Synthesis of 3- and 29-substituted celastrol derivatives and structure-activity relationship studies of their cytotoxic activities

Article abstract of DOI:10.1016/j.bmcl.2017.05.083

A series of 3-carbamate and 29-ester celastrol derivatives (compounds 1–26) were designed and synthesized. These analogues were evaluated for their cytotoxic activities against several cancer cell lines. Cytotoxicity data revealed that the properties of substituents and substitution position had important influence on cytotoxic activity. Modification of C-3 hydroxyl with size-limited groups did not reduce the activity obviously. The introduction of polarity group like piperazine could improve the solubility. Compound 23 was chosen to further evaluate anti-tumor efficacy in vivo. It showed higher inhibition rate and better safety than celastrol during in vivo experiment by intragastric administration. The preliminary antitumor studies of compound 23 in vivo showed that it might be promising for the development of new antitumor agents.

Full text of DOI:10.1016/j.bmcl.2017.05.083

Accepted Manuscript
Synthesis of 3- and 29-substituted celastrol derivatives and structure-activity
relationship studies of their cytotoxic activities
Wei-Guang Shan, Han-Guang Wang, Yan Chen, Rui Wu, Yan-Tao Wen, Li-
Wen Zhang, You-Min Ying, Jian-Wei Wang, Zha-Jun Zhan
PII:
S0960-894X(17)30580-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.05.083
BMCL 25028
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
1 April 2017
25 May 2017
26 May 2017
Please cite this article as: Shan, W-G., Wang, H-G., Chen, Y., Wu, R., Wen, Y-T., Zhang, L-W., Ying, Y-M., Wang,
J-W., Zhan, Z-J., Synthesis of 3- and 29-substituted celastrol derivatives and structure-activity relationship studies
of their cytotoxic activities, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/
j.bmcl.2017.05.083
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Bioorganic & Medicinal Chemistry Letters
Synthesis of 3- and 29-substituted celastrol derivatives and structure-activity
relationship studies of their cytotoxic activities
a
a
a
a
b
b
Wei-Guang Shan , Han-Guang Wang , Yan Chen , Rui Wu , Yan-Tao Wen , Li-Wen Zhang , You-Min
a
a
a, 
Ying , Jian-Wei Wang , and Zha-Jun Zhan
a
College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou, 310014, P. R. China
The Children’s Hospital, Zhejiang University School of Medicine, Hangzhou, 310003, P. R. China
b
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A series of 3-carbamate and 29-ester celastrol derivatives (compounds 1–26) were designed and
synthesized. These analogs were evaluated for their cytotoxic activities against several cancer
cell lines. Cytotoxicity data revealed that the properties of substituents and substitution position
had important influence on cytotoxic activity. Modification of C-3 hydroxyl with size-limited
groups did not reduce the activity obviously. The introduction of polarity group like piperazine
could improve the solubility. Compound 23 was chosen to further evaluate anti-tumor efficacy
in vivo. It showed higher inhibition rate and better safety than celastrol during in vivo
experiment by intragastric administration. The preliminary antitumor studies of compound 23 in
vivo showed that it might be promising for the development of new antitumor agents.
Available online
Keywords:
celastrol
C-3 hydroxyl derivatives
cytotoxicity
in vivo activity
intragastric administration
2
016 Elsevier Ltd. All rights reserved.
Celastrol is a natural quinone methide triterpenoid found in the extracts of Trypterygium wilfordii and shows anti-inflammatory, anti-
1
–4
proliferative, and anti-obesity activity. In researches of celastrol on tumor cells, celastrol has been found to induce multiple biological
5
, 6
7
8, 9
10
effects， such as Hsp90 inhibition , proteasome inhibition , NF-B pathway perturbation , topoisomerase Ⅱ inhibition , and heat
1
1, 12, 13
shock response activation
. It shows considerable potential to
become an anti-tumor chemical entity.
Celastrol has been proved a special Hsp90 inhibitor, which
shows cytotoxic activity. Inhibition of Hsp90 induces degradation
of the misfolded clients and disrupts different pathways in
1
4, 15
development of tumors
. Previous studies showed that celastrol
could disrupt the Hsp90-Cdc37 interaction and the quinone
methide moiety is essential for its cytotoxic activity against tumor
2, 6, 7, 18
cell lines.
Disruption of the Hsp90-Cdc37 interaction leads
to the degradation of the obutncogenic kinases and may bring
1
6, 17
Figure 1. Synthesis of compound 1–18.
promising anticancer effects.
Remarkable stereospecific
conjugate addition on this moiety may be the major contribution to
1
9
the protein target selectivity.
2
0-24
Some structure modifications of celastrol were reported
, but chemical modification about C-3 hydroxyl has rarely been studied
before. The exact target(s) and the C-3 structure-activity relationship (SAR) of celastrol remains undefined. Meanwhile, low water
2
5, 26
solubility and obvious toxicity hindered its application.
Because of the structure of A ring, C-3 hydroxyl forms a hydrogen bond with the neighboring carbonyl group and conjugate with the
quinone methide moiety. Modification on this position may cause subtle changes on the molecule. In this study, we elaborated a series
of derivatives in order to investigate the structure-activity relationship and improve the drug-like properties of celastrol. Meanwhile,
oral administration was tested and found improved the compliance.
—
——

Corresponding author. Zha-Jun Zhan. Tel.: +86-571-88871075; fax: +86-571-88320913; E-mail: zjnpr@zjut.edu.cn

Compounds 1–18 were readily synthesized by nucleophilic reaction between celastrol and organic halides in DMF at room
temperature, using NaHCO as basic (Figure 1). The activity of these compounds were evaluated by MTT method with several tumor
3
cell lines.
Then, compound 12, because of its better antitumor activity， was chosen to be modified in order to adjust the chemical
environment of C-3 hydroxyl which may affect the electron density of the quinone methide moiety. New pharmacophores were added
on the structure to alter the activity of these compounds and improve the solubility. Therefore, a series of carbamates (compounds 19–
2
6) of compound 12 were synthesized by one-pot reaction with BTC and chosen amine (Scheme 1). The polarity of these compounds
was enhanced, which is benefit for solubility and bioavailability. The in-vitro activity of these compounds remained as potent as
celastrol, shown in Table 2.
Table 1. Structure of celastrol analogues 1–26
1
2
1
2
Compound
R
R
Compound
R
R
1
3
H
Celastrol
H
H
H
H
14
15
16
H
H
H
1
2
CH
3
2
CH
CH
3
2
3
4
5
H
H
H
CH
2
CH
CH
3
17
18
19
H
H
3 2
CH(CH )
CH
2
CH
2
CH
2
CH
3
6
7
H
H
CH(CH
3
)CH
2
CH
3
20
21
CH
2
CH(CH3)
2
8
9
H
H
H
CH
CH
CH
2
2
2
CH
2
CH
2
2
CH
2
CH
3
2
22
23
24
CH
2
CH
CH
2
CH
CH
3
1
0
1
CH=CH
2
1
H
H
CH
2
COOCH
2
CH
3
25
26
1
2
Total 26 compounds 1–26 were tested for their cytotoxicity against several cell lines include (A549, Bel7402, SGC7901, Hela,
HepG2) by MTT assay employing celastrol as positive control. The IC50 values for the inhibition of proliferation are shown in Table 2.
The 29- ester derivatives 1–18 initially synthesized showed
comparable cytotoxic activity with celastrol. In addition, they were
easier to be synthesized and purified. These results indicated that it
was suitable for improving the cytotoxicity to introduce the alkyl
group at the carboxyl. This might be caused by better cell
permeability. And compound 12 showed slightly better activity
than the others. So, our present work focused on exploring some
modifications on the hydroxyl to improve the polarity and solubility
of the compounds and studying their effects on cytotoxicity.
Therefore, compounds 19–26 were synthesized and tested for their
cytotoxicity.
o
From Table 2, we found that most of our carbamates of celastrol
showed potent activity as well as their precursor. However, the
activity of derivatives with piperazine (compounds 23–26) were
Scheme 1. Reagents and conditions: (a) Et
3 2 2
to rt; (b) Et N, DMAP, CH Cl , 0 C to rt, 12 h.
3
N, DCM, 0 C
o
better than that with aniline (compound 19), Piperidine (compounds 20, 21) and morpholine (compound 22). The substitution of
piperazine and morpholine by piperidine and aniline weakened the activity, which might indicate that nitrogen atom and oxygen atom
here are favorable as hydrogen bond acceptor but the alkyl substituents and hydrophobic groups not. Nitrogen atom is more suitable
than oxygen atom here and the groups on it make difference to the activity. The activity get lower when the substituents get bigger.

Meanwhile, we observed a significant loss of cytotoxicity when the size of substituent group on C-3 hydroxyl become as big as
compound 25 which was about 8–10 carbons length we estimated. However, when the size was smaller than that, it did not cause
obvious change of the activity. It indicated a threshold about the size on this position, which located in front of C-3 hydroxyl. This
encouraged us a lot, because it could be a new space for us to put in some new pharmacophore and groups, which might affect the
activity and pharmacokinetic property of celastrol, and the stereo structure here may improve the selectivity. The carbamates was
introduced nitrogen and oxygen atoms as new hydrogen bond acceptors and hydrophilic center. It might also be a basis for further
research and helpful to identify the target of celastrol.
Table 2. In-vitro activity of celastrol analogues 1–26
IC50(M/L)
Compound
A549 Bel7402 SGC7901
Hela
0.84
0.32
0.83
1.38
1.75
0.79
0.36
0.41
0.56
1.84
0.31
0.61
0.46
1.05
0.87
1.32
0.41
0.42
1.12
6.36
2.82
4.27
1.55
2.31
1.59
18.01
1.35
HepG2
0.58
0.78
0.49
0.28
0.32
1.05
0.39
0.34
0.44
1.74
0.31
0.76
0.80
0.43
0.66
1.95
0.44
0.87
0.37
4.25
7.43
8.19
1.36
2.61
4.61
9.90
5.79
Celastrol
2.12
1.03
1.44
2.15
3.41
1.36
1.93
1.07
2.30
9.31
1.49
3.89
2.33
3.12
3.07
7.70
2.01
3.25
3.78
14.85
12.07
15.03
9.35
5.18
5.91
8.85
7.20
0.44
1.16
0.92
0.41
0.43
1.01
0.59
0.53
0.81
2.21
0.63
0.30
0.20
0.63
0.52
0.86
0.24
0.37
0.51
4.07
3.22
5.05
1.56
1.02
2.44
5.17
4.23
0.87
0.63
0.24
0.47
0.47
0.73
0.36
0.24
0.46
1.58
0.33
0.67
0.31
0.70
0.81
0.81
0.51
0.31
0.30
4.31
3.04
4.73
1.78
1.28
2.88
11.72
3.64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
Due to the results of in-vitro activity, compound 23, which showed potent activity and was easily prepared, was chosen to further
evaluate anti-tumor efficacy in vivo.
During preliminary experiment, high fatality rate was always observed in intraperitoneal injection of celastrol, which indicated high
toxic reaction. In case of this, intragastric administration was tried as alternative options and showed good tolerance. The reason
remained unknown and further experiment was carried on in our lab.
The anti-tumor activity of compound 23 in vivo was evaluated at doses of 3, 6, and 12 mg/kg in nude mice bearing A549 xenografts
as the animal model in comparison with celastrol. Compound 23 demonstrated a dose-dependent inhibition of A549 tumor and good
tumor inhibitory activity with 70.96% inhibition at a dose of 12 mg/kg, which showed significantly better effect than celastrol at a dose
of 12 mg/kg (P<0.05). Meanwhile, slight weight loss (26.5g to 25.5g in average) was observed in the celastrol group but weight gain
(25.8g to 26.9g in average) was observed in compound 23 group, which may indicate the improvement in safety. The detailed
experimental data are shown in Table 3.
In conclusion, a series of celastrol derivatives 1–26 including novel C-3 derivatives were synthesized and evaluated for their growth
inhibition activities against several tumor cell lines. The SAR information collected so far suggested that the type and size of the
carbamate substituents at the C-3 hydroxyl were important factors for cytotoxic activity in-vitro. Compounds maintained potent
cytotoxic activities in five tumor cell lines. C-3 hydroxyl could be a potential position to introduce stereo selective groups to improve

the selectivity. The in vivo antitumor studies of compound 23 showed that it might be promising for the development of new antitumor
agents and suitable for oral administration that showed better compliance.
Table 3. In vivo anti-tumor efficacy of compound 23 against non-small cell lung cancer A549 xenografts
3
Body weight
Tumor volume (mm )
X ± SD
Dose
Mice (n)
Relative TV
(X ± SD)
Group
(g)
Weight (mg)
Inhibition(%)
(
mg/kg)
Initial/end
20/20
Initial/end
25.1/25.5
26.8/26.5
25.9/27.0
25.8/26.9
26.5/25.5
Initial
End
control
120.46 ± 58.41
128.31 ± 41.84
123.58 ± 51.75
112.19 ± 40.97
118.33 ± 50.74
175.19 ± 107.17
99.81 ± 57.98
81.43 ± 76.56
57.15 ± 47.08
94.28 ± 71.63
1.57 ± 0.85
0.76 ± 0.32
0.63 ± 0.38
0.55 ± 0.48
0.84 ± 0.54
172.54 ± 31.34
*
2
2
2
3
3
3
3
6
15/15
92.94 ± 11.64
46.13 ± 6.75
57.46 ± 12.47
*
15/15
73.40 ± 21.51
*
**
12
12
15/15
50.11 ± 13.73
70.96 ± 7.96
*
Celastrol
15/15
65.60 ± 16.33
61.98 ± 9.46
a
*
*
The in vivo experiment was carried out in the nude mice bearing A549 xenografts and the compounds were intragastricly given.
P < 0.01 versus control group.
*
P < 0.05 versus celastrol group
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Synthesis of 3- and 29-substituted celastrol
derivatives and structure-activity
relationship studies of their cytotoxic
activities
Leave this area blank for abstract info.
a
a
a
a
b
b
a
Wei-Guang Shan , Han-Guang Wang , Yan Chen , Rui Wu , Yan-Tao Wen , Li-Wen Zhang , You-Min Ying ,
a
a,
Jian-Wei Wang , and Zha-Jun Zhan 
a
College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou, 310014, P. R. China
The Children’s Hospital, Zhejiang University School of Medicine, Hangzhou, 310003, P. R. China
b