Design, synthesis, and evaluation of novel heteroaryldihydropyrimidine derivatives as non-nucleoside hepatitis B virus inhibitors by exploring the solvent-exposed region

Article abstract of DOI:10.1111/cbdd.13512

In continuation of our efforts toward the discovery of potent non-nucleoside hepatitis B virus (HBV) inhibitors with novel structures, we have explored the solvent-exposed protein region of heteroaryldihydropyrimidine derivatives. Herein, the morpholine ring of GLS4 was replaced with substituted sulfonamides and triazoles to generate novel non-nucleoside HBV inhibitors with desirable potency. In in vitro biological evaluation, several derivatives showed good anti-HBV DNA replication activity compared to lamivudine. In particular, compound II-1 displayed the most potent activity against HBV DNA replication (IC₅₀?=?0.35?±?0.04?μM). The preliminary structure–activity relationships of the new compounds were summarized, which may help in discovering more potent anti-HBV agents via rational drug design.

Full text of DOI:10.1111/cbdd.13512

DR JI YU (Orcid ID : 0000-0001-6868-1218)
PROFESSOR XINYONG LIU (Orcid ID : 0000-0002-5833-3807)
Article type
: Research Article
Design, Synthesis and Evaluation of Novel Heteroaryldihydropyrimidines
Derivatives as Non-nucleoside Hepatitis B Virus Inhibitors by Exploring
the Solvent-exposed Region
Ji Yu,† Xiaowei Guo,| Samuel Desta,† Shuo Zhang,† Jian Zhang,† Xiao Ding,† Xiaohong
Liang,*,| Haiyong Jia,*,§ Xinyong Liu*,† and Peng Zhan*,†
†
Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of
Education), School of Pharmaceutical Sciences, Shandong University, 44 West Culture Road,
250012 Jinan, Shandong, PR China
|
Department of Immunology, Key Laboratory for Experimental, Teratology of Ministry of
Education, Shandong Provincial Key Laboratory of Infection and Immunology, Shandong
University School of Medicine, Jinan 250012, Shandong Province, PR China
§ School of Pharmacy, Weifang Medical University, 261053 Weifang, Shandong, PR China
AUTHOR INFORMATION
Corresponding Authors
*Xiaohong, Liang.: e-mail, liangxiaohong@sdu.edu.cn.
*Haiyong, Jia.: e-mail, 502378774@163.com; phone, 15063389092.
*Xinyong, Liu.: e-mail, xinyongl@sdu.edu.cn; phone, 086-531-88380270.
*Peng, Zhan.: e-mail, zhanpeng1982@sdu.edu.cn; phone, 086-531-88382005
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13512
This article is protected by copyright. All rights reserved.

ABSTRACT
In continuation of our efforts toward the discovery of potent non-nucleoside HBV
inhibitors with novel structures, we have explored the solvent-exposed protein region of
heteroaryldihydropyrimidines (HAPs) derivatives. Herein, the morpholine ring of GLS4 was
replaced with substituted sulfonamides and triazoles to generate novel non-nucleoside HBV
inhibitors with desirable potency. In in vitro biological evaluation, several derivatives showed
good anti-HBV DNA replication activity compared to lamivudine. In particular, compound
II-1 displayed the most potent activity against HBV DNA replication (IC50 = 0.35 ± 0.04 µM).
The preliminary structure-activity relationships (SARs) of the new compounds were
summarized, which may help in discovering more potent anti-HBV agents via rational drug
design.
KEYWORDs: HBV, capsid, HAP, sulfonamide, triazoles, protein-solvent interface
INTRODUCTION
Chronic hepatitis B (CHB), a global therapeutic problem caused by the hepatitis B virus
(HBV), infects over 250 million people worldwide especially in Asia-Pacific regions. About
one third of these individuals develop life-threatening diseases such as liver failure, cirrhosis
and hepatocellular carcinoma (HCC) with over 650,000 deaths every year.1 Currently
therapies are limited to the use of interferons (interferon-alpha and pegylated interferon) and
nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs): lamivudine (3TC), entecavir
(ETV), telbivudine (LdT), adefovir dipivoxil (ADV), tenofovir disoproxil (TDF) and
tenofovir alafenamide (TAF) (Figure 1).2 Both therapies can reduce HBV DNA and
normalize liver enzymes. However, they can’t eradicate chronic HBV infection, as viral
covalently closed circular DNA (cccDNA) yet remains.3 Furthermore, interferons are poorly
tolerated and expensive, and the usage of NRTIs results in long-term treatment and
drug-resistance.4 As none of these inhibitors can cure HBV infections, there is always an
urgent need to develop more effective and safety drugs with novel chemical structures and
mechanisms of action.
This article is protected by copyright. All rights reserved.

(Figure 1)
HBV capsid protein plays essential roles in key steps of HBV life cycle such as reverse
transcription of pregenomic RNA (pgRNA), formation and intracellular trafficking of relaxed
circular DNA and subviral particle formation, which has been a novel drug target in recent
years.5 Currently, great progress have been made in the development of HBV capsid
inhibitors (or effectors), as exemplified by several drug candidates, including DVR-23,
AT-130, Bay 41-4109 and GLS4 (Figure 2).6
(Figure 2)
Heteroaryldihydropyrimidines (HAPs) were identified as novel HBV capsid assembly
inhibitors, which can prevent the normal assembly of core protein and lead to a formation of
misassembled capsid.7,8 What’s more, the misassembled capsid was then degraded by host
proteasomes.9 In 2001, Bayer company firstly reported Bay 41-4109 (IC50 = 124.25 nM), a
potential HAP compound that prevent HBV infections by reducing HBV viral DNA in the
liver and plasma. However, it was found out to be hepatotoxic at high doses (CC50 = 35 µM).
After years of structural optimization based on bioisosterism and computer-aided drug design,
GLS4 (IC50 = 62.24 nM, CC50 = 115 µM), with improved activity and decreased toxicity was
discovered and is currently in the phase II clinical stage. In addition, GLS4 also had potent
anti-HBV activity against adefovir-resistant strains with IC50 of 12 nM.10,11
Even so, the poor water solubility and oral bioavailability of GLS4 limited the chance of
being developed into a market drug, therefore, in the past several years, systematic structural
modifications have performed on this core structure.9,12 In this article, we have tried to
employ rational drug design strategies to explore the chemically diverse space of GLS4, with
the aim to enhance anti-HBV activity and improve water solubility and in vitro safety profiles.
To obtain some enlightenment, detailed SARs of existing HAPs were summarized as follows:
1-position of HAPs: the hydrogen of pyrrimidine is necessary for the activity as
substituted alkyl groups are not favored at this position. Interestingly, the fused ring analogs
could moderate to excellent potency; 2-position of HAPs: high-polarity rings significantly
reduce activity, thiazolyl is the most suitable substituent at this position; 4-position of HAPs:
phenyls bearing fluorine, chlorine and bromine are well tolerated while polar and bulky
This article is protected by copyright. All rights reserved.

groups are not favored; 5-position of HAPs: polar and large groups are not favored, methyl
or ethyl ester appear to be the most favorable groups; 6-position of HAPs: polar and
hydrophilic groups (such as carboxyl) at this position are favored. They could significantly
reduce liposolubility of the compounds, and form hydrogen bond with critical residues
nearby the protein-solvent interface such as Ser121 and Ser141.12,13
(Figure 3)
Sulfonamide groups possess several unique physicochemical properties. They are able to
modulate solubility and acid-base property and improve binding affinity through hydrogen
bond interactions, that are extensively used in drug design.14,15 Besides, with the development
of structural biology, we have investigated some X-ray crystal structures of HBV core protein
in complex with 4-methyl HAPs (PDB ID: 5GMZ) and sulfamoylbenzamides (PDB ID:
5T2P), discovering sulfonamide may be well tolerated at the solvent-exposed region, as it
overlaps pretty well with the carboxyl pharmacophore (Figure 3).12 Meanwhile, considering
the poor in vivo instability of GLS4, we replaced the morpholine ring with substituted
triazoles to improve the metabolic stability and anti-HBV activity. Herein, we report the
design, synthesis and in vitro biological evaluation of novel anti-HBV HAP-sulfonamide and
HAP-triazole derivatives (Figure 4).
(Figure 4)
CHEMISTRY
The synthetic routes of HAP derivatives I-(1-18) and II-(1-10) were illustrated in
Scheme 1 and 2. The key intermediate 2, prepared from the commercially available
2-thiazolecarboxamidine hydrochloride (1), 2-bromo-4-fluorobenzaldehyde and ethyl
acetoacetate by classic “Biginelli reaction” was converted to intermediate 3 with NBS and
CCl4 by free radical reaction. Using acetone as solvent, the intermediate 3 was reacted with
NaN3 to give intermediate 4 by nucleophilic substitution reaction,16 which was further
reduced to intermediate 5 via one-step “Staudinger reaction”.17 Finally, target compounds
I-(1-18) were obtained from intermediate 5 by easy condensation reaction with DCM, Et3N
This article is protected by copyright. All rights reserved.

and different substituted sulfonyl chloride as raw materials (Scheme 1), and target
compounds II-(1-10) were obtained from intermediate 4 by CuAAC reaction (Scheme 2).
Scheme 1. Reagents and Conditions: (i) 2-bromo-4-fluorobenzaldehyde, ethyl acetoacetate, CH3COONa, EtOH,
80 °C; (ii) NBS, CCl4, 50 °C; (iii) NaN3, acetone, r.t.; (iv) PPh3, THF, H2O, r.t.; (v) for I-(1~3) and I-(5~18):
substituted sulfonyl chloride, Et3N, DCM, r.t.; for I-4: cyclopropanesulfonyl chloride, Et3N, DMF, 90 °C.
Scheme 2. Reagents and Conditions: (i) CuSO4·5H2O, Sodium ascorbate, H2O, THF, r.t.
This article is protected by copyright. All rights reserved.

RESULTS AND DISCUSSION
The target compounds were evaluated for their cytotoxicity, inhibitory effect on HBV
DNA replication and HBsAg and HBeAg secretion in HepG2.2.15 cells (human HBV
transgenic hepatocellular carcinoma cells) via cell counting kit-8 (CCK-8), PCR and standard
ELISA method, respectively. The biological results were represented as inhibition
percentages, IC50 values, CC50 values and selectivity index (SI) values (determined as the
CC50/IC50 values).18,19
The cytotoxicity of HAP-sulfonamide derivatives I-(1~18) were measured by cell
counting kit-8 (CCK-8) method in HepG2.2.15 cell line. As shown in Table 1, most of these
analogues exhibited low toxicity under the concentration of 20 µM (Inhibition percentages <
50%). It was shown that phenyl group bearing one or more methyl improved the toxicity of
the inhibitors; for instance, I-8, I-9, I-10 and I-17 with the inhibition percentages of 43.1 ±
3.3%, 45.1 ± 2.7%, 43.3 ± 0.9% and 45.2 ± 6.2%, respectively. When the substituent had
short aliphatic chain (methyl, ethyl or propyl), the corresponding inhibitors showed no
toxicity at the tested concentration (I-1, I-2 and I-3).
The cytotoxicity of HAP-triazole derivatives were also illustrated in Table 2. We
identified that substitution of large group at R2 improved the toxicity of the inhibitors (II-8:
97.8 ± 0.2%; II-9: 73.2 ± 3.8%). Phenyl substitution of R2 made no contribution to the
toxicity (II-5) while phenyl group bearing -NH2 exhibited high toxicity, especially -NH2 on
meta- and para- positions (II-2 and II-6).
The inhibitory effects on HBV DNA replication of HAP-sulfonamide derivatives were
screened in the HepG2.2.15 cell line using GLS4 and 3TC as the positive controls. In the
first-round screen, following the treatment of cells with the tested compounds at 20 µM for 8
days, the extracellular HBV DNA were collected and then quantified by real-time FQ-PCR.
As shown in Table 1, some of HAP-sulfonamide derivatives such as I-5, I-11, I-13, I-15 and
I-17 showed better inhibitory activities (72.6 ± 1.3%, 80.6 ± 0.7%, 80.1 ± 4.2%, 78.4 ± 0.6%
and 76.7 ± 8.0%, respectively) against HBV DNA replication compared to 3TC (72.3 ±
15.0%). Primary SARs were summarized as follows：
This article is protected by copyright. All rights reserved.

First, we introduced a short aliphatic chain at R1 position, and obtained activity in the
following sequence: propyl > methyl > ethyl (I-3 > I-1 > I-2). Next, R1 replacement by
heterocycles indicated the order of anti-HBV DNA replication activity as follows:
thiophene > benzene > naphthalene > pyridine (I-5 > I-7 > I-18 > I-6). In addition, SAR of
the substituent group at benzene ring was studied. The results suggested that strong
electron-withdrawing group (NO2) appeared to be the most favorable group (I-11 and I-13),
closely followed by other substituents in the following sequence: OCH3 > CH3 > F > CN
(I-15 > I-8 > I-14 > I-16). More specifically, the substitution pattern in the benzene ring
played an essential role in maintaining anti-HBV activity. Ortho- and para- substituents were
favored and showed similar activity (I-8 vs I-10, I-11 vs I-13) whereas meta-position
counterparts (I-9 and I-12) dropped the activity.
The inhibitory effects on HBV DNA replication of HAP-triazole derivatives (20 µM)
were illustrated in Table 2. To investigate the effects of different aromatic substituents in the
position of R2, six derivatives II-(1-6) were synthesized and assessed for anti-HBV activity.
Compared to II-3 (73.1 ± 3.3%) and II-4 (76.5 ± 8.3%), compound II-5 (81.9 ± 5.9%)
showed better activity, which suggested that phenyl was superior to pyridine and thiophene.
However, II-5 lost its activity when the concentration dropped to 5 µM (-0.7%). Furthermore,
Ortho- substituent was found to be more preferable than meta- and para- substituents (II-1 >
II-2 > II-6). In addition, large substituents on position R2 was studied (II-(8-10)). Compound
II-9 showed anti-HBV actitivity (82.0 ± 4.4%) but also had high cytotoxicity (73.2 ± 3.8%).
This article is protected by copyright. All rights reserved.

Table 1. Anti-HBV activity, cytotoxicity of HAP-sulfonamide derivatives I-(1~18) and positive drug
GLS4 and 3TC.a
Inhibition percentages (%)
Compounds
R1
DNA
HepG2.2.15 Cell
I-1
I-2
I-3
21.2 ± 15.5
-13.4 ± 14.4
0.4 ± 1.4
33.9 ± 1.1
66.5 ± 5.2
14.9 ± 14.3
I-4
I-5
I-6
I-7
23.9 ± 5.9
72.6 ± 1.3
19.5 ± 4.3
61.8 ± 2.9
20.9 ± 8.5
43.9 ± 3.4
22.7 ± 7.8
36.7 ± 11.9
I-8
I-9
65.3 ± 2.8
57.1 ± 7.2
67.0 ± 3.7
43.1 ± 3.3
45.1 ± 2.7
43.3 ± 0.9
I-10
This article is protected by copyright. All rights reserved.

I-11
80.6 ± 0.7
-35.6 ± 0.3
I-12
I-13
69.8 ± 3.7
28.8 ± 8.0
43.9 ± 4.9
80.1 ± 4.2
I-14
I-15
I-16
60.5 ± 7.0
78.4 ± 0.6
19.6 ± 15.9
17.3 ± 17.5
25.3 ± 14.3
13.7 ± 9.1
I-17
I-18
76.7 ± 8.0
47.8 ± 5.7
45.2 ± 6.2
14.8 ± 12.7
GLS4
3TC
94.6 ± 0.04
72.3 ± 15.0
-0.5 ± 0.01
3.7 ± 5.7
a
HepG2.2.15 cells were cultured in the presence of HAP-sulfonamide derivatives, GLS4 and 3TC at 20
µM for 8 days, and then extracellular HBV DNA levels were quantified by real-time FQ-PCR, cytotoxicity
were quantified by CCK-8.
Table 2. Anti-HBV activity, cytotoxicity of HAP-triazole derivatives II-(1~10) and positive drug
GLS4 and 3TC.a
This article is protected by copyright. All rights reserved.

Inhibition percentages (%)
Compounds
R2
DNA
HepG2.2.15 Cell
II-1
86.2 ± 4.8
12.2 ± 16.8
II-2
II-3
II-4
II-5
78.7 ± 2.5
73.1 ± 3.3
76.5 ± 8.3
81.9 ± 5.9
-0.7
60.7 ± 4.4
66.5 ± 4.5
14.4 ± 11.0
8.4 ± 12.1
6.3 ± 5.8
II-5
(5 µM)
II-6
II-7
75.9 ± 4.8
78.9 ± 4.8
59.1 ± 5.6
1.7 ± 7.3
II-8
NDb
97.8 ± 0.2
II-9
82.0 ± 4.4
9.6 ± 0.5
73.2 ± 3.8
II-10
41.3 ± 11.4
GLS4
3TC
91.8 ± 0.8
74.9 ± 4.7
38.2 ± 22.7
-2.3 ± 1.6
a HepG2.2.15 cells were cultured in the presence of HAP-triazole derivatives, GLS4 and 3TC at 20 µM for
8 days, and then extracellular HBV DNA levels were quantified by real-time FQ-PCR, cytotoxicity were
quantified by CCK-8.
b ND: not determinded.
This article is protected by copyright. All rights reserved.

The activity of compound I-11, I-13 and II-1 were further evaluated in HepG2.2.15 cells
in a dose-dependent manner (50 µM, 5 µM, 0.5 µM, 0.05 µM, 0.005 µM) to analyze their
IC50 and CC50 values. Among them, II-1 showed the best in vitro anti-HBV DNA activity
(IC50 = 0.35 ± 0.04 µM), which was 1.8-fold higher than that of 3TC (IC50 = 0.62 ± 0.31 µM).
The potency of I-13 (IC50 = 0.76 ± 0.12 µM) was close to 3TC, being 4.5-fold higher than
I-11 (IC50 = 3.52 ± 0.64 µM) (Table 3). All these three compounds had no cytotoxicity in the
tested concentration (CC50 > 50 µM).
Table 3. Anti-HBV DNA replication activity, cytotoxicity and selectivity indices of HAP derivatives
(I-11, I-13 and II-1) and positive drug GLS4 and 3TC.
DNA
a
CC50
b
Compounds
IC50
SIc
(mol/L)
(mol/L)
I-11
I-13
> 50
> 50
> 50
> 50
> 50
3.52 ± 0.64
> 14.2
0.76 ± 0.12
0.35 ± 0.04
0.051 ± 0.001
0.62 ± 0.31
> 65.8
> 143
II-1
GLS4
3TC
> 980.4
> 80.6
a IC50: Concentrations of compounds achieving 50% inhibition of DNA replication.
b CC50: Concentrations of compounds required to reduce the viability of HepG2.2.15 cells by 50%.
c SI: selectivity index, the ratio of CC50/IC50.
This article is protected by copyright. All rights reserved.

Compound II-1 was further evaluated for its inhibitory effect on HBsAg and HBeAg
secretion in HepG2.2.15 cells in a dose-dependent manner (50 µM, 5 µM, 0.5 µM, 0.05 µM,
0.005 µM). However, as illustrated in Table 4, II-1 doesn’t have anti-HBsAg and HBeAg
secretion activity (IC50 > 50 µM). Since GLS4 only had moderate inhibitory values (IC50 =
14.8 ± 0.75 and 6.1 ± 0.07 µM, respectively), we presumed that HAPs don’t act directly on
antigen secretion.
Table 4. Anti-HBsAg and HBeAg secretion activity, cytotoxicity and selectivity indices of II-1 and
positive drug GLS4 and 3TC.
HBsAg
HBeAg
a
Compounds
IC50
IC50
SIb
SI
(mol/L)
(mol/L)
II-1
GLS4
3TC
> 50
—
1.5
—
> 50
—
3.7
—
14.8 ± 0.75
> 100
6.1 ± 0.07
> 100
a IC50: Concentrations of compounds achieving 50% inhibition of DNA replication.
b SI: selectivity index, the ratio of CC50/IC50.
MOLECULAR MODELING STUDIES
II-1, the best compound in cell-based HBV infection assay, was docked into the HBV
capsid (PDB ID: 5e0i) using Sybyl-X 2.0 to study its binding mode. Default parameters were
used as described in the Sybyl-X 2.0 manual unless otherwise specified, and the result was
displayed by PyMOL.
(Figure 5)
As is shown in Figure 5, the results indicated that the Br, F-substituted phenyl region
occupied a hydrophobic pocket, surrounded by Pro25, Asp29, Leu30, Thr33, Trp102 and
Ile105 (chain B). The 2-thiazolyl group interacted with Phe23 (chain B) and Thr128 (chain C),
being coplanar with the dihydropyrimidine core as previously reported.12 Interestingly, the
designed triazole fragment nearby the protein-solvent interface interacted with some crucial
This article is protected by copyright. All rights reserved.

residues, which might contribute to the anti-HBV activity. Two N atoms of triazole ring can
form a hydrogen bond with Thr128 (chain C). Besides, the “-NH2” of phenyl ring also
interacted with Ser121 (chain C) by hydrogen bonding. In addition, the benzene ring was
likely to interacted with Pro134 (chain C) by Van der Waals' force.
PHYSICOCHEMICAL PROPERTIES PREDICTION
Furthermore, some physicochemical properties of the II-1 were calculated using free
online software (http://www.molinspiration.com/). As shown in Table 5, active compounds
II-1 conformed well to the Lipinski’s rule of five, which could be considered as a promising
lead compound for further development.
Table 5. Prediction of physicochemical propertiesa of II-1.
Compd. nViol
II-1
MW
Natoms miLogP nON nOHNH Nrotb
37 3.40
TPSA
MV
1
582.46
9
3
8
120.33 443.18
a nViol: no. of violations; natoms: no. of atoms; miLogP: molinspiration predicted LogP; MW: molecular
weight; nON: no. of hydrogen bond acceptors; nOHNH: no. of hydrogen bond donors; nrotb: no. of
rotatable bonds; TPSA: topological polar surface area; MV: molar volume.
CONCLUSION
In conclusion, novel HAP-sulfonamide and HAP-triazole derivatives were designed and
synthesized as non-nucleoside anti-HBV inhibitors. All the designed compounds were
evaluated for their bioactivities against HBV DNA replication. Among them, compound II-1
was found to be the most potent, with IC50 value of 0.35 ± 0.04 µM, which was 1.8-fold
higher than 3TC (IC50 = 0.62 ± 0.31 µM). The anti-HBeAg and HBsAg secretion activity
(IC50 > 50 µM) of II-1 indicated that it doesn’t act directly on antigen secretion. Furthermore,
molecular modeling study carried out to predict the interactions between II-1 and HBV
capsid suggested that the H-bond interaction formed by “-NH2” of phenyl ring and Ser121
This article is protected by copyright. All rights reserved.

was important in the binding of this compound. The results of some physicochemical
properties of II-1 like miLogP measured showed II-1 conformed well to the Lipinski’s rule of
five. The preliminary SARs of the new compounds were summarized, which may help
discovering more potent anti-HBV agents with diverse structures.
EXPERIMENTAL SECTION
Chemistry. All melting points were measured with a micromelting point apparatus and
1
are uncorrected. H NMR and 13C NMR spectra were performed on a Bruker AV-400
spectrometer (Bruker BioSpin, Switzerland) or Bruker AV-300 spectrometer (Bruker BioSpin,
Switzerland) using tetramethylsilane (TMS) as internal reference and DMSO-d6 or CDCl3 as
solvent. Chemical shifts were expressed in δ units (ppm) and J values were presented in hertz
(Hz). Mass spectra were taken on a LC Autosampler Device: Standard G1313 instrument.
TLC was performed on Silica Gel GF254 for TLC (Merck) and spots were visualized by
irradiation with UV light (254 nm). Flash column chromatography was performed on column
packed with Silica Gel 60 (200-300 mesh). Solvents were reagent grade and, when necessary,
were purified and dried by standard methods. Rotary evaporators served in concentration of
the reaction solutions at reduced pressure.
General procedure for the synthesis of intermediate 2. To the mixture solution of
2-thiazolecarboxamidine hydrochloride (0.50 g, 3.05 mmol) in ethanol (50 mL),
2-bromo-4-fluorobenzaldehyde (0.93 g, 4.60 mmol) and ethyl acetoacetate (600 µL, 4.60
mmol) were added. The resulting mixture was stirred for 6 h at 80 °C. The reaction mixture
was cooled and the solvent was evaporated under reduced pressure. The residue was taken up
with water (60 mL) and extrated with EtOAc (25 mL × 3), and the organic phase was washed
with saturated sodium chloride (25 mL), dried over anhydrous Na2SO4, evaporated under
reduced pressure to give the corresponding crude product. The residue was dissolved in a
small amount EtOAc and purified by column chromatography on silica gel using PE-EA
system to give intermediate 2 in good yield.
This article is protected by copyright. All rights reserved.

Ethyl
4-(2-bromo-4-fluorophenyl)-6-methyl-2-(thiazol-2-yl)-1,4-dihydropyrimidine-5-carboxylate
(2). Yellow solid, yield 58%, mp: 158–160 °C; 1H NMR (400 MHz, CDCl3) δ 7.81 (d, J = 2.8
Hz, 1H), 7.46 (s, 1H), 7.38–7.28 (m, 2H), 6.97 (t, J = 8.2 Hz, 1H), 6.15 (s, 1H), 4.05 (q, J =
7.1 Hz, 2H), 2.53 (s, 3H), 1.13 (t, J = 7.1 Hz, 3H); EI-MS: 424.3 [M+H]+.
General procedure for the synthesis of intermediate 3. To the mixture solution of
intermediate 2 (0.50 g, 1.17 mmol) in CCl4 (50 mL), NBS (0.22 g, 1.24 mmol) were added
slowly. The resulting mixture was stirred for 2 h at 50 °C. The reaction mixture was cooled
and the solvent was evaporated under reduced pressure. The residue was added with water
(60 mL) and extrated with EtOAc (25 mL × 3), and the organic phase was washed with
saturated sodium chloride (25 mL), dried over anhydrous Na2SO4, evaporated under reduced
pressure to give the corresponding crude product. The residue was dissolved in a small
amount EtOAc and purified by column chromatography on silica gel using PE-EA system to
give intermediate 3 in good yield.
Ethyl
4-(2-bromo-4-fluorophenyl)-6-(bromomethyl)-2-(thiazol-2-yl)-1,4-dihydropyrimidine-5-carbo
xylate (3). Yellow solid, yield 59%, mp: 125–127 °C; 1H NMR (400 MHz, CDCl3) δ 7.84 (d,
J = 3.1 Hz, 1H), 7.52 (s, 2H), 7.44–7.35 (m, 1H), 7.32 (dd, J = 8.1, 2.6 Hz, 1H), 7.02 (t, J =
8.0 Hz, 1H), 6.09 (s, 1H), 4.94 (d, J = 8.9 Hz, 1H), 4.61 (s, 1H), 4.09 (d, J = 7.0 Hz, 2H), 1.16
(t, J = 7.1 Hz, 3H); EI-MS: 502.2 [M+H]+.
General procedure for the synthesis of intermediate 4. To the mixture solution of
intermediate 3 (0.50 g, 1.00 mmol) in acetone (45 mL) were added with NaN3 (0.13 g, 2.00
mmol). The resulting mixture was stirred overnight at room temperature. The solvent was
evaporated under reduced pressure. The residue was taken up with water (60 mL) and
extrated with EtOAc (25 mL × 3), and the organic phase was washed with saturated sodium
chloride (25 mL), dried over anhydrous Na2SO4, evaporated under reduced pressure to give
the corresponding crude product. The residue was dissolved in a small amount EtOAc and
choromatographed on silica gel using PE-EA system to give intermediate 4 in good yield.
This article is protected by copyright. All rights reserved.

Ethyl
6-(azidomethyl)-4-(2-bromo-4-fluorophenyl)-2-(thiazol-2-yl)-1,4-dihydropyrimidine-5-carbox
1
ylate (4). Yellow solid, yield 80%, mp: 126–128 °C; H NMR (400 MHz, CDCl3) δ 8.64 (s,
1H), 7.85 (d, J = 3.1 Hz, 1H), 7.55 (d, J = 3.1 Hz, 1H), 7.48–7.37 (m, 1H), 7.35–7.29 (m, 1H),
7.10–6.92 (m, 1H), 6.29–6.02 (m, 1H), 4.97 (s, 1H), 4.60 (d, J = 2.6 Hz, 1H), 4.17–4.00 (m,
2H), 1.13 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 165.78, 165.03, 163.31, 162.71,
162.36, 162.09, 160.80, 160.22, 154.83, 150.00, 143.92, 143.54, 143.10, 142.75, 139.59,
137.74 (d, J = 3.5 Hz), 130.80, 130.72, 130.61, 124.92, 123.38, 122.07 (d, J = 9.7 Hz), 120.23
(dd, J = 24.4, 17.0 Hz), 115.83, 115.62, 115.18, 114.97, 106.27, 98.60, 77.37, 77.06, 76.74,
60.70, 60.32, 58.37, 51.91 (d, J = 2.0 Hz), 49.79, 14.07 (d, J = 5.7 Hz); EI-MS: 465.4
[M+H]+.
General procedure for the synthesis of intermediate 5. To the mixture solution of THF
(30 mL) and H2O (10 mL) were added with intermediate 4 (0.37 g, 0.80 mmol) and PPh3
(0.21 g, 0.80 mmol). The resulting mixture was stirred for 4 h at 35 °C. Upon completion of
the reaction, the mixture was taken up with water (60 mL) and extracted with EtOAc (25 mL
× 3), and the organic phase was washed with saturated sodium chloride (25 mL), dried over
anhydrous Na2SO4, evaporated under reduced pressure to give the corresponding crude
product. The residue was dissolved in a small amount EtOAc and purified by column
chromatography, using PE-EA as eluent to give intermediate 5 in good yield.
Ethyl
6-(aminomethyl)-4-(2-bromo-4-fluorophenyl)-2-(thiazol-2-yl)-1,4-dihydropyrimidine-5-carbo
xylate (5). Yellow solid, yield 37%; 1H NMR (400 MHz, DMSO) δ 7.99 (t, J = 5.5 Hz, 1H),
7.93 (d, J = 3.1 Hz, 1H), 7.55 (dd, J = 8.6, 2.6 Hz, 1H), 7.37 (dd, J = 8.6, 6.3 Hz, 1H), 7.21
(td, J = 8.5, 2.6 Hz, 1H), 5.98 (d, J = 11.9 Hz, 1H), 4.76 (d, J = 89.1 Hz, 3H), 4.00 (d, J =
13.5 Hz, 2H), 3.94 (q, J = 7.0 Hz, 2H), 1.05 (t, J = 7.1 Hz, 3H); EI-MS: 439.4 [M+H]+.
General procedure for the synthesis of target compounds I-(1-3) and I-(5-18). To the
mixture solution of intermediate 5 (0.10 g, 0.23 mmol) in DCM (10 mL) were added with
substituted sulfonyl chloride (0.46 mmol) and Et3N (60 µL, 0.46 mmol). The resulting
mixture was stirred for 5 h at room temperature. Upon completion of the reaction, the mixture
was added to water (50 mL) and extrated with EtOAc (20 mL × 3), and the organic phase was
This article is protected by copyright. All rights reserved.

washed with saturated sodium chloride (25 mL), dried over anhydrous Na2SO4, evaporated
under reduced pressure to give the corresponding crude product. The residue was dissolved in
a small amount EtOAc and purified by column chromatography using PE-EA as eluent.
Crystallization of this material from DCM-Hexane gave the target compounds I-(1~3) and
I-(5~18) in good yield.
Ethyl
4-(2-bromo-4-fluorophenyl)-6-(methylsulfonamidomethyl)-2-(thiazol-2-yl)-1,4-dihydropyrimi
1
dine-5-carboxylate (I-1). Yellow solid, yield 59%, mp: 132–134 °C; H NMR (400 MHz,
DMSO) δ 9.35 (s, 1H), 8.01 (d, J = 3.1 Hz, 1H), 7.95 (d, J = 3.0 Hz, 1H), 7.71 (s, 1H), 7.58
(dd, J = 8.6, 2.4 Hz, 1H), 7.44 (dd, J = 8.6, 6.3 Hz, 1H), 7.23 (td, J = 8.5, 2.5 Hz, 1H), 6.02 (s,
1H), 4.56 (d, J = 4.1 Hz, 2H), 3.97 (q, J = 7.0 Hz, 2H), 3.05 (s, 3H), 1.05 (t, J = 7.1 Hz, 3H);
EI-MS: 517.5 [M+H]+; HRMS m/z C18H18BrFN4O4S2: calcd 515.9937, found 517.0009
[M+H]+.
Ethyl
4-(2-bromo-4-fluorophenyl)-6-(ethylsulfonamidomethyl)-2-(thiazol-2-yl)-1,4-dihydropyrimidi
1
ne-5-carboxylate (I-2). Yellow solid, yield 57%, mp: 149–151 °C; H NMR (400 MHz,
DMSO) δ 9.35 (s, 1H), 8.01 (d, J = 3.1 Hz, 1H), 7.95 (d, J = 3.1 Hz, 1H), 7.74 (t, J = 6.1 Hz,
1H), 7.58 (dd, J = 8.6, 2.4 Hz, 1H), 7.44 (dd, J = 8.6, 6.2 Hz, 1H), 7.23 (td, J = 8.4, 2.4 Hz,
1H), 6.02 (s, 1H), 4.61–4.48 (m, 2H), 3.97 (q, J = 7.0 Hz, 2H), 3.16 (q, J = 6.9 Hz, 2H), 1.25
(t, J = 7.2 Hz, 3H), 1.05 (t, J = 7.1 Hz, 3H); EI-MS: 530.2 [M+H]+.
Ethyl
4-(2-bromo-4-fluorophenyl)-6-(propylsulfonamidomethyl)-2-(thiazol-2-yl)-1,4-dihydropyrimi
1
dine-5-carboxylate (I-3). Yellow solid, yield 80%, mp: 69–70 °C; H NMR (400 MHz,
DMSO) δ 9.37 (s, 1H), 8.02 (d, J = 3.0 Hz, 1H), 7.96 (d, J = 3.0 Hz, 1H), 7.74 (t, J = 6.1 Hz,
1H), 7.58 (dd, J = 8.5, 2.3 Hz, 1H), 7.44 (dd, J = 8.5, 6.3 Hz, 1H), 7.26–7.20 (m, 1H), 6.02 (s,
1H), 4.56 (qd, J = 16.7, 6.2 Hz, 2H), 3.97 (q, J = 6.9 Hz, 2H), 3.13 (dt, J = 12.1, 6.2 Hz, 2H),
1.71 (dt, J = 13.3, 6.7 Hz, 2H), 1.05 (t, J = 7.1 Hz, 3H), 0.97 (t, J = 7.4 Hz, 3H); EI-MS:
544.2 [M+H]+; HRMS m/z C20H22BrFN4O4S2: calcd 544.0250, found 545.0323 [M+H]+.
This article is protected by copyright. All rights reserved.

Ethyl
4-(2-bromo-4-fluorophenyl)-2-(thiazol-2-yl)-6-((thiophene-2-sulfonamido)methyl)-1,4-dihydr
1
opyrimidine-5-carboxylate (I-5). Yellow solid, yield 52%, mp: 160–162 °C; H NMR (400
MHz, DMSO) δ 9.20 (s, 1H), 8.55 (s, 1H), 8.03 (d, J = 3.1 Hz, 1H), 7.99 (d, J = 5.0 Hz, 1H),
7.96 (d, J = 3.1 Hz, 1H), 7.68–7.64 (m, 1H), 7.57 (dd, J = 8.5, 2.5 Hz, 1H), 7.38 (dd, J = 8.6,
6.2 Hz, 1H), 7.23 (dd, J = 8.4, 2.2 Hz, 1H), 7.20–7.17 (m, 1H), 5.96 (s, 1H), 4.61 (d, J = 16.6
Hz, 1H), 4.47–4.37 (m, 1H), 3.93 (q, J = 6.9 Hz, 2H), 1.01 (t, J = 7.1 Hz, 3H); EI-MS: 585.1
[M+H]+; HRMS m/z C21H18BrFN4O4S3: calcd 583.9658, found 584.9730 [M+H]+.
Ethyl
4-(2-bromo-4-fluorophenyl)-6-((pyridine-3-sulfonamido)methyl)-2-(thiazol-2-yl)-1,4-dihydro
pyrimidine-5-carboxylate (I-6). Yellow solid, yield 75%, mp: 77–80 °C; 1H NMR (400 MHz,
DMSO-d6) δ 9.30 (s, 1H), 8.98 (d, J = 2.4 Hz, 1H), 8.80 (d, J = 4.8 Hz, 1H), 8.54 (t, J = 6.2
Hz, 1H), 8.19 (t, J = 6.6 Hz, 1H), 8.02 (d, J = 3.2 Hz, 1H), 7.95 (d, J = 3.2 Hz, 1H),
7.58–7.54 (m, 2H), 7.35 (dd, J = 8.7, 6.1 Hz, 1H), 7.22–7.18 (m, 1H), 5.90 (s, 1H), 4.64 (dd,
J = 16.2, 6.4 Hz, 1H), 4.36 (dd, J = 16.2, 5.9 Hz, 1H), 3.92 (q, J = 6.9 Hz, 2H), 1.00 (t, J =
7.0 Hz, 3H); EI-MS: 580.1 [M+H]+.
Ethyl
4-(2-bromo-4-fluorophenyl)-6-(phenylsulfonamidomethyl)-2-(thiazol-2-yl)-1,4-dihydropyrimi
1
dine-5-carboxylate (I-7). Yellow solid, yield 23%, mp: 136–139 °C; H NMR (400 MHz,
DMSO) δ 9.20 (s, 1H), 8.35 (t, J = 6.2 Hz, 1H), 8.03 (d, J = 3.0 Hz, 1H), 7.96 (d, J = 3.0 Hz,
1H), 7.83 (d, J = 7.4 Hz, 2H), 7.68–7.61 (m, 1H), 7.59–7.53 (m, 3H), 7.35 (dd, J = 8.5, 6.3
Hz, 1H), 7.21 (td, J = 8.4, 2.4 Hz, 1H), 5.90 (s, 1H), 4.55 (dd, J = 16.7, 6.6 Hz, 1H), 4.30 (dd,
J = 16.8, 5.9 Hz, 1H), 3.91 (q, J = 7.1 Hz, 2H), 0.98 (t, J = 7.1 Hz, 3H); EI-MS: 579.2
[M+H]+.
Ethyl
4-(2-bromo-4-fluorophenyl)-6-((2-methylphenylsulfonamido)methyl)-2-(thiazol-2-yl)-1,4-dihy
dropyrimidine-5-carboxylate (I-8). Yellow solid, yield 37%, mp: 136–138 °C; 1H NMR (400
MHz, DMSO) δ 9.22 (s, 1H), 8.26 (t, J = 6.2 Hz, 1H), 8.03 (d, J = 3.1 Hz, 1H), 7.96 (d, J =
3.1 Hz, 1H), 7.82 (d, J = 7.7 Hz, 1H), 7.58–7.49 (m, 2H), 7.36 (t, J = 7.6 Hz, 1H), 7.33–7.26
(m, 2H), 7.20 (td, J = 8.4, 2.6 Hz, 1H), 5.82 (s, 1H), 4.66 (dd, J = 16.7, 6.7 Hz, 1H), 4.27 (dd,
This article is protected by copyright. All rights reserved.

J = 16.7, 5.8 Hz, 1H), 3.91 (q, J = 7.1 Hz, 2H), 2.55 (s, 3H), 1.00 (t, J = 7.1 Hz, 3H); EI-MS:
593.2 [M+H]+.
Ethyl
4-(2-bromo-4-fluorophenyl)-6-((3-methylphenylsulfonamido)methyl)-2-(thiazol-2-yl)-1,4-dihy
dropyrimidine-5-carboxylate (I-9). Yellow solid, yield 59%, mp: 145–147 °C; 1H NMR (400
MHz, DMSO) δ 9.17 (s, 1H), 8.28 (t, J = 6.1 Hz, 1H), 8.02 (d, J = 2.9 Hz, 1H), 7.95 (d, J =
2.9 Hz, 1H), 7.60 (d, J = 12.5 Hz, 2H), 7.55 (dd, J = 8.4, 2.1 Hz, 1H), 7.45–7.38 (m, 2H),
7.33 (dd, J = 8.3, 6.0 Hz, 1H), 7.23–7.16 (m, 1H), 5.88 (s, 1H), 4.65 (dd, J = 16.7, 6.9 Hz,
1H), 4.25 (dd, J = 16.7, 5.7 Hz, 1H), 3.93 (q, J = 7.1 Hz, 2H), 2.30 (s, 3H), 1.00 (t, J = 7.0 Hz,
3H); EI-MS: 593.2 [M+H]+.
Ethyl
4-(2-bromo-4-fluorophenyl)-6-((4-methylphenylsulfonamido)methyl)-2-(thiazol-2-yl)-1,4-dihy
1
dropyrimidine-5-carboxylate (I-10). Yellow solid, yield 51%, mp: 78–82 °C; H NMR (400
MHz, DMSO) δ 9.17 (s, 1H), 8.25 (s, 1H), 8.02 (d, J = 3.1 Hz, 1H), 7.96 (d, J = 3.1 Hz, 1H),
7.69 (d, J = 8.2 Hz, 2H), 7.56 (dd, J = 8.5, 2.4 Hz, 1H), 7.35 (m, 3H), 7.20 (td, J = 8.6, 2.5 Hz,
1H), 5.90 (s, 1H), 4.55 (d, J = 15.8 Hz, 1H), 4.26 (d, J = 16.8 Hz, 1H), 3.91 (q, J = 7.1 Hz,
2H), 2.36 (s, 3H), 0.99 (t, J = 7.1 Hz, 3H); EI-MS: 593.2 [M+H]+.
Ethyl
4-(2-bromo-4-fluorophenyl)-6-((2-nitrophenylsulfonamido)methyl)-2-(thiazol-2-yl)-1,4-dihydr
1
opyrimidine-5-carboxylate (I-11). Yellow solid, yield 70%, mp: 89–92 °C; H NMR (400
MHz, DMSO) δ 9.43 (s, 1H), 8.42 (t, J = 5.5 Hz, 1H), 8.05–7.93 (m, 4H), 7.82 (td, J = 7.5,
1.4 Hz, 1H), 7.75 (dd, J = 11.8, 6.8 Hz, 1H), 7.58–7.53 (m, 1H), 7.31 (dd, J = 8.6, 6.2 Hz,
1H), 7.20 (td, J = 8.6, 2.5 Hz, 1H), 5.88 (s, 1H), 4.81 (dd, J = 16.3, 6.0 Hz, 1H), 4.47 (dd, J =
16.3, 5.2 Hz, 1H), 3.93 (q, J = 7.1 Hz, 2H), 1.03 (t, J = 7.1 Hz, 3H); EI-MS: 624.2 [M+H]+.
Ethyl
4-(2-bromo-4-fluorophenyl)-6-((3-nitrophenylsulfonamido)methyl)-2-(thiazol-2-yl)-1,4-dihydr
1
opyrimidine-5-carboxylate (I-12). Yellow solid, yield 70%, mp: 163–166 °C; H NMR (400
MHz, DMSO) δ 9.25 (s, 1H), 8.60 (s, 1H), 8.54 (s, 1H), 8.41 (d, J = 8.2 Hz, 1H), 8.20 (d, J =
7.9 Hz, 1H), 7.96 (d, J = 3.0 Hz, 1H), 7.93 (d, J = 3.0 Hz, 1H), 7.79 (t, J = 8.0 Hz, 1H), 7.54
(dd, J = 8.4, 2.4 Hz, 1H), 7.31 (dd, J = 8.6, 6.2 Hz, 1H), 7.19 (td, J = 8.4, 2.9 Hz, 1H), 5.80 (s,
This article is protected by copyright. All rights reserved.

1H), 4.83 (d, J = 16.2 Hz, 1H), 4.29 (d, J = 16.2 Hz, 1H), 3.90 (q, J = 7.1 Hz, 2H), 1.02 (t, J
= 7.1 Hz, 3H); EI-MS: 624.2 [M+H]+; HRMS m/z C23H19BrFN5O6S2: calcd 622.9944, found
624.0021 [M+H]+.
Ethyl
4-(2-bromo-4-fluorophenyl)-6-((4-nitrophenylsulfonamido)methyl)-2-(thiazol-2-yl)-1,4-dihydr
1
opyrimidine-5-carboxylate (I-13). Yellow solid, yield 49%, mp: 98–101 °C; H NMR (400
MHz, DMSO) δ 9.24 (s, 1H), 8.64 (s, 1H), 8.32 (d, J = 8.7 Hz, 2H), 8.06 (d, J = 8.7 Hz, 2H),
7.99 (d, J = 3.0 Hz, 1H), 7.93 (d, J = 3.1 Hz, 1H), 7.54 (td, J = 8.2, 2.3 Hz, 1H), 7.34 (dd, J =
8.5, 6.3 Hz, 1H), 7.20 (dd, J = 8.2, 6.5 Hz, 1H), 5.88 (s, 1H), 4.73 (dd, J = 16.2, 5.0 Hz, 1H),
4.35 (dd, J = 16.1, 4.1 Hz, 1H), 3.94 (q, J = 7.1 Hz, 2H), 1.01 (t, J = 7.0 Hz, 3H); EI-MS:
624.2 [M+H]+.
Ethyl
4-(2-bromo-4-fluorophenyl)-6-((4-fluorophenylsulfonamido)methyl)-2-(thiazol-2-yl)-1,4-dihy
1
dropyrimidine-5-carboxylate (I-14). Yellow solid, yield 36%, mp: 82–84 °C; H NMR (400
MHz, DMSO) δ 9.21 (s, 1H), 8.37 (s, 1H), 8.02 (d, J = 3.0 Hz, 1H), 7.96 (d, J = 3.0 Hz, 1H),
7.89 (dd, J = 8.2, 5.4 Hz, 2H), 7.56 (dd, J = 8.4, 2.2 Hz, 1H), 7.37 (dt, J = 10.1, 7.6 Hz, 3H),
7.24–7.18 (m, 1H), 5.92 (s, 1H), 4.59 (d, J = 16.1 Hz, 1H), 4.31 (d, J = 16.6 Hz, 1H), 3.93 (q,
J = 7.1 Hz, 2H), 1.00 (t, J = 7.0 Hz, 3H); EI-MS: 597.2 [M+H]+.
Ethyl
4-(2-bromo-4-fluorophenyl)-6-((4-methoxyphenylsulfonamido)methyl)-2-(thiazol-2-yl)-1,4-di
1
hydropyrimidine-5-carboxylate (I-15). Yellow solid, yield 57%, mp: 142–144 °C; H NMR
(400 MHz, DMSO) δ 9.18 (s, 1H), 8.17 (t, J = 6.1 Hz, 1H), 8.02 (d, J = 3.1 Hz, 1H), 7.95 (d,
J = 3.1 Hz, 1H), 7.74 (d, J = 8.8 Hz, 2H), 7.56 (dd, J = 8.6, 2.5 Hz, 1H), 7.35 (dd, J = 8.7, 6.2
Hz, 1H), 7.20 (td, J = 8.5, 2.5 Hz, 1H), 7.06 (d, J = 8.8 Hz, 2H), 5.91 (s, 1H), 4.54 (dd, J =
16.8, 6.6 Hz, 1H), 4.25 (dd, J = 16.9, 5.7 Hz, 1H), 3.92 (q, J = 7.1 Hz, 2H), 3.82 (s, 3H), 1.00
(t, J = 7.1 Hz, 3H); EI-MS: 609.1 [M+H]+.
Ethyl
4-(2-bromo-4-fluorophenyl)-6-((4-cyanophenylsulfonamido)methyl)-2-(thiazol-2-yl)-1,4-dihy
1
dropyrimidine-5-carboxylate (I-16). Yellow solid, yield 58%, mp: 77–80 °C; H NMR (400
MHz, DMSO) δ 9.22 (s, 1H), 8.58 (s, 1H), 7.99 (m, 5H), 7.89 (d, J = 8.2 Hz, 1H), 7.56 (dd, J
This article is protected by copyright. All rights reserved.

= 8.4, 2.5 Hz, 1H), 7.34 (dd, J = 8.5, 6.3 Hz, 1H), 7.21 (td, J = 8.5, 2.4 Hz, 1H), 5.89 (s, 1H),
4.69 (d, J = 16.2 Hz, 1H), 4.34 (d, J = 16.1 Hz, 1H), 3.93 (q, J = 7.1 Hz, 2H), 1.01 (t, J = 7.0
Hz, 3H); EI-MS: 606.1 [M+H+2]+.
Ethyl
4-(2-bromo-4-fluorophenyl)-2-(thiazol-2-yl)-6-((2,4,6-trimethylphenylsulfonamido)methyl)-1,
1
4-dihydropyrimidine-5-carboxylate (I-17). Yellow solid, yield 49%, mp: 105–109 °C; H
NMR (400 MHz, CDCl3) δ 7.87 (d, J = 3.0 Hz, 1H), 7.57 (d, J = 3.0 Hz, 1H), 7.45 (s, 1H),
7.30 (dd, J = 8.1, 2.5 Hz, 1H), 7.18 (dd, J = 8.6, 6.0 Hz, 1H), 6.97 (td, J = 8.3, 2.5 Hz, 1H),
6.91 (s, 2H), 6.20 (t, J = 5.5 Hz, 1H), 5.91 (d, J = 1.7 Hz, 1H), 4.38 (qd, J = 16.3, 5.7 Hz, 2H),
4.03 (q, J = 7.1 Hz, 2H), 2.67 (s, 6H), 2.29 (s, 3H), 1.10 (t, J = 7.1 Hz, 3H); EI-MS: 623.4
[M+H+2]+.
Ethyl
4-(2-bromo-4-fluorophenyl)-6-((naphthalene-2-sulfonamido)methyl)-2-(thiazol-2-yl)-1,4-dihy
1
dropyrimidine-5-carboxylate (I-18). Yellow solid, yield 48%, mp: 145–148 °C; H NMR
(400 MHz, DMSO) δ 9.21 (s, 1H), 8.48 (s, 1H), 8.43 (d, J = 6.1 Hz, 1H), 8.12 – 8.01 (m, 3H),
7.99 (d, J = 3.1 Hz, 1H), 7.93 (d, J = 3.0 Hz, 1H), 7.84–7.80 (m, 1H), 7.69 (m, 2H), 7.53 (dd,
J = 8.5, 2.4 Hz, 1H), 7.29 (dd, J = 8.5, 6.3 Hz, 1H), 7.12 (td, J = 8.5, 2.2 Hz, 1H), 5.78 (s,
1H), 4.62 (dd, J = 16.7, 6.6 Hz, 1H), 4.33 (dd, J = 16.7, 5.8 Hz, 1H), 3.87 (q, J = 7.1 Hz, 2H),
0.93 (t, J = 7.1 Hz, 3H); EI-MS: 629.2 [M+H]+; HRMS m/z C27H22BrFN4O4S2: calcd
628.0250, found 629.0324 [M+H]+.
General procedure for the synthesis of target compounds I-4. To the mixture solution of
intermediate 5 (0.10 g, 0.23 mmol) in DMF (10 mL) were added with cyclopropanesulfonyl
chloride (30 µL, 0.28 mmol) and Et3N (60 µL, 0.46 mmol). The resulting mixture was stirred
for 5 h at 90 °C. Upon completion of the reaction, the mixture was added with water (50 mL)
and extrated with EtOAc (20 mL × 3), and the organic phase was washed with saturated
sodium chloride (25 mL), dried over anhydrous Na2SO4, evaporated under reduced pressure
to give the corresponding crude product. The residue was dissolved in a small amount EtOAc
and purified by column chromatography on silica gel using PE-EA system. Crystallization of
this material from DCM-Hexane gave the target compound I-4 in good yield.
This article is protected by copyright. All rights reserved.

Ethyl
4-(2-bromo-4-fluorophenyl)-6-(cyclopropanesulfonamidomethyl)-2-(thiazol-2-yl)-1,4-dihydro
1
pyrimidine-5-carboxylate (I-4). Yellow solid, yield 16%, mp: 158–161 °C; H NMR (400
MHz, DMSO) δ 9.29 (s, 1H), 8.01 (d, J = 2.8 Hz, 1H), 7.95 (d, J = 2.7 Hz, 1H), 7.83 (t, J =
5.9 Hz, 1H), 7.57 (d, J = 6.5 Hz, 1H), 7.47–7.40 (m, 1H), 7.23 (t, J = 8.2 Hz, 1H), 6.02 (s,
1H), 4.63 (ddd, J = 22.8, 17.0, 6.3 Hz, 2H), 3.98 (q, J = 6.9 Hz, 2H), 2.70 (s, 1H), 1.05 (t, J =
7.1 Hz, 3H), 1.01–0.84 (m, 4H); EI-MS: 543.1 [M+H]+.
General procedure for the synthesis of lead compounds GLS4. To the mixture solution
of intermediate 3 (0.50 g, 1.00 mmol) in DMF (45 mL), morpholine (173 µL, 2.00 mmol)
was added. The resulting mixture was stirred overnight at room temperature. The residue was
added with water (100 mL) and extrated with EtOAc (25 mL × 3), and the organic phase was
washed with saturated sodium chloride (25 mL), dried over anhydrous Na2SO4, evaporated
under reduced pressure to give the corresponding crude product. The residue was dissolved in
a small amount EtOAc and purified by column chromatography on silica gel using PE-EA
system to give GLS4 in good yield.16
Ethyl
4-(2-bromo-4-fluorophenyl)-6-(morpholinomethyl)-2-(thiazol-2-yl)-1,4-dihydropyrimidine-5-
carboxylate (GLS4). Yellow solid, yield 79%, mp: 124–127 °C; 1H NMR (400 MHz, DMSO)
δ 9.69 (s, 1H), 8.04 (d, J = 3.1 Hz, 1H), 7.95 (d, J = 2.5 Hz, 1H), 7.57 (dd, J = 8.5, 1.9 Hz,
1H), 7.40 (dd, J = 8.6, 6.2 Hz, 1H), 7.22 (td, J = 8.5, 2.4 Hz, 1H), 6.04 (s, 1H), 4.03–3.85 (m,
4H), 3.68 (t, J = 4.2 Hz, 4H), 2.55 (t, J = 7.2 Hz, 4H), 1.06 (t, J = 7.1 Hz, 3H); EI-MS: 509.4
[M+H]+.
General procedure for the synthesis of target compounds II-(1-10). To the mixture
solution of intermediate 4 (0.20 g, 0.43 mmol) in THF (6 mL) and water (6 mL) were added
with CuSO4·5H2O (0.01g, 0.043 mmol), Sodium ascorbate (0.025g, 0.13 mmol) and different
acetylenes (0.86 mmol). The resulting mixture was stirred overnight at room temperature.
Water (100 mL) was added to the residue and extrated with EtOAc (25 mL × 3), and the
This article is protected by copyright. All rights reserved.

organic phase was washed with saturated sodium chloride (25 mL), dried over anhydrous
Na2SO4 and evaporated under reduced pressure to give the corresponding crude product. The
residue was dissolved in a small amount EtOAc and purified by column chromatography on
silica gel using PE-EA system to give II-(1-10) in good yield.
Ethyl
6-((4-(2-aminophenyl)-1H-1,2,3-triazol-1-yl)methyl)-4-(2-bromo-4-fluorophenyl)-2-(thiazol-2
-yl)-1,4-dihydropyrimidine-5-carboxylate (II-1). Yellow solid, yield: 73%, mp: 184–187 °C;
1H NMR (400 MHz, CDCl3) δ: 8.08 (s, 1H), 7.79 (d, J = 3.1 Hz, 1H), 7.52 (s, 1H), 7.47 (d, J
= 3.1 Hz, 1H), 7.43 (dd, J = 7.7, 1.3 Hz, 1H), 7.34 (ddd, J = 9.3, 8.4, 4.2 Hz, 2H), 7.16–7.07
(m, 1H), 7.03 (td, J = 8.3, 2.5 Hz, 1H), 6.77 (dd, J = 6.6, 5.9 Hz, 1H), 6.72 (dd, J = 11.6, 4.3
Hz, 1H), 6.17 (t, J = 15.1 Hz, 1H), 5.94 (dd, J = 39.1, 15.3 Hz, 2H), 5.54 (s, 2H), 4.14 (q, J =
13
7.2 Hz, 2H), 1.17 (t, J = 7.1 Hz, 3H) ; C NMR (100 MHz, CDCl3): 165.02, 162.10 (d, J =
252.8 Hz), 161.77, 152.47, 150.09, 147.97, 145.20, 143.95, 137.62, 130.77 (d, J = 8.8 Hz),
128.78, 127.74, 125.06, 122.04 (d, J = 9.7 Hz), 121.84, 120.37 (d, J = 24.7 Hz), 117.24,
116.67, 115.85 (d, J = 21.1 Hz), 114.17, 106.48, 60.92, 52.12, 52.00, 14.08; EI-MS: 582.3
[M+H]+.
Ethyl
6-((4-(3-aminophenyl)-1H-1,2,3-triazol-1-yl)methyl)-4-(2-bromo-4-fluorophenyl)-2-(thiazol-2
-yl)-1,4-dihydropyrimidine-5-carboxylate (II-2). Yellow solid, yield: 75%, mp: 148–151 °C;
1H NMR (400 MHz, CDCl3) δ: 8.02 (s, 1H), 7.78 (d, J = 3.1 Hz, 1H), 7.54 (s, 1H), 7.45 (d, J
= 3.1 Hz, 1H), 7.41–7.28 (m, 3H), 7.23–7.15 (m, 2H), 7.03 (td, J = 8.3, 2.5 Hz, 1H),
6.73–6.58 (m, 1H), 6.17 (d, J = 32.5 Hz, 1H), 5.92 (q, J = 15.4 Hz, 2H), 4.13 (q, J = 7.1 Hz,
2H), 3.77 (s, 2H), 1.16 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3): 165.04, 162.08 (d, J
= 252.8 Hz), 161.82, 152.60, 150.06, 147.41, 146.88, 143.93, 137.66 (d, J = 3.4 Hz), 132.04,
130.80 (d, J = 8.8 Hz), 129.71, 125.07, 123.28 , 122.03 (d, J = 9.7 Hz), 121.70, 120.34 (d, J =
24.7 Hz), 116.10, 115.82 (d, J = 21.1 Hz), 114.69, 112.34, 106.33, 60.90, 52.12, 52.00, 14.08;
EI-MS: 582.3 [M+H]+.
This article is protected by copyright. All rights reserved.

Ethyl
4-(2-bromo-4-fluorophenyl)-6-((4-(pyridin-2-yl)-1H-1,2,3-triazol-1-yl)methyl)-2-(thiazol-2-yl
)-1,4-dihydropyrimidine-5-carboxylate (II-3). Yellow solid, yield: 74%, mp: 194–197 °C; 1H
NMR (400 MHz, CDCl3) δ: 8.59 (d, J = 4.8 Hz, 1H), 8.39 (s, 1H), 8.22 (d, J = 7.9 Hz, 1H),
7.84–7.71 (m, 2H), 7.53 (s, 1H), 7.43 (d, J = 3.1 Hz, 1H), 7.38 (dd, J = 8.6, 5.9 Hz, 1H), 7.32
(dd, J = 6.6, 4.0 Hz, 1H), 7.22 (ddd, J = 7.5, 4.9, 1.1 Hz, 1H), 7.04 (td, J = 8.3, 2.5 Hz, 1H),
6.13 (d, J = 2.4 Hz, 1H), 5.95 (dd, J = 31.5, 15.3 Hz, 2H), 4.14 (q, J = 7.1 Hz, 2H), 1.16 (t, J =
7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3): 164.97, 162.08 (d, J = 252.7 Hz), 161.83, 152.44,
150.77, 150.12, 149.41, 148.02, 143.87, 137.69 (d, J = 3.5 Hz), 136.81, 130.81 (d, J = 8.8 Hz),
125.04, 123.71, 122.61, 122.02 (d, J = 9.7 Hz), 120.45, 120.27, 120.20, 115.85 (d, J = 21.1
Hz), 106.58, 60.91, 52.31, 51.99, 14.07; EI-MS: 568.4 [M+H]+.
Ethyl
4-(2-bromo-4-fluorophenyl)-2-(thiazol-2-yl)-6-((4-(thiophen-2-yl)-1H-1,2,3-triazol-1-yl)meth
yl)-1,4-dihydropyrimidine-5-carboxylate (II-4). Yellow solid, yield: 76%, mp: 108–110 °C;
1H NMR (400 MHz, CDCl3) δ: 7.99 (s, 1H), 7.79 (d, J= 3.1 Hz, 1H), 7.52 (s, 1H), 7.47 (d, J=
3.1 Hz, 1H), 7.44–7.39 (m, 1H), 7.39–7.27 (m, 3H), 7.08 (dd, J = 5.0, 3.6 Hz, 1H), 7.04 (td,
J= 8.3, 2.6 Hz, 1H), 6.13 (s, 1H), 5.91 (q, J= 15.5 Hz, 2H), 4.21–4.07 (m, 2H), 1.17 (t, J= 8.3
Hz, 3H); 13C NMR (100 MHz, CDCl3): 165.01, 162.11 (d, J= 252.8 Hz), 161.77, 152.38,
150.07, 143.96, 142.38, 137.61 (d, J = 3.6 Hz), 133.53, 130.78 (d, J = 8.8 Hz), 127.64 (d, J =
10.3 Hz), 125.04, 124.72, 123.91, 122.04 (d, J = 9.7 Hz), 121.28, 120.37 (d, J = 24.6 Hz),
115.84 (d, J = 21.0 Hz), 106.41, 60.93, 52.14, 52.00, 14.07; EI-MS: 573.4 [M+H]+.
Ethyl
4-(2-bromo-4-fluorophenyl)-6-((4-phenyl-1H-1,2,3-triazol-1-yl)methyl)-2-(thiazol-2-yl)-1,4-d
1
ihydropyrimidine-5-carboxylate (II-5). Yellow solid, yield: 71%, mp: 109–112 °C; H NMR
(400 MHz, CDCl3) δ: 8.07 (s, 1H), 7.90 (d, J = 1.3 Hz, 1H), 7.88 (s, 1H), 7.78 (d, J = 3.1 Hz,
1H), 7.54 (s, 1H), 7.48–7.29 (m, 6H), 7.03 (td, J = 8.3, 2.6 Hz, 1H), 6.13 (s, 1H), 5.93 (dd, J =
13
36.0, 15.4 Hz, 2H), 4.14 (q, J = 7.1 Hz, 2H), 1.17 (t, J = 7.1 Hz, 3H); C NMR (101 MHz,
CDCl3): 165.04, 162.09 (d, J = 252.8 Hz), 161.81, 152.55, 150.06, 147.33, 137.65 (d, J = 3.5
This article is protected by copyright. All rights reserved.

Hz), 131.14, 130.80 (d, J = 8.8 Hz), 128.91, 128.81, 127.89, 125.84, 125.75, 125.01, 122.05
(d, J = 9.7 Hz), 121.66, 120.36 (d, J = 24.6 Hz), 115.82 (d, J = 21.1 Hz), 106.41, 60.91, 52.13,
52.01, 14.08; EI-MS: 567.4 [M+H]+.
Ethyl
6-((4-(4-aminophenyl)-1H-1,2,3-triazol-1-yl)methyl)-4-(2-bromo-4-fluorophenyl)-2-(thiazol-2
-yl)-1,4-dihydropyrimidine-5-carboxylate (II-6). Yellow solid, yield: 52%, mp: 123–127 °C;
1H NMR (400 MHz, CDCl3) δ: 7.93 (s, 1H), 7.78 (d, J = 3.0 Hz, 1H), 7.68 (d, J = 8.2 Hz, 2H),
7.52 (s, 1H), 7.45 (d, J = 2.8 Hz, 1H), 7.40–7.34 (m, 1H), 7.32 (dd, J = 8.1, 2.5 Hz, 1H), 7.02
(t, J = 9.1 Hz, 1H), 6.74 (d, J = 8.5 Hz, 2H), 6.17 (d, J = 29.7 Hz, 1H), 5.90 (q, J = 15.2 Hz,
13
2H), 4.13 (q, J = 7.1 Hz, 2H), 3.78 (s, 2H), 1.17 (t, J = 7.1 Hz, 3H); C NMR (100 MHz,
CDCl3): 165.05, 162.08 (d, J = 252.6 Hz), 161.88, 152.71, 150.04, 147.66 (s), 146.26, 143.91,
137.64, 130.81 (d, J = 8.9 Hz), 126.96, 125.01, 121.98, 121.74, 120.35, 120.33 (d, J = 24.6
Hz), 115.81 (d, J = 21.1 Hz), 115.27, 106.37, 60.89, 52.08, 51.99, 14.08; EI-MS: 582.3
[M+H]+.
Ethyl
6-((1H-1,2,3-triazol-1-yl)methyl)-4-(2-bromo-4-fluorophenyl)-2-(thiazol-2-yl)-1,4-dihydropyr
imidine-5-carboxylate (II-7). Yellow solid, yield: 72%, mp: 115–119 °C; 1H NMR (400 MHz,
CDCl3) δ: 7.85 (d, J = 0.7 Hz, 1H), 7.80 (d, J = 3.1 Hz, 1H), 7.75 (s, 1H), 7.52 (s, 1H), 7.49
(d, J = 3.1 Hz, 1H), 7.39–7.28 (m, 2H), 7.03 (td, J = 8.3, 2.6 Hz, 1H), 6.13 (d, J = 2.4 Hz, 1H),
13
5.95 (s, 1H), 5.90 (s, 1H), 4.13 (q, J = 7.1 Hz, 2H), 1.15 (t, J = 7.1 Hz, 3H); C NMR (100
MHz, CDCl3): 165.00, 162.08 (d, J = 252.8 Hz), 161.81, 152.58, 150.03, 143.97, 137.64 (d, J
= 3.5 Hz), 133.35, 130.76 (d, J = 8.8 Hz), 125.23, 124.90, 122.03 (d, J = 9.6 Hz), 120.34 (d, J
= 24.6 Hz), 115.80 (d, J = 21.1 Hz), 106.44, 60.88, 51.96, 51.85, 14.05; EI-MS: 491.4
[M+H]+.
Ethyl
4-(2-bromo-4-fluorophenyl)-6-((4-(1-hydroxypentyl)-1H-1,2,3-triazol-1-yl)methyl)-2-(thiazol-
2-yl)-1,4-dihydropyrimidine-5-carboxylate (II-8). Yellow solid, yield: 61%, mp: 79–83 °C;
This article is protected by copyright. All rights reserved.

1H NMR (400 MHz, CDCl3) δ: 7.78 (d, J = 3.1 Hz, 1H), 7.76 (s, 1H), 7.46 (d, J = 2.9 Hz, 1H),
7.39–7.25 (m, 2H), 7.02 (td, J = 8.3, 2.3 Hz, 1H), 6.13 (s, 1H), 5.93 (dd, J = 15.4, 7.7 Hz, 1H),
5.82 (dd, J = 15.4, 7.5 Hz, 1H), 4.93 (t, J = 6.5 Hz, 1H), 4.11 (q, J = 7.1 Hz, 2H), 2.60 (s, 1H),
1.99–1.84 (m, 2H), 1.63–1.28 (m, 4H), 1.15 (t, J = 7.1 Hz, 3H), 0.88 (t, J = 7.1 Hz, 3H);
EI-MS: 577.5 [M+H]+.
Ethyl
4-(2-bromo-4-fluorophenyl)-6-((4-((4-methylbenzamido)methyl)-1H-1,2,3-triazol-1-yl)methyl
)-2-(thiazol-2-yl)-1,4-dihydropyrimidine-5-carboxylate (II-9). Yellow solid, yield: 47%, mp:
85–89 °C; 1H NMR (400 MHz, CDCl3) δ: 7.93 (s, 1H), 7.72 (d, J = 8.1 Hz, 2H), 7.70 (d, J =
3.1 Hz, 1H), 7.30 (ddd, J = 15.0, 7.8, 5.2 Hz, 3H), 7.20 (d, J = 7.9 Hz, 2H), 7.15 (s, 1H), 7.00
(td, J = 8.3, 2.5 Hz, 1H), 6.11 (s, 1H), 5.89 (dd, J = 40.6, 15.7 Hz, 2H), 4.76 (d, J = 5.2 Hz,
2H), 4.11 (q, J = 7.1 Hz, 2H), 2.37 (s, 3H), 1.14 (t, J = 7.1 Hz, 3H); EI-MS: 638.3 [M+H]+.
Ethyl
4-(2-bromo-4-fluorophenyl)-2-(thiazol-2-yl)-6-((4-((2,4,6-trimethylphenylsulfonamido)methyl
)-1H-1,2,3-triazol-1-yl)methyl)-1,4-dihydropyrimidine-5-carboxylate (II-10). Yellow solid,
yield: 61%, mp: 96–99 °C; 1H NMR (400 MHz, CDCl3) δ: 7.81 (d, J = 3.1 Hz, 1H), 7.69 (s,
1H), 7.51 (d, J = 3.1 Hz, 2H), 7.36–7.29 (m, 2H), 7.04 (td, J = 8.3, 2.5 Hz, 1H), 6.95 (s, 2H),
6.12 (d, J = 2.5 Hz, 1H), 5.05 (t, J = 6.0 Hz, 1H), 4.25 (d, J = 6.1 Hz, 2H), 4.12 (d, J = 7.1 Hz,
3H), 2.65 (s, 6H), 2.31 (s, 3H), 1.15 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3): 164.94,
162.09 (d, J = 252.9 Hz), 161.70, 152.26, 150.06, 144.00, 142.94, 142.34, 139.11, 137.63 (d,
J = 3.5 Hz), 133.50, 132.01, 130.77 (d, J = 8.8 Hz), 125.08, 124.14, 122.02 (d, J = 9.7 Hz),
120.35 (d, J = 24.7 Hz), 115.85 (d, J = 21.1 Hz), 106.36, 60.89, 58.31, 51.97, 38.34, 22.98,
20.95, 14.05; EI-MS: 702.4 [M+H]+.
In Vitro Anti-HBV Assay.19 HepG2.2.15 cell lines are HBV-transfected hepatoma cell
lines, which can stably secrete HBsAg, HBeAg, HBV DNA, HBV RNA, cccDNA and intact
virus particles for a long time. It is the most widely used HBV drug screening cell model.
HepG2.2.15 cell lines were obtained from the cell bank of the Chinese Academy of Sciences.
This article is protected by copyright. All rights reserved.

The cell lines were maintained in MEM medium supplemented with 10% fetal bovine serum,
100 U/mL penicillin, 100 µg/mL streptomycin, and 0.38 mg/mL of G418. Designed
compounds were evaluated their in-vitro biological activities on the HepG2.2.15 cell line.
After adding compounds, the HBV particles and antigens in the cell culture supernatant will
change. Cytotoxicity was tested using CCK-8 method (CCK-8; Dojindo, Tokyo, Japan).
Anti-HBV DNA replication activities were tested by real-time PCR assay. The anti-HBV
antigen secretion activities were tested by enzyme linked immunosorbent assay (ELISA;
Autobio Diagnostics Co., Ltd, China).
Toxicity measurements.20 Cytotoxicity of tested compounds to HepG2.2.15 cells were
assessed via CCK-8 method. Five different doses of tested compounds were added to 96-well
tissue culture plates with 4000 cells in every well. Untreated cells with media alone were
used as controls. The culture mediumwas replaced with a fresh one on day 4. After 8-day in
triplicate of cell culture, 10% CCK-8 solution was added 0.5 h before the end of culture. OD
absorbance values at 450 nm were collected by microplate reader (Bio-Rad, model 550) and
the cell death percent was calculated.
Real time fluorescent PCR.18 HepG2.2.15 cells were cultured in triplicate of 96-well
tissue culture plates with five different doses of tested compounds. The culture mediumwas
replaced with a fresh one on day 4 during the 8-day experiment. Untreated cells with media
alone were used as controls. On day 8, the supernatants of HepG2.2.15 cell were collected,
which were quantified their HBV DNA using PCR-fluorescent probing (Quantitative
diagnostic kit for HBV DNA). Briefly, 100 µL of the supernatants were added into the
extraction buffer, boiled for 10 min and centrifuged for 10 min, and then proper aliquots were
used for the fluorescent probing PCR. After PCR was run, the results were analyzed.
Inhibiting the secretion of HBeAg and HBsAg.21 HepG2.2.15 cells were cultured in
triplicate of 96-well tissue culture plates with five different doses of tested compounds. The
culture mediumwas replaced with a fresh one on day 4 during the 8-day experiment.
Untreated cells with media alone were used as controls. On day 8, aspirate cell supernatant
This article is protected by copyright. All rights reserved.

and detect content of HBeAg and HBsAg by using diagnostic kit (Autobio Diagnostics Co.,
Ltd, China). The absorbance (A) of tested compounds were determined on microtiter plate
ELISA reader. Drug inhibition ratio = (A normal cell group - A experimental group)/(A
normal cell group - A blank group) × 100%.
Molecular Docking. Molecular modeling was performed with the Tripos molecular
modeling package Sybyl-X 2.0. All the molecules for docking were built using standard bond
lengths and angles from Sybyl-X 2.0/Base Builder. The molecule (II-1) was optimized by
using the Tripos force field for 1000 generations two times or more until the minimized
conformers of the ligand were the same. The protein was prepared by removing the water
molecules and ligand and adding polar hydrogen atoms. The published crystal structure of the
liganded HBV capsid complex (PDB codes: 5e0i) was retrieved from the Protein Data Bank.
The flexible docking method (Surflex-Dock) docks the ligand automatically into the
ligand-binding site of the receptor by using a protocol-based approach and an
empirically-derived scoring function. The protocol is a computational representation of a
putative ligand that binds to the intended binding site and is a unique and essential element of
the docking algorithm. The scoring function in Surflex-Dock, containing hydrophobic, polar,
repulsive, entropic, and solvation terms, was trained to estimate the dissociation constant (Kd)
expressed in −log(Kd)2. Surflex-Dock default settings were used for other parameters, such as
the maximum number of rotatable bonds per molecule (set to 100) and the maximum number
of poses per ligand (set to 20). During the docking procedure, all of the single bonds in
residue side-chains inside the defined HBV capsid binding pocket were regarded as rotatable
or flexible, and the ligand was allowed to rotate at all single bonds and move flexibly within
the tentative binding pocket. The atomic charges were recalculated using the Kollman
all-atom approach for the protein and the GasteigereHückel approach for the ligand. The
binding interaction energy was calculated, including van der Waals, electrostatic, and
torsional energy terms defined in the Tripos force field. The structure optimization was
performed for 10,000 generations using a genetic algorithm, and the 20-best-scoring
ligand-protein complexes were kept for further analysis. The −log(Kd)2 values of the
20-best-scoring complexes, representing the binding affinities of ligand with HBV capsid,
This article is protected by copyright. All rights reserved.

encompassed a wide range of functional classes (10−2-10−9). Hence, only the highest-scoring
3D structural model of ligand-bound HBV capsid was chosen to define the binding
interaction.
.
Funding
Financial support from the National Natural Science Foundation of China (NSFC Nos.
81273354, 81573347), Key Project of NSFC for International Cooperation (No.
81420108027), Young Scholars Program of Shandong University (YSPSDU, No.
2016WLJH32), the Fundamental Research Funds of Shandong University (No. 2017JC006),
Key research and development project of Shandong Province (No. 2017CXGC1401), Key
Laboratory of Chemical Biology (Ministry of Education) Open Projects Fund (No.
CB-201715) and Major Project of Science and Technology of Shandong Province (No.
2015ZDJS04001) is gratefully acknowledged.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
HAPs, heteroaryldihydropyrimidines; SARs, structure-activity relationships; CHB, Chronic
hepatitis B; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; NRTIs,
nucleoside/nucleotide reverse transcriptase inhibitors; 3TC, lamivudine; ETV, entecavir; LdT,
telbivudine; ADV, adefovir dipivoxil; TDF, tenofovir disoproxil; TAF, tenofovir alafenamide;
cccDNA, covalently closed circular DNA; pgRNA, pregenomic RNA; FDA, Food and Drug
Administration; IC50, the concentration causing 50% inhibition of antiviral activity; CC50,
50% cytotoxicity concentration; SI, selection index.
This article is protected by copyright. All rights reserved.

FIGURE LEGENDS
Figure 1. The structures of six NRTIs approved by US FDA for HBV therapy.
Figure 2. The structures of four HBV capside inhibitors.
Figure 3. 4-methyl HAPs (green, PDB ID: 5GMZ) and sulfamoylbenzamides (yellow, PDB ID: 5T2P)
bind in Hepatitis B virus core protein Y132A mutant. The result was displayed by PyMOL.
Figure 4. The design of target compounds.
Figure 5. (a) Predicted binding mode of compound II-1 in HBV capsid (PDB ID: 5e0i); (b) Surface
presentation of II-1 located toward a solvent exposure region. Hydrogen bonds are indicated with dashed
lines in red, and all the non-polar hydrogen atoms are omitted for clarity.
REFERENCES
(1) Schinazi, R. F.; Ehteshami, M.; Bassit, L.; Asselah, T. Towards HBV curative therapies.
Liver International. 2018, 38, 102−114.
(2) Lin, C.; Yang, H.; Kao, J. Hepatitis B virus: new therapeutic perspectives. Liver
International: Official Journal of the International Association for the Study of the Liver.
2016, 36, 85−92.
(3) Tajiri, K.; Shimizu, Y. New horizon for radical cure of chronic hepatitis B virus
infection. World Journal of Hepatology. 2016, 8, 863−873.
(4) De Clercq, E. Current treatment of hepatitis B virus infections. Reviews in Medical
Virology. 2015, 25, 354–365.
(5) Liaw, Y.; Chu, C. Hepatitis B virus infection. Seminars in Fetal & Neonatal Medicine.
2009, 373, 582–592.
(6) Liu, N.; Zhao, F.; Zhan, P.; Liu, X. Review of Small Synthetic Molecules Targeting
HBV Capsid Assembly. Medicinal Chemistry. 2015, 11, 710−716.
This article is protected by copyright. All rights reserved.