Synthesis of hemslecin A derivatives: A new class of hepatitis B virus inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.01.024

A series of hemslecin A derivatives were synthesized and evaluated for their anti-hepatitis B virus (HBV) activities, namely, inhibiting the secretion of hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and HBV DNA replication on HepG 2.2.15 cells. Most of the derivatives showed enhanced anti-HBV activities, of which compounds A1-A7, B5, C and E exhibited significant activities inhibiting HBV DNA replication with IC₅₀ values of 2.8-11.6 μM, comparable to that of the positive control, tenofovir. Compounds A1-A3, A5, B5, and C displayed low cytotoxicities, which resulted in high SI values of 89.7, 55.6, 77.8, >83.4, >55.8, and >150.5, respectively.

Full text of DOI:10.1016/j.bmcl.2013.01.024

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1201–1205
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of hemslecin A derivatives: A new class of hepatitis B virus
inhibitors
Rui-Hua Guo a, Chang-An Geng a, Xiao-Yan Huang a, Yun-Bao Ma a, Quan Zhang a, Li-Jun Wang a,
Xue-Mei Zhang a, Rong-Ping Zhang b, , Ji-Jun Chen a,
⇑
⇑
a State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, PR China
b Kunming Medical University, Kunming, Yunnan 650500, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of hemslecin A derivatives were synthesized and evaluated for their anti-hepatitis B virus (HBV)
activities, namely, inhibiting the secretion of hepatitis B surface antigen (HBsAg), hepatitis B e antigen
(HBeAg), and HBV DNA replication on HepG 2.2.15 cells. Most of the derivatives showed enhanced
anti-HBV activities, of which compounds A1–A7, B5, C and E exhibited significant activities inhibiting
Received 22 August 2012
Revised 23 December 2012
Accepted 7 January 2013
Available online 16 January 2013
HBV DNA replication with IC50 values of 2.8–11.6 lM, comparable to that of the positive control, tenofo-
vir. Compounds A1–A3, A5, B5, and C displayed low cytotoxicities, which resulted in high SI values of
89.7, 55.6, 77.8, >83.4, >55.8, and >150.5, respectively.
Keywords:
Hemslecin A
Derivatives
Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.
Hepatitis B virus
Hepatitis B virus (HBV) infection is a major health problem
worldwide. It is estimated that more than 2 billion people have
been infected with HBV, of which 350 million live with HBV
chronic infection.1,2 Chronic HBV infection concomitant with liver
damage, cirrhosis of liver, and hepatocellular carcinoma leads to
1 million deaths per year.3 HBV vaccines have made great achieve-
ments in preventing new infections, but are ineffective for HBV
carriers.4 Currently, interferons and HBV reverse transcriptase
inhibitors (lamivudine, adefovir dipivoxil, entecavir, telbivudine,
tenofovir disoproxil fumarate) are the main treatment agents for
HBV infection.5,6 However, their usage is limited due to low re-
sponse rate, serious side effects or drug resistance.7,8 Thus, the cur-
rent therapies for HBV remain unsatisfactory, and new anti-HBV
agents are urgently needed.
Natural products with various skeletons and diverse biological
activities are considered as important sources in drug discovery.
Presently, many naturally originated compounds have been re-
ported with promising anti-HBV activities and different mecha-
nism of action compared to nucleoside analogs.9–18
Many plants of the genus Hemsleya are traditionally used for
treating hepatitis, sore throat, pelvic inflammatory disease, enteri-
tis, etc. in China.19 Hemslecin A (1, Fig. 1), a cucurbitane-type ter-
pene, widely present in this genus has been used to cure
inflammatory diseases in clinical practice.20 Our recent investiga-
tion revealed that hemslecin A exhibited activity against HBV
DNA replication (IC50 = 11.2 lM, SI = 5.8) based on anti-HBV assay
on HepG 2.2.15 cell line in vitro. Thus, with hemslecin A as the
starting substrate, a series of derivatives via chemical modification
on hydroxyl groups at 2, 3, 16-position, C-5(6) double bond, and C-
25 were synthesized and evaluated for their anti-HBV activity in
order to further study the structure–activity relationships.
The presence of free hydroxyl groups allowed us to prepare es-
ter derivatives of compound 1 in order to evaluate the influence of
ester side chain on their anti-HBV activities. Treatment of com-
pound 1 with various anhydrides in pyridine at room temperature
(rt) gave mono- or di-acylated derivatives A1–A7. Compound 1
was treated with various anhydrides or carboxylic acids in the
presence of 4-dimethylaminopyridine (DMAP) to afford triacylated
compounds B1–B5 (Scheme 1). Epoxidation of compound 1 with
1.2 equiv m-chloroperoxybenzoic acid (mCPBA) yielded derivative
C without affecting the carbonyl and hydroxyl groups, which was
further converted to derivatives D1–D5. The epoxy group was
O
HO
25
OAc
O
H
OH
H
HO
HO
2
3
5
6
⇑
Corresponding authors. Tel./fax: +86 871 5223265.
E-mail addresses: zhrpkm@163.com (R.-P. Zhang), chenjj@mail.kib.ac.cn (J.-J.
1
Chen).
Figure 1. Structure of hemslecin A.
0960-894X/$ - see front matter Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.01.024

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R.-H. Guo et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1201–1205
determined as b-oriented by ROESY experiment, in which no corre-
lation between dH 2.48 [H-6] and 2.28 [H-8, b] was observed. How-
ever, treatment of compound 1 with 2.5 equiv mCPBA afforded
compound E. Derivatives F1–F3 were obtained by epoxidation of
triacylated compounds B1, B4 and B5 with 2.5 equiv mCPBA in
CH2Cl2. Connections among 3-chlorobenzoyl and C-5 was deter-
mined by HMBC experiment, in which no correlation between H-
6 and carbonyl group was detected. The ROESY spectrum showed
no correlation between H-6 and H-8. Thus, the structures of com-
pounds E and F1–F3 were assigned as showed in Scheme 2. 25-
Deacylated compound 2 was isolated from dried rhizomes of
Hemsleya chinensis, which was treated with ethoxyacetic acid to
produce derivatives G and H in order to assay the influence of acyl-
ation at C-25 on their anti-HBV activity (Scheme 3).
hydroxyl groups of compound 1 on anti-HBV activity, compounds
A1–A7 were synthesized and tested for their anti-HBV activities
and cytotoxicities, and the results were shown in Table 1. Deriva-
tives A1–A7 exhibited significant inhibition against HBV DNA rep-
lication with IC50 values in the range of 2.8–9.8 lM. Compounds
A1, A4, and A7 with acylation of hydroxyl group at C-3 position
exhibited significant inhibition against HBV DNA replication
(IC50 = 2.8, 3.1, 4.7
Acetylated derivative A1 (CC50 = 253.5
ionylated derivative A4 (CC50 = 100.3 M, SI = 31.9) showed low
cytotoxicity. Replacement of acyl moiety with butyryl (compound
A7, CC50 = 6.3 M) led to high cytotoxicity, which demonstrated
lM, respectively).
lM, SI = 89.7) and prop-
l
l
that the cytotoxicity of the derivatives increased with the acyl
chain lengthened. Meanwhile, compound A4 showed moderate
The anti-HBV activity and cytotoxicity of the synthesized hem-
slecin A derivatives were evaluated in HepG 2.2.15 cells, and ten-
ofovir, a clinical anti-HBV agent, was used as positive control.
The anti-HBV results were summarized in Table 1. The anti-HBV
activity of each compound was expressed as the concentration of
compound that achieved 50% inhibition (IC50) of HBsAg, HBeAg,
and HBV DNA replication. The cytotoxicity of each compound
was expressed as the concentration of compound required to de-
stroy 50% (CC50) of the HepG 2.2.15 cells. The selectivity index
(SI), one of the important pharmaceutical parameter, was deter-
mined as the ratio of CC50 value to IC50 value. The bioactivity of
each compound was evaluated by the combination of its IC50 val-
ues and SI.
activity against the secretion of HBsAg (IC50 = 18.4
and HBeAg (IC50 = 17.0 M, SI = 5.9). Compounds A3 and A6 with
acylation of hydroxyl group at C-2 position exhibited high activity
against HBV DNA replication (IC50 = 4.7 M, 6.1 M, respectively).
Propionyl derivative (A3) appeared to be less toxic compared to
butyryl derivative (A6) (CC50 = 126.0 vs CC50 = 12.4 M),
lM, SI = 5.5)
l
l
l
l
M
l
resulting in a relatively high SI value (77.8 vs 2.0). Moreover, com-
pound A3 showed inhibitory potency to the secretion of HBsAg
(IC50 = 15.6
cylated compounds A2 and A5 exhibited high activities against
HBV DNA replication with IC50 values of 9.8 (SI = 55.6),
7.2 M (SI >83.4). Furthermore, compound A5 showed the highest
activity against the secretion of HBsAg (IC50 = 9.9 M). These re-
lM, SI = 8.1) and HBeAg (IC50 = 14.4 lM, SI = 8.7). Dia-
lM
l
l
Compound 1 showed moderate activity against HBV DNA repli-
cation (IC50 = 11.4 lM), with obvious toxicity (CC50 = 66.2 lM),
which led to a low SI value (SI = 5.8). To explore the influence of
sults indicated that mono- or di-acylation of hydroxyl group(s) at
C-2 or (and) C-3 position resulted in enhancement of anti-HBV
activity.
O
O
HO
HO
OAc
O
H
OH
OAc
O
H
OH
H
a
HO
HO
H
R1O
R2O
1
A1-A7
b
R1
R2
O
O
O
HO
A1
A2
H
O
OAc
R
O
O
H
O
R
O
H
O
O
O
H
O
R
O
A3
A4
B1-B6
H
O
R
O
CH3
A5
B1
B2
B3
O
H
H
O
A6
A7
B4
B5
O
O
B6
OMe
Scheme 1. Reagents and conditions: (a) for A1 and A2: (CH3CO)2O, pyridine, rt, 8–30%; for A3–A5: (CH3CH2CO)2O, pyridine, rt, 7–51%; for A6 and A7: (CH3CH2CH2CO)2O,
pyridine, rt, 14–77%; (b) for B1–B3: (RCO)2O, DMAP, pyridine, rt, 84–92%; for B4–B6: RCOOH, DCC, DMAP, CH2Cl2, rt, 90–93%.

R.-H. Guo et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1201–1205
O
1203
O
HO
HO
OAc
OAc
O
H
OH
O
H
O
R
O
O
R
O
H
H
CH3
D1
b
HO
HO
O
O
R
D2
D3
R
O
O
D1-D5
O
D4
D5
C
O
O
O
HO
a
HO
OAc
OAc
c
O
H
OH
O
H
OH
H
H
HO
HO
HO
HO
O
OH
E
O
1
O
d
O
HO
HO
Cl
OAc
R
O
O
O
H
O
OAc
O
R
O
O
H
O
H
O
e
O
O
R
H
O
O
R
O
R
R
O
B1, B4, B5
OH
O
R
CH3
R
F1
F2
CH3
B1
O
B4
B5
O
O
Cl
F3
O
F1-F3
Scheme 2. Reagents and conditions: (a) mCPBA (1.2 equiv), CH2Cl2, rt, 75%; (b) for D1–D5: (RCO)2O, DMAP, pyridine, rt, 81–90%; for D4, D5: RCOOH, DCC, DMAP, CH2Cl2, rt,
90–93%; (c) mCPBA (2.5 equiv), CH2Cl2, rt, 89%; (d) for B1: (CH3CO)2O, DMAP, pyridine, rt, 88%; for B4, B5: RCOOH, DCC, DMAP, CH2Cl2, rt, 93%; (e) mCPBA (2.5 equiv), CH2Cl2,
rt, 55–82%.
O
HO
OH
O
O
H
O
O
O
H
O
O
O
O
O
O
HO
OH
a
G
O
H
OH
H
+
HO
HO
O
O
HO
O
O
2
O
O
H
O
O
O
H
O
O
O
O
O
H
Scheme 3. Reagents and conditions: (a) C2H5OCH2COOH, DCC, DMAP, CH2Cl2, rt, 24–80%.
The subseries of triacylated compounds B1–B6 were linked
with different ester chains at C-2, C-3, and C-16 of compound 1.
As shown in Table 1, these compounds were non-cytotoxic, which
revealed that the acylation of hydroxyl groups at C-2, C-3, C-16 de-
creased cytotoxicity, exhibited inhibitory activity on HBV DNA rep-
lication with IC50 values of 17.3
114.9 M (>4.5), 10.0 M (>55.8), 51.2
the secretion of HBsAg and HBeAg, except for compound B3 which
lM (SI >46.4), 19.0
lM (SI >34.2),
l
l
lM (>14.0), but inactive to

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R.-H. Guo et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1201–1205
Table 1
Anti-HBV activity, cytotoxicity, and selectivity index of compounds 1, 2 and A–H
a
No.
CC50
(l
M)
HBsAg
HBeAg
HBV DNA
b
IC50
(l
M)
SIc
IC50
(
lM)
SI
IC50
(l
M)
SI
1
66.2
>66.2
189.5
22.4
15.6
18.4
9.9
>12.4
>6.3
>807.1
>649.9
18.0
>522.4
>560.3
>718.9
>1745.0
>960.9
768
>617.6
>1111.3
>633.3
24.0
<1.0
1.3
24.3
8.1
5.5
>60.0
<1.0
<1.0
—
—
>33.8
—
—
—
>66.2
>253.5
46.7
14.4
17.0
<1.0
<1.0
11.7
8.7
5.9
>1.0
<1.0
<1.0
—
—
—
—
—
—
—
—
—
>2.0
>1.9
—
11.9
>3.2
—
11.4
2.8
9.8
4.7
3.1
7.2
6.1
4.7
17.3
19.0
>608.0
114.9
10.0
51.2
11.6
5.8
A1
A2
A3
A4
A5
A6
A7
B1
B2
B3
B4
B5
B6
C
D1
D2
D3
D4
D5
E
253.5
546.2
126.0
100.3
>596.4
12.4
89.7
55.6
77.8
31.9
>83.4
2.0
596.4
>12.4
>6.3
>807.1
>649.9
>608.0
>522.4
>560.3
>718.9
>1745.0
>960.9
>1667.0
401.3
591.7
>633.3
30.9
327.2
>1744.8
>710
6.3
1.3
>807.1
>649.9
>608.0
>522.4
>560.3
>718.9
>1745.0
>960.9
>1667.0
>617.6
>1111.3
>633.3
366.5
>1047.1
>1744.8
>710.4
60.6
184.5
>1452.1
>1740.0
>46.6
>34.2
—
>4.5
>55.8
>14.0
>150.5
—
—
—
—
—
50.9
—
—
—
1.9
2.2
>17.8
>3571.4
—
—
>2.2
—
—
>960.9
>1667.0
>617.6
>1111.3
>633.3
7.2
>1047.1
>1744.8
>710.4
31.4
—
15.3
>5.9
—
F1
F2
F3
2
G
H
178.0
>1744.8
>710.4
>60.6
101.3
225.4
—
—
<1.0
1.8
>6.4
>1.1
>60.6
<1.0
<1.0
—
>184.5
>1452.1
1160.2
84.6
81.4
0.49
Tenofovird
1572.1
>1.5
a
b
c
CC50: 50% cytotoxic concentration.
IC50: 50% inhibitory concentration.
SI (selectivity index) = CC50/IC50
.
d
Tenofovir: an antiviral agent used as positive control.
lost the activity against HBV DNA replication but showed suppres-
following conclusions could be made: (a) Mono- or di-acylation
of hydroxyl group(s) at C-2 or (and) C-3 resulted in enhancement
of anti-HBV activity. (b) The acylation of hydroxyls at C-2, C-3, C-
16 decreased cytotoxicity. (c) Epoxide of double bond at C-5(6) de-
creased the cytotoxicity and maintained activity against HBV DNA
replication. (d) Acylation of the hydroxyl group at C-25 of hemsle-
cin A was an important feature in conferring anti-HBV activity.
sant potency against the secretion of HBsAg (IC50 = 18.0
>33.8).
lM, SI
Compound
(IC50 = 11.4 M) showed obviously decreased cytotoxicity, and
similar activity against HBV DNA replication (IC50 = 11.6 M), lead-
C derived from epoxidation of compound 1
l
l
ing to a high SI value (>150.5). This suggested that the epoxy group
was an important feature in conferring low cytotoxicity. Unfortu-
nately, triacylated derivativies D1–D5 lost anti-HBV activity. After
opening the epoxide ring, compound E possessed inhibitory po-
Acknowledgments
tency to the secretion of HBsAg (IC50 = 24.0
(IC50 = 30.9 M, SI = 11.9) and the replication of HBV DNA
(IC50 = 7.2 M, SI = 50.9). Compounds F1–F3 were non-cytotoxic
l
M, SI >15.3), HBeAg
The work was supported by grants from the National Natural
Science Foundation of China for Distinguished Young Scholars
(No. 81025023). The authors are grateful to the staff of the analyt-
ical group of the State Key Laboratory of Phytochemistry and Plant
Resources in West China, Kunming Institute of Botany, Chinese
Academy of Sciences, for measurements of all spectra.
l
l
but showed a significant reduction of anti-HBV activity.
The effects of different kinds of substituents to the C-25 were also
studied. Deacetylation of compound 1 (IC50 = 11.4
sponding compound 2 (IC50 = 31.4 M) reduced the activity against
HBV DNA replication. Compounds (IC50 = 84.6 M) and
(IC50 = 81.4 M) showed a considerable reduction of activity against
HBV DNA replication compared with compound B5 (IC50 = 10.0 M).
lM) to corre-
l
G
l
H
Supplementary data
l
l
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
01.024.
These findings suggested that the presence of acetyl at C-25 played
an important role in maintaining anti-HBV activity.
In summary, a series of derivatives were synthesized via chem-
ical modifications on hydroxyl groups at C-2, C-3, C-16, C-5(6) dou-
ble bond and C-25 position of hemslecin A and evaluated for their
anti-HBV activity and cytotoxicities in vitro. Fifteen derivatives
showed potent activity against HBV DNA replication. Ten com-
pounds (A1–A7, B5, C, and E) showed significant activity against
HBV DNA replication with IC50 values in the range of 2.8–
11.6 lM. Among them, compounds A1–A3, A5, B5, and C had low
cytotoxicity, resulting in high SI values of 89.7, 55.6, 77.8, >83.4,
>55.8, and >150.5, respectively. Based on the above results, the
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