Synthesis, biological evaluation and molecular modeling study of 2-amino-3,5-disubstituted-pyrazines as Aurora kinases inhibitors

Article abstract of DOI:10.1016/j.bmc.2020.115351

Serine/threonine protein kinases Aurora A, B, and C play essential roles in cell mitosis and cytokinesis, and a number of Aurora kinase inhibitors have been evaluated in the clinic. Herein we report the synthesis and their antiproliferation of 3,5-disubstituted-2-aminopyrazines as kinases inhibitors. Amongst, 4-((3-amino-6- (3,5-dimethylisoxazol-4-yl)pyrazin-2-yl)oxy)-N-(3-chlorophenyl) benzamide (12Aj) exhibited the strongest antiproliferative activities against U38, HeLa, HepG2 and LoVo cells with IC₅₀ values were 11.5 ± 3.2, 1.34 ± 0.23, 7.30 ± 1.56 and 1.64 ± 0.48 μM, as well as inhibited Aurora A and B with the IC₅₀ values were 90 and 152 nM, respectively. Molecular docking studies indicated that 12Aj appeared to form stable hydrogen bonds with either Aurora A or Aurora B. Furthermore, 12Aj arrested HeLa cell cycle in G2/M phase by regulating protein levels of cyclinB1 and cdc2. In addition, the bioinformatics prediction further revealed that 12Aj possessed good drug likeness using SwissADME. These results suggested that 12Aj was worthy of future development of potent anticancer agents as pan-Aurora kinases.

Full text of DOI:10.1016/j.bmc.2020.115351

Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, biological evaluation and molecular modeling study of 2-amino-3,
5-disubstituted-pyrazines as Aurora kinases inhibitors
⁎
Yong-Xin Boa,1, Rong Xiangb,1, Yu Xua, Shu-Yi Haoa, Xing-Rong Wanga, Shi-Wu Chena,
a School of Pharmacy, Lanzhou University, Lanzhou 730000, PR China
b Department of Medicinal, The Second Clinical Medical College of Northwest Minzu University & The Second Provincial People’s Hospital of Gansu Province, Lanzhou
730000, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Serine/threonine protein kinases Aurora A, B, and C play essential roles in cell mitosis and cytokinesis, and a
number of Aurora kinase inhibitors have been evaluated in the clinic. Herein we report the synthesis and their
antiproliferation of 3,5-disubstituted-2-aminopyrazines as kinases inhibitors. Amongst, 4-((3-amino-6- (3,5-di-
methylisoxazol-4-yl)pyrazin-2-yl)oxy)-N-(3-chlorophenyl) benzamide (12Aj) exhibited the strongest anti-
Anti-tumor
Aurora kinase
Pyrazines
Cell cycle
proliferative activities against U38, HeLa, HepG2 and LoVo cells with IC50 values were 11.5
3.2,
1.34
0.23, 7.30
1.56 and 1.64
0.48 μM, as well as inhibited Aurora A and B with the IC50 values were
90 and 152 nM, respectively. Molecular docking studies indicated that 12Aj appeared to form stable hydrogen
bonds with either Aurora A or Aurora B. Furthermore, 12Aj arrested HeLa cell cycle in G2/M phase by regulating
protein levels of cyclinB1 and cdc2. In addition, the bioinformatics prediction further revealed that 12Aj pos-
sessed good drug likeness using SwissADME. These results suggested that 12Aj was worthy of future develop-
ment of potent anticancer agents as pan-Aurora kinases.
1. Introduction
680 (Tozasertib),15 AMG90016 and PHA-739358 (Danusertib)17 (Figure
1). However, the majority of which focus on hematopoietic cancers. A
The Aurora kinase family is a subfamily of serine/threonine kinases
that is essential for the regulation of centrosome maturation, mitotic
spindle formation, chromosome segregation and cytokinesis during
mitosis.1,2 The family includes three kinases designated as Aurora A, B,
and C, which are very closely related in the kinase domain sequence.
However, these kinases have quite different and nonoverlapping func-
tions during mitosis.3 Aurora A plays an important role in centrosome
maturation, spindle assembly, meiotic maturation, and metaphase I
spindle orientation,4,5 while Aurora B is a member of the chromosomal
passenger complex, critical for chromosome condensation, chromosome
orientation on the mitotic spindle and the spindle-assembly checkpoint,
as well as the final stages of cytokinesis.6 Aurora C has similar location
as Aurora B and leads to the formation of multinucleated cells.7
The potential roles of Aurora kinases in regulating cell mitosis and
tumorigenesis make them attractive targets for anticancer therapy.8,9
Some subtype-selective or pan-Aurora kinase inhibitors have been dis-
covered and moved forward in clinical trials,10 such as Aurora A in-
hibitor MLN-8237 (Alisertib)11 and ENMD-2076,12 Aurora B inhibitor
AZD-1152 (Barasertib)13 and AT9283,14 and pan-Aurora inhibitors VX-
possible explanation for limited clinical responses in the treatment of
solid tumors is the likely need for sustained exposures through a
number of cell cycles (for Aurora B inhibitors) or time in mitosis (for
Aurora A inhibitors) to elicit a maximum therapeutic response. At such
sustained exposures, mechanism-based toxicity may become dose-lim-
iting.18
In our previous publications, some 2,4-diaminopyrimidines and 2,4-
disubstituted phthalazinones showed Aurora inhibitory activities, and
more potent antiproliferations against tumor cell lines compared with
the VX-680.19–22 Pyrazine as a skeletal structure widely applied in
protein kinases inhibitors, such as Nek223,24, CHK125,26, PI3K27,28 and
MNK1/229. Therefore, in continuation of our efforts on scaffold hop-
ping strategy towards Aurora kinase inhibitors, a novel series of 3,5-
disubstituted-2-aminopyrazines were designed by replacement of pyr-
imidine with 2-aminopyrazine fragment (Figure 2). Herein, we report
the synthesis of 3,5-disubstituted-2-aminopyrazines, as well as their
activities on antiproliferation toward cancer cell lines, Aurora kinase
inhibition and cell cycle arrest.
⁎ Corresponding author.
E-mail address: chenshw@lzu.edu.cn (S.-W. Chen).
1 These authors made equal contributions to this work.
https://doi.org/10.1016/j.bmc.2020.115351
0968-0896/©2020ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:Yong-XinBo,etal.,Bioorganic&MedicinalChemistry,https://doi.org/10.1016/j.bmc.2020.115351

Y.-X. Bo, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
Fig. 1. Representative Aurora kinase inhibitors.
Fig. 2. Design of target compounds.
2. Results and discussion
Simultaneously, 4-aminomethylbenzamide (8) was prepared from
4-aminomethyl-benzoic acid (6) as shown in Scheme 2. Boc protection
of the amino group of 6 would give intermediate 7, which reacted with
4-chloroaniline under tri(dimethylamino)benzotriazol-1-yloxyphos
phonium hexafluorophosphate (BOP), and disposed with solution of
trifluoroacetic acid in the dichloromethane to yield compound 8.
The synthesis of derivatives 12Aa–n, 12Bg–n, 13A,B and 16A,B
based on the 2-aminopyrazine scaffolds is shown in Scheme 3. The 3-
bromine displacement of 2-amino-3,5-dibromopyrazine (9) with inter-
mediates 5a–n or 8 in N-methyl pyrrolidone (NMP) in the presence of
K2CO3 provided 10a–n or 11.23 Subsequent Suzuki cross-coupling re-
actions of 10a–n or 11 with 3,5-dimethylisoxazole-4-boronic acid or 1-
methylpyrazole-4-boronic acid pinacol ester under the catalysis of Pd
(dppf)2Cl2·CH2Cl2 gave the target compounds 12Aa–n, 12Bg–n and
13A,B.20,23 Besides, compounds 16A,B were also prepared from 9 by
the similar procedures including displacement, amidation and
2.1. Chemistry
The general synthetic route for intermediates 5a–n is illustrated in
Scheme 1. Specifically, 5a–j were directly obtained by reacting p-hy-
droxylbenzoic acid (3) with various amines in acetone under conden-
sing agent 1-ethyl-(3-dimethyl aminopropyl) carbodiimide hydro-
chloride (EDCI). For intermediates 5k–n, compound 3 was firstly
reacted with acetic anhydride in the presence of 4-methylbenzene-
sulfonic acid (TsOH), and further treated with thionyl chloride to afford
4-(chlorocarbonyl)phenyl acetate (4). After amidation of compound 4
with 3-chloropyridin-2-amine (or 4-chloropyridin-2-amine, or 5-chlor-
opyridin-2-amine, or 6-chloropyridin-2-amine), removal of the pro-
tecting group provided p-hydroxybenzamides 5k–n in the methanol
solution of potassium hydroxide.
Scheme 1. Synthesis of compounds 5a–n. Reagents and conditions: (i) amines, EDCI, acetone, 60 °C, 3 h; (ii) a: Ac2O, TsOH, 85 °C, 1 h; b: SOCl2, DCM, r.t., 2 h; (iii) a:
amines, Et3N, DCM, r.t., 4 h; b: KOH, MeOH, 75 °C, 2 h.
2

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Scheme 2. Synthesis of compound 8. Reagents
and conditions: (i) Boc2O, NaOH, H2O/EtOH
(1:1), r.t., 5 h; (ii) a: 4-chloroaniline, BOP, Et3N,
DCM, r.t., overnight; b: CF3COOH, DCM, r.t., 1 h.
palladium-catalyzed Suzuki cross-coupling reaction. The newly syn-
thesized compounds were characterized by physicochemical and spec-
tral means, and both analytical and spectral data of all the compounds
were in full agreement with the proposed structures.
activities toward tumor cells in aforementioned MTT assay, were se-
lected to identify their inhibition activities against Aurora kinases by
Kinase-Glo luminescent kinase assay in vitro,30 with VX-680 as reference
compound. As showed in Table 2, these compounds showed moderate
Aurora inhibitory activities, and less compared with VX-680. Amongst,
compound 12Aj exhibited the most potent inhibition on Aurora A and
Aurora B with the IC50 values were 90 and 152 nM, respectively. These
results suggested that this series of compounds inhibited cancer cell
proliferation at least in part by targeting Aurora kinases.
2.2. Biological activity
2.2.1. In vitro antiproliferation activities of target compounds
The in vitro antiproliferation activities of target compounds 12Aa–n,
12Bg–n 13A,B and 16A,B were evaluated in a panel of four human
tumor cell lines (Human malignant glioblastoma U87, cervical carci-
noma HeLa, hepatic carcinoma HepG2 and human colorectal adeno-
carcinoma LoVo cells) by MTT assay after treatment for 72 h, with VX-
680 as a reference compound,20 and the results are summarized in
Table 1. Most target compounds showed moderate to better anti-
proliferation activities in the four human tumor cell lines, although
some of them were less compared with VX-680. Notably, compounds
with different substitutions at C-3 position of pyrazine showed various
effects in regards to their antiproliferations, compounds 12Aa–n and
12Bg–n with p-hydroxylbenzoic chains showed more potent anti-
proliferations than those of compounds 13A,B and 16A,B with 4-ami-
nomethylbenzoic or 4-mercaptobenzoic chains at the C-3 position of
pyrazine. However, compounds 12Aa–n and 12Bg–n, in which the C-5
position of pyrazine were substituted with 3,5-dimethylisoxazole or 1-
methylpyrazole, exhibited no obvious differences in their anti-
proliferations. Furthermore, target compounds with various p-hydro-
xylbenzoic amides (such as cyclopentylamine, morpholine, 1-methyl-
piperazine, aniline, or chloroanilines) except chloropyridines illustrated
potent antiproliferative activities. Among the synthetic target com-
pounds, compound 12Aj showed the strongest growth-inhibitory ac-
2.2.3. Compound 12Aj blocks phosphorylation of Aurora kinases in HeLa
cells
Aurora A is activated by phosphorylation of Thr288, and Aurora B is
activated by phosphorylation of Thr232.31 To further identification the
Aurora kinases were targets of compound 12Aj, the phosphorylation of
Aurora A on Thr288 and Aurora B on Thr232 were investigated in HeLa
cells treated with various concentrations of compound 12Aj (0, 1, 5 and
10 μM) for 24 h by western blot.32 As shown in Fig. 3, compound 12Aj
obviously decreased the expression level of Aurora A and B, as well as
reduced phosphorylation of Aurora A on Thr288 (p-Thr288) and Aurora
B on Thr232 (p-Thr232) in a dose-dependent manner. These results
suggested that compound 12Aj is a potential Aurora kinases inhibitor.
2.2.4. Molecular docking of compound 12Aj binding Aurora kinase
To gain insight into the interaction of compound 12Aj with Aurora
A and Aurora B, docking simulation was performed using Glide module
(Glide, version 6.7, Schrödinger, LLC, New York, NY, 2015) of
Schrödinger Suite (Maestro, version 10.1, Schrödinger, LLC, New York,
NY, 2015).33 All the figures displaying the docking results were ob-
tained using the scientific software Pymol.34 The detailed interactions
of compound 12Aj with Aurora A (PDB code: 3H0Z) and Aurora B (PDB
code: 4C2V) were shown in Fig. 4. For Aurora A, the ligand 12Aj ap-
peared to form stable hydrogen bonds with several amino acid residues
of Aurora A kinase. Notably, the amino and nitrogen atom on the nu-
clear formed two stable hydrogen-bond interactions with Glu211 and
Ala213 in the hinge region, respectively (Fig. 4A). Besides, the amine
group in the side chain established a hydrogen bond with Glu260 and
the terminal chlorobenzenecould form π interaction with Tyr219. For
Aurora B, the amino and nitrogen atom on the nuclear formed two
tivities with the IC50 values were 11.5
3.2, 1.34
0.23,
7.30
1.56 and 1.64
0.48 μM for the U87, HeLa, HepG2 and LoVo
tumor cell lines, respectively.
2.2.2. Aurora kinases inhibitory activities of the selected compounds
Although pyrazines show various protein kinases inhibitory activ-
ities, few 3,5-disubstituted-2-aminopyrazines was reported to inhibit
the Aurora kinases. Thus, compounds 12Ad, 12Ae, 12Af, 12Ag, 12Ai,
12Aj, 12Bg, 12Bj and 12Bl, which exhibited better antiproliferative
Scheme 3. Synthesis of target compounds
12, 13 and 16. Reagents and conditions: (i)
5a–n (for 10a–n), K2CO3, NMP, 100 °C, 6 h;
(ii) 3,5-dimethylisoxazole-4-boronic acid
(for 12Aa–n, 13A and 16A) or 1-methyl-
pyrazole-4-boronic acid pinacol ester (for
12Bg–n,
13B
and
16B),
Pd
(dppf)2Cl2·CH2Cl2, 2 M Na2CO3, dioxane,
100 °C,
4 h; (iii) 8 (for 11), KOH,
dioxane:H2O = 2:1, 100 °C, 48 h; (iv) 4-
mercaptobenzoic acid, KOH, EtOH, 80 °C,
12 h; (v) a: SOCl2, DCM, r.t., 2 h; b: 4-
chloroaniline, Et3N, DCM, r.t., 4 h.
3

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Table 1
The in vitro antiproliferations of target compounds.
Compounds
X
NR1R2
(IC50, μM)a,b
U87
HeLa
HepG2
LoVo
12Aa
12Ab
12Ac
O
O
O
47.8
43.3
57.6
3.2
4.1
5.2
32.1
17.8
9.97
4.3
3.5
2.4
6.88
43.2
12.8
1.02
4.6
1.7
17.5
5.1
> 100
25.2
4.2
2.6
12Ad
O
40.4
4.7
26.1
3.1
5.42
2.8
21.6
12Ae
12Af
12Ag
12Ah
O
O
O
O
27.2
21.5
26.6
60.5
3.3
2.2
2.3
5.6
14.1
8.03
10.5
81.3
2.7
15.4
1.4
2.5
2.1
10.2
1.6
1.33
1.7
17.6
7.69
1.53
1.22
11.8
5.74
6.3
> 100
> 100
12Ai
12Aj
O
O
15.5
11.5
2.1
3.2
2.23
1.34
0.65
0.23
14.4
7.30
1.6
1.92
1.64
0.12
0.48
1.56
12Ak
O
64.1
5.4
25.5
2.7
35.7
2.6
3.4
39.3
5.1
12Al
O
O
45.6
70.8
3.6
5.6
19.2
37.9
1.7
3.8
35.6
9.18
1.12
12Am
> 100
> 100
12An
O
45.6
3.5
31.9
3.3
27.2
3.7
14.8
1.5
12Bg
12Bh
O
O
17.7
44.8
0.8
5.2
12.3
73.0
1.1
6.8
11.6
66.9
1.3
5.6
9.21
41.4
0.76
2.4
12Bi
12Bj
O
O
35.8
21.7
3.3
1.2
3.44
4.46
0.42
0.41
11.9
21.2
1.4
1.8
3.08
24.5
0.43
2.2
12Bk
O
54.8
4.6
38.2
3.2
44.2
3.9
2.4
48.6
4.7
12Bl
O
O
22.2
47.9
2.1
4.7
21.5
40.8
1.6
3.8
24.2
6.16
26.6
1.12
2.4
12Bm
> 100
12Bn
O
37.6
2.6
36.1
2.2
21.7
1.8
16.5
1.6
13A
13B
16A
16B
CH2NH
> 100
> 100
> 100
> 100
14.5
> 100
42.1
38.4
42.7
9.5
> 100
20.1
> 100
43.6
CH2NH
3.5
2.7
4.6
1.6
3.3
2.6
3.6
S
S
–
45.2
49.2
> 100
12.8
> 100
13.6
VX-680
–
3.4
1.8
2.3
1.6
a
b
IC50 values are presented as the means
MTT method drug exposure for 72 h.
SD of triplicate experiments.
4

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Table 2
have good permeating cell membranes.38 From data depicted in
Table 3, compounds 12Aj and VX-680 had TPSA with 116.16 and
127.37 Å2, respectively, which revealed that either 12Aj or VX-680
could permeate cell membranes. Simultaneously, consensus lipophili-
city (LogPO/W) of compounds 12Aj and VX-680 were 3.56 and 2.95,
respectively, that showed similar lipophilicity. Next, predictions of
water solubility (LogS) of the compounds revealed that the compounds
12Aj as well as VX-680 had moderate water solubility. Pharmacoki-
netics studies indicated that compound 12Aj and VX-680 had high GI
absorption (gastrointestinal absorption) and could not permeate BBB
(blood brain barrier). Based on these data, it could be concluded that
the compounds were suitable for oral administration. From the skin
permeation (LogKP), we infered that compounds 12Aj possessed similar
permeability with VX-680. Finally, the compound 12Aj obeyed the
Lipinski's rule of five. Together, compound 12Aj, similar to VX-680,
follow Lipinski’s rule of 5 and possessed moderate to good % human
oral absorption.
The in vitro kinase inhibitions of selected compounds.
Compounds
(IC50, nM)a
Compounds
(IC50, nM)a
Aurora A
Aurora A
Aurora B
Aurora B
12Ad
12Ae
12Af
12Ag
12Ai
230
172
143
220
458
320
360
189
538
260
12Aj
12Bg
12Bi
90
152
164
175
272
15.7
220
190
658
1.05
12Bj
VX680
a
The IC50 values are the means of two experiments.
stable hydrogen-bond interactions with Glu171 and Ala173 in the hinge
region, respectively. In addition, the amine group in the side chain
established a hydrogen bond with Glu177 and the terminal chlor-
obenzene formed π interaction with Tyr179 (Fig. 4C). Additionally, the
methyl isoxazole of compound 12Aj could well occupy the hydrophobic
pocket of Aurora A and Aurora B (Fig. 4B and D). The model would also
be helpful for us to further understand the pyrazine analogues were
potent Aurora kinases inhibitors.
3. Conclusions
Aurora kinases have been of interest as potential therapeutic targets
in oncology. Here we described a series of 3,5-disubstituted-2-amino-
pyrazine Aurora kinases inhibitors that exerted their antiproliferation
activities in human tumor cell lines. We specifically demonstrated that
compound 12Aj has the potential to inhibit Aurora A and Aurora B in
HeLa cells. Treatment of HeLa cells with compounds 12Aj also resulted
in G2/M accumulation by regulating the expression of Cyclin B1 and
cdc2. Furthermore, the SwissADME prediction showed that compound
12Aj exhibited good drug likeness. These results suggested that these
compounds have potential anticancer activities for further development
as Aurora inhibitors.
2.2.5. Cell cycle arrest of compound 12Aj
Many studies have shown that Aurora kinase inhibitor VX-680 in-
duces cell cycle arrest in the G2/M phase.15 Thus, we also investigated
the effects of 12Aj on cell cycle progression using fluorescence-acti-
vated cell sorting analysis of HeLa cells stained with propidium io-
dide.20 As shown in Figure 5, treatment of HeLa cells with 12Aj resulted
in a dose-dependent accumulation of cells in the G2/M phase with a
concomitant decrease in the population of G1 phase cells. The per-
centages of cells in G2/M phase arrest after treatment with 1, 5 and
10 μM of compound 12Aj for 24 h were 21.0%, 30.1% and 34.9%,
respectively, compared with 10.5% in untreated cultures. These results
demonstrated that 12Aj interfered with cell proliferation by arresting
the cell cycle in G2/M.
4. Experiment
4.1. Chemistry
Cyclin B1 and cdc2 are members of the cell cycle-related protein
and regulate the progression of cells into and out of M phase.35
Therefore, we used western blot to measure protein expression levels of
cyclinB1 and cdc2 in HeLa cells following treatment with compound
12Aj (0, 1, 5 and 10 μM) for 24 h, VX-680 as reference compound.20 As
shown in Fig. 6, treatment of HeLa cells with 12Aj resulted in an ap-
parent decrease of Cyclin B1 and cdc2, demonstrating that compound
12Aj can interfere with cell cycle progression. This conclusion is con-
sistent with the cell cycle analysis results.
4.1.1. General procedures
All starting materials and reagents were purchased commercially
and used without further purified, unless otherwise stated. All reactions
were monitored by thin layer chromatograph (TLC) on silica gel GF254
(0.25 mm thick). Column chromatography (CC) was performed on
Silica Gel 60 (230–400 mesh, Qingdao Ocean Chemical Ltd., China).
HPLC analysis using a UltiMate300 DAD HPLC system equipped with a
PU-2089 Plus quaternary gradient pump and a UV-2075 Plus UV–vis
detector, using an Alltech Kromasil C18 column with dimensions of
250 mm × 4.6 mm and 5 μm particle size. 1H NMR and 13C NMR
spectra were recorded with an Agilent-NMR-Inova 600 spectrometer
with TMS as an internal standard, all chemical shift values are reported
as ppm. Mass spectra were recorded on an Esquire6000 (ESI-ION TRAP)
spectrometer with ESI source as ionization, respectively.
2.2.6. Drug-likeness of compound 12Aj
The computational prediction of important physicochemical de-
scriptors related to absorption, distribution, metabolism, and excretion
(ADME) properties represents a cost-effective strategy to filter out
molecules at early stages of drug-discovery process.36 Here, we did in
silico physicochemical prediction for compound 12Aj in comparison to
VX-680 using SwissADME web service37, and all results are summarized
in Table 3.
4.1.2. General procedure for synthesis of 4-hydroxybenzamides (5a–j)
To a solution of 4-hydroxybenzoic acid (3, 690 mg, 5 mmol) in
acetone (10 mL) at room temperature was added n-propylamine
Firstly, topological polar surface area (TPSA) used for optimization
of drug's ability to permeate cell, molecule with TPSA less than 140 Å2
Fig. 3. Western blot for inhibition of p-T288 (A) and p-T232 (B) with various concentrations of compound 12Aj and VX680 in HeLa cells for 24 h.
5

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Fig. 4. Molecular binding modes of compound 12Aj to Aurora A (PDB code: 3H0Z) and Aurora B (PDB code: 4C2V). (A) Predicted binding mode for 12Aj to Aurora
A. (B) Surface of 12Aj and Aurora A. (C) Predicted binding mode for 12Aj to Aurora B. (D) Surface of 12Aj and Aurora B.
followed by EDCI (1.15 g, 6.0 mmol). After stirring the reaction mixture
for 12 h at 70 °C, the mixture was concentrated under vacuo, added
water (10 mL), extracted with ethyl acetate (3 × 10 mL). The combined
organic layers were washed with brine, dried over anhydrous sodium
sulfate, and concentrated under vacuo. The crude product was then
purified by column chromatography using dichloromethane/acetone
(20:1) to yield the pure 5a.
Similar procedure as that described for 5a gave pure 5b–j.
4.1.2.1. N-Propyl-4-hydroxybenzamide (5a). Light brown liquid; yield:
90%; 1H NMR (600 MHz, DMSO‑d6) δ 9.94 (s, 1H), 8.20 (s, 1H), 7.70 (d,
J = 8.4 Hz, 2H), 6.78 (d, J = 8.4 Hz, 2H), 3.19–3.15 (m, 2H),
1.52–1.48 (m, 2H), 0.87 (t, J = 7.2 Hz, 3H).
Fig. 5. Effect of 12Aj and VX-680 on cell cycle progression. (A) Control HeLa cells. (B) HeLa cells treated with 1 μM 12Aj for 24 h. (C) HeLa cells treated with 5 μM
12Aj for 24 h. (D) HeLa cells treated with 10 μM 12Aj for 24 h. (E) HeLa cells treated with 10 μM VX-680 for 24 h.
6

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Fig. 6. Western blotting analysis of expression of cyclin B1 and cdc2 in HeLa cells treated with 12Aj (0, 1, 5 and 10 μM) or VX-680 (10 μM) for 24 h.
Table 3
J = 8.4 Hz, 2H).
Prediction of physicochemical properties for ‘‘drug-likeness” for compounds
12Aj and VX-680.
4.1.2.9. N-(4-Chlorophenyl)-4-hydroxybenzamide (5i). Light yellow
solid; yield: 76%; 1H NMR (600 MHz, DMSO‑d6) δ 10.11 (s, 1H),
10.10 (s, 1H), 7.85 (d, J = 9.0 Hz, 2H), 7.80 (d, J = 9.0 Hz, 2H), 7.39
(d, J = 8.4 Hz, 2H), 6.87 (d, J = 7.8 Hz, 2H).
Models
12Aj
VX-680
Molecular weight
Num. H-bond acceptors
Num. H-bond donors
Num. rotatable bonds
TPSA (≤140 Å2)
Consensus LogPO/W
LogS (ESOL)
435.86 g/mol
464.59 g/mol
6
5
2
3
6
8
4.1.2.10. N-(3-Chlorophenyl)-4-hydroxybenzamide (5j). Yellow solid;
yield: 51%; 1H NMR (600 MHz, DMSO‑d6) δ 10.15 (s, 1H), 10.14 (s,
1H), 7.96 (s, 1H), 7.85 (d, J = 8.4 Hz, 2H), 7.70 (d, J = 8.4 Hz, 1H),
7.35 (t, J = 7.8 Hz, 1H), 7.13 (d, J = 7.8 Hz, 1H), 6.88 (d, J = 9.0 Hz,
2H).
116.16 Å2
3.56
127.37 Å2
2.95
−5.01
High
−4.72
High
GI absorption
BBB permeant
No
No
LogKP (skin permeation)
PAINS
−6.53 cm/s
0 alert
0.55
−6.71 cm/s
0 alert
0.55
4.1.3. General procedure for synthesis of 4-hydroxybenzamides (5k–n)
A solution of 4-hydroxybenzoic acid (3, 1.4 g, 10 mmol) in acetic
anhydride (1.6 g, 16.0 mmol) and p-methylbenzenesulfonic acid
(80 mg, 0.4 mmol) was allowed to stir at 85℃ for 1 h. After completion
of the reaction, the reaction mixture was quenched by addition of water
and separated in ice-bath. The crude product was then purified by re-
crystallization (ethanol-water) to provide 4-acetoxybenzoic acid (1.5 g,
yield 83%).
Bioavailability score
Lipinski
Yes; 0 violation
Yes; 0 violation
4.1.2.2. N-Cyclopropyl-4-hydroxybenzamide (5b). Colorless oil liquid;
yield: 64%; 1H NMR (600 MHz, DMSO‑d6) δ 10.12 (s, 1H), 9.95 (s,
1H), 7.87 (d, J = 8.4 Hz, 2H), 6.88 (d, J = 8.4 Hz, 2H), 2.85–2.84 (m,
1H), 0.71–0.68 (m, 2H), 0.59–0.56 (m, 2H).
To a solution of 4-acetoxybenzoic acid (1.0 g, 5.6 mmol) in dried
DCM (10 mL), thionyl chloride (660 mg, 5.6 mmol) was added drop-
wise, and the reaction mixture was stirred for 2 h at room temperature.
After completion of the reaction, the mixture was concentrated under
vacuo to give 4-(chlorocarbonyl)phenyl acetate (4), which was used
further without purification.
4.1.2.3. N-Cyclopentyl-4-hydroxybenzamide (5c). White solid; yield:
58%; 1H NMR (600 MHz, DMSO‑d6)
δ 9.87 (s, 1H), 7.96 (d,
J = 7.8 Hz, 1H), 7.68 (d, J = 9.0 Hz, 2H), 6.74 (d, J = 9.0 Hz, 2H),
4.17–4.14 (m, 1H), 1.84–1.80 (m, 2H), 1.65–1.64 (m, 2H), 1.48–1.45
(m, 4H).
To a solution of 2-amino-6-chloropyridine (2.1 mmol) in dried DCM
(10 mL) were added Et3N (0.4 mL, 3.7 mmol) followed by dropwise
addition of a solution of 4 (0.4 g, 2.3 mmol) in dried DCM (5 mL). After
stirring the reaction mixture for 4 h at room temperature, the mixture
was concentrated under vacuo, and the crude product was purified by
column chromatography using DCM/acetone (20:1) to yield pure 4-((6-
chloropyridin-2-yl)carbamoyl)phenyl acetate.
4.1.2.4. N-Cyclohexyl-4-hydroxybenzamide (5d). White solid; yield:
64%; 1H NMR (600 MHz, DMSO‑d6)
δ 9.87 (s, 1H), 7.88 (d,
J = 7.8 Hz, 1H), 7.68 (d, J = 8.4 Hz, 2H), 6.74 (d, J = 9.0 Hz, 2H),
3.69–3.68 (m, 1H), 1.76 (t, J = 6.0 Hz, 2H), 1.69 (t, J = 7.8 Hz, 2H),
1.58 (s, 1H), 1.29–1.22 (m, 4H), 1.10–1.07 (m, 1H).
To a mixture of 4-((6-chloropyridin-2-yl)carbamoyl)phenyl acetate
(2 mmol) in MeOH (10 mL) were added KOH (22 mg, 0.2 mmol), and
the reaction mixture was stirred for 2 h at 75℃. After completion of the
reaction, 1 M aqueous hydrochloric acid was added dropwise to adjust
pH = 3. A precipitate was obtained, and washed with water. The crude
product was purified by column chromatography using DCM/MeOH
(20:1) to give N-(6-chloropyridin-2-yl)-4-hydroxybenzamide (5k).
Similar procedure as that described for 5k gave pure 5l–n.
4.1.2.5. (4-Hydroxyphenyl)(morpholino)methanone (5e). White solid;
yield: 80%; 1H NMR (600 MHz, DMSO‑d6) δ 9.90 (s, 1H), 7.27 (d,
J = 8.4 Hz, 2H), 6.80 (d, J = 8.4 Hz, 2H), 3.41 (s, 4H), 3.48 (s, 4H).
4.1.2.6. (4-Hydroxyphenyl)(4-methylpiperazin-1-yl)methanone
(5f). White solid; yield: 68%; 1H NMR (600 MHz, DMSO‑d6) δ 9.90 (s,
1H), 7.24 (d, J = 8.4 Hz, 2H), 6.79 (d, J = 8.4 Hz, 2H), 3.41 (s, 4H),
2.29 (s, 4H), 2.18 (s, 3H).
4.1.3.1. N-(6-Chloropyridin-2-yl)-4-hydroxybenzamide
(5k). White
4.1.2.7. 4-Hydroxy-N-phenylbenzamide (5g). White solid; yield: 71%;
1H NMR (600 MHz, DMSO‑d6) δ 10.08 (s, 1H), 9.97 (s, 1H), 7.85 (d,
J = 9.0 Hz, 2H), 7.75 (d, J = 7.2 Hz, 2H), 7.32 (t, J = 7.8 Hz, 2H),
7.07–7.06 (m, 1H), 6.86 (d, J = 9.0 Hz, 2H).
solid; yield: 75%; 1H NMR (600 MHz, DMSO‑d6) δ 11.11 (s, 1H), 8.19
(d, J = 8.4 Hz, 1H), 8.08 (d, J = 7.8 Hz, 2H), 7.90 (t, J = 7.8 Hz, 1H),
7.28 (d, J = 8.4 Hz, 1H), 7.28 (d, J = 9.0 Hz, 2H).
4.1.2.8. N-(2-Chlorophenyl)-4-hydroxybenzamide (5h). Colourless oil
liquid; yield: 72%; 1H NMR (600 MHz, DMSO‑d6) δ 10.12 (s, 1H),
9.74 (s, 1H), 7.87 (d, J = 8.4 Hz, 2H), 7.60 (t, J = 8.4 Hz, 1H),
7.54–7.52 (m, 1H), 7.38–7.35 (m, 1H), 7.28–7.25 (m, 1H), 6.87 (d,
4.1.3.2. N-(5-Chloropyridin-2-yl)-4-hydroxybenzamide (5l). White solid;
yield: 66%; 1H NMR (600 MHz, DMSO‑d6) δ 10.99 (s, 1H), 8.42 (s, 1H),
8.20 (d, J = 9.0 Hz, 1H), 8.04 (d, J = 8.4 Hz, 2H), 7.94 (d, J = 9.0 Hz,
1H), 7.25 (d, J = 9.0 Hz, 2H).
7

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4.1.3.3. N-(4-Chloropyridin-2-yl)-4-hydroxybenzamide
(5m). White
98% (MeOH:H2O = 70:30, 0.5 mL/min, tR = 8.46 min); 1H NMR
(600 MHz, DMSO‑d6) δ 8.27 (d, J = 7.8 Hz, 1H), 7.90 (d, J = 8.4 Hz,
2H), 7.78 (s, 1H), 7.30 (d, J = 8.4 Hz, 2H), 6.78 (s, 2H), 4.22–4.18 (m,
1H), 2.29 (s, 3H), 2.07 (s, 3H), 1.87–1.85 (m, 2H), 1.67–1.65 (m, 2H),
1.49–1.45 (m, 4H); 13C NMR (150 MHz, DMSO‑d6) δ 165.3, 165.0,
158.0, 155.0, 145.9, 145.2, 134.6, 131.3, 128.7 (2C), 127.7, 121.3
(2C), 112.5, 50.9, 32.0 (2C), 23.6 (2C), 11.8, 11.0. ESI-MS m/z 394.2
for [M+H].
solid; yield: 71%; 1H NMR (600 MHz, DMSO‑d6) δ 10.78 (s, 1H), 8.36
(d, J = 5.4 Hz, 1H), 8.28 (s, 1H), 7.94 (d, J = 9.0 Hz, 2H), 7.28 (d,
J = 4.8 Hz, 1H), 6.86 (d, J = 9.0 Hz, 2H).
4.1.3.4. N-(3-Chloropyridin-2-yl)-4-hydroxybenzamide (5n). Colourless
oil liquid; yield: 70%; 1H NMR (600 MHz, DMSO‑d6) δ 10.80 (s, 1H),
8.47 (d, J = 4.8 Hz, 1H), 8.07 (d, J = 3.0 Hz, 1H), 8.05 (d, J = 9.0 Hz,
2H), 7.41 (t, J = 5.4 Hz, 1H), 7.32 (d, J = 9.0 Hz, 2H).
4.1.5.4. 4-((3-Amino-6-(3,5-dimethylisoxazol-4-yl)pyrazin-2-yl)oxy)- N-
cyclohexylbenzamide (12Ad). White solid; Yield: 58%; HPLC Purity:
95% (MeOH:H2O = 70:30, 0.5 mL/min, tR = 9.05 min); 1H NMR
(600 MHz, DMSO‑d6) δ 8.18 (d, J = 8.4 Hz, 1H), 7.91 (d, J = 8.4 Hz,
2H), 7.79 (s, 1H), 7.30 (d, J = 9.0 Hz, 2H), 6.77 (s, 2H), 3.74–3.72 (m,
1H), 2.29 (s, 3H), 2.07 (s, 3H), 1.80 (s, 2H), 1.72 (s, 2H), 1.61–1.58 (m,
1H), 1.31–1.28 (m, 4H), 1.13–1.10 (m, 1H); 13C NMR (150 MHz,
DMSO‑d6) δ 165.3, 164.5, 158.0, 155.0, 145.9, 145.2, 134.7, 131.3,
128.7 (2C), 127.7, 121.3 (2C), 112.6, 48.3, 32.4 (2C), 25.2, 24.9 (2C),
11.8, 11.0. ESI-MS m/z 408.2 for [M+H].
4.1.4. Synthesis of 4-(aminomethyl)-N-(4-chlorophenyl)benzamide 8
To a solution of 4-aminomethylbenzoic acid (6, 750 mg, 5 mmol) in
EtOH (7.5 mL) and water (7.5 mL) were added (Boc)2O (1.31 g,
6 mmol), following the reaction mixture was stirred for 5 h at room
temperature. The reaction mixture was concentrated under vacuo to
give 7, which was used further without purification.
To a solution of 7 (1.04 g, 4 mmol), 4-chloroaniline (0.51 g,
4 mmol) in dry DCM (10 mL) were added BOP (300 mg, 5.0 mmol) and
NEt3 (0.8 mL, 7.5 mmol) following the reaction mixture was stirred for
overnight at room temperature. The reaction mixture was added tri-
fluoroacetic acid (0.91 g, 8 mmol), and stirred for another 1 h. The
reaction mixture was concentrated under vacuo, and purified by silica
gel chromatography using a solution of DCM/acetone (20:1) to yield
white solid; yield: 75%; 1H NMR (600 MHz, DMSO‑d6) δ 10.40 (s, 1H),
8.29 (s, 2H), 8.01 (d, J = 8.4 Hz, 2H), 7.83 (d, J = 8.4 Hz, 2H), 7.61 (d,
J = 8.4 Hz, 2H), 7.42 (d, J = 9.0 Hz, 2H), 4.14 (s, 2H).
4.1.5.5. (4-((3-Amino-6-(3,5-dimethylisoxazol-4-yl)pyrazin-2-yl)oxy)
phenyl) (morpholino)methanone (12Ae). Light yellow solid; Yield: 48%;
HPLC Purity: 97% (MeOH:H2O = 70:30, 0.5 mL/min, tR = 6.72 min);
1H NMR (600 MHz, acetone‑d6) δ 7.82 (s, 1H), 7.54 (d, J = 8.4 Hz, 2H),
7.34 (d, J = 9.0 Hz, 2H), 6.16 (d, J = 6.6 Hz, 1H), 3.67 (s, 4H), 3.58 (s,
4H), 2.34 (s, 3H), 2.14 (s, 3H); 13C NMR (150 MHz, DMSO‑d6) δ 168.5,
165.3, 157.9, 153.8, 146.1, 145.1, 134.4, 132.1, 128.6 (2C), 127.8,
121.9 (2C), 112.4, 65.9 (2C), 46.1 (2C), 11.8, 11.0. ESI-MS m/z 394.2
for [M−H].
4.1.5. General synthetic procedure of compounds 12Aa–n and 12Bg–n
To a solution of 2-amino-3,5-dibromopyrazine (9, 253 mg, 1 mmol),
4-hydroxybenzamides (5a–n, 1.2 mmol) in NMP (6 mL) was added
K2CO3 (276 mg, 2.0 mmol). The resulting mixture was refluxed for 6 h
at 100 °C under nitrogen gas. After completion of the reaction, the re-
action mixture was concentrated under vacuo, and purified by silica gel
chromatography using a solution of DCM/acetone (20:1) to yield pure
10a–n.
4.1.5.6. (4-((3-Amino-6-(3,5-dimethylisoxazol-4-yl)pyrazin-2-yl)oxy)
phenyl) (4-methylpiperazin-1-yl)methanone (12Af). Yellow solid; Yield:
55%; HPLC Purity: 98% (MeOH:H2O
= 70:30, 0.5 mL/min,
tR = 6.88 min); 1H NMR (600 MHz, DMSO‑d6) δ 7.79 (s, 1H), 7.43
(d, J = 9.0 Hz, 2H), 7.29 (d, J = 8.4 Hz, 2H), 6.77 (s, 2H), 3.32 (brs,
8H), 2.27 (s, 3H), 2.18 (s, 3H), 2.05 (s, 3H); 13C NMR (150 MHz,
DMSO‑d6) δ 168.4, 165.3, 157.9, 153.7, 146.1, 145.2, 134.4, 132.5,
128.4 (2C), 127.8, 121.9 (2C), 120.9, 114.8, 112.4, 54.4, 45.4 (2C),
11.8, 11.0. ESI-MS m/z 409.2 for [M+H].
To a mixture of 2-amino-5-dibromo-3-sustitutedpyrazines (10a–n,
0.5 mmol) and 3,5-dimethylisoxazole-4-boronic acid (or 1-methylpyr-
azole-4-boronic acid pinacol ester, 0.6 mmol) in dioxane (10 mL) were
added Pd(dppf)2Cl2·CH2Cl2 (40.8 mg, 0.05 mmol) and 2 M Na2CO3
solution (0.05 mL, 0.1 mmol). The resulting mixture was refluxed for
4 h at 100 °C under nitrogen gas. The reaction mixture was con-
centrated under vacuo, and purified by column chromatography using a
solution of DCM/MeOH (20:1) to yield compound 12Aa–n and 12Bg–n.
4.1.5.7. 4-((3-Amino-6-(3,5-dimethylisoxazol-4-yl)pyrazin-2-yl)oxy)- N-
phenylbenzamide (12Ag). White solid; Yield: 72%; HPLC Purity: 95%
(MeOH:H2O = 70:30, 0.5 mL/min, tR = 9.29 min); 1H NMR (600 MHz,
DMSO‑d6) δ 10.25 (s, 1H), 8.05 (d, J = 9.0 Hz, 2H), 7.83 (s, 1H), 7.78
(d, J = 7.8 Hz, 2H), 7.41 (d, J = 8.4 Hz, 2H), 7.36 (d, J = 7.2 Hz, 2H),
7.10 (s, 1H), 6.83 (s, 2H), 2.33 (s, 3H), 2.12 (s, 3H); 13C NMR
(150 MHz, DMSO‑d6) δ 165.3, 164.5, 157.9, 155.5, 145.8, 145.2,
139.0, 134.8, 131.2, 129.1 (2C), 128.4 (2C), 127.8, 123.5, 121.3 (2C),
120.3 (2C), 92.5, 11.7, 10.9. ESI-MS m/z 402.2 for [M+H].
4.1.5.1. 4-((3-Amino-6-(3,5-dimethylisoxazol-4-yl)pyrazin-2-yl)oxy)- N-
propylbenzamide (12Aa). White solid; Yield: 72%; HPLC Purity: 95%
(MeOH:H2O = 70:30, 0.5 mL/min, tR = 7.60 min); 1H NMR (600 MHz,
DMSO‑d6) δ 8.44 (t, J = 5.4, 1H), 7.89 (d, J = 7.2 Hz, 2H), 7.79 (s,
1H), 7.31 (d, J = 7.2 Hz , 2H), 6.78 (s, 2H), 3.21–3.18 (m, 2H), 2.29 (s,
3H), 2.07 (s, 3H), 1.53–1.49 (m, 2H), 0.87 (t, J = 7.2 Hz, 3H); 13C NMR
(150 MHz, DMSO‑d6) δ 165.4, 165.3, 158.0, 155.1, 145.9, 145.2, 134.6,
131.2, 128.6 (2C), 127.8, 121.4 (2C), 112.5, 40.9, 22.3, 11.8, 11.4,
11.0. ESI-MS: m/z 368.2 for [M+H].
4.1.5.8. 4-((3-Amino-6-(3,5-dimethylisoxazol-4-yl)pyrazin-2-yl)oxy)- N-
(2-chlorophenyl)benzamide (12Ah). White solid; Yield: 60%; HPLC
Purity: 97% (MeOH:H2O = 70:30, 0.5 mL/min, tR = 10.12 min); 1H
NMR (600 MHz, DMSO‑d6) δ 10.04 (s, 1H), 8.06 (d, J = 8.4 Hz, 2H),
7.81 (s, 1H), 7.59 (d, J = 7.2 Hz, 1H), 7.54 (d, J = 7.2 Hz, 1H), 7.40 (d,
J = 7.8 Hz, 2H), 7.37 (t, J = 8.4 Hz, 1H), 7.28 (t, J = 7.8 Hz, 1H), 6.79
(s, 2H), 2.31 (s, 3H), 2.10 (s, 3H); 13C NMR (150 MHz, DMSO‑d6) δ
165.4, 164.6, 158.0, 155.8, 145.8, 145.3, 135.0, 134.9, 130.4, 129.5
(2C), 129.2 (2C), 128.5, 127.8, 127.4 (2C), 121.6 (2C), 112.6, 11.8,
11.0. ESI-MS m/z 436.2 for [M + H].
4.1.5.2. 4-((3-Amino-6-(3,5-dimethylisoxazol-4-yl)pyrazin-2-yl)oxy)- N-
cyclopropylbenzamide (12Ab). White solid; Yield: 74%; HPLC Purity:
95% (MeOH:H2O = 70:30, 0.5 mL/min, tR = 7.00 min); 1H NMR
(600 MHz, DMSO‑d6) δ 8.44 (d, J = 4.2 Hz, 1H), 7.89 (d, J = 8.4 Hz,
2H), 7.81 (s, 1H), 7.32 (d, J = 8.4 Hz, 2H), 6.79 (s, 2H), 2.85–2.84 (m,
1H), 2.29 (s, 3H), 2.07 (s, 3H), 0.71–0.68 (m, 2H), 0.59–0.56 (m, 2H);
13C NMR (150 MHz, DMSO‑d6) δ 166.6, 165.3, 158.0, 155.1, 145.9,
145.2, 134.7, 130.9, 129.0, 128.6 (2C), 121.3 (2C), 114.6, 23.0, 11.8,
11.0, 5.68 (2C). ESI-MS m/z 366.2 for [M+H].
4.1.5.9. 4-((3-Amino-6-(3,5-dimethylisoxazol-4-yl)pyrazin-2-yl)oxy)- N-
(4-chlorophenyl)benzamide (12Ai). White solid; Yield: 77%; HPLC
Purity: 95% (MeOH:H2O = 70:30, 0.5 mL/min, tR = 12.40 min); 1H
NMR (600 MHz, DMSO‑d6) δ 10.35 (s, 1H), 8.05 (d, J = 9.0 Hz, 2H),
7.83 (s, 1H), 7.82 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 8.4 Hz, 4H), 6.80 (s,
4.1.5.3. 4-((3-Amino-6-(3,5-dimethylisoxazol-4-yl)pyrazin-2-yl)oxy)-N-
cyclopentylbenzamide (12Ac). White solid; Yield: 66%; HPLC Purity:
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NMR (150 MHz, DMSO‑d6) δ 164.7, 156.0, 145.0, 144.9, 139.1, 135.5,
132.2, 131.1, 130.6, 129.1 (2C), 128.4 (2C), 127.3, 123.5, 120.3 (2C),
120.2 (2C), 119.8, 38.4. ESI-MS m/z 387.2 for [M+H].
2H), 2.33 (s, 3H), 2.11 (s, 3H); 13C NMR (150 MHz, DMSO‑d6) δ 165.4,
164.7, 158.0, 155.7, 145.8, 145.2, 138.1, 134.8, 131.0, 129.2 (2C),
128.4 (2C), 127.8, 127.2, 121.8 (2C), 121.5 (2C), 112.5, 11.8, 11.0.
ESI-MS m/z 436.2 for [M+H].
4.1.5.16. 4-((3-Amino-6-(1-methyl-1H-pyrazol-4-yl)pyrazin-2-yl)oxy) -N-
(2-chlorophenyl)benzamide (12Bh). White solid; Yield: 60%; HPLC
Purity: 97% (MeOH:H2O = 70:30, 0.5 mL/min, tR = 8.47 min); 1H
NMR (600 MHz, DMSO‑d6) δ 10.06 (s, 1H), 8.08 (d, J = 8.4 Hz, 2H),
8.04 (s, 1H), 7.85 (s, 1H), 7.68 (s, 1H), 7.61 (d, J = 6.6 Hz, 1H), 7.56 (t,
J = 7.2 Hz, 1H), 7.42 (d, J = 8.4 Hz, 2H), 7.40 (d, J = 7.8 Hz, 1H),
7.31 (t, J = 7.8 Hz, 1H), 6.52 (s, 2H), 3.80 (s, 3H); 13C NMR (150 MHz,
DMSO‑d6) δ 164.6, 156.3, 145.0, 135.5, 135.1, 132.3, 131.1, 129.7,
129.6, 129.5 (2C), 129.3 (2C), 128.4 (2C), 127.4 (2C), 120.3 (2C),
119.8, 38.5. ESI-MS m/z 421.2 for [M+H].
4.1.5.10. 4-((3-Amino-6-(3,5-dimethylisoxazol-4-yl)pyrazin-2-yl)oxy)- N-
(3-chlorophenyl)benzamide (12Aj). White solid; Yield: 66%; HPLC
Purity: 96% (MeOH:H2O = 70:30, 0.5 mL/min, tR = 12.63 min); 1H
NMR (600 MHz, DMSO‑d6) δ 10.40 (s, 1H), 8.06 (d, J = 8.4 Hz, 2H),
7.98 (s, 1H), 7.84 (s, 1H), 7.72 (d, J = 8.4 Hz, 1H), 7.43 (d, J = 9.0 Hz,
2H), 7.39 (t, J = 8.4 Hz, 1H), 7.17 (d, J = 8.4 Hz, 1H), 6.82 (s, 2H),
2.33 (s, 3H), 2.12 (s, 3H); 13C NMR (150 MHz, DMSO‑d6) δ 165.4,
164.9, 158.0, 155.8, 145.8, 145.2, 140.6, 134.9, 132.8, 130.8, 130.2,
129.3 (2C), 127.8, 123.3, 121.5 (2C), 119.7, 118.6, 112.5, 11.8, 11.0.
ESI-MS m/z 436.2 for [M+H].
4.1.5.17. 4-((3-Amino-6-(1-methyl-1H-pyrazol-4-yl)pyrazin-2-yl)oxy)- N-
(4-chlorophenyl)benzamide (12Bi). White solid; Yield: 72%; HPLC
Purity: 95% (MeOH:H2O = 70:30, 0.5 mL/min, tR = 10.58 min); 1H
NMR (600 MHz, DMSO‑d6) δ 10.38 (s, 1H), 8.05 (d, J = 9.0 Hz, 2H),
8.04 (s, 1H), 7.84 (s, 1H), 7.83 (d, J = 9.0 Hz, 2H), 7.67 (s, 1H), 7.42
(d, J = 7.8 Hz, 4H), 6.53 (s, 2H), 3.79 (s, 3H); 13C NMR (150 MHz,
DMSO‑d6) δ 164.9, 156.2, 145.0, 138.1, 135.5, 131.1, 130.4, 129.7,
129.3, 128.5 (2C), 128.4 (2C), 127.3, 121.8 (2C), 120.3 (2C), 119.8,
114.9, 38.5. ESI-MS m/z 421.1 for [M+H].
4.1.5.11. 4-((3-Amino-6-(3,5-dimethylisoxazol-4-yl)pyrazin-2-yl)oxy)- N-
(6-chloropyridin-2-yl)benzamide (12Ak). Yellow solid; Yield: 42%;
HPLC Purity: 97% (MeOH:H2O
=
70:30, 0.5 mL/min,
tR = 7.67 min); 1H NMR (600 MHz, DMSO‑d6) δ 11.16 (s, 1H), 8.21
(d, J = 7.8 Hz, 1H), 8.12 (d, J = 7.8 Hz, 2H), 7.91 (t, J = 8.4 Hz, 1H),
7.84 (s, 1H), 7.39 (d, J = 9.0 Hz, 2H), 7.28 (d, J = 7.8 Hz, 1H), 6.86 (s,
2H), 2.34 (s, 3H), 2.13 (s, 3H); 13C NMR (150 MHz, DMSO‑d6) δ 165.4,
158.0, 156.1, 152.3, 147.9, 145.7, 145.3, 141.5, 134.9, 130.0, 129.8
(2C), 127.8, 121.3 (2C), 119.5, 113.3 (2C), 112.5, 11.8, 11.0. ESI-MS
m/z 437.2 for [M+H].
4.1.5.18. 4-((3-Amino-6-(1-methyl-1H-pyrazol-4-yl)pyrazin-2-yl)oxy)- N-
(3-chlorophenyl)benzamide (12Bj). White solid; Yield: 71%; HPLC
Purity: 99% (MeOH:H2O = 70:30, 0.5 mL/min, tR = 10.05 min); 1H
NMR (600 MHz, DMSO‑d6) δ 10.40 (s, 1H), 8.06 (d, J = 9.6 Hz, 2H),
8.04 (s, 1H), 7.98 (t, J = 1.8 Hz, 1H), 7.84 (s, 1H), 7.72 (d, J = 8.4 Hz,
1H), 7.67 (s, 1H), 7.42 (d, J = 9.0 Hz, 2H), 7.39 (t, J = 8.4 Hz, 1H),
7.38 (s, 1H), 7.17 (d, J = 8.4 Hz, 1H), 6.53 (s, 2H), 3.80 (s, 3H); 13C
NMR (150 MHz, DMSO‑d6) δ 165.0, 156.3, 145.0, 140.6, 135.5, 132.9,
132.3, 131.1, 130.3, 130.2, 129.3 (2C), 127.3, 123.2, 120.3 (2C),
119.8, 119.6, 118.6, 114.9, 38.5. ESI-MS m/z 421.2 for [M+H].
4.1.5.12. 4-((3-Amino-6-(3,5-dimethylisoxazol-4-yl)pyrazin-2-yl)oxy)-N-
(5-chloropyridin-2-yl)benzamide (12Al). Yellow solid; Yield: 44%; HPLC
Purity: 95% (MeOH:H2O = 70:30, 0.5 mL/min, tR = 10.66 min); 1H
NMR (600 MHz, DMSO‑d6) δ 11.03 (s, 1H), 8.45 (d, J = 2.4 Hz, 1H),
8.24 (d, J = 8.4 Hz, 1H), 8.11 (d, J = 9.0 Hz, 2H), 7.97 (dd, J = 9.0,
3.0 Hz, 1H), 7.84 (s, 1H), 7.39 (d, J = 9.0 Hz, 2H), 6.81 (s, 2H), 2.34 (s,
3H), 2.13 (s, 3H); 13C NMR (150 MHz, DMSO‑d6) δ 165.4, 165.3, 158.0,
156.0, 150.9, 146.2, 145.8, 145.3, 137.8, 134.9, 130.2, 129.8 (2C),
127.8, 125.5, 121.4 (2C), 115.8, 112.5, 11.8, 11.1. ESI-MS m/z 437.1
for [M+H].
4.1.5.19. 4-((3-Amino-6-(1-methyl-1H-pyrazol-4-yl)pyrazin-2-yl)oxy)- N-
(6-chloropyridin-2-yl)benzamide (12Bk). Yellow solid; Yield: 37%;
4.1.5.13. 4-((3-Amino-6-(3,5-dimethylisoxazol-4-yl)pyrazin-2-yl)oxy)- N-
HPLC Purity: 95% (MeOH:H2O
=
70:30, 0.5 mL/min,
(4-chloropyridin-2-yl)benzamide (12Am). Yellow solid; Yield: 46%;
tR = 9.05 min); 1H NMR (600 MHz, DMSO‑d6) δ 11.13 (s, 1H), 8.21
(d, J = 7.8 Hz, 1H), 8.12 (d, J = 8.4 Hz, 2H), 8.06 (s, 1H), 7.92 (t,
J = 8.4 Hz, 1H), 7.86 (s, 1H) , 7.69 (s, 1H), 7.37 (d, J = 8.4 Hz, 2H),
7.28 (d, J = 7.2 Hz, 1H), 6.57 (s, 2H), 3.81 (s, 3H); 13C NMR (150 MHz,
DMSO‑d6) δ 165.5, 156.6, 152.3, 147.9, 145.1, 144.8, 141.5, 135.5,
132.5, 131.2, 129.8 (2C), 129.3, 127.4, 120.0 (2C), 119.8, 119.4,
113.2, 38.5. ESI-MS m/z 422.2 for [M+H].
HPLC Purity: 96% (MeOH:H2O
=
70:30, 0.5 mL/min,
tR = 10.16 min); 1H NMR (600 MHz, DMSO‑d6) δ 11.08 (s, 1H), 8.37
(d, J = 4.8 Hz, 1H), 8.28 (d, J = 1.8 Hz, 1H), 8.09 (d, J = 9.0 Hz, 2H),
7.81 (s, 1H), 7.37 (d, J = 9.0 Hz, 2H), 7.29 (dd, J = 5.4, 1.8 Hz, 1H),
6.78 (s, 2H), 2.31 (s, 3H), 2.10 (s, 3H); 13C NMR (150 MHz, DMSO‑d6) δ
165.6, 165.4, 158.0, 156.1, 153.3, 149.3, 145.7, 145.3, 143.9, 134.9,
130.0, 129.7 (2C), 127.8, 121.4 (2C), 119.7, 114.1, 112.5, 11.8, 11.0.
ESI-MS m/z 437.1 for [M+H].
4.1.5.20. 4-((3-Amino-6-(1-methyl-1H-pyrazol-4-yl)pyrazin-2-yl)oxy)-N-
(5-chloropyridin-2-yl)benzamide (12Bl). Yellow solid; Yield: 41%; HPLC
Purity: 98% (MeOH:H2O = 70:30, 0.5 mL/min, tR = 9.73 min); 1H
NMR (600 MHz, DMSO‑d6) δ 10.99 (s, 1H), 8.45 (d, J = 2.4 Hz, 1H),
8.25 (d, J = 8.4 Hz, 1H), 8.12 (d, J = 8.4 Hz, 2H), 8.05 (s, 1H), 7.98 (d,
J = 8.4 Hz, 1H), 7.85 (s, 1H) , 7.69 (s, 1H), 7.39 (d, J = 9.0 Hz, 2H),
6.53 (s, 2H), 3.80 (s, 3H); 13C NMR (150 MHz, DMSO‑d6) δ 165.4,
156.6, 150.9, 146.2, 145.1, 144.8, 137.8, 135.5, 132.4, 131.2, 129.8
(2C), 129.5, 127.4, 125.4, 120.1 (2C), 119.8, 115.7, 38.5. ESI-MS m/z
422.1 for [M+H].
4.1.5.14. 4-((3-Amino-6-(3,5-dimethylisoxazol-4-yl)pyrazin-2-yl)oxy) -N-
(3-chloropyridin-2-yl)benzamide (12An). Yellow solid; Yield: 37%;
HPLC Purity: 97% (MeOH:H2O
=
70:30, 0.5 mL/min,
tR = 8.42 min); 1H NMR (600 MHz, DMSO‑d6) δ 11.12 (s, 1H), 8.40
(d, J = 5.4 Hz, 1H), 8.31 (d, J = 1.8 Hz, 1H), 8.12 (d, J = 7.8 Hz, 2H),
7.84 (s, 1H), 7.40 (d, J = 8.4 Hz, 2H), 7.32 (t, J = 5.4 Hz, 1H), 6.82 (s,
2H), 2.34 (s, 3H), 2.13 (s, 3H); 13C NMR (150 MHz, DMSO‑d6) δ 165.6,
165.4, 158.0, 156.1, 153.3, 149.3, 145.7, 145.3, 143.8, 134.9, 130.0,
129.7 (2C), 127.8, 121.4 (2C), 119.7, 114.1, 112.5, 11.8, 11.0. ESI-MS
m/z 437.2 for [M+H].
4.1.5.21. 4-((3-Amino-6-(1-methyl-1H-pyrazol-4-yl)pyrazin-2-yl)oxy)- N-
(4-chloropyridin-2-yl)benzamide (12Bm). Yellow solid; Yield: 45%;
4.1.5.15. 4-((3-Amino-6-(1-methyl-1H-pyrazol-4-yl)pyrazin-2-yl)oxy) -N-
phenylbenzamide (12Bg). White solid; Yield: 76%; HPLC Purity: 99%
(MeOH:H2O = 70:30, 0.5 mL/min, tR = 7.81 min); 1H NMR (600 MHz,
DMSO‑d6) δ 10.25 (s, 1H), 8.05 (d, J = 9.0 Hz, 2H), 8.04 (s, 1H), 7.84
(s, 1H), 7.79 (d, J = 7.2 Hz, 2H), 7.67 (s, 1H), 7.41 (d, J = 7.8 Hz, 2H),
7.37 (t, J = 7.2 Hz, 2H), 7.10 (s, 1H), 6.54 (s, 2H), 3.79 (s, 3H); 13C
HPLC Purity: 97% (MeOH:H2O
=
70:30, 0.5 mL/min,
tR = 6.04 min); 1H NMR (600 MHz, DMSO‑d6) δ 11.05 (s, 1H), 8.37
(d, J = 5.4 Hz, 1H), 8.29 (s, 1H), 8.10 (d, J = 8.4 Hz, 2H), 8.03 (s, 1H),
7.83 (s, 1H) , 7.66 (s, 1H), 7.36 (d, J = 8.4 Hz, 2H), 7.30 (d, J = 5.4 Hz,
1H), 6.50 (s, 2H), 3.77 (s, 3H); 13C NMR (150 MHz, DMSO‑d6) δ 165.7,
156.7, 153.4, 149.3, 145.1, 144.8, 143.9, 135.6, 132.5, 131.2, 129.8
9

Y.-X. Bo, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
(2C), 129.3, 127.4, 120.1 (2C), 119.8, 119.7, 114.0, 38.5. ESI-MS m/z
added into the above mixture, and the reaction mixture was stirred for
4 h at room temperature. After completion of the reaction, the reaction
mixture was concentrated under vacuo, and the crude product was
422.1 for [M+H].
4.1.5.22. 4-((3-Amino-6-(1-methyl-1H-pyrazol-4-yl)pyrazin-2-yl)oxy)- N-
purified by silica gel chromatography using a solution of di-
(3-chloropyridin-2-yl)benzamide (12Bn). Yellow solid; Yield: 35%;
chloromethane/acetone (20:1) to yield pure 15.
HPLC Purity: 96% (MeOH:H2O
=
70:30, 0.5 mL/min,
To a mixture of 15 (0.5 mmol), 3,5-dimethylisoxazole-4-boronic
acid (or 1-methylpyrazole-4-boronic acid pinacol ester, 0.6 mmol) in
dioxane (10 mL) were added Pd(dppf)2Cl2·CH2Cl2 (40.8 mg,
0.05 mmol) and 2 M Na2CO3 solution (0.05 mL, 0.1 mmol). The re-
sulting mixture was refluxed for 4 h at 100 °C under nitrogen gas. After
completion of the reaction, the reaction mixture was concentrated
under vacuo, and the crude product was purified by silica gel chro-
matography using a solution of DCM/MeOH (20:1) to yield pure com-
pounds 16A,B.
tR = 9.08 min); 1H NMR (600 MHz, DMSO‑d6) δ 11.09 (s, 1H), 8.40
(d, J = 5.4 Hz, 1H), 8.32 (s, 1H), 8.13 (d, J = 8.4 Hz, 2H), 8.06 (s, 1H),
7.86 (s, 1H) , 7.69 (s, 1H), 7.39 (d, J = 9.0 Hz, 2H), 7.32 (t, J = 3.0 Hz,
1H), 6.53 (s, 2H), 3.80 (s, 3H); 13C NMR (150 MHz, DMSO‑d6) δ 165.6,
156.7, 153.4, 149.3, 145.1, 144.8, 143.8, 135.5, 132.5, 131.2, 129.8
(2C), 129.3, 127.4, 120.0 (2C), 119.8, 119.7, 114.0, 38.5. ESI-MS m/z
422.2 for [M+H].
4.1.6. General synthetic procedure of compounds 13A,B
To a solution of 2-amino-3,5-dibromopyrazine (9, 253 mg, 1 mmol),
4-aminomethylbenzamide 8 (1.2 mmol) in dioxane (5 mL) and water
(5 mL) was added KOH (276 mg, 2.0 mmol). The resulting mixture was
refluxed for 48 h at 100℃ under nitrogen gas. After completion of the
reaction, the reaction mixture was concentrated under vacuo, and
purified by column chromatography using DCM/acetone (20:1) to yield
pure 11.
4.1.7.1. 4-((3-Amino-6-(3,5-dimethylisoxazol-4-yl)pyrazin-2-yl)thio)- N-
(4-chlorophenyl)benzamide (16A). Brown solid; Yield: 42%; HPLC
Purity: 99% (MeOH:H2O = 70:30, 0.5 mL/min, tR = 6.73 min); 1H
NMR (600 MHz, DMSO‑d6) δ 10.44 (s, 1H), 8.00 (s, 1H), 7.98 (d,
J = 8.4 Hz 2H), 7.82 (d, J = 8.4 Hz, 2H), 7.63 (d, J = 8.4 Hz, 2H), 7.42
(d, J = 9.0 Hz, 2H), 6.70 (s, 2H), 2.27 (s, 3H), 2.04 (s, 3H); 13C NMR
(150 MHz, DMSO‑d6) δ 165.8, 164.9, 158.1, 151.5, 138.7, 138.0, 137.1,
134.4, 134.2, 133.4 (2C), 133.1, 128.6 (4C), 127.4, 121.9 (2C), 112.7,
11.8, 10.9. ESI-MS m/z 452.1 for [M+H].
To a mixture of 11 (0.5 mmol), 3,5-dimethylisoxazole-4-boronic
acid (84.6 mg, 0.6 mmol) or 1-methylpyrazole-4-boronic acid pinacol
ester (124.8 mg, 0.6 mmol) in dioxane (10 mL) were added Pd
(dppf)2Cl2·CH2Cl2 (40.8 mg, 0.05 mmol) and 2 M Na2CO3 solution
(0.05 mL, 0.1 mmol). The resulting mixture was refluxed for 4 h at
100℃ under nitrogen gas. After completion of the reaction, the reaction
mixture was concentrated under vacuo, and purified by column chro-
matography using DCM/MeOH (20:1) to yield target compounds
13A,B.
4.1.7.2. 4-((3-Amino-6-(1-methyl-1H-pyrazol-4-yl)pyrazin-2-yl)thio)-N-
(4-chlorophenyl)benzamide (16B). Brown solid; Yield: 37%; HPLC
Purity: 97% (MeOH:H2O = 70:30, 0.5 mL/min, tR = 6.24 min); 1H
NMR (600 MHz, DMSO‑d6) δ 10.33 (s, 1H), 8.28 (s,1H), 7.69 (s, 1H),
7.91 (s, 1H), 7.87 (d, J = 7.8 Hz, 2H), 7.79 (d, J = 8.4 Hz, 2H), 7.40 (d,
J = 9.0 Hz, 2H), 7.34 (d, J = 8.4 Hz, 2H), 6.60 (s, 2H), 3.89 (s, 3H); 13
C
NMR (150 MHz, DMSO‑d6) δ 164.9, 151.6, 143.8, 141.4, 138.0, 134.7,
133.9, 132.0, 130.5, 128.4 (4C), 127.1 (2C), 127.0, 121.7 (2C), 121.7,
117.9, 39.0. ESI-MS m/z 437.1 for [M+H].
4.1.6.1. 4-(((3-Amino-6-(3,5-dimethylisoxazol-4-yl)pyrazin-2-yl)amino)
methyl)- N-(4-chlorophenyl)benzamide (13A). Yellow solid; Yield: 70%;
HPLC Purity: 95% (MeOH:H2O = 70:30, 0.5 mL/min, tR = 12.25 min);
1H NMR (600 MHz, DMSO‑d6) δ 10.27 (s, 1H), 7.90 (d, J = 8.4 Hz, 2H),
7.81 (d, J = 7.2 Hz, 2H), 7.46 (d, J = 7.8 Hz, 2H), 7.40 (d, J = 7.2 Hz,
2H), 7.32 (s, 1H), 6.91 (s, 1H), 6.19 (s, 2H), 4.67 (d, J = 6.0 Hz, 2H),
2.35 (s, 3H), 2.15 (s, 3H); 13C NMR (150 MHz, DMSO‑d6) δ 165.3,
164.8, 158.2, 144.2, 142.8, 141.8, 138.1, 132.9, 129.7, 128.4 (2C),
127.7 (2C), 127.3, 127.1, 126.7 (2C), 121.8 (2C), 113.7, 43.7, 11.9,
11.1. ESI-MS m/z 449.2 for [M+H].
4.2. Biology
4.2.1. Antiproliferation assays
Cells were incubated at 37 °C in a 5% CO2 atmosphere. The U38,
HeLa, HepG2 and LoVo cells were seeded in 96-well plates at a density
of 5 × 103 cells/well in 150 μL of the medium with 10% FBS for 24 h.
The target compounds 12, 13, 16 and VX-680 were dissolved and di-
luted to various concentrations using culture medium, and then added
them to the well and incubated at 37 °C for 72 h, then the media was
aspirated, and 10 μL of 5 mg/mL MTT solution diluted in serum-free
media was added to each well. After 4 h, the supernatant was discarded
and 100 μL of DMSO was added to each well. The mixture was shaken
on an oscillator and measured at 490 nm using universal microplate
reader (Infinite M200 Pro, Tecan Inc.). IC50 values were determined
from a log plot of percent of control versus concentration. All samples
were tested in triplicate.
4.1.6.2. 4-(((3-Amino-6-(1-methyl-1H-pyrazol-4-yl)pyrazin-2-yl)amino)
methyl)- N-(4-chlorophenyl)benzamide (13B). Yellow solid; Yield: 65%;
HPLC Purity: 97% (MeOH:H2O = 70:30, 0.5 mL/min, tR = 10.11 min);
1H NMR (600 MHz, DMSO‑d6) δ 10.28 (s, 1H), 7.90 (d, J = 6.6 Hz, 2H),
7.89 (s, 1H), 7.80 (d, J = 8.4 Hz, 2H), 7.69 (s, 1H), 7.55 (d, J = 8.4 Hz,
2H), 7.47 (s, 1H), 7.40 (d, J = 9.0 Hz, 2H), 6.85 (s, 1H), 5.92 (s, 2H),
4.68 (d, J = 6.0 Hz, 2H), 3.81 (s, 3H); 13C NMR (150 MHz, DMSO‑d6) δ
165.4, 144.5, 142.2, 143.2, 141.7, 138.1, 135.5, 133.0, 132.3, 129.6,
128.4 (2C), 127.6 (2C), 127.5 (2C), 127.0, 121.7 (2C), 121.4, 43.6,
38.5. ESI-MS m/z 434.2 for [M+H].
4.2.2. Kinase-Glo luminescent kinase assay
The synthetic compounds and reference compound were diluted to
six concentrations in the PBS and then added 5 µL to the 50 µL reaction
mixture (40 mM Tris, pH = 7.4, 10 mM MgCl2, 1 mM DTT, 0.1 mg/ml
BSA, 10 µM ATP, 0.2 μg/ml Kinase and 100 µM Kemptide acetate salt),
and then the kinase reactions were incubated for 30 min at 37 °C. Then
we used Kinase-Glo luminescence kinase assay kit tested luminescent
signal of the reaction mixture and calculated the IC50 using Prism Graph
Pad software.
4.1.7. General synthetic procedure of compounds 16A,B
To a solution of 2-amino-3,5-dibromopyrazine (9, 1.0 g, 4 mmol)
and 4-mercaptobenzoic acid (670 mg, 4.4 mmol) in EtOH (10 mL) was
added KOH (450 mg, 8 mmol), and the resulting mixture was refluxed
for 12 h at 80 °C. After completion of the reaction, to the reaction
mixture was added water and extracted with ethyl acetate, the organic
layer was coupled and dried over anhydrous sodium sulfate. The mix-
ture was concentrated under vacuo to give 14, which was used without
further purification.
4.2.3. Western blot analysis
To a solution of 14 (650 mg, 2 mmol) in dried DCM (10 mL), thionyl
chloride (2.2 mmol) was added dropwise, and the reaction continued
for 4 h at room temperature. Followed 4-chloroaniline and Et3N were
HeLa cells were treated with 12Aj (0, 1, 5 and 10 μM) or VX-680
(10 μM) for 24 h. Followed the cells were washed and lysed in lysis
buffer, total proteins were achieved by centrifuging and the
10

Y.-X. Bo, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
concentrations were determined by using BCA protein assay kit
(Beyotime, Jiangsu, China). For western blot analysis, equal amounts of
proteins (20–30 μg) were separated on 12% SDS-PAGE gels and trans-
ferred to polyvinylidine difluoride (PVDF) membranes (Millipore
Corporation, USA). The blot was blocked in blocking buffer (5% non-fat
dry milk in TBST) for 2 h at room temperature, and then incubated with
dilute solution (1:500–1:1000) of the antibody against Aurora A and
Aurora B (Abcam), phospho-Aurora A (Thr288) and phospho-Aurora B
(Thr232) (Cell Signaling Technology), cyclinB1 and cdc2 (BioLegend),
and β-actin (ZSGB-BIO) in blocking buffer overnight at 4℃, respec-
tively. The blot was then incubated with appropriate secondary anti-
body (1:5000–1:10,000 dilution), β-Actin was used as a loading control.
The protein bands were visualized using the Gel Imaging System
(ChemDoc-It610, UVP, USA).
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmc.2020.115351.
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4.3. Molecular docking study
The docking simulation was performed using the Glide module of
Schrödinger. Before docking, the ligand 12Aj was prepared firstly using
the LigPrep module in Schrödinger. The X-ray crystal structures of
Aurora (http://www.rscb.org./pdb, Aurora A PDB code: 3H0Z and
Aurora B PDB code: 4C2V) were derived from the PDB Database and
minimized using the Protein Preparation Wizard module in the
Schrödinger Suite.33 The crystal waters were removed and polar hy-
drogen was added to the two proteins. Then, a grid was generated at the
active site of the prepared protein structure using the Grid Generation
module of Schrödinger. The Glide docking module of Schrödinger was
used to investigate the interactions between the synthesized compounds
and Aurora kinases. Each docking process was performed in 250,000
energy evaluation with 10 conformations kept and the most favorable
pose of each compound was displayed. And all the figures displaying
the docking results were obtained using the scientific software Pymol.
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tumor growth in aggressive B-cell lymphomas. Int J Cancer. 2012;130:2997–3005.
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Declaration of Competing Interest
The authors declare that they have no known competing financial
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