Discovery of 4-((7H-Pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrazole-3-carboxamide (FN-1501), an FLT3- and CDK-Kinase Inhibitor with Potentially High Efficiency against Acute Myelocytic Leukemia

Article abstract of DOI:10.1021/acs.jmedchem.7b01261

A series of 1-H-pyrazole-3-carboxamide derivatives have been designed and synthesized that exhibit excellent FLT3 and CDK inhibition and antiproliferative activities. A structure-activity-relationship study illustrates that the incorporation of a pyrimidine-fused heterocycle at position 4 of the pyrazole is critical for FLT3 and CDK inhibition. Compound 50 (FN-1501), which possesses potent inhibitory activities against FLT3, CDK2, CDK4, and CDK6 with IC₅₀ values in the nanomolar range, shows antiproliferative activities against MV4-11 cells (IC₅₀: 0.008 μM), which correlates with the suppression of retinoblastoma phosphorylation, FLT3, ERK, AKT, and STAT5 and the onset of apoptosis. Acute-toxicity studies in mice show that compound 50 (LD₅₀: 186 mg/kg) is safer than AT7519 (32 mg/kg). In MV4-11 xenografts in a nude-mouse model, compound 50 can induce tumor regression at the dose of 15 mg/kg, which is more efficient than cytarabine (50 mg/kg). Taken together, these results demonstrate the potential of this unique compound for further development into a drug applied in acute-myeloid-leukemia (AML) therapeutics.

Full text of DOI:10.1021/acs.jmedchem.7b01261

Subscriber access provided by Universitaetsbibliothek | Johann Christian Senckenberg
Article
Discovery of 4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N-
(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrazole-3-
carboxamide (FN-1501), as a FLT3/CDKs kinase inhibitor with
potential high efficiency against acute myelocytic leukemia (AML)
Yue Wang, Yanle Zhi, Qiaomei Jin, Shuai Lu, Guowu Lin, Haoliang Yuan, Taotao Yang, Zhanwei Wang,
Chao Yao, Jun Ling, Hao Guo, Tonghui Li, Jianlin Jin, Baoquan Li, Li Zhang, Yadong Chen, and Tao Lu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01261 • Publication Date (Web): 22 Jan 2018
Downloaded from http://pubs.acs.org on January 23, 2018
Just Accepted
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Discovery of 4-((7H-pyrrolo[2,3-d]pyrimidin-4-
yl)amino)-N-(4-((4-methylpiperazin-1-
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yl)methyl)phenyl)-1H-pyrazole-3-carboxamide
FN-1501), as a FLT3/CDKs kinase inhibitor with
(
potential high efficiency against acute myelocytic
leukemia (AML)
Yue Wang ,ꢀYanle zhi†, , Qiaomei Jin , Shuai Lu , Guowu Lin , Haoliang Yuan , Taotao Yang , Zhanwei
†
,
⋚
,
⊥
⋚
,
⊥
†
†
†,‡
†
†
†
†
†
†
†
†
†
†
,†
Wang , Chao Yao , Jun Ling , Hao Guo , Tonghui Li , Jianlin Jin , Baoquan Li , Li Zhang , Yadong Chen* , Tao Lu
,†,
⋚
*
.
†
School of Sciences, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China.
State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing
10009, PR China.
⋚
2
‡
Department of Structural Biology, University of Pittsburgh, School of medicine, Pittsburgh, PA 1526, U.S.A.,
current address.
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ABSTRACT
A series of 1-H-pyrazole-3-carboxamide derivatives have been designed and synthesized, exhibiting
excellent FLT3/CDKs inhibition and anti-proliferative activity. Structure-activity relationship study illustrates
that the incorporation of a pyrimidine fused heterocycle at position 4 of the pyrazole is critical for FLT3/CDKs
inhibition. Compound 50 (FN-1501), possessing potent inhibitory activities against FLT3, CDK2, CDK4 and
CDK6 with IC50 values in the nanomolar range, shows anti-proliferative activity against MV4-11 cells (IC50:
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.008 μM), which correlates with the suppression of retinoblastoma phosphorylation, FLT3, ERK, AKT, STAT5
and the onset of apoptosis. The acute toxicity studies in mice show that compound 50 (LD : 186 mg/kg) is
50
safer than AT7519 (32 mg/kg). In MV4-11 xenografts in nude mice mode, compound 50 can induce tumor
regression at the dose of 15 mg/kg, which is more efficient than Cytarabine (50 mg/kg). Taken together, these
results demonstrate the potential of this unique compound for further development into drug applied in
acute myeloid leukemia (AML) therapeutics.
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INTRODUCTION
AML is a heterogeneous disease of the hematopoietic progenitor cell, characterized by a block in
1
differentiation and uncontrolled proliferation . More than one quarter of a million adults throughout the
world are diagnosed with AML annually. Induction chemotherapy with Cytarabine (ara-C)
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and Anthracycline (most often Daunorubicin) are usually performed as the first-line treatment of AML .
Although some improvement has been apparent among younger patients during the last four decades, only
approximately 35% of those patients entered on clinical trials are cured of their disease, but older people who
2
are not able to withstand intensive chemotherapy can only survive 5-10 months . Thus there is an unmet
need for effective therapeutics.
Fms-like receptor tyrosine kinase 3 (FLT3) is a Class III receptor tyrosine kinase (RTK), which acts as an
3
important regulatory molecule in hematopoiesis and is overexpressed in most cases of acute leukemia . FLT3
pathway plays an important role in the growth and differentiation of hematopoietic progenitor cells
4, 5
(Hematopoietic progenitor cells, HPCs) . Many studies have shown that AML and other hematologic cancers
6-10
are closely related to abnormal FLT3 pathway . As other RTKs, FLT3 receptor dimerized when binding to the
FLT3 ligand, resulting in autophosphorylation and activation of downstream signaling pathways, such as
RAS/MEK, PI3K/AKT/mTOR and JAK/STAT. These pathways play important roles in regulating cell cycle, cell
apoptosis and cell differentiation. However, mutations of FLT3 lead to the hyperactivation of FLT3 in the
11
absence of ligand binding and activation of downstream signaling pathways . About 30% of AML patients
harbor some form of FLT3 mutation, constitutively activating internal tandem duplication (ITD) mutations (of
1–100 amino acids) in the juxtamembrane domain (in approximately 25% of AML patients) invariably present
the greatest clinical challenge. FLT3 has always been an important target for the treatment of AML, with a lot
of small molecule FLT3 inhibitors (Quizartinib , Sorafenib , Midostaurin , Lestaurtinib , Crenolanib) marketed
12
or under investigation . Although some of these compounds have shown initial therapeutic benefits,
responses have been transient and relapse occurs within a few weeks, partially due to insufficient target
1
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coverage, activation of parallel pathways, and acquisition of resistance mutations . Midostaurin (PKC412; N-
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benzoylstaurosporin) is a multi-targeted tyrosine kinase inhibitor (TKI) against FLT3, VEGFR2, KIT, PDGFR and
PKC-a. Midostaurin was approved and first launched in 2017 in the U.S. for the treatment of adult patients
with newly diagnosed acute myeloid leukemia, in combination with chemotherapy. The success of
Midostaurin indicates that inhibition of FLT3 and other antitumor target can improve the antitumor efficiency.
Cyclin-Dependent Kinases (CDKs) are a family of 20 serine/threonine protein kinases that are generally
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classified as regulators of the cell cycle (CDK1, 2, 4, 6) or transcription (CDK7, 8, 9, 11, 20) . The uncontrolled
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5-17
regulation of CDK activity has been identified as a hallmark of cancer
. Indeed, oncogenic alterations of
CDKs, cyclins and other components of the Rb pathway have been reported in more than 90% of human
18-20
cancers
. For example, overexpression of cyclin E or cyclin A results in CDK2 hyperactivation in several
human malignancies, including AML, lung cell carcinoma, melanoma, osteosarcoma, ovarian carcinoma,
2
1
pancreatic neoplasia and sarcomas . Cyclin D2 and cyclin D3 belong to D-type cyclins, binding and activating
CDK4/CDK6, play important roles in hematologic malignancies. Mutations or overexpression of CDK4, cyclin D,
as well as reduced levels of p27Kip1 have also been found in various tumors, indicating that deregulation at
2
2-24
checkpoints throughout the cell cycle contributes to the process of tumorigenesis
. These studies suggest
that the CDK family has been considered as one of the most important targets for therapeutic intervention in
18, 25-
cancer, including hematologic malignancies. CDK inhibitors (AT-7519, Flavopiridol, AMG-925, Palbociclib)
32
33
are currently in clinical development in various solid tumors and hematopoietic malignances . However,
34
most of CDKs inhibitors were discontinued during phase II trials because of severe toxicity or limited clinical
35
activity as monotherapy .
In fact, activation of FLT3 downstream signal transduction pathway is an extremely important process in
promoting cell proliferation, which requires the cooperation of CDKs. Therefore, inhibitor of FLT3 and CDKs
can arrest anti-mitotic signaling pathways and cell cycle simultaneously, which may exert synergistic effect to
1
8, 28, 36
increase the anti-leukemic efficiency
. We have reported a series of compounds with moderate CDK2
37-40
activity
. Among these compounds, 26 (Figure 1) has a certain inhibitory activity against CDK2 (IC : 318.21
50
nM) and FLT3 (IC : 42.60 nM) and shows moderate activity in MV4-11 (IC : 0.45 μM). Starting from 26, our
50
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group has explored the biological properties of variously substituted 1-H-pyrazole-3-carboxamide derivatives
with the aim to find new active compounds and pharmacophores as the potent FLT3/CDKs inhibitors. Among
these compounds, compound 50 was found to be the most active in vitro and in vivo, which also possesses
good pharmacokinetic properties and low toxicity. The report presents the discovery of 50 as a highly efficient
multi-target FLT3/CDKs kinase inhibitor suitable for further evaluation.
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Figure 1. Molecular docking analysis of compound 26 to CDK2 kinase and FLT3 kinase. (A) Chemical structure
of 26. (B) Compound 26 docked into CDK2 kinase (PDB code 2VU3). (C) Compound 26 docked into homology
18
model of FLT3 .
CHEMISTRY
Scheme 1. Synthetic routes of compounds 27-62
Br
a
R1
b
R1
c
NO2
70 - 95 %
NO2
80 - 95 %
NH2
1
1
2: R = morpholine
8: R = morpholine
1
1
3: R = 1-methylpiperazine
9: R = 1-methylpiperazine
1
1
4: R = piperazine
10: R = piperazine
1
1
5: R = 1-methyl-1,4-diazepane
11: R = 1-methyl-1,4-diazepane
1
1
6
7
: R = diethylamine
12: R = diethylamine
1
1
: R = cyclopropan amine
13: R = cyclopropan amine
R1
O
R1
O
R1
R2
NO2
NH2
NH
O
HN
d
e
N
N
N
H
H
H
N
N
N
NH
NH
1
1
1
4: R = morpholine
20: R = morpholine
2
7-57 as in Table 1
57-62 as in Table 2
1
1
1
5: R = 1-methylpiperazine
21: R = 1-methylpiperazine
1
1
16: R = piperazine
22: R = piperazine
1
1
17: R = 1-methyl-1,4-diazepane
23: R = 1-methyl-1,4-diazepane
1
1
18: R = diethylamine
24: R = diethylamine
1
1
19: R = cyclopropan amine
25: R = cyclopropan amine
.
Reagents and conditions: (a) relative amine, Et N, CH Cl ; (b) FeO(OH)/C, 80% NH NH H O, 95% EtOH; (c) 4-
3
2
2
2
2
2
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Nitropyrazole-3-carboxylic acid, EDC HCl, HOBt, DMF, r.t; (d) FeO(OH)/C, 80% NH NH H O, 95% EtOH; (e) R -
2
2
2
Cl, AcOH/H O, 50 °C
2
The synthesis of compound 27-62 was depicted in Scheme 1. In total, 35 pyrazole-carboxamide
derivatives were prepared (Table 1 and Table 2). Compounds 27-62 were prepared in a similar fashion. 4-
Nitrobenzyl bromide was used as the starting material and compound 2-7 were prepared through a
nucleophilic substitution reaction. Reduction of compound 2-7 with 80% hydrazine hydrate gave compound 8-
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13 and after coupling of 4-nitropyrazole-3-carboxylic acid with EDC HCl and HOBt , compound 14-19 were
obtained. Compound 20-25 were obtained via the reduction of the nitro-group in compound 14-19. All target
compounds (27-62) were prepared by ammonolysis of appropriate chlorides with 20-25.
RESULTS AND DISCUSSION
40
In our previous studies , a series of pyrazole-3-carboxamide compounds were synthesized. Recently, it has
been identified that compound 26 inhibits FLT3 and CDK2 with IC of 42.6 nM and 318 nM, respectively. With
50
the aid of CADD (Computer Aided Drug Design), we found that the hydrophobic cavity of CDK2 formed by
ASP145, VAL18, PHE80 and LYS33 (corresponding to ASP757, PHE691, PHE627 and LYS650 in FLT3) are wide
enough to accommodate a larger hydrophobic group. Guided by our previous studies, we kept the 1-H-
pyrazole-3-carboxamide skeleton and the hydrophobic phenyl ring linked to 3-carboxamide (The binding
modes were shown in Figure 1). In position 4 of pyrazole, the benzoyl group was cyclized to give a
pyrimidobenzene ring structure, and then various binary aromatic heterocycles were incorporated to study
the effects of the hydrophobic group on kinase inhibitory activity. In solvent accessible region, different
hydrophilic fragments were introduced, leading to a series of 1-H-pyrazole-3-carboxamide derivatives. All
derivatives were tested for their inhibitory potency toward recombinant human CDK2/Cyclin A1 and FLT3 and
their antiproliferative effects on MV4-11 cells (human acute monocytic leukemia cell line).
Structure-Activity Relationship (SAR) Studies
The results of the kinase inhibition assays are listed in Table 1 and Table 2 with AT-7519 and Sorafenib as
positive controls. Minimizing the diversity of the substituents in the phenyl ring enabled us to investigate how
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varying the substituents at the C4-position of the pyrazole will affect the activity against CDK2 and FLT3. As is
shown in Table 1, both CDK2 and FLT3 affinity were increased slightly compared with the parent compound
when the benzoyl in 26 were replaced by quinolone (compound 27, 42) or isoquinoline (compound 28,
4
4) .This may benefit from the edge-to-face aromatic-aromatic interaction between the pyridine and PHE80
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in CDK2 and PHE691 in FLT3 and the hydrophobic interaction formed by the phenyl ring with the ASP145 in
CDK2 and ASP698 in FLT3, respectively (Figure 2).
Figure 2. Binding mode analysis of compound 42 to CDK2 kinase and FLT3 kinase. (A) Compound 42 docked
18
into CDK2 kinase (PDB code 2VU3). (B) Compound 42 docked into homology model of FLT3 .
CDK2 and FLT3 inhibitory activities were lost when the hydrogen at position 2 of quinoline was replaced by
trifluoromethyl (33), probably because trifluoromethyl prevented the edge-to-face interaction between the
aromatic ring and PHE80. Further introduction of different substituents to isoquinoline or quinoline yield
analogues 29-30 and 44-46. Electron withdrawing groups such as bromine in 29 and 46 can improve the
affinity to FLT3. However, compounds 30, 44 and 46, substituted with electron-donating groups such as
methoxyl were slightly less active against both CDK2 and FLT3 when compared to 27 and 42.
Interestingly, introduction of a nitrogen to the pyridine ring of 27 yielded analogues 31, which showed
significant increase of inhibitory activities against CDK2 and FLT3 (IC : 5.30 nM and 0.588 nM, respectively).
50
Replacement of the morpholine (31) to N-methylpiperazine (47) also retained excellent activity against CDK2
IC : 3.17 nM) and FLT3 (IC : 8.27 nM). The inhibitory activity was approximately 10 times more potent than
(
50
50
that of AT7519 and Sorafenib. Addition of the chlorine atom into 2 position of pyrimidine (32) resulted in
decrease of affinity to CDK2 when compared with 31, supporting the hypothesis that substituent in C2
position of pyrimidine or pyridine might prevent edge-to-face interaction between the aromatic ring and
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PHE80 (CDK2). However, introduction of a chlorine atom has no impact on the inhibitory activity of 32 against
FLT3. We speculate that the distance between the pyrimidine ring and gatekeeper residues PHE675 of FLT3
are far enough to accommodate the chlorine atom.
Table 1. Structures and biological evaluation of target compounds
0
1
2
3
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9
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(
% cell growth
(% enzyme activity at 0.123 µM)
inhibition at 1µM)
2
a
Compounds
A
R
IC50(nM)
c
IC (µM)
50
CDK2
FLT3
MV4-11
b
AT7519
57.20 ± 1.31
nd
0.232 ± 0.003
Sorafenib
nd
15.48
0.004 ± 0.0002
0.45 ± 0.009
26
27
O
O
318.21 ± 13.5
42.60 ± 1.6
(57.67%)
nd
(40.75%)
nd
(41.97%)
nd
(
50.40%)
nd
2
8
9
O
O
287.19 ± 11.4
24.15 ± 1.8
42.38 ± 4.3
16.90 ± 0.5
(
93.66%)
nd
2
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(
74.99%)
(22.20%)
nd
3
3
0
1
O
O
97.4 ± 2.1
nd
5.30 ± 0.32
58.63 ± 1.6
0.588 ± 0.019
0.958 ± 0.023
0.023 ± 0.003
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
(91.10%)
32
33
34
O
O
O
nd
(
99.7%)
(83.22%)
nd
(4.7%)
nd
nd
（
53.11%）
29.8 ± 2.4
3.36 ± 0.6
3.78 ± 0.3
nd
(98.49%)
3
3
3
5
6
7
O
O
0.623 ± 0.04
nd
7.25 ± 0.28
2.06 ± 0.05
0.021 ± 0.003
O
O
O
3.30 ± 0.01
9.85 ± 0.91
1.08 ± 0.04
1.56 ± 0.02
7.63 ± 0.65
1.37 ± 0.06
0.034 ± 0.004
0.015 ± 0.002
0.011 ± 0.003
38
3
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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
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5
5
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5
5
6
40
O
O
11.64 ± 1.56
7.34 ± 0.24
6.15 ± 0.87
0.035 ± 0.008
0.030 ± 0.005
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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8
9
0
1
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9
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2
3
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9
0
41
42
43
3.52 ± 0.15
(
26.51%)
nd
(27.72%)
nd
(98.79%)
nd
NCH
NCH
NCH
3
3
3
(
60.47%)
nd
(24.49%)
nd
(66.46%)
nd
(97.24%)
nd
44
45
5.74 ± 0.73
697 ± 23.05
4.84 ± 0.21
197 ± 24.71
(22.60%)
nd
NCH
NCH
3
3
(32.45%)
(10.45%)
nd
(64.40%)
nd
4
6
7
nd
4
NCH
NCH
NCH
3
3
3
8.27 ± 1.32
5.85 ± 0.61
16.5 ± 0.85
3.17 ± 0.57
0.60 ± 0.05
2.65 ± 0.24
0.017 ± 0.004
0.019 ± 0.004
48
(94.69%)
nd
49
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0
1
NCH
3
2.47 ± 0.06
0.27 ± 0.01
0.008 ± 0.001
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
NCH
NCH
NCH
NCH
NCH
NCH
9.32 ± 0.27
1.90 ± 0.03
1.74 ± 0.04
4.49 ± 0.19
3.89 ± 0.24
1.87 ± 0.16
0.47 ± 0.01
0.576 ± 0.01
0.384 ± 0.012
0.167 ± 0.003
2.31 ± 0.32
0.023 ± 0.003
>0.00015
3
3
3
3
3
3
52
53
54
55
0.017 ± 0.002
0.021 ± 0.004
0.027 ± 0.001
0.029 ± 0.001
5
6
0.101 ± 0.02
a
In the presence of 10 μM ATP with 0.123 μM compound, the values are the mean ± SD from three
b
c
independent experiments. nd: not determined. n = 3.
The pyrimidine in the fused heterocycle seems to be optimal to form aromatic-aromatic interaction with
the gatekeeper PHE80 in CDK2. To further investigate the hydrophobic interaction of aromatic rings with the
hydrophobic cavity, the phenyl ring of the fused heterocycle was then replaced by a 5-membered aromatic
heterocycles. Modifications to the phenyl ring resulted in analogies 35-39 and 48-56, among which the
thienopyrimidine analogues (35, 48) retained the same affinity with CDK2 and FLT3 when compared to (31,
4
7). Even, substituting 7 or/and 8 hydrogen of thieno[2,3-d]pyrimidine with methyl (39, 53, 54), ethyl (37, 56)
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
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3
3
3
3
4
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4
4
4
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5
5
5
5
5
5
5
6
or isopropyl(38, 55) also maintained strong inhibitory activities against CDK2 and FLT3. Besides, changing the
position of the sulfur atom (51) did not affect kinase activities. In order to obtain additional SAR information,
we replaced the thiazole ring with the furan (49) and pyrrole (36, 50) based on the bioisosterism. The
pyrrolopyrimidine analogue 50 exhibited similar kinase inhibitory potency to that of 48 for both CDK2 and
FLT3. But the furan substituted compound 49 showed a decreasing inhibitory efficiency against CDK2. Overall，
introduction of pyrrolopyrimidine or thienopyrimidine is important for kinase inhibit activity, especially for
FLT3.
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
Although the 1-methylpiperazine or morpholine stretched to hydrophilic region conferred good potency for
either CDK2 or FLT3 inhibitory activities, there had been no evidence to clarify which hydrophilic group would
be optimal in consideration of potency and physical properties. Thus, a series of piperazine replacements was
investigated using both the thieno[2,3-d]pyrimidine and the 7H-pyrrolo[2,3-d]pyrimidine as the hydrophobic
segments. All these compounds are listed in Table 2.
Table 2. Optimization of the hydrophilic group in the solvent accessible area
(
% cell
(
% Enzyme
activity at
growth
inhibition at
1
Compounds
R
B
0.123µM)
LogP CLogP TPSA
b
1
µM)
a
IC (nM)
5
0
IC (µM)
50
CDK2
.08 ±
.18
FTL3
0.34 ±
0.03
MV4-11
2
57
S
0.019 ± 0.004 2.65
2.80
105
0
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.59 ±
.02
.05 ±
.42
5.36 ±
0.98
5
5
8
9
S
S
0.007 ± 0.001 3.95
0.013 ± 0.004 3.19
3.97
3.02
93
0
2
1.05 ±
0.11
102
0
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
3
.03 ±
.11
6
6
6
0
1
2
S
0.32 ± 0.047 ± 0.006 3.14
.03
3.31
2.32
3.48
98
0
0
4
.49 ±
.72
1.03 ±
0.05
NH
0.053 ± 0.006 1.22
0.012 ± 0.004 2.51
118
105
0
0
.13 ±
.03
NH 13.6 ±
.12
0
1
a
In the presence of 10 μM ATP with 0.123 μM compound, the values are the mean ± SD from three
b
independent experiments. n = 3.
It is noteworthy that modifications in the hydrophilic region can maintain the inhibitory activity of the
compounds against CDK2 and FLT3. However, 7H-pyrrolo[2,3-d]pyrimidine derivatives (Table 2, 61-62) led to a
decrease in cell-based activity compared to thieno[2,3-d]pyrimidine derivatives (Table 2, 57-58). These may
be caused by the decrease of compounds’ lipophilicity which influences the permeability of compounds.
Further studies have shown that introducing thieno[2,3-d]pyrimidine led to high clearance rates in liver
microsomes. Introduction of diethylamine group (62, Table 2) maintained cell potency with acceptable
lipophilicity, but showed poor CDK2 activity. Taken together, optimizing the hydrophilic region did not
significantly improve the efficiency of the compounds. Hence, introducing of the fused heterocycle that
contains pyrimidine/pyridine structure to pyrazole is suggested to be the most critical factor determining
potency of pyrazole-carboxamide derivatives towards FLT3 and CDKs.
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6
Kinase Inhibition Profile of compound 48
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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8
9
0
1
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0
1
2
3
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8
9
0
1
2
3
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5
6
7
8
9
0
Figure 3. (A) Distribution of inhibited kinases of compound 48 within the human kinome. The color code for
inhibition is indicated. Residual enzymatic activity was determined in single dose duplicate at a compound
concentration of 10 μM. The ATP concentration was 10 μM. Staurosporine served as a positive control. The
experiment was performed by Reaction Biology Corp. using a P33-radiolabel assay. (B) Inhibitory activity of
compound 48 against selected kinases using the P33-Radiolabeled assay.
Among the series of compounds mentioned above, one promising compound 48 was tested against a panel
of 339 kinases by Reaction Biology Corporation at a single concentration of 10 μM (Figure 3A). On the basis of
kinase activity result, 28 kinases were selected for further assays (Figure 3B, Table S1). Compound 48
displayed highest inhibitory activity against CDK2/CyclinA1 (IC : 9.47 nM) and FLT3 (IC : 0.438 nM). It should
50
50
be noted that 48 also has inhibitory activities against CDK4/cyclinD1 (IC : 5.57 nM) and CDK6/cyclinD1 (IC :
50
50
4
.5 nM), followed by CDK9/cyclinK (IC : 38.9 nM), FLT1/VEGFR1 (IC : 19.8 nM), KDR/VEGFR2 (IC : 35.1 nM),
50 50 50
ALK (IC : 94.1nM) and CDK1/cyclinE (IC : 96 nM). Therefore, compound 48 is identified to be a multiple
50
50
kinases inhibitor, inhibition of multiple pathways simultaneously by compound 48 is beneficial to improve its
antitumor efficiency and overcome drug resistant.
Inspired by the kinase profiling result, five representative CDK2 and FLT3 inhibitors 35, 48, 49, 50 and 51
were further evaluated for inhibitory activities against CDK4/cyclin D1, CDK6/cyclin D1 other than
CDK2/cyclinA and FLT3 (Table 3). All selected compounds showed high inhibitory activities against these
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kinases with IC lower than 10 nM, suggesting that these compounds may be even more potent CDKs and
50
FLT3 inhibitors. (ie. 50: CDK2, IC : 2.47 nM; CDK4, IC : 0.85 nM; CDK6, IC : 1.96 nM; FLT3, IC : 0.28 nM).
50
50
50
50
Table 3. Inhibitory activities of compound 35, 48, 49, 50 and 51 toward CDKs and FLT3 kinases
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
Compounds IC (nM)
50
Kinases
3
5
48
49
50
51
CDK2/cyclin A
3.36±0.26 5.85±0.58 16.5±0.76 2.47±0.21 9.32±0.45
CDK4/cyclin D1 2.40±0.27 3.33±0.24 5.67±0.30 0.85±0.28 3.10±0.34
CDK6/cyclin D1 3.88±0.17 4.24±0.23 5.09±0.44 1.96±0.08 4.11±0.29
FLT3
0.62±0.06 0.60±0.04 2.56±0.11 0.28±0.01 0.47±0.02
a
In the presence of 10 μM ATP with 0.123 μM compound, the values are the mean ± SD from three
independent experiments.
In Vitro Cell Assays
Table 4. In vitro growth inhibitory activities of selected compounds
a
Cell type GI (µM)
50
compounds
AT-7519
MGC803 RS4;11
MCF-7
HCT-116 NCI-H82
0.26±0.13 0.27±0.14
0.58±0.11 0.29±0.06
0.21±0.01 0.20±0.04
0.21±0.02 0.17±0.05
0.16±0.05 0.13±0.01
0.37±0.04 0.05±0.01
0.11±0.02
0.61±0.07
3
4
5
5
8
0
0.62±0.09 0.37±0.02 0.17±0.02
2.84±0.25 0.09±0.04 0.11±0.02
a
n = 3.
All of target compounds (27-62) were evaluated against human leukemia cell line MV4-11 (FLT3 ) using
ITD
the MTT assay after 72 h incubation (see Table 1 and Table 2). Compounds 31, 36-40, 47-48, 50-62 showed
significant antiproliferative activities against MV4-11, with IC50 values ranging from 0.0015 to 0.035 μM.
Almost all of these derivatives proved to be more antiproliferative than 26, which further certify the
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1
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1
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1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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4
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5
5
5
5
5
5
6
rationality of our design strategy. Compounds 35, 48 and 50 were selected to evaluate their activity against a
panel of cell lines that represent different tumor types, including gastric, leukemia, breast cancer, colorectal
cancer. The growth of all cell lines were inhibited by selected compounds and the 50% growth inhibition (GI )
50
values range from 51 to 620 nM (Table 4).
0
1
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1
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1
2
3
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0
1
2
3
4
5
6
7
8
9
0
With these findings, we submitted 35, 48, 50 to the Developmental Therapeutics Program at NCI
(http://dtp.nci.nih.gov) to test their selectivity against the NCI60 cancer cell lines. In this screen, compounds
35, 48 and 50 exhibit potent inhibit activity on all 60 cancer cell lines (Figure S1, S2, S3 in Supporting
Information), with GI <0.5 μmol/L (Growth inhibition of 50 %: calculated from [(Ti-Tz)/(C-Tz)] x 100 = 50,
50
which is the drug concentration resulting in a 50% reduction in the net protein increase (as measured by SRB
staining) in control cells during the drug incubation). For the great majority of the lines, LC (concentration of
50
drug resulting in a 50% reduction in the measured protein at the end of the drug treatment as compared to
that at the beginning. Indicating a net loss of cells following treatment is calculated from [(Ti-Tz)/Tz] x 100 = -
50) was >100 μmol/L (mean LC >400 μmol/L). In comparison, the lethal concentration of flavopiridol (mean
50
GI : 0.074 μmol/L against NCI60 cell lines), one of a potent inhibitor of CDKs, reached submicromolar
50
4
2
concentrations (LC : 0.904 μmol/L) .
50
Table 5. Pharmacokinetics of selected compounds in liver microsomes of multiple species
Compounds
35
50
2a
R
0.9793
0.138
165.1
0.8963
0.118
352.4
0.3209
1.717
13.3
b
HML
RLM
T1/2 (h)
c
CL (mL/min/kg)
int
2
R
0.5008
1.032
40.3
T1/2 (h)
CL (mL/min/kg)
int
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R
0.9056
0.12
0.9465
0.66
MLM
DLM
CLM
T1/2 (h)
CL (mL/min/kg)
765.1
0.9899
0.025
1327.7
0.9929
0.182
170.9
138.6
0.9908
0.257
129.3
0.9051
0.855
36.5
int
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
2
R
T1/2 (h)
CL (mL/min/kg)
int
2
R
T1/2 (h)
CL (mL/min/kg)
int
a
b
2
R is the correlation coefficient of the linear regression for the determination of kinetic constant
c
T1/2 is half life, C is the intrinsic clearance
Lint
Pharmacokinetics of 35 and 50 in Liver Microsomes of Multiple Species
The preliminary metabolic stability study of the compound 35 and 50 were performed by using human
liver microsomes (HML), rat liver microsomes (RLM), mouse liver microsomes (MLM), dog liver microsomes
(
DLM) and monkey liver microsomes(CLM) (Table 5). The terminal t1/2 was moderate in human liver
microsomes, rat liver microsomes, mouse liver microsomes and monkey liver microsomes (ranged between
.025 and 1.7 h). Compound 50 was metabolically more stable (t1/2 ranged between 0.25 and 1.7 h) than 35
0
(t1/2 shorter than 0.18 h). Given the drastic metabolic instability in the liver microsomes experiment,
compound 35 was not considered for further study. Some other studies have demonstrated that compounds
4
3, 44
with pyrimidothiophene ring are metabolically unstable
been considered for further investigation.
. Therefore, this series of compounds had not
Molecular Modeling of Compound 50 with CDK2 and FLT3
Compound 50 showed optimal FLT3/CDKs inhibitory activities, and importantly, it is metabolically stable.
Hence, the binding mode of compound 50 within CDK2 and FLT3 were elucidated using a docking model
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(
Figure 4). As is shown in Figure 4A, compound 50 binds to the ATP-binding site of CDK2 in an orientation
similar to that of AT-7519 (Figure 4B). The pyrazole-3-carboxamide skeleton of compound 50 forms three
conserved hydrogen bonds with the hinge region of CDK2, one between the N1 of pyrazole and GLU81, and
the other two between the NH of formamide, N2 of pyrazole and LEU83 (Figure 4A).
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The fused heterocycle substituent has complementary packing and hydrophobic interactions with LYS33,
VAL18, ASP145, ALA144 and the hydrophobic residue of the ILE10 side chain. The pyrrole NH forms additional
polar interactions with the side chains of ASP145. A face-to-face interaction with the adjacent aromatic
planes is observed between the pyrimidine and the kinase gatekeeper residue, PHE80. Phenyl ring occupied
the small hydrophobic cavity between hinge region and the hydrophilic group. The N-methylpiperazine group
sits in a solvent accessible area which is mainly hydrophobic but lined with several polar residues at the edge
(
LYS89, ASP86, LEU298, GLN85).
Figure 4. Binding mode analysis of compound 50 to CDK2 kinase and FLT3 kinase. (A) Compound 50 docked
into CDK2 kinase (PDB code 2VU3). (B) Overlapping of compound 50 (green) and AT7519 (gray) complexed
18
with CDK2. (C) Compound 50 docked into homology model of FLT3 . (D) Chemic structure of compound 50.
In Figure 4B, we found that compound 50 binds to FLT3 in a similar fashion to its binding mode to CDK2,
with the core pyrazole-3-carboxamide skeleton forming two conserved hydrogen bonds in the hinge region of
FLT3. One is between the N1 of pyrazole and GLU692, the other one is between the N2 of pyrazole and
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CYS694. The 7H-pyrrolo[2,3-d]pyrimidine substituent of 50 occupies the hydrophobic pocket and the N-
methylpiperazine group extends into solvent accessible area (Figure 4C). In general, the docking results
further confirm the rationality of our design strategy.
In Vivo Effects of Compound 50 In s.c. MV4-11 Tumor Xenografts
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On the basis of a desirable set of in vitro properties, compound 50 was finally selected for further in vivo
evaluation. We proceeded to test the antitumor efficacy of compound 50 in MV4-11 cell inoculated xenograft
mouse.
Figure 5. The antitumor efficacy of 50 in MV4-11 xenograft model. Female nu/nu mice bearing established
MV4-11 tumor xenografts were treated by intravenous injection (iv, QD) with 50 at 15, 30, or 40 (mg/kg)/d or
Cytarabine at 50 (mg/kg)/d or vehicle. (A) Body weight and (B) tumor size measurements from MV4-11
xenograft mice after 50 or Cytarabine administration. Initial body weight and tumor size was set as 100%. (C)
Representative photographs of tumors in each group after 15, 30, or 40 (mg/kg)/d of 50 or 50 (mg/kg)/d of
Cytarabine or vehicle treatment. (D) Comparison of the final tumor weight in each group after 22-day
treatment period. Numbers in columns indicate the mean tumor weight in each group. ns, p > 0.05, (∗) p <
0
.05, (∗∗) p < 0.01.
In the MV4-11 model, when the tumor grew to a volume of 200 mm , the mice were grouped and treated
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intravenous once daily with 15, 30, or 40 mg/kg/d compound 50 for 21 days. Another two groups were
recruited, one group for negative control (only vehicle) and another group treated intravenous once daily with
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0 mg/kg/d Cytarabine as a positive control for 21 days. The group injected with 50 mg/kg/d Cytarabine only
reduced the tumor growth rate, and the tumor growth inhibition was 61.94% on the 21th day. While injected
with 50 by 15 mg/kg/d induced tumor regression and the PTR (percent tumor regression) rate was -6.11% on
the 21th day (Figure 5). Meanwhile, groups injected with 50 by 30 and 40 mg/kg/d significantly induce tumor
regression and the PTR on the 21th day were 78.95% and 81.33% (Figure5B, 5C and 5D), respectively.
Furthermore, it was well tolerated with no overt signs of toxicity or changes in body weight (Figure 5A).
Experiments in rats demonstrated that 50 did have significant anti-tumor activity in vivo compared with
Cytarabine which is used as a first-line chemotherapy drug against AML.
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Toxicity Studies
Inspired by the excellent antitumor activity of compound 50, we assessed its cytotoxicity on normal
lymphocytes cells. Compound 50 shows little cytotoxicity to normal lymphocytes cells, which are comparable
with AC220 (IC :0.492 μM) or AT7519 (IC :0.554 μM). To further evaluate the safety of 50 in vivo, we
50
50
conducted additional LD tests for AT-7519 and 50, and groups of ICR mice (half male and half female) were
50
administered with compound 50 or AT7519 for a single dose by intravenous injection. The survival of the mice
was monitored and recorded over 12 days (Table S2). The LD values of AT7519 are 32 mg/kg. However, the
50
LD50 values of 50 were 185.67 mg/kg as is shown in Table 6. Compound 50 exhibited significantly reduced
toxicity compared with AT7519.
Table 6. Median Lethal Dose (LD ) Values of 50 and AT7519
50
Compounds
AT7519
LD (mg/kg)
95% confidence interval (μmol/kg)
5
0
32.00
28.20-36.32
5
0
185.67
158.57-208.12
Cellular Mode of Action
To characterize the mode of cell death induced by compound 50, biparametric flow cytometric analysis
with annexin-V/propidium iodide (PI) was performed. Inducing apoptosis is considered as one of the major
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strategies for antitumor drug development . Flow cytometry was used to investigate whether the anti-
proliferative effect of compound 50 was caused by activation of cellular apoptosis in MV4-11 cells.
Quantitative analysis of early apoptotic cells and advanced apoptotic/necrotic cells was determined via an
Annexin V-FITC/ PI assay.
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MV4-11 cells were stimulated with either 0.2-2 μM of compound 50 for 24 h, and DMSO was used as a
negative control. Sorafenib was used as positive control. As shown in Figure 6, apoptosis induced by
compound 50 was much greater than the control, and MV4-11 cells underwent both early apoptosis (Annexin
+
-
+
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V /PI , lower right) and advanced apoptosis/necrosis (Annexin V /PI , upper right). The results showed that
compound 50 could effectively induce cell apoptosis starting from 0.2 μM with an enhanced effect at higher
concentrations. The apoptosis ratios of compound 50 measured at different concentrations were 24.35% (0.2
μM), 41.98% (0.5 μM), 46.34% (1 μM), 78.30% (2 μM) and the apoptotic effect increases in a dose-dependent
manner. From these results, we can conclude that 50 caused effective apoptotic effect in MV4-11 cell line,
which shows equivalent efficiency as Sorafenib (75.32% at 2 μM).
Figure 6. MV4-11 cells were treated with compound 50 for 24 h and analysed by Annexin V/PI staining. The
percentage of cells undergoing apoptosis was defined as the sum of early apoptosis, advanced apoptosis and
necrotic cells. UR (upper right): advanced apoptotic cells and necrotic cells labeled with PI and Annexin, LL
(
lower left): fully viable cells, LR (lower right): early apoptotic cells labeled with Annexin V but not with PI. (A)
DMSO control; (B) 0.2 µM compound 50; (C) 0.5 µM compound 50; (D) 1 µM compound 50; (E) 2 µM
compound 50; (F) 2 µM Sorafenib; (G) Percentage of apoptosis cells.
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Figure 7. Compound 50 treatment of MV4-11 and HCT116 cells lead to cancer cell growth inhibition, cell cycle
arrest. (A) Effect of compound 50 on MV4-11 cell cycle kinetics. Compound 50 at the concentrations shown
was added to MV4-11 cells for 24 h prior to fixation, staining with propidium iodide (PI), and flow cytometric
analysis. (B) Effect of compound 50 on HCT-116 cell cycle.
Furthermore, compound 50 was measured in MV4-11 and HCT-116 cell lines to consider if the
antiproliferative activity is consistent with FLT3 and CDK2 signaling inhibition. The antiproliferative activity of
compound 50 was verified by flow cytometry analysis of MV4-11 and the colon HCT-116 cell line, through
double staining with propidium iodide and 5-bromo-20-deoxyuridine (BrdU). As is shown in Figure 7,
compound 50 arrested cell cycle progression of this cell line into the G0/G1 phase in a dose-dependent
manner, which is consisted with Sorafenib (Figure 7A) in MV4-11 cell line. Treatment with compound 50
mainly caused G2/M phase arrest in the HCT-116 cell lines (Figure 7B), which would be related to the potent
inhibition of CDKs. Overall, compound 50 arrested the cell cycle of two human cancer cell lines as efficiency as
AT-7519 and Sorafenib, and both cell lines displayed similar patterns as the positive control against cell-cycle
blockade.
We then treated MV4-11 cell line with increasing doses of compound 50 for 24 h and immune blotted
the lysates with antibodies against relative proteins involved in FLT3 mediated signaling pathway (Figure 8A).
Treatment of MV4-11 cells with compound 50 for 24h significantly decrease the phosphorylation of FLT3 and
its downstream mediators STAT5, ERK and AKT’s phosphorylation than Sorafenib. As shown in Figure 8B,
Ser807/811
compound 50 inhibited Rb
phosphorylation in a dose-dependent manner and its efficiency was equal
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to AT7519 at 1μM. These findings reveal that compound 50 acts as a highly efficient FLT3 and CDKs inhibitor.
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Figure 8. . Western blot analysis. (A) MV4-11 cells were treated with compound 50 or Sorafenib for 24 h and
phosphorylation of FLT3, STAT5, ERK and AKT protein were analyzed by immune blotting. (B) MV4-11 cells
were treated for 24 h with compound 50 or AT7519 and phosphorylation of Rb protein was analyzed by
immune blotting.
CONCLUSIONS
Starting from compound 26, we have discovered a series of N-(4-methylphenyl)-1-H-pyrazole-3-
carboxamide derivatives as potent FLT3/CDKs inhibitors. The structure-activity relationship analysis
demonstrated that substitution with fused heterocycle that contains pyrimidine/pyridine structure in position
4
of the 1-H-pyrazole-3-carboxamide moiety was considered to be key strategic group to improve CDK2 and
FLT3 inhibitory activity and antiproliferative activity. Compound 50 showed nanomolar range CDKs and FLT3
inhibition and significant cytotoxic effect on NCI60 cancer cell lines (IC50 < 1μM). In addition, 50 exhibited
higher metabolic stability in liver microsomal metabolism essay. In vivo study of 50 in MV4-11 xenograft
model showed higher tumor efficacy at a dosage of 15.0 (mg/kg)/d than Cytarabine (50.0 (mg/kg)/d), and
furthermore, it induces tumor regression when increasing the doses to 30.0 (mg/kg)/d or 40.0 (mg/kg)/d.
Currently compound 50 is in phase I clinical trials for cancer (In the United States and China) and it may
represent a promising drug with therapeutic potential for the AML.
EXPERMENTAL SECTION
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General Procedures
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 (Beijing
Tech Instrument Co., Ltd.); NMR spectra were recorded on Bruker AVANCE AV-600 spectrometer (800 MHz for
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1H, 150 MHz for C) or Bruker AVANCE AV-300 spectrometer (300 MHz for 1H, 75 MHz for C); Mass spectra
were obtained on the Agilent 1100 LC / MSD mass spectrometer (Agilent, USA) and Q-tofmicro MS
(
micromass company). All reactions were monitored by TLC (Merck Kieselgel GF254) and spots were
visualized with UV light or iodine. The purity of biologically evaluated compounds was >95% as determined by
HPLC.
General Procedure A for Synthesis of Compound 2-7
4-Nitrobenzyl bromide (46.3 mmol) was dissolved in dichloromethane (100 mL). The solution was added to
the mixture of relative amine (47.0 mmol) and triethylamine (70.3 mmol) in dichloromethane (20 ml). The
reaction mixture was stirred at r.t. for 24 h and was extracted with dichloromethane (100 ml × 3). After
removal of the solvent, the residue was crystallized from ethanol, giving yellow powder. Compounds 2-7 were
used for further reaction without purification.
.
To a suspension of compounds 2-7 (36.2 mmol) in 95% ethanol (100 ml), 85% NH NH H O (362 mmol),
2
2
2
40, 45,
95% ethanol (100 ml) and iron (III) oxide hydroxide (FeO(OH)/C, 2.0 g) were added and heated to reflux
46
.
When TLC analysis showed complete conversion of the starting material, the reaction mixture was filtrated
through Cellit 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.
4-(morpholinomethyl)aniline (8). Compound 8 was prepared according to general procedure A on 36.2
mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound (6.0 g, 31
+
1
mmol, yield 86%). [M+H] : 193. H-NMR (DMSO-d ): 2.32 (s, 4 H), 3.24 (s, 4 H), 3.51 (s, 2 H), 4.92 (s, 2 H), 6.53
6
(d, J = 8.4 Hz, 2 H), 6.90 (d, 2 H, J = 8.4 Hz).
4-((4-methylpiperazin-1-yl)methyl)aniline (9). Compound 9 was prepared according to general procedure
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A on 36.2 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound
+
1
(
7.3 g, 34.2 mmol Yield 94%). [M+H] : 206. H-NMR (300 MHz, DMSO-d ): 2.11 (s, 3 H), 2.37 (br, 8 H), 3.50 (s,
6
2
H), 4.01 (s, 2 H), 7.54 (d, 2 H, J = 8.7 Hz), 8.23 (d, 2 H, J = 8.7 Hz).
Tert-butyl-4-(4-aminobenzyl)piperazine-1-carboxylate (10). Compound 10 was prepared according to
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general procedure A on 36.2 mmol scale. Purification by column chromatography (10% MeOH/DCM) yield the
+
1
title compound (8.2 g, 28.1 mmol Yield 78%). [M+H] : 292. H-NMR (300 MHz, DMSO-d ): 1.38 (s, 9 H), 2.24-
6
2.26 (m, 4 H), 3.26 (s, 4 H), 3.41 (s, 2 H), 4.92 (s, 2 H), 6.48-6.51 (d, 2 H, J = 8.7 Hz), 6.89-6.92 (d, 2 H, J = 8.7
Hz).
4
-((4-methyl-1,4-diazepan-1-yl)methyl)aniline (11). Compound 11 was prepared according to general
procedure A on 36.2 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title
+
1
compound (4.9 g, 23.1 mmol Yield 65%). [M+H] : 220. H-NMR (DMSO-d ): 1.64−1.69 (m, 2 H), 1.81−1.96 (m,
6
4
H), 2.09−2.15 (m, 2 H), 4.59 (d, 2 H, J = 6.72 Hz), 4.77 (m, 1 H, J = 7.05Hz), 7.28(d, 2 H, J = 8.22 Hz), 7.49 (d, 2
H, J = 8.22 Hz), 8.26 (s, 1 H),8.83 (t, 1 H, J = 6.72 Hz).
4-((diethylamino)methyl)aniline (12). Compound 12 was prepared according to general procedure A on
3
1
3
6.2 mmol scale. Purification by column chromatography (3 % MeOH/DCM) yield the title compound (2.9 g,
+
1
7.1 mmol Yield 46%). [M+H] : 179. H-NMR (DMSO-d ): 1.04−1.09 (t, 6 H), 2.61-2.69 (q, 4 H, J = 6.72 Hz),
6
.65 (s, 2 H), 5.10 (br, 2 H), 6.52-6.55 (d, 2 H, J = 8.31 Hz), 7.03-7.07 (d, 2 H, J = 8.22 Hz).
4
-((cyclopropylamino)methyl)aniline (13). Compound 13 was prepared according to general procedure A
on 36.2 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound (3.1
+
1
g, 19.8 mmol Yield 54%). [M+H] : 163. H-NMR (DMSO-d ): 0.05-0.14 (m, 4 H), 1.76−1.83 (m, 2 H), 3.00 (s, 1
6
H), 4.64 (d, 2 H, J = 6.72 Hz), 6.26-6.29 (d, J = 8.22 Hz, 2 H), 6.71-6.75 (d, 2 H, J = 8.22 Hz).
General Procedure B for Synthesis of Compound 20-25
The mixture of compound 8-13 (1 eq., 18.5 mmol), 4-Nitro-1H-pyrazole-3-acid (1.1equiv, 20.4mmol), EDC
(
1.2equiv, 22.2mmol), HOBT (1.2equiv, 22.2mmol) in DMF (50 ml) was stirred for 24 hours. The ice water (100
ml) added to the reaction mixture. A large amount of yellow solid precipitation (compound 3a-f) was
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acquired. 14-19 were used without further purification. Compounds 20-25 were reduced by the same process
of compound 8-13, then the resulting compound 20-25 were purified by column chromatography on silica gel,
eluted with the appropriate solvent.
4-amino-N-(4-(morpholinomethyl)phenyl)-1H-pyrazole-3-carboxamide (20). Compound 20 was prepared
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according to general procedure B on 18.5 mmol scale. Purification by column chromatography (5%
+
1
MeOH/DCM) yield the title compound (3.6 g, 12.0 mmol Yield 65%). [M+H] : 302. H-NMR (300 MHz, DMSO-
d6) δ: 2.51 (s, 4 H), 3.42 (s, 2 H), 3.61 (s, 4 H), 4.71 (s, 2 H), 7.21-7.26 (m, 3 H), 7.72-7.58 (d, 2 H, J = 8.4 Hz), 9.7
(
s, 1 H), 12.7 (s, 1 H);
-amino-N-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrazole-3-carboxamide (21). Compound 21
was prepared according to general procedure B on 18.5 mmol scale. Purification by column chromatography
4
+
1
(
5% MeOH/DCM) yield the title compound (4.1 g, 15.4 mmol Yield 76%). [M+H] : 315. H-NMR (300 MHz,
DMSO-d ) δ: 2.13 (s, 3 H), 2.38 (s, 8 H), 3.43 (s, 2 H), 4.71 (s, 2 H), 7.11-7.24 (m, 3 H), 7.71-7.81 (d, 2 H, J = 8.4
6
Hz), 9.72 (s, 1 H), 12.71 (s, 1 H).
Tert-butyl-4-(4-(4-amino-1H-pyrazole-3-carboxamido)benzyl)piperazine-1-carboxylate (22). Compound
2
2 was prepared according to general procedure B on 18.5 mmol scale. Purification by column
+
1
chromatography (5% MeOH/DCM) yield the title compound (1.8 g, 4 mmol Yield 24%). [M+H] : 401. H-NMR
300 MHz, DMSO-d ) δ: 1.39 (s, 9 H), 2.31 (s, 4 H), 3.43 (s, 2 H), 4.68 (s, 2 H), 7.17-7.20(d, 2 H, J = 7.71 Hz),
(
6
7
.22 (s, 1 H), 7.72-7.74 (d, 2 H, J = 7.95 Hz), 9.70 (s, 1 H), 12.75 (s, 1 H).
-amino-N-(4-((4-methyl-1,4-diazepan-1-yl)methyl)phenyl)-1H-pyrazole-3-carboxamide (23). Compound
3 was prepared according to general procedure B on 18.5 mmol scale. Purification by column
4
2
+
1
chromatography (5% MeOH/DCM) yield the title compound (1.3 g, 3.9 mmol Yield 21%). [M+H] : 329.3. H-
NMR (DMSO-d ): 1.66−1.73 (m, 2 H), 2.24 (s, 2 H), 2.51−2.64 (m, 8 H), 3.38 (s, 2 H), 4.73 (br, 2 H), 7.14 (s, 1 H),
6
7
.21-7.24 (d, 2 H, J = 8.37 Hz), 7.68-7.71 (d, 2 H, J = 8.40 Hz), 9.94 (s, 1H).
-amino-N-(4-((diethylamino)methyl)phenyl)-1H-pyrazole-3-carboxamide (24). Compound 24 was
4
prepared according to general procedure B on 18.5 mmol scale. Purification by column chromatography (5%
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+
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MeOH/DCM) yield the title compound (2.0 g, 6.8 mmol Yield 37%). [M+H] : 288. H-NMR (300 MHz, DMSO-
d6) δ: 1.06 (s, 6 H), 2.52 (s, 2 H), 2.61-2.69 (m, 4 H), 4.70 (d, 2 H, J = 6.72 Hz), 7.18 (s, 1 H, J = 7.05 Hz), 7.32 (d,
2
H, J = 8.22 Hz), 7.76 (d , 2 H, J = 8.22 Hz), 9.76 (s, 1 H ), 12.79 (s, 1 H).
-amino-N-(4-((cyclopropylamino)methyl)phenyl)-1H-pyrazole-3-carboxamide (25). Compound 25 was
prepared according to general procedure B on 18.5 mmol scale. Purification by column chromatography (5%
4
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
+
1
MeOH/DCM) yield the title compound (0.9 g, 3.3 mmol Yield 18%). [M+H] : 272. H-NMR (300 MHz, DMSO-
d6) δ: 0.34-0.36 (m, 4 H), 2.05 (s, 2 H), 3.69 (s, 2 H), 4.70 (s, 2 H), 7.17-7.26 (m, 3 H), 7.69-7.70 (d, 2 H, J = 4.5
Hz), 9.61 (s, 1 H), 12.74 (s, 1 H).
General Procedure C for Synthesis of Compound 27-62
Compound 20-25 (1 equiv.) and corresponding chorides (1.2 equiv.) in AcOH/H O:1/1 (20 ml) was heated to
2
50 °C. When TLC analysis showed complete conversion of the starting material, sodium hydroxide (2 equiv.)
was added to the mixture. Then, the reaction mixture was extracted with ethyl acetate. After removal of the
solvent, the residue was purified by column chromatography and the corresponding compound was obtained.
N-(4-(morpholinomethyl)phenyl)-4-(quinolin-4-ylamino)-1H-pyrazole-3-carboxamide (27). Compound 27
was prepared according to general procedure C on 0.5 mmol scale. Purification by column chromatography
(
5% MeOH/DCM) yield the title compound (77 mg, 0.18 mmol Yield 36%). yellow solid; m.p 175-279 °C. HPLC
1
analysis: retention time =5.168 min; peak area, 98.43%. H-NMR (300 MHz, DMSO-d ) δ: 2.35 (m, 4 H,), 3.42
6
(s, 2 H), 3.57 (m, 4 H) , 7.00 (d, 1 H, J = 4.8 Hz), 7.25-7.28 (d, J = 7.9 Hz, 2 H), 7.62 (t, J = 8.0Hz, 1 H), 7.69-7.72
(d, 2 H, J = 7.9 Hz), 7.76 (d, 2 H, J = 8.0 Hz), 7.90 (d, 1 H, J = 8.1 Hz), 8.13 (d, 1 H, J = 8.0 Hz), 8.23 (s ,1 H), 8.6
13
(
d, 1 H, J = 4.8 Hz), 9.9 (br, 1 H); C-NMR (75 MHz, DMSO-d ) δ: 25.08, 53.10, 62.03, 66.18, 100.71; 118.90,
6
1
2125.24, 125.87, 129.12, 129.31, 132.05, 132.95, 137.33, 145.56, 148.21, 150.78, 161.97, 175.20; HRMS-EI
+
m/z [M+H] calcd for C H N O : 429.2039, found: 429.2049.
2
4 25 6 2
4
-(isoquinolin-1-ylamino)-N-(4-(morpholinomethyl)phenyl)-1H-pyrazole-3-carboxamide (28). Compound 28
was prepared according to general procedure C on 0.5 mmol scale. Purification by column chromatography
(
5% MeOH/DCM) yield the title compound ( 116 mg, 0.27 mmol Yield 54%). white solid; m.p 285-287 °C. HPLC
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2
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2
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2
2
2
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3
3
3
3
3
3
3
3
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4
4
4
4
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5
5
5
5
5
5
5
5
5
5
6
1
analysis: retention time =4.109 min; peak area,100.00%. H NMR (300 MHz, DMSO-d ) δ: 2.38 (s, 4 H), 3.46 (s,
6
2
8
H), 3.59 (s, 4 H) , 7.19 (d, 1 H, J = 5.8 Hz) , 7.31 (d, 2 H, J = 8.1 Hz) , 7.75 (dd, 2 H, J = 9.1 Hz) , 7.85 (d, 3 H, J =
.1 Hz) , 8.03 (d, 1 H, J = 6.7 Hz) , 8.13 (d, 1 H, J = 5.7 Hz) , 8.66 (s, 1 H) , 10.30 (d, 2 H, J = 6.5 Hz) , 13.42 (s, 1
13
H); C-NMR (150 MHz, DMSO-d ) δ: 53.09, 62.01, 66.15, 112.04, 117.67, 119.39, 120.40, 120.87, 121.18,
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
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5
6
7
8
9
0
1
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7
8
9
0
1
25.84, 127.27, 127.36, 129.47, 130.38, 132.01, 136.68, 137.34, 141.24, 150.48, 163.39; HRMS-EI m/z [M+H]
calcd for C H N O : 429.2039, found: 429.2049.
+
24
24
6
2
4-((6-bromoquinolin-4-yl)amino)-N-(4-(morpholinomethyl)phenyl)-1H-pyrazole-3-carboxamide (29).
Compound 29 was prepared according to general procedure C on 0.5 mmol scale. Purification by column
chromatography (5% MeOH/DCM) yield the title compound ( 65 mg, 0.13 mmol Yield 26%). yellow solid; m.p
1
2
2
31-235 °C. HPLC analysis: retention time =4.360 min; peak area, 99.49%. H-NMR (300 MHz, DMSO-d ) δ:
6
.35 (s, 4 H), 3.43 (s, 2 H), 3.57 (s, 4 H), 6.83-6.86 (d, 1 H, J=5.13 Hz), 7.24-7.27 (d, 2 H, J= 8.25 Hz), 7.73-7.76
(
d, 2 H, J =8.16 Hz), 7.84 (s, 1 H), 8. 28 (s, 1 H), 8.47 (s, 1 H), 8.52 (s, 1 H), 9.38 (s, 1 H), 10.23 (s, 1 H), 13.65 (s,
13
1 H); C-NMR (150 MHz, DMSO-d ) δ: 53.16, 62.05, 66.22, 101.52, 118.03, 120.27, 120.48, 122.84, 123.51,
6
123.89, 129.34, 131.51, 132.44, 132.98, 136.30, 137.36, 146.50, 146.91, 151.30, 161.65; HRMS-EI m/z [M+H]
+
calcd for C H BrN O : 507.1144, found: 507.1143.
24 24 6 2
4
-((7-Methoxyquinolin-4-yl)amino)-N-(4-(morpholinomethyl)phenyl)-1H-pyrazole-3-carboxamide (30).
Compound 30 was prepared according to general procedure C on 0.5 mmol scale. Purification by column
chromatography (5% MeOH/DCM) yield the title compound ( 49 mg, 0.11 mmol Yield 21%). yellow solid; m.p
1
1
2
85-187 °C. HPLC analysis: retention time = 3.516 min; peak area, 100.00%. H-NMR (300 MHz, DMSO-d ) δ:
6
.34 (s, 4 H), 3.42 (s, 2 H), 3.57 (s, 4 H), 3.91 (s, 3 H), 6.88 (d, 1 H, J = 4.2 Hz), 7.28 (s, 4 H), 7.75 (d, 1 H, J =7.5
13
Hz), 8.02 (d, 1 H, J =8.7 Hz), 8.35 (s, 1 H), 8.49 (d, 1 H, J = 4.2 Hz), 9.64 (s, 1 H), 10.31 (s, 1 H), 13.6 (s, 1 H); C-
NMR (75 MHz, DMSO-d ) δ: 21.20, 53.01, 55.27, 61.95, 66.10, 99.63, 107.64, 113.18, 117.31, 120.20, 120.51,
6
1
+
21.66, 125.31, 129.17, 133.08, 133.79, 137.00, 145.94, 149.60, 150.74, 160.00, 162.04; HRMS-EI m/z [M+H]
calcd for C H N O : 459.2145, found: 459.2134.
25
27
6
3
N-(4-(Morpholinomethyl)phenyl)-4-(quinazolin-4-ylamino)-1H-pyrazole-3-carboxamide (31). Compound 31
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was prepared according to general procedure C on 0.5 mmol scale. Purification by column chromatography
(
5% MeOH/DCM) yield the title compound ( 120 mg, 0.28 mmol Yield 56%). yellow solid; m.p 215-217 °C.
1
HPLC analysis: retention time = 4.234 min; peak area, 99.18%. H-NMR (300 MHz, DMSO-d ) δ: 2.33 (s, 4 H),
6
3
7
.17 (s, 2 H), 3.41 (s, 2H), 3.56 (s, 4 H), 7.27-7.29 (d, 2 H, J = 7.86 Hz), 7.71-7.73 (t, 1 H), 7.79-7.88 (m, 4 H),
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
13
.98-8.00 (1 H, d, J = 7.98 Hz), 8.64 (s, 1 H), 8.73 (s, 1 H), 10.45 (s, 1 H), 10.63 (s, 1 H); C-NMR (150 MHz,
DMSO-d ) δ: 53.23, 62.15, 66.30, 114.65, 120.95, 121.04, 123.94, 127.27, 128.25, 129.29, 132.87, 133.29,
6
+
133.44, 137.04, 149.21, 154.91, 155.29, 163.00; HRMS-EI m/z [M+H] calcd for C H N O : 430.1991, found:
2
3 24 7 2
4
30.2006.
-((2-chloroquinazolin-4-yl)amino)-N-(4-(morpholinomethyl)phenyl)-1H-pyrazole-3-carboxamide (32).
4
Compound 32 was prepared according to general procedure C on 0.5 mmol scale. Purification by column
chromatography (5% MeOH/DCM) yield the title compound ( 54 mg, 0.12 mmol Yield 23%). yellow solid; m.p
1
2
35-237 °C. HPLC analysis: retention time = 3.758 min; peak area, 99.00%. H-NMR (300 MHz, DMSO-d ) δ:
6
2.36 (s, 4 H), 3.45(s, 2 H), 3.60 (s, 4 H), 7.28-7.31(d, 2 H, J = 8.28 Hz), 7.72-7.81(m, 4 H), 7.90-7.95 (s, 1 H, J =
13
7.35 Hz), 8.0-8.1(d, 1 H, J = 8.01 Hz), 8.47 (s, 1 H), 10.42 (s, 1 H), 10.87(s, 1 H), 13.62 (s, 1 H); C-NMR (75 MHz,
DMSO-d ) δ: 162.86, 156.82, 156.23, 150.36, 136.82, 134.21, 133.42, 129.13, 127.45, 127.18, 122.83, 121.58,
6
+
1
20.87, 113.19, 66.17, 62.00, 53.10; HRMS-EI m/z [M+H] calcd for C H ClN O : 464.1602, found: 464.1602.
23
23
7 2
N-(4-(morpholinomethyl)phenyl)-4-((2-(trifluoromethyl)quinolin-4-yl)amino)-1H-pyrazole-3-carboxamide
33). Compound 33 was prepared according to general procedure C on 0.5 mmol scale. Purification by
(
column chromatography (5% MeOH/DCM) yield the title compound (46 mg, 0.09 mmol Yield 19%). yellow
1
solid; m.p 215-218 °C. HPLC analysis: retention time = 4.386 min; peak area, 100.00%. H-NMR (300 MHz,
DMSO-d ) δ: 2.45 (s, 4 H), 3.42 (s, 2 H), 3.63 (s, 4 H), 7.18-7.20 (d, 1 H, J = 5.73 Hz), 7.34-7.36 (d, 2 H, J = 6.63
6
Hz), 7.73-7.76 (m, 2 H), 8.01-8.04 (d, 1 H, J = 6.57 Hz), 8.12-8.14 (d, 1 H, J = 5.73 Hz), 8.66 (s, 1 H), 10.28 (s, 1
1
3
H), 10.33 (s, 1 H), 13.48 (s, 1 H); C-NMR (75 MHz, DMSO-d ) δ: 51.99, 52.82, 65.86, 111.89, 117.55, 119.26,
6
1
20.71, 121.05, 125.70, 127.10, 127.20, 129.23, 129.49, 130.20, 136.56, 137.33, 141.09, 150.37,163.26;
+
HRMS-EI m/z [M+H] calcd for C H F N O : 497.1913, found: 497.1921.
2
5 24 3 6 2
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