Discovery of novel quinazolinone derivatives as potential anti-HBV and anti-HCC agents

Article abstract of DOI:10.1016/j.ejmech.2020.112581

As a continuation of earlier works, a series of novel quinazolinone derivatives (5a-s) were synthesized and evaluated for their in vitro anti-HBV and anti-hepatocellular carcinoma cell (HCC) activities. Among them, compounds 5j and 5k exhibited most potent inhibitory effect on HBV DNA replication in both drug sensitive and resistant (lamivudine and entecavir) HBV strains. Interestingly, besides the anti-HBV effect, compound 5k could significantly inhibit the proliferation of HepG2, HUH7 and SK- cells, with IC₅₀ values of 5.44, 6.42 and 6.75 μM, respectively, indicating its potential anti-HCC activity. Notably, the in vitro anti-HCC activity of 5k were more potent than that of positive control 5-fluorouracil and sorafenib. Further studies revealed that compound 5k could induce HepG2 cells apoptosis by dose-dependently upregulating Bad and Bax expression and decreasing Bcl-2 and Bcl-xl protein level. Considering the potent anti-HBV and anti-HCC effect, compound 5k might be a promising lead to develop novel therapeutic agents towards HBV infection and HBV-induced HCC.

Full text of DOI:10.1016/j.ejmech.2020.112581

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Discovery of novel quinazolinone derivatives as potential anti-HBV and anti-HCC
agents
Jingying Qiu, Qingqing Zhou, Yinpeng Zhang, Mingyu Guan, Xin Li, Yueting Zou,
Xuan Huang, Yali Zhao, Wang Chen, Xiaoke Gu
PII:
S0223-5234(20)30553-5
DOI:
https://doi.org/10.1016/j.ejmech.2020.112581
EJMECH 112581
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 September 2019
Revised Date: 10 June 2020
Accepted Date: 11 June 2020
Please cite this article as: J. Qiu, Q. Zhou, Y. Zhang, M. Guan, X. Li, Y. Zou, X. Huang, Y. Zhao, W.
Chen, X. Gu, Discovery of novel quinazolinone derivatives as potential anti-HBV and anti-HCC agents,
European Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112581.
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Discovery of novel quinazolinone derivatives as potential anti-HBV and
anti-HCC agents
Jingying Qiu a, b, #, Qingqing Zhou b, #, Yinpeng Zhang b, Mingyu Guan a, Xin Li a, Yueting Zou b,
Xuan Huang a, Yali Zhao a, Wang Chen b, Xiaoke Gu a,
a
Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical
University, Xuzhou 221004, People’s Republic of China
b
Department of Pharmaceutical Analysis, School of Pharmacy, Xuzhou Medical University,
Xuzhou 221004, People’s Republic of China
Graphical Abstract
O
N
O
N
N
O
O
O
S
B
N
N
N
A
SAR exploration
S
S
5k
C
LC5f
Inhibiting HBV DNA replication with IC50 values of 0.58 M;
Inhibiting HepG2, HUH7 and SK-Hep-1 cells proliferation with
IC50 values of 5.44, 6.42 and 6.75 M, respectively.
Compound 5k exhibited more potent in vitro anti-HBV and anti-HCC activities than lead
compound LC5f and positive control 5-fluorouracil and sorafenib, respectively.
Corresponding authors. Tel.: +86-516-83262137; fax: +86-516-83262630; e-mail: gu_xk@xzhmu.edu.cn (X. G.)
# These two authors contributed equally to this work.

Discovery of novel quinazolinone derivatives as potential anti-HBV and anti-HCC
agents
Jingying Qiu a, b, #, Qingqing Zhou b, #, Yinpeng Zhang b, Mingyu Guan a, Xin Li a, Yueting Zou b, Xuan
Huang a, Yali Zhao a, Wang Chen b, Xiaoke Gu a,
a Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical University,
Xuzhou 221004, People’s Republic of China
b
Department of Pharmaceutical Analysis, School of Pharmacy, Xuzhou Medical University, Xuzhou
221004, People’s Republic of China
Abstract
As a continuation of earlier works, a series of novel quinazolinone derivatives (5a-s) were synthesized
and evaluated for their in vitro anti-HBV and anti-hepatocellular carcinoma cell (HCC) activities. Among
them, compounds 5j and 5k exhibited most potent inhibitory effect on HBV DNA replication in both drug
sensitive and resistant (lamivudine and entecavir) HBV strains. Interestingly, besides the anti-HBV effect,
compound 5k could significantly inhibit the proliferation of HepG2, HUH7 and SK- cells, with IC50 values
of 5.44, 6.42 and 6.75 μM, respectively, indicating its potential anti-HCC activity. Notably, the in vitro
anti-HCC activity of 5k were more potent than that of positive control 5-fluorouracil and sorafenib. Further
studies revealed that compound 5k could induce HepG2 cells apoptosis by dose-dependently upregulating
Bad and Bax expression and decreasing Bcl-2 and Bcl-xl protein level. Considering the potent anti-HBV
and anti-HCC effect, compound 5k might be a promising lead to develop novel therapeutic agents towards
HBV infection and HBV-induced HCC.
Keywords: Anti-HBV agents; Quinazolinone derivatives; Non-nucleoside; Anti-HCC activity.
1. Introduction
Hepatitis B virus (HBV) infection is a worldwide threat to public health. According to the World Health
Organization (WHO), more than 257 million people are living with HBV infection globally [1, 2]. Up to 30%
HBV carriers may develop progressive chronic liver disease, including hepatitis, fibrosis, cirrhosis and
Corresponding authors. Tel.: +86-516-83262137; fax: +86-516-83262630; e-mail: gu_xk@xzhmu.edu.cn (X. G.)
# These two authors contributed equally to this work.
1

even hepatocellular carcinoma (HCC) [3-6]. It was also reported that the majority of HCC cases was
associated with HBV-infection [7, 8]. Up to now, several anti-HBV drugs are available in clinic, such as
nucleos(t)ides-based reverse transcriptase inhibitors and interferons, however, the clinical cure rate for
chronic HBV infection are not satisfactory, mainly because of the viral resistance and drug adverse effects
[9-13]. Therefore, it is of great importance to develop novel anti-HBV agents with diverse structure and
action mechanism to treat HBV infection and related diseases [14, 15].
Recently, a series of quinazolinones derivatives were prepared as potential anti-HBV agents by us [16].
Among them, compound LC5f could significant inhibit HBV DNA replication in wild-type and drug
resistant HBV strains with IC50 values of 0.71 and 0.84 μM, respectively (Figure 1). Previous structure
activity relationship study indicated that the introduction of diethylamine ethoxy substituents in ring A
could significantly affect the anti-HBV activity. This led us to expect that the modification of compound
LC5f would improve the anti-HBV activity. In this study, in order to further investigate the effect of
substitutions on anti-HBV activity comprehensively, we prepared novel quinazolinones derivatives by
introducing various alkoxy substituents (ie. dimethylamino ethoxy, diethylamine ethoxy or dimethylamino
propoxy) in ring A of LC5f, replacing thiophenyl (ring B of LC5f) with furanyl or phenyl by bioisosterism
principle, and/or introducing methoxy or acetyl in ring C of LC5f, respectively (Figure 1). Thus, nineteen
compounds were synthesized, and their anti-HBV activities were subsequently evaluated in wild-type and
polymerase drug resistant HBV strains. In addition, considering that HBV infection is a major global cause
of HCC [17, 18], the in vitro anti-HCC activities of the target compounds were also investigated in the
present work.
Figure 1. Chemical structures of LC5f and target compounds
2. Chemistry
The target compounds, quinazolinone derivatives 5a-s, were prepared according to the following
2

synthetic procedures (Scheme 1). Briefly, various methylamine derivatives was firstly treated with CS2 and
toluene sulfonyl chloride to afford intermediate 1. Chloromethylation intermediate 2 was prepared by
reacting benzene or benzene derivatives with paraformaldehyde in the presence of hydrochloric acid.
Methyl 2-amino-4-hydroxybenzoate was condensed with the isothiocyanate intermediate 1 to give
intermediate 3, which was subsequently reacted with intermediate 2 to prepare intermediate 4. Finally, the
target compounds 5a-s were prepared by alkylating intermediate 4 with 2-dimethylaminoethyl chloride
hydrochloride, 2-diethylaminoethyl chloride hydrochloride or 3-dimethylaminopropyl chloride
hydrochloride, respectively.
Scheme 1. Synthetic route of target compounds 5a-s. Reagents and conditions: a) TEA, THF, 0 °C; b) TsCl, 0 °C; c) HCl,
80 °C; d) TEA, EtOH, reflux; e) K2CO3, 1,4-dioxane, 90 °C; f) 2-dimethylaminoethyl chloride hydrochloride,
2-diethylaminoethyl chloride hydrochloride or 3-dimethylaminopropyl chloride hydrochloride, K2CO3, 1,4-dioxane, 90 °C.
3. Results and discussion
3.1 Virological assays of anti-HBV activities in vitro
3.1.1 Inhibitory effect on HBV DNA replication
The intrinsic cytotoxicity of the target compounds were firstly investigated in HBV-infected HepG2
2.2.15 cells by MTT assay. Data showed that most of the CC50 values of the target compounds were more
than 10 μM (Table 1). Next, the in vitro anti-HBV activity of the target compounds was determined in
HepG2 2.2.15 cells according to previous method [19, 20]. Briefly, HepG2 2.2.15 cells were treated with
target compounds at the concentration of 0.8 μM (a non-toxic concentration far below CC50) for 6 days, and
3

then the extra cellar HBV DNA levels were measured by real time PCR (RT-PCR) assay. LC5f and
lamivudine (3TC) were selected as the reference and positive controls, respectively. As shown in Table 1,
the intracellular HBV DNA levels in cells was decreased to some extent, especially in 5e, 5f, 5g, 5j, 5k, 5q
and 5r treated group, indicating their significant anti-HBV activity. Clearly, 5f, 5j, 5k, 5q and 5r were the
most potent ones, and their the inhibition rate on HBV DNA replication was 67.07%, 67.45%, 67.31%,
65.53% and 64.51%, respectively, which was much higher than that of reference control LC5f (with an
inhibition rate of 53.59%).
Table 1. Intrinsic cytotoxicity and inhibitory effect of the target compounds 5a-s on HBV DNA levela
HepG2 2.2.15
a CC50 Inhibition rate (%)
R1
R2
R3
R4
Compds
5a
(μM)
(0.8 μM)
OCH3
COCH3
CH2CH2CH2N(CH3)2
9.63
0.16
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
CH2CH2CH2N(CH3)2
CH2CH2N(CH3)2
>10
>10
>10
>10
>10
>10
8.38
>10
>10
21.65
20.68
45.19
54.24
67.07
52.63
34.38
21.69
67.45
5b
5c
5d
5e
5f
COCH3
COCH3
CH2CH2N(CH2CH3)2
CH2CH2CH2N(CH3)2
CH2CH2N(CH3)2
COCH3
H
H
H
H
H
CH2CH2N(CH2CH3)2
CH2CH2CH2N(CH3)2
CH2CH2N(CH3)2
5g
5h
5i
H
CH2CH2N(CH2CH3)2
5j
4

H
H
CH2CH2CH2N(CH3)2
CH2CH2N(CH3)2
>10
>10
>10
>10
>10
>10
>10
>10
>10
67.31
15.64
4.93
5k
5l
OCH3
OCH3
OCH3
OCH3
OCH3
H
COCH3
COCH3
CH2CH2N(CH2CH3)2
CH2CH2N(CH3)2
5m
5n
5o
5p
5q
5r
5s
H
H
H
H
H
H
11.83
9.80
CH2CH2N(CH2CH3)2
CH2CH2CH2N(CH3)2
CH2CH2N(CH3)2
3.66
65.53
64.51
42.84
H
CH2CH2N(CH2CH3)2
CH2CH2CH2N(CH3)2
H
>10
53.59
84.42
LC5f
3TC
>100
a CC50 represents the dose of compound required to cause 50% death of the HepG2 2.2.15 cells.
To further confirm the anti-HBV activity, the dose-dependent inhibitory effect of active compounds 5e,
5f, 5g, 5j, 5k, 5q and 5r on HBV DNA replication were subsequently determined in HepG2 2.2.15 cells by
RT-PCR assay as mentioned above. As shown in Table 2, the IC50 values of the tested compounds on HBV
DNA replication were in range of 0.58-0.79 μM, confirming their promising anti-HBV activity. Among
them, 5j and 5k were the most potent ones, and their IC50 values on HBV DNA replication was 0.63 and
0.58 μM, respectively. Notably, 5j and 5k had the largest SI values, indicating their relative safety profiles.
Table 2. Anti-HBV activities and SI values of target compounds 5e, 5f, 5g, 5j, 5k, 5q and 5r
DNA replication
a IC50 (μM)
0.79
b SI
Compds
>12.66
>13.89
>12.99
>15.87
>17.24
5e
5f
0.72
0.77
5g
5j
0.63
0.58
5k
5

0.68
0.66
0.77
<0.1
>14.71
>15.15
>12.99
>1000
5q
5r
LC5f
3TC
a
b
IC50 represents the dose of compound required to cause 50% inhibition of DNA replication. SI means selectivity index,
which was calculated as the CC50 (shown in Table 1)/IC50 (shown in Table 2) value.
Subsequently, we determined the inhibitory effect of 5j and 5k on the production of HBsAg in
HBV-infected 2.2.15 cells according to our previous method [21]. Unfortunately, compounds 5j, 5k and
lamivudine displayed relatively poor inhibitory effect on HBsAg production with an inhibitory rate of
13.64%, 4.55% and 9.09%, respectively.
According to the bioactivity results shown in Tables 1 and 2, structure and anti-HBV activity
relationship could be preliminarily drawn as follows: chemical structure of ring B in the quinazolinone
derivatives significantly affect the anti-HBV activity. When the thiophenyl was replaced with a furanyl by
bioisosterism principle, the anti-HBV activity was obviously increased (5e vs. 5a; 5h vs. 5b; 5j vs. LC5f).
In sharp contrary, after changing the thiophenyl with a phenyl, the anti-HBV activity was remarkably
decreased (5p vs. 5b). In addition, methoxyl at position-2 and acetyl at position-5 on ring C of the
quinazolinone derivatives might play an adverse effect on the anti-HBV activities. When the methoxy or
acetyl group was removed, the anti-HBV activity was generally increased (5j vs. 5g; 5k vs. 5h; 5q vs. 5n;
5r vs. 5o; 5s vs. 5p; 5g vs. 5d; 5f vs. 5c; 5o vs. 5m), which was consistent with our previous findings [16].
3.1.2 Anti-HBV effect of 5j and 5k against drug resistant HBV strain
Considering that drug resistance is one of the major limitations of the nucleos(t)ide therapy, we next
evaluated whether 5j and 5k could inhibit HBV DNA replication in lamivudine and entecavir-resistant
HBV strain. The inhibitory effect on HBV DNA replication in wild-type (WT) strain was served as the
control group. As shown in Figure 2, both nucleoside drugs lamivudine (3TC, 100 μM) and entecavir (ETV,
10 μM) could significantly decrease the HBV DNA levels in WT strain with replication rates of 90.00%
and 96.90%, respectively. In sharp contrast, such anti-HBV effect was dramatically attenuated in drug
resistant strain, for the inhibition rate on HBV DNA replication were only 15.69% and 18.92%,
6

respectively. Interestingly, both compound 5j and 5k displayed significant anti-viral effect against
lamivudine and entecavir-resistant mutant HBV strain, and the HBV DNA replication inhibition rates on
resistant strain were comparable with that on wild-type strain (58.89% and 64.03% vs. 60.64% and 61.07%,
respectively).
Figure 2. Inhibitory effect of 5j and 5k on HBV DNA replication in wild-type and drug resistant HBV strains. Briefly, the
full genome of wide-type or plasmids containing lamivudine and entecavir-resistant (LERS) mutant (rtL180M + rtM204V +
rtT184L) were transiently transfected into HepG2 2.2.15 cells, and the cells were then incubated in the presence or absence of
lamivudine (3TC, 100 μM), entecavir (ETV, 10 μM), 5j (0.8 μM) or 5k (0.8 μM) for 72 h, respectively. Finally, the
replicative intermediate levels of HBV DNA in both strains was determined using RT-PCR. **P < 0.01 represents significant
difference from the WT HBV group.
3.2 In vitro anti-HCC activities of the target compounds
3.2.1 Inhibitory effect on HCC cells proliferation
As mentioned above, HBV infection is one of the leading cause for HCC, and we have confirmed that
the newly synthetic quinazolinones derivatives displayed potent anti-HBV activities. Therefore, we were
interested to see whether these compounds could have an anti-HCC activity. Firstly, we determined the
anti-proliferation effect of the target compounds on HepG2 cells according to previous methods [22-24].
Classic anti-cancer drug 5-fluorouracil (5-Fu) and sorafenib were served as the positive control,
respectively. As shown in Table 3, compounds 5a, 5b, 5e, 5h and 5k could obviously inhibit HepG2 cells
proliferation with IC50 values of 8.98, 8.81, 9.40, 6.76, and 5.44 μM, respectively. Notably, compounds 5h
and 5k were the most potent ones, and their IC50 values were even lower than that of positive control 5-Fu
7

(IC50 = 8.69 μM) and sorafenib (IC50 = 7.67 μM), indicating their potent anti-HCC activity in vitro.
Table 3. In vitro anti-HCC activity of the target compounds 5a-s
HepG2
HepG2
HepG2
IC50 (μM)
IC50 (μM)
IC50 (μM)
Compds
Compds
Compds
8.98
8.81
6.76
17.54
17.32
5.44
>20
11.79
>20
5a
5b
5c
5d
5e
5f
5h
5i
5o
5p
11.33
16.99
9.40
5j
5q
>20
5k
5l
5r
18.97
>20
>20
5s
>100
18.98
8.69
7.67
5m
5n
5-Fu
sorafenib
>20
5g
To further confirm the anti-HCC activity, the in vitro anti-proliferation activity of compound 5k against
two other human HCC cell lines (HUH7 and SK-Hep-1 cell lines) was subsequently evaluated by MTT
assay. As shown in Table 4, compound 5k could also significantly inhibit HUH7 and SK-Hep-1 cells
proliferation, with IC50 values of 6.42 and 6.75 μM, respectively. Notably, the IC50 values of 5k were lower
than that of positive control sorafenib (IC50 = 7.92 and 9.64 μM, respectively), confirming its potential
anti-HCC effect.
Table 4. In vitro anti-HCC activity of the target compounds 5k
in vitro inhibition of HCC proliferation (IC50, μM)
HUH7
6.42
SK-Hep-1
6.75
Compds
5k
7.92
9.64
sorafenib
3.2.2 Preliminary anti-HCC mechanism of the target compound 5k
To understand the preliminary mechanism of anti-HCC effect, we firstly determined the morphology
changes of HepG2 cells treated with 5k by Hoechst 33342 staining under fluorescent microscopy [22]. As
shown in Figure 3a, after treatment with 5k (5, 10 or 20 μM) for 48 h, the morphology of HepG2 cells was
significantly changed (ie. nucleus fragmentation and chromatin condensation), indicating the cell apoptosis.
8

Next, we performed the Annexin V/PI assay to further evaluate the apoptosis induction ability of 5k by
flow cytometry [25-27]. As shown in Figure 3b and 3d, after the incubation with 5k (5, 10 or 20 μM) for 48
h, apoptotic HepG2 cells at early (Annexin-V+/PI−) and late (Annexin-V+/PI+) stages were significantly
increased to 17.95%, 37.98% and 71.98%, respectively.
It is well-known that Bcl-2 family proteins, including pro-apoptotic proteins (ie. Bax and Bad) and
anti-apoptotic proteins (ie. Bcl-2 and Bcl-xl), play a significant role in HCC development. In addition, the
expression of Bcl-2 is a poor predictor for HCC prognosis [28, 29], and Bcl-2 family proteins are also
related to chronic active hepatitis and cirrhosis [30]. Therefore, we next determined whether 5k could affect
the expression of apoptosis-related proteins by western blot assay [31]. As shown in Figure 3c and 3e, 5k
could dose-dependently upregulate Bad and Bax expression and decrease Bcl-2 and Bcl-xl protein level.
These results indicated that the apoptosis induction capacity of 5k was achieved, at least partly, by
interfering with the expression of apoptosis-related proteins.
Figure 3. Apoptosis induction effect of 5k in HepG2 cells. (a) Alterations of morphology and nuclear changes (white
9

arrow marked cells). HepG2 cells were incubated with 5k (5, 10 or 20 μM) for 48 h, and then stained by Hoechst 33342 and
visualized by fluorescence microscopy; (b) HepG2 cells were incubated in the absence or presence of 5k (5, 10 or 20 μM) for
48 h, and then cells were collected and stained with Annexin V/PI to analyze early apoptotic (Annexin-V+/PI−) and late
apoptotic (Annexin-V+/PI−) cell fractions by flow cytometric analysis; (c) Effect of 5k on apoptosis-related proteins was
determined by western blotting assay. HepG2 cells were treated with 5k (5, 10 or 20 μM) for 48 h, and then harvested and
lysed to detect the expression of Bad, Bax, Bcl-2 and Bcl-xl; (d) The percentage of cell distribution; (e) Density ratios of Bad,
Bax, Bcl-2 and Bcl-xl to GADPH.
4. Conclusions
In summary, nineteen quinazolinone derivatives (5a-s) were obtained and their in vitro anti-HBV and
anti-HCC activities were subsequently evaluated. Among them, compounds 5j and 5k exhibited potent
anti-HBV activities in vitro with IC50 values of 0.63 and 0.58 μM, respectively. In addition, compounds 5j
and 5k could also obviously inhibit the HBV DNA replication in lamivudine and entecavir-resistant HBV
stains. Interestingly, besides the anti-HBV activities, compound 5k possessed significant anti-HCC effect
against HepG2, HUH7 and SK-Hep-1 cells, with IC50 values of 5.44, 6.42 and 6.75 μM, respectively.
Notably, the in vitro anti-HCC activities of 5k were more potent than that of positive control 5-fluorouracil
and sorafenib. Preliminary anti-HCC mechanism studies showed that compound 5k could induce HepG2
cells apoptosis by dose-dependently upregulating Bad and Bax expression and decreasing Bcl-2 and Bcl-xl
protein level. Considering the potent anti-HBV and anti-HCC effect, compound 5k might be a promising
lead to develop novel therapeutic agents towards HBV infection and HBV-induced HCC, and warranted
further research.
5. Experimental protocols
5.1. Chemical analysis
The synthesized products in the presnet work were purified by column chromatography using 200-300
mesh silica gel 60 or thin layer chromatography (TLC) using silica gel 60 F254 plates (250 mm; Qingdao
Ocean Chemical Company, China). Melting points of the target compounds were determined by using a
model YRT-3 apparatus and are uncorrected. Subsequently, the structure of the target compounds were
10

analyzed by 1H NMR and 13C NMR (JEOL, 400 MHz), IR (Shimadzu, FTIR-8400S) and MS (Agilent 6460
and Agilent 6530 QTOF spectrometer) routinely. The purity of target compounds was determined by
high-performance liquid chromatography (HPLC, see the Supplementary Material). Individual compounds
with a purity of >95% were used for subsequent experiments. All solvents were reagent grade and, when
necessary, were purified and dried by standards methods.
5.1.1. General synthetic procedure of target compounds 5a-s
CS2 (2.3 mL, 30 mmol) was added dropwise to the mixture of methylamine derivative (30 mmol) and
triethylamine (12 mL) in THF (25 mL) at 0 °C within 30 min. After stirring for 1 h at room temperature, the
mixture was then cooled down to 0 °C and the toluene sulfonyl chloride (6 g, 31.5 mmol) were added in.
After stirring for another 1 h at room temperature, the mixture was diluted with 5% HCl (100 mL) and
petroleum ether, sequentially washed with water, saturated NaHCO3 solution and brine, dried with
anhydrous Na2SO4 and evaporated in vacuo to obtain compound 1. HCl (10.5 mL) was added to the
mixture of benzene or benzene derivatives (6.65 mmol) and paraformaldehyde (11.97 mmol), and then
stirred for 10 h at 60 °C. After cooling down to room temperature, the mixture was then dispersed with
water and ethyl acetate, and sequentially washed with water, 5% HCl, saturated NaHCO3 solution and brine,
dried with anhydrous Na2SO4. The solvent was then removed by evaporation to give compound 2. Methyl
2-amino-4-hydroxybenzoate (0.01 mol) and isothiocyanate derivatives (0.01 mol) was heated in the
presence of triethylamine and ethanol at reflux temperature for 4 h. After cooling down to room
temperature, the mixture was filtered to give intermediate 3, which was subsequently reacted with
intermediate 2 (0.01 mmol) in the presence of K2CO3 and 1,4-dioxane under reflux for 10 h. Then, the
mixture was cooled down to room temperature, dissolved with ethyl acetate, sequentially washed with
water, 5% HCl, saturated NaHCO3 solution and brine, dried with anhydrous Na2SO4. Removal of the
solvent gave a residue, which was then recrystallized in ethanol to obtain intermediate 4. Finally, the target
compounds 5a-s were prepared by alkylating intermediate 4 (5 mmol) with 2-dimethylaminoethyl chloride
hydrochloride, 2-diethylaminoethyl chloride hydrochloride or 3-dimethylaminopropyl chloride
hydrochloride (6 mmol) in the presence of 1, 4-dioxane and K2CO3, respectively. Spectra of the target
compounds were shown in the Supplementary Material.
11

5.1.1.1.
quinazolin-4(3H)-one (5a)
The title compound
2-((5-acetyl-2-methoxybenzyl)thio)-6-(3-(dimethylamino)propoxy)-3-(thiophen-2-ylmethyl)
was
obtained
starting
from
thiazol-2-ylmethanamine,
1-(4-methoxyphenyl)ethan-1-one, methyl 2-amino-4-hydroxybenzoate and 3-dimethylaminopropyl
1
chloride hydrochloride. As a yellow powder, yield: 10%; m.p.97.1-100.1 °C. Analytical data for 5a: H
NMR (400 MHz, CDCl3, δ ppm): 8.25 (d, J = 2.4 Hz, 1H, Ar-H), 7.90 (dd, J = 8.4 Hz, 2.4 Hz, 1H, Ar-H),
7.58-7.61 (m, 2H, Ar-H), 7.32 (dd, J = 8.8 Hz, 2.8 Hz, 1H, Ar-H), 7.21 (d, J = 3.2 Hz, 1H, Ar-H), 7.20 (dd,
J = 5.2 Hz, 1.2 Hz, 1H, Ar-H), 6.89-6.93 (m, 2H, Ar-H), 5.42 (s, 2H, CH2), 4.55 (s, 2H, CH2), 4.09 (t, J =
7.2 Hz, 2H, OCH2), 3.96 (s, 3H, OCH3), 2.48-2.52 (m, 5H, CH2, CH3), 2.29 (s, 6H, N(CH3)2), 1.98-2.02 (m,
2H, CH2); 13C NMR (100 MHz, CDCl3, δ ppm): 196.74, 161.64, 161.57, 156.98, 153.11, 142.28, 137.37,
132.09, 130.30, 129.88, 128.74, 127.69, 126.42, 126.22, 125.30, 125.15, 120.09, 110.12, 107.27, 66.68,
56.33, 56.00, 45.34(2C), 42.49, 31.35, 27.19, 26.47; IR (KBr, cm-1): v 2945.62, 1674.42, 1600.92, 1577.77,
1550.77, 1492.90, 1473.62, 1269.16, 1238.30, 1222.87, 1122.57, 1022.27, 946.88, 831.07; ESI-MS: m/z [M
+ H]+ 538.2; ESI-HRMS (TOF): m/z [M + H]+ calcd for C28H32N3O4S2, 538.1834, found 538.1844. Purity:
95.4%.
5.1.1.2.
6-(3-(dimethylamino)propoxy)-2-((2-methoxybenzyl)thio)-3-(thiophen-2-ylmethyl)quinazolin-4(3H)-one
(5b)
The title compound was obtained starting from thiazol-2-ylmethanamine, anisole, methyl
2-amino-4-hydroxybenzoate and 3-dimethylaminopropyl chloride hydrochloride. As a yellow oil, yield:
27%. Analytical data for 5b: 1H NMR (400 MHz, CDCl3, δ ppm): 7.60 (d, J = 2.8 Hz, 1H, Ar-H), 7.56 (d, J
= 8.8 Hz, 1.6 Hz, 1H, Ar-H), 7.52 (dd, J = 8.0 Hz, 1.6 Hz, 1H, Ar-H), 7.19-7.31 (m, 4H, Ar-H), 6.87-6.92
(m, 3H, Ar-H), 5.44 (s, 2H, CH2), 4.58 (s, 2H, CH2), 4.11 (t, J = 6.4 Hz, 2H, OCH2), 3.89 (s, 3H, CH3),
2.56 (t, J = 6.4 Hz, 2H, NCH2), 2.34 (s, 6H, N(CH3)2), 2.03-2.06 (m, 2H, OCH2CH2); 13C NMR (100 MHz,
CDCl3, δ ppm): 161.69, 157.81, 156.81, 153.70, 142.43, 137.45, 131.19, 129.23, 128.76, 127.78, 126.40,
126.20, 124.99, 124.76, 120.52, 120.05, 110.65, 107.22, 66.57, 56.28, 55.63, 45.17(2C), 42.50, 31.73,
27.02; IR (KBr, cm-1): v 2943.37, 1670.31, 1549.24, 1489.74, 1467.76, 1354.03, 1246.02, 1145.72, 1107.14;
12

ESI-MS: m/z [M + H]+ 496.3; ESI-HRMS (TOF): m/z [M + H]+ calcd for C26H30N3O3S2, 496.1729, found
496.1714. Purity: 95.4%.
5.1.1.3.
2-((5-acetyl-2-methoxybenzyl)thio)-6-(2-(dimethylamino)ethoxy)-3-(furan-2-ylmethyl)quinazolin-4(3H)-one
(5c)
The
title
compound
was
obtained
starting
from
furan-2-ylmethanamine,
1-(4-methoxyphenyl)ethan-1-one, methyl 2-amino-4-hydroxybenzoate and 2-dimethylaminoethyl chloride
hydrochloride. As a brown powder, yield: 20%; m.p.169.4-172.9 °C. Analytical data for 5c: 1H NMR (400
MHz, CDCl3, δ ppm): 8.26 (d, J = 2.4 Hz, 1H, Ar-H), 7.92 (dd, J = 8.8 Hz, 2.4 Hz, 1H, Ar-H), 7.64 (d, J =
9.2 Hz, 1H, Ar-H), 7.60 (d, J = 2.8 Hz, 1H, Ar-H), 7.39 (dd, J = 8.8 Hz, 2.8 Hz, 1H, Ar-H), 7.34 (dd, J =
1.6 Hz, 0.8 Hz,, 1H, Ar-H), 6.94 (d, J = 8.8 Hz, 1H, Ar-H), 6.38 (dd, J = 3.2 Hz, 0.8 Hz, 1H, Ar-H), 6.30
(dd, J = 3.2 Hz, 2.0 Hz, 1H, Ar-H), 5.32 (s, 2H, CH2), 4.56 (s, 2H, CH2), 4.19 (t, J = 5.6 Hz, 2H, OCH2),
3.96 (s, 3H, CH3), 2.84 (t, J = 5.6 Hz, 2H, NCH2), 2.51 (s, 6H, CH3), 2.40 (s, 6H, N(CH3)2); 13C NMR (100
MHz, CDCl3, δ ppm): 196.73, 161.66, 161.56, 156.74, 153.69, 148.95, 142.44, 132.05, 130.26, 129.91,
127.73, 125.41, 125.35, 119.99, 110.51, 110.13, 109.71, 107.24, 66.07, 58.03, 55.98, 45.66 (2C), 40.65,
31.35, 26.45; IR (KBr, cm-1): v 3330.36, 2924.07, 1673.39, 1600.92, 1577.77, 1550.77, 1427.32, 1357.89,
1270.17, 1226.73, 1157.29, 1076.28, 1014.56, 824.89; ESI-MS: m/z 508.4 [M + H]+; ESI-HRMS (TOF):
m/z [M + H]+ calcd for C27H30N3O5S, 508.1906, found 508.1903. Purity: 96.3%.
5.1.2.4.
2-((5-acetyl-2-methoxybenzyl)thio)-6-(2-(diethylamino)ethoxy)-3-(furan-2-ylmethyl)quinazolin-4(3H)-one
(5d)
The
title
compound
was
obtained
starting
from
furan-2-ylmethanamine,
1-(4-methoxyphenyl)ethan-1-one, methyl 2-amino-4-hydroxybenzoate and 2-diethylaminoethyl chloride
1
hydrochloride. As a yellow oil, yield: 19%. Analytical data for 5d: H NMR (400 MHz, CDCl3, δ ppm):
8.26 (d, J = 2.4 Hz, 1H, Ar-H), 7.92 (dd, J = 8.4 Hz, 2.4 Hz, 1H, Ar-H), 7.63 (d, J = 8.8 Hz, 1H, Ar-H),
7.60 (d, J = 3.2 Hz, 1H, Ar-H), 7.33-7.36 (m, 2H, Ar-H), 6.93 (d, J = 8.8 Hz, 1H, Ar-H), 6.39 (d, J = 3.2 Hz,
1H, Ar-H), 6.30 (dd, J = 3.2 Hz, 1.6 Hz, 1H, Ar-H), 5.31 (s, 2H, CH2), 4.56 (s, 2H, CH2), 4.14 (t, J = 6.0
Hz, 2H, OCH2), 3.96 (s, 3H, CH3), 2.93 (t, J = 6.0 Hz, 2H, NCH2), 2.63-2.69 (m, 4H, N(CH2CH3)2), 2.51 (s,
13

3H, CH3), 1.09 (t, J = 6.8 Hz, 6H, N(CH2CH3)2); 13C NMR (100 MHz, CDCl3, δ ppm): 196.66, 161.63,
161.53, 156.95, 153,52, 148.96, 142.40, 142.24, 132.02, 130.24, 129.85, 127.64, 125.35, 125.29, 120.00,
110.50, 110.10, 109.67, 107.28, 66.98, 55.95, 51.56, 47.88 (2C), 40.61, 31.31, 26.42, 11.91 (2C); IR (KBr,
cm-1): v 2931.80, 1670.31, 1600.92, 1546.91, 1488.71, 1354.03, 1259.91, 1149.11, 1014.56, 821.68;
ESI-MS: m/z [M + H]+ 536.5; ESI-HRMS (TOF): m/z [M + H]+ calcd for C29H34N3O5S, 536.2219, found
536.2215. Purity: 95.1%.
5.1.1.5.
2-((5-acetyl-2-methoxybenzyl)thio)-6-(3-(dimethylamino)propoxy)-3-(furan-2-ylmethyl)quinazolin-4(3H)-o
ne (5e)
The
title
compound
was
obtained
starting
from
furan-2-ylmethanamine,
1-(4-methoxyphenyl)ethan-1-one, methyl 2-amino-4-hydroxybenzoate and 3-dimethylaminopropyl
1
chloride hydrochloride. As a yellow powder, yield: 10%; m.p.96.1-97.2 °C. Analytical data for 5e: H
NMR (400 MHz, DMSO, δ ppm): 8.21 (d, J = 2.4 Hz, 1H, Ar-H), 7.93 (dd, J = 8.8 Hz, 2.4 Hz, 1H, Ar-H),
7.66 (d, J = 9.6 Hz, 1H, Ar-H), 7.57 (d, J = 0.8 Hz, 1H, Ar-H), 7.43-7.45 (m, 2H, Ar-H), 7.14 (d, J = 8.8 Hz,
1H, Ar-H), 6.40 (dd, J = 3.2 Hz, 1.6 Hz, 1H, Ar-H), 6.36 (d, J = 3.2 Hz, 1H, Ar-H), 5.24 (s, 2H, CH2), 4.51
(s, 2H, CH2), 4.10 (t, J = 2.4 Hz, 2H, OCH2), 3.93 (s, 3H, OCH3), 2.49 (s, 3H, CH3), 2.38 (t, J = 3.2 Hz, 2H,
NCH2), 2.16 (s, 6H, N(CH3)2), 1.87-1.90 (m, 2H, CH2); 13C NMR (100 MHz, DMSO, δ ppm): 196.70,
161.69, 160.77, 157.14, 153.96, 149.19, 143.21, 141.85, 131.84, 130.87, 129.81, 128.20, 125.23, 124.98,
119.93, 111.31, 111.21, 109.50, 107.55, 69.92, 56.71, 56.07, 45.67 (2C), 40.44, 31.25, 27.22, 26.92; IR
(KBr, cm-1): v 3362.17, 2723.49, 1600.92, 1554.63, 1485.63, 1337.89, 1267.09, 1153.43, 1018.41, 825.53,
786.96; ESI-MS: m/z 522.4 [M + H]+ ; ESI-HRMS (TOF): m/z [M + H]+ calcd for C28H32N3O5S, 522.2063,
found 522.2058. Purity: 95.3%.
5.1.1.6. 6-(2-(dimethylamino)ethoxy)-3-(furan-2-ylmethyl)-2-((2-methoxybenzyl)thio)quinazolin-4(3H)-one
(5f)
The title compound was obtained starting from furan-2-ylmethanamine, anisole, methyl
2-amino-4-hydroxybenzoate and 2-dimethylaminoethyl chloride hydrochloride. As a grey powder, yield:
26%; m.p.97.1-98.5 °C. Analytical data for 5f: 1H NMR (400 MHz, CDCl3, δ ppm): 7.60 (d, J = 2.8 Hz, 1H,
Ar-H), 7.56 (d, J = 9.2 Hz, 1H, Ar-H), 7.51 (dd, J = 7.6 Hz, 1.6 Hz, 1H, Ar-H), 7.37 (dd, J = 8.8 Hz, 2.8 Hz,
14

1H, Ar-H), 7.32 (dd, J = 2.0 Hz, 0.8 Hz, 1H, Ar-H), 7.22-7.26 (m, 1H, Ar-H), 6.86-6.89 (m, 2H, Ar-H),
6.38 (d, J = 3.2 Hz, 1H, Ar-H), 6.28 (dd, J = 3.2 Hz, 1.6 Hz, 1H, Ar-H), 5.31 (s, 2H, CH2), 4.56 (s, 2H,
CH2), 4.15 (t, J = 5.2 Hz, 2H, OCH2), 3.86 (s, 3H, OCH3), 2.76 (t, J = 5.2 Hz, 2H, NCH2), 2.34 (s, 6H,
N(CH3)2); 13C NMR (100 MHz, CDCl3, δ ppm): 161.76, 157.79, 156.76, 154.13, 149.05, 142.49, 142.41,
131.16, 129.18, 127.76, 125.40, 124.87, 120.51, 119.92, 111.64, 110.48, 109.70, 107.06, 66.25, 58.24,
55.62, 45.86 (2C), 40.65, 31.68; IR (KBr, cm-1): v 2927.94, 2828.65, 1678.07, 1546.91, 1502.05, 1457.93,
1244.52, 1107.14, 1029.99, 752.24; ESI-MS: m/z 466.3 [M+H]+; ESI-HRMS (TOF): m/z [M + H]+ calcd
for C25H28N3O4S, 466.1801, found 466.1796. Purity: 95.4%.
5.1.2.7.
6-(2-(diethylamino)ethoxy)-3-(furan-2-ylmethyl)-2-((2-methoxybenzyl)thio)quinazolin-4(3H)-one (5g)
The title compound was obtained starting from furan-2-ylmethanamine, anisole, methyl
2-amino-4-hydroxybenzoate and 2-diethylaminoethyl chloride hydrochloride. As a yellow oil, yield: 21%.
Analytical data for 5g: 1H NMR (400 MHz, CDCl3, δ ppm): 7.60 (d, J = 3.2 Hz, 1H, Ar-H), 7.57 (d, J = 8.8
Hz, 1H, Ar-H), 7.52 (dd, J = 8.0 Hz, 2.0 Hz, 1H, Ar-H), 7.31-7.34 (m, 2H, Ar-H), 7.24-7.29 (m, 1H, Ar-H),
6.87-6.91 (m, 2H, Ar-H), 6.39 (dd, J = 3.2 Hz, 0.8 Hz, 1H, Ar-H), 6.29 (dd, J = 3.2 Hz, 2.0 Hz, 1H, Ar-H),
5.33 (s, 2H, CH2), 4.57 (s, 2H, CH2), 4.16 (t, J = 6.0 Hz, 2H, OCH2), 3.88 (s, 3H, CH3), 2.95 (t, J = 5.6 Hz,
2H, NCH2), 2.65-2.71 (m, 4H, N(CH2CH3)2), 1.10 (t, J = 7.2 Hz, 6H, N(CH2CH3)2); 13C NMR (100 MHz,
CDCl3, δ ppm): 161.75, 157.79, 156.83, 154.06, 149.07, 142.40(2C), 131.16, 129.18, 127.72, 125.22,
124.87, 120.50, 119.97, 110.64, 110.48, 109.69, 107.22, 66.88, 55.61, 51.56, 47.88 (2C), 40.65, 31.68,
11.89 (2C); IR (KBr, cm-1): v 2966.52, 2935.66, 1678.07, 1549.24, 1491.79, 1465.90, 1438.90, 1346.31,
1246.02, 1157.29, 1107.14, 1029.99, 752.24; ESI-MS: m/z 494.3 [M + H]+; ESI-HRMS (TOF): m/z [M +
H]+ calcd for C27H32N3O4S, 494.2114, found 494.2109. Purity: 99.0%.
5.1.1.8.
6-(3-(dimethylamino)propoxy)-3-(furan-2-ylmethyl)-2-((2-methoxybenzyl)thio)quinazolin-4(3H)-one (5h)
The title compound was obtained starting from furan-2-ylmethanamine, anisole, methyl
2-amino-4-hydroxybenzoate and 3-dimethylaminopropyl chloride hydrochloride. As a yellow oil, yield:
54%. Analytical data for 5h: 1H NMR (400 MHz, CDCl3, δ ppm): 7.58 (d, J = 2.8 Hz, 1H, Ar-H), 7.56 (d, J
= 9.2 Hz, 1H, Ar-H), 7.50 (dd, J = 6.8 Hz, 1.6 Hz, 1H, Ar-H), 7.23-7.31 (m, 3H, Ar-H), 6.87 (m, 2H, Ar-H),
15

6.37 (d, J = 3.2 Hz, 1H, Ar-H), 6.28 (dd, J = 2.8 Hz, 0.8 Hz, 1H, Ar-H), 5.31 (s, 2H, CH2), 4.56 (s, 2H,
CH2), 4.10 (t, J = 6.4 Hz, 2H, OCH2), 3.87 (s, 3H, OCH3), 2.63 (t, J = 7.2 Hz, 2H, NCH2), 2.37 (s, 6H,
13
N(CH3)2), 2.05-2.09 (m, 2H, CH2); C NMR (100 MHz, CDCl3, δ ppm): 160.65, 157.75, 156.91, 153.99,
149.08, 142.341, 142.31, 131.13, 129.17, 127.72, 124.97, 124.78, 120.49, 119.96, 110.63, 110.51, 109.64,
107.28, 66.76, 56.36, 55.55, 45.48(2C), 40.63, 31.68, 27.38; IR (KBr, cm-1): v 2939.52, 1678.07, 1546.16,
1489.74, 1467.16, 1432.28, 1346.31, 1246.02, 1157.29, 1107.14, 1029.99, 833.25, 752.24; ESI-MS: m/z
480.3 [M + H]+; ESI-HRMS (TOF): m/z [M + H]+ calcd for C26H30N3O4S, 480.1957, found 480.1952.
Purity: 99.3%.
5.1.1.9. 2-(benzylthio)-6-(2-(dimethylamino)ethoxy)-3-(furan-2-ylmethyl)quinazolin-4(3H)-one (5i)
The title compound was obtained starting from furan-2-ylmethanamine, (chloromethyl)benzene, methyl
2-amino-4-hydroxybenzoate and 2-dimethylaminoethyl chloride hydrochloride. As a yellow powder, yield:
30%; m.p.91.2-92.8 °C. Analytical data for 5i: 1H NMR (400 MHz, CDCl3, δ ppm): 7.60 (d, J = 2.8 Hz, 1H,
Ar-H), 7.52 (d, J = 8.8 Hz, 1H, Ar-H), 7.45 (d, J = 6.8 Hz, 2H, Ar-H), 7.24-7.36 (m, 5H, Ar-H), 6.38 (d, J =
2.8 Hz, 1H, Ar-H), 6.29 (dd, J = 3.2 Hz, 2.0 Hz, 1H, Ar-H), 5.32 (s, 2H, CH2), 4.52 (s, 2H, CH2), 4.14 (t, J
= 5.6 Hz, 2H, OCH2), 2.76 (t, J = 5.6 Hz, 2H, NCH2), 2.34 (s, 6H, N(CH3)2); 13C NMR (100 MHz, CDCl3,
δ ppm): 161.65, 156.93, 153.33, 148.91, 142.49, 142.35, 136.52, 129.48 (2C), 128.69 (2C), 127.76, 127.68,
125.45, 120.03, 110.54, 109.79, 107.07, 66.36, 58.27, 45.92 (2C), 40.71, 36.90; IR (KBr, cm-1): v 2939.52,
2768.12, 1676.47, 1553.35, 1492.81, 1350.17, 1226.73, 1225.03, 1157.29, 1076.26, 1028.04, 833.25,
786.96, 702.09; ESI-MS: m/z 436.3 [M + H]+; ESI-HRMS (TOF): m/z [M + H]+ calcd for C24H26N3O3S,
436.1695, found 436.1689. Purity: 98.8%.
5.1.1.10. 2-(benzylthio)-6-(2-(diethylamino)ethoxy)-3-(furan-2-ylmethyl)quinazolin-4(3H)-one (5j)
The title compound was obtained starting from furan-2-ylmethanamine, (chloromethyl)benzene, methyl
2-amino-4-hydroxybenzoate and 2-diethylaminoethyl chloride hydrochloride. As a white powder, yield:
1
20%; m.p.92.7-94.3 °C. Analytical data for 5j: H NMR (400 MHz, DMSO, δ ppm): 7.59 (d, J = 8.8 Hz,
2H, Ar-H), 7.46-7.50 (m, 3H, Ar-H), 7.42 (dd, J = 8.8 Hz, 3.2 Hz, 1H, Ar-H), 7.30-7.34 (m, 2H, Ar-H),
7.27 (d, J = 7.2 Hz, 1H, Ar-H), 6.41 (dd, J = 3.2 Hz, 2.0 Hz, 1H, Ar-H), 6.37 (d, J = 2.8 Hz, 1H, Ar-H),
5.27 (s, 2H, CH2), 4.52 (s, 2H, CH2), 4.11 (t, J = 6.0 Hz, 2H, OCH2), 2.80 (t, J = 6.0 Hz, 2H, NCH2),
2.52-2.58 (m, 4H, N(CH2CH3)2), 0.97 (t, J = 7.2 Hz, 6H, N(CH2CH3)2); 13C NMR (100 MHz, CDCl3, δ
16

ppm): 161.63, 157.01, 153.26, 148.94, 142.48, 142.25, 136.52, 129.48 (2C), 128.68 (2C), 127.72, 127.68,
125.27, 120.08, 110.55, 109.79, 107.23, 67.04, 51.61, 47.92 (2C), 40.70, 36.90, 12.00 (2C); IR (KBr, cm-1):
v 2966.52, 2927.15, 1680.57, 1550.27, 1486.66, 1350.17, 1226.73, 1157.29, 1072.42, 827.97, 700.75;
ESI-MS: m/z 464.3 [M + H]+; ESI-HRMS (TOF): m/z [M + H]+ calcd for C26H30N3O3S, 464.2008, found
464.2001. Purity: 98.0%.
5.1.1.11. 2-(benzylthio)-6-(3-(dimethylamino)propoxy)-3-(furan-2-ylmethyl)quinazolin-4(3H)-one (5k)
The title compound was obtained starting from furan-2-ylmethanamine, (chloromethyl)benzene, methyl
2-amino-4-hydroxybenzoate and 3-dimethylaminopropyl chloride hydrochloride. As a yellow powder,
yield: 30%; m.p.89.2-91.3 °C. Analytical data for 5k: 1H NMR (400 MHz, CDCl3, δ ppm): 7.59 (d, J = 2.8
Hz, 1H, Ar-H), 7.52 (d, J = 8.8 Hz, 1H, Ar-H), 7.46 (d, J = 8.4 Hz, 1H, Ar-H), 7.25-7.33 (m, 5H, Ar-H),
6.38 (d, J = 3.2 Hz, 1H, Ar-H), 6.29 (dd, J = 3.2 Hz, 2.0 Hz, 1H, Ar-H), 5.32 (s, 2H, CH2), 4.52 (s, 2H,
CH2), 4.09 (t, J = 6.4 Hz, 2H, OCH2), 2.46 (t, J = 6.8 Hz, 2H, NCH2), 2.60 (s, 6H, N(CH3)2), 1.97-2.00 (m,
2H, OCH2CH2); 13C NMR (100 MHz, CDCl3, δ ppm): 161.65, 157.05, 153.23, 148.93, 142.48, 142.23,
136.53, 129.48 (2C), 128.69 (2C), 127.73, 127.68, 125.07, 120.10, 110.53, 109.76, 107.35, 66.77, 56.36,
45.47 (2C), 40.70, 36.90, 27.36; IR (KBr, cm-1): v 2943.37, 2761.96, 1677.49, 1547.19, 1489.74, 1350.17,
1226.73, 1157.29, 1103.28, 833.25, 702.09; ESI-MS: m/z 450.3 [M + H]+; ESI-HRMS (TOF): m/z [M +
H]+ calcd for C25H28N3O3S, 450.1851, found 450.1846. Purity: 99.3%.
5.1.1.12. 2-((5-acetyl-2-methoxybenzyl)thio)-3-benzyl-6-(2-(dimethylamino)ethoxy)quinazolin-4(3H)-one
(5l)
The title compound was obtained starting from phenylmethanamine, 1-(4-methoxyphenyl)ethan-1-one,
methyl 2-amino-4-hydroxybenzoate and 2-dimethylaminoethyl chloride hydrochloride. As a yellow oil,
yield: 7%. Analytical data for 5l: 1H NMR (400 MHz, CDCl3, δ ppm): 8.23 (d, J = 2.4 Hz, 1H, Ar-H), 7.89
(dd, J = 8.4 Hz, 2.0 Hz, 1H, Ar-H), 7.66 (d, J = 8.8 Hz, 1H, Ar-H), 7.60 (d, J = 2.8 Hz, 1H, Ar-H),
7.21-7.53 (m, 6H, Ar-H), 6.89 (d, J = 8.8 Hz, 1H, Ar-H), 5.33 (s, 2H, CH2), 4.51 (s, 2H, CH2), 4.16 (t, J =
5.6 Hz, 2H, OCH2), 3.90 (s, 3H, OCH3), 2.79 (t, J = 5.6 Hz, 2H, NCH2), 2.49 (s, 3H, CH3), 2.36 (s, 6H,
N(CH3)2); 13C NMR (100 MHz, CDCl3, δ ppm): 196.78, 162.05, 161.52, 156.83, 154.11, 142.47, 135.68,
132.02, 130.25, 129.79, 128.60 (2C), 127.74, 127.68, 127.60 (2C), 125.47, 125.28, 119.94, 110.12, 107.15,
66.23, 58.15, 55.94, 47.42, 45.81(2C), 31.30, 26.47; IR (KBr, cm-1): v 2924.07, 1677.49, 1600.54, 1550.27,
17

1489.74, 1353.28, 1260.94, 1168.60, 1024.96, 827.97, 714.09; ESI-MS: m/z 518.3 [M+H]+; ESI-HRMS
(TOF): m/z [M + H]+ calcd for C29H32N3O4S, 518.2114, found 518.2102. Purity: 95.0%.
5.1.1.13.
2-((5-acetyl-2-methoxybenzyl)thio)-3-benzyl-6-(2-(diethylamino)ethoxy)quinazolin-4(3H)-one
(5m)
The title compound was obtained starting from phenylmethanamine, 1-(4-methoxyphenyl)ethan-1-one,
methyl 2-amino-4-hydroxybenzoate and 2-diethylaminoethyl chloride hydrochloride. As a yellow powder,
1
yield: 21%; m.p.116.9-118.7 °C. H NMR (400 MHz, CDCl3, δ ppm): 7.91 (dd, J = 8.8 Hz, 2.4 Hz, 1H,
Ar-H), 7.66 (d, J = 9.2 Hz, 1H, Ar-H), 7.60 (d, J = 3.2 Hz, 1H, Ar-H), 7.24-7.38 (m, 7H, Ar-H), 6.91 (d, J =
8.8 Hz, 1H, Ar-H), 5.34 (s, 2H, CH2), 4.51 (s, 2H, CH2), 4.15 (t, J = 6.0 Hz, 2H, OCH2), 3.92 (s, 3H,
OCH3), 2.94 (t, J = 6.0 Hz, 2H, NCH2), 2.65-2.70 (m, 4H, N(CH2CH3)2), 2.51 (s, 3H, CH3), 1.09 (t, J = 7.2
Hz, 6H, N(CH2CH3)2); 13C NMR (100 MHz, CDCl3, δ ppm): 196.78, 162.08, 161.53, 156.97, 154.02,
142.38, 135.71, 132.06, 130.23, 129.83, 128.60 (2C), 127.67(2C), 127.64 (2C), 125.37, 125.35, 120.01,
110.11, 107.26, 66.94, 55.94, 51.54, 47.87 (2C), 47.43, 31.30, 26.48, 11.90(2C); IR (KBr, cm-1): v 2965.11,
1673.39, 1600.92, 1546.91, 1489.05, 1357.89, 1262.99, 1228.11, 1170.65, 1026.13, 829.39; ESI-MS: m/z
546.2 [M + H]+; ESI-HRMS (TOF): m/z [M + H]+ calcd for C31H36N3O4S, 546.2427, found 546.2421.
Purity: 95.5%.
5.1.1.14. 3-benzyl-6-(2-(dimethylamino)ethoxy)-2-((2-methoxybenzyl)thio)quinazolin-4(3H)-one (5n)
The title compound was obtained starting from phenylmethanamine, anisole, methyl
2-amino-4-hydroxybenzoate and 2-dimethylaminoethyl chloride hydrochloride. As a yellow oil, yield: 43%.
Analytical data for 5n: 1H NMR (400 MHz, CDCl3, δ ppm): 7.57-7.62 (m, 2H, Ar-H), 7.49 (dd, J = 7.6 Hz,
2.0 Hz, 1H, Ar-H), 7.22-7.39 (m, 9H, Ar-H), 5.35 (s, 2H, CH2), 4.53 (s, 2H, CH2), 4.17 (t, J = 4.8 Hz, 2H,
13
OCH2), 3.84 (s, 3H, OCH3), 2.80 (t, J = 4.2 Hz, 2H, NCH2), 2.38 (s, 6H, N(CH3)2); C NMR (100 MHz,
CDCl3, δ ppm): 162.13, 157.75, 156.76, 154.64, 142.61, 135.78, 131.13, 129.15(2C), 128.57(2C), 127.79,
127.70, 127.63, 125.41, 124.87, 120.48, 119.93, 110.64, 107.04, 66.24, 58.23, 55.59, 47.49, 45.84(2C),
31.65; IR (KBr, cm-1): v 2935.66, 1674.21, 1546.91, 1489.74, 1354.03, 1244.52, 1165.00, 1107.14, 1029.99,
834.13, 752.24; ESI-MS: m/z 476.2 [M + H]+; ESI-HRMS (TOF): m/z [M + H]+ calcd for C27H30N3O3S,
476.2008, found 476.1998. Purity: 96.3%.
5.1.1.15. 3-benzyl-6-(2-(diethylamino)ethoxy)-2-((2-methoxybenzyl)thio)quinazolin-4(3H)-one (5o)
18

The title compound was obtained starting from phenylmethanamine, anisole, methyl
2-amino-4-hydroxybenzoate and 2-diethylaminoethyl chloride hydrochloride. As a yellow oil, yield: 47%.
Analytical data for 5o: 1H NMR (400 MHz, CDCl3, δ ppm): 7.60 (d, J = 2.8 Hz, 1H, Ar-H), 7.59 (d, J = 8.8
Hz, 1H, Ar-H), 7.49 (dd, J = 7.6 Hz, 1.6 Hz, 1H, Ar-H), 7.23-7.36 (m, 7H, Ar-H), 6.86-6.90 (m, 2H, Ar-H),
5.35 (s, 2H, CH2), 4.53 (s, 2H, CH2), 4.16 (t, J = 6.0 Hz, 2H, OCH2), 3.85 (s, 3H, OCH3), 2.96 (t, J = 5.2
Hz, 2H, NCH2), 2.66-2.71 (m, 4H, N(CH2CH3)2), 1.10 (t, J = 7.2 Hz, 6H, N(CH2CH3)2); 13C NMR (100
MHz, CDCl3, δ ppm): 162.14, 157.75, 156.85, 154.56, 142.52, 135.80, 131.13, 129.16, 128.57(2C), 127.76,
127.71(2C), 127.63, 125.24, 124.85, 120.49, 119.97, 110.65, 107.20, 66.87, 55.59, 51.56, 45.84 (2C), 47.49,
31.66, 11.87(2C); IR (KBr, cm-1): v 2933.31, 2831.50, 1674.21, 1546.91, 1489.74, 1462.04, 1357.89,
1244.52, 1165.00, 1107.14, 1029.99, 752.24; ESI-MS: m/z 504.2 [M + H]+; ESI-HRMS (TOF): m/z [M +
H]+ calcd for C29H34N3O3S, 504.2321, found 504.2309. Purity: 95.1%.
5.1.1.16. 3-benzyl-6-(3-(dimethylamino)propoxy)-2-((2-methoxybenzyl)thio)quinazolin-4(3H)-one (5p)
The title compound was obtained starting from phenylmethanamine, anisole, methyl
2-amino-4-hydroxybenzoate and 3-dimethylaminopropyl chloride hydrochloride. As a yellow oil, yield:
1
50%. Analytical data for 5p: H NMR (400 MHz, CDCl3, δ ppm): 7.57-7.60 (m, 2H, Ar-H), 7.49 (dd, J =
7.6 Hz, 1.6 Hz, 1H, Ar-H), 7.23-7.34 (m, 7H, Ar-H), 6.96-6.89 (m, 2H, Ar-H), 5.35 (s, 2H, CH2), 4.53 (s,
2H, CH2), 4.11 (t, J = 6.0 Hz, 2H, OCH2), 3.85 (s, 3H, OCH3), 2.55 (t, J = 6.8 Hz, 2H, NCH2), 2.33 (s, 6H,
N(CH3)2), 2.02-2.06 (m, 2H, OCH2CH2); 13C NMR (100 MHz, CDCl3, δ ppm): 162.11, 157.75, 156.91,
154.52, 142.47, 135.82, 131.13, 129.14, 128.56(2C), 127.72(3C), 127.62, 125.04, 124.89, 120.47, 120.01,
110.64, 107.30, 66.75, 56.38, 55.59, 47.46, 45.46(2C), 31.64, 27.35; IR (KBr, cm-1): v 2935.66, 2837.89,
1674.21, 1546.91, 1495.89, 1467.16, 1357.89, 1244.52, 1168.86, 1107.14, 1029.99, 752.24; ESI-MS: m/z
490.3 [M + H]+; ESI-HRMS (TOF): m/z [M + H]+ calcd for C28H32N3O3S, 490.2164, found 490.2156.
Purity: 95.9%.
5.1.1.17. 3-benzyl-2-(benzylthio)-6-(2-(dimethylamino)ethoxy)quinazolin-4(3H)-one (5q)
The title compound was obtained starting from phenylmethanamine, (chloromethyl)benzene, methyl
2-amino-4-hydroxybenzoate and 3-dimethylaminopropyl chloride hydrochloride. As a white powder, yield:
16%; m.p.102.4-105.7 °C. Analytical data for 5q: 1H NMR (400 MHz, CDCl3, δ ppm): 7.61 (d, J = 2.8 Hz,
1H, Ar-H), 7.56 (d, J = 8.8 Hz, 1H, Ar-H), 7.42 (d, J = 8.0 Hz, 1H, Ar-H), 7.38 (dd, J = 9.2 Hz, 3.2 Hz, 1H,
19

Ar-H), 7.24-7.33 (m, 8H, Ar-H), 5.36 (s, 2H, CH2), 4.49 (s, 2H, CH2), 4.20 (t, J = 5.2 Hz, 2H, OCH2), 2.84
(t, J = 5.2 Hz, 2H, NCH2), 2.41 (s, 6H, N(CH3)2); 13C NMR (100 MHz, CDCl3, δ ppm): 162.06, 156.75,
153.93, 142.55, 136.51, 135.63, 129.45(2C), 128.66(4C), 127.85, 127.74, 127.65(3C), 125.37, 120.03,
107.18, 66.00, 58.02, 47.53, 45.63(2C), 36.85; IR (KBr, cm-1): v 2933.31, 2554.71, 2449.60, 1670.35,
1540.01, 1489.05, 1357.89, 1222.87, 1168.86, 1029.99, 840.96, 705.95; ESI-MS: m/z 446.2 [M + H]+;
ESI-HRMS (TOF): m/z [M + H]+ calcd for C26H28N3O2S, 446.1902, found 446.1894. Purity: 95.3%.
5.1.1.18. 3-benzyl-2-(benzylthio)-6-(2-(diethylamino)ethoxy)quinazolin-4(3H)-one (5r)
The title compound was obtained starting from phenylmethanamine, (chloromethyl)benzene, methyl
2-amino-4-hydroxybenzoate and 2-diethylaminoethyl chloride hydrochloride. As a white powder, yield:
1
25%; m.p.94.2-97.8 °C. Analytical data for 5r: H NMR (400 MHz, CDCl3, δ ppm): 7.60 (d, J = 3.2 Hz,
1H, Ar-H), 7.55 (d, J = 8.8 Hz, 1H, Ar-H), 7.42 (d, J = 8.4 Hz, 2H, Ar-H), 7.23-7.34 (m, 9H, Ar-H), 5.35 (s,
2H, CH2), 4.48 (s, 2H, CH2), 4.16 (t, J = 6.0 Hz, 2H, OCH2), 2.95 (t, J = 6.0 Hz, 2H, NCH2), 2.65-2.71 (m,
4H, N(CH2CH3)2), 1.10 (t, J = 7.2 Hz, 6H, N(CH2CH3)2); 13C NMR (100 MHz, CDCl3, δ ppm): 162.07,
156.94, 153.81, 142.42, 136.53, 135.67, 129.46(2C), 128.67(2C), 128.65(2C), 127.78, 127.73, 127.66(3C),
125.26, 120.07, 107.28, 66.81, 51.51, 47.87(3C), 47.52, 36.85, 11.80(2C); IR (KBr, cm-1): v 2966.52,
2931.80, 1670.31, 1549.24, 1485.63, 1454.33, 1357.89, 1276.88, 1226.73, 1168.86, 1029.99, 834.13,
702.09; ESI-MS: m/z 474.2 [M + H]+; ESI-HRMS (TOF): m/z [M + H]+ calcd for C28H32N3O2S, 474.2215,
found 474.2207. Purity: 96.5%.
5.1.1.19. 3-benzyl-2-(benzylthio)-6-(3-(dimethylamino)propoxy)quinazolin-4(3H)-one (5s)
The title compound was obtained starting from phenylmethanamine, (chloromethyl)benzene, methyl
2-amino-4-hydroxybenzoate and 3-dimethylaminopropyl chloride hydrochloride. As a white powder, yield:
1
36%; m.p.93.7-96.7 °C. Analytical data for 5s: H NMR (400 MHz, CDCl3, δ ppm): 7.61 (d, J = 2.8 Hz,
1H, Ar-H), 7.54 (d, J = 9.2 Hz, 1H, Ar-H), 7.38-7.40 (m, 2H, Ar-H), 7.21-7.32 (m, 9H, Ar-H), 5.33 (s, 2H,
CH2), 4.47 (s, 2H, CH2), 4.09 (t, J = 6.4 Hz, 2H, OCH2), 2.45 (t, J = 7.2 Hz, 2H, NCH2), 2.25 (s, 6H,
13
N(CH3)2), 1.96-1.99 (m, 2H, CH2); C NMR (100 MHz, CDCl3, δ ppm): 162.02, 157.14, 153.71, 142.33,
136.56, 135.75, 129.47(2C), 128.67(2C), 128.65(2C), 127.78, 127.70(3C), 127.66, 125.10, 120.15, 107.39,
66.88, 56.41, 47.50, 45.64(2C), 36.86, 27.54; IR (KBr, cm-1): v 3379.29, 2939.46, 2757.86, 1670.31,
1616.35, 1577.77, 1485.43, 1428.18, 1355.33, 1275.30, 1221.95, 1165.00, 1103.28, 1072.42, 983.70,
20

948.98, 833.25, 779.24, 563.21; ESI-MS: m/z 460.2 [M + H]+; ESI-HRMS (TOF): m/z [M + H]+ calcd for
C27H30N3O2S, 460.2059, found 460.2049. Purity: 95.2%.
5.2. Biological assays
The anti-HBV activities and intrinsic cytotoxicity of the target compounds were determined in HepG2
2.2.15 cell line, a stably transfected cell line containing HBV genome on a plasmid. The cell line was
provided by 302 military hospital of china, and cultivated in DMEM medium supplemented with 10% fetal
bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.38 mg/mL of G418. The anti-HCC
activities and preliminary anti-HCC action mechanism of the target compounds were evaluated in HepG2
cell line.
5.2.1. Cytotoxicity assay
The intrinsic cytotoxicity and in vitro anti-HCC activities of the target compounds were measured by
MTT assay. Briefly, HepG2 2.2.15 (used to determine the intrinsic cytotoxicity) or HepG2 (used to
evaluate the in vitro anti-HCC effect) cells were seeded in 96-well culture plates and cultured for 24 h to
maintain the cells in exponential growth phase. Then, cells were cultured for another 72 h in the presence
or absence of the test compounds (in 0.2 mL culture medium/well), respectively. Finally, the cell viability
was measured by analyzing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
absorbance at 490 nm, as calculated from the absorbance relative to untreated cells at 24 h after MTT
treatment.
5.2.2. In vitro anti-HBV activity assay.
The vitro anti-HBV activity of the target compounds was evaluated by detecting their inhibitory effect on
HBV DNA replication according to previous method [19, 20]. Briefly, HepG2 2.2.15 cells (2×105
cells/well) were seeded in 24 well culture plates and cultured for another 24 h to recover from the
trypsinization. After that, the culture medium was replaced with a fresh medium containing the test
compound or 3TC. Subsequently, medium was changed every 2 days. After 6 days incubation, intracellular
HBV DNA was extracted, and quantified by real time PCR.
HepG2 2.2.15 cells, transiently transfected with plasmids containing lamivudine and entecavir-resistant
mutant (rtL180M + rtM204V + rtT184L), were used to evaluate the antiviral efficacy of the target
21

compounds against nucleoside analogue-resistant HBV.
5.2.3. Hoechst 33342 Staining
HepG2 cells (5 × 104 cells/well) were seeded in six-well plates, and then incubated in the presence of 5k
(5, 10 or 20 μM) for 48 h. After the incubation, the cells were gently washed with PBS, fixed in 4%
paraformaldehyde for 30 min, and subsequently stained with Hoechst 33342 (20 μg/mL, KeyGen, Nanjing,
China) at room temperature for 15 min in the darkness. Finally, morphological changes of cells was
observed using fluorescence microscopy (Olympus, BX41).
5.2.4. Cell Apoptosis Analysis
Annexin V staining was performed with an apoptosis assay kit (KeyGen, Nanjing, China). Briefly,
HepG2 cells were incubated with or without 5k at the indicated concentrations (5, 10 or 20 μM) for 48 h,
and then gently washed twice with PBS, centrifuged, and reincubated in annexin V binding buffer (500 μL).
The cells were then harvested, washed, and stained with 5 μL annexin V-FITC and 5 μL propidium iodide
(PI) for 15 min in the darkness. Subsequently, apoptosis was analyzed by a FACS Calibur flow cytometer
(Becton-Dickinson, San Jose, CA, USA).
5.2.5. Western blotting analysis of apoptotic proteins Bad, Bax, Bcl-2 and Bcl-xl
HepG2 cells lysates (40 μg protein/lane) from each group were separated SDS-PAGE and then
transferred to polyvinyl difluoride membranes by electroblotting. After incubation in blocking solution (5%
nonfat milk), the blots were incubated with primary antibodies at 4°C overnight, respectively. Primary
antibodies were mouse anti-Bad, Bax, Bcl-2 and Bcl-xl (Santa Cruz Biotechnology, USA). The blots were
subsequently washed with 1× Tris-Buffered Saline Tween-20 (TBST) solution, incubated with secondary
antibodies for 2 h, and washed with 1× TBST solution. The blots were detected using an enhanced
chemiluminescence system, and imaged using a G:BOX chemiXR5 digital imaging system. The protein
bands' relative intensities were analyzed by Gel-Pro32 software.
Acknowledgements
This work was supported by the grants from the National Natural Science Foundation of China (NSFC
No. 81703359), and the Natural Science Foundation of Jiangsu province (No. BK20181151 and
BK20171179), the Six Talent Peaks Project in Jiangsu Province (Grant No. 2017-YY-039) and Jiangsu
22

Provincial 333 High-level Talents Cultivation Project (BRA201927) and Postgraduate Research & Practice
Innovation Program of Jiangsu Province (No. KYCX19_2251). We thank to Public Experimental Research
Center of Xuzhou Medical University for providing chemical structure analysis.
References and notes
[1] Y. Hutin, M. Nasrullah, P. Easterbrook, B.D. Nguimfack, E. Burrone, F. Averhoff, M. Bulterys,
Access to Treatment for Hepatitis B Virus Infection - Worldwide, 2016, Mmwr-Morbidity and
Mortality Weekly Report, 67 (2018) 773-777.
[2] J.W. Ward, A.R. Hinman, What Is Needed to Eliminate Hepatitis B Virus and Hepatitis C Virus as
Global Health Threats, Gastroenterology., 156 (2019) 297-310.
[3] T.-Y. Li, Y. Yang, G. Zhou, Z.-K. Tu, Immune suppression in chronic hepatitis B infection
associated liver disease: A review, World J. Gastroentero, 25 (2019) 3527-3537.
[4] L.Y. Liang, G.L.-H. Wong, Unmet need in chronic hepatitis B management, Clinical and molecular
hepatology, 25 (2019) 172-180.
[5] Z.Q. Wang, Challenges and Opportunities in Hepatitis B Research, Acs Infectious Diseases, 5
(2019) 652-654.
[6] Q.H. Yang, J.F. Kang, L.Y. Zheng, X.J. Wang, N. Wan, J. Wu, Y. Qiao, P.F. Niu, S.Q. Wang, Y.M.
Peng, Q.D. Wang, W.Q. Yu, J.B. Chang, Synthesis and Biological Evaluation of 4-Substituted
Fluoronucleoside Analogs for the Treatment of Hepatitis B Virus Infection, J. Med. Chem., 58
(2015) 3693-3703.
[7] A.J. Sanyal, S.K. Yoon, R. Lencioni, The Etiology of Hepatocellular Carcinoma and Consequences
for Treatment, Oncologist, 15 (2010) 14-22.
[8] A. Arzumanyan, H.M.G.P.V. Reis, M.A. Feitelson, Pathogenic mechanisms in HBV- and
HCV-associated hepatocellular carcinoma, Nat. Rev. Cancer, 13 (2013) 123-135.
[9] C.L. Lin, J.H. Kao, Review article: novel therapies for hepatitis B virus cure - advances and
perspectives, Aliment. Pharm. Therap, 44 (2016) 213-222.
[10] A.S. Lok, F. Zoulim, G. Dusheiko, M.G. Ghany, Hepatitis B cure: From discovery to regulatory
approval, J. Hepatol., 67 (2017) 847-861.
23

[11] T. Pan, Y. Ding, L. Wu, L. Liang, X. He, Q. Li, C. Bai, H. Zhang, Design and synthesis of
aminothiazole based Hepatitis B Virus (HBV) capsid inhibitors, Eur. J. Med. Chem., 166 (2019)
480-501.
[12] O. Sari, S. Boucle, B.D. Cox, T. Ozturk, O.I. Russell, L. Bassit, F. Amblard, R.F. Schinazi,
Synthesis of sulfamoylbenzamide derivatives as HBV capsid assembly effector, Eur. J. Med.
Chem., 138 (2017) 407-421.
[13] H.Y. Jia, F.X. Bai, N. Liu, X.H. Liang, P. Zhan, C.H. Ma, X.M. Jiang, X.Y. Liu, Design, synthesis
and evaluation of pyrazole derivatives as non-nucleoside hepatitis B virus inhibitors, Eur. J. Med.
Chem., 123 (2016) 202-210.
[14] H. Jia, F. Bai, N. Liu, X. Liang, P. Zhan, C. Ma, X. Jiang, X. Liu, Design, synthesis and
evaluation of pyrazole derivatives as non-nucleoside hepatitis B virus inhibitors, Eur. J. Med.
Chem., 123 (2016) 202-210.
[15] H.Y. Jia, Y. Song, J. Yu, P. Zhan, D. Rai, X.H. Liang, C.H. Ma, X.Y. Liu, Design, synthesis and
primary biological evaluation of the novel 2-pyridone derivatives as potent non-nucleoside HBV
inhibitors, Eur. J. Med. Chem., 136 (2017) 144-153.
[16] J. Qiu, W. Chen, Y. Zhang, Q. Zhou, J. Chen, L. Yang, J. Gao, X. Gu, D. Tang, Assessment of
quinazolinone derivatives as novel non-nucleoside hepatitis B virus inhibitors, Eur. J. Med. Chem.,
176 (2019) 41-49.
[17] C. de Martel, D. Maucort-Boulch, M. Plummer, S. Franceschi, World-wide relative contribution
of hepatitis B and C viruses in hepatocellular carcinoma, Hepatology., 62 (2015) 1190-1200.
[18] S.L. Chan, V.W.S. Wong, S.K. Qin, H.L.Y. Chan, Infection and Cancer: The Case of Hepatitis B,
J. Clin. Oncol., 34 (2016) 83-90.
[19] S.-H. Guan, M. Lu, P. Grünewald, M. Roggendorf, G. Gerken, J.F. Schlaak, Interferon-alpha
response in chronic hepatitis B-transfected HepG2.2.15 cells is partially restored by lamivudine
treatment, World J. Gastroentero, 13 (2007) 228-235.
[20] L. Yang, L.P. Shi, H.J. Chen, X.K. Tong, G.F. Wang, Y.M. Zhang, W.L. Wang, C.L. Feng, P.L. He,
F.H. Zhu, Y.H. Hao, B.J. Wang, D.L. Yang, W. Tang, F.J. Nan, J.P. Zuo, Isothiafludine, a novel
non-nucleoside compound, inhibits hepatitis B virus replication through blocking pregenomic
24

RNA encapsidation, Acta Pharmacol. Sin., 35 (2014) 410-418.
[21] J.Y. Qiu, B.X. Xu, Z.M. Huang, W.D. Pan, P.X. Cao, C.X. Liu, X.J. Hao, B.A. Song, G.Y. Liang,
Synthesis and biological evaluation of Matijing-Su derivatives as potent anti-HBV agents, Bioorg.
Med. Chem., 19 (2011) 5352-5360.
[22] W.L. Li, F.J. Xu, W. Shuai, H.H. Sun, H. Yao, C. Ma, S.T. Xu, H.Q. Yao, Z.Y. Zhu, D.H. Yang,
Z.S. Chen, J.Y. Xu, Discovery of Novel Quinoline-Chalcone Derivatives as Potent Antitumor
Agents with Microtubule Polymerization Inhibitory Activity, J. Med. Chem., 62 (2019) 993-1013.
[23] S. Ji, S.N. Tang, K. Li, Z.W. Li, W.F. Liang, X. Qiao, Q. Wang, S.W. Yu, M. Ye, Licoricidin
inhibits the growth of SW480 human colorectal adenocarcinoma cells in vitro and in vivo by
inducing cycle arrest, apoptosis and autophagy, Toxicol. Appl. Pharm., 326 (2017) 25-33.
[24] T. Zhang, W. Wu, D. Li, T. Xu, H. Zhu, D. Pan, S. Zhu, Y. Liu, Anti-oxidant and anti-apoptotic
effects of luteolin on mice peritoneal macrophages stimulated by angiotensin II, Int.
Immunopharmacol., 20 (2014) 346-351.
[25] Q.F. Ye, Y.C. Zhang, X.Q. Peng, Z. Long, Y.Z. Ming, L.Y. He, Silencing Notch-1 induces
apoptosis and increases the chemosensitivity of prostate cancer cells to docetaxel through Bcl-2
and Bax, Oncology Letters, 3 (2012) 879-884.
[26] Y.D. Hu, K.K. Yu, G. Wang, D.P. Zhang, C.J. Shi, Y.H. Ding, D. Hong, D. Zhang, H.Q. He, L.
Sun, J.N. Zheng, S.Y. Sun, F. Qian, Lanatoside C inhibits cell proliferation and induces apoptosis
through attenuating Wnt/beta-catenin/c-Myc signaling pathway in human gastric cancer cell,
Biochem. Pharmacol., 150 (2018) 278-290.
[27] D.H. Li, P. Hu, S.T. Xu, C.Y. Fang, S. Tang, X.Y. Wang, X.Y. Sun, H. Li, Y. Xu, X.K. Gu, J.Y. Xu,
Lasiokaurin derivatives: synthesis, antimicrobial and antitumor biological evaluation, and
apoptosis-inducing effects, Arch. Pharm. Res., 40 (2017) 796-806.
[28] W. Hu, J. Fu, S.-X. Lu, L.-L. Liu, R.-Z. Luo, J.-P. Yun, C.Z. Zhang, Decrease of
Bcl-xL/Bcl-2-associated death promoter in hepatocellular carcinoma indicates poor prognosis, Am.
J. Cancer Res., 5 (2015) 1805-1813.
[29] X.-F. Zhang, X. Yang, H.-L. Jia, W.-W. Zhu, L. Lu, W. Shi, H. Zhang, J.-H. Chen, Y.-F. Tao, Z.-X.
Wang, J. Yang, L.-X. Wang, M. Lu, Y. Zheng, J. Zhao, Q.-Z. Dong, L.-X. Qin, Bcl-2 expression is
25

a poor predictor for hepatocellular carcinoma prognosis of andropause-age patients, Cancer
Biology & Medicine, 13 (2016) 459-468.
[30] M. Bonolami, A. Kotsafti, C. Carlotto, R. Cardin, F. Fannati, Bcl-2, Bax and Bad mRNA
expression in chronic active hepatitis, cirrhosis, and hepatocellular carcinoma, Gastroenterology.,
130 (2006) A427-A428.
[31] Q. Jiang, D.F. Pan, Y. Yang, Y. Hu, L. Fang, P.P. Shang, Y. Xia, D.Y. Li, Luteolin Regulates
Macrophage Polarization via the PI3K/Akt Pathway to Inhibit the Apoptosis Stimulated by
Angiotensin II, Current Pharmaceutical Biotechnology, 19 (2018) 428-437.
26

Highlights
1. Quinazolinone derivatives were synthesized and evaluated as anti-HBV and anti-HCC agents.
2. 5j and 5k exhibited potent anti-HBV activity against wild and resistant HBV strains.
3. 5k exhibited more potent in vitro anti-HCC activity than 5-fluorouracil and sorafenib.
4. 5k induced HepG2 cells apoptosis by regulating the expression of Bcl-2 family proteins.

Declaration of Interest Statement
The authors declared that there was no conflicts of interest to this work. We declare
that we do not have any commercial or associative interest that represents a conflict of
interest in connection with the work submitted.