation, including
3
proliferation, survival, adhesion, and chemotaxis. Constitutive activation of the cKIT kinase is
4,5
critical in the pathogenesis of GISTs. Due to the critical role of cKIT kinase to the
tumorigenesis of GISTs, it has been extensively explored as an important drug discovery target
for antiꢀGIST therapy.
Currently there are two cKIT kinase inhibitors approved for the clinical use for GISTs.
6
Compound 1 (Imatinib , Figure 1) was the first type II kinase inhibitor approved as the firstꢀline
treatment for advanced GIST. Unfortunately, approximately 14% of patients are initially
7,8
insensitive to compound 1, furthermore, 46% to 67% of the responding patients will develop
9
ꢀ11
resistance through acquisition of a secondary mutation in the cKIT kinase domain,
e.g. the
ATPꢀbinding pocket mutants V654A and T670I, within 2 years of compound 1 treatment. The
T670 residue located at the gatekeeper position in cKIT kinase provides one of the key hydrogen
bonds for the binding of 1. Therefore, mutations at this position have a profound effect for
12
compound 1’s binding. In addition, experimental evidence supports that cKIT T670I mutant
1
3
will drive a more aggressive earlier metastasis and shorter progressionꢀfree survival.
Compound 2 (Sunitinib), a type I kinase inhibitor, was approved by FDA for the treatment of
patients with Imatinibꢀresistant GIST in 2006. Because compound 2 does not occupy the deep
ACS Paragon Plus Environment

Page 5 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
hydrophobic pocket generated by DFG motif flipꢀout as type II inhibitor compound 1 does, there
is enough space to accommodate the more bulky amino acid residue Isoleucine associated with
1
2
the T670I mutation without affecting 2’s binding. Moreover, a panel of small molecule type II
kinase inhibitors have been reported to bear cKIT kinase inhibitory activity such as 3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
4
15
16
(Nilotinib), 4 (Masitinib), 5 (Sorafenib), etc. Among them compound 3 and 4 significantly
lost activity against T670I mutant, while compound 5 exhibited moderate inhibitory activity.
Here, we reported our medicinal chemistry effort that started from a hit compound 6 to the
discovery of a new quinoline scaffold based type II cKIT kinase inhibitor compound 35
(
CHMFLꢀKITꢀ8140), which displayed potent in vitro and in vivo activities against cKIT
gatekeeper T670I mutant (Figure 2).
Figure 1. Chemical structures of the representative cKIT kinase inhibitors.
Figure 2. Schematic illustration of discovery of compound 35 (CHMFLꢀKITꢀ8140).
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 59
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
RESULTS AND DISCUSSION
Structure-Activity Relation (SAR) Exploration
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
During highꢀthroughput screening of our inꢀhouse generated kinase inhibitors, we obtained a
quinoline scaffold based compound 6 which exhibited moderate antiꢀproliferative activity against
BaF3ꢀTELꢀcKIT cell line (GI : 0.4 µM) and BaF3ꢀTELꢀcKITꢀT670I cell line (GI : 2.7 µM).
5
0
50
However, the compound demonstrated a good selectivity window against the parental BaF3 cell
line (GI : >10 µM). Therefore, we decided to use 6 as the starting point for further medicinal
5
0
chemistry modification. In order to get a view of the structural basis for the SAR exploration, we
first made a homology model of cKIT T670I mutant based on the cKIT wt Xꢀray crystal
structure (PDB ID: 1T46) and examined the detailed binding information of compounds 1 and 6
with cKIT wt/T670I (Figure 3). In the cKIT wt, compound 1 forms four key hydrogen bonds:
one in the hinge binding area between the pyridine nitrogen of 1 and Cys673; one in the linker
moiety between the aminopyrimidine nitrogen of 1 and the gatekeeper residue Thr670; two
canonical hydrogen bonds between the Glu640 in the cꢀHelix and Asp810 in the DFG motif with
the amide bond of 1 (Figure 3A). The mutation of gatekeeper residue Thr670 to the more bulky
Ile670 results in loss of one of the key hydrogen bonds and meanwhile the newly generated steric
hindrance impeded compound 1’s binding (Figure 3B). In comparison, in our model compound 6
forms three key hydrogen bonds with cKIT wt, i.e. one in the hinge binding area and two in the
DFG motif and cꢀHelix area (Figure 3C). Compound 6 has an Oꢀlinked phenyl ring in the
gatekeeper area and lacks the hydrogen bond formed between Thr670 with compound 1.
However, the Oꢀlinked phenyl ring in 6 will be likely to generate enough space to accommodate
the more bulky Ile670 if it can orientate into a proper direction, which will make it possible to
overcome the T670I mutation (Figure 3D). In addition, the CF3 group in 6 might form
ACS Paragon Plus Environment

Page 7 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
hydrophobic interactions with Leu647, Ile653, Leu783 and Ile808 in cKIT, which could be
beneficial to the binding (Figure 3E). Based on the analysis and the typical type II kinase
1
7
inhibitor design approach, compound 6 was divided into four parts: the quinoline hinge binding
part, the Oꢀbridged phenyl linker part, the amide mediated hydrogen bonding part and the tail
part that occupies the DFG shifting created hydrophobic pocket (Figure 3F). Since the quinoline
occupied hinge binding part is required for binding and the Oꢀbridged phenyl linker part is
necessary for overcoming cKITꢀT670I mutation, we decided to keep them unchanged. We
envisioned that variation of the Hꢀbonding area (R1) and the tail part (R2, R3) might alter the
orientation of the Oꢀbridged phenyl linker moiety to achieve higher binding affinity and better
selectivity against cKITꢀT670I kinase mutant.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 59
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 3. Schematic illustration of SAR exploration rationale. (A) Binding mode of compound 1
with cKIT wt (PDB ID: 1T46). (B) Binding mode of compound 1 with cKIT T670I homology
model (generated based on PDB ID: 1T46). (C) Binding mode of compound 6 with cKIT wt
(PDB ID: 1T46, docking model). (D) Binding mode of compound 6 with cKIT T670I homology
model (generated based on PDB ID: 1T46, docking model). (E) Illustration of the hydrophobic
interaction between the CF3 group of compound 6 and Leu647, Ile653, Leu783 and Ile808. (F)
Illustration of the chemical modification strategy of compound 6.
As the starting point, we first explored the SAR of the hydrogen bonding part (R1). BaF3ꢀ
TELꢀcKIT, BaF3ꢀTELꢀcKITꢀT670I and parental BaF3 cells were used to monitor the cKIT wt
ACS Paragon Plus Environment

Page 9 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
and cKITꢀT670I kinase inhibitory activities. Replacement of the amide group in compound 6
with urea group (7) significantly increased the activity both to cKIT wt (GI : 0.12 ꢁM) and
50
cKIT T670I (GI : 0.071 ꢁM), meanwhile showed a selectivity ratio of approximately 12 against
5
0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
parental BaF3 cells (GI : 1.5 ꢁM) (Table 1). However, switching the amide group to a much
5
0
larger group, i.e., N,N'ꢀdimethylcyclopropaneꢀ1,1ꢀdicarboxamide (8), led to the complete loss of
activity to both the cꢀKIT wt and cKITꢀT670I (GI : >10 ꢁM). This might be due to the
5
0
interaction of the cyclopropane substituent with the Glu640, which abolished the critical
hydrogen bonds in this area for the type II binding. We then explored the R2 moiety in the tail
part by fixing the R1 as the uera. Removal of the trifluoromethyl group (9) also caused complete
activity loss to cKIT wt and cKITꢀT670I (GI : >10 ꢁM), which indicated that the hydrophobic
50
interaction between the CF3 group and the hydrophobic environment formed by Leu647, Ile653
Leu783 and Ile808 was important for the binding. In addition, replacement of the phenyl group
with a pyridine group (10) resulted in significant activity loss in BaF3ꢀTELꢀcKITꢀT670I cells
(GI : ＞10 ꢁM). These results indicated that the urea group at the R1 position and the
50
(trifluoromethyl)benzyl group at R2 were preferred for better activity.
a
Table 1. SAR Exploration Focused on the R1/R2 positons
BaF3ꢀTELꢀcKIT BaF3ꢀTELꢀcKITꢀT670I
BaF3
Compd
R1
R2
(
GI : µM)
(GI : µM)
(GI : µM)
5
0
50
50
6
0.4±0.011
2.7±0.058
＞10
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 59
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
7
8
9
0.12±0.016
＞10
0.071±0.0011
＞10
1.5±0.17
＞10
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
＞10
＞10
＞10
10
0.34±0.01
＞10
＞10
N
a
All GI values were obtained by triplet testing.
5
0
On the basis of compound 7, we next focused SAR exploration on the R3 position, which
presumably occupies the DFGꢀout shifting generated hydrophobic pocket and usually will
provide the selectivity and higher binding affinity for the type II inhibitors (Table 2). Shifting the
propionyl group from 4ꢀposition (7) to 3ꢀposition (11) led to about 13ꢀfold activity loss in BaF3ꢀ
TELꢀcKITꢀT670I cells (GI : 0.071ꢁM versus 0.92 ꢁM). Replacement of the propionyl group in
5
0
7
with acetyl group (12) retained the activities against BaF3ꢀTELꢀcKIT cells and BaF3ꢀTELꢀ
cKITꢀT670I cells (GI : 0.11 ꢁM and 0.046 ꢁM, respectively), meanwhile displayed better
5
0
selectivity against parental BaF3 cells (GI : 7.1 ꢁM). Installment of larger groups such as
5
0
dimethylbutyl (13), glycine (14) and alanine (15) all led to about 10ꢀfold loss of activity against
cKIT T670I mutant. However, the N,Nꢀdimethylglycine (16) gained back the activity against
cKIT T670I mutant (GI : 0.053 ꢁM) and displayed better activity against cKIT wt (GI : 0.038
5
0
50
ꢁ
M) as well as better selectivity to parental BaF3 cells (GI : 2.9 ꢁM) compared to 7.
50
Introduction of cyclohexene (17) and pyridine groups (18) both led to about 5ꢀ10 fold activity
loss, while tetrahydropyran group (19) gained back the activity against both cKIT wt (GI : 0.087
5
0
ACS Paragon Plus Environment

Page 11 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
ꢁ
M) and cKIT T670I (GI : 0.083 ꢁM). NꢀMethyl piperidine (20) and Nꢀethyl piperidine (21)
5
0
retained similar potency. NꢀAcyl (22), Nꢀcyclopropanecarbonyl (23) and Boc (24) substituents
caused 2ꢀ3 fold activity loss, and 2ꢀmethyl piperidine group (25) resulted in about 10ꢀfold
activity loss against cKIT T670I. Increasing the length or size of the piperidineꢀderived
substituents either narrowed down the selectivity window to parental BaF3 cells (26 and 27) or
decreased the activity against cKIT T670I (28 and 29). Interestingly, ethyl linked morpholine
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(30) showed impressive activities against cKIT wt (GI : 0.042 ꢁM) and cKIT T670I (GI :
50 50
0.059 ꢁM), meanwhile kept a good selectivity window to parental BaF3 cells (GI : 3.6 ꢁM).
5
0
Switching the amide moiety to sulfonamide derivatives with different aliphatic chains such as
methyl (31), ethyl (32) and propyl (33) did not improve the antiꢀproliferative efficacy against
cKIT T670I mutant either. However, introduction of the cyclopropyl group (34) started to gain
back the activity against BaF3ꢀTELꢀcKITꢀT670I cells (GI : 0.063 ꢁM). Increasing the size of
5
0
R3 to Nꢀ(piperidinꢀ4ꢀylmethyl)propionamide (35) enhanced the activity against cKIT wt (GI50:
0.057 ꢁM) meanwhile retained the activity against cKIT T670I and remarkably improved the
selectivity window to parental BaF3 cells (GI : >10 ꢁM). Unfortunately, further size increase
5
0
(36) resulted in obvious activity loss compared to compound 35.
a
Table 2. SAR Exploration Focused on the R3 Position
BaF3ꢀTELꢀcKIT BaF3ꢀTELꢀcKITꢀT670I
GI : µM) (GI : µM)
BaF3
Compd
R3
(
(GI : µM)
5
0
50
50
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 59
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
1
1
1
2
0.28±0.025
0.11±0.016
0.92±0.04
1.3±0.17
7.1±0.38
0.046±0.001
0.34±0.025
0.58±0.02
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
1
3
4
0.43±0.01
0.13±0.01
＞10
5±1.0
15
16
0.12±0.011
0.48±0.011
3±0.15
0.038±0.001
0.053±0.001
2.9±0.17
1
7
0.76±0.0057
0.02±0.001
0.76±0.1
0.32±0.025
0.083±0.021
1.1±0.47
7±0.74
18
1
2
2
9
0
1
0.087±0.0015
2.9±0.056
0.16±0.01
0.081±0.006
0.087±0.008
1.7±0.1
0.039±0.041
1.3±0.11
2
2
0.059±0.001
0.23±0.0001
3.4±1.2
ACS Paragon Plus Environment

Page 13 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
2
3
0.13±0.0057
0.12±0.02
0.8±0.084
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N
N
Boc
24
25
0.13±0.001
0.46±0.021
0.17±0.05
0.51±0.15
4.0±0.32
N
O
0.65±0.076
2
6
0.12±0.0001
0.33±0.015
0.037±0.009
0.2±0.011
0.48±0.006
0.87±0.068
27
2
2
3
8
9
0
0.44±0.03
0.19±0.021
0.042±0.003
0.26±0.052
0.2±0.02
7.6±0.61
1.2±0.11
3.6±0.89
0.059±0.006
3
3
1
2
3.9±0.023
3.8±0.02
＞10
0.034±0.002
0.33±0.029
6.2±0.38
3
3
3
4
0.059±0.003
0.14±0.021
0.95±0.04
4.2±0.36
0.063±0.002
1.9±0.058
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 59
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
35
0.057±0.0021
0.25±0.06
0.063±0.001
＞10
3
6
0.46±0.01
4.4±1.37
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
a
All GI values were obtained by triplet testing.
5
0
Since compound 35 exhibited the best activity and selectivity profile, we then studied its
binding modes with cKIT wt and T670I mutant by molecular modeling. In the cKIT wt (PDB
ID: 1T46), compound 35 adopted a canonical type II binding mode. As expected, the quinoline
nitrogen formed a hydrogen bond with the cKIT residue Cys673 in the hinge binding area
(
Figure 4A). The two NHs in the urea moiety formed two hydrogen bonds with Glu640 in the cꢀ
Helix, and the carbonyl formed a hydrogen bond with the Asp810 in the DFG motif. The tail part
occupied the hydrophobic pocket generated by the DFGꢀout shift. In addition, a hydrogen bond
was formed between the NH of the amide and the Ile789 in cKIT kinase. In the homology model
of cKIT T670I mutant, compound 35 adopted the similar type II binding mode. The three
hydrogen bonds formed via the urea moiety caused the Oꢀbridged phenyl moiety in 35 to orient
to an angle that provided enough space for the bulky residue Isoleucine, which could explain its
potency against T670I mutant (Figure 4B).
ACS Paragon Plus Environment

Page 15 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Figure 4. Molecular modeling analysis of the binding modes of cKIT wt/T670I with compound
35. (A) Binding mode of compound 35 with cKIT wt (PDB ID: 1T46). (B) Binding mode of
compound 35 with cKIT T670I homology model (generated based on PDB ID: 1T46).
Biochemical and Cellular Property Evaluation
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
We further examined the activity of compound 35 against a panel of cKIT mutants in the
TEL engineered BaF3 systems as well as intact GIST cancer cell lines (Table 3). The results
demonstrated that besides cKIT wt and cKIT T670I, compound 35 was also effective against
cKIT L567P (GI : 0.023 ꢁM), cKIT N822K (GI : 0.04 ꢁM), and cKITT670I/V559D (GI :
5
0
50
50
0
.073 ꢁM). However, compound 1 was only potent against cKIT L567P (GI : 0.01 ꢁM).
50
Compound 2 was more potent against these mutants but it also affected the parental BaF3 cells
growth (GI : 1.6 µM) which indicated its multiple target feature. In addition, compound 35 is
5
0
about 10ꢀfold and 2ꢀfold more potent than compounds 1 and 2 against cKIT wt, respectively.
Compound 35 displayed similar antiꢀproliferative effects to compound 2 against cKIT wt
expressing GIST cancer cell line GISTꢀT1 (GI : 0.004 ꢁM) and is about 7ꢀfold more potent than
5
0
compound 1 (GI : 0.027 ꢁM). 35 also exhibited similar potency to compound 2 against cKIT
5
0
T670I expressing GIST cancer cell line GISTꢀ5R (GI : 0.026 ꢁM) and was about 300ꢀfold more
5
0
active than compound 1 (GI : 8.3 ꢁM).
50
Table 3. Antiꢀproliferative Effects of Compounds 1, 2 and 35 against a Variety of BaF3ꢀTELꢀ
a
cKIT Isogenic Cells and GIST Intact Cell Lines
Compd. 1
Compd. 2
Compd. 35
Cell line
GI (ꢁM)
GI (ꢁM)
GI (ꢁM)
5
0
50
50
BaF3
＞10
1.6±0.15
＞10
BaF3ꢀTELꢀcKIT
0.59±0.050
0.11±0.040
0.057±0.002
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 59
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
BaF3ꢀTELꢀcKITꢀT670I
BaF3ꢀTELꢀcKITꢀV654A
BaF3ꢀTELꢀcKITꢀL567P
BaF3ꢀTELꢀcKITꢀN822K
BaF3ꢀTELꢀcKITꢀD816V
BaF3ꢀTELꢀcKITꢀT670IꢀV559D
BaF3ꢀTELꢀcKITꢀV559DꢀV654A
GISTꢀT1 (cKIT wt)
9.6±0.32
0.70±0.015
0.01±0.001
0.36±0.006
＞10
0.005±0.0001
0.063±0.001
0.32±0.040
0.006±0.0003
0.003±0.0006
0.11±0.018
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
0.023±0.001
0.04±0.001
0.42±0.0057
0.007±0.001
0.006±0.0006
0.005±0.0002
0.021±0.0001
0.67±0.015
5.2±1.1
0.073±0.004
0.36±0.0001
0.004±0.0026
0.026±0.006
0.70±0.16
0.027±0.0011
8.3±0.32
GISTꢀ5R (cKITꢀT670I)
a
All GI values were obtained by triple testing.
5
0
We also used ADPꢀGlo based biochemical activity assay with the purified kinase proteins to
confirm the inhibition activities of compound 35 against cKIT and cKITꢀT670I kinases. The
results showed that 35 inhibited cKIT wt kinase with an IC50 of 33 nM, and inhibited cKIT
T670I kinase with an IC of 99 nM (Figure 5).
5
0
ACS Paragon Plus Environment

Page 17 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Figure 5. ADPꢀGlo biochemical characterization of compound 35 against cKIT wt/T670I
kinases.
To better understand compound 35’s selectivity, we examined its kinome wide selectivity
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
18
profile with KinomeScan technology. The results demonstrated that compound 35 bore a good
selectivity (S score (1) = 0.03) in a panel of 468 kinases and mutants at 1 ꢀM concentration.
Besides high binding affinity to cKIT and cKITꢀT670I kinases, it also displayed strong binding
against CDKL2, CDKL3, CSF1R, DDR1, FLT3, FLT4, LOK, RET and PDGFRβ kinases
(
percent activity remaining less than 1% at 1 ꢁM 35) (Figure 6 and Supplemental Table 1).
Given the fact that KinomeScan is a binding assay and sometimes cannot really reflect the
compound’s inhibitory activity, we then used TEL transformed BaF3 system to further test the
onꢀtarget activity and selectivity of 35 against other potential offꢀtargets revealed by
KinomeScan assay (Table 4). The data showed that compound 35 also potently inhibited
PDGFRβ (GI : 0.005 ꢀM) and displayed apparent activity against FLT3 (GI : 0.099 ꢀM),
5
0
50
FLT4 (GI : 0.24 ꢀM), CSF1R (GI : 0.11 ꢀM) as well as RET (GI : 0.12 ꢀM). This is not
5
0
50
50
surprising since cKIT, FLT3, CSF1R and PDGFR kinases all belong to the type III receptor
tyrosine kinase family and the ATP binding pocket of these kinases are highly conserved.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 59
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. Kinome wide selectivity profiling of compound 35. (A) KinomeScan profiling of
compound 35 at a concentration of 1 ꢁM against 468 kinases and mutants. (B) Kinases that
remained activity less than 1% of control in the presence of 1 µM 35.
Table 4. Antiꢀproliferative Effects of Compound 35 against KinaseTargets Revealed from the
a
KinomeScan Profiling in Isogenic BaF3 Cell Lines
Cell line
GI (ꢁM)
50
BaF3ꢀTELꢀCSF1R
BaF3ꢀTELꢀFLT3
BaF3ꢀTELꢀFLT4
BaF3ꢀTELꢀRET
0.11±0.006
0.099±0.0011
0.24±0.01
0.12±0.0057
0.005±0.0007
BaF3ꢀTELꢀPDGFRβ
a
All GI values were obtained by triple testing.
5
0
ACS Paragon Plus Environment

Page 19 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
We next investigated compound 35’s effects on the cKIT mediated signaling pathways in
cKIT wt and cKIT T670I driven GIST cell lines GISTꢀT1 and GISTꢀ5R, respectively (Figure 7).
As the results demonstrated, compound 35 completely blocked cKIT pY703, pY719, pY823
autoꢀphosphorylation sites in GISTꢀT1 cells at the concentration of 100 nM, and also remarkably
inhibited downstream signaling mediators pAKT (T308, S473), pS6 (S235/236), pERK
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(T202/204) (EC50 less than 100 nM). Compound 1 displayed similar effect on the signaling
pathways, which further proved 35’s cKIT kinase inhibitory activity. In the Imatinibꢀresistant
cell line GISTꢀ5R (cKIT T670I), 35 also significantly affected cKIT pY703, pY719, pY823
autoꢀphosphorylation sites and downstream mediator phosphorylation (EC less than 100 nM).
50
Not surprisingly, compound 1’s inhibitory effect was much weaker even at the concentration of 1
ꢁM. In addition, compound 35 started to induce doseꢀdependent cell apoptotic death (by
examining the cleaved PARP) in both GISTꢀT1 and GISTꢀ5R cells from the concentration of 30
nM at 24 h, while at the same time point compound 1 did not cause apparent apoptosis in both
cells (Figure 8).
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 59
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 7. Effect of compounds 1, 2 and 35 on cKIT mediated signaling pathways in GISTꢀT1
and GISTꢀ5R cancer cell lines.
Figure 8. Effect of compounds 1, 2 and 35 on induction of apoptosis in GISTꢀT1 and GISTꢀ5R
cell lines.
In Vivo PK/PD Evaluation.
ACS Paragon Plus Environment

Page 21 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
The PK properties of compound 35 were evaluated using rats model (Table 5). With the
intravenous injection compound 35 exhibited a T1/2 of 2.04 h and Cmax of 2563.2 ng/mL.
However, in the oral administration there was almost no absorption, which prevented 35 from the
oral application in the animal model. We also tested the solubility of compound 35 and the data
showed that in the PBS buffer (pH=7.4) it was 22.8±0.2 ꢀg/mL (about 35 ꢀM) while in the pure
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
water it was 50.8±4.7 ꢀg/mL (about 78 ꢀM). This indicated that 35 should be fully dissolved at
the concentrations we used in the in vitro studies.
Table 5. Pharmacokinetic Study of Compound 35 on Sprague Dawley Rats
Cmax
AUC(0ꢀt)
AUC(0ꢀ∞)
MRT (0ꢀ∞)
Vz
L/kg)
CLz
(L/h/kg)
T1/2 (h)
2.04
Tmax (h)
0.017
0.0
(
(ng/mL) (ng/mL*h) (ng/mL*h)
(h)
iv
1 mg/kg
mean
2563.2
825.4
377.2
100.1
384.9
105.0
7.91
3.11
2.76
0.89
0.78
0.11
SD
0
.86
(
n = 3)
We finally tested the antitumor efficacy of compound 35 in BaF3ꢀTELꢀcKITꢀT670I cells
inoculated xenograft mouse model. Based on the PK data, we chose the IP injection method. The
results demonstrated that none of the 25, 50 and 100 mg/kg/day treatment affected the mice body
weight (Figure 9A). During 10 days of continuous treatment, compound 35 doseꢀdependently
inhibited the growth of the BaF3ꢀTELꢀcKITꢀT670I tumor progression and a dosage of 100
mg/kg/day exhibited 47.7% TGI (tumor growth inhibition) (Figure 9BꢀD). The
immunohistochemistry stain showed that in the tumors the inhibitor doseꢀdependently inhibited
the tumor cell proliferation (examined by Kiꢀ67 staining) and induced apoptosis (examined by
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 59
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
TUNEL staining) (Figure 9E). Compared to compound 1, which has been shown that it could
promote but not suppress the cKIT T670I tumor growth, compound 35 demonstrated a proof of
1
9
concept that a type II kinase inhibitor could effectively suppress the tumor progression.
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

Page 23 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Figure 9. Compound 35’s antitumor efficacy in BaF3ꢀTELꢀcKITꢀT670I xenograft model.
Female nu/nu mice bearing established BaF3ꢀTELꢀcKITꢀT670I tumor xenografts were treated
with compound 35 at 25, 50,100 mg/kg/d or vehicle. Daily IP administration was initiated when
3
BaF3ꢀTELꢀcKITꢀT670I tumors had reached a size of 200−400 mm . Each group contained seven
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
animals. Data, mean ± SEM. (A) Body weight and (B) tumor size measurements from BaF3ꢀ
TELꢀcKITꢀT670I xenograft mice after compound 35 administration. Initial body weight and
tumor size were set as 100%. (C) Representative photographs of tumors in each group after 25,
50, 100 (mg/kg)/d compound 35 or vehicle treatment. (D) Comparison of the final tumor weight
in each group after 10ꢀday treatment period of 35. Numbers in columns indicate the mean tumor
weight in each group. ns, p > 0.05, (∗) p < 0.05, (∗∗) p < 0.01. (E) Representative micrographs of
hematoxylin and eosin (HE), Kiꢀ67, and TUNEL staining of tumor tissues of 35 treatment groups
in comparison with the vehicle group. Note the specific nuclear staining of cells with
morphology consistent with proliferation and apoptosis (E, red arrow).
CHEMISTRY
The synthesis of urea compounds 7, 9, 11-36 started from nucleophilic substitution of 4ꢀ
chloroꢀ6,7ꢀdimethoxyquinoline with 3ꢀaminophenol which afforded compound 37 (Scheme 1).
Bocꢀprotected 3ꢀ or 4ꢀaminopiperidine analogs (39) reacted with chloronitrobenzene derivatives
(38) to provide 40 in two steps. Urea formation from the two precursors with triphosgene
afforded compound 41. Acidic deprotection of Boc group (42) and amide bond formation with
corresponding acyl chlorides or carboxylic acids furnished the target compounds.
a
Scheme 1. Synthesis of Compounds 7, 9, 11-36
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 59
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
a
Reagents and conditions: (a) tBuOK, K CO , DMSO, 100 °C, 12 h; (b) K CO , DMF, 100 °C, 8
2
3
2
3
h; (c) H , 10% Pd/C, MeOH, rt, 3 h; (d) triphosgene, Et N, DMAP, DCM, 0 °C to rt, 1 h; (e) 4 M
2
3
HCl in ethyl acetate, rt, 1 h; (f) for 7, 9, 11-13 and 31-35: acyl chloride, Et N, DMF, ꢀ50 °C, 5
3
min; for 16-24, 26ꢀ30 and 36: carboxylic acid, HATU, Et N, DMF, rt, overnight; for 14ꢀ
3
1
5 and 25: (i) carboxylic acid, HATU, Et N, DMF, rt, overnight; (ii) 4 M HCl in ethyl acetate, rt,
3
1 h.
ACS Paragon Plus Environment

Page 25 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Compound 10 was prepared via a similar approach in different reaction order (Scheme 2). The
nucleophilic substitution of trifluoromethyl substituted chloronitrobenzene (38b) and Bocꢀ
protected 4ꢀaminopiperidine (39b) was followed by Bocꢀdeprotection (44) and connection with
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
7 through triphosgene. Hydrogenation of the nitro group in 45 provided the amine moiety,
which then readily reacted with the propionyl chloride.
a
Scheme 2. Synthesis of Compound 10
a
Reagents and conditions: (a) K CO , DMF, 100 °C, 8 h; (b) 4 M HCl in ethyl acetate, rt, 1 h; (c)
2
3
37, triphodgene, Et N, DMAP, DCM, 0 °C to rt, 1 h; (d) H , 10% Pd/C, MeOH, rt, 8 h; (e)
3
2
propionyl chloride, Et N, DMF, ꢀ50 °C, 5 min.
3
The synthesis of compound 8 that bears a cyclopropaneꢀ1,1ꢀdicarboxamide moiety at the R1
position was achieved by amidation of 37 with 1ꢀ(methoxycarbonyl)cyclopropanecarboxylic
acid, which was followed by methyl ester hydrolysis of 47 and coupling reaction of 48 with 40d.
Final Boc deprotection and acylation afforded compound 8 (Scheme 3).
a
Scheme 3. Synthesis of Compound 8
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 59
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
a
Reagents and conditions: (a) HATU, DIPEA, DMF, rt, overnight; (b) 1.0 M NaOH, MeOH,
reflux, 2 h; (c) 40d, HATU, DIPEA, DMF, rt, overnight; (d) 4 M HCl in ethyl acetate, rt, 1 h; (e)
propionyl chloride, Et N, DMF, ꢀ50 °C, 5 min.
3
CONCLUSIONS
In summary, we have discovered a new quinoline scaffold based highly potent type II kinase
inhibitor compound 35, which possessed strong inhibitory activities against both cKIT wt GIST
cancer cells (GISTꢀT1) and cKIT T670I gatekeeper mutant cells (GISTꢀ5R). The compound
strongly inhibited cKIT mediated signaling pathways and doseꢀdependently induced apoptosis.
Although 35 displayed good antiꢀtumor efficacies in the cKIT T670I mutant cells mediated
xenograft mouse models, the poor oral PK profile prevented it from further development. Further
medicinal chemistry effort is needed to improve the PK profile of this series of compounds. We
believe compound 35 will be a useful pharmacological tool for exploration of the cKIT kinase
gatekeeper T670I mutant mediated pathology in GISTs.
ACS Paragon Plus Environment

Page 27 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
EXPERIMENTAL SECTION
Chemistry. All reagents and solvents were purchased from commercial sources and used as
1
13
obtained. H NMR and C NMR spectra were recorded with a Bruker 400 NMR spectrometer
and referenced to deuterium dimethyl sulfoxide (DMSOꢀd ) or deuterium chloroform (CDCl ).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
6
3
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 ꢁm). The purities of all
compounds were determined to be above 95% by HPLC.
Compounds 7-13 and 31-35 were prepared following the synthetic procedure of 7.
Nꢀ(1ꢀ(4ꢀ(3ꢀ(3ꢀ((6,7ꢀDimethoxyquinolinꢀ4ꢀyl)oxy)phenyl)ureido)ꢀ2ꢀ
(trifluoromethyl)phenyl)piperidinꢀ4ꢀyl)propionamide (7) To a solution of 42d (30.0 mg, 0.057
mmol), Et N (0.039 mL, 0.171 mmol) in anhydrous DMF (0.5 mL) at ꢀ50 °C under Ar was
3
added propionyl chloride (6.3 mg, 0.0682mmol). After being stirred at ꢀ50 °C for 5 min., the
reaction mixture was quenched with water and warmed up to room temperature. The mixture
was diluted with ethyl acetate and washed with water and brine. The organic phase was dried
with MgSO , filtered, and concentrated under vacuum to give the residue which was purified by
4
silica gel flash chromatography with dichloromethane/methanol (10:1) to afford the title
1
compound 7 as a white solid (8.5 mg, 25%). H NMR (400 MHz, DMSOꢀd ) δ 9.02 (s, 2H), 8.51
6
(
d, J = 5.2 Hz, 1H), 7.88 (d, J = 2.2 Hz, 1H), 7.77 (d, J = 7.8 Hz, 1H), 7.60 – 7.53 (m, 2H), 7.50
d, J = 8.9 Hz, 2H), 7.47 – 7.39 (m, 2H), 7.28 (d, J = 8.0 Hz, 1H), 6.90 (dd, J = 8.0, 1.9 Hz, 1H),
(
6.55 (d, J = 5.2 Hz, 1H), 3.95 (s, 6H), 3.70 – 3.64 (m, 1H), 2.87 – 2.84 (m, 2H), 2.79 – 2.74 (m,
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 59
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
2
H), 2.07 (q, J = 7.6 Hz, 2H), 1.80 – 1.77 (m, 2H), 1.52 – 1.46 (m, 2H), 1.00 (t, J = 7.6 Hz, 3H).
1
3
C NMR (101 MHz, DMSOꢀd ) δ 172.55, 159.90, 154.98, 152.99, 149.86, 149.32, 147.26,
6
141.95, 137.14, 130.96, 125.78, 123.45, 115.73, 114.52, 110.86, 108.34, 104.14, 99.52, 56.16,
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
+
53.23, 45.73, 32.77, 29.03, 10.44. LCꢀMS (ESI, m/z): calcd for C H F N O [M+H] :
33
35
3
5
5
638.2590; found: 638.2593.
Nꢀ(3ꢀ((6,7ꢀdimethoxyquinolinꢀ4ꢀyl)oxy)phenyl)ꢀNꢀ(4ꢀ(4ꢀpropionamidopiperidinꢀ1ꢀyl)ꢀ3ꢀ
1
(trifluoromethyl)phenyl)cyclopropaneꢀ1,1ꢀdicarboxamide (8) (white solid，19% yield). H NMR
(400 MHz, DMSOꢀd ) δ 10.23 (s, 1H), 10.18 (s, 1H), 8.51 (d, J = 5.1 Hz, 1H), 8.03 (d, J = 5.6
6
Hz, 1H), 7.80 – 7.75 (m, 2H), 7.66 (d, J = 1.0 Hz, 1H), 7.61 (d, J = 8.4 Hz, 1H), 7.51 (dd, J =
12.8, 7.2 Hz, 2H), 7.41 (d, J = 0.5 Hz, 1H), 7.09 (d, J = 4.0 Hz, 1H), 7.00 (d, J = 7.6 Hz, 1H),
6.55 (d, J = 5.2 Hz, 1H), 3.96 (s, 3H), 3.93 (s, 3H), 3.73 – 3.64 (m, 1H), 2.86 – 2.74 (m, 4H),
2.09 – 2.04 (m, 2H), 1.82 – 1.76 (m, 2H), 1.57 – 1.44 (m, 4H), 1.00 (t, J = 7.4 Hz, 3H). LCꢀMS
+
(ESI, m/z): calcd for C H F N O [M+H] : 706.2852; found: 706.2867.
3
7
39
3
5
6
Nꢀ(1ꢀ(4ꢀ(3ꢀ(3ꢀ((6,7ꢀdimethoxyquinolinꢀ4ꢀyl)oxy)phenyl)ureido)phenyl)piperidinꢀ4ꢀ
1
yl)propionamide (9) (white solid, 29 % yield). H NMR (400 MHz, DMSOꢀd ) δ 9.25 (s, 1H),
6
8
.88 (s, 1H), 8.51 (d, J = 4.9 Hz, 1H), 7.74 (d, J = 7.6 Hz, 1H), 7.56 (s, 1H), 7.51 (s, 1H), 7.44 –
.35 (m, 2H), 7.27 (t, J = 7.4 Hz, 3H), 6.85 (t, J = 9.1 Hz, 3H), 6.55 (d, J = 5.0 Hz, 1H), 3.95 (s,
7
6H), 3.73 – 3.60 (m, 1H), 3.54 – 3.50 (m, 2H), 2.72 – 2.66 (m, 2H), 2.05 – 2.00 (m, 2H), 1.81 –
1.78 (m, 2H), 1.52 – 1.42 (m, 2H), 0.99 (t, J = 7.5 Hz, 3H). LCꢀMS (ESI, m/z): calcd for
+
C H N O [M+H] : 570.2716; found: 570.2733.
32
36
5
5
Nꢀ(4ꢀ(4ꢀ(3ꢀ(3ꢀ((6,7ꢀdimethoxyquinolinꢀ4ꢀyl)oxy)phenyl)ureido)piperidinꢀ1ꢀyl)ꢀ3ꢀ
1
(trifluoromethyl)phenyl)propionamide (10) (white solid, 45 % yield). H NMR (400 MHz,
DMSOꢀd ) δ 10.11 (s, 1H), 8.78 (s, 1H), 8.50 (d, J = 5.0 Hz, 1H), 8.00 (d, J = 1.5 Hz, 1H), 7.78
6
ACS Paragon Plus Environment

Page 29 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
(
d, J = 7.2 Hz, 1H), 7.59 – 7.47 (m, 2H), 7.41 (s, 1H), 7.39 – 7.31 (m, 1H), 7.19 (d, J = 7.8 Hz,
1
3
1
H), 6.86 – 6.75 (m, 1H), 6.54 (d, J = 5.0 Hz, 1H), 6.48 (d, J = 6.1 Hz, 1H), 3.96 (s, 6H), 3.65 –
.57 (m, 1H), 2.94 – 2.86 (m, 2H), 2.83 – 2.73 (m, 2H), 2.33 (dd, J = 14.5, 7.1 Hz, 2H), 1.95 –
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
13
.87 (m, 2H), 1.59 – 1.47 (m, 2H), 1.08 (t, J = 7.5 Hz, 3H). C NMR (101 MHz, DMSOꢀd ) δ
6
172.69, 159.96, 155.01, 154.90, 153.05, 149.32, 147.00, 143.01, 136.92, 130.75, 125.99, 123.90,
117.35, 115.84,114.86, 113.37, 110.03, 108.36, 104.21, 99.55, 56.19, 56.14, 52.91, 33.16, 29.93,
+
1
0.00. LCꢀMS (ESI, m/z): calcd for C H F N O [M+H] : 638.2590; found: 638.2599.
3
3
35
3
5
5
Nꢀ(1ꢀ(4ꢀ(3ꢀ(3ꢀ((6,7ꢀdimethoxyquinolinꢀ4ꢀyl)oxy)phenyl)ureido)ꢀ2ꢀ
trifluoromethyl)phenyl)piperidinꢀ3ꢀyl)propionamide (11) (yellow solid, 41% yield). H NMR
400 MHz, DMSOꢀd ) δ 9.34 (s, 2H), 8.51 (d, J = 4.5 Hz, 1H), 7.89 (s, 1H), 7.59 (s, 3H), 7.52 (s,
1
(
(
6
1H), 7.47 – 7.36 (m, 3H), 7.31 (d, J = 6.9 Hz, 2H), 6.89 (d, J = 7.2 Hz, 1H), 6.56 (d, J = 4.5 Hz,
1
2
1
H), 3.96 (d, J = 5.3 Hz, 6H), 3.86 – 3.83 (m, 1H), 3.10 – 3.07 (m, 1H), 2.96 – 2.92 (m, 1H),
.80 – 2.75 (m, 1H), 2.62 – 2.57 (m, 1H), 2.07 (q, J = 7.3 Hz, 2H), 1.80 – 1.72 (m, 2H), 1.61 –
.52 (m, 1H), 1.33 – 1.29 (m, 1H), 0.98 (t, J = 7.4 Hz, 3H). LCꢀMS (ESI, m/z): calcd for
+
C H F N O [M+H] : 638.2590; found: 638.2591.
33
35
3
5
5
Nꢀ(1ꢀ(4ꢀ(3ꢀ(3ꢀ((6,7ꢀdimethoxyquinolinꢀ4ꢀyl)oxy)phenyl)ureido)ꢀ2ꢀ
trifluoromethyl)phenyl)piperidinꢀ4ꢀyl)acetamide (12) (white solid, 40% yield). H NMR (400
1
(
MHz, DMSOꢀd ) δ 9.56 (s, 2H), 8.56 – 8.48 (m, 1H), 7.88 (d, J = 10.2 Hz, 2H), 7.53 (dt, J =
6
16.7, 4.3 Hz, 4H), 7.46 – 7.39 (m, 2H), 7.29 (dd, J = 7.6, 1.1 Hz, 1H), 6.89 (dd, J = 7.8, 1.1 Hz,
1H), 6.57 (d, J = 5.1 Hz, 1H), 3.96 (s, 6H), 3.75 – 3.62 (m, 1H), 2.88 – 2.85 (m, 2H), 2.81 – 2.70
(m, 2H), 1.81 (s, 3H), 1.80 – 1.77 (m, 2H), 1.57 – 1.45 (m, 2H). LCꢀMS (ESI, m/z): calcd for
+
C H F N O [M+H] : 624.2434; found: 624.2452.
32
33
3
5
5
ACS Paragon Plus Environment