Optimization and Synthesis of Pyridazinone Derivatives as Novel Inhibitors of Hepatitis B Virus by Inducing Genome-free Capsid Formation

Article abstract of DOI:10.1021/acsinfecdis.6b00159

The capsid of hepatitis B virus (HBV) plays a vital role in virus DNA replication. Targeting nucleocapsid function has been demonstrated as an effective approach for anti-HBV drug development. A high-throughput screening and mechanism study revealed the h

Full text of DOI:10.1021/acsinfecdis.6b00159

Subscriber access provided by UB + Fachbibliothek Chemie | (FU-Bibliothekssystem)
Article
Optimization and Synthesis of Pyridazinone Derivatives as Novel
Inhibitors of Hepatitis B Virus by Inducing Genome-free Capsid Formation
Dong Lu, Feifei Liu, Weiqiang Xing, Xiankun Tong, Lang Wang, Yajuan
Wang, Limin Zeng, Chunlan Feng, Li Yang, Jian-Ping Zuo, and Youhong Hu
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.6b00159 • Publication Date (Web): 18 Dec 2016
Downloaded from http://pubs.acs.org on December 19, 2016
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
ACS Infectious Diseases is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 25
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
Optimization and Synthesis of Pyridazinone
Derivatives as Novel Inhibitors of Hepatitis B
Virus by Inducing Genomeꢀfree Capsid
Formation
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Dong Lu,
Yajuan Wang,
Youhong Hu*,
‖
,
§
,
‡
Feifei Liu,
‖
,
†
,
‡
Weiqiang Xing,
§
Xiankun Tong,
† Lang Wang,§
†
,
‡
Limin Zeng,
§
Chunlan Feng,
†
Li Yang, *, Jianping Zuo,*,†
†
§
§
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, 555 ZuChongZhi Road, Shanghai, 201203, China
†
Laboratory of Immunopharmacology, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, 555 ZuChongZhi Road, Shanghai, 201203, China
‡University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049,
China
*
To whom all correspondences should be addressed:
Youhong Hu, Ph.D, Fax: +86ꢀ2150805896. Eꢀmail: yhhu@simm.ac.cn;
Jianping Zuo, Ph.D, Fax: +86ꢀ2150806701. Eꢀmail: jpzuo@simm.ac.cn;
Li Yang, Ph.D, Fax: +86ꢀ2150806701. Eꢀmail: yangli@simm.ac.cn;
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 2 of 25
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ABSTRACT: The capsid of Hepatitis B virus (HBV) plays a vital role in the virus DNA
replication. Targeting nucleocapsid function has been demonstrated as an effective
approach for antiꢀHBV drugs development. The highꢀthroughput screening and
mechanism study revealed the hit compound 4a as an HBV assembly effectors (AEfs),
which could inhibit HBV replication by inducing the formation of HBV DNAꢀfree
capsids. The subsequent SARs study and drugꢀlike optimization resulted in the discovery
of the lead candidate 4r, with potent antiviral activity (IC50 = 0.087±0.002 µM), low
cytotoxicity (CC50 = 90.6±2.06 µM), sensitivity to nucleosideꢀanalog resistant HBV
mutants and synergistic effect with nucleosideꢀanalogs in HepG2.2.15 cell.
KEYWORDS: Pyridazinone, HBV DNAꢀfree capsids, drugꢀlike optimization, SARs
study, lead candidate
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
INTRODUCTION
Hepatitis B virus (HBV), the prototype of the Hepadnaviridae family, is a small,
circular, enveloped, and partially doubleꢀstranded DNA virus harboring only four
overlapping reading frames that encode for precore/core, polymerase, envelop and X
1
ꢀ3
proteins. Chronic HBV infection remains a common public health issue worldwidely,
which is strongly associated with cirrhosis, liver failure, and hepatocellular carcinoma.
4ꢀ10
Indeed, approximately 240 million people around the world are chronically infected
1
1
and contributes to 300,000 deaths annually.
Currently, treatments for HBV included interferon (interferonꢀα and pegylated
ACS Paragon Plus Environment

Page 3 of 25
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
interferonꢀα) and nucleoside analogues (i.e. lamivudine, telbivudine, adefovir, tenofovir,
2
entecavir and clevudine. Figure 1). Interferonꢀα enhances the innate immune response by
triggering a complex intracellular cascade from the JAKꢀSTAT pathway, which restricts
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
7
, 12ꢀ14
viral dissemination.
Due to the low response rate (20ꢀ30%), the need for
subcutaneous injections and the high frequency of adverse effects, the application of
15ꢀ18
interferon is limited.
Nucleoside analogues (Figure 1) are orally effective and well
tolerated through directly inhibiting HBV by targeting viral DNA polymerase. However,
emergence of drugꢀresistance and relapse are frequent after longꢀterm therapy with all the
1
9ꢀ23
nucleoside analogues beside tenofovir.
More recently, nonꢀnucleos(t)ide inhibitors
are demonstrated equally effective against wildꢀtype and nucleos(t)ide inhibitorsꢀresistant
2
4ꢀ26
viruses.
Thus, new nonꢀnucleos(t)ide agents targeting different stages in the viral life
cycle are expected and drug combinations may contribute to treatment of HBV infection.
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 4 of 25
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Figure 1. Structures of approved HBV inhibitors.
During the life cycle of HBV, the capsid plays vital roles in pregenomic RNA (pgRNA)
8
, 27ꢀ29
packaging and reverse transcription progress.
The process of nucleocapsid
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
formation is exquisitely timed and regulated, and any disruption could have significant
3
0
effects on virus replication. In the last decade, assembly effectors (AEfs), involving
3
1ꢀ35
29, 36ꢀ39
heteroaryldihydropyrimidines
and phenylpropenamides
, targeting the
encapsidation step have been developed (Figure 2). Heteroaryldihydropyrimidines,
including Bay41ꢀ4109, prevent the proper formation of viral core particles and inhibit
normal icosahedral capsid formation in vitro. Phenylpropenamides, such as ATꢀ130,
increase capsid assembly rate with disrupting the capsid tertiary and quaternary
2
9
structure. Importantly, previous research has demonstrated that either of them is active
24 ,40
against the main lamivudineꢀ and adefovirꢀresistant mutants,
which suggest that
targeting nucleocapsid functions may represent an efficient approach to the development
of novel HBV inhibitors to prevent and combat drug resistance.
In the highꢀthroughput screening of our inꢀhouse collection of compounds for
antiꢀHBV
activity,
compound
Nꢀ(4ꢀ(1ꢀ(4ꢀchlorobenzyl)ꢀ5ꢀmethylꢀ6ꢀoxoꢀ1,6ꢀdihydropyridazinꢀ3ꢀyl)phenyl)acetamide
(4a, Figure 2) was identified as a modestly potent HBV inhibitor with the primary SARs
(
Table S1ꢀS2 in SI). Moreover, the subsequent mechanism exploration indicated that
compound 4a is an HBV assembly effector (AEf), which inhibits HBV replication by
ACS Paragon Plus Environment

Page 5 of 25
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
4
1
inducing genomeꢀfree capsid formation. In consideration of its novel structural scaffold,
4
2ꢀ50
differing from those of all reported HBV inhibitors,
here we studied further the
structureꢀactivity relationships (SARs) and drugꢀlike optimization of the related class of
compounds to find the lead candidate.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. Structures of Bay41ꢀ4109, ATꢀ130 and compound 4a as assembly effectors
(AEfs).
CHEMISTRY
The synthetic route of compounds 4a-4z was depicted in Scheme 1.
5
1
6
ꢀChloroꢀ4ꢀmethylpyridazinꢀ3(2H)ꢀone (2) was synthesized by the reported procedure.
Nꢀ benzylation of pyridazinone 2 with the 1ꢀchloroꢀ4ꢀ(chloromethyl)benzene at the
presence of cesium carbonate in DMF afforded the key intermediates 3. Then, coupling
of 3 with the corresponding boronic acids or esters by a Suzuki reaction produced the
target compounds 4a-4z.
a
Scheme 1. Synthesis of compounds 4a-4z
a
0
Reagents and conditions: (a) AcOH, reflux; (b) 1ꢀchloroꢀ4ꢀ(chloromethyl)benzene, Cs CO , DMF, 50 C; (c) the
2
3
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 6 of 25
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
corresponding boronic acids or esters, Pd(dppf) Cl , K PO , 1,4ꢀdioxane/H O=4/1, 90 C.
2
2
3
4
2
RESULTS AND DISCUSSION
52
As described previously,
the potential antiꢀHBV activity and cytotoxicity of the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
synthesized pyridazinone analogues, together with the reference antiꢀHBV agent
lamivudine (3TC) and ATꢀ130, were evaluated in HepG2.2.15 cells. The results are
summarized in Tables 1ꢀ3.
Initially, the screening for the inꢀhouse libraries result showed that 4ꢀchlorobenzyl
attached to the N atom of pyridazinone core and 4ꢀmethyl group of pyridazinone are
crucial for antiꢀHBV activity, any modification on these two groups led to the loss of
activity (Table S1ꢀS2). Thus, the further structural optimization should be achieved on
the 6ꢀphenyl (A ring) of pyridazinone. Considering the low hydrophilicity of compound
4
a, the further modification on the 6ꢀphenyl (A ring) of pyridazinone was achieved by
introducing various hydrophilic heterocycles to increase the solubility and investigate the
SARs. The antiꢀHBV activity and cytotoxicity of analogues 4b-4h were summarized in
Table 1. The pyridine analogues 4b and 4c, with IC vales of 6.31±0.34 µM and 7.35±
50
1
.28 µM respectively, exhibited comparable antiꢀHBV activities and cytotoxicity
(CC50 >100 µM), indicating that pyridine ring was an ideal moiety to replace the
ꢀphenyl of pyridazinone. Oppositely, replacement of 6ꢀphenyl with pyrimidyl, thiazolyl
6
or pyrazinyl (4d-4f) led to the loss of antiviral activity. The derivatives with quinoline or
1Hꢀpyrrolo[2,3ꢀb]pyridine moiety (4g-4h) possessed the moderate activities. However,
they had pronounced cytotoxicity (CC = 47.6±3.2 and 11.1±0.9 µM, respectively),
5
0
ACS Paragon Plus Environment

Page 7 of 25
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
resulting in a relatively narrow selectivity index (SI =7.0 and 1.1, respectively). It is
noteworthy that
Table 1: SAR Study on A ring of pyridazinone core
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
b
c
d
compd
A ring
IC (µM)
CC (µM)
SI
cLogP
3.50
5
0
50
4a
1.14±0.21
>100
>90.9
4b
6.31±0.34
7.35±1.28
45.48±7.33
>100
>100
>15.6
>13.8
>2.2
ꢀꢀ
2.86
2.86
1.88
2.88
2.09
4.25
3.40
ꢀꢀ
4c
4d
>100
e
4e
NA
>100
4f
26.72±2.55
6.85±0.76
7.11±0.42
0.36±0.08
0.91±0.41
>100
>3.7
7.0
4
g
h
47.6±3.2
11.1±0.9
>1000
>20
4
1.6
3TC
>2778
ATꢀ130
>22.0
ꢀꢀ
a
b
IC50 is 50% inhibitory concentration of cytoplasmic HBVꢀDNA replication. CC50 is 50% cytotoxicity
c
d
concentration in HepG2 2.2.15 cells. Selectivity index(SI=CC /IC ). cLogP is the calculated logarithm
50
50
e
of the partition coefficient in octanol/water using the ChemBioDraw Ultra 12.0 program. NA= not active at
concentration of CC .
5
0
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 8 of 25
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
When the fluorine atom was introduced to 2 position of pyridine ring (4i), the
antiꢀHBV activity increased dramatically with IC value of 0.45 ± 0.07 µM against HBV
5
0
DNA replication. The computerized docking result (Figure S1) showed that the F atom of
i could formed two hydrogen bonds with Trp102 and Ser106 residues of capsid protein,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
which may contribute to the improvement of the activity. However, the antiꢀHBV activity
of compound was lost when the fluoro group was moved to 4 position (4j). Moreover,
two analogues 4k and 4l, with fluorine atom at 5 or 6 positions of pyridine ring, showed
good antiꢀHBV activity with their IC50 values of 1.80 ± 1.56 and 1.90 ± 0.57 µM,
respectively, which is slightly more potent than compound 4b with no substituent on the
pyridine ring. Incorporation of electronꢀdonating group (4m, 4n) or strong
electronꢀwithdrawing group (4q) at 2 position of pyridine, led to the loss of antiꢀHBV
activity. In addition, compound 4o, with a methyl group, or compound 4p, with a cyano
group, also exhibited moderate antiꢀHBV activity. The above results indicated that the
substituent with F at 2 position of the pyridine ring is optimal for antiꢀHBV activity.
Table 2: SAR Study of substituents on pyridine moiety.
a
b
c
d
compd
R
2ꢀF
4ꢀF
IC (µM)
CC (µM)
SI
cLogP
3.05
5
0
50
4i
0.45±0.07
95.4±5.31
10.4±0.29
212
ꢀꢀ
e
4j
NA
3.05
ACS Paragon Plus Environment

Page 9 of 25
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
5ꢀF
4
k
1.80±1.56
>100
>100
>55.5
>52.6
ꢀꢀ
3.05
3.05
2.55
3.08
3.06
2.66
3.86
ꢀꢀ
4l
6ꢀF
1.90 ±0.57
e
4m
2ꢀNH2
2ꢀOMe
2ꢀMe
NA
>100
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
e
4
n
NA
46.7±15.1
57.3±26.3
>100
ꢀꢀ
4o
4.88±0.39
4.64±0.65
11.7
>21.6
ꢀꢀ
4
4
p
q
2ꢀCN
2ꢀCF3
e
NA
>100
3TC
0.36±0.08
0.91±0.41
>1000
>20
>2778
ATꢀ130
>22.0
ꢀꢀ
a
b
IC50 is 50% inhibitory concentration of cytoplasmic HBVꢀDNA replication. CC50 is 50% cytotoxicity
c
d
concentration in HepG2 2.2.15 cells. Selectivity index(SI=CC /IC ). cLogP is the calculated logarithm
50
50
e
of the partition coefficient in octanol/water using the ChemBioDraw Ultra 12.0 program. NA= not active
at concentration of CC .
5
0
The further optimization and SAR exploration based on compound 4i were carried out
to explore the effects of substituents at 6 position of the 2,6ꢀdisubstituted derivatives,
which retained the F at 2 position. Encouragingly, all the 2,6ꢀdisubstituted derivatives
exhibited significant potency against HBV DNA replication, except for the analogues 4y
and 4z, with bulky group at 6 position, which decreased or lost the activities. 2,6ꢀDifluoro
analogue (4r), combined with the substitution of 4i and 4l, showed the most potent
antiꢀHBV activity, with an IC value of 0.087±0.002 µM against HBV DNA replication
5
0
and excellent SI data = 1041 (CC = 90.6±2.06 µM). However, compound 4s, with the
50
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 10 of 25
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
introduction of a methoxyl group, resulted in a remarkable decreasing in antiꢀHBV
potency. Although the analogues 4t-4w showed comparable antiꢀHBV activities, the
cytotoxicity also increased dramatically compared to 4r. In addition, when the amino
group of compound 4t was disubstituted, the activity of compound 4x was also
decreased.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 3: SAR Study of 2,6ꢀdisubstituted derivatives
a
b
c
d
compd
R
F
IC (µM)
CC (µM)
SI
cLogP
3.21
3.82
2.77
3.55
3.58
2.93
5
0
50
4r
0.087±0.002
1.22±0.03
0.30±0.14
0.54±0.33
0.22±0.01
0.27±0.02
90.6±2.06
70.7±14.4
44.1±13.7
84.5±7.97
28.4±2.66
6.67±0.05
1041
58.0
147
4s
OMe
NH2
4t
4u
Me
92.0
129
4v
4w
24.7
4
x
y
0.94±0.05
2.10±0.13
20.9±4.81
21.9±3.08
2.96±0.22
>1000
22.2
10.4
ꢀꢀ
3.76
4.82
3.04
ꢀꢀ
4
e
4z
NA
3TC
0.36±0.08
0.91±0.41
>2778
>22.0
ATꢀ130
>20
ꢀꢀ
ACS Paragon Plus Environment

Page 11 of 25
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
a
b
IC50 is 50% inhibitory concentration of cytoplasmic HBVꢀDNA replication. CC50 is 50% cytotoxicity
c
d
concentration in HepG2 2.2.15 cells. Selectivity index(SI=CC /IC ). cLogP is the calculated logarithm
50
50
e
of the partition coefficient in octanol/water using the ChemBioDraw Ultra 12.0 program. NA= not active
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
at concentration of CC .
5
0
The antiꢀHBV activity and cytotoxicity of 4r were furtherly examined by using various
HepG2 hepatomaꢀderived cell lines that stably replicate HBV: HepG2.2.15 (ayw serotype,
GenBank accession number: U95551), HepG2.117 (ayw serotype, GenBank accession
number: U95551), and HepGTo2.45 (adw serotype, GenBank accession number:
X02763.1). The result showed that 4r also inhibit HBV DNA synthesis effectively in
other HBVꢀproducing cell lines, with IC values similar to those observed in HepG2.2.15
5
0
cells (Table 4).
a
Table 4: AntiꢀHBV activity and cytotoxicity of 4r, ATꢀ130 and 3TC
Cells HepG2.2.15(ayw) HepG2.117(ayw) HepGTo2.45(adw2)
IC (µM) CC (µM) IC (µM) CC (µM) IC (µM) CC (µM)
5
0
50
50
50
50
50
4
r
0.087±0.002 90.6±2.06 0.082±0.011 >100
0.034±0.005 >100
0.015±0.003 >20
0.20±0.03
ATꢀ130 0.91±0.41
3TC 0.36±0.08
>20
>1000
0.63±0.02
0.029±0.011 >1000
>20
>1000
a
HepG2.2.15 and HepG2.117 cells carry genotype D HBV (ayw serotype, GenBank accession number: U95551), and
HepGTo2.45 cells contain HBV genotype A2 genome (adw2 subtype; GenBank accession number: X02763.1).
Subsequently, the study on the mechanism revealed that compound 4r remained
displaying the same mode of action as compound 4a, which could induce the formation
st
of genomeꢀfree capsids (Figure 3A, 1 panel). Southern blotting analysis of 4r
demonstrated that the compound could inhibit the intracellular HBV DNA replication
nd
intermediates (RC and SS HBV DNA) in a doseꢀdependent manner (Figure 3A, 2
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 12 of 25
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
panel). Moreover, the active pyridazinones to accumulate DNAꢀfree capsids were
correlated with their antiviral activities. Compound 4a, 4i, 4r, which exhibited potent
antiꢀHBV activities, could interfere with nucleocapsid assembly correspondingly.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Oppositely, compound 4n with no HBV activity had no effect on capsid assembly (Figure
3B).
A:
B:
Figure 3: (A) 4r induced the accumulation of HBV DNAꢀfree capsids and inhibited
ACS Paragon Plus Environment

Page 13 of 25
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
intracellular HBV DNA replication intermediates. (B) The assembly effects of a set of
pyridazinones.
The emergency of drug resistant strains is the main problem associated with the current
small molecule antiviral HBV medications. It is thus extremely necessary that new
antiꢀHBV inhibitors should be evaluated for activity against drug resistant HBV
mutations, as this would be a desirable feature. Compound 4r was tested against the
major class of drug resistant HBV strain, such as 3TC/ETVꢀdualꢀresistant mutant
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(rtL180M/M204V). The result showed that the 3TC/ETVꢀresistant HBV mutants showed
resistance to 3TC and ETV but remained sensitive to 4r treatment. Compound 4r could
inhibit the various forms (RC and SS HBV DNA) of nucleosideꢀanalog resistant HBV
mutant in a doseꢀdependent manner, which confirmed the possible utility in patients who
have developed drug resistant strains (Figure 4).
Figure 4: 4r doseꢀdependently inhibited the replication of 3TC/ETVꢀresistant HBV
mutant.
Finally, we evaluated the multidrug treatment of compound 4r with nucleoside analogs
entecavir (ETV). HepG2.2.15 cells were cultured in 96ꢀwell plates and treated with 4r
and/or ETV at various concentrations for 8 days. HBV replication intermediates were
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 14 of 25
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
detected by qPCR. The results were analyzed using the medianꢀeffect/combination index
5
3
(CI)ꢀisobologram equation. For each effect level, combination index (CI) values were
then calculated according to the general combination index equation for n chemical
combination at x% inhibition: where n(CI)x is the combination index for n chemicals at
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
54
x% inhibition. As shown in Table 5 and Figure 5, the compound 4r exhibited
synergistic effect (CI<1) by coꢀtreatment with entecavir (ETV) in HepG2.2.15 cells.
Table 5: Doseꢀeffect relationship parameters and mean combination index (CI) values of
4
r and entecavir (ETV)
a
Two chemicals combination (nM)
CI Value
5
0
2CI50=
+
4
r (D)1
ETV (D)2
10.4
0
0
3
1
6
.24
.98
.91
5.6
2.5
0.98
0.83
0.33
0.42
0.96
8.68
3.02
2.56
2.52
a
CI50 is the combination index for two chemicals (4r & ETV) at 50% inhibition; D is the dose of chemical, D50 is the
effective dose of a chemical which caused a 50% inhibition of HBV replication ((D )1=86.9 nM & (D )2=10.6 nM).
50
50
ACS Paragon Plus Environment

Page 15 of 25
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
Figure 5: 4r showed obvious synergistic effect (CI<1) by coꢀtreatment with entecavir
(ETV) in HepG2.2.15 cells.
CONCLUSIONS
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In conclusion, considering the low hydrophilicity of compound 4a, the further
drugꢀlike modification on the 6ꢀphenyl (A ring) of pyridazinone was achieved by
introducing various hydrophilic heterocycles to increase the solubility and investigate the
SARs. A series of novel pyridazinone derivatives based on hit 4a was synthesized and
evaluated for their in vitro antiꢀHBV DNA replication and cytotoxicity. The Most of
compounds exhibited moderate to good antiꢀHBV activity. The most promising
compound 4r displayed the potent antiꢀHBV activity, low cytotoxicity, sensitivity to
nucleosideꢀanalog resistant HBV mutants and synergistic effect with nucleosideꢀanalogs.
Moreover, the subsequent mechanistic study presented that compound 4r remained
displaying the same mode of action as compound 4a, which could induce the formation
of genomeꢀfree capsids. Overall, the potent activity, the synthetic ease, the unique mode
of action for 4r as an attractive lead candidate, is worthy for further investigation on the
treatment of HBV infection.
ABBREVIATIONS
HBV, hepatitis B virus; 3TC, Lamivudine; ETV, entecavir; NA, not active; SI, selectivity
index; RC, relaxed circular HBV DNA; SS, singleꢀstranded HBV DNA; CI, combination
index; DMF, N,NꢀDimethylformamide; Cs CO , cesium carbonate; Pd(dppf) Cl ,
2
3
2
2
1
,1'ꢀBis(diphenylphosphino)ferroceneꢀpalladium(II)dichloride dichloromethane complex;
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 16 of 25
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
K PO , potassium phosphate.
3
4
AUTHOR INFORMATION
Corresponding Author
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
*
Y. H.: Eꢀmail: yhhu@simm.ac.cn.
*J. Z.: Eꢀmail: jpzuo@simm.ac.cn.
L. Y.: Eꢀmail: yangli@simm.ac.cn.
*
Author Contributions
‖
These authors D.L. and F. L.contributed equally and are considered as coꢀfirst authors to
this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This study was supported by a grant from the National Science Fund (81225022,
81322049 and 31570166), the National Program on Key Basic Research Project (973
Program) (2013CB911104), and Institutes for Drug Discovery and Development, Chinese
Academy of Sciences (No.CASIMM0120162013). We thank Dr. Michael Nassal
(Internal Medicine II / Molecular Biology, University Hospital Freiburg, Germany) and
Prof. Dr. Mengji Lu (Institute of Virology, University Hospital Essen, University
Duisburg Essen, Essen, Germany) for providing HepG2.117 cell line.
SUPPORTING INFORMATION
Initial SAR study of pyridazinone derivatives; docking study of 4i with capsid protein;
ACS Paragon Plus Environment

Page 17 of 25
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
synthetic procedures and characterization data of target compounds; biological assay
methods. This information is available free of charge via the Internet at
http://pubs.acs.org/.
REFERENCES
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1.
Tiollais, P., Charnay, P., Vyas, G.N. (1981) Biology of hepatitis B virus. Science,
2
2
13, 406ꢀ411.
.
Kim, K. H., Kim, N. D., Seong, B. L. (2010) Discovery and Development of
AntiꢀHBV Agents and Their Resistance. Molecules, 15, 5878ꢀ5908.
Qiu, L. P., Chen, L., Chen, K. P. (2014) Antihepatitis B therapy: a review of current
medications and novel small molecule inhibitors. Fundam Clin Pharmacol, 28, 364–381
4. Robinson, W. S. (1994) Molecular events in the pathogenesis of
hepadnavirusꢀassociated hepatocellular carcinoma. Annu Rev Med, 45, 297ꢀ323.
Kremsdorf, D., Soussan, P., PaterliniꢀBrechot, P., Brechot, C. (2006) Hepatitis B
3
.
5
.
virusꢀrelated hepatocellular carcinoma: Paradigms for viralꢀrelated human carcinogenesis.
Oncogene, 25, 3823ꢀ3833.
6
.
Brechot, C. (2004) Pathogenesis of hepatitis B virusꢀrelated hepatocellular
carcinoma: Old and new paradigms. Gastroenterology, 127, S56ꢀS61.
Bhattacharya, D., Thio, C. L. (2010) Review of Hepatitis B Therapeutics. Clin
Infect Dis, 51, 1201ꢀ1208
7
.
8.
Dienstag, J. L. (2008) Hepatitis B virus infection. N Engl J Med, 359, 1486–1500.
9
.
Ganem, D., Prince, A. M. (2004) Hepatitis B virus infectionꢀnatural history and
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 18 of 25
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
clinical consequences. N Engl J Med, 350, 1118ꢀ1129.
10. Shepard, C. W., Simard, E. P., Finelli, L., Fiore, A. E., Bell, B. P. (2006) Hepatitis B
virus infection: epidemiology and vaccination. Epidemiol Rev, 28, 112ꢀ125.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1. Naghavi, M., Wang, H.D., Lozano, R., Davis, A. (2015) Global, regional, and
national ageꢀsex specific allꢀcause and causeꢀspecific mortality for 240 causes of death,
990ꢀ2013: a systematic analysis for the Global Burden of Disease Study 2013.
1
Lancet, 385, 117ꢀ171.
12. Darnell, J. E., Kerr, I. M., Stark, G. R. (1994) JakꢀSTAT pathways and
transcriptional activation in response to IFNs and other extracellular signaling proteins.
Science, 264, 1415–1421.
13. Feld, J. J., Hoofnagle, J. H. (2005) Mechanism of action of interferon and ribavirin
in treatment of hepatitis C. Nature, 436, 967ꢀ972
1
4. Sato, K., Mori, M. (2010) Current and Novel Therapies for Hepatitis B Virus
Infection. Mini Rev Med Chem, 10, 20ꢀ31.
5. Degasperi, E., Viganò, M., Aghemo, A., Lampertico, P., Colombo, M. (2013)
1
PegIFNꢀα2a for the treatment of chronic hepatitis B and C: a 10ꢀyear history. Expert Rev
Anti Infect Ther, 11, 459ꢀ474.
1
6. Fung, J., Lai, C.L., Seto, W.K., Yuen, M. F. (2011) Nucleoside/nucleotide analogues
in the treatment of chronic hepatitis B. J Antimicrob Chemother, 66, 2715–2725.
17. Kumar, R., Nath, M., Tyrrell, D. L. (2002) Design and synthesis of novel
5
ꢀsubstituted acyclic pyrimidine nucleosides as potent and selectiveinhibitors of hepatitis
ACS Paragon Plus Environment

Page 19 of 25
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
B Virus. J Med Chem, 45, 2032ꢀ2040.
18. Suzuki, F., Suzuki, Y., Tsubota, A., Akuta, N., Someya, T., Kobayashi, M., Saitoh,
S., Arase, Y., Ikeda, K., Kumada, H. (2002) Mutations of polymerase,precore and core
promoter gene in hepatitis B virus during 5ꢀyear lamivudine therapy. J Hepatol, 37,
824ꢀ830.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
9. Chan, H. L., Tsang, S. W., Hui, Y., Leung, N. W., Chan, F. K., Sung, J. J. (2002) The
role of lamivudine and predictors of mortality in severe flareꢀup of chronic hepatitis B
with jaundice. J Viral Hepat, 9, 424–428.
2
0. Lai, C. L., Dienstag, J., Schiff, E., Leung, N. W., Atkins, M., Hunt, C., Brown, N.,
Woessner, M., Boehme, R., Condreay, L. (2003) Prevalence and Clinical Correlates of
YMDD Variants during Lamivudine Therapy for Patients with Chronic Hepatitis B. Clin
Infect Dis, 36, 687ꢀ696.
2
1. Papatheodoridis, G. V., Deutsch, M. (2008) Resistance issues in treating chronic
hepatitis B. Future Microbiol, 3, 525ꢀ538.
2. Yotsuyanagi, H., Koike, K (2007) Drug resistance in antiviral treatment for
2
.
infections with hepatitis B and C viruses. J Gastroenterol, 42, 329ꢀ335.
23. Karayiannis, P. (2003) Hepatitis B virus: old, new and future approaches to antiviral
treatment. J Antimicrob Chemother, 51, 761–785.
2
4. Billioud, G., Pichoud, C., Puerstinger, G., Neyts, J., Zoulim, F. (2011) The main
Hepatitis B virus (HBV) mutants resistant to nucleoside analogs are susceptible in vitro
to nonꢀnucleoside inhibitors of HBV replication. Antiviral Res, 92, 271–276.
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 20 of 25
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
5. Korba, B. E., Montero, A. B., Farrar, K., Mukerjee, S., Ayers, M. S., Rossignol, J. F.
(2008) Nitazoxanide, tizoxanide and other thiazolides are potent inhibitors of hepatitis B
virus and hepatitis C virus replication. Antiviral Res, 77, 56–63.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
6. Bader, T., Korba, B. (2010) Simvastatin potentiates the antiꢀhepatitis B virus
activity of FDAꢀapproved nucleoside analogue inhibitors in vitro. Antiviral Res, 86,
2
2
41–245.
7. Julie, L., Fabien, Z. (2011) The life cycle of hepatitis B virus and antiviral targets,
Future Virol, 6, 599–614.
8. Tan, Z., Maguire, M. L., Loeb, D. D., Zlotnick, A. (2013) Genetically altering the
2
thermodynamics and kinetics of hepatitis B virus capsid assembly has profound effects
on virus replication in cell culture. J Virol, 87, 3208ꢀ3216.
2
9. Katen, S. P., Tan, Z., Chirapu, S. R., Finn, M. G., Zlotnick, A. (2013) Assembly
directed antivirals differentially bind quasiꢀequivalent pockets to modify HBV capsid
tertiary and quaternary structure. Structure, 21, 1406ꢀ1416.
3
0. Tan, Z., Pionek, K., Unchwaniwala, N., Maguire, M. L., Loeb, D. D, Zlotnick, A.
2015) The interface between HBV capsid proteins affects selfꢀassembly, pgRNA
packaging, and reverse transcription. J Virol, 89, 3275ꢀ84.
1. Weber, O., Schlemmer, K. H., Hartmann, E., Hagelschuer, I., Paessens, A., Graef, E.,
(
3
Deres, K., Goldmann, S., Niewoehner, U., Stoltefuss, J., Haebich, D.,
RuebsamenꢀWaigmann, H., Wohlfeil, S. (2002) Inhibition of human hepatitis B virus
(HBV) by a novel nonꢀnucleosidic compound in a transgenic mouse model. Antiviral Res,
ACS Paragon Plus Environment

Page 21 of 25
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
5
4, 69–78.
32.
Bourne, C., Lee, S., Venkataiah, B., Lee, A., Korba, B., Finn, M. G., Zlotnick.
(2008) A. SmallꢀMolecule Effectors of Hepatitis B Virus Capsid Assembly Give
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Insight into Virus Life Cycle. J Virol, 82, 10262–10270.
33. Zlotnick, A., Ceres, P., Singh, S., Johnson, J. M. (2002) Small Molecule Inhibits and
Misdirects Assembly of Hepatitis B Virus Capsids. J Virol, 76, 4848–4854.
3
4. Stray, S. J., Bourne, C. R., Punna, S., Lewis, W. G., Finn, M. G., Zlotnick, A.
(2005) A heteroaryldihydropyrimidine activates and can misdirect hepatitis B virus
capsid assembly. Proc Natl Acad Sci U S A,102, 8138–8143.
3
5. Deres, K., Schröder, C. H., Paessens, A., Goldmann, S., Hacker, H. J, Weber, O.,
Krämer, T., Niewöhner, U., Pleiss, U., Stoltefuss, J., Graef, E., Koletzki, D., Masantschek,
R. N, Reimann, A., Jaeger, R., Gross, R., Beckermann, B., Schlemmer, K. H, Haebich, D.,
RübsamenꢀWaigmann, H
DrugꢀInduced Depletion of Nucleocapsids. Science, 299, 893ꢀ895.
6. Perni, R. B., Conway, S. C., Ladner, S. K., Zaifert, K., Otto, M. J., King, R. W.
2010) Phenylpropenamide Derivatives as Inhibitors of Hepatitis B Virus Replication.
Bioorg Med Chem Lett, 10, 2687ꢀ2690.
7. King, R. W., Ladner, S. K., Miller, T. J., Zaifert, K., Perni, R. B., Conway, S. C.,
. (2003) Inhibition of Hepatitis B Virus Replication by
3
(
3
Otto, M. J. (1998) Inhibition of Human Hepatitis B Virus Replication by ATꢀ61, a
’
Phenylpropenamide Derivative, Alone and in Combination with (ꢀ) betaꢀLꢀ2 ,
’
’
3
ꢀDideoxyꢀ3 ꢀThiacytidine. Antimicrob Agents Chemother, 42, 3179–3186.
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 22 of 25
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
8. Feld, J. J., Colledge, D., Sozzi, V., Edwards, R., Littlejohn, M., Locarnini, S. A.
(2007) The phenylpropenamide derivative ATꢀ130 blocks HBV replication at the level of
viral RNA packaging. Antiviral Res, 76, 168–177.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
9. Wang, P., Naduthambi, D., Mosley, R. T., Niu, C., Furman, P. A., Otto, M. J., Sofia,
M. J. (2011) Phenylpropenamide derivatives: Antiꢀhepatitis B virus activity of the Z
isomer, SAR and the search for novel analogs. Bioorg Med Chem Lett, 21, 4642–4647.
4
0. Delaney, W. E., Edwards, R., Colledge, D., Shaw, T., Furman, P., Painter, G.,
Locarnini, S. (2002) Phenylpropenamide Derivatives ATꢀ61 and ATꢀ130 Inhibit
Replication of WildꢀType and LamivudineꢀResistant Strains of Hepatitis B Virus In Vitro.
Antimicrob Agents Chemother, 46, 3057–3060.
41. Wang, Y. J., Lu, D., Xu, Y. B., Xing, W. Q., Tong, X. K., Wang, G. F., Feng, C. L.,
He, P. L., Yang, L., Tang, W., Hu, Y. H., Zuo, J. P. (2015) A novel pyridazinone derivative
inhibits hepatitis B virus replication by inducing genomeꢀfree capsid formation.
Antimicrob Agents Chemother, 59, 7061 –7072.
4
2. Li, Y. F., Wang, G. F., He, P. L., Huang, W. G., Zhu, F. H., Gao, H. Y., Tang, W.,
Luo, Y., Feng, C. L., Shi, L. P., Ren, Y. D., Lu, W., Zuo, J. P (2006) Synthesis and
AntiꢀHepatitis B Virus Activity of Novel Benzimidazole Derivatives. J Med Chem, 49,
.
4
4
790ꢀ4794.
3. Stachulski, A. V., Pidathala, C., Row, E. C., Sharma, R., Berry, N. G., Iqbal, M.,
Bentley, J., Allman, S. A., Edwards, G., Helm, A., Hellier, J., Korba, B. E., Semple, J. E,
Rossignol, J. F. (2011) Thiazolides as Novel Antiviral Agents. 1. Inhibition of Hepatitis B
ACS Paragon Plus Environment

Page 23 of 25
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
Virus Replication. J Med Chem, 54, 4119–4132.
44. Zhang, F., Wang, G. (2014) A review of nonꢀnucleoside antiꢀhepatitis B virus agents.
Eur J Med Chem, 75, 267ꢀ281.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4
5. Hassan, H. H. (2009) Current Chemical Approaches Directed Toward New and
Effective Therapeutic Agents Against Chronic Hepatitis Infections. Curr Org Chem, 13,
3
4
79ꢀ406.
6. Geng, C. A., Wang, L. J., Guo, R. H., Chen, J. J. (2013) SmallꢀMolecule Inhibitors
for the Treatment of Hepatitis B Virus Documented in Patents. Mini Rev Med Chem, 13,
7
4
49ꢀ776.
7. Zhan, P., Jiang, X., Liu, X. (2010) Naturally Occurring and Synthetic Bioactive
Molecules as Novel NonꢀNucleoside HBV Inhibitors. Mini Rev Med Chem, 10, 162ꢀ171.
8. Kaushik, S., Gupta, S. P., Sharma, P. K. (2010) Design and Development of
4
AntiꢀHepatitis B Virus Agents. Curr Med Chem, 17, 3377ꢀ3392.
49. Yu, W., Goddard, C., Clearfield, E., Mills, C., Xiao, T., Guo, H., Morrey, J. D.,
Motter, N. E., Zhao, K., Block, T. M., Cuconati, A., Xu, X. (2011) Design, Synthesis, and
Biological Evaluation of Triazoloꢀpyrimidine Derivatives as Novel Inhibitors of Hepatitis
B Virus Surface Antigen (HBsAg) Secretion. J Med Chem, 54, 5660–5670.
5
0. Lv, Z., Sheng, C., Wang, T., Zhang, Y., Liu, J., Feng, J., Sun, H., Zhong, H., Niu, C.,
Li, K. (2010) Design, Synthesis, and Antihepatitis B Virus Activities of Novel
2ꢀPyridone Derivatives. J Med Chem, 53, 660–668.
5
1. Himmelsbach, F., Eckhardt, M., Hamilton, B. S., SchulerꢀMetz, A., Zhuang, L.
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 24 of 25
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Inhibitors of 11BetaꢀHydroxysteroid Dehydrogenase 1. US 20120108578A1.
52. Tong, X. K., Qiu, H., Zhang, X., Shi, L. P., Wang, G. F., Ji, F. H., Ding, H. Y.,
Tang, W., Ding, K., Zuo, J. P. (2010) WSS45, a sulfated alphaꢀDꢀglucan, strongly
interferes with Dengue 2 virus infection in vitro. Acta pharmacol Sin, 31, 585ꢀ592.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
3. Chou, T.C., Talalay, P. (1983) Analysis of combined drug effects: a new look at a
very old problem. Trends Pharmacol Sci, 4, 450ꢀ454.
4. RodeaꢀPalomares, I., Petre, A.L., Boltes, K, Leganés, F., PerdigónꢀMelón, J.A.,
Rosal, R., FernándezꢀPiñas F. (2010) Application of the combination index
CI)ꢀisobologram equation to study the toxicological interactions of lipid regulators in
5
(
two aquatic bioluminescent organisms. Water Res, 44, 427ꢀ438.
ACS Paragon Plus Environment

Page 25 of 25
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
For Table of Contents Use Only
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Optimization and Synthesis of Pyridazinone
Derivatives as Novel Inhibitors of Hepatitis B
Virus by Inducing Genomeꢀfree Capsid
Formation
Dong Lu,
Yajuan Wang,
Youhong Hu*,
‖
,
§
,
‡
Feifei Liu,
‖
,
†
,
‡
Weiqiang Xing,
§
Xiankun Tong,
† Lang Wang,§
†
,
‡
Limin Zeng,
§
Chunlan Feng,
†
Li Yang, *, Jianping Zuo,*,†
†
§
ACS Paragon Plus Environment