Identification of Pyrrolo[2,3-d]pyrimidine-Based Derivatives as Potent and Orally Effective Fms-like Tyrosine Receptor Kinase 3 (FLT3) Inhibitors for Treating Acute Myelogenous Leukemia

Article abstract of DOI:10.1021/acs.jmedchem.9b00223

A series of pyrrolo[2,3-d]pyrimidine derivatives were prepared and optimized for cytotoxic activities against FLT3-ITD mutant cancer cells. Among them, compound 9u possessed nanomolar FLT3 inhibitory activities and subnanomolar inhibitory activities against MV4-11 and Molm-13 cells. It also showed excellent inhibitory activities in FLT3-ITD-D835V and FLT3-ITD-F691L cells which were resistant to quizartinib. Furthermore, 9u exhibited over 40-fold selectivity toward FLT3 relative to c-Kit kinase, which might reduce myelosuppression toxicity. Cellular assays demonstrated that 9u inhibited phosphorylated FLT3 and downstream signaling factors and also induced cell cycle arrest in the G₀/G₁ stage and apoptosis in MV4-11 and Molm-13 cells. Oral administration of 9u at 10 mg/kg could achieve rapid tumor extinction in the MV4-11 xenograft model and significantly inhibit the tumor growth in the MOLM-13 xenograft model with a tumor growth inhibitory rate of 96% without obvious toxicity. Additionally, 9u demonstrated high bioavailability (F = 59.5%) and suitable eliminated half-life time (T_1/2 = 2.06 h), suggesting that 9u may be a potent candidate for treating acute myelogenous leukemia.

Full text of DOI:10.1021/acs.jmedchem.9b00223

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Article
Identification of Pyrrolo[2,3-d]pyrimidine-based Derivatives as
Potent and Orally Effective Fms-like Tyrosine Receptor Kinase
3
(FLT3) Inhibitors for Treating Acute Myelogenous Leukemia
Xue Yuan, Yong Chen, Wanhua Zhang, Jun He, Lei Lei, Minghai Tang,
Jiang Liu, Muzhou Li, Caixia Dou, Tao Yang, Linyu Yang, Shengyong Yang,
Yuquan Wei, Aihua Peng, Ting Niu, Mingli Xiang, Haoyu Ye, and LiJuan Chen
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00223 • Publication Date (Web): 02 Apr 2019
Downloaded from http://pubs.acs.org on April 3, 2019
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Peng, Aihua; State Key Laboratory of Biotherapy/Collaborative
Innovation Center of Biotherapy and Cancer Center, West China Hospital
of Sichuan University
Niu, Ting; Department of Hematology and Research Laboratory of
Hematology, West China Hospital of Sichuan University
Xiang, Mingli; State Key Laboratory of Biotherapy/Collaborative
Innovation Center of Biotherapy and Cancer Center, West China Hospital
of Sichuan University
Ye, Haoyu; State Key Laboratory of Biotherapy and Cancer Center, West
China Hospital, Sichuan University, and Collaborative Innovation Center
for Biotherapy,
Chen, LiJuan; State Key Laboratory of Biotherapy, West China Medical
School
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Identification of Pyrrolo[2,3-d]pyrimidine-based Derivatives as Potent and
Orally Effective Fms-like Tyrosine Receptor Kinase 3 (FLT3) Inhibitors for
Treating Acute Myelogenous Leukemia
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a,§
a,§
b,§
a,§
a
a
Xue Yuan , Yong Chen , Wanhua Zhang , Jun He , Lei Lei , Minghai Tang ,
a
a
a
a
a
a
Jiang Liu , Muzhou Li , Caixia Dou , Tao Yang , Linyu Yang , Shengyong Yang ,
a
a
b
a
a,
Yuquan Wei , Aihua Peng , Ting Niu , Mingli Xiang , Haoyu Ye *, and Lijuan
Chena,*
a
State Key Laboratory of Biotherapy and Cancer Center, National Clinical Research
Center for Geriatrics; b Department of Hematology and Research Laboratory of
Hematology, West China Hospital of Sichuan University, Chengdu, 610041, China.
§
These authors contributed equally and should be considered as co-first authors
Abstract
A series of pyrrolo[2,3-d]pyrimidine derivatives were prepared and optimized for
cytotoxic activities against FLT3-ITD mutants cancer cells. Among them, compound
9
u possessed of nanomolar FLT3 inhibitory activity and subnanomolar inhibitory
activities against MV4-11 and Molm-13 cells. It also showed excellent inhibitory
activities in FLT3-ITD-D835V and FLT3-ITD-F691L cells which were resistant to
quizartinib. Furthermore, 9u exhibited over 40-fold selectivity toward FLT3 relative
to c-Kit kinase, which might reduce myelosuppression toxicity. Cellular assays
demonstrated that 9u inhibited phosphorylated FLT3 and downstream signaling
factors and also induced cell cycle arrest in G /G stage and apoptosis in MV4-11 and
0
1
Molm-13 cells. Oral administration of 9u at 10 mg/kg could achieve rapid tumor
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extinction in MV4-11 xenograft model and significantly inhibited the tumor growth in
MOLM-13 xenograft model with TGI of 96% without obvious toxicity. Additionally,
9
u demonstrated high bioavailability (F=59.5%) and suitable eliminated half-life time
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(T =2.06 h), suggesting that 9u may be a potent candidate for treating AML.
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Introduction
Acute myelogenous leukemia (AML) belongs to an invasive hematologic disorder
characterized by blocked differentiation and deregulated proliferation of
1, 2
hematopoietic precursor cells . Fms-like tyrosine receptor kinase 3 (FLT3) plays an
essential role in hematopoiesis and leukemogenesis. The most frequent mutations in
the FLT3 receptor gene are internal tandem duplications (FLT3-ITD), which account
for approximately 25% in AML patients. Mutations in tyrosine kinase domain
(
FLT3-TKD) occupy close to 5%, mostly at the activation loop residue D8353.
FLT3-ITD and FLT3-TKD mutations are activated, causing ligand-independent FLT3
receptor signaling transduction, and facilitating survival and proliferation of
cytokine-independent AML cell, resulting in poor prognosis, higher recurrence, and
4
-8
low overall survival rate .
Recently decades, plenty of FLT3 inhibitors have been studied and applied in clinical
trials. The first-generation FLT3 inhibitors are multiple tyrosine receptor kinase
9
10
11
inhibitors (RTKs), such as lestaurtinib , midostaurin (PKC-412) , and sorafenib ,
and could enhance anti-leukemia efficacy but lack specificity for FLT3, which may
cause off-target toxicities. Midostaurin (PKC-412) blocks FLT3 autophosphorylation
and induces growth arrest and apoptosis which has been approved by FDA for
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treating newly diagnosed AML patients12-14. Quizartinib (AC220) , as a
second-generation specific FLT3 inhibitor, is in phase III clinical trial as monotherapy
in refractory or relapsed AML patients (NCT02039726). However, the occurance of
FLT3-TKD mutations, most commonly at D835 and F691, is a mechanism of
acquired resistance to quizartinib15, 16. To overcome drug-resistance mutations, a
novel, selective, and irreversible FLT3 inhibitor, FF-10101, which is in phase I
clinical trial in US in refractory or relapsed AML patients (NCT03194685), has
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favorable therapeutic effects against AML cells with FLT3 mutations . Although the
first- and second-generation inhibitors achieve potent efficacy in AML, off-target
toxicities such as myelosuppression caused by simultaneous inhibition of FLT3 and
c-Kit18-20, and FLT3-TKD mutations remain a big challenge for treating AML. For
example, the selectivity margin of FLT3 vs c-Kit is only 10-fold, 3-fold and 20-fold
for FF-10101, quizartinib and midostaurin respectively, which is in low scope of
therapeutic window17, 21. Thereby, improvement of the selectivity of FLT3 vs c-Kit
could be a good strategy to decrease potential myelosuppression with maintance of
potent FLT3 inhibition effects.
O
N
O
O
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O
H
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HN
N
S
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H
CN
O
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O
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N
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N
H
N
H
H
H
Quizartinib (AC220)
Midostaurin (PKC-412)
FF-10101
Figure 1. Illustration of representative FLT3 inhibitors.
Qingsong Liu group made great efforts focused on the development of FLT3
inhibitors in treating AML19,
22,
23. They developed one FLT3 inhibitor
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(CHMFL-FLT3-213) using pyrazolo[3,4-d]pyrimidine as core pharmacophore using a
structure-guided drug design methodology. CHMFL-FLT3-213 potently interfered
FLT3-ITD mediated signal pathways and showed highly potent anti-tumor effect in
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FLT3-ITD positive AML . However, the low selectivity scope of FLT3 vs c-Kit
(0.9-fold) and relatively low bioavailability may affect effectiveness in clinical trial.
On the basis of their studies, we further studied the binding mode of
CHMFL-FLT3-213 with FLT3 kinase, and found that pyrazolo[3,4-d]pyrimidine of
CHMFL-FLT3-213 can be replaced by pyrrolo[2,3-d]pyrimidine. The positions of
urea-linked benzene ring and ethylmorpholine can be exchanged while the binding
capacity with FLT3 kinase can be retained. Hence, we adopted
pyrrolo[2,3-d]pyrimidine as the core skeleton and simplified functional group with
retained key functional group and tried to improve the selectivity of FLT3 vs c-Kit
and bioavailability. Numerous compounds were designed, prepared, and evaluated for
biochemical effects. Among them, 9u showed efficient inhibitory effects in
FLT3-ITD positive cancer cells and two secondary mutation transformed BaF3 cells
including FLT3-ITD-D835V and FLT3-ITD-F691L (Figure 2). 9u had higher
selectivity toward FLT3 relative to c-Kit than quizartinib and CHMFL-FLT3-213.
More importantly, 9u possessed good pharmacokinetics and favorable safety profiles.
9u could achieve complete tumor remission at low dosage without obvious observed
toxicities. All the results indicated that 9u was an excellent drug candidate for treating
AML.
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Key functional group
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N O
O
NH
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NH2
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NH2
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Replace pharmacophore
Simplify functional group
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N
3N
2
N
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N
N7
N1
NH
O
N
CHMFL-FLT3-213
Compound 9u
HN
O
Figure 2. Structural explaination of development of compound 9u.
Results and Discussions
Rational Strategy Based on Computer Aided Design
We first investigated the similarities and differences of binding mode between
compound 9a with pyrrolo[2,3-d]pyrimidine and reference compound
CHMFL-FLT3-213 with FLT3 kinase. As
illustrated
in
Figure
3A,
CHMFL-FLT3-213 formed three canonical hydrogen bonds between the urea moiety
with Glu661 and Asp829 residues. Two hydrogen bonds formed between the amino
and pyrimidine with Cys694 and Cys695, and pyrazol didn’t form hydrogen bonds
with ambient amino acid residues. The hydrophobic pocket was occupied by the
tert-butyl substituted isoxazole. When urea-linked benzene ring moiety was moved to
C-5 position and ethylmorpholine was moved to N-7 position of pyrrole, compound
9
a adopted a similar docking mode with FLT3 kinase (Figure 3B). 9a still formed
three canonical hydrogen bonds by the urea moiety with the residues of Glu661 and
Asp829. Amino of pyrimidine formed hydrogen with Glu692 and π-π stacking was
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formed between the linking phenyl with Phe691 and Phe830 residues. When
CHMFL-FLT3-213 and 9a were placed together, they adopted highly similar
alignment and were well embedded in the pocket (Figure 3C). Ispired by the
encouraging design outcomes, we continued to design and synthesize a series of
compounds and evaluate their biochemical activities in vitro and in vivo.
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Figure 3. Docking model using FLT3 kinase (PDB code: 4XUF). (A)
CHMFL-FLT3-213 was docked into the FLT3 kinase. (B) 9a was docked into the
FLT3 kinase. (C) Alignment of CHMFL-FLT3-213 and 9a into the FLT3 kinase.
In Vitro Cell Growth Inhibitory Effects and Enzymatic Inhibitory Activities
against FLT3 Kinase. The introduction of 3-amino-5-tert-butylisoxazole group to
aniline could improve the potency for FLT33, 16, 24. Thus we attempted to keep the key
functional group and compounds 9a-ae were synthesized. MV4-11 and Molm-13 cells
are both FLT3-ITD positive cells, which can reflect the antitumor activities of
compounds against FLT3 mutant cells. Thereby we primarily evaluated the inhibitory
activities of compounds 9a-ae in MV4-11 and Molm-13 cells. As expected, all the
compounds showed potent inhibitory activities against MV4-11 and Molm-13 cells
and the IC values were below 10 nM (Table 1). 9a with ethylmorpholine showed
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comparable inhibitory activity to quizartinib. 9f with extended carbon chain of
morpholine enhanced the inhibitory activity with IC50 values of 0.069 nM and
0
.193nM in MV4-11 and Molm-13 cells respectively. Introduction with large
substituents at N-7 postion such as benzyl (9o), cyclopentane (9n) and
-ethyl-4-(methylsulfonyl)piperazine (9r) significantly decreased the inhibitory
1
effects. Interestingly, compounds with short nonsubstitutive aliphatic chains, such as
9g-k, 9p, 9s-v, exhibited stronger inhibitory activities at the subnamolar level or even
the picomole level. Surprisingly, 9b without substitution at N-7 positon showed the
strongest inhibitory effect with IC < 1 pM against Molm-13. These compounds were
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further evaluated IC values against FLT3 kinase. Several compounds showed potent
5
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inhibitory activities against FLT3 with average IC50 below 10 nM and displayed
2
5
comparable effects with quizartinib (IC50 = 7.4 nM) . These results were in
accordance with our previous rational drug design and these potent compounds should
be further evaluated to obtain the optimal drug candidate.
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Table 1. IC Values of Compounds 9a-ae in MV4-11, Molm-13, and FLT3 Kinase
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and Quizartinib as Positive Control
O
N O
HN
N
H
NH2
N
N
N
R
b
IC ,
5
0
a
IC ± SD , nM
5
0
nM
Compd
9a
R
MV4-11
Molm-13
FLT3
N
O
0.329 ± 0.035
1.153 ± 0.096
20
9b
9c
H
0.043 ± 0.007
0.107 ± 0.010
< 0.001
6
Boc
0.081 ± 0.025
25
N
N
N Boc
NH
NDc
9
9
d
2.536 ± 0.193
1.751 ± 0.010
0.069 ± 0.015
4.435 ± 0.364
2.56 ± 0.513
0.193 ± 0.027
9
e
N
9
f
9
O
N
9
g
0.152 ± 0.010
0.311 ± 0.084
0.134 ± 0.016
0.014 ± 0.003
17
7
9
h
Methyl
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i
Ethyl
0.476 ± 0.057
0.100 ± 0.045
0.403 ± 0.091
0.144 ± 0.017
0.971 ± 0.121
0.736 ± 0.010
7
9
j
Isopropyl
Propyl
20
15
9
k
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
9l
n-butyl
n-hexyl
2.179 ± 0.281
5.133 ± 2.010
2.160 ± 0.063
1.425 ± 0.306
3.771 ± 0.468
1.511 ± 0.038
34
105
32
9
m
9
n
9o
1.756 ± 0.198
2.495 ± 0.225
86
9
p
q
0.876 ± 0.131
0.140 ± 0.001
1.946 ± 0.052
16
31
OH
O
N
9
1.734 ± 0.049
O
9
r
N
O
O
N S
O
14.173 ± 0.620 10.601 ± 0.110
26
9
s
0.149 ± 0.042
1.468 ± 0.022
0.089 ± 0.001
0.049 ± 0.010
0.117 ± 0.010
0.022 ± 0.003
21
37
7
9
t
9u
9v
1.753 ± 0.134
1.061 ± 0.055
38
113
30
9w
9x
10.325 ± 1.255 2.767 ± 0.116
1.746 ± 0.219
1.025 ± 0.097
9y
2.940 ± 0.282
1.788 ± 0.615
0.602 ± 0.068
2.222 ± 0.184
2.855 ± 0.016
0.262 ± 0.041
69
70
13
9
z
9aa
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5
5
5
5
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9
ab
ac
ad
3.923 ± 0.384
5.842 ± 0.221
1.145 ± 0.072
2.046 ± 0.210
26
64
9
9
N
9
0.032 ± 0.007 0.038 ± 0.010
0
1
2
3
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5
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8
9
0
1
2
3
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9
0
1
2
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8
9
0
1
2
3
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9
0
1
2
3
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7
8
9
0
9ae
0.376 ± 0.041
0.452 ± 0.177
0.520 ± 0.030
1.885 ± 0.220
6
N
quizartinib
7.4
a
IC , compound concentration required to inhibit tumor cell proliferation by 50%,
5
0
data are expressed as the mean ± SD from the dose-response curves of at least three
b
independent experiments. IC values for enzymatic inhibition of FLT3 kinase; data
5
0
are expressed from the dose-response curves of at least two independent experiments.
c
ND, not detected.
Although compound 9b showed potent anti-proliferative activity against Molm-13
with IC < 1 pM, which was more than 100-times active than quizartinib. However,
5
0
9
b showed unsatisfactory bioavailability and anti-tumor activity in vivo (data was not
shown). The bioavailability and anti-tumor activity of other two promising
compounds 9a and 9f in vivo were also unsatisfactory (data was not shown). Very
luckly, 9u with allylic substitution obtained satisfactory bioavailability and
anti-proliferation effect both in MV4-11 and Molm-13 cells (Table 1). It is well
known that introduction of key functional groups (such as halogen atom) is an
important structural modification strategy to improve metabolic stability and
2
6
bioavailability of lead compounds . We attempted to introduce F atom to C-3
position of phenyl in selected coumpounds and 13a-h were synthesized and evaluated
for inhibitory activities in MV4-11 and Molm-13 cells. The data were illustrated in
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Table 2. Although F-substitued analogues maintained potent inhibitory activities in
MV4-11 and Molm-13 cells, they decreased efficacy in comparision with
corresponding analogues without F atom. Worsemore, F-substitued analogues
decreased inhibitory activities against FLT3 kinase except for 13a with IC of 4 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
5
0
Since wild-type FLT3 is essential for the proliferation of normal primitive
2
7
hematopoietic cells , all the compounds were further evaluated the inhibitory
activities in RS4;11 (acute lymphoblastic leukemia, FLT3 wt) and HL-60
(promyelocytic leukemia cell, FLT3 wt). These compounds showed very weak
inhibitory activities against RS4;11 and HL-60, and the IC values were > 1000 nM
5
0
(Table S2 and S3), indicating that these compounds possessed of excellent selectivity
between FLT3 wt and FLT3-ITD mutants (MV4-11, Molm-13).
Table 2. IC Values of Compounds 13a-h in MV4-11, Molm-13, and FLT3 Kinase
5
0
and Quizartinib as Positive Control
O
N O
HN
F
N
H
NH2
N
N
N
R
a
b
IC ± SD , nM
IC , nM
5
0
50
Compd
R
MV4-11
Molm-13
FLT3
1
3a
H
0.131 ± 0.035
2.336 ± 0.022
0.007 ± 0.001
4
1
3b
0.650 ± 0.038
1.450 ± 0.050
17
51
13c
3.223 ± 0.067
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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
3d
3e
3f
13g
3h
quizartinib
0.434 ± 0.084
1.977 ± 0.100
1.343 ± 0.157
0.389 ± 0.047
0.580 ± 0.010
1.550 ± 0.180
0.730 ± 0.120
46
32
49
19
O
O
1
1
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
N
0.422 ± 0.057
1
2.208 ± 0.298
0.452 ± 0.177
2.330 ± 0.190
1.885 ± 0.220
22
N
7.4
a
IC , compound concentration required to inhibit tumor cell proliferation by 50%,
5
0
data are expressed as the mean ± SD from the dose-response curves of at least three
b
independent experiments. IC values for enzymatic inhibition of FLT3 kinase; data
5
0
are expressed from the dose-response curves of at least two independent experiments.
Patients with FLT3-ITD mutations which relapsed while treated with quizartinib
usually had point mutations in activation loop (most frequently at D835) or
28
“
gatekeeper” domain . F691L is a hotspot for drug resistance to FLT3 inhibitors
which situated at the “gatekeeper” location in FLT3 kinase16, 29. These secondary
mutations conferring resistance in kinase domain will pose a significant clinical
challenge, which prompted us to identify new FLT3 inhibitors against FLT3 resistant
30
variants . FLT3-ITD, FLT3-ITD-F691L and D835V transformed BaF3 cells were
established to evaluate inhibitory activities of all the compounds. On the basis of the
characteristics of inhibitory activities in MV4-11 and Molm-13 cells and IC values
50
of FLT3 kinase of the above compounds, all the compounds were further evaluated
the inhibitory activities against BaF3-FLT3-ITD cell. As shown in Table 3, all the
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compounds strongly inhibited proliferation of BaF3-FLT3-ITD with IC values at the
5
0
nanomolar level, but showed great difference between BaF3-FLT3-ITD-F691L and
BaF3-FLT3-ITD-D835V. Most compounds still exhibited potent inhibitory effects
against these two mutational cells which were resistant to quizartinib and a majority
of these compounds were below 100 nM (IC ). 9b, 9j, 9p, 9u, 9aa, 9ad, and 13a
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
showed considerable and equivalent potent effects against FLT3-ITD-F691L and
FLT3-ITD-D835V cells. Conclusion can be drawn that these compounds could
overcome two crucial point mutations which conferred resistance to quizartinib.
Considering of cytotoxicity and FLT3 kinase assay, 9b, 9j, 9p, 9u, 9aa, 9ad, and 13a
were further selected out and evaluated their bioavailability in SD rats.
Table 3. IC50 Values of Compounds 9a-ae and 13a-h against FLT3 Mutants
Transformed BaF3 Cells and Quizartinib as Positive Control
a
IC ±SD , nM
5
0
Compd
BaF3-ITD
BaF3-ITD-D835V BaF3-ITD-F691L
9
a
1.07 ± 0.05
0.16 ± 0.04
1.28 ± 0.19
8.95 ± 0.81
8.67 ± 0.32
0.99 ± 0.10
1.62 ± 0.15
1.44 ± 0.09
0.92 ± 0.13
5.30 ± 0.57
2.72 ± 0.24
52.38 ± 1.66
4.83 ± 0.25
34.81 ± 6.75
2.35 ± 0.40
8.92 ± 1.18
> 500
9
b
9
c
12.39 ± 1.26
385.4 ± 82.2
155.67 ± 9.54
61.84 ± 8.11
170.23 ± 12.40
46.25 ± 3.05
23.09 ± 2.31
12.19 ± 1.86
41.49 ± 2.69
9
d
9e
9f
> 200
32.12 ± 3.52
85.02 ± 8.00
27.92 ± 0.71
28.53 ± 3.93
12.56 ± 0.52
72.09 ± 3.96
9g
9h
9i
9
j
9
k
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1
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3
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5
5
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5
5
5
5
5
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6
9
l
5.69 ± 0.80
20.71 ± 1.98
7.25 ± 0.79
8.45 ± 0.07
1.73 ± 0.03
3.10 ± 0.47
3.59 ± 0.24
0.95 ± 0.12
3.21 ± 0.78
0.92 ± 0.01
4.92 ± 1.04
8.67 ± 0.29
5.09 ± 0.96
5.95 ± 0.18
6.06 ± 0.72
1.66 ± 0.04
6.18 ± 0.69
8.65 ± 0.02
0.97 ± 0.07
1.60 ± 0.06
0.25 ± 0.04
3.04 ± 0.26
5.41 ± 0.53
2.48 ± 0.16
5.57 ± 0.27
4.60 ± 0.33
1.27 ± 0.09
4.26 ± 0.33
4.94 ± 0.20
111.5 ± 0.2
208.7 ± 31.7
252.7 ± 27.8
90.87 ± 0.83
133.5 ± 22.3
13.63 ± 1.82
28.23 ± 4.31
85.99 ± 13.22
28.65 ± 0.70
98.6 ± 12.1
12.99 ± 0.87
91.8 ± 0.7
9
m
187.0 ± 11.9
36.80 ± 5.57
95.40 ± 6.43
14.74 ± 0.91
28.50 ± 1.86
166.5 ± 4.4
50.14 ± 1.89
173.0 ± 5.2
20.71 ± 2.32
175.6 ± 11.4
305.3 ± 31.7
93.7 ± 9.3
9
n
9
o
p
q
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
9
9
9
r
9
s
9
t
9
u
9
v
9
w
216.5 ± 6.0
57.79 ± 8.30
200.3 ± 5.7
228.1 ± 16.9
13.88 ± 0.77
51.17 ± 6.26
147.9 ± 6.8
3.61 ± 0.01
19.60 ± 1.33
10.71 ± 1.48
82.43 ± 4.92
50.25 ± 2.16
16.08 ± 0.86
39.39 ± 2.57
42.66 ± 2.76
9.84 ± 0.58
31.46 ± 1.58
> 1000
9
9
x
y
139.8 ± 10.2
185.3 ± 8.5
21.15 ± 1.09
86.43 ± 5.68
361.1 ± 21.3
9.17 ± 0.48
24.64 ± 0.44
18.01 ± 0.87
51.85 ± 5.17
84.41 ± 4.24
102.5 ± 5.7
69.70 ± 5.97
55.37 ± 4.21
86.69 ± 1.24
111.5 ± 10.9
> 1000
9z
9aa
9ab
9ac
9ad
9
ae
3a
3b
3c
3d
3e
3f
3g
3h
quizartinib
1
1
1
1
1
1
1
1
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a
IC , compound concentration required to inhibit tumor cell proliferation by 50%,
5
0
data are expressed as the mean ± SD from the dose-response curves of at least three
independent experiments.
1
1
1
1
1
1
1
1
1
1
2
2
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2
2
2
2
2
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5
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5
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5
5
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0
1
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9
0
1
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0
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0
As shown in Table 4, 13a with F-substitution showed poor pharmacokinetic (PK)
properties than 9b, suggesting that halogen contributed worse effect to PK properties.
9p with hydroxyl group significantly promoted the clearance of drug. Compound 9ad
wasn’t further explored due to its high clearance rate and low bioavailability. Among
them, compound 9u demonstrated excellent PK properties with low clearance (1.22
L/h/kg), reasonable half-life time (2.06 h), and highest oral exposure (59.5%). These
PK properties showed strong correlation with bioavailability predicted by
3
1
swissADME tool (Figure S1 and S2) .
Table 4. Oral Pharmacokinetic Study of the Selected Compounds in SD Ratsa
Parameter
Tmax (h)
T (h)
9 b
9j
9p
9u
9aa
0.83
2.78
1.70
40.0
9ad
0.44
3.7
13a
0.88
2.54
3.38
25.6
0.28
6.22
0.89
22.5
0.50
4.22
0.31
2.77
0.75
2.06
1.22
59.5
1/2
CL (L/h/kg)
F (%)
15.31 48.07
12.1 9.8
9.17
11.2
a
Dosage, 5 mg/kg po; Tmax, time for peak concentration; T , elimination half-life
1/2
period; Vss, distribution volume; CL, plasma clearance; F (%), percent oral
bioavailability.
Kinase Selectivity Profiling of Compound 9u. To further understand the selectivity
of compound 9u, we then evaluated its wide kinome selectivity profile. The results
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6
indicated that compound 9u possessed a relatively good selectivity in a panel of more
than 400 kinases and mutants at 100 nM (Table S1). The S (10) selectivity score,
which is calculated by dividing the number of nonmutant kinases which are ≤ 10% of
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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8
9
0
1
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9
0
1
2
3
4
5
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7
8
9
0
3
2
control can be determined by the total number of nonmutant kinases . From the
nonmutant kinases there were 38 hits, giving the selectivity of 0.17 (Figure 4).
Because receptor tyrosine kinase inhibitors usually target many other targets which
share homologous sequences. Thereby the inhibitory activities of 9u against hit
targets were further evaluated the IC values and the data were shown in Table S4.
5
0
Most importantly, 9u showed good selectivity of FLT3 to c-Kit, and its selectivity
range of FLT3 vs c-Kit was > 40-fold (FLT3, IC = 7 nM; c-Kit, IC > 300 nM),
5
0
50
much better than quizartinib and CHMFL-FLT3-213, indicating that 9u possessed of
better safety window. Additionally, Hck, Ret, Lyn, Lck, Mer, Met, and Tie2 kinases
have been proved as promising targets for treating leukemia33-39. Fibroblast growth
factor receptor (FGFR) is also overexpressed in leukemia cells and could be one
promising druggable target for treating leukemia40-42. 9u also strongly inhibited
PDGFRα mutants at D842V and V561D and Abl (T315I), which might overcome
imatinib-resistant mutants43, 44
.
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1
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0
1
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9
0
1
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5
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7
8
9
0
1
2
3
4
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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
Figure 4. Wide kinome selectivity profiling of compound 9u with Eurofins
KinaseProfile platform. Tests were conducted at 0.1 μM of compound 9u. Treespot
image was mapped by TREEspotTM software tool provided by DiscoverX corporation.
Clinically relevant FLT3-resisitant mutants indicated poor prognosis and the
dissociation constants (K ) of 9u against a panel of FLT3-ITD/TKD mutations were
d
evaluated by DiscoverX Kinomescan technology. As displayed in Table 5, 9u binds
with high affinity to wt FLT3 and FLT3 mutants. In particular, 9u displayed nice
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1
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1
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2
2
2
2
2
2
2
2
3
3
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3
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6
binding affinity with drug-resistance mutants such as D835 mutants. As for the
secondary ITD-TKD mutants, 9u also showed considerable binding affinity for
FLT3-ITD-D835V and FLT3-ITD-F691L mutants (K : 41.26 nM, 48.47 nM,
d
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
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
respectively). The high affinity for those mutants confering dramatical resistance to
FLT3 inhibitory monotherapy made 9u very promising for clinical applications.
Table 5. Dissociation Constants of Compound 9u against Wt and Mutant FLT3
Kinasesa
Mutant type
FLT3 wt
K , nM
d
4.37
FLT3(D835V)
FLT3(ITD)
7.04
20.52
41.26
48.47
2.37
FLT3(ITD, D835V)
FLT3(ITD, F691L)
FLT3(N841I)
FLT3(R834Q)
FLT3(D835H)
FLT3(D835Y)
FLT3(K663Q)
a
10.15
5.14
7.78
2.48
The biochemical tests were provided by DiscoverX. All the data were obtained by
duplicated test.
Molecular Docking of Compounds into FLT3 kinase. In order to further
understand the different activities of compounds 9b, 9u, and 13a against FLT3 kinase,
we performed the molecular docking into FLT3. The active pocket of FLT3 kinase is
narrow and long with a small opening at the bottom of the pocket to be communicated
with the outside (PDB code 4XUF). Docking of 9b into the FLT3 kinase (DFG-out
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inactive conformation) indicated that compound 9b was well embedded in the pocket,
and the nitrogen atom of pyrimidine formed hydrogen bond with Cys694 residue
(Figure 5C). Amino of pyrimidine formed one hydrogen bond with carbonyl of
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Glu692 residue. Additionally, the π-π stacking was formed between the linking
phenyl with Phe691 and Phe830 residues. Canonical hydrogen bonds were formed
between Glu661 and Asp829 by urea linkage. The hydrophobic pocket was occupied
by the tert-butyl substituted isoxazole. Through these actions, 9b occupied the active
pocket well and restricted conformational changes of the FLT3 active pocket, thereby
inhibiting FLT3 kinase activity. The docking pattern of compound 9u was highly
similar to 9b, with one hydrogen bond loss between N at pyrimidine with Cys694,
which led to slight decline of inhibitory activities against FLT3 and tumor cells
(Figure 5A and 5B). However, the introduction of F atom to benzene ring (13a)
blocked the hydrogen bond action between N-H of urea linkage and Glu661, which
caused slight efficacy decrease in comparison to 9b (Figure 5D).
Figure 5. (A, B): Mimetic binding mode of compound 9u (purple stick) within FLT3
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protein; (C): Mimetic binding mode of compound 9b (purple stick) within FLT3
protein; (D): Mimetic binding mode of compound 13a (purple stick) within FLT3
protein. For clarity, nitrogen, oxygen and fluorine atoms are dispalyed in blue, red,
and green, respectively.
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Cell Cycle Arrest Assays and Apoptosis Assays by Flow Cytometry. MAPK and
STAT pathways are crucial regulators of cell proliferation and their inhibition induce
4
5
cell cycle arrest in the G /G phase . Cell cycle analyses in MV4-11 and Molm-13
1
0
cells treated with compound 9u were conducted. As shown in Figure 6A, 9u strongly
induced cell cycle arrest with G /G percentage of 53% even at 1 nM compared to
1
0
vehicle in Molm-13 cell. The percentage of G /G cells populations increased from
1
0
53% to 94% after treatment with 9u at 1 nM, 3 nM, and 10 nM. As expected, similar
results were observed at the same concentration in MV4-11 cells. We next conducted
apoptosis assays to confirm that compound 9u could induce cell death. As exhibited
in Figure 6C, the total apoptosis accounted for 34% when Molm-13 cells were treated
with 9u at 10 nM. Moreover, the apoptosis effects were strengthening as the dose
increased. As expected, similar results were observed at the same concentration in
MV4-11 cells. Obviously, compound 9u blocked the AML cells in G stage, which
1
led to decreased S-phase and G -phase percentage and also induced AML cells
2
apoptosis.
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Figure 6. (A, B): Molm-13 and MV4-11 cells were incubated with compound 9u,
respectively. (C, D): Molm-13 and MV4-11 cells were incubated with compound 9u,
respectively.
Western Blot of Signaling Pathways for FLT3 Kinase. To further investigate
whether the anti-tumor activities were relevant to inhibition of FLT3 and its
downstream signal factors, we then examined the signaling proteins in Molm-13 and
MV4-11 cells treated with compound 9u by western blot and conducted densitometric
quantification by ImageJ software. As the results showed (Figure 7), compound 9u
blocked FLT3 autophosphorylation at Tyr591 at 10 nM completely, which exhibited
comparable inhibition effects with quizartinib. Notably, 9u at 3 nM abolished
phosphorylation of STAT5 at Y694, which was a direct substrate of the oncogenic
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FLT3-ITD variant. Phosphorylation ERK at T202/Y204, one key component of
MAPK signaling pathway, was obviously reduced upon treatment with compound 9u.
Similar results were obtained in MV4-11 cells when treated with compound 9u
(Figure 7).
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Figure 7. Compound 9u inhibited phosphorylation of FLT3 and downstream
signaling proteins STAT5 and ERK after treatment for 2 h.
Anti-tumor Efficacy in AML Xenograft Models. We then evaluated the anti-tumor
efficacy of compound 9u in Molm-13 and MV4-11 cells inoculated xenograft mouse
models (Table 7). As shown in Figure 8A and 8B, 9u significantly and
dose-dependent inhibited the tumor growth both in Molm-13 and MV4-11 cells
inoculated xenograft models. Oral administration of 9u at 1 mg/kg showed potent
antitumor effect and tumor growth inhibitory rate (TGI) was 77.9% in Molm-13
bearing mice model. Moreover, 9u exhibited comparable antitumor activity with
quizartinib (AC220) at dosage of 3 mg/kg. No obvious toxicity was observed during
the treatment stage in all the experiment groups. Not surprisingly, 9u also showed
excellent anti-tumor activities in MV4-11 xenograft model (Table 6 and Figure 8B).
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Treatment with 9u at 3 mg/kg achieved one-third tumor regression on day 10. Both
9
u and quizartinib treatment groups achieved rapid and complete tumor extinction on
day 8. Mice from control, 1 mg/kg, and 3 mg/kg groups were sacrificed on day 14 and
tumors were extracted out and photographed subsequently. 9u and quizartinib at 10
mg/kg groups continued to investigate the recurrence of tumor. After continuous
treatment for prolonged 4 days, both 9u and quizartinib groups were stopped
treatment on day 18. However, two groups both recurred quickly at day 24 and 26,
respectively. These results suggested that leukemic stem cells may lead to tumor
recurrence due to the complex regulatory mechanisms, such as incomplete and and
discontinuous inhibiton of FLT317, 46. We next determined to evaluate higher dosage
for delaying or preventing tumor recurrence.
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Table 6. Tumor Growth Inhibition of 9u in Vivo.
Tumor
Treatment
Tumor
models
Survivors
(day)
mass
change(
%)
Cmpd
Dosage
Schedulea
Route
(mg/kg)
9
9
9
u
u
u
QD×10
QD×10
QD×10
QD×10
QD×14
QD×14
QD×18
QD×18
1
po
po
po
po
po
po
po
po
6/6
6/6
6/6
6/6
6/6
6/6
6/6
6/6
77.9
94.5
96.0
98.2
70.5
98.2
100
3
10
3
Molm-13
quizartinib
9
9
u
u
1
3
MV4-11
9u
10
3
quizartinib
100
a
QD, per day.
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Figure 8. Anti-tumor activities of 9u in Molm-13 and MV4-11 xenograft models.
Conclusion
FLT3-ITD/TKD resistant mutants conferred poor prognosis and higher relapsed rate
after treatment with FLT3 inhibitors. Overcoming FLT3 mutants made it a promising
drug design strategy for treating AML. Combination of advantages and disadvantages
of clinical trials and applications of FLT3 inhibitors, we designed and synthesized a
series of novel FLT3 kinase inhibitors. Compound 9u possessed low nanomolar FLT3
kinase inhibitory activities and lower-dose anti-AML activities. It showed relatively
good kinases selectivity and high binding affinity to FLT3 mutants. Compound 9u
also showed potent antiproliferation activities against MV4-11 and Molm-13 cells. It
also showed excellent potent antiproliferation activities against FLT3-ITD-D835V
and F691L cells which were resistant to quizartinib. Furthermore, 9u had higher
selectivity toward FLT3 relative to c-Kit than quizartinib, which might lower
myelosuppression toxicity. Cellular assays demonstrated that 9u inhibited
phosphorylated FLT3 and downstream signaling factors and also induced cell cycle
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arrest in G /G stage and apoptosis in MV4-11 and Molm-13 cells. Compound 9u,
0
1
with high bioavailability, strongly suppressed tumor growth in Molm-13 model and
achieved rapid tumor regression at 10 mg/kg in MV4-11 model via oral
administration. All the reported results suggested that 9u may be a potent candidate
for treating AML.
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Experimental Section
Chemistry. All the chemical solvents and reagents, which were analytically pure
without further purification, were commercially available. TLC was performed on
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1
0.20 mm Silica Gel 60 F254 plates (Qingdao Haiyang Chemical, China). H NMR and
13C NMR spectra were recorded on Bruker Avance 400 spectrometer (Bruker
Company, Germany), using TMS as an internal standard. Chemical shifts were given
in ppm (parts per million). Mass spectra were recorded on Q-TOF Priemier mass
spectrometer (Micromass, Manchester, UK). The purity of each compound (> 95%)
was determined on an Waters e2695 series LC system (column, Xtimate C18, 4.6 mm
×
150 mm, 5 μm; mobile phase, methanol (70%)/H O (30%); low rate, 1.0 mL/min;
2
o
UV wavelength, 254 - 400 nM; temperature, 25 C; injection volume, 10 μL).
The general procedure to prepare compounds 9a-ae has been shown in Scheme 1 and
2
. 4-chloro-7H-pyrrolo[2,3-d]pyrimidine (1) was aminated with ammonium
hydroxide to obtain 2. Iodine was added to C-5 position of compound 2 by NIS to
obtain 3. Then N-H of 3 was protected by Ts group to give 4. By Suzuki coupling
reaction with 4-aminophenylboronic acid, 4 was converted to 5. Under strong alkali
condition, Ts group was removed from 5 to give 6. 3-amino-5-tert-butylisoxazole was
acylated by triphosgene with itself to give the key intermediate 8. N-H of compound 6
was substituted by appropriate halide to give 7a-ae. Then via ammonolysis 7a-ae
were converted to target molecules 9a-ae.
Scheme 1. Synthesis of Intermediate 8a
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O
N
O
O
N
N
O
a
H N
2
N
H
N
H
8
1
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0
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9
0
a
o
Reagent and condition: (a) Triphosgene, NEt , THF, 65 C, 5 h.
3
Scheme 2. Syntheses of Compounds 9a-aea
NH2
Cl
N
NH2
NH2
NH2
NH2
I
I
a
b
c
d
N
N
N
N
N
N
N
N
N
N
N
N
N
N
H
H
H
Ts
Ts
1
2
3
4
5
O
N O
NH2
NH2
NH
NH
NH2
NH2
NH2
e
f
g
N
N
N
8
N
N
N
N
N
N
H
R
R
6
7a-ae
9a-ae
a
o
Reagents and conditions: (a) NH ·H O, high pressure, 130 C, 4 h; (b) NIS, THF, rt,
3
2
o
1 h; (c) TsCl, DMAP, NEt , CH CN, 80 C, 2 h; (d) 4-aminophenylboronic acid,
3
3
o
(
dppf)PdCl , K CO , dioxane/EtOH/H O = 7/3/4(v/v/v), 80 C, 2 h; (e) KOH, H O, rt,
2 2 3 2 2
o
o
1 h; (f) Appropriate halide, Cs CO , 80 C, 1 h; (g) Intermediate 8, CH CN, 80 C, 1
2
3
3
h.
Scheme 3. Syntheses of Compounds 13a-ha
O
N
O
NH2
NH2
NH2
HN
F
F
F
F
N
H
NH2
NH2
NH2
NH2
NH2
I
a
b
c
d
N
N
N
N
N
8
N
N
N
N
N
N
N
N
N
N
Ts
Ts
H
R
R
4
10
11
12a-h
13a-h
a
Reagents and conditions: (a) 4-amino-3-fluorobenzeneboronic acid pinacol ester,
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