Assessment of quinazolinone derivatives as novel non-nucleoside hepatitis B virus inhibitors

Article abstract of DOI:10.1016/j.ejmech.2019.05.014

Hepatitis B virus (HBV)infection is a worldwide public health issue. Search for novel non-nucleoside anti-HBV agents is of great importance. In the present study, a series of quinazolinones derivatives (4a-t and 5a-f)were synthesized and evaluated as novel anti-HBV agents. Among them, compounds 5e and 5f could significantly inhibit HBV DNA replication with IC₅₀ values of 1.54 μM and 0.71 μM, respectively. Interestingly, the selective index values of 5f was higher than that of lead compound K284–1405, suggesting 5f possessed relatively safety profile than K284–1405. Notably, 5e and 5f exhibited remarkably anti-HBV activities against lamivudine and entecavir resistant HBV strain with IC₅₀ values of 1.90 and 0.84 μM, confirming their effectiveness against resistant HBV strain. In addition, molecular docking studies indicated that compounds 5e and 5f could well fit into the dimer-dimer interface of HBV core protein dominated by hydrophobic interactions. Notably, their binding modes were different from the lead compound K284–1405, which may be attributed to the additional substituent groups in the quinazolinone scaffold. Taken together, 5e and 5f possessed novel chemical structure and potent anti-HBV activity against both drug sensitive and resistant HBV strains, thus warranting further research as potential non-nucleoside anti-HBV candidates.

Full text of DOI:10.1016/j.ejmech.2019.05.014

Accepted Manuscript
Assessment of quinazolinone derivatives as novel non-nucleoside hepatitis B virus
inhibitors
Jingying Qiu, Wang Chen, Yinpeng Zhang, Qingqing Zhou, Jing Chen, Lihua Yang,
Jian Gao, Xiaoke Gu, Daoquan Tang
PII:
S0223-5234(19)30423-4
DOI:
https://doi.org/10.1016/j.ejmech.2019.05.014
EJMECH 11324
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 November 2018
Revised Date: 9 February 2019
Accepted Date: 6 May 2019
Please cite this article as: 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,
European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.05.014.
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ACCEPTED MANUSCRIPT
Assessment of quinazolinone derivatives as novel non-nucleoside hepatitis B
virus inhibitors
Jingying Qiua, b, #, Wang Chenb, #, Yinpeng Zhangb, Qingqing Zhoub, Jing Chena, Lihua Yangb, Jian
Gaoa, *, Xiaoke Gua, *, Daoquan Tanga, b,
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
Compound 5f exhibited significantly anti-HBV activity against wild and drug (lamivudine and
entecavir) resistant HBV strains, and could well fit into the dimer-dimer interface of HBV core
protein dominated by hydrophobic interactions.
Corresponding authors. Tel.: +86-516-83262138; fax: +86-516-83262630; e-mail: tdq993@hotmail.com (D. Q.)
Tel.: +86-516-83262137; fax: +86-516-83262630; e-mail: gu_xk@163.com (X. G.)
Tel.: +86-516-83262137; fax: +86-516-83262630; e-mail: gaojian@xzhmu.edu.cn (J. G.)
# These two authors contributed equally to this work.

ACCEPTED MANUSCRIPT
Assessment of quinazolinone derivatives as novel non-nucleoside hepatitis B
virus inhibitors
Jingying Qiua, b, #, Wang Chenb, #, Yinpeng Zhangb, Qingqing Zhoub, Jing Chena, Lihua Yangb, Jian
Gaoa, *, Xiaoke Gua, *, Daoquan Tanga, b,
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
Hepatitis B virus (HBV) infection is a worldwide public health issue. Search for novel
non-nucleoside anti-HBV agents is of great importance. In the present study, a series of
quinazolinones derivatives (4a-t and 5a-f) were synthesized and evaluated as novel anti-HBV
agents. Among them, compounds 5e and 5f could significantly inhibit HBV DNA replication with
IC50 values of 1.54 µM and 0.71 µM, respectively. Interestingly, the selective index values of 5f
was higher than that of lead compound K284-1405, suggesting 5f possessed relatively safety
profile than K284-1405. Notably, 5e and 5f exhibited remarkably anti-HBV activities against
lamivudine and entecavir resistant HBV strain with IC50 values of 1.90 and 0.84 µM, confirming
their effectiveness against resistant HBV strain. In addition, molecular docking studies indicated
that compounds 5e and 5f could well fit into the dimer-dimer interface of HBV core protein
dominated by hydrophobic interactions. Notably, their binding modes were different from the lead
compound K284-1405, which may be attributed to the additional substituent groups in the
quinazolinone scaffold. Taken together, 5e and 5f possessed novel chemical structure and potent
anti-HBV activity against both drug sensitive and resistant HBV strains, thus warranting further
research as potential non-nucleoside anti-HBV candidates.
Keywords: Anti-HBV agents; Synthesis; Quinazolinone derivatives; HBV core protein
1. Introduction
Hepatitis B virus (HBV) infection, a worldwide health threat, may cause acute, chronic hepatitis,
Corresponding authors. Tel.: +86-516-83262138; fax: +86-516-83262630; e-mail: tdq993@hotmail.com (D. Q.)
Tel.: +86-516-83262137; fax: +86-516-83262630; e-mail: gu_xk@163.com (X. G.)
Tel.: +86-516-83262137; fax: +86-516-83262630; e-mail: gaojian@xzhmu.edu.cn (J. G.)
# These two authors contributed equally to this work.
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liver cirrhosis, liver fibrosis or hepatocelullar carcinoma (HCC) [1-3]. Statistically, there are about
350 million people worldwide are chronically infected by HBV [4, 5]. Despite the availability of
the effective prophylactic vaccine, HBV infection and complications still result in 686000 deaths
per year [6-8]. Currently, interferons (IFNs) and nucleos(t)ides-based reverse transcriptase
inhibitors have been approved for the treatment of HBV infection. Unfortunately, due to the
limitations and side effects of the current treatment, the clinical cure rate for patients with chronic
HBV infection is not satisfactory [9-11]. For IFNs therapy, parenteral administration is mandatory,
and IFNs usually results in adverse effect, such as “flu-like” symptoms [12]. For nucleos(t)ide
therapy, patients are required to take drugs for life to prevent HBV viral rebound [13]. What's
more frustrating is that HBV can develop resistance to nucleos(t)ide drugs [14, 15]. Therefore,
there is a clear and urgent need to develop anti-HBV agents with novel structure and mode of
action.
It is well-known that HBV core protein performs crucial roles at HBV virus life cycle via
affecting the encapsidation of pregenomic RNA, the maturation and maintenance of the stable
HBV virion and blocking virion secretion [16]. Consequently, HBV core protein is considered as a
promising target for the development of novel, safe and effective anti-HBV agents [17, 18]. The
latest research confirmed that several compounds can prevent encapsidation of pgRNA by
modulate the kinetics/thermodynamics of HBV core protein aggregation, thus inhibiting HBV
DNA synthesis and infectious virion production [7, 19-22]. Recently, the crystal structure of the
HBV core protein in complex with
a
heteroaryldihydropyrimidine (HAP) derivate
NVR-010-001-E2 was reported. Meanwhile, the mode of action of this compound with HBV core
protein was described in detail [23]. Based on these research findings, our group obtained a
compound with quinazolinone scaffold (compound K284-1405, Figure 1) as a new HBV core
protein inhibitor by molecular docking based virtual screening. Preliminary anti-HBV activity
studies showed that compound K284-1405 displayed potential anti-HBV activity with an IC50
value on HBV DNA replication of 4.07 µM, and the selective index (SI) values was more than
17.57. Herein, to obtain novel anti-HBV agents with more potent anti-HBV activities, we firstly
synthesized a series novel quinazolinones derivatives (Figure 1), and then evaluated their
anti-HBV activities against both drug sensitive and resistant HBV strains in the present work. In
addition, the mode of binding between the active compounds and HBV core protein were also
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proposed by docking studies in this context.
Figure 1. Chemical structures of lead compound K284-1405 and the quinazolinone derivatives.
2. Chemistry
Synthetic procedures of the novel quinazolinone derivatives 4a-t and 5a-f were illustrated in
Scheme 1. Briefly, 2-thiophenemethylamine was reacted with CS2 and toluene sulfonyl chloride in
THF to obtain compound 1. Compound 2 was obtained by the chloromethylation reaction with
benzene or benzene derivatives and paraformaldehyde was selected as the raw materials. Methyl
anthranilate or methyl anthranilate derivatives was condensed with 2-(isothiocyanatomethyl)
thiophene to give intermediate 3, which was then reacted with compound 2 mentioned above in
the presence of K2CO3 and 1,4-dioxane to obtain target compounds 4a-t, respectively. Compound
4f, 4m, or 4t was alkylated with 2-dimethylaminoethyl chloride hydrochloride or
2-diethylaminoethyl chloride hydrochloride to provide alkoxyl substituted derivative 5a-f,
respectively.
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Scheme 1. Synthetic route of target compounds 4a-t and 5a-f. Reagents and conditions: a) TEA, THF, 0 °C; b)
TsCl, 0 °C; c) HCl, 80 °C; d) TEA, EtOH, reflux; e) 1,4-dioxane, K2CO3, 90 °C; f) 2-dimethylaminoethyl chloride
hydrochloride or 2-diethylaminoethyl chloride hydrochloride, 1,4-dioxane, K2CO3, 90 °C.
3. Results and discussion
3.1. Biological evaluation
The intrinsic cytotoxicity of the target compounds against HepG2 2.2.15 cells was
determined by MTT. The dose of the test compound that results in 50% death of HepG2 2.2.15
cells was defined as CC50. The anti-HBV activities of the target compounds were assessed in
HepG2 2.2.15 cells using real time quantitative PCR (RT-PCR) assay. The concentration of the
test compound that inhibits HBV DNA replication by 50% was defined as IC50. Selectivity index
(SI) was calculated as the CC50/IC50 value. Classic anti-HBV agent lamivudine (3TC) was selected
as the positive control.
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3.1.1. Cytotoxicity of the target compounds
Firstly, the intrinsic cytotoxicity of the target compounds against HepG2 2.2.15 cells was
measured by MTT assay. Briefly, HepG2 2.2.15 cells were incubated with various concentrations
of target compounds 4a-t and 5a-f for 72 h, respectively. Then the cell viability was determined
by MTT assay. As seen from Table 1, most of the test compounds display little (CC50 > 100 µM)
or slight (CC50 = 8.76-89.04 µM) intrinsic cytotoxicity in vitro, suggesting that the target
compounds possessed a relatively safety profile.
3.1.2. Effect of the target compounds on HBV DNA replication
Subsequently, the inhibitory effect of the target compounds 4a-t and 5a-f on HBV DNA
replication was screened in the HepG2 2.2.15 cell line by RT-PCR assay. Compound K284-1405
and lamivudine (3TC) were used as the lead compound and positive control, respectively.
Specifically, HepG2 2.2.15 cells was treated with the test compounds at the non-toxic dose (4 µM)
for 6 days, then the extracellular HBV DNA levels were quantified by RT-PCR. As shown in
Table 1, at non-toxic dose (4 µM) most test compounds can inhibit HBV DNA replication to some
extent. Among them, some of the target compounds displayed more potent anti-HBV activity than
lead compound K284-1405, for their inhibition rate on HBV DNA replication was much higher
than that of K284-1405 (47.08%). Obviously, compounds 5f was the most potent ones with a
HBV DNA inhibition rate of 98.42%, respectively, which was comparable with the positive
control 3TC.
Table 1. Inhibitory effect of the target compounds 4a-t and 5a-f on HBV DNA replication
CC50
Inhibition rate (%)
Compounds
R1
R2
R3
(µM)
(4 µM)
OCH3
OCH3
OCH3
COCH3
COCH3
COCH3
6-F
6-Cl
6-Br
89.04
74.62
>100
30.67
40.74
45.96
4a
4b
4c
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OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
COCH3
6-I
81.13
>100
>100
17.99
8.76
45.41
56.15
3.26
4d
COCH3
6-CH3
4e
COCH3
6-OH
4f
COCH3
6-OCH2CH2N(CH3)2
33.88
41.96
76.27
79.61
63.27
64.15
65.87
69.43
0.59
5a
COCH3
6-OCH2CH2N(CH2CH3)2
5b
H
H
H
>100
>100
>100
>100
>100
>100
>100
14.70
10.14
>100
>100
>100
>100
>100
>100
>100
21.13
15.79
71.51
>100
4g
6-F
4h
H
6-Cl
4i
H
6-Br
4j
H
6-I
4k
H
6-CH3
4l
H
6-OH
4m
H
6-OCH2CH2N(CH3)2
53.89
75.97
11.03
9.53
5c
H
6-OCH2CH2N(CH2CH3)2
5d
H
H
4n
H
H
6-F
4o
H
H
6-Cl
16.06
16.72
1.87
4p
H
H
6-Br
4q
H
H
6-I
4r
H
H
6-CH3
6-OH
13.79
5.62
4s
H
H
4t
5e
H
H
6-OCH2CH2N(CH3)2
6-OCH2CH2N(CH2CH3)2
H
83.28
98.42
47.08
94.31
H
H
5f
OCH3
COCH3
K284-1405
3TC
To further confirm the anti-HBV activity, the inhibitory effect on HBV DNA replication of
active compounds 4g, 4h and 5d-f at different concentrations was subsequently determined by
RT-PCR assay. As shown in Table 2, all the tested compounds could significantly inhibit HBV
DNA replication with IC50 values in range of 0.71-2.81 µM. Clearly, 5e and 5f exhibited most
potent anti-HBV activity with IC50 values of 1.54 and 0.71 µM, respectively. In addition, the SI
value of compound 5f was 22.24, which was higher than that of lead compound K284-1405 (SI:
17.57), indicating that 5f possessed relatively safety profile than K284-1405.
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Table 2. Anti-HBV activity and SI values of target compounds 4g, 4h and 5d-f
DNA replication
Compounds
CC50 (µM)
IC50 (µM)
2.81
SI
>100
>100
10.14
21.13
15.79
71.51
>100
>35.59
>40.48
5.25
4g
2.48
4h
1.93
5d
5e
1.54
13.72
22.24
17.57
>1000
0.71
5f
4.07
K284-1405
3TC
<0.1
Based on the data shown in Tables 1-2, the structure activity relationship (SAR) can be drawn
as follows: Acetyl substituent in ring B was not necessary for the anti-HBV activity. When the
acetyl substituent was removed, the anti-HBV activity was all increased (4g vs. K284-1405; 4a-f
vs. 4h-m, Table 1). In sharp contrary, the methoxy group in ring B was crucial for the anti-HBV
activity of the target compounds. After removing the methoxy group, the anti-HBV activity was
remarkably decreased (4n-t vs. 4g-m, Table 1). Among them, compounds 4n-t were inactive at a
test concentration of 4 µM，confirming that the methoxy substituent played a significant role in the
anti-HBV activity. In addition, the anti-HBV activity was significantly weakened when
introducing a hydroxy substituent in ring A (4f, 4m, 4t vs. K284-1405), suggesting that the
introduction of a meta substituent might be not conducive to the anti-HBV activity. Interestingly,
when the hydroxy substituent was replaced with dimethylamino ethoxy substituent or
diethylamine ethoxy, the anti-HBV activity was significantly increased (4f vs. 5a-b; 4m vs. 5c-d;
4t vs. 5e-f). One plausible explanation is that introduction of dimethylamino ethoxy substituent or
diethylamine ethoxy group might form an additional interactions with HBV core protein, thus
increasing the affinity of the target compound with HBV core protein. However, the precise SAR
remains further investigation when more quinazolinone derivatives are available in the near future.
3.1.3. Anti-HBV activity of compounds 5e and 5f against polymerase drug resistant HBV strain
As mentioned above, one of the main limitations of the nucleos(t)ide therapy is the emergence
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of drug resistance. Therefore, it is of great interest to investigate whether the active compounds 5e
and 5f exhibit anti-HBV activities against drug resistant HBV strains. HepG2 cells, transiently
transfected with lamivudine and entecavir resistant strain mutant (rtL180M + rtM204V + rtT184L)
(LERS) was served as an evaluation model. Lamivudine and entecavir were used as reference
drugs. After the treatment of HepG2 cells (LERS HBV) or wild-type strain with target compounds
5e (4 µM), 5f (4 µM), lamivudine (100 µM) or entecavir (10 µM) respectively for 72 h, the level
of HBV DAN replicative intermediate was examined by RT-PCR. As shown in Figure 2,
lamivudine and entecavir displayed potent anti-HBV activity against wild-type strain with an
inhibition rate on HBV DNA replication of 90.67% and 98.07%. As was expected, lamivudine and
entecavir resistant strain was indeed resistant to both lamivudine and entecavir, and their
inhibition rate of HBV DNA replication was only 17.73% and 22.58%, respectively. Interestingly,
both compound 5e and 5f exhibited potent anti-HBV activity against resistant HBV cell strain.
Notably, their inhibition rates on HBV DNA replication were respectively 80.91% and 93.99%,
which was comparable with that on wild-type HBV cell strain (81.95% and 96.09%, respectively).
Figure 2. Anti-HBV activities of 5e and 5f against lamivudine and entecavir resistant HBV strain. Briefly, the
full genome of wide-type or lamivudine and entecavir resistant HBV were transiently transfected into HepG2 cells.
Then the cells were treated with entecavir (10 µM), lamivudine (100 µM), 5e (4 µM) or 5f (4 µM) for 72 h,
respectively. Finally, HBV DNA replicative intermediate level was determined by RT-PCR.
In order to confirm the anti-HBV activity against resistant HBV strain, we further determined
the IC50 values for inhibition of HBV DNA replication and the SI values of compounds 5e and 5f
in lamivudine and entecavir resistant HBV strain. Data in Table 3 showed that the IC50 values of
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5e and 5f were 1.90 and 0.84 µM, respectively, confirming their potent inhibitory effect on HBV
DNA replication against drug resistant strain. Meanwhile, SI values of compound 5f was 21.40,
indicating 5f possessed relatively safety profile.
Table 3. Anti-HBV activities of compounds 5e and 5f against lamivudine and entecavir resistant HBV strain
DNA replication
Compounds
CC50 (µM)
IC50 (µM)
1.90
SI
26.72
17.98
>100
14.06
21.40
<1
5e
5f
0.84
3TC
>100
3.2. Molecular docking
It is well-known that HBV core protein performs multiple essential roles at different stages of
HBV virus life cycle, which is regarded as an excellent target for developing novel, efficacious
and safety anti-HBV agents. Therefore, we preliminarily investigated the binding interactions of
the lead compound K284-1405 and its two analogs 5e and 5f with HBV core protein by molecular
docking, and compound NVR-010-001-E2 was also studied for comparison (Figure 3). It can be
seen that all three compounds fitted well into the dimer-dimer interface of HBV core protein, and
the bindings were almost dominated by the hydrophobic interactions in light of the fact that the
binding interface composed of a large number of hydrophobic residues (Figure 3). In detail, each
compound was accommodated into a well-defined hydrophobic groove which consisted of
residues Phe23, Phe24, Pro25, Asp29, Leu30, Thr33, Leu37, Trp102, Ile105, Ser106, Thr109,
Phe110, Tyr118, Ile139, Leu140 and Ser141 in one protein subunit (Figure 3A-D). Meanwhile,
each compound was also covered by a "lid" formed by the C-terminal helix of the other protein
subunit (Figure 3E-H). Notably, compared with compounds K284-1405 and NVR-010-001-E2,
compounds 5e and 5f had additional substituent groups in quinazolinone scaffold, which inserted
into a new hydrophobic pocket composed of the residues (Ser121, Pro138 and Phe156) in the lid.
It is likely that the main difference of binding patterns between compounds 5e (or 5f) and
NVR-010-001-E2 was in the lid rather than the hydrophobic groove. Moreover, the binding
affinities of compounds 5e and 5f were stronger than those of K284-1405 and NVR-010-001-E2,
with the corresponding docking scores of 7.03, 8.42, 6.58 and 6.36, respectively.
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Figure 3. Molecular docking predicted binding modes of compounds K284-1405, 5e and 5f. The crystal structure
of NVR-010-001-E2 interacted with HBV core protein is used for comparison. (A-D) Compounds K284-1405, 5e,
5f and NVR-010-001-E2 sit in the hydrophobic groove of the contact domain (gray surface) on one dimer and the
lid is removed for clarity. (E-H) Binding surface to the lid, with the contact domain removed for clarity.
4. Conclusions
In summary, twenty-six quinazolinone derivatives (4a-t and 5a-f) were synthesized and
evaluated as novel non-nucleoside anti-HBV agents. Among them, compounds 5e and 5f could
significantly inhibit HBV DNA replication with IC50 values of 1.54 and 0.71 µM, respectively,
thus displaying potent anti-HBV activities. Notably, 5e and 5f also possessed remarkably
anti-HBV activities against lamivudine and entecavir resistant HBV strain with IC50 values of 1.90
and 0.84 µM, confirming their effectiveness against resistant HBV strain. In addition, molecular
docking studies showed that compounds 5e and 5f could well fit into the dimer-dimer interface of
HBV core protein by hydrophobic interactions. Considering the novel chemical structure and
potent anti-HBV activity against both wild and resistant HBV strains, 5e and 5f might be
warranted for the further investigation as novel non-nucleoside anti-HBV candidates.
5. Experimental protocols
5.1. Chemical analysis
All of the synthesized compounds were purified by column chromatography on silica gel 60
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(Qingdao Ocean Chemical Company, China). Melting points of individual compounds were
determined on a model YRT-3 apparatus and uncorrected. 1H NMR (400 MHz) and 13C NMR (100
MHz) spectra were performed on a JEOL (400 MHz) spectrometer in CDCl3 or DMSO-d6
(Representative NMR spectra were shown in supplementary material). MS detection was recorded
using an Agilent G6125BA single quadrupole mass spectrometer. All solvents were reagent grade
and, when necessary, were purified and dried by standards methods.
5.1.1. General procedure for the synthesis of compounds 1-4
CS2 (2.3 mL, 30 mmol) was added dropwise to the mixture of 2-thiophenemethylamine (30
mmol) and triethylamine (12 mL) in THF (25 mL) at 0 °C within 30 min. After stirring at room
temperature for 1 h, the mixture was cooled down to 0 °C and then add the toluene sulfonyl
chloride (6 g, 31.5 mmol). After stirring at room temperature for 1 h, the mixture was dispersed in
5% HCl (100 mL) and petroleum ether, washed sequentially with water, saturated NaHCO3
solution and brine, dried with anhydrous Na2SO4 and evaporated in vacuo to give compound 1.
HCl (10.5 mL) was added to the mixture of benzene or benzene derivatives (6.65 mmol) and
paraformaldehyde (11.97 mmol). After stirring at 60 °C for 10 h, the mixture was cooled down to
room temperature and then the mixture was dispersed in water and ethyl acetate, washed
sequentially with water, 5% HCl, saturated NaHCO3 solution and brine, dried with anhydrous
Na2SO4. Removal of the solvent gave a residue to afford compound 2. A solution of methyl
anthranilate or methyl anthranilate derivatives (0.01 mol) and 2-(isothiocyanatomethyl)thiophene
(1.55 g, 0.01 mol) in the mixture of triethylamine and ethanol was heated at reflux temperature for
4 h. The reaction mixture was cooled down to room temperature and then filtered to give
intermediate 3, which was then reacted with compound 2 (0.01 mmol) mentioned above in the
presence of K2CO3 and 1,4-dioxane. After reflux for 10 h, the mixture was cooled down to room
temperature and dissolved in ethyl acetate, washed sequentially with water, 5% HCl, saturated
NaHCO3 solution and brine, dried with anhydrous Na2SO4. Removal of the solvent gave a residue
which was recrystallized from ethanol to obtain target compound 4, respectively.
5.1.1.1. 2-((5-acetyl-2-methoxybenzyl)thio)-6-fluoro-3-(thiophen-2-ylmethyl)quinazolin-4(3H)-one
(4a)
1
As a yellow powder, yield: 17%; m.p.158.3-162.8 °C. Analytical data for 4a: H NMR (400
MHz, CDCl3) δ ppm: 8.24 (d, J = 2.0 Hz, 1H, Ar-H), 8.00 (s, 1H, Ar-H), 7.89 (dd, J = 8.4 Hz, 2.4
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Hz, 1H, Ar-H), 7.84 (dd, J = 8.4, 3.2 Hz, 1H, Ar-H), 7.68 (dd, J = 8.8, 4.8 Hz, 1H, Ar-H), 7.44 (m,
1H, Ar-H), 7.20 (m, 1H, Ar-H), 6.91 (m, 2H, Ar-H), 5.42 (s, 2H, CH2), 4.56 (s, 2H, CH2), 3.96 (s,
3H, CH3), 2.50 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3) δ ppm: 196.65, 162.64, 161.58, 155.27,
144.18, 136.98, 132.01, 130.48, 129.93, 128.87, 128.40, 126.47, 126.31, 125.07, 123.35, 123.11,
112.06, 111.83, 110.11, 56.01, 42.52, 31.56, 26.39; ESI-MS: m/z 454.9 [M+H]+.
5.1.1.2.
2-((5-acetyl-2-methoxybenzyl)thio)-6-chloro-3-(thiophen-2-ylmethyl)quinazolin-4(3H)-one (4b)
1
As a white powder, yield: 16%; m.p.170.3-173.6 °C. Analytical data for 4b: H NMR (400
MHz, DMSO, δ ppm): 8.21 (s, 1H, Ar-H), 8.04 (s, 1H, Ar-H), 7.93 (d, J = 8.8 Hz, 1H, Ar-H), 7.88
(d, J = 8.4 Hz, 1H, Ar-H), 7.73 (d, J = 8.4 Hz, 1H, Ar-H), 7.44 (d, J = 5.2 Hz, 1H, Ar-H),
7.13-7.18 (m, 2H, Ar-H), 6.97 (t, 1H, Ar-H), 5.36 (s, 2H, CH2), 4.55 (s, 2H, CH2), 3.95 (s, 3H,
CH3), 2.48 (s, 3H, CH3); 13C NMR (100 MHz, DMSO, δ ppm): 201.44, 167.58, 166.48, 164.82,
162.05, 150.70, 142.23, 140.39, 136.69, 135.74, 136.48, 134.62, 133.76, 133.47, 131.85, 130.76,
129.45, 125.22, 116.12, 61.51, 41.07, 36.06, 31.68; ESI-MS: m/z 470.9 [M+H]+.
5.1.1.3.
2-((5-acetyl-2-methoxybenzyl)thio)-6-bromo-3-(thiophen-2-ylmethyl)quinazolin-4(3H)-one (4c)
1
As a yellow powder, yield: 15%; m.p.143.3-150.6 °C. Analytical data for 4c: H NMR (400
MHz, CDCl3, δ ppm): 8.33 (d, J = 2.4 Hz, 1H, Ar-H), 8.23 (d, J = 2.0 Hz, 1H, Ar-H), 8.00 (s, 1H,
Ar-H), 7.88 (dd, J = 8.8 Hz, 2.0 Hz, 1H, Ar-H), 7.78 (dd, J = 8.4, 2.0 Hz, 1H, Ar-H), 7.55 (d, J =
8.8 Hz, 1H, Ar-H), 7.20 (m, 1H, Ar-H), 6.91 (m, 2H, Ar-H), 5.41 (s, 2H, CH2), 4.55 (s, 2H, CH2),
3.96 (s, 3H, CH3), 2.50 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3, δ ppm): 196.64, 162.65,
161.57, 160.53, 156.63, 146.23, 137.80, 136.87, 132.02, 130.51, 129.93, 129.68, 128.91, 127.96,
126.41, 124.94, 120.84, 119.04, 110.12, 56.01, 42.55, 31.61, 26.40; ESI-MS: m/z 514.8 [M+H]+.
5.1.1.4. 2-((5-acetyl-2-methoxybenzyl)thio)-6-iodo-3-(thiophen-2-ylmethyl)quinazolin-4(3H)-one
(4d)
1
As a white powder, yield: 16%; m.p.170.3-177.6 °C. Analytical data for 4d: H NMR (400
MHz, CDCl3, δ ppm): 8.54 (s, 1H, Ar-H), 8.23 (br, 1H, Ar-H), 7.96 (d, J = 8.4 Hz, 1H, Ar-H), 7.89
(d, J = 6.8 Hz, 1H, Ar-H), 7.42 (d, J = 8.8 Hz, 1H, Ar-H), 7.20 (br, 2H, Ar-H), 6.90-6.93 (m, 2H,
Ar-H), 5.41 (s, 2H, CH2), 4.56 (s, 2H, CH2), 3.96 (s, 3H, CH3), 2.51 (s, 3H, CH3); 13C NMR (100
MHz, CDCl3, δ ppm): 196.67, 161.57, 160.29, 156.83, 146.69, 143.37, 136.88, 135.98, 132.04,
12

ACCEPTED MANUSCRIPT
130.51, 129.93, 128.90, 128.00, 126.48, 126.35, 124.95, 121.16, 116.31, 110.12, 56.02, 42.56,
31.61, 26.41; ESI-MS: m/z 563.2 [M+H]+.
5.1.1.5.
2-((5-acetyl-2-methoxybenzyl)thio)-6-methyl-3-(thiophen-2-ylmethyl)quinazolin-4(3H)-one (4e)
1
As a white powder, yield: 6%; m.p.189.5-191.0 °C. Analytical data for 4e: H NMR (400
MHz, CDCl3, δ ppm): 8.25 (d, J = 2.4 Hz, 1H, Ar-H), 7.98 (d, J = 8.4 Hz, 1H, Ar-H), 7.90 (dd, J =
8.8 Hz, 2.4 Hz, 1H, Ar-H), 7.61 (d, J = 8.4 Hz, 1H, Ar-H), 7.54 (dd, J = 8.4 Hz, 2.0 Hz, 1H, Ar-H),
7.17-7.20 (m, 2H, Ar-H), 6.88-6.92 (m, 2H, Ar-H), 5.41 (s, 2H, CH2), 4.58 (s, 2H, CH2), 3.95 (s,
3H, CH3), 2.49 (s, 3H, CH3), 2.43 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3, δ ppm): 196.76,
161.70, 161.56, 154.87, 145.36, 137.36, 136.16, 136.04, 132.13, 130.31, 129.87, 128.69, 126.56,
126.41, 126.19, 125.84, 125.22, 119.16, 110.13, 56.00, 42.40, 31.45, 26.47, 21.33; ESI-MS: m/z
450.9 [M+H]+.
5.1.1.6.
2-((5-acetyl-2-methoxybenzyl)thio)-6-hydroxy-3-(thiophen-2-ylmethyl)quinazolin-4(3H)-one (4f)
1
As a yellow powder, yield: 12%; m.p.218.5-220.0 °C. Analytical data for 4f: H NMR (400
MHz, CDCl3, δ ppm): 8.20 (d, J = 2.4 Hz, 1H, Ar-H), 7.91 (dd, J =8.8, 2.4 Hz, 1H, Ar-H), 7.57 (d,
J = 8.8 Hz, 1H, Ar-H), 7.38-7.40 (m, 2H, Ar-H), 7.28 (dd, J = 8.8 Hz, 3.2 Hz, 1H, Ar-H), 7.15 (dd,
J = 7.6 Hz, 1.2 Hz, 1H, Ar-H), 7.12 (d, J = 8.8 Hz, 1H, Ar-H), 6.94 (dd, J = 4.8 Hz, 3.2 Hz, 1H,
Ar-H), 5.35 (s, 2H, CH2), 4.51 (s, 2H, CH2), 3.95 (s, 3H, CH3), 2.48 (s, 3H, CH3); 13C NMR (100
MHz, CDCl3, δ ppm): 196.52, 161.67, 160.87, 156.18, 152.37, 140.79, 138.01, 131.85, 130.77,
129.78, 128.67, 128.09, 126.93, 126.89, 125.05, 124.82, 120.21, 111.18, 109.77, 55.31, 49.15,
31.23, 26.86; ESI-MS: m/z 452.9 [M+H]+.
5.1.1.7. 2-((2-methoxybenzyl)thio)-3-(thiophen-2-ylmethyl)quinazolin-4(3H)-one (4g)
1
As a white powder, yield: 6%; m.p.134.4-136.6 °C. Analytical data for 4g: H NMR (400
MHz, CDCl3, δ ppm): 8.22 (br, 1H, Ar-H), 7.68 (br, 1H, Ar-H), 7.62 (br, 1H, Ar-H), 7.51 (br, 1H,
Ar-H), 7.37 (br, 1H, Ar-H), 7.23-7.26 (m, 3H, Ar-H), 6.90 (br, 3H, Ar-H), 5.44 (s, 2H, CH2), 4.60
(s, 2H, CH2), 3.89 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3, δ ppm): 161.76, 157.81, 156.44,
147.46, 137.34, 134.55, 131.21, 129.28, 128.78, 127.18, 126.42, 126.20, 126.12, 125.77, 124.66,
120.53, 119.47, 110.65, 55.63, 42.45, 31.84; ESI-MS: m/z 395.1 [M+H]+.
5.1.1.8. 6-fluoro-2-((2-methoxybenzyl)thio)-3-(thiophen-2-ylmethyl)quinazolin-4(3H)-one (4h)
13

ACCEPTED MANUSCRIPT
1
As a white powder, yield: 23%; m.p.116.1-122.8 °C. Analytical data for 4h: H NMR (400
MHz, CDCl3, δ ppm): 7.86 (dd, J = 8.4 Hz, 2.8 Hz, 1H, Ar-H), 7.61 (dd, J = 8.8 Hz, 4.8 Hz, 1H,
Ar-H), 7.49 (d, J = 7.2 Hz, 1H, Ar-H), 7.39-7.44 (m, 1H, Ar-H), 7.18-7.29 (m, 3H, Ar-H),
6.87-6.92 (m, 3H, Ar-H), 5.43 (s, 2H, CH2), 4.57 (s, 2H, CH2), 3.89 (s, 3H, CH3); 13C NMR (100
MHz, CDCl3, δ ppm): 157.82, 155.78, 144.28, 137.06, 131.16, 129.35, 128.93, 128.49, 128.40,
126.46, 126.32, 124.48, 123.19, 122.95, 120.53, 112.05, 111.81, 110.68, 55.63, 42.53, 31.89;
ESI-MS: m/z 413.2 [M+H]+.
5.1.1.9. 6-chloro-2-((2-methoxybenzyl)thio)-3-(thiophen-2-ylmethyl)quinazolin-4(3H)-one (4i)
1
As a white powder, yield: 25%; m.p.114.8-122.9 °C. Analytical data for 4i: H NMR (400
MHz, CDCl3, δ ppm): 8.18 (d, J = 2.4 Hz, 1H, Ar-H), 7.61 (dd, J = 8.8 Hz, 2.4 Hz, 1H, Ar-H),
7.54 (d, J = 8.8 Hz, 1H, Ar-H), 7.47 (d, J = 8.0 Hz, 1H, Ar-H), 7.28 (d, J = 6.4 Hz, 1H, Ar-H),
7.19-7.22 (m, 2H, Ar-H), 6.87-6.91 (m, 3H, Ar-H), 5.42 (s, 2H, CH2), 4.57 (s, 2H, CH2), 3.89 (s,
3H, CH3); 13C NMR (100 MHz, CDCl3, δ ppm): 156.01, 153.05, 152.20, 141.24, 132.20, 130.17,
126.48, 126.43, 124.64, 124.22, 123.06, 121.71, 121.62, 119.60, 115.78, 115.68, 105.91, 105.52,
50.88, 37.79, 27.19; ESI-MS: m/z 428.8 [M+H]+.
5.1.1.10. 6-bromo-2-((2-methoxybenzyl)thio)-3-(thiophen-2-ylmethyl)quinazolin-4(3H)-one (4j)
1
As a white powder, yield: 25%; m.p.134.2-139.3 °C. Analytical data for 4j: H NMR (400
MHz, CDCl3, δ ppm): 8.35 (dd, J = 6.8 Hz, 2.8 Hz, 1H, Ar-H), 7.75 (dd, J = 8.4 Hz, 2.0 Hz, 1H,
Ar-H), 7.47-7.49 (m, 2H, Ar-H), 7.20-7.30 (m, 3H, Ar-H), 6.87-6.93 (m, 3H, Ar-H), 5.42 (s, 2H,
CH2), 4.57 (s, 2H, CH2), 3.89 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3, δ ppm): 159.91, 157.94,
157.66, 146.20, 138.25, 137.47, 131.52, 129.91, 129.03, 128.91 (2C), 127.19, 127.08, 123.96,
120.79, 120.77, 118.68, 111.58, 56.13, 42.94, 31.90; ESI-MS: m/z 472.8 [M+H]+.
5.1.1.11. 6-iodo-2-((2-methoxybenzyl)thio)-3-(thiophen-2-ylmethyl)quinazolin-4(3H)-one (4k)
1
As a gray powder, yield: 7%; m.p.126.6-131.7 °C. Analytical data for 4k: H NMR (400
MHz, CDCl3, δ ppm): 8.55 (s, 1H, Ar-H), 7.93 (d, J = 8.0 Hz, 1H, Ar-H), 7.48 (d, J = 8.0 Hz, 2H,
Ar-H), 7.35 (d, J = 8.0 Hz, 1H, Ar-H), 7.28 (d, J = 8.0 Hz, 1H, Ar-H), 7.20-7.22 (m, 2H, Ar-H),
6.87-6.92 (m, 3H, Ar-H), 5.42 (s, 2H, CH2), 4.58 (s, 2H, CH2), 3.89 (s, 3H, CH3); 13C NMR (100
MHz, CDCl3, δ ppm): 160.31, 157.82, 157.40, 146.73, 143.19, 136.95, 135.95, 131.19, 129.40,
128.98, 128.03, 126.46, 126.38, 124.35, 121.14, 120.55, 110.68, 89.46, 55.64, 42.56, 31.99;
ESI-MS: m/z 520.8 [M+H]+.
14

ACCEPTED MANUSCRIPT
5.1.1.12. 2-((2-methoxybenzyl)thio)-6-methyl-3-(thiophen-2-ylmethyl)quinazolin-4(3H)-one (4l)
1
As a white powder, yield: 32%; m.p. 108.1-110.2 °C. Analytical data for 4l: H NMR (400
MHz, CDCl3, δ ppm): 8.02 (s, 1H, Ar-H), 7.50-7.56 (m, 3H, Ar-H), 7.27 (d, J = 8.0 Hz, 1H, Ar-H),
7.18-7.22 (m, 2H, Ar-H), 6.86-6.90 (m, 3H, Ar-H), 5.44 (s, 2H, CH2), 4.61 (s, 2H, CH2), 3.88 (s,
3H, CH3); 13C NMR (100 MHz, CDCl3, δ ppm): 161.76, 157.82, 155.42, 145.47, 137.48, 136.00,
135.86, 131.22, 129.26, 128.72, 126.67, 126.39, 126.18, 125.91, 124.74, 120.54, 119.17, 110.65,
55.63, 42.43, 31.85, 21.34; ESI-MS: m/z 408.9 [M+H]+.
5.1.1.13. 6-hydroxy-2-((2-methoxybenzyl)thio)-3-(thiophen-2-ylmethyl)quinazolin-4(3H)-one (4m)
As a yellow powder, yield: 28%; m.p. 113.0-115.0 °C. Analytical data for 4m: 1H NMR (400
MHz, DMSO, δ ppm): 7.50 (d, J = 8.8 Hz, 1H, Ar-H), 7.50 (dd, J = 7.2 Hz, 1.6 Hz, 1H, Ar-H),
7.37-7.38 (m, 2H, Ar-H), 7.21-7.27 (m, 2H, Ar-H), 7.10 (d, J = 1.6 Hz, 1H, Ar-H), 6.99 (d, J = 8.1
Hz, 1H, Ar-H), 6.82-6.91 (m, 3H, Ar-H), 5.30 (s, 2H, CH2), 4.44 (s, 2H, CH2), 3.79 (s, 3H, CH3);
13C NMR (100 MHz, DMSO, δ ppm): 160.90, 157.93, 156.27, 152.70, 140.81, 138.14, 131.40,
129.76, 128.68, 128.19, 127.03, 126.95, 124.94, 124.41, 120.78, 120.17, 111.59, 109.79, 55.45,
42.65, 31.56; ESI-MS: m/z 410.9 [M+H]+.
5.1.1.14. 2-(benzylthio)-3-(thiophen-2-ylmethyl)quinazolin-4(3H)-one (4n)
1
As a white powder, yield: 17%; m.p.99.1-101.3 °C. Analytical data for 4n: H NMR (400
MHz, DMSO, δ ppm): 8.25 (dd, J = 8.8 Hz, 1.2 Hz, 1H, Ar-H), 7.71 (t, 1H, Ar-H), 7.59 (d, J = 7.6
Hz, 1H, Ar-H), 7.46-7.48 (m, 2H, Ar-H), 7.28-7.40 (m, 4H, Ar-H), 7.20-7.23 (m, 2H, Ar-H), 6.93
(t, 1H, Ar-H), 5.46 (s, 2H, CH2), 4.56 (s, 2H, CH2); 13C NMR (100 MHz, DMSO, δ ppm): 162.56,
157.14, 146.94, 146.68, 141.15, 134.38, 133.83, 131.09, 130.44, 128.55, 128.26 (2C), 128.00 (3C),
127.76 (2C), 126.77, 43,83, 25.42; ESI-MS: m/z 364.9 [M+H]+.
5.1.1.15. 2-(benzylthio)-6-fluoro-3-(thiophen-2-ylmethyl)quinazolin-4(3H)-one (4o)
1
As a white powder, yield: 8%; m.p.157.2-167.1 °C. Analytical data for 4o: H NMR (400
MHz, CDCl3, δ ppm): 7.87 (dd, J = 8.4 Hz, 2.8 Hz, 1H, Ar-H), 7.58 (dd, J = 8.8 Hz, 4.8 Hz, 1H,
Ar-H), 7.47 (d, J = 6.8 Hz, 2H, Ar-H), 7.28-7.47 (m, 4H, Ar-H), 7.21-7.25 (m, 2H, Ar-H), 6.92 (t,
1H, Ar-H), 5.45 (s, 2H, CH2), 4.55 (s, 2H, CH2); 13C NMR (100 MHz, CDCl3, δ ppm): 160.46,
155.81, 144.21, 137.59, 137.06, 129.96 (2C), 129.44, 129.36, 128.99 (3C), 127.99, 127.19, 127.13,
124.17, 123.93, 111.80, 43.00, 36.30; ESI-MS: m/z 382.9 [M+H]+.
5.1.1.16. 2-(benzylthio)-6-chloro-3-(thiophen-2-ylmethyl)quinazolin-4(3H)-one (4p)
15

ACCEPTED MANUSCRIPT
As a yellow powder, yield: 18%; m.p.139.6-145.5 °C. Analytical data for 4p: 1H NMR (400
MHz, CDCl3, δ ppm): 8.20 (d, J = 2.0 Hz, 1H, Ar-H), 7.62 (dd, J = 8.8 Hz, 2.4 Hz, 1H, Ar-H),
7.52 (d, J = 8.8 Hz, 1H, Ar-H), 7.46 (d, J = 7.2 Hz, 2H, Ar-H), 7.28-7.35 (m, 3H, Ar-H), 7.22 (d, J
= 4.4 Hz, 2H, Ar-H), 6.92 (t, 1H, Ar-H), 5.45 (s, 2H, CH2), 4.55 (s, 2H, CH2); 13C NMR (100 MHz,
DMSO, δ ppm): 160.09, 157.10, 145.93, 137.49, 136.96, 135.64, 130.74, 129.97 (2C), 129.00
(3C), 128.81, 128.02, 127.22, 127.14, 125.99, 120.48, 43.06, 36.36; ESI-MS: m/z 437.0 [M+K]+.
5.1.1.17. 2-(benzylthio)-6-bromo-3-(thiophen-2-ylmethyl)quinazolin-4(3H)-one (4q)
1
As a white powder, yield: 16%; m.p.145.3-147.6 °C. Analytical data for 4q: H NMR (400
MHz, CDCl3, δ ppm): 8.35 (d, J = 2.0 Hz, 1H, Ar-H), 7.76 (dd, J = 8.0 Hz, 2.0 Hz, 1H, Ar-H),
7.45 (d, J = 6.8 Hz, 3H, Ar-H), 7.21-7.34 (m, 5H, Ar-H), 6.92 (t, 1H, Ar-H), 5.44 (s, 2H, CH2),
4.54 (s, 2H, CH2); 13C NMR (100 MHz, DMSO, δ ppm): 159.95, 157.22, 146.18, 138.34, 137.48,
136.94, 129.98 (2C), 129.08, 129.00 (3C), 128.95, 128.02, 127.22, 127.14, 120.85, 118.80, 43.07,
36.37; ESI-MS: m/z 465.1 [M+Na]+.
5.1.1.18. 2-(benzylthio)-6-iodo-3-(thiophen-2-ylmethyl)quinazolin-4(3H)-one (4r)
1
As a white powder, yield: 13%; m.p.144.6-145.2 °C. Analytical data for 4r: H NMR (400
MHz, CDCl3, δ ppm): 8.56 (d, J = 1.6 Hz, 1H, Ar-H), 7.93 (dd, J = 8.4 Hz, 2.0 Hz, 1H, Ar-H),
7.45 (d, J = 6.8 Hz, 2H, Ar-H), 7.27-7.34 (m, 4H, Ar-H), 7.20-7.22 (m, 2H, Ar-H), 6.91 (t, 1H,
Ar-H), 5.43 (s, 2H, CH2), 4.54 (s, 2H, CH2); 13C NMR (100 MHz, DMSO, δ ppm): 159.73, 157.17,
146.44, 143.80, 137.51, 136.93, 135.20, 129.98 (2C), 129.00 (3C), 128.71, 128.02, 127.22, 127.13,
121.06, 91.27, 43.03, 36.37; ESI-MS: m/z 490.8 [M+H]+.
5.1.1.19. 2-(benzylthio)-6-methyl-3-(thiophen-2-ylmethyl)quinazolin-4(3H)-one (4s)
1
As a white powder, yield: 18%; m.p.151.9-152.7 °C. Analytical data for 4s: H NMR (400
MHz, CDCl3, δ ppm): 8.04 (s, 1H, Ar-H), 7.46-7.50 (m, 4H, Ar-H), 7.21-7.33 (m, 5H, Ar-H), 6.91
(br, 1H, Ar-H), 5.46 (s, 2H, CH2), 4.56 (s, 2H, CH2), 2.45 (s, 3H, CH3); 13C NMR (100 MHz,
CDCl3, δ ppm): 161.73, 154.54, 145.53, 137.44, 136.43, 136.03 (2C), 129.53 (2C), 128.74 (3C),
127.75, 126.61, 126.48, 126.27, 126.03, 119.31, 42.52, 36.97, 21.37; ESI-MS: m/z 378.9 [M+H]+.
5.1.1.20. 2-(benzylthio)-6-hydroxy-3-(thiophen-2-ylmethyl)quinazolin-4(3H)-one (4t)
1
As a white powder, yield: 15%; m.p.213.8-215.0 °C. Analytical data for 4t: H NMR (400
MHz, CDCl3, δ ppm): 10.01 (s, 1H, OH), 7.45-7.47 (m, 3H, Ar-H), 7.39 (dd, J = 5.2 Hz, 1.2 Hz,
1H, Ar-H), 7.35 (d, J = 3.6 Hz, 1H, Ar-H),7.19-7.30 (m, 4H, Ar-H), 7.12 (d, J = 3.6 Hz, 1H, Ar-H),
16

ACCEPTED MANUSCRIPT
6.92 (dd, J = 5.2 Hz, 3.6 Hz, 1H, Ar-H), 5.33 (s, 2H, CH2), 4.48 (s, 2H, CH2); 13C NMR (100 MHz,
CDCl3, δ ppm): 160.88, 156.22, 152.29, 140.79, 138.10, 137.29, 129.92 (2C), 128.96 (2C), 128.72,
128.26, 127.91, 127.08, 127.04, 124.92, 120.23, 109.75, 42.76, 36.16; ESI-MS: m/z 381.2
[M+H]+.
5.1.2. General procedure for the synthesis of compounds 5a-f
Finally, Compound 4f, 4m or 4t (0.01 mol) was alkylated with 2-dimethylaminoethyl chloride
hydrochloride or 2-diethylaminoethyl chloride hydrochloride (0.012 mol) in the presence of
K2CO3 and 1,4-dioxane to provide alkoxyl substituted derivative 5a-f. The crude product was
further purified via column chromatography to produce the title compound, respectively.
5.1.2.1.
2-((5-acetyl-2-methoxybenzyl)thio)-6-(2-(dimethylamino)ethoxy)-3-(thiophen-2-ylmethyl)quinazoli
n-4(3H)-one (5a)
1
As a white powder, yield: 10%; m.p.70.2-72.8 °C. Analytical data for 5a: H NMR (400
MHz, CDCl3, δ ppm): 8.23 (d, J = 2.4 Hz, 1H, Ar-H), 7.89 (dd, J = 8.8 Hz, 2.4 Hz, 1H, Ar-H),
7.61 (d, J =9.2 Hz, 1H, Ar-H), 7.57 (d, J = 2.8 Hz, 1H, Ar-H), 7.35 (dd, J = 8.8 Hz, 2.8 Hz, 1H,
Ar-H), 7.18-7.25 (m, 2H, Ar-H), 6.88-6.92 (m, 2H, Ar-H), 5.41 (s, 2H, CH2), 4.54 (s, 2H, CH2),
4.32 (t, 2H, OCH2), 3.95 (s, 3H, CH3), 3.09 (t, 2H, NCH2), 2.59 (s, 6H, CH3), 2.49 (s, 3H, CH3);
13C NMR (100 MHz, CDCl3, δ ppm): 196.77, 161.58, 161.53, 155.96, 153.68, 142.72, 137.23,
132.06, 130.36, 129.87, 128.77, 127.97, 126.45, 126.25, 125.20, 125.00, 120.06, 110.12, 107.58,
64.71, 57.13, 56.01, 44.67 (2C), 42.52, 31.41, 26.46; ESI-MS: m/z 524.3 [M+H]+.
5.1.2.2.
2-((5-acetyl-2-methoxybenzyl)thio)-6-(2-(diethylamino)ethoxy)-3-(thiophen-2-ylmethyl)quinazolin
-4(3H)-one (5b)
As a yellow oil, yield: 9%. Analytical data for 5b: 1H NMR (400 MHz, CDCl3, δ ppm): 8.24
(d, J = 2.4 Hz, 1H, Ar-H), 7.91 (dd, J = 8.4 Hz, 2.0 Hz, 1H, Ar-H), 7.61 (m, 2H, Ar-H), 7.34 (dd, J
= 8.4 Hz, 3.2 Hz, 1H, Ar-H), 7.18-7.22 (m, 2H, Ar-H), 6.89-6.93 (m, 2H, Ar-H), 5.42 (s, 2H, CH2),
4.55 (s, 2H, CH2), 4.16 (t, 2H, OCH2), 3.96 (s, 3H, CH3), 2.94 (t, 2H, NCH2), 2.65-2.70 (m, 4H,
N(CH2CH3)2), 2.50 (s, 3H, CH3), 1.09 (t, 6H, N(CH2CH3)2); 13C NMR (100 MHz, CDCl3, δ ppm):
196.76, 162.64, 161.66, 161.57, 156.86, 153.18, 142.34, 137.35, 132.09, 130.30, 129.89, 128.73,
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127.69, 126.43, 126.21, 125.28, 120.07, 110.13, 107.25, 66.70, 56.00, 51.45, 47.83 (2C), 42.50,
31.37, 26.46, 11.72 (2C); ESI-MS: m/z 552.3 [M+H]+.
5.1.2.3.
6-(2-(dimethylamino)ethoxy)-2-((2-methoxybenzyl)thio)-3-(thiophen-2-ylmethyl)quinazolin-4(3H)-
one (5c)
1
As a yellow powder, yield: 25%; m.p.82.5-83.2 °C. Analytical data for 5c: H NMR (400
MHz, DMSO, δ ppm): 7.57 (d, J = 8.8 Hz, 1H, Ar-H), 7.45-7.47 (m, 2H, Ar-H), 7.38-7.41 (m, 2H,
Ar-H), 7.22-7.26 (m, 1H, Ar-H), 7.11 (d, J = 3.2 Hz, 1H, Ar-H), 6.99 (d, J = 8.0 Hz, 1H, Ar-H),
6.91 (dd, J = 5.2 Hz, 3.6 Hz, 1H, Ar-H), 6.84 (t, 1H, Ar-H), 5.33 (s, 2H, CH2), 4.45 (s, 2H, CH2),
4.15 (t, 2H, OCH2), 3.80 (s, 3H, CH3), 2.69 (t, 2H, NCH2), 2.24 (s, 6H, CH3); 13C NMR (100 MHz,
DMSO, δ ppm): 160.87, 157.94, 156.90, 153.97, 142.04, 137.94, 131.46, 129.82, 128.82, 128.32,
127.05 (2C), 125.34, 124.30, 120.79, 119.92, 111.60, 107.65, 66.59, 57.90, 56.13, 45.85 (2C),
42.77, 31.64; ESI-MS: m/z 482.2 [M+H]+.
5.1.2.4.
6-(2-(diethylamino)ethoxy)-2-((2-methoxybenzyl)thio)-3-(thiophen-2-ylmethyl)quinazolin-4(3H)-o
ne (5d)
1
As a yellow oil, yield: 25%. Analytical data for 5d: H NMR (400 MHz, DMSO, δ ppm):
7.57 (d, J = 8.8 Hz, 1H, Ar-H), 7.45-7.47 (m, 2H, Ar-H), 7.37-7.39 (m, 2H, Ar-H), 7.24 (t, 1H,
Ar-H), 7.11 (d, J = 2.4 Hz, 1H, Ar-H), 6.99 (d, J = 8.0 Hz, 1H, Ar-H), 6.91 (dd, J = 5.2 Hz, 3.6 Hz,
1H, Ar-H), 6.85 (t, 1H, Ar-H), 5.33 (s, 2H, CH2), 4.45 (s, 2H, CH2), 4.10 (t, 2H, OCH2), 3.80 (s,
3H, CH3), 2.80 (t, 2H, NCH2), 2.55 (m, 4H, N(CH2CH3)2), 0.95 (t, 6H, N(CH2CH3)2); 13C NMR
(100 MHz, DMSO, δ ppm): 162.85, 160.87, 157.93, 156.97, 153.94, 141.99, 137.93, 131.46,
129.83, 128.82, 128.32, 127.06, 125.32, 124.28, 120.79, 119.91, 111.59, 107.66, 56.13, 55.45,
51.61, 47.52 (2C), 42.77, 31.64, 12.25 (2C); ESI-MS: m/z 510.3 [M+H]+.
5.1.2.5. 2-(benzylthio)-6-(2-(dimethylamino)ethoxy)-3-(thiophen-2-ylmethyl)quinazolin-4(3H)-one
(5e)
As a white powder, yield: 13%; m.p.75.8-77.2 °C. Analytical data for 5e: 1H NMR (400 MHz,
CDCl3, δ ppm): 7.55 (d, J = 8.8 Hz, 1H, Ar-H), 7.45-7.47 (m, 3H, Ar-H), 7.37-7.40 (m, 2H, Ar-H),
7.25-7.30 (m, 2H, Ar-H), 7.19-7.23 (m, 1H, Ar-H), 7.13 (dd, J = 3.6 Hz, 1.2 Hz, 1H, Ar-H), 6.92
(dd, J = 5.2 Hz, 3.2 Hz, 1H, Ar-H), 5.35 (s, 2H, CH2), 4.50 (s, 2H, CH2), 4.14 (t, 2H, OCH2), 2.67
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(t, 2H, NCH2), 2.20 (s, 6H, CH3); 13C NMR (100 MHz, DMSO, δ ppm): 160.87, 156.98, 153.48,
141.96, 137.91, 137.22, 129.95 (2C), 128.97(2C), 128.83, 128.32, 127.94, 127.10 (2C), 125.38,
119.97, 107.63, 66.65, 57.90, 45.89 (2C), 42.87, 36.20; ESI-MS: m/z 452.3 [M+H]+.
5.1.2.6. 2-(benzylthio)-6-(2-(diethylamino)ethoxy)-3-(thiophen-2-ylmethyl)quinazolin-4(3H)-one
(5f)
1
As a yellow powder, yield: 13%; m.p.108.8-110.2 °C. Analytical data for 5f: H NMR (400
MHz, CDCl3, δ ppm): 7.55 (d, J = 8.8 Hz, 1H, Ar-H), 7.45-7.48 (m, 3H, Ar-H), 7.36-7.40 (m, 2H,
Ar-H), 7.26-7.30 (m, 2H, Ar-H), 7.20-7.24 (m, 1H, Ar-H), 7.13 (d, J = 2.4 Hz, 1H, Ar-H), 6.92 (dd,
J = 5.2 Hz, 3.6 Hz, 1H, Ar-H), 5.35 (s, 2H, CH2), 4.50 (s, 2H, CH2), 4.08 (t, 2H, OCH2), 2.76 (t,
2H, NCH2), 2.49-2.54 (m, 4H, N(CH2CH3)2), 0.94 (t, 6H, CH3); 13C NMR (100 MHz, CDCl3, δ
ppm): 165.63, 161.84, 158.19, 146.67, 142.68, 141.97, 134.70 (2C), 133.73 (2C), 133.59, 133.06,
132.70, 131.85(2C), 130.09, 124.74, 112.40, 72.20, 56.42, 52.27 (2C), 47.62, 40.97, 17.17 (2C);
ESI-MS: m/z 480.3 [M+H]+.
5.2. Biological assays
HepG2 cell lines, provided by 302 military hospital of China, were 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 cytotoxicity and anti-HBV activities of the target
compounds were evaluated on HepG2 2.2.15 cell line, which was a stably transfected cell line
containing HBV genome on a plasmid. Antiviral efficacy against nucleoside analogue-resistant
HBV was conducted on HepG2 cell line transiently transfected with plasmids containing
drug-resistant mutant (rtL180M + rtM204V + rtT184L).
5.2.1. Cytotoxicity assay
The cytotoxicity of the target compounds against HepG2 2.2.15 cells was determined by MTT
assay. Briefly, HepG2 2.2.15 cells were seeded in 96-well culture plates and then cultured for 24 h.
The exponentially growing cells were incubated with test compounds (in 0.2 mL culture
medium/well) for 72 h, respectively. The cytotoxicity was determined by analyzing MTT
(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) absorbance at 490 nm, as
determined from the absorbance relative to untreated cells.
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5.2.2. Determination the effect on HBV DNA replication
HepG2 2.2.15 cells (2×105 cells/well) were seeded in 24 well culture plates and cultured for 24
h. Then, culture medium was replaced by assay medium containing the test compound or positive
control 3TC at the indicated concentrations. The medium was changed every 2 days. After 6 days
incubation, the intracellular HBV DNA was extracted according to the method described
previously [24], and the HBV DNA levels were quantified by RT-PCR.
5.2.3. Antiviral efficacy against nucleoside analogue-resistant HBV strain [25]
HepG2 cells, transiently transfected respectively with plasmids containing drug-resistant mutant
(rtL180M + rtM204V + rtT184L) were selected as an evaluation model [26]. Briefly, HepG2 cells
(2×105) were plated into 24-well plates. After 24 incubation, the supernatant was discarded,
followed by PBS wash. The cells were then incubated with fresh medium containing 5e (4 µM), 5f
(4 µM), lamivudine (100 µM) or Entecavir (10 µM), respectively. After 72 h incubation, HBV
DNA replicative intermediate was extracted according to the method mentioned above [24], and
the level was quantified by RT- PCR.
5.3. Molecular docking
Surflex molecular docking module in Sybyl X-2.1 (SYBYL_X2.1 is availabe from Tripos
Associates Inc., S Hanley Rd., St. Louis, MO 631444, USA) was used for conducting molecular
docking based virtual screening and investigating the possible binding interactions between HBV
core protein and its inhibitors. The crystal structure of HBV core protein complex with
NVR-010-001-E2 (PDB ID: 5E0I) was obtained from the RCSB Protein Data Bank [23]. The
ChemDiv database, a commercially available small molecule database from TopScience Co.
(Shanghai, China), was consulted as a screening library. Because only 2D structural information
was available, the compounds in the ChemDiv database were preprocessed by the dbtranslate
module in Sybyl-X2.1. The space where ligand NVR-010-001-E2 placed was selected as the
binding site of inhibitor, and all water molecules were removed. The missing hydrogen atoms
were added by biopolymer module during protein preparation, the docking parameters were kept
as default values. The binding affinity of inhibitors were evaluated by docking score.
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Acknowledgements
This work was supported by the grants from the National Natural Science Foundation of China
(NSFC No. 81703359 and 21708033), the Natural Science Foundation of Jiangsu province (No.
BK20181151 and BK20171179), the Foundation for Jiangsu Six Talent Peaks Program (Grant No.
2017-YY-039) and Jiangsu Provincial 333 High-level Talents Cultivation Project.
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Highlights
1. Novel quinazolinone derivatives were synthesized and evaluated as anti-HBV agents.
2. 5e and 5f exhibited potent anti-HBV activity against wild and resistant HBV strains.
3. 5e and 5f could well fit into the dimer-dimer interface of HBV core protein.