Anti-HBV agents. Part 1: Synthesis of alisol A derivatives: A new class of hepatitis B virus inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.07.012

A series of alisol A derivatives were synthesized and evaluated for their anti-hepatitis B virus (HBV) activities and cytotoxicities in vitro. The preliminary investigation demonstrates that simple modifications of the parent structure of alisol A can produce a number of potentially important derivatives against HBV. The most active anti-HBV compound 6a showed high activities against the secretion of HBV surface antigen (IC₅₀ = 0.024 mM), HBV e antigen (IC₅₀ = 0.028 mM) and remarkable selective indices (SI_HBsAg > 108, SI_HBeAg > 93), which was selected for further evaluation as a novel HBV inhibitor.

Full text of DOI:10.1016/j.bmcl.2008.07.012

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4647–4650
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Anti-HBV agents. Part 1: Synthesis of alisol A derivatives: A new class
of hepatitis B virus inhibitors
Quan Zhang a,c, Zhi-Yong Jiang a, Jie Luo a, Pi Cheng a,c, Yun-Bao Ma a, Xue-Mei Zhang a,
Feng-Xue Zhang b, 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, Lanhei Road, Kunming 650204, PR China
b Institute of Tropical Medicine Sciences, Guangzhou University of Traditional Chinese Medicines, Guangzhou, PR China
c 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:
A series of alisol A derivatives were synthesized and evaluated for their anti-hepatitis B virus (HBV) activ-
ities and cytotoxicities in vitro. The preliminary investigation demonstrates that simple modifications of
the parent structure of alisol A can produce a number of potentially important derivatives against HBV.
The most active anti-HBV compound 6a showed high activities against the secretion of HBV surface anti-
gen (IC50 = 0.024 mM), HBV e antigen (IC50 = 0.028 mM) and remarkable selective indices (SIHBsAg > 108,
SIHBeAg > 93), which was selected for further evaluation as a novel HBV inhibitor.
Received 21 May 2008
Revised 1 July 2008
Accepted 5 July 2008
Available online 10 July 2008
Keywords:
Anti-HBV activity
Alisol A
Ó 2008 Elsevier Ltd. All rights reserved.
Derivatives
Synthesis
Hepatitis B virus (HBV) infection is still a major world health
problem despite the availability of an effective vaccine.1 Approxi-
mately 350 million people are chronically infected with the virus
worldwide, including 1.25 million in the U.S. and more than 200
million in China. HBV infection can persist for the life of the host,
often leading to severe consequences such as liver failure, fibrosis,
cirrhosis and hepatocellular carcinoma (HCC).2 Worldwide deaths
from liver cancer caused by HBV infection probably exceed 1 mil-
lion per year.3 Currently, there is no effective treatment that com-
pletely eliminates the infection in all chronically infected patients.
The approved clinical chemotherapeutic agents (i.e., lamivudine,
adefovir dipivoxil, entecavir, telbivudine) inhibit the virus replica-
tion by targeting the viral DNA polymerase, and after long-term
treatment development of drug-resistant virus becomes problem-
ited diverse biological activities, such as anti-complementary,12,13
anti-allergic,14 anti-HIV reverse transcriptase15, and other activi-
ties.16–22 Recently, we reported that protostane-type triterpenes
exhibit significant in vitro antiviral activity against HBV.23 To the
best of our knowledge, it was the first report of protostane-type tri-
terpenes possessing anti-HBV activities. In view of their novel
structural template, which differs from those of all reported anti-
HBV agents, we were interested to further study the structure–
activity relationships of the related class of compounds. Alisol A
(1, Fig. 1),24 a protostane-type triterpene, comprises 0.05–0.1% of
rhizomes of Alisma orientalis Juzep.16 and carries four hydroxyl
groups providing possibilities for more diverse library. Thus, with
compound 1 as the starting substrate, we synthesized a series of
atic.4 Interferon-
a (IFN-a) is also clinically useful for HBV infection,
but it has substantial side effects as well. The side effects of inter-
feron and the viral resistance of nucleoside analogues make the
current treatment regimens far from satisfactory.5–7 To circumvent
the existing therapeutic difficulties, novel compounds with unique
modes of actions are urgently needed.
Natural products as the most consistently successful sources in
drug discovery may offer more opportunities to find drugs or lead
compounds.8–11 Protostane-type triterpenoids are natural product
molecules mainly found in Alisma genus and most of them exhib-
OH
25
23
24
12
OH
13
HO
1
17
OH
11
9
8
H
3
4
O
6
H
1
* Corresponding author. Tel./fax: +86 871 5223265.
E-mail address: chenjj@mail.kib.ac.cn (J.-J. Chen).
Figure 1. Structure of compound 1.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.07.012

4648
Q. Zhang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4647–4650
alisol A derivatives and investigated their biological activities as
potential anti-HBV agents.
nopyridine (DMAP) to afford compounds 2a–e. Compounds 2a–e
were subjected to dehydration by SOCl2 in pyridine to give 3a–e
in moderate yields25 (Scheme 1).
Epoxidation of compound 1 with m-chloroperoxybenzoic acid
(mCPBA) in CH2Cl2 at room temperature gave compound 4 without
affecting the 3-carbonyl and hydroxyl groups26 (Scheme 2). Com-
pound 4 was reacted with acetic anhydride or carboxylic acids in
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 biological activities. Compound 1 was
treated with acetic anhydride or with carboxylic acids in the pres-
ence of N,N0-dicyclohexylcarbodiimide (DCC) and 4-dimethylami-
O
O
O
O
OH
R1
O
R1
O
O
O
OH
R1
OH
b
R1
HO
H
OH
O
O
O
O
a
R1
R1
H
H
H
O
O
O
H
H
3a-e
2a-e
1
R1
CH3
R1
3a
3b
3c
3d
2a
2b
2c
2d
CH3
CH2CH3
CH2CH3
CH(CH3)2
CH(CH3)2
3e
2e
Scheme 1. Reagents and conditions: (a) for 2a: (CH3CO)2O, pyridine, rt, 86%; for 2b–e: R1COOH, DCC, DMAP, CH2Cl2, rt, 68–85%; (b) SOCl2, pyridine, rt, 59–75%.
O
OH
OH
R1
O
O
OH
a
OH
b
OH
O
R1
O
HO
H
HO
H
OH
OH
O
O
O
R1
H
H
H
O
O
O
H
5a-e
1
4
R1
CH3
c
O
O
5a
5b
5c
5d
R1
O
CH2CH3
O
R1
CH(CH3)2
O
O
O
R1
H
5e
O
H
6a-e
R1
6a CH3
CH2CH3
6b
6c
6d
CH(CH3)2
6e
Scheme 2. Reagents and conditions: (a) mCPBA, CH2Cl2, rt, 88%; (b) for 5a: (CH3CO)2O, pyridine, rt, 72%; for 5b–e: R1COOH, DCC, DMAP, CH2Cl2, rt, 55–87%; (c) SOCl2,
pyridine, rt, 55–72%.

Q. Zhang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4647–4650
4649
Table 1
the presence of DCC and DMAP to give compounds 5a–e, which
were followed by dehydration with SOCl2 in pyridine to afford
compounds 6a–e.
Anti-HBV activity, cytotoxicity and selectivity index of compounds 1–8a
b
Compound
CC50 (mM)
HBsAgc
HBeAgd
IC50 (mM) SI
<0.030
Treatment of compound 1 with 4-methylbenzenesulfonohyd-
razide afforded compound 7, which was reduced with NaBH3CN
to give compound 8. Furthermore, C-3 keto was converted to C-3
thioketo for further evaluation whether a carbonyl function at C-
3 was necessary for biological activity. As shown in Scheme 3,
refluxing of 2a with Lawensson’s reagent in toluene did not give
the C-3 thioketo derivative but gave compound 3a, which could
be confirmed by additional signals of a double bond (dC 140.5, C-
25; dC 114.6, C-26) in 13C NMR spectrum of 3a. Additionally, the
yield (82%) of conversion of 2a to 3a was better than that reported
previously (75%).25
As a preliminary study in the developing protostane-type trit-
erpenoids against HBV agents, a series of alisol A derivatives were
prepared and tested for their cytotoxicities and potential anti-HBV
activities, namely the ability 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,27 in order to better understand the structural
requirements for the biological effect. The results are 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 relatively low selectivity index
(1.6). The subseries of derivatives 2a–e have different patterns of
substitution on C-11, C-23 and C-24. As shown in Table 1, these
compounds were non-cytotoxic but decreased inhibition to secre-
tion of HBsAg. For derivatives 3a–e that resulted from dehydration
of compounds 2a–e, compound 3a can inhibit the secretion of
HBsAg (IC50 = 0.028 mM, SI = 18) and HBeAg (IC50 = 0.029 mM,
SI = 18), which indicated that the hydroxyl group at C-25 of alisol
A might be a good target for further lead optimization.
Little change in inhibition to secretion of HBsAg was observed
with the epoxidation of 1 (IC50 = 0.039 mM) to the corresponding
4 (IC50 = 0.045 mM), whereas compound 4 had a high SI (9.6), sug-
gesting that the epoxy group is an important feature in the confer-
ring relatively low cytotoxicity. Tri-acyl derivatives of compound 4
were non-cytotoxic. But unfortunately, compounds 5a–e lost sup-
pressant properties on the secretion of HBsAg and HBeAg. For
dehydrated compounds 6a–e, the most active compound 6a exhib-
e
IC50 (mM)
SIf
1.6
—
—
>1.3
—
—
18
—
—
—
—
9.6
—
—
—
—
—
>108
>16
—
—
—
<1.0
<1.0
2.5
124
2a24
2b
2c
2d
2e
3a25
3b
3c
3d
3e
425
5a
5b
5c
5d
5e
6a
6b
6c
0.062
>2.0
>1.4
>2.2
>1.5
>1.4
0.52
>1.2
>1.6
>2.3
>1.8
0.43
>2.0
>1.2
>1.9
>1.7
>1.3
>2.6
>2.5
>1.3
>1.6
>1.6
<0.029
<0.080
30
0.039
>2.0
>1.4
1.7
>1.5
>1.4
0.028
>12
>1.6
>2.3
>1.8
0.045
>2.0
>1.2
>1.9
>1.7
>1.3
0.024
0.16
>1.3
>1.6
>1.6
0.029
0.080
12
>2.4
>2.0
>1.4
0.9
—
—
>2.4
—
—
18
—
—
—
—
>1.5
>1.4
0.029
>12
>1.6
>2.3
>1.8
1.1
>2.0
>1.2
>1.9
1.1
1.0
0.028
>2.5
>>1.3
>1.6
0.39
—
—
—
>1.5
>1.3
>93
—
—
—
—
6d
6e
7
>1.6
0.029
5.1
26
<1.0
<0.016
1.2
8
3TCg
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
3TC: lamivudine, an antiviral agent used as positive control.
.
g
ited high activities against secretion of HBsAg (IC50 = 0.024 mM)
and HBeAg (IC50 = 0.028 mM), which was more potent than the po-
sitive control drug lamivudine (IC50HBsAg = 12 mM, IC50HBeAg
= 26 mM). Meanwhile it had low cytotoxicity (CC50 > 2.6 mM),
resulting in high selectivity index (SIHBsAg > 108, SIHBeAg > 93).
Compound 6b showed suppressant properties on the secretion of
HBsAg (IC50 = 0.16 mM, SI > 16), while lost activity against secre-
tion of HBeAg. Above result further demonstrated the importance
of functionality at the position of C-25 to potent anti-HBV activity.
OH
OH
OH
OH
OH
OH
HO
OH
HO
OH
HO
H
OH
a
b
H
H
H
O
TsHNN
H
H
7
8
1
c
OAc
OAc
OAc
OH
d
AcO
H
AcO
H
OAc
H
H
O
O
2a
3a
Scheme 3. Reagents and conditions: (a) TsNHNH2, ethanol, reflux, 82%; (b) NaBH3CN, TsOH, DMF/sulfolane (1:1), 120 °C, 48%; (c) (CH3CO)2O, pyridine, rt, 86%; (d)
Lawensson’s reagent, toluene, reflux, 82%.

4650
Q. Zhang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4647–4650
Compounds
7 (IC50 = 0.029 mM) and 8 (IC50 = 0.080 mM)
References
exhibited similar inhibitory potency to the secretion of HBsAg
with that of compound 1 (IC50 = 0.039 mM), indicating that a car-
bonyl function at C-3 might not be necessary for biological
activity.
1. Kao, J. H.; Chen, D. S. Lancet Infect. Dis. 2002, 2, 395.
2. Park, N. H.; Song, I. H.; Chung, Y. H. Postgrad. Med. J. 2006, 82, 507.
3. Parkin, D. M.; Pisani, P.; Ferlay, J. Int. J. Cancer 1999, 80, 827.
4. Lim, Y. S.; Suh, D. J. J. Korean Med. Sci. 2004, 19, 489.
5. Cooksley, W. G. Semin. Liver Dis. 2004, 24, 45.
In summary, a series of alisol A derivatives were synthesized
and assayed for their anti-HBV activity and cytotoxicity in vitro,
using lamivudine as positive control. Our investigation demon-
strates that simple modifications of the parent structure of alisol
A can produce a number of potentially important derivatives.
Based on the above results, the following conclusions could be
made: (a) the acylation of hydroxyl group at 11, 23, 24-position
was found to decrease cytotoxicity; (b) epoxy group at C-13 (17)
is an important feature in the conferring relatively low cytotox-
icity; (c) hydroxyl group at C-25 of alisol A might be a good tar-
get for further lead optimization; and (d) a carbonyl function at
C-3 might not be necessary for biological activity. However, re-
sults from more extensive investigation using a great number
of derivatives are necessary for structure–activity relationship
study for the design and ultimate synthesis of more effective ali-
sol A-derived anti-HBV agents. Further investigation on protos-
tane-type triterpene compounds as promising HBV inhibitors
are ongoing in our laboratory and the results will be reported
in due course.
6. Liaw, Y. F.; Leung, N. W.; Chang, T. T.; Guan, R.; Tai, D. I.; Ng, K. Y.; Chien, R.
N.; Dent, J.; Roman, L.; Edmundson, S.; Lai, C. L. Gastroenterology 2000, 119,
172.
7. Suzuki, F.; Suzuki, Y.; Tsubota, A.; Akuta, N.; Someya, T.; Kobayashi, M.; Saitoh,
S.; Arase, Y.; Ikeda, K.; Kumada, H. J. Hepatol. 2002, 37, 824.
8. Xie, L.; Takeuchi, Y.; Cosentino, L. M.; Lee, K. H. Bioorg. Med. Chem. Lett. 1998, 8,
2151.
9. Sun, I. C.; Wang, H. K.; Kashiwada, Y.; Shen, J. K.; Cosentino, L. M.; Chen, C. H.;
Yang, L. M.; Lee, K. H. J. Med. Chem. 1998, 41, 4648.
10. Ding, P. L.; Liao, Z. X.; Huang, H.; Zhou, P.; Chen, D. F. Bioorg. Med. Chem. Lett.
2006, 16, 1231.
11. Yan, X. H.; Lin, L. P.; Ding, J.; Guo, Y. W. Bioorg. Med. Chem. Lett. 2007, 17, 2661.
12. Matsuda, H.; Tomohiro, N.; Yoshikawa, M.; Kubo, M. Biol. Pharm. Bull. 1998, 21,
1317.
13. Lee, S. M.; Kim, J. H.; Zhang, Y.; An, R. B.; Min, B. S.; Joung, H.; Lee, H. K. Arch.
Pharmacol. Res. 2003, 26, 463.
14. Yoshikawa, M.; Tomohiro, N.; Murakami, T.; Ikebata, A.; Matsuda, H.; Matsuda,
H.; Kubo, M. Chem. Pharm. Bull. 1999, 47, 524.
15. Rukachaisirikul, V.; Pailee, P.; Hiranrat, A.; Tuchinda, P.; Yoosook, C.; Kasisit, J.;
Taylor, W. C.; Reutrakul, V. Planta Med. 2003, 69, 1141.
16. Murata, T.; Imai, Y.; Hirata, T.; Miyamoto, M. Chem. Pharm. Bull. 1970, 18,
1347.
17. Hikino, H.; Iwakawa, T.; Oshima, Y.; Nishikawa, K.; Murata, T. Shoyakugaku
Zasshi 1982, 36, 150.
18. Kato, T.; Tomita, M.; Takigawa, M.; Iwasaki, H.; Hirukawa, T.; Yamahara, J. Bull.
Chem. Soc. Jpn. 1994, 67, 1394.
19. Yoshikawa, M.; Murakami, T.; Ikebata, A.; Ishikado, A.; Murakami, N.;
Yamahara, J.; Matsuda, H. Chem. Pharm. Bull. 1997, 45, 756.
20. Makino, B.; Kobayashi, M.; Kimura, K.; Ishimatsu, M.; Sakakibara, I.; Higuchi,
M.; Kubo, M.; Sasaki, H.; Okada, M. Planta Med. 2002, 68, 226.
21. Huang, Y. T.; Huang, D. M.; Chueh, S. C.; Teng, C. M.; Guh, J. H. Cancer Lett. 2006,
231, 270.
22. Wang, C.; Zhang, J. X.; Shen, X. L.; Wan, C. K.; Tse, A K. W.; Fong, W. F. Biochem.
Pharmacol. 2004, 68, 843.
23. Jiang, Z. Y.; Zhang, X. M.; Zhang, F. X.; Liu, N.; Zhao, F.; Zhou, J.; Chen, J. J. Planta
Med. 2006, 72, 951.
24. Murata, T.; Shinohara, M.; Hirata, T.; Kamiya, K.; Nishikawa, M.; Miyamoto, M.
Tetrahedron Lett. 1968, 9, 103.
Acknowledgements
The authors are grateful to the staff of the analytical 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. This work was supported
by National Natural Science Foundation of China (NSFC No.
30672522), Xibu Zhiguang Joint-Scholarship and guided project
of Chinese Academy of Sciences (No. KSCX1-YW-R-24).
25. Yoshikawa, M.; Hatakeyama, S.; Tanaka, N.; Fukuda, Y.; Yamahara, J.;
Murakami, N. Chem. Pharm. Bull. 1993, 41, 1948.
Supplementary data
26. Nakajima, Y.; Satoh, Y.; Katsumata, M.; Tsujiyama, K.; Ida, Y.; Shoji, J.
Phytochemistry 1994, 36, 119.
27. Cheng, P.; Ma, Y. B.; Yao, S. Y.; Zhang, Q.; Wang, E. J.; Yan, M. H.; Zhang, X. M.;
Zhang, F. X.; Chen, J. J. Bioorg. Med. Chem. Lett. 2007, 19, 5316.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.07.012.