Design, synthesis, biological evaluation of 6-(2-amino-1H-benzo[d]imidazole-6-yl)quinazolin-4(3H)-one derivatives as novel anticancer agents with Aurora kinase inhibition

Article abstract of DOI:10.1016/j.ejmech.2020.112108

Aurora A kinase, a member of the Aurora kinase family, is frequently overexpressed in various human cancers. In addition, Overexpression of Aurora A kinase is associated with drug resistance and poor prognosis in many cancers including breast cancer. Therefore, Aurora A kinase has been considered as an attractive anticancer target for the treatment of human cancers. Herein, A series of 6-(2-amino-1H-benzo[d]imidazole-6-yl)quinazolin-4(3H)-one derivatives were designed, synthesized, and evaluated as Aurora A kinase inhibitors. The cell-based cytotoxicity assays showed that compound 16h was the most potent cytotoxic agent against all tested cancer cells and had a lower IC₅₀ value than ENMD-2076 against MDA-MB-231 cells. Meanwhile, Aurora A kinase assay and Western blot analysis showed that 16h inhibited Aurora A kinase with an IC₅₀ value of 21.94 nM and suppressed the phosphorylation of Histone H3 on Ser10 and Aurora A kinase on Thr288, which were consistent with the activation of Aurora A kinase. Accordingly, 16h caused aberrant mitotic phenotypes and obvious G2/M phase arrest in MDA-MB-231 cells and induced caspase-dependent apoptosis in MDA-MB-231 cells. These results demonstrated that 16h is a potential candidate for the development of anticancer agents targeting Aurora A kinase.

Full text of DOI:10.1016/j.ejmech.2020.112108

Journal Pre-proof
Design, synthesis, biological evaluation of 6-(2-amino-1H-benzo[d]imidazole- 6-
yl)quinazolin-4(3H)-one derivatives as novel anticancer agents with Aurora kinase
inhibition
Chengcheng Fan, Ting Zhong, Huarong Yang, Ying Yang, Daoping Wang, Xiaosheng
Yang, Yongnan Xu, Yanhua Fan
PII:
S0223-5234(20)30075-1
DOI:
https://doi.org/10.1016/j.ejmech.2020.112108
EJMECH 112108
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 October 2019
Revised Date: 11 January 2020
Accepted Date: 28 January 2020
Please cite this article as: C. Fan, T. Zhong, H. Yang, Y. Yang, D. Wang, X. Yang, Y. Xu, Y. Fan,
Design, synthesis, biological evaluation of 6-(2-amino-1H-benzo[d]imidazole- 6-yl)quinazolin-4(3H)-
one derivatives as novel anticancer agents with Aurora kinase inhibition, European Journal of Medicinal
Chemistry (2020), doi: https://doi.org/10.1016/j.ejmech.2020.112108.
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Design, synthesis, biological evaluation of 6-(2-amino-1H-benzo[d]imidazol-
6-yl)quinazolin-4(3H)-one derivatives as novel anticancer agents with Aurora
kinase inhibition
Chengcheng Fan b,c, Ting Zhong a,b, Huarong Yang b, Ying Yang a,b, Daoping Wang a,b,
Xiaosheng Yang a,b, Yongnan Xu c, , Yanhua Fan a,b,
a State Key Laboratory for Functions and Applications of Medicinal Plants, Guizhou
Medical University, Guiyang 550014, China.
b
The Key Laboratory of Chemistry for Natural Products of Guizhou Province and
Chinese Academy of Sciences, Guiyang 550014, China.
c
Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of
Education, Shenyang Pharmaceutical University, Shenyang 110016, China.
Corresponding author.
Corresponding author.
Email addresses: fanyhkyem@163.com; ynxu@syphu.edu.cn.

Design, synthesis, biological evaluation of 6-(2-amino-1H-benzo[d]imidazol-
6-yl)quinazolin-4(3H)-one derivatives as novel anticancer agents with Aurora
kinase inhibition
Chengcheng Fan b,c, Ting Zhong a,b, Huarong Yang b, Ying Yang a,b, Daoping Wang a,b,
Xiaosheng Yang a,b, Yongnan Xu c, , Yanhua Fan a,b,
a State Key Laboratory for Functions and Applications of Medicinal Plants, Guizhou
Medical University, Guiyang 550014, China.
b
The Key Laboratory of Chemistry for Natural Products of Guizhou Province and
Chinese Academy of Sciences, Guiyang 550014, China.
c
Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of
Education, Shenyang Pharmaceutical University, Shenyang 110016, China.
Abstract
Aurora A kinase, a member of the Aurora kinase family, is frequently
overexpressed in various human cancers. In addition, Overexpression of Aurora A
kinase is associated with drug resistance and poor prognosis in many cancers
including breast cancer. Therefore, Aurora A kinase has been considered as an
attractive anticancer target for the treatment of human cancers. Herein, A series of
6-(2-amino-1H-benzo[d]imidazol-6-yl)quinazolin-4(3H)-one
derivatives
were
Corresponding author.
Corresponding author.
Email addresses: fanyhkyem@163.com; ynxu@syphu.edu.cn.

designed, synthesized, and evaluated as Aurora A kinase inhibitors. The cell-based
cytotoxicity assays showed that compound 16h was the most potent cytotoxic agent
against all tested cancer cells and had a lower IC50 value than ENMD-2076 against
MDA-MB-231 cells. Meanwhile, Aurora A kinase assay and Western blot analysis
showed that 16h inhibited Aurora A kinase with an IC50 value of 21.94 nM and
suppressed the phosphorylation of Histone H3 on Ser10 and Aurora A kinase on
Thr288, which were consistent with the activation of Aurora A kinase. Accordingly,
16h caused aberrant mitotic phenotypes and obvious G2/M phase arrest in
MDA-MB-231 cells and induced caspase-dependent apoptosis in MDA-MB-231 cells.
These results demonstrated that 16h is a potential candidate for the development of
anticancer agents targeting Aurora A kinase.
Key word: Aurora A kinase; 6-(1H-benzo[d]imidazol-6-yl)quinazolin-4(3H)-one
derivatives; cell cycle arrest; cell apoptosis
1. Introduction
The Aurora kinases (including Aurora A, B and C) are essential regulators of
diverse cell cycle events in all eukaryotic organisms. Dysfunction of the Aurora
kinases prevents cells from maintaining a stable chromosome content and contributes
to tumorigenesis [1-2]. Aurora A kinase, which regulates maturation, mitotic spindle
formation, and cytokinesis during mitosis, is frequently overexpressed in various
human cancers [3-7], including colon, breast, pancreatic and ovarian tumors [8-12].
The inhibition of Aurora A kinase leads to mitotic catastrophe in cancer cells, which

eventually induces cell apoptosis [13-14]. Therefore, therapeutic inhibition of Aurora
A kinase as an anticancer regime shows great promise because of the critical role of
Aurora A kinase in division and proliferation of cancer cells [15]. In addition,
overexpression of Aurora A kinase confers resistance to anticancer agents [16-18],
including the epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI),
cisplatin, Taxol and BRAF inhibitor, while the combination of Aurora A kinase
inhibitors and these anticancer agents can delay the onset of drug resistances [19-22].
Hence, the development of Aurora A kinase inhibitors with minimal side effects and
good pharmacological properties is an attractive platform for designing cancer
therapeutics. In fact, numerous Aurora kinase inhibitors are currently under clinical
trials including Aurora A specific inhibitors (Fig.1) [23-28]. However, the preclinical
development of some Aurora A kinase inhibitors was limited by a narrow safety
margin or other side effects [28]. Therefore, it’s still urgent to develop new safe and
effective Aurora A kinase inhibitors.
Fig. 1. Aurora A kinase inhibitors in clinical trials and P9J.

In this study, we intend to prepare a novel class of compounds that displays
selective inhibitory for Aurora A kinase. Based on the binding mode of Aurora A and
its specific inhibitors, the quinazolin-4(3H)-one and benzo[d]imidazole scaffolds were
selected as a putative building block. As shown in Fig. 2, the aminopyrazole of P9J (a
specific Aurora A inhibitor) formed key hydrogen bonding interactions with Ala213 in
the hinge region of Aurora A, which are essential for maintaining the kinase activity
and observed in most of the Aurora A kinase inhibitor complex structures [23-25]. As
with the aminopyrazole of P9J, replacement the phthalazin-1-one moiety with
quinazolin-4(3H)-one also formed hydrogen bonding interactions with Ala213.
Quinazolin-4(3H)-one moiety is existing in nature products, such as febrigufine and
isofebrifugine, and its derivatives were found to have distinctive biological functions
[30]. Because quinazolin-4(3H)-one derivatives have various heterocyclic moieties
and showed diverse bioactivities, it is possible to construct the modified and
functional quinazolin-4(3H)-one-containing fragments as initial building block
[30-33]. In addition, the benzo[d]imidazole, from which several Aurora kinase
inhibitors had been built (e.g. AT-9238), has been proven to be a moiety with high
efficiency against Aurora kinase [28, 34]. Based on these observations, a novel hybrid
benzo[d]imidazole-quinazolinone was designed, synthesized and evaluated as Aurora
A kinase inhibitors. In order to improve the kinase inhibitory activity, compounds
were modified to further improve the physicochemical properties and binding affinity
based on ligand-receptor interaction models of Aurora A kinase. Ultimately, a series of
6-(2-amino-1H-benzo[d]imidazol-6-yl)quinazolin-4(3H)-one
derivatives
were

obtained and identified. Furthermore, to examine the structure-activity relationships
of these derivatives, structure modification was performed by introduction of various
substituents at the C2-, N3- of quinazolin-4(3H)-one and the amino from
1H-benzo[d]imidazol-2-amine. Biological evaluation showed that compound 16h
displayed the best anticancer and Aurora A kinase inhibitor properties. Additionally,
molecular docking studies, cell cycle distribution and cell apoptosis analysis were
performed to explore the underlying anticancer mechanism of 16h. These results
demonstrated that 16h is a promising candidate for the development of Aurora A
kinase inhibitors.
Fig. 2. Design of new scaffolds as Aurora A kinase inhibitors and the modification in the schematic active site of
Aurora A kinase.
2. Results and discussion
2.1. Chemistry
The substituted quinazolin-4(3H)-one intermediates were synthesized from
2-amino-5-bromobenzoic acid. As shown in Scheme 1, three general methods A, B
and C were used to synthesize the substituted quinazolin-4(3H)-one moiety [32-33].

Scheme 1. (i) formamide, 130-135 ℃, 4 h; (ii) alkyl halides, NaH, DMF, ambient temperature, overnight; (iii)
triethyl orthoformate, I2, EtOH, reflux, 6 h; (iv) chloroacetonitrile, Na, MeOH, ambient temperature, 2 h; (v)
amines, K2CO3, EtOH, reflux, overnight; (vi) alkyl halides, NaH, DMF, ambient temperature, overnight.
The alkylation of quinazolin-4(3H)-ones has been reported to afforded
N3-substituted products or a mixture of N3- and O4-substituted products. However,
only N3-substituted quinazolin-4(3H)-ones were obtained in this study according to
the reported conditions [32]. After the quinazolin-4(3H)-ones moieties were
constructed, the first step was to convert the C6-bromo substituent of the
quinazolin-4(3H)-ones into the 4,4,5,5-tertramethyl-1,3,2-dioxaborolane, and these
intermediates were then used to prepare the final products via a Suzuki Coupling

Reaction with 1H-benzo[d]imidazole moiety. As shown in Scheme 2, even though the
preparation of quinazolin-4(3H)-one borate esters 7e afforded high yields, the
homo-coupling byproduct (product 9) rather than the desired cross-coupling product 8
was finally obtained. Then, we tried to convert compound 10c to corresponding borate
esters 10 via Miyaura reaction. Unfortunately, 10 was not gained as we had expected.
Scheme 2.: (i) bis(pinacolato)diborane, Pd(dppf)Cl2, AcOK, Dioxane, 100 ºC, 2 h; (ii) Pd(dppf)Cl2, K2CO3 ,
Dioxane/H2O, 100 ℃, 20 h; (iii) CNBr, MeOH/H2O, 60 ℃, 5 h; (iv) Propionyl chloride, Et3N, THF, 0 ℃to ambient
temperature, 5 h; (v) bis(pinacolato)diborane, Pd(dppf)Cl2, AcOK, dioxane, 100 ºC, 24 h.
To obtain the final products bearing the 6-(2-amino-1H-benzo[d]imidazol-6-
yl)quinazolin-4(3H)-one as skeleton and verify our conjecture that the quinazolin-
4(3H)-one fragments are much more reactive than the N-(6-bromo-1H-
benzo[d]imidazol-2-yl)amide fragments, an alternative synthetic strategy was applied,
as shown in the Scheme 3. Critical intermediate 13 were prepared via a
Suzuki-Miyaura Cross-Coupling reaction. 6-bromoquinazolin-4(3H)-ones was treated

with 2-nitro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline, which was
obtained from 4-bromo-2-nitroaniline, to afford intermediate 13, and Pd(dppf)Cl2 was
chosen as a highly efficient catalyst. Interestingly, the yields of the borate esters from
the Miyaura reactions are more than 90%, and the cross-coupling reactions afford the
crucial intermediates 13 in good yields (above 80%). Pd/C was utilized as catalyst
for the reduction of the nitro group and yielded corresponding o-phenylenediamine
compounds 14 with nearly full conversion [35-37]. Subsequently, compounds 15 were
obtained by treating compounds 14 with BrCN using a direct one-step synthetic
method to produce the corresponding 6-(2-amino-1H-benzo[d]imidazol-6-yl)-
quinazolin-4(3H)-ones [37-38]. Finally, the treatment of compounds 15 with different
acyl chlorides resulted in target compounds 16. Various substituents were introduced
at the C2- and N3-positions of the quinazolin-4(3H)-one scaffold. Various amide, urea
and carbamate derivatives were also synthesized to further explore the
structure-activity relationship. All compounds are shown in Table 1.
Scheme 3. (i) bis(pinacolato)diborane, Pd(dppf)Cl2, AcOK, dioxane, 100 ºC,
2 h; (ii) substituted
quinazolin-4(3H)-one, Pd(dppf)Cl2, K2CO3 ,dioxane/H2O, 100 ℃, 5 h; (iii) NH2NH2·H2O, Pd/C, MeOH, 80 ℃, 2 h;
(iv) CNBr, MeOH/H2O, 60 ℃, 5 h; (v) Acyl chloride, Et3N, THF, 0 ℃to ambient temperature, overnight.

Table 1. Cytotoxic activity (IC50, µM) a against cancer cell lines.
Comp.
16a
R1
R2
R3
MDA-MB-231
PC3
SH-SY5Y
H
>20
>20
>20
H
>20
>20
>20
>20
16b
16c
H
H
13.5±0.18
18.13±0.28
>20
>20
>20
16d
16e
0.94±0.14
1.65±0.23
1.08±0.19
>20
>20
5.49±0.42
16f
H
H
H
H
H
H
H
H
>20
>20
>20
16g
16h
16i
0.38±0.08
1.23±0.09
3.85±0.28
1.22±0.23
>20
1.09±0.24
6.57±1.02
6.54±1.08
3.21±0.25
>20
0.77±0.12
4.02±0.98
4.19±0.79
1.68±0.16
>20
16j
16k
16l
>20
>20
>20
16m
16n
4.85±0.69
6.45±0.21
3.92±0.24
H
1.76±0.17
8.13±0.92
2.52±0.31
16o

H
H
1.61±0.21
10.2±1.56
11.2±1.08
16.5±2.89
7.33±0.67
14.2±2.11
16p
16q
H
H
>20
>20
>20
>20
>20
>20
16r
16s
16t
H
-
0.55±0.10
0.48±0.09
1.67±0.12
0.67±0.11
1.23±0.19
NT
ENMD-
2076
-
-
a IC50 values are the mean of triplicate measurements. NT: not tested.
2.2. Biological evaluation
2.2.1. Cytotoxicity
To test the cytotoxicity of the synthesized compounds, an MTT assay was
employed to measure their anticancer activities against various human cancer cell
lines, including breast cancer MDA-MB-231, prostate cancer PC3, and neuroblastoma
SH-SY5Y cells. As shown in Table 1, most of the synthesized compounds displayed
potent antiproliferative activity against the three cancer cell lines. Among them, 16h
showed the strongest inhibitory potency against MDA-MB-231 cells with an IC50
value of 0.38 µM. In addition, the proliferation of cells treated with 16h for 48 h was
inhibited in a dose-dependent manner, and some typical morphological features of
apoptotic cells, such as shrinkage and blebbing, were observed in the cells treated
with 16h above 1.0 µM (Fig. 3A). Colony formation was also significantly inhibited
by 16h (Fig. 3B), which is consistent with the growth-inhibitory activity results from

the MTT assay. According to the results of the MTT assay, compared with 16h,
compounds 16e and 16f, containing a methyl group at N3 and a substituent at C2,
showed weaker activities against all the tested cancer cell lines. This indicated that the
replacement of the morpholinoethyl moiety at N3 with a methyl group might decrease
the anticancer activity. In addition, the replacement of morpholino ethyl with
(1H-indol-2-yl) ethyl (16t) can decrease both the anticancer activity and the water
solubility. The introduction of an electron-withdrawing carbonyl in the
morpholinoethyl side chain significantly reduced the activity (16a, 16b). Extended the
alkyl side chain of the propenamide at the 2-position of the benzimidazole (16j) or
increased the steric hindrance due to the introduction of a macrocyclic substituent
(16p-16s) may diminished the anticancer activity. Although replacement of the
propenamide with groups containing carbamate or urea fragments (16m-16o) further
improved the water solubility, the activities were decreased. The replacement of the
propenamide with ethyl sulfonamide, an electron withdrawing group, at the
2-substituent of benzimidazole (16a and 16g) also significantly reduced the anticancer
activity.

Fig. 3. The effects of 16h on cell proliferation. (A) The cell proliferation and morphologic changes after 16h
treatment for 48 h. (B) 16h inhibited the capacity of colony formation of MDA-MB-231 cells. 500 cells were
plated in 6 wells plates and incubated for 12 h before treatment with DMSO or 16h at indicated concentration for
72 h. Then cells were washed once with PBS and continue to culture with full-growth medium for 10 days, change
the culture medium to fresh ones every three days. Cells were stained with 0.1% crystal violet after fixing with
100% methanol.
2.2.2. 16h inhibited Aurora kinase activity
To elucidate whether the antiproliferative activity of the synthesized compounds
is associated with Aurora kinase, an Aurora kinase assay was conducted to test the
kinase inhibitory activity of 16h against Auroras A and B. As shown in Table 2, 16h
exhibited excellent inhibitory activities against Aurora A kinase with an IC50 value of
21.94 nM, but much lower activity against Aurora B kinase with an IC50 value of
273.18 nM. In addition, Western blot analysis was used to further examine the effects
of 16h on the expression of phospho-Aurora A at Thr288 which is a critical residue
for activating phosphorylation events in its catalytic domain. The phospho-Histone

H3 at Ser10 was also detected, a downstream target of Aurora kinase. As depicted in
Fig. 4, upon treatment with 16h for 24 h, a dose-dependent and significant reduction
in the phosphorylation of Aurora A and Histone H3 was observed in MDA-MB-231
cells, which is consistent with the effects of other inhibitors of Aurora kinase [39-40].
Table 2. Aurora kinase inhibition by 16t and 16h.
Compound
16t
Aurora A kinase inhibition (IC50) a
Aurora B kinase inhibition (IC50) a
329.81 nM
27.12 nM
21.94 nM
15.28 nM
18.09 nM
16h
273.18 nM
ENMD-2076
Staurosporine
293.72 nM
26.13 nM
a IC50 values are the mean of triplicate measurements.
Fig. 4. Aurora A kinase inhibition by 16h. (A) 16h decreased the phospho-Aurora A kinase at Thr288. (B) 16h
decreased the phospho-Histone H3 at Ser10. Cells were exposed to DMSO or 16h at indicated concentrations for
24 h and then were collected for Western blot analysis. The changes in corresponding protein expression levels
were quantified using Image J. Each bar represents the mean ± SEM (n = 3); *P < 0.05 or **P < 0.01 was
considered statistically significant compared with the corresponding control values.
To predict the binding model of the active compounds with Aurora A kinase,
molecular docking simulation was performed using Autodock 4.2. According to the
results of the molecular docking study, 16h showed the best docking score and
binding affinities with Aurora A kinase. The docking information of 16h with Aurora
A kinase was analyzed. Analysis of the protein-ligand interactions in the co-crystal

with the ligand of Aurora A showed that the common interaction patterns include the
following: the nitrogen atom of the morpholine ring, the benzimidazoles and the
amide side chains of 16h formed conserved hydrogen bonds with amino acid residues
Ala213 in the kinase hinge region, the main chain NH of Thr217 and the catalytic
lysine of Lys162. Compared with 16t and P9J (the co-crystal ligand of Aurora A), 16h
formed two extra hydrogen bones with Thr217 and Lys162 in the active site to further
stabilize the interactions with Aurora A. In addition, the quinazolinone fragment
formed hydrophobic interactions including π-π stacked and π-alkyl interactions with
residues including Leu139, Ala160, Leu194, Leu210, Ala213 and leu263 of Aurora A
(Fig. 5). In summary, the molecular docking study also predicted the potential
inhibitory activity of 16h against Aurora A.
Fig.5. Proposed binding mode of compound 16h and in the active site of Aurora A kinase (PDB ID code:3P9J).
(A) The 2D binding model of 16t and Aurora A kinase. (B) The 2D binding model of 16h and Aurora A kinase. (C)
The 2D binding model of the co-crystal ligand and Aurora A kinase. (D) The 3D binding conformation of 16h
(blue), 16t (purple) and the co-crystal ligand (yellow) in the active site of Aurora A kinase. (E) Structural overlay

of the complexes: 16h (blue), 16t (purple) and co-crystal ligand of Aurora A kinase (yellow) in the ligand-binding
pocket. The figure was generated using Accelrys Discovery Studio Visualizer 4.5.
2.2.3 16h induced G2/M cell cycle arrest
Aurora kinases are critical regulators throughout the cell cycle and Aurora A is
usually responsible for mediation the G2/M transition [40-41]. To further confirm the
effects of 16h on Aurora A kinase, the cell cycle distribution of MDA-MB-231 cells
was examined after treatment with 16h, the most highly active compounds, for 24 h.
As shown in Fig. 6, significant G2/M transition arrest was observed in MDA-MB-231
cells treated with 16h. The Fraction of cells in the G2/M phase was dose-dependently
increased by the treatment with 16h, and the population of cells in the G2/M phase
was markedly increased to 76.19% in 5µM 16h-treated cells compared to 16.62% in
untreated cells.
Fig 6. 16h induced cell cycle arrest at G2/M phase. 16h altered cell cycle distribution in MDA-MB-231 cells.
Cells were exposed to DMSO or 16h at indicated concentrations for 24 h and then were collected for DNA content
analysis by flow cytometric analysis as experiment.
2.2.4 16h induced apoptosis of MDA-MB-231 cells
To determine whether the antiproliferative activity of 16h was accompanied by
enhanced cancer cell apoptosis, cell apoptosis was detected by a flow cytometry assay

after staining with an Annexin V-FITC apoptosis detection kit. Cells treated with 16h
at 2.0 µM and 4.0 µM displayed significant increases in the percentage of
Annexin-V-positive cells, from 6.42% in the vehicle control to 19.86% for 2.0 µM
and 30.74% for 4.0 µM 16h-treated cells. The changes in apoptotic proteins were
further evaluated, including cleaved caspase 3/9 and cleaved-PARP. As expected, 16h
significantly increased the levels of all three cleaved proteins (Fig. 7). Hence, these
observations demonstrated that compound 16h induced obvious apoptosis in
MDA-MB-231 cells in a concentration-dependent manner.
Fig.7. 16h induced apoptosis in MDA-MB-231 cells. (A) Cell apoptosis was analyzed by flow cytometric analysis
after Annexin V-FITC/PI staining. Cells were collected and centrifuged at 1500 rpm for 10min after compound
16h treatment at the indicated concentrations for 48 h. (B-D) The effect of 16h on the induction of apoptosis was
also analyzed by Western blot in MDA-MB-231 cells. The changes in corresponding protein expression levels
were quantified using Image J. Each bar represents the mean ± SEM (n = 3). *P < 0.05 or **P < 0.01 was
considered statistically significant compared with the corresponding control values.
3. Conclusions

In the present study, a series of 6-(1H-benzo[d]imidazol-6-yl)-3-quinazolin-
4(3H)-one derivatives were synthesized and biologically evaluated. Among them,
compound 16h exhibited the strongest antiproliferative activity against the tested
cancer cells lines. Further mechanistic studies showed that 16h exhibited
antiproliferative activity and induced G2/M phase cell cycle arrest and cell apoptosis
via Aurora kinase inhibition and the mitochondrial apoptotic pathway.
4. Experimental
4.1. Chemistry
All reagents and solvents were commercially available and were used without
1
further purification. H NMR and 13C NMR spectra were recorded on a Bruker
AVANCE NEO 600 MHz NMR spectrometer and the NMR spectra was generated
using Mestrenova 12.0 as processing software. Spectra were recorded at 25 using
dimethyl sulfoxide-d6 and methanol-d4 as solvents with tetramethylsilane (TMS) as an
internal standard. All chemical shifts are reported in ppm (δ), and coupling constants
(J) are in hertz (Hz). All the melting points were determined on a Beijing micro
melting-point apparatus, and the values are uncorrected. High-resolution exact mass
measurements were performed using electrospray ionization (positive mode) on a
quadrupole time-of-flight (QTOF) mass spectrometer (microTOF-Q, Bruker Inc.).
1
13
The structures of compounds 16a to 16t were confirmed based on H and C NMR
spectroscopy, HR-MS and element analysis. The HMBC and HMQC data of
compound 16h were used to characterize the series of compounds, although several
carbon signals were undetectable in the 13C NMR spectra of some derivatives.

4.1.1. Preparation of 6- bromo-N3-substituted quinazolin-4(3H)-ones, Method A.
A mixture of 2-amino-5-bromobenzoic acid (2.16 g, 10 mmol) and formamide
(1.80 g, 40 mmol) was heated at 130-135 °C. After the mixture had been stirred for 4
h, water (30 mL) was added. The reaction mixture was cooled to 60 °C, and additional
water 20 mL was added to the mixture. After stirring the mixture for another 30 min,
the precipitated product was separated by vacuum filtration. The crude products were
recrystallized from ethanol to give 6-bromoquinazolin-4(3H)-one 2 in yields of
80-95%. To a solution of compound 2 (1.125 g, 5 mmol) in anhydrous DMF 15 mL
under argon was added NaH (60% dispersion in mineral oil, 0.24 g, 6 mmol) in one
portion. The reaction mixture was stirred at room temperature for 3 min, and then
2-chloro-1-morpholinoethan-1-one (1.145 g, 7 mmol) was added dropwise. The
reaction mixture was stirred at room temperature overnight and then partitioned
between EtOAc (100 mL) and brine (100 mL). The organic layer was washed with
more brine (100 mL). The combined aqueous washings were extracted with EtOAc
(3×75 mL). The combined AcOEt extracts were washed with brine (2×100 mL), dried
(Na2SO4), and concentrated in vacuo. Purification by column chromatography eluting
with EtOAc / PE, afforded 6-bromo-3-(2-morpholino-2-oxoethyl)quinazolin-
4(3H)-one 6a as a white solid (1.21 g, 3.44 mmol, 68.8 % yield). ESI-MS: m/z 352.0,
354.0 [M+H]+.
Compound 6b - 6c was synthesized according to the procedure described in 6a.
4.1.2. 6-bromo-3-(2-(piperidin-1-yl)ethyl)quinazolin-4(3H)-one (6b).
Yellow solid, 62.5% yield. ESI-MS: m/z 336.0, 338.0 [M+H]+.

4.1.3
4.1.4
．
6-bromo-3-methylquinazolin-4(3H)-one (6c).
White solid, 73.3% yield. ESI-MS: m/z 260.9, 262.9 [M+Na]+.
Preparation of 6-bromo-N3-substituted quinazolin-4(3H)-ones, Method B.
The mixture of 2-amino-5-bromobenzoic acid (2.16 g, 10 mmol), triethyl
．
orthoformate (1.93 g, 13 mmol), 2-morpholinoethan-1-amine (1.69 g, 13 mmol),
iodine (0.025 g, 0.1 mmol) and anhydrous ethanol 30 (mL) was refluxed under argon
atmosphere for 4 - 6 h, then concentrated under vacuum to give a residue which was
dissolved in ethyl acetate (100 mL). The ethyl acetate solution was washed with 1 N
aqueous sodium hydroxide (50 mL×3) and brine (50 mL×3), dried over anhydrous
sodium sulfate and concentrated to give 6-bromo-3-(2-morpholinoethyl)quinazolin-
4(3H)-one 6d as yellow solid (2.755 g, 8.15 mmol, 81.5% yield). ESI-MS: m/z 338.0,
340.0 [M+H]+.
Compounds 6e - 6g were synthesized according to the procedure described in 6d.
4.1.5. 6-bromo-3-butylquinazolin-4(3H)-one (6e).
White solid, 81.1% yield. ESI-MS m/z: 281.0, 283.0 [M+H]+.
4.1.6. 3-(2-(1H-indol-3-yl) ethyl)-6-bromoquinazolin-4(3H)-one (6f).
White solid, 65.0% yield. ESI-MS: m/z 368.0, 370.0 [M+H]+.
4.1.7. 6-bromo-3-(3-morpholinopropyl)quinazolin-4(3H)-one (6g).
White solid, 71.3% yield. ESI-MS: m/z 352.2, 354.2 [M+H]+.
4.1.8. Preparation of 6- bromo-C2, N3-substituted quinazolin-4(3H)-ones, Method C.
To a flask containing sodium (69 mg, 3 mmol) was added anhydrous MeOH (10
mL), then chloroacetonitrile (3.40 g, 45 mmol) with a syringe via a rubber septum,

and the solution was stirred at ambient temperature for about 40 min under argon. A
solution of 2-amino-5-bromobenzoic acid (3.24 g, 15 mmol) in anhydrous MeOH (30
mL) was then added. The reaction mixture was stirred at this temperature for about 2
h under argon. The precipitate was collected by filtration, washed by MeOH (15 mL)
and H2O (10 mL), and MeOH (15mL), then the white filter residue was dried under
vacuum at room temperature to obtain the 6-bromo-2-(chloromethyl)quinazolin-
4(3H)-one 4 as pure white solid. A solution of 4 (1.37 g, 5 mmol) in EtOH (25 mL)
was added morpholine (0.52 g, 6 mmol) slowly in a three-necked flask and stirred for
30 minutes at room temperature, then K2CO3 (1.38 g, 10 mmol) was added to the
bottle in batches. After that, the mixture were heated to reflux and stirred overnight.
After the reaction completed (monitored by TLC), The crude reaction mixture was
cooled and EtOH were removed under reduced pressure and add water (100 ml),
extracted with ethyl acetate (3×100 ml), the organic layer was washed with water
(3×100 ml), dried with Na2SO4 and evaporated, the residue was purified through a
column chromatography on silica with EtOAc/PE to afford white solid,
6-bromo-2-(morpholinomethyl)quinazolin-4(3H)-one. With this intermediate in hand,
6-bromo-3-methyl-2-(morpholinomethyl)quinazolin-4(3H)-one 6h was obtained from
compound 5 which was treated with CH3I under the condition described in Method B,
as white solid, 63% yield of three steps. 1H NMR (600 MHz, DMSO-d6) δ 8.23 (d, J
= 2.3 Hz, 1H), 7.97 (dd, J = 8.7, 2.3 Hz, 1H), 7.64 (d, J = 8.7 Hz, 1H), 5.75 (s, 2H),
3.65 (t, J = 4.6 Hz, 4H), 3.51 (s, 3H), 2.56-2.49 (m, 4H). ESI-MS: m/z 360.0, 362.0
[M+Na]+

Compound 6i was synthesized according to the procedure described in 6h.
4.1.9. 6-bromo-3-methyl-2-((4-propionylpiperazin-1-yl)methyl)quinazolin-4(3H)-one
(6i)
White solid, 61.2% yield. ESI-MS: m/z 415.0, 417.0 [M+Na]+.
4.1.10. 2-nitro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (12)
A solution of the 4-bromo-2-nitroaniline (1.63 g, 7.5 mmol), KOAc (2.21 g, 22.5
mmol), bis(pinacolato)diborane (3.81 g, 15 mmol) and Pd(dppf)2Cl2 (0.28 g, 0.38
mmol) in anhydrous 1,4-dioxane (30 ml) under argon was stirred at 100
for 2 h.
1,4-dioxane was removed under reduced pressure and add water (100 ml), extracted
with ethyl acetate (3×100 ml), the organic layer was washed with water (2×100 ml),
dried with Na2SO4 and evaporated to give as a yellow solid and used for next step
without another purification (>90% yield).
4.1.11. General procedure of preparation of compounds 6-(4-amino-3-nitrophenyl)-
quinazolin-4(3H)-ones (13).
A solution of 2-nitro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline 12
(1.32 g, 5 mmol), 6-bromoquinazolin-4(3H)-ones 6 (5 mmol) , and K2CO3 (2.07 g, 15
mmol) , 1,4-dioxane/water 25 mL [V(dioxane):V(water) = 4:1] mixture as solvent
was heated to 100
and stirred for 15 min. Pd(dppf)Cl2 (0.18 g, 0.25 mmol) was
added, and the mixture was continued stirred at this temperature under argon for
another 4-6 h. 1, 4-dioxane and water was removed under reduced pressure and the
residue was purified through a column chromatography on silica with
dichloromethane/methanol to afford bright yellow solid.

4.1.12. 6-(4-amino-3-nitrophenyl)-3-(2-morpholino-2-oxoethyl)quinazolin-4(3H)-one
(13a).
Yellow solid, 86.0% yield. 1H NMR (600 MHz, DMSO-d6) δ 8.31 (d, J = 2.3 Hz,
1H), 8.26 (s, 1H), 8.15 (dd, J = 8.5, 2.3 Hz, 1H), 7.82 (d, J = 1.8 Hz, 1H), 7.79 (d, J =
8.5 Hz, 1H), 7.59 (dd, J = 8.2, 1.8 Hz, 1H), 7.37 (d, J = 8.2 Hz, 1H), 7.09 (bs, 2H),
4.98 (s, 2H), 3.69, 3.48 (t, J = 4.9, 4H), 3.63-3.57 (m, 4H). ESI-MS: m/z 432.1
[M+Na]+.
4.1.13. 6-(4-amino-3-nitrophenyl)-3-(2-(piperidin-1-yl)ethyl)quinazolin-4(3H)-one
(13b).
1
Yellow solid, 81.3% yield. H NMR (600 MHz, DMSO-d6) δ 8.31 (s, 1H), 8.18
(d, J = 2.3 Hz, 1H), 8.00 (dd, J = 8.5, 2.3 Hz, 1H), 7.65-7.63 (m, 1H), 7.61 (d, J = 8.4
Hz, 1H), 7.39 (d, J = 8.3 Hz, 1H), 7.33 (dd, J = 8.3, 1.8 Hz, 1H), 7.09 (s, 2H), 4.30 (t,
J = 6.7 Hz, 2H), 3.20 (s, 4H), 2.80 (s, 2H), 2.32 (t, J = 7.6 Hz, 2H), 1.64 (d, J = 6.8
Hz, 4H). ESI-MS: m/z 416.2 [M+Na]+.
4.1.14. 6-(4-amino-3-nitrophenyl)-3-methylquinazolin-4(3H)-one (13c).
Yellow solid, 82.1% yield. 1H NMR (600 MHz, DMSO-d6) 8.37 (s, 1H), 8.35 (d,
J = 2.3 Hz, 1H), 8.12 (dd, J = 8.7, 2.3 Hz, 1H), 7.81 (bs, 1H), 7.74 (d, J = 8.5 Hz, 1H),
7.55 (d, J = 8.3 Hz, 1H), 7.50-7.48 (dd, J = 8.3, 1.8 Hz, 1H), 7.10 (s, 2H), 3.53 (s, 3H),
ESI-MS: m/z 297.0 [M+H]+.
4.1.15. 6-(4-amino-3-nitrophenyl)-3-(2-morpholinoethyl)quinazolin-4(3H)-one (13d).
Yellow solid, 81.1% yield. 1H NMR (600 MHz, DMSO-d6) δ 8.34 (s, 1H), 8.33
(d, J = 2.2 Hz, 1H), 8.13 (dd, J = 8.5, 2.2 Hz, 1H), 7.81 (d, J = 1.5 Hz, 1H), 7.76 (d, J

= 8.5 Hz, 1H), 7.59 (dd, J = 8.2, 1.7 Hz, 1H), 7.36 (d, J = 8.2 Hz, 1H), 7.08 (s, 2H),
4.12 (t, J = 6.1 Hz, 2H), 3.53 (t, J = 4.6 Hz, 4H), 2.63 (t, J = 6.1 Hz, 2H), 2.45 (bs,
4H). ESI-MS: m/z 418.2 [M+Na]+.
4.1.16. 6-(4-amino-3-nitrophenyl)-3-butylquinazolin-4(3H)-one (13e)
Yellow solid, 82.3% yield. ESI-MS: m/z 361.1 [M+Na]+.
4.1.17. 3-(2-(1H-indol-3-yl)ethyl)-6-(4-amino-3-nitrophenyl)quinazolin-4(3H)-one
(13f).
Yellow solid, 89.4% yield. ESI-MS: m/z 448.1 [M+Na]+.
4.1.18.
6-(4-amino-3-nitrophenyl)-3-(3-morpholinopropyl)quinazolin-4(3H)-one
(13g).
Yellow solid, 80.3% yield. ESI-MS: m/z 432.2 [M+Na]+.
4.1.19. 6-(4-amino-3-nitrophenyl)-3-methyl-2-(morpholinomethyl)quinazolin-
4(3H)-one (13h).
Yellow solid, 82.8% yield. ESI-MS: m/z 418.1 [M+Na]+.
4.1.20. 6-(4-amino-3-nitrophenyl)-3-methyl-2-((4-propionylpiperazin-1-yl)methyl)
quinazolin-4(3H)-one (13i).
Yellow solid, 88.9% yield. ESI-MS: m/z 473.2 [M+Na]+.
4.1.21. Hydrogenation of nitroarenes.
For a typical run, a solution of 6-(4-amino-3-nitrophenyl)-3-(2-morpholino-
2-oxoethyl)quinazolin-4(3H)-one (1.975 g, 5 mmol) and Pd/C 0.118 g (10% on
Carbon, wetted with ca.55% water) in 30 mL MeOH was stirred at ambient
temperature for 15 min, then the hydrazine hydrate (2.50 g, 50 mmol) was added at

this temperature and was stirred for another 15 min. The reaction mixture was heated
to reflux and stirred about 2 h. After the desired reaction time, the mixture was
filtrated with Celite, the remaining hydrazine hydrate was removed under reduced
pressure to offered corresponding 6-(3, 4-diaminophenyl)-3-(2-morpholino-2-
oxoethyl)quinazolin-4(3H)-one 14a which was used in the next step directly without
another purification.
Compounds 14b-i were synthesized according to the procedure described in 14a.
4.1.22. Preparation of 6-(2-amino-1H-benzo[d]imidazol-6-yl)-3-(2-morpholino-2-
oxoethyl)quinazolin-4(3H)-one (15a).
The 14a (1.14 g, 3 mmol) was dissolved in a 1:1 mixture of methanol (15 mL)
and water (15 mL). The reaction mixture was treated with CNBr (0.95 g, 9 mmol) and
heated at 60 °C for 2 h. After cooling to room temperature, methanol was removed
under reduced pressure and the remaining reaction mixture was basified with 1M
NaOH aq. to pH=8, then extracted with n-butanol (3×30 mL). The combined organic
fractions were washed with water (2×50 mL), brine (2×50 mL), dried over MgSO4
and the solvent was removed under reduced pressure, the residue was purified through
a column chromatography on silica with dichloromethane/methanol/ammonia system
to give the pure title compound 15a, as white solid, 65.0% yield. ESI-MS (Neg.): m/z
403.1 [M-H]-.
Compounds 15b-h were synthesized according to the procedure described in 15a.
4.1.23. 6-(2-amino-1H-benzo[d]imidazol-6-yl)-3-(2-(piperidin-1-yl)ethyl)quinazolin-
4(3H)-one (15b).

White solid keeping under argon, 60.2% yield. ESI-MS (Neg.): m/z 387.2 [M-H]-.
4.1.24. 6-(2-amino-1H-benzo[d]imidazol-6-yl)-3-methylquinazolin-4(3H)-one (15c).
White solid keeping under argon, 63.2% yield. ESI-MS (Neg.): m/z 290.1 [M-H]-.
4.1.25. 6-(2-amino-1H-benzo[d]imidazol-6-yl)-3-(2-morpholinoethyl)quinazolin-
4(3H)-one (15d).
White solid keeping under argon, 63.1% yield. ESI-MS (Neg.): m/z 389.1 [M-H]-.
4.1.26. 6-(2-amino-1H-benzo[d]imidazol-6-yl)-3-butylquinazolin-4(3H)-one (15e).
White solid keeping under argon, 64.1% yield. ESI-MS (Neg.): m/z 332.1 [M-H]-.
4.1.27. 3-(2-(1H-indol-3-yl)ethyl)-6-(2-amino-1H-benzo[d]imidazol-6-yl)quinazolin-
4(3H)-one (15f).
White solid keeping under argon, 59.6% yield. ESI-MS (Neg.): m/z 419.1 [M-H]-.
4.1.28 6-(2-amino-1H-benzo[d]imidazol-6-yl)-3-(3-morpholinopropyl)quinazolin-
4(3H)-one (15g).
White solid keeping under argon, 63.7% yield. ESI-MS (Neg.): m/z 403.2 [M-H]-.
4.1.29.
6-(2-amino-1H-benzo[d]imidazol-6-yl)-3-methyl-2-(morpholinomethyl)-
quinazolin-4(3H)-one (15h).
White solid keeping under argon, 61.5% yield. ESI-MS (Neg.): m/z 389.1 [M-H]-.
4.1.30. 6-(2-amino-1H-benzo[d]imidazol-6-yl)-3-methyl-2-((4-propionylpiperazin-1-
yl)methyl)quinazolin-4(3H)-one (15i).
White solid keeping under argon, 59.5% yield. ESI-MS (Neg.): m/z 444.2 [M-H]-.
4.1.31. N-(6-(3-(2-morpholino-2-oxoethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)-
1H-benzo[d]imidazol-2-yl)ethanesulfonamide (16a).

A solution of 15a (0.404 g, 1 mmol) and triethylamine (0.152 g, 1.5 mmol) in
anhydrous tetrahydrofuran (THF, 20 mL) was stirred for 45 min. Subsequently, a
solution of ethanesulfonyl chloride (0.128 g, 1 mmol) in THF (5 mL) was added drop
wisely to the reaction mixture under ice cold conditions using a dropping funnel and
the contents were stirred further for 2 h at ambient temperature. Completion of the
reaction was monitored by TLC. The slurry was pulled into saturated salt water 50 mL
and extracted with dichloromethane (50 mL×3)，the combined organic fractions were
washed with saturated NaHCO3 aqueous solution three times, dried over MgSO4 and
the solvent was removed under reduced pressure to obtain a solid residue which was
purified through a column chromatography on silica with dichloromethane /methanol
to afford 16a, as a white solid (0.274 g, 0.552 mmol, 55.2% yield), mp 237-239 ℃. 1H
NMR (600 MHz, DMSO-d6) δ 8.31 (d, J = 2.3 Hz, 1H), 8.26 (s, 1H), 8.15 (dd, J = 8.5,
2.3 Hz, 1H), 7.82 (d, J = 1.8 Hz, 1H), 7.79 (d, J = 8.5 Hz, 1H), 7.59 (dd, J = 8.2, 1.8
Hz, 1H), 7.37 (d, J = 8.2 Hz, 1H), 7.09 (s, 2H), 4.98 (s, 2H), 3.78 (q, J = 7.2 Hz, 2H),
3.69 (t, J = 4.7 Hz, 2H), 3.64-3.57 (m, 4H), 3.48 (t, J = 4.9 Hz, 2H), 1.22 (t, J = 7.2
13
Hz, 3H). C NMR (151 MHz, DMSO-d6) δ 165.68, 160.72, 153.68, 148.74, 147.34,
143.14, 139.81, 133.46, 132.29, 131.79, 128.45, 124.00, 123.32, 122.27, 116.87,
110.56, 66.48, 66.44, 48.53, 47.10, 45.24, 42.54, 8.00. HRMS (ESI): calcd. for
C23H25O5N6S [M+H]+ m/z 497.1602, found 497.1589; C23H24O5N6NaS [M+Na]+ m/z
519.1421, found 519.1414.
Compounds 16b-t were synthesized according to the procedure described in 16a.
4.1.32. N-(6-(3-(2-morpholino-2-oxoethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)-1H-

benzo[d]imidazol-2-yl)propionamide (16b).
1
White solid, 51.7% yield, mp 289-291 . H NMR (600 MHz, DMSO-d6) δ
12.16 (s, 1H), 11.58 (s, 1H), 8.33 (s, 1H), 8.26 (s, 1H), 8.20 – 8.14 (1H), 7.84 – 7.78
(2H), 7.59 – 7.51 (2H), 4.98 (s, 2H), 3.69 (bs, 2H), 3.60 (bs, 4H), 3.48 (bs, 2H), 2.47
(q, J =7.3 Hz, 2H), 1.22 (t, J =7.03 Hz, 2H). 13C NMR (151 MHz, DMSO-d6) δ
165.72, 165.65, 160.76, 160.63, 149.33, 148.62, 148.15, 147.19, 137.75, 133.46,
128.74, 124.13, 122.38, 66.47, 66.43, 47.13, 45.23, 42.54, 29.23, 9.64. HRMS (ESI):
calcd. for C24H25O4N6 [M+H]+ m/z 461.1932, found 461.1919; C24H24O4NaN6
[M+Na]+ m/z 483.1751, found 483.1740.
4.1.33.
N-(6-(3-methyl-4-oxo-3,4-dihydroquinazolin-6-yl)-1H-benzo[d]imidazol-2-
yl)propionamide (16c).
White solid, 53.5% yield, mp 248-250 . 1H NMR (600 MHz, DMSO-d6) δ
12.16 (s, 1H), 11.58 (s, 1H), 8.37 (s, 1H), 8.34 (d, J = 2.2 Hz, 1H), 8.12 (d, J = 8.8 Hz,
1H), 7.81 (bs, 1H), 7.74 (d, J = 8.4 Hz, 1H), 7.55 (d, J = 8.3 Hz, 1H), 7.49 (d, J = 8.3
Hz, 1H), 3.53 (s, 3H), 2.47 (q, J = 7.6 Hz, 2H), 1.14 (t, J = 7.5 Hz, 3H). 13C NMR
(151 MHz, DMSO) δ 173.81, 161.25, 148.53, 148.05, 147.27, 140.31, 133.16, 132.60,
128.27, 123.16, 122.23, 120.72, 34.06, 29.23, 9.64. HRMS (ESI): calcd. for
C19H18O2N5 [M+H]+ m/z 348.1455, found 348.1451; C19H17O2NaN5 [M+Na]+ m/z
370.1274, found 370.1262.
4.1.34.
N-(6-(3-methyl-4-oxo-3,4-dihydroquinazolin-6-yl)-1H-benzo[d]imidazol-2-
yl)ethanesulfonamide (16d).
White solid, 53.5% yield, mp 230-232 . 1H NMR (600 MHz, DMSO-d6) δ 8.38