Design, synthesis, biological activity evaluation of 3-(4-phenyl-1H-imidazol-2-yl)-1H-pyrazole derivatives as potent JAK 2/3 and aurora A/B kinases multi-targeted inhibitors

Article abstract of DOI:10.1016/j.ejmech.2020.112934

In this study, a series of 3-(4-phenyl-1H-imidazol-2-yl)-1H-pyrazole derivatives were designed, synthesized, and evaluated for their biological activities. Upon performing kinase assays, most of the compounds exhibited potent inhibition against JAK2/3 and Aurora A/B with the IC₅₀ values ranging from 0.008 to 2.52 μM. Among these derivatives, compound 10e expressed the most moderate inhibiting activities against all the four kinases with the IC₅₀ values of 0.166 μM (JAK2), 0.057 μM (JAK3), 0.939 μM (Aurora A), and 0.583 μM (Aurora B), respectively. Moreover, most of the derived compounds exhibited potent cytotoxicity against human chronic myeloid leukemia cells K562 and human colon cancer cells HCT116, while compound 10e expressed antiproliferative activities against K562 (IC₅₀₌6.726 μM). According to western blot analysis, compound 10e down-regulated the phosphorylation of STAT3, STAT5, Aurora A, and Aurora B in a dose-dependent manner in K562 and HCT116 cells. Cell cycle analysis revealed that compound 10e inhibited the proliferation of cells by inducing cell cycle arrest in the G2 phase. The molecular modeling suggested that compound 10e could maintain a binding mode similar to the binding mode of AT9832, a common JAK 2/3 and Aurora A/B kinases multi-target kinase inhibitor. Therefore, compound 10e might be a potential agent for cancer therapy deserving further research.

Full text of DOI:10.1016/j.ejmech.2020.112934

Journal Pre-proof
Design, Synthesis, Biological Activity Evaluation of 3-(4-Phenyl-1H-Imidazol-2-yl)-1H-
Pyrazole Derivatives as Potent JAK 2/3 and Aurora A/B Kinases Multi-targeted
Inhibitors
You-Guang Zheng, Jin-An Wang, Long Meng, Xin Pei, Ling Zhang, Lin An, Cheng-Lin
Li, Ying-Long Miao
PII:
S0223-5234(20)30906-5
DOI:
https://doi.org/10.1016/j.ejmech.2020.112934
EJMECH 112934
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 August 2020
Revised Date: 6 October 2020
Accepted Date: 9 October 2020
Please cite this article as: Y.-G. Zheng, J.-A. Wang, L. Meng, X. Pei, L. Zhang, L. An, C.-L. Li, Y.-L.
Miao, Design, Synthesis, Biological Activity Evaluation of 3-(4-Phenyl-1H-Imidazol-2-yl)-1H-Pyrazole
Derivatives as Potent JAK 2/3 and Aurora A/B Kinases Multi-targeted Inhibitors, European Journal of
Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112934.
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18Design,
Synthesis,
Biological
Activity
Evaluation
of
3-(4-Phenyl-1H-Imidazol-2-yl)-1H-Pyrazole Derivatives as Potent JAK
2/3 and Aurora A/B Kinases Multi-targeted Inhibitors
You-Guang Zhenga, b*, Jin-An Wangc, Long Menga, b, Xin Peia, b, Ling Zhanga, b, Lin
Ana, b, Cheng-Lin Lia, b, Ying-Long Miaoc.
aCollege of Pharmacy, Xuzhou Medical University, Xuzhou 221004, PR China;
bJiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou
Medical University, Xuzhou 221004, PR China;
c
Center for Computational Biology and Department of Molecular Biosciences, University of
Kansas, Lawrence, KS 66047, USA
.
Youguang Zheng, Jin-An Wang and Long Meng contributed equally to this work.
ABSTRACT
In this study, a series of 3-(4-phenyl-1H-imidazol-2-yl)-1H-pyrazole derivatives were
designed, synthesized, and evaluated for their biological activities. Upon performing
kinase assays, most of the compounds exhibited potent inhibition against JAK2/3 and
Aurora A/B with the IC50 values ranging from 0.008 to 2.52 µM. Among these
derivatives, compound 10e expressed the most moderate inhibiting activities against
all the four kinases with the IC50 values of 0.166 µM (JAK2), 0.057 µM (JAK3),
0.939 µM (Aurora A), and 0.583 µM (Aurora B), respectively. Moreover, most of the
derived compounds exhibited potent cytotoxicity against human chronic myeloid
leukemia cells K562 and human colon cancer cells HCT116, while compound 10e
expressed antiproliferative activities against K562 (IC50 = 6.726µM). According to
western blot analysis, compound 10e down-regulated the phosphorylation of STAT3,
STAT5, Aurora A, and Aurora B in a dose-dependent manner in K562 and HCT116
cells. Cell cycle analysis revealed that compound 10e inhibited the proliferation of
cells by inducing cell cycle arrest in the G2 phase. The molecular modeling suggested
that compound 10e could maintain a binding mode similar to the binding mode of
AT9832, a common JAK 2/3 and Aurora A/B kinases multi-target kinase inhibitor.
Therefore, compound 10e might be a potential agent for cancer therapy deserving
further research.

Keywords: JAK2/3, Aurora kinases, pyrazole derivatives, multi-targeted inhibitors
1. Introduction
The Janus kinases (JAKs) family, consisting of four structurally related kinases, JAK1,
JAK2, JAK3, and TYK2, regulates the JAK/STAT signaling pathway [1, 2]. The
JAK/STAT pathway plays a vital role in the oncogenesis in the immune system [3],
and mediates signaling by cytokines, which control survival, proliferation, and
differentiation of several cell types, such as human chronic myeloid leukemia K562
cells [4-6], human colorectal cancer HCT-116 cells [7-9] and other cancer cells
[10-14]. In recent years, the JAK kinase inhibitors that target the JAK/STAT pathway,
such as Ruxolitinib, has been approved by FDA [15], extensively studied [16-18] and
provided a great value for the treatment of myeloproliferative neoplasias and beyond
[19]. In addition, some JAK inhibitors have been developed and introduced into
clinical trials, such as AZD1480 [20], Fedratinib [21], AT9283 [22] and so on (Fig.1).
Fig.1 Chemical structures of JAK inhibitors.
Aurora kinases belong to serine/threonine kinases that are involved in chromosome
condensation, spindle dynamics, kinetochore-microtubule interactions, chromosome
orientation, and establishment of the metaphase plate during mitosis [23]. The family
includes three kinases designated as Aurora A, B, and C, which possess a very similar
sequence, particularly in the carboxy-terminal catalytic domain [24]. Overexpression
of aurora kinases in several human tumors has made them appealing targets for the
development of anticancer therapies [23-26]. Over the past decades, some Aurora
kinase inhibitors have been developed for the treatment of cancer, such as VX-680
[27], AT9283 [22], CYC-116 [28], AZD-1152 [29], AMG-900 [30], PHA-739358
[31], MLN8054 [32], MLN8237 [33] and ENMD2076 [34] ( Fig.2).

Fig.2 Chemical structures of Aurora inhibitors
Although recent studies have identified a significant number of single-targeted
selective kinase inhibitors and proven their efficacy in treatment for specific cancers
[35-37], the mortality rate caused by cancer remains high. Therefore, the
multi-targeted kinase inhibitors could exhibit more potent therapeutic effects by
synergistic effects as well as by the combination of various antitumor mechanisms
[38]. Meanwhile, studies have also confirmed that the Aurora kinase and JAKs
multi-target inhibitors can inhibit the growth of tumor cells more effectively than the
selective aurora kinase inhibitors [39]. AT9283 is a multi-target kinase inhibitor,
which can effectively inhibit the activities of Aurora A, Aurora B, JAK2, JAK3, and
ABL1 kinases [40]. According to the reported crystal structure study, AT9283
complexed with Aurora-A by forming hydrogen-bonds to the backbone carbonyl
groups of Glu211, as well as to the backbone NH and carbonyl groups of Ala213 of
the protein hinge region [40].
In this paper, retaining the hydrogen-bonded pharmacophore of AT9283, a series of
3-(4-phenyl-1H-imidazol-2-yl)-1H-pyrazole derivatives were rationally designed and
synthesized as JAK 2/3 and Aurora A/B kinases multi-target inhibitors.

O
HN
N
HN
N
N
N
R1
H
N
H
H
HN
N
HN
N
AT9283
N
R2
O
Fig.3 Design of novel s3-(4-phenyl-1H-imidazol-2-yl)-1H-pyrazole derivatives
2. Results and discussion
2.1. Chemistry
As depicted in Scheme 1, N-protection of 4-nitropyrazol-3-carboxylic acid (1) was
performed by treating with DHP reagent to obtain compound (2). Compound (6) was
synthesized from compound (3) by bromomethylation and N-alkylation, followed by
bromination of ketones at α-position. Compound (2) reacted with compound (6) to
produce compound (7), which was treated with ammonium acetate and acetic acid to
afford compound (8). Then after, compounds (8) were reduced to its amino
derivatives (9) using H2, and Pd/C. The target compounds (10) were prepared by
reacting compound (9) with various benzoic acids or chloropyrimidines.
Scheme 1. The synthesis of compounds 10a-10j. Reagents and conditions: (a) DHP, PTSA, THF;
(b) NBS, AIBN, Acetonitrile; (c) K2CO3, KI, Acetonitrile; (d) Br2, CH3COOH, HBr; (e) K2CO3,
KI, Acetonitrile; (f) CH3COONH4, CH3COOH, Reflux; (g) H2, Pd/C, CH3OH; (h) standard amide
and aminopyrimidine coupling methods (for specific reagents, see the experimental section)
The overall synthetic route of compounds 18a starting from 4-hydroxyacetophenone
(11), has been outlined in Scheme 2. Compounds (17) were obtained upon sequential

O-alkylation, N-alkylation, bromination of ketones at α position, followed by
esterification, cyclization, and reduction. Further, the target compounds (18a) were
prepared by treating compound (17) with different chloropyrimidines.
Scheme 2. The synthesis of compounds 18a. Reagents and conditions: (a) Trimethylene
chlorobromide, K2CO3, KI, Acetonitrile; (b)Morpholine, K2CO3, KI, Acetonitrile; (c) Br2,
CH3COOH, HBr; (d) Compound 2, K2CO3, KI, Acetonitrile; (e) CH3COONH4, CH3COOH,
Reflux; (f) H2, Pd/C, CH3OH; (g) Chlor pyrimidine, NaI, DIPEA, DMF.
2.2. Biological activities
The target compounds were evaluated for their enzymatic activities against Aurora A,
Aurora B, JAK2, and JAK3. Additionally, the in vitro antiproliferative effects of the
targeted compounds against human chronic myeloid leukemia cells K562 and human
colon cancer cells HCT116 were also carried out. The results were summarized in
Table 1.
2.2.1. In vitro inhibition activities studies of kinases
As shown in Table 1, most of the compounds (10a-10j) exhibited potent inhibition
against JAK2/3 and Aurora A/B with the IC50 values ranging from 0.008 to 2.52 µM.
Among these derivatives, compound 10e expressed the most moderate inhibiting
activities against all the four kinases with the IC50 values, 0.166 µM (JAK2), 0.057
µM (JAK3), 0.939 µM (Aurora A), and 0.583 µM (Aurora B), respectively.
Compounds (10b, 10d, 10e, and 10i) containing –Cl and–OCH3 groups at the benzene

ring of R2 exhibited selective inhibiting activities against JAKs (approximately
20-fold selectivity), while compound 10c bearing a nitro group at the benzene ring of
R2 showed pan-inhibiting activities against JAK2/3 and Aurora A/B. In addition,
replacement of the benzoyl group of R2 with a pyrimidine group (compound 10j)
increased the inhibition activities against JAK2/3 and Aurora A (JAK2 IC50
0.008µM, JAK3 IC50 = 0.023µM, and Aurora A IC50 = 0.145µM).
=
Table 1 Enzymatic activities and In vitro anti-proliferative activities in tumor cell
lines of compounds 10a-10j.
IC50(µM)
Compd
10a
R1
R2
Aurora Aurora
JAK2 JAK3
K562 HCT116
A
B
NT
NT
NT
NT
>10
>10
>10
10b
0.014
0.024
0.359
0.466
2.514
0.939
NT
>1
0.65
NT
5.652
10c
10d
10e
10f
10g
10h
1.178
0.094
0.166
NT
0.569
0.064
0.057
NT
4.34
2.63
6.726
NT
5.21
>10
0.583
NT
15.054
NT
NT
NT
1.523
2.055
NT
>10
>10
NT
NT
NT
>10
1.592

10i
10j
0.016
0.008
0.013
0.023
1.132
0.145
NT
2.503
>10
NT
10.048
0.748
>10
AT9283
0.0022 0.0012 0.026
0.062
0.09
NT (Not Test)
2.2.2. Antiproliferation assay
Most of our target compounds were tested against human chronic myeloid leukemia
cell lines (K562) and human colorectal cancer cell line (HCT116), in which
overexpression of JAKs and Aurora kinases remains prominent [40-43]. The results of
the antiproliferative activities are shown in Table 1 and Table 2. Most compounds
exhibit potent cytotoxicity against both of these tumor cell lines. In K562 cell lines,
compounds 10b-10e (IC50 values of 2.630–6.726 µM) having the morpholine ring at
the side chain shown more potent antiproliferation activity in comparison to the
compounds (10 g and 10 h) with piperidine ring (IC50 values >10 µM). It was noted,
compounds having –Cl, –OCH3, and–NO2 groups at the benzene ring (10b-10e)
exhibited significant improvement to their proliferative inhibition; compound 10c and
10d that contained electron-withdrawing groups (Cl and NO2) expressed slightly
higher K562 proliferative inhibition (the IC50 = 4.34 µM, 2.63 µM, respectively)
compare to 10b having an electron-donating group (CH3O-).
We also synthesized one compound bearing longer side chain at 4-position of the
benzene ring (18a). As indicated in Table 2, Extension of the side chain (compound
18a) displayed no significant change to cytotoxicity against K562 and HCT116.
Compound 10e, which expressed the most potent inhibition on kinases and cancer
cells, was selected for further study.
Table 2 Enzymatic activities and In vitro anti-proliferative activities in tumor cell
lines of compounds 18a.

IC50(µM)
Compd
18a
R3
Aurora Aurora
JAK2 JAK3
K562 HCT116
A
B
NT
NT
NT
NT
9.88
16.14
0.09
AT9283
0.0022 0.0012 0.026
0.062
0.748
NT (Not Test)
In addition, in vitro anti-proliferative activities of compound 10e against human
normal colon epithelial cells (HCoEpiC) and human umbilical vein endothelial cells
(HUVEC) had been tested (Table 3). The result showed that 10e has significantly
lower toxic effect on HCoEpiC (IC50=31.509µM) and HUVEC (IC50=28.978µM) than
AT9283 (IC50=2.367µM, 1.793µM, respectively).
Table 3 In vitro anti-proliferative activities of compound 10e against normal cells
(IC50 µM)
Compound
10e
HCoEpiC
31.509
2.367
HUVEC
28.978
1.793
AT9283
2.2.3 Compound 10e blocks JAKs and Aurora kinases phosphorylation in K562 and
HCT116 cell
Western blot analysis was performed to evaluate the performance of compound 10e in
a cellular context according to the published research works [44-47]. The
phosphorylation of Aurora A, Aurora B, STAT3, and STAT5 was investigated in
K562 and HCT116 cells by treating with various concentrations of compound 10e
(control, 1, 10, and 20 µM). The results demonstrated that multi-target kinase
inhibitor 10e also led to decreased levels of p-STAT3 and STAT5, which could be
correlated with JAKs inhibition, indicating the inhibition of intracellular JAK-STATs

pathway. Furthermore, compound 10e down-regulated the phosphorylation of Aurora
A and Aurora B in a dose-dependent manner in K562 and HCT116 cells, respectively
(Fig. 4). According to the above results, compound 10e could be considered as a
potential JAKs and Aurora kinases inhibitor.
Fig. 4 Western blot analysis of p-Aurora A, p-Aurora B, p-STAT3 and p-STAT5 with
various concentrations of compound 10e in K562 and HCT116 cells. GAPDH was
used as a loading control.
2.2.4 Cell cycle arrest
As per the previous reports, the JAK-STATs pathway and Aurora kinases could play
important roles in controlling cell cycle progression [48, 49]. AT9283, which is a
multi-target kinase inhibitor, induces common cancer cell-cycle arrest at the G2 phase
[50, 51]. Accordingly, we tested the effects of 10e on cell-cycle progression in K562
and HCT116 cells using flow cytometry. As shown in Fig. 5, a dose-dependent G2
phase transition arrest was observed in K562 and HCT116 cells when treated with 10e.

Exposure of compound 10e in K562 cell at 10 µM concentration resulted in 16.27%
of cell arrest at the G2 phase, compared with 7.73% in untreated cultures and 14.99%
in AT9283 (10 µM) treated cultures (Fig. 5A). On the other hand, exposure of
compound 10e in HCT116 cells at 10 µM resulted in 16.16% of cell arrest at the G2
phase, while the values for cell arrest in untreated cultures and in AT9283 (10 µM)
treated cultures were 10.46% and 25.19%, respectively (Fig. 5B).
Fig. 5 compound 10e induced cell cycle arrest at G2 phase in K562 and HCT116
cells.
2.3 Binding mechanism of 10e to the JAK2, JAK3, Aurora A and Aurora B

Considering the above-mentioned integrated molecular modeling approach, RMSDs
of kinase backbone atoms (Fig S3) and ligand heavy atoms (Fig S4) of the initial
structures were calculated to assess the stability of the complex. In comparison to
AT9832, the binding of 10e induced large fluctuations of kinases (Fig S3), indicating
that the binding of 10e was less stable than AT9832, and also the stability of 10e in
these kinases was less than that of AT9832 (Fig S4). The above results were very
consistent with our experimental data, which represented the lower binding affinities
of 10e (Table 4). These results encouraged us to explore the binding mechanism of
10e further, by analyzing the MD trajectories.
To explore the binding mechanism of 10e to kinases, MM-GBSA was employed to
calculate the binding affinities of both 10e and AT9832 with these kinases. The
information on binding free energy obtained from MM-GBA calculations is given in
Tables 4. The calculated differences of binding free energy between AT9832 and 10e
to JAK2, JAK3, Aurora A, and Aurora B were –2.85, –3.54, –1.71, and –1.61
kcal/mol, which were very consistent with the corresponding experimental data (-2.57,
–2.30, –2.13 and –1.33 kcal/mol, respectively) (Table 4). The calculated difference of
binding free energy between AT9832 and 10e correlated properly to the experimental
data (R2 = 0.48, Fig S5). Thus, our current analysis of binding free energies could be
considered as reliable.
As shown in Table 4, the van der Waals interactions (∆Evdw), electrostatic
interactions (∆Eele), and nonpolar solvation energies (∆Gsurf) were favorable for the
binding of the ligands to kinases, while polar solvation energies (∆Ggb) heavily
impaired the bindings. The van der Waals interaction was the main driving force for
the binding of the inhibitors to kinases, which was consistent with the results of our
previous simulations[52]. However, the electrostatic interactions (∆Eele+∆Ggb)
between AT9832 and 10e seemed to differ mostly. The differences of electrostatic
interaction between AT9832 and 10e to the JAK2, JAK3, Aurora A, and Aurora B
were –11.48, –2.57, –1.62, and –0.61 kcal/mol. Thus, less electrostatic interactions of
10e largely impaired the binding affinity of 10e.

Table 4 Binding free energies of ligands (10e and AT9283) to JAK2, JAK3, Aurora
A and Aurora B calculated using MM-GBSA method. The unit for binding free
energy is kcal/mol.
∆Gexpa
∆Gcalb
-42.28
-45.13
-38.35
-41.88
-40.72
-42.43
-41.31
-42.92
∆Evdw
-52.70
-44.92
-42.65
-43.45
-43.40
-43.48
-45.05
-47.41
∆Eele
-16.46
-27.3
∆Egb
33.08
32.44
32.44
34.20
31.52
37.19
36.89
33.35
∆Esurf
-6.20
-5.34
-4.99
-5.16
-5.19
-5.2
∆∆Gcalc
∆∆Gexpd
∆Eele+∆Egb
JAK2
(2W1I)
JAK3
-9.30
10e
AT9283
10e
-2.57
-2.85
-11.48
-11.87
-9.94
-23.14
-27.47
-23.63
-30.92
-26.13
-23.19
-2.30
-2.13
-1.33
-3.54
-1.71
-1.61
-2.57
-1.62
-0.61
(3ZEP)
Aurora A
(2W1G)
Aurora B
(4AF3)
AT9283
10e
-12.24
-8.27
AT9283
10e
-10.40
-8.55
-5.2
AT9283
-9.89
-5.71
a The experimental values were derived from the experimental ki values using the
equation ∆Gexp = −RTlnIC50.
b ∆Gcal = ∆Eele + ∆Evdw + ∆Ggb + ∆Gsurf.
c∆∆Gcal=∆Gcal(AT9283)- ∆Gcal(10e).
d∆∆Gexp=∆Gexp(AT9283)- ∆Gexp(10e).
Next, snapshots from the last 150 ns trajectories were used for clustering to obtain a
representative binding pose. As shown in Fig 6, the hydrogen bonds formed between
the hinge region of the kinases and the compounds (AT9832 and 10e) were the main
interactions between ligands and kinases. For most of the kinases (JAK3, Aurora A,
and Aurora B), the residues in the hinge could form three important hydrogen bonds
between the ligands and the kinases. However, smaller distance and larger occupancy
of these hydrogen bonds were identified in AT9832 than that of 10e (Fig 6 and Table
S1), suggesting that the interactions of AT9832 to the kinases were stronger than that
of 10e to the same kinases. In the case of JAK2, AT9832 formed three strong
hydrogen bonds with the residues E930 and L932 in the hinge region with distances

of 2.8, 3.0, and 2.8 Å (Fig 6A), respectively. However, 10e could form only one
hydrogen bond between the residues E930 in the hinge region with a distance of 3.2 Å
(Fig 6B). 10e could form two other weaker hydrogen bonds to residues G993 and
S936 of JAK2 (Fig 6B and Table S1). In summary, the current molecular modeling
suggested that the 10e could maintain a binding mode similar to AT9832, although it
has a less binding affinity.

Fig 6 Binding pose of ligands (AT9283 and 10e) to JAK2 (2W1I), JAK3 (3ZEP),
Aurora A (2W1G) and Aurora B (4AF3), respectively. The important hydrogen bonds
were plotted as red lines. The ligands and key residues were displayed in stick modes.
The carbon atoms of AT9283 and 10e were colored in green and yellow, respectively.

3. Conclusions
In summary, we designed and synthesized 3-(4-phenyl-1Himidazol-2-yl)-1H-pyrazole
derivatives as potent JAK 2/3 and aurora A/B kinases multi-targeted inhibitors.
Most of the compounds exhibited potent inhibition against JAK2/3 and Aurora A/B
with the IC50 values ranging from 0.008 to 2.52 µM. Among these derivatives,
compound 10e expressed the most moderate inhibiting activities against all the four
kinases with the IC50 values, 0.166 µM (JAK2), 0.057 µM (JAK3), 0.939 µM (Aurora
A), and 0.583 µM (Aurora B), respectively. Additionally, anti-proliferation assays
shown that the compounds having a morpholine ring at the side chain (10b-10e)
exhibited higher anti-proliferative activities against K562 and HTC116. In addition,
10e has significantly lower toxic effect on human normal cells than JAK 2/3 and
Aurora A/B kinases multi-target kinase inhibitor (AT9832). Furthermore, compound
10e down-regulated the phosphorylation of STAT3, STAT5, Aurora A, and Aurora B
in K562 and HCT116 cells. Cell cycle analysis revealed that compound 10e inhibited
the proliferation of cells by inducing cell cycle arrest in the G2 phase. The molecular
modeling suggested that the 10e could maintain a binding mode similar to AT9832,
although it had a less binding affinity. Overall, compound 10e is a potential anticancer
agent that exhibits potent JAK 2/3 and aurora A/B kinases multi-targeted inhibitors.
4. Experimental
4.1. Chemistry
Unless otherwise noted, all reagents were purchased from commercial sources and
used without further purification. All compounds were routinely monitored by
thin-layer chromatography with silica gel GF-254 glass plates and viewed under UV
light at 254 nm. The 1H-NMR and 13C-NMR spectra were determined in DMSO-d6 on
a JNM-ECZ400s/L spectrometer. Chemical shifts (δ) were reported in parts per
million (ppm) relative to tetramethylsilane (TMS), which was used as an internal
standard. The mass spectra (MS) were obtained from Agilent 1100LC/MS
Spectrometry Services. HR-MS was obtained using a Q-tof high resolution mass
spectrometer. The purity was obtained using Agilent 1260 HPLC instrument with
ZORBAX SB-C18 chromatographic column. Melting point was obtained using X-4A

melting point analyzer.
4.1.1 4-nitro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-3-carboxylic acid (2)
To a solution of 4-nitropyrazole-3-carboxylicacid (5.0 g，31.8 mmol) in THF (40 mL)
was added 3,4-dihydro-2H-pyran (DHP, 8.04 g，95.5 mmol) and p-toluenesulfonic
acid monohydrate (PTSA, 0.30 g，0.15 mmol). The reaction mixture was stirred at RT
and monitored by thin layer chromatography. After completion of the reaction, the
solvent was removed under vacuum and the product was dissolved in water (30 mL),
and washed with acetic ether (3×20 mL). The organic layer was washed with saline
(50 mL) and was dried (anhydrous sodium sulfate). The solvent was removed under
vacuum and the crude product was purified by chromatographic column to afford 7.29
g compound 2. Light yellow oil, yield: 95%.1H NMR (400 MHz, CDCl3-d6) δ (ppm)：
8.50 (s, 1H), 5.48 (d, J = 8.0 Hz, 1H), 4.11 (d, J = 12.0 Hz, 1H), 3.74 (t, J = 12.0 Hz,
1H), 2.29 (d, J = 11.8 Hz, 1H), 1.99 (s, 1H), 1.87 (d, J =8.0 Hz, 1H), 1.68 (s, 3H).
4.1.2 1-(4-(bromomethyl)phenyl)ethan-1-one (4)
The mixture of 4'-methylacetophenone (10 mL, 74.5 mmol), NBS(14.6 g, 82.0 mmol),
o
AIBN(1.23 g, 7.49 mmol) and 80 mL acetonitrile was stirred at 90 C under argon
atmosphere for 4 h, then concentrated under vacuum to give a residue which was
dissolved in water (30 mL) and washed with dichloromethane (3×50 mL). The
dichloromethane solution was washed with 1 M hydrochloric acid solution (50 mL),
saturated sodium bicarbonate solution and brine (3×50 mL), dried over anhydrous
sodium sulfate and concentrated. The crude product was recrystallized from ethyl
acetate (25.5 mL) and petroleum ether (255 mL) to afford 15.1g pure product. White
solid, yield: 95%. 1H NMR (400 MHz, CDCl3-d6) δ (ppm)：7.92 (d, J = 8.1 Hz, 2H),
7.47 (d, J = 8.0 Hz, 2H), 4.49 (s, 2H), 2.60 (s, 3H).
4.1.3 General procedure for the preparation of the compounds (5)
To a solution of compound 4 (31.8 mmol) in acetonitrile (30 mL) was added
potassium carbonate (23.5 mmol), catalytic amount of potassium iodide and different
secondary amines (23.5 mmol). The reaction mixture was stirred at 90 oC for 4h, then
concentrated under vacuum to give a residue which was purified by chromatographic
column.

4.1.3.1 1-(4-(morpholinomethyl)phenyl)ethan-1-one (5a)
Yield: 90%. 1H NMR (400 MHz, CDCl3-d
) δ (ppm)：7.88 (d, J = 7.9 Hz, 2H), 7.41 (d,
6
J = 7.8 Hz, 2H), 3.68 (s, 4H), 3.52 (s, 2H), 2.56 (s, 3H), 2.42 (s, 4H). HR-MS
(calculated for C13H18NO2 (M+H)+ 220.1337; found 220.1334) (Figure 44).
4.1.3.2 1-(4-(piperidin-1-ylmethyl)phenyl)ethan-1-one (5b)
Yield: 90%. 1H NMR (400 MHz, CDCl3-d
) δ (ppm)：7.89 (d, J = 8.1 Hz, 2H), 7.41 (d,
6
J = 8.0 Hz, 2H), 3.51 (s, 2H), 2.57 (s, 3H), 2.37 (s, 4H), 1.54 – 1.60 (m, 4H), 1.43 (d,
J = 4.0 Hz, 2H). HR-MS (calculated for C14H20NO (M+H)+ 218.1545; found
218.1542)
4.1.3.2 1-(4-(pyrrolidin-1-ylmethyl)phenyl)ethan-1-one (5c)
Yield: 84%. 1H NMR (400 MHz, CDCl3-d
) δ (ppm)：7.88 (d, J = 7.9 Hz, 2H), 7.41 (d,
6
J = 7.9 Hz, 2H), 3.65 (s, 2H), 2.57 (s, 3H), 2.50 (s, 4H), 1.77 (s, 4H). HR-MS
(calculated for C13H18NO (M+H)+ 204.1388; found 204.1381).
4.1.4 General procedure for the preparation of the compounds (6)
To a solution of compound 5 (0.48mmol) in 48% HBr solution (2 mL) was added 0.96
o
mL bromine (0.5M in acetic acid) at 60 C. After completion of the reaction, the
solvent was removed under vacuum and the product was used for the next step
without being purified.
4.1.4 General procedure for the preparation of the compounds (7)
To a solution of compound 2 (10 mmol), potassium carbonate (10 mmol) and catalytic
amount of potassium in acetonitrile (10 mL) was added compound 6 (10 mmol) at RT.
The mixture was stirred overnight, then filtered. The filtrate was removed under
vacuum and the crude product was purified by chromatographic column to afford
compound 7.
4.1.4.1
4-nitro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrrole-3-carboxylate (7c)
Yield: 63%. 1H NMR (400 MHz, CDCl3-d
) δ (ppm)：8.42 (s, 1H), 7.91 (d, J = 7.6 Hz,
2-oxo-2-(4-(pyrrolidin-1-ylmethyl)phenyl)ethyl
6
2H), 7.53 (d, J = 7.6 Hz, 2H), 5.61 (s, 2H), 5.46 (d, J = 9.2 Hz, 1H), 4.09 (d, J = 11.6
Hz, 1H), 3.79 (s, 2H), 3.70 – 3.75 (m, 1H), 2.65 (s, 4H), 2.23-2.27 (m, 1H), 1.85 –
2.03 (m, 6H), 1.68 (s, 3H). HR-MS (calculated for C22H27N4O6 (M+H)+ 443.1930;

found 443.1921).
4.1.5 General procedure for the preparation of the compounds (8)
To a solution of compound 7 (10 mmol) in acetic acid (30 mL) was added ammonium
o
acetate (120 mmol). The mixture was stirred 130 C for 4 h. Then the solvent was
removed under vacuum and the residue was poured slowly into saturated sodium
bicarbonate solution while stirring constantly. The solid formed was filtered off and
purified by chromatographic column to afford compound 8.
4.1.5.1 4-(4-(2-(4-nitro-1H-pyrazol-3-yl)-1H-imidazol-4-yl)benzyl)morpholine (8a)
1
Yellow solid, Yield: 46.2%. H NMR (400 MHz, DMSO-d
6
) δ (ppm)：8.59 (s, 1H),
7.78 (s, 3H), 7.29 (d, J = 4.0 Hz, 2H), 3.55 (s, 4H), 3.44 (s, 2H), 2.34 (s, 4H). HR-MS
(calculated for C17H19N6O3 (M+H)+ 355.1518; found 355.1512)
4.1.6 General procedure for the preparation of the compounds (9)
A solution of compound 8 (4 mmol) and 10% Pd/C (1.3mmol) in 40mL methanol
o
under hydrogen was stirred at 40 C for 12 h. Then the mixture was cool to RT, then
filtered. The filtrate was removed under vacuum and the crude product was used for
next step without being purified.
4.1.7 General procedure for the preparation of the target compounds (10a-10i)
To a solution of compound 9 (1.67 mmol) in dichloromethane (10 mL) was added
triethylamine (2 mmol), then added the solution of acyl chloride (2 mmol) in
o
dichloromethane (5 mL) drop by drop at 0 C. The mixture was heated to RT and
stirred for 4h. After completion of the reaction, the solvent was removed under
vacuum and the residue was dissolved in water (10 mL) and extracted with acetic
ether (3×20 mL). The acetic ether solution was washed with brine (50 mL), dried
over anhydrous sodium sulfate and concentrated. The crude product was purified by
chromatographic column to afford the target compounds.
4.1.7.1
N-(3-(4-(4-(morpholinomethyl)phenyl)-1H-imidazol-2-yl)-1H-pyrazol-4-yl)benzamide
(10a)

1
While solid, yield 31.3%, Mp 220.7-221.3oC. H NMR (400 MHz, DMSO-d6) δ
(ppm)： 11.55 (s, 1H), 8.29 (s, 1H), 8.04 (d, J = 7.6 Hz, 2H), 7.81 (d, J = 7.4 Hz, 2H),
7.59-7.67 (m, 4H), 7.33 (d, J = 7.4 Hz, 2H), 3.56 (s, 4H), 3.46 (s, 2H), 2.34 (s,
4H);13C NMR (100 MHz, DMSO-d6) δ (ppm)：163.23, 142.61, 140.34, 134.55,
134.42, 132.56, 131.95, 129.89, 129.51, 127.43, 127.30, 124.95, 124.72, 120.16,
120.03, 66.49, 62.62, 53.44; HR-MS (calculated for C24H25N6O2 (M+H)+ 429.2039;
found 429.2028); purity: 96.84%.
4.1.7.2
4-methoxy-N-(3-(4-(4-(morpholinomethyl)phenyl)-1H-imidazol-2-yl)-1H-pyrazol-4-yl
)benzamide (10b)
1
While solid, yield 14.5 %, Mp 283.6-284.0oC. H NMR (400 MHz, DMSO-d6) δ
(ppm)：12.98 (s, 1H), 12.97 (s, 1H), 11.42 (s, 1H), 8.26 (s, 1H), 7.99 (d, J = 8.8
Hz,2H), 7.81 (d, J = 8.4 Hz, 2H), 7.67 (s, 1H), 7.35 (d, J = 8.0 Hz, 2H), 7.12 (d, J =
13
8.8 Hz, 2H), 3.87 (s, 3H), 3.55 (t, J = 4.0 4H), 3.47 (s, 2H), 2.35 (s, 4H); C NMR
(100 MHz, DMSO-d6) δ (ppm)：162.91, 162.67, 140.39, 136.61, 133.44, 129.86,
129.28, 129.12, 127.97, 126.70, 124.76, 120.41, 114.67, 113.64, 109.79, 66.77, 62.74,
56.05, 53.71; HR-MS (calculated for C25H27N6O3 (M+H)+ 459.2144; found 459.2137);
purity:97.83%.
4.1.7.3
4-methoxy-N-(3-(4-(4-(morpholinomethyl)phenyl)-1H-imidazol-2-yl)-1H-pyrazol-4-yl
)benzamide (10c)
1
Yellow solid, yield 26.6 %, Mp 273.8-274.1oC. H NMR (400 MHz, DMSO-d6) δ
(ppm)：13.09 (s, 1H), 12.96 (s, 1H), 11.71 (s, 1H), 8.39 (d, J = 8.4 Hz, 1H), 8.32 (s,
1H), 8.24 (d, J = 8.4 Hz, 1H), 7.78 (d, J = 7.6 Hz, 2H), 7.67 (s, 1H), 7.32 (d, J = 7.6
13
Hz, 2H), 3.56 (s, 4H), 3.46 (s, 2H), 2.35 (s, 4H); C NMR (100 MHz, DMSO-d6) δ
(ppm)：161.63, 149.99, 142.41, 140.47, 140.09, 136.64, 133.32, 132.17, 129.77,
128.98, 124.86, 124.61, 120.47, 119.79, 113.71, 66.51, 62.90, 53.72; HR-MS
(calculated for C24H24N7O4 (M+H)+ 474.1890; found 474.1881); purity: 95.72%.
4.1.7.4
4-chloro-N-(3-(4-(4-(morpholinomethyl)phenyl)-1H-imidazol-2-yl)-1H-pyrazol-4-yl)b

enzamide (10d)
1
While solid, yield 23.6 %, Mp 296.7-297.1oC. H NMR (400 MHz, DMSO-d6) δ
(ppm)：11.52 (s, 1H), 8.28 (s, 1H), 8.03 (d, J = 8.4 Hz, 2H), 7.78 (d, J = 8.0 Hz,2H),
7.66 (d, J = 8.0 Hz, 3H), 7.35 (d, J = 8.0 Hz, 2H), 3.56 (s, 4H), 3.47(s,2H), 2.35 (s,
4H); 13C NMR (100 MHz, DMSO-d6 ) δ (ppm)：162.28, 142.65, 140.45, 137.35,
133.44, 133.28, 131.98, 129.91, 129.55. 129.32, 129.21, 124.79, 120.24, 120.00,
113.62, 66.63, 62.80, 53.67; HR-MS (calculated for C24H24ClN6O2 (M+H)+ 463.1649;
found 463.1639) ; purity: 96.33%.
4.1.7.5
3-chloro-N-(3-(4-(4-(morpholinomethyl)phenyl)-1H-imidazol-2-yl)-1H-pyrazol-4-yl)b
enzamide (10e)
1
While solid, yield 27.3 %, Mp 266.4-266.8oC. H NMR (400 MHz, DMSO-d6) δ
(ppm)：11.47 (s, 1H), 8.31 (s, 1H), 8.00 (s, 2H), 7.63-7.82 (m, 5H), 7.32 (d, J=4.8 Hz,
2H), 3.55 (s, 4H), 3.45 (s, 2H), 2.34 (s, 4H); 13C NMR (100 MHz, DMSO-d6) δ (ppm)：
161.84, 142.63, 140.50, 136.44, 134.33, 133.40, 132.44, 132.04, 131.54, 129.89,
126.92, 126.46, 125.02, 124.71, 120.34, 119.86, 113.74, 66.49, 62.71, 53.65; HR-MS
(calculated for C24H24ClN6O2 (M+H)+ 463.1649; found 463.1630); purity: 95.26%.
4.1.7.6
4-nitro-N-(3-(4-(4-(pyrrolidin-1-ylmethyl)phenyl)-1H-imidazol-2-yl)-1H-pyrazol-4-yl)
benzamide (10f)
1
Yellow solid, yield 23.6 %. H NMR (400 MHz, DMSO-d6) δ (ppm)：13.10 (s, 1H),
11.60 (s, 1H), 10.78 (s, 1H), 8.43 (d, J = 8.4 Hz, 2H), 8.37 (s, 1H), 8.29 (d, J = 8.0 Hz,
2H), 7.93 (d, J = 8.0 Hz, 2H), 7.84 (s, 1H), 7.64 (d, J = 8.0 Hz, 2H), 4.37(d, J = 4.4
13
Hz, 2H), 3.08 (s, 2H), 2.50 (s, 2H), 2.03 (s, 1H), 1.89 (s, 1H); C NMR (100 MHz,
DMSO-d6) δ (ppm)：161.65, 149.98, 142.62, 140.14, 140.07, 134.53, 132.13, 130.73,
129.00, 125.00, 124.79, 124.42, 120.72, 119.89, 114.34, 58.36, 53.38, 23.30; HR-MS
(calculated for C24H24N7O3 (M+H)+ 458.1949; found 458.1937).
4.1.7.7
4-methoxy-N-(3-(4-(4-(piperidin-1-ylmethyl)phenyl)-1H-imidazol-2-yl)-1H-pyrazol-4-

yl)benzamide (10g)
1
While solid, yield 18.3 %, Mp 242.7-243.4oC. H NMR (400 MHz, DMSO-d6) δ
(ppm)：11.42 (s, 1H), 8.26 (s, 1H), 8.00 (d, J = 8.4 Hz, 2H), 7.81 (d, J = 8.0 Hz, 2H),
7.66 (s, 1H), 7.34 (d, J = 7.6 Hz, 2H), 7.11 (d, J = 8.4 Hz, 2H), 3.86 (s, 3H), 3.46 (s,
2H), 2.34 (s, 4H), 1.48 (s, 4H), 1.36 (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ (ppm)：
162.90, 162.61, 142.75, 140.42, 133.38, 131.79, 129.81, 129.30, 129.18, 126.73,
124.70, 120.38, 119.82, 114.65, 113.58, 62.63, 56.04, 54.30, 25.99, 24.45; HR-MS
(calculated for C26H29N6O2 (M+H)+ 457.2352; found 457.2346); purity: 97.20%.
4.1.7.8
4-chloro-N-(3-(4-(4-(piperidin-1-ylmethyl)phenyl)-1H-imidazol-2-yl)-1H-pyrazol-4-yl
)benzamide (10h)
1
While solid, yield 14.2 %, Mp 292.8-293.1oC. H NMR (400 MHz, DMSO-d6) δ
(ppm)：11.43 (s, 1H), 10.66 (s, 1H), 8.34 (s, 1H), 8.01 – 8.05 (m, 2H), 7.96 (d, J = 8.0
Hz, 2H), 7.84 (s, 1H), 7.76 (d, J = 8.0 Hz, 1H), 7.71 (d, J = 8.0 Hz, 1H), 7.66 (d, J =
7.6 Hz, 2H), 4.25 (s, 2H), 3.33 (s, 3H), 2.83 (s, 2H), 1.67-1.78 (m, 5H), 1.36 (s, 1H);
13C NMR (100 MHz, DMSO-d6) δ (ppm)：161.15, 142.20, 139.22, 135.67, 134.79,
133.70, 131.67, 131.24, 130.86, 126.20, 125.76, 124.20, 119.69, 119.16, 114.04,
58.60, 51.35, 22.05, 21.35; HR-MS (calculated for C25H26ClN6O (M+H)+ 461.1856;
found 461.1850); purity: 99.50%.
4.1.7.9
3-chloro-N-(3-(4-(4-(piperidin-1-ylmethyl)phenyl)-1H-imidazol-2-yl)-1H-pyrazol-4-yl
)benzamide (10i)
1
While solid, yield 19.4%, Mp 259.9-261.3oC. H NMR (400 MHz, DMSO-d6) δ
(ppm)：11.43 (s, 1H), 10.68 (s, 1H), 8.34 (s, 1H), 8.01 – 8.05 (m, 2H), 7.96 (d, J = 8.0
Hz, 2H), 7.84 (s, 1H), 7.76 (d, J = 8.0 Hz, 1H), 7.71 (d, J = 8.0 Hz, 1H), 7.66 (d, J =
7.6 Hz, 2H), 4.25 (s, 2H), 3.29 (s, 2H), 2.84 (s, 2H), 1.78 (s, 4H), 1.67 (s, 1H), 1.36 (s,
1H); 13C NMR (100 MHz, DMSO-d6) δ (ppm)：161.15, 142.20, 139.22, 135.67,
134.79, 133.70, 131.67, 131.24, 130.86, 126.20, 125.76, 124.41, 124.20, 119.69,
119.48, 119.26, 114.04, 58.60, 51.35, 22.05, 21.35; HR-MS (calculated for

C25H26ClN6O (M+H)+ 461.1856; found 461.1836); purity: 97.01%.
4.1.8
6-chloro-2-methyl-N-(3-(4-(4-(morpholinomethyl)phenyl)-1H-imidazol-2-yl)-1H-pyra
zol-4-yl)pyrimidin-4-amine (10j)
The mixture of compound 9 (0.32 g, 1.0 mmol), 4,6-dichloro-2-methylpyrimidine
(0.13 g, 0.83 mmol), sodium iodide (0.15g, 1.0 mmol), N,N-diisopropylethylamine
(0.13 g, 1.0 mmol) and 3 mL DMF was stirred at 90 oC under argon atmosphere for 4
h. Then the mixture was cool to RT, poured in ice water (30 mL) and stirred for 30
min. The solid formed was filtered off and purified by chromatographic column to
afford 0.131g compound 10j.
While solid, yield 29.5%, Mp 287.2-287.7oC.1H NMR (400 MHz, DMSO-d
) δ (ppm)：
6
10.02 (s, 1H), 8.37 (s, 1H), 7.84 (d, J = 7.2 Hz, 1H), 7.78 (d, J = 8.0 Hz, 1H), 7.63 (s,
1H), 7.30 (d, J = 7.6 Hz, 2H), 6.90 (s, 1H), 3.55 (s, 4H), 3.44 (s, 2H), 2.46 – 2.47(m,
3H), 2.34 (s, 4H); 13C NMR (100 MHz, DMSO-d6) δ (ppm)：168.46, 168.37, 164.49,
160.37, 142.33, 140.52, 136.27, 133.40, 129.88, 129.77, 124.90, 120.98, 120.11,
113.48, 66.51, 62.93, 53.71, 25.87; HR-MS (calculated for C22H24ClN8O (M+H)+
451.1761; found 451.1753); purity: 99.64%.
4.1.9 1-(4-(3-chloropropoxy)phenyl)ethan-1-one (12)
To the solution of 4'-hydroxyacetophenone (1.36 g, 10 mmol) and
1-bromo-3-chloropropane (1.56 g, 10 mmol) in 10mL acetonitrile was added
potassium carbonate (1.38g, 10 mmol) and catalytic amount of potassium. The
o
mixture as stirred at 85 C for 4 h, then was cool to RT and filtered. The filtrate was
removed under vacuum and the crude product was purified by chromatographic
column to afford 2.02 g compound 12 (yield 95.2%).1H NMR (400 MHz, CDCl3-d
) δ
6
(ppm)：δ 7.91 (d, J = 7.8 Hz, 2H), 6.91 (d, J = 7.9 Hz, 2H), 4.16 (t, J = 6.0 Hz, 2H),
3.73 (t, J = 5.6 Hz, 2H), 2.53 (s, 3H), 2.21 – 2.27 (m, 2H); HR-MS (calculated for
C11H14O2Cl (M+H)+ 213.0682; found 213.0677).
4.1.10 1-(4-(3-morpholinopropoxy)phenyl)ethan-1-one (13)
To a solution of compound 12 (2.12 g, 10 mmol) and morpholine (0.87 g, 10 mmol)
in acetonitrile (10 mL) was added potassium carbonate (1.38g, 10 mmol), catalytic

o
amount of potassium iodide. The reaction mixture was stirred at 85 C for 4h, then
concentrated under vacuum and the residue was purified by chromatographic column
to give 2.2g compound 13 (yield 83.6 %).1H NMR (400 MHz, CDCl3-d
) δ (ppm)：
6
7.89 (d, J = 8.1 Hz, 2H), 6.90 (d, J =12 Hz, 2H), 3.99 - 4.08 (m, 1H), 3.63 - 3.70 (m,
3H), 2.48 – 2.52 (m, 3H), 2.39-2.45 (m, 4H), 1.91 - 2.00 (m, 2H); HR-MS (calculated
for C15H22NO3 (M+H)+ 264.1599; found 264.1596) (Figure 56).
4.1.11 2-bromo-1-(4-(3-morpholinopropoxy)phenyl)ethan-1-one (14)
To a solution of compound 13 (2.63 g, 10 mmol) in 48% HBr solution (20 mL) was
o
added 20 mL solution of bromine in acetic acid(0.5M) at 60 C. After completion of
the reaction, the solvent was removed under vacuum and the product was used for the
next step without being purified.
4.1.12
2-(4-(3-morpholinopropoxy)phenyl)-2-oxoethyl
4-nitro-1H-pyrazole-3-carboxylate (15)
To a solution of compound 2 (2.41g, 10 mmol), potassium carbonate (1.38g, 10 mmol)
and catalytic amount of potassium in acetonitrile (10 mL) was added compound 14
(4.21g, 10 mmol) at RT. The mixture was stirred overnight, then filtered. The filtrate
was removed under vacuum and the crude product was purified by chromatographic
column to afford 3.26g compound 15 (yield 65.0%).1H NMR (400 MHz, CDCl3-d
) δ
6
(ppm)：8.40 (s, 1H), 7.90 (d, J = 8.0 Hz, 2H), 6.94 (d, J = 8.0 Hz, 2H), 5.57 (s, 2H),
5.45 (d, J = 8.0 Hz, 1H), 4.09 (t, J = 8.0 Hz, 3H), 3.71 (s, 5H), 2.53 (t, J = 8.0 Hz, 3H),
2.48 (s, 3H), 2.20 – 2.25 (m, 1H), 1.87 – 2.01 (m, 4H), 1.66 (s, 3H); HR-MS
(calculated for C24H31N4O8 (M+H)+ 503.2142; found 503.2130).
4.1.13
2-(4-(3-morpholinopropoxy)phenyl)-2-oxoethyl
4-nitro-1H-pyrazole-3-carboxylate (16)
To a solution of compound 15 (5.02g, 10 mmol) in acetic acid (25 mL) was added
o
ammonium acetate (9.24g, 120 mmol). The mixture was stirred 125 C for 4 h. Then
the solvent was removed under vacuum and the residue was poured slowly into
saturated sodium bicarbonate solution while stirring constantly. The solid formed was
filtered off and used for next step without being purified.
4.1.14
2-(4-(3-morpholinopropoxy)phenyl)-2-oxoethyl

4-nitro-1H-pyrazole-3-carboxylate (17)
A solution of compound 16 (1.59g, 4 mmol) and 10% Pd/C (0.14g, 1.3mmol) in
o
40mL methanol under hydrogen was stirred at 40 C for 12 h. Then the mixture was
cool to RT, then filtered. The filtrate was removed under vacuum and the crude
product was used for next step without being purified.
4.1.15 General procedure for the preparation of the target compounds (18a)
The mixture of compound 17 (1.0 mmol), chlor pyrimidine derivative (0.83 mmol),
sodium iodide (0.15g, 1.0 mmol), N,N-diisopropylethylamine (0.13 g, 1.0 mmol) and
5 mL DMF was stirred at 90 oC under argon atmosphere for 4 h. Then the mixture was
cool to RT, poured in ice water (50 mL) and stirred for 30 min, and extracted with
ethyl acetate (3×40 mL). The combined organic layer was washed with brine (2×100
mL) and dried (Na2SO4). The solvent was removed under vacuum and the product
was purified by chromatographic column.
4.1.15.1
6-chloro-5-methyl-2-(methylthio)-N-(3-(5-(4-(3-morpholinopropoxy)
phenyl)-1H-imidazol-2-yl)-1H-pyrazol-4-yl) pyrimidin-4-amine (18a)
1
While solid, yield 24.0%, Mp 245.3-245.9oC. H NMR (400 MHz, DMSO-d
6
) δ
(ppm)：δ 10.38 (s, 1H), 8.30 (s, 1H), 7.74 (d, J = 8.0 Hz, 2H), 7.55 (s, 1H), 6.94 (d, J
= 8.0 Hz, 2H), 4.01 (t, J = 4.0 Hz, 2H), 3.56 (s, 4H), 2.53 (s, 5H), 2.28 – 2.40(m, 7H),
1.87 (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ (ppm)：168.76, 158.08, 156.58,
143.25, 142.40, 140.58, 127.31, 126.61, 126.21, 120.54, 119.88, 115.10, 112.58,
107.52, 66.58, 66.28, 55.36, 53.82, 26.32, 14.34, 13.04; HR-MS (calculated for
C25H30N8O2SCl (M+H)+ 541.1901; found 541.1897); purity: 99.72%.
4.2. Biological evaluation
4.2.1 In vitro kinases assays
Theses assays were carried out as described previously[53]. All of the enzymatic
reactions were conducted at 30℃for 40 minutes. The 50µl reaction mixture contains
40 mM Tris, pH 7.4, 10 mM MgCl2, 0.1 mg/ml BSA, 1 mM DTT, 10 µM ATP, Kinase
and the enzyme substrate. The compounds were diluted in 10% DMSO and 5µl of the
dilution was added to a 50µl reaction so that the final concentration of DMSO is 1%
in all of reactions. The assay was performed using Kinase-Glo Plus luminescence

kinase assay kit. It measures kinase activity by quantitating the amount of ATP
remaining in solution following a kinase reaction. The luminescent signal from the
assay is correlated with the amount of ATP present and is inversely correlated with the
amount of kinase activity. The IC50 values were calculated using nonlinear regression
with normalized dose-response fit using Prism GraphPad sofeware.
4.2.2 In vitro anti-proliferative assays
The cytotoxicity of each compound was determined using CCK-8 assay. Cells were
seeded in 96-well plates and then treated with different concentrations of drugs. After
72 h of incubation, CCK-8 staining was performed to determine the OD value and
calculate the inhibition rate. The IC50 values were calculated by using LOGIT method.
4.2.3 Western blot
Western blot analysis was performed according to previously published researches
[44-47]. Cells were treated with compounds at different concentrations for 48h, then
harvested and washed in ice-cold PBS, added cold RIPA buffer. The cells were
centrifuged and the supernatant was collected as whole cell lysate. Protein
concentration was determined by BCA method. Western blot analyses were conducted
after separation by SDS/PAGE electrophoresis and transfer to nitrocellulose filter
membrane. After blocking the membranes with 5% fat-free milk, primary antibody
o
was added, shocked at 4 C for overnight and washed with TBST. Then second
antibody was added, shocked for 1h, washed with TBST. GAPDH was used as a
loading control. The protein bands were visualized using the G: BOX chemiXR5.
4.2.4 Cell cycle assays
Cells were treated with compounds at different concentrations for 48h and washed
o
with PBS twice. 5×105 cells were collected, added 100 µL RNase A at 37 C for 30
o
min, stained with propidium iodide (400µL) at 4 C for 30 min and then analyzed
using flow cytometry.
4.3 Binding mechanism explored by molecular modeling
In order to explore the binding mechanism of our active compound 10e, an integrated
molecular modeling approach, which combines the docking calculation and molecular
dynamics (MD) simulations, was performed (Fig S1). Initial ligand-protein complex

structures were obtained using the Autodock Vina. The best binding pose obtained
from AutoDock Vina was selected for further refinement using MD simulations. The
final binding pose was identified by clustering the trajectories from MD simulations.
AutoDock Vina (version 1.1.2) [54], which is a widely used molecular docking and
virtual screening application , was selected to predict the binding pose of our active
compound MZ-1E to the JAK2, JAK3, Aurora A and Aurora B, respectively. The
compound AT9832 was chosen as reference. Corresponding X-ray structures of JAK2,
JAK3, Aurora A and Aurora B were download from PDB with ID of 2W1I[55],
3ZEP[56], 2W1G[55] and 4AF3[57], respectively. All non-protein atoms including
water and ligand in the X-ray structures were deleted and polar hydrogen atoms were
added using MGLTools (version 1.5.6) [58]. The center of the ligand in the X-ray
structures was used as target binding site grid center for docking. SMILES of 10e and
AT9832 were converted to the PDBQT format using Open Babel (version
2.3.2) [59] with the following options: --gen3d and -p 7.0. Then, docking calculations
were performed with AutoDock Vina to obtain the initial binding pose. The
exhaustiveness parameter was set to 30. In order to validate the parameters and
software, the compound AT9832 with experimental structures was docked into JAK2
and AURKA, respectively. The root mean square deviations (RMSDs) of the AT9832
between the calculated and crystal determined one are very small (below 1.0 Å) (Fig
S2), suggesting our docking protocol is reliable.
Then, the best docking models of each system were used as initial structure for
MD simulations. The missing hydrogen atoms were added using tleap in AMBER18
[60]. The ff14SB force field [61] and GAFF[62] were employed to model the protein
and ligand, respectively. The kinase-ligand complex was solvated in a cubic box of
TIP3P water molecules [63] with a minimum distance of 10 Å between the protein
surface and box edge. The system was first energy minimized with 2 kcal/(mol Å2)
harmonic position restraints applied on the heavy atoms of protein and ligand. Then,
the system was heated from 0 to 300 K in the constant volume and temperature (NVT)
canonical ensemble for 1 ns, imposing position restraints of 2.0 kcal/(mol Å2) on the

heavy atoms of protein and ligand. The system was further equilibrated in the
isothermal−isobaric (NPT) ensemble for 1 ns, reducing the position restraints to 1.0
kcal/(mol Å2). Finally, 300 ns production MD simulations were carried out in the
NPT ensemble without any constraints. All the simulations were performed using the
GPU version of AMBER18 [60]. The bond lengths of hydrogen–heavy atoms were
constrained using the SHAKE algorithm [64]. A distance cutoff of 9.0 Å was used for
the van der Waals and short-range electrostatic interactions. Long-range electrostatic
interactions were computed with the particle-mesh Ewald summation method [65]. A
2-fs integration time step was used in all MD simulations.
The MD simulations became stable after 150 ns as there were small fluctuations in
the protein backbone RMSDs from 150 ns to 300 ns (Fig S3). Thus, snapshots were
extracted from the last 150ns MD trajectories to obtain refined binding pose and
binding free energy (∆G) calculation with MM/GBSA approach. The ∆G in the
MM/GBSA approach was calculated according to equation (1),
∆Gꢀꢁꢂꢃꢁꢂꢄ = ∆H − T∆S = ∆ꢅꢆꢇꢆ + ∆ꢅꢈꢃꢉ + ∆ꢊꢄꢀ + ∆ꢊꢋꢌꢍꢎ − ꢏ∆ꢐ (1)
where ∆Evdw and ∆Eele were the non-bonded van der Waals and electrostatic
interaction energies between the ligands and kinases in the gas phase, respectively.
The ∆Ggb and ∆Gsurf were the polar and non-polar interaction energies between the
ligand and protein, respectively. The ∆Ggb was computed using the pbsa program in
Amber 18[60]. The dielectric constants of solvent and solute were set to 80.0 and 1.0,
respectively. The OBC solvation model[66] was used in this study. The ∆Gsurf term
was calculated as:
∆ꢊꢋꢌꢍꢎ = ꢑ ∗ ꢐꢒꢐꢒ + ꢓ (2)
where SASA was the solvent accessible surface area as computed using the MSMS
program [67]. The ꢑ and ꢓ coefficients were set to 0.0072 kcal/(mol Å2) and 0
kcal/mol, respectively. The fluctuation of entropy is quite large, thus we do not
include this energy term in the current study.
Acknowledgment
The authors are grateful for the financial support by National Natural Science
Foundation of China (81602951); Natural Science Foundation of Jiangsu Province

(BK20130216); Six talent peaks project in Jiangsu Province(2016-SWYY-062);
Science and technology project of Xuzhou City (KC16SG255).
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