SAR study of celastrol analogs targeting Nur77-mediated inflammatory pathway

Article abstract of DOI:10.1016/j.ejmech.2019.05.009

Nur77, an orphan member of the nuclear receptor superfamily, plays an important role in the regulation of inflammatory processes. Our previous work found that celastrol, a pentacyclic triterpene, bound to Nur77 to inhibit inflammation in a Nur77-dependent

Full text of DOI:10.1016/j.ejmech.2019.05.009

Accepted Manuscript
SAR study of celastrol analogs targeting Nur77-mediated inflammatory pathway
Zi-Wen Chen, Duo Zhang, Si-Wei Yan, Chao-Chao Hu, Zhen-Fei Huang, Zhuo-Er
Li, Shuang-Zhou Peng, Xiao-Tong Li, Yi Zhu, Hong-Yu Yu, Bao-Huan Lian, Qi Kang,
Mingyu Li, Zhi-Ping Zeng, Xiao-Kun Zhang, Ying Su
PII:
S0223-5234(19)30418-0
DOI:
https://doi.org/10.1016/j.ejmech.2019.05.009
EJMECH 11319
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 26 February 2019
Revised Date: 6 May 2019
Accepted Date: 6 May 2019
Please cite this article as: Z.-W. Chen, D. Zhang, S.-W. Yan, C.-C. Hu, Z.-F. Huang, Z.-E. Li, S.-Z.
Peng, X.-T. Li, Y. Zhu, H.-Y. Yu, B.-H. Lian, Q. Kang, M. Li, Z.-P. Zeng, X.-K. Zhang, Y. Su, SAR study
of celastrol analogs targeting Nur77-mediated inflammatory pathway, European Journal of Medicinal
Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.05.009.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
SAR study of celastrol analogs targeting Nur77-mediated inflammatory
pathway
a, 1
a, 1
a, 1
a
a
a
Zi-Wen Chen , Duo Zhang , Si-Wei Yan , Chao-Chao Hu , Zhen-Fei Huang , Zhuo-Er Li , Shuang-Zhou
a
a
a
a
a
a
a
a
Peng , Xiao-Tong Li , Yi Zhu , Hong-Yu Yu , Bao-Huan Lian , Qi Kang , Mingyu Li , Zhi-Ping Zeng , Xiao-
a, *
a, b, *
Kun Zhang and Ying Su
a
School of Pharmaceutical Science, Fujian Provincial Key Laboratory of Innovative Drug Target Research, Xiamen
University, Fujian, 361002, China
b
Sanford Burnham Prebys Medical Discovery Institute, 10901 N. Torrey Pines Road, La Jolla, CA 92037, USA
1
These authors contributed equally to this work.
*
To whom correspondence should be addressed:
Email: ysu@sbpdiscovery.org (Y. Su) or to xkzhang@xmu.edu.cn (X.-K. Zhang)
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Abstract
Nur77, an orphan member of the nuclear receptor superfamily, plays an important role in the
regulation of inflammatory processes. Our previous work found that celastrol, a pentacyclic triterpene,
bound to Nur77 to inhibit inflammation in a Nur77-dependent manner. Celastrol binding to Nur77
promotes Nur77 translocation from nucleus to cytoplasm, resulting in clearance of inflamed mitochondria
and then alleviation of inflammation. Here, we report the design, synthesis, SAR study and biological
evaluation of a series of celastrol analogues. A total of 24 celastrol derivatives were made. Compound 3a
with a K of 0.87
µ
M was found to be less toxic than celastrol and could be a hit molecule for further
d
optimization.
Key words：Nur77, celastrol, anti-inflammatory, celastrol derivatives, SAR
1
. Introduction
Nur77 (also known as TR3, NGFI-B, and NR4A1), an orphan member of the nuclear receptor
superfamily, is known to play an integral role in many different cellular processes especially the
inflammatory responses [1-3]. Though, Nur77 possesses common structural features shared by the NR
family: an activation function 1 (AF-1), a DNA binding domain (DBD), a ligand binding domain (LBD),
and a ligand-dependent transcriptional activation domain (AF-2), from N-terminal to C-terminal [4], its
binding pocket in the LBD is shown to be incapable of binding ligands [5]. Consistently not endogenous
ligands have been identified for Nur77 and Nur77 remains to be an orphan receptor. However, in recent
years, ligands that bind to the surface regions of the LBD of Nur77 have been found and reported to
mediate the biological functions of Nur77 [6-13].
Our lab recently reported the identification of celastrol as a submicromolar ligand of Nur77 [12].
Celastrol is a potent anti-inflammatory agent isolated from the root of Tripterygium wilfordii, a traditional
Chinese medicine also known as “Thunder God Vine”. Traditionally, “Thunder God Vine” has been used
to treat rheumatoid arthritis, lupus and other auto-immune diseases [13-17]. As the key bioactive
molecule of Thunder God Vine, celastrol has been studied widely and recognized to be involved in
multiple pathways and to have the potential for therapeutic applications [18-20]. We found that celastrol
binds to Nur77 with an affinity of 292 nM and demonstrated in vitro and in animal models that the anti-
inflammatory effects of celastrol requires its binding to Nur77, which therefore identifies Nur77 as a
direct intracellular target of celastrol. Furthermore, using multiple approaches we demonstrated and
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confirmed that celastrol binds to Nur77 via a reversible covalent bond resulted from Michael addition
reaction at C551 in Nur77. In addition, we showed the formation of this covalent bond requires specific
noncovalent interactions with Nur77 to position celastrol in close proximity to the -SH group of C551.
Such specific noncovalent interactions render celastrol’s binding selectivity, providing opportunity to
develop celastrol-derived agents that can selectively bind Nur77 to modulate its anti-inflammatory effect
[
21].
Here we report our design, synthesis, SAR study and biological evaluation of 24 celastrol analogs.
Binding activities of the synthesized compounds were studied to understand the structure-activity
relationship. Compounds were also evaluated for their anti-inflammatory effects. Compounds 3a and 1e
displayed similar anti-inflammatory effects as celastrol and were selected for further biological
evaluation. 3a was found to be less toxic when its toxicity was assessed in comparison to celastrol in
Zebrafish model. It is worth noting that celastrol and analogs have been widely studied for their anti-
cancer potentials [22-24], but less studied for their anti-inflammatory applications. This study will
provide insights into the optimization of celastrol derivatives selectively targeting Nur77 for anti-
inflammatory therapy.
2
. Chemistry
The synthetic routes of the proposed celastrol derivatives of formula I and II (Fig. 1) were outlined in
Schemes 1-3. First, derivatives with modifications to the carboxylic acid or hydroxyl groups were
introduced (Scheme 1, 1a - 1g, 2a - 2c). Treating celastrol with DAST (Diethylaminosulfurtrifluoride,
(
CH CH ) NSF ) in dichloromethane at -78 °C for 1 h, gave the product 1a in moderate yield 58%.
3 2 2 3
Esterification of celastrol with various alkyl iodides at room temperature catalyzed by NaHCO yielded
3
corresponding derivatives in moderate yield (87% for 1b, 78% for 1d and 79% for 1f). Under the
catalysis of PyBOP (Benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate) and DIPEA
(
N,N-diisopropylethylamine), treatment of celastrol with three different primary amines (methylamine,
ethylamine and propylamine) generated the products 1c (72% yield), 1e (48% yield) and 1g (31% yield).
a - 2c were obtained by treating 1b with methyl iodide, bromoethane and 1,1-Difluoro-2-iodoethane
CHCH I) respectively, catalyzed by K CO (2c, 57% yield) or by both K CO and TBAB
Tetrabutylammonium bromide) (2a, 54% yield and 2b, 85% yield).
a was obtained in 99% yield by treating celastrol with NaBH in all deuterated methanol.
2
(
(
F
2
2
2
3
2
3
3
4
Conjugation of nitromethane to celastrol with the assistance of TBAF (Tetrabutylammonium fluoride)
formed 3b in 87% yield. Michael addition of celastrol with acetone in the present of catalytic amount
hydrochloric acid (12 mol/L) resulted in product 3c (51% yield, Scheme 1, step ix). As indicated in
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Scheme 1, catalyzed by MeONa celastrol could react with heterocyclic reagent pyrrole to afford product
3
d (82% yield, Scheme 1, step x). Catalyzed by 5 mol% AlCl ·6H O dimethylphosphonate group was
3 2
introduced to C-6 position of celastrol, producing compound 3e with a moderate yield 55% (Scheme 1,
step xi). Products 4a and 4b were prepared as described in Scheme 1, steps xii and xiii: first, celastrol was
2 2 3
protected by benzyl group PhCH Br with the help of K CO and triethylamine (TEA) in DMF solution,
giving the intermediate compound; after purification using flash column chromatography, the
intermediate compound continues to react with MeMgBr (1 mol/L) in THF at 0 °C for 1 h to obtain
compound 4a in 56% yield. The two benzyl protective groups were removed from 4a by hydrogen gas
and Pd/C at room temperature for 12 h, yielding the product 4b in 87% yield. 5c was obtained according
to Scheme 2: the two hydroxyl groups of 3b were protected by 2,2-dimethoxypropane to afford
2 2 2
compound 5a in 63% yield. Subsequently, the nitro group of 5a was reduced by Pd/C and H NNH ·H O
in EtOH to give compound 5b in 36% yield. Removing the protecting group from compound 5b in the
presence of catalyst In(OTf) and under the microwave condition (120 °C for 3 h) afforded 5c in 77%
3
yield [25]. Treatment of compound 3a with 2,2-dimethoxypropane for 12 h in the presence of catalytic
2
acid p-TsOH·H O afforded 6a in moderate yield (57%). 6b was obtained by oxidizing 6a using PDC and
dimethylpyrazole in DCM for 12 h at room temperature. Using In(OTf) as a catalyst, the protective
3
group acetonides were removed from compound 6b efficiently in CH CN/H O (10:1 v/v) under the
3
2
microwave irradiation, and 6c was obtained (37%). Compounds 7a and 7f were synthesized by reducing
compounds 1b and 2a with 10 equiv. NaBH in deuterated MeOH (step vii in Scheme 1), respectively
78% yield for 7f and 99% for 7a). 7a was methylated by dimethyl sulfate to afford compound 7e (24%
4
(
yield, step xiv). Compound 7g was obtained in 66% yield by using another methylating reagent - CH3I
under the same condition (step xv). Subsequently, compound 7e, 7f and 7g were hydrolyzed by KOH
under 100 °C in mixed dioxane/water (1:1 v/v) solvent to obtain 7b, 7c and 7d in high yields (step xvi in
Scheme 1). All synthesized compounds were structurally elucidated by different spectrometric methods,
1
13
including H NMR (600 MHz), C NMR (151 MHz), and high-resolution mass spectra (HR-MS) (see the
spectra in the supporting information).
ꢀ
ꢀ
Fig. 1. Chemical structures of celastrol and its analogs
ꢀ
ꢀ
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Scheme 1. Reagents and conditions: i) DAST, DCM, -78 � , 1 h; ii) Alkyl iodides, NaHCO , DMF, r. t., 12 h; iii)
3
Various amines, DMF, PyBOP, DIPEA, 0 � 30 min and then r. t. for 2 h; iv) CH I, K CO , TBAB, THF, 20 mol%
3
2
3
Dioxane, 70 � , 12 h; v) CH
DMF, 50 � , 24 h. vii) NaBH
HCl (12 mol/L), r. t., 12 h; x) MeONa, pyrrole, r. t., 5 min; xi) AlCl ·6H O, HPO(OCH ) , DCM, r. t., 6 h; xii)
3
CH
2
Br, K
2
CO
3
, TBAB, THF, 20 mol% Dioxane, 70 � , 12 h; vi) F
2
CHCH
2
I, K
2 3
CO ,
4
, CD
3
OD, r. t., 30 min; viii) CH
3
NO , TBAF, THF, r. t., 1 h; ix) CH
2
3
COCH
3
, 1 drop
3
2
3 2
PhCH Br, DMF, K CO , Et N, 80 � , 2.5 h, 68% ; Then MeMgBr, THF, 0 � , 1 h, 83% yield, (Two Steps: 56%);
2
2
3
3
xiii) Pd/C, H , MeOH, r. t., 12 h; xiv) (CH O) SO , K CO , acetone, 70 � , 12 h; xv) MeI, K CO , acetone, 70 � , 12
2
3
2
2
2
3
2
3
h; xvi) KOH, dioxane/H O = 1:1 (v/v), 100 � , 4 h, then, naturally cool to r. t. for additional 10 min.
2
ꢀ
Scheme 2. Reagents and conditions: i) (MeO) CMe , p-TsOH, DCM, r. t., overnight, 63% yield; ii) Pd/C,
2
2
H NNH ·H O, EtOH, 75 � , overnight, 36% yield; iii) In(OTf) , MeCN/water = 10:1 (v/v), microwave, 120 � , 3 h,
2
2
2
3
7
7% yield.
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ꢀ
Scheme 3. Reagents and conditions: i) (MeO) CMe , p-TsOH, DCM, r. t., overnight, 57% yield; ii) PDC, 3,5-
2
2
dimethylpyrazole, DCM, r. t., overnight, 48% yield; iii) In(OTf) , MeCN/water = 10:1 (v/v), microwave. 120 � , 1 h,
3
3
7% yield.
ꢀ
3
. Results and Discussion
3
.1 Design of celastrol derivatives
We recently reported the binding characterization of celastrol to Nur77 [21]. We demonstrated that
celastrol binds to Nur77-LBD via specific noncovalent interactions and a reversible covalent bond. We
also showed that covalent bond is formed through Michael addition reaction between celastrol and C551
in Nur77. Furthermore, we developed a binding model of celastrol to Nur77 by using molecular modeling
and mutagenesis approaches. In this predicted model (Fig. 2A), celastrol fits well to a groove formed by
loop H7-H8, helices 8, 9, 10 and loop H9-H10; the hydroxyl group on ring A and the carboxyl on ring E
form H-bonds with D499 and Q547 respectively; the central portion of the celastrol molecule is
hydrophobic, making Van Der Waals interactions with many hydrophobic residues; C-6 atom where the
nucleophilic addition occurs is positioned next to the -SH group of C551. These modeling results
prompted us to investigate the effects of different substituents on rings A, B and E. First, we designed and
synthesized derivatives 1a-g and 2a-c to explore the importance of the hydroxyl and the carboxyl groups.
Our previous studies revealed that celastrol binding to Nur77 requires its interaction with Nur77 first
via specific noncovalent interactions. We found that due to these specific noncovalent interactions,
compound 3a of which the C-6 atom is reduced and is not capable of nucleophilic addition can still bind
to Nur77. Therefore, secondly, we designed and synthesized compounds 3b-e and 7b-g to study the role
of substituents at C-6 and the importance of the hydroxyls on ring A in the noncovalent interactions
between celastrol derivatives and Nur77.
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A
B
ꢀ
Fig. 2. Modeling studies. (A) Modeling study of celastrol binding to Nur77-LBD. The protein is displayed as surface
representation and celastrol is shown as green sticks. The contributing hydrophobic residues are colored in pale
green. The polar residues Q547 and D499 are colored in blue and in orange, respectively. (B) Binding comparison
between compound 1d (gray sticks) and celastrol.
3
.2 Fluorescence binding studies
The binding affinities of the synthesized celastrol derivatives to Nur77 were evaluated by a
fluorescence quenching assay. The compounds were titrated into the cuvette filled with a 3 mL Nur77-
LBD protein solution (1 µmol/L) with an increasing concentration from 0.1 µmol/L to 5 µmol/L. The
fluorescence intensity excited by Nur77-LBD was quenched due to the interaction between the protein
and compounds. Using the fluorescence quenching assay, the dissociation constant of celastrol was
evaluated as 0.32 µM [21], which is close to the K of 0.29 µM reported previously by the Surface
d
Plasmon Resonance (SPR) binding analysis [12]. Thus, Celastrol was used as the positive control.
Binding affinity results (Fig. S1) for compounds 1b-g made from the esterification and amidation of
the -COOH at position 20 (Fig. 1) are summarized in Table 1. Overall these compounds’ binding activities
3
are not significantly different from celastrol. Replacing the -OH of carboxylic acid with -OCH or -
NHCH3 slightly weakened the binding. However, when larger groups (R1 = OCH(CH3)2 or
NHCH CH CH ) were used the weakening effects were reduced and some improvement was observed
2
2
3
for compound 1d of which R = OCH CH . These observations suggested that when substituting the -
1
2
3
OH group in the carboxyl group, small groups such as methyl amide and methoxy are not the ideal ones
for binding improvement, which is supported by the result of 1a and is consistent with information
gathered from our previous modeling study. Our predicted model shows there is a small hydrophobic
channel below the -OH group and alkyl chains with moderate length (1d) would fit well (Fig. 2B).
In the predicted model, the hydroxyl group at position 3 forms a H-bond with D499 (see Fig. 2A). It
is anticipated that disruption of this H-bond would drastically weaken the binding to Nur77. To evaluate
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the contribution of this H-bond, compounds 2a-2c, of that the ability of H-bond formation was disabled at
position 3 were synthesized and indeed the binding results (Table 1) showed several-fold reduction in
their binding affinities.
Table 1. Binding and biological results of 1a-g and 2a-c.
Binding affinity
µM)
Inhibition of TNFα-induced IκB-α
Compounds
Celastrol
R1
R2
(
degradation
c
OH
F
H
H
0.32
***
c
1
a
0.72 ± 0.01
0.96 ± 0.03
0.80 ± 0.31
0.22 ± 0.08
0.83 ± 0.09
0.52 ± 0.05
0.27 ± 0.12
2.94 ± 0.79
4.13 ± 0.39
3.30 ± 0.21
***
c
1
b
OCH3
NHCH3
H
***
c
1
c
H
***
c
1
d
OCH CH3
H
***
2
c
1
e
NHCH CH3
H
***
2
c
1f
OCH(CH3)2
H
***
c
1
2
g
a
NHCH CH CH
H
***
2
2
3
a
OCH3
Methyl
Ethyl
NS
a
2
b
OCH3
OCH3
NS
a
2
c
CH CHF2
NS
2
a
NS: no significant;
c
:*P<0.05, **P<0.01, ***P<0.001
Reduction of the quinone methide motif in celastrol affords a hydrolyzed celastrol incapable of
undergoing Michael addition. Though losing the ability to make covalent bond interaction with Nur77,
the reduced celastrol can still bind to Nur77 via noncovalent bond interaction [21]. For example,
compound 3a can bind to Nur77 with a K of 0.87 µM, indicating strong noncovalent bond interaction
d
with the protein. This inspired us to synthesize compounds listed in Table 2 to study the impact of
different substituents at C-6 position on the noncovalent interactions between compounds and Nur77.
Binding affinity results (Fig. S2) shows that any substituents at C-6 position weakened the interactions
between the reduced celastrol (3a) and Nur77. Replacing H with substituents (4b, 5c, 3d and 3e) ranging
from small group such as methyl to sterically bulky dimethyl phosphonate group demolished the binding
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although carbonyl (6c) and medium-sized substituents (3b and 3c) did not show the same impact. 6c, 3b
and 3c displayed binding affinity towards Nur77 though weaker in comparison to 3a (Table 2). These
results implied that size was not the only factor playing roles in the interactions with Nur77 protein. Our
model showed that R554 is about 4.8
Å away from the C-6 (Fig. 2) and would form H-bonds with the H-
bond donors, the carbonyl and nitro groups in 6c, 3b and 3c, which explained why 6c and the medium-
sized substituents 3b and 3c were different from 4b and 5c.
Compound 3a possesses 2 hydroxyl groups on the A ring and our model showed that there was not
much empty space around the hydroxyl groups (Fig. 3A) and the hydroxyl group at C-3 position could
form a H-bond with D599 similar to celastrol (Fig. 3A). Therefore, we decided to design compounds 7b-d
to evaluate the importance of these 2 hydroxyl groups for the noncovalent interactions. Binding results
(
Fig. S3) showed that methylation of one or both of the hydroxyl groups (7b-d) led to loss of the binding
activity. Consistently, the same results were observed for compounds 7e-g.
In summary, studies on the binding affinities of the aforementioned 24 celastrol derivatives helped us
gain some understanding of the SAR, which is summarized in Fig. 4. Binding affinity is not very sensitive
to the modification to the -COOH at C-20. However, modification to C-6 with groups larger than H is in
general not favorable. In addition, the hydroxyl groups on the A-ring of the reduced celastrol or the
hydroxyl group on the A-ring of celastrol are important to the binding.
Table 2. Binding and biological results of 3b-e, 4b, 5c and 6c.
Bond type between R
C-6 atom
3
and
Inhibition of TNFα-induced IκB-α
Compounds
R
3
Binding affinity (µM)
degradation
c
3
3
3
3
3
4
5
6
a
b
c
H
Single Bond
Single Bond
Single Bond
Single Bond
Single Bond
Single Bond
Single Bond
Double Bond
0.87
***
a
CH
CH COCH
Pyrrolyl
2
NO
2
5.56 ± 0.15
3.80 ± 0.31
NS
a
2
3
2
NS
b
a
d
e
NB
NS
b
a
PO(OCH
CH
CH NH
3
)
NB
NS
b
a
b
c
3
NB
NS
b
a
2
2
NB
NS
a
c
O
2.69 ± 0.07
NS
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a
bNS: no significant;
NB: no binding;
c
:*P<0.05, **P<0.01, ***P<0.001
Table 3. Binding and biological results of 7b-7g.
O
R1
H
O
R
R
4
5
O
Inhibition of TNFα-induced IκB-
Compounds
R1
R4
R5
Binding affinity (µM)
0.87
α degradation
c
3
7
7
7
7
7
7
a
b
c
d
e
f
OH
OH
H
H
H
***
b
a
CH3
H
NB
NS
b
a
OH
CH3
CH3
H
NB
NS
b
a
OH
CH3
CH3
H
NB
NS
b
a
OCH3
OCH3
OCH3
NB
NS
b
c
CH3
CH3
NB
*
b
a
g
CH3
NB
NS
a
bNS: no significant;
NB: no binding;
c
:*P<0.05, **P<0.01, ***P<0.001
Fig. 3. Modeling studies of 3a. (A) Docking mode of compound 3a, compound was shown as sticks while
binding site in surface presentation; (B) Superimpose compound 3a (pink sticks) with celastrol (green sticks)
at the binding site.
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Fig. 4. Summary of the structure-activity relationship
3
.3 Evaluation of the anti-inflammatory effects of celastrol derivatives
Celastrol could antagonize the effects of inflammatory cytokine TNFα on inducing IκBα degradation
in a Nur77-dependent manner [12, 26]. Therefore, we asked if the synthesized celastrol derivatives
possessed such an anti-inflammatory effect. HepG2 cells treated with TNFα in the presence or absence of
celastrol or analogs were analyzed by Western blot for the level of IκBα (Fig. 5). Consistent with
previous observation [12, 26], TNFα-induced downregulation of IκBα level was largely prevented when
cells were cotreated with celastrol. Analysis of the effect of celastrol analogs revealed a close correlation
between their anti-inflammatory effects and binding affinities. Compounds (2a-c, 3b-e and 4b, 5c, 6c)
that either failed to bind or bound weakly to Nur77 exhibited weak anti-inflammatory effect in
comparison to celastrol (also see Tables 1–3), whereas compounds (1a-g and 3a) that bind Nur77 with a
K of less than 1 µM could strongly antagonize the effect of TNFα on inducing IκBα degradation (also
d
see Tables 1–2).
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A
B
C
D
E
Fig. 5. Western blot analysis of the anti-inflammatory effect of celastrol derivatives. Lysates from HepG2 cells
treated with compound (2 µM) for 1 hr and TNFα (20 ng/mL) for 30 min were analyzed by Western blotting. The
grey processing was carried out using imageJ. All the bar graphs represent mean ± SEM of three independent
experiments. Ns, not significant, *P<0.05, **P<0.01, ***P<0.001 vs the TNFα group, ###P<0.001 vs the control
group (Student’s test).
3
.4 Compound binding promotes Nur77 interactions with TRAF2 and p62/SQSTM1
We previously showed that Nur77 mediates the anti-inflammatory effect of celastrol by translocating
to mitochondria where it interacts with tumor necrosis factor receptor-associated factor 2 (TRAF2) and
the autophagic adaptor p62/SQSTM1, a process that results in the elimination of damaged mitochondria
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via autophagy. Thus, we asked if aforementioned celastrol analogs that displayed both good binding
affinity and anti-inflammatory effect could act through a similar mechanism. Compounds 3a and 1e were
selected for such an evaluation. To examine whether they promoted the interaction between Nur77 and
p62/SQSTM1, HepG2 cells transfected with GFP-Nur77 and mCherry-p62/SQSTM1 were treated with
TNFα together with celastrol or compound 3a or 1e. Confocal microscopy analysis showed that GFP-
Nur77 resided exclusively in the nucleus in control cells. However, when cells were treated with TNFα
and celastrol, GFP-Nur77 could be found in the cytoplasm, colocalizing extensively with p62/SQSTM1
(
Fig. 6A). Similar results were obtained when cells were treated with TNFα and compound 3a or 1e. To
study the effect on Nur77 interaction with TRAF2, HepG2 cells transfected with Myc-Nur77 and Flag-
TRAF2 were treated TNFα and celastrol or compounds 3a or 1e and analyzed by co-immunoprecipitation
(
coIP) assay (Fig. 6B). Our results showed that treatment of cells with compound 3a or 1e could promote
the interaction between Nur77 and TRAF2, similar to the effect of celastrol. Thus, like celastrol 3a and 1e
could promote the interactions between Nur77 and TRAF2 and p62/SQSTM1, respectively (Fig. 6B).
Collectively, compounds 3a and 1e can inhibit inflammatory response by promoting the interactions of
Nur77 with TRAF2 and p62/SQSTM1.
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A
B
Fig. 6. Compounds 3a and 1e promote Nur77 interaction with TRAF2 and p62. (A) Representative images
show colocalization of transfected GFP-Nur77 with mCherry-p62 in HepG2 cells after treatment with
celastrol, 3a, 1e and TNFα, examined by immunostaining; (B) HepG2 cells transfected with Myc-Nur77 and
Flag-TRAF2 or Flag-p62 were treated with celastrol, 3a or 1e (2 µM) and TNFα (20 ng/mL) for 1 hr and
analyzed by coIP.
3
.5 Toxicity assessment of compounds 3a and 1e using in vitro assays and Zebrafish model
Celastrol is well known for its toxicity, which is one of the key factors limiting its clinical
translation[27]. For anti-inflammatory agents, their apoptotic actions can lead to unwanted side effects.
Thus, first we performed in vitro assays to assess the apoptotic effects of 1e and 3a. In the PARP cleavage
assay, 3a was less active, however 1e showed stronger PARP cleavage effect than celastrol (Fig. 7A). The
effect of 1e and 3a on cell death was also assessed using flow cytometry-based Annexin V/ Propidium
iodide (PI) apoptosis assay. 3a consistently showed to induce less cell death than 1e and celastrol (Fig.
7
B). Fig.7B showed that more than 10% of HepG2 cells were apoptotic when treated with 2 µM of
celastrol or 1e for 10 hours, while only 3.12% of cells were apoptotic when treated with 2 µM of 3a.ꢀꢀ
We then evaluated the toxicity of both 3a and celastrol in zebrafish model (Fig. 7C-E). Fig. 7C
showed that celastrol killed zebrafish embryo as much as 3 times more than 3a when zebrafish embryo
was treated with 0.5 µM of celastrol or 3a for 24 hours. When the treatment time was prolonged to 72
hours, the death rate of zebrafish embryo caused by celastrol increased to near 100%, whereas the effect
of 3a did not change significantly. When the compound concentration was increased to 1.25 µM, their
difference in toxicity was more pronounced with 90% of the zebrafish embryo killed by celastrol in 24
hours, whereas, the toxicity of 3a at 1.25 µM stayed comparable to its corresponding one at 0.5 µM. In
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addition, the impact of 3a on the malformation of zebrafish was studied. Fig. 7D showed that celastrol
had a much higher impact on the malformation of zebrafish than 3a. Fig. 7E showed visually under the
same conditions, 3a had less effect than celastrol on the death rate and malformation of zebrafish either at
a concentration of 1.25 µM for 24 hours or at a concentration of 0.5 µM for 72 hours. In summary, our
data showed that 3a was less toxic than celastrol and could be a better anti-inflammatory agent as a probe
molecule or a hit molecule for further optimization.
A
B
Fig. 7.ꢀ Toxicityꢀ studies.ꢀ (A) Western blot analysis for apoptosis effect of 3a, 1e and celastrol, HepG2 cells
treated with celastrol, 3a, 1e (2 µM) and TNFα (20 ng/mL) for 10 hr were analyzed by WB, the apoptosis
effect was detected through cleaved-PARP; (B) Flow cytometry analysis of 3a and 1e, Q1-UL: necrotic cells,
Q1-UR: viable apoptotic cells, Q1-LR: non-viable apoptotic cells, Q1-LL: normal cells; HepG2 cells treated
with celastrol，3a or 1e (2 µM) and TNFα (20 ng/mL) for 10 hr were stained by FITC Annexin V and
Propidium Iodide Staining Solution, and then analyzed by CytoFLEX; (C-E) Embryotoxicity and
developmental toxicity assay, 20 zebrafish embryos per condition at 2 hpf were exposed to celastrol or 3a
at the concentrations of 0.5 µM, 1 µM and 1.25 µM and 0.1% DMSO served as control. C) The statistics of
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2
4 hpf and 72 hpf mortality rate; D) The statistics of 72 hpf malformation rate in 0.5 µM group; E)
Morphology of 24 hpf embryo or 72 hpf zebrafish larvae treated with celastrol or 3a
.
4
. Conclusion
In conclusion, we described the synthesis and SAR studies and biological evaluation of 24 celastrol
analogs as anti-inflammatory agents by targeting the orphan nuclear receptor Nur77. Our results validated
a proposed binding model of celastrol and provide insight into the design of improved celastrol analogs.
Compound 3a, a reduction product of celastrol, having comparable binding affinity and biological effects
to celastrol, displayed remarkable improvement in cell toxicity, offering a hit molecule for further
optimization for anti-inflammatory application.
5
5
5
. Experimental section
.1 Chemistry
.1.1. General methods
Celastrol was purchased from Chengdu Pufei De Biotech Co., Ltd. All commercially available
reagents and solvent were purchased from Energy Chemical (Shanghai, China) and used without further
1
13
purification unless otherwise stated. H and C spectra were recorded in CDCl
3 6
or DMSO-d (dimethyl
sulfoxide-d ) on a Bruker AV600 spectrometer (Switzerland) with tetramethylsilane (TMS) as an internal
6
standard. Chemical shifts are expressed in δ (ppm) units downfield from TMS. All coupling constants (J -
values) were reported in Hertz (Hz). Signals are described as follow: s, singlet; br. s., broad signal; d,
1
doublet; t, triplet; q, quartet; m, multiplet. Chemical shifts of common trace H-NMR impurities (ppm):
2 6 6 3
H O: 3.29-3.4 in DMSO-d , DMSO-d : 2.50, CDCl : 7.27. Mass spectra were recorded on a Q-Exactive-
LC/MS/MS ThermoFisher instrument (America) with a HESI, APCI or nanoscale ESI ion source and
high-resolution MS (HRMS) on Orbitrap. The samples for HR-MS analysis were prepared as described:
Compounds were dissolved in HPLC purity methyl alcohol individually, to obtain a 10 µg/mL solution of
compound, and then 10 µL of 10 µg/mL sample was injected to Q-Exactive LC-MS apparatus for
analysis.
Flash column chromatography was carried out with 300-400 mesh silica gel using Biotage Isolera
Prime (Sweden). The melting points (Mp.) were determined using a SGW® X-4 Micro Melting Point
apparatus (Shanghai INESA Physico-Optical Instrument Co.,Ltd., China). All tested compounds have a
purity ≥ 95%.
5
.1.2. (9β,13α,14β,20α)-3-Hydroxy-9,13-dimethyl-2-oxo-24,25,26-trinoroleana-1(10),3,5,7-tetraen-29-
oyl fluoride (1a) CAS: 2172373-26-9
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Compound 1a was synthesized according to procedure i in Scheme 1. 50 mg (0.11 mmol) celastrol was
dissolved in 2 mL DCM and stirred in a closed 25-mL tube. After 150 µL (10 equiv) DAST was added,
the solution was kept to react in -78 � for 1 h. Cold water (10 mL) was added to quench the reaction, and
subsequently the aqueous phase was separated and extracted with DCM (15 mL * 3). The organic layers
were combined and dried with anhydrous sodium sulfate. Finally, compound was purified by flash
column chromatography on silica gel (Ethyl acetate/Hexane = 1:4). Orange red solid, 29.2 mg, yield:
2
D
4
1
5
7
8%. Mp: 169.0 - 171.0 � . [α]
6
= -168.6 (c 0.1, MeOH). H NMR (600 MHz, DMSO-d ) δ 8.73 (s, 1H),
.06 (dd, J = 1.28, 6.97 Hz, 1H), 6.38 (d, J = 1.28 Hz, 1H), 6.35 (d, J = 7.34 Hz, 1H), 2.23 (d, J = 12.65
Hz, 1H), 2.17 - 2.21 (m, 1H), 2.09 (s, 3H), 1.97 (br. s., 1H), 1.88 - 1.94 (m, 1H), 1.81 - 1.87 (m, 1H), 1.77
-
1.81 (m, 1H), 1.69 - 1.74 (m, 1H), 1.65 - 1.69 (m, 1H), 1.60 - 1.65 (m, 1H), 1.58 (d, J = 7.89 Hz, 1H),
1
.56 (d, J = 4.95 Hz, 1H), 1.49 - 1.54 (m, 1H), 1.46 - 1.49 (m, 1H), 1.41 - 1.46 (m, 1H), 1.38 (s, 3H), 1.29
13
(
s, 3H), 1.22 (s, 3H), 1.08 (s, 3H), 0.94 - 1.01 (m, 1H), 0.56 (s, 3H) ppm; C NMR (151 MHz, DMSO-d )
6
δ 178.0, 167.1, 167.6 (d, J = 369.7 Hz, 1 C), 162.8, 146.4, 132.9, 127.0, 120.2, 118.1, 117.2, 44.3, 43.3,
4
1.9, 40.2, 39.2, 38.7, 37.9, 35.8, 33.8, 32.7, 31.2, 29.9, 29.5, 29.3, 29.3, 27.9, 21.4, 18.9, 10.1 ppm;
+
+
3
HRMS (ESI): m/z calcd for C29H37FNaO [M+Na] : 475.2619, found: 475.2626.
5
.1.3. (9β,13α,14β,20α)-3-Hydroxy-9,13-dimethyl-2-oxo-24,25,26-trinoroleana-1(10),3,5,7-tetraen-29-oic
acid methyl ester (1b) CAS: 1258-84-0
Compound 1b was synthesized according to procedure ii in Scheme 1. 50 mg (0.11 mmol) celastrol was
dissolved in 3 mL DMF and stirred in a 25-mL round-bottom flask, and then 94 mg (0.66 mmol) CH
and 28 mg (0.33 mmol) NaHCO were added in the solution. After reacting for 12 h at room temperature,
.1 mol/L HCl (5 mL) and distilled water (5 mL) were added to quench the reaction. Subsequently, the
3
I
3
0
aqueous phase was separated and extracted with ethyl acetate (15 mL) three times. The organic layers
were combined and dried with anhydrous sodium sulfate. Compound was purified by flash column
chromatography on silica gel (Ethyl acetate/Hexane = 1:8). Orange solid, 44.9 mg, yield: 87%. Mp:
2
D
4
1
1
98.8-200.2 � , [α]
= -266.6 (c 0.1, MeOH). H NMR (600 MHz, DMSO-d ) δ 8.72 (s, 1 H), 7.07 (dd, J
6
=
7.15, 1.10 Hz, 1 H), 6.39 (d, J = 1.28 Hz, 1 H), 6.35 (d, J = 7.15 Hz, 1 H), 3.48 (s, 3 H), 2.31 (d, J =
1
5.77 Hz, 1 H), 2.17 - 2.22 (m, 1 H), 2.09 (s, 3 H), 2.06 (d, J = 14.12 Hz, 1 H), 1.95 (td, J = 13.98, 3.76
Hz, 1 H), 1.78 - 1.86 (m, 1 H), 1.61 - 1.72 (m, 4 H), 1.50 - 1.59 (m, 3 H), 1.41 - 1.46 (m, 1 H), 1.38 (s, 3
H), 1.30 - 1.35 (m, 1 H), 1.21 (s, 3 H), 1.12 (s, 3 H), 1.07 (s, 3 H), 0.91 (d, J = 14.31 Hz, 1 H), 0.44 (s, 3
13
H) ppm; C NMR (151 MHz, DMSO-d ) δ 178.0, 177.9, 167.8, 162.9, 146.4, 133.1, 126.9, 120.2, 118.1,
6
117.3, 51.4, 44.5, 43.6, 42.0, 39.8, 38.8, 37.8, 36.0, 34.4, 32.9, 32.2, 31.3, 30.3, 30.1, 29.4, 29.2, 28.1,
+
+
2
1.4, 18.0, 10.1 ppm; HRMS (ESI): m/z calcd for C H NaO [M+Na] : 487.2819; found: 487.2816.
30 40 4
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5
.1.4. (9β,13α,14β,20α)-3-Hydroxy-N,9,13-trimethyl-2-oxo-24,25,26-trinoroleana-1(10),3,5,7-tetraen-29-
amide (1c) CAS: 1105067-36-4
Compound 1c was synthesized according to procedure iii in Scheme 1. To a solution of celastrol (50 mg,
0
.11 mmol) in 2 mL DMF, PyBOP (68.7 mg, 0.132 mmol) and DIPEA (28.4 mg, 0.22 mmol) were added.
The reaction was cooled down to 0 � for 30 min, and then transferred to room temperature. After that, the
reactant CH NH (33 wt% in absolute ethanol, 55 µL, 0.33 mmol) was added and the solution was stirred
for another 2 h. Finally, the mixture was quenched with water (15 mL) and extracted with ethyl acetate (3
15 mL). After washed by saturated brine (3 * 10 mL). The organic layers were combined and dried with
anhydrous sodium sulfate. Compound was purified by flash column chromatography on silica gel (Ethyl
3
2
*
2
D
4
acetate/Hexane = 1:4). Red solid, 36.0 mg, yield: 72%. Mp: 218.7-220.9 � . [α]
= -156.6 (c 0.1, MeOH).
1
H NMR (600 MHz, DMSO-d ) δ 8.71 (s, 1H), 7.55 (q, J = 3.97 Hz, 1H), 7.07 (d, J = 7.15 Hz, 1H), 6.39
6
(
s, 1H), 6.34 (d, J = 7.34 Hz, 1H), 2.43 (d, J = 4.22 Hz, 3H), 2.38 (d, J = 15.41 Hz, 1H), 2.18 (d, J = 9.90
Hz, 1H), 2.09 (s, 3H), 2.05 (d, J = 14.12 Hz, 1H), 1.94 - 2.01 (m, 1H), 1.79 - 1.85 (m, 1H), 1.73 - 1.79 (m,
H), 1.61 - 1.67 (m, 2H), 1.56 - 1.60 (m, 1H), 1.54 (s, 2H), 1.50 (d, J = 7.89 Hz, 1H), 1.41 (d, J = 13.94
Hz, 1H), 1.37 (s, 3H), 1.26 - 1.33 (m, 1H), 1.20 (s, 3H), 1.08 (s, 3H), 1.02 (s, 3H), 0.86 (d, J = 12.65 Hz,
1
1
3
1
1
2
H), 0.49 (s, 3H) ppm; C NMR (151 MHz, DMSO-d ) δ 177.9, 177.5, 168.3, 163.0, 146.4, 133.2, 126.8,
6
20.1, 117.9, 117.2, 44.5, 44.0, 42.0, 38.8, 37.7, 36.1, 34.8, 33.2, 33.0, 31.4, 30.8, 30.2, 29.4, 28.9, 28.2,
+
6.0, 21.4, 17.4, 10.1 (One was buried in solvent peaks) ppm; HRMS (ESI): m/z calcd for C30H41NNaO
3
+
[
M+Na] :486.2979, found: 486.2982.
5
.1.5. (9β,13α,14β,20α)-3-Hydroxy-9,13-dimethyl-2-oxo-24,25,26-trinoroleana-1(10),3,5,7-tetraen-29-oic
acid ethyl ester (1d) CAS: 1105067-39-7
Compound 1d was synthesized according to procedure ii in Scheme 1 with the steps mentioned above
(
e.g.: 1b) and purified by flash column chromatography on silica gel (Ethyl acetate/Hexane = 1:8).
Starting with 50 mg celastrol, 41.4 mg product was obtained. Orange solid, yield: 78%. Mp: 133.7-
2
D
4
1
1
7
36.1 � . [α]
6
= -81.34 (c 0.1, MeOH). H NMR (600 MHz, DMSO-d ) δ 8.72 (s, 1H), 7.07 (dd, J = 1.01,
.06 Hz, 1H), 6.38 (d, J = 1.10 Hz, 1H), 6.34 (d, J = 7.15 Hz, 1H), 3.86 - 3.95 (m, 2H), 2.33 (d, J = 15.59
Hz, 1H), 2.20 (d, J = 11.19 Hz, 1H), 2.08 (s, 3H), 2.03 - 2.07 (m, 1H), 1.89 - 1.97 (m, 1H), 1.78 - 1.86 (m,
1
1
1
1
2
H), 1.61 - 1.71 (m, 4H), 1.50 - 1.57 (m, J = 6.60, 6.60 Hz, 3H), 1.40 - 1.45 (m, 1H), 1.37 (s, 3H), 1.30 -
.35 (m, 1H), 1.21 (s, 3H), 1.13 (t, J = 7.20 Hz, 3H), 1.11 (s, 3H), 1.06 (s, 3H), 0.90 (d, J = 14.12 Hz,
13
H), 0.47 (s, 3H) ppm; C NMR (151 MHz, DMSO-d ) δ 178.0, 177.4, 167.9, 163.0, 146.5, 133.3, 126.9,
6
20.2, 118.1, 117.4, 59.9, 44.5, 43.7, 42.0, 40.0, 39.7, 38.9, 37.8, 36.0, 34.4, 32.9, 32.4, 31.4, 30.2, 29.4,
+
4
+
9.2, 28.2, 21.4, 18.1, 13.9, 10.1 ppm; HRMS (ESI): m/z calcd for C31
H
42NaO
[M+Na] : 501.2975;
found: 501.2966.
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5
.1.6.
(9β,13α,14β,20α)-N-ethyl-3-Hydroxy-9,13-dimethyl-2-oxo-24,25,26-trinoroleana-1(10),3,5,7-
tetraen-29-amide (1e) CAS: 1810867-89-0
Compound 1e was synthesized according to procedure iii in Scheme 1 with the same steps as described
for 1b and purified by flash column chromatography on silica gel (Ethyl acetate/Hexane = 1:8). Starting
2
D
4
with 50 mg celastrol, compound (25.2 mg) was obtained. Red solid, yield: 48%. Mp: 134.6-136.5 � . [α]
1
=
6
-262.6 (c 0.1, MeOH). H NMR (600 MHz, DMSO-d ) δ 8.71 (s, 1H), 7.55 (t, J = 5.32 Hz, 1H), 7.07
(
(
dd, J = 1.10, 6.97 Hz, 1H), 6.39 (d, J = 1.10 Hz, 1H), 6.33 (d, J = 7.15 Hz, 1H), 2.89 - 3.02 (m, 2H), 2.40
d, J = 15.59 Hz, 1H), 2.19 (d, J = 8.62 Hz, 1H), 2.09 (s, 3H), 2.07 (dd, J = 1.00, 6.79 Hz, 1H), 1.93 -
1
1
1
.99 (m, 1H), 1.76 - 1.86 (m, 2H), 1.63 (d, J = 7.70 Hz, 2H), 1.55 - 1.58 (m, 1H), 1.54 (br. s., 1H), 1.48 -
.52 (m, 1H), 1.42 (d, J = 14.31 Hz, 1H), 1.37 (s, 3H), 1.28 (dt, J = 3.94, 13.98 Hz, 1H), 1.22 (br. s., 1H),
.20 (s, 3H), 1.04 - 1.06 (s, 3H), 1.02 (s, 3H), 0.91 (t, J = 7.24 Hz, 3H), 0.82 - 0.87 (m, 1H), 0.54 (s, 3H)
13
ppm; C NMR (151 MHz, DMSO-d ) δ 177.9, 176.7, 168.3, 163.0, 146.4, 133.2, 126.8, 120.0, 117.9,
6
1
2
5
17.2, 44.5, 44.0, 42.0, 39.3, 38.8, 37.7, 36.1, 34.9, 33.6, 33.3, 33.0, 31.4, 30.5, 30.2, 29.4, 28.8, 28.2,
+
3
+
1.4, 17.8, 14.5, 10.1 ppm; HRMS (ESI): m/z calcd for C31
H
43NNaO
[M+Na] :500.3135, found:
00.3138.
5
.1.7. (9β,13α,14β,20α)-3-Hydroxy-9,13-dimethyl-2-oxo-24,25,26-trinoroleana-1(10),3,5,7-tetraen-29-oic
acid, 1-methylethyl ester (1f) CAS: 1105067-40-0
Compound 1f was synthesized according to procedure ii in Scheme and purified by flash column
chromatography on silica gel (Ethyl acetate/Hexane = 1:8). Starting with 50 mg celastrol, compound
2
D
4
1
(
43.3 mg) was obtained, Orange solid, yield: 79%. Mp: 105.1-107.8 � . [α]
= -246.6 (c 0.1, MeOH). H
NMR (600 MHz, DMSO-d ) δ 8.73 (s, 1H), 7.07 (d, J = 6.60 Hz, 1H), 6.38 (s, 1H), 6.35 (d, J = 6.97 Hz,
6
1
3
4
1
H), 4.70 (td, J = 6.17, 12.43 Hz, 1H), 2.33 (d, J = 15.59 Hz, 1H), 2.22 (d, J = 12.47 Hz, 1H), 2.09 (s,
H), 2.05 (d, J = 12.10 Hz, 1H), 1.94 (dt, J = 3.12, 15.59 Hz, 1H), 1.78 - 1.86 (m, 1H), 1.60 - 1.71 (m,
H), 1.50 - 1.58 (m, 3H), 1.43 (d, J = 13.94 Hz, 1H), 1.38 (s, 3H), 1.29 - 1.36 (m, 1H), 1.21 (br. s., 3H),
.14 (d, J = 6.24 Hz, 3H), 1.09 (br. s., 3H), 1.08 (d, J = 6.24 Hz, 3H), 1.07 (br. s., 3H), 0.90 (d, J = 13.57
13
Hz, 1H), 0.51 (s, 3H) ppm; C NMR (151 MHz, DMSO-d
6
) δ 178.0, 176.8, 167.8, 163.0, 146.5, 133.3,
1
2
4
26.9, 120.1, 118.1, 117.3, 66.9, 44.5, 43.6, 42.0, 39.7, 38.9, 37.7, 36.0, 34.3, 33.0, 32.3, 31.3, 30.2, 29.9,
+
+
9.3, 29.0, 28.1, 21.4, 21.3, 21.3, 18.2, 10.1 ppm; HRMS (ESI): m/z calcd for C H O [M+H] :
32
45
4
93.3312, found: 493.3315.
5
.1.8. (9β,13α,14β,20α)-N-n-propyl-3-Hydroxy-9,13-dimethyl-2-oxo-24,25,26-trinoroleana-1(10),3,5,7-
tetraen-29-amide (1g) CAS: 2027514-22-1
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Compound 1g was synthesized according to procedure iii in Scheme 1 like compound 1c. It was purified
by flash column chromatography on silica gel (Ethyl acetate/Hexane = 1:8). Red solid, 17.2 mg, yield:
2
D
4
1
3
7
1
1%. Mp: 122.3-124.4 � . [α]
= -231.4 (c 0.1, MeOH). H NMR (600 MHz, DMSO-d ) δ 8.72 (s, 1H),
6
.56 (t, J = 5.50 Hz, 1H), 7.08 (dd, J = 1.01, 7.06 Hz, 1H), 6.40 (d, J = 0.92 Hz, 1H), 6.35 (d, J = 7.34 Hz,
H), 2.89 - 2.97 (m, 1H), 2.78 - 2.85 (m, 1H), 2.42 (d, J = 15.41 Hz, 1H), 2.19 (d, J = 9.17 Hz, 1H), 2.11
(
br. s., 1H), 2.09 (s, 3H), 1.93 - 1.99 (m, 1H), 1.76 - 1.86 (m, 2H), 1.62 - 1.65 (m, J = 7.30 Hz, 2H), 1.57 -
1
1
7
1
3
.60 (m, 1H), 1.53 - 1.56 (m, 2H), 1.49 - 1.53 (m, 1H), 1.43 (d, J = 13.75 Hz, 1H), 1.38 (s, 3H), 1.31 -
.36 (m, 2H), 1.28 - 1.30 (m, 1H), 1.21 (s, 3H), 1.06 (s, 3H), 1.04 (s, 3H), 0.85 - 0.88 (m, 1H), 0.77 (t, J =
13
.43 Hz, 3H), 0.54 (s, 3H) ppm; C NMR (151 MHz, DMSO-d ) δ 177.9, 176.9, 168.3, 163.0, 146.4,
6
33.2, 126.8, 120.0, 117.9, 117.2, 44.5, 44.0, 42.0, 40.8, 39.4, 38.9, 37.7, 36.1, 34.9, 33.4, 33.0, 31.5, 30.5,
+
0.2, 29.5, 28.8, 28.2, 21.9, 21.5, 17.7, 11.5, 10.1 ppm; HRMS (ESI): m/z calcd for C32H45NNaO
3
+
[
M+Na] : 514.3292, found: 514.3297.
5
.1.9. (9β,13α,14β,20α)-3-Methoxy-9,13-dimethyl-2-oxo-24,25,26-trinoroleana-1(10),3,5,7-tetraen-29-
oic acid methyl ester (2a) CAS: 1365906-42-8
3
Compound 2a was obtained according to procedure iv in Scheme 1. (50 mg, 0.108 mmol) 1b and CH I
(
153.4 mg, 1.08 mmol) were dissolved in 2 mL THF, then 10 mol% TBAB (Tert-butyl ammonium
bromide) (3.2 mg, 0.011 mmol), 20 mol% Dioxane (400 µL) and K CO (60 mg, 0.43 mmol) were added
2
3
to the solution. The mixture was stirred at 70 � for 12 h. The mixture was quenched with 1 mol/L HCl
a.q. (5 mL), extracted with ethyl acetate (3 * 15 mL). The organic layers were combined and dried with
anhydrous sodium sulfate. Compound was purified by flash column chromatography on silica gel (Ethyl
2
D
4
acetate/Hexane = 1:8). Yellow solid, 27.7 mg, yield: 54%. Mp: 204.7-207.0 � . [α]
= -69.4 (c 0.1,
1
MeOH). H NMR (600 MHz, CHLOROFORM-d) δ 6.99 (d, J = 6.60 Hz, 1H), 6.45 (s, 1H), 6.31 (d, J =
6
2
.97 Hz, 1H), 3.85 (s, 3H), 3.56 (s, 3H), 2.42 (d, J = 15.77 Hz, 1H), 2.22 (s, 3H), 2.17 - 2.21 (m, 1H),
.10 - 2.16 (m, 1H), 2.05 (dt, J = 3.67, 14.03 Hz, 1H), 1.86 - 1.91 (m, 1H), 1.81 - 1.86 (m, 1H), 1.78 (dd,
J = 3.76, 13.48 Hz, 1H), 1.68 - 1.73 (m, 1H), 1.66 - 1.68 (m, 1H), 1.61 - 1.66 (m, 1H), 1.58 (d, J = 8.62
Hz, 1H), 1.53 - 1.57 (m, 1H), 1.50 (dd, J = 3.85, 14.67 Hz, 1H), 1.46 (s, 3H), 1.38 (dt, J = 4.40, 14.03 Hz,
13
1
H), 1.26 (s, 3H), 1.18 (s, 3H), 1.10 (s, 3H), 0.97 (d, J = 14.12 Hz, 1H), 0.54 (s, 3H) ppm; C NMR (151
MHz, CHLOROFORM-d) δ 180.7, 178.7, 170.3, 162.5, 150.3, 134.3, 132.1, 127.2, 123.5, 117.9, 60.1,
5
1
1.6, 45.1, 44.3, 42.4, 40.4, 39.2, 38.2, 36.4, 34.8, 33.6, 32.7, 31.6, 30.8, 30.5, 29.9, 29.6, 28.6, 21.9, 18.4,
+
4
+
0.8 ppm; HRMS (ESI): m/z calcd for C H O [M+H] : 479.3156; found: 479.3155.
3
1
43
5
.1.10. (9β,13α,14β,20α)-3-Ethoxy-9,13-dimethyl-2-oxo-24,25,26-trinoroleana-1(10),3,5,7-tetraen-29-oic
acid methyl ester (2b)
2
019-05-12
page 20 of 37

ACCEPTED MANUSCRIPT
Compound 2b was obtained according to procedure v in Scheme 1. (50 mg, 0.108 mmol) 1b and
CH
3 2
CH Br (117.7 mg, 1.08 mmol) were dissolved in 2 mL THF, and then 10 mol% TBAB (Tert-butyl
ammonium bromide) (3.2 mg, 0.011 mmol), 20 mol% Dioxane (400 µL) and K CO (60 mg, 0.43 mmol)
2
3
were added to the solution. After stirred at 70 � for 10 h, the mixture was quenched with 1 mol/L HCl a.q.
5 mL), and then extracted with ethyl acetate (3 * 15 mL). The organic layers were combined and dried
with anhydrous sodium sulfate. Compound was purified by flash column chromatography on silica gel
(
2
4
(
Ethyl acetate/Hexane = 1:8). Yellow solid, 44.8 mg, yield: 85%. Mp: 141.3-142.9 � . [α]
D
= -62.6 (c 0.1,
MeOH). H NMR (600 MHz, CHLOROFORM-d) δ 6.95 (d, J = 6.79 Hz, 1H), 6.41 (s, 1H), 6.29 (d, J =
.97 Hz, 1H), 4.02 - 4.21 (m, 2H), 3.56 (s, 3H), 2.42 (d, J = 15.96 Hz, 1H), 2.22 (s, 3H), 2.19 (d, J =
1.19 Hz, 1H), 2.12 (dd, J = 2.20, 13.75 Hz, 1H), 2.05 (dt, J = 3.67, 14.03 Hz, 1H), 1.86 - 1.92 (m, 1H),
1
6
1
1
1
3
1
1
3
.82 - 1.86 (m, 1H), 1.78 (dd, J = 3.48, 13.39 Hz, 1H), 1.68 - 1.72 (m, 1H), 1.65 - 1.68 (m, 1H), 1.62 -
.65 (m, 1H), 1.58 (d, J = 8.44 Hz, 1H), 1.53 - 1.57 (m, 1H), 1.49 (dd, J = 4.68, 14.58 Hz, 1H), 1.45 (s,
H), 1.37 - 1.41 (m, 1H), 1.34 (t, J = 7.06 Hz, 3H), 1.26 (s, 3H), 1.18 (s, 3H), 1.10 (s, 3H), 0.97 (d, J =
1
3
3.94 Hz, 1H), 0.55 (s, 3H) ppm; C NMR (151 MHz, CHLOROFORM-d) δ 181.0, 178.7, 169.5, 162.3,
49.4, 133.7, 132.0, 127.4, 123.6, 117.9, 68.1, 51.6, 45.0, 44.3, 42.2, 40.4, 39.2, 38.2, 36.4, 34.8, 33.5,
+
2.7, 31.6, 30.8, 30.5, 29.9, 29.6, 28.6, 21.9, 18.4, 15.8, 11.0 ppm; HRMS (ESI): m/z calcd for C H O
4
3
2
45
+
[
M+H] : 493.3312; found: 493.3311.
5
.1.11. (9β,13α,14β,20α)-3-(2,2-difluoroethoxy)-9,13-dimethyl-2-oxo-24,25,26-trinoroleana-1(10),3,5,7-
tetraen-29-oic acid methyl ester (2c)
Compound 2c was obtained according to procedure vi in Scheme 1. (100 mg, 0.215 mmol) 1b was
dissolved in 2 mL DMF, and then 1,1-difluoro-2-iodoethane (F
and K CO (59.34 mg, 0.43 mmol) were added to the solution. The mixture was stirred at 50 � for 24 h (1
day). The mixture was diluted in 50 mL ethyl acetate, and then washed with saturated lithium chloride a.q.
3 * 25 mL) and saturated brine (50 mL). The organic layers were combined and dried with anhydrous
sodium sulfate. Compound was purified by flash column chromatography on silica gel (Ethyl
2 2
CHCH I, 413 mg, 195 µL, 2.15 mmol)
2
3
(
2
4
acetate/Hexane = 1:8). Yellow solid, 64.5 mg, yield: 57%. Mp: 189.3-191.5 � . [α]
D
= -23.4 (c 0.1,
MeOH). H NMR (600 MHz, CHLOROFORM-d) δ 7.03 (dd, J = 1.01, 7.06 Hz, 1H), 6.42 (d, J = 1.10 Hz,
H), 6.32 (d, J = 7.15 Hz, 1H), 6.00 - 6.20 (m, 1H), 4.26 - 4.35 (m, 2H), 3.56 (s, 3H), 2.42 (d, J = 15.77
Hz, 1H), 2.24 (s, 3H), 2.18 - 2.23 (m, 1H), 2.14 (dd, J = 2.57, 13.75 Hz, 1H), 2.03 - 2.09 (m, 1H), 1.87 -
1
1
1
1
1
1
.92 (m, 1H), 1.82 - 1.87 (m, 1H), 1.79 (dd, J = 5.41, 13.66 Hz, 1H), 1.69 - 1.73 (m, 1H), 1.66 - 1.69 (m,
H), 1.62 - 1.66 (m, 1H), 1.58 (d, J = 8.44 Hz, 1H), 1.55 (dd, J = 4.13, 6.51 Hz, 1H), 1.50 (dd, J = 4.31,
5.13 Hz, 1H), 1.46 (s, 3H), 1.35 - 1.41 (m, 1H), 1.27 (s, 3H), 1.18 (s, 3H), 1.10 (s, 3H), 0.98 (d, J =
1
3
4.12 Hz, 1H), 0.55 (s, 3H) ppm; C NMR (151 MHz, CHLOROFORM-d) δ 180.0, 178.7, 170.8, 162.8,
2
019-05-12
page 21 of 37

ACCEPTED MANUSCRIPT
1
4
48.5, 134.9, 131.8, 126.9, 123.4, 118.0, 114.4 (t, J = 239.90 Hz), 70.6 (t, J = 28.61 Hz), 51.6, 45.1, 44.3,
2.5, 40.4, 39.3, 38.2, 36.4, 34.8, 33.6, 32.7, 31.6, 30.9, 30.5, 29.9, 29.6, 28.6, 21.9, 18.4, 10.7 ppm;
+
4
+
HRMS (ESI): m/z calcd for C H F O [M+H] : 529.3124; found: 529.3124.
32
43
2
5
.1.12. (9β,13α,14β,20α)-2,3-Dihydroxy-9,13-dimethyl-24,25,26-trinoroleana-1(10),3,5,7-tetraen-29-oic
acid (3a) CAS: 193957-88-9
Compound 3a was obtained as white solid according to procedure vii in Scheme 1. 50 mg (0.11 mmol)
celastrol was dissolved in 2 mL CD OD and stirred in a 25-mL flask, and then 44 mg (1.1 mmol, 10
3
equiv) NaBH was added. The mixture was kept at room temperature for 30 min. After that, 0.1 mol/L
4
HCl (3 mL) was added to quench the reaction, and subsequently the aqueous phase was separated and
extracted with DCM (15 mL) three times. The organic layers were combined and dried with anhydrous
sodium sulfate. Compound was purified by removing the solvent under vacuum conditions. White solid,
2
D
4
1
4
1
6
1
1
1
9.7 mg, yield: 99%. Mp: 178.3-180.1 � . [α]
= -4.0 (c 0.1, MeOH). H NMR (600 MHz, DMSO-d ) δ
6
2.05 (br. s., 1H), 8.80 (s, 1H), 7.82 (s, 1H), 6.61 (s, 1H), 5.72 (dd, J = 1.74, 6.14 Hz, 1H), 3.18 (dd, J =
.24, 20.54 Hz, 1H), 2.91 (dd, J = 1.47, 19.99 Hz, 1H), 2.34 (d, J = 15.59 Hz, 1H), 2.04 (d, J = 13.57 Hz,
H), 2.01 (s, 3H), 1.94 - 2.00 (m, 2H), 1.86 (dt, J = 5.00, 13.80 Hz, 1H), 1.79 (dt, J = 6.50, 13.80 Hz, 1H),
.63 - 1.69 (m, 1H), 1.59 - 1.63 (m, 1H), 1.53 - 1.59 (m, 1H), 1.43 - 1.51 (m, 3H), 1.36 - 1.41 (m, 1H),
.29 (dt, J = 4.40, 13.70 Hz, 1H), 1.22 (s, 3H), 1.17 (s, 3H), 1.11 (s, 3H), 1.05 (s, 3H), 0.85 (d, J = 13.94
13
Hz, 1H), 0.66 (s, 3H) ppm; C NMR (151 MHz, DMSO-d ) δ 179.5, 149.2, 143.1, 140.6, 139.4, 123.1,
6
1
2
4
20.1, 117.7, 108.2, 43.8, 43.3, 39.4, 37.1, 36.6, 36.1, 34.4, 34.1, 34.1, 32.4, 31.4, 30.2, 30.1, 29.8, 29.5,
+
+
4
8.4, 27.3, 22.7, 18.1, 11.5 ppm; HRMS (ESI): m/z calcd for C29H40NaO [M+Na] : 475.2819, found:
75.2823.
5
1
.1.13.
(6β,9β,13α,14β,20α)-2,3-Dihydroxy-9,13-dimethyl-6-(nitromethyl)-24,25,26-trinoroleana-
(10),3,5,7-tetraen-29-oic acid (3b) CAS: 1350630-32-8
Compound 3b was obtained according to procedure viii in Scheme 1. Nitromethane (14.04 mg, 0.23
mmol) and 51.6 mg (0.115 mmol) celastrol were dissolved in 1 mL THF, and then 1 M solution of TBAF
in THF (600 µL, 0.6 mmol) was added. The mixture was stirred at room temperature for 1 h. After
quenched with water (20 mL), the solution was extracted with ethyl acetate (3 * 20 mL). The organic
layers were combined and dried with anhydrous sodium sulfate. Compound was purified by flash column
chromatography on silica gel (Ethyl acetate/Hexane = 1:2). Light brown solid, 49.0 mg, yield: 87%. Mp:
2
D
4
1
1
99.3-202.4 � . [α]
= -80.0 (c 0.1, MeOH). H NMR (600 MHz, DMSO-d ) δ 12.08 (br. s., 1H), 9.14 (br.
6
s., 1H), 8.11 (br. s., 1H), 6.69 (s, 1H), 5.62 (d, J = 6.05 Hz, 1H), 4.77 (dd, J = 3.94, 11.28 Hz, 1H), 4.10 -
.15 (m, 1H), 4.07 (d, J = 11.19 Hz, 1H), 2.33 (d, J = 15.59 Hz, 1H), 2.15 (s, 3H), 2.00 - 2.08 (m, 2H),
4
2
019-05-12
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ACCEPTED MANUSCRIPT
1
2
1
1
3
C
.92 - 1.99 (m, 1H), 1.79 (dt, J = 6.14, 13.98 Hz, 1H), 1.63 - 1.71 (m, 2H), 1.60 (dd, J = 8.25, 15.77 Hz,
H), 1.46 - 1.53 (m, 2H), 1.40 (s, 3H), 1.30 - 1.36 (m, 1H), 1.22 - 1.30 (m, 2H), 1.17 (s, 3H), 1.10 (s, 3H),
1
3
.05 (s, 3H), 0.82 - 0.89 (m, 1H), 0.62 (s, 3H) ppm; C NMR (151 MHz, DMSO-d ) δ 179.5, 153.3,
6
44.4, 141.4, 140.6, 120.6, 120.5, 117.6, 109.0, 80.9, 43.9, 43.6, 39.4, 37.4, 37.0, 36.3, 36.2, 35.8, 35.3,
4.5, 32.4, 31.4, 30.2, 30.0, 29.8, 29.4, 28.5, 22.1, 18.0, 11.1 ppm; HRMS (ESI): m/z calcd for
-
6
−
30
H
40NO
[M] : 510.2861; found: 510.2856.
5
1
.1.14.
(6β,9β,13α,14β,20α)-2,3-Dihydroxy-9,13-dimethyl-6-(2-oxopropyl)-24,25,26-trinoroleana-
(10),3,5,7-tetraen-29-oic acid (3c) CAS: 1643802-69-0
Compound 3c was obtained according to procedure ix in Scheme 1. 50 mg (0.11 mmol) celastrol was
dissolved in 4 mL acetone and then stirred in a 25-mL flask. After that, 1 drop concentrated HCl (12
mol/L) was added as catalyst and the mixture was kept at room temperature for 12 h. After the reaction
finished, the solvent was removed under vacuum conditions. Compound was purified by flash column
chromatography on silica gel (Ethyl acetate/Hexane = 1:4). White solid, 28.8 mg, yield: 51%. Mp: 121.6-
2
4
1
1
7
22.7 � . [α]
.91 (s, 1H), 6.63 (s, 1H), 5.72 (d, J = 6.24 Hz, 1H), 3.75 (ddd, J = 2.84, 6.10, 10.87 Hz, 1H), 2.67 (dd, J
2.75, 16.14 Hz, 1H), 2.31 - 2.36 (m, 1H), 2.29 (d, J = 11.00 Hz, 1H), 2.10 (s, 3H), 2.03 - 2.05 (m, 1H),
D
6
= -72.6 (c 0.1, MeOH). H NMR (600 MHz, DMSO-d ) δ 12.06 (br. s., 1H), 8.91 (s, 1H),
=
2
1
1
1
1
3
.03 (s, 3H), 2.00 (d, J = 13.76 Hz, 1H), 1.93 - 1.98 (m, 1H), 1.74 - 1.82 (m, 1H), 1.66 - 1.72 (m, 1H),
.61 - 1.65 (m, 1H), 1.58 - 1.61 (m, 1H), 1.52 - 1.58 (m, 1H), 1.48 (d, J = 8.44 Hz, 1H), 1.42 - 1.47 (m,
H), 1.39 (s, 3H), 1.33 - 1.38 (m, 1H), 1.28 - 1.33 (m, 1H), 1.22 - 1.28 (m, 1H), 1.15 (s, 3H), 1.10 (s, 3H),
1
3
6
.04 (s, 3H), 0.79 - 0.90 (m, 1H), 0.62 (s, 3H) ppm; C NMR (151 MHz, DMSO-d ) δ 207.5, 179.5,
49.4, 143.4, 141.2, 140.0, 126.1, 121.6, 119.6, 108.6, 59.8, 51.3, 43.9, 43.3, 39.4, 37.3, 36.5, 36.4, 36.3,
5.1, 34.5, 32.5, 32.4, 31.4, 30.3, 30.2, 30.0, 29.9, 29.4, 28.6, 18.0, 11.2 ppm; HRMS (ESI): m/z calcd for
-
5
-
C H O [M] : 507.3116; found: 507.3109.
3
2
43
5
1
.1.15.
(6β,9β,13α,14β,20α)-2,3-Dihydroxy-9,13-dimethyl-6-(1H-pyrrol-3-yl)-24,25,26-trinoroleana-
(10),3,5,7-tetraen-29-oic acid (3d)
Compound 3d was obtained according to procedure x in Scheme 1. 50 mg (0.11 mmol) celastrol was
dissolved in 1 mL pyrrole in a 10-mL closed tube, and then 20 µL sodium methoxide was added. The
mixture was stirred at room temperature for 5 minutes. When the reaction was finished, 1 mol/L HCl a. q.
(
5 mL) was added to stop the reaction and then subsequently the aqueous phase was separated and
extracted with ethyl acetate (20 mL) three times. The organic layers were combined and dried with
anhydrous sodium sulfate. Then the solvent was removed in vacuo, giving a crude mixture, which was
purified by flash chromatography on silica column (Hexane/Ethyl acetate/AcOH = 4:1:0.005) to give the
2
019-05-12
page 23 of 37

ACCEPTED MANUSCRIPT
2
D
4
1
pure product. Wine red solid, yield: 82%. Mp: >300 � . [α]
= -58.0 (c 0.1, MeOH). H-NMR (600 MHz,
DMSO-d ) δ 12.06 (br. s., 1H), 10.38 (d, J = 1.83 Hz, 1H), 8.89 (s, 1H), 7.83 (s, 1H), 6.66 (s, 1H), 6.51 (d,
6
J = 1.65 Hz, 1H), 6.00 (d, J = 6.60 Hz, 1H), 5.75 (q, J = 2.63 Hz, 1H), 5.12 (d, J = 0.55 Hz, 1H), 4.52 (d,
J = 6.24 Hz, 1H), 2.33 (d, J = 15.41 Hz, 1H), 2.04 (d, J = 15.59 Hz, 1H), 1.99 - 2.01 (m, 1H), 1.95 - 1.99
(
m, 1H), 1.87 (s, 3H), 1.77 - 1.82 (m, 1H), 1.73 - 1.77 (m, 1H), 1.61 - 1.67 (m, 1H), 1.55 - 1.61 (m, 1H),
1
1
1
1
3
C
.52 - 1.55 (m, 1H), 1.48 - 1.52 (m, 1H), 1.47 (d, J = 8.07 Hz, 1H), 1.39 (dd, J = 3.39, 13.85 Hz, 1H),
.28 - 1.33 (m, 1H), 1.25 - 1.28 (m, 1H), 1.24 (s, 3H), 1.10 (s, 3H), 1.06 (s, 3H), 1.04 (s, 3H), 0.86 (d, J =
1
3
1.37 Hz, 1H), 0.67 (s, 3H) ppm; C-NMR (151 MHz, DMSO-d ) δ 179.5, 147.6, 143.5, 140.8, 140.3,
6
34.5, 124.8, 121.5, 121.1, 115.8, 108.3, 106.7, 103.4, 43.8, 43.2, 39.4, 39.2, 37.2, 36.5, 36.4, 35.1, 34.9,
4.5, 32.4, 31.4, 30.2, 30.1, 29.8, 29.5, 28.4, 22.0, 18.1, 11.4 ppm; HRMS (ESI): m/z calcd for
-
4
-
33
H
42NO
[M] : 516.3119; found: 516.3118.
5
.1.16.
(6β,9β,13α,14β,20α)-2,3-Dihydroxy-9,13-dimethyl-6-(dimethoxyphosphoryl)-24,25,26-
trinoroleana-1(10),3,5,7-tetraen-29-oic acid (3e)
Compound 3e was obtained as white solid according to procedure xi in Scheme 1. 50 mg (0.11 mmol)
celastrol, and 123 mg dimethyl phosphonate (1.1 mmol) were dissolved in 2 mL dichloromethane and
stirred in a 50-mL closed tube, and then 2.7 mg (0.1 equiv, 5 mol%) AlCl ·6H O was added as a catalyst.
3
2
The mixture was kept at room temperature for 6 h. After the reaction finished, saturated sodium chloride
solution (15 mL) was added to quench the reaction, and then subsequently the aqueous phase was
separated and extracted with ethyl acetate (15 mL) three times. The organic layers were combined and
dried with anhydrous sodium sulfate. Compound was purified by flash column chromatography on silica
24
gel (Ethyl acetate/Hexane = 1:2). White solid, 34.2 mg, yield: 55%. Mp: 199.0-201.4 � . [α] = -91.34 (c
D
1
0
1
.1, MeOH). H NMR (600 MHz, DMSO-d ) δ 12.04 (br. s., 1H), 9.04 (br. s., 1H), 7.97 (s, 1H), 6.66 (s,
6
H), 5.64 (dd, J = 3.21, 6.33 Hz, 1H), 4.19 (dd, J = 6.05, 29.34 Hz, 1H), 3.51 (d, J = 8.99 Hz, 3H), 3.49
(
d, J = 8.80 Hz, 3H), 2.32 (d, J = 15.59 Hz, 1H), 2.13 (d, J = 0.92 Hz, 3H), 2.04 (d, J = 13.20 Hz, 1H),
1
1
1
1
.98 - 2.01 (m, 1H), 1.92 - 1.98 (m, 1H), 1.81 (dt, J = 5.87, 13.85 Hz, 1H), 1.60 - 1.64 (m, 1H), 1.58 -
.60 (m, J = 7.90 Hz, 2H), 1.57 (s, 3H), 1.54 - 1.56 (m, 1H), 1.51 - 1.54 (m, 1H), 1.49 (d, J = 8.07 Hz,
H), 1.43 (d, J = 5.32 Hz, 1H), 1.40 (d, J = 4.95 Hz, 1H), 1.28 (dt, J = 4.40, 13.66 Hz, 1H), 1.18 (s, 3H),
13
6
.10 (s, 3H), 1.06 (s, 3H), 0.86 (d, J = 12.84 Hz, 1H), 0.61 (s, 3H) ppm; C NMR (151 MHz, DMSO-d )
δ 179.5, 150.6 (d, J = 12.10 Hz, 1 C), 144.1, 141.1 (d, J = 3.30 Hz, 1 C), 140.5 (d, J = 6.60 Hz, 1 C),
1
6
3
21.3 (d, J = 4.40 Hz, 1 C), 117.9 (d, J = 7.70 Hz, 1 C), 114.6 (d, J = 12.10 Hz, 1 C), 109.3, 52.8 (d, J =
.60 Hz, 1 C), 52.3 (d, J = 6.60 Hz, 1 C), 43.9, 38.8, 37.9, 37.4, 36.4, 34.6, 33.6, 33.5, 32.4, 31.5, 31.0,
31
0.2, 30.0 (d, J = 13.20 Hz, 1 C), 29.5, 28.8, 22.1, 21.6 (d, J = 6.60 Hz, 1 C), 18.0, 14.0, 12.6 ppm; P
2
019-05-12
page 24 of 37

ACCEPTED MANUSCRIPT
+
+
NMR (243 MHz, DMSO-d
6
7
) δ 27.3 ppm; HRMS (ESI): m/z calcd for C31H45NaO P [M+Na] : 583.2795,
found: 583.2794.
ꢀ
5
1
.1.17.
(6β,9β,13α,14β,20α)-2-Hydroxy-3-(phenylmethoxy)-6,9,13-trimethyl-24,25,26-trinoroleana-
(10),3,5,7-tetraen-29-oic acid benzyl ester (4a) CAS: 2095711-55-8
Compound 4a was obtained according to procedure xii in Scheme 1 (two steps). To a solution of celastrol
300 mg, 0.67 mmol) in DMF (6 mL) K CO (184 mg, 1.33 mmol), PhCH Br (350 µL, 2.94 mmol) and
(
2
3
2
Et N (200 µL) were added sequentially. The reaction was heated at 80 � for 2.5 hours. After quenched by
3
ice-water (30 mL), the mixture was exacted by ethyl acetate (50 mL) and then washed by pure-water (30
mL) and brine (30 mL). The organic layers were combined and dried with anhydrous sodium sulfate and
then concentrated in vacuo. The intermediate product was purified by flash column chromatography on
silica gel (Ethyl acetate/Hexane = 1:4). Yellow solid, 285.6 mg, yield: 68%.
To a solution of intermediate product (300 mg, 0.45 mmol) in THF (10 mL), 3 M MeMgBr in THF
solution (3.3 mL, 10 mmol) was added at 0 � . The reaction was stirred at 0 � for 1 hour. The reaction
was quenched by addition of pure-water (30 mL) and extracted with ethyl acetate (20 mL) three times.
The organic layers were combined and dried with anhydrous sodium sulfate and then concentrated in
vacuo. Our compound was purified by flash column chromatography on silica gel (Ethyl acetate/Hexane
2
D
4
=
1:8) to get the product 4a. Pale yellow solid, 254.6 mg, yield: 83%. Mp: 103.7-105.0 � . [α] = -30.0 (c
1
0
1
7
.1, MeOH). H NMR (600 MHz, DMSO-d
6
) δ 9.00 (s, 1H), 7.50 (d, J = 1.10 Hz, 1H), 7.47 - 7.49 (m,
H), 7.38 - 7.40 (m, 1H), 7.36 - 7.38 (m, 1H), 7.34 - 7.36 (m, 1H), 7.33 - 7.34 (m, J = 1.10, 1.10 Hz, 1H),
.32 (d, J = 6.97 Hz, 1H), 7.28 - 7.30 (m, 2H), 6.73 (s, 1H), 5.73 (d, J = 6.24 Hz, 1H), 4.97 (d, J = 12.47
Hz, 1H), 4.91 (s, 1H), 4.89 (s, 1H), 4.84 (d, J = 11.00 Hz, 1H), 3.35 - 3.40 (m, 1H), 2.36 (d, J = 15.59 Hz,
1
1
1
1
6
H), 2.12 (s, 3H), 2.09 (d, J = 13.75 Hz, 1H), 1.98 (dt, J = 3.85, 13.94 Hz, 1H), 1.92 (d, J = 12.84 Hz,
H), 1.75 - 1.84 (m, 1H), 1.66 (dd, J = 8.07, 15.96 Hz, 1H), 1.57 - 1.63 (m, 1H), 1.55 (d, J = 2.75 Hz, 1H),
.50 - 1.53 (m, 1H), 1.49 (d, J = 8.07 Hz, 1H), 1.46 (br. s., 1H), 1.45 (d, J = 2.93 Hz, 1H), 1.40 (s, 3H),
.38 (d, J = 9.54 Hz, 1H), 1.33 - 1.37 (m, 1H), 1.25 - 1.30 (m, 1H), 1.16 (s, 3H), 1.14 (s, 3H), 1.08 (d, J =
13
.97 Hz, 3H), 1.06 (s, 3H), 0.89 (d, J = 13.94 Hz, 1H), 0.49 (s, 3H) ppm; C NMR (151 MHz, DMSO-d )
6
δ 177.1, 148.0, 147.4, 144.5, 142.6, 138.2, 135.9, 128.4 (2 C), 128.3, 128.2 (2 C), 128.0, 127.9 (2 C),
1
3
27.8 (2 C), 127.6, 126.8, 124.6, 110.3, 73.2, 65.6, 43.8, 43.1, 39.8, 37.2, 36.9, 36.8, 36.4, 34.8, 34.3,
2.4, 31.5, 31.3, 30.1, 30.0, 29.9, 29.4, 28.5, 22.2, 22.0, 17.8, 11.5 ppm; HRMS (ESI): m/z calcd for
+
4
+
C H NaO [M+Na] : 669.3914; found: 669.3911.
4
4
54
5
2
.1.18. (6β,9β,13α,14β,20α)-2,3-Dihydroxy-6,9,13-trimethyl-24,25,26-trinoroleana-1(10),3,5,7-tetraen-
9-oic acid (4b) CAS: 2095711-56-9
2
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ACCEPTED MANUSCRIPT
Compound 4b was obtained according to procedure xiii in Scheme 1. To a solution of 4a (130 mg, 0.20
mmol) in MeOH (6 mL), 20% Pd/C (26 mg) was added. The solution was hydrogenated with a balloon of
hydrogen, and then kept at room temperature for 12 hours. After filtered by celite, the filterate was
concentrated in vacuo. The residue was purified by flash column chromatography on silica gel (Ethyl
24
acetate/Hexane = 1:4). Gray solid, 78.6 mg, yield: 87%. Mp: 139.3-140.8 � . [α] = -79.4 (c 0.1, MeOH).
D
1
H NMR (600 MHz, DMSO-d
d, J = 5.87 Hz, 1H), 3.35 - 3.39 (m, 1H), 2.33 (d, J = 15.59 Hz, 1H), 2.06 (s, 3H), 2.02 (d, J = 17.06 Hz,
H), 1.94 - 1.99 (m, 1H), 1.76 - 1.86 (m, 1H), 1.65 - 1.73 (m, 1H), 1.60 - 1.65 (m, 1H), 1.59 (d, J = 7.52
Hz, 1H), 1.52 - 1.57 (m, 1H), 1.48 - 1.51 (m, 1H), 1.47 - 1.48 (m, 1H), 1.44 - 1.47 (m, 1H), 1.40 (br. s.,
6
) δ 12.06 (br. s., 1H), 8.82 (br. s., 1H), 7.83 (br. s., 1H), 6.61 (s, 1H), 5.73
(
1
1
6
1
3
H), 1.39 (s, 3H), 1.28 - 1.32 (m, 1H), 1.25 (d, J = 10.09 Hz, 1H), 1.17 (s, 3H), 1.10 (s, 3H), 1.07 (d, J =
13
.79 Hz, 3H), 1.05 (s, 3H), 0.86 (d, J = 6.42 Hz, 1H), 0.64 (s, 3H) ppm; C NMR (151 MHz, DMSO-d ) δ
6
79.5, 147.7, 143.0, 141.0, 139.5, 128.1, 124.5, 119.7, 108.6, 43.9, 43.1, 37.3, 36.9, 36.5, 36.5, 35.1, 34.5,
2.4, 31.4, 31.4, 30.9, 30.1, 30.0, 29.9, 29.4, 28.5, 22.3, 22.1, 17.9, 11.3 ppm; HRMS (ESI): m/z calcd for
-
4
-
C H O [M] : 465.3010; found: 465.3010.
3
0
41
5
.1.19. (6β,9β,13α,14β,20α)-2,3-[(1-methylethylidene)bis(oxy)]-9,13-dimethyl-6-(nitromethyl)-24,25,26-
trinoroleana-1(10),3,5,7-tetraen-29-oic acid (5a) CAS: 2095710-94-2
Compound 5a was obtained according to procedure i in Scheme 2. To a solution of 3b (337 mg, 0.66
mmol) in DCM (4 mL) was added (MeO) CMe (3.24 mL, 2.75 g, 26.4 mmol) followed by p-TsOH·H O
2
2
2
(
(
18.9 mg, 0.132 mmol). The reaction was stirred at 35 � overnight. The solution was diluted with DCM
50 mL), and then washed with sat. NaHCO a. q. (50 mL) followed by brine (50 mL). The final solution
3
was dried with anhydrous sodium sulfate and concentrated in vacuo. The crude product was purified by
flash column chromatography on silica gel (Ethyl acetate/Hexane = 1:4). White solid, 228.0 mg, yield:
2
D
4
1
6
1
3%. Mp: 174.5-176.2 � . [α]
= -92.6 (c 0.1, MeOH). H NMR (600 MHz, CHLOROFORM-d) δ 6.67 (s,
H), 5.72 (d, J = 6.24 Hz, 1H), 4.54 (dd, J = 4.03, 11.37 Hz, 1H), 4.21 - 4.27 (m, 1H), 4.11 (t, J = 11.19
Hz, 1H), 2.39 (d, J = 15.77 Hz, 1H), 2.24 (s, 3H), 2.08 - 2.14 (m, 1H), 2.02 - 2.08 (m, 1H), 1.95 (dt, J =
3
3
1
.94, 14.17 Hz, 1H), 1.82 - 1.89 (m, 1H), 1.76 - 1.81 (m, 1H), 1.70 - 1.74 (m, 1H), 1.69 (s, 3H), 1.65 (s,
H), 1.60 - 1.64 (m, 1H), 1.55 - 1.60 (m, 1H), 1.51 (d, J = 7.89 Hz, 1H), 1.47 (s, 3H), 1.40 - 1.44 (m, 1H),
.38 (d, J = 16.32 Hz, 1H), 1.34 (d, J = 4.58 Hz, 1H), 1.28 - 1.32 (m, 1H), 1.21 (s, 3H), 1.12 (s, 3H), 1.04
13
(
s, 3H), 0.84 - 0.89 (m, 1H), 0.61 (s, 3H) ppm; C NMR (151 MHz, CHLOROFORM-d) δ 183.9, 154.4,
1
3
46.5, 144.4, 143.5, 122.4, 117.4, 117.4, 114.2, 103.0, 80.8, 44.2, 44.1, 40.2, 38.1, 37.6, 36.5, 36.3, 36.0,
5.8, 34.5, 32.7, 31.5, 30.5, 30.5, 30.1, 29.5, 28.7, 26.1, 26.0, 22.1, 18.8, 11.1 ppm; HRMS (ESI): m/z
-
6
-
calcd for C H NO [M] : 550.3174; found: 550.3174.
33
44
2
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ACCEPTED MANUSCRIPT
5
2
.1.20. (6β,9β,13α,14β,20α)-2,3-[(1-methylethylidene)bis(oxy)]-9,13-dimethyl-6-(aminomethyl)-24,25,
6-trinoroleana-1(10),3,5,7-tetraen-29-oic acid (5b) CAS: 2095710-95-3
Compound 5b was obtained according to procedure ii in Scheme 2. To a solution of 5a (300 mg, 0.54
mmol) in EtOH (10 mL) was added Pd/C (54 mg) followed by H NNH ·H O (270 mg, 5.4 mmol). The
2
2
2
reaction was stirred at 75 � overnight. The solution was filtered, and the filtrate was concentrated in
vacuo. Compound was purified by flash column chromatography on silica gel (CH Cl /MeOH = 15:1).
= -83.3 (c 0.1, DCM). H NMR (600 MHz,
DMSO-d ) δ 6.68 (br. s., 1H), 5.91 (d, J = 5.87 Hz, 1H), 3.45 (br. s., 2H), 2.85 (d, J = 11.55 Hz, 1H), 2.34
2
2
2
D
4
1
White solid, 102.1 mg, yield: 36%. Mp: 209.2-211.7 � . [α]
6
(
br. s., 1H), 2.32 (br. s., 1H), 2.10 (s, 3H), 2.04 - 2.09 (m, 1H), 2.02 (d, J = 9.54 Hz, 1H), 1.94 - 2.00 (m,
1
7
1
H), 1.74 - 1.86 (m, 1H), 1.63 - 1.68 (m, 1H), 1.62 (s, 3H), 1.59 (br. s., 3H), 1.56 (br. s., 1H), 1.49 (d, J =
.15 Hz, 1H), 1.43 - 1.47 (m, 1H), 1.39 - 1.43 (m, 1H), 1.38 (s, 3H), 1.29 - 1.36 (m, 1H), 1.25 - 1.29 (m,
H), 1.22 - 1.25 (m, 1H), 1.20 (s, 3H), 1.09 (s, 3H), 1.06 (s, 3H), 0.86 (d, J = 7.89 Hz, 1H), 0.63 (s, 3H)
13
ppm; C NMR (151 MHz, METHANOL-d ) δ 185.8, 155.7, 147.8, 145.8, 144.5, 131.0, 125.5, 119.0,
4
1
3
5
18.5, 115.3, 104.0, 47.5, 46.3, 45.5, 42.4, 39.5, 39.4, 38.4, 37.5, 37.3, 37.0, 36.5, 32.3, 32.3, 32.0, 31.9,
+
+
4
1.6, 30.3, 26.5, 26.3, 22.5, 19.3, 11.3; HRMS (ESI): m/z calcd for C33H48NO [M+H] : 522.3578; found:
22.3582.
5
1
.1.21.
(6β,9β,13α,14β,20α)-2,3-Hydroxy-9,13-dimethyl-6-(aminomethyl)-24,25,26--trinoroleana-
(10),3,5,7-tetraen-29-oic acid (5c)
Compound 5c was obtained according to procedure iii in Scheme 2. To a solution of 5b (70 mg, 0.14
mmol) in CH CN/Water mixed solvent (3.3 mL, 10:1 v/v) was added In(OTf) (78 mg, 0.14 mmol). The
3
3
reaction was stirred under microwave conditions at 120 � for 3 h. After quenched with water (5 mL), the
mixture was extracted with ethyl acetate (3 * 10 mL), and then washed with 5 mL brine. The organic
layers were combined and dried with anhydrous sodium sulfate. Compound was purified by flash column
chromatography on silica gel (CH
2
Cl
2
/MeOH = 10:1). Pale yellow solid, 49.7 mg, yield: 77%. Mp: 229.1-
2
D
4
1
2
1
1
2
33.4 � . [α]
= -66.0 (c 0.1, MeOH). H NMR (600 MHz, DMSO-d ) δ 11.90 (br. s., 1H), 9.03 (br. s,
6
H), 8.06 (br. s., 1H), 7.68 (br. s, 2H), 6.66 (s, 1H), 5.87 (d, J = 6.24 Hz, 1H), 3.59 (ddd, J = 3.58, 5.91,
1.23 Hz, 1H), 2.95 (dd, J = 3.39, 12.20 Hz, 1H), 2.40 (t, J = 11.83 Hz, 1H), 2.33 (d, J = 15.59 Hz, 1H),
.10 (s, 3H), 2.03 - 2.07 (m, 1H), 2.02 (d, J = 3.85 Hz, 1H), 1.94 - 2.00 (m, 1H), 1.77 - 1.86 (m, 1H), 1.71
(
dd, J = 5.78, 12.20 Hz, 1H), 1.64 - 1.68 (m, 1H), 1.63 (d, J = 5.32 Hz, 1H), 1.60 - 1.62 (m, 1H), 1.55 -
1
.59 (m, 1H), 1.50 (d, J = 8.07 Hz, 1H), 1.44 - 1.49 (m, 1H), 1.40 - 1.44 (m, 1H), 1.39 (s, 3H), 1.29 (dt, J
13
=
4.58, 13.66 Hz, 1H), 1.23 (s, 3H), 1.11 (s, 3H), 1.07 (s, 3H), 0.85 - 0.90 (m, 1H), 0.62 (s, 3H) ppm; C
NMR (151 MHz, DMSO-d ) δ 179.5, 152.5, 144.1, 141.4, 140.3, 122.4, 120.2, 118.3, 108.9, 45.3, 43.9,
6
2
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ACCEPTED MANUSCRIPT
4
3.8, 39.4, 37.3, 36.8, 36.6, 36.4, 35.3, 35.1, 34.5, 32.4, 31.4, 30.2, 30.0, 29.8, 29.4, 27.8, 21.4, 18.0, 11.5
+
+
4
ppm; HRMS (ESI): m/z calcd for C30H44NO [M+H] : 482.3265; found: 482.3251.
5
1
.1.22.
(9β,13α,14β,20α)-2,3-[(1-methylethylidene)bis(oxy)]-9,13-dimethyl-24,25,26-trinoroleana-
(10),3,5,7-tetraen-29-oic acid (6a) CAS: 2095710-88-4
Compound 6a was obtained according to procedure i in Scheme 3. To a solution of 3a (150 mg, 0.33
mmol) in DCM (2 mL) was added (MeO) CMe (407 µL, 345.1 mg, 3.3 mmol) followed by p-TsOH·H
6.3 mg, 0.033 mmol). The reaction was stirred at room temperature overnight. The solution was diluted
2
2
2
O
(
with DCM (40 mL), washed with saturated sodium bicarbonate (2 * 20 mL) and brine (50 mL). After that,
the organic phase was dried with anhydrous sodium sulfate and concentrated in vacuo. The residue was
purified by flash column chromatography on silica gel (Ethyl acetate/Hexane = 1:10). White solid, 93.6
2
D
4
1
mg, yield: 57%. Mp: 164.2-166.4 � . [α]
= -20.6 (c 0.1, MeOH). H NMR (600 MHz, DMSO-d ) δ 11.98
6
(
br. s., 1H), 6.63 (s, 1H), 5.72 (dd, J = 1.47, 6.79 Hz, 1H), 3.21 (dd, J = 6.42, 20.36 Hz, 1H), 2.94 (d, J =
1
2
1
1
9.62 Hz, 1H), 2.34 (d, J = 15.59 Hz, 1H), 2.05 - 2.08 (m, 1H), 2.02 - 2.05 (m, 1H), 2.02 (s, 3H), 1.95 -
.00 (m, 1H), 1.80 - 1.85 (m, 1H), 1.75 - 1.80 (m, 1H), 1.63 - 1.69 (m, 1H), 1.60 (s, 3H), 1.59 (s, 3H),
.58 (d, J = 8.25 Hz, 1H), 1.52 - 1.57 (m, 1H), 1.46 - 1.51 (m, 3H), 1.36 - 1.42 (m, 1H), 1.25 - 1.32 (m,
H), 1.24 (s, 3H), 1.18 (s, 3H), 1.10 (s, 3H), 1.04 (s, 3H), 0.87 (d, J = 12.65 Hz, 1H), 0.67 (s, 3H) ppm;
13
C NMR (151 MHz, DMSO-d ) δ 179.4, 149.0, 144.8, 142.9, 141.2, 124.8, 117.1, 116.5, 113.9, 101.6,
6
4
2
3.7, 43.2, 39.1, 37.0, 36.7, 36.5, 34.4, 34.0, 33.7, 32.4, 31.3, 30.1, 30.0, 29.7, 29.4, 28.4, 26.9, 25.7 (2 C),
-
-
43 4
2.6, 18.0, 11.4 ppm; HRMS (ESI): m/z calcd for C32H O [M] : 491.3167; found: 491.3172.
5
1
.1.23. (9β,13α,14β,20α)-2,3-[(1-methylethylidene)bis(oxy)]-9,13-dimethyl-6-oxo-24,25,26-trinoroleana-
(10),3,5,7-tetraen-29-oic acid (6b) CAS: 2095710-97-5
Compound 6b was obtained according to procedure ii in Scheme 3. To a solution of 6a (80 mg, 0.16
mmol) in DCM (2 mL) was added 3, 5-dimethylpyrazole (30.7 mg, 0.32 mmol) followed by PDC (60.2
mg, 0.16 mmol). The reaction was stirred at room temperature overnight. The solution was filtered
through celite and concentrated in vacuo. The residue was purified by flash column chromatography on
24
silica gel (Ethyl acetate/Hexane = 1:10). White solid, 39.7 mg, yield: 48%. Mp: 223.1-225.8 � . [α] = -
D
1
7
2
1
1
3
7.4 (c 0.1, MeOH). H NMR (600 MHz, CHLOROFORM-d) δ 6.71 (s, 1H), 6.24 (s, 1H), 2.55 (s, 3H),
.38 - 2.42 (m, 1H), 2.15 - 2.19 (m, 1H), 2.11 - 2.15 (m, 1H), 1.96 - 2.05 (m, 1H), 1.88 - 1.94 (m, 1H),
.83 - 1.87 (m, 1H), 1.77 - 1.83 (m, 1H), 1.70 (s, 3H), 1.67 (s, 3H), 1.65 (br. s., 1H), 1.60 - 1.64 (m, 1H),
.52 - 1.60 (m, 3H), 1.51 (s, 3H), 1.45 (d, J = 14.86 Hz, 1H), 1.34 (dt, J = 4.13, 14.26 Hz, 1H), 1.26 (s,
13
H), 1.14 (s, 3H), 1.08 (s, 3H), 0.90 - 0.94 (m, 1H), 0.64 (s, 3H) ppm; C NMR (151 MHz,
CHLOROFORM-d) δ 187.5, 183.5, 171.8, 153.3, 150.0, 145.2, 125.6, 123.1, 119.6, 118.2, 102.3, 44.7,
2
019-05-12
page 28 of 37