Evaluation of anti-HCV activity and SAR study of (+)-lycoricidine through targeting of host heat-stress cognate 70 (Hsc70)

Article abstract of DOI:10.1016/j.bmcl.2013.02.089

The anti hepatitis C virus (HCV) activity of (+)-lycoricidine (1) was evaluated for the first time in this letter, yielding an EC₅₀ value of 0.55 nmol/mL and an selection index (SI) value of 12.72. Further studies indicated that 1 induced this effect by down-regulating host heat-stress cognate 70 (Hsc70) expression. In addition, 20 derivatives were designed and synthesised to investigate the basic structure-activity relationship (SAR) of the title compound. Several of these derivatives exhibit a good inhibition of HCV, such as compound 3 (EC₅₀ = 0.68 nmol/mL, SI = 33.86), compound 2d (EC₅₀ = 15 nmol/mL, SI = 12) and compound 5 (EC₅₀ = 33 nmol/mL, SI >10.91). Meanwhile, the experimental data suggest that the modification of certain groups of (+)-lycoricidine can reduce the cytotoxicity of the compounds.

Full text of DOI:10.1016/j.bmcl.2013.02.089

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2679–2682
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Evaluation of anti-HCV activity and SAR study of (+)-lycoricidine
through targeting of host heat-stress cognate 70 (Hsc70)
Duo-Zhi Chen a,b,c, Jian-Dong Jiang d, Ke-Qing Zhang c, Hong-Ping He a, Ying-Tong Di a, Yu Zhang a,
Jie-Yun Cai a,b, Lei Wang a,b, Shun-Lin Li a, Ping Yi e, Zong-Gen Peng d, , Xiao-Jiang Hao a,
⇑
⇑
a State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Yunnan, PR China
b University of Chinese Academy of Sciences, Beijing 100039, PR China
c Laboratory for Conservation and Utilization of Bio-Resources, and Key Laboratory of Microbial Diversity in Southwest China, Ministry of Education, Yunnan University, Yunnan,
Kunming 650091, PR China
d Laboratory of Antiviral Research, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences/Peking Union Medical College, Beijing 100050, China
e Key Laboratory of Natural Pharmaceutical & Chemical Biology of Yunnan Province, School of Science, Honghe University, Mengzi 661100, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
The anti hepatitis C virus (HCV) activity of (+)-lycoricidine (1) was evaluated for the first time in this let-
ter, yielding an EC50 value of 0.55 nmol/mL and an selection index (SI) value of 12.72. Further studies
indicated that 1 induced this effect by down-regulating host heat-stress cognate 70 (Hsc70) expression.
In addition, 20 derivatives were designed and synthesised to investigate the basic structure–activity rela-
tionship (SAR) of the title compound. Several of these derivatives exhibit a good inhibition of HCV, such as
compound 3 (EC50 = 0.68 nmol/mL, SI = 33.86), compound 2d (EC50 = 15 nmol/mL, SI = 12) and compound
5 (EC50 = 33 nmol/mL, SI >10.91). Meanwhile, the experimental data suggest that the modification of cer-
tain groups of (+)-lycoricidine can reduce the cytotoxicity of the compounds.
Received 2 December 2012
Revised 15 February 2013
Accepted 20 February 2013
Available online 1 March 2013
Keywords:
(+)-Lycoricidine
Heat-stress cognate 70 (Hsc70)
Down-regulators
Ó 2013 Elsevier Ltd. All rights reserved.
Structure–activity relationship (SAR)
Anti-HCV activity
There are 150 million people infected with the hepatitis C virus
(HCV) worldwide, and more than 80% of them will progress into
chronic hepatitis.1–3 At present, typical treatment for hepatitis C
focuses on the inhibition of NS protease,4–7 which involves PEGy-
cells can reduce the level of Hsc70 packaging into HCV particles,
and therefore inhibit HCV replication in the next infection cycle.15
(+)-Lycoricidine (1, Fig. 1), a benzylphenethylamine alkaloid
mainly isolated from the Amaryllidaceae family, has attracted con-
siderable interest due to its promising cytotoxic activities.16–21 It is
interesting, however, that (+)-lycoricidine and other lycoris alka-
loids also exhibit broad-spectrum capabilities against a series of
RNA viruses,22 which suggests that these alkaloids do not directly
affect the virus but act on the host cell.
lated interferon-a (IFN-a) plus the nucleoside analogue ribavirin.
However, this treatment also carries side effects and yields a poor
sustained virological response (SVR).8–10 Therefore, the exploration
of new drugs, especially ones that involve new mechanisms by
which to inhibit HCV proliferation, strongly attracts many
researchers.
Previous studies implied that human heat shock cognate 70
(Hsc70, or heat shock protein A8), a member of the heat shock pro-
tein 70 (Hsp70) family, could be a new target to inhibit HCV. Hsc70
is a cytoplasmic adenosine triphosphate (ATP)-binding protein
with 646 amino acids.11–13 A study by Parent et al. demonstrated
that host Hsc70 was a part of the HCV particle and that it modu-
lated HCV infectivity, as well as lipid droplet-dependent virus re-
lease, after HCV entered host cells.14 Additionally, we have
reported that down-regulation of this protein in HCV-infected host
Thus, (+)-lycoricidine and two other lycoris alkaloids, 6 and 7
(Fig. 1), were initially evaluated for both the inhibition of HCV
and the expression of host Hsc70 in Huh-7.5 cells. Under treatment
with compound 1, at the concentration of 0.1 lg/mL, Hsc70
expression in host cells was reduced by 28%. Simultaneously, the
replication of HCV decreased dramatically to 6%. The correlation
between Hsc70 expression and HCV replication was clear from
western blot analysis (Fig. 2). Furthermore, 1 also exhibited an
inhibitory activity on Hsc70 mRNA expression with 91.4% inhibi-
tion at the concentration of 10
lg/mL and 83.1% inhibition at the
concentration of 100 g/mL. According to the results above, it
l
could be speculated that 1 might inhibit HCV replication by sup-
pressing Hsc70 expression.
⇑
Corresponding authors. Tel./fax: +86 10 63010984 (Z.-G.P.); tel./fax: +86 871
5223070 (X.-J.H.).
(+)-Lycoricidine (1) exhibited
a strong inhibitory activity
E-mail addresses: pumcpzg@126.com (Z.-G. Peng), haoxj@mail.kib.ac.cn (X.-J.
against HCV expression, but it exhibited obvious cytotoxicity, as
Hao).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.02.089

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D.-Z. Chen et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2679–2682
OH
H
OH
N
OH
2
HO
HO
3
OH
OH
1
H
H
10
7
4
10b
10a
9
8
O
O
O
O
O
O
4a
5NH
H
H
N
6
6a
O
7
6
1
Figure 1. Structures of compounds 1 ((+)-lycoricidine), 6 and 7.
Hsc70
HCV
Actin
Comp.
Control
1.00
6 (0.1)
1.01
6 (1.0)
0.68
6 (10)
0.60
6 (100)
0.67
7 (0.1)
0.78
7 (1.0)
0.79
1 (0.1)
0.72
Hsc70/actin
HCV/actin
1.00
1.20
1.16
1.22
0.10
1.86
0.20
0.06
(µg /mL)
Figure 2. Intracellular Hsc70 protein concentration decreased dose-dependently in Huh-7.5 cells either untreated or treated with 1 (0.1
and 7 (0.1, 1.0 g/mL) for 24 h.
lg/mL), 6 (0.1, 1.0, 10, 100 lg/mL)
l
well. Therefore, further exploration of this compound’s active sites
was necessary and valuable. As shown in Fig. 3, the structure mod-
ifications of 1 were initiated with the transformation of substitu-
ents at the 2-, 3- and 4-positions, as well as the double bond
between the 1- and 10b-positions. Compounds 2–5 were designed
and synthesised as four key intermediates (Fig. 3) and then modi-
fied these four compounds to afford other 16 derivatives (Fig. 4).
All these 20 compounds were evaluated for their anti-HCV activi-
ties (Table 1).
The SAR analysis first concentrated on the double bond between
the 1- and 10b-positions, which contributes to a large conjugated
system within 1. Hydrogenation of this double bond would notably
affect the electronic cloud distribution within the compound and
would have an impact on the bioactivities. The comparison of 3
with 1 indicated that the compound without the double bond
exhibited higher CC50 and SI values. Specifically, 3 had a similar
EC50 value (0.68 nmol/mL) to that of 1, but its CC50 value
(23 nmol/mL) was significantly (threefold) greater than that of 1.
Similar results can also be observed in the comparisons of 2 with
4, 2c–d with 4a–b and 3a–c with 1b–d, respectively. Therefore, it
can be deduced that the double bond between the 1- and 10b-posi-
tions is crucial to the anti-HCV activity: reduction of this bond
might reduce the cytotoxicity of the compounds and improve their
SI values.
Ether linkage of propylidene was achieved on the hydroxyl
groups at the 3- and 4-positions of 1. Such conversion may influ-
ence the formation of hydrogen bonds between the compound
and its target protein. These results showed that the CC50 of 2
was much higher than that of 1, that 4 exhibited a lower cytotox-
icity than 3 and that both 2 and 4 lost their inhibitory activities to a
certain extent. These results suggested that the ether linkage at the
3-, 4-position affected both the cytotoxicity and the anti-HCV
activity but was most pronounced in its effects by increasing the
CC50 for the title compound.
OH
OH
H
2
3
OH
OH
O
O
1
H
4a
5NH
10
7
4
10b
10a
9
8
O
O
O
O
a
NH
6
6a
O
1
O
2
d
b
OH
OH
H
OH
OH
OH
OH
H
O
O
O
O
H
NH
NH
O
3
c
OH
H
5
O
O
O
O
H
NH
O
4
When the hydroxyl groups of the 3- and 4-positions were re-
placed by propylidene, the substitution at the 2-position influ-
enced the bioactivities of the compounds. Comparing 2 with its
Figure 3. Synthesis of the intermediates 2–5. Reagents and conditions: (a) 2,2-
dimethoxypropane, p-TSA, DMF, 12 h, rt, 85%; (b) 10% Pb–C, H2, Cl2CH2/CH3OH,
24 h, rt, 82%; (c) 2,2-dimethoxypropane, p-TSA, DMF, 20 h, rt, 90%. (d) LiAlH4, THF,
À10 °C, 4 h, 45%.

D.-Z. Chen et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2679–2682
2681
Table 1
OR1
H
OR1
Inhibition of HCV (+)-lycoricidine derivatives
OR2
OR3
OR2
OR3
Compd.
HCV
H
CC50 (nmol/mL)
EC50 (nmol/mL)
SI
O
O
O
O
H
O
NR4
NR4
1
7.2 0.48
33 3.36
180 9.34
252 5.59
120 9.98
170 10.16
>300
160 10.49
280 12.36
180 10.95
250 6.43
200 9.94
23 1.46
>300
0.55 0.07
>0.98
22 1.79
>83
12.72
<33.8
8.2
<3.01
1.62
9.09
>4.76
2.13
1a
1b
1c
1d
2
2a
2b
2c
2d
2e
2f
O
3, R1 = R2 = R3 = R4 = H
1, R1 = R2 = R3 = R4 = H
74 7.91
11 0.85
63 3.51
75 5.41
93 6.88
15 1.05
40 4.82
65 4.77
0.68 0.07
54 4.34
43 2.30
12 0.64
>11
3a, R1 = R2 = R3 = R4 = CH3
3b, R1 = R2 = R3 = R4 = CH2CH3
1a, R1 = R2 = R3 = Ac , R4 = H
1b, R1 = R2 = R3 = R4 = CH3
1c, R1 = R2 = R3 = R4 = CH2CH3
3.01
12.00
6.25
3c, R1 = R2 = R3 = R4 =
OR1
3.08
3
33.82
>5.56
>6.98
3.17
< 25.46
2.17
3.06
3.03
>10.91
>24000
3a
3b
3c
4
4a
4b
4c
5
>300
1d R1 = R2 = R3 = R4 =
OR1
38 1.89
280 9.28
250 14.55
260 13.74
270 15.27
>360
O
H
92 3.85
85 3.85
89 4.04
33 4.47
0.250 (U/mL)
O
O
O
O
H
O
NR2
H
O
O
O
Intron A
> 6000 (U/mL)
NR2
4, R1 = R2 = H
O
4a, R1 = R2 = CH3
derivatives (2a, 2b), in which the 2-hydroxyl was acetylated (2a) or
silylated (2b), revealed that the EC50 values increased significantly
from 11 nmol/mL to 63 nmol/mL (2a) and 75 nmol/mL (2b),
respectively. Coincidentally, compound 4c, in which the 2-position
was also acetylated, exhibited a much weaker anti-HCV activity
than that of 4. In this sense, a hydroxyl group at the 2-position
may play an important role in inhibiting HCV replication.
The hydroxyl groups at the 2-, 3-, and 4-positions, as well as the
amide group at the 5-position, each could form hydrogen bonds
with target proteins. Acylation or etherification of these groups
would significantly affect the cytotoxicity of the compounds. The
respective comparisons of 1 with 1a–d and 3 with 3a–c, indicated
that 1 and 3 exhibited much higher cytotoxicities. Additionally, the
analogue modifications decreased the ability to inhibit the replica-
tion of HCV to varying degrees, which suggested that these modi-
fications influence not only the cytotoxicity but also the anti-HCV
activity. It was notable that compound 2d had a good inhibitory
2, R1 = R2 = H
4b, R1 = R2 = CH2CH3
4c, R1 = Ac, R2 = H
2a, R1 = Ac , R2 = H
2b, R1 = TBDMS , R2 = H
2c, R1 = R2 = CH3
2d, R1 = R2 = CH2CH3
2e, R1 = R2 =
OH
OH
OH
H
O
O
NH
2f, R1 = R2 = CH2Ph
5
Figure 4. Structures of (+)-lycoricidine derivatives.
Figure 5. The cytotoxicity of (+)-lycoricidine derivatives. The CC50 values of 2a, 3a, 3b and 5 were greater than 300.

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D.-Z. Chen et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2679–2682
activity, with an EC50 value of 15 nmol/mL and an SI value of 12.0;
thus, we deduced that the ethyl substituents at the 2- and 5-posi-
tions with propylidene at the 3-, 4-position might together im-
prove anti-HCV activity of compounds.
The electron-withdrawing carbonyl group of the 6-position was
crucial to the bioactivities of (+)-lycoricidine, as it can connect to
peptide chains through H-bonds and can contribute to the conju-
gated system of 1. Furthermore, the reduction derivative 5 also
showed a higher CC50 value, which suggested that the amide group
might be another functional group that can affect the cytotoxicity
of (+)-lycoricidine.
This study was the first evaluation of the anti-HCV activity of
(+)-lycoricidine (1) and its derivatives. The results indicated that
compound 1 inhibited the replication of HCV in HCV-infected
Huh-7.5 cells by suppressing host Hsc70 expression, which was
different from the mechanism of nucleosides. Based on this finding,
20 (+)-lycoricidine derivatives were further synthesised to evaluate
their anti-HCV activities. The SAR analysis revealed that the conju-
gated system of 1 was crucial to its bioactivities. Reduction of the
double bond between the 1- and 10b-positions, or of the carbonyl
group at the 6-position, may reduce the cytotoxicity and improve
the SI of the compound. Additionally, acyl or ether substitutions
at the 2-, 3-, 4- and 5-positions were introduced to prevent hydro-
gen bonding with the groups’ respective host proteins and were
shown to be efficacious, as demonstrated by the increased CC50 va-
lue of the derivatives (Fig. 5). However, these modifications also re-
duced the inhibitory activity of the derivatives. Furthermore, it
should be noted that compound 2d exhibited relatively good
anti-HCV activity, which suggested that certain ether substituents
at 2-, 3-, 4- and 5-positions may improve the SI values of
compounds.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
02.089.
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Acknowledgments
This research was supported financially by the National Natural
Science Foundation of China (30830114 and 31270404) and the
Ministry of Science and Technology (2009CB522300).