Discovery of 1-(4-(4-Amino-3-(4-(2-morpholinoethoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)phenyl)-3-(5-(tert-butyl)isoxazol-3-yl)urea (CHMFL-FLT3-213) as a Highly Potent Type II FLT3 Kinase Inhibitor Capable of Overcoming a Variety of FLT3 Kinase Mutan

Article abstract of DOI:10.1021/acs.jmedchem.7b00840

FLT3-ITD mutant has been observed in about 30% of AML patients and extensively studied as a drug discovery target. On the basis of our previous study that ibrutinib (9) exhibited selective and moderate inhibitory activity against FLT3-ITD positive AML cel

Full text of DOI:10.1021/acs.jmedchem.7b00840

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Article
Discovery of 1-(4-(4-Amino-3-(4-(2-morpholinoethoxy)phenyl)-1H-pyrazolo[3,4-
d] pyrimidin-1-yl)phenyl)-3-(5-(tert-butyl)isoxazol-3-yl)urea (CHMFL-
FLT3-213) as a Highly Potent Type II FLT3 Kinase Inhibitor Capable of
Overcoming a Variety of FLT3 Kinase Mutants in FLT3-ITD Positive AML
Aoli Wang, Xixiang Li, Cheng Chen, Hong Wu, Ziping Qi, Chen Hu, Kailin Yu, Jiaxin Wu, Juan Liu,
Xiaochuan Liu, Zhenquan Hu, Wei Wang, Wenliang Wang, Wenchao Wang, Li Wang, Beilei Wang,
Qingwang Liu, Lili Li, Jian Ge, Tao Ren, Shanchun Zhang, Ruixiang Xia, Jing Liu, and Qingsong Liu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00840 • Publication Date (Web): 28 Sep 2017
Downloaded from http://pubs.acs.org on September 28, 2017
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Discovery
of
1ꢀ(4ꢀ(4ꢀAminoꢀ3ꢀ(4ꢀ(2ꢀ
morpholinoethoxy)phenyl)ꢀ1Hꢀpyrazolo[3,4ꢀd]
pyrimidinꢀ1ꢀyl)phenyl)ꢀ3ꢀ(5ꢀ(tertꢀ
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butyl)isoxazolꢀ3ꢀyl)urea (CHMFLꢀFLT3ꢀ213) as
a Highly Potent Type II FLT3 Kinase Inhibitor
Capable of Overcoming a Variety of FLT3
Kinase Mutants in FLT3ꢀITD Positive AML
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,2,7
1,2,7
1,3,7
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1,2,7
1,3,7
Aoli Wang , Xixiang Li , Cheng Chen , Hong Wu , Ziping Qi , Chen Hu
,
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,3
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Kailin Yu , Jiaxin Wu , Juan liu , Xiaochuan Liu , Zhenquan Hu , Wei Wang ,
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Wenliang Wang , Wenchao Wang , Li Wang , Beilei Wang , Qingwang Liu , Lili
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5*
1,2*
Li , Jian Ge , Tao Ren , Shanchun Zhang , Ruixiang Xia , Jing Liu , Qingsong
1,2,3,4*
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 230031, P. R. China
3. University of Science and Technology of China, Hefei, Anhui 230036, P. R. China
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. Precision Targeted Therapy Discovery Center, Institute of Technology Innovation,
Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, Anhui
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30088, P. R. China
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. Department of Hematology, the First Affiliated Hospital of Anhui Medical
University, Hefei, Anhui 230022, P. R. China
6
. Hefei Cosource Medicine Technology Co. LTD., 358 Ganquan Road, Hefei, Anhui
2
30031, P. R. China
7. These authors contribute equally
ABSTRACT
FLT3ꢀITD mutant has been observed in about 30% of AML patients and extensively
studied as a drug discovery target. Based on our previous study that Ibrutinib (9)
exhibited selective and moderate inhibitory activity against FLT3ꢀITD positive AML
cells, through a structureꢀguided drug design approach, we have discovered a new type II
FLT3 kinase inhibitor, compound 14 (CHMFLꢀFLT3ꢀ213), which exhibited highly
potent inhibitory effects against FLT3ꢀITD mutant and associated oncogenic mutations
(including FLT3ꢀD835Y/H/V, FLT3ꢀITDꢀD835Y/I/N/A/G/Del and FLT3ꢀITDꢀF691L).
In the cellular context 14 strongly affected FLT3ꢀITD mediated signaling pathways and
induced apoptosis by arresting cell cycle into G0/G1 phase. In the in vivo studies 14
demonstrated an acceptable bioavailability (F=19%) and significantly suppressed the
tumor growth in MV4ꢀ11 cell inoculated xenograft model (15 mg/kg/d, TGI=97%)
without exhibiting obvious toxicity. Compound 14 might be a potential drug candidate
for FLT3ꢀITD positive AML.
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INTRODUCTION
Acute myeloid leukemia (AML) is one of the most common and lethal malignancies
among children and young adults. Epidemiologic studies show that AML accounts for at
least 3% of all human cancers, causing over 250 thousand cancerꢀrelated deaths annually.
Fmsꢀlike tyrosine receptor kinase 3 (FLT3) plays an important role in normal
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hematopoiesis and leukemogenesis and is expressed in most AML blasts. Mutation in
the FLT3 gene is the most frequent genetic alteration in AML and is well known as an
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important driver mutation for the development of myeloid malignancies.
The most prevalent FLT3 mutant is the internal tandem duplication alteration
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FLT3-ITD) . Other important mutations occur in the tyrosine kinase domain (TKD),
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typically at the activation loop (AL) residue D835. These mutations are present in
approximately 30% of all AML patients and lead to a far worse prognosis. Therefore,
targeting FLT3 kinase is one of the current major efforts to fight against AML.
Over the last decade, many agents have been investigated as FLT3 inhibitors and tested
in clinical trials. Based on the binding modes, these inhibitors could be classified as type
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I or type II depending on whether the compound binds to the “DFGꢀin” active kinase
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conformation (type I) or the “DFGꢀout” inactive kinase conformation (type II). As
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shown in Figure 1, compounds 1 (Sunitinib) , 2 (Gilteritinib) , 3 (Crenolanib) and 4
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(Midostaurin) are type I inhibitors and recently compound 4 received a breakthrough
therapy designation from the FDA for newly diagnosed FLT3ꢀITD positive AML.
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Compounds 5 (Pexidartinib) , 6 (Ponatinib) , 7 (Quizartinib) and 8 (Sorafenib) are
type II inhibitors bearing FLT3 kinase activity and currently they are at different stages of
clinical evaluation for AML.
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In our previous studies, we found that BTK kinase inhibitor 9 (Ibrutinib), which has
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been approved for the treatment of mantle cell lymphoma (MCL) and chronic
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lymphocytic leukemia (CLL) , exhibited selective and moderate inhibitory activity
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against FLT3ꢀITD mutant positive AML. Based on this observation, we have
discovered a potent and selective type I FLT3 kinase inhibitor 10 (CHMFLꢀFLT3ꢀ122)
that displayed impressive in vitro and in vivo activities against FLT3ꢀITD positive
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AML. However, compound 10 could not overcome the secondary mutations in the
FLT3 kinase domain, e.g. ITDꢀF691L, which is located at the “gatekeeper” position in
FLT3 that is analogous to residues in other kinases that are hotspots for drugꢀresistance to
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tyrosine kinase inhibitors (TKIs). Therefore, we continue to explore this pharmacophore
and medicinal chemistry efforts led to the discovery of a novel type II FLT3 kinase
inhibitor compound 14 (CHMFLꢀFLT3ꢀ213), which displayed potent in vitro and in vivo
activities against FLT3ꢀITD and a variety of mutants including gatekeeper FLT3ꢀITDꢀ
F691L, FLT3ꢀITDꢀD835Y as well as FLT3ꢀD835Y/H/V mutants (Figure 2).
Figure 1. Chemical structures of representative FLT3 kinase inhibitors.
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Figure 2. Schematic illustration of discovery of compound 14.
RESULTS AND DISCUSSION
Design Rationale and StructureꢀActivity Relationship (SAR) Exploration
We first examined the binding mode of 9 with FLT3 kinase to understand the
structural resistance mechanism. The result showed that the pyrazolopyrimidine core
formed two hydrogen bonds with Cys694 and Glu692 respectively. The gatekeeper
residue Phe691 positioned in parallel with the Oꢀlinked benzene ring which formed
partial πꢀπ interaction and strengthened the binding (Figure 3A). However, when Phe691
is mutated to leucine (F691L), the beneficial πꢀπ interaction was lost and steric
interaction between the benzene ring and leucine was introduced which weakened the
binding (Figure 3B). This was confirmed by the observation that the antiproliferative
efficacy (GI ) of compound 9 significantly reduced from 0.12 ꢀM (BaF3ꢀFLT3ꢀITD
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cell) to 2.5 ꢀM (BaF3ꢀFLT3ꢀITDꢀF691L cell) (Table 1). In order to overcome this
gatekeeper mutation induced drug resistance, such steric interaction should be avoided.
This made us turn to the type II inhibitors because usually for type II inhibitors an amide
or urea bond is required at this position to form the canonical hydrogen bonds with the
amino acid residues located in the cꢀHelix and this would shift this moiety away from the
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interaction with leucine (F691L). We also noticed that the pyrazolopyrimidine could form
the hinge binding if it was flipped 180 degree, although in this case it will only form two
hydrogen bonds with Cys694 but not with Cys694 and Glu692. Hence we reasoned that
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introduction of proper type II binding elements at R position (Table 1) would be able to
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Based on this postulation, we prepared compounds 11 and 12 which bear the urea
linkage at R (Table 1). Further testing showed that 11 exhibited weak activity against
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BaF3ꢀFLT3ꢀITD cell (GI : 0.96 ꢀM) and MV4ꢀ11 cell (GI : 0.65 ꢀM). When changing
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the cyclohexane ring to benzene ring for the connection of the amide (12), it gained
potent antiproliferative efficacy against BaF3ꢀFLT3ꢀITD (GI : 0.001 ꢀM). In addition, it
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antiproliferative effect against FLT3ꢀITD positive AML cells MV4ꢀ11 (GI : 0.021 ꢀM).
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Although compound 12 still could not overcome the FLT3ꢀITDꢀF691L mutation, its
potent inhibitory activity against FLT3ꢀITD indicated that type II binding might be
achieved and this encouraged us to study in detail of this new class of compounds.
Docking of 12 into the DFGꢀout inactive conformation of FLT3 kinase (PDB ID: 4XUF)
showed that the aminopyrimidine moiety formed two hydrogen bonds with Cys694 in the
hinge binding area (Figure 3C). As expected, the newly introduced urea linkage formed
two canonical hydrogen bonds with Glu661 in the cHelix and Asp829 in the DFG motif.
The tꢀbutyl substituted isoxazole occupied the hydrophobic pocket generated by the
DFGꢀmotif shift. In this binding mode, the diphenyl ether directed to the solvent exposed
area near the hinge binding area.
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Figure 3. Design rationale of new FLT3 kinase inhibitor. (A) Docking of 9 into the
active conformation of FLT3 wt kinase (homology model built from PDB ID: 4RT7 and
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LCD). (B) Docking of 9 into the active conformation of FLT3ꢀF691L kinase (site
mutation from FLT3 wt homology model). (C) Docking of 12 into the DFGꢀout inactive
conformation of FLT3 wt kinase (PDB ID: 4XUF). (D) Docking of 12 into the active
conformation of FLT3ꢀF691L kinase (site mutation from PDB ID: 4XUF).
When the gatekeeper residue Phe691 is mutated to Leu691, the new leucine residue
was away from the urea and should not be the hindrance for binding. However, the partial
πꢀπ interaction was lost and this could partially explain why compound 12 did not display
inhibitory effect against FLT3ꢀITDꢀF691L mutant (Figure 3D). In addition, the polar
environment formed by Asp698 and Asn701 surrounded the hydrophobic nonpolar
diphenyl ether moiety in the hinge binding area, which may also disfavor this binding.
Therefore, in order to further gain activity against FLT3ꢀITDꢀF691L, we removed one
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benzene ring from the R moiety (13, Table 1), which significantly improved its activity
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GI : 0.001 ꢀM for MV4ꢀ11 cell) meanwhile kept a good selectivity window to parental
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BaF3 cell (GI : >10 ꢀM). This result indicated that our postulation of binding analysis
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was reasonable and we next further explored the SAR on the basis of this type II binding
mode.
Given the fact that R moiety (Table 1) is exposed to the solvent area and more
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hydrophilic group would be preferred, we then installed Oꢀlinked morpholine (14) into
compound 13, which led to potent inhibition activity against FLT3ꢀITD (GI s:
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0.0003ꢀM for BaF3ꢀFLT3ꢀITD and MV4ꢀ11 cells) and FLT3ꢀITDꢀF691L (GI : 0.002
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M) meanwhile kept good selectivity over parental BaF3 cell (GI : >10 ꢀM). Switching
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the connection site of the benzene ring at R from –para to –meta position (15) retained
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the activity against FLT3ꢀITD but lost about 40ꢀfold activity against FLT3ꢀITDꢀF691L.
Switching the connection site of the urea linkage at R from –para to –meta position (16)
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resulted in significant activity loss to both FLT3ꢀITD and F691L mutants. Interestingly,
changing the tertꢀbutyl group at R position that occupied the hydrophobic pocket
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generated by the DFG motif shift to a methyl group (17) caused over 150ꢀfold activity
loss against FLT3ꢀITD. Removal of the tertꢀbutyl group (18) resulted in further activity
loss. Replacement of the isoxazole with thiazole (19) retained good inhibitory activity
against FLT3ꢀITD (GI : 0.021ꢀM) but lost activity to F691L mutant (GI : 5.6 ꢀM).
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Replacement of the tertꢀbutyl isoxazole to nꢀbutyl benzene (21) also retained good
activity against FLT3ꢀITD (GI : 0.024 ꢀM) but lost activity to F691L mutant (GI : 8.7
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started to gain activity to F691L mutant (GI : 0.63 ꢀM), although it was still much less
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potent compared to 14. A larger hydrophobic moiety 1,2,3ꢀtrimethoxybenzene (23) also
retained activity to FLT3ꢀITD (GI : 0.013 ꢀM) but lost activity to F691L mutant (GI :
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.8 ꢀM). Although introduction of a methyl group at the -ortho position of urea linkage
(
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ꢀM), shifting the methyl group to ꢀmeta position (25) displayed good activity to both
FLT3ꢀITD (GI : 0.002 ꢀM) and F691L mutants (GI : 0.013 ꢀM). Elongation of the Oꢀ
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linked morpholine with one more carbon at R position (26) retained activity to FLT3ꢀ
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morpholine to piperidine (27) resulted in potent inhibition activity against FLT3ꢀITD
GI : <0.0003 ꢀM) and F691L mutants (GI : 0.023 ꢀM). Installation of the hydrophilic
(
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morpholine directly into the -para position of the benzene ring at R position (28), or via
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a carbonyl linkage (29), or a propionamide linkage (30) all retained potent activities to
FLT3ꢀITD but lost potency to F691L mutant. Attachment of hydrophilic groups such as
piperazine (31), methyl piperazine (32), ethyl piperazine (33), ethanol piperazine (34) and
ethyl piperidine (35) at R position all resulted in high potencies against both FLT3ꢀITD
1
and F691L mutants, despite that they all displayed some activities against parental BaF3
cells (GI s: 1.0ꢀ3.3 ꢀM). These results indicated that a polar hydrophilic group was
5
0
preferred to achieve both FLT3ꢀITD and gatekeeper F691L mutant activities.
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Table 1. SAR Exploration at R /R Positions by Examination of the Antiproliferation
1
2
a
Efficacies against FLT3ꢀITD/F691L Transformed BaF3 and MV4ꢀ11 Cells
10
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13
14
15
16
17
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19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
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37
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BaF3ꢀFLT3ꢀ
ITD
BaF3ꢀFLT3ꢀ
ITDꢀF691L
Parental
BaF3
MV4ꢀ11
Compd.
R1
R2
(GI : ꢀM)
50
(
GI : ꢀM)
(GI : ꢀM)
(GI : ꢀM)
5
0
50
50
9
0.12±0.005
0.96±0.072
0.001±0.0003
<0.0003
2.5±0.1
2.6±0.086
7.9±0.056
0.11±0.023
0.002±0.0009
0.082±0.005
4.6±0.28
>10
5.2±0.1
>10
0.33±0.011
0.65±0.029
0.021±0.004
0.001±0.0001
<0.0003
1
1
1
1
2
3
>10
14
15
<0.0003
>10
0.002±0.0001
0.87±0.032
>10
0.001±0.0005
0.56±0.04
1
6
6.9±0.1
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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
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5
5
5
5
5
6
1
1
7
8
0.056±0.008
0.24±0.015
0.021±0.004
0.17±0.03
>10
>10
>10
0.069±0.007
0.26±0.055
0.038±0.0068
0.26±0.015
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
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5.4±0.15
5.6±0.15
>10
19
20
6.2±0.61
>10
NH
O
NH
S
N
F
F
F
2
2
1
2
0.024±0.001
0.002±0.0006
8.7±0.21
>10
0.063±0.0006
0.002±0.0006
0.63±0.01
6.5±0.35
2
2
2
2
2
3
4
5
6
7
0.013±0.002
0.046±0.008
0.002±0.0001
<0.0003
3.8±0.11
1.4±0.058
>10
0.042±0.006
0.12±0.005
<0.0003
>10
0.013±0.001
0.77±0.0067
0.023±0.003
5.5±0.15
6.4±0.28
7.2±0.36
0.001±0.0005
0.001±0.0005
<0.0003
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9
2
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9
<0.0003
<0.0003
0.16±0.005
>10
>10
0.0014±0.0005
0.002±0.0001
0.001±0.0005
<0.0003
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12
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14
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25
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1.2±0.034
0.17±0.011
0.008±0.001
0.10±0.006
0.013±0.003
30
31
32
0.002±0.0001
<0.0003
>10
2.8±0.15
3.3±0.11
1.9±0.058
<0.0003
<0.0003
3
3
3
3
4
5
<0.0003
<0.0003
<0.0003
0.008±0.0006 2.5±0.078
0.019±0.0025 1.0±0.072
<0.0003
<0.0003
<0.0003
a
All GI values were obtained by triplet testing (±SD)
.
50
Since compound 14 displayed best potencies against FLT3ꢀITD and FLT3ꢀITDꢀ
F691L gatekeeper mutants meanwhile exhibited best selectivity to parental BaF3 cells,
we then further examined 14’s activity against a panel of FLT3 mutants transformed
BaF3 cells (Table 2). Not surprisingly, 14 displayed potent inhibitory activities against
FLT3 wt, FLT3ꢀITD associated mutants including gatekeeper F691L,
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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
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5
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D835Y/I/N/A/G/del as well as gain of function mutants including FLT3ꢀD835Y/V/H.
Compared to wellꢀestablished type II FLT3 inhibitor 7, 14 was more potent against
FLT3ꢀITDꢀF691L/D835Y. Interestingly, neither 14 nor 7 were potent to FLT3ꢀK663Q
mutant.
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. Antiproliferative Effects of Compounds 14 and 7 Against a Panel of FLT3
a
wt/mutants Transformed BaF3 Cells
Compd. 14
GI : ꢁM)
Compd. 7
Cell line
(
(GI : ꢁM)
5
0
50
Parental BaF3
＞10
＞10
BaF3ꢀTELꢀFLT3 wt
BaF3ꢀFLT3ꢀITD
<0.0003
<0.0003
<0.0003
<0.0003
BaF3ꢀFLT3ꢀITDꢀF691L
BaF3ꢀFLT3ꢀITDꢀD835Y
BaF3ꢀFLT3ꢀITDꢀD835I
BaF3ꢀFLT3ꢀITDꢀD835N
BaF3ꢀFLT3ꢀITDꢀD835G
BaF3ꢀFLT3ꢀITDꢀD835A
BaF3ꢀFLT3ꢀITDꢀD835del
BaF3ꢀFLT3ꢀD835Y
BaF3ꢀFLT3ꢀD835V
BaF3ꢀFLT3ꢀD835H
BaF3ꢀFLT3ꢀK663Q
0.002±0.0009
0.002±0.0008
0.056±0.008
0.001±0.0005
<0.0003
0.021±0.002
0.028±0.002
0.056±0.018
<0.0003
<0.0003
0.0093±0.00006
0.046±0.0032
0.012±0.003
0.003±0.0001
<0.0003
<0.0003
0.012±0.0015
0.002±0.0001
0.011±0.0005
<0.0003
3.5±0.46
0.37±0.02
a
All GI values were obtained by triple testing (±SD)
.
5
0
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Compound 14’s Binding Mode
In order to better understand compound 14’s binding mechanism, we then docked it
1
9
into FLT3 wt Xꢀray structure (PDB ID: 4XUF) (Figure 4A). The modeling results
showed that it adopted a typical type II binding mode with FLT3 kinase. The
aminopyrimidine formed two hydrogen bonds with Cys694 in the hinge binding area.
The urea moiety formed two signature type II binding hydrogen bonds with Gly661 in the
cHelix and Asp829 in the DFG motif. The tertꢀbutyl isoxazole occupied the hydrophobic
pocket generated by the DFG motif shift. In addition, the morpholine moiety that directed
into solvent exposed area formed a hydrogen bond with the Leu616 backbone carbonyl
located in the Pꢀloop after being protonated. This may explain why compound 14 was
more potent than 12 which is because the latter compound lacks such hydrogen bond. In
the F691L mutant model (site mutation from PDB ID: XUF) compound 14 adopted a
similar type II binding mode (Figure 4B). In this binding model, three hydrogen bonds
formed by the urea moiety turned the benzene ring away from this position, which
generated enough space to accommodate the Leu691 residue. This may explain why
compound 14 was still sensitive to the F691L mutant. The hydrogen bond between the
nitrogen atom in the morpholine and Leu616 played critical roles for the binding because
compounds like 31ꢀ35 that bore a similar nitrogen atom at this position all displayed
potent inhibition activity to both FLT3ꢀITD and F691L mutants. However, compounds
28ꢀ30 which lack this hydrogen bond formation capability lost activities to F691L
mutant. In addition, when a methyl group was introduced to the -ortho position of this
benzene ring (24), a hindrance interaction between Leu691 or Asp829 disfavored the
binding (Figure 4C). However, when the methyl group was introduced to the –meta
10
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14
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40
41
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1
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1
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1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
4
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position, there was enough space to accommodate it, which explained why compound 25
was still potent to both FLT3ꢀITD and F691L mutants (Figure 4D). Comparably,
compound 7 adopted a similar type II binding mode except for the hinge region, where
the sulfur atom in the imidazobenzothiazole ring forms a weak hydrogen bond with the
backbone nitrogen of Cys694. In addition, the weak π ꢀπ stacking interactions between
Tyr693 in the hinge region and the imidazobenzothiazole ring also increased the binding
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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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
affinity.
Figure 4. Binding mode examination of compound 14. (A) Docking of 14 into the DFGꢀ
out conformation of FLT3 kinase (PDB ID: 4XUF). (B) Docking of 14 into the homology
model of DFGꢀout conformation of FLT3ꢀF691L mutant (site mutation from PDB ID:
4XUF). (C) Docking of 24 into the homology model of DFGꢀout conformation of FLT3ꢀ
F691L mutant (Green and blue color represents two possible directions of methyl group
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respectively). (D) Docking of 25 into the homology model of DFGꢀout conformation of
FLT3ꢀF691L mutant.
Compound 14’s Selectivity Profiling
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
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45
46
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In order to further understand compound 14’s selectivity, we then examined its
20
kinome wide selectivity profile with KINOMEScan technology . The results showed
that 14 possessed a good selectivity (S score (1) = 0.04) in a panel of 468 kinases and
mutants at 1 ꢀM concentration (Figure 5A and supplemental Table 1). Not surprisingly,
1
4 displayed strong binding affinities against FLT3 wt and FLT3 (ITD) as well as a
number of mutants including FLT3 D835H/K663Q/R834Q. In addition, it presented
strong binding activities to CDK11, CDK7, CDK8, CDKL2, CIT, CSF1R, DDR1,
DDR2, FLT4, KIT, LCK, LOK, MKNK2, PDGFRA, PDGFRB, and RET kinases
(
percent activity remaining less than 1% at 1 ꢁM) (Figure 5B). Given the fact that
KINOMEScan is a binding assay and may not fully reflect the inhibitory activities, we
then used Invitrogen’s Z’Lyte based and ADPꢀGlo based biochemical activity assays to
further confirm these potential targets (Figure 5C). The results demonstrated that 14
inhibited FLT3 wt (IC : 91 nM), FLT3ꢀITD (IC : 9 nM), CDK11 (IC : 36 nM), CDK8
5
0
50
50
(IC : 48 nM), DDR1 (IC : 63 nM), DDR2 (IC : 61 nM), LCK (IC : 425 nM), FLT4
5
0
50
50
50
(
IC : 141 nM), PDGFRα (IC : 426 nM), PDGFRβ (IC : 606 nM), RET (IC : 32 nM),
5
0
50
50
50
KIT (IC : 83 nM) and CSF1R (IC : 154 nM) kinases, respectively. We also used ADPꢀ
50
50
Glo based biochemical activity assays to test positive compound 7 and the results showed
that 7 inhibited FLT3 wt (IC : 30.4 nM) as well as FLT3ꢀITD (IC : 16.8 nM). Moreover,
5
0
50
1
3
7’s selectivity profile has been published and the results demonstrated that 7 also
showed strong binding constants against DDR1 (Kd: 81 nM), DDR2 (Kd: 690 nM),
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1
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1
1
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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
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5
5
5
5
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FLT4 (Kd: 41 nM), PDGFRα (Kd: 11 nM), PDGFRβ (Kd: 7.7 nM), RET (Kd: 9.9 nM),
KIT (Kd: 4.8 nM), CSF1R (Kd: 12 nM) and FLT1 (Kd: 41 nM) kinases. We then used
the TEL transformed BaF3 system to further confirm the onꢀtarget activity and selectivity
against other potential offꢀtargets revealed by KINOMEScan binding assay (Table 3).
The data showed that 14 could potently inhibite PDGFRα (GI <0.0003ꢁM), PDGFRβ
0
1
2
3
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7
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9
0
1
2
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9
0
1
2
3
4
5
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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
(
GI <0.0003 ꢁM), KIT (GI = 0.001 ꢁM) as well as RET (GI50 = 0.04 ꢁM) kinase
50 50
transformed BaF3 cells. This is not surprising because cKIT, FLT3 and PDGFR kinases
all belong to the type III receptor tyrosine kinase family and the ATP binding pocket of
these kinases are highly conserved. In addition, CSF1R, DDR1/2, FLT4 and LCK kinase
transformed BaF3 cells were not potently inhibited which indicated that 14 might not
really potently inhibit those kinases in cell. In comparison, compound 7 displayed more
potent activities than compound 14 against CSF1R, DDR1, DDR2, FLT4 and LCK
kinases. Both of them potently inhibited cKIT and PDGFRα/β kinases besides FLT3ꢀITD
kinase. In addition, compound 14 was more potent against RET kinase than compound 7
(Table 3).
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57
58
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Figure 5. Kinome wide selectivity profiling of compound 14 with DiscoveRx
KINOMEScan technology. Measurements were performed at a concentration of 1 ꢁM of
the inhibitor. The affinity was defined with respect to a DMSO control. (A) Treespot
demonstration of 14’s selectivity in 468 kinase targets. (B) Other targets that
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9
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1
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2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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4
4
4
4
4
4
4
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4
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5
5
5
5
5
5
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demonstrated strong binding to 14 with a percent control number less than 1. (C) ADPꢀ
a
Glo assay and Invitrogen's biochemical characterization of 14. ( The biochemical tests
b
used ADPꢀGlo assay (Promega, Madison, WI). The biochemical tests were provided by
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
Invitrogen (Carlsbad, CA, USA). All data was obtained by triplet testing (±SD)
.
Table 3. Antiproliferative Efficacy of Compounds 14 and 7 Against a Panel of Kinase
a
Transformed BaF3 Cells
Cell line
Compd. 14 (GI : ꢁM)
Compd. 7 (GI : ꢁM)
5
0
50
Parental BaF3
＞10
＞10
BaF3ꢀTELꢀCSF1R
BaF3ꢀTELꢀDDR1
BaF3ꢀ TELꢀDDR2
BaF3ꢀTELꢀFLT4
BaF3ꢀTELꢀcKIT
BaF3ꢀTELꢀLCK
BaF3ꢀTELꢀPDGFRα
BaF3ꢀTELꢀPDGFRβ
BaF3ꢀTELꢀRET
0.29±0.026
＞10
0.024±0.003
0.12±0.02
0.11±0.02
0.27±0.06
0.017±0.001
0.4±0.06
6.4±0.23
2.0±0.056
0.001±0.0007
3.7±0.1
<0.0003
<0.0003
<0.0003
0
.0044±0.0001
0.04±0.002
0.13±0.01
a
All GI values were obtained by triple testing (±SD)
.
5
0
Compound 14’s In Cell Activity Evaluation
We next tested compound 14 against a panel of established AML cancer cell lines.
The results showed that it could selectively and strongly inhibit the proliferation of
FLT3ꢀITD positive AML cell lines such as MOLM13, MOLM14 and MV4ꢀ11 (GI s:
5
0
<0.0003 ꢀM) meanwhile exhibited good selectivity over FLT3 wt expressing cell lines
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such as U937 (GI : 4.2 ꢀM), SKMꢀ1 (GI : 8.9 ꢀM), NB4 (GI : 5.4 ꢀM), OCIꢀAMLꢀ2
5
0
50
50
(GI : 8.5 ꢀM), HLꢀ60 (GI : 1.1 ꢀM) as well as CMK (GI : 1.1 ꢀM). The well
5
0
50
50
established type II FLT3 inhibitor compound 7 also displayed similar potencies against
FLT3ꢀITD positive cell lines but for FLT3ꢀwt expressing cell lines it bore narrower
selectivity window compared to compound 14 (Table 4).
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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
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55
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57
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Table 4. Antiproliferative Effects of Compounds 14 and 7 Against a Panel of AML
a
Cancer Cell Lines
Cell line
Compd. 14 (GI : ꢁM)
Compd. 7 (GI : ꢁM)
5
0
50
MOLMꢀ13 (FLT3ꢀITD)
MOLMꢀ14 (FLT3ꢀITD)
MV4ꢀ11 (FLT3ꢀITD)
U937 (FLT3ꢀwt)
<0.0003
<0.0003
<0.0003
<0.0003
1.2±0.1
<0.0003
<0.0003
4.2±0.057
8.9±0.11
5.4±0.15
8.5±0.11
1.1±0.057
1.1±0.058
SKMꢀ1 (FLT3ꢀwt)
NB4 (FLT3ꢀwt)
1.3±0.058
7.3±0.15
>10
OCIꢀAMLꢀ2 (FLT3ꢀwt)
HLꢀ60 (FLT3ꢀwt)
CMK (FLT3ꢀwt)
0.13±0.035
0.12±0.0056
a
All GI values were obtained by triple testing (±SD)
.
50
We then examined compound 14’s effects on the FLT3 mediated signaling pathways
in MOLMꢀ13, MOLMꢀ14 and MV4ꢀ11 cells (Figure 6A). As the results demonstrated,
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compound 14 completely blocked FLT3 autoꢀphosphorylation at Tyr589/591 site at the
concentration of 1 nM, and also remarkably inhibited the downstream signaling
mediators pSTAT5 (Tyr694), pERK (Thr202/Tyr204) and pAKT (Thr308/Ser473)(EC50
less than 10 nM). In addition, the expression of cꢀMyc was also significantly down
regulated. Compound 14 could also effectively arrest cell cycle into the G0/G1 phase
starting from a concentration of 10 nM in these cells (Figure 6B). Furthermore, apparent
apoptosis was observed by examining the cleavage of PARP and caspaseꢀ3 proteins
(Figure 6C). Comparably, similar doseꢀresponsive results were observed for positive
control compound 7 (Supplemental Figure 1).
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Figure 6. Cellular effects of compound 14 in FLT3ꢀITD positive AML cell lines. (A)
Effects of 14 on FLT3 mediated signaling pathways. (B) Effects of 14 on cell cycle
progression. (C) Effects of 14 on apoptosis. All data were obtained by duplicate runs and
only one run was shown as representative.
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In Vivo PK/PD Evaluation.
We next evaluated compound 14 and 7’s PK properties in rats following intravenous
and oral administration (Table 5). Upon oral administration it showed a halfꢀlife (T ) of
1
/2
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3.61 h and a bioavailability (F) of 18.8 %, which indicated that 14 was suitable for oral
application in the following animal efficacy study. In comparison, compound 7 exhibited
much better PK profile than compound 14 regarding to the AUC, Cmax and
bioavailability.
a
Table 5. Pharmacokinetic Study of Compounds 14 and 7 on Sprague Dawley Rats
I.V.
(1 mg/mg)
P.O.
(10 mg/mg)
I.V.
(1 mg/mg)
P.O.
(10 mg/mg)
Compd. 14
Compd. 7
AUC(0ꢀt)
AUC(0ꢀt)
5
5
19.81±165.64
978.32±482.72
1928±754
1973±785
728±117
0.03±0
14467±6599
14970±6980
1142±495
8.00±3.46
3.73±0.15
4078±1539
756±284
(ng/mL·h)
(ng/mL·h)
AUC(0ꢀ∞)
ng/mL·h)
AUC(0ꢀ∞)
(ng/mL·h)
24.69±170.77 990.719±489.86
(
Cmax
ng/mL)
Cmax
(ng/mL)
839.39±111.18
0.017±0
83.50±50.90
6.66±1.15
3.61±0.46
60.82±26.87
11.56±4.45
7.22±0.12
18.8
(
Tmax (h)
T1/2 (h)
Tmax (h)
T1/2 (h)
3.34±1.22
9.09±0.72
4.38±0.24
3532±1244
561±208
5.83±1.47
/
Vz (mL/kg)
Vz (mL/kg)
CLz
(mL/h/kg)
CLz
(mL/h/kg)
2
.07±0.78
MRT(0ꢀ∞) (h)
F (%)
3.13±0.70
/
MRT(0ꢀ∞) (h)
F (%)
10.11±1.27
75.9
a
All testing data were obtained from three independent mice. (±SD)
The antitumor efficacy of compound 14 was tested in MV4ꢀ11 cell (which bears
exclusive FLT3ꢀITD mutation genes) inoculated xenograft mouse model. The results
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demonstrated that dosages of 3.75, 7.5 and 15 mg/kg/day through oral administration did
not significantly affect the mice body weight (Figure 7A). During 28 days of continuous
treatment, 14 doseꢀdependently suppressed the MV4ꢀ11 tumor progression and a dosage
of 15 mg/kg/day exhibited the TGI (tumor growth inhibition) of 97% (Figure 7BꢀD).
However, compound 7 showed better activity at a dosage of 10 mg/kg/day with 100%
TGI, which reflected its superior PK profile than compound 14. Immunohistochemistry
stain results demonstrated that 14 could dose dependently inhibit cancer cell proliferation
(Kiꢀ67 stain) and induce apoptosis (TUNEL stain) in the tumor tissues (Figure 7E). In
addition, immunoblotting analysis of the extracted tumor cells from the tumor tissues
after drug treatment showed that FLT3 mediated signaling pathways were potently
inhibited, which further confirmed that compound 14 exhibited its antiꢀtumor efficacy
through FLT3 onꢀtarget effect (Figure 7F). Since the clinically advanced FLT3 inhibitor
compound 7 is known to prevent the tumor regrowth upon stopping the treatment, we
also examined this trend for compound 14. At 25 mg/kg/day, tumors appeared to regrow
after stop of 14 treatment. In contrast, the tumors only slightly regrow after day 38 (17
days post last dose) with 50 mg/kg dose of 14. Moreover, the 100 mg/kg/day dose of 14
completely inhibited tumor regrowth in the 50 days observation (Figure 7G).
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Figure 7. Compound 14’s antitumor efficacy in MV4ꢀ11 cell (FLT3ꢀITD) inoculated
xenograft mouse model. Female nu/nu mice bearing established MV4ꢀ11 tumor
xenografts were treated with 14 at 3.75, 7.5 and 15 mg/kg/d dosage or 7 at 10 m/kg/d or
vehicle. Daily oral administration was initiated when MV4ꢀ11 tumors had reached a size
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3
of 200 to 400 mm . Each group contained 5ꢀ7 animals. Data, mean ± SEM. mpk,
mg/kg/d. (A) Body weight and (B) Tumor size measurements from MV4ꢀ11 xenograft
mice after 28 days compound administration. Initial body weight and tumor size were set
as 100%. (C) Representative photographs of tumors in each group after 0, 3.75, 7.5 and
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5 mg/kg/d 14 or 10 m/kg/d 7 treatments. (D) Comparison of the final tumor weights in
each group after 28ꢀday treatment period. Numbers in columns indicate the mean tumor
weight in each group. (∗∗∗∗) p<0.0001. (E) Representative micrographs of hematoxylin
and eosin (HE), Kiꢀ67 and TUNEL staining of tumor tissues with 14 treatment groups in
comparison with the vehicle treatment group. Note the specific nuclear staining of cells
with morphology consistent with proliferation and apoptosis (E, blue arrows). (F) Doseꢀ
responsive immunoblotting of the signaling pathway with the cells extracted from tumor
tissues of the experimental animal after the 14 and 7 treatments. (G) Tumor progression
examination after stopping compound 14 treatment.
CHEMISTRY
Compounds 14ꢀ27 were prepared as shown in Scheme 1. The synthesis started from Oꢀ
esterification between R ꢀsubstituted alcohol and methanesulfonyl chloride, which
1
generated intermediates 36a−c. Then Oꢀalkylation between 36a−c and 4ꢀ or 3ꢀ(4,4,5,5ꢀ
tetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)phenol provided intermediates 37a−d. Nucleophilic
substitution
of
3ꢀiodoꢀ1Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ4ꢀamine
with
substituted
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fluoronitrobenzene afforded intermediates 38aꢀd. Suzuki coupling between 38aꢀd and
pinacol boronic ester 37aꢀd followed by hydrogenation of the nitro group yielded
compounds 39aꢀd, which then reacted with substituted amines to afford the target
molecules.
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a
Scheme 1. Synthesis of Compounds 14ꢀ27
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a
o
Reagents and conditions: (a) methanesulfonyl chloride, DIEPA, THF, 0 Cꢀrt, overnight,
o
yield: 87.3%; (b) K CO , DMF, 70 C, 8 h, yield: 74.3ꢀ75.6%; (c) for 38a, cꢀd, K CO ,
2
3
2
3
o
o
DMF, 100 C, overnight; for 38b, K CO , DMF, 120 C, overnight, yield: 80.1ꢀ82.5%;
2
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(
d) 37aꢀd, Pd(PPh ) , K CO , 1,4ꢀdioxane/H O, 90 °C, 12 h; (e) 10 % Pd/C, H , MeOH,
3 4 2 3 2 2
rt, 4 h, twoꢀstep yield: 85.4ꢀ87.8%; (f) triphosgene, Et N, CH Cl , ꢀ10 to 25 °C, 16 h,
3
2
2
yield: 48.9ꢀ55.3%.
Similarly, Pdꢀcatalyzed Suzuki coupling between 38a and a series of substituted
pinacol boronic esters following by hydrogenation reaction generated intermediates 40aꢀ
h. Then compounds 40aꢀh or 41 reacted with 5ꢀ(tertꢀbutyl)isoxazolꢀ3ꢀamine to yield the
desired products as shown in Scheme 2. Compound 31 was prepared following the same
synthetic procedure except that the Boc group was deprotected under acidic condition as
the last step (Scheme 3). Compound 34 was obtained from nucleophilic substitution of 31
with 2ꢀiodoethanol.
a
Scheme 2. Synthesis of Compounds 11ꢀ13, 28ꢀ30, 32ꢀ33 and 35
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