Design and synthesis of 4-(Heterocyclic substituted amino)-1h-pyrazole-3-carboxamide derivatives and their potent activity against acute myeloid leukemia (AML)

Article abstract of DOI:10.3390/ijms20225739

Fms-like receptor tyrosine kinase 3 (FLT3) has been emerging as an attractive target for the treatment of acute myeloid leukemia (AML). By modifying the structure of FN-1501, a potent FLT3 inhibitor, 24 novel 1H-pyrazole-3-carboxamide derivatives were designed and synthesized. Compound 8t showed strong activity against FLT3 (IC₅₀: 0.089 nM) and CDK2/4 (IC₅₀: 0.719/0.770 nM), which is more efficient than FN-1501(FLT3, IC₅₀: 2.33 nM; CDK2/4, IC₅₀: 1.02/0.39 nM). Compound 8t also showed excellent inhibitory activity against a variety of FLT3 mutants (IC₅₀ < 5 nM), and potent anti-proliferative effect within the nanomolar range on acute myeloid leukemia (MV4-11, IC₅₀: 1.22 nM). In addition, compound 8t significantly inhibited the proliferation of most human cell lines of NCI60 (GI₅₀ < 1 μM for most cell lines). Taken together, these results demonstrated the potential of 8t as a novel compound for further development into a kinase inhibitor applied in cancer therapeutics.

Full text of DOI:10.3390/ijms20225739

International Journal of
Molecular Sciences
Article
Design and Synthesis of 4-(Heterocyclic Substituted
Amino)-1H-Pyrazole-3-Carboxamide Derivatives and
Their Potent Activity against Acute Myeloid
Leukemia (AML)
Yanle Zhi 1,2,3, Zhijie Wang 4, Chao Yao 4, Baoquan Li 4, Hao Heng 4, Jiongheng Cai 4, Li Xiang 2,
Yue Wang 4, Tao Lu 4,* and Shuai Lu 4,
*
1
School of Pharmacy, Henan University of Chinese Medicine, Zhengzhou 450046, China;
15311050167@stu.cpu.edu.cn
2
3
4
School of Pharmacy, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China;
16401607@stu.cpu.edu.cn
Collaborative Innovation Center for Respiratory Disease Diagnosis, Treatment & Chinese Medicine
Development of Henan Province, Henan University of Chinese Medicine, Zhengzhou 450046, China
School of Science, China Pharmaceutical University, 639 Longmian Avenue, Nanjing 211198, China;
3119050171@stu.cpu.edu.cn (Z.W.); 15211050685@stu.cpu.edu.cn (C.Y.); 15221051005@stu.cpu.edu.cn (B.L.);
dg1924026@smail.nju.edu.cn (H.H.); 3319051009@stu.cpu.edu.cn (J.C.); 1020001155@cpu.edu.cn (Y.W.)
Correspondence: lutao@cpu.edu.cn (T.L.); lu_shuai@cpu.edu.cn (S.L.); Tel.: +86-25-83271555 (T.L.);
+86-25-86185153 (S.L.)
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Received: 28 October 2019; Accepted: 13 November 2019; Published: 15 November 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Fms-like receptor tyrosine kinase 3 (FLT3) has been emerging as an attractive target for
the treatment of acute myeloid leukemia (AML). By modifying the structure of FN-1501, a potent
FLT3 inhibitor, 24 novel 1H-pyrazole-3-carboxamide derivatives were designed and synthesized.
Compound 8t showed strong activity against FLT3 (IC50: 0.089 nM) and CDK2/4 (IC50: 0.719/0.770 nM),
which is more efficient than FN-1501(FLT3, IC50: 2.33 nM; CDK2/4, IC50: 1.02/0.39 nM). Compound 8t
also showed excellent inhibitory activity against a variety of FLT3 mutants (IC50 < 5 nM), and potent
anti-proliferative effect within the nanomolar range on acute myeloid leukemia (MV4-11, IC50: 1.22
nM). In addition, compound 8t significantly inhibited the proliferation of most human cell lines of
NCI60 (GI50 < 1 µM for most cell lines). Taken together, these results demonstrated the potential of 8t
as a novel compound for further development into a kinase inhibitor applied in cancer therapeutics.
Keywords: 1H-pyrazole-3-carboxamide; AML; Cyclin-dependent kinases; fms-like receptor tyrosine
kinase 3; protein kinase inhibitors; anti-cancer
1. Introduction
Acute myeloid leukemia (AML) is a malignant hematopoietic disease, characterized by
uncontrolled proliferation of hematopoietic progenitor cells of the myeloid lineage within the bone
marrow [1]. Fms-like receptor tyrosine kinase 3 (FLT3) represents a promising target for treatment of
AML [
(ALL) [
and tend to have a negative prognostic effect [
been identified in AML patients, internal-tandem duplications (ITDs) and tyrosine kinase domain (TKD)
point mutations [ ]. Mutation of FLT3 causes ligand-independent autophosphorylation and constitutive
2
4
,
3
]. High expression of FLT3 is a common feature of AML and acute lymphoblastic leukemia
]. Furthermore, FLT3 mutations occur in approximately 30% of new diagnosed AML patients
]. Two major classes of activating FLT3 mutations have
5,6
7
activation of downstream pathways, including RAS/MEK, PI3K/AKT/mTOR, and JAK/STAT. Excessive
activation of these pathways always results in uncontrolled cell proliferation [8,9].
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www.mdpi.com/journal/ijms

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As shown in Figure 1, various small molecular FLT3 inhibitors have been developed [7,10–12].
As a first-generation FLT3 inhibitor, midostaurin was first approved by FDA (in April 2017) for the
treatment of newly diagnosed FLT3-mutant AML in combination with chemotherapy [13]. However,
other first-generation FLT3 inhibitors were characterized by low clinical efficacy and significant
toxicity. Despite initial optimism, response rates and response duration of those inhibitors were
limited in patients with relapsed or refractory AML, due to the FLT3 mutation and lack of sustained
FLT3 inhibition in vivo [14–16]. Subsequently, a series of second-generation FLT3 inhibitors were
developed and achieved significant therapeutic effects. Up to now, the second-generation FLT3
inhibitors gilteritinib (approved by FDA) and quizartinib (approved in Japan) have been used for the
treatment of relapsed or refractory AML [17]. Crenolanib also entered Phase III clinical research in
2017 (For treatment of Newly Diagnosed FLT3 Mutated AML). As shown in Table 1, although high
selectivity against FLT3 was achieved for these second-generation FLT3 inhibitors, they still inhibit
other targets that are also associated with the tumorigenesis [14,18,19]. Therefore, new agents with
potent and sustained inhibition of FLT3 and the related signal pathways were noted to be beneficial to
the suppression of tumor cell proliferation and overcoming drug resistance.
Cyclin-dependent kinases (CDKs) are a family of serine/threonine protein kinases that are known
to play a vital role in cell cycle regulation and modulating the transcription activity [20]. Cell cycle
dysregulation, resulting from aberrant mitogenic signaling and leading to uncontrolled proliferation,
is one of the hallmarks of cancer [21]. Thus, inhibitors that simultaneously block FLT3 and CDKs
could synergistically improve the response rate and duration in the treatment of AML. For instance,
FN-1501 (Figure 1) is a FLT3 and CDKs inhibitor that we have reported, showing significant anti-AML
activity [22]. In this paper, we further modified the structure of FN-1501 by optimizing the moieties
that bind to the hydrophobic zone and hydrophilic regions in FLT3, and then a series of compounds
with better FLT3/CDKs inhibitory activities were discovered.
Figure 1. Chemical structures of several Fms-like receptor tyrosine kinase 3 (FLT3) inhibitors in clinical
trials for treatment of acute myeloid leukemia (AML).

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Table 1. FLT3 tyrosine kinase inhibitors under clinical investigation.
Phases of
Development
(AML)
FLT3 Inhibitors
Selectivity
Generation
Targets
Sunitinib
(Type I)
Lestaurtinib
(Type I)
Midostaurin
(Type I)
Sorafenib
(Type II)
FN-1501
(Type I)
Quizartinib
(Type II)
Gilteritinib
(Type II)
c-KIT, KDR, PDGFR,
and FLT3
Mutant and wild-type
FLT3, JAK2, and FLT3
FLT3, FLT3-ITD and
FLT3-TKD
Non-selective
Non-selective
Non-selective
Non-selective
Non-selective
Selective
First-generation
First-generation
First-generation
First-generation
First-generation
Second-generation
Second-generation
Phase II
Phase II
Launched
Phase II
Phase I 1
RAF-1, VEGFR, PDGFR,
c-KIT and FLT3
FLT3, CDKs
PDGFR, c-KIT, FLT3,
CSF-1R and RET
Launched (In
Japan)
Selective
FLT3 and AXL
Launched
Crenolanib
(Type II)
Selective
Second-generation FLT3 and PDGFR α/β
Phase III
1
Currently, under Phase I clinical trial for the advanced solid tumor in USA.
2. Results and Discussion
2.1. Chemistry
Coupling 4-nitropyrazole-3-carboxylic acid with a series of amine, followed by the reduction
reaction, generated the intermediates 2a 2c. Compounds 3a 3c were then obtained by the substitution
of 2a 2c with 4-chloro-7H-pyrrolo[2,3-d]pyrimidine (Scheme 1). Boc group was removed from 3c to
produce 3d in the last step.
–
–
–
Scheme 1. Synthesis of compounds 3a–3d.
–8t were prepared as shown in Scheme 2. Intermediates 4a and 4b were prepared
Compounds 8a
by coupling p-nitrobenzoic acid with the corresponding amines. Nucleophilic substitution of
the corresponding amines with 5-fluoro-2-nitropyridine or 1-fluoro-4-nitrobenzene afforded the
intermediates 4c
Intermediates 5a
reduction reaction, to yield the intermediate products 7a
reacted with the appropriate chlorides to yield the desired products 8a
–
–
4h. Then hydrogenation of the nitro group of 4a
5h were reacted with 4-nitro-1H-pyrazole-3-carbonyl to give 6a
7h. Intermediates 7a
8g and 8r. Intermediates 7c
–
4h yielded intermediates 5a
6h, followed by the
7b and 7e 7h were
–5h.
–
–
–
–
–
and 7d were reacted with the appropriate chlorides under the appropriate temperature (initially 50
◦C), and the Boc group were then removed by increasing the temperature to 70 ◦C when 7c and 7d
vanished (TLC detection), to yield the desired products 8h–8q, 8s, and 8t (Scheme 2).

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Scheme 2. Synthesis of compounds 8a–8t.
2.2. Structure-Activity Relationship Study
The enzymatic inhibitory activities of the target compounds were evaluated by CDK2, CDK4, and
FLT3 kinase activity assays, and the cell-growth inhibitory potency against AML cell line MV4-11 were
further evaluated for selected compounds (8a–8t). The results were summarized in Tables 2–4.
Table 2. Structures and biological activities of compounds 3a–3d.
H
N
N
N
N
R1
N
N
H
NH
H
O
IC50 (nM) 1
CDK4
R 1
Cpd.
CDK2
FLT3
FN-1501
2.33 ± 0.02
1.02 ± 0.16
0.39 ± 0.07
3a
0.20 ± 0.01
4.31 ± 0.91
34.13 ± 0.94
54.24 ± 1.26
5.10 ± 0.46
5.83 ± 0.74
3b
3c
32.81 ± 1.34
87.07 ± 1.26
88.76 ± 1.06
3d
63.21 ± 0.91
77.37 ± 1.10
74.30 ± 1.21
1
In the presence of 10 µM ATP, the values are the mean ± SD from three independent experiments.

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Table 3. Structures and biological activities of compounds 8a–8j.
Structure
R 2
IC50 (nM) 1
CDK4
IC50 (nM) 2
Cpd.
R 1
A
CDK2
FLT3
MV4-11
FN-1501
8a
H
H
NH
2.33 ± 0.02
1.02 ± 0.16
0.39 ± 0.07
9 ± 0.27
S
S
10.39 ± 0.41
8.42 ± 0.52
32.99 ± 0.94
17.81 ± 0.89
19.18 ± 0.18
33.10 ± 0. 17
35.21 ± 0.83
8b
H
30.14 ± 0.99
8c
H
H
S
3.51 ± 0.19
2.41 ± 0.21
5.32 ± 0.31
0.176 ± 0.09
0.262 ± 0.01
4.28 ± 0.35
9.5 ± 0.01
8d
NH
2.32 ± 0.014
8e
8f
H
H
NH
S
5.49 ± 0.42
31.7 ± 0.55
3.74 ± 0.16
0.282 ± 0.013
51.035 ± 0.88
67.28 ± 1.09
10.605 ± 0.24
1.19 ± 0.09
2.71 ± 0.31
8.07 ± 0.21
38.3 ± 1.21
54.15 ± 1.73
16.02 ± 0.43
7.3 ± 0.33
8g
8h
H
H
NH
NH
1.945 ± 0.013
0.038 ± 0.001
8i
8j
H
H
S
24.53 ± 0.57
9.64 ± 0.46
9.165 ± 0.33
17.79 ± 0.82
3.24 ± 0.14
2.81 ± 0.11
27.21 ± 0.43
21.35 ± 0.56
NH
1
In the presence of 10
µ
M ATP, the highest test concentration is 1
µ
M. The values are the mean
±
SD from three
SD from three
2
independent experiments. The highest test concentration is 1
independent experiments.
µ
M, the values are the mean
±

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Table 4. Structures and biological activities of compounds 8k–8t.
Structure
A
IC50 (nM) 1
CDK4
IC50 (nM) 2
Cpd.
R 1
R 2
CDK2
FLT3
MV4-11
8k
8l
N
N
N
315.21 ± 2.30
22.3 ± 0.92
23.11 ± 0.55
17.53 ± 0.88
6.03 ± 0.16
51.09 ± 1.34
156.54 ± 3.22
133.37 ± 1.74
13.83 ± 0.37
23.69 ± 0.65
133.50 ± 1.64
143.50 ± 1.21
8m
8n
8o
N
N
98.72 ± 1.33
109.21 ± 1.01
4.85 ± 0.20
1.88 ± 0.09
8.28 ± 0.12
19.92 ± 0.74
45.44 ± 1.07
1.81 ± 0.023
8p
8q
8r
8s
8t
N
86.36 ± 1.36
3.81 ± 0.26
123.37 ± 1.09
24.95 ± 0.29
4.36 ± 0.19
7.45 ± 0.19
20.23 ± 0.35
3.80 ± 0.10
27.04 ± 0. 31
458.32 ± 9.20
3.28 ± 0.19
9.13 ± 0.11
1.22 ± 0.06
N
508.94
±
10.33
CH
CH
CH
9.29 ± 0.64
5.43 ± 0.41
0.82 ± 0.003
0.089 ± 0.013
0.719 ± 0.064
0.770 ± 0.007
1
In the presence of 10
µ
M ATP, the highest test concentration is 1
µ
M. The values are the mean
±
SD from three
SD from three
2
independent experiments. The highest test concentration is 1
independent experiments.
µ
M. The values are the mean
±

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As shown in Table 2, compounds 3a and 3b were synthesized to evaluate if the hydrophilic
groups were necessary for the inhibitory activity against CDK2/4 and FLT3. Their activities against
CDK4 largely decreased compared with FN-1501, which confirmed that the hydrophilic group was
important for binding to CDK4. Furthermore, replacement of benzene and pyridine rings (3a and 3b
)
with piperidine (3c 3d) caused reduction of their activity against CDK2/4 and FLT3. This suggested
,
that both hydrophilic group and aromatic-ring structure were necessary for the compounds to inhibit
CDK2/4 and FLT3.
In order to find out the optimal groups in the hydrophilic region of ATP-binding site, a series
of substitutions were introduced to the benzene ring (8a–8j, as shown in Table 3). A decrease of
kinase inhibitory activity was observed when fixing the N-methylpiperazine or morpholine to benzene
by carbonyl group (8a and 8b) compared with FN-1501. Bulkier groups (such as homopiperazine),
when directly connected to the benzene ring, generally had no obvious influence on the activities of
compunds 8c and 8d against CDK2/CDK4 and FLT3. Changing the N-methylhomopiperazine (8c and
8d) to meta-position (8e and 8f) in the benzene ring caused an obvious decrease in the inhibitory
activities against CDK4 and the antiproliferative activity against MV4-11. Similarly, replacement
of homopiperazine (8d) with morpholine (8g) caused a 10-fold decrease of IC50 value against FLT3
compared with FN-1501. However, replacement of NH by S in the hydrophobic ring structure has
little effect on their kinase inhibitory activity. Moreover, compound 8h with a single piperazine
group showed improved inhibitory effects against CDK2 and FLT3, probably benefiting from the
extra intermolecular interaction between the secondary amine of piperazine with CDK2 (GLU8)/FLT3
(ASN701) (Figure 2A,B). Compound 8h also exhibited high inhibitory activity against MV4-11 (IC50:
7.3 nM). However, the derivatives 8i and 8j consisted of the bridged-piperazine rings displayed weaker
inhibitory effects against CDK2/4 and FLT3, when compared with compound 8h. Overall, a piperazine
ring that directly attaches to the para position of the benzene ring is beneficial in elevating the inhibitory
activity against CDK2/4 and FLT3.
Figure 2. Binding mode analysis of compound 8h bound to cyclin-dependent kinase 2 (CDK2) (PDB
code 2VU3) and FLT3 (homology model). Stick model of 8h in CDK2 (A) and FLT3 (B); Surface model
of 8h in CDK2 (C) and FLT3 (D), Red circle: the deep hydrophobic pocket
We next turned our attention to the aromatic linker that connected the piperazine ring and the
structure bound in the hinge area. As shown in Table 4, replacement of the benzene ring with 2-pyridine
(8k, 8l) significantly decreased their inhibitory activities against CDK2, and attenuated the inhibitory
activities against CDK4 and FLT3 moderately. Molecular modeling showed that an unoccupied space
existed in the deep hydrophobic pocket (Figure 2C,D), suggesting that the additional hydrophobic
substitution was beneficial to the inhibitory activity against CDK2/4 and FLT3. Therefore, we retained

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the pyridine ring as aromatic linker and introduced different groups in the deep hydrophobic pocket.
When the five-member ring in the bicycle system was replaced by benzene ring (8m), the kinase
inhibitory activities were basically maintained compared with 8l. Further alteration with the saturated
five-member ring (8q), led to overall reduction in kinase and cell inhibitory activity. However,
compounds 8n, 8o, and 8p, which contained the additional bulkier hydrophobic groups in different
size in the deep hydrophobic pocket, showed improved inhibitory activities against CDK4 and FLT3
compared with 8k. Unfortunately, these compounds exhibited weaker anti-proliferative effects on
MV4-11 cells (IC50 value: 0.045–0.35 µM). It was presumed that the introduction of pyridine ring may
reduce the permeability of the entire molecule, and thus decrease their activity to MV4-11. Therefore,
benzene ring was reserved as the aromatic linker.Combining the bulkier hydrophobic substituents
in deep pocket and the benzene as aromatic linker, compounds 8r–8t were designed (as shown in
Table 4). Generally, compounds 8r and 8s showed enhanced CDK2/4 and FLT3 inhibitory activities
as well as the antiproliferative potency against MV4-11 cells, compared with compound 8n. As the
optimal compound, 8t exhibited the sub-nanomolar IC50 values against CDK2 (0.719 nM), CDK4
(0.770 nM), and FLT3 (0.0890 nM), and consistently strong anti-proliferative activity in MV4-11 cells
(IC50: 1.22 nM). Thus, the step-by-step structural optimization demonstrated that the combination of
piperazine in the hydrophilic pocket, benzene ring as the aromatic linker, and the bulkier fused ring
in the deep hydrophobic pocket were beneficial for kinase inhibitory activity and anti-proliferative
activity to MV4-11.
2.3. Molecular Modeling of Compound 8t with CDK2 and FLT3
Compound 8t showed optimal FLT3/CDK2/CDK4 inhibitory activities. Hence, the binding mode
of compound 8t with CDK2 and FLT3 were elucidated using a docking model. Since 8t is a type I
FLT3 inhibitor, we used our homology model structure of “DFG-in” FLT3 [22]. As shown in Figure 3,
compound 8t binded to the ATP-binding site of CDK2 and FLT3 in an orientation similar to FN-1501 [22].
The pyrazole-3-carboxamide skeleton of compound 8t formed three conserved hydrogen bonds with
the hinge region of CDK2 and FLT3 respectively (Figure 3). The aromatic heterocycle moiety occupied
the hydrophobic pocket and the piperazine group extended to solvent accessible area. The difference
was that the NH of piperazine formed a hydrogen bond with GLU85 in CDK2 and it also formed
a hydrogen bond with ASN701 in FLT3. The cyclopentane extended to ribose zones which were
not occupied by FN-1501. This binding mode was beneficial to improving the inhibitory activity of
8t against CDK2 and FLT3. In general, the docking results further confirmed the rationality of our
design strategy.
Figure 3. Binding mode analysis of compounds 8t in CDK2 (
(B, homology model).
A, PDB code 2VU3) and FLT3

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2.4. Kinase Profiling
In order to investigate the kinase profile vulnerable to compound 8t, its enzymatic inhibitory effects
against 32 kinases were tested (Table S1), which were the representative kinase drug targets. Among
those kinases (Table 5), compound 8t showed significant inhibitory activities against CDKs and FLT3,
except for CDK1 that is considered not reasonable as an anti-tumor target [20]. In addition, compound
8
t exhibited inhibitory activities against KDR/VEGFR2, ERK7, FLT1, FLT4, and GSK3β (Table 5),
which were related to the tumorigenesis. These data indicated that compound 8t is a highly potent
pan-kinase inhibitor with the prominent inhibitory potency against CDKs and FLT3. Furthermore,
compound 8t potently inhibited eight FLT3 mutants (IC50 values less than 5 nM) that are correlated
to the drug resistance [23,24]. Compound 8t also showed significant inhibitory activity against the
FLT3 (ITD)-F691L mutation (IC50: 0.6 nM), which led to the drug resistance to FLT3 inhibitors, such as
quizartinib. Accordingly, in the BaF3 cells that are transformed with FLT3-ITD-F691L, compound 8t
showed the improved antiproliferative activity over quizartinib (Table S2).
Table 5. Inhibition of compound 8t against CDKs, FLT3s, and other kinases.
IC50 (nM)
Kinase
Compound 8t
CDK1/cyclin B
CDK2/cyclin A
CDK3/cyclin E
CDK4/cyclin D1
CDK5/p35
CDK6/cyclin D1
CDK7/cyclin H
CDK9/cyclin K
ERK7/MAPK15
FLT1/VEGFR1
138.03 ± 1.24
2.56 ± 0.31
6.88 ± 0.25
0.78 ± 0.04
9.64 ± 0.81
0.59 ± 0.09
21.31 ± 1.01
14.20 ± 0.82
9.57 ± 0.23
12.41 ± 0.19
0.035 ± 0.01
0.75 ± 0.04
0.63 ± 0.09
1.38 ± 0.11
4.32 ± 0.23
0.94 ± 0.09
1.59 ± 0.14
0.66 ± 0.08
0.60 ± 0.01
4.16 ± 0.19
11.99 ± 1.20
8.32 ± 0.54
FLT3
FLT3 (D835Y)
FLT3 (F594_R595insR)
FLT3 (F594_R595insREY)
FLT3 (ITD)-NPOS
FLT3 (ITD)-W51
FLT3 (R595_E596insEY)
FLT3 (Y591-V592insVDFREYEYD)
FLT3 (ITD)-F691L
FLT4/VEGFR3
GSK3β
KDR/VEGFR2
2.5. In vitro Cell Assays
With these findings, we submitted compound 8t to National Cancer Institute (NCI) to evaluate
their antitumor efficacy against 60 human cancer cell lines. As shown in Table 6, compound 8t
exhibited anti-proliferative activities against a variety of cancer cell lines, which was consistent with
its multi-kinase inhibition potency, indicating that 8t has the potential of further development as a
powerful anti-tumor agent for various human cancers, including AML.

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Table 6. In vitro antiproliferative activity of compound 8t from NCI60 screening.
Panel
Cell Line
GI50 (µM)
Panel
Cell Line
GI50 (µM)
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
0.22
1.15
0.12
0.08
0.89
0.06
0.14
0.06
0.37
0.04
0.32
0.26
0.14
0.06
0.21
0.12
1.20
0.11
0.22
0.26
0.12
0.17
0.72
0.27
0.39
0.24
0.05
0.11
0.18
0.30
LOX IMVI
MALME-3M
M14
0.20
0.24
0.12
0.11
1.16
0.25
1.13
0.48
1.09
0.12
0.23
0.22
0.27
0.36
1.48
0.26
0.36
0.14
0.16
0.08
0.39
0.45
0.47
0.40
0.16
1.93
0.10
1.02
0.59
0.42
Leukemia
MDA-MB-435
SK-MEL-2
SK-MEL-5
SK-MEL-28
UACC-257
UACC-62
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
786-0
Melanoma
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
SW-620
KM12
SF-268
SF-295
SF-539
Non-Small Cell
Lung Cancer
Ovarian
Cancer
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
MCF7
Colon Cancer
Renal Cancer
Breast Cancer
CNS Cancer
SNB-19
SNB-75
U251
PC-3
DU-145
MDA-MB-231
HS 578T
BT-549
T-47D
MDA-MB-468
Prostate Cancer
2.6. Cellular Mode of Action
To characterize the mode of cellular effects induced by compound 8t, flow cytometry was
performed in MV4-11 cell line. Since compound 8t had strong inhibitory activity against FLT3 and
CDK, sorafenib with potent inhibitory activity against FLT3 and pan-CDK inhibitor AT-7519 were
selected as the positive controls [25,26]. As detected by annexin V staining, not only was the apoptosis
triggered, but also a dose-dependent increase in the percentage of apoptotic and dead cells was seen
(Figure 4). In the presence of vehicle alone for 24 h, only 4.7% of the cells underwent apoptosis,
while treatment with compound 8t at 2 µM for 24 h led to an apoptosis rate up to 51.36%.
To further investigate whether the antitumor activities were relevant to the inhibition of FLT3 and
CDK2, we then examined the relative signaling proteins in MV4-11 cells treated with compound 8t by
western blot. According to the MV4-11 cell-based western blot assays, the phosphorylation of the FLT3
was weakened by compound 8t in a dose-dependent manner, and eliminated when the compound
concentration was increased to 1
sorafenib. As downstream signal pathways of FLT3, the phosphorylation of STAT5/AKT/ERK were also
completely blocked at 1 M. Compound 8t also inhibited the phosphorylation of the retinoblastoma
protein (Rb), a key downstream factor of CDK2/4, in a dose-dependent manner. At the concentration
of 1 M, compound 8t exhibited comparable inhibitory effects against the phosphorylation of Rb with
µM (Figure 5), which exhibited more potent inhibition effects than
µ
µ
AT-7519. In all, the anti-proliferative activity of compound 8t was associated with the inhibition of
FLT3 and CDK2/4.

Int. J. Mol. Sci. 2019, 20, 5739
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Figure 4. MV4-11 cells were treated with compound 8t for 24 h and then analyzed by AnnexinV/PI
staining. The percentage of cells undergoing apoptosis was defined as the sum of early apoptosis,
advanced apoptosis and necrotic cells. (
8t at 0.5 M; ( ) compound 8t at 1 M; (
2 µM; (H) Quantification of apoptotic cells.
A
) DMSO control; (
B
µ
) compound 8t at 0.2
M; ( ) Sorafenib at 2
µ
M; (
M; (
C
) compound
µ
D
µ
E) compound 8t at 2
F
µ
G) AT-7519 at
Figure 5. MV4-11 cells were treated with compound 8t, AT-7519, or sorafenib for 4 h, and the
phosphorylation of FLT3, STAT5, ERK, AKT, and Rb protein was analyzed by immunoblotting.
3. Materials and Methods
Unless otherwise specified, reagents were purchased from commercial suppliers and used without
further purification. Melting points were determined by X-4 digital display micro-melting point
apparatus (Tech Instrument Co., Ltd., Beijing, China); NMR spectra were recorded on Bruker AVANCE
AV-600 spectrometer (600 MHz for 1H, 150 MHz for 13C) or Bruker AVANCE AV-300 spectrometer
(300 MHz for 1H, 75 MHz for 13C); Mass spectra were obtained on the Agilent 1100 LC/MSD mass
spectrometer (Agilent, Santa Clara, CA, USA). All reactions were monitored by TLC (Merck Kieselgel
GF254, Merck, Kenilworth, NJ, China) and spots were visualized with UV light or iodine. The purity
of biologically evaluated compounds was >95% as determined by HPLC.

Int. J. Mol. Sci. 2019, 20, 5739
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3.1. Procedure A For the Synthesis of Compounds 4a and 4b
The mixture of appropriate amine (18.5 mmol), p-nitrobenzoic acid (20.4 mmol), EDC (22.2 mmol),
HOBT (22.2 mmol) in DMF (30 mL) was stirred for 24 h. The ice water (100 mL) was added to the
reaction mixture. A large amount of yellow solid precipitation (compounds 4a and 4b) was acquired.
Compounds 4a and 4b were used without further purification.
3.2. Procedure B For the Synthesis of Compounds 4c–4h
Fluorobenzene or fluoropyridine (46.3 mmol) and K2CO3 (69.5 mmol) were dissolved in DMSO
(50 mL). The reaction mixture was stirred at r.t. for 30 min and then amine (69.5 mmol) was added.
The reaction mixture was stirred at 70 ◦C for 5 h. The ice water (500 mL) was added to the reaction
mixture. A large amount of yellow solid precipitation (compounds 4c–4h) was acquired. Compounds
4c–4h were used for further reaction without purification.
3.3. Procedure C For the Synthesis of Compounds 2a–2c, 5a–5h, and 7a–7h
To a suspension of compounds 1a
–
1c
,
4a
–
4h
,
6c
–
6h, or 4a
–
4h (26.2 mmol) in 95% ethanol (100 mL),
.
85% NH2NH2 H2O (262 mmol), 95% ethanol (100 mL), and iron (III) oxide hydroxide (FeO(OH)/C, 0.5
g) were added and heated to reflux. When TLC analysis showed complete conversion of the starting
material, the reaction mixture was filtrate through Celite® and the filtrate was concentrated in vacuum.
The crude product was purified by silica gel column chromatography (DCM/MeOH) to yield the title
compound as white solid.
3.4. Procedure D for the Synthesis of Compounds 1a–1c and 6a–6h
4-nitro-1H-pyrazole-3-carboxylic acid (4.19 g, 13.94 mmol) was dissolved in 20 mL THF,
DMF (0.5 mL) and oxalyl chloride (1.78 mL, 20.91 mmol) were added at 0 ◦C, the resultant mixture was
stirred at room temperature for 60 min. After the mixture was concentrated in vacuo, the residue was
dissolved in pyridine (20 mL), and the solution was added dropwise into the solution of 7 (dissolved
in 20 mL pyridine) at 0 ◦C. The solution was stirred for 6 h at 25 ◦C. Upon completion of the reaction,
the solvent was removed on a rotary evaporator. Then water (100 mL) was added, and the mixture
was basified using 10% NaOH until pH 8~9. The solid precipitation was filtered to give the crude
product, which was used for next step without further purification.
3.5. Procedure E for the Synthesis of Compounds 3a–3c, 8a–8g, and 8r
Compounds 2a–2c, 7a–7b, or 7e–7h (10 mmol) were reacted with corresponding chlorides
(12 mmol) in AcOH/H2O:1/1 (10 mL) at 50 ◦C. When TLC analysis showed complete conversion of the
starting material, 10% NaOH was added and the pH was adjusted to 8–9. The precipitate was collected
and purified by silica gel column chromatography with DCM/MeOH (30/1) to yield the compounds
3a–3c, 8a–8g and 8r.
3.6. Procedure F for the Synthesis of Compounds 3d, 8h–8q and 8s–8t
Compounds 3c
,
7c
–
7d, or 7g (10 mmol) were reacted with corresponding chlorides (12 mmol)
7c 7d, or 7g,
in AcOH/H2O:1/1 (10 mL) at 50 ◦C. TLC analysis showed complete conversion of 2c
,
–
increasing the reaction temperature to 70 ◦C (5 h) to the cleavage of t-butylcarbamoyl group. Upon
completion of the reaction, 10% NaOH was added and the pH was adjusted to 8–9. The precipitate
was collected and purified by silica gel column chromatography with DCM/MeOH (30/1) to yield the
compounds 3d, 8h–8q, and 8s–8t.
Detailed synthetic process and structural characterization were provided in the
Supplementary Materials.

Int. J. Mol. Sci. 2019, 20, 5739
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3.7. Kinase Inhibition Assay
Activities of kinases were determined using Hot-SpotSM kinase assay which was performed by
Reaction Biology Corp. (Malvern PA, USA) as described previously [27].
3.8. Cell Growth Inhibition Assay
The human AML cell line MV4-11 was purchased from the American Type Culture Collection
(ATCC) (Manassas, VA, USA). MV4-11 was cultured in IMDM media (Corning, Crown Bioscience Inc.,
Taicang, China) with 10% FBS and supplemented with 2% L-glutamine and 1% pen/strep. The MV4-11
cell line was maintained in culture media at 37 ◦C with 5% CO2. The effects of target compounds
on MV4-11 proliferation was performed by Crown Bioscience Inc. Cells were grown in 96-well
culture plates (10,000/well). The compounds of various concentrations were added into the plates.
Cell proliferation was determined after treatment with compounds for 72 h. Cell viability was
measured using the CellTiter-Glo assay (Promega, Crown Bioscience Inc., Taicang, China) according to
the manufacturer’s instructions, and luminescence was measured in a multilabel reader (Envision2014,
PerkinElmer, Crown Bioscience Inc., Taicang, China). Data were normalized to the control group
(DMSO) and represented by the mean of three independent measurements with standard error of
<20%. IC50 values were calculated using Prism 5.0 (GraphPad Software, San Diego, CA, USA).
Analysis of pFLT3, pSTAT5, pERK, pAKT, and pRb in vitro: to determine the levels of pFLT3,
pSTAT5, pERK, pAKT, and pRb, cells were seeded in a 6-well cell culture plate at a density of 400,000
cells per well for MV4-11 in a total volume of 1800
µL and incubated overnight in medium containing
10% fetal bovine serum (Life Technologies, Rockville, MD, USA). Then 200
µL of serially diluted
compounds were added to each well the next day. Cell lysates were harvested after 4 h and pFLT3,
pSTAT5, pERK, pAKT, or pRb were quantified using assay kits for FLT3/Phospho-FLT3Tyr589/591,
pSTAT5 (Tyr694)/total STAT5, pRb (Ser807)/total Rb (Nanjin keyGen Biotech Company, Nanjing, China)
following the manufacturer’s protocols.
3.9. Cell Apoptosis Assay
The apoptosis of MV4-11 cells was determined by Annexin V-FITC/PI assay. Annexin V binds to
phosphatidylserine, which is exposed on the cell membrane and is one of the earliest indicators of
cellular apoptosis. PI (Propidium Iodide) is used as a DNA stain for both flow cytometry to evaluate
cell viability or DNA content in cell cycle analysis and microscopy to visualize the nucleus and other
DNA containing organelles. It can be used to differentiate necrotic, apoptotic, and normal cells.
Cells (2
×
105) were seeded in 6-well plate and were treated with varying concentrations of inhibitor for
24 h. MV4-11 cells were collected and incubated with FITC-conjugated Annexin V (Nanjing keyGen
Biotech Company, Nanjing, China). The nuclei were then counterstained with PI. After the dual
staining, the cells were screened by a FAC Scan flow cytometer (FACS Calibun, Becton Dickinson,
Nanjing, China). The upper left corner of the quadrant represents debris, lower left are live cells,
upper right are advanced apoptotic or necrotic cells and lower right are apoptotic cells.
3.10. Molecular Modeling
Compounds 8h and 8t were prepared by the protein preparation wizard in Maestro with standard
settings. Grids of CDK2 and FLT3 were generated using Glide, version 10.2, following the standard
procedure recommended by Schrodinger. Then 8h and 8t were docked into CDK2 (PDB code: 2VU3)
and FLT3 with DFG-in conformation as previous reported [22].
4. Conclusions
In summary, a series of 1H-pyrazole-3-carboxamides derivatives were designed and synthesized.
The step-by-step structural optimization demonstrated that the combination of piperazine in the
hydrophilic pocket, a benzene ring as aromatic linker, and a bulkier fused ring in the deep hydrophobic

Int. J. Mol. Sci. 2019, 20, 5739
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pocket can significantly increase the inhibitory activity of these kinds of compounds against CDK2/4
and FLT3. Among these compounds, compound 8t showed significant potency against CDKs and
FLT3, almost 10 times more powerful than FN-1501. Compound 8t also exhibited significant inhibitory
activity against various FLT3 mutations, especially against FLT3 (ITD)-F691L, indicating its potential
to overcome drug resistance caused by FLT3 mutation. Compound 8t showed potent anti-proliferative
activity against a variety of cancer cell lines including MV4-11 cells, and inhibited phosphorylation of
CDK and FLT3 pathways in a dose-dependent manner. These results demonstrated the potential of
this compound (8t) for further development as a promising agent for treatment of AML as well as
other cancers.
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/22/
5739/s1.
Author Contributions: Y.Z., Z.W., C.Y., B.L., H.H., J.C., Y.W. and L.X. participated in synthesis, purification, and
characterization of the chemical compounds and molecular modeling; writing—original draft preparation, Y.Z.;
writing—review and editing, S.L.; supervision, Y.W.; project administration, S.L.; funding acquisition, S.L., T.L.
and Y.Z.
Funding: This research was funded by the National Natural Science Foundation of China (81502925), the Natural
Science Foundation of Jiangsu Province (SBK2016020485), the Fundamental Research Funds for the Central
Universities (2016ZZD005) and QingLan Project from Jiangsu Province for financial support, the key project (NO.
192102310408) from the Department of Science and Technology, Henan Province. Li Xiang and Jiongheng Cai
appreciate the College Students Innovation Project for the R&D of Novel Drugs (J1310032) from the National
Found for Fostering Talents of Basic Science (NFFTBS).
Acknowledgments: We also express our gratitude to Xiazhong Ren, Huajun Yang, Chunlan Dong (Crown
Bioscience Corporation) and Jamie Planck (RBC, Pennsylvania, USA) for their helpful support in biological
evaluation. We are grateful to Feng Zhang of Nanjing University of Chinese Medicine and Huifang Li of Chemical
Computing Group Inc. for the critical reading and careful language modification.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
FLT3
AML
ALL
ITD
TKD
CDK
Fms-like receptor tyrosine kinase 3
acute myeloid leukemia
acute lymphoblastic leukemia
internal-tandem duplication
tyrosine kinase domain
cyclin-dependent kinases
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