Design, synthesis and biological evaluation of C(6)-modified celastrol derivatives as potential antitumor agents

Article abstract of DOI:10.3390/molecules190710177

New six C6-celastrol derivatives were designed, synthesized, and evaluated for their in vitro cytotoxic activities against nine human cancer cell lines (BGC-823, H4, Bel7402, H522, Colo 205, HepG2 and MDA-MB-468). The results showed that most of the compo

Full text of DOI:10.3390/molecules190710177

Molecules 2014, 19, 10177-10188; doi:10.3390/molecules190710177
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Design, Synthesis and Biological Evaluation of C(6)-Modified
Celastrol Derivatives as Potential Antitumor Agents
Kaiyong Tang 1,2, Qingqing Huang 1, Jafeng Zeng 2, Guangming Wu 2, Jinwen Huang 2,
Junfang Pan 2,*and Wei Lu 1,*
1
Institute of Drug Discovery and Development, Shanghai Engineering Research Center of Molecular
Therapeutics and New Drug Development, East China Normal University, Shanghai 200062,
China; E-Mails: tangkaiyong@huatuodrug.com (K.T.); qqhuang@sat.ecnu.edu.cn (Q.H.)
2
Shanghai Hotmed Sciences Co., Ltd., Shanghai 201201, China;
E-Mails: zengjiafeng@huatuodrug.com (J.Z.); wuguangming@huatuodrug.com (G.W.);
huangjinwen@hotmail.com (J.H.)
* Authors to whom correspondence should be addressed: E-Mails: jfpan@huatuodrug.com (J.P.);
wlu@chem.ecnu.edu.cn (W.L.); Tel.: +86-21-5080-2099 (J.P.); Fax: +86-21-5080-2101 (J.P.).
Received: 7 May 2014; in revised form: 2 July 2014 / Accepted: 4 July 2014 /
Published: 14 July 2014
Abstract: New six C6-celastrol derivatives were designed, synthesized, and evaluated for
their in vitro cytotoxic activities against nine human cancer cell lines (BGC-823, H4,
Bel7402, H522, Colo 205, HepG2 and MDA-MB-468). The results showed that most of
the compounds displayed potent inhibition against BGC823, H4, and Bel7402, with IC50s
of 1.84–0.39 μM. The best compound NST001A was tested in an in vivo antitumor assay
on nude mice bearing Colo 205 xenografts, and showed significant inhibition of tumor
growth at low concentrations. Therefore, celastrol C-6 derivatives are potential drug
candidates for treating cancer.
Keywords: celastrol C-6 derivatives; antitumor activity; in vitro; in vivo
1. Introduction
Celastrol (tripterine, 1, Figure 1), a quinone methide triterpene, was extracted from the Traditional
Chinese Medicine “Thunder of God Vine” (Tripterygium wilfordii Hook F.). Recently, abundant
research has indicated that celastrol is involved with several novel molecular targets, such as Hsp90-Cdc37,

Molecules 2014, 19
10178
NF-κB/IKKβ, VEGFR1, and VEGFR2 [1,2], which suggest its development as an antitumor,
antifungal, antioxidant and anti-inflammatory agent. Celastrol is also known to inhibit the proliferation
of various tumor cells, including those of gliomas [3], hepatocellular carcinomas [4] and prostate
cancer [5]. In addition, it potently inhibited the proteasome and induced apoptosis [5–7], suggesting it
might serve as a potential cancer treatment.
Although celastrol is very effective toward many molecular targets, it has some deficiencies, such
as poor stability, poor water solubility and high toxicity, which hinder its further application and thus
has attracted intensive interest from medicinal scientists. Some structure modifications were reported
in order to improve solubility or reduce toxicity, a simple strategy being the esterification or
amidification of 20-carboxylic acid of celastrol, such as seen in compounds 2–5 (Figure 1), that lead to
decreased potency [8–10]. Some analogues with modified A/B rings at the C-2, 3 positions (6–10,
Figure 1) were reported as the inducers of heat shock response [8–10]. However, the mechanism of
action underlying the anti-inflammatory and antiproliferative effects of celastrol and most analogues is
not yet fully understood.
Figure 1. Structures of celastrol and its analogues.
O
H
O
R
O
XR
29
20
O
H
O
R
19
18
21
22
E
12
C
17
11
9
13
H
O
O
1
A
14
O
O
R'
R'
O
2
10
5
D
16
R'
R'
8
15
B
7
3
HO
4
6
Celastrol (1) XR = OH
pristimerin (2) XR = OCH3
3 XR = OBn
4 XR = Obutyl
5 XR = NR'R''
6 R = R' = H
pristomerol (7) R = CH3, R' = H
8 R = H, R' = acetyl
10 R = acetyl, R' = ethyl
9 R = CH3, R' = acetyl
Our company, Shanghai Hotmed Sciences Co. Ltd., is one of the biggest suppliers of celastrol in
China, In a previous study [11], we patented some water soluble analogues with improved potency
modified at the C-6 position of celastrol. NST001, a C(6)-sulfonated analogues derived from the
reaction of celastrol and bisulfate sodium (Scheme 1), was selected for animal test evaluation, in which
it displayed good activity on PC-3 tumor-bearing nude mice (i.p. 3 mg/kg, dl/3.5 w) and the toxicity
was decreased by compared with celastrol [11]. These promising results suggested the C-6 position of
celastrol as a potentially useful modification site. In order to explore more the chemical space and
obtain potential drug candidates with improved pharmacologic profiles and low toxicity, six novel
C(6)-modified celastrol analogues were prepared and are reported herein.
2. Results and Discussion
2.1. Synthesis of Celastrol C-6 Analogues (Scheme 1)
NST001 was a white solid recrystallized from ethanol/water binary solution. Acetylation of the two
hydroxyl groups of NST001 affords NST001A. NST6A-A was synthesized from the reaction of

Molecules 2014, 19
10179
celastrol and acetone catalyzed by 1 N HCl, followed by acetylation. NST6A (B–D) were obtained
from the reaction of celastrol and mercaptans (or thiophenols), followed by acetylation. All compounds
1
13
were identified by H-NMR, C-NMR, IR, LC-ESI-MS, [α], and the HPLC purity of all compounds
was over 98.0%.
Scheme 1. Synthetic scheme for the designed compounds.
Reagents and conditions: (i) For NST001: NaHSO3, MeOH, RT, 1.5 h, N2; For NST6A: acetone,
1 N HCl (cat.), RT, N2; For NST6A~D: RSH, MeOH, RT, N2; (ii) Ac2O, Py, RT, N2; or
(CH3CH2CO)2O, Py, RT, N2.
2.2. Activity of Celastrol Analogues against Human Cancer Cell Lines
To evaluated the antiproliferative effects of celastrol analogues on human cancer cells, all
compounds were screened against a variety of human cancer cell lines including the human gastric
cancer cell line BGC-823, and the human liver cancer cell lines H4 and Bel7402. As shown in Table 1,
compounds bearing a C–S bond showed similar or better inhibition than celastrol (1), however, the
compound bearing a C–C bond at the C-6 position (NST6A-A) completely lost its activity. To
investigate the inhibition on more sensitive human colon cancer cell line Colo 205, another
cytotoxicity assay was conducted at concentrations of 0.1, 10 μg/mL, respectively. As shown in Figure 2,
the compound NST6A-A displayed decreased cytotoxicity at a concentration of 10 μg/mL. The
cytotoxicity of other analogues with C(6)-sulfided celastrol showed good dose-response relationships.
Among them, sodium bisulfate modifications (NST001, NST001A, NST001B) showed prospective
effects against Colo 205 growth.
Table 1. IC50 values (μM) on human cancer cell lines.
IC50 (μM)
Compounds
BGC823
3.73
H4
2.09
0.91
1.37
1.12
>150
0.37
0.35
0.46
Bel7402
1.55
Celastrol (1)
NST001
0.77
1.17
NST001A
NST001B
NST6A-A
NST6A-B
NST6A-C
NST6A-D
0.49
1.73
0.74
2.39
>150
0.47
>150
0.45
0.42
0.46
0.50
0.54

Molecules 2014, 19
10180
As NST001 and NST001A showed the most potent inhibition at both concentrations, these two
compounds were selected to identify precise IC50 values on the human non-small cell lung cell line
H522, human colon cancer cell line Colo 205, human hepatocellular liver carcinoma cell line HepG2,
human breast adenocarcinoma cell line MDA-MB-468 and human gastric cancer cell line BGC823,
respectively. The results are summarized in Table 2. NST001A was more potent than NST001 in
anti-tumor profile except for the BGC823 cell line. Interestingly, Colo 205 cells are more sensitive to
NST001A than the other four cancer cell types, and that compound shows good selectivity to Colo 205
cells with potency increased about 8- times compared to the parent compound NST001.
Figure 2. The inhibitory rate (at concentrations of 0.1, 10 μg/mL for 72 h) on Colo 205
growth. The results are expressed as the percentage of that of the treat cells.
100
80
60
40
20
0
0.1μg/mL
10μg/mL
Table 2. IC50 values (μM) of NST001 and NST001A on human cancer cell lines.
IC50 (μM) for 72 h
Compounds
H522 Colo 205 HepG2 MDA-MB-468 BGC823
NST001A
NST001
0.39
0.47
0.06
0.51
0.29
0.68
0.33
0.89
1.53
0.77
2.3. In Vivo Anti-Tumor Activity Evaluation
As shown above, NST001A was more potent than the other C6-derivatives and the most active
compound on Colo 205 cells. Thus, female nude mice loaded Colo 205 xenografts were used for
in vivo anti-tumor activity evaluation of the most potent compound NST001A. The body weights of
the nude mice were monitored and their tumor sizes were measured and recorded. The rate of
inhibition of the different groups against Colo 205 solid tumors in female nude mice are shown in
Figure 3. After treatment, there were no apparent body weight loss during the treatment in the negative
control group injected with saline solution (Model), and the negative control group induced an average
tumor weight of 0.99 ± 0.25 g, the T/C (%) value treated with cisplatin (4 mg/kg, i.p., qd × 20) was
49.32% with about 6 g body weight lost. The NST001A-treated group showed similar activity as
cisplatin, the T/C (%) value of NST001A ranged from 54.00% to 77.25% without obvious body weight
loss. These results proved that NST001A was a potential antitumor drug candidate with lower toxicity
compared with cisplatin.

Molecules 2014, 19
Figure 3. Effects of NST001A on the growth of Colo 205 xenografts in nude mice.
10181
Colo 205
Colo 205
**
**
**
***
Colo 205
Colo 205
NST001A
All date are means ± SD: * p < 0.05, ** p < 0.01, *** p < 0.001 when compared with model.
Tumor size and body weight were measured and clinical symptoms were observed at day 0, 5, 8, 13,
16, 20. The tumor volume (TV) was calculated according to the formula: TV = (1/2 × length × width2),
and the relative tumor volume (RTV) was calculated according to the formula: RTV = TVt/TV0, where
TVt is the tumor volume at day t, and TV0 is the tumor volume at the first treatment. The relative
growth rate of the tumor T/C (%) calculated as follows:
[0066] T/C (%) = RTVt/RTVc × 100%T/C (%) = (TRTV/CRTV) × 100%
where the TRTV is the treatment group relative tumor volume; CRTV is the control group relative tumor
volume. Data are expressed as means ± SD. Statistical evaluations were analyzed with one-way
ANOVA (t-test). p < 0.05 was considered statistically significant.
2.4. Preliminary Non-Clinical Safety Evaluation
The in vivo anti-cancer evaluation showed that NST001A was a potential antitumor compound, and
safer than cisplatin (4 mg/kg). Thus, NST001A along with NST001 were evaluated for preliminary
non-clinical safety evaluation on female nude mice through injection in the tail vein for seven days
(i.v., qd × 7). The results are shown in Figure 4. After loading for 7 days, the body weight of the
control group ranged from 17.0 g up to 19.0 g, and all the test groups lost a certain weight. During the
experiment, the injection site of NST001 group showed successive swelling and fracture, but this
phenomenon was not found in the NST001A groups. Polypnea was found in one mouse in the test with
NST001A (40 mg/kg) and disappeared after drug withdrawal. After delivery for 7 days, all mice were

Molecules 2014, 19
10182
put to death, and no obvious tissue lesions were found in any of the groups. These results suggested
that NST001A was safer than NST001.
Figure 4. Tumor growth after systemic application of different drug loaded systems.
Model
Cisplatin(4mg/kg)
**
NST001A(6mg/mk)
**
NST001A(12mg/mk)
NST001A(24mg/mk)
**
All date are means ± SD: ** p < 0.01, when compared with model.
3. Experimental Section
3.1. General Information
Celastrol was extracted from the Traditional Chinese Medicine (Tripterygium wilfordii Hook F.) by
ourselves. Silica gel FCP200-300 mesh was used for column chromatography. All chemicals were
1
purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). H-NMR and 13C-NMR
spectra were collected in CDCl3 and DMSO-d6 at 25 °C on a Bruker AV-400 spectrometer. All
chemical shifts are reported in the standard δ notation of parts per million using the peak of residual
proton signals of CDCl3 or DMSO-d6 as an internal reference. Electrospray ionization (ESI) analyses
were performed by using a Thermo LTQ (LC-ESI-MS/MS). Melting point analyses were performed by
using a digital micro-melting point apparatus. All compounds were confirmed by 1H-NMR, 13C-NMR,
IR, LC-ESI-MS, [α], and the HPLC purity of all compounds was over 98.0%. All the in vitro and
in vivo tests were carried out in accordance with guidelines evaluated and approved by the Shanghai
Institute of Materia Medica Chinese Academy of Sciences (SIMM). All animal experiments were
performed under specific pathogen-free conditions in accordance with institutional guidelines.
3.2. Synthesis
Sodium
(6bS,8aS,11R,12aR,12bS,14aS)-2,3-diacetoxy-11-carboxy-4,6b,8a,11,12b,14a-hexamethyl-
5,6b,7,8,8a,9,10,11,12,12a,12b,13,14,14a-tetradecahydropicene-5-sulfonate (NST001). To a stirred
solution of celastrol (100 mg, 0.22 mmol) in methanol (5 mL) was added a solution of sodium
bisulfate (27 mg, 0.26 mmol in 2 mL water) under nitrogen. After 1.5 h, the reaction solution
concentrated and dried under vacuum at 40 °C, recrystallization from ethanol/water afforded the white
compound NST001 (103 mg, 84% yield). HPLC purity > 98.0% (mobile phase 78:22 (v/v) mixture of

Molecules 2014, 19
10183
1
methanol and 0.005% H3PO4). H-NMR (DMSO-d6): δ 12.01 (s, 1H), 8.81 (s, 1H), 7.62 (s, 1H), 6.58
(s, 3H), 5.81 (d, J = 6.2 Hz, 1H), 4.49 (d, J = 6.2 Hz, 1H), 2.21 (s, 3H), 1.62 (s, 3H), 1.18 (s, 3H), 1.09
(s, 3H), 1.05 (s, 3H), 0.59 (s, 3H).
Sodium
(6bS,8aS,11R,12aR,12bS,14aS)-2,3-diacetoxy-11-carboxy-4,6b,8a,11,12b,14a-hexamethyl-
5,6b,7,8,8a,9,10,11,12,12a,12b,13,14,14a-tetradecahydropicene-5-sulfonate (NST001A). To
a
solution of NST001 (100 mg, 0.18 mmol) in pyridine (3 mL) was added acetic anhydride (0.1 mL) at
room temperature under nitrogen. After stirring for 3 h, the mixture was quenched with ethanol (5 mL).
The solvent was removed under reduced pressure, the residue was purified by gel filtration
chromatography (anhydrous ethanol) to afford the title compound as a white solid (80 mg, 69% yield).
HPLC purity > 98.0% (mobile phase 60:40 (v/v) mixture of acetonitrile and 0.005% H3PO4).
Mp > 160 °C. α2D0 −94.76° (MeOH, c = 0.2). 1H-NMR (DMSO-d6): δ 7.01 (s, 1H), 5.83 (d, J = 6.1 Hz,
1H), 4.60 (d, J = 6.0 Hz, 1H), 2.50 (s, 1H), 2.29 (s, 2H), 2.24 (d, J = 6.7 Hz, 5H), 2.08 (s, 1H), 1.99 (dd,
J = 23.8, 12.5 Hz, 3H), 1.83 (dd, J = 21.7, 15.3 Hz, 1H), 1.67 (s, 2H), 1.63–1.36 (m, 8H), 1.28 (s, 1H),
1.20 (s, 3H), 1.09 (s, 3H), 1.05 (s, 2H), 0.85 (d, J = 13.8 Hz, 1H), 0.61 (s, 2H). 13C-NMR (DMSO-d6):
δ 179.9, 168.9, 168.7, 149.0, 148.6, 141.3, 138.5, 130.5, 129.8, 118.5, 117.3, 60.1, 56.5, 44.3, 44.1,
38.8, 37.8, 36.9, 36.6, 35.1, 34.4, 32.9, 31.9, 30.6, 30.5, 29.9, 29.1, 21.6, 20.9, 20.6, 19.0, 18.6, 13.8.
IR (KBr) 3447.9, 2944.5, 2873.1, 1768.8, 1466.0, 1374.3, 1228.7, 1194.0, 1158.3, 1039.7 cm−1.
LC-ESI-MS (−) m/z (%) = 615.41 [M−1Na+]− (100%), 1231.86 [2M−2Na+]2− (10%).
Sodium
(6bS,8aS,11R,12aR,12bS,14aS)-11-carboxy-4,6b,8a,11,12b,14a-hexamethyl-2,3-bis-
(propionyloxy)-5,6b,7,8,8a,9,10,11,12,12a,12b,13,14,14a-tetradecahydropicene-5-sulfonate (NST001B).
To a solution of NST001 (100 mg, 0.18 mmol) in pyridine (3 mL) was added propionic anhydride
(0.12 mL) at room temperature under nitrogen. After stirred for 3 h, the mixture was quenched with
ethanol (5 mL). The solvent was removed under reduced pressure, the residue was purified by gel
filtration chromatography (anhydrous ethanol) to afford the title compound as a white solid (86 mg,
71.6% yield). HPLC purity > 98.0% (mobile phase 55:45 (v/v) mixture of acetonitrile and 0.005% H3PO4).
mp > 150 °C. α2D0 −98.21° (MeOH, c = 0.2). 1H-NMR (DMSO-d6): δ 7.01 (s, 1H), 5.83 (d, J = 6.2 Hz,
1H), 4.60 (d, J = 6.1 Hz, 1H), 2.66–2.45 (m, 3H), 2.36 (d, J = 14.7 Hz, 1H), 2.24 (s, 1H), 2.10 (d,
J = 12.8 Hz, 1H), 2.06–1.93 (m, 1H), 1.82 (d, J = 5.7 Hz, 1H), 1.68 (s, 2H), 1.40 (d, J = 15.0 Hz, 4H),
1.22 (d, J = 14.1 Hz, 2H), 1.17–1.07 (m, 4H), 1.05 (s, 3H), 0.83 (d, J = 11.8 Hz, 1H), 0.64 (s, 2H).
13C-NMR (DMSO-d6): δ 180.6, 172.1, 171.9, 149.0, 148.7, 141.3, 138.3, 130.4, 129.7, 118.3, 117.2,
60.0, 56.3, 44.3, 44.0, 38.7, 37.8, 37.0, 36.7, 35.2, 34.5, 33.2, 32.6, 31.9, 30.6, 30.4, 30.2, 30.0, 29.1,
27.1, 26.9, 21.6, 18.5, 13.8, 9.6, 9.4. IR (KBr): 2969.5, 2944.6, 2873.1, 1762.0, 1559.5, 1457.3, 1155.4,
1040.6 cm−1. LC-ESI-MS (−) m/z (%) = 643.45 [M−1Na+]− (100%), 1288.09 [2M−2Na+]2− (5%).
(2R,4aS,6aS,12bS,14aS,14bR)-10,11-diacetoxy-2,4a,6a,9,12b,14a-hexamethyl-8-(2-oxopropyl)-1,2,3,
4,4a,5,6,6a,8,12b,13,14,14a,14b-tetradecahydropicene-2-carboxylic acid (NST6A-A). To a solution
of celastrol (130 mg, 0.29 mmol) in acetone (10 mL) was added 1 drop of 1 N HCl. The mixture was
stirred overnight under nitrogen, then the solution concentrated under vacuum. The residue was
redissolved in pyridine (0.4 mL), and acetic anhydride (2 mL) were added. The mixture was stirred
overnight under nitrogen. The reaction was quenched with 3 mL ice water, then the mixture was

Molecules 2014, 19
10184
extracted with ethyl acetate, concentrated under vacuum. The residue was purified by silica gel
(petroleum ether/acetone) as a pale solid (123 mg, 72% yield). HPLC purity > 98.0% (mobile phase
87:13 (v/v) mixture of acetonitrile and 0.005% H3PO4); mp 146.3–147.5 °C; α2D0 −92.16° (MeOH,
1
c = 0.2); H-NMR (CDCl3): δ 7.00 (s, 1H), 5.85 (d, J = 6.0 Hz, 1H), 4.25–3.86 (m, 1H), 2.36 (t,
J = 11.1 Hz, 1H), 2.32 (s, 3H), 2.29 (s, 3H), 2.15 (s, 3H), 2.09 (m, 4H), 1.88–1.57 (m, 5H), 1.53 (d,
J = 4.3 Hz, 1H), 1.50 (s, 3H), 1.42 (dd, J = 16.3, 5.1 Hz, 3H), 1.27 (s, 3H), 1.20 (s, 3H), 1.10 (s, 3H),
1.06 (s, 3H), 0.93–0.83 (m, 4H), 0.66 (s, 3H); 13C-NMR (CDCl3): δ 207.2, 184.1, 168.5, 168.2, 149.7,
148.2, 140.8, 138.7, 134.1, 127.4, 121.3, 117.3, 53.7, 51.4, 44.2, 43.8, 40.1, 37.6, 36.6, 35.4, 34.5, 33.0,
32.6, 31.5, 30.8, 30.4, 30.1, 29.4, 28.8, 22.6, 22.4, 20.7, 20.4, 18.6, 12.1; IR (KBr) 2941.6, 2872.1,
1768.8, 1465.0, 1369.5, 1213.3, 1188.2 cm−1; LC-ESI-MS (−) m/z (%) = 591.3 [M−1H+]− (5%),
1183.3 [2M−1H+]− (100%), 1775.8 [3M−1H+]− (60%).
(2R,4aS,6aS,12bS,14aS,14bR)-10,11-diacetoxy-2,4a,6a,9,12b,14a-hexamethyl-8-(p-tolylthio)-1,2,3,4,4a,
5,6,6a,8,12b,13,14,14a,14b-tetradecahydropicene-2-carboxylic acid (NST6A-B). To a solution of
celastrol (100 mg, 0.22 mmol) in methanol (3 mL), p-thiocresol (40 mg, 0.32 mmol) was added, the
reation was stirred for 1 h at room temperature under nitrogen, then the reaction mixture was
concentrated under reduced pressure, and the residue was redissolved in pyridine (0.5 mL) and acetic
anhydride (4 mL) was added, the reaction was stirred overnight under nitrogen. Then quenched by ice
water, extracted with ethyl acetate, concentrated under vacuum and purified by chromatography on
silica gel (petroleum ether/acetone) as a white solid (97 mg, 67% yield). HPLC purity > 98.0% (mobile
phase 80:20 (v/v) mixture of acetonitrile and 0.005% H3PO4). mp 109.4–110.3 °C. α2D0 −69.82°
(MeOH, c = 0.2). 1H-NMR (CDCl3) δ 7.34 (d, J = 7.9 Hz, 1H), 7.11 (d, J = 7.8 Hz, 1H), 7.01 (s, 1H),
5.70 (d, J = 6.1 Hz, 1H), 4.75 (d, J = 6.0 Hz, 1H), 2.34 (s, 2H), 2.32 (s, 1H), 2.30 (s, 1H), 2.28 (s, 1H),
1.47 (s, 1H), 1.32 (d, J = 11.9 Hz, 3H), 1.25 (s, 3H), 1.22 (s, 1H), 1.09 (s, 2H), 1.04 (s, 1H), 0.96 (t,
13
J = 7.4 Hz, 1H), 0.87 (d, J = 7.1 Hz, 1H), 0.60 (s, 3H); C-NMR (DMSO-d6): δ 179.8, 168.9, 168.7,
152.0, 149.7, 142.1, 139.1, 138.0, 133.7, 130.9, 130.2, 130.1, 129.6, 119.9, 117.8, 44.8, 44.1, 44.1,
38.2, 37.6, 36.7, 35.0, 34.8, 34.1, 32.7, 31.8, 30.6, 30.4, 29.9, 29.8, 29.5, 28.8, 22.3, 21.2, 20.9, 20.5,
18.6, 12.3; IR (KBr) 2924.2, 2870.2, 1772.7, 1696.5, 1490.1, 1369.5, 1210.4, 1188.2 cm−1. LC-ESI-MS (−)
m/z (%) = 657.1 [M−1H+]− (6%), 1315.2 [2M−1H+]− (100%), 1927.8 [3M−1H+]− (65%).
(2R,4aS,6aS,12bS,14aS,14bR)-10,11-diacetoxy-8-((4-aminophenyl)thio)-2,4a,6a,9,12b,14a-hexamethyl-
1,2,3,4,4a,5,6,6a,8,12b,13,14,14a,14b-tetradecahydropicene-2-carboxylic acid (NST6A-C).
A
solution of celastrol (100 mg, 0.22 mmol) and 4-acetamidothiophenol (50 mg, 0.30 mmol) in methanol
(3 mL) was stirred for 1hat room temperature under nitrogen, the solvent was removed under reduced
pressure. The residue was redissolved in acetic anhydride (4 mL) and pyridine (0.5 mL) was added and
stirred overnight under nitrogen. The reaction solution was quenched by ice water, extracted with ethyl
acetate, concentrated under vacuum and purified by chromatography on silica gel (petroleum
ether/acetone) as a white solid (84 mg, 54% yield). HPLC purity > 98.0% (mobile phase 80:20 (v/v)
mixture of acetonitrile and 0.005% H3PO4); mp 157–160 °C; α2D0 −66.23° (MeOH, c = 0.2).
1H-NMR (CDCl3) δ 7.76 (d, J = 22.2 Hz, 1H), 7.37–7.21 (m, 4H), 7.01 (s, 1H), 5.59 (d, J = 6.1 Hz,
1H), 4.51 (d, J = 6.0 Hz, 1H), 3.47 (d, J = 2.9 Hz, 1H), 2.39 (s, 1H), 2.34 (d, J = 7.0 Hz, 7H), 2.30 (s,
3H), 2.12 (t, J = 7.4 Hz, 4H), 2.02 (d, J = 13.1 Hz, 2H), 1.88–1.54 (m, 6H), 1.50 (s, 1H), 1.47 (s, 3H),

Molecules 2014, 19
10185
1.43–1.29 (m, 4H), 1.25 (s, 2H), 1.21 (s, 3H), 1.14 (s, 3H), 1.05 (s, 3H), 0.94–0.82 (m, 1H), 0.53 (s, 3H);
13C-NMR (DMSO-d6): δ 180.6, 179.8, 168.9, 168.7, 151.9, 149.7, 142.0, 139.9, 139.1, 135.0, 130.1,
129.6, 127.4, 119.9, 119.7, 117.8, 60.0, 45.2, 44.1, 44.1, 38.2, 37.7, 36.7, 35.0, 34.8, 34.1, 32.7, 31.8,
30.6, 30.4, 30.0, 29.8, 28.8, 24.5, 22.3, 20.9, 20.5, 18.6, 12.4; IR (KBr) 2940.6, 2871.2, 1775.5, 1700.2,
1590.3, 1524.8, 1369.5, 1360.8, 1213.3 cm−1. LC-ESI-MS (−) m/z (%) = 700.9 [M−1H+]− (7%), 746.0
[M+2Na+−1H+]+ (55%), 1402.1 [2M−1H+]− (15%), 1447.5 [2M+2Na+−1H+]+ (100%).
(2R,4aS,6aS,12bS,14aS,14bR)-8-((2-acetamido-2-carboxyethyl)thio)-10,11-diacetoxy-2,4a,6a,9,12b,14a-
hexamethyl-1,2,3,4,4a,5,6,6a,8,12b,13,14,14a,14b-tetradecahydropicene-2-carboxylic acid (NST6A-D). A
solution of celastrol (200 mg, 0.44 mmol) and acetylcysteine (91 mg, 0.56 mmol) methanol (5 mL),
was stirred at room temperature under nitrogen. After 1 h, the solvent was removed under reduced
pressure, and the residue was redissolved in acetic anhydride (0.15 mL) and pyridine (3 mL) was
added, the reaction was stirred for 1 h under nitrogen, then quenched by ice water, extracted with ethyl
acetate, concentrated under vacuum and purified by chromatography on silica gel (ethyl acetate) as a
pale solid (81 mg, 26% yield). HPLC purity > 98.0% ( mobile phase 70:30 (v/v) mixture of acetonitrile
and 0.005% H3PO4); mp 187–189 °C; α2D0 −111.01° (MeOH, c = 0.2); 1H-NMR (DMSO-d6) δ 8.14–7.78
(m, 1H), 7.09 (s, 1H), 6.09 (dd, J = 24.2, 5.7 Hz, 1H), 4.71 (d, J = 5.4 Hz, 1H), 4.38 (s, 1H), 4.27 (s,
1H), 3.56–3.53 (m, 1H), 3.44 (dd, J = 13.8, 6.9 Hz, 1H), 3.24 (d, J = 9.5 Hz, 1H), 3.06 (d, J = 5.5 Hz,
1H), 2.76 (dd, J = 13.3, 8.2 Hz, 1H), 2.50 (s, 1H), 2.33 (d, J = 16.1 Hz, 2H), 2.24 (s, 1H), 2.19 (d,
J = 9.8 Hz, 1H), 2.16–1.92 (m, 2H), 1.86 (s, 1H), 1.83 (s, 1H), 1.63 (d, J = 9.2 Hz, 2H), 1.55–1.36 (m, 3H),
1.25 (d, J = 24.4 Hz, 3H), 1.10 (s, 2H), 1.05 (d, J = 9.1 Hz, 2H), 0.87 (d, J = 11.2 Hz, 1H), 0.64 (s, 2H);
13C-NMR (DMSO-d6): δ 179.9, 172.8, 169.2, 169.0, 168.9, 168.7, 152.0, 151.6, 149.5, 149.4, 141.8,
141.7, 138.9, 131.4, 131.2, 129.4, 129.4, 120.3, 119.8, 117.5, 117.4, 56.4, 54.6, 53.8, 45.7, 44.3, 44.2,
38.1, 38.1, 37.6, 36.8, 35.0, 34.8, 34.2, 34.2, 34.0, 33.9, 32.8, 31.8, 31.7, 30.6, 30.4, 29.9, 29.9,
28.6, 23.2, 23.2, 22.6, 22.5, 22.4, 22.3, 21.7, 20.9, 20.5, 19.0, 18.8, 12.3, 12.1; IR (KBr) 2939.6,
2871.2, 1771.69, 1374.3, 1213.3 cm−1; LC-ESI-MS (−) m/z (%) = 696.4 [M−1H+]− (8%), 1393.5
[2M−1H+]− (100%).
3.3. Activity of Celastrol Analogues against Human Cancer Cell Lines
In vitro culture, BGC-823, H4, Bel7402, H522, Colo 205, HepG2, MDA-MB-468 cells grown to
logarithmic growth phase were collected, and centrifuged at 1,000 rpm for 5 min. The cells were
seeded at a density of 3.5 × 104/mL in 96-well plate (100 μL/well), incubator (37 °C, 5% CO2) for 24 h,
the test drugs at serial dilutions were added, the negative control group was added to a final
concentration of 0.5% DMSO, After the incubator for another 72 h, added to each well of 5 mg/mL MTT
(20 μL/well) and cultured for 4 h, then DMSO was added in (150 μL/well), shaking for 5 min and the
absorbance were measured at 492 nm and 620 nm. The growth inhibition rate was calculated by the
software GraphPad Prism.
3.4. Anti-Tumor Activity Evaluation and Preliminary Non-Clinical Safety Evaluation in Vivo
BALB/c nude mice (16–17 g), female, 4–6 weeks, supplied by Shanghai SLAC Laboratory Animal
Co. Ltd. (Shanghai, China, License No:SCXK (Hu)2007-0005). Cisplatin for injection were supplied

Molecules 2014, 19
10186
by Qilu-pharma (Jinan, China), Normal saline were supplied by Jiangsu Shihuan Bioengineering Co.,
Ltd (Wuxi, China), human colon cancer cell Colo 205 was purchased from ATCC.
For the in vivo anti-tumor activity evaluation, the exuberant growth of human colon cancer Colo
205 tumor tissue was cut into 1.5 mm3 tumor fragments, and inoculated into nude mice subcutaneously
under aseptic conditions. When tumors reached an average volume of 100–150 mm3, the nude mice
were randomly divided into five groups according to the experimental plan (Table 3).
Table 3. The experimental plan of tumor bearing nude mice treated with NST001A.
Dosage
N.O. of
Route of
Groups
Defined Daily Dose
Mice
Administration
(mg/kg) (mL/10 g)
Model
12
6
0.2 mL
4
i.p.
i.p.
i.p.
i.p.
i.p.
qd × 20
3 times/week
qd × 20
Cisplatin (4 mg/kg)
NST001A (6 mg/kg)
NST001A (12 mg/kg)
NST001A (24 mg/kg)
6
6
0.1
6
12
24
qd × 20
6
qd × 20
Female nude mice were used for preliminary non-clinical safety evaluation of NST001A. The
compound was soluble in normal saline for injecting in the tail vein for seven days (i.v., qd × 7). The
results are shown in Table 4 and Figure 5.
Table 4. The change of nude mice bodyweight of different drug loaded systems.
N.O. of Mice
Body Weight (g)
Group
Initial
Final
Initial
Final
control
3
3
3
3
3
3
3
3
17.0 ± 0.00
17.33 ± 0.58
17.33 ± 0.58
17.0 ± 0.00
19.00 ± 0.00
NST001 (20 mg/kg)
NST001A (20 mg/kg)
NST001A (40 mg/kg)
16.33 ± 1.15 *
17.33 ± 0.58 **
15.67 ± 0.58 ***
* p < 0.05, ** p < 0.01, *** p < 0.001 when compared with model.
Figure 5. Effects of different drug loaded system on the body weight of nude mice.
**
*
***
All data are means ± SD: * p < 0.05, ** p < 0.01, *** p < 0.001 when compared with control.

Molecules 2014, 19
10187
4. Conclusions
In summary, we have synthesized three kinds of C(6)-modified celastrol derivatives and their
anticancer activity were assayed at the cell level. Comparing with the parent compound celastrol, the
in vitro cytotoxicity potency of C(6)-sulfonted and C(6)-sulfided celastrol derivatives increased about
5–8 times against the human cancer cell BGC823, H4, Bel7401, however, for the C(6)–carbonated
celastol derivatives, the potency dropped dramatically. Moreover, 2,3-acetylation was more effective
than propionylation. The best compound, NST001A, which shows good selectivity towards Colo 205
cell with a 0.06 μM IC50 value, was selected for in vivo evaluation in nude mice bearing xenografts of
human colon cancer. The results indicated that NST001A can potently inhibit the growth of Colo 205
xenografts in nude mice with a better safety profile compared with NST001.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/19/7/10177/s1.
Acknowledgments
This study was supported by the National Science and Technology Major Project (2011ZX11401-026)
in China.
Author Contributions
K.T., J.P., W.L. and J.Z. designed research; K.T., J.H. and G.W. performed research and analyzed
the data; K.T., and Q.H. wrote the paper. All authors read and approved the final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
References
1. Sreeramulu, S.; Gande, S.L.; Gobel, M.; Schwalbe, H. Molecular mechanism of inhibition of the
human protein complex Hsp90-Cdc37, a kinome chaperone-cochaperone, by triterpene celastrol.
Angew. Chem. Int. Ed. Engl. 2009, 48, 5853–5855.
2. Kannaiyan, R.; Shanmugam, M.K.; Sethi, G. Molecular targets of celastrol derived from Thunder
of God Vine: Potential role in the treatment of inflammatory disorders and cancer. Cancer Lett.
2011, 303, 9–20.
3. Zhou, Y.X.; Huang, Y.L. Antiangiogenic effect of celastrol on the growth of human glioma:
An in vitro and in vivo study. Chin. Med. J. (Engl.) 2009, 122, 1666–1673.
4. Jiang, H.L.; Jin, J.Z.; Wu, D.; Xu, D.; Lin, G.F.; Yu, H.; Ma, D.Y.; Liang, J. Celastrol exerts
synergistic effects with PHA-665752 and inhibits tumor growth of c-Met-deficient hepatocellular
carcinoma in vivo. Mol. Biol. Rep. 2013, 40, 4203–4209.

Molecules 2014, 19
10188
5. Yang, H.; Chen, D.; Cui, Q.C.; Yuan, X.; Dou, Q.P. Celastrol, a triterpene extracted from the
Chinese “Thunder of God Vine,” is a potent proteasome inhibitor and suppresses human prostate
cancer growth in nude mice. Cancer Res. 2006, 66, 4758–4765.
6. Zhang, T.; Hamza, A.; Cao, X.; Wang, B.; Yu, S.; Zhan, C.G.; Sun, D. A novel Hsp90 inhibitor to
disrupt Hsp90/Cdc37 complex against pancreatic cancer cells. Mol. Cancer Ther. 2008, 7, 162–170.
7. Trott, A.; West, J.D.; Klaic, L.; Westerheide, S.D.; Silverman, R.B.; Morimoto, R.I.; Morano, K.A.
Activation of heat shock and antioxidant responses by the natural product celastrol:
Transcriptional signatures of a thiol-targeted molecule. Mol. Biol. Cell 2008, 19, 1104–1112.
8. Westerheide, S.D.; Bosman, J.D.; Mbadugha, B.N.; Kawahara, T.L.; Matsumoto, G.; Kim, S.;
Gu, W.; Devlin, J.P.; Silverman, R.B.; Morimoto, R.I. Celastrols as inducers of the heat shock
response and cytoprotection. J. Biol. Chem. 2004, 279, 56053–56060.
9. Klaic, L.; Morimoto, R.I.; Silverman, R.B. Celastrol analogues as inducers of the heat shock
response. Design and synthesis of affinity probes for the identification of protein targets.
ACS Chem. Biol. 2012, 7, 928–937.
10. Sun, H.; Xu, L.; Yu, P.; Jiang, J.; Zhang, G.; Wang, Y. Synthesis and preliminary evaluation
of neuroprotection of celastrol analogues in PC12 cells. Bioorg. Med. Chem. Lett. 2010, 20,
3844–3847.
11. Zeng, J.F.; Pan, J.F.; Li, B.Y.; Zhu, Q.; Fang, T.; Ni, H.Y. Inventor A Water-Soluble
Triterpenephenol Compound Having Antitumor Activities and the Preparation Method Thereof.
WIPO Patent Application WO/2009/067891, 4 June 2009.
Sample Availability: Samples of the compounds NST001, NST001A, NST001B, NST6A-A are
available from the authors.
© 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).