Design, synthesis and biological evaluation of seco-A-pentacyclic triterpenoids-3,4-lactone as potent non-nucleoside HBV inhibitors

Article abstract of DOI:10.1016/j.bmcl.2018.03.076

A series of seco-A-pentacyclic triterpenoids-3,4-lactone were synthesized and the anti-HBV activities were evaluated in vitro. Several compounds inhibited the secretion of HBV antigen and the replication of HBV DNA in micromolar level. Compounds D7 and D10, seco-A-oleanane-3,4-lactone, suppressed the HBeAg secretion with IC₅₀ values of 0.14 μM and 0.86 μM respectively, and the inhibitory activities were also confirmed by detecting the fluorescence intensity of FITC-labeled monoclonal mouse HBeAg antibody via flow cytometry. Compounds D7 and D10 as well as B4, ring-A cleaved 3,30-dioic acid, also displayed remarkable inhibition on both HBV DNA replication at the concentration of 25 μM and HBV cccDNA (covalently closed circularDNA) replication with IC₅₀ values of 33.5 μM, 32.7 μM and 12.3 μM respectively.

Full text of DOI:10.1016/j.bmcl.2018.03.076

Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and biological evaluation of seco-A-pentacyclic
triterpenoids-3,4-lactone as potent non-nucleoside HBV inhibitors
a
b
a
a
a
a,c,
⇑
Zhijian Li , Qingxi Min , Haoji Huang , Ruixuan Liu , Yongyan Zhu , Quanhong Zhu
a
School of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510515, PR China
Integrated Hospital of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510315, PR China
Guangdong Provincial Key Laboratory of Chinese Medicine Pharmaceutics, Guangzhou 510515, PR China
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of seco-A-pentacyclic triterpenoids-3,4-lactone were synthesized and the anti-HBV activities
were evaluated in vitro. Several compounds inhibited the secretion of HBV antigen and the replication
of HBV DNA in micromolar level. Compounds D7 and D10, seco-A-oleanane-3,4-lactone, suppressed
the HBeAg secretion with IC50 values of 0.14 lM and 0.86 lM respectively, and the inhibitory activities
were also confirmed by detecting the fluorescence intensity of FITC-labeled monoclonal mouse HBeAg
Received 1 February 2018
Revised 26 March 2018
Accepted 28 March 2018
Available online xxxx
antibody via flow cytometry. Compounds D7 and D10 as well as B4, ring-A cleaved 3,30-dioic acid, also
Keywords:
displayed remarkable inhibition on both HBV DNA replication at the concentration of 25
l
M and HBV
seco-A-triterpenoid-3,4-lactone
HBeAg secretion inhibition
HBV cccDNA replication
cccDNA (covalently closed circularDNA) replication with IC50 values of 33.5
respectively.
l
M, 32.7
l
M and 12.3 lM
Ó 2018 Elsevier Ltd. All rights reserved.
Hepatitis B virus (HBV), one of the DNA viruses, represents a
major global health problem with an estimated 2 billion carriers
worldwide. The HBV carriers are strongly associated with liver
for the treatment of hepatitis in south China, and exhibited much
stronger anti-HBV activities than their co-exiting precursors.11
Therefore, we speculated that seco-ring-A-lactone in the triter-
penoids may be a potential pharmacophore which was able to turn
1
diseases like acute and chronic hepatitis, cirrhosis, hepatocellular
carcinoma, and even liver failure, which cause as many as
7
the rigid skeleton into flexible one and get more binding sites for
80,000 deaths per year.2 Up to now, there are seven HBV inhibi-
,3
the anti-HBV activities.
14,15
In this manuscript, several triter-
tors approved by FDA, including the immune system modulator
interferon- and viral DNA polymerase inhibitor nucleotides. How-
ever, the low curing rate, serious side effects, severe drug resis-
penoids with antiviral activities such as oleanolic acid (OA, Fig. 1)
and glycyrrhetinic acid (GA, Fig. 1), approved by China Food and
Drug Administration for auxiliary treatment of chronic hepatitis
a
1
6–18
tance and rebound phenomena restrict the drugs’ clinical
B and gastritis,
as well as ursolic acid (UA) were modified to
4
–6
application.
the corresponding seco-ring-A-lactone derivatives and their anti-
HBV activities were evaluated.
Natural products with various skeletons and diverse biological
activities are considered as important sources in drug discovery.
The naturally occurring seco-A-triterpenoids, highly oxidized
metabolites among the triterpenes, often showed significant bio-
logical activities, such as anti-tumor, anti-inflammatory, anti-der-
matophytic and anti-HBV, although they are relatively rare
among secondary metabolites.7–11 Moreover, the triterpene deriva-
tives with the structure of seco-ring-A have been designed, synthe-
sized and assessed for anti-HCV12 and anti-glycosidase activities.
In our previous work, several seco-ring-A-friedelanes-3,4-lacone
were first isolated from Viola diffusa Ging, a kind of medicinal herb
The synthetic route of compounds A2–D10 was shown in
Schemes 1 and 2. C-3 hydroxyl groups of pentacyclic triterpenoids
were first oxidized to C-3 ketones (A2–C2) by Jones reagent respec-
tively. And then Baeyer-Villiger reaction was successfully per-
formed using 3-chloroperbenzoid acid (m-CPBA) to yield seco-A-
3,4-lactones A3, B3, C3, which were subsequently converted to
the ring A cleaved 3,28-dioic acids A4 and B4 by the hydrolysis
of lactones. It was found that C-12/C-13 double bond of OA was
more preferentially oxidized than 3-ketone in the synthesis of
ring-fusion compound, and thus two intermediates D3 and D6,
13
1
2,16-lactone and 10,11-a,b-unsaturated ketone of OA were pro-
duced first. The corresponding seco-A-triterpenoids D4 and D7
were acquired via the second Baeyer-Villiger oxidation on the
intermediates. In order to synthesize the seco-A-3,4-lactone of
⇑
Corresponding author at: School of Traditional Chinese Medicine, Southern
Medical University, Guangzhou 510515, PR China.
E-mail address: zqh@smu.edu.cn (Q. Zhu).
https://doi.org/10.1016/j.bmcl.2018.03.076
0
960-894X/Ó 2018 Elsevier Ltd. All rights reserved.
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2
Z. Li et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Fig. 1. Structure of ursolic acid, glycyrrhetinic acid, oleanolic acid and friedelin.
2 2 3 2 2
Scheme 1. Reagents and conditions: (a) Jones reagent, CH Cl , acetone, room temperature, 1 h; (b) m-CPBA, LiCO , CH Cl , room temperature, 24 h; (c) 5% KOH-MeOH, room
temperature, 44 h.
Scheme 2. Reagents and conditions: (a) Jones reagent, CH
2
Cl
2
, acetone, room temperature, 1 h; (b) m-CPBA, CH
2
2 3 2 2
Cl , room temperature, 8 h; (c) m-CPBA, LiCO , CH Cl , room
temperature, 24 h; (d) 5% KOH-MeOH, room temperature, 44 h; (e) NBS, CH
2
2
Cl , 0 °C, 4 h; (f) zinc powder, 5% CH
3
COOH-CHCl , room temperature, 48 h.
3
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Z. Li et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
3
OA, the C-12/C-13 double was protected firstly by bromine (D8),
and then followed by oxidization (D9) and deprotection, the target
compound (D10) were successfully obtained in the end.
HepG 2.2.15 cells are derived from the clonal cells of Hep G2
cells transfected with a plasmid containing hepatitis B virus
(HBeAg), core particles, and virions.21 Since HepG2.2.15 cell con-
tained the whole HBV genome, some procedures of the HBV prop-
agation such as nucleic acid translation and transcription, antigenic
secretion, viral core assembly, can be perfectly reproduced like the
hepatocytes in hepatitis B patient. HepG2.2.15 cells have been
commonly used at present for the anti-HBV activity evaluation of
1
9,20
(
HBV) DNA and could steadily secrete spherical and filamentous
2
2
forms of hepatitis B surface antigen (HBsAg), hepatitis B e antigen
the potent compounds or candidate drugs in vitro.
Table 1
Anti-HBV activities of synthesized compounds.
Compound
CC50a
(
lM)
HBsAg
IC50b
HBeAg
(
l
M)
SIc
IC50b
(l
M)
SIc
A1
A3
A4
B1
B3
B4
C1
C2
D1
D4
D5
D7
D8
D9
D10
1537.8
405.7
461.3
650.0
546.2
2043.0
1787.4
481.7
731.2
540.5
452.4
583.1
100.4
484.6
477.0
876.2
1357.1
284.3
405.7
509.8
170.6
259.4
1206.5
765.4
1364.5
286.5
340.4
321.1
882.7
304.7
360.3
917.4
1.13
1.43
1.13
1.27
3.20
7.87
1.48
0.63
0.53
1.57
1.32
1.81
0.11
1.59
1.32
0.95
896.7
176.5
384.5
805.5
81.9
1.72
2.30
1.20
0.81
6.67
7.95
1.43
0.61
0.90
3.03
4.04
>200
0.10
5.21
>200
0.61
257.1
1247.1
789.5
813.6
149.1
111.7
0.14
924.6
92.3
0.86
3
TC
1436.1
a
b
c
CC50, concentration required to reduce the viability of HepG 2.2.15 cells by 50%.
IC50, concentration of compound required for 50% inhibition of HBeAg or HBsAg secretion.
SI, selectivity index, the ratio of CC50/IC50
.
Fig. 2. Inhibitory effects of D7 on HBeAg secretion determined using FITC-labeled monoclonal mouse HBeAg antibody: (a) control group; (b) 6.25
M.
l
M; (c) 12.5
lM; and (d) 25
l
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4
Z. Li et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Fig. 3. Inhibitory effects of D10 on HBeAg secretion determined using FITC-labeled monoclonal mouse HBeAg antibody: (a) control group; (b) 6.25
M.
lM; (c) 12.5 lM; and (d)
25 l
by 50% (CC50). The anti-HBV activity of each compound was
expressed as the concentration of compound that achieved 50%
inhibition (IC50) of HBsAg, HBeAg, and HBV cccDNA replication.
The selectivity index (SI), one of the important pharmaceutical
parameter, was determined as the ratio of CC50 value to IC50 value.
The bioactivity of each compound was evaluated by the combina-
tion of its IC50 values and SI.
Seven compounds A3, B3, C2, D4, D7, D9 and D10, possessing
the structure of seco-A-triterpenoid-3,4-lactone, improved the
inhibitory effects against the secretion of both HBsAg and HBeAg
compared to A1, B1, C1, and D1. The hydrolysis products A4, B4,
the ring-A cleaved acids, showed less inhibitory activity compared
to the unhydrolyzed lactones. Compounds D7 and D10 displayed
the most promising suppression against the HBeAg secretion with
IC50 values of 0.14
consistent with our previous study.
lM and 0.86 lM respectively. The results was
1
1
The inhibitory effects of compounds D7 and D10 on HBeAg
secretion inhibition were further observed by flow cytometry. As
shown in Figs. 2 and 3, after treatment with different concentra-
Fig. 4. Inhibitory effects of seco-A-triterpenoids on HBV DNA level.
tions (6.25 lM, 12.5 lM, and 25 lM) of compound D7 and D10
for 3 days, the cells without staining of FITC-labeled monoclonal
mouse HBeAg antibody were increased from 0.5% (control) to
7
.7%, 7.8%, 33.8%, and 6.2%, 10.6%, 34.0% respectively due to the
The cytotoxicity of the compounds was measured by MTT assay
decrease of HBeAg secretion, demonstrating that D7 and D10 can
effectively suppress the secretion of HBeAg, which can induce T
cells to attack hepatocytes, causing liver damage.
After the treatment of the HepG 2.2.15 cells with the test
compounds at the concentration of 25 M for 9 days, the cellular
in HepG2.2.15 cell line, and the inhibitory effects on the secretion
of HBsAg and HBeAg were determined by ELISA and flow cytome-
try. The suppressions on HBV DNA and cccDNA replication were
screened by real-time PCR using lamivudine (3TC) as positive con-
trol. The results were summarized in the Table 1 and Figs. 2–5. The
cytotoxicity of each compound was expressed as the concentration
of compound required to reduce the viability of HepG 2.2.15 cells
2
3–25
l
HBV DNA levels were quantified by real-time PCR. Fig. 4 showed
that compounds B3, B4, D4, D5, D7 and D10 significantly dis-
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Z. Li et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
5
Fig. 5. Inhibitory effects of seco-A-triterpenoids on HBV cccDNA level.
played the suppressing potency on HBV DNA replication com-
pared to the parent compounds. It was exciting that the inhibi-
tory rate of compounds B4, D7 and D10 were 91.40% ± 2.59%,
of 0.14 lM and 0.86 lM respectively. Compounds D7 and D10 as
well as B4, ring-A cleaved 3,30-dioic acid, displayed remarkable
inhibition on both HBV DNA replication at the concentration of
8
2.64% ± 3.68% and 81.62% ± 5.78% respectively at the concentra-
tion of 25 M, which were closer to that of the positive control
TC 92.80% ± 3.41%. The inhibition of compounds B4, D7 and
D10 on HBV cccDNA was further investigated by real-time PCR,
and the IC50 values were 12.3 Μ (SI = 166.5), 33.5 M (SI =
7.4) and 32.7 M (SI = 14.5) respectively, which were slightly
less than that of the positive control 3TC 4.1 M (SI = 213.6)
Fig. 5). It was very interesting B4 showed low cytotoxicity and
25
32.7
l
M and HBV cccDNA replication with IC50 values of 33.5
lM,
l
l
M and12.3 M respectively.
l
3
Acknowledgment
l
l
1
l
This work was supported by the grants from the Natural Science
Foundation of Guangdong Province, China (No. 2017A030313775).
l
(
low activity in HBeAg and HBsAg secretion inhibition while high
activity in HBV DNA replication which was the same as the pos-
itive control 3TC.
A. Supplementary data
The anti-HBV activity of oleanane type derivatives was gener-
ally better than that of the ursane type and friedeiane type. The
activities of derivatives with C12/C13 double bond B3, B4, D7,
D10 were better than compounds D4, D5, D8 and D9. And when
the double bond of oleanane type derivatives was blocked by the
stereo-hindrance (ursane type derivatives) or oxidized by NBS/m-
CPBA, the derivatives’ activities declined. Moreover the inhibition
on HBV DNA replication of hydrolysis products A4, B4, D5, was
slightly higher than A3, B3, D4, revealing that both unhydrolyzed
lactones and their ring-A cleaved acids possessed the inhibitory
activities on HBV DNA replication.
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.bmcl.2018.03.076.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
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