Design, synthesis and anti-HBV activity of NVR3-778 derivatives

Article abstract of DOI:10.1016/j.bioorg.2019.103363

NVR3-778, one of the most advanced capsid assembly modulators (CAMs), is currently in phase II clinical trial for the treatment of HBV infection. In this study, we reported the first structure optimization of NVR3-778. Compound 2d was found to exhibit more potent anti-HBV activity (IC₅₀: 0.25 μM), lower cytotoxicity (CC₅₀: 10.68 μM) and higher selectivity index (SI: 40.72) than NVR3-778 (IC₅₀: 0.33 μM; CC₅₀: 5.14 μM; SI: 18.36) in vitro, and also display similar inhibitory effect on the assembly of HBV capsids as NVR3-778. Molecular docking further suggested that compound 2d might form a stronger interaction with core protein. Moreover, compound 2d also showed acceptable pharmacokinetic profiles. Currently compound 2d was selected as a new lead for further modifications, and studies to determine the in vivo anti-HBV studies of 2d will begin soon.

Full text of DOI:10.1016/j.bioorg.2019.103363

Journal Pre-proofs
Design, Synthesis and anti-HBV activity of NVR3-778 derivatives
Kai Lv, Shuo Wu, Wenyan Li, Yunhe Geng, Meng Wu, Jinming Zhou,
Yuhuan Li, Qiang Gao, Mingliang Liu
PII:
S0045-2068(19)31254-4
DOI:
https://doi.org/10.1016/j.bioorg.2019.103363
YBIOO 103363
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To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
1 August 2019
6 October 2019
12 October 2019
Please cite this article as: K. Lv, S. Wu, W. Li, Y. Geng, M. Wu, J. Zhou, Y. Li, Q. Gao, M. Liu, Design,
Synthesis and anti-HBV activity of NVR3-778 derivatives, Bioorganic Chemistry (2019), doi: https://doi.org/
10.1016/j.bioorg.2019.103363
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Design, Synthesis and anti-HBV activity of NVR3-778
derivatives
Kai Lv a, 1, Shuo Wu a, 1, Wenyan Li b, Yunhe Geng a, b, Meng Wu,a Jinming Zhou,c
Yuhuan Li a, *, Qiang Gao a, Mingliang Liu a, *
a Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union
Medical College, Beijing 100050, China
b College of Chemistry & Material Science, Hebei Normal University, Shijiazhuang 050024, China
c Key Laboratory of the Ministry of Education for Advanced Catalysis Materials, Department of
Chemistry, Zhejiang Normal University, Jinhua, 321004, China
* Corresponding author.
E-mail addresses: lmllyx@126.com (M. Liu), yuhuanlibj@126.com (Y. Li).
1 These authors contributed equally to this work.
Abstract
NVR3-778, one of the most advanced capsid assembly modulators (CAMs), is currently
in phase II clinical trial for the treatment of HBV infection. In this study, we reported the
first structure optimization of NVR3-778. Compound 2d was found to exhibit more potent
anti-HBV activity (IC50: 0.25 µM), lower cytotoxicity (CC50, 10.68 µM) and higher
selectivity index (SI, 40.72) than NVR3-778 (IC50, 0.33 µM; CC50, 5.14 µM; SI: 18.36) in
vitro, and also display similar inhibitory effect on the assembly of HBV capsids as NVR3-
778. Molecular docking further suggested that compound 2d might form a stronger
interaction with core protein. Moreover, compound 2d also showed acceptable
pharmacokinetic profiles. Currently compound 2d was selected as a new lead for further
modifications, and studies to determine the in vivo anti-HBV studies of 2d will begin soon.
Keywords: NVR3-778; structure modification; sulfamoylbenzamides; anti-HBV activity;
capsid assembly modulators.
1. Introduction
The safe and effective prophylactic Hepatatis B virus (HBV) vaccine has been
available since 1982, and vaccination dramatically reduced new HBV infections among
children[1]. However, HBV infection remains a major health problem. The World Health
Organization (WHO) estimated that approximately 257 million people are living with

chronic HBV, and 0.88 million died from HBV related complications (including cirrhosis
and hepatocellular carcinoma) worldwide in 2015 [2]. Currently, the treatment of chronic
HBV mainly relies on pegylated interferon-alpha (PEG-INFα) and nucleos(t)ide analogues,
such as lamivudine, tenofovir, and entecavir [3]. These treatments can slow the progression
of cirrhosis, reduce incidence of liver cancer, and improve long-term survival, but seldom
result in cure as they do not eradicate the covalently closed circular DNA (cccDNA), which
provides for viral persistence [4]. Therefore, at present, long-term (potentially lifelong)
therapy is required for the majority of patients. As such, there is an urgent need for
discovery of new drugs which could suppress HBV viral load and decrease cccDNA
formation.
F
F
F
H
H
B
H
B
B
O
O
O
N
N
F
F
F
F
N
F
F
S
S
S
A
N
N
N
A
F
A
O
O
O
C
O
O
H
O
H
OH
NVR3-778 (phase II)
DVR-23
AB-423 (phase I)
Figure 1. Structures of DVR-23, AB-423 and NVR3-778
In recent years, sulfamoylbenzamides (SBAs), a novel class of HBV agents as capsid
assembly modulators (CAMs) are expected to significantly decrease the formation of
cccDNA and cure HBV infection [5]. Since the discovery of the early SBA lead DVR-23
in 2013 by Blumberg Institute [6], various optimization efforts were conducted by
industrial and academia [7]. Currently, multi-SBAs have entered clinical trials such as AB-
423 (phase I, Arbutus Biopharma Inc and Blumberg Institute) [8], NVR3-778 (phase II,
Novira Therapeutics and Janssen Pharmaceutical, Figure 1) [9, 10] and JNJ6379 (phase II,
structure not disclosed, Janssen Pharmaceutical) [11]. In fact, although NVR3-778 [12] and
AB423 [8] have advanced into clinical trials for years, their mystical structures were just
uncovered in 2018. Inspired by the promising anti-HBV activity and disclosed structures
[13, 14], we intend to design, and synthesis new SBAs based on the structure feature of
NVR3-778, which were expected to explore structure-activity relationships (SAR) of
SBAs, and identify alternative candidate as anti-HBV agents.
2. Results and discussion
2.1. Structure modification and SAR

Although the structure of NVR3-778 was just disclosed in 2018 [9], the SBAs have
been a hot spot in the anti-HBV research area since the discovery of DVR-23 in 2013. A
lot of SBAs related patents have been applied [15-19], mainly revolving around the
modification of C segment of NVR3-778 (seen in figure 1). In addition, two papers from
Vandyck [11] and Schinazi [12] also reported the SAR of the SBAs, suggesting that the
ring A and B (seen in figure 1) might be important to the anti-HBV activity of these SBA
analogues. Moreover, based on the recently reported crystal structure of NVR3-778 and
core protein (PDB code: 5T2P), the benzamide oxygen of NVR3-778 forms essential
hydrogen bonds with the side chains of Trp102 (Chain B) and Thr128 (Chain C), and the
4-hydoxyl-piperidyl group is fully accessible to solvent [8, 20]. Thus, we focused on the
modification of 4-hydoxyl-piperidine moiety of NVR3-778 in this research.
As shown in Table 1, the hydroxyl group on the piperidine ring of NVR3-778 was
replaced with various functional groups such as amine, ester, oxime et al. They were
synthesized and evaluated for their anti-HBV activity and cytotoxicity against HepAD38
cell line. Compared to NVR3-778, most of them show decreased anti-HBV activity (1a-f,
IC50s: 0.75 - >2). More precisely, the introduction of amine or ester functional groups to
the piperidine ring diminished the anti-HBV activity significantly (1a-c, IC50 > 2 µM). To
our delight, compounds 1g-h with ketone and hydroxylamine functional group displayed
comparable anti-HBV activity (IC50s: 0.21-0.30 µM) to NVR3-778 (IC50: 0.28 µM).
Additionally, compounds 1g-h also showed slightly less cytotoxic to HepAD38 cells
(CC50s: 7.41 µM) and higher selectivity index (SIs, 24.70 - 35.29) than NVR3-778 (CC50:
5.77 µM; SI, 18.36).
Table 1. Structures and activity of new SBAs (Series 1)
F
H
O
N
F
F
S
W
O
O
F
IC50 CC50
(µM) (µM)
Compd.
1a
W group
SI
-
N
NH2
>2
96.15
O
O
N
1b
>2
138.67
-

O
O
N
N
N
1c
1d
1e
>2
>200
-
O
OH
1.02 12.83
0.75 66.67
1.05 >200
12.58
88.89
O
NH2
O
1f
>190.48
N
N
OH
1g
0.21 7.41
0.30 7.41
0.28 5.14
35.29
24.70
18.36
N
N
N
N
O
1h
OH
NVR3-778
Changes were subsequently focused on the piperidine ring of NVR3-778, whereas the
hydroxyl functional group was maintained. As shown in table 2, the piperidine ring of
NVR3-778 was exchanged by fused/bridged/spiro-heterocycle, amino-cyclohexyl,
phenylamino or opening rings. Among them, compounds 2a-c with fused/bridged/spiro-
heterocycle exhibited good anti-HBV activity (IC50s, 0.25-0.48 µM) and also significantly
decreased cytotoxicity (CC50s, 22.22-155.21 µM). Interestingly, the cis-aminocyclohexan-
1-ol substituted analogue 2d (IC50, 0.25 µM) was significantly more active than its trans-
diastereomer 2e (IC50 > 2 µM). N-methylation of the amide of 2d, as in 2f, resulted in
reduced anti-HBV activity. Introduction of opening rings or phenol also led to lose potency
(2g-i, IC50 > 2 µM).
Table 2. Structures and activity of new SBAs (Series 2)
F
H
N
O
F
F
S
W
O
O
F
Compd.
W group
IC50
(µM)
0.30
CC50
(µM)
22.22
SI
2a
2b
2c
74.07
30.51
323.35
N
OH
OH
N
N
0.35
0.48
10.68
155.21
OH
OH
2d
0.25
10.68
42.72
HN

2e
2f
>2
>200
22.22
-
HN
OH
OH
1.46
15.21
N
Me
HN
2g
2h
>2
>2
12.83
66.67
-
-
OH
OH
OH
HN
2i
-
NH
>2
10.68
5.14
OH
NVR3-778
0.33
15.58
According the SAR findings discussed above, the hydroxyl group on piperidine of
NVR3-778 might be replaced by ketone or hydroxylamine; the fused/ spiro/ bridged -
heterocycle ring could be an alternative selection of piperidine. We therefore installed the
ketone or oximes groups to the hydroxyl position of 2a-c, resulting in SBAs 3a-e as shown
in table 3. In accordance with the above SAR, introduction of ketone and hydroxylamine
maintained the potent anti-HBV activity (3a-b and 3d-e, IC50s: 0.22-0.54 µM). In contrast,
compound 3c with methoxylamine showed decreased anti-HBV activity with IC50 of > 2
µM. In addition, the cytotoxicity of 3a-b and 3d-e (CC50, 7.41-18.74) were all lower than
that of NVR3-778.
Table 3. Structures and activity of new SBAs (Series 3)
F
H
O
N
F
F
S
W
O
O
F
Compd.
W group
IC50 (µM)
CC50
(µM)
18.74
SI
3a
3b
0.39
0.54
48.05
13.72
N
N
O
OH
N
7.41
O
N
3c
>2
>200
-
N
N
3d
0.44
0.22
0.31
9.54
7.41
5.14
21.68
33.68
16.58
O
3e
N
O
NVR3-778

2.2 Detection of the effect of compounds on capsids assembly by a particle gel assay
We and others have demonstrated that type I CAMs such as Bay 41-4109 can abolish
capsid formation while type II CAMs may not change the amounts of capsids but induce
the assembly of capsids with faster electrophoresis mobility [21-22]. Thus, capsid
electrophoresis mobility shift can used as a diagnostic marker of compounds that target
core protein assembly and predicts sensitivity of HBV strains to specific type II CAMs [21-
22]. To further determine the anti-HBV activity of the novel compounds, the effects of
these compounds on capsids at a concentration of 1 μM were then examined by a particle
gel assay. As shown in Figure 2, NVR3-778 treatment reduced the amounts of slow-
migrating capsids but significantly increased the amounts of fast-migrating capsids, which
is consistent with the results of other SBAs [21-22]. Compounds with relatively good anti-
HBV activities (1g-h, 2a-b, 2d, 3a-3b and 3d-3e) showed similar capsids electrophoresis
mobility alteration characteristics, while compounds with IC50 more than 2 µM (1a-c, 2e,
2g-2i, and 3c) exhibited similar capsids electrophoresis migration profile to the mock
treated (NC) group. These results indicated that the synthesized novel compounds with
good antiviral activities also have inhibitory effect on the assembly of HBV capsids.
Figure 2. Particle gel assay of capsids assembled in cells treated with the indicated
compounds. HepAD38 cells were culture in the absence of tetracycline and mock treated
(NC) or treated with the indicated compounds at a concentration of 1 μM for 6 days. The
total amounts of capsids were separated by a particle gel assay in a 1.5% agarose gel

electrophoresis, transferred onto a nylon membrane, and detected by a rabbit polyclonal
antibody against core protein.
2.3. Pharmacokinetic profiles of selected SBAs
SBAs 1h, 2a-d, 3a and 3d with potent anti-HBV activity were selected and further
evaluated for their in vivo pharmacokinetic (PK) profiles in ICR mouse after a single oral
administration of 50 mg/kg. As shown in table 4, except for compound 2d, the AUC0-ts of
these SBAs were more than 10 times lower than that of NVR3-778. The introduction of
ketone (1h, 3a, 3d) resulted in dramatically decreased AUC0-t probably due to the
ketalization of ketone in vivo. Compound 2d showed acceptable PK profiles with AUC0-t
of 232025 h.ng/mL and T1/2 of 7.79 h although the AUC0-t of 2d was lower than that of
NVR3-778. Currently, studies to determine the in vivo anti-HBV activity of 2d will begin
soon.
Table 4. In vivo PK profiles of selected SBAs in mice at 50 mg/Kg
Compd.
T1/2
(h)
Tmax
(h)
Cmax
(ng/mL)
AUC0-t
MRT
(h)
7.68±1.91
2.65±0.34
2.58±0.53
1.43±0.73
(h.ng/mL)
311±44.7
10132± 3448
3051±224
503±81
1h
2a
2b
2c
2d
3a
3d
4.97±1.59 1.50±2.17 70.1±5.8
3.54 ±1.77 0.33±0.14 5406 ± 2203
2.18±0.65 0.33±0.14 1226±167
1.71±0.59 0.25±0
7.79±0.77 0.83±0.28 38247±10310 232025±46728 9.78±3.19
846±270
10.9±5.04 0.25±0
0.62±0.24 0.25±0
819±301
110±42
1425±246
80.6±31.0
13.01±5.93
0.88±0.23
NVR3-778 8.14±0.95 1.08±0.88 66090±17219 455931±56864 10.71±3.71
2.4. Molecular docking study of compound 2d
The capsid inhibitors bound to the core protein dimer-dimer interface were widely
reported [8, 20, 23-24], which indicted that compound 2d might also bound to the same
site. Thus, we predicted the binding mode of compound 2d in the core protein dimer-dimer
interface (PDB code: 5T2P) through molecular docking with CDOCK module of
Discovery Studio 3.5. The docked model of the compound 2d chemical structure was
compared with NVR3-778 which was an intrinsic ligand from the X-ray structure (PDB
code: 5T2P). As the chemical structure of compound 2d was similar to that of NVR3-778,
the binding model of compound 2d also showed a closed interaction pattern with NVR3-

778 (Figure 3A). The ligand-receptor interaction analysis showed that the central amide of
NVR3-778 forms a crucial hydrogen bond with the Trp102 of core protein (Figure 3B),
while compound 2d forms a hydrogen bond with Trp102 through its central amide as well
as an additional hydrogen bond with Leu140 through its sulfanilamido radical (Figure 3C),
which indicates compound 2d forms a stronger interaction with core protein. What is more,
comparing to NVR3-778, the more flexible sulfanilamido radical of compound 2d is
supposed to form a more suitable binding mode with the hydrophobic pocket around
Leu140 at dimer-dimer interface of core protein, thus strengthen its interaction with core
protein.
Figure 3 Binding mode of compound 2d and X-ray structure of core protein: (A)
comparison of the predicted binding model of compound 2d and NVR3-778 in the core
protein X-ray structure (PDB code: 5T2P) , the surface of the capsid protein is in yellow,
the NVR3-778 is in purple stick and compound 2d is in blue stick; (B) The detailed
interactions between NVR3-778 and core protein at the dimer-dimer interface site; (C) The

predicted binding mode of compound 2d and core protein at the dimer-dimer interface site.
3. Chemistry
Detailed synthetic pathways to target compounds 1-3 are outlined in scheme 1.
Electrophilic substitution of 4-flurobenzoic acid with chlorsulfonic acid gave sulfuryl
chloride 5, which was refluxed with thionyl chloride to yield compound 6. Refluxing
compound 6 and 3,4,5-trifluoroaniline in toluene formed the key intermediate 7 as the
reported procedure. [17] Coupling compound 7 with various amines afforded target
compounds 1-3. For the preparation of 1a, the Boc-group on piperdin-4-amine has to be
subsequently removed by TFA in DCM.
Cl
O
S
O
S
O
O
O
S
F
F
O
F
F
F
Cl
O
Cl
c
b
a
F
F
NH
HOOC
ClOC
HOOC
6
5
4
7
W
O
S
O
F
O
d
F
F
NH
F
1-3
Regents and conditions: a) ClSO3H, reflux, 3h, 71% yield; b) SOCl2, 80 oC, 85% yield; c)
toluene, 3,4,5-trifluoroaniline, reflux, 83% yield; d) i) Et3N, CH3CN, rt, 40-89% yields; ii)
For compound 1a, then TFA, DCM, 75% yield.
Scheme 1. Synthesis of the target compounds 1-3
4. Conclusion
In summary, multi-series of NVR3-778 analogues were designed and synthesized as
new anti-HBV agents in this study. Structural modifications and SAR studies focused on
the 4-hydoxyl-piperidine moiety of NVR3-778. The alternative replacement of hydroxyl
group and piperidine ring were investigated respectively. In addition, a series of SBAs was
further designed and synthesized according the SAR findings above. Compared to NVR3-
778, SBAs 1g-h, 2a-d, 3a and 3d-e were found to have comparable anti-HBV activity (IC50:
0.25-0.48 µM), lower cytotoxicity and higher selectivity index. Moreover, the particle gel
assay proved that these compounds have inhibitory effect on the assembly of HBV capsids.

However, most of them displayed decreased AUC0-t in PK evaluation. Among them,
compound 2d exhibited acceptable PK profiles with AUC0-t of 232025 h.ng/mL, and T1/2
of 7.79 h. In addition, molecular docking further suggested that compound 2d might forms
a stronger interaction with core protein. Based on above results, compound 2d was selected
as a new lead for further modifications, and studies to determine the in vivo anti-HBV
studies of 2d will begin soon.
Funding Sources
This work is supported by CAMS Innovation Fund for Medical Science (CAMS-2018-
I2M-3-004), the National Mega-project for Innovative Drugs (2018ZX09711001-007-002,
2018ZX09101003-003-003), National Natural Science Foundation of China (81621064)
and the Fundamental Research Funds for the Central Universities (3332019007).
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Hight Lights
1. 2d exhibited potent anti-HBV activity as NVR3-778.
2. 2d displayed lower cytotoxicity and higher selectivity index than NVR3-778.
3. 2d showed acceptable PK properties.
4. 2d displayed similar inhibitory effect on the assembly of HBV capsids as NVR3-778.
5. Docking suggested that 2d might form a stronger interaction with core protein.

Graphical Abstract
Title: Design, Synthesis and anti-HBV activity of NVR3-778 derivatives
Kai Lv a, 1, Shuo Wu a, 1, Wenyan Li b, Yunhe Geng a, b, Meng Wu,a Jinming Zhou,c Yuhuan Li a, *, Qiang Gao a,
Mingliang Liu a, *
a Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical
College, Beijing 100050, China
b College of Chemistry & Material Science, Hebei Normal University, Shijiazhuang 050024, China
c Key Laboratory of the Ministry of Education for Advanced Catalysis Materials, Department of Chemistry,
Zhejiang Normal University, Jinhua, 321004, China
* Corresponding author.
E-mail addresses: lmllyx@126.com (M. Liu), yuhuanlibj@126.com (Y. Li).
1 These authors contributed equally to this work.
F
S
F
S
OH
H
H
O
O
N
F
F
N
F
F
N
N
O
O
O
O
H
OH
F
F
2d
NVR3-778 (phase II)
Anti-HBV activity IC50: 0.33 µM
CC50: 5.14 µM SI: 15.58
Anti-HBV activity IC50: 0.25 µM
CC50: 10.68 µM SI:
40.72
T1/2: 8.14 h AUC0-t: 455931 h.ng/mL
T1/2: 7.79 h AUC0-t: 232025
h.ng/mL
We report herein the design, synthesis and anti-HBV activity of NVR3-778 derivatives. Results revealed
that 2d not only exhibited potent anti-HBV activity (IC50: 0.25 µM), but also low cytotoxicity (CC50: 10.68
µM), good selectivity index (SI: 40.72), and acceptable PK profiles. Further modifications and in vivo anti-
HBV studies of 2d will begin soon.