Synthesis and antitumor activity of α,β-unsaturated carbonyl moiety- containing oleanolic acid derivatives targeting PI3K/AKT/mTOR signaling pathway

Article abstract of DOI:10.1016/j.bioorg.2020.104036

Oleanolic acid (OA) and its semi-synthetic derivatives have been reported to have a wide range of biological activities. The introduction of electrophilic Michael acceptor group can increase the reactivity of OA to cellular targets and thus improve the an

Full text of DOI:10.1016/j.bioorg.2020.104036

Journal Pre-proofs
Synthesis and antitumor activity of α,β-unsaturated carbonyl moiety- contain-
ing oleanolic acid derivatives targeting PI3K/AKT/mTOR signaling pathway
Shi-Sheng Wang, Qiao-Li Zhang, Peng Chu, Ling-Qi Kong, Guang-Zhe Li,
Yue-Qing Li, Li Yang, Wei-Jie Zhao, Xiu-Han Guo, Ze-Yao Tang
PII:
S0045-2068(20)31333-X
DOI:
https://doi.org/10.1016/j.bioorg.2020.104036
YBIOO 104036
Reference:
To appear in:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
19 January 2020
24 April 2020
19 June 2020
Please cite this article as: S-S. Wang, Q-L. Zhang, P. Chu, L-Q. Kong, G-Z. Li, Y-Q. Li, L. Yang, W-J. Zhao, X-
H. Guo, Z-Y. Tang, Synthesis and antitumor activity of α,β-unsaturated carbonyl moiety- containing oleanolic
acid derivatives targeting PI3K/AKT/mTOR signaling pathway, Bioorganic Chemistry (2020), doi: https://
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Title: Synthesis and antitumor activity of ,β-unsaturated carbonyl moiety-
containing oleanolic acid derivatives targeting PI3K/AKT/mTOR signaling
pathway
Graphic Abstract
Highlights

21 new oleanolic acid derivatives containing ,β-unsaturated carbonyl were synthesized.
Most synthesized compounds exhibited potent antiproliferative activities against MCF-7,
HepG2 and HeLa cells.



Compound 3d displayed the most potent inhibitory activity in MCF-7 cells with an
IC50 of 0.77ꢀμM.
Compound 3d inhibited the cells migration, arrested cell cycle at S phase and targeted
PI3K/AKT/mTOR signaling pathway in MCF-7 cells.
1

Synthesis and antitumor activity of ,β-unsaturated carbonyl moiety-
containing oleanolic acid derivatives targeting PI3K/AKT/mTOR signaling
pathway
Shi-Sheng Wang1, Qiao-Li Zhang1, Peng Chu2, Ling-Qi Kong1, Guang-Zhe Li1, Yue-Qing
Li1, Li Yang1, Wei-Jie Zhao1, Xiu-Han Guo1*, Ze-Yao Tang2*
1. Department of Pharmacy, School of Chemical Engineering, Dalian University of
Technology, Dalian 116024, China
2. Department of Pharmacology, Dalian Medical University, Dalian 116044, China
*Corresponding author: kakumicin@dlut.edu.cn (Xiu-Han Guo); tangzeyao@163.com
(Ze-Yao Tang).
Abstract
Oleanolic acid (OA) and its semi-synthetic derivatives have been reported to have a wide
range of biological activities. The introduction of electrophilic Michael acceptor group can
increase the reactivity of OA to cellular targets and thus improve the anti-tumor activity. In
this work, a series of novel α,β-unsaturated carbonyl derivatives of OA were designed and
synthesized. Their in vitro cytotoxic activity against MCF-7, HepG2 and HeLa cells were
tested. Most derivatives exhibited improved cell growth inhibitory activity, especially for
3d with an IC50 of 0.77ꢀμM in MCF-7 cells. Moreover, 3d inhibited the migration of MCF-7
and HeLa cells at the concentration of 4 M. Flow cytometric analysis revealed that 3d
induced cell apoptosis and S phase arrest in a concentration-dependent manner. Western
blotting experiment demonstrated that 3d inhibited the phosphorylation of AKT and mTOR.
These results suggest that this series of OA derivatives bearing exocyclic methylene ketone
pharmacophore are promising anticancer agents as potential PI3K/AKT/mTOR pathway
inhibitors.
Keywords: Oleanolic acid derivatives, anticancer, PI3K/AKT/mTOR signaling pathway, cell
apoptosis
induction,
S
phase
arrest
2

1. Introduction
Pentacyclic triterpenoids are widely distributed in the plant kingdom. Oleanolic acid
(OA, Fig.1) is one of the most well-known pentacyclic triterpenes[1,2], which has a wide
range of biological activities including anti-inflammatory, hepatoprotective, antitumor,
antiviral, antidiabetic, antimicrobial, antiparasitic and analgesic effects[3–5]. In the past
decades, a large number of OA derivatives have been synthesized and explored for their
antitumor[6], anti-angiogenic[7], anti-diabetic[8], antiviral[9], anti-inflammatory[10] and
other activities. The antitumor and anti-inflammatory effects of OA have received the most
attention and many semi-synthetic OA derivatives were reported to have improved activity. In
the previous investigation of OA derivatives as anticancer agents in our laboratory, a series of
Mannich base derivatives (SZC014, SZC015, SZC017, Fig.1) were synthesized and exhibited
significant antitumor activities through various mechanisms, including inducing apoptosis in
cancer cells, inhibiting AKT or NF-κB signaling pathway, promoting ROS production in a
variety of cancer cell lines[11–17].
According to literatures on the modification of OA, the introduction of electrophilic
Michael acceptor in rings A and C can increase the reactivity of OA to cellular targets, and
thus improves the antitumor activity[18]. Some Michael acceptor derivatives of OA have
entered clinical trials for cancer , such as 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid
(CDDO) and it methyl ester CDDO-Me[19,20] (Fig. 1), which possess an α-cyano-substituted
α,β-unsaturated ketone (CUK) in ring A and an α,β-unsaturated ketone in ring C, respectively,
and display potent anti-cancer, anti-inflammatory and anti-diabetic nephropathy (DN)
activities. The CUK moiety in ring A can react with thiol(s) in Keap1 and IKKβ through
Michael addition[21,22].
During the past decade, covalent targeting has been more and more widely used in the
fields of drug design and molecular biology, especially in oncology[23,24]. Targeted covalent
inhibitors are typically designed from reversible ligands by introducing an electrophilic
covalent reactive group, such as ,-unsaturated ketones/aldehydes/esters/amides, that reacts
with a variety of thiols in cellular proteins via Michael addition[25–27]. However, the
selectivity and safety remain challenging in the design of targeted covalent inhibitors. For
example, in a phase III clinical trial, CDDO-Me caused heart failure in patients with stage 4
3

DN, which resulted in the termination of the clinical trial[28]. The highly reactive CUK
moiety in CDDO-Me was considered to be responsible for the cytotoxic effects through
depletion of mitochondrial glutathione[29]. Therefore, the proper reactivity and selectivity of
an electrophilic covalent reactive group is crucial for designing an effective and safety
covalent drug.
The compounds containing ,-unsaturated carbonyl groups are widely distributed in
biologically active molecules that have been used successfully in cancer therapeutics due to
the ability to react with cellular thiols. In this study, with exocyclic methylene ketone as a
Michael acceptor moiety, a series of novel ,-unsaturated carbonyl-containing OA
analogues were designed and synthesized. Their in vitro anticancer activities were
investigated by MTT assay and flow cytometry analysis, and the effect on the
PI3K/AKT/mTOR signaling pathway was validated with western blotting analysis.
Fig.1. The chemical structures of oleanolic acid and its derivatives.
2. Results and discussion
2.1. Chemistry
The conjugated α,β-unsaturated carbonyl moiety is considered to be one of the most
important functional groups as Michael acceptors in covalent drug discovery[25]. In order to
improve the binding of OA derivatives with macromolecular targets, we here introduced
exocyclic methylene ketone at 2, 3-position of the A ring as the Michael acceptor, followed
by amidation of 28-COOH. As described in scheme 1, 3-oxo oleanolic acid (1) was first
synthesized from OA by the oxidization of 3-OH to a carbonyl group with Jone’s regent.
4

Compound 1 was then converted to 2-methylene 3-oxo oleanolic acid (2) via Mannich
reaction with paraformal dehyde and dimethylamine hydrochloride and subsequent Huffman
elimination under alkaline condition. Finally, compound 2 underwent chlorination of
28-COOH and amidation with different amines to yield seven new derivatives 3a-3g with
different sizes of side chains.
Scheme 1. Synthetic route to compounds 3a-3g. Reagents and conditions: (a) Jone’s reagent, acetone/
CH2Cl2(1:1), 0℃,90%. (b) i. paraformaldehyde, dimethylamine hydrochloride, DMF,100℃; ii.NaHCO3(aq),
100℃,1h, 61%. (c) oxalyl chloride, DMF/ CH2Cl2,rt. (d) amine compounds,DIEA,CH2Cl2,rt,26%-90%.
1,2,3-triazole is a kind of essential nitrogen heterocyclic moiety which is widely applied
in the construction of drug molecules, especially in the field of anticancer drugs[30]
1,2,3-triazole derivatives have attracted much attention in the field of medicinal chemistry due
to their broad range of pharmacological properties and facile synthesis via click
chemistry[31]. For further optimizing the substituents at C-28, we synthesized a series of
novel OA derivatives (4a-4n) containing ,-unsaturated carbonyls at ring A and
benzene-substituted 1,2,3-triazole moieties at C-28. By a Cu-catalyzed click reaction,
compounds 4a~4n were synthesized with compound 3c and aromatic azides (5a-5n), which
were prepared from their corresponding anilines by diazotization with sodium nitrite under
acidic conditions followed by displacement with sodium azide (Scheme.2).
5

Scheme 2. Synthetic route to compounds 4a-4n. Reagents and conditions: (a) sodium ascorbate, CuSO4,
BuOH/H2O(1:1),room temperature(rt),overnight,41%-77%. (b) i. H2O/HCl(1:1),NaNO2(aq),0℃,2h; ii.NaN3(aq),
0℃,1h.
2.2. Biology
2.2.1. Cytotoxicity assay
To evaluate the antitumor activity of the synthesized OA derivatives, we tested their
inhibitory effects on the cell proliferation of MCF-7, HepG2 and HeLa cells by MTT assay
using CDDO-Me as a positive control. As shown in table 1, compounds 3c-3f displayed more
potent inhibitory activity than the precursor compound 2. Among them, compounds 3c and 3d
with the smaller size of substituent at C-28 showed stronger inhibitory activity against MCF-7
proliferation with IC50 of 1.04 and 0.77 μM, respectively. Compared with 3e, compound 3f
increased the cytotoxic effects whereas 3g significantly decreased the cytotoxic effects,
suggesting the position of methoxyl group at benzene ring is important for anti-proliferative
effects of OA derivatives. Moreover, compound 3a with a maleimide moiety at C-28 as a
Michael acceptor showed weakest anti-proliferative effects. A long chain substituent at C-28
as in compound 3b is also unfavorable to anti-proliferative effects.
Another series of OA derivatives 4a-4n, which contain substituted phenyl 1,2,3-triazole
moiety, showed almost equivalently high antiproliferative activity against three cancer cell
lines. Compared respectively with 3e-3g, compounds 4a, 4l and 4j showed better inhibitory
activity, indicating that the insertion of 1,2,3-triazole increased their anticancer activities.
However, the substituents on the benzene ring, regardless of electron-withdrawing groups (-F,
6

-Cl, -NO2) or electron-donating groups (-CH3, -OCH3), displayed little effects on their
antitumor activity. Among these triazole -containing OA derivatives, 4a and 4f exhibited very
strong anti-proliferative effects against HeLa cells with IC50 values of 1.14 and 1.12 M,
respectively, while 4k exhibited most potent anti-proliferative effects against HepG2 cells
with IC50 of 0.94 M. As expected, all these compounds 2, 3a-3g and 4a-4n showed much
lower cytotoxicity than the positive control CDDO-Me, which might be attributed to the
relatively weak eletrophilicity of ,-unsaturated ketone compared with CUK moiety of
CDDO-Me.
Table.1. In vitro anti-proliferative effects of OA derivatives against MCF-7, HepG2 and HeLa cells
Entry
Compds
IC50 (μM)a
HepG2
MCF-7
7.13±0.63
14.01±0.67
4.66±0.10
1.04±0.07
0.77±0.03
2.81±0.13
1.46±0.05
7.44±0.38
1.94±0.15
2.86±0.25
2.58±0.27
2.22±0.13
3.36±0.14
1.40±0.17
2.45±0.13
2.09±0.13
2.00±0.15
1.69±0.04
1.52±0.04
1.29±0.04
2.95±0.11
1.93±0.05
0.67±0.02
HeLa
1
2
2
12.83±3.15
37.63±3.28
12.66±1.88
3.13±0.88
4.57±1.33
6.98±0.30
3.47±0.36
7.73±0.11
2.38±0.08
1.42±0.08
1.73±0.06
2.42±0.12
3.61±0.09
3.90±0.31
3.42±0.13
1.92±0.10
1.62±0.02
1.41±0.06
0.94±0.01
1.38±0.05
6.54±0.22
1.97±0.16
0.26±0.02
6.16±0.48
>50
3a
3
3b
16.23±0.98
2.08±0.56
1.23±0.03
2.00±0.16
2.67±0.23
11.60±0.10
1.14±0.31
1.36±0.34
2.78±0.03
1.32±0.24
2.29±0.23
1.12±0.12
1.25±0.13
2.32±0.29
1.71±0.21
2.19±0.10
1.82±0.08
1.60±0.10
2.67±0.41
1.84±0.09
-
4
3c
5
3d
6
3e
7
3f
8
3g
9
4a
10
11
12
13
14
15
16
17
18
19
20
21
22
23
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
CDDO-Me
aIC50: The concentration that causes 50% inhibition of cell proliferation. Data are expressed as the means±SD
from triplicate determination from three independent experiments.
2.2.2 . Compound 3d inhibited cell migration of MCF-7 and HeLa cells
To investigate the effects of these OA derivatives on migration inhibition in tumor cells,
7

we used the in vitro scratch assay to measure the effect of compound 3d on the migration
ability of MCF-7 and HeLa cells. As shown in Fig.2, the migration rates of both MCF-7 and
HeLa cells after 24 h from the scratch in 3d (4μM) group were much smaller than those in the
control group. These results indicated that 3d significantly inhibited the migration of MCF-7
and HeLa cells in addition to their cell proliferation.
Fig.2. Compound 3d inhibited migration of MCF-7 and HeLa cells. MCF-7 cells and HeLa cells were scratched
by pipette and treated with 3d (4M). After 24 h from the scratch, the migrated cells were photographed.(A)
Compound 3d inhibited MCF-7 cells migration.(B) Compound 3d inhibited HeLa cells migration. (C) and (D)
illustrate the percentage of wound closure in untreated control cells and 3d treated cells at 24h. Values are given
as the means ± SD of each group of cells from three individual experiments. ***P < 0.001
2.2.3. Compound 3d induced apoptosis of MCF-7 cells
The apoptosis of MCF-7 cells treated with various concentrations of 3d for 24 h was
examined. The cells were harvested and stained with Annexin V-FITC and propidium iodide
(PI), and the percentages of apoptotic cells were determined by flow cytometry analysis. It
was observed that treatment with 3d induced apoptosis of MCF-7 cells in a dose-dependent
manner. When treated with 2, 4 and 8 μM of 3d for 24 h, the percentages of apoptotic cells
were 5.44%, 19.02% and 57.59% (Q2 + Q3), respectively (Fig.3), while the vehicle control
group treated with DMSO contained 2.71% apoptotic cells. This result demonstrated that the
anti-proliferative activity observed in 3d-treated MCF-7 cells involved with the cell apoptosis
8

induction.
Fig.3. Flow cytometric evaluation of apoptosis based on Annexin V/PI double labeling. (A) Apoptosis of MCF-7
cells treated with 3d (2,4,8 μM) for 24 h before harvested and analyzed by flow cytometry.(B) Apoptosis rate is
displayed through the histogram. It was observed that treatment with 3d induced apoptosis in MCF-7 cells in a
dose-dependent manner. Results are representative images or expressed as the means ± SD of three individual
experiments. ***P < 0.001, ****P < 0.0001.
2.2.4. Target Prediction of compound 3d in its anticancer action
Although the anticancer effects of OA and its derivatives have been extensively
investigated by many researchers, the definite targets and underlying mechanisms remain
ambiguous until now. In this study, we used Pharma DB database in Discovery Studio
software to predict the target of compound 3d (FitValue≥0.9), and as a result, a total of 103
proteins were obtained. Then all proteins were input into DAVID 6.8, a bioinformatics
analysis tool, to predict the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway
[32]. The results were displayed in a bubble chart (Fig.4), and it was found that these enriched
proteins affect cells through multiple pathways, including PI3K/AKT, TNF pathway and cell
cycle.
9

Fig.4. KEGG pathway of all predicted target proteins for compound 3d. The top 40 most significant canonical
signaling pathways are displayed.
The protein-protein interaction (PPI) of the predicted proteins was then analyzed using
the STRING database [33] and 15 most important protein interactions were identified (Fig.5).
The further KEGG pathway analysis of these 15 proteins suggested that these pathways were
related to the cell cycle and cell apoptosis (Table 2).
Fig.5. Protein interaction network construction and analysis using the cytohubba plug-in in cytoscape 3.6.0
statistical software, to determine the most important 15 protein interactions.
10

Table.2.KEGG pathway analysis of key proteins
Term
Count
PValue
3.00E-04
2.52E-03
4.21E-03
6.78E-03
2.10E-02
Genes
Cell cycle
4
3
3
3
3
TTK, MDM2, CHEK1, CDK2
MDM2, CHEK1, CDK2
AR, AURKA, CDK2
p53 signaling pathway
Oocyte meiosis
Proteoglycans in cancer
PI3K-AKT signaling pathway
MAPK14, ESR1, MDM2
HSP90AA1, MDM2, CDK2
2.2.5. Compound 3d induced S phase arrest in MCF-7 cells
Cell cycle arrest is often observed during programmed cell death. Enlightened by the
KEGG pathway analysis of key proteins, the effect of 3d on the cell cycle of MCF-7 cells was
determined by flow cytometric experiments. Upon treatment with 3d at 2, 4 and 8 μM for 24
h, the percentage of cells at the different phases of cell circle was determined as shown in
Fig.6. A dose-dependent accumulation of cells was observed at the S phase, increasing from
40.0% in the untreated cells to 50.8% in the cells treated with 8 M 3d. It is worth noting that
the low concentration group at 2 M of 3d exhibited an unremarkable increase in the S phase
cell proportion (43.0%), suggesting that some other mechanisms might be involved in
inhibiting cell growth beside the S phase arrest.
Fig.6. Compound 3d induced S phase arrest in MCF-7 cells. MCF-7 cells were treated with 3d at 2, 4, 8μM
concentration for 24 h prior to analysis. The cell cycle was evaluated by propidium iodide staining. For the
analysis, only live cells were selected. Untreated cells were taken as a control. (A) Effects of compound 3d on
the cell-cycle distribution in MCF-7 cells. (B) Bar graph proportions of cells in G1, S and G2/M phases. Results
are representative images or expressed as the mean ± SD of three individual experiments.
11

2.2.6. Inhibitory effect of 3d on the PI3K/AKT/mTOR Signaling Pathways
PI3K/AKT/mTOR signaling pathway plays an important role in occurrence and
progression of tumor and drug resistance by inducing survival, differentiation and
vascularization, and thus becomes an attractive target for cancer treatment [34–36]. Previous
studies demonstrated that OA derivative SZC015 displayed anti-cancer activity by inhibiting
the PI3K/AKT/mTOR signaling pathway in human MCF-7 cells and other cancer cell
lines[37]. The KEGG pathway analysis suggested that 3d might exert its anticancer activity
via PI3K/AKT/mTOR pathway. The effects of 3d on the PI3K/AKT/mTOR signaling
pathway were explored by western blotting. As shown in Fig.7, treatment with 3d
downregulated the levels of phosphorylation-AKT (p-AKT) and phosphorylation-mTOR
(p-mTOR) in MCF-7 cells, which are known mediators of cell proliferation and inhibition of
apoptosis. Moreover, the p-AKT/AKT ratio and p-mTOR level were significantly decreased
in 3d treatment groups in a concentration-dependent (Fig.7C and 7D) and time-dependent
manner (Fig. 7E and 7F).
Fig.7. Effect of 3d on PI3K/AKT/mTOR signaling pathway by western blot assay. (A) the changes of proteins
expression in MCF-7 cells treated with 3d (2, 4, and 8μM) for 12 h. (B) the changes of proteins expression in
MCF-7 cells treated with 3d (4μM) for 0,6,12 and 24 h. The statistical analysis of p-AKT/AKT ratio (C, E) and
the expression of p-mTOR (D, F) in MCF-7 cells. Results are representative images or expressed as the mean ±
SD of three individual experiments. **P < 0.01, ***P < 0.001.
12

3. Conclusion
In summary, we designed and synthesized a series of OA derivatives containing
α,β-unsaturated carbonyl moiety and various amide substituents, and evaluated their
anticancer activity against MCF-7, HepG2 and HeLa cell lines. Most derivatives showed
much more potent anti-proliferative activity than OA, especially for 3d against MCF-7 cells
(IC50=0.77 μM) and 4k against HepG2 cells (IC50=0.94 μM). Moreover, 3d can significantly
arrest the cell cycle of MCF-7 cells in the S phase and induce apoptosis of MCF-7 cells with
57.59% of total apoptosis rate. Besides inhibiting cell proliferation, 3d can significantly
inhibit the migration of MCF-7 and HeLa cells at concentration of 4 M. In the preliminary
study of anticancer mechanism of 3d, the target prediction by KEGG analysis supplied
multiple signaling pathways including PI3K/AKT pathway. Western blotting analysis
validated that 3d blocks phosphorylation of AKT and mTOR in MCF-7 cells, thus inactivates
the PI3K/AKT/mTOR pathway, suggesting 3d might be a potential PI3K/AKT/mTOR
pathway inhibitor with the ability to induce apoptosis and suppress cell migration. Other
related pathways and protein targets are presumably involved in the anticancer effect of this
series of OA derivatives and will be further investigated in the future.
4. Experimental
4.1 Chemical synthesis
In the synthesis of derivatives of OA, all commercially available reagents, solvents and
chemicals were used without further purification. The synthetic reactions were carried out at
ambient temperature and the products were purified by column chromatography (silica gel;
1
petroleum ether/ethyl acetate) to afford pure compounds. H and 13C NMR spectra were
recorded on a Bruker Avance Ⅱ 400 MHz spectrometer, using TMS as an internal standard.
Chemical shifts are given as δ values in parts per million (ppm) and coupling constants (J) are
given in hertz (Hz). HRMS data were obtained on a LTQ Orbitrap XL instrument operating in
multimode. Thin-layer chromatographic (TLC) separations were carried out on precoated
silica gel plates. The purity of final compounds was determined to be >95% by HPLC
analysis.
4.1.1. 3-Oxo-olean-12-en-28-oic acid（1）
10.0 g (21.9 mmol) of OA was added to 500 mL dichloromethane: acetone (v:v 1:1) and
13

dissolved by stirring at 0 °C, and then Jones reagent was added dropwise to the mixture. The
reaction mixture was continuously stirred at 0 °C for 30 minutes. The reaction solution was
concentrated and 300 mL cooling NaHCO3 and 300 mL EtOAc were added to produce a
precipitation which was filtrated and dried. The crude products were recrystallized using
methanol at 0 °C to yield a white solid (9.0 g, yield 90%). 1H NMR (400 MHz, CDCl3) δ 5.30
(t, J = 3.4 Hz, 1H), 2.84 (dd, J = 13.8, 4.2 Hz, 1H), 2.55( m, 1H), 2.36 (m,1H), 1.15 (s, 3H),
1.08 (s, 3H), 1.05 (s, 3H), 1.03 (s, 3H), 0.93 (s, 3H), 0.91 (s, 3H), 0.81 (s, 3H) ppm. 13C NMR
(101 MHz, CDCl3) δ 217.72, 184.03, 143.64, 122.40, 55.30, 47.43, 46.89, 46.58, 45.81,
41.02, 39.27, 39.10, 36.80, 34.14, 33.80, 33.05, 32.40, 32.15, 30.68, 27.67, 26.44, 25.84,
23.56, 23.48, 22.89, 21.44, 19.54, 17.00, 15.01, 14.12 ppm .
4.1.2. 2-methylene-3-Oxo-olean-12-en-28-oic acid（2）
Compound 1 2.5 g (5.5 mmol) was dissolved in 100 mL anhydrous DMF, to which
dimethylamine hydrochloride 624 mg (7.7 mmol), paraformaldehyde 231 mg and acetic acid
(0.3 mL) were added. The reaction mixture was stirred at 100℃ for 1.5 h. The mixture was
cooled, adjusted to pH 8 with saturated aqueous NaHCO3, and stirred at 100 ℃ for 1 h. The
reaction solutionwas cooled, poured into brine and extracted with EtOAc. The organic phase
was washed with saturated aqueous NaHCO3 and brine and dried over anhydrous Na2SO4.
After filtration and evaporation under reduced pressure, the yielded crude product was then
purified with silica column chromatography (petroleum ether: ethyl acetate=5:1) to obtain
1
compound 2 (1.6 g, yield 61%) as a white solid. HPLC: Purity 99.8%. mp: 228-232℃. H
NMR (400 MHz, CDCl3) δ 5.99 (t, J = 2.1 Hz, 1H), 5.32 (t, J = 3.4 Hz, 1H), 5.16 (s, 1H),
2.85 (dd, J = 13.6, 4.1 Hz, 1H), 2.64 (d, J = 15.2 Hz, 1H), 2.13 (d, J = 15.1 Hz, 1H), 1.16 (s,
3H), 1.13 (s, 3H), 1.04 (s, 3H), 0.94 (s, 3H), 0.93 (s, 3H), 0.91 (s, 3H), 0.81 (s, 3H) ppm. 13C
NMR (101 MHz, CDCl3) δ 207.52, 184.68, 143.77, 142.09, 123.94, 122.52, 54.27, 46.89,
45.96, 45.56, 41.96, 41.19, 39.32, 36.99, 34.84, 33.97, 33.23, 32.54, 32.14, 28.38, 27.83,
27.08, 25.92, 25.45, 23.71, 23.60, 23.07, 22.86, 20.25, 16.97, 15.08 ppm.
4.1.3. General procedure for synthesis of compounds 3a-3g
Compound 2 (0.2 mmol) was added to 1.5 ml of dry CH2Cl2, 2 drops of DMF, and 0.6
mmol of oxalyl chloride, After being stirred under room temperature for 1 h, the reaction
mixture was concentrated. Then anhydrous CH2Cl2 (2 mL), amine compounds (0.3 mmol)
and DIEA (0.3 mmol) were added and the reaction was carried out at room temperature and
monitored by TLC. Finally the reaction mixture was concentrated and purified by silica gel
14

column chromatography to yield 3a-3g (35%-92%).
4.1.4. N-(maleimide-1-ethyl)-2-methylene-3-oxo-olean-12-en-28-amide (3a)
Yield 56%,
white solid. HPLC: Purity 97.5%. HRMS (ESI Positive) m/z: calcd for C37H53N2O4 [M+H]+:
1
589.4005; found:589.4006. H NMR (400 MHz, CDCl3) δ6.71(s, 2H), 6.16(s, 1H), 5.97(s,
1H), 5.42 (t, J = 3.4 Hz, 1H), 5.15(s, 1H), 3.78 – 3.59 (m, 5H), 3.06 (ddd, J = 13.3, 7.8, 4.2
Hz, 1H), 2.61 (d, J = 15.1 Hz, 1H), 2.51 (d, J = 12.5 Hz, 1H), 2.11 (d, J = 15.1 Hz, 1H), 1.15
(s, 3H), 1.11 (s, 3H), 1.04 (s, 3H), 0.91 (s, 3H), 0.89 (s, 6H), 0.73 (s, 3H) ppm. 13C NMR (101
MHz, CDCl3) δ 207.32, 178.52, 170.84, 144.88, 141.88, 134.25(2C), 123.79, 122.61, 58.43,
54.06, 46.75, 46.29, 45.95, 45.33, 42.12, 41.88, 39.22, 38.81, 37.36, 36.79, 34.16, 33.04,
32.28, 31.58, 30.78, 28.26, 27.28, 25.69, 23.70, 23.57, 23.47, 22.78, 20.13, 18.47, 16.41,
15.00 ppm.
4.1.5.
N-1-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl-2-methylene-3-oxo-olean-12-en-
28-amide (3b) Yield 40%, white solid. HPLC: Purity 99.1%. mp: 55-58℃. HRMS (ESI
Positive) m/z: calcd for C39H63N4O5 [M+H]+ 667.4798; found: 667.4808. 1H NMR (400 MHz,
CDCl3) δ 6.32 (s, 1H), 6.00 (s, 1H), 5.42 (s, 1H), 5.16 (s, 1H), 3.67(s, 8H), 3.53(s, 4H),
3.39(s, 2H), 2.62(m, 2H), 2.14 (d, J = 15.2 Hz, 1H), 1.19 (s, 3H), 1.14 (s, 3H), 1.07 (s, 3H),
0.92 (s, 9H), 0.84 (s, 3H) ppm. 13C NMR (101 MHz, CDCl3) δ 207.26, 177.97, 144.67,
141.90, 123.69, 122.46, 70.76, 70.66, 70.63, 70.30, 70.11, 69.72, 54.10, 50.68, 46.80, 46.66,
46.42, 45.91, 45.31, 42.28, 42.21, 39.30, 39.09, 36.74, 34.16, 33.02, 32.66, 31.80, 30.74,
28.26, 27.34, 25.57, 23.72, 23.60, 22.73, 20.14, 16.62, 15.01 ppm.
4.1.6. N-propargyl-2-methylene-3-oxo-olean-12-en-28-amide (3c) Yield 92%, white solid.
HPLC: Purity 99.6%. mp: 135-140℃. HRMS (ESI Positive) m/z: calcd for C34H50NO2
1
[M+H]+ 504.3842; found: 504.3839. H NMR (400 MHz, CDCl3) δ 6.10 (t, J = 4.7 Hz, 1H),
6.00 (s, 1H), 5.47 (t, J = 3.3 Hz, 1H), 5.17 (s, 1H), 4.09 – 3.90 (m, 2H), 2.64 (d, J = 15.1 Hz,
1H), 2.58 (dd, J = 12.8, 3.3 Hz, 1H), 2.22 (t, J = 2.5 Hz, 1H), 2.15 (d, J = 15.0 Hz, 1H), 1.20
(s, 3H), 1.14 (s, 3H), 1.07 (s, 3H), 0.94 (s, 3H), 0.92(s, 3H),0.91(s, 3H), 0.86 (s, 3H) ppm. 13C
NMR (101 MHz, CDCl3) δ 207.47, 178.06, 144.91, 142.01, 123.89, 122.98, 79.78, 71.76,
54.19, 50.94, 46.93, 46.71, 46.52, 46.06, 45.42, 42.37, 39.43, 36.87, 34.21, 33.12, 32.44,
31.84, 30.87, 29.48, 28.38, 27.38, 25.71, 24.01, 23.71, 23.69, 22.88, 20.25, 16.85, 15.15 ppm.
4.1.7. N-allyl-2-methylene-3-oxo-olean-12-en-28-amide (3d) Yield 56%, light yellow solid.
15

HPLC: Purity 99.6%. mp: 121-125℃. HRMS (ESI Positive) m/z: calcd for C34H52NO2
[M+H]+ 506.3998; found: 506.3986. 1H NMR (400 MHz, CDCl3) δ 5.98 (dd, J = 8.5, 4.1 Hz,
2H), 5.84 (ddd, J = 16.1, 10.9, 5.8 Hz, 1H), 5.43 (t, 1H), 5.15 (dt, J = 13.1, 11.4 Hz, 3H), 4.01
(dt, J = 13.6, 5.7 Hz, 1H), 3.65 (dt, J = 10.4, 4.8 Hz, 1H), 2.63 (d, J = 15.2 Hz, 1H), 2.14 (d, J
= 15.0 Hz, 1H), 1.20 (s, 3H), 1.14 (s, 3H), 1.07 (s, 3H), 0.94 (s, 3H), 0.92 (s, 3H),0.91(s, 3H),
13
0.82 (s, 3H) ppm. C NMR (101 MHz, CDCl3) δ 207.36, 178.08, 148.16, 145.14, 141.98,
134.50, 123.83, 122.67, 116.45, 54.18, 46.89, 46.83, 46.52, 46.02, 45.41, 42.48, 42.38, 42.15,
39.40, 36.85, 34.27, 33.11, 32.66, 31.83, 30.85, 28.38, 27.41, 25.70, 23.96, 23.69, 22.85,
20.24, 16.74, 15.11 ppm.
4.1.8. N-phenyl-2-methylene-3-oxo-olean-12-en-28-amide (3e)
Yield 36%, gray solid.
HPLC: Purity 95.6%. mp: 199-201℃. HRMS (ESI Positive) m/z: calcd for C37H52NO2
1
[M+H]+ 542.3998;found:542.3997. H NMR (400 MHz, CDCl3) δ 7.68 (s, 1H), 7.48 (d, J =
7.8 Hz, 2H), 7.30 (t, J = 7.9 Hz, 2H), 7.08 (t, J = 7.4 Hz, 1H), 5.99 (s, 1H), 5.59 (t, J = 3.3 Hz,
1H), 5.17 (s, 1H), 2.71 (dd, J = 12.4, 2.9 Hz, 1H), 2.65 (d, J = 15.1 Hz, 1H), 2.15 (d, J = 15.6
Hz, 1H), 1.23 (s, 3H), 1.12 (s, 3H), 1.04 (s, 3H), 0.94 (s, 3H), 0.92(s, 3H), 0.91 (s, 3H), 0.78
13
(s, 3H) ppm. C NMR (101 MHz, CDCl3) δ 207.29, 176.36, 145.12, 141.86, 138.01, 128.96
(2C), 124.13, 123.76, 123.02, 119.77(2C), 54.06, 47.26, 46.82, 46.75, 45.92, 45.31, 42.74,
42.43, 39.30, 36.73, 34.20, 32.98, 32.48, 31.73, 30.78, 28.26, 27.38, 25.60, 24.13, 23.75,
23.57, 22.72, 20.06, 16.62, 15.06 ppm.
4.1.9. N-p-methoxyphenyl-2-methylene-3-oxo-olean-12-en-28-amide (3f) Yield 45%, white
solid.HPLC: Purity 96.7%. mp:200-203℃. HRMS (ESI Positive) m/z: calcd for C38H54NO3
[M+H]+ 572.4104; found: 572.4099. 1H NMR (400 MHz, CDCl3) δ 7.57 (s, 1H), 7.38 (d, J =
9.0 Hz, 2H), 6.84 (d, J = 9.0 Hz, 2H), 5.99 (s, 1H), 5.57 (t, J = 3.2 Hz, 1H), 5.17 (s, 1H), 3.78
(s, J = 3.1 Hz, 3H), 2.69 (dd, J = 13.1, 3.6 Hz, 1H), 2.64 (d, J = 15.1 Hz, 1H), 2.15 (d, J =
15.2 Hz, 1H), 1.23 (s, 3H), 1.13 (s, 3H), 1.04 (s, 3H), 0.95 (s,3H), 0.94 (s,3H), 0.91 (s, 3H),
13
0.79 (s, 3H) ppm. C NMR (101 MHz, CDCl3) δ 207.38, 176.22, 156.41, 145.29, 142.00,
131.35, 123.86, 123.03, 121.70(2C), 114.23(2C), 55.61, 54.21, 47.20, 46.95, 46.91, 46.05,
45.46, 42.85, 42.55, 39.45, 36.87, 34.35, 33.13, 32.64, 31.88, 30.92, 28.41, 27.52, 25.74,
24.21, 23.87, 23.73, 22.85, 20.21, 16.84, 15.18 ppm.
4.1.10. N-o-methoxyphenyl-2-methylene-3-oxo-olean-12-en-28-amide (3g) Yield 35%, gray
solid. HPLC: Purity 99.7%. mp: 145-150℃. HRMS (ESI Positive) m/z: calcd for C38H54NO3
16

[M+H]+ 572.4104; found: 572.4082. 1H NMR (400 MHz, CDCl3) δ 8.49 (dd, J = 8.0, 1.5 Hz,
1H), 8.43 (s, 1H), 7.01 (td, J = 7.7, 1.5 Hz, 1H), 6.93 (td, J = 7.8, 1.1 Hz, 1H), 6.86 (d, J = 8.0
Hz, 1H), 5.99 (s, 1H), 5.55 (t, J = 3.2 Hz, 1H), 5.16 (s, 1H), 3.89 (s, 3H), 2.75 (dd, J = 12.4,
2.9 Hz, 1H), 2.64 (d, J = 15.1 Hz, 1H), 2.15 (d, J = 15.1 Hz, 1H), 1.22 (s, 3H), 1.12 (s, 3H),
13
1.03 (s, 3H), 0.95 (s,3H), 0.94 (s,3H), 0.88 (s, 3H), 0.70 (s, 3H) ppm. C NMR (101 MHz,
CDCl3) δ 207.47, 176.44, 148.10, 144.27, 142.11, 128.18, 123.79, 123.39, 123.37, 121.37,
119.76, 109.85, 55.72, 54.28, 48.01, 47.00, 46.09, 45.49, 42.70, 42.35, 39.45, 36.89, 36.22,
34.43, 33.19, 32.84, 31.90, 30.92, 28.40, 27.58, 25.85, 24.28, 23.80, 23.76, 22.86, 20.23,
16.12, 15.14 ppm.
4.1.11.General procedure for synthesis of compounds 4a-4n
To a solution of compound 3c (100.0 mg, 0.20 mmol) in t-BuOH/H2O (v:v 1:1, 4 mL),
sodium ascorbate (32.0 mg, 0.16 mmol) and CuSO4·5H2O (20.2 mg, 0.08 mmol) were added
at room temperature. To this mixture, aryl azide (1.0 mmol) was added and the reaction
mixture was stirred at 35℃ for overnight. The crude mixture was extracted with CH2Cl2 (3 ×
20 ml) and the combined organic layer was dried over Na2SO4 and purified through column
chromatography to offer 4a–4n.
4.1.12. N-((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)-2-methylene-3-oxo-olean-12-en-28-amide
(4a) Yield 60%,white solid. HPLC: Purity 95.6%. mp: 181-186℃. HRMS (ESI Positive)
1
m/z: calcd for C40H55N4O2 [M+H]+ 623.4325; found: 623.4304. H NMR (400 MHz, CDCl3)
δ 8.02 (s, 1H), 7.72 (d, J = 7.6 Hz, 2H), 7.51 (t, J = 7.5 Hz, 2H), 7.44 (d, J = 7.3 Hz, 1H), 6.74
(s, 1H), 5.98 (s, 1H), 5.45 (s, 1H), 5.15 (s, 1H), 4.61 (dd, J = 14.8, 4.6 Hz, 1H), 4.48 (dd, J =
14.8, 4.9 Hz, 1H), 2.66 (d, J = 12.0 Hz, 1H), 2.61 (d, J = 15.2 Hz, 1H), 2.10 (d, J = 14.8 Hz,
1H), 1.16 (s, 3H), 1.11 (s, 3H), 1.02 (s, 3H), 0.91 (s, 6H), 0.80 (s, 3H), 0.54 (s, 3H) ppm. 13C
NMR (101 MHz, CDCl3) δ 207.32, 178.33, 145.54, 144.38, 141.99, 137.14, 129.85(2C),
128.87, 123.79, 122.98, 121.00, 120.53(2C), 54.16, 53.55, 46.84, 46.64, 46.42, 45.97, 45.39,
42.23, 39.31, 36.80, 35.03, 34.24, 33.12, 32.83, 31.86, 30.84, 28.35, 27.40, 25.67, 23.91,
23.70, 23.59, 22.79, 20.16, 16.40, 14.97 ppm.
4.1.13. N-((1-(2-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-2-methylene-3-oxo-olean-12-en-
28-amide (4b) Yield 48%,white solid. HPLC: Purity 99.3%. mp: 188-191℃. HRMS (ESI
1
Positive) m/z: calcd for C40H54FN4O2 [M+H]+ 641.4231; found: 641.4221. H NMR (400
MHz, CDCl3) δ 8.06 (s, 1H), 7.95-7.87 (m, 1H), 7.48-7.40 (m, 1H), 7.32 (t, J = 7.6 Hz, 2H),
17

6.66 (s, 1H), 5.98 (s, 1H), 5.46 (s, 1H), 5.15 (s, 1H), 4.62 (dd, J = 14.9, 5.0 Hz, 1H), 4.51 (dd,
J = 15.0, 5.1 Hz, 1H), 2.11 (d, J = 15.2 Hz, 1H), 1.17 (s, 3H), 1.11 (s, 3H), 1.03 (s, 3H), 0.91
13
(s, 6H), 0.83 (s, 3H) ppm. C NMR (101 MHz, CDCl3) δ 207.46, 178.40, 154.86, 152.37,
144.53, 142.07, 130.48 (d, J = 7.7 Hz), 125.39, 125.35, 125.04, 123.86, 123.11, 117.25 (d, J =
20.0 Hz), 54.24, 46.93, 46.75, 46.53, 46.05, 45.46, 42.39, 42.34, 39.39, 36.87, 35.07, 34.30,
33.17, 32.77, 31.91, 30.91, 29.86, 28.42, 27.45, 25.73, 24.03, 23.75, 23.67, 22.86, 20.24,
16.39, 15.03 ppm.
4.1.14. N-((1-(4-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-2-methylene-3-oxo-olean-12-en-
28-amide (4c) Yield 48%,white solid. HPLC: Purity 97.8%. mp:189-193℃. HRMS (ESI
1
Positive) m/z: calcd for C40H54FN4O2 [M+H]+ 641.4231; found: 641.4210. H NMR (400
MHz, CDCl3) δ 7.98 (s, 1H), 7.74-7.66 (m, 2H), 7.25-7.18 (m, 2H), 6.69 (t, J = 5.4 Hz, 1H),
5.99 (s, 1H), 5.45 (t, J = 3.2 Hz, 1H), 5.16 (s, 1H), 4.61 (dd, J = 15.0, 5.4 Hz, 1H), 4.45 (dd, J
= 15.0, 5.5 Hz, 1H), 2.11 (d, J = 15.2 Hz, 1H), 1.17 (s, 3H), 1.12 (s, 3H), 1.03 (s, 3H), 0.91 (s,
13
6H), 0.84 (s, 3H), 0.56 (s, 3H) ppm. C NMR (101 MHz, CDCl3) δ 207.22, 178.37, 162.46
(d, J = 248.8 Hz), 145.67, 144.34, 141.91, 133.37 (d, J = 3.1 Hz), 123.79, 122.91, 122.43
(2C,d, J = 8.6 Hz), 121.22, 116.76 (2C,d, J = 23.2 Hz), 54.09, 46.78, 46.58, 46.36, 45.93,
45.33, 42.17, 39.26, 36.77, 34.98, 34.18, 33.08, 32.76, 31.81, 30.79, 28.28, 27.34, 25.64,
23.85, 23.65, 23.54, 22.76, 20.11, 16.35, 14.94 ppm.
4.1.15. N-((1-(2-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-2-methylene-3-oxo-olean-12-en-
28-amide (4d) Yield 45%, light green solid. HPLC: Purity 95.5%. mp: 178-183℃. HRMS
(ESI Positive) m/z: calcd for C40H54ClN4O2 [M+H]+ 657.3935;found:657.3925. 1H NMR (400
MHz, CDCl3) δ 8.01 (s, 1H), 7.62-7.55 (m, 2H), 7.49-7.40 (m, 2H), 6.69 (t, J = 5.2 Hz, 1H),
5.98 (s, 1H), 5.47 (t, J = 2.9 Hz, 1H), 5.16 (s, 1H), 4.62 (dd, J = 15.0, 5.3 Hz, 1H), 4.52 (dd, J
= 15.1, 5.3 Hz, 1H), 2.11 (d, J = 15.4 Hz, 1H), 1.18 (s, 3H), 1.12 (s, 3H), 1.04 (s, 3H), 0.91 (s,
13
6H), 0.87 (s, 3H), 0.62 (s, 3H) ppm. C NMR (101 MHz, CDCl3) δ 207.41, 178.51, 144.63,
144.56, 142.04, 135.03, 131.00, 130.92, 128.68, 128.03, 127.81, 124.81, 123.84, 123.09,
54.22, 46.92, 46.79, 46.49, 46.05, 45.45, 42.33, 42.30, 39.39, 36.87, 35.18, 34.30, 33.14,
32.70, 31.87, 30.88, 28.38, 27.41, 25.71, 24.08, 23.72, 23.68, 22.86, 20.24, 16.52, 15.13 ppm.
4.1.16. N-((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-2-methylene-3-oxo-olean-12-en-
28-amide (4e) Yield 59%, white solid. HPLC: Purity 97.9%. mp: 203-207℃. HRMS (ESI
1
Positive) m/z: calcd for C40H54ClN4O2 [M+H]+ 657.3935; found: 657.3924. H NMR (400
18

MHz, CDCl3) δ 7.99 (s, 1H), 7.67 (d, J = 8.8 Hz, 2H), 7.49 (d, J = 8.8 Hz, 2H), 6.66 (t, J =
4.8 Hz, 1H), 5.99 (t, J = 2.0 Hz, 1H), 5.45 (t, J = 3.3 Hz, 1H), 5.16 (s, 1H), 4.60 (dd, J = 15.0,
5.4 Hz, 1H), 4.45 (dd, J = 15.0, 5.5 Hz, 1H), 2.67-2.58 (m, 2H), 2.11 (d, J = 15.1 Hz, 1H),
1.17 (s, 3H), 1.11 (s, 3H), 1.03 (s, 3H), 0.90 (d, J = 1.8 Hz, 6H), 0.83 (s, 3H) ppm. 13C NMR
(101 MHz, CDCl3) δ 207.31, 178.48, 145.83, 144.43, 141.97, 135.63, 134.68, 130.04(2C),
123.86, 123.00, 121.69(2C), 121.03, 54.17, 46.86, 46.65, 46.44, 46.00, 45.40, 42.25, 39.32,
36.83, 35.03, 34.23, 33.12, 32.80, 31.86, 30.85, 28.33, 27.40, 25.68, 23.96, 23.70, 23.61,
22.82, 20.17, 16.42, 15.00, 14.24 ppm.
4.1.17. N-((1-(2-nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl)-2-methylene-3-oxo-olean-12-en-
28-amide (4f) Yield 65%,light yellow solid. HPLC: Purity 97.5%. mp:188-191℃. HRMS
(ESI Positive) m/z: calcd for C40H54N5O4 [M+H]+ 668.4176; found: 668.4166. 1H NMR (400
MHz, CDCl3) δ 8.07 (d, J = 8.0 Hz, 1H), 7.78 (t, J = 7.6 Hz, 1H), 7.70 (t, J = 7.8 Hz, 1H),
7.59 (d, J = 7.7 Hz, 1H), 6.72 (t, J = 4.8 Hz, 1H), 5.98 (s, 1H), 5.47 (s, 1H), 5.16 (s, 1H), 4.62
(dd, J = 15.0, 5.2 Hz, 1H), 4.48 (dd, J = 15.0, 5.0 Hz, 1H), 2.63 (dd, J = 9.5, 5.7 Hz, 2H), 2.12
(d, J = 15.0 Hz, 1H), 1.18 (s, 3H), 1.12 (s, 3H), 1.04 (s, 3H), 0.91 (s, 6H), 0.89-0.88 (m, 3H),
13
0.66 (s, 3H) ppm. C NMR (101 MHz, CDCl3) δ 207.35, 178.56, 145.36, 144.54, 144.43,
141.97, 133.75, 130.81, 130.22, 127.73, 125.64, 124.30, 123.73, 123.02, 54.12, 46.81, 46.68,
46.37, 45.95, 45.37, 42.21, 42.14, 39.31, 36.78, 35.06, 34.19, 33.05, 32.59, 31.74, 30.78,
28.28, 27.32, 25.64, 23.97, 23.63, 23.57, 22.78, 20.14, 16.45, 15.00 ppm.
4.1.18. N-((1-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl)-2-methylene-3-oxo-olean-12-en-
28-amide (4g) Yield 77%,light yellow solid. HPLC: Purity 98.3%. mp: 216-225℃. HRMS
(ESI Positive) m/z: calcd for C40H54N5O4 [M+H]+ 668.4176; found: 668.4167. 1H NMR (400
MHz, CDCl3) δ 8.42 (d, J = 9.1 Hz, 2H), 8.16 (s, 1H), 7.97 (d, J = 9.1 Hz, 2H), 6.68 (t, J =
5.5 Hz, 1H), 5.99 (t, J = 2.1 Hz, 1H), 5.46 (t, J = 3.2 Hz, 1H), 5.17 (s, 1H), 4.63 (dd, J = 15.0,
5.5 Hz, 1H), 4.45 (dd, J = 15.0, 5.4 Hz, 1H), 2.66-2.58 (m, 2H), 2.12 (d, J = 15.2 Hz, 1H),
1.18 (s, 3H), 1.12 (s, 3H), 1.02 (s, 3H), 0.91 (s, 6H),0.90 (s, 6H), 0.85 (s, 3H), 0.59 (s, 3H)
ppm. 13C NMR (101 MHz, CDCl3) δ 207.15, 178.58, 147.25, 146.45, 144.37, 141.86, 141.22,
125.57(2C), 123.80, 122.93, 121.16, 120.47(2C), 54.05, 46.77, 46.54, 46.38, 45.92, 45.31,
42.19, 42.15, 39.25, 36.76, 34.97, 34.14, 33.03, 32.72, 31.77, 30.77, 28.23, 27.32, 25.63,
23.89, 23.61, 23.54, 22.76, 20.09, 16.36, 14.94 ppm.
4.1.19. N-((1-(2-methylphenyl)-1H-1,2,3-triazol-4-yl)methyl)-2-methylene-3-oxo-olean-12-en-
19

28-amide (4h) Yield 41%,yellow solid. HPLC: Purity 96.4%. mp: 170-175℃. HRMS (ESI
Positive) m/z: calcd for C41H57N4O2 [M+H]+ 637.4482; found: 637.4467. 1H NMR (400 MHz,
CDCl3) δ 7.76 (s, 1H), 7.43-7.34 (m, 2H), 7.34-7.29 (m, 2H), 6.71 (t, J = 5.4 Hz, 1H), 5.99 (s,
1H), 5.47 (t, J = 3.1 Hz, 1H), 5.16 (s, 1H), 4.62 (dd, J = 15.0, 5.4 Hz, 1H), 4.50 (dd, J = 15.0,
5.3 Hz, 1H), 2.69 – 2.60 (m, 2H), 2.22 (s, 3H), 2.12 (d, J = 15.3 Hz, 1H), 1.18 (s, 3H), 1.12 (s,
3H), 1.05 (s, 3H), 0.91 (s, 6H), 0.87 (s, 3H), 0.64 (s, 3H) ppm. 13C NMR (101 MHz, CDCl3) δ
207.25, 178.33, 144.37, 141.86, 140.20, 136.42, 133.41, 131.54, 129.85, 126.83, 125.85,
124.12, 123.73, 122.92, 54.05, 46.75, 46.61, 46.33, 45.90, 45.29, 42.16, 42.11, 39.23, 36.72,
35.04, 34.13, 33.00, 32.58, 31.73, 30.73, 28.22, 27.27, 25.57, 23.89, 23.57, 23.52, 22.71,
20.07, 17.90, 16.44, 14.97 ppm.
4.1.20. N-((1-(4-methylphenyl)-1H-1,2,3-triazol-4-yl)methyl)-2-methylene-3-oxo-olean-12-en-
28-amide (4i) Yield 51%,white solid. HPLC: Purity 99.4%. mp: 172-177℃. HRMS (ESI
Positive) m/z: calcd for C41H57N4O2 [M+H]+ 637.4482; found:637.4465. 1H NMR (400 MHz,
CDCl3) δ 7.95 (s, 1H), 7.58 (d, J = 8.4 Hz, 2H), 7.30 (d, J = 8.3 Hz, 2H), 6.68 (t, 1H), 5.98 (s,
1H), 5.45 (t, J = 3.2 Hz, 1H), 5.15 (s, 1H), 4.60 (dd, J = 14.9, 5.2 Hz, 1H), 4.47 (dd, J = 14.9,
5.4 Hz, 1H), 2.69-2.56 (m, 2H), 2.41 (s, 3H), 2.10 (d, J = 15.1 Hz, 1H), 1.16 (s, 3H), 1.11 (s,
3H), 1.02 (s, 3H), 0.91 (s, 6H), 0.81 (s, 3H), 0.54 (s, 3H) ppm. 13C NMR (101 MHz, CDCl3) δ
207.35, 178.30, 145.35, 144.34, 141.96, 138.98, 134.83, 130.30(2C), 123.82, 122.95, 120.98,
120.43(2C), 54.12, 46.80, 46.61, 46.38, 45.94, 45.36, 42.19, 39.27, 36.77, 35.00, 34.22,
33.11, 32.79, 31.83, 31.67, 30.83, 28.34, 27.37, 25.65, 23.87, 23.68, 23.57, 22.76, 22.74,
21.17, 20.14, 16.37, 14.95, 14.22 ppm.
4.1.21.
N-((1-(2-methoxylphenyl)-1H-1,2,3-triazol-4-yl)methyl)-2-methylene-3-oxo-olean-
12-en-28-amide (4j) Yield 57%,light yellow solid. HPLC: Purity 96.8%. mp: 146-152℃.
1
HRMS (ESI Positive) m/z: calcd for C41H57N4O3 [M+H]+ 653.4431; found:653.4417. H
NMR (400 MHz, CDCl3) δ 8.09 (s, 1H), 7.75 (d, J = 7.0 Hz, 1H), 7.46-7.39 (m, 1H), 7.09 (t, J
= 7.5 Hz, 2H), 6.70 (t, J = 5.1 Hz, 1H), 5.98 (s, 1H), 5.46 (t, J = 3.0 Hz, 1H), 5.15 (s, 1H),
4.61 (dd, J = 15.0, 5.3 Hz, 1H), 4.51 (dd, J = 14.9, 5.3 Hz, 1H), 3.88 (s, 3H), 2.64 (m, 2H),
2.11 (d, J = 15.0 Hz, 1H), 1.17 (s, 3H), 1.11 (s, 3H), 1.03 (s, 3H), 0.91 (s, 6H), 0.83 (s, 3H),
13
0.56 (s, 3H) ppm. C NMR (101 MHz, CDCl3) δ 207.33, 178.16, 151.07, 144.34, 144.07,
141.95, 130.16, 126.31, 125.38, 124.64, 123.75, 123.00, 121.21, 112.28, 55.96, 54.12, 46.80,
46.63, 46.37, 45.93, 45.35, 42.22, 42.20, 39.26, 36.76, 35.03, 34.22, 33.08, 32.67, 31.83,
30.80, 28.30, 27.35, 25.62, 23.90, 23.65, 23.56, 22.76, 20.15, 16.30, 14.96 ppm.
20

4.1.22.
N-((1-(3-methoxylphenyl)-1H-1,2,3-triazol-4-yl)methyl)-2-methylene-3-oxo-olean-
12-en-28-amide (4k) Yield 43%,red solid. HPLC: Purity 98.8%. mp:118-124℃. HRMS
(ESI Positive) m/z: calcd for C41H57N4O3 [M+H]+ 653.4431; found: 653.4417. 1H NMR (400
MHz, CDCl3) δ 7.99 (s, 1H), 7.40 (t, J = 8.1 Hz, 1H), 7.31 (s, 1H), 7.24 (d, J = 7.9 Hz, 1H),
6.96 (dd, J = 8.3, 2.0 Hz, 1H), 6.65 (s, 1H), 5.98 (s, 1H), 5.45 (s, 1H), 5.15 (s, 1H), 4.60 (dd, J
= 14.9, 4.8 Hz, 1H), 4.47 (dd, J = 14.9, 4.8 Hz, 1H), 3.87 (s, 3H), 2.68-2.57 (m, 2H), 2.10 (d,
J = 15.0 Hz, 1H), 1.16 (s, 3H), 1.11 (s, 3H), 1.02 (s, 3H), 0.91 (s, 6H), 0.82 (s, 3H), 0.55 (s,
13
3H) ppm. C NMR (101 MHz, CDCl3) δ 207.37, 178.33, 160.71, 145.43, 144.34, 141.96,
138.10, 130.61, 123.80, 122.95, 121.09, 114.62, 112.42, 106.41, 55.71, 54.12, 46.81, 46.60,
46.39, 45.94, 45.35, 42.20, 39.27, 36.77, 34.95, 34.20, 33.09, 32.79, 31.83, 30.81, 28.32,
27.36, 25.63, 23.89, 23.66, 23.56, 22.74, 20.13, 16.37, 14.94, 14.28 ppm.
4.1.23.
N-((1-(4-methoxylphenyl)-1H-1,2,3-triazol-4-yl)methyl)-2-methylene-3-oxo-olean-
12-en-28-amide (4l) Yield 57%,white solid. HPLC: Purity 99.8%. mp: 115-121℃. HRMS
(ESI Positive) m/z: calcd for C41H57N4O3 [M+H]+ 653.4431; found: 653.4417. 1H NMR (400
MHz, CDCl3) δ 7.92 (s, 1H), 7.61 (d, J = 8.6 Hz, 2H), 7.00 (d, J = 8.6 Hz, 2H), 6.71 (s, 1H),
5.98 (s, 1H), 5.45 (s, 1H), 5.15 (s, 1H), 4.59 (dd, J = 14.9, 5.0 Hz, 1H), 4.47 (dd, J = 14.9, 5.2
Hz, 1H), 3.85 (s, 3H), 2.63 (m, 2H), 2.11 (d, J = 15.0 Hz, 1H), 1.16 (s, 3H), 1.11 (s, 3H), 1.02
13
(s, 3H), 0.91 (s, 6H), 0.82 (s, 3H), 0.55 (s, 3H) ppm. C NMR (101 MHz, CDCl3) δ 207.32,
178.30, 159.94, 145.27, 144.36, 141.96, 130.55, 123.78, 122.96, 122.13(2C), 121.06, 114.84,
55.71, 54.13, 53.51, 46.81, 46.62, 46.39, 45.95, 45.37, 42.21, 39.28, 36.78, 35.00, 34.21,
33.09, 32.76, 31.83, 30.81, 28.32, 27.37, 25.64, 23.90, 23.67, 23.57, 22.76, 20.14, 16.37,
14.97 ppm.
4.1.24.
N-((1-(2-methyl-4-nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl)-2-methylene-3-oxo-
Yield 72%,light yellow solid. HPLC: Purity 98.3%. mp:
olean-12-en-28-amide (4m)
178-183℃. HRMS (ESI Positive) m/z: calcd for C41H56N5O4 [M+H]+ 682.4332;found:
1
682.4320. H NMR (400 MHz, CDCl3) δ 8.28 (d, J = 1.9 Hz, 1H), 8.20 (dd, J = 8.6, 2.0 Hz,
1H), 7.91 (s, 1H), 7.55 (d, J = 8.6 Hz, 1H), 6.73 (s, 1H), 5.99 (s, 1H), 5.48 (s, 1H), 5.16 (s,
1H), 4.63 (dd, 1H), 4.49 (dd, J = 11.2 Hz, 1H), 2.63 (m, 2H), 2.41 (s, 3H), 2.13 (d, J = 15.0
Hz, 1H), 1.99 (dd, J = 8.6, 2.5 Hz, 2H), 1.19 (s, 3H), 1.13 (s, 3H), 1.05 (s, 3H), 0.92 (s,
3H),0.91 (s, 3H), 0.89 (s, 3H), 0.64 (s, 3H) ppm. 13C NMR (101 MHz, CDCl3) δ 207.21,
178.67, 148.01, 144.47, 141.88, 141.14, 135.16, 126.91, 126.73, 123.85, 123.01, 122.27,
21

54.10, 46.82, 46.65, 46.42, 45.97, 45.35, 42.25, 42.16, 39.31, 36.81, 35.08, 34.18, 33.05,
32.66, 31.81, 30.80, 28.27, 27.33, 25.66, 23.98, 23.64, 23.61, 22.81, 20.14, 18.73, 16.52,
15.05 ppm.
4.1.25.
N-((1-(2,3-difluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-2-methylene-3-oxo-olean-
12-en-28-amide (4n) Yield 66%,light yellow solid. HPLC: Purity 96.4%. mp:118-124℃.
1
HRMS (ESI Positive) m/z: calcd for C40H53F2N4O2 [M+H]+ 659.4137;found: 659.4125. H
NMR (400 MHz, CDCl3) δ 8.13 (d, J = 2.9 Hz, 1H), 7.73 (ddd, J = 8.7, 5.8, 3.1 Hz, 1H), 7.28
(dt, J = 10.1, 4.1 Hz, 1H), 7.17 – 7.10 (m, 1H), 6.73 (t, J = 5.4 Hz, 1H), 5.98 (t, J = 2.0 Hz,
1H), 5.45 (t, J = 3.3 Hz, 1H), 5.15 (s, 1H), 4.62 (dd, J = 15.0, 5.4 Hz, 1H), 4.50 (dd, J = 15.0,
5.4 Hz, 1H), 2.65 (dd, J = 21.9, 9.1 Hz, 2H), 2.11 (d, J = 14.8 Hz, 1H), 1.96 (dd, J = 8.8, 3.4
Hz, 2H), 1.17 (s, 3H), 1.12 (s, 3H), 1.03 (s, 3H), 0.91 (s, 6H), 0.84 (s, 3H), 0.56 (s, 3H) ppm.
13C NMR (101 MHz, CDCl3) δ 207.21, 178.25, 171.09, 158.59 (dd, J = 245.8, 2.4 Hz),
149.22 (dd, J = 247.2, 3.2 Hz), 145.46, 144.30, 141.85, 125.70 (dd, J = 12.1, 10.7 Hz),
123.77, 122.85, 118.16 (dd, J = 22.9, 9.0 Hz), 116.61 (dd, J = 24.0, 8.0 Hz), 111.61 (d, J =
28.3 Hz), 60.35, 54.02, 46.71, 46.51, 46.31, 45.85, 45.25, 42.11, 39.17, 36.68, 34.82, 34.09,
32.98, 32.62, 31.71, 30.70, 28.20, 27.24, 25.54, 23.78, 23.54, 23.46, 22.65, 21.01, 20.03,
16.19, 14.82, 14.18 ppm.
4.2.Biological assays
4.2.1. Cell Culture
MCF-7, HeLa and HepG2 cells were cultured in DMEM growth medium, supplemented
with 10% FBS and 1% Penicillian-Streptomycin. All of the cell lines were grown at 37℃ in a
5% CO2 atmosphere.
4.2.2. MTT assay
The compounds were dissolved in DMSO and diluted to the required concentration with
the culture medium when used. The cells harvested from the exponential phase were
equivalently seeded into a 96-well plate (3000 cells/well) and treated with or without different
concentrations of the test compounds for 48 h. The MTT (20 μL, 5 mg/mL) was added into
each well, the cells were incubated for additional 4 h, and the resulting formazan crystals were
dissolved in 200 μL of DMSO and the absorbance was read at 570 nm. Experiments were
conducted in triplicate.
22

4.2.3. Apoptosis analysis
MCF-7 cells were incubated in six-well plates(3.5×105 cells/well) and treated with 3d at
concentrations of 0,2,4,8 μM for 24 h, respectively. The cells were collected, washed with
PBS, and stained with FITC-Annexin-V and PI and incubated for 15 min. Apoptosis was
determined by flow cytometry. Annexin-V positive, propidium iodide negative cells were
considered early apoptotic and Annexin-V positive, propidium iodide positive cells were
considered late apoptotic.
4.2.4. Cell cycle analysis
MCF-7 cells were treated with 3d at concentrations of 0,2,4,8 μM for 24 h, respectively.
The cells were harvested, fixed with 75% ethanol for 4 h at -20℃, and incubated with
PI/RNase staining buffer for 30 min at room temperature. The DNA content in different
groups of cells was assessed by flow cytometry.
4.2.5. Cell migration scratch assay
For wound healing assay, 2 ml MCF-7 or HeLa cells with a density of 5×105 cells/well
were placed in 6-well plate and incubated at 37 ℃in a humidified atmosphere with 5% CO2
for 12-24 h and grown to confluency. Then we used 20 μL pipette to scratch cell monolayers
to create a cell-free zone. Washed each well 3 times with PBS buffer and incubated using
serum-free media or 3d to each well, and then incubated at 37 ℃ with 5% CO2 for 24 h.
Photographed at 0 h, 24 h under the microscope to observe cell migration.
4.2.6 .Network pharmacological analysis
The Pharma DB database in Discovery Studio software was used to predict the target of
compound 3d, and the result with a FitValue greater than 0.9 was selected. In order to further
determine the most likely target protein, we imported the predicted protein target into the
string database(https://string-db.org/) to define species for humans, got the protein interaction
relationship, saved the results in TSV format, and import Cytoscape 3.6.0 statistical software
to draw interaction networks. First analyzed the protein interaction network as a whole, then
used the cytohubba plug-in in Cytoscape statistical software to determine the 15 most
important protein interaction relationships, and selected the maximum cluster centrality
(MCC) for the calculation method. KEGG analysis was performed on the protein input the
23

DAVID 6.8(https://david.ncifcrf.gov) .
4.2.7. Western blotting analysis
MCF-7 Cells at a density of 4 ×105 cells/3 mL were seeded onto 6-well plates. After
incubation for 24 h, 3d at various concentrations were added. After treatment, cells were
collected and lysed in RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1%
NP-40,0.5% sodium deoxycholate, 0.1% SDS), and centrifuged at 13,000 r for 15 min in the 4
℃ centrifuge. Supernatants were collected, and the total protein concentration was quantified
with a bicinchoninic acid (BCA) assay kit. The protein concentration was determined, equal
amounts of proteins were loaded onto SDS-PAGE gels, and separated proteins were
transferred to PVDF membranes. After blocking with 5% BSA at room temperature for 2 h,
the membranes were incubated with primary antibodies against AKT,p-AKT,mTOR and
p-mTOR, a monoclonal antibody against β-actin was used as a protein loading control. The
membranes were washed three times with TBST buffer for 30 min, 10 min at a time, then
incubated with HRP-conjugated secondary antibody for 2 h. After being washed with the
TBST buffer again, membranes were scanned with the Odyssey Infrared Imaging System.
Conflicts of interest
The authors declare that there are no conflicts of interest.
Acknowledgements
This study was supported by the National Natural Science Foundation of China (Grant No.
21506024) and Natural Science Foundation of Liaoning Province (2019-MS-060).
Reference
[1]
J. Pollier, A. Goossens, Oleanolic acid, Phytochemistry. 77 (2012) 10–15.
https://doi.org/10.1016/j.phytochem.2011.12.022.
[2]
X.L. Wan, Y.F. Lu, S.F. Xu, Q. Wu, J. Liu, Oeanolic acid protects against the hepatotoxicity of
D-galactosame plus endotoxin in mice, Biomed. Pharmacother. 93 (2017) 1040–1046.
https://doi.org/10.1016/j.biopha.2017.07.045.
[3]
J. Liu, Y.F. Lu, Q. Wu, S.F. Xu, F.G. Shi, C.D. Klaassen, Oleanolic acid reprograms the liver to
protect against hepatotoxicants, but is hepatotoxic at high doses, Liver Int. 39 (2019) 427–439.
https://doi.org/10.1111/liv.13940.
24

[4]
[5]
[6]
[7]
M. Masullo, C. Pizza, S. Piacente, Oleanane derivatives for pharmaceutical use: a patent review
(2000-2016), Expert Opin. Ther. Pat. 27 (2017) 237–255.
https://doi.org/10.1080/13543776.2017.1253680.
M.K. Shanmugam, X. Dai, A.P. Kumar, B.K.H. Tan, G. Sethi, A. Bishayee, Oleanolic acid and its
synthetic derivatives for the prevention and therapy of cancer: Preclinical and clinical evidence,
Cancer Lett. 346 (2014) 206–216. https://doi.org/10.1016/j.canlet.2014.01.016.
M.W. Pertino, C. Lopez, C. Theoduloz, G. Schmeda-Hirschmann, 1,2,3-triazole-substituted
oleanolic acid derivatives: Synthesis and antiproliferative activity, Molecules. 18 (2013) 7661–7674.
https://doi.org/10.3390/molecules18077661.
H.E. Elsayed, M.R. Akl, H.Y. Ebrahim, A.A. Sallam, E.G. Haggag, A.M. Kamal, K.A. El Sayed,
Discovery, optimization, and pharmacophore modeling of oleanolic acid and analogues as breast
cancer cell migration and invasion inhibitors through targeting Brk/Paxillin/Rac1 axis, Chem. Biol.
Drug Des. 85 (2015) 231–243. https://doi.org/10.1111/cbdd.12380.
[8]
[9]
J.J. Ramírez-Espinosa, M.Y. Rios, P. Paoli, V. Flores-Morales, G. Camici, V.D. La Rosa-Lugo, S.
Hidalgo-Figueroa, G. Navarrete-Vázquez, S. Estrada-Soto, Synthesis of oleanolic acid derivatives:
In vitro, in vivo and in silico studies for PTP-1B inhibition, Eur. J. Med. Chem. 87 (2014) 316–327.
https://doi.org/10.1016/j.ejmech.2014.09.036.
X. Han, L.L. Si, Y.Y. Shi, Z.B. Fan, S.X. Wang, Z.Y. Tian, M. Li, J.Q. Sun, P.X. Jiao, F.X. Ran,
Y.M. Zhang, D.M. Zhou, S.L. Xiao, Synthesis and in vitro anti-influenza virus evaluation of novel
sialic acid (C-5 and C-9)-pentacyclic triterpene derivatives, Molecules. 22 (2017) 1–17.
https://doi.org/10.3390/molecules22071018.
[10] L. Fu, Q.X. Lin, E.O. Onyango, K.T. Liby, M.B. Sporn, G.W. Gribble, Design, synthesis, and
biological activity of second-generation synthetic oleanane triterpenoids, Org. Biomol. Chem. 15
(2017) 6001–6005. https://doi.org/10.1039/c7ob01420a.
[11] L.X. Rui, S.Y. Shu, W.J. Jun, C.Z. Mo, S.Z. Wu, L.S. Min, L. Yuan, P.J. Yong, S.Z. Cheng, W.S.
Sheng, T.Z. Yao, The dual induction of apoptosis and autophagy by SZC014, a synthetic oleanolic
acid derivative, in gastric cancer cells via NF-κB pathway, Tumor Biol. 37 (2016) 5133–5144.
https://doi.org/10.1007/s13277-015-4293-2.
[12] L. Gao, Z. Xu, Y. Wang, B. Sun, Z. Song, B. Yang, X. Liu, Y. Lin, J. Peng, G. Han, S. Wang, Z.
Tang, Anticancer effect of SZC017, a novel derivative of oleanolic acid on human gastric cancer
25

cells, Oncol. Rep. 35 (2016) 1101–1108. https://doi.org/10.3892/or.2015.4447.
[13] Y. Song, L. Gao, Z. Tang, H. Li, B. Sun, P. Chu, E. Qaed, X. Ma, J. Peng, S. Wang, M. Hu, Z.
Tang, Anticancer effect of SZC015 on pancreatic cancer via mitochondria-dependent apoptosis and
the constitutive suppression of activated nuclear factor κB and STAT3 in vitro and in vivo, J. Cell.
Physiol. 234 (2018) 777–788. https://doi.org/10.1002/jcp.26892.
[14] L. Gao, Y. Wang, Z. Xu, X. Li, J. Wu, S. Liu, P. Chu, Z. Sun, B. Sun, Y. Lin, J. Peng, G. Han, S.
Wang, Z. Tang, SZC017, a novel oleanolic acid derivative, induces apoptosis and autophagy in
human breast cancer cells, Apoptosis. 20 (2015) 1636–1650.
https://doi.org/10.1007/s10495-015-1179-0.
[15] P. Chu, H. Li, R. Luo, A. Ahsan, E. Qaed, A. Shopit, X. Ma, Y. Lin, J. Peng, J. Zhang, S. Wang, Z.
Tang, Oleanolic acid derivative SZC014 inhibit cell proliferation and induce apoptosis of human
breast cancer cells in a ROS-dependent way, Neoplasma. 64 (2017) 681–692.
https://doi.org/10.4149/neo_2017_505.
[16] Y. Song, L. Kong, B. Sun, L. Gao, P. Chu, A. Ahsan, E. Qaed, Y. Lin, J. Peng, X. Ma, J. Zhang, S.
Wang, Z. Tang, Induction of autophagy by an oleanolic acid derivative, SZC017, promotes
ROS-dependent apoptosis through Akt and JAK2/STAT3 signaling pathway in human lung cancer
cells, Cell Biol. Int. 41 (2017) 1367–1378. https://doi.org/10.1002/cbin.10868.
[17] B. Sun, L. Gao, A. Ahsan, P. Chu, Y. Song, H. Li, Z. Zhang, Y. Lin, J. Peng, Z. Song, S. Wang, Z.
Tang, Anticancer effect of SZC015 on lung cancer cells through ROS-dependent apoptosis and
autophagy induction mechanisms in vitro, Int. Immunopharmacol. 40 (2016) 400–409.
https://doi.org/10.1016/j.intimp.2016.09.026.
[18] M.B. Sporn, K.T. Liby, M.M. Yore, L. Fu, J.M. Lopchuk, G.W. Gribble, New synthetic
triterpenoids: Potent agents for prevention and treatment of tissue injury caused by inflammatory
and oxidative stress, J. Nat. Prod. 74 (2011) 537–545. https://doi.org/10.1021/np100826q.
[19] T. Honda, G.W. Gribble, N. Suh, H.J. Finlay, B.A. V. Rounds, L. Bore, F.G. Favaloro, Y. Wang,
M.B. Sporn, Novel synthetic oleanane and ursane triterpenoids with various enone functionalities in
ring A as inhibitors of nitric oxide production in mouse macrophages, J. Med. Chem. 43 (2000)
1866–1877. https://doi.org/10.1021/jm000008j.
[20] T. Honda, B. V. Rounds, L. Bore, H.J. Finlay, F.G. Favaloro, N. Suh, Y. Wang, M.B. Sporn, G.W.
Gribble, Synthetic oleanane and ursane triterpenoids with modified rings A and C: A series of
26

highly active inhibitors of nitric oxide production in mouse macrophages, J. Med. Chem. 43 (2000)
4233–4246. https://doi.org/10.1021/jm0002230.
[21] M.H.L. Wong, H.K. Bryan, I.M. Copple, R.E. Jenkins, P.H. Chiu, J. Bibby, N.G. Berry, N.R.
Kitteringham, C.E. Goldring, P.M. O’Neill, B.K. Park, Design and Synthesis of Irreversible
Analogues of Bardoxolone Methyl for the Identification of Pharmacologically Relevant Targets and
Interaction Sites, J. Med. Chem. 59 (2016) 2396–2409.
https://doi.org/10.1021/acs.jmedchem.5b01292.
[22] A. Cleasby, J. Yon, P.J. Day, C. Richardson, I.J. Tickle, P.A. Williams, J.F. Callahan, R. Carr, N.
Concha, J.K. Kerns, H. Qi, T. Sweitzer, P. Ward, T.G. Davies, Structure of the BTB domain of
Keap1 and its interaction with the triterpenoid antagonist CDDO, PLoS One. 9 (2014) 15–21.
https://doi.org/10.1371/journal.pone.0098896.
[23] M. Gehringer, S.A. Laufer, Emerging and Re-Emerging Warheads for Targeted Covalent Inhibitors:
Applications in Medicinal Chemistry and Chemical Biology, J. Med. Chem. 62 (2019) 5673–5724.
https://doi.org/10.1021/acs.jmedchem.8b01153.
[24] S. Ray, A.S. Murkin, New Electrophiles and Strategies for Mechanism-Based and Targeted
Covalent Inhibitor Design, Biochemistry. (2019). https://doi.org/10.1021/acs.biochem.9b00293.
[25] M. Hossain, U. Das, J.R. Dimmock, Recent advances in α,β-unsaturated carbonyl compounds as
mitochondrial toxins, Eur. J. Med. Chem. 183 (2019).
https://doi.org/10.1016/j.ejmech.2019.111687.
[26] E.H. Krenske, R.C. Petter, K.N. Houk, Kinetics and Thermodynamics of Reversible Thiol
Additions to Mono- and Diactivated Michael Acceptors: Implications for the Design of Drugs That
Bind Covalently to Cysteines, J. Org. Chem. 81 (2016) 11726–11733.
https://doi.org/10.1021/acs.joc.6b02188.
[27] G. Akçay, M.A. Belmonte, B. Aquila, C. Chuaqui, A.W. Hird, M.L. Lamb, P.B. Rawlins, N. Su, S.
Tentarelli, N.P. Grimster, Q. Su, Inhibition of Mcl-1 through covalent modification of a
noncatalytic lysine side chain, Nat. Chem. Biol. 12 (2016) 931–936.
https://doi.org/10.1038/nchembio.2174.
[28] D. De Zeeuw, T. Akizawa, Bardoxolone methyl in type 2 diabetes and stage 4 chronic kidney
disease, N. Engl. J. Med. 369 (2013) 2492–2503. https://doi.org/10.1056/NEJMoa1306033.
[29] Z. Huang, Y. Mou, X. Xu, D. Zhao, Y. Lai, Y. Xu, C. Chen, P. Li, S. Peng, J. Tian, Y. Zhang,
27

Novel Derivative of Bardoxolone Methyl Improves Safety for the Treatment of Diabetic
Nephropathy, J. Med. Chem. 60 (2017) 8847–8857. https://doi.org/10.1021/acs.jmedchem.7b00971.
[30] E. Bonandi, M.S. Christodoulou, G. Fumagalli, D. Perdicchia, G. Rastelli, D. Passarella, The
1,2,3-triazole ring as a bioisostere in medicinal chemistry, Drug Discov. Today. 22 (2017) 1572–
1581. https://doi.org/10.1016/j.drudis.2017.05.014.
[31] G. Wei, W. Luan, S. Wang, S. Cui, F. Li, Y. Liu, Y. Liu, M. Cheng, A library of
1,2,3-triazole-substituted oleanolic acid derivatives as anticancer agents: Design, synthesis, and
biological evaluation, Org. Biomol. Chem. 13 (2015) 1507–1514.
https://doi.org/10.1039/c4ob01605j.
[32] M. Kanehisa, The KEGG resource for deciphering the genome, Nucleic Acids Res. 32 (2004) 277D
– 280. https://doi.org/10.1093/nar/gkh063.
[33] C. von Mering, L.J. Jensen, B. Snel, S.D. Hooper, M. Krupp, M. Foglierini, N. Jouffre, M.A.
Huynen, P. Bork, STRING: Known and predicted protein-protein associations, integrated and
transferred across organisms, Nucleic Acids Res. 33 (2005) 433–437.
https://doi.org/10.1093/nar/gki005.
[34] G. Hoxhaj, B.D. Manning, The PI3K–AKT network at the interface of oncogenic signalling and
cancer metabolism, Nat. Rev. Cancer. (2019). https://doi.org/10.1038/s41568-019-0216-7.
[35] J. Luo, B.D. Manning, L.C. Cantley, Targeting the PI3K-Akt pathway in human cancer: Rationale
and promise, Cancer Cell. 4 (2003) 257–262. https://doi.org/10.1016/S1535-6108(03)00248-4.
[36] S. Noorolyai, N. Shajari, E. Baghbani, S. Sadreddini, B. Baradaran, The relation between
PI3K/AKT signalling pathway and cancer, Gene. 698 (2019) 120–128.
https://doi.org/10.1016/j.gene.2019.02.076.
[37] J. Wu, C. Yang, C. Guo, X. Li, N. Yang, L. Zhao, H. Hang, S. Liu, P. Chu, Z. Sun, B. Sun, Y. Lin,
J. Peng, G. Han, S. Wang, Z. Tang, SZC015, a synthetic oleanolic acid derivative, induces both
apoptosis and autophagy in MCF-7 breast cancer cells, Chem. Biol. Interact. 244 (2016) 94–104.
https://doi.org/10.1016/j.cbi.2015.11.013.
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