Design, synthesis and antitumor evaluation of novel celastrol derivatives

Article abstract of DOI:10.1016/j.ejmech.2019.04.050

On the basis of the hybridization strategy of natural products, a total of 32 novel celastrol hybrids were designed, synthesized and evaluated for their antitumor activities. Most of these derivatives exihibited significant antiproliferative activities compared to celastrol, among which compound 29 displayed the strongest inhibitory capability [IC₅₀ = 0.15 ± 0.03 μM (A549),0.17 ± 0.03 μM (MCF-7), 0.26 ± 0.02 μM (HepG2)], which exhibited equal or superior anti-cancer activities in comparison to 2-cyano-3,12-dioxoolean-1,9 (11)-dien-28-oic acid methyl ester (CDDO-Me). The mechanism of pharmacological research indicated that 29 possessed the ability to disrupt Hsp90-Cdc37 complex which was stronger than celastrol. Meanwhile, compound 29 could induce abnormal regulation of clients (p-Akt and Cdk4) of Hsp90 and cell cycle arrest at G₀/G₁ phase in a concentration-dependent manner. In addition, compound 29 could also induce cell apoptosis through the death receptor pathway on A549 cells. Taken together, our results demonstrated that 29 might be a promising novel candidate for further druggability research.

Full text of DOI:10.1016/j.ejmech.2019.04.050

Accepted Manuscript
Design, synthesis and antitumor evaluation of novel celastrol derivatives
Manyi Xu, Na Li, Zihao Zhao, Zhixian Shi, Jianbo Sun, Li Chen
PII:
S0223-5234(19)30368-X
DOI:
https://doi.org/10.1016/j.ejmech.2019.04.050
EJMECH 11285
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 December 2018
Revised Date: 13 April 2019
Accepted Date: 17 April 2019
Please cite this article as: M. Xu, N. Li, Z. Zhao, Z. Shi, J. Sun, L. Chen, Design, synthesis and antitumor
evaluation of novel celastrol derivatives, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.04.050.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, synthesis and antitumor evaluation of novel celastrol
derivatives
Manyi Xu, Na Li, Zihao Zhao, Zhixian Shi, Jianbo Sun*, and Li Chen**
State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry,
School of Traditional Chinese Pharmacy, China Pharmaceutical University, 24 Tong Jia Xiang,
Nanjing 210009, People's Republic of China
* Corresponding author: Tel: +86 83271415, E-mail addresses: sjbcpu@gmail.com (J. Sun)
** Corresponding author: Tel: +86 83271447, chenli627@cpu.edu.cn (L. Chen).
Keywords: Celastrol, ferulic acid, apoptosis, anti-tumor, hybridization
ABSTRACT
On the basis of the hybridization strategy of natural products, a total of 32 novel
celastrol hybrids were designed, synthesized and evaluated for their antitumor
activities. Most of these derivatives exihibited significant antiproliferative activities
compared to celastrol, among which compound 29 displayed the strongest inhibitory
capability [IC50 = 0.15 ± 0.03 µM (A549),0.17 ± 0.03 µM (MCF-7), 0.26 ± 0.02 µM
(HepG2)], which exhibited equal or superior anti-cancer activities in comparison to
2-cyano-3,12-dioxoolean-1,9(11)-dien-28-oic acid methyl ester (CDDO-Me). The
mechanism of pharmacological research indicated that 29 possessed the ability to
disrupt Hsp90-Cdc37 complex which was similar to celastrol. Meanwhile, compound
29 could induce abnormal regulation of clients (p-Akt and Cdk4) of Hsp90 and cell
cycle arrest at G0/G1 phase in a concentration-dependent manner. In addition,
compound 29 could also induce cell apoptosis through the death receptor pathway on
A549 cells. Taken together, our results demonstrated that 29 might be a promising
novel candidate for further druggability research.

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1. Introduction
Celastrol (CE) (Fig.1), a pentacyclic triterpenoid obtained from the root bark of
Tripterygium wilfordii Hook F.[1-2], possesses an unique quinone methide moiety
exhibiting multiple pharmacological activities, especially for its widely investigated
anti-cancer activity[3]. Zhang et al.[4] pointed out that the anti-cancer property of
celastrol mainly due to the disruption of the interaction between heat shock protein
90 (Hsp90) and its molecular co-chaperone, cell division cycle protein 37 (Cdc37), so
as to prevent a variety of downstream client proteins from achieving stable
conformations, cell transportation and transmembrane. Most of these client proteins
are oncogenic proteins, such as protein kinase B (Akt), cyclin-dependent kinases 4
(Cdk4), Raf family proteins and MEK1/2[5].
Ferulic acids (FA, 4-hydroxy-3-methoxycinnamic acid), a class of phenolic acid
widely existed in fruits, vegetables, and Chinese medicinal herbs such as Angelica
sinensis, Cimicifuga racemosa and Ligusticum chuanxiong[6-8]. FA has been proved to
possess potential therapeutic effects for the advantages of anti-oxidation,
anti-diabetes, anti-inflammatory, neuroprotection, etc[9]. It is worth mentioning that
FA and its derivatives also showed promising anticarcinogenic efficacy in vivo and
vitro[10-15]. Silva and Batista[16] have reviewed hundreds of naturally occurring
compounds containing feruloyl moieties with various biological activities. As a
naturally oleanolic acid derivative bearing a trans-feruloyl unit at C-27 position,
uncarinic acid A[17] (Fig.1) showed potent cytotoxicity against A549 (human lung
cancer cell lines) and MCF-7 (human breast cancer cell lines) with EC50 values 4.6
µg/ml and 7.7 µg/ml, respectively. Li et al.[18] also introduced various feruloyl
moieties into glycyrrhetinic acid (GA) and obtained a series of glycyrrhetinic acid
derivatives (Fig.1), which displayed enhanced anti-cancer activities. Therefore,
feruloyl moieties can be used as a promising fragment in in anti-cancer agents design.
Molecular hybridization, a strategy which aims to fuse more than one
pharmacophoric group to obtain new hybrids with improved bioactivities[19-20] is
widely used in drug design. In order to develop novel anticarcinogenic agents, many

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hybrids have been reported via this strategy based on natural products[21-24]. Thus,
combined with the strategy and analysis above, we designed and synthesized
thirty-two (1-32) novel celastrol hybrids in which 29-carboxyl group was modified
by methyl ferulate and its derivatives with different linkers. All hybrids were assayed
in vitro for their anti-proliferative activities against A549, MCF-7, HepG2 cancer
cells. Among them, compound 29 showed strongest cytotoxicity on the above three
cancer cells. Furthermore, disruption of Hsp90-Cdc37 complex, apoptosis, cell cycle
arrest, regulation of Hsp90 clients of 29 were assessed. Additionally, related proteins
of exogenous apoptotic pathway of 29 were preliminarily evaluated by western blot.
Fig.1 The structure of celastrol, and several examples of feruloyl moiety-containing
natural and synthetic antitumor agents.
2. Results and discussion
2.1. Chemistry
The synthetic routes of 32 target compounds (1-32) are depicted in Scheme 1.
The different hydroxy aryl aldehyde (a1-7 and d1-2) were treated with methyl
cyanoacetate for 12 h at 100 ℃ to get the α-cyano substituted methyl cinnamate
derivatives b1-7 and e1-2, respectively[25]. Different kinds of benzene acrylic acid
derivatives a8 or d3-4 were under esterification reaction to get compounds b8 and e3-4.
Then the intermediates were obtained via coupling b1-10 or e1-4 with
trans-1,4-dibromo-2-butene catalyzed by K2CO3 and Bu4N+Br- or alkyl bromides
catalyzed by K2CO3[26]. Subsequently, the target compounds 1-32 were synthesized
by connecting intermediates (c1-22 or f1-10) to C-29 carboxyl group of celastrol with
NaHCO3 in DMF[27]. The substituent groups R1-R6 of 1-32 were listed in Table 1 &
Table 2, respectively.

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Scheme 1. Reagents and conditions: i) Methyl cyanoacetate, NH4OAc, tolune, 100
reflux, 12 h; ii) CH3OH, H2SO4, 70 , reflux, 12 h; iii) alkyl bromides, K2CO3,
acetone, 50 , reflux, 7-8 h or trans-1,4-dibromo-2-butene, K2CO3, Bu4N+Br-,
acetone, 50 , reflux,7 h; iv) celastrol, NaHCO3, DMF, 60 , reflux, 7 h.
,

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Table 1 Structures of target compounds 1-22.
X
O
O
R1
O
R3
R2
H
R4
O
COOCH3
HO
1-22
Cpd.
1
Intermediates
b1, c1
X
R1
H
R2
H
R3
H
R4
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
H
(CH2)3
(CH2)3
(CH2)3
(CH2)3
(CH2)3
(CH2)2
(CH2)3
(CH2)4
2
b2, c2
H
CH3
OCH3
F
H
3
b3, c3
H
H
4
b4, c4
H
H
5
b5, c5
CH3
OCH3
OCH3
OCH3
OCH3
F
H
H
6
b6, c6
H
H
7
b6, c7
H
H
8
b6, c8
H
H
9
b6, c9
CH2CH=CHCH2
(CH2)3
H
H
10
11
12
13
14
15
16
17
18
19
20
21
22
b7, c10
b8, c11
b8, c12
b8, c13
b8, c14
b9, c15
b9, c16
b9, c17
b9, c18
b10,c19
b10, c20
b10, c21
b10, c22
H
H
(CH2)2
OCH3
OCH3
OCH3
OCH3
H
H
OCH3
OCH3
OCH3
OCH3
H
(CH2)3
H
H
(CH2)4
H
H
CH2CH=CHCH2
(CH2)2
H
H
H
H
(CH2)3
H
H
H
H
(CH2)4
H
H
H
H
CH2CH=CHCH2
(CH2)2
H
H
H
H
OCH3
OCH3
OCH3
OCH3
H
H
H
(CH2)3
H
H
H
(CH2)4
H
H
H
CH2CH=CHCH2
H
H
H

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Table 2 Structures of target compounds 23-32.
Cpd.
Intermediates
X
R5
R6
23
24
25
26
27
28
29
30
31
32
e1, f1
e2, f2
e2, f3
e2, f4
e2, f5
e3, f6
e4, f7
e4, f8
e4, f9
e4, f10
(CH2)3
(CH2)2
(CH2)3
(CH2)4
H
CN
CN
CN
CN
CN
H
OCH3
OCH3
OCH3
CH2CH=CHCH2 OCH3
(CH2)3
(CH2)2
(CH2)3
(CH2)4
H
OCH3
OCH3
OCH3
H
H
H
CH2CH=CHCH2 OCH3
H

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2.2. Biological activities
2.2.1. In vitro cytotoxic activity
The synthesized compounds (1-5, 7, 10, 16, 20, 23, 25, 28, 30) with linker
bearing three carbon atoms were preliminarily evaluated their inhibitory effects in
vitro against MCF-7 by MTT assay at 1.0 µM, adopting celastrol as positive control.
According to the data listed in Table 3, we selected compounds whose inhibition rate
exhibited over 50% for further research to discuss their inhibitory activities by
replacing different linkers. Additionally, we found that derivatives containing
benzene ring with a methoxy group showed best inhibitory ability. Thus, we
synthesized four more compounds (11-14) to explore whether the number of methoxy
group made any sense. As shown in Table 3, eight target compounds (7, 11, 15, 20,
24-25, 29-30) displayed superior inhibitory activity (≥ 60%) compared to celastrol.
The preliminary structure and activity relationships (SARs) could be concluded
as follows: (1) Various linkers had certain influence on potency. Hybrids with
unsaturated alkyl possessed higher anti-proliferative activity than those with saturated
butyl linker, but lower than two or three carbons. Compounds with 2 carbons
exhibited stronger anti-cancer capability than 3 carbons (11 and 12, 15 and 16, 24 and
25, 29 and 30), while 6 and 7, 19 and 20 showed reversed results. (2) Under the
condition that the compounds had the same linker, methoxy group exhibited the best
effect on the activities as the substituent group in benzene of ferulic acid derivatives
compared with the R1 analogs 1 (-H), 5 (-CH3), 7 (-OCH3), and 10 (-F). However, the
R2 analogs 1 (-H), 2 (-CH3), 3 (-OCH3), 4 (-F) possessed similar less cytotoxicity. (3)
Compared with 11-14 and 15-18 and 19-22, it was interesting to find out compounds
with one methoxy group displayed the best cytotoxicity except for the situation that
compound with one methoxy group(19) had the least cytotoxicity while those without
(15) or with two (11) methoxy groups are equipotent. (4) The presence of CN group
had little effect on the activity of the compounds, e.g. 1 and 16, 6-9 and 19-22, 23 and
28, 24-27 and 29-32. (The CN group was designed to investigate whether it had the
function to increase the electrophilicity of the unsaturated methyl cinnamate group

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like CDDO-Me. However, we only treated it as a common substituent group due to
the experimental results.)
To comprehensively evaluate the cytotoxicity of synthesized compounds, the
IC50 values (concentration of compound required to reduce 50% of cell viability) of
these compounds against three human cancer cells lines (A549, MCF-7, HepG2)
were then measured using MTT assay (Table 4). We used CDDO-Me as a positive
control, which was studied in phase III clinical trail[28-30]. Evidently, eight selected
products were proved to show superior cytotoxicity against A549, MCF-7 and
HepG2 cells than celastrol. Particularly, compound 29 displayed the strongest
cytotoxcity with IC50 values of 0.15 µM, 0.17 µM, and 0.26 µM on A549, MCF-7
and HepG2, respectively. Meanwhile, 29 showed equal or better anti-proliferative
activity compared to CDDO-Me. Thus, 29 was selected for further investigation.
Moreover, A549 cell lines were chosen for subsequent experiments as they were the
most sensitive to 29.
Table 3 Preliminary inhibitory effects of the tested compounds on MCF-7 cells.
Cpd. Inhibition rate (%) Cpd. Inhibition rate (%) Cpd. Inhibition rate (%)
at 1.0 µMa
22.07
19.83
11.04
8.36
at 1.0 µMa
at 1.0 µMa
46.97
79.69
65.91
27.42
45.59
32.96
87.70
67.42
40.09
49.18
60.21
1
2
12
13
14
15
16
17
18
19
20
21
22
8. 61
23
24
25
26
27
28
29
30
31
32
CEb
7. 52
3
18.01
63.89
50.47
0.94
4
5
21.95
59.91
66.45
13.75
25.67
18.84
60.30
6
7
5.29
8
27.70
78.53
15.93
32.50
9
10
11
aMTT methods, cells were incubated with corresponding compounds at a
concentration of 1.0 µM for 48 h. Values are mean of three independent experiments.
bPositive control.
Table 4 IC50 values of eight compounds on three human cancer cell lines(A549,
MCF-7, HepG2)
Cytotoxicity IC50 (µM)a
Cpd.

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A549
MCF-7
HepG2
7
11
15
20
24
25
29
30
CE
0.66 ± 0.09 0.65 ± 0.31 0.75 ± 0.15
1.07 ± 0.42 0.88 ± 0.21 0.89 ± 0.22
0.60 ± 0.13 0.67 ± 0.12 0.60 ± 0.13
0.56 ± 0.21 0.45 ± 0.11 0.53 ± 0.12
0.46 ± 0.02 0.30 ± 0.04 0.37 ± 0.03
0.78 ± 0.06 0.64 ± 0.11 0.71 ± 0.15
0.15 ± 0.03 0.17 ± 0.03 0.26 ± 0.02
0.70 ± 0.05 0.67 ± 0.15 0.65 ± 0.22
1.28 ± 0.24 1.06 ± 0.17 1.33 ± 0.13
CDDO-Meb 0.36 ± 0.18 0.35 ± 0.07 0.26 ± 0.09
aMTT methods, cells were incubated with corresponding compounds for 48 h. IC50
(µM) values (means ± SD, n = 3).
bPositive control.
2.2.2. 29 disrupted the Hsp90-Cdc37 Interaction in vitro
To make a thorough inquiry about the function of 29 on the interaction of
Hsp90-Cdc37, immunoprecipitation experiment had been carried out with DMSO as
negative control and CE as positive control. We treated A549 cells with 29 or CE at
the concentration of 5.0 µM for 6 h, respectively, followed by the pull-down of
Hsp90α with γ-phosphate-linked ATP-Sepharose. As shown in Fig. 2(A-a) and (A-b),
there were no non-specific proteins that affected the results of the experiment.
Furthermore, it was proved that when Hsp90α was pulled down, 29 could decrease
more amount of Cdc37 distinctly compared to CE (Fig. 2B). Thus, these results
indicated that 29 possessed stronger function to disrupt the interaction between
Hsp90 and Cdc37 than CE.
Fig. 2 29 disrupted the Hsp90-Cdc37 interaction in A549 cells. A549 cells were

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treated with 5.0 µM 29 or CE for 6 h, respectively. 29 decreased the amount of
Cdc37 associated with Hsp90. A: (a) Expression of Cdc37 after Hsp90α was pulled
down;(b) Expression of Hsp90α after Hsp90α was pulled down. IgG is to exclude
interference from non-specific proteins. B: Grayscale analysis was presented by
means of the density ratios of proteins treated with 29 or CE to those untreated. Data
were expressed as the mean ± SD (n = 3).
2.2.3. 29 regulated the Hsp90-Cdc37 client proteins and apoptosis-related proteins
Immunoprecipitation assay has proved 29 as a Hsp90-Cdc37 disruptor, which
could result in degradation of downstream client proteins. Once the biological
behaviour of these clients playing a key role in the oncogenic process was hindered,
they might trigger the apoptosis-related proteins to induce apoptosis. Thus, we
detected these proteins with specific antibodies in 29-treated A549 cells at different
concentrations. As depicted in Fig. 3, it was observed that 29 could down-regulate
p-Akt and Cdk4 in a concentration-dependent manner. Moreover, compared to CE,
the expression level of the clients induced by 29 at the same concentration was much
lower, which indicated that 29 exhibited stronger inhibitory activity. The levels of
Bax, a pro-apoptotic protein of Bcl-2 family, were positively related to concentration.
Instead, Bcl-2 displayed the opposite behavior as an anti-apoptotic protein.
Additionally, the amount of activated ultimate apoptosis-executing protein caspase-3
got higher as the concentration increased. All these results were consistent with what
we have designed and visually represented by grayscale analysis in Fig. 3B.
Considering, 29 had inhibition on the clients of Hsp90 and regulate apoptosis-related
proteins to induce cell apoptosis, whose effect was stronger than celastrol to some
extent.

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Fig. 3 Effects of 29 on clients of Hsp90-Cdc37 and apoptosis-related proteins in
A549 cells. (A) The western blot results revealed the expression levels of Hsp90,
Cdk4, p-Akt, cleaved caspase-3, Bax, Bcl-2 in A549 cells untreated (control) and
treated with 29 for 8 h at different concentrations (0.5, 1.0, 2.0 µM). CE (2.0 µM)
was used as a positive control. β-actin was used as a loading control. (B) Grayscale
analysis was presented by means of the density ratios of proteins to β-actin. Data
were expressed as the mean ± SD (n = 3).
2.2.4. Effects of 29 on cell-cycle distribution
Cdk4 is a key regulator protein of cell cycle. As mentioned above, 29 could
cause a decrease in the expression level of Cdk4 after destroying Hsp90-Cdc37
complex. To test whether the anti-proliferative activity was due to cell cycle arrest,
A549 cells were treated with compound 29 at three different concentrations (0.06,
0.18, and 0.27 µM) for 12 and 24 h and untreated cells as the control. The analysis
was performed by flow cytometry using a Fluorescence-Activated Cell Sorter (FACS)
after labelling with propidium iodide (PI). As shown in Fig. 4, the results indicated
that 29 arrested cell cycle at G0/G1 phase. Thus, cell cycle arrest may be one of the
mechanisms of the anti-proliferation activity of 29. The analysis lead to a
concentration-dependent accumulation of cells in the G0/G1 phase with a concomitant
increase after both 12 and 24 h of treatment. Moreover, from the results illustrated in
Fig. 4E, the proportions of cells treated after 24h had a negligible accumulation than
the proportions of cells treated after 12h at the same concentration. Thus, these
results suggest that growth inhibition of the A549 cells proliferation by 29 may be

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related to induction of G0/G1 phase arrest.
Fig. 4 Cell cycle analysis of 29 in A549 cells. (A) and (C) Cells were treated with 29
at 0.06, 0.18 and 0.27 µM for 12 h, harvested, stained with PI, and then analyzed by
flow cytometry. (B) and (D) Cells were treated with 29 at 0.06, 0.18 and 0.27 µM for
24 h, harvested, stained with PI, and then analyzed by flow cytometry. (E)
Time-dependent effects of 29 on cell cycle were investigated at three different
concentrations (0.06, 0.18, and 0.27 µM).
2.2.5. 29 induced apoptosis by the extrinsic death receptor pathway
To further explore the apoptotic mechanism induced by 29, the levels of related

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proteins in the extrinsic signaling pathway were analyzed. It is well-known that cell
apoptosis is complex. One of its main mechanisms is extrinsic pathway, which is also
called Fas/Fas-L death receptor pathway[31]. Caspase-8 is an initiating apoptotic
protease, which is in response to extracellular apoptosis-inducing ligands after being
activated[32]. PARP, a cleavage substrate for caspase, is another core member in cell
apoptosis[33]. Consequently, we evaluated the effects of 29 on Fas, Fas-L, cleaved
caspase-8 and cleaved PARP, adopting β-actin as an internal reference. As shown in
Fig. 5, the expressions of all tested proteins were performed in a
concentration-dependent manner. These findings proved that 29 could induce
apoptosis in an extrinsic death receptor pathway. Moreover, it was worth mentioned
that the band of cleaved PARP treated with 29 at 2.0 µM got unusually conspicuous
while those which were induced by CE at the same condition or untreated were
almost unobserved. The results could indicate to some extent that PARP was more
sensitive to 29, in other words, 29 might target mainly at PARP to cause cell death.
However, the specific mechanism is intricate, which deserves our in-depth research.
Fig. 5 Effects of 29 on extrinsic death receptor pathway proteins in A549 cells. (A)
The western blot results revealed the expression levels of Fas, Fas-L, cleaved
caspase-8, cleaved PARP in A549 cells untreated (control) and treated with 29 for 8 h
at different concentrations (0.5, 1.0, 2.0 µM). CE (2.0 µM) was used as a positive
control. β-actin was used as a loading control. (B) Grayscale analysis was presented
by means of the density ratios of proteins to β-actin. Data were expressed as the mean
± SD (n = 3).
2.2.6. Effects of 29 on cell apoptosis

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On the basis of the experiments above, 29 was witnessed to induce cell apoptosis.
In order to provide more convincing evidence, we analyzed A549 cells treated with
different concentrations of 29 by flow cytometry after Annexin V-FITC/PI double
staining. As observed in Fig. 6, the total numbers of apoptotic cells grew with the
increasing consistency. Particularly, late apoptosis was conspicuous. The greatest
consequent of 29 was at 1.2 µM with 11.3 % early and 52.2 % late apoptotic cells
while those are 4.1 % and 4.2 % separately in the control condition. Interestingly, 0.4
µM 29-incubated cells had similar effects on those dealt with 2.0 µM CE. In other
words, the potency of anti-tumor of 29 was approximately five times stronger
compared to the parent compound. Hence, our structural modification design for
celastrol CE could promote the apoptosis of A549.
Fig. 6 Apoptosis analysis of 29 and CE in A549 cells. Cells were treated with CE
(2.0 µM) and compound 29 at distinct concentrations (0.2, 0.4, 0.6, 0.8, 1.2 µM) for
24 h, analyzed by flow cytometry. The results were presented in the form of a column
chart. The figures were representative of three independent experiments and the
values were expressed as means ± SD.
2.2.7. Morphological analysis of A549 treated with 29
It’s reported that apoptotic cells acquire morphological characteristics, such as
cell shrinkage, nuclear fragmentation, zeiosis and apoptotic bodies formation[34]. To
corroborate the effect of compound 29 on induction of apoptosis, cells treated with

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compound 29 for 48 h at different concentrations (0.06, 0.09, and 0.18 µM) were
observed by fluorescence microscopy after stained with Hoechst 33342. As depicted
in Fig. 7, with the concentration increased, A549 cells showed typical apoptotic
features such as cell shrinkage and nuclear fragmentation. Moreover, the amount of
cells was obviously decreased, which indicated that there was
a
concentration-dependent effect of 29 on cell apoptosis. These morphological
observations further confirmed our hypothesis that compound 29 induces apoptosis in
A549 cells.
Fig. 7 Fluorescence microscopy images of A549 cells stained by Hoechst 33342.
A549 cells were treated with the 29 at indicated concentrations (0.06, 0.09, and 0.18
µM) for 48 h.
3. Conclusion
In summary, based on the hybridization strategy of developing novel anti-tumor
drugs, a series of celastrol hybrids were designed and synthesized. Some derivatives
performed promoted anti-proliferative activity on three human cancer cell lines
(A549, MCF-7, HepG2) in vitro. Among them, compound 29 showed the strongest
anti-tumor activity (IC50 = 0.15-0.26 µM). The SAR of all target products was also
discussed. Further bio-assay proved that 29 retained the ability to disrupt the
interaction of Hsp90 and Cdc37 compared to celastrol. Meanwhile, it could
down-regulate Hsp90 client proteins, followed by triggering cell apoptosis. Studies
on mechanism suggested that 29 induced apoptosis in A549 cells by activating
Fas/Fas-L, caspase-8 and PARP through extrinsic signaling pathway. Moreover, 29
arrested cell cycle at G0/G1 phase, which was considered as another main mechanism
of cell death. Taken together, as a promising candidate for cancer therapy, compound

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29 is worth further research to obtain more details.
4. Experimental
4.1.Chemistry
All reagents were available from commercial suppliers and used directly unless
otherwise stated. Routine thin-layer chromatography (TLC) was performed to
monitor reactions on silica gel plates with an ultraviolet lamp. Column
1
chromatography was carried out on silica gel (200-300 mesh). H NMR and 13C
NMR spectra were all recorded on a Bruker AVANCE instrument at 25 . The
molecular weights were detected on HP 1100LC/MSD spectrometer.
4.1.1. Synthesis of intermediates and target compounds
4.1.1.1. General procedures for the preparation of Compound
4.1.1.1.1. General procedures for Synthesizing Compounds b1-7, e1-2
NH4OAc (0.5 eq, 4.0 mmol, 308.0 mg) and hydroxy aryl aldehyde a1-7 or d1-2 (0.9 eq,
7.2 mmol) were dissolved in a solution of methyl cyanoacetate (1.0 eq, 8.0 mmol,
706 µl) in tolune (45 ml).The mixture was refluxed at 100 for 12 h by stirring. The
reaction mixture was filtered and then the crude product was purified by column
chromatography using petroleum ether : ethyl acetate (EtOAc) = 2:1 (v/v).
4.1.1.1.2. General procedures for Synthesizing Compounds b8, e3-4
Sinapic acid (a8) or 3-hydroxycinnamic acid (d3) or isoferulic acid (d4) (1.0 eq, 5.0
mmol) was dissolved in methanol (50 ml), stirred under the catalysis of H2SO4
(1.0-2.0 ml) at 70
for 12 h. On cooling, the solvent was removed and drops of
Na2CO3 were then added to the residue until the pH was greater or equal to 7.0. The
resulting solution was extracted with EtOAc (3×20 ml). The combined organic layer
was washed with NaCl solution (2×20 ml), dried with Na2SO4, and subsequently
solvent was evaporated to acquire the crude product b8 or e3-4.
4.1.1.1.3. General procedures for Synthesizing Compounds c1-22, f1-10
4.1.1.1.3.1. General procedures for Synthesizing Compounds c1-8, c10, c11-13, c15-17
,
c19-21, f1-4, f6-9
Compound b1-7 or methyl sinapate (b8) or methyl 4-hydroxycinnamate (b9) or methyl

ACCEPTED MANUSCRIPT
ferulate (b10) or compound e1-2 or methyl 3-hydroxycinnamate (e3) or isoferulic acid
methyl ester (e4) (1.0 eq, 1.2 mmol) and K2CO3 (3.0 eq, 3.6 mmol) were successively
dissolved in anhydrous acetone (8 ml). A solution of different alkyl bromides (5.0 eq,
6.0 mmol) in anhydrous acetone was added and this reaction mixture was stirred at
50 for 7-8 h. The reaction mixture was concentrated under vaccum and the residue
was dissolved in dichloromethane. The organic layer was washed with 5% aqueous
NaHCO3 solution (60 ml) and aqueous saturated NaCl solution (30 ml). After drying
over anhydrous Na2SO4, the dichloromethane was removed in vaccum. The crude
materials was purified by column chromatography on silica gel [petroleum ether
/ethyl acetate=6:1 (v/v)] .
4.1.1.1.3.2. General procedures for Synthesizing Compounds c9, c14, c18, c22, f5, f10
Methyl sinapate (b8) or methyl 4-hydroxycinnamate (b9) or methyl ferulate (b10) or
compound e2 or isoferulic acid methyl ester (e4) (1.0 eq, 0.5 mmol) , K2CO3 (2.0 eq,
1.0 mmol, 138.0 mg), Bu4N+Br- (0.06 eq, 0.03 mmol, 9.67 mg) and
trans-1,4-dibromo-2-butene (3.0 eq, 1.5 mmol, 321.0 mg) were added into anhydrous
acetone. The reaction mixture was refluxed for 7 h by stirring. The acetone was
removed under reduced pressure and the residue was extracted with ethyl acetate (60
ml). The combined organic layers were dried over sodium sulfate, filtered, and the
solvent was evaporated. The crude materials was purified by column chromatography
on silica gel [petroleum ether /ethyl acetate=10:1 (v/v)] to afford pure intermediates
c9, c14, c18, c22, f5, f10.
4.1.1.1.4. General procedures for Synthesizing Target Compounds 1-32
A mixture of celastrol (1.0 eq, 0.1 mmol, 45.1 mg), c1-22 or f1-10 (3.0eq, 0.3 mmol)
and NaHCO3 (5.0 eq, 0.5 mmol, 42.0 mg) in anhydrous DMF (6 ml) was stirred at
60 ℃ under reflux for 6 h. The reaction solution was washed with DCM (100 ml)
for four times. The combined organic layers were washed by aqueous saturated NaCl
solution (30 ml), then dried over sodium sulfate, filtered, and the solvent was
evaporated. The crude product was purified by column chromatography [petroleum
ether /ethyl acetate=3:1 (v/v)].

ACCEPTED MANUSCRIPT
4.1.1.1.4.1. Compound 1. Obtained from celastrol and c1. Orange red powder, 40.5%
yield. Analytical data for 1: 1H-NMR (300 MHz, CDCl3, TMS), δ ppm: 0.53 (3H, s),
1.10 (3H, s), 1.19 (3H, s), 1.25 (3H, s), 1.44 (3H, s), 2.24 (3H, s), 3.94 (3H, s),
4.05-4.23 (4H, m), 6.30 (1H, d, J = 7.1 Hz), 6.50 (1H, s), 6.96 (2H, d, J = 8.9 Hz),
13
7.05 (1H, d, J = 7.0 Hz), 8.00 (2H, d, J = 8.8 Hz), 8.22 (1H, s); C NMR (75 MHz,
CDCl3) δC: 178.3, 178.2, 170.0, 164.7, 163.6, 163.0, 154.7, 146.0, 134.0, 133.8,
127.4 (C×2), 124.4, 119.5, 118.2, 117.2, 116.3, 115.1 (C×2), 98.9, 64.7, 60.9, 53.2,
45.0, 44.2, 42.9, 40.5, 39.4, 38.2, 36.3, 34.8, 33.4, 32.8, 31.6, 30.9, 30.5, 29.8, 29.7,
28.4, 28.1, 21.6, 18.6, 10.3; HRMS (ESI) calculated for C43H52NO7 [M + H]+
694.3744, found 694.3753.
4.1.1.1.4.2. Compound 2. Obtained from celastrol and c2. Orange red powder, 38.8%
yield. Analytical data for 2: 1H-NMR (300 MHz, CDCl3, TMS), δ ppm: 0.54 (3H, s),
1.10 (3H, s), 1.19 (3H, s), 1.25 (3H, s), 1.44 (3H, s), 2.23 (3H, s), 2.45 (3H, s), 3.94
(3H, s), 4.01-4.14 (3H, m), 4.17-4.23 (1H, m), 6.31 (1H, d, J = 7.2 Hz), 6.51 (1H, s),
6.78 (1H, s), 6.81 (1H, d, J = 8.8 Hz), 7.04 (1H, d, J = 7.1 Hz), 8.34 (1H, d, J = 8.8
13
Hz), 8.52 (1H, s); C NMR (75 MHz, CDCl3) δC: 178.3, 178.2, 169.9, 164.7 (C×2),
162.3, 156.5, 152.1, 146.0, 134.1, 131.0, 127.4 (C×2), 123.3, 119.5, 118.2, 117.2
(C×2), 112.3, 99.9, 64.5, 60.9, 53.2, 45.0, 44.2, 42.9, 40.5, 39.3, 38.2, 36.3, 34.8, 33.4,
32.8, 31.6, 30.9, 30.5, 29.8, 29.7, 28.5, 28.2, 21.6, 20.1, 18.6, 10.3; HRMS (ESI)
calculated for C44H54NO7 [M + H]+ 708.3900, found 708.3895.
4.1.1.1.4.3. Compound 3. Obtained from celastrol and c3. Orange red powder, 39.6%
yield. Analytical data for 3: 1H-NMR (300 MHz, CDCl3, TMS), δ ppm: 0.45 (3H, s),
1.10 (3H, s), 1.19 (3H, s), 1.22 (3H, s), 1.42 (3H, s), 2.25 (3H, s), 3.75 (3H, s), 3.93
(3H, s), 4.01-4.06 (1H, m), 4.11-4.22 (3H, m), 6.20 (1H, d, J = 7.1 Hz), 6.36 (1H, d, J
= 2.2 Hz), 6.50 (1H, s), 6.55 (1H, dd, J = 9.0 Hz, 2.2 Hz), 7.00 (1H, d, J = 7.0 Hz),
13
8.41 (1H, d, J = 8.9 Hz), 8.73 (1H, s); C NMR (75 MHz, CDCl3) δC: 178.3 (C×2),
170.0, 165.0, 164.7, 164.1, 148.9, 146.0, 143.3, 134.3, 131.2, 127.3, 119.4, 118.1,
117.3, 116.9, 114.1, 105.6 (C×2), 98.8, 64.5, 60.7, 55.8, 45.0, 44.1, 42.9, 40.5, 39.2,
38.2, 36.3, 35.0, 33.2, 33.0, 31.6, 30.9, 30.5, 29.7, 29.6, 28.4, 28.0, 21.6, 20.1, 18.6,

ACCEPTED MANUSCRIPT
10.3; HRMS (ESI) calculated for C44H54NO8 [M + H]+ 724.3849, found 724.3855.
4.1.1.1.4.4. Compound 4. Obtained from celastrol and c4. Orange red powder, 41.2%
yield. Analytical data for 4: 1H-NMR (300 MHz, CDCl3, TMS), δ ppm: 0.52 (3H, s),
1.10 (3H, s), 1.18 (3H, s), 1.24 (3H, s), 1.44 (3H, s), 2.23 (3H, s), 3.94 (3H, s),
4.03-4.18 (4H, m), 6.29 (1H, d, J = 7.4 Hz), 6.51 (1H, s), 6.73 (1H, d, J = 8.9 Hz),
13
6.78 (1H, d, J = 8.9 Hz), 7.03 (1H, d, J = 7.3 Hz), 7.06 (1H, s), 8.50 (1H, s); C
NMR (75 MHz, CDCl3) δC: 178.3 (C×2), 170.0, 164.7 (C×2), 158.3, 157.5, 153.1,
146.0, 134.1, 130.3, 127.4, 119.5, 118.2, 117.2, 116.9, 111.6, 106.6, 102.2, 98.9, 65.3,
60.8, 53.3, 45.0, 44.2, 42.9, 40.5, 39.4, 38.2, 36.3, 34.8, 33.5, 32.8, 31.6, 30.9, 30.5,
29.7, 29.6, 28.5, 28.0, 21.6, 18.6, 10.3; HRMS (ESI) calculated for C43H51FNO7 [M +
H]+ 712.3650, found 712.3646.
4.1.1.1.4.5. Compound 5. Obtained from celastrol and c5. Orange red powder, 35.1%
yield. Analytical data for 5: 1H-NMR (300 MHz, CDCl3, TMS), δ ppm: 0.50 (3H, s),
1.10 (3H, s), 1.19 (3H, s), 1.23 (3H, s), 1.43 (3H, s), 2.13 (3H, s), 2.26 (3H, s), 3.94
(3H, s), 4.07-4.21 (4H, m), 6.25 (1H, d, J = 7.1 Hz), 6.50 (1H, s), 6.90 (1H, d, J = 8.6
Hz), 7.07 (1H, d, J = 7.2 Hz), 7.79 (1H, s), 7.93 (1H, d, J = 8.6 Hz), 8.19 (1H, s); 13C
NMR (75 MHz, CDCl3) δC: 178.3, 178.2, 170.0, 164.6, 163.7, 153.6, 152.0, 146.0,
134.0, 130.9, 127.4 (C×2), 124.8, 119.5, 118.4, 118.2, 117.2, 115.7, 114.1, 100.6,
66.0, 60.8, 53.4, 45.0, 44.2, 42.9, 40.5, 39.4, 38.2, 36.3, 34.8, 33.5, 32.8, 31.6, 30.9,
30.5, 29.8, 29.7, 28.5, 28.2, 21.6, 20.1, 18.5, 10.3; HRMS (ESI) calculated for
C44H54NO7 [M + H]+ 708.3900, found 708.3904.
4.1.1.1.4.6. Compound 6. Obtained from celastrol and c6. Orange red powder, 37.5%
yield. Analytical data for 6: 1H-NMR (300 MHz, CDCl3, TMS), δ ppm: 0.53 (3H, s),
1.11 (3H, s), 1.21 (3H, s), 1.25 (3H, s), 1.43 (3H, s), 2.21 (3H, s), 3.92 (3H, s), 3.95
(3H, s), 4.26-4.34 (3H, m), 4.38-4.51 (1H, m), 6.31 (1H, d, J = 7.2 Hz), 6.42 (1H, s),
6.93 (1H, d, J = 8.4 Hz), 7.00 (1H, d, J = 7.1 Hz), 7.41 (1H, dd, J = 8.5 Hz, 2.1 Hz),
13
7.80 (1H, d, J = 2.2 Hz), 8.18 (1H, s); C NMR (75 MHz, CDCl3) δC: 178.3 (C×2),
169.7, 164.6, 163.6, 154.9, 152.7, 149.7, 146.0, 133.9, 127.6, 127.4, 125.1, 119.5,
118.1, 117.2 (C×2), 112.9, 112.3, 99.3, 66.9, 62.5, 56.0, 45.0, 44.2, 42.9, 40.6, 39.4,

ACCEPTED MANUSCRIPT
38.2, 36.4, 34.8, 33.4, 32.8, 31.6, 30.8, 30.5, 29.8, 29.6, 28.5, 21.6, 18.6, 10.3; HRMS
(ESI) calculated for C43H52NO8 [M + H]+ 710.3693, found 710.3688.
4.1.1.1.4.7. Compound 7. Obtained from celastrol and c7. Orange red powder, 36.8%
yield. Analytical data for 7: 1H-NMR (300 MHz, CDCl3, TMS), δ ppm: 0.50 (3H, s),
1.10 (3H, s), 1.19 (3H, s), 1.24 (3H, s), 1.44 (3H, s), 2.24 (3H, s), 3.82 (3H, s), 3.95
(3H, s), 4.04-4.12 (1H, m), 4.17-4.21 (3H, m), 6.28 (1H, d, J = 7.2 Hz), 6.49 (1H, s),
6.92 (1H, d, J = 8.6 Hz), 7.07 (1H, d, J = 7.0 Hz), 7.49 (1H, d, J = 8.5 Hz), 7.75 (1H,
13
s), 8.20 (1H, s); C NMR (75 MHz, CDCl3) δC: 178.3, 178.2, 169.8, 164.6, 163.6,
155.0, 153.1, 149.5, 146.0, 134.1, 127.8, 127.4, 124.6, 119.5, 118.2, 117.2, 116.4,
112.1, 111.8, 98.8, 65.3, 60.9, 55.8, 45.0, 44.2, 42.9, 40.5, 39.3, 38.2, 36.3, 34.9, 33.3,
32.8, 31.6, 30.9, 30.5, 29.7, 29.6, 28.4, 28.0, 21.6, 18.5, 10.3; HRMS (ESI) calculated
for C44H54NO8 [M + H]+ 724.3849, found 724.3852.
4.1.1.1.4.8. Compound 8. Obtained from celastrol and c8. Orange red powder, 39.2%
yield. Analytical data for 8: 1H-NMR (300 MHz, CDCl3, TMS), δ ppm: 0.49 (3H, s),
1.10 (3H, s), 1.19 (3H, s), 1.23 (3H, s), 1.43 (3H, s), 2.23 (3H, s), 3.82 (3H, s), 3.95
(3H, s), 4.06-4.11 (1H, m), 4.17-4.19 (3H, m), 6.28 (1H, d, J = 6.8 Hz), 6.50 (1H, s),
6.92 (1H, d, J = 8.6 Hz), 7.08 (1H, d, J = 6.9 Hz), 7.49 (1H, d, J = 8.5 Hz), 7.76 (1H,
13
s), 8.22 (1H, s); C NMR (75 MHz, CDCl3) δC: 178.3, 178.2, 170.0, 164.7, 163.7,
155.0, 153.2, 149.5, 146.0, 134.2, 127.9, 127.1, 124.5, 119.5, 118.1, 117.2, 116.4,
112.0, 111.8, 98.8, 68.5, 64.0, 56.0, 53.2, 45.0, 44.3, 42.9, 40.4, 39.4, 38.3, 36.4, 34.9,
33.5, 32.8, 31.6, 30.9, 30.5, 29.8, 29.7, 25.7, 25.3, 21.6, 18.5, 10.3; HRMS (ESI)
calculated for C45H56NO8 [M + H]+ 738.4006, found 738.4004.
4.1.1.1.4.9. Compound 9. Obtained from celastrol and c9. Orange red powder, 40.3%
yield. Analytical data for 9: 1H-NMR (300 MHz, CDCl3, TMS), δ ppm: 0.57 (3H, s),
1.12 (3H, s), 1.21 (3H, s), 1.28 (3H, s), 1.46 (3H, s), 2.21 (3H, s), 3.95 (6H, s),
4.46-4.51 (2H, m), 4.68-4.69 (2H, m), 5.99 (2H, m), 6.36 (1H, d, J = 7.2 Hz), 6.55
(1H, d, J = 1.4 Hz), 6.91 (1H, d, J = 8.5 Hz), 7.02 (1H, dd, J = 7.1 Hz, 1.4 Hz), 7.45
13
(1H, dd, J = 2.1 Hz, 8.6 Hz), 7.83 (1H, d, J = 2.1 Hz), 8.20 (1H, s); C NMR (75
MHz, CDCl3) δC:177.8 (C×2), 170.0, 164.6, 163.6, 155.0, 152.6, 149.5, 146.0, 134.0,

ACCEPTED MANUSCRIPT
128.5, 128.0, 127.7, 127.4, 124.8, 119.4, 118.1, 117.1, 116.4, 112.4, 111.8, 90.0, 68.5,
63.8, 56.1, 45.0, 44.2, 42.9, 40.5, 39.4, 38.3, 36.3, 34.8, 33.5, 32.8, 31.6, 30.8, 30.5,
29.8, 29.6, 28.7, 21.8, 18.7, 10.3; HRMS (ESI) calculated for C45H54NO8 [M + H]+
736.3849, found 736.3853.
4.1.1.1.4.10. Compound 10. Obtained from celastrol and c10. Orange red powder,
38.1% yield. Analytical data for 10: 1H-NMR (300 MHz, CDCl3, TMS), δ ppm: 0.55
(3H, s), 1.10 (3H, s), 1.19 (3H, s), 1.26 (3H, s), 1.45 (3H, s), 2.23 (3H, s), 3.95 (3H,
s), 4.07-4.15 (1H, m), 4.19-4.22 (3H, m), 6.32 (1H, d, J = 7.2 Hz), 6.49 (1H, s), 7.01
(1H, d, J = 8.8 Hz), 7.04 (1H, s), 7.05 (1H, d, J = 7.0 Hz), 7.77 (1H, d, J = 8.9 Hz),
8.17 (1H, s); 13C NMR (75 MHz, CDCl3) δC: 178.3, 178.2, 170.0, 164.7, 163.2, 153.4,
152.1, 146.0, 134.1, 130.8, 127.4 (C×2), 123.3, 119.5, 118.1, 117.2 (C×2), 116.3,
112.4, 100.0, 64.5, 60.9, 53.2, 45.0, 44.2, 42.9, 40.5, 39.3, 38.2, 36.3, 34.8, 33.4, 32.8,
31.6, 30.9, 30.5, 29.8, 29.7, 28.5, 28.2, 21.6, 20.1, 18.6, 10.3; HRMS (ESI) calculated
for C43H51FNO7 [M + H]+ 712.3650, found 712.3638.
4.1.1.1.4.11. Compound 11. Obtained from celastrol and c11. Orange red powder,
33.8% yield. Analytical data for 11: 1H-NMR (300 MHz, CDCl3, TMS), δ ppm: 0.57
(3H, s), 1.11 (3H, s), 1.20 (3H, s), 1.26 (3H, s), 1.41 (3H, s), 2.22 (3H, s), 3.84 (6H,
s), 4.10-4.17 (1H, m), 4.21-4.26 (3H, m), 6.34 (1H, d, J = 7.3 Hz), 6.35 (1H, s), 6.40
(1H, d, J = 16.1 Hz), 6.77 (2H, s), 7.01 (1H, d, J = 7.4 Hz), 7.65 (1H, d, J = 16.0 Hz);
13C NMR (75 MHz, CDCl3) δC: 178.3 (C×2), 170.0, 167.5, 164.8, 153.5 (C×2), 146.0,
144.9, 139.0, 134.0, 130.1, 127.4, 119.5, 118.1, 117.2, 117.0, 105.2 (C×2), 70.0, 64.0,
56.0 (C×2), 51.7, 45.1, 44.3, 42.9, 40.4, 39.5, 38.2, 36.4, 34.7, 33.4, 32.6, 31.6, 30.9,
30.6, 29.7, 29.3, 28.6, 21.6, 18.5, 10.3; HRMS (ESI) calculated for C43H55O9 [M +
H]+ 715.3846, found 715.3854.
4.1.1.1.4.12. Compound 12. Obtained from celastrol and c12. Orange red powder,
37.8% yield. Analytical data for 12: 1H-NMR (300 MHz, CDCl3, TMS), δ ppm: 0.57
(3H, s), 1.10 (3H, s), 1.19 (3H, s), 1.27 (3H, s), 1.46 (3H, s), 2.21 (3H, s), 3.80 (3H,
s), 3.85 (6H, s), 3.90-3.95 (1H, m), 4.02-4.10 (2H, m), 6.35 (1H, d, J = 6.9 Hz), 6.36
(1H, d, J = 15.8 Hz), 6.51 (1H, s), 6.74 (2H, s), 7.00 (1H, d, J = 6.9 Hz), 7.59 (1H, d,

ACCEPTED MANUSCRIPT
J = 15.8 Hz); 13C NMR (75 MHz, CDCl3) δC: 178.3, 178.2, 170.0, 167.4, 164.6,
153.6 (C×2), 146.0, 144.9, 138.9, 134.0, 130.0, 127.4, 119.5, 118.1, 117.1, 117.0,
105.2 (C×2), 70.0, 61.5, 56.1 (C×2), 51.7, 45.0, 44.3, 42.9, 40.4, 39.4, 38.2, 36.4,
34.8, 33.4, 32.8, 31.6, 30.8, 30.5, 29.8, 29.6, 29.2, 28.6, 21.6, 18.5, 10.3; HRMS (ESI)
calculated for C44H57O9 [M + H]+ 729.4003, found 729.3997.
4.1.1.1.4.13. Compound 13. Obtained from celastrol and c13. Orange red powder,
38.3% yield. Analytical data for 13: 1H-NMR (300 MHz, CDCl3, TMS), δ ppm: 0.57
(3H, s), 1.11 (3H, s), 1.19 (3H, s), 1.27 (3H, s), 1.45 (3H, s), 2.22 (3H, s), 3.82 (3H,
s), 3.86 (6H, s), 3.92-3.96 (1H, m), 4.01-4.09 (2H, m), 6.35 (1H, d, J = 6.9 Hz), 6.36
(1H, d, J = 15.8 Hz), 6.51 (1H, s), 6.74 (2H, s), 7.02 (1H, d, J = 6.9 Hz), 7.62 (1H, d,
J = 15.9 Hz); 13C NMR (75 MHz, CDCl3) δC: 178.3 (C×2), 170.0, 167.4, 164.7, 153.6
(C×2), 146.0, 144.9, 139.2, 134.1, 129.8, 127.4, 119.5, 118.1, 117.1, 116.9, 105.2
(C×2), 72.7, 64.2, 56.1 (C×2), 51.7, 45.1, 44.3, 42.9, 40.4, 39.5, 38.2, 36.4, 34.8, 33.5,
32.8, 31.6, 30.8, 30.6, 29.8, 29.7, 28.6, 26.7, 25.0, 21.6, 18.5, 10.3; HRMS (ESI)
calculated for C45H59O9 [M + H]+ 743.4159, found 743.4159.
4.1.1.1.4.14. Compound 14. Obtained from celastrol and c14. Orange red powder,
41.1% yield. Analytical data for 14: 1H-NMR (300 MHz, CDCl3, TMS), δ ppm: 0.56
(3H, s), 1.11 (3H, s), 1.19 (3H, s), 1.27 (3H, s), 1.45 (3H, s), 2.22 (3H, s), 3.83 (3H,
s), 3.88 (6H, s), 4.36-4.48 (2H, m), 4.54-4.56 (2H, m), 5.80-5.87 (1H, m), 5.97-6.07
(1H, m), 6.35 (1H, d, J = 6.6 Hz), 6.37 (1H, d, J = 16.1 Hz), 6.50 (1H, s), 6.75 (2H, s),
13
7.02 (1H, d, J = 6.7 Hz), 7.63 (1H, d, J = 16.1 Hz); C NMR (75 MHz, CDCl3) δC:
178.3, 177.9, 170.0, 167.4, 164.7, 153.6 (C×2), 146.0, 144.9, 139.3, 134.0, 130.0,
129.8, 127.5, 127.4, 119.5, 118.1, 117.0, 116.9, 105.1 (C×2), 73.0, 64.1, 56.1 (C×2),
51.7, 45.1, 44.3, 42.9, 40.4, 39.4, 38.2, 36.4, 34.8, 33.4, 32.8, 31.6, 30.8, 30.5, 29.8,
29.6, 28.7, 21.7, 18.5, 10.3; HRMS (ESI) calculated for C45H57O9 [M + H]+ 741.4003,
found 741.4004.
4.1.1.1.4.15. Compound 15. Obtained from celastrol and c15. Orange red powder,
38.4% yield. Analytical data for 15: 1H-NMR (300 MHz, CDCl3, TMS), δ ppm: 0.57
(3H, s), 1.10 (3H, s), 1.21 (3H, s), 1.26 (3H, s), 1.45 (3H, s), 2.23 (3H, s), 3.82 (3H,

ACCEPTED MANUSCRIPT
s), 4.13-4.27 (3H, m), 4.31-4.38 (1H, m), 6.33 (1H, d, J = 6.7 Hz), 6.37 (1H, d, J =
15.9 Hz), 6.51 (1H, s), 6.88 (2H, d, J = 8.7 Hz), 7.02 (1H, d, J = 6.8 Hz), 7.50 (2H, d,
J = 8.7 Hz), 7.66 (1H, d, J = 16.0 Hz); 13C NMR (75 MHz, CDCl3) δC: 178.3 (C×2),
169.8, 167.8, 164.7, 160.2, 146.0, 144.4, 134.0, 129.8 (C×2), 127.6, 127.4, 119.6,
118.1, 117.1, 115.6, 114.9 (C×2), 65.8, 62.9, 51.6, 45.0, 44.2, 42.9, 40.5, 39.4, 38.2,
36.4, 34.8, 33.6, 32.8, 31.6, 30.7, 30.5, 29.8, 29.7, 28.6, 21.6, 18.6, 10.3; HRMS (ESI)
calculated for C41H51O7 [M + H]+ 655.3635, found 655.3622.
4.1.1.1.4.16. Compound 16. Obtained from celastrol and c16. Orange red powder,
40.7% yield. Analytical data for 16: 1H-NMR (300 MHz, CDCl3, TMS), δ ppm: 0.52
(3H, s), 1.10 (3H, s), 1.19 (3H, s), 1.25 (3H, s), 1.44 (3H, s), 2.24 (3H, s), 3.82 (3H,
s), 4.01-4.09 (3H, m), 4.16-4.24 (1H, m), 6.28 (1H, d, J = 7.0 Hz), 6.35 (1H, d, J =
16.0 Hz), 6.51 (1H, s), 6.86 (2H, d, J = 8.7 Hz), 7.02 (1H, d, J = 6.9 Hz), 7.47 (2H, d,
J = 8.7 Hz), 7.67 (1H, d, J = 16.0 Hz); 13C NMR (75 MHz, CDCl3) δC: 178.3, 178.2,
169.8, 167.8, 164.7, 160.5, 146.0, 144.5, 134.0, 129.8 (C×2), 127.4, 127.2, 119.5,
118.1, 117.1, 115.3, 114.7 (C×2), 64.3, 61.0, 51.6, 45.0, 44.2, 42.9, 40.5, 39.4, 38.2,
36.3, 34.8, 33.4, 32.8, 31.6, 30.9, 30.5, 29.8, 29.7, 28.5, 28.3, 21.6, 18.6, 10.3; HRMS
(ESI) calculated for C42H53O7 [M + H]+ 669.3791, found 669.3792.
4.1.1.1.4.17. Compound 17. Obtained from celastrol and c17. Orange red powder,
39.1% yield. Analytical data for 17: 1H-NMR (300 MHz, CDCl3, TMS), δ ppm: 0.57
(3H, s), 1.11 (3H, s), 1.20 (3H, s), 1.27 (3H, s), 1.46 (3H, s), 2.22 (3H, s), 3.81 (3H,
s), 3.93-3.97 (3H, m), 4.00-4.09 (1H, m), 6.31 (1H, d, J = 7.0 Hz), 6.33 (1H, d, J =
16.0 Hz), 6.53 (1H, s), 6.87 (2H, d, J = 8.6 Hz), 7.03 (1H, d, J = 7.2 Hz), 7.46 (2H, d,
J = 8.6 Hz), 7.66 (1H, d, J = 16.1 Hz); 13C NMR (75 MHz, CDCl3) δC: 178.3 (C×2),
170.0, 167.8, 164.7, 160.7, 146.0, 144.6, 134.1, 129.7 (C×2), 127.4, 127.1, 119.5,
118.2, 117.1, 115.2, 114.8 (C×2), 67.5, 64.1, 51.6, 45.0, 44.3, 42.9, 40.4, 39.4, 38.2,
36.4, 34.8, 33.5, 32.8, 31.6, 30.9, 30.5, 29.8, 29.7, 28.6, 26.9, 25.9, 21.6, 18.5, 10.3;
HRMS (ESI) calculated for C43H55O7 [M + H]+ 683.3948, found 683.3948.
4.1.1.1.4.18. Compound 18. Obtained from celastrol and c18. Orange red powder,
34.4% yield. Analytical data for 18: 1H-NMR (300 MHz, CDCl3, TMS), δ ppm: 0.57

ACCEPTED MANUSCRIPT
(3H, s), 1.11 (3H, s), 1.21 (3H, s), 1.27 (3H, s), 1.45 (3H, s), 2.21 (3H, s), 3.81 (3H,
s), 4.43-4.49 (2H, m), 4.56 (2H, m), 5.94 (2H, s), 6.34 (1H, d, J = 16.1 Hz), 6.35 (1H,
d, J = 6.9 Hz), 6.52 (1H, s), 6.89 (2H, d, J = 8.6 Hz), 7.02 (1H, d, J = 7.2 Hz), 7.48
(2H, d, J = 8.6 Hz), 7.66 (1H, d, J = 16.0 Hz); 13C NMR (75 MHz, CDCl3) δC: 178.3,
177.9, 170.0, 167.8, 164.7, 160.2, 146.0, 144.5, 134.1, 129.8 (C×3), 128.5 (C×2),
127.5, 127.4, 119.4, 118.1, 115.4, 115.0 (C×2), 67.6, 64.0, 51.6, 45.0, 44.2, 42.9, 40.4,
39.4, 38.2, 36.3, 34.7, 33.5, 32.8, 31.6, 30.8, 30.5, 29.8, 29.6, 28.6, 21.6, 18.6, 10.3;
HRMS (ESI) calculated for C43H53O7 [M + H]+ 681.3791, found 681.3791.
4.1.1.1.4.19. Compound 19. Obtained from celastrol and c19. Orange red powder,
38.3% yield. Analytical data for 19: 1H-NMR (300 MHz, CDCl3, TMS), δ ppm: 0.53
(3H, s), 1.10 (3H, s), 1.21 (3H, s), 1.25 (3H, s), 1.43 (3H, s), 2.22 (3H, s), 3.83 (3H,
s), 3.89 (3H, s), 4.22-4.30 (3H, m), 4.35-4.45 (1H, m), 6.30 (1H, d, J = 7.2 Hz), 6.36
(1H, d, J = 15.9 Hz), 6.46 (1H, s), 6.88 (1H, d, J = 8.6 Hz), 6.99 (1H, s), 7.00 (1H, d,
J = 7.1 Hz), 7.08 (1H, d, J = 8.4 Hz), 7.64 (1H, d, J = 15.9 Hz); 13C NMR (75 MHz,
CDCl3) δC: 178.3 (C×2), 169.8, 167.8, 164.5, 150.4, 149.5, 146.0, 144.8, 134.0, 127.5,
127.4, 122.3, 119.5, 118.1, 117.1, 115.5, 112.2, 110.1, 65.2, 61.0, 51.7, 45.0, 44.2,
42.9, 40.4, 39.3, 38.2, 36.3, 34.9, 33.3, 32.9, 31.7, 30.9, 30.5, 29.8, 29.6, 28.3, 21.7,
18.5, 10.3; ESI/HRMS (m/z) HRMS (ESI) calculated for C42H53O8 [M + H]+
685.3740, found 685.3744.
4.1.1.1.4.20. Compound 20. Obtained from celastrol and c20. Orange red powder,
37.4% yield. Analytical data for 20: 1H-NMR (300 MHz, CDCl3, TMS), δ ppm: 0.49
(3H, s), 1.10 (3H, s), 1.19 (3H, s), 1.24 (3H, s), 1.43 (3H, s), 2.25 (3H, s), 3.77 (3H,
s), 3.83 (3H, s), 4.01-4.14 (3H, m), 4.19-4.27 (1H, m), 6.25 (1H, d, J = 7.2 Hz), 6.36
(1H, d, J = 15.9 Hz), 6.50 (1H, s), 6.85 (1H, d, J = 8.3 Hz), 7.02 (1H, s), 7.02 (1H, d,
J = 7.2 Hz), 7.11 (1H, d, J = 8.3 Hz), 7.67 (1H, d, J = 15.9 Hz); 13C NMR (75 MHz,
CDCl3) δC: 178.3, 178.2, 169.8, 167.7, 164.6, 150.4, 149.5, 146.0, 144.8, 134.0,
127.5, 127.4, 122.4, 119.5, 118.1, 117.0, 115.5, 112.2, 110.2, 65.3, 61.1, 51.6, 45.0,
44.2, 42.9, 40.4, 39.3, 38.2, 36.3, 34.9, 33.3, 32.9, 31.6, 30.9, 30.5, 29.7, 29.6, 28.4,
28.2, 21.7, 18.5, 10.3; ESI/HRMS (m/z) HRMS (ESI) calculated for C43H55O8 [M +

ACCEPTED MANUSCRIPT
H]+ 699.3897, found 699.3900.
4.1.1.1.4.21. Compound 21. Obtained from celastrol and c21. Orange red powder,
39.2% yield. Analytical data for 21: 1H-NMR (300 MHz, CDCl3, TMS), δ ppm: 0.56
(3H, s), 1.11 (3H, s), 1.19 (3H, s), 1.27 (3H, s), 1.46 (3H, s), 2.23 (3H, s), 3.82 (3H,
s), 3.89 (3H, s), 3.93-3.99 (3H, m), 4.02-4.10 (1H, m), 6.33 (1H, d, J = 16.0 Hz), 6.35
(1H, d, J = 7.3 Hz), 6.53 (1H, s), 6.85 (1H, d, J = 8.4 Hz), 7.03 (1H, d, J = 7.2 Hz),
7.04 (1H, s), 7.09 (1H, d, J = 8.3 Hz), 7.65 (1H, d, J = 15.9 Hz); 13C NMR (75 MHz,
CDCl3) δC: 178.3, 178.2, 169.9, 167.7, 164.7, 150.4, 149.5, 146.0, 144.8, 134.1,
127.5, 127.4, 122.5, 119.5, 118.1, 117.1, 115.5, 112.6, 110.1, 68.4, 64.1, 55.9, 51.6,
45.0, 44.3, 42.9, 40.4, 39.4, 38.2, 36.3, 34.8, 33.5, 32.8, 31.6, 30.9, 30.8, 30.5, 29.8,
28.5, 25.8, 25.2, 21.6, 18.5, 10.2; HRMS (ESI) calculated for C44H57O8 [M + H]+
713.4053, found 713.4053.
4.1.1.1.4.22. Compound 22. Obtained from celastrol and c22. Orange red powder,
36.0% yield. Analytical data for 22: 1H-NMR (300 MHz, CDCl3, TMS), δ ppm: 0.55
(3H, s), 1.11 (3H, s), 1.19 (3H, s), 1.26 (3H, s), 1.44 (3H, s), 2.21 (3H, s), 3.81 (3H,
s), 3.91 (3H, s), 4.39-4.54 (2H, m), 4.62-4.63 (2H, m), 5.86-6.06 (2H, m), 6.32 (1H, d,
J = 7.8 Hz), 6.33 (1H, d, J = 15.7 Hz), 6.51 (1H, s), 6.84 (1H, d, J = 8.0 Hz), 7.01
(1H, d, J = 8.5 Hz), 7.06 (1H, s), 7.08 (1H, d, J = 8.6 Hz), 7.64 (1H, d, J = 16.0 Hz);
13C NMR (75 MHz, CDCl3) δC: 178.3, 177.8, 170.0, 167.7, 164.7, 150.4, 149.9,
146.0, 144.8, 134.1, 128.4, 128.0, 127.7, 127.4, 122.4, 119.5, 118.1, 117.1, 115.6,
112.8, 110.0, 68.5, 63.9, 55.9, 51.7, 45.0, 44.2, 42.9, 40.4, 39.4, 38.2, 36.3, 34.7, 33.4,
32.8, 31.6, 30.8, 30.5, 29.8, 29.6, 28.6, 21.6, 18.6, 10.3; HRMS (ESI) calculated for
C44H55O8 [M + H]+ 711.3897, found 711.3902.
4.1.1.1.4.23. Compound 23. Obtained from celastrol and f1. Orange red powder,
38.1% yield. Analytical data for 23: 1H-NMR (300 MHz, CDCl3, TMS), δ ppm: 0.54
(3H, s), 1.10 (3H, s), 1.19 (3H, s), 1.25 (3H, s), 1.44 (3H, s), 2.23 (3H, s), 3.89 (3H,
s), 4.07 (3H, s), 4.17-4.23 (1H, m), 6.32 (1H, d, J = 6.9 Hz), 6.52 (1H, s), 6.91 (1H, d,
J = 8.0 Hz), 7.03 (2H, s), 7.15 (1H, d, J = 7.9 Hz), 7.29 (1H, d, J = 6.9 Hz), 8.19 (1H,
13
s); C NMR (75 MHz, CDCl3) δC: 178.3 (C×2), 170.0, 164.7 (C×2), 159.1, 155.3,

ACCEPTED MANUSCRIPT
146.0, 134.1, 132.5, 127.4, 120.8, 119.5, 118.1, 117.1, 116.9, 114.6, 113.4, 98.9, 64.4,
61.0, 53.5, 45.0, 44.2, 42.9, 40.5, 39.4, 38.2, 36.3, 34.8, 33.4, 32.9, 31.6, 30.9, 30.5,
29.8, 29.7, 28.5, 28.2, 21.6, 18.6, 10.3; HRMS (ESI) calculated for C43H52NO7 [M +
H]+ 694.3744, found 694.3747.
4.1.1.1.4.24. Compound 24. Obtained from celastrol and f2. Orange red powder,
37.4% yield. Analytical data for 24: 1H-NMR (300 MHz, CDCl3, TMS), δ ppm: 0.55
(3H, s), 1.10 (3H, s), 1.22 (3H, s), 1.25 (3H, s), 1.41 (3H, s), 2.22 (3H, s), 3.92 (3H,
s), 3.94 (3H, s), 4.26-4.28 (2H, m), 4.32-4.37 (2H, m), 6.32 (1H, d, J = 7.2 Hz), 6.34
(1H, s), 6.96 (1H, d, J = 8.7 Hz), 7.00 (1H, d, J = 7.4 Hz), 7.50 (1H, d, J = 8.6 Hz),
7.77 (1H, s), 8.12 (1H, s); 13C NMR (75 MHz, CDCl3) δC: 178.4, 177.9, 170.0, 164.7,
163.7, 154.9, 154.2, 148.6, 146.0, 134.0, 128.2, 127.4, 124.3, 119.5, 118.1, 117.0
(C×2), 112.5, 111.2, 98.8, 68.7, 64.0, 56.0, 51.6, 45.0, 44.2, 42.9, 40.4, 39.4, 38.2,
36.3, 34.8, 33.4, 32.8, 31.6, 30.8, 30.5, 29.8, 29.7, 28.6, 21.7, 18.6, 10.3; HRMS (ESI)
calculated for C43H52NO8 [M + H]+ 710.3693, found 710.3692.
4.1.1.1.4.25. Compound 25. Obtained from celastrol and f3. Orange red powder,
36.8% yield. Analytical data for 25: 1H-NMR (300 MHz, CDCl3, TMS), δ ppm: 0.46
(3H, s), 1.09 (3H, s), 1.18 (3H, s), 1.22 (3H, s), 1.41 (3H, s), 2.25 (3H, s), 3.79 (3H,
s), 3.94 (3H, s), 4.00-4.08 (1H, m), 4.14-4.18 (2H, m), 4.23-4.31 (1H, m), 6.23 (1H, d,
J = 7.0 Hz), 6.45 (1H, s), 6.84 (1H, d, J = 8.5 Hz), 7.02 (1H, d, J = 7.0 Hz), 7.44 (1H,
d, J = 8.6 Hz), 7.79 (1H, s), 8.17 (1H, s); 13C NMR (75 MHz, CDCl3) δC: 178.4,
178.3, 170.0, 164.7, 163.7, 154.9, 154.2, 148.6, 146.0, 134.1, 128.3, 127.3, 124.3,
119.5, 118.1, 117.0 (C×2), 112.5, 111.2, 98.7, 65.0, 60.9, 55.9, 53.2, 45.0, 44.2, 42.9,
40.5, 39.3, 38.2, 36.3, 34.9, 33.3, 32.9, 31.6, 30.9, 30.5, 29.7, 29.6, 28.5, 28.1, 21.7,
18.5, 10.3; HRMS (ESI) calculated for C44H54NO8 [M + H]+ 724.3849, found
724.3852.
4.1.1.1.4.26. Compound 26. Obtained from celastrol and f4. Orange red powder,
35.1% yield. Analytical data for 26: 1H-NMR (300 MHz, CDCl3, TMS), δ ppm: 0.57
(3H, s), 1.11 (3H, s), 1.19 (3H, s), 1.27 (3H, s), 1.45 (3H, s), 2.22 (3H, s), 3.94 (6H,
s), 4.04-4.15 (4H, m), 6.36 (1H, d, J = 7.2 Hz), 6.51 (1H, s), 6.93 (1H, d, J = 8.4 Hz),

ACCEPTED MANUSCRIPT
13
7.02 (1H, d, J = 7.1 Hz), 7.47 (1H, d, J = 8.6 Hz), 7.80 (1H, s), 8.18 (1H, s); C
NMR (75 MHz, CDCl3) δC: 178.3 (C×2), 170.0, 164.7, 163.7, 155.1, 154.2, 148.6,
145.8, 134.1, 128.1, 127.4, 124.4, 119.5, 118.1, 117.0 (C×2), 113.0, 111.2, 98.7, 68.5,
64.2, 56.1, 53.2, 45.1, 44.3, 42.9, 40.4, 39.4, 38.2, 36.4, 34.8, 33.5, 32.8, 31.6, 30.8,
30.6, 29.8, 29.7, 28.6, 25.8, 25.3, 21.6, 18.5, 10.3; HRMS (ESI) calculated for
C45H56NO8 [M + H]+ 738.4006, found 738.4004.
4.1.1.1.4.27. Compound 27. Obtained from celastrol and f5. Orange red powder,
38.1% yield. Analytical data for 27: 1H-NMR (300 MHz, CDCl3, TMS), δ ppm: 0.55
(3H, s), 1.10 (3H, s), 1.19 (3H, s), 1.26 (3H, s), 1.44 (3H, s), 2.20 (3H, s), 3.93 (3H,
s), 3.96 (3H, s), 4.41-4.54 (2H, m), 4.68 (2H, s), 6.00 (2H, s), 6.35 (1H, d, J = 7.0 Hz),
6.49 (1H, s), 6.96 (1H, d, J = 8.5 Hz), 7.01 (1H, d, J = 6.9 Hz), 7.50 (1H, d, J = 8.2
13
Hz), 7.79 (1H, s), 8.18 (1H, s); C NMR (75 MHz, CDCl3) δC: 178.3, 177.9, 170.0,
164.7, 163.6, 155.0, 148.0, 146.0, 134.1, 128.6, 128.4, 128.0, 127.4, 124.4, 119.5,
118.1, 117.1, 116.3, 113.3, 111.2, 98.8, 68.6, 64.0, 56.2, 53.2, 45.0, 44.2, 42.9, 40.4,
39.4, 38.2, 36.3, 34.8, 33.4, 32.8, 31.6, 30.8, 30.5, 30.2, 29.8, 29.6, 28.6, 21.7, 18.6,
10.3; HRMS (ESI) calculated for C45H54NO8 [M + H]+ 736.3849, found 736.3842.
4.1.1.1.4.28. Compound 28. Obtained from celastrol and f6. Orange red powder,
41.6% yield. Analytical data for 28: 1H-NMR (300 MHz, CDCl3, TMS), δ ppm: 0.53
(3H, s), 1.09 (3H, s), 1.19 (3H, s), 1.24 (3H, s), 1.43 (3H, s), 2.23 (3H, s), 3.82 (3H,
s), 4.04-4.09 (3H, m), 4.15-4.23 (1H, m), 6.29 (1H, d, J = 7.1 Hz), 6.43 (1H, d, J =
16.0 Hz), 6.50 (1H, s), 6.89 (1H, d, J = 8.1 Hz), 7.01 (1H, s), 7.02 (1H, d, J = 8.0 Hz),
7.12 (1H, d, J = 7.3 Hz), 7.28 (1H, t, J = 7.9 Hz), 7.66 (1H, d, J = 16.0 Hz); 13C NMR
(75 MHz, CDCl3) δC: 178.3, 178.2, 169.9, 167.4, 164.7, 160.5, 159.0, 146.0, 144.8,
135.7, 134.2, 130.0, 127.4, 120.9, 119.5, 118.1 (C×2), 117.2, 116.7, 113.3, 64.3, 61.0,
51.6, 45.0, 44.2, 42.9, 40.5, 39.4, 38.2, 36.3, 34.8, 33.4, 32.8, 31.6, 30.9, 30.5, 29.8,
29.7, 28.5, 28.3, 21.6, 18.6, 10.3; HRMS (ESI) calculated for C42H53O7 [M + H]+
669.3791, found 669.3790.
4.1.1.1.4.29. Compound 29. Obtained from celastrol and f7. Orange red powder,
42.1% yield. Analytical data for 29: 1H-NMR (300 MHz, CDCl3, TMS), δ ppm: 0.53

ACCEPTED MANUSCRIPT
(3H, s), 1.10 (3H, s), 1.22 (3H, s), 1.24 (3H, s), 1.41 (3H, s), 2.21 (3H, s), 3.82 (3H,
s), 3.88 (3H, s), 4.24 (2H, m), 4.28-4.29 (1H, m), 4.35 (1H, m), 6.27 (1H, d, J = 16.1
Hz), 6.31 (1H, d, J = 7.2 Hz), 6.38 (1H, s), 6.89 (1H, d, J = 8.2 Hz), 6.99 (2H, m),
13
7.13 (1H, d, J = 8.1 Hz), 7.59 (1H, d, J = 15.8 Hz); C NMR (75 MHz, CDCl3) δC:
178.3 (C×2), 169.8, 167.6, 164.6, 152.0, 148.2, 146.0, 144.4, 133.9, 127.3 (C×2),
123.7, 119.5, 118.1, 117.0, 115.6, 113.5, 111.9, 67.6, 63.2, 56.0, 51.6, 45.0, 44.3, 42.9,
40.5, 39.5, 38.2, 36.4, 34.8, 33.5, 32.8, 31.6, 30.8, 30.5, 29.8, 29.6, 28.6, 21.6, 18.5,
10.3; HRMS (ESI) calculated for C42H53O8 [M + H]+ 685.3740, found 685.3745.
4.1.1.1.4.30. Compound 30. Obtained from celastrol and f8. Orange red powder,
36.9% yield. Analytical data for 30: 1H-NMR (300 MHz, CDCl3, TMS), δ ppm: 0.49
(3H, s), 1.10 (3H, s), 1.19 (3H, s), 1.24 (3H, s), 1.42 (3H, s), 2.24 (3H, s), 3.78 (3H,
s), 3.82 (3H, s), 4.01-4.13 (3H, m), 4.21-4.29 (1H, m), 6.27 (1H, d, J = 7.3 Hz), 6.31
(1H, d, J = 15.9 Hz), 6.47 (1H, s), 6.81 (1H, d, J = 8.3 Hz), 7.01 (1H, d, J = 7.0 Hz),
7.03 (1H, s), 7.10 (1H, d, J = 8.2 Hz), 7.64 (1H, d, J = 16.0 Hz); 13C NMR (75 MHz,
CDCl3) δC: 178.3 (C×2), 170.0, 167.7, 164.7, 151.2, 148.4, 146.0, 144.8, 134.1, 127.1
(C×2), 122.9, 119.5, 118.1 (C×2), 115.4, 111.6, 111.2, 65.2, 61.1, 55.8, 51.7, 45.0,
44.2, 42.9, 40.4, 39.3, 38.2, 36.3, 34.9, 33.3, 32.9, 31.6, 30.9, 30.5, 29.7, 29.6, 28.5,
28.3, 21.6, 18.5, 10.3; HRMS (ESI) calculated for C43H55O8 [M + H]+ 699.3897,
found 699.3901.
4.1.1.1.4.31. Compound 31. Obtained from celastrol and f9. Orange red powder,
38.2% yield. Analytical data for 31: 1H-NMR (300 MHz, CDCl3, TMS), δ ppm: 0.57
(3H, s), 1.11 (3H, s), 1.19 (3H, s), 1.27 (3H, s), 1.45 (3H, s), 2.22 (3H, s), 3.81 (3H,
s), 3.89 (3H, s), 3.93-4.00 (1H, m), 4.05-4.09 (3H, m), 6.31 (1H, d, J = 16.0 Hz), 6.35
(1H, d, J = 7.4 Hz), 6.52 (1H, s), 6.87 (1H, d, J = 8.3 Hz), 7.01 (1H, d, J = 7.3 Hz),
7.06 (1H, s), 7.11 (1H, d, J = 8.4 Hz), 7.64 (1H, d, J = 16.1 Hz); 13C NMR (75 MHz,
CDCl3) δC:178.3 (C×2), 170.0, 167.7, 164.7, 151.9, 148.5, 146.0, 144.8, 134.1, 127.4,
127.3, 122.8, 119.5, 118.1, 117.1, 115.4, 111.6, 111.4, 68.6, 64.2, 56.0, 51.6, 45.0,
44.2, 42.9, 40.4, 39.4, 38.2, 36.4, 34.8, 33.5, 32.8, 31.6, 30.8, 30.5, 29.8, 29.7, 28.6,
26.0, 25.2, 21.6, 18.5, 10.3; HRMS (ESI) calculated for C44H57O8 [M + H]+ 713.4053,