Discovery of penipanoid C-inspired 2-(3,4,5-trimethoxybenzoyl)quinazolin-4(3H)-one derivatives as potential anticancer agents by inhibiting cell proliferation and inducing apoptosis in hepatocellular carcinoma cells

Article abstract of DOI:10.1016/j.ejmech.2021.113671

Hepatocellular carcinoma (HCC) is the most common form of liver cancer and the fourth leading cause of cancer-related death worldwide. First-line drugs such as sorafenib provide only a modest benefit to HCC patients. In this study, the gram-scale synthesis of 2-benzoylquinazolin-4(3H)-one skeleton was achieved successfully via the I₂/DMSO catalytic system. A series of penipanoid C-inspired 2-(3,4,5-trimethoxybenzoyl)quinazolin-4(3H)-one derivatives was synthesized and evaluated for their cytotoxic activities against four cancer cell lines, HepG2, Bel-7402, A549, and U251. Among these compounds, 4a was the most effective one with IC₅₀ values of 1.22 μM and 1.71 μM against HepG2 and Bel-7402 cells, respectively. Mechanistic studies showed that 4a inhibited hepatocellular carcinoma cell proliferation via arresting cell cycle. Additionally, 4a induced HepG2 cells apoptosis by inducing reactive oxygen species production and elevating the expression of apoptosis-related proteins. More importantly, 4a displayed significant in vivo anticancer effects in the HepG2 xenograft models. This suggests that 4a is a promising lead compound with the potential to be developed as a chemotherapy agent for hepatocellular carcinoma.

Full text of DOI:10.1016/j.ejmech.2021.113671

European Journal of Medicinal Chemistry 224 (2021) 113671
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Discovery of penipanoid C-inspired 2-(3,4,5-trimethoxybenzoyl)
quinazolin-4(3H)-one derivatives as potential anticancer agents by
inhibiting cell proliferation and inducing apoptosis in hepatocellular
carcinoma cells
,
b
,
,
b
,
,
,
, b
Chao-Jie Wang a 1, Xinxin Guo a 1, Rui-Qin Zhai a b, Changning Sun a b, Guokai Xiao a
,
Jin Chen a b, Mei-Yan Wei a c, Chang-Lun Shao a *, Yuchao Gu a
,
,
,
b,
, b, **
a Key Laboratory of Marine Drugs, The Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao, 266003,
People's Republic of China
b Laboratory for Marine Drugs and Bioproducts of Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao, 266237, People's
Republic of China
c College of Food Science and Engineering, Ocean University of China, Qingdao, 266003, People's Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Hepatocellular carcinoma (HCC) is the most common form of liver cancer and the fourth leading cause of
cancer-related death worldwide. First-line drugs such as sorafenib provide only a modest benefit to HCC
patients. In this study, the gram-scale synthesis of 2-benzoylquinazolin-4(3H)-one skeleton was achieved
Received 1 April 2021
Received in revised form
22 June 2021
Accepted 24 June 2021
Available online 30 June 2021
successfully via the I2/DMSO catalytic system.
A series of penipanoid C-inspired 2-(3,4,5-
trimethoxybenzoyl)quinazolin-4(3H)-one derivatives was synthesized and evaluated for their cytotoxic
activities against four cancer cell lines, HepG2, Bel-7402, A549, and U251. Among these compounds, 4a
was the most effective one with IC50 values of 1.22 mM and 1.71 mM against HepG2 and Bel-7402 cells,
Keywords:
respectively. Mechanistic studies showed that 4a inhibited hepatocellular carcinoma cell proliferation via
arresting cell cycle. Additionally, 4a induced HepG2 cells apoptosis by inducing reactive oxygen species
production and elevating the expression of apoptosis-related proteins. More importantly, 4a displayed
significant in vivo anticancer effects in the HepG2 xenograft models. This suggests that 4a is a promising
lead compound with the potential to be developed as a chemotherapy agent for hepatocellular
carcinoma.
Hepatocellular carcinoma
Penipanoid C
Quinazoline
Apoptosis
Cell cycle
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
patients [2]. Therefore, there is an urgent task for researchers to
discover novel molecules for HCC patients. Marine natural products
Liver cancer remains a global health challenge, with an esti-
mated incidence of >1 million cases by 2025 [1]. Hepatocellular
carcinoma (HCC) is the most common form of liver cancer and the
fourth leading cause of cancer-related death worldwide [1]. First-
line drugs such as sorafenib provide only a modest benefit to HCC
are an alternative resource for new drugs used to combat major
diseases [3]. Several marine drugs have been successfully mar-
keted, including the anticancer drug ET743 (Yondelis®) [3].
Quinazoline is a core structure in a large variety of compounds
that exhibited a diverse biological activities, such as anticancer [4],
anti-inflammatory [5], anti-tuberculosis [6], anti-pulmonary
fibrosis [7], and anti-viral [8]. Especially, a large number of quina-
zoline derivatives have been studied for their anticancer activities
over the years. In our previous research, a series of bioactive natural
products were isolated from marine-derived fungi [9‒12]. The
quinazoline-containing natural alkaloid, penipanoid C (Fig. 1) was
reported first from Penicillium paneum SD-44 [13], and re-isolated
from the organic crude extraction of the marine-derived
* Corresponding author. Key Laboratory of Marine Drugs, The Ministry of Edu-
cation of China, School of Medicine and Pharmacy, Ocean University of China,
Qingdao, 266003, People's Republic of China.
** Corresponding author. Key Laboratory of Marine Drugs, The Ministry of Edu-
cation of China, School of Medicine and Pharmacy, Ocean University of China,
Qingdao, 266003, People's Republic of China.
E-mail addresses: shaochanglun@163.com (C.-L. Shao), guych@126.com (Y. Gu).
These authors contributed equally to this work.
1
https://doi.org/10.1016/j.ejmech.2021.113671
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

C.-J. Wang, X. Guo, R.-Q. Zhai et al.
European Journal of Medicinal Chemistry 224 (2021) 113671
Fig. 1. Structures of compound A, BPR0L075, BNC-105P and penipanoid C.
Penicillum sp. CHNSCLM-0019 fungus in our laboratory.
synthesized and evaluated for their cytotoxic activities against four
cancer cell lines, including hepatocellular carcinoma HepG2/Bel-
7402 cells, human lung cancer cells A549, and human glioma cells
U251. The most effective compound, 4a, notably inhibited the
proliferation of hepatocellular carcinoma cells. Additionally, the
roles and mechanisms of 4a in anticancer activity were investigated
in both hepatocellular carcinoma cellular and mouse xenograft
models. The results indicated that 4a is a potentially promising lead
compound for hepatocellular carcinoma therapy.
The structural skeleton of penipanoid C is 2-benzoylquinazolin-
4(3H)-one. The gram-scale synthesis of the skeleton of penipanoid
C and the anticancer activity of its derivatives has not been reported
before. Therefore, our research aims to synthesize and functionalize
the 2-benzoylquinazolin-4(3H)-one skeleton at gram-scale fol-
lowed by anticancer activity evaluation of the derivatives. How to
perform the structural modification? It has to be noticed that the
structural skeleton of penipanoid C is similar to some anticancer
agents, such as compound A [14], BPR0L075 [15], and BNC-105P
[16] (Fig. 1). Moreover, the 3,4,5-trimethoxyphenyl (TMP) moiety
is an essential active group for these anticancer agents [14e16].
Inspired by this, the 4-hydroxyphenyl group in penipanoid C was
replaced with a TMP group, followed by structural modifications at
different sites to obtain a series of 2-(3,4,5-trimethoxybenzoyl)-
quinazolin-4(3H)-one derivatives (Fig. 2).
2. Results and discussion
2.1. Chemistry
Two classic methods based on oxidative coupling reaction were
reported to synthesize 2-benzoylquinazolin-4(3H)-one derivatives.
Firstly, the Wu group reported an I2/DMSO promoted oxidative
coupling reaction between 2-aminobenzamides with acetophe-
nones to provide various 2-benzoylquinazolin-4(3H)-ones [17].
Secondly, the Nguyen group demonstrated recently a new method
to access 2-benzoylquinazolin-4(3H)-ones utilizing sulfur [18]. The
advantage of Wu's reaction strategy is the rapid synthesis of the
target product in an atom- and step-economic fashion. However,
when there is no substituent on the benzene ring of 2-
aminobenzamide, the by-product 2-benzoyl-6-iodoquinazolin-
4(3H)-one analogue is easily produced and difficult to remove. The
advantage of Nguyen's reaction strategy is the wide range of sub-
strate applicability, while the disadvantage is the long reaction
time. Neither reaction strategy has studied the gram-scale syn-
thesis of the 2-benzoylquinazolin-4(3H)-one skeleton.
The gram-scale synthesis of 2-benzoylquinazolin-4(3H)-one
skeleton was successfully realized via the I2/DMSO catalytic system,
and then
trimethoxybenzoyl)-quinazolin-4(3H)-one
a
series of penipanoid C-inspired 2-(3,4,5-
derivatives were
The optimization studies of the synthesis of penipanoid C at
100 mg level based on oxidative coupling reaction were executed
(Table 1). Initially, we tried the reaction of 2-aminobenzamide 1a
with 40-hydroxyacetophenone 2a in the presence of 300 mol %
SeO2, 400 mol % H2SO4, in 4 mL of DMSO, at 100 ꢀC. To our disap-
pointment, the reaction was significantly less effective (Table 1,
entry 1). In the absence of the H2SO4, no desired product was
detected (Table 1, entry 2). The catalyst Se, was also studied and
unfortunately, no good yields of penipanoid C were obtained
(Table 1, entries 3e5). The formation of penipanoid C was observed
when Nguyen's reaction method was used (23e26%, Table 1, entries
Fig. 2. Design strategy for novel 2-(3,4,5-trimethoxybenzoyl)quinazolin-4(3H)-one
derivatives as anticancer agents.
2

C.-J. Wang, X. Guo, R.-Q. Zhai et al.
European Journal of Medicinal Chemistry 224 (2021) 113671
Table 1
Optimization of reaction for the synthesis of penipanoid C.
entry
cat.(mol %)
additive (mol %)
solvent
Temp (ꢀC)
Time (h)
Yield (%)c
1a
2a
3a
4a
5a
6a
7a
8b
SeO2 (300)
SeO2 (300)
Se (400)
Se (400)
Se (400)
S8 (400)
S8 (400)
I2 (110)
H2SO4 (400)
e
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
100
100
110
130
160
100
110
110
24
24
48
48
48
24
24
2
<5
0
0
0
0
23
26
48
N-methylpiperidine (100)
H2SO4 (400)
CH3COOH (400)
N-methylpiperidine (100)
N-methylpiperidine (100)
e
a
A mixture of 2-aminobenzamide 1a (1 mmol), 40-hydroxyacetophenone 2a (1 mmol), catalyst, additive, were heated in DMSO (4 mL).
b
A mixture of 40-hydroxyacetophenone 2a (1 mmol) and I2 (1.1 mmol) in DMSO (2 mL) was stirred at 110 ꢀC, and then 2-aminobenzamide 1a (1 mmol in 2 mL DMSO) was
added slowly dropwise to the mixture during 0.5 h.
c
Isolated yield.
Table 2
Gram-scale synthesis of penipanoid C derivatives (3aꢁ3i)a and their cytotoxic activities in vitrob.
entry
Product
R1
Mass (g)
Yield (%)c
IC50 (mM)
1
2
3
4
5
6
7
8
9
3a
3b
3c
3d
3e
3f
3g
3h
3i
4-OH
1.22
2.47
2.62
2.78
3.21
1.83
1.92
1.94
1.86
31
60
64
71
77
37
39
42
40
>50
>50
>50
>50
>50
>50
>50
>50
>50
4-OCH3
2-OCH3
4-F
4-Cl
4-OCF3
2-OCF3
4-CF3
2-CF3
a
Reaction condition: A mixture of 2aꢁ2i (14.69 mmol) and I2 (16.16 mmol) in DMSO (10 mL) was stirred at 110 ꢀC, and then 2-aminobenzamide 1a (14.69 mmol in 15 mL
DMSO) was added slowly dropwise to the mixture during 7.5 h.
b
HepG2 cell line.
Isolated yield.
c
6e7). Finally, the reaction worked even better with Wu's reaction
conditions (48%, Table 1, entry 8).
With the optimized conditions (Table 1, entry 8) in hand, the
gram-scale synthesis of penipanoid C was carried out. Preliminary
experiments showed that the addition rate of compound 1a was
critical for the gram-scale reaction and affected the production of
electron-donating or halogen group gave better yields (Table 2,
entries 2e5). On the other side, acetophenone analogues o- or p-
substituted by an electron-withdrawing group lowered the yields
(Table 2, entries 1 and 6e9).
After completing the gram-level synthesis of 2-
benzoylquinazolin-4(3H)-one skeleton,
a series of 2-(3,4,5-
by-product
2-(4-hydroxybenzoyl)-6-iodoquinazolin-4(3H)-one
trimethoxybenzoyl)quinazolin-4(3H)-one derivatives were syn-
thesized (Scheme 1). The synthesis of 4aꢁ4u is similar to that of
penipanoid C. Compound 4a was reacted with the appropriate
halogenated compounds in the presence of K2CO3 to give the target
products 5aꢁ5c and 8aꢁ8d. Treatment of 4a with BOP, DBU, and
suitable alcohols in refluxing acetonitrile produced 6aꢁ6b. Com-
pound 4a was chlorinated by phosphorus oxychloride to afford 9 in
85% yield.
(data not shown); the gram-scale synthesis of penipanoid C gave a
yield of 31% at an optimal addition rate of 4.44 mg/min (Table 2,
entry 1). Therefore, the applicability of the scope of diversely ace-
tophenone analogues 2bꢁ2i was explored. The reactions pro-
ceeded smoothly for the corresponding penipanoid C derivatives
(3bꢁ3i) in acceptable to moderate isolated yields (Table 2,
37e77%). Generally, acetophenone analogues p-substituted by an
3

C.-J. Wang, X. Guo, R.-Q. Zhai et al.
European Journal of Medicinal Chemistry 224 (2021) 113671
Scheme 1. Synthesis of 2-(3,4,5-trimethoxybenzoyl)quinazolin-4(3H)-one derivatives. Reagents and conditions: (i) I2, DMSO, 110 ꢀC, 1.5 h; (ii) R3X (X ¼ I or Br), K2CO3, DMF, 80 ꢀC,
12 h; (iii) R4OH, BOP, DBU, CH3CN, 85 ꢀC, 48 h; (iv) 7aꢁ7d, K2CO3, DMF, 80 ꢀC, 12 h; (v) POCl3, 100 ꢀC, 1 h.
2.2. Biological assays
8bꢁ8d, and 9 were evaluated for their cytotoxic activity against
another three cancer cell lines (Bel-7402, A549, and U251) with
Adriamycin as positive control (Table 3). The results showed that 4a
was the most effective compound against all four cancer cell lines
2.2.1. Cytotoxic activity of 2-(3,4,5-trimethoxybenzoyl)quinazolin-
4(3H)-one derivatives and structure-activity relationships analysis
The results of the titled compounds (4aꢁ4u, 5aꢁ5c, 6aꢁ6b,
with the IC50 values ranging from 1.22 to 5.48
Compounds 3a‒3i showed no apparent inhibitory effects
against HepG2 cells (IC50 > 50 M, Table 2). However, 4a exhibited
good inhibitory activity against HepG2 with the IC50 value of
mM.
8aꢁ8d, 9) against HepG2 cancer cells at 2.5
mM are present in Fig. 3.
The compounds with the inhibition rate of more than 10% were
m
considered acceptable. Therefore, 4a, 4m, 4q, 4s, 5aꢁ5c, 6aꢁ6b,
4

C.-J. Wang, X. Guo, R.-Q. Zhai et al.
European Journal of Medicinal Chemistry 224 (2021) 113671
Fig. 3. In vitro cytotoxic activity of 2-(3,4,5-trimethoxybenzoyl)quinazolin-4(3H)-one derivatives against HepG2 cells. HepG2 cells were treated with the compounds at 2.5
then inhibition rates were determined by the MTT assay at 72 h. Inhibition rates are presented as mean standard error of mean, n ¼ 5.
mM, and
Table 3
The cytotoxicity data of 2-(3,4,5-trimethoxybenzoyl)quinazolin-4(3H)-one derivatives in cancer cell linesa.
Compounds
IC50
(mmol/L)
HepG2
Bel-7402
A549
U251
NCIeH1299
HCT116
4a
4m
4q
4s
5a
5b
5c
6a
6b
8b
8c
8d
1.22 0.12
7.45 0.41
9.15 0.21
10.62 0.38
8.19 0.19
14.10 0.35
4.97 0.17
15.55 1.03
7.45 0.41
7.07 0.16
10.47 0.32
7.51 0.16
10.92 0.25
2.21 0.16
1.71 0.18
4.91 0.19
7.1 0.31
3.88 0.22
7.27 0.19
15.42 0.87
12.73 0.68
20.26 1.08
10.52 0.63
7.66 0.28
>100
7.27 0.19
7.66 0.31
47.13 2.21
4.62 0.21
7.09 0.34
2.19 0.28
5.48 0.29
>100
19.16 0.85
13.69 0.79
>100
12.99 0.91
12.82 1.07
10.57 0.63
>100
27.46 1.45
26.96 1.72
15.93 0.89
>100
13.94 2.26
NTb
5.47 0.11
NTb
NTb
NTb
4.13 0.16
6.34 0.12
10.70 0.56
5.40 0.24
8.29 0.24
4.91 0.19
4.86 0.15
8.88 0.32
8.15 0.33
6.88 0.32
1.28 0.12
NTb
NTb
NTb
NTb
NTb
NTb
NTb
NTb
NTb
NTb
NTb
NTb
NTb
NTb
NTb
NTb
NTb
NTb
9
NTb
NTb
Adriamycin
0.157 0.07
NTb
NTb
a
Each experiment was repeated at least three times independently.
NT means not tested.
b
1.22
m
M (Table 3), which suggested that the TMP moiety in 4a is
It had to be noticed that 4a, 4q, 4s, 5a, 5c, 8b, and 8c exhibited
stronger proliferation inhibition effects on hepatocellular carci-
noma HepG2 and Bel-7402 cell lines than non-hepatocellular car-
cinoma cell lines of A549 and U251. Therefore, the cytotoxic activity
of 4a was further evaluated against two other non-hepatocellular
carcinoma cell lines, including human lung cancer cell
necessary for the cytotoxic activity. The activity decreased obvi-
ously when the halogen/electron-donating groups were
substituted at the 6-position (Fig. 3, 4c/4h/4l/4r vs 4a). Compound
4a exhibited better inhibitory activity than 3-substituted com-
pounds 5aꢁ5c and 8aꢁ8d against four cancer cell lines (Table 3),
this indicated that the substitute at 3-position (the NH group) of 4a
may play a critical role in the cytotoxic activity. A summary of the
structureeactivity relationships was shown in Fig. 4.
NCIeH1299 (IC50 13.94
(IC50 5.47 M). Compound 4a showed strong cytotoxic activity
against HepG2 and Bel-7402 cells with IC50 values lower than 2 M.
mM) and human colon cancer cell HCT116
m
m
5

C.-J. Wang, X. Guo, R.-Q. Zhai et al.
European Journal of Medicinal Chemistry 224 (2021) 113671
Fig. 4. Structureeactivity relationships of 2-(3,4,5-trimethoxybenzoyl)quinazolin-4(3H)-one derivatives as anticancer agents.
2.2.2. Compound 4a inhibits colony formation of hepatocellular
carcinoma cells
Table 4
The cytotoxicity data of compound 4a in cancer cell lines and normal cell lines.
Colony formation assay was used to evaluate the proliferation
and survival abilities of a single adherent cell [19,20]. Compound 4a
was chosen to test its inhibitory effects on colony formation assay.
HepG2 and Bel-7402 cells were treated with 4a at concentrations of
Compound
IC50
HepG2
1.22 0.12
(
m
mol/L)
SI
L-O2
4a
6.01 0.31
4.9
SI: selectivity index (IC50 on L-O2 cells/IC50 on HepG2 cells).
0.2, 0.4, 0.8, 1.6, and 3.2
Fig. 5, 1.6 M of 4a suppressed the colony formation of HepG2 and
Bel-7402 cells effectively. Moreover, 3.2 M of 4a almost eliminated
the colony formation. Compound 4a displayed strong anti-
proliferation effects on hepatocellular carcinoma cells. Therefore,
the mechanisms of anticancer activity of 4a in hepatocellular car-
cinoma cells were further investigated.
mM for 10 days, respectively. As shown in
m
m
But, 4a displayed at least two times decreased potency against four
non-hepatocellular carcinoma cell lines (IC50 3.88e13.94 M).
These results suggested that 4a exhibited stronger inhibition of the
proliferation of hepatocellular carcinoma cell lines than non-
hepatocellular carcinoma cell lines. Compound 4a was further
tested against normal L-O2 cells (Table 4).
m
Fig. 5. Colony formations of HepG2 and Bel-7402 cells were inhibited by compound 4a. (A) HepG2 and Bel-7402 cells were cultured for 10 days with varying concentrations of
compound 4a (0, 0.2, 0.4, 0.8, 1.6, 3.2 M) and the colonies were fixed and dyed with 0.1% crystal violet. (B) The relative colony numbers were shown in histograms. Error bars are
m
the SD, n ¼ 3. Statistical significance of differences in mean values are *p < 0.05 and ***p < 0.001.
6

C.-J. Wang, X. Guo, R.-Q. Zhai et al.
European Journal of Medicinal Chemistry 224 (2021) 113671
Fig. 6. Compound 4a induced cell cycle arrest in HepG2 and Bel-7402 cells. (A) HepG2 and Bel-7402 cells were incubated respectively with varying concentrations of compound 4a
(0, 0.8, 1.6, 3.2
m
M) and analyzed by flow cytometry. (B) Histograms displaying the percentage of cell cycle distribution. Error bars are the SD, n ¼ 3. Statistical significance of
differences in mean values are *p < 0.05, **p < 0.01 and ***p < 0.001.
2.2.3. Compound 4a blocks the cell cycle and regulates the
expression of proliferation-related proteins
Cell cycle is the basis of cell life activity and controls the entry of
stationary phase cells into the proliferative phase [21,22]. A cell-
cycle cytotoxicity assay was performed by treating HepG2 and
Bel-7402 cells at various concentrations of 4a (0, 0.8, 1.6, and
pathways mediating the cell proliferation response [23,24], the
effects of 4a on AKT and ERK1/2 phosphorylation were examined by
Western blot. Treatment of HepG2 cells with 4a resulted in the
down-regulation of phosphorylation levels of AKT and ERK1/2 in a
dose-dependent manner (Fig. 7). Treatment of Bel-7402 cells with
4a resulted in the down-regulation of phosphorylation levels of
AKT and the up-regulation of phosphorylation levels of ERK1/2 in a
dose-dependent manner (Fig. 7). These results suggested that the
inhibition of the AKT signaling pathway might be the reason for cell
cycle arresting of hepatocellular carcinoma cells by 4a.
3.2
mM) for 48 h, respectively. However, after incubating with
compound 4a at 3.2
mM for 48 h, most of the HepG2 cells died,
making the result unavailable. The cell cycle of HepG2 cells was
significantly arrested at the S and G2/M phase. The percentage of
cells in the S stage increased from 28.23% to 35.65% (Fig. 6). The
percentage of cells in the G2/M stage increased from 24.89% to
35.18% (Fig. 6). The cell cycle of Bel-7402 cells was arrested at the
G2/M phase, the percentage of cells in the G2/M stage increased
from 25.20% to 41.71% (Fig. 6). These results indicated that 4a
induced cell cycle arrest in both HepG2 and Bel-7402 cells.
2.2.4. Compound 4a induces apoptosis of HepG2 cells
HepG2 cells were cultured in a DMEM medium containing
different concentrations of 4a (0, 0.8, 1.6, 3.2 mM) for 48 h. Hoechst
33342 staining was used to assess nuclear changes in HepG2 cells.
The chromatin was markedly shrunk after incubating with 4a
(Fig. 8A), indicating that the cells were undergoing apoptosis. The
Since PI3K/AKT and MAPK/ERK1/2 are important signaling
7

C.-J. Wang, X. Guo, R.-Q. Zhai et al.
European Journal of Medicinal Chemistry 224 (2021) 113671
Fig. 7. Compound 4a regulates the expression and activity of AKT and ERK1/2. (A) Western blot for AKT, p-AKT, ERK and p-ERK1/2 in HepG2 and Bel-7402 cells after incubated with
the indicated concentrations of compound 4a (0, 1.6, 3.2 M) for 48 h. (B) The histograms are the quantification of AKT and ERK phosphorylation levels detected by Western blot in
(A) (Control group set to 1). Error bars are the SD, n ¼ 3. Statistical significance of differences in mean values are *p < 0.05, **p < 0.01 and ***p < 0.001.
m
apoptotic induction effects of 4a were monitored by the Muse cell
analyzer. The results showed that 4a induced a dose-dependent
increase (7.80e46.25%) of apoptosis of both early and late stages
in HepG2 cells (Fig. 8B&C). Subsequently, Western blot analysis was
carried out to examine the expression of apoptosis-related proteins
(Fig. 8D&E). The expression of Cleaved-PARP and Cleaved-Caspase3
was significantly increased in HepG2 cells treated by 4a. These data
strongly demonstrated that 4a could play anticancer roles by
inducing cell apoptosis.
established by the subcutaneous inoculation of HepG2 cells. Pre-
liminary experiments showed that 4a (5 mg/kg/d) could slightly
but significantly inhibit tumor growth (data not shown). Therefore,
4 and 8 mg/kg of 4a per day were used to assess its anticancer
activity in vivo. The tumor volume (Fig. 10A) and body weight of
nude mice (Fig.10B) were monitored for 20 days. The low dose of 4a
(4 mg/kg) only slightly inhibited tumor growth inhibition
(TGI ¼ 19.11%) compared with the vehicle group (Fig. 10A). But, the
high dose of compound 4a (8 mg/kg) achieved significant tumor
growth inhibition with a tumor growth inhibition (TGI) value of
67.57% (Fig. 10A). During the 20 days of treatment, all the animals in
different treatment groups were kept at relatively stable body
weight (Fig. 10B), suggesting the low toxicity of 4a. After 20 days of
drug treatment, the tumors were dissected and weighed
(Fig. 10C&D), and the results were consistent with the tumor
growth curves. Overall, these findings indicated that 4a effectively
inhibited cancer growth in vivo and represented a promising drug
candidate for hepatocellular carcinoma.
2.2.5. Compound 4a induced reactive oxygen species generation
Reactive oxygen species (ROS) are chemically reactive molecules
with essential functions in living organisms [25]. Excessive
amounts of ROS cause oxidative damage to cells, and many
chemotherapeutic drugs induce cancer cell apoptosis by generating
ROS [26]. Therefore, the intracellular ROS production using fluo-
rescence probe DCFH-DA was detected after treatment with
different concentrations of 4a, and then the accumulation of ROS
was observed in a dose-dependent (Fig. 9). These data suggested
that 4a induced HepG2 cells apoptosis through the elevation of ROS
levels.
3. Conclusion
In summary, this study has reported the gram-scale synthesis of
2-benzoylquinazolin-4(3H)-one skeleton utilizing the I2/DMSO
catalytic system and then identified penipanoid C-inspired anti-
cancer agents that inhibit cell proliferation and induce apoptosis in
hepatocellular carcinoma cells. Compound 4a, notably inhibited the
proliferation of hepatocellular carcinoma HepG2/Bel-7402 cells.
2.2.6. Compound 4a suppresses the growth of xenograft tumors in
nude mice
Due to the remarkable antiproliferative and apoptosis induction
activities of 4a in vitro, its anticancer activity in vivo was assessed
utilizing a mouse hepatic carcinoma cancer xenograft model
8

C.-J. Wang, X. Guo, R.-Q. Zhai et al.
European Journal of Medicinal Chemistry 224 (2021) 113671
Fig. 8. Compound 4a induced apoptosis of HepG2 cells. (A) HepG2 cell morphological changes after incubation with compound 4a. (B) Different concentrations of compound 4a
(0.8, 1.6 and 3.2
M) induced apoptosis in HepG2 cells. (C) Histograms display the percentage of cell distribution. Error bars are the SEM, n ¼ 3. Statistical significance of differences
in mean values are *p < 0.05, **p < 0.01 and ***p < 0.001. (D) Western blot of apoptosis-related proteins in HepG2 cells after incubated with the indicated concentrations of
compound 4a (0, 1.6, 3.2
m
m
M) for 48 h. (E) Quantification of Cleaved-PARP, Cleaved-Caspase3 is shown (Control group set to 1). Error bars are the SD, n ¼ 3. Statistical significance of
differences in mean values are *p < 0.05, **p < 0.01 and ***p < 0.001.
Fig. 9. Compound 4a increased levels of ROS in HepG2 cells. The ROS levels of HepG2 cells were detected after incubated with different concentrations of compound 4a (0, 0.8, 1.6,
3.2 mM).
9

C.-J. Wang, X. Guo, R.-Q. Zhai et al.
European Journal of Medicinal Chemistry 224 (2021) 113671
Fig. 10. Compound 4a inhibits HepG2 xenograft growth in vivo. The mice bearing HepG2 xenograft tumors with the volumes of 100e200 mm3 were randomly divided into 3 groups
with 5 mice in each group. Mice were treated intraperitoneally with 4a (4 mg/kg and 8 mg/kg) and control (15% N-Methyl pyrrolidone, 25% Solutol HS-15, 60% saline) once every
days for 20 days. (A) The tumor volumes were examined twice a week. (B) Body weight changes of mice during treatment. (C) Visible tumor formation and photographs of
representative tumors removed from mice at 20 days after initiation of treatment. (D) Compound 4a treatment resulted in significantly lower tumor weight compared control. Error
bars are the SEM, n ¼ 3. Statistical significance of differences in mean values are *p < 0.05, **p < 0.01 and ***p < 0.001.
The SAR results indicated that (1) the TMP moiety is necessary for
the anticancer activity, (2) the halogen/electron-donating group
substituted at the 6-position should be restricted, (3) the NH group
at the 3-position is beneficial to anticancer activity. Mechanistic
studies showed that 4a inhibited hepatocellular carcinoma cell
proliferation via arresting cell cycle. Additionally, compound 4a
induced HepG2 cancer cells apoptosis by inducing reactive oxygen
species production and the expression of apoptosis-related pro-
teins. Compound 4a displayed significant in vivo anticancer effects
in the HepG2 xenograft models, suppressing tumor growth by
67.57% at a dose of 8 mg/kg by ip injection every day for 20 days.
Overall, these results demonstrated that 4a is a potentially prom-
ising lead for hepatocellular carcinoma therapeutics.
spectra were recorded on a Bruker 150 MHz, 125 MHz and 100 MHz
spectrometer, respectively (Bruker Company). HRMS data were
obtained using an APEX IV Fourier-Transform Mass Spectrometry
(Bruker Company).
4.1.1. General procedures to synthesize 3aꢁ3i
I2 (1.1 eq) was added to a mixture of 2aꢁ2i (1 eq) and DMSO
(10 mL). The solution was stirred at 110 ꢀC, then 2-aminobenzamide
(2.00 g, 14.69 mmol, 1 eq) in 15 mL DMSO was added dropwise to
the mixture during 7.5 h. After the complete disappearance of the
starting material, a sodium thiosulfate solution (1 g Na2S2O3 in
60 mL water) was added dropwise to the reaction system. Filtrating
and drying, the residue was purified by silica gel chromatography
(petroleum ether/EtOAc 4:1) to yield the desired compounds.
4. Experimental section
4.1. Chemistry
4.1.1.1. 2-(4-hydroxybenzoyl)quinazolin-4(3H)-one
(3a). Yellow
Chemicals and solvents were purchased from commercial sup-
pliers and were used without further purification. Thin-layer
chromatography (TLC) was performed on silica gel 60 F254 (Qing-
dao Marine Chemical Ltd.). Column chromatography was per-
formed over silica gel (200e300 mesh, Qingdao Marine Chemical
Ltd.). 1H NMR spectra were recorded on a Bruker 600 MHz and
400 MHz spectrometer, respectively (Bruker Company). 13C NMR
solid, 1.22 g, yield 31%; 1H NMR (400 MHz, DMSO‑d6, J in Hz) dH
12.59 (s, 1H), 10.77 (s, 1H), 8.20 (dd, J ¼ 8.0, 1.5 Hz, 1H), 8.14e8.03
(m, 2H), 7.89 (ddd, J ¼ 8.5, 7.1, 1.6 Hz, 1H), 7.78 (dd, J ¼ 8.3, 1.2 Hz,
1H), 7.63 (ddd, J ¼ 8.0, 7.2, 1.2 Hz, 1H), 7.12e6.62 (m, 2H). 13C NMR
(100 MHz, DMSO‑d6) dC 185.1, 163.6, 161.1, 149.9, 147.3, 134.8, 133.8,
128.2, 126.0, 125.2, 122.7, 115.5. HRESIMS m/z calcd for C15H11N2O3þ
267.0764 [MþH]þ, found 267.0768.
10

C.-J. Wang, X. Guo, R.-Q. Zhai et al.
European Journal of Medicinal Chemistry 224 (2021) 113671
4.1.1.2. 2-(4-(methoxy)benzoyl)quinazolin-4(3H)-one
(3b).
4.1.1.9. 2-(2-(trifluoromethyl)benzoyl)quinazolin-4(3H)-one
(3i).
White solid, 2.47 g, yield 60%; 1H NMR (400 MHz, DMSO‑d6, J in Hz)
dH 12.61 (s, 1H), 8.30e8.11 (m, 3H), 7.89 (ddd, J ¼ 8.5, 7.2, 1.6 Hz, 1H),
7.78 (dd, J ¼ 8.2, 1.2 Hz, 1H), 7.65 (ddd, J ¼ 8.1, 7.2, 1.2 Hz, 1H),
7.17e7.07 (m, 2H), 3.89 (s, 3H). 13C NMR (100 MHz, DMSO‑d6) dC
185.8,164.8,161.5, 150.0,147.7,135.2,133.9,128.8,128.7, 127.1,126.5,
123.2, 114.6, 56.2. HRESIMS m/z calcd for C16H13N2Oþ3 281.0921
[MþH]þ, found 281.0925.
White solid, 1.86 g, yield 40%; 1H NMR (600 MHz, DMSO‑d6, J in Hz)
dH 12.84 (s, 1H), 8.18 (dd, J ¼ 7.9, 1.6 Hz, 1H), 7.89 (dd, J ¼ 6.7, 2.4 Hz,
1H), 7.86e7.83 (m, 1H), 7.82e7.78 (m, 3H), 7.67e7.59 (m, 2H). 13
C
NMR (125 MHz, DMSO‑d6) dC 189.8, 161.3, 148.3, 147.3, 135.6 (q, 3JC-
2
¼ 2 Hz), 135.3, 132.5, 131.9, 130.5, 129.9, 129.2, 127.4 (q, JC-
F
¼ 32.5 Hz),127.0 (q, 3JC-F ¼ 5 Hz),126.6,124.2 (q, 1JC-F ¼ 271.25 Hz),
F
123.6. HRESIMS m/z calcd for C16H10F3N2Oþ2 319.0689 [MþH]þ,
found 319.0695.
4.1.1.3. 2-(2-(methoxy)benzoyl)quinazolin-4(3H)-one
(3c).
White solid, 2.62 g, yield 64%; 1H NMR (600 MHz, DMSO‑d6, J in Hz)
dH 12.69 (s, 1H), 8.16 (dd, J ¼ 7.9, 1.6 Hz, 1H), 7.83 (ddd, J ¼ 8.4, 7.1,
1.6 Hz, 1H), 7.66e7.60 (m, 3H), 7.61 (ddd, J ¼ 8.2, 7.1, 1.2 Hz, 1H),
7.19e7.16 (m, 1H), 7.14 (td, J ¼ 7.5, 1.0 Hz, 1H), 3.63 (s, 3H). 13C NMR
(150 MHz, DMSO‑d6) dC 189.5, 161.6, 159.7, 151.3, 148.1, 135.4, 135.3,
131.0, 128.7, 128.5, 126.6, 125.8, 123.1, 121.3, 113.5, 56.8. HRESIMS m/
z calcd for C16H13N2Oþ3 281.0921 [MþH]þ, found 281.0926.
4.1.2. General procedures to synthesize 4aꢁ4u
I2 (362.25 mg, 1.43 mmol, 1.1 eq) was added to a mixture of 2j
(272.77 mg, 1.3 mmol, 1 eq) and DMSO (3 mL). The solution was
stirred at 110 ꢀC, then 1aꢁ1u (1 eq) in 3 mL DMSO was added
dropwise to the mixture during 1 h. After the complete disap-
pearance of the starting material, a sodium thiosulfate solution
(0.2 g Na2S2O3 in 12 mL water) was added dropwise to the reaction
system. Filtration and drying, the residue was purified by silica gel
chromatography to yield the desired products 4aꢁ4u.
4.1.1.4. 2-(4-fluorobenzoyl)quinazolin-4(3H)-one (3d). White solid,
2.78 g, yield 71%; 1H NMR (600 MHz, DMSO‑d6, J in Hz) dH 12.65 (s,
1H), 8.28e8.23 (m, 2H), 8.18 (dd, J ¼ 7.9, 1.6 Hz, 1H), 7.89e7.83 (m,
1H), 7.75 (d, J ¼ 8.1 Hz, 1H), 7.63 (t, J ¼ 7.5 Hz, 1H), 7.40 (t, J ¼ 8.8 Hz,
4.1.2.1. 2-(3,4,5-trimethoxybenzoyl)quinazolin-4(3H)-one
(4a).
White solid, 293 mg, yield 66%; 1H NMR (400 MHz, DMSO‑d6, J in
Hz) dH 12.65 (s, 1H), 8.22 (dd, J ¼ 7.9, 1.5 Hz, 1H), 7.90 (ddd, J ¼ 8.5,
7.0, 1.6 Hz, 1H), 7.83 (dd, J ¼ 8.3, 1.3 Hz, 1H), 7.66 (ddd, J ¼ 8.1, 7.1,
1.3 Hz,1H), 7.58 (s, 2H), 3.84 (s, 6H), 3.81 (s, 3H). 13C NMR (100 MHz,
DMSO‑d6) dC 185.9, 161.6, 153.0, 149.4, 147.6, 143.5, 135.2, 129.4,
129.0, 128.9, 126.5, 123.4, 109.2, 60.8, 56.6. HRESIMS m/z calcd for
2H). 13C NMR (125 MHz, DMSO‑d6) dC 186.0, 166.1 (d, JC-
1
¼ 252.5 Hz), 164.3, 149.3, 147.5, 135.2, 134.6 (d, 3JC-F ¼ 10 Hz), 131.2
F
(d, 4JC-F ¼ 2.5 Hz), 129.1, 128.8, 126.5, 123.4, 116.2 (d, 2JC-F ¼ 22.5 Hz).
HRESIMS m/z calcd for C15H10FN2Oþ2 269.0721 [MþH]þ, found
269.0725.
C
18H15N2Oꢁ5 339.0986 [MꢁH]ꢁ, found 339.0984.
4.1.1.5. 2-(4-chlorobenzoyl)quinazolin-4(3H)-one(3e). White solid,
3.21 g, yield 77%; 1H NMR (600 MHz, DMSO‑d6, J in Hz) dH 12.64 (s,
1H), 8.20e8.15 (m, 3H), 7.86 (ddd, J ¼ 8.4, 7.1, 1.5 Hz, 1H), 7.75 (dd,
J ¼ 8.1, 1.1 Hz, 1H), 7.65e7.61 (m, 3H). 13C NMR (150 MHz, DMSO‑d6)
dC 186.5, 161.6, 149.1, 147.6, 139.8, 135.3, 133.4, 133.3, 129.3, 129.2,
129.0, 126.6, 123.5. HRESIMS m/z calcd for C15H10ClN2Oþ2 285.0425
[MþH]þ, found 285.0432.
4.1.2.2. 5-Fluoro-2-(3,4,5-trimethoxybenzoyl)quinazolin-4(3H)-one
(4b). Yellow solid, 239 mg, yield 51%; 1H NMR (400 MHz, DMSO‑d6,
J in Hz) dH 12.69 (s, 1H), 7.86 (td, J ¼ 8.2, 5.5 Hz, 1H), 7.62 (dd, J ¼ 8.2,
1.0 Hz, 1H), 7.54 (s, 2H), 7.40 (ddd, J ¼ 11.1, 8.2, 1.1 Hz, 1H), 3.83 (s,
6H), 3.81 (s, 3H). 13C NMR (125 MHz, DMSO‑d6) dC 185.4, 160.5 (d,
2
1JC-F ¼ 261.25 Hz), 158.6, 152.6, 149.7 (d, JC-F ¼ 68.75 Hz), 143.3,
135.4 (d, 3JC-F ¼ 11.25 Hz), 128.8, 124.4, 114.8 (d, 2JC-F ¼ 20 Hz), 112.3
3
(d, JC-F ¼ 6.25 Hz), 108.7, 60.35, 56.22. HRESIMS m/z calcd for
C
18H14FN2Oꢁ5 357.0892 [MꢁH]ꢁ, found 357.0894.
4.1.1.6. 2-(4-(trifluoromethoxy)benzoyl)quinazolin-4(3H)-one (3f).
White solid, 1.83 g, yield 37%;1H NMR (600 MHz, DMSO‑d6, J in Hz)
dH 12.65 (s, 1H), 8.32e8.28 (m, 2H), 8.18 (dd, J ¼ 7.9, 1.5 Hz, 1H), 7.86
(ddd, J ¼ 8.5, 7.1, 1.6 Hz, 1H), 7.76 (dd, J ¼ 8.3, 1.2 Hz, 1H), 7.66e7.61
(m, 1H), 7.55e7.52 (m, 2H). 13C NMR (150 MHz, DMSO‑d6) dC 186.2,
4.1.2.3. 6-Fluoro-2-(3,4,5-trimethoxybenzoyl)quinazolin-4(3H)-one
(4c). White solid, 267 mg, yield 57%; 1H NMR (400 MHz, DMSO‑d6, J
in Hz) dH 12.83 (s, 1H), 8.03 (dd, J ¼ 8.0, 1.4 Hz, 1H), 7.78 (ddd,
J ¼ 10.4, 8.0, 1.4 Hz, 1H), 7.68 (s, 2H), 7.67e7.62 (m, 1H), 3.84 (s, 6H),
3.81 (s, 3H). 13C NMR (150 MHz, DMSO‑d6) dC 184.8, 160.5, 157.2 (d,
161.6, 152.7, 149.0, 147.6, 135.3, 134.1, 133.5, 129.3, 129.0, 126.6,
1
123.5, 120.5 (q, JC-F
C
¼
255 Hz). HRESIMS m/z calcd for
16H10F3N2Oþ3 335.0638 [MþH]þ, found 335.0643.
3
1JC-F ¼ 253.5 Hz), 152.5, 149.3, 143.2, 136.5, 128.9 (d, JC-F ¼ 6 Hz),
2
128.8, 124.9, 121.8, 120.2 (d, JC-F ¼ 18 Hz), 109.0, 60.3, 56.1. HRE-
SIMS m/z calcd for C18H14FN2Oꢁ5 357.0892 [MꢁH]ꢁ, found 357.0890.
4.1.1.7. 2-(2-(trifluoromethoxy)benzoyl)quinazolin-4(3H)-one (3g).
White solid, 1.92 g, yield 39%; 1H NMR (600 MHz, DMSO‑d6, J in Hz)
dH 12.86 (s, 1H), 8.18 (dd, J ¼ 7.9, 1.5 Hz, 1H), 7.90 (dd, J ¼ 7.7, 1.8 Hz,
1H), 7.82 (ddd, J ¼ 8.4, 7.2, 1.6 Hz, 1H), 7.77 (td, J ¼ 7.9, 1.8 Hz, 1H),
4.1.2.4. 7-Fluoro-2-(3,4,5-trimethoxybenzoyl)quinazolin-4(3H)-one
(4d). White solid, 312 mg, yield 67%; 1H NMR (400 MHz, DMSO‑d6, J
in Hz) dH 12.77 (s, 1H), 8.26 (dd, J ¼ 8.8, 6.1 Hz, 1H), 7.64 (dd, J ¼ 9.9,
2.5 Hz, 1H), 7.54 (s, 2H), 7.53e7.46 (m, 1H), 3.83 (s, 6H), 3.80 (s, 3H).
7.67e7.59 (m, 2H), 7.56 (t, J ¼ 7.6 Hz, 1H), 7.52 (d, J ¼ 8.4 Hz, 1H). 13
C
NMR (125 MHz, DMSO‑d6) dC 187.4, 161.4, 148.9, 147.5, 146.9, 135.3,
134.8, 132.5, 129.8, 129.5, 128.9, 127.8, 126.6, 123.4, 121.4, 120.3 (q,
1JC-F ¼ 256.25 Hz). HRESIMS m/z calcd for C16H10F3N2Oþ3 335.0638
[MþH]þ, found 335.0646.
1
13C NMR (100 MHz, DMSO‑d6) dC 185.6, 166.0 (d, JC-F ¼ 250 Hz),
160.8, 156.6, 152.8, 150.5, 143.4, 129.3 (d, 3JC-F ¼ 9 Hz), 128.9, 120.1,
2
2
117.0 (d, JC-F ¼ 23 Hz), 113.7 (d, JC-F ¼ 21 Hz), 108.9, 60.5, 56.4.
HRESIMS m/z calcd for C18H16FN2Oþ5 359.1038 [MþH]þ, found
359.1039.
4.1.1.8. 2-(4-(trifluoromethyl)benzoyl)quinazolin-4(3H)-one
(3h).
White solid, 1.94 g, yield 42%; 1H NMR (600 MHz, DMSO‑d6, J in Hz)
dH 12.68 (s, 1H), 8.32 (d, J ¼ 8.1 Hz, 2H), 8.19 (dd, J ¼ 8.0, 1.6 Hz, 1H),
7.93 (d, J ¼ 8.3 Hz, 2H), 7.86 (ddd, J ¼ 8.4, 7.1, 1.5 Hz, 1H), 7.75 (dd,
J ¼ 8.2, 1.2 Hz, 1H), 7.64 (ddd, J ¼ 8.1, 7.1, 1.2 Hz, 1H). 13C NMR
(125 MHz, DMSO‑d6) dC 186.9, 161.6, 148.7, 147.5, 138.3, 135.4, 133.5
4.1.2.5. 8-Fluoro-2-(3,4,5-trimethoxybenzoyl)quinazolin-4(3H)-one
(4e). Yellow solid, 273 mg, yield 59%; 1H NMR (400 MHz, DMSO‑d6,
J in Hz) dH 12.84 (s, 1H), 8.03 (dd, J ¼ 7.9, 1.4 Hz, 1H), 7.84e7.75 (m,
1H), 7.68 (s, 2H), 7.65 (dt, J ¼ 8.0, 4.0 Hz, 1H), 3.85 (s, 6H), 3.81 (s,
2
3
1
(q, JC-F ¼ 27.5 Hz), 132.2, 129.5, 129.1, 126.6, 125.8 (q, JC-
3H). 13C NMR (150 MHz, DMSO‑d6) dC 184.8, 160.4, 157.2 (d, JC-
¼ 3.75 Hz), 124.3 (q, 1JC-F ¼ 226.25 Hz), 123.6. HRESIMS m/z calcd
¼ 253.5 Hz), 152.5, 152.5, 149.2, 143.2, 136.5 (d, JC-F ¼ 19.5 Hz),
2
F
F
for C16H10F3N2Oþ2 319.0689 [MþH]þ, found 319.0695.
129.0 (d, 3JC-F ¼ 9 Hz), 128.8, 124.9, 121.8 (d, 4JC-F ¼ 4.5 Hz), 120.2 (d,
11

C.-J. Wang, X. Guo, R.-Q. Zhai et al.
European Journal of Medicinal Chemistry 224 (2021) 113671
2JC-F ¼ 12 Hz), 109.0, 60.3, 56.1. HRESIMS m/z calcd for C18H14FN2O5ꢁ
357.0892 [MꢁH]ꢁ, found 357.0890.
4.1.2.13. 7-Bromo-2-(3,4,5-trimethoxybenzoyl)quinazolin-4(3H)-one
(4 m). Yellow solid, 409 mg, yield 75%; 1H NMR (400 MHz,
DMSO‑d6, J in Hz) dH 12.83 (s, 1H), 8.12 (d, J ¼ 8.5 Hz, 1H), 8.05 (d,
J ¼ 1.9 Hz, 1H), 7.81 (dd, J ¼ 8.5, 1.9 Hz, 1H), 7.53 (s, 2H), 3.84 (s, 6H),
3.81 (s, 3H). 13C NMR (150 MHz, DMSO‑d6) dC 185.7, 161.1, 152.8,
150.6,148.7, 143.4,131.5, 130.7,128.9,128.4,128.2,122.3,108.9, 60.5,
56.4. HRESIMS m/z calcd for C18H16BrN2Oþ5 419.0237 [MþH]þ, found
419.0234.
4.1.2.6. 6,7-Difluoro-2-(3,4,5-trimethoxybenzoyl)quinazolin-4(3H)-
one (4f). White solid, 277 mg, yield 57%; 1H NMR (400 MHz,
DMSO‑d6, J in Hz) dH 12.91 (s, 1H), 8.13 (dd, J ¼ 10.3, 8.6 Hz, 1H), 7.97
(dd, J ¼ 11.2, 7.3 Hz, 1H), 7.53 (s, 2H), 3.84 (s, 6H), 3.80 (s, 3H). 13
C
1
NMR (150 MHz, DMSO‑d6) dC 185.3, 160.1, 154.0 (dd, JC-
¼ 253.5 Hz, 1JC-F ¼ 15 Hz), 152.6, 149.9, 149.6 (dd, 1JC-F ¼ 249 Hz,
F
1JC-F ¼ 13.5 Hz), 145.3 (d, 3JC-F ¼ 10.5 Hz), 143.3, 128.7, 120.5 (d, 3JC-
4.1.2.14. 8-Bromo-2-(3,4,5-trimethoxybenzoyl)quinazolin-4(3H)-one
(4n). White solid, 245 mg, yield 45%; 1H NMR (400 MHz, DMSO‑d6,
J in Hz) dH 12.84 (s, 1H), 8.22 (t, J ¼ 6.5 Hz, 2H), 7.74 (s, 2H), 7.56 (t,
J ¼ 8.0 Hz, 1H), 3.88 (s, 6H), 3.82 (s, 3H). 13C NMR (150 MHz, pyri-
dine-d5) dC 185.6,162.4,153.8,146.4,144.9,138.7, 135.5, 130.3,129.8,
126.8, 126.4, 124.8, 123.5, 110.8, 61.1, 56.8. HRESIMS m/z calcd for
¼ 6 Hz), 116.5 (d, 2JC-F ¼ 18 Hz), 113.7 (d, 2JC-F ¼ 18 Hz), 108.8, 60.4,
F
56.2. HRESIMS m/z calcd for C18H15F2N2Oþ5 377.0944 [MþH]þ, found
377.0940.
4.1.2.7. 5-Chloro-2-(3,4,5-trimethoxybenzoyl)quinazolin-4(3H)-one
(4g). White solid, 241 mg, yield 50%; 1H NMR (400 MHz, DMSO‑d6, J
in Hz) dH 12.68 (s, 1H), 7.82e7.71 (m, 2H), 7.64 (dd, J ¼ 7.6, 1.4 Hz,
1H), 7.55 (s, 2H), 3.83 (s, 6H), 3.81 (s, 3H). 13C NMR (100 MHz,
DMSO‑d6) dC 185.4, 159.6, 152.7, 150.0, 149.9, 143.4, 134.7, 132.8,
130.8, 128.9, 128.0, 120.1, 108.8, 60.5, 56.4. HRESIMS m/z calcd for
C
18H16BrN2Oþ5 419.0237 [MþH]þ, found 419.0237.
4.1.2.15. 6-Nitro-2-(3,4,5-trimethoxybenzoyl)quinazolin-4(3H)-one
(4o). Yellow solid, 273 mg, yield 55%; 1H NMR (400 MHz, DMSO‑d6,
J in Hz) dH 8.87 (d, J ¼ 2.7 Hz, 1H), 8.59 (dd, J ¼ 8.9, 2.7 Hz, 1H), 8.00
(d, J ¼ 9.0 Hz, 1H), 7.52 (s, 2H), 3.83 (s, 6H), 3.81 (s, 3H). 13C NMR
(150 MHz, DMSO‑d6) dC 185.6, 160.8, 152.7, 152.3, 146.0, 143.5, 130.1,
128.62, 128.55, 123.3, 121.9, 108.8, 60.4, 56.3. HRESIMS m/z calcd for
C
18H16ClN2Oþ5 375.0742 [MþH]þ, found 375.0741.
4.1.2.8. 6-Chloro-2-(3,4,5-trimethoxybenzoyl)quinazolin-4(3H)-one
(4h). White solid, 312 mg, yield 64%; 1H NMR (400 MHz, DMSO‑d6,
J in Hz) dH 12.86 (s, 1H), 8.15 (d, J ¼ 2.5 Hz, 1H), 7.93 (dd, J ¼ 8.7,
2.5 Hz, 1H), 7.85 (d, J ¼ 8.7 Hz, 1H), 7.55 (s, 2H), 3.84 (s, 6H), 3.81 (s,
3H). 13C NMR (100 MHz, DMSO‑d6) dC 185.6, 160.5, 152.7, 149.6,
146.1, 143.3, 135.0, 133.0, 130.8, 129.0, 125.2, 124.4, 108.9, 60.5, 56.3.
HRESIMS m/z calcd for C18H16ClN2Oþ5 375.0742 [MþH]þ, found
375.0741.
C
18H14N3Oꢁ7 384.0837 [MꢁH]ꢁ, found 384.0836.
4.1.2.16. 7-Nitro-2-(3,4,5-trimethoxybenzoyl)quinazolin-4(3H)-one
(4p). Yellow solid, 254 mg, yield 51%; 1H NMR (400 MHz, DMSO‑d6,
J in Hz) dH 13.12 (s, 1H), 8.51 (d, J ¼ 2.2 Hz, 1H), 8.43 (d, J ¼ 8.6 Hz,
1H), 8.34 (dd, J ¼ 8.7, 2.2 Hz, 1H), 7.53 (s, 2H), 3.83 (overlapped, 9H).
13C NMR (150 MHz, DMSO‑d6) dC 185.8, 160.7, 152.8, 151.5, 148.0,
143.5, 128.8, 128.4, 127.5, 123.3, 122.0, 108.9, 60.5, 56.4. HRESIMS m/
z calcd for C18H14N3Oꢁ7 384.0837 [MꢁH]ꢁ, found 384.0839.
4.1.2.9. 7-Chloro-2-(3,4,5-trimethoxybenzoyl)quinazolin-4(3H)-one
(4i). White solid, 304 mg, yield 63%; 1H NMR (400 MHz, DMSO‑d6, J
in Hz) dH 12.83 (s, 1H), 8.20 (d, J ¼ 8.5 Hz, 1H), 7.90 (s, 1H), 7.68 (s,
1H), 7.53 (s, 2H), 3.84 (s, 6H), 3.81 (s, 3H). 13C NMR (150 MHz,
DMSO‑d6) dC 185.5, 152.6, 150.4, 148.4, 143.3, 139.3, 128.7, 128.6,
128.0, 127.5, 121.8, 108.8, 60.3, 56.2. HRESIMS m/z calcd for
4.1.2.17. 5-Methoxy-2-(3,4,5-trimethoxybenzoyl)quinazolin-4(3H)-
one (4q). White solid, 173 mg, yield 36%; 1H NMR (400 MHz,
DMSO‑d6, J in Hz) dH 12.26 (s,1H), 7.76 (t, J ¼ 8.1 Hz,1H), 7.55 (s, 2H),
7.32 (d, J ¼ 8.0 Hz, 1H), 7.16 (d, J ¼ 8.3 Hz, 1H), 3.90 (s, 3H), 3.83 (s,
6H), 3.80 (s, 3H). 13C NMR (100 MHz, DMSO‑d6) dC 185.2, 159.8,
159.1, 152.5, 149.7, 149.3, 147.2, 143.1, 135.0, 133.8, 128.8, 120.1, 112.2,
110.2, 108.6, 60.3, 56.2, 56.1. HRESIMS m/z calcd for C19H19N2O6þ
371.1238 [MþH]þ, found 371.1234.
C
18H16ClN2Oþ5 375.0742 [MþH]þ, found 375.0740.
4.1.2.10. 8-Chloro-2-(3,4,5-trimethoxybenzoyl)quinazolin-4(3H)-one
(4j). White solid, 212 mg, yield 44%; 1H NMR (400 MHz, DMSO‑d6, J
in Hz) dH 12.84 (s, 1H), 8.18 (dd, J ¼ 8.0, 1.4 Hz, 1H), 8.07 (dd, J ¼ 7.8,
1.4 Hz, 1H), 7.77 (s, 2H), 7.64 (t, J ¼ 7.9 Hz, 1H), 3.87 (s, 6H), 3.82 (s,
3H). 13C NMR (125 MHz, DMSO‑d6) dC 184.2, 160.8, 152.4, 149.0,
143.9, 143.2, 134.8, 132.0, 129.0, 128.7, 125.2, 124.8, 109.1, 60.3, 56.1.
HRESIMS m/z calcd for C18H14ClN2Oꢁ5 373.0597 [MꢁH]ꢁ, found
373.0595.
4.1.2.18. 6-Methoxy-2-(3,4,5-trimethoxybenzoyl)quinazolin-4(3H)-
one (4r). White solid, 227 mg, yield 47%; 1H NMR (400 MHz,
DMSO‑d6, J in Hz) dH 12.55 (s, 1H), 7.80 (d, J ¼ 8.9 Hz, 1H), 7.61 (s,
1H), 7.60 (s, 2H), 7.50 (dd, J ¼ 9.0, 3.0 Hz, 1H), 3.92 (s, 3H), 3.84 (s,
6H), 3.80 (s, 3H). 13C NMR (100 MHz, DMSO‑d6) dC 185.1, 160.9,
159.4, 153.8, 152.4, 146.7, 142.9, 141.3, 130.4, 129.2, 124.1, 108.8,
106.3, 60.3, 56.1, 55.9. HRESIMS m/z calcd for C19H19N2Oþ6 371.1238
[MþH]þ, found 371.1235.
4.1.2.11. 5-Bromo-2-(3,4,5-trimethoxybenzoyl)quinazolin-4(3H)-one
(4k). Yellow solid, 344 mg, yield 63%; 1H NMR (400 MHz, DMSO‑d6,
J in Hz) dH 12.70 (s, 1H), 7.85 (dd, J ¼ 7.7, 1.3 Hz, 1H), 7.78 (dd, J ¼ 8.1,
1.3 Hz, 1H), 7.69 (t, J ¼ 7.9 Hz, 1H), 7.55 (s, 2H), 3.83 (s, 6H), 3.81 (s,
3H). 13C NMR (150 MHz, pyridine-d5) dC 186.2, 161.1, 153.8, 150.9,
150.1, 144.9, 135.6, 134.9, 130.3, 129.5, 123.5, 122.6, 122.1, 110.4, 61.1,
56.7. HRESIMS m/z calcd for C18H16BrN2Oþ5 419.0237 [MþH]þ, found
419.0235.
4.1.2.19. 7-Methoxy-2-(3,4,5-trimethoxybenzoyl)quinazolin-4(3H)-
one (4s). White solid, 351 mg, yield 73%; 1H NMR (400 MHz,
DMSO‑d6, J in Hz) dH 12.53 (s, 1H), 8.11 (d, J ¼ 8.8 Hz, 1H), 7.52 (s,
2H), 7.31e7.14 (m, 2H), 3.91 (s, 3H), 3.84 (s, 6H), 3.81 (s, 3H). 13C
NMR (150 MHz, DMSO‑d6) dC 185.7, 164.3, 160.7, 152.6, 149.7, 149.5,
143.1,129.0,127.6,117.7,116.2,109.7,108.7, 60.3, 56.2, 55.9. HRESIMS
m/z calcd for C19H17N2Oꢁ6 369.1092 [MꢁH]ꢁ, found 369.1089.
4.1.2.12. 6-Bromo-2-(3,4,5-trimethoxybenzoyl)quinazolin-4(3H)-one
(4l). Yellow solid, 266 mg, yield 49%; 1H NMR (400 MHz, DMSO‑d6,
J in Hz) dH 12.86 (s, 1H), 8.29 (d, J ¼ 2.4 Hz, 1H), 8.04 (dd, J ¼ 8.6,
2.4 Hz, 1H), 7.77 (d, J ¼ 8.7 Hz, 1H), 7.55 (s, 2H), 3.83 (s, 6H), 3.81 (s,
3H). 13C NMR (100 MHz, DMSO‑d6) dC 185.5, 160.3, 152.7, 149.7,
146.4, 143.3, 137.7, 130.8, 128.9, 128.3, 124.7, 121.3, 108.9, 60.5, 56.3.
HRESIMS m/z calcd for C18H16BrN2Oþ5 419.0237 [MþH]þ, found
419.0234.
4.1.2.20. 8-Methoxy-2-(3,4,5-trimethoxybenzoyl)quinazolin-4(3H)-
one (4t). White solid, 31 mg, yield 6%; 1H NMR (400 MHz, CDCl3, J
in Hz) dH 10.32 (s, 1H), 8.25 (s, 2H), 7.95 (dd, J ¼ 7.9, 1.2 Hz, 1H), 7.58
(t, J ¼ 8.1 Hz, 1H), 7.29 (dd, J ¼ 8.2, 1.2 Hz, 1H), 4.00 (overlapped,
12H). 13C NMR (150 MHz, DMSO‑d6) dC 185.3, 159.8, 159.2, 152.5,
149.7, 149.3, 143.2, 135.1, 128.9, 120.1, 112.3, 110.3, 108.7, 60.3, 56.2,
12

C.-J. Wang, X. Guo, R.-Q. Zhai et al.
European Journal of Medicinal Chemistry 224 (2021) 113671
56.2. HRESIMS m/z calcd for C19H17N2Oꢁ6 369.1092 [MꢁH]ꢁ, found
1.4 Hz, 1H), 7.44 (s, 2H), 4.71 (q, J ¼ 7.1, 2H), 3.94 (s, 3H), 3.86 (s, 6H),
1.52 (td, J ¼ 7.1, 1.6 Hz, 3H). 13C NMR (100 MHz, CDCl3) dC 190.5,
167.4, 158.4, 152.8, 150.4, 143.2, 134.1, 130.3, 128.5, 128.3, 123.7,
116.5, 108.7, 63.7, 60.9, 56.3, 14.3. HRESIMS m/z calcd for
369.1090.
4.1.2.21. 6,7-Dimethoxy-2-(3,4,5-trimethoxybenzoyl)quinazolin-
4(3H)-one (4u). White solid, 106 mg, yield 20%; 1H NMR (400 MHz,
CDCl3, J in Hz) dH 10.37 (s, 1H), 7.83 (s, 2H), 7.70 (s, 1H), 7.22 (s, 1H),
4.03 (s, 3H), 4.01 (s, 3H), 3.97 (s, 3H), 3.94 (s, 6H). 13C NMR
(100 MHz, CDCl3) dC 183.8, 160.3, 155.2, 152.7, 151.1, 145.2, 143.9,
143.4,128.8, 117.1, 109.5, 109.3, 105.8, 61.0, 56.5, 56.4, 56.3. HRESIMS
m/z calcd for C20H21N2Oþ7 401.1343 [MþH]þ, found 401.1341.
C
20H21N2Oþ5 369.1445 [MþH]þ, found 369.1440.
4.1.4.2. (4-Isopropoxyquinazolin-2-yl)
(3,4,5-trimethoxyphenyl)
methanone (6b). White solid, 21 mg, yield 19%; 1H NMR (400 MHz,
CDCl3, J in Hz) dH 8.41 (t, J ¼ 6.8 Hz, 1H), 8.01 (d, J ¼ 5.7 Hz, 2H),
7.94e7.83 (m, 2H), 7.69e7.65 (m, 1H), 3.99 (overlapped, 10H), 1.26
(overlapped, 6H). 13C NMR (150 MHz, CDCl3) dC 183.3, 160.7, 152.7,
147.4, 146.1, 144.1, 134.9, 129.5, 129.2, 128.5, 126.9, 123.2, 109.6, 61.0,
56.3, 31.2, 29.6. HRESIMS m/z calcd for C21H23N2Oþ5 383.1601
[MþH]þ, found 383.1595.
4.1.3. General procedures to synthesize 5aꢁ5c
The compound 4a (80 mg, 235.07 mmol, 1 eq) was added to a
stirred solution of DMF (4 mL). After compound 4a was dissolved,
K2CO3 (49 mg, 352.6 mol, 1.5 eq) and appropriate halogenated
m
compound (2 eq) were added to the solvent. The reaction mixture
was heated to 80 ꢀC. After the reaction was complete (monitored by
TLC), the solvent was removed under reduced pressure, the solid
was filtered and washed with acetone then with water to yield the
pure products.
4.1.5. General procedures to synthesize 8aꢁ8d
K2CO3 (49 mg, 352.6 mmol, 1.5 eq) was added to a stirred solu-
tion of compound 4a (80 mg, 235.07
m
mol, 1 eq) and 7aꢁ7d (1.5 eq)
in DMF (4 mL). The reaction mixture was heated to 80 ꢀC for 12 h.
The mixture was cooled, separated by filtration and purified by
chromatography on silica gel using CH2Cl2 to obtain 8aꢁ8d.
4.1.3.1. 3-Methyl-2-(3,4,5-trimethoxybenzoyl)quinazolin-4(3H)-one
(5a). White solid, 11 mg, yield 13%; 1H NMR (400 MHz, DMSO‑d6, J
in Hz) dH 8.24 (dd, J ¼ 8.0, 1.6 Hz, 1H), 7.87 (ddd, J ¼ 8.5, 7.1, 1.6 Hz,
1H), 7.71 (dd, J ¼ 8.3, 1.2 Hz, 1H), 7.64 (ddd, J ¼ 8.2, 7.1, 1.2 Hz, 1H),
7.38 (s, 2H), 3.814 (s, 6H), 3.808 (s, 3H), 3.41 (s, 3H). 13C NMR
(100 MHz, DMSO‑d6) dC 187.4, 161.4, 153.5, 152.1, 146.7, 144.4, 135.1,
129.1, 128.5, 127.9, 126.7, 122.0, 108.7, 60.8, 56.8, 32.2. HRESIMS m/z
calcd for C19H19N2Oþ5 355.1288 [MþH]þ, found 355.1282.
4.1.5.1. 3-((5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)methyl)-2-
(3,4,5-trimethoxybenz- oyl)quinazolin-4(3H)-one (8a). White solid,
21 mg, yield 18%; 1H NMR (400 MHz, CDCl3, J in Hz) dH 8.39 (dd,
J ¼ 8.0, 1.5 Hz, 1H), 7.88e7.78 (m, 4H), 7.63 (ddd, J ¼ 8.2, 6.9, 1.4 Hz,
1H), 7.44e7.40 (m, 2H), 7.37 (s, 2H), 5.78 (s, 2H), 3.93 (s, 3H), 3.85 (s,
6H). 13C NMR (100 MHz, CDCl3) dC 186.7, 164.6, 161.9, 161.0, 153.0,
149.6, 145.9, 144.5, 138.4, 135.3, 129.4, 129.0, 128.6, 128.3, 128.2,
127.4, 121.7, 121.6, 108.8, 61.1, 56.4, 38.0. HRESIMS m/z calcd for
4.1.3.2. 3-Allyl-2-(3,4,5-trimethoxybenzoyl)quinazolin-4(3H)-one
(5b). White solid, 15 mg, yield 17%; 1H NMR (400 MHz, CDCl3, J in
Hz) dH 8.38 (dd, J ¼ 7.9, 1.5 Hz, 1H), 7.84e7.72 (m, 2H), 7.59 (ddd,
J ¼ 8.2, 7.0, 1.4 Hz, 1H), 7.27 (s, 2H), 5.88 (ddt, J ¼ 16.5, 10.7, 5.9 Hz,
1H), 5.18e5.10 (m, 2H), 4.75 (dt, J ¼ 5.9,1.4 Hz, 2H), 3.96 (s, 3H), 3.86
(s, 6H). 13C NMR (100 MHz, CDCl3) dC 186.6, 161.3, 153.4,151.0, 146.4,
144.7, 134.9, 131.7, 129.1, 128.4, 128.0, 127.3, 122.1, 119.6, 108.5, 61.2,
56.6, 46.6. HRESIMS m/z calcd for C21H21N2Oþ5 381.1445 [MþH]þ,
found 381.1440.
C
27H22ClN4Oþ6 533.1222 [MþH]þ, found 533.1219.
4.1.5.2. 3-((5-(4-fluorophenyl)-1,3,4-oxadiazol-2-yl)methyl)-2-
(3,4,5-trimethoxybenz- oyl)quinazolin-4(3H)-one (8b). White solid,
13 mg, yield 11%; 1H NMR (400 MHz, CDCl3, J in Hz) dH 8.40 (dd,
J ¼ 8.0, 1.5 Hz, 1H), 7.92e7.77 (m, 4H), 7.64 (ddd, J ¼ 8.2, 6.9, 1.4 Hz,
1H), 7.37 (s, 2H), 7.13 (t, J ¼ 8.7 Hz, 2H), 5.79 (s, 2H), 3.93 (s, 3H), 3.85
(s, 6H). 13C NMR (100 MHz, CDCl3) dC 186.8, 165.10 (d, JC-
1
¼ 252 Hz), 164.7, 161.9, 161.1, 153.2, 149.7, 146.0, 144.7, 135.4, 129.4
F
3
4
(d, JC-F ¼ 8.1 Hz), 129.1, 128.7, 128.4, 127.6, 121.7, 119.7 (d, JC-
4.1.3.3. 3-(Prop-2-yn-1-yl)-2-(3,4,5-trimethoxybenzoyl)quinazolin-
4(3H)-one (5c). White solid, 8 mg, yield 9%; 1H NMR (400 MHz,
CDCl3, J in Hz) dH 8.39 (dd, J ¼ 8.0, 1.6 Hz, 1H), 7.83e7.74 (m, 1H),
7.75 (d, J ¼ 8.5 Hz, 1H), 7.60 (ddd, J ¼ 7.5, 6.8,1.3 Hz,1H), 7.36 (s, 2H),
5.08 (d, J ¼ 2.5 Hz, 2H), 3.97 (s, 3H), 3.87 (s, 6H), 2.11 (t, J ¼ 2.5 Hz,
1H). 13C NMR (100 MHz, CDCl3) dC 186.3, 160.6, 153.0, 149.8, 145.9,
144.3, 135.0, 129.0, 128.6, 128.1, 127.3, 121.7, 108.6, 78.1, 73.6, 61.1,
56.4, 32.7. HRESIMS m/z calcd for C21H19N2Oþ5 379.1288 [MþH]þ,
found 379.1285.
2
¼ 3 Hz), 116.6 (d, JC-F ¼ 22 Hz), 116.4, 109.0, 61.2, 56.5, 38.0.
F
HRESIMS m/z calcd for C27H22FN4Oþ6 517.1518 [MþH]þ, found
517.1513.
4.1.5.3. 3-((5-(p-tolyl)-1,3,4-oxadiazol-2-yl)methyl)-2-(3,4,5-
trimethoxybenzoyl)quin-azolin-4(3H)-one (8c). White solid, 17 mg,
yield 15%; 1H NMR (400 MHz, CDCl3, J in Hz) dH 8.40 (dd, J ¼ 7.9,
1.4 Hz, 1H), 7.87e7.71 (m, 4H), 7.67e7.59 (m, 1H), 7.36 (d, J ¼ 1.1 Hz,
2H), 7.23 (d, J ¼ 7.9 Hz, 2H), 5.80 (s, 2H), 3.91 (s, 3H), 3.84 (s, 6H),
2.39 (s, 3H). 13C NMR (100 MHz, CDCl3) dC 186.7, 165.6, 161.4, 161.0,
153.0, 149.7, 145.9, 144.5, 142.6, 135.2, 129.7, 128.9, 128.6, 128.2,
127.4, 126.9, 121.6, 120.4, 108.8, 61.0, 56.4, 37.8, 21.6. HRESIMS m/z
calcd for C28H25N4Oþ6 513.1769 [MþH]þ, found 513.1769.
4.1.4. General procedures to synthesize 6a‒6b
To a room temperature solution of compound 4a (100 mg,
293.83
mmol, 1eq) and BOP (169 mg, 381.98
mmol, 1.3 eq) in MeCN
(4 mL) were added DBU (67 mg, 440.75
mmol, 1.5 eq). Then
appropriate alcohol (4 mL) was added to the vial. The reaction
mixture was stirred at 85 ꢀC for 2 days. After the reaction was
complete (monitored by TLC), the solvent was removed under
reduced pressure, the solid was filtered and washed with dimethyl
sulfoxide then with water to yield the pure product.
4.1.5.4. 3-((5-(furan-2-yl)-1,3,4-oxadiazol-2-yl)methyl)-2-(3,4,5-
trimethoxybenzoyl)q-uinazolin-4(3H)-one (8d). White solid, 31 mg,
yield 27%; 1H NMR (400 MHz, CDCl3, J in Hz) dH 8.40 (dd, J ¼ 7.9,
1.5 Hz, 1H), 7.88e7.77 (m, 2H), 7.68e7.54 (m, 2H), 7.36 (s, 2H), 7.05
(dd, J ¼ 3.5, 0.8 Hz, 1H), 6.55 (dd, J ¼ 3.5, 1.8 Hz, 1H), 5.78 (s, 2H),
3.93 (s, 3H), 3.86 (s, 6H). 13C NMR (100 MHz, CDCl3) dC 186.7, 161.0,
160.9, 158.1, 153.0, 149.6, 145.9, 144.4, 138.7, 135.2, 128.9, 128.5,
128.3, 127.4, 121.6, 114.6, 112.2, 108.8, 61.0, 56.4, 37.8. HRESIMS m/z
calcd for C25H21N4Oþ7 489.1405 [MþH]þ, found 489.1399.
4.1.4.1. (4-Ethoxyquinazolin-2-yl)
(3,4,5-trimethoxyphenyl)meth-
anone (6a). White solid, 54 mg, yield 50%; 1H NMR (400 MHz,
CDCl3, J in Hz) dH 8.26 (dd, J ¼ 8.3, 1.6 Hz, 1H), 8.05 (dd, J ¼ 8.4,
1.5 Hz, 1H), 7.89 (ddd, J ¼ 8.4, 6.9, 1.5 Hz, 1H), 7.66 (ddd, J ¼ 8.3, 7.0,
13

C.-J. Wang, X. Guo, R.-Q. Zhai et al.
European Journal of Medicinal Chemistry 224 (2021) 113671
4.1.6. Preparation of target compound 9
4.2.4. Western blot analysis
Compound 4a (80 mg, 235.07
m
mol, 1 eq) was added to phos-
The cells were washed with pre-chilled PBS and then resus-
pended in cell lysis buffer to prepare HepG2 and Bel-7402 cell ex-
tracts. The cell lysate was separated on a 12.5% SDS-PAGE gel. After
the separation process, the protein was transferred to the PVDF
membrane (#IPVH00010, Merck Millipore) and incubated with the
antibodies of interest at 4 ꢀC overnight. Cleaved caspases-3 (1:1000,
#CST9661, Cell Signaling Technology), cleaved PARP (1:1000,
#CST5625, Cell Signaling Technology), Tubulin (1:1000, #CST2148,
Cell Signaling Technology), AKT (1:1000, #CST9272, Cell Signaling
Technology), P-AKT (1:1000, #CST4060S, Cell Signaling Technol-
ogy), ERK1/2 (1:1000, #CST4695, Cell signaling Technology), P-
ERK1/2 (1:1000, #CST4370S, Cell Signaling Technology). Then, the
membranes were washed and incubated with horseradish peroxi-
dase (HRP) conjugated anti-rabbit or anti-mouse secondary anti-
bodies for 1 h. A chemiluminescence imaging system was used to
capture protein bands.
phorus oxychloride (0.5 mL). The reaction mixture was heated to
100 ꢀC for 1 h. The mixture was cooled, the solvent was removed
under reduced pressure, and the solid was filtered and washed with
dimethyl sulfoxide then with water to yield the pure product.
4.1.6.1. (4-Quinazolin-2-yl) (3,4,5-trimethoxyphenyl)methanone (9).
White solid, 72 mg, yield 85%; 1H NMR (400 MHz, DMSO‑d6, J in Hz)
dH 8.21 (dd, J ¼ 8.0,1.4 Hz,1H), 7.89 (ddd, J ¼ 8.5, 7.0,1.5 Hz,1H), 7.82
(dd, J ¼ 8.3,1.2 Hz,1H), 7.65 (ddd, J ¼ 8.2, 7.1, 1.3 Hz,1H), 7.57 (s, 2H),
3.83 (s, 6H), 3.80 (s, 3H). 13C NMR (100 MHz, DMSO‑d6) dC 185.9,
161.6, 153.0, 149.4, 147.6, 143.5, 135.3, 129.4, 129.0, 128.8, 126.5,
123.3, 109.2, 60.8, 56.6. HRESIMS m/z calcd for C18H16ClN2O4þ
359.0793 [MþH]þ, found 359.0790.
4.2. Materials for biological studies
4.2.5. Cell apoptosis analysis
Roswell Park Memorial Institute (RPMI) 1640, Dulbecco's
modified Eagle's medium (DMEM) and Fetal bovine serum (FBS)
were purchased from Gibco company. 6-well plates and 96-well
plates were purchased from Costar company. HepG2/Bel-7402
(human hepatic carcinoma), A549 (human lung carcinoma), U251
(human glioma carcinoma), NCIeH1299 (human lung carcinoma),
HCT116 (human colon carcinoma) cell strains were purchased from
EnoGene company.
HepG2 and Bel-7402 cells were treated with different concen-
trations of compound 4a or DMSO for 48 h. Muse™ Annexin V &
Dead Cell assay kit (Muse TM Cell Analyzer, Millipore (catalog no.
MCH100105)) was used to determine the cell apoptosis by the
Muse cell analyzer.
4.2.6. Hoechst 33342 stain
The cells were seeded on a 6-well cell culture plate and the
negative control DMSO and different concentrations of compound
4a were added, then the cells were placed in a 37 ꢀC cell incubator
4.2.1. Cell proliferation assay
for 48 h. The cells were incubated with 10 mg/mL Hoechst 33342
All compounds were dissolved in DMSO. HepG2, Bel-7402,
A549, U251, NCIeH1299, and HCT116 were seeded into 96-well
plates (3 ꢂ 103 per well). The cells were cultured overnight in
DMEM containing 10% FBS, and then negative control (0.1% DMSO)
and different concentrations of compounds were added respec-
tively, and then incubated at 37 ꢀC for 72 h. The antiproliferative
effects of the compounds were tested by the MTT assay. The 10%
MTT (5 mg/mL, PBS) reagent was added to the medium and the cells
were incubated at 37 ꢀC for another 4 h. Then, supernatant was
dye (Beyotime Biotechnology, China) in darkness at 37 ꢀC for
30 min and observed under a fluorescent microscopy.
4.2.7. Intracellular ROS measurement
The cells were incubated with different concentrations of
compound 4a for 48 h. Then, the cells were washed and treated
with 10 mM DCFH-DA (Beyotime Biotechnology, China) at 37 ꢀC for
20 min in the dark. The fluorescence was checked with a fluores-
cent microscopy.
discarded, 100 m
L of DMSO was added, and incubated at 37 ꢀC for
10 min. Then the absorbance of the cells was measured at 570 nm.
Through MTT measurement, the growth inhibition rate of the
compound is calculated as (AceAs)/(AcꢁAb) ꢂ 100% (Ac, absor-
bance of control wells; As, absorbance of sample wells; Ab,
Absorbance of blank wells). The IC50 calculator was used to calcu-
late the IC50 value corresponding to the concentration that caused
50% inhibition of cell proliferation.
4.2.8. Tumor nude mice model
Female BALB/c nude mice (14e17 g) were inoculated with
HepG2 cells (1 ꢂ 106 per mouse). When the tumor volume reached
100e200 mm3, the mice were randomly divided into three groups.
Taking injection of (15% N-methylpyrrolidone, 25% Solutol HS-15,
60% saline) as the solvent control group, the compound 4a was
dissolved at 4 mg/kg and 8 mg/kg and injected into mice intra-
peritoneally every day for 20 days. Use a vernier caliper to measure
the tumor size and use the following formula to calculate the tumor
volume (TV): TV (mm3) ¼ width2 ꢂ (length/2). The inhibition rates
of tumor growth (IRT) were calculated as follows:
IRT ¼ 100% ꢂ [(M1ꢁM0)ꢁ(M2ꢁM0)]/(M1ꢁM0) (M0: Tumor vol-
umes before administration; M1: Tumor volumes in the control
group; M2: Tumor volumes in the drug treatment group).
4.2.2. Colony formation assay
HepG2 and Bel-7402 cells were seeded in a 6-well plate at
1000 cells/well. The cells were cultured overnight in DMEM con-
taining 10% FBS. Compound 4a was added according to the con-
centration gradient, and the cells were placed in 37 ꢀC cell
incubator containing 5% CO2 for 10 days. Then, the cells were fixed
with 4% paraformaldehyde for 15 min, stained with crystal violet
for 30 min. Then, the pictures were taken and colony numbers were
counted. The clone formation rate relative to the solvent control
group was calculated.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
4.2.3. Cell cycle arrest analysis
HepG2 and Bel-7402 cells were treated with different concen-
trations of compound 4a or DMSO for 48 h. Then, a Cell Cycle and
Apoptosis Analysis Kit (Yeasen Biotech Co., Ltd.) was used to
determine the phase of the cell cycle by flow cytometry on an
FC500 cytometer (Beckman Coulter).
Acknowledgments
We thank Prof. Yu-Cheng Gu at Syngenta Jealott's Hill Interna-
tional Research Centre for his proofreading of the manuscript. This
14

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work was supported by the Program of National Natural Science
Foundation of China (Nos. U1706210, 81871868, 41776141, and
41322037), the Program of Natural Science Foundation of Shandong
Province of China (No. JQ201510), the Fundamental Research Funds
for the Central Universities (Nos. 201841004 and 202042011), the
Marine S&T Fund of Shandong Province for Pilot National Labora-
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2018SDKJ0403-2 and 2015ASKJ02), the Taishan Scholars Program,
China (No. tsqn20161010).
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Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113671.
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