Anti-HBV agents. Part 2: Synthesis and in vitro anti-hepatitis B virus activities of alisol A derivatives

Article abstract of DOI:10.1016/j.bmcl.2009.02.122

Chemical modifications were performed on hydroxyl groups at C-11,23,24,25 positions and C-13(17) double bond of alisol A for structure-activity relationship study. Forty-one derivatives of alisol A were synthesized and assayed for their in vitro anti-hepa

Full text of DOI:10.1016/j.bmcl.2009.02.122

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2148–2153
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Anti-HBV agents. Part 2: Synthesis and in vitro anti-hepatitis B virus activities
of alisol A derivatives
a
Quan Zhang a,b, Zhi-Yong Jiang a, , Jie Luo , Ji-Feng Liu a,b, Yun-Bao Ma a, Rui-Hua Guo a,b
,
*
Xue-Mei Zhang a, Jun Zhou a, 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 Graduate School of Chinese Academy of Sciences, Beijing 100039, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Chemical modifications were performed on hydroxyl groups at C-11,23,24,25 positions and C-13(17)
double bond of alisol A for structure–activity relationship study. Forty-one derivatives of alisol A were
synthesized and assayed for their in vitro anti-hepatitis B virus (HBV) activities and cytotoxicities. Of
them, 14 compounds were active against HBV surface antigen (HBsAg) and HBV e antigen (HBeAg) secre-
tion in HepG 2.2.15 cells, and the most promising compound 25 exhibited high activities against secre-
tion of HBsAg (IC50 = 0.028 mM), HBeAg (IC50 = 0.027 mM) and remarkable selective indices (SIHBsAg >90,
SIHBeAg >93).
Received 6 January 2009
Revised 19 February 2009
Accepted 27 February 2009
Available online 4 March 2009
Keywords:
Anti-HBV activity
Protostane-type triterpene
Alisol A
Ó 2009 Elsevier Ltd. All rights reserved.
Derivatives
Chemical modification
Various efforts are currently underway to develop therapeutic
agents to arrest the replication of the hepatitis B virus (HBV).
Although several nucleoside inhibitors (i.e., lamivudine, adefovir
dipivoxil, entecavir, telbivudine) have been approved by the FDA
and used clinically, adverse side effects and development of drug
resistant virus have been reported.1 Therefore, compounds pos-
sessing potent anti-HBV activity with novel modes of action are ur-
gently needed to add to the existing anti-HBV therapies.
Nowadays, the development of new anti-HBV agents is focused
on discovering diverse compounds with either novel structures
or a new mechanism of action. So far, non-nucleosides have also
been reported to inhibit HBV infection.2–11 Nevertheless, the
search for compounds with novel anti-HBV target and mechanism
is still needed.
Discovery of novel plant-derived natural products as potential
new lead compounds for anti-HBV agents as well as the modifica-
tion of these new lead compounds is continuing goals of our
laboratory. Previously, we first reported protostane-type triter-
penes possess anti-HBV activities.12 Our efforts to improve the
anti-HBV activity of alisol A (1, Fig. 1), a naturally occurring protos-
tane-type triterpene, led us to discover 11,23,24-tri-O-acetyl-
25-anhydroalisol A (2) and its related compounds [for example,
11,23,24-tri-O-acetyl-13b,17b-epoxy-25-anhydroalisol A (3)] with
high activities against secretion of HBsAg and HBeAg, and our
OAc
OAc
OH
25
23
24
O
12
OH
O
AcO
H
13
AcO
H
OAc
HO
1
17
OAc
OH
11
9
8
H
H
H
3
4
O
O
6
H
3
2
1
Figure 1. Structures of compounds 1–3.
* Corresponding authors. Tel./fax: + 86 871 5223265 (J.-J.C).
E-mail addresses: jiangzy@mail.kib.ac.cn (Z.-Y. Jiang), chenjj@mail.kib.ac.cn (J.-J. Chen).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.02.122

Q. Zhang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2148–2153
2149
Table 1
a
Structures, anti-HBV activity, cytotoxicity and selectivity index of compounds 1–45
O
O
O
R1
OH
O
O
R1
O
O
OR2
R1
R1
OH
HO
H
O
OH
O
O
O
O
R1
R1
H
H
H
O
O
O
H
H
1
4—31
2, 32—36
O
O
R1
R1
O
O
OH
O
O
OR2
R1
R1
O
O
O
OH
O
O
O
O
O
O
HO
OH
R1
R1
H
H
H
O
O
O
H
H
H
37
3, 38—42
43—45
HBsAgc
HBeAgd
b
Compounds
CC50 (mM)
R1
R2
IC50 (mM)
SIf
IC50(mM)
SI
e
1g
2g
3g
/
/
/
/
0.062
0.52
>2.6
0.039
0.028
0.024
1.6
18
>108
>2.4
0.029
0.028
<0.030
18
>93
Me
Me
4
5
H
H
>1.2
>1.2
>1.2
>1.2
—
—
>1.2
>1.2
—
—
6
7
8
9
H
H
H
H
>1.1
>1.3
>0.90
>0.6
>1.1
>1.3
>0.90
>0.6
—
—
—
—
>1.1
>1.3
>0.90
>0.6
—
—
—
—
10
11
H
H
>0.85
>0.90
>0.85
>0.90
—
—
>0.85
>0.90
—
—
12
13
14
H
H
H
>1.0
>1.1
>0.8
>1.0
>1.1
>0.80
—
—
—
>1.0
>1.1
>0.8
—
—
—
15
H
>0.81
>0.81
—
>0.81
—
(continued on next page)

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Q. Zhang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2148–2153
Table 1 (continued)
b
Compounds
CC50 (mM)
>0.90
HBsAgc
HBeAgd
R1
R2
H
IC50 (mM)
SIf
—
IC50(mM)
>0.9
SI
—
e
16
>0.90
>0.65
Cl
17
H
H
H
0.65
<1.0
—
>0.65
>1.5
>1.2
<1.0
—
Br
I
18
>1.5
>1.2
>1.5
>1.2
Cl
19
—
—
Cl
Cl
20
21
H
H
>1.3
>1.0
0.95
>1.0
>1.3
—
>1.3
>1.0
—
—
O2N
22
23
24
25
H
H
>1.3
>0.95
0.40
>2.5
>1.3
—
>1.3
—
O
>0.95
0.14
—
>0.95
>0.4
—
O
H
2.9
>90
—
MeO
MeO
MeO
0.028
0.027
>93
O
26
27
H
>2.6
>2.3
0.40
0.20
>6.5
>11
2.0
>1.3
>8.7
EtO
EtO
EtO
0.26
O
28
29
H
H
0.051
0.27
<0.038
0.29
>1.3
<1.0
>0.051
0.88
<1.0
<1.0
AcO
AcHN
O
30
31
H
H
>1.5
>1.5
1.2
>1.2
>4.7
0.14
>11
>16
N
O
O
0.32
0.094
O
N
H
32
33
/
/
>1.2
>1.2
—
—
>1.2
—
—
>0.92
>0.92
>0.92
34
/
>2.8
0.068
>42
0.12
>24
MeO

Q. Zhang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2148–2153
2151
Table 1 (continued)
HBsAgc
HBeAgd
b
Compounds
CC50 (mM)
R1
R2
/
IC50 (mM)
SIf
IC50(mM)
0.03
SI
e
35
>1.2
0.020
>60
>40
EtO
36
/
<0.032
0.43
1.1
<0.032
0.045
0.90
—
<0.032
1.1
—
AcO
37g
38
/
/
9.6
1.2
<1.0
<1.0
H
>1.1
MeO
MeO
EtO
MeO
39
40
41
42
43
44
45
>0.80
0.25
>1.4
0.25
>1.3
>1.8
0.11
0.13
0.25
0.12
0.044
0.020
>7.3
1.9
>1.6
>0.25
0.32
>0.25
>1.3
>1.8
—
O
H
<1.0
>4.4
<1.0
—
EtO
>5.6
2.1
EtO
O
H
/
AcO
MeO
EtO
>29
>90
/
—
/
/
>1.6
31
0.051
18
>31
1.7
>1.6
38
—
AcO
3TCh
/
0.82
a
All values are the mean of two independent experiments.
CC50: 50% cytotoxic concentration.
HBsAg: HBV surface antigen.
HBeAg: HBV e antigen.
IC50: 50% effective concentration.
b
c
d
e
f
SI (selective index) = CC50/IC50
Data from Ref. 13.
.
g
h
3TC: Lamivudine, an antiviral agent used as positive control.
investigations revealed two important structure–activity relation-
ships (SARs). Both acylation of hydroxyl groups at 11,23,24-posi-
tion and epoxidation of C-13(17) double bond are important
feature in the conferring relatively low cytotoxicity of compound
3, and a double bond at C-25(26) is crucial for the high potency
of compounds 2 and 3,13 which encouraged us to synthesize addi-
tional analogues. Herein, we report the synthesis, anti-HBV activ-
ity, and SARs of these protostane-type triterpenes in detail.
Previous study showed that acyl groups at C-11,23,24 hydroxyls
of alisol A derivatives could decrease cytotoxicity.13 A series of es-
ter derivatives of compound 1 were synthesized to further evaluate
the influence of ester side chains on the biological properties. Com-
pound 1 was treated with carboxylic acids in the presence of N’,N’-
dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine
(DMAP) to afford acyl derivatives 4–31 (Table 1). Compounds 4,
7, 24, 26, 28 were subjected to dehydration by refluxing with
Lawesson’s reagent, commonly used in organic synthesis as a thia-
tion agent,14–16 in toluene to give compounds 32–36 in good yields
without affecting the 3-carbonyl (Scheme 1).13 Epoxidation of
compound 1 with m-chloroperoxybenzoic acid (mCPBA) in CH2Cl2
at room temperature gave compound 37. Treatment of compound
37 with carboxylic acids in the presence of DCC and DMAP afforded
compounds 38–42. Compounds 38, 40, 42 were further converted
to corresponding dehydrated compounds 43–45 by refluxing with
Lawesson’s reagent in toluene in good yields (Scheme 2).
The synthesized alisol A derivatives were tested for their cyto-
toxicities and potential anti-HBV activities, namely the abilities
to inhibit the secretion of HBV surface antigen (HBsAg) and HBV
e antigen (HBeAg) in HBV-infected 2.2.15 cells using lamivudine
(3TC, a clinically popular anti-HBV agent) as a positive control.17
The anti-HBV activity of each compound was expressed as the con-
centration of compound that achieved 50% inhibition (IC50) to the
secretion of HBsAg and HBeAg. The cytotoxicity of each compound
was expressed as the concentration of compound required to kill
50% (CC50) of HepG 2.2.15 cells. The selectivity index (SI), a major
pharmaceutical parameter that estimates possible future clinical
development, was determined as the ratio of CC50 to IC50. The bio-
activity of each compound was evaluated by the combination of its
IC50 and SI. The results were summarized in Table 1.
The parent compound 1 showed inhibitory potency to the
secretion of HBsAg (IC50 = 0.039 mM), but appeared toxic
(CC50 = 0.062 mM), which led to a relative low SI (1.6). The sub-
series of derivatives 4–27 had different patterns of substitution
on C-11, C-23, C-24 and C-25. As shown in Table 1, these com-
pounds were non-cytotoxic, which revealed that the acylation of
hydroxyl groups at 11,23,24-position decreased cytotoxicity, and

2152
Q. Zhang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2148–2153
O
O
R1
R1
OH
O
O
O
O
OR2
OH
R1
R1
HO
H
OH
O
O
O
O
O
O
a
R1
b
R1
H
H
H
O
O
O
H
H
1
32-36
4-31
R1
32
33
34
O
35
O
O
36
O
Scheme 1. Reagents and conditions: (a) R1COOH, DCC, DMAP, CH2Cl2, rt, 54–95%; (b) Lawesson’s reagent, toluene, reflux, 63–83%.
O
R1
OH
OH
O
O
OR2
OH
O
OH
R1
O
O
HO
H
HO
H
OH
O
OH
O
O
a
b
R1
H
H
H
O
O
c
H
38-42
37
1
O
R1
R2
H
R1
O
38
39
O
O
R1
O
O
O
O
O
O
O
O
O
R1
H
40
41
H
O
H
O
43-45
43
R1
O
O
O
42
H
O
44
45
O
O
O
Scheme 2. Reagents and conditions: (a) mCPBA, CH2Cl2, rt, 88%; (b) R1COOH, DCC, DMAP, CH2Cl2, rt, 66%–82%; (c) Lawesson’s reagent, toluene, reflux, 72–84%.
most of these compounds showed a significant reduction of anti-
HBV activity, except that compounds 24 (IC50 = 0.14 mM, SI = 2.9)
and 26 (IC50 = 0.40 mM, SI >6.5) exhibited moderate activity
against the secretion of HBsAg suggesting that short straight
chains with 2’-oxygen atom at C-11, C-23 and C-24 groups ap-
peared to improve inhibitory activities against the secretion of
HBsAg compared to saturated carbon side chains (7 vs 26). An
important advance was obtained when the C-25 hydroxyl groups
of compounds 24 and 26 were acylated with corresponding car-
boxylic acids. The tetra-methoxyacetyl derivative 25 and the tet-
ra-ethoxyacetyl analogue 27 showed more highly potent
activities against secretion of HBsAg (IC50 = 0.028 mM, 0.20 mM,
respectively), HBeAg (IC50 = 0.028 mM, 0.26 mM, respectively)
and much higher SI values (SIHBsAg >90, SIHBeAg >93; SIHBsAg
>11, SIHBeAg >8.7, respectively) than those of corresponding tri-
acyl derivatives (24 vs 25, 26 vs 27). Compound 25 was more
potent against the secretion of HBsAg and HBeAg than com-
pound 27, which indicated that the longer terminal alkyl chain
reduced anti-HBV activity. To our surprise, compounds 39 and
41 obtained by the epoxidation from compounds 25 and 27,
respectively, showed a significant decrease in inhibition to the
secretion of HBsAg and HBeAg.

Q. Zhang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2148–2153
2153
To further explore the influence of side chains with 2’-hetero-
atom at C-11, C-23 and C-24 on anti-HBV activity. Compounds
28–31 were synthesized and evaluated for their anti-HBV activities
and cytotoxicities. Compound 28 showed high activity against the
secretion of HBsAg (IC50 <0.038 mM), whereas an increase in cyto-
toxicity was also noted (CC50 = 0.051 mM). As reported previously,
epoxidation of C-13(17) double bond decrease cell cytotoxicity.13
Compound 28 was converted to compound 42 which showed low-
er cell cytotoxicity (CC50 = 0.25 mM). However, activity against the
secretion of HBsAg also decreased (IC50 = 0.12 mM, SI = 2.1). For 2’-
N-substituted derivatives 29–31, compounds 30 and 31 exhibited
show high activity against the secretion of HBsAg, but can not
inhibit the secretion of HBeAg. Finally, epoxide group leads to
the decrease of suppressant property on the secretion of HBsAg
and HBeAg: compounds 39 and 41 are less potent than com-
pounds 25 and 27, respectively.
Based on these structure–activity relationships, further optimi-
zation and biological evaluation on protostane-type triterpene
compounds as promising HBV inhibitors are ongoing in our labora-
tory and the results will be reported in due course.
Acknowledgements
highly inhibitory potency to the secretion of HBeAg (IC50
=
0.14 mM, SI >11; IC50 = 0.094 mM, SI >16, respectively). But unfor-
tunately, both compounds showed low activity against the secre-
tion of HBsAg.
This work was supported by National Natural Science Founda-
tion of China (NSFC No. 30672522), Xibu Zhiguang Joint-Scholar-
ship of Chinese Academy of Sciences, the External Cooperation
Program of Chinese Academy of Sciences (No. GJHZ200818), and
CAS–Croucher Foundation (CAS-CF07/08.SC03). The authors are
grateful to the staff of the analytical group of the State Key Labora-
tory of Phytochemistry and Plant Resources in West China, Kun-
ming Institute of Botany, Chinese Academy of Sciences, for
measurements of all spectra.
Among dehydrated compounds 2 and 32–36, compound 2
showed IC50 of 0.028 and 0.029 mM on HBsAg and HBeAg
secretion, respectively, which led to high SI values (SIHBsAg = 18,
SIHBeAg = 18). Replacement of the acetyl moieties with more bulky
substituents (32, 33) led to loss of anti-HBV activity. Interestingly,
potent inhibitory activities against the secretion of HBsAg and
HBeAg were found to be retained with alkyloxyacetyl (or acetoxy-
acetyl) derivatives (34–36). The results indicated that chain length
may not be the only one factor for potent anti-HBV activity. Com-
pounds 43–45 that resulted from dehydration of compounds 38,
40, 42 showed low cell cytotoxicities, and their IC50 values on inhi-
bition of HBsAg secretion were 0.044, 0.020, and 0.051 mM, respec-
tively (SI >29, >90, >31, respectively). Noteworthy is that an
epoxide functionality at C-13(17) of compounds 43–45 caused
the loss of suppressant property on the secretion of HBeAg.
In summary, a series of new alisol A analogues were synthe-
sized and examined for their in vitro anti-HBV activities and
cytotoxicities and 14 tested compounds were active against
HBV in HepG 2.2.15 cells. Based on the above structure and
activity relationship results, the following conclusion can be
drawn: (1) the present investigation indicates that the double
bond at C-25(26) of alisol A analogues is crucial for potent
anti-HBV activity; (2) for dehydrated derivatives, the anti-HBV
activity largely depends on the size and character of the substit-
uents on the 11,23,24-ester moieties. As the alkyl chain is
lengthened, anti-HBV activity of the derivatives decreased. How-
ever, chain length may not be the only one factor, because the
alkyloxyacetyl (or acetoxyacetyl) derivatives (34–36 and 43–45)
exhibited high potent anti-HBV activity; (3) acylation of the hy-
droxyl at C-25 of alisol A is important for the high potency of
compounds 25 and 27; (4) the role of epoxide functionality at
C-13(17) of alisol A derivatives is ambiguous. In some analogues,
epoxide group retains the inhibitory potency to the secretion of
HBsAg and HBeAg: compounds 2 and 3 show similar activity. For
other analogues, epoxide functionality retains the inhibitory po-
tency to the secretion of HBsAg, but causes the loss of suppres-
sant property on the secretion of HBeAg: compounds 43–45
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.02.122.
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