Synthesis of celastrol derivatives as potential non-nucleoside hepatitis B virus inhibitors

Article abstract of DOI:10.1111/cbdd.13746

A series of para-quinone methide (pQM) moiety and C-20- modified derivatives of celastrol were synthesized and evaluated for their inhibitory effect on the secretion of HBsAg and HBeAg as well as the inhibitory effect against HBV DNA replication. The results suggested that amidation of C-20 carboxylic group could generate derivatives with good anti-HBV profile, among them compound 14 showed the best inhibitory activity on the secretion of HBsAg (IC₅₀?=?11.9?μμ) and HBeAg (IC₅₀?=?13.1?μμ) with SI of 3.3 and 3.0, respectively. In addition, 14 also showed potent inhibitory effect against HBV DNA replication (48.5?±?15.1%, 25?μM). This is, to our knowledge, the first report of celastrol derivatives as potential non-nucleoside HBV inhibitors.

Full text of DOI:10.1111/cbdd.13746

DR GONGXI LU (Orcid ID : 0000-0002-4470-1189)
Article type
: Research Letter
Synthesis of celastrol derivatives as potential non-nucleoside hepatitis B
virus inhibitors
He Zhang a,* and Gongxi Lu a,*
a Beijing BeiqinBiotech Co. LtD., Xinggu economic development zone, Beijing, 101200, PR.China
Corresponding author, e-mail: gxlu_bbq@yeah.net (G. Lu), zhanghe_bbqzh@tom.com (H. Zhang)
Running Head
Celastrol derivatives as anti-hepatitis B virus agents
Abstract
A series of para-quinone methide (pQM) moiety and C-20 modified derivatives of celastrol were synthesized
and evaluated for their inhibitory effect on the secretion of HBsAg and HBeAg as well as the inhibitory effect
against HBV DNA replication. The results suggested that amidation of C-20 carboxylic group could generate
derivatives with good anti-HBV profile, among them compound 14 showed the best inhibitory activity on the
secretion of HBsAg (IC50 = 11.9 µΜ) and HBeAg (IC50 = 13.1 µΜ) with SI of 3.3 and 3.0, respectively. In
addition, 14 also showed potent inhibitory effect against HBV DNA replication (48.5 ± 15.1%, 25µM). This is,
to our knowledge, the first report of celastrol derivatives as potential non-nucleoside HBV inhibitors.
Keywords
Tripterygium wilfordii Hook, F., Celastrol, Hepatitis virus B, Inhibitor
1. Introduction
Viral hepatitis type B is a serious infectious disease caused by hepatitis B virus (HBV) (Dienstag, 2008). It is
reported by the World Health Organization (WHO) that more than 2 billion people have been infected with HBV
in their lives (Shepard, et al., 2006). Due to its characters of high incidence, long course and difficulty to cure,
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
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HBV infection has become a major threat to the public health. Especially in China, HBV infection has become a
serious issue with about 28 million people have chronic hepatitis B (CHB), and 90 million HBV carriers (Jia, et
al., 2017). Worse yet, long-term development of Hepatitis B can lead to acute or chronic viral hepatitis, severe
hepatitis, liver cirrhosis (LC) and hepatocellular carcinoma (HCC) (Rehermann, et al., 2005). Every year, more
than 1 million people death from HBV infection relating diseases such as liver failure, cirrhosis, and
hepatocellular carcinoma. Currently, drugs that used for the treatment of HBV infection include interferon,
immunomodulators, and DNA polymerase inhibitors. Although, interferon has dual effects of
immunomodulation and antivirus, the need for parenteral administration, the effective only to 30%-40% people,
and the adverse side effects greatly limited its clinical application (Chan, et al., 2005; Liu, et al., 2014).
Mechanistically, nucleotide HBV DNA polymerase inhibitors such as lamivudine, adefovir, entecavir and
tenofovir exert their antivirus activity though targeting viral DNA polymerase, and thus would result in the
development of drug-resistant virus after long-term treatment (Zhang, et al., 2014), which make them not ideal.
Therefore, the therapies for HBV remain unsatisfactory, and the development of anti-HBV agents with novel
structures and mechanisms of action is of the top priority
Tripterygium wilfordii Hook, F. (TWHF), also known as Lei Gong Teng or Thunder God Vine, is a vine-like
medicinal plant whose extracts have been used to treat autoimmune and inflammatory diseases such as
rheumatoid arthritis (RA) for centuries in traditional Chinese medicine (TCM) (Goldbach-Mansky, 2009; Zhou, et
al., 2018). It is reported that celastrol (Hou, et al., 2020a), triptolide (Kupchan, et al., 1972), triptonide (Kupchan, et
al., 1972), 15-hydroxytriptolide (Niu, et al., 2015), triptriolide (Wang, et al., 2019; Yang, et al., 2018; Yang, et al.,
2019) and triptophenolide (He, et al., 2016) are the major bioactive component of TWHF (Figure 1). From a
structural point of view, celastrol is a pentacyclic triterpenoid that decorated with a bioactive para-quinone
methide (pQM) moiety. Previous studies have reported that pQM could interact with DNA (Huang, et al., 2016)
or target proteins residues by π-π stacking (Duan, et al., 2013), hydrophobic interactions, hydrogen bonds and/or
covalent addition (Zhao, et al., 2015). Therefore, celastrol has been shown to be effective against various human
diseases via interacting with many different cellular targets. It is reported that celastrol could suppress the NF-
κB activation by interacting with IKK and showed anti-inflammatory and anti-cancer activities (Lee, et al., 2006).
Additionally, it could disrupt the interaction of Hsp90 and CDC37 through binding to the C-terminal domain of
Hsp90. It could also induce apoptosis in multiple cancer cells by activating c-Jun N-terminal kinase and
suppressing PI3K/Akt signaling pathways (Kannaiyan, et al., 2011). Recently, celastrol was reported to have the
ability to increase leptin sensitivity, thus has the potential to be developed as a anti-obesity agent (Kyriakou, et al.,
2011; Liu, et al., 2011). Taken together, celastrol should be a promising bioactive natural product for new drug
discovery. However, unlike its relative, triptolide, on which lots of total synthesis and structural modifications
have conducted in the past two decades (Chen, et al., 2012; Hou, et al., 2019b; Kaloun, et al., 2016; Liu, et al., 2018;
Ning, et al., 2018; Patil, et al.,2015; Wang, et al., 2017; Xu, et al., 2014a; Xu, et al., 2014b; Xu, et al., 2014c; Xu, et al.,
2014d; Xu, et al., 2019; Xu, et al., 2017; Zhang, et al., 2019; Zhou, et al., 2012), and some triptolide derivatives
have already entered clinic for the treatment of challenging cancer and/or rheumatoid arthritis (RA) (Carter, et al.,
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2012; Pao, et al., 2019; Patil, et al., 2019; Wang, et al., 2012;; Zhou et al., 2005). So far, there are only one total
synthesis (Camelio, et al., 2015) and several chemical modifications of celastrol have been reported (Figueiredo,
et al., 2017a; Jiang, et al., 2016; Kyriakou, et al., 2018; Li, et al., 2015; Li, et al., 2018; Pang, et al., 2018; Shan, et al.,
2017; Tang, et al., 2015; Zhang, et al., 2018; Zhu, et al., 2017), which are largely focused on its anticancer activity.
In order to further explore the promising multiple biological activities of celastrol and with the aim to find new
anti-HBV agents with novel chemical structure and mechanism of action, herein we reported the synthesis and
anti-HBV activity evaluation of a series of pQM moiety and C-20 modified celastrol derivatives (Figure 2).
COOH
Me
Me
Me
O
O
O
O
O
O
Me
Me
Me
Me
O
H
O
H
O
H
HO
O
OH
Me
H
Me
OH
Me
O
Me
Me
O
O
Me
celastrol
O
O
HO
O
O
Me
tripdiolide
triptonide
triptolide
OH Me
Me
Me
OH
Me
O
O
O
O
OH
Me
OH
Me
Me
OH
Me
H
O
HO
O
O
OH
OH
O
Me
Me
H
Me
O
O
O
O
H
H
O
O
O
O
triptophenolide
15-hydroxytriptolide
16-hydroxytriptolide
triptriolide
Figure 1. Representative natural products isolated form TWHF
COOH
COOMe
20
CH2OH
COOMe
Me
COOH
20
20
Me
Me
Me
Me
Me
E
Me
H
Me
Me
4
H
Me
Me
4
H
Me
Me
H
Me
H
Me
1
1
1
C
HO
2
MeO
O
MeO
MeO
O
D
2
2
Me
Me
B
Me
Me
A
Me
5
5
3
5
HO
MeO
MeO
HO
3
3
3
4
Me
6
6
6
Me
Me
O2
Me
Me
1. Celastrol
4
5
3
2
N
Me
O
O
O
O
O
O
O
O
Me
COOMe
Me
20
20
20
20
20
Me
Me
Me
Me
Me
Me
H
Me
Me
H
Me
H
Me
Me
H
Me
Me
H
Me
O
O
O
O
O
Me
Me
Me
Me
Me
HO
HO
HO
HO
HO
Me
Me
Me
Me
Me
8
10
9
7
6
Me
N
H
H
H
N
O
N
O
Me
N
O
O
N
O
Me
Me
Me
20
20
20
20
20
Me
Me
Me
Me
Me
Me
Me
H
Me
Me
H
Me
Me
H
Me
Me
H
Me
Me
H
Me
O
O
O
O
O
Me
Me
Me
Me
Me
HO
HO
HO
HO
HO
Me
Me
Me
Me
Me
11
13
15
14
12
HO
O
HO
O
HO
O
O
N
O
N
O
20
Me
Me
Me
20
Me
H
Me
Me
H
Me
Me
Me
H
Me
Me
HO
HO
HO
HO
HO
HO
Me
Me
Me
H
Me
Me
Me
H
Me
6
6
O
6
O
Me
Me
HO
HO
F
OMe
HN
20
HN
19
Me
Me
HN
18
16
17
Figure 2. Celastrol derivatives 2-20.
2. Result and discussion
The syntheses to access the target celastrol derivatives 2-20 was depicted in Scheme 1. Firstly, in order to
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probe the effect of the para-quinone methide (pQM) moiety on anti-HBV activity, A-ring aromatic derivative 1-
4 were synthesized by reducing of celastrol 1 in the presence of sodium borohydride (NaBH4), and thus provided
derivative 2 in almost quantitative yield (Figueiredo, et al., 2017b). Methylation of the phenolic C-3 hydroxyl
group along with the C-20 carboxylic acid group gave ester 3, which was reduced by lithium aluminum hydride
(LAH) provided C-20 hydroxymethyl substituted derivative 4. Directly methylation of the C-3 hydroxyl group
and the C-20 carboxylic acid group of celastrol in the presence of sodium hydride and methyl iodide afforded
derivative 5. AlCl3-Catalyzed Friedel-Crafts alkylation of the C-6 moiety of celastrol with various indoles
afforded derivatives 18-20 in moderate yield. These derivatives can be used to probe the influence of the pQM
moiety on the anti-HBV activity as well as the substituent effect of C-6. Finally, in order to probe the substituent
properties of the C-20 carboxylic acid group, esters derivatives 6-10 and amide derivatives 11-17 were
synthesized via alkylation of the C-20 carboxylic acid group with various alkyl halides, and/or condensation of
the C-20 carboxylic acid group with various amines, respectively.
COOH
COOMe
COOH
Me
Me
Me
b
a
Me
H
Me
Me
H
Me
Me
H
Me
HO
HO
MeO
MeO
O
Me
Me
c
Me
HO
Celastrol (1)
Me
Me
Me
2
3
d
e
o
r
g
f
R
O
HO
O
COOMe
CH2OH
Me
20
Me
Me
Me
H
Me
Me
H
Me
Me
Me
H
Me
Me
Me
Me
H
O
HO
HO
HO
HO
MeO
MeO
Me
Me
Me
6
Me
HO
Me
R
6-17
Me
5
4
18-20
Scheme 1. Synthesis of celastrol derivatives. a) NaBH4, MeOH, r.t.; b) MeI, K2CO3, DMF, r.t.; c) LAH, THF, r.t.; d) MeI, NaH,
DMF, r.t.; e) alkyl iodides, NaHCO3, DMF, r.t; f) amines, HATU, DIPEA, DMF, r.t.; g) indoles, AlCl3·6H2O (5 mol%), DCM, r.t..
All the synthesized celastrol derivatives were tested for their potential anti-HBV activity, namely inhibiting
the secretion of HBsAg (HBV surface antigen, which indicates current hepatitis B infection), and HBeAg (HBV
e antigen, which is an indicator of active viral replication. It means the person infected with Hepatitis B can
likely transmit the virus onto another person), and also their inhibitory effect on HBV DNA replication in HepG
2.2.15 cells using lamivudine (3 TC, a clinically popular anti-HBV agent) as a positive control. The anti-HBV
activity of the celsatrol derivatives was expressed as the concentration of derivative required for 50% inhibition
(IC50) of HBsAg or HBeAg secretion. The cytotoxicity of each celastrol derivatives was defined as the
concentration of derivative required to kill 50% (CC50) of the HepG 2.2.15 cells. While, the selectivity index
(SI), a major pharmaceutical parameter that indicates possible future clinical development, was defined as the
ratio of CC50 to IC50.
As shown in Table 1, celastrol 1 was more effective on the inhibiting the secretion of HBsAg and HBeAg
than that of positive control 3TC with the IC50 values of 12.7 and 15.1 µΜ, respectively. However, it showed
very poor selectivity index (SI = 0.4 and 0.3 for HBsAg and HBeAg, respectively) and inhibitory rate against
HBV DNA replication (25.9 ± 3.8%). After aromatization of the ring-A, the related derivatives 2-4 all lost their
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inhibitory activity on the secretion of HBsAG and HBeAg as well as their cytotoxic activity, except for
compound 4, which only showed moderate inhibitory activity against the secretion of HBsAg and HBeAg with
IC50 values of 68.4 µΜ and 72.5 µΜ, respectively. Esterification of the C-20 carboxylic group, the resulting
derivatives (6-10) generally showed comparable inhibitory activity and cytotoxic activity as celastrol, except of
compound 5 (SI of 1.1 and 1.2) which showed a little increased selectivity than celastrol. These results suggested
that esterification of the C-20 carboxylic group could retain the cytotoxic activity of celastrol, however, as listed
in Table 1, the antivirus activity are poor. Gratefully, amidation of the C-20 carboxylic group with either primary
amines or secondary amines, the resulting derivative generally showed increased selectivity (SI). Among all the
tested amide derivatives, C-20 dimethylcarbamoyl substituted compound 14 showed the best inhibitory profile
on the secretion of HBsAg (IC50 = 9.2 µΜ) and HBeAg (IC50 = 10.0 µΜ) with SI of 3.3 and 3.0, respectively. In
addition, compound 14 also showed highest inhibition rate against HBV DNA replication (48.5 ± 15.1%) among
all the tested derivatives. These results suggested that amidaiton of the C-20 carboxylic group could generate
celastrol derivative with good inhibitory activity and selectivity for the anti-HBV activity. This is probably due
to the introduction of the basic nitrogen atom, which could interaction with the relative protein target the
responsible for anti-HBV activity. Disappointedly, the introduction of the indoles at C-6 position of celastrol
destroyed dramatically the inhibitory activity on the secretion of HBsAg and HBeAg, among the tested
derivative, only derivative 19 showed moderate inhibitory activity, this is probably due to the aromatization of
the A-ring that lead to the destroy of the pQM moiety, which is generally recognized as the key bioactive
structural element for the various bioactivity of celastrol.
According to the experimental results mentioned above, preliminary structure-activity relationship (SAR)
were summarized as followed: (1) the para-quinone methide (pQM) moiety of celastrol play a key role in
retaining its cytotoxicity as well anti-HBV activity, aromatization of the ring-A or introduction of substituents
on C-6 would exert negative effect on anti-HBV activity; (2) Esterification of the C-20 carboxylic group could
retain the cytotoxic activity of celastrol, but poor anti-HBV activity; (3) Amidation of the C-20 carboxylic group
could generated derivatives with good anti-HBV activity. In general, these preliminary SAR results would
provide valuable information for further modification of celastrol to find more potent anti-HBV derivatives.
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Table 1. Anti-HBV activity of derivatives 2-20.
a
HBsAg
IC50b(µM)
HBeAg
IC50b(µM)
CC50
Inhibition rate (%)d
Compd
SIc
0.4
---
SIc
0.3
---
(µM)
(25 µM)
25.9±3.8%
---
1
2
5.2±0.9
>100
12.7±2.2
>100
15.1±3.0
>100
3
>100
91±
---
>100
---
---
4
79.0±17.4
23.5±4.7
17.1±1.8
6.3±2.3
4.1±1.2
3.6±0.6
7.7±3.9
15.6±4.4
12.9±4.5
29.3±6.3
39.1±8.6
17.1±9.0
12.4±3.3
11.3±1.1
74.0±14.9
69.0±8.8
91.0±12.5
68.4±21.3
22.1±6.2
19.4±5.1
23.1±5.4
11.5±3.1
9.2±1.7
15.1±3.5
18.4±5.1
21.4±5.4
15.9±2.3
11.9±2.6
24.1±6.1
33.1±6.3
15.7±3.2
>100
---
72.5±12.3
19.9±5.5
29.8±3.7
25.4±5.0
17.3±4.4
10.0±3.5
14.4±4.4
20.3±3.9
29.7±6.0
17.2±3.8
13.1±3.3
21.7±7.7
25.2±5.9
14.6±1.7
>100
---
---
5
1.1
0.9
0.3
0.4
0.4
0.5
0.8
0.6
1.8
3.3
0.7
0.4
0.7
---
1.2
0.3
0.2
0.2
0.4
0.5
0.8
0.4
1.7
3.0
0.8
0.5
0.8
---
10.5±4.1%
9.8±1.5%
---
6
7
8
---
9
---
10
11
12
13
14
15
16
17
18
19
20
3TCe
---
13.2±5.0%
17.2±7.4%
35.2±8.7%
48.5±15.1%
25.9±9.4%
---
26.0±8.6%
---
67.3±5.4
>100
---
64.4±9.6
97.9±11.9
---
---
---
---
---
493.5±65.4 177.4±33.3 2.8 192.1±45.1 2.7
89.1±22.4%
aCC50: CC50 is 50% cytotoxicity concentration in HepG2 2.2.15 cells.
bIC50: Concentration of compound required for 50% inhibition of HBsAg (HBV surface antigen) or HBeAg (HBV e antigen) secretion.
cSI: Selective index, the ratio of CC50 /IC50.
dInhibitory effect of celastrol on HBV DNA level.
e Lamivudine (3TC) was used as positive control.
3. Conclusion
In summary, we have designed and synthesized a series of para-quinone methide (pQM) moiety and C-20
modified celastrol derivatives and evaluated their inhibitory activity on the secretion of HBsAg and HBeAg for
the first time. The present results suggested that the aromatization of the A-ring would exert negative influence
on the inhibitory activity. Esterification of the C-20 carboxylic group could generate derivatives with increasing
inhibitory activity, but decreasing the selectivity. Amidation of the C-20 carboxylic group could give derivatives
with increased inhibitory profile. Among all the tested derivatives, 14 showed the best inhibitory activity on the
secretion of HBsAg (IC50 = 11.9 µΜ) and HBeAg (IC50 = 13.1 µΜ) with SI of 3.3 and 3.0, respectively. In
addition, 14 also showed potent inhibitory activity against HBV DNA replication (48.5 ± 15.1%, 25 µM). Over
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all, these results indicated that derivative 14 might be used as a starting point for the discovery of non-nucleoside
anti-HBV agents. It is also important to address the exact molecular target, through which derivative 14 exerts
its anti-HBV activity, the related studies are currently ongoing in our laboratory and will be disclosed in the due
course.
Conflict of interest
The authors of this manuscript declare no conflict of interests.
SUPPORTING INFORMATION
Additional supporting information may be found online in the Supporting Information section at the end of the
article.
Data Availability Statement
No additional data are available.
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