Discovery of Potent and Orally Effective Dual Janus Kinase 2/FLT3 Inhibitors for the Treatment of Acute Myelogenous Leukemia and Myeloproliferative Neoplasms

Article abstract of DOI:10.1021/acs.jmedchem.9b01348

Herein, we describe the design, synthesis, and structure-activity relationships of a series of unique 4-(1H-pyrazol-4-yl)-pyrimidin-2-amine derivatives that selectively inhibit Janus kinase 2 (JAK2) and FLT3 kinases. These screening cascades revealed that 18e was a preferred compound, with IC₅₀ values of 0.7 and 4 nM for JAK2 and FLT3, respectively. Moreover, 18e was a potent JAK2 inhibitor with 37-fold and 56-fold selectivity over JAK1 and JAK3, respectively, and possessed an excellent selectivity profile over the other 100 representative kinases. In a series of cytokine-stimulated cell-based assays, 18e exhibited a higher JAK2 selectivity over other JAK isoforms. The oral administration of 60 mg/kg of 18e could significantly inhibit tumor growth, with a tumor growth inhibition rate of 93 and 85% in MV4-11 and SET-2 xenograft models, respectively. Additionally, 18e showed an excellent bioavailability (F = 58%), a suitable half-life time (T_1/2 = 4.1 h), a satisfactory metabolic stability, and a weak CYP3A4 inhibitory activity, suggesting that 18e might be a potential drug candidate for JAK2-driven myeloproliferative neoplasms and FLT3-internal tandem duplication-driven acute myelogenous leukemia.

Full text of DOI:10.1021/acs.jmedchem.9b01348

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Article
Discovery of Potent and Orally Effective Dual JAK2/
FLT3 Inhibitors for the Treatment of Acute Myelogenous
Leukemia and Myeloproliferative Neoplasms
Tao Yang, Mengshi Hu, Wenyan Qi, Zhuang Yang, Minghai Tang, Jun He, Yong Chen,
Peng Bai, Xue Yuan, Chufeng Zhang, Kongjun Liu, Yulin Lu, Mingli Xiang, and LiJuan Chen
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01348 • Publication Date (Web): 31 Oct 2019
Downloaded from pubs.acs.org on November 3, 2019
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Discovery of Potent and Orally Effective Dual JAK2/FLT3 Inhibitors for the
Treatment of Acute Myelogenous Leukemia and Myeloproliferative Neoplasms
a,§
a,§
a,§
a,§
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Tao Yang , Mengshi Hu , Wenyan Qi , Zhuang Yang , Minghai Tang , Jun He ,
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Yong Chen , Peng Bai , Xue Yuan , Chufeng Zhang , Kongjun Liu , Yulin Lu ,
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Mingli Xiang , and Lijuan Chen *
a
State Key Laboratory of Biotherapy and Cancer Center, National Clinical Research
Center for Geriatrics, West China Hospital of Sichuan University, Chengdu, 610041,
China.
§
These authors contributed equally and should be considered as co-first authors
Abstract
Herein, we describe the design, synthesis, and structure-activity relationships of a
series of unique 4-(1H-pyrazol-4-yl)-pyrimidin-2-amine derivatives that selectively
inhibit JAK2 and FLT3 kinases. These screening cascades revealed that 18e was a
preferred compound, with IC values of 0.7 nM and 4 nM for JAK2 and FLT3,
5
0
respectively. Moreover, 18e was a potent JAK2 inhibitor with 37-fold and 56-fold
selectivity over JAK1 and JAK3, respectively, and possessed an excellent selectivity
profile over the other 100 representative kinases. In a series of cytokine-stimulated
cell-based assays, 18e exhibited a higher JAK2 selectivity than other JAK isoforms.
The oral administration of 18e at 60 mg/kg could significantly inhibit tumor growth,
with a tumor growth inhibitory rate of 93% and 85% in MV4-11 and SET-2 xenograft
models, respectively. Additionally, 18e showed an excellent bioavailability (F = 58%),
a suitable half-life time (T1/2 = 4.1 h), a satisfactory metabolic stability and a weak
CYP3A4 inhibitory activity, suggesting that 18e might be a potential drug candidate
for JAK2-driven myeloproliferative neoplasms and FLT3-ITD driven acute
myelogenous leukemia.
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Introduction
Janus kinase 2 (JAK2) is an intracellular non-receptor tyrosine kinase that belongs to
the JAK family kinases (JAK1, JAK2, JAK3, and TYK2). The JAK-signal transducer
and activator of transcription (JAK - STAT) pathway mediates signaling by cytokines,
which controls the survival, proliferation and differentiation of a variety of cells.1-4
JAK2 phosphorylation, downstream STAT phosphorylation and activation of gene
transcription ultimately result in increased proliferation, differentiation, and survival
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of erythroid and myeloid cells. Among the four JAK subtypes, JAK2 was proven to
be critical for the growth and progression of hematologic malignancies, especially for
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myeloproliferative neoplasms. Activating JAK2 fusion proteins have been also
described in chronic myelogenous leukemia (CML),7, 8 acute myelocytic leukemia
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AML), and acute lymphoblastic leukemia (ALL). Currently, a number of pan JAK
and selective JAK inhibitors, such as ruxolitinib, a JAK1/2 inhibitor, used for
myelofibrosis,11, 12 tofacitinib, a JAK1/JAK3 inhibitor used for rheumatoid arthritis,13
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and fedratinib, a JAK2/FLT3 inhibitor used for myelofibrosis in Aug 2019, have
been used clinically.
FMS-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase that plays a crucial
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role in the development of hematopoietic progenitor cells. Up to 20-30% of patients
with AML have been shown to have an activated FLT3 internal tandem duplication
(ITD) mutation, which is a high-risk factor for disease relapse.16-18 To date, several
19
small-molecule inhibitors against FLT3, such as midostaurin (PKC412, approved
for AML by the FDA),20, 21, quizartinib (AC220, approved for AML in Japan in
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2
2019) and gilteritinib (ASP2215, approved for AML by the FDA in 2018) have been
developed.23 Although positive outcomes were obtained for these FLT3 inhibitors
during clinical trials, most patients experienced rapid drug-resistant relapse, which has
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become a major challenge in treating AML.20, 24 The phosphorylation of JAK2 was
already found to increase in primary samples of AML, and in vitro studies have
shown that the negative regulators of the JAK signal are downregulated in FLT3-TKI
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FLT3 tyrosine kinase inhibitor) resistant AML cells with FLT3-ITD.25-27 These
(
discoveries indicate that targeting JAK2 may provide an opportunity to prevent the
formation of leukemic cells and eliminate FLT3 mono-inhibition, which may improve
treatment outcomes in AML patients. Pacritinib, a dual FLT3/JAK2 inhibitor, has
been used in clinical trials to treat AML (phase 1), myelofibrosis (phase 3) and
lymphoma (phase 2).28, 29 However, pacritinib is a macrocycle compound with a
moderate enzymatic activity for JAK2 and FLT3 (IC values = 23 nM and 22 nM,
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respectively) and a poor oral bioavailability (F = 10% in rats). As both JAK2 and
FLT3 play vital functions in hematologic neoplasms and the existing development is
still unable to meet medical needs, the development of better candidate drugs is
urgently needed.
Though extensive studies on the binding mode of Pacritinib with JAK2 and FLT3, we
found that it was crucial to keep the 4-substituent pyrimidine-2-amine pharmacophore
30
of pacritinib to maintain the ATP domain binding group for JAK2 (Figure 1A). For
the hydrophobic group of pacritinib, we designed pyrazol instead of phenyl to
increase the enzymatic activity for JAK2 and FLT3. Then, we introduced a series of
hydrophile groups in the benzene ring to improve the oral bioavailability. Accordingly,
we used computer aided drug design to simulate the shape of the designed compound
(named molecule A) in the JAK2 and FLT3 proteins (Figure 1B and 1C). The docking
results clearly indicated that the pyrazol and hydrophile groups formed hydrogen
bonds with the surrounding amino acid residues such as Leu932, Cys694 and Asp939,
suggesting increased protein affinity. Based on our hypothesis, a series of compounds
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were synthesized and their cell inhibition activities and enzymatic inhibition activities
were evaluated. Herein, we report the design and synthesis of
a
4-(1H-pyrazol-4-yl)-pyrimidin-2-amine derivatives as potent JAK2 and FLT3
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inhibitors. A series of structure modification led to the discovery of
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-((1-(2-Fluoro-4-((4-(1-isopropyl-1H-pyrazol-4-yl)-5-methylpyrimidin-2-yl)amino)p
henyl)piperidin-4-yl)(methyl)amino)ethan-1-ol (18e). 18e showed good cell potency,
excellent kinase activity, good pharmacokinetic profiles and favorable safety. More
importantly, 18e could significantly inhibit the tumor growth without obvious death
both in the MV4-11 and SET-2 xenograft tumor models. In this paper, we described
our efforts toward identifying potent, highly selective, and metabolically stable dual
JAK2/FLT3 inhibitors for the treatment of AML and myeloproliferative neoplasms.
Figure 1. (A). Schematic showing plans for splitting the loop of Parcritinib; (B).
Docking result of designed compound (molecule A) in the JAK2 protein (PDB code:
2
XA4); (C). Docking result of designed compound (molecule A) in the FLT3 protein
PDB code: 4RT7)
Results and Discussion
(
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In Vitro Structure−Activity Analysis. In the beginning, compounds 12a-q were
synthesized. These compounds were tested to determine their ability to inhibit the
recombinant FLT3 kinase and JAK2 kinase and antiproliferative activities against the
FLT3-ITD positive acute myeloid leukemia cell lines of MV4-11 and Molm-13 were
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evaluated. It is clear that introduction of an F atom at the C-2 position of R (12a, 12c,
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2f) resulted in significantly decreased inhibitory activities compared to the C-3
position substituted analogues (12b, 12d, 12g) in the MV4-11 and Molm-13 cells.
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However, no substitution at the C-3 position of R (12e) exhibited a slightly
improvement in antitumor activity in the MV4-11 cells compared with 12b (Table 1).
Considering that the electronegativity of fluorine atom can acidify the NH bridge
between the pyrimidine and benzene ring and increase the strength of hydrogen bond
between it and amino acid residues, there is still a substituted F at the C-3 position of
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R . We next focused on the modification of the extended substituents at the R
position of pyrazole (Table 2). High steric resistance rings such as cyclopentyl (12i
and 12j) resulted in significantly decreased activities against the MV4-11 and
Molm-13 cells. In contrast, 12k and 12l with piperidyl substituted exhibited an
improved activity in the MV4-11 but a decreased activity in the Molm-13 cells. When
1
the R was substituted with extended side chain, 12h retained a moderate activity both
1
in the MV4-11 and Molm-13 cells. Furthermore, when the R substituent was replaced
with an isopropyl group, 12m exhibited an excellent activity both in JAK kinases and
in the MV4-11 cells and a desirable selectivity for JAK1 and JAK3. Since isopropyl
1
group substituent at the R position could remain relatively high inhibitory activities
both in kinases and the MV4-11 cells, we further investigated the effect of the R2
2
substituent. Compared with 12m and 12p, when an F atom at the R position was
replaced with methyl, 12p significantly increased inhibitory activities toward the
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Molm-13 cells and possessed a relatively good selectivity for JAK2. Therefore, a
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methyl group was chosen as the R substituent. However, 12p had relatively low
inhibitory activities against MV4-11 and Molm-13 cells, more modification should be
investigated.
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Table 1. Structures and in Vitro Biological Activities of Compounds 12a-g
R5
N 4
F
N
1
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
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4
0
1
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9
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N
N
N R1
N
R4
2
H
IC50 a ± SEM (nM)
% control @100 nM
b
Substituents
R4
Compd
R1
R5
MV4-11
Molm-13
JAK1
JAK2
JAK3
FLT3
1
2a
2b
12c
2d
2e
12f
2g
n-Pr
n-Pr
n-Pr
n-Pr
i-Pr
i-Pr
i-Pr
2-F
3-F
2-F
3-F
NS c
2-F
3-F
NCH3
NCH3
8100±128
57±17
9700±700
ND
48
ND
19
ND
69
ND
14
1
490±140
>10000
410±58
540±17
ND
NCH CH
4200±860
61±24
ND
59
ND
27
ND
52
ND
15
2
3
3
1
NCH CH
2
1
NCH3
27±14
49
15
41
11
NCH CH
8300±150
27±18
ND
57
ND
27
ND
68
ND
12
2
3
3
1
NCH CH
410±46
2
a
IC50 = compound concentration required to inhibit tumor cell proliferation by 50%; data are expressed as the mean ± SEM from the
b
c
dose-response curves of at least three independent experiments. % control = kinase activity remained. NS = none substituent
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Table 2. Structures and in Vitro Biological Activities of Compounds 12h-q
R5
N
F
R2
N
0
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N
N
N R1
N
H
IC50 a ± SEM (nM)
% control @100 nM
b
Substituents
R2
Compd
R1
R5
MV4-11
Molm-13
JAK1
JAK2
JAK3
FLT3
1
2h
F
F
NCH3
110 ± 34
190 ± 45
ND
ND
ND
ND
12i
NCH3
240 ± 24
180 ± 49
> 10
68
19
32
13
1
2j
F
NCH CH
7800 ± 890
ND
ND
ND
ND
2
3
N
N
12k
F
F
NCH3
NCH3
26 ± 11
57 ± 12
940 ± 69
ND
ND
ND
ND
1
2l
850 ± 130
113
39
77
16
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12m
i-Pr
i-Pr
F
F
NCH3
31 ± 20
250 ± 56
590 ± 32
62
54
17
10
66
44
17
10
N
1
2n
36 ± 21
N
1
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1
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3
3
4
4
4
4
4
4
4
0
1
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9
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1
2o
i-Pr
F
N
26 ± 9.9
42 ± 35
100 ± 11
36
8
47
10
1
1
2p
2q
i-Pr
i-Pr
Me
Me
NCH3
120 ± 15
35
3
40
6
NCH CH
1100 ± 250
4000 ± 110
ND
ND
ND
ND
2
3
a
IC50 = compound concentration required to inhibit tumor cell proliferation by 50%; data are expressed as the mean ± SEM from the
b
dose-response curves of at least three independent experiments. % control = kinase activity remained.
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We noticed that 12o possessing piperidyl maintained a superior activity against the
MV4-11 cells and certain selectivity over JAK1 and JAK3. Therefore, we speculated
that moving N out of the piperazine ring might increase the antiproliferative activity.
We docked a newly designed compound (molecule B) into the JAK2 and FLT3
protein crystals (Figure 2), and the results suggested that the piperidine amino of
molecule B could form strong hydrogen bonds with the amino acid residues of
Asp939 and Asp829, respectively. Hence, we synthesized compounds 18a-d, and the
results were summarized in Table 3. As expected, 18b and 18d with the piperidine
amine group had good antiproliferative activities and kinase activities, suggesting that
the piperidine amine group played a vital role on the cellular activity. 18d substituted
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with a methyl group at the R position increased the MV4-11 cell activity by an order
2
of magnitude than 18b with an F atom at the R position, suggesting that the methyl
2
group was the suitable R substituent. However, we noticed that the kinase selectivity
of 18d over JAK1 and JAK3 was slightly decreased.
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2
3
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9
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Figure 2. (A). Docking result of designed compound (molecule B) in the JAK2
protein (PDB code: 2XA4); (B). Docking result of designed compound (molecule B)
in the FLT3 protein (PDB code: 4RT7)
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Table 3. Structures and in Vitro Biological Activities of Compounds 18a-d
R6
N
F
R2
N
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
2
3
4
5
6
N
N
N R1
N
H
IC50 a ± SEM (nM)
% control @100 nM
b
Substituents
R2
Compd
18a
R1
R6
MV4-11
Molm-13
JAK1
JAK2
JAK3
FLT3
F
F
N(CH3)2
N(CH3)2
4500 ± 1200
> 10
ND
ND
ND
ND
1
8b
8c
18d
i-Pr
i-Pr
i-Pr
32 ± 26
160 ± 67
5.4 ± 2.5
540 ± 28
840 ± 110
100 ± 13
57
ND
17
6
ND
5
48
ND
23
9
ND
8
1
Me
Me
CH CH OH
2 2
N(CH3)2
a
IC50 = compound concentration required to inhibit tumor cell proliferation by 50%; data are expressed as the mean ± SEM from the
b
dose-response curves of at least three independent experiments. % control = kinase activity remained.
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Hoping to improve the kinase selectivity toward the JAK family, we continued to
modify the piperidine amine structure based on 18d. At first, the hydrophilic side
8
chains (18e-g) and hydrophobic side chains (18h-j) were introduced to the R position.
1
1
1
1
1
1
1
1
1
1
2
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2
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2
2
2
2
2
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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
Obviously, when the side chain length was maintained at 3~4 atoms, 18e, 18f and 18i
had excellent activities both in the cells and kinases. Meanwhile, as the hydroxyl
group was alkylated (18k), the kinase selectivity decreased apparently, suggesting the
8
hydroxyl group played a certain role in the kinase activity. When the R substituent
was replaced by a series of heterocycle groups, such as cyclopropane (18l), furan
(18m) and morpholine (18n, 18q, 18r), these compounds almost lost kinase
selectivity for JAK2. Interestingly, compounds with hydrophilic substituents such as
piperidine (18o) and piperazine (18p) still maintained the kinase selectivity toward
JAK2 and had good activities against the MV4-11 and Molm-13 cells. Considering
that 18p had a better cellular activity and kinase selectivity than 18o, we chose 18p as
7
one of the candidates in the next study. When the methyl group at the R position was
replaced by longer chains (18q-r) or H (18s), the cellular activity decreased sharply.
To further investigate the kinase activity and selectivity, 18e, 18f, 18i and 18p were
studied to determine the IC values toward the JAK family and FLT3 (Table 5).
5
0
Meanwhile, the pharmacokinetic properties and safety the CYP3A4 activities were
also evaluated (Table 6).
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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
Table 4. Structures and in Vitro Biological Activities of Compounds 18e-s
R8
N
R7
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
N
F
N
N
N
N
N
H
IC50 a ± SEM (nM)
% control @100 nM
b
Substituents
R8
Compd
R7
MV4-11
Molm-13
72 ± 6.3
82 ± 2.4
> 10000
47 ± 8.4
24 ± 10
JAK1
25
JAK2
JAK3
36
FLT3
1
8e
8f
8g
18h
8i
8j
8k
Me
Me
Me
Me
Me
Me
Me
5.3 ± 0.89
8.3 ± 0.82
> 1000
0
5
4
7
HO
1
22
31
HO
HO
1
ND
24
ND
3
ND
9
ND
6
7.3 ± 0.60
6.4 ± 2.5
70 ± 36
1
27
4
33
7
1
210 ± 140
28 ± 3.6
26
18
9
44
6
O
1
7.3 ± 1.6
19
28
5
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1
8l
8m
8n
Me
Me
8.1 ± 3.7
31 ± 16
70 ± 15
20
23
15
10
31
36
4
6
O
1
30 ± 9.5
18 ± 5.6
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1
1
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2
2
2
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2
2
2
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3
3
3
3
4
4
4
4
4
4
4
0
1
2
3
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9
0
1
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9
0
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2
3
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8
9
0
1
2
3
4
5
6
O
1
Me
Me
Me
Et
89 ± 68
170 ± 57
50 ± 7.8
150 ± 38
20
21
24
23
23
4
37
38
44
33
5
6
5
7
N
N
1
8o
12 ± 7.8
4.3 ± 0.45
43 ± 5.9
N
1
8p
5
N
O
O
18q
11
N
N
1
8r
8s
n-Pr
210 ± 67
200 ± 97
210 ± 89
20
8
9
8
HO
1
H
550 ± 110
ND
ND
ND
ND
a
IC50 = compound concentration required to inhibit tumor cell proliferation by 50%; data are expressed as the mean ± SEM from the
b
dose-response curves of at least three independent experiments. % control = kinase activity remain.
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6
As shown in Table 5, 18e, 18f, 18i, 18p all demonstrated potent antiproliferative
activities against the MV4-11 and Molm-13 cells (IC <100 nM), potent inhibitory
5
0
activities for JAK2 and FLT3 and good selectivity over JAK1 and JAK3. Among the
four compounds, 18e had the best kinase activities toward JAK2 and FLT3 and a
suitable selectivity toward JAK1 and JAK3. Further in vivo pharmacokinetic studies
and CYP3A4 inhibition activities of the four compounds were summarized in Table 6.
As shown in Table 6, the four compounds showed no inhibition against CYP3A4 even
above 100 uM. 18e had a high volume of distribution at steady state with a terminal
half-life of 4.1 h and relatively rapid absorption (tmax = 1.5 h). In addition, 18e
exhibited the best oral bioavailability (F = 58%) among them. Based on the above
results, we chose 18e as a candidate compound for the next study.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 5. In Vitro Profiling of Preferred Compounds
Kinase IC50 a (nM)
Selectivity (fold)
Compd
JAK1
26
JAK2
JAK3 FLT3
JAK2/JAK1
JAK2/JAK3
1
8e
8f
18i
8p
0.7
1
39
23
4
9
37
47
38
40
56
23
19
34
1
47
76
2
39
15
11
1
121
3
103
a
The biochemical tests were provided by Eurofins Discovery. All data were obtained
by double testing.
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Table 6. In Vivo Profiling of Preferred Compounds
ADME Parameter a
CYP3A4 IC50
Compd
CLogP
Cmax (ng/mL)
102 ± 34
tmax (h)
1.5 ± 0.4
4.1 ± 3.6
2.3 ± 1.5
2.1 ± 0.9
T (h)
1/2
AUC (ng·h/mL)
F (%)
58
(μM)
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
0
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0
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9
0
1
2
3
4
5
6
0‑t
1
8e
8f
18i
8p
4.1 ± 1.3
7.8 ± 2.1
5.9 ± 2.3
16 ± 3.2
2552 ± 379
1510 ± 1097
304.8 ± 109
2265 ± 198
>100
>100
>100
>100
4.40
4.62
6.00
4.46
1
88.9 ± 55.3
56.5 ± 12.9
2.3 ± 0.6
43
31
1
45
a
5
mg/kg po (0.25% DMSO, 1% tween 80, saline). Cmax: peak concentration of drug tmax (h) : time to peak; T1/2 : end elimination half-life;
AUC : area under the concentration-time curve; F : bioavailability.
0
‑t
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Kinase Selectivity Profiling of 18e. To further understand the selectivity of 18e, we
then examined its kinome selectivity profile with KinaseProfile technology developed
by Eurofins Discovery. A panel of 100 related kinases was tested at a 0.1 μM
concentration of 18e (Table S1). 18e displayed strong binding affinities with JAK2
and FLT3 (Figure 3). 18e also exhibited a potent inhibitory activity against the FLT3
0
1
2
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9
0
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1
2
3
4
5
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7
8
9
0
D835Y mutation with IC values of 4nM (Table 7). In addition, 18e possessed
50
relatively good selectivity in a panel of 100 kinases at 100 nM concentration except
for CDK4/6, Lck and Lyn (Table 7). Since the pharmacologic inhibition of the FLT3
internal tandem duplication (ITD) led to the downregulation of cyclin D2 and D3,
which was followed by retinoblastoma protein (pRb) dephosphorylation and G1
31
cell-cycle arrest, the inhibition of CDK4/6 may contribute synergistically to AML
treatment.31-33 Additionally, Lck and Lyn kinases have been proved as promising
targets for treating leukemia.34, 35 Hence, we can conclude that 18e is highly selective
to JAK2 and FLT3.
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8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Kinome wide selectivity profiling of compound 18e with KinaseProfile
assay by Eurofins Discovery. Measurements were performed at a concentration of 0.1
μM of the inhibitor in duplicate. The % percent control means remaining active kinase
percentage. The affinity was defined with respect to a DMSO control. Treespot image
was mapped with TREEspotTM software tool provided by DiscoverX corporation.
Table 7. Data in Enzymatic Assays for 18e
a
Kinase
IC (nM)
50
CDK4/cyclinD3
CDK6/cyclinD3
Flt1
18
66
22
4
Flt3(D835Y)
Flt3
4
Flt4
8
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JAK1
JAK2
JAK3
Lck
26
0.7
39
27
17
0
1
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0
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0
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0
1
2
3
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8
9
0
Lyn
a
The biochemical tests were provided by Eurofins Discovery. All data were obtained
by double testing.
The JAK2 Selectivity of 18e in Cell-based Assays. Due to the complexity of the
cooperative property of JAK kinases, the cellular potency and selectivity of 18e for
each individual kinase were characterized in human peripheral blood mononuclear
cells (hPBMCs), which can be stimulated by different cytokines. For instance,
IL-2-induced STAT5 phosphorylation is mediated by JAK1 and JAK3,36
IL-6-induced STAT3 phosphorylation is considered to be triggered by JAK1 and
JAK2,37 and GM-CSF-induced STAT5 phosphorylation is dependent on JAK2.38
Accordingly, the potency and selectivity of 18e from a cellular context were assessed
in total lymphocytes in hPBMCs by flow cytometry. As shown in Table 8 and
Supplementary Figure S2, 18e inhibited the IL-6-induced phosphorylation of STAT3
and the IL-2-induced phosphorylation of STAT5 with IC values of 94.2 nM and
50
112.3 nM, respectively. Meanwhile, 18e also inhibited the phosphorylation of STAT5,
which was elicited by GM-CSF, with an IC value of 10.4 nM, and demonstrated an
5
0
acceptable selectivity over the IL-2 and IL-6 stimulated pathway.
To further determine the selectivity of 18e for the inhibition of different JAK isotypes
within cells, we used a series of cytokine-stimulated cell-based assays. The cell lines
were preincubated with 18e and treated with different JAK1 heterodimeric or JAK2
homodimeric complexes as signaling cytokines. All the results were obtained by a
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western blot and quantified by examining the grayscale intensity levels. To assess
JAK2 signaling, we stimulated TF-1 cells via IL-3 and measured the levels of
phosphorylated STAT5 (Figure S3A). In this system, 18e concentration-dependent
inhibited the phosphorylation of JAK2 with an IC value of 269.9 nM. Compared
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
with the JAK1 and JAK3 assays (IL-6 phosphorylation of STAT3 in TF-1 cells and
IL-4 phosphorylation of STAT6, respectively) (Figure S3B, C), 18e exhibited a
relatively weak ability to inhibit STAT and JAK phosphorylation. The data from these
in vitro enzyme and cell-based assays indicate that 18e inhibits JAK2 rather than
JAK1 or JAK3.
Table 8. Cellular Potency of 18e in Total Lymphocytes in Human Peripheral Blood
Mononuclear Cells
Cytokine
IL-2
JAK
JAK1/JAK3
JAK1
pSTAT
pSTAT5
pSTAT3
pSTAT5
pSTAT3
pSTAT5
pSTAT6
Cells
PBMC
PBMC
PBMC
TF-1
IC (nM)
50
112.3
94.2
IL-6
GM-CSF
IL-6
JAK2
10.4
JAK1
692.1
269.9
578.4
IL-3
JAK2
TF-1
IL-4
JAK1/JAK3
THP-1
Molecular Docking Study of 18e. To better understand how 18e interacted with
JAK2 and FLT3, molecular docking was performed. As expected, compound 18e
docked into the cavity of the JAK2 protein (PDB code: 2XA4) by independently
forming hydrogen bonds between the crucial residues Asp939 and Leu932, and a
salt-bridge interaction was found to occur between thr methylamine of piperidine and
the Asp939 residues. Similarly, a network of hydrogen bonds was found between 18e
with receptor residues such as Met665 and Asp829 in the FLT3 protein. The benzene
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ring of 18e was found to interact via cation-π stacking interactions with the Phe830
residue (Figure 4). The 50 ns molecular dynamics simulation result showed that the
complex of 18e and FLT3 achieved a balance after approximately 10 and had a
RMSD < 2 Å (Figure S1).
0
1
2
3
4
5
6
7
8
9
0
1
2
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1
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8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. (A) Molecular docking of 18e in the cavity of JAK2 protein (PDB code:
2
XA4); (B) Molecular docking of 18e in the cavity of FLT3 protein (PDB code:
4RT7)
Pharmacological Safety and in Vivo Anticancer Activity of 18e. 18e was further
tested on a panel of tumor cell lines to determine the antiproliferative effects and
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toxicity to normal cell lines (Table S2, 3), and pacritinib was selected as a positive
control. As shown in Table S2, 18e possessed potent inhibitory activities against
Ba/F3-JAK2V617F cells and SET-2 cells (over expressing JAK2V617F) with IC values
5
0
1
1
1
1
1
1
1
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1
1
2
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2
2
2
2
2
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3
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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
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8
9
0
1
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7
8
9
0
1
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9
0
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8
9
0
of 145 nM and 270 nM, respectively. In contrast, pacritinib had only IC values of
5
0
1
061.3nM for Ba/F3-JAK2V617F and 915.7 nM for SET-2 cells. Similar results were
obtained in MV4-11 cells, which are FLT3-ITD positive cells. In addition, 18e
inhibited the growth of a panel of solid tumors cells (NCl-H460, HCT116,
MDA-MB-231, and MDA-MB-453). These results are consistent with the strong in
vitro kinase activities of 18e to JAK2 and FLT3, and indicated that 18e had a potential
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extra anti-cancer activity. The normal human cell lines were not sensitive to 18e,
indicating its low toxicity to normal cells. 18e was further applied to in vitro CYP450
and hERG channel inhibitory screens to assess the potential impact of 18e on drug
-drug interactions and cardiac responsibility. 18e exhibited good safety profile with an
IC value of 28 μM against hERG and IC values over 50 μM against six common
5
0
50
CYP450 isoforms. Liver microsomal metabolism experiments revealed that
metabolism of 18e in various liver microsome was relatively stable (Table 9). Based
on the above results, 18e was further evaluated in vivo studies.
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Table 9. Biopharmaceutical Profiles of 18e
inhibition of CYP450 (IC μM)
CYP1A2
4.4
CYP2C9
CYP2C19
CYP2D6 CYP2E1
CYP3A4
5
0,
0
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78.1
74.7
>100
>100
>100
species
human
rat
dog
mice
monkey
Liver microsomal metabolism
T (min)
1
/2
2
88.8
770.0
187.3
0.0037
385.0
0.0018
1386.0
0.0005
-1
-1
Cl (mg ·ml·min )
int
0
.0024
0.0009
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Western Blot of Signaling Pathways for JAK2 and FLT3 kinase. To evaluate
whether the observed antiproliferative activity of 18e was derived from the inhibition
of JAK2 and FLT3 kinase, we then examined the signaling proteins in the two cell
lines expressing endogenous JAK2V617F (SET-2) and FLT3-ITD (MV4-11),
respectively.27, 40, 41 As shown in Figure 5 (A and B), the p-STAT5 and p-JAK2 signal
intensities decreased sharply or even vanished at 0.2 μM in MV4-11. 18e could also
down-regulate p-FLT3 in a dose-dependent manner from 0.008 to 1 μM. Similar
potencies for the inhibition of JAK2 signaling were achieved in the SET-2 cell line.
These results showed that 18e was a potent JAK2 and FLT3 dual target inhibitor.
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Figure 5. (A and B): MV4-11 and SET-2 cells were treated with 18e for 2 h at 0.008,
0
.04, 0.2, 1 μM, respectively. The cells were harvested for preparation of whole-cell
protein lysates and subsequent western blot analysis with the indicated antibodies.
Cell Cycle Analysis and Apoptosis Assays Performed by Flow Cytometry. As
JAK2 and FLT3 signaling have key roles in important functional responses such as
cell proliferation and survival.28, 42, 43 the effects of 18e on the cell cycle and apoptosis
were investigated. As shown in Figure 6A, 18e strongly induced cell cycle arrest with
a G1/G0 percentage of 85% at 100 nM compared to the vehicle in the MV4-11 cell.
We next confirmed that the phenotypic observation of cell death induced by 18e was
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due to apoptosis. As exhibited in Figure 6B, the treatment with 18e had a
dose-dependent effect on the induction of apoptosis in the MV4-11 cells. It was
evident that 18e blocked the tumor cells during G0/G1-phase progression, which
resulted in decreased S-phase and G2-phase populations and induced tumor cell
apoptosis.
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Figure 6. (A): Cell cycle effects of 18e on MV4-11 cells. Cells were treated with
increasing concentrations of 18e for 24 h, harvested, fixed, and stained with
propidium iodide prior to flow cytometric analysis. (B): Effects of 18e on the
induction of apoptosis.
In vivo Xenograft and Allograft Model Experiments. We then evaluated the
antitumor efficacy of 18e in the MV4-1 and SET-2 cell-inoculated xenograft
NOD/SCID mouse models. The administration, dosing schedules, route and results
were presented in Table 10. The oral administration of 18e at 30 and 60 mg/kg
exhibited significant antitumor effects and the tumor growth inhibitory rates (TGI)
were respective 58% and 93% in the MV4-11-bearing mice model (Figures 7A, 7B).
To further investigate the therapeutic effects of 18e on JAK2-driven malignancy in
vivo, a xenograft model using the subcutaneous injection of SET-2 cells was
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established. As expected, the 18e-treated group at 30 and 60 mg/kg administrations
significantly inhibited tumor growth with the TGI of 66% and 85%, respectively
(Figures 7D, 7E). In addition, no obvious body weight changes were observed during
the treatment period (Figures 7C, 7F).
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To investigate the effect of 18e on myeloproliferative neoplasms (MPNs), we further
established a Ba/F3-JAK2V617F allograft model by tail vein injection. This model
represented a JAK2-driven myeloproliferative disease characterized by typical
symptoms such as splenomegaly and hepatomegaly. In this model, fedratinib, a dual
JAK2 and FLT3 inhibitor, was chosen as a positive control because it was approved
for myelofibrosis by the FDA in Aug 2019 and showed much better JAK2/FLT3
inhibitory activities and bioavailability than pacritinib, The treatments started 4 days
after the cells were injected and continued for 20 consecutive days at a dose of 60
mg/kg p.o. q.d. At the end of study, the vehicle control mice exhibited splenomegaly
and hepatomegaly (∼7-fold and 1.4-fold, respectively). 18e significantly improved the
disease symptoms, with a 59.5% and 94.3% normalization of the spleen and liver
weight, respectively (Figure 7H, 7I, 7G). In comparison, fedratinib had only
respective 24.7% and 69.8% improvement of the spleen and liver weight. These
results indicate that 18e has a good therapeutic effect, suggesting that 18e has
potential clinical value in the development of myelofibrosis drug.
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Table 10. Summary of tumor growth inhibition of compound 18e.
Administrationb
Route
Tumor
model
Survivors Inhibition rate
Schedulea
Dose(mg/kg)
(day)
6/6
(%)
58
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MV4-11
QD×14
PO
PO
60
6/6
93
3
6
0
0
6/6
66
SET-2
QD×18
6/6
85
a
QD, every day
Figure 7. (A) Tumor volume changes following the treatment of 18e at doses of 30
mg/kg (po) and 60 mg/kg; (B) Tumor weight of each mouse on day 14; (C) Average
body weights for 18e-treated mice groups; (D) Tumor volume changes following the
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treatment of 18e at doses of 30 mg/kg (po) and 60 mg/kg (po) in the SET-2 xenograft
model; ( (E) Tumor weight of each mouse on day 18; (F) Average body weights for
18e-treated mice groups; (H) Inhibition of the spleen growth in the Ba/F-JAK2V617F
allograft model; (I) Spleen weight of each mouse on day 25; (G) Inhibition of the
liver growth in the Ba/F-JAK2V617F allograft model; *, P<0.05 **, P<0.01 ***,
P<0.001.
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Conclusion
Based on the progress made in clinical research by combining JAK2 inhibitors with
FLT3 inhibitors, our groups designed and synthesized a number of dual target
inhibitors for JAK2 and FLT3. We designed 4-substituent pyrimidine-2-amine
pharmacophores to append JAK2 and FLT3 inhibition. Among these compounds, 18e
was the optimal compound and had a potent inhibition activity for JAK2 and FLT3
with an IC of 0.7 nM and 4 nM, respectively. The kinome profile confirmed that 18e
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had weak inhibition on other kinases, except of JAK2 and FLT3. In addition, the
cellular assays also verified that 18e possessed a desirable selectivity toward JAK1
and JAK3. Compound 18e also exhibited potent inhibition activities for hematologic
malignancies cells, such as MV4-11, Molm-13 and SET-2. Furthermore, the western
blot confirmed that the antitumor activities were indeed on-target. Compound 18e
could also stimulate cell cycle arrest in the G /G phase and induce tumor cell
0
1
apoptosis. Most importantly, 18e possessed excellent pharmacokinetic properties in
vivo. In established MV4-11 and SET-2 xenograft models, 18e could inhibit tumor
growth, and the TGI reached 93% and 85% when 18e was orally administered at a 60
mg/kg dose. Additionally, in a model of a JAK2V617F-driven disease, 18e normalized
the splenomegaly and hepatomegaly symptoms when administered with a relatively
low dosage. We speculated that the potent antitumor activities of 18e were due to dual
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