Synthesis and biological activity of glycyrrhetinic acid derivatives as antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2019.06.029

Glycyrrhetinic acid (GA) had been the star anticancer lead compound and appealed to many scientists all over the world; however, its antitumor activity was not potent enough. To improve GA's cytoxicity and explore the effect of bonding mode on antitumor activity, 32 compounds including GA-OH series (GO, esters in C-3 position) and GA-NH₂ series (GN, with amide linkages in C-3 position) had been designed and synthesized. All the compounds were screened for in vitro cytotoxicity against A549, HepG2, MCF-7, Hela and MDCK cell lines. As a result, all the de-protected (without Boc group) derivatives showed much stronger cytotoxic activity than GA, and surprisingly enough, all the GN series of the compounds were more potent than GO series against various tumor cells. Among them, the compound 26 (amide linkages in C-3 position) exhibited stronger antitumor activity against A549 cell line (IC₅₀ = 2.109 ± 0.11 μM) than the positive drug cisplatin (IC₅₀ = 9.001 ± 0.37 μM). Further studies indicated that compound 26 could induce A549 apoptosis via nuclei fragmentation. The detection of apoptosis and cell cycle analysis indicated that compound 26 could induce the early apoptosis and prevent A549 cells transition from S to G2 phase. Furthermore, the structure-activity relationships were briefly discussed. Among which, current study displayed amide linkages in C-3 position could effectively enhance GA cytotoxicity, providing a new modification strategy for further study.

Full text of DOI:10.1016/j.ejmech.2019.06.029

Accepted Manuscript
Synthesis and biological activity of glycyrrhetinic acid derivatives as antitumor agents
Fei Zhou, Gao-Rong Wu, De-Sheng Cai, Bing Xu, Meng-Meng Yan, Tao Ma, Wen-Bo
Guo, Wen-Xi Zhang, Xue-Mei Huang, Xiao-hui Jia, Yu-Qin Yang, Feng Gao, Peng-
Long Wang, Hai-Min Lei
PII:
S0223-5234(19)30555-0
DOI:
https://doi.org/10.1016/j.ejmech.2019.06.029
EJMECH 11432
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 May 2019
Revised Date: 2 June 2019
Accepted Date: 10 June 2019
Please cite this article as: F. Zhou, G.-R. Wu, D.-S. Cai, B. Xu, M.-M. Yan, T. Ma, W.-B. Guo, W.-X.
Zhang, X.-M. Huang, X.-h. Jia, Y.-Q. Yang, F. Gao, P.-L. Wang, H.-M. Lei, Synthesis and biological
activity of glycyrrhetinic acid derivatives as antitumor agents, European Journal of Medicinal Chemistry
(2019), doi: https://doi.org/10.1016/j.ejmech.2019.06.029.
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Synthesis and biological activity of
glycyrrhetinic acid derivatives as antitumor
agents
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Fei Zhou, Gao-Rong Wu, De-Sheng Cai, Bing Xu, Meng-Meng Yan, Tao
Ma, Wen-Bo Guo, Wen-Xi Zhang, Xue-Mei Huang, Xiao-hui Jia, Yu-Qin
Yang, Feng Gao, Peng-Long Wang**, Hai-Min Lei*.
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School of Chinese Pharmacy, Beijing University of Chinese Medicine,
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Beijing 100102, China
* Corresponding author.
** Corresponding author.
E-mail addresses: wpl581@126.com (P.-L. Wang), hm_lei@126.com
(H.-M. Lei).
Abstract: Glycyrrhetinic acid (GA) had been the star anticancer lead
compound and appealed to many scientists all over the world; however,
its antitumor activity was not potent enough. To improve GA’s cytoxicity
and explore the effect of bonding mode on antitumor activity, 32
compounds including GA-OH series (GO, esters in C-3 position) and
GA-NH series (GN, with amide linkages in C-3 position) had been
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designed and synthesized. All the compounds were screened for in vitro

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cytotoxicity against A549, HepG2, MCF-7, Hela and MDCK cell lines.
As a result, all the de-protected (without Boc group) derivatives showed
much stronger cytotoxic activity than GA, and surprisingly enough, all
the GN series of the compounds were more potent than GO series against
various tumor cells. Among them, the compound 26 (amide linkages in
C-3 position) exhibited stronger antitumor activity against A549 cell line
(IC = 2.109 ± 0.11 µM) than the positive drug cisplatin (IC = 9.001
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± 0.37 µM). Further studies indicated that compound 26 could induce
A549 apoptosis via nuclei fragmentation. The detection of apoptosis and
cell cycle analysis indicated that compound 26 could induce the early
apoptosis and prevent A549 cells transition from S to G2 phase.
Furthermore, the structure-activity relationships were briefly discussed.
Among which, current study displayed amide linkages in C-3 position
could effectively enhance GA cytotoxicity, providing a new modification
strategy for further study.
Keywords: Glycyrrhetinic; amino acid; antitumor; bonding mode; amide
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1. Introduction
In recent years, increasing effort had been made to find new antitumor
compounds, and natural products always appealed to most scientists [1-3].
Among them, pentacyclic triterpenoids were largely found in medicinal

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plants and possessed a wide range of pharmacological activities [4-6].
Glycyrrhetinic acid (GA), the aglycone of glycyrrhizin found in the roots
of licorice [7], had become a star anticancer compound as thousands of
research papers published were carried around it. GA had been gotten into
the focus of worldwide scientific interest because of its advantages such
as being obtained easily and cheaply, good stability, high biosecurity and
antiproliferative activity against various cancer cell lines. Nowadays, due
to the influence of cancer, it was very common that people became poor
or even went bankrupt in many developing countries. With the
advantages of GA-like drugs, it had the prospect of developing affordable
chemotherapy drugs that were acceptable for the poor people [8-12].
However, the weak antitumor activity of GA was a big weakness when
compared to other triterpenes like triptolide and betulinic acid [13,14]. All
the advantages mentioned and desire to improve the inadequacies of GA
had made it meaningful to be modified for the discovery of potential
antitumor compounds [15,16].
According to many other researches and previous work performed by
our group, introduction of protected or de-protected amino acid could
increase the antitumor activity as well as water solubility [17-19].
Structure activity relationship indicated that esterification at C-30 could
enhance the activity of GA, and among methyl ester, ethyl ester and
benzyl (Bn) ester at C-30, benzyl ester showed the best antitumor

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activities [20,21]. In addition, according to the reports that bonding mode
of the compound might strongly affect the activity, and amide linkages
not only improved activity of compound but also more stable than esters
in metabolism [22,23]. Based on the above, to improve GA’s cytotoxicity
and to explore the effect of bonding mode on antitumor activity, 32 GA
derivatives including GO series and GN series had been designed and
synthesized. The cytotoxic activity of all compounds was screened
against A549 (human lung cancer), MCF-7 (human mammary cancer),
HepG2 (human hepatocellular carcinoma), Hela (human cervical cancer)
and MDCK (Madin-Darby canine kidney) cell lines in vitro. Moreover,
fluorescence staining observations and flow cytometric analyses were
performed to observe the preliminary antitumor mechanisms of the most
selectivity compound. In addition, the structure-activity relationships of
the two series of GA derivatives were briefly discussed. Notably, current
study displayed amide linkages in C-3 position could effectively enhance
the antitumor activity, providing a new modification strategy for the
following study of GA.
2. Results and Discussion
2.1 Chemistry
The designed derivatives were prepared following the procedures in
Schemes 1-3. In Scheme 1, GA derivative GA-BN were synthesized

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through a combination of GA and benzyl bromide (Bn-Br) containing
K CO at 85℃ in N,N-dimethylformamide (DMF). Then the intermediate
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GA-BN was further reacted with N-protected-L-amino acid in dry
dichloromethane (DCM) in the presence of
1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI)
and 4-dimethylaminopyridine (DMAP) to yield compounds 1-8.
Subsequently, deprotection was performed with trifluoroacetic acid (TFA)
in dry DCM at 0℃ to afford compounds 9-16.
In Scheme 2, the intermediate GO-BN was obtained through the
oxidation with CrO /H SO in acetone, the two epimers of GN-BN was
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00 obtained by Borch reduction with sodium cyanoborohydride and
01 ammonium acetate in methanol [22]. Considering that the change of
02 absolute stereochemistry in C3-NH didn’t evidently improve the
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03 antitumor activity which was reported by Czuk’s group [24,25], we only
04 chose the main product 3β-GN-BN to synthesize the GA derivatives.
05 After 3β-GN-BN was produced, as shown in Scheme 3, compounds
06 17-24 were obtained through the combination of 3β-GN-BN and
07 N-protected-L-amino acid under catalyzed by EDCI and
08 1-Hydroxybenzotriazole (HOBt) and N,N-diisopropylethylamine (DIPEA)
09 in dry DCM, and deprotection was performed with TFA in dry DCM at 0 ℃
10 to yield compounds 25-32. All reactions were carried out as detailed in
11 the experimental section, and the structures of all target derivatives were

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12 confirmed by spectral ( H NMR, C NMR and HRMS) analysis.
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14 (Insert Scheme 1.)
15 (Insert Scheme 2.)
16 (Insert Scheme 3.)
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18 2.2 Biology
19 2.2.1 Cytotoxicity assay
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The cytotoxicity of glycyrrhetinic acid derivatives in vitro was
21 evaluated on four tumor cell lines (A549: human non-small-cell lung
22 cancer; MCF-7: human mammary cancer; HepG2: human hepatocellular
23 carcinoma; Hela: human cervical cancer cell) using the the
24 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
25 assay. In addition, their toxicity was tested using one normal cell line
26 (MDCK: Madin-Darby canine kidney) cells. The IC values of these
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28 amino acid derivatives seemed not improved apparently, but all of the
29 de-protection GA amino acid derivatives were more potent against four
30 tumor cell lines than GA. The cytotoxicity detection also revealed that
31 most of GA amino acids derivatives, especially the amide linkages
32 derivatives (GN series), exhibited better antitumor activities than the
33 positive drug cisplatin (DDP). For A549 cell lines, compound 26

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34 exhibited stronger antitumor activities (IC =2.109±0.11µM) than DDP
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35 (IC =9.001±0.37µM), and was over 18 times better than GA (IC >
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36 40µM); For HepG2 cell line, compound 28 (IC =2.143±0.09µM) was
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37 over 16 times better than GA (IC > 40µM) and stronger than DDP
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38 (IC =3.908 ± 0.17µM). Among the derivatives, four most potent
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39 compounds and DDP were listed and calculated their average IC values
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40 against four cancer cell lines as shown in Fig.1. Compound 26 showed
41 the strongest antitumor activity (average IC =2.268µM) which was
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42 threefold than the positive drug DDP (average IC =7.392µM)
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In the meantime, it could be obviously seen that the amide linkages
44 derivatives (GN series) exhibited better antiproliferative activities against
45 the cell lines than ester linkages derivatives (GO series). As shown in
46 Fig.2, the IC values of all GN series were lower than GO series on A549
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47 cells, especially for derivatives connected with phenylalanine and leucine,
48 which in separate improved 5.64 times and 6.98 times, respectively.
49 Among them, compound 26 was the most potent one against A549 cells.
50 As described above, introduction of de-protected amino acid (without
51 Boc group) could enhance antitumor activities and turning hydroxyl of
52 C-3 into amino made all derivatives obtain better antitumor activities,
53 Considering compound 26 had the lowest average of IC values, we
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54 chose it for further analysis to learn the mechanism of growth inhibition
55 on A549 cells.

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57 (Insert Table 1.)
58 (Insert Figure 1.)
59 (Insert Figure 2.)
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61 2.2.2 Analyses of apoptosis
62 2.2.2.1 Morphological detection of apoptosis using DAPI staining
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To further investigate the mechanism of apoptosis inducted by
64 compound 26 on A549 cell line, DAPI staining was performed after
65 treating A549 cells with 0, 1, 3 and 5 µM of compound 26 for 72 h. As
66 shown in Fig.3, control groups of A549 cells showed intact cell bodies
67 with clear round nuclei; while with increasing concentration of compound,
68 the formation of apoptotic bodies appeared and A549 cells showed
69 condensed chromatin, nuclear fragmentation and weak fluorescence. In
70 summary, these results indicated that compound 26 could induced
71 apoptosis in A549 cells.
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73 (Insert Figure 3.)
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75 2.2.2.2 Detection of apoptosis using Annexin V-FITC/PI staining
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To substantiate the ability of compound 26 to induce apoptosis,
77 further studies were carried out using Annexin V-FITC/PI staining

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78 technique in A549 cells. As performed in Fig.4, when treated with
79 different concentrations (0, 1, 3, and 5 µM) of compound 26, the early
80 apoptosis ratios increased from 3.0% of the control to 5.9%, 26.5%,
81 44.8%, respectively. These results indicated that compound 26 had the
82 potential to induce A549 cell early apoptosis.
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84 (Insert Figure 4.)
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86 2.2.2.3Cell cycle analysis using PI staining
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To analyze the cell cycle, A549 cells were stained with PI apoptosis
88 detection kit As shown in Fig.5, with the increasing concentration of
89 compound 26 (0, 1, 3, and 5 µM), the percentage of A549 cells in S phase
90 increased dramatically (from 31.41% to 53.21%), which evidently
91 indicated that compound 26 was able to induce cell cycle arrest in the S
92 phase.
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94 (Insert Figure 5.)
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96 3. Conclusions
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In this study, 32 Glycyrrhetinic acid derivatives had been designed
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98 and synthesized and all of them were characterized by H NMR, C
99 NMR, HRMS. In addition, all these compounds were tested for cytotoxic

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00 activity against MCF-7, A549, Hela, HepG2 and MDCK cell lines by
01 standard MTT assay. From the obtained results, it could be known that all
02 of the de-protection GA amino acid derivatives were more active against
03 four tumor cell lines than GA, while those protection GA amino acid
04 derivatives did not behave obvious antitumor activities. Meanwhile, what
05 surprised us was that the amide linkages derivatives (GN series) were
06 more potent against those tumor cells than ester linkages derivatives (GO
07 series), which proved the bonding mode of GA derivatives indeed
08 effectively affected the antitumor activity. Among them, compound 26
09 exhibited a wide range of antitumor activity and was better than cisplatin
10 against A549 cells whose IC was 2.109±0.11µM and 9.001±0.37µM
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11 respectively. Through DAPI staining, it was confirmed that compound 26
12 could induce A549 apoptosis via nuclei fragmentation. The detection of
13 apoptosis and cell cycle analysis proved that compound 26 could induce
14 cell cycle arrest at the S phase. In summary, the introduction of amino
15 and the formation of amide bond could effectively enhance the antitumor
16 activity of GA, although that the selectivity of the compound was not
17 improved, this kind of compounds with significantly increased activity
18 were suitable for the prodrug design, the strategy of our group by
19 introducing targeted pentapeptide which could effectively change the
20 selective
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22 cells[26]. Furthermore, these results in this manuscript might prove
23 meaningful for the chemical modification of GA and the further
24 researches on GN series would be carried out on the following research.
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26 4. Experimental section
27 4.1 Chemistry
28
Reagents were obtained from commercial suppliers and used without
29 further purification. All melting points were determined on a micro
30 melting point apparatus and were uncorrected. TLC was performed on
31 silica gel coated aluminum sheets (Qingdao Haiyang Chemical Co.,
32 Qingdao, China) and visualized in UV light (254 nm). NMR spectra were
33 recorded on a Bruker 400 spectrometer (Bruker, Germany) with
34 tetramethylsilan (TMS) as an internal standard; chemical shifts δ were
35 given in ppm and coupling constants J in Hz. HR-MS were acquired
36 using a Thermo Scientific TM LTQ Orbitrap XL hybrid FTMS instrument
37 (Thermo Technologies, New York, NY, USA).
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39 4.1.1 Preparation of Benzyl protected glycyrrhetinic acid (GA-BN)
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Glycyrrhetinic acid (1 equiv.) was dissolved in DMF (40 mL), Benzyl
41 bromide (1.2 equiv.) and anhydrous K CO (3 equiv.) were added. The
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42 mixture was stirred at 80 ℃ for 3 h. After completion of the reaction (as
43 monitored by TLC), the solvent was condensed to less than 20 mL under

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44 reduced pressure, and then 100 mL water was added while the mixture
45 was stirred, precipitated substances in water were filtered and dried them
46 in the oven. Purification was performed by flash chromatography.
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48 4.1.2 Preparation of Benzyl protected 3-aminoglycyrrhetinic acid
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To a solution of Benzyl protected glycyrrhetinic acid (1 equiv.) in
51 CH COCH (20 mL), a solution of chromium trioxide in dilute sulfuric
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52 acid (10 mL.) was added drop by drop. The reaction mixture was stirred
53 at 0 ℃ for 1 h. After completion of the reaction (as monitored by TLC),
54 the reaction mixture was diluted with 50 mL DCM, then successively
55 washed with water and brine (20 mL each), dried over anhydrous sodium
56 sulfate and filtered, and the solvent was evaporated under vacuum.
57 Purification was performed by flash chromatography to give GO-BN as
58 white solid.
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To a solution of the intermediate (GO-BN) (1 equiv.) and
60 CH COONH (10 equiv.）in CH OH (100 mL), NaCNBH (1.5 equiv.)
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61 was added and the reaction mixture was stirred for 12 h at room
62 temperature. The reaction mixture was concentrated, H O (100 mL) was
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63 added. The pH of the mixture was adjusted to 9 with NaOH solution (1M),
64 and filtered to give crude product. The crude product was purified using
65 flash chromatography to give Benzyl protected 3β-aminoglycyrrhetinic

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66 acid as white solid.
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68 4.1.3. General procedure for esterification at C3-OH of Benzyl
69 protected glycyrrhetinic acid (method A)
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The corresponding intermediate (GA-BN) (1 equiv.) was dissolved in
71 dry DCM (25 mL), then DMAP (0.2 equiv.), EDCI (1.5 equiv.) and the
72 protected amino acid with Boc group (1.2 equiv.) were added. The
73 mixture was stirred at room temperature for 12 h. After completion of the
74 reaction (as monitored by TLC), the reaction mixture was diluted with 50
75 mL DCM, then successively washed with water and brine (20 mL each),
76 dried over anhydrous sodium sulfate and filtered, and the solvent was
77 evaporated under vacuum. Purification was performed by flash
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78 chromatography.
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80 4.1.4. General procedure for de-protection (method B)
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TFA (2 mL) was added slowly to the solution of the Boc-protected
82 compound in dry DCM (6 mL). The mixture was stirred at 0 ℃ for 4 h.
83 After completion of the reaction (as monitored by TLC), the solvent was
84 removed under reduced pressure and washed with a saturated sodium
85 bicarbonate solution (10 mL). The aqueous layer was extracted with
86 DCM (3×25 mL) and the combined organic extracts were dried over
87 anhydrous sodium sulfate, filtrated and evaporated. Then the crude

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88 product was purified by flash chromatography.
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90 4.1.5. General procedure for esterification at C3-NH2 of Benzyl
91 protected 3-aminoglycyrrhetinic acid (method C)
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The corresponding intermediate (GN-BN) (1 equiv.) was dissolved in
93 dry DCM (25 mL), then HOBt (1.5 equiv.), EDCI (1.5 equiv.), DIPEA
94 (2.5 equiv.) and the protected amino acid with Boc group (1.2 equiv.)
95 were added. The mixture was stirred at room temperature for 12 h. After
96 completion of the reaction (as monitored by TLC), the reaction mixture
97 was diluted with 50 mL DCM, then successively washed with water and
98 brine (20 mL each), dried over anhydrous sodium sulfate and filtered, and
99 the solvent was evaporated under vacuum. Purification was performed by
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04 4.1.3.1.Benzyl
05 3β-(N-Boc-glycyl)-11-oxo-olean-12-en-30-oate(Compound
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06 According to Method A, compound 1 was obtained as white powder;
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07 yield: 79.6%; m.p.: 89.8 ℃ , [a] =+140 (c 0.3 mg/mL, MeOH); H NMR
D
08 (400MHz, CDCl ): δ (ppm) 7.40-7.30 (m, 5H, H-Ar), 5.55 (s, 1H, H-12),
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09 5.18, 5.10 (d, each, 1H, Bn-CH , J = 12.4 Hz), 4.59 (dd, 1H, H-3, J =
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10 11.6 Hz, 4.8 Hz), 3.90 (m, 2H, Gly-CH NH), 2.80 (dt, 1H, H-1, J = 13.6
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11 Hz, 3.6 Hz), 2.33 (s, 1H, H-9), 2.05-1.91, 1.80-1.51, 1.42-1.35, 1.31-1.18,
12 1.04-0.96, 0.80-0.77 (19H, methylene and methine of triterpenoid
13 structure), 1.45 (brs, 9H, Boc-CH ), 1.34 (s, 3H, H-27), 1.16-1.15 (br, 6H,
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14 H-29 and H-25), 1.10 (s, 3H, H-26), 0.87 (brs, 6H, H-23 and H-24), 0.73
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15 (s, 3H, H-28). C NMR (100MHz, CDCl ): δ (ppm) 200.1 (C-11), 176.3
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16 (C-13), 170.3(Gly-COO), 169.3(C-13), 155.8 (Boc-COO), 136.3 (Car),
17 128.8 (C ), 128.8 (C ), 128.6 (C-12), 128.4 (C ), 128.3 (C ), 128.3 (C ),
ar
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18 82.1 (C-3), 80.0 (Boc-q.C), 66.4 (Bn-CH ), 61.8 (C-9), 55.1 (C-5), 48.4
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19 (C-18), 45.5 (C-8), 44.1 (C-20), 43.3 (C-14), 42.8 (Gly-CH NH), 41.2
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20 (C-19), 38.9 (C-1), 38.3 (C-4), 37.8 (C-22), 37.0 (C-10), 32.8 (C-7), 31.9
21 (C-17), 31.3 (C-21), 28.6 (C-29), 28.5 (C-23), 28.4 (Boc-CH ), 28.2
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22 (C-28), 26.6 (C-16), 26.5 (C-15), 23.7 (C-2), 23.4 (C-27), 18.8 (C-26),
23 17.5 (C-6), 16.8 (C-24), 16.5 (C-25). HRMS (ESI) m/z: 740.4481
+
24 [M+Na] , calcd. for C H NNaO 740.4502.
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26 4.1.3.2.Benzyl
27 3β-(N-Boc-L-alanyl)-11-oxo-olean-12-en-30-oate(Compound
2).
28 According to Method A, compound 2 was obtained as white powder;
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29 yield: 65.4%; m.p.: 192.4 ℃ , [a] =+140 (c 0.3 mg/mL, MeOH); H NMR
D
30 (400MHz, CDCl ): δ (ppm) 7.40-7.29 (m, 5H, H-Ar), 5.55 (d, 1H, H-12,
3
31 J =8.8 Hz), 5.18, 5.09 (d, each, 1H, Bn-CH , J = 12.0 Hz), 4.94 (m, 1H,
2
32 H-3), 4.11 (m, 1H, Ala-CHNH), 3.64 (m, 1H, Ala-CHNH), 2.78-2.66 (br,

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33 1H, H-1), 2.35 (s, 1H, H-9), 2.07-1.91, 1.83-1.48, 1.42-1.39, 1.30-1.25,
34 1.03-0.96, 0.86-0.84 (19H, methylene and methine of triterpenoid
35 structure), 1.44 (s, 9H, Boc-CH ), 1.36-1.32 (br, 6H, H-27 and -CH of
3
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36 Alanine), 1.16-1.12 (br, 6H, H-29 and H-25), 1.10 (s, 3H, H-26), 0.89 (s,
1
3
37 3H, H-24), 0.78 (m, 3H, H-23), 0.73 (s, 3H, H-28). C NMR (100MHz,
38 CDCl ): δ (ppm) 200.1(C-11), 176.3 (C-30), 173.2 (Ala-COO), 169.3
3
39 (C-13), 155.2 (Boc-COO), 136.3 (C ), 128.8 (C ), 128.8 (C ), 128.6
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40 (C-12), 128.4 (C ), 128.3 (C ), 128.3 (C ), 81.8 (C-3), 79.8 (Boc-q.C),
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41 66.4 (Bn-CH ), 61.8 (C-9), 55.1 (C-5), 49.70 (Ala-CHNH), 48.4 (C-18),
2
42 45.5 (C-8), 44.1 (C-20), 43.3 (C-14), 41.2 (C-19), 38.9 (C-1), 38.4 (C-4),
43 37.8 (C-22), 37.0 (C-10), 32.8 (C-7), 31.9 (C-17), 31.3 (C-21), 28.6
44 (C-29), 28.5 (C-23), 28.4 (Boc-CH ), 28.2 (C-28), 26.6 (C-16), 26.5
3
45 (C-15), 23.7 (C-2), 23.4 (C-27), 19.1 (Ala-CH ), 18.8 (C-26), 17.5 (C-6),
3
+
46 16.9 (C-24), 16.5 (C-25). HRMS (ESI) m/z: 754.4686 [M+Na] , calcd.
47 for C H NNaO 754.4659.
45
65
7
48
49 4.1.3.3.Benzyl
50 3β-(N-Boc-L-phenylalanine)-11-oxo-olean-12-en-30-oate(Compound 3).
51 According to Method A, compound 3 was obtained as white powder;
1
52 yield: 66.9%; m.p.: 99.0 ℃ , [a] =+132 (c 0.3 mg/mL, MeOH); H NMR
D
53 (400MHz, CDCl ): δ (ppm) 7.40-7.27, 7.25-7.15 (br, 10H, H-Ar), 5.54 (s,
3
54 1H, H-12), 5.19, 5.10 (d, each, 1H, Bn-CH , J = 12.0 Hz), 4.90 (d, 1H,
2

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55 Phe-CHNH, J = 8.4 Hz), 4.54 (m, 2H, Phe-CHNH and H-3), 3.12 (m, 1H,
56 Phe-CHH′), 3.03 (m, 1H, Phe-CHH′), 2.81 (dt, 1H, H-1, J = 13.6 Hz, 3.6
57 Hz), 2.32 (s, 1H, H-9), 2.08-1.90, 1.84-1.43, 1.32-1.18, 1.05-0.95, 0.76
58 (19H, methylene and methine of triterpenoid structure), 1.40 (brs, 9H,
59 Boc-CH ), 1.34 (s, 3H, H-27), 1.16 (s, 3H, H-29), 1.14 (s, 3H, H-25),
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60 1.10 (s, 3H, H-26), 0.82 (s, 3H, H-24), 0.80 (s, 3H, H-23), 0.73 (s, 3H,
1
3
61 H-28). C NMR (100MHz, CDCl ): δ (ppm) 200.1(C-11), 176.3 (C-30),
3
62 171.9 (Phe-COO), 169.2 (C-13), 155.2 (Boc-COO), 136.3 (Phe-Car),
63 136.2 (Bn-C ), 129.5 (Phe-C ), 129.5 (Phe-C ), 128.8 (Bn-C ), 128.8
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64 (Bn-C ), 128.7 (Bn -C ), 128.6 (C-12), 128.5 (Phe -C ), 128.5 (Phe-C ),
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65 128.4 (Bn-C ), 128.4 (Bn-C ), 127.1 (Phe-C ), 82.3 (C-3), 79.9
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66 (Boc-q.C), 66.4 (Bn-CH ), 61.8 (C-9), 55.2 (C-5), 54.8 (Phe-CHNH),
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67 48.4 (C-18), 45.5 (C-8), 44.1 (C-20), 43.3 (C-14), 41.2 (C-19), 38.9 (C-1),
68 38.6 (C-4), 38.2 (Phe-CH ), 37.8 (C-22), 37.0 (C-10), 32.8 (C-7), 31.9
2
69 (C-17), 31.3 (C-21), 28.6 (C-29), 28.5 (C-23), 28.4(Boc-CH ), 28.2
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70 (C-28), 26.6 (C-16), 26.5 (C-15), 23.6 (C-2), 23.4 (C-27), 18.8 (C-26),
71 17.5 (C-6), 16.8 (C-24), 16.5 (C-25). HRMS (ESI) m/z: 830.4996
+
72 [M+Na] , calcd. for C H NNaO 830.4972.
5
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69
7
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74 4.1.3.4.Benzyl
75 3β-(N-Boc-L-proline)-11-oxo-olean-12-en-30-oate(Compound
4).
76 According to Method A, compound 4 was obtained as white powder;

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77 yield: 75.7%; m.p.: 102.5 ℃ , [a] =+148 (c 0.3 mg/mL, MeOH); H NMR
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78 (400MHz, CDCl ): δ (ppm) 7.41-7.29 (m, 5H, H-Ar), 5.55 (d, 1H, H-12,
3
79 J = 5.2 Hz), 5.18, 5.10 (d, each, 1H, Bn-CH , J = 12.4 Hz), 4.53 (m, 1H,
2
80 H-3), 4.31 (m, 1H, Pro-CH), 3.48 (m, 2H, Pro-CH ), 2.79 (m, 1H, H-1),
2
81 2.33 (s, 1H, H-9), 2.21 (m, 1H, Pro-CHH′), 2.04-1.52, 1.42-1.36,
82 1.32-1.18, 1.05-0.93, 0.83-0.78 (22H, methylene and methine of
83 triterpenoid structure and proline), 1.45-1.42 (br, 9H, Boc-CH ), 1.32 (s,
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84 3H, H-27), 1.16 (brs, 6H, H-29 and H-25), 1.10 (s, 3H, H-26), 0.88 (m,
1
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85 6H, H-23 and H-24), 0.73 (s, 3H, H-28). C NMR (100MHz, CDCl ): δ
3
86 (ppm) 200.1 (C-11), 176.3 (C-30), 173.0 (Pro-COO), 169.3 (C-13), 154.0
87 (Boc-COO), 136.3 (C ), 128.8 (C ), 128.8 (C ), 128.6 (C-12), 128.5
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88 (C ), 128.4 (C ), 128.4 (C ), 81.2 (C-3), 80.1 (Boc-q.C), 66.4 (Bn-CH ),
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89 61.8 (C-9), 59.6 (Pro-CH), 55.2 (C-5), 48.4 (C-18), 46.4 (Pro-CH ), 45.5
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90 (C-8), 44.1 (C-20), 43.3 (C-14), 41.2 (C-19), 38.8 (C-1), 38.3 (C-4), 37.8
91 (C-22), 37.0 (C-10), 32.8 (C-7), 31.9 (C-17), 31.3 (C-21), 28.7 (Pro-CH2),
92 28.6 (C-29), 28.5 (C-23), 28.4 (Boc-CH ), 28.3 (C-28), 26.6 (C-16), 26.5
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93 (C-15), 24.5 (Pro-CH ), 23.6 (C-2), 23.4 (C-27), 18.8 (C-26), 17.5 (C-6),
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94 16.9 (C-24), 16.5 (C-25). HRMS (ESI) m/z: 780.4837 [M+Na] , calcd.
95 for C H6 NNaO 780.4815.
47
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96
97 4.1.3.5.Benzyl
98 3β-(N-Boc-L-sarcosine)-11-oxo-olean-12-en-30-oate(Compound
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99 According to Method A, compound 5 was obtained as white powder;
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00 yield: 81.2%; m.p.: 82.6 ℃ , [a] =+140 (c 0.3 mg/mL, MeOH); H NMR
D
01 (400MHz, CDCl ): δ (ppm) 7.40-7.30 (m, 5H, H-Ar), 5.54 (s, 1H, H-12),
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02 5.18, 5.10 (d, each, 1H, Bn-CH , J = 12.0 Hz), 4.57 (m, 1H, H-3), 3.90
2
03 (m, 2H, Sar-CH ), 2.94 (m, 3H, Sar-CH ), 2.80 (m, 1H, H-1), 2.33 (s, 1H,
2
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04 H-9), 2.07-1.89, 1.83-1.52, 1.38, 1.32-1.18, 1.06-0.95, 0.83-0.78 (19H,
05 methylene and methine of triterpenoid structure), 1.45-1.43 (br, 9H,
06 Boc-CH ), 1.34 (s, 3H, H-27), 1.16 (brs, 6H, H-29 and H-25), 1.10 (s, 3H,
3
13
07 H-26), 0.87 (m, 6H, H-23 and H-24), 0.73 (s, 3H, H-28). C NMR
08 (100MHz, CDCl ): δ (ppm) 200.1 (C-11), 176.3 (C-30), 169.9 (Sar-COO),
3
09 169.3 (C-13), 155.6 (Boc-COO), 136.3 (C ), 128.8 (C ), 128.8 (C ),
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10 128.6 (C-12), 128.5 (C ), 128.4 (C ), 128.4 (C ), 81.7 (C-3), 80.3
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11 (Boc-q.C), 66.4 (Bn-CH ), 61.8 (C-9), 55.1 (C-5), 51.5 (Sar-CH ), 48.4
2
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12 (C-18), 45.5 (C-8), 44.1 (C-20), 43.3 (C-14), 41.2 (C-19), 38.9 (C-1),
13 38.2 (C-4), 37.8 (C-22), 37.0 (C-10), 35.8 (Sar-CH ), 32.8 (C-7), 31.9
3
14 (C-17), 31.3 (C-21), 28.6 (C-29), 28.5 (C-23), 28.4 (Boc-CH3),
15 28.3(C-28), 26.6 (C-16), 26.5 (C-15), 23.7 (C-2), 23.4 (C-27), 18.8
16 (C-26), 17.5 (C-6), 16.9 (C-24), 16.5 (C-25). HRMS (ESI) m/z: 754.4682
+
17 [M+Na] , calcd. for C H NNaO 754.4659.
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19 4.1.3.6.Benzyl
20 3β-(N-Boc-L-leucine)-11-oxo-olean-12-en-30-oate(Compound
6).

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21 According to Method A, compound 6 was obtained as white powder;
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22 yield: 77.8%; m.p.: 166.1 ℃ , [a] =+132 (c 0.3 mg/mL, MeOH); H NMR
D
23 (400MHz, CDCl ): δ (ppm) 7.40-7.31 (m, 5H, H-Ar), 5.54 (s, 1H, H-12),
3
24 5.18, 5.10 (d, each, 1H, Bn-CH , J = 12.0 Hz), 4.88 (m, 1H, Leu-CHNH),
2
25 4.55 (dd, 1H, H-3, J = 12.0 Hz, 4.8 Hz), 4.29 (m, 1H, Leu-CHNH), 2.82
26 (dt, 1H, H-1, J = 13.6 Hz, 3.6 Hz), 2.33 (s, 1H, H-9), 2.05-1.92, 1.84-1.48,
27 1.42-1.36, 1.32-1.18, 1.07-0.98, 0.83-0.77 (22H, methylene and methine
28 of triterpenoid structure and leucine), 1.44 (brs, 9H, Boc-CH ), 1.34 (s,
3
29 3H, H-27), 1.16 (brs, 6H, H-29 and H-25), 1.10 (s, 3H, H-26), 0.95-0.94
30 (br, 6H, Leu-CH ), 0.89-0.87 (m, 6H, H-23 and H-24), 0.73 (s, 3H, H-28).
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31
C NMR (100MHz, CDCl ): δ (ppm) 200.1 (C-11), 176.3 (C-30), 173.3
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32 (Leu-COO), 169.2 (C-13), 155.5 (Boc-COO), 136.3 (C ), 128.8 (C ),
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33 128.8 (C ), 128.6 (C-12), 128.5 (C ), 128.4 (C ), 128.4 (C ), 81.8 (C-3),
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34 79.8 (Boc-q.C), 66.4 (Bn-CH ), 61.8 (C-9), 55.2 (C-5), 52.7 (Leu-CHNH),
2
35 48.4 (C-18), 45.5 (C-8), 44.1 (C-20), 43.3 (C-14), 42.2 (Leu-CH ), 41.2
2
36 (C-19), 38.9 (C-1), 38.3 (C-4), 37.8 (C-22), 37.0 (C-10), 32.8 (C-7), 31.9
37 (C-17), 31.3 (C-21), 28.6 (C-29), 28.5 (C-23), 28.4 (Boc-CH ), 28.3
3
38 (C-28), 26.6 (C-16), 26.5 (C-15), 25.0 (Leu-CH), 23.7 (C-2), 23.4 (C-27),
39 23.1 (Leu-CH ), 18.8 (C-26), 17.5 (C-6), 16.9 (C-24), 16.5 (C-25).
3
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40 HRMS (ESI) m/z: 796.5153 [M+Na] , calcd. for C H NNaO 796.5128.
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41

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42 4.1.3.7.Benzyl
43 3β-(N-Boc-L-isoleucine)-11-oxo-olean-12-en-30-oate(Compound
7).
44 According to Method A, compound 7 was obtained as white powder;
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45 yield: 70.9%; m.p.: 162.8 ℃ , [a] =+132 (c 0.3 mg/mL, MeOH); H NMR
D
46 (400MHz, CDCl ): δ (ppm) 7.40-7.28 (m, 5H, H-Ar), 5.54 (s, 1H, H-12),
3
47 5.18, 5.09 (d, each, 1H, Bn-CH , J = 12.0 Hz), 5.01 (m, 1H, Ile-CHNH),
2
48 4.56 (dd, 1H, H-3, J = 11.6 Hz, 5.2 Hz), 4.26 (m, 1H, Ile-CHNH), 2.81
49 (dt, 1H, H-1, J = 13.6 Hz, 3.2 Hz), 2.33 (s, 1H, H-9), 2.08-1.87, 1.84-1.46,
50 1.42-1.37, 1.30-1.18, 1.07-0.98, 0.83-0.77 (22H, methylene and methine
51 of triterpenoid structure and isoleucine), 1.44 (brs, 9H, Boc-CH ), 1.34 (s,
3
52 3H, H-27), 1.15 (brs, 6H, H-29 and H-25), 1.10 (s, 3H, H-26), 0.96-0.91
53 (m, 6H, Ile-CH ), 0.89-0.88 (m, 6H, H-23 and H-24), 0.73 (s, 3H, H-28).
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54
C NMR (100MHz, CDCl ): δ (ppm) 200.1 (C-11), 176.3 (C-30), 172.2
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55 (Ile-COO), 169.2 (C-13), 155.8 (Boc-COO), 136.3 (C ), 128.8 (C ),
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56 128.8 (C ), 128.6 (C-12), 128.5 (C ), 128.4 (C ), 128.4 (C ), 82.1 (C-3),
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57 79.8 (Boc-q.C), 66.4 (Bn-CH ), 61.8 (C-9), 58.6 (Ile-CHNH), 55.2 (C-5),
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58 48.4 (C-18), 45.5 (C-8), 44.1 (C-20), 43.3 (C-14), 41.2 (C-19), 38.9 (C-1),
59 38.2 (C-4), 38.1 (Ile-CH), 37.8 (C-22), 37.0 (C-10), 32.8 (C-7), 31.9
60 (C-17), 31.3 (C-21), 28.6 (C-29), 28.5 (C-23), 28.4 (Boc-CH ), 28.2
3
61 (C-28), 26.6 (C-16), 26.5 (C-15), 24.9 (Ile-CH ), 23.7 (C-2), 23.4 (C-27),
2
62 18.8 (C-26), 17.5 (C-6), 17.0 (C-24), 16.5 (C-25), 15.9 (Ile-CH CH ),
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63 11.8 (Ile-CH CH). HRMS (ESI) m/z: 796.5156 [M+Na] , calcd. for
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64 C H NNaO 796.5128.
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71
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66 4.1.3.8.Benzyl
67 3β-(N-Boc-L-methionine)-11-oxo-olean-12-en-30-oate(Compound 8).
68 According to Method A, compound 8 was obtained as white powder;
1
69 yield: 66.9%; m.p.: 152.9 ℃ , [a] =+120 (c 0.3 mg/mL, MeOH); H NMR
D
70 (400MHz, CDCl ): δ (ppm) 7.41-7.29 (m, 5H, H-Ar), 5.54 (s, 1H, H-12),
3
71 5.18, 5.10 (d, each, 1H, Bn-CH , J = 12.0 Hz), 4.57 (dd, 1H, H-3, J =
2
72 11.6 Hz, 4.8 Hz), 4.46-4.34 (br, 1H, Met-CHNH), 2.80 (dt, 1H, H-1, J =
73 13.6 Hz, 3.2 Hz), 2.55 (m, 2H, Met-CH S), 2.33 (s, 1H, H-9), 2.26-2.11
2
74 (m, 2H, Met-CH CH), 2.10 (s, 3H, Met-CH ), 2.04-1.92, 1.79-1.53,
2
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75 1.42-1.37, 1.32-1.18, 1.07-0.96, 0.83-0.76 (19H, methylene and methine
76 of triterpenoid structure), 1.44 (brs, 9H, Boc-CH ), 1.34 (s, 3H, H-27),
3
77 1.16 (brs, 6H, H-29 and H-25), 1.10 (s, 3H, H-26), 0.89-0.87 (m, 6H,
1
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78 H-23 and H-24), 0.73 (s, 3H, H-28). C NMR (100MHz, CDCl ): δ (ppm)
3
79 200.1 (C-11), 176.3 (C-30), 172.1 (Met-COO), 169.3 (C-13), 155.5
80 (Boc-COO), 136.3 (C ), 128.8 (C ), 128.8 (C ), 128.6 (C-12), 128.5
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81 (C ), 128.4 (C ), 128.4 (C ), 82.3 (C-3), 80.1 (Boc-q.C), 66.4 (Bn-CH ),
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82 61.8 (C-9), 55.1 (C-5), 53.4 (Met-CHNH), 48.4(C-18), 45.5 (C-8), 44.1
83 (C-20), 43.3 (C-14), 41.2 (C-19), 38.8 (C-1), 38.3 (C-4), 37.8 (C-22),
84 37.0 (C-10), 32.8 (C-7), 31.9 (C-17), 31.3 (C-21), 30.2 (Met-CH CH),
2

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85 28.6 (C-29), 28.5 (C-23), 28.4 (Boc-CH ), 28.3 (C-28), 28.2 (Met-CH S),
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86 26.6 (C-16), 26.5 (C-15), 23.7 (C-2), 23.4 (C-27), 18.8 (C-26), 17.5 (C-6),
87 17.0 (C-24), 16.5 (C-25), 15.6 (Met-CH ). HRMS (ESI) m/z: 814.4712
3
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88 [M+Na] , calcd. for C H NNaO S 814.4692.
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89
90 4.1.4.1.Benzyl 3β-(glycyl)-11-oxo-olean-12-en-30-oate(Compound 9).
91 According to Method B, compound 9 was obtained as white powder;
1
92 yield: 79.8%; m.p.: 189.0 ℃ , [a] =+144 (c 0.3 mg/mL, MeOH); H NMR
D
93 (400MHz, CDCl ): δ (ppm) 7.40-7.30 (m, 5H, H-Ar), 5.54 (s, 1H, H-12),
3
94 5.18, 5.09 (d, each, 1H, Bn-CH , J = 12.0 Hz), 4.58 (dd, 1H, H-3, J =
2
95 11.6 Hz, 4.8 Hz), 3.44 (s, 2H, Gly-CH ), 2.80 (dt, 1H, H-1, J = 13.6 Hz,
2
96 3.6 Hz), 2.36 (s, 1H, H-9), 2.04-1.92, 1.84-1.37, 1.32-1.18, 1.05-0.95,
97 0.83-0.76 (19H, methylene and methine of triterpenoid structure), 1.34 (s,
98 3H, H-27), 1.15 (brs, 6H, H-29 and H-25), 1.10 (s, 3H, H-26), 0.87 (brs,
1
3
99 6H, H-23 and H-24), 0.73 (s, 3H, H-28). C NMR (100MHz, CDCl ): δ
3
00 (ppm) 200.1 (C-11), 176.3 (C-30), 173.9 (Gly-COO), 169.2 (C-13), 136.3
01 (C ), 128.7 (C ), 128.7 (C ), 128.6 (C-12), 128.4 (C ), 128.3 (C ), 128.3
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02 (C ), 81.6 (C-3), 66.4 (Bn-CH ), 61.8 (C-9), 55.1 (C-5), 48.4 (C-18), 45.5
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03 (C-8), 44.1 (Gly-CH ), 44.1(C-20), 43.3 (C-14), 41.2 (C-19), 38.9 (C-1),
2
04 38.3 (C-4), 37.8(C-22), 37.0 (C-10), 32.8 (C-7), 31.9 (C-17), 31.3 (C-21),
05 28.5 (C-29), 28.4 (C-23), 28.2 (C-28), 26.6 (C-16), 26.5 (C-15), 23.7
06 (C-2), 23.4 (C-27), 18.8 (C-26), 17.5 (C-6), 16.8 (C-24), 16.5 (C-25).

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07 HRMS (ESI) m/z: 618.4159 [M+H] , calcd. for C H NO 618.4158.
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09 4.1.4.2.Benzyl
3β-(L-alanyl)-11-oxo-olean-12-en-30-oate(Compound
10 10). According to Method B, compound 10 was obtained as white powder;
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11 yield: 71.7%; m.p.: 199.1 ℃ , [a] =+164 (c 0.3 mg/mL, MeOH); H NMR
D
12 (400MHz, CDCl ): δ (ppm) 7.42-7.28 (m, 5H, H-Ar), 5.54 (s, 1H, H-12),
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13 5.18, 5.09 (d, each, 1H, Bn-CH , J = 12.0 Hz), 4.56 (dd, 1H, H-3, J =
2
14 11.6 Hz, 4.8 Hz), 3.52 (q, 1H, Ala-CH, J = 7.2 Hz), 2.79 (dt, 1H, H-1, J =
15 13.6 Hz, 3.6 Hz), 2.34 (s, 1H, H-9), 2.08-1.89, 1.80-1.41, 1.31-1.18,
16 1.05-0.95, 0.81-0.79 (19H, methylene and methine of triterpenoid
17 structure), 1.37-1.34 (br, 6H, H-27 and Ala-CH ), 1.16 (brs, 6H, H-29 and
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18 H-25), 1.10 (s, 3H, H-26), 0.89 (s, 3H, H-24), 0.87 (s, 3H, H-23), 0.73 (s,
13
19 3H, H-28). C NMR (100MHz, CDCl ): δ (ppm) 200.1 (C-11), 176.4
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20 (C-30), 176.3 (Ala-COO), 169.2 (C-13), 136.3 (C ), 128.7 (C ), 128.7
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21 (C ), 128.6 (C-12), 128.4 (C ), 128.3 (C ), 128.3 (C ), 81.2 (C-3), 66.3
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22 (Bn-CH ), 61.8 (C-9), 55.1 (C-5), 50.5 (Ala-CH), 48.3 (C-18), 45.5 (C-8),
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23 44.1 (C-20), 43.3 (C-14), 41.2 (C-19), 38.9 (C-1), 38.4 (C-4), 37.8 (C-22),
24 37.0 (C-10), 32.8 (C-7), 31.9 (C-17), 31.3 (C-21), 28.5 (C-29), 28.4
25 (C-23), 28.2 (C-28), 26.6 (C-16), 26.5 (C-15), 23.7 (C-2), 23.4 (C-27),
26 20.9 (Ala-CH ), 18.8 (C-26), 17.5 (C-6), 16.9 (C-24), 16.5 (C-25). HRMS
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27 (ESI) m/z: 632.4277 [M+H] , calcd. for C H NO 632.4315.
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29 4.1.4.3.Benzyl
30 3β-(L-phenylalanine)-11-oxo-olean-12-en-30-oate(Compound
11).
31 According to Method B, compound 11 was obtained as white powder;
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32 yield: 80.9%; m.p.: 192.8 ℃ , [a] =+140 (c 0.3 mg/mL, MeOH); H NMR
D
33 (400MHz, CDCl ): δ (ppm) 7.40-7.28, 7.24-7.18 (br, 10H, H-Ar), 5.54 (s,
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34 1H, H-12), 5.19, 5.10 (d, each, 1H, Bn-CH , J = 12.4 Hz), 4.56 (dd, 1H,
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35 H-3, J = 11.6 Hz, 4.8 Hz), 3.74 (m, 1H, Phe-CHNH ), 3.14 (dd, 1H,
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36 Phe-CHH′, J = 13.6 Hz, 4.4 Hz), 2.82 (m, 1H, H-1), 2.78 (m, 1H,
37 Phe-CHH′), 2.34 (s, 1H, H-9), 2.05-1.91, 1.81-1.41, 1.32-1.18, 1.08-0.96,
38 0.78 (19H, methylene and methine of triterpenoid structure), 1.35 (s, 3H,
39 H-27), 1.16 (brs, 6H, H-29 and H-25), 1.10 (s, 3H, H-26), 0.86 (s, 3H,
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40 H-24), 0.80 (s, 3H, H-23), 0.73 (s, 3H, H-28). C NMR (100MHz,
41 CDCl ): δ (ppm) 200.1 (C-11), 176.3 (C-30), 174.8 (Phe-COO), 169.2
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42 (C-13), 137.6 (Phe-C ), 136.3(Bn-C ), 129.5 (Phe-C ), 129.5 (Phe-C ),
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43 128.8(Bn-C ), 128.8(Bn-C ), 128.7(Bn-C ), 128.6 (C-12), 128.5
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44 (Phe-C ), 128.5 (Phe-C ), 128.4(Bn-C ), 128.4(Bn-C ), 126.9 (Phe-C ),
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45 81.6 (C-3), 66.4 (Bn-CH ), 61.8 (C-9), 56.4 (Phe-CHNH ), 55.2 (C-5),
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46 48.4 (C-18), 45.5 (C-8), 44.1 (C-20), 43.3 (C-14), 41.2 (C-19), 38.9 (C-1),
47 38.6 (C-4), 38.3 (Phe-CH ), 37.8 (C-22), 37.0 (C-10), 32.8 (C-7), 31.9
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48 (C-17), 31.3 (C-21), 28.6 (C-29), 28.4 (C-23), 28.2 (C-28), 26.6 (C-16),
49 26.5 (C-15), 23.7 (C-2), 23.4 (C-27), 18.8 (C-26), 17.5 (C-6), 16.9 (C-24),

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50 16.5 (C-25). HRMS (ESI) m/z: 708.4593 [M+H] , calcd. for C H NO
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62
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51 708.4628.
52
53 4.1.4.4.Benzyl 3β-(L-proline)-11-oxo-olean-12-en-30-oate(Compound
54 12). According to Method B, compound 12 was obtained as white powder;
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55 yield: 70.8%; m.p.: 152.9 ℃ , [a] =+164 (c 0.3 mg/mL, MeOH); H NMR
D
56 (400MHz, CDCl ): δ (ppm) 7.40-7.28 (m, 5H, H-Ar), 5.54 (s, 1H, H-12),
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57 5.18, 5.09 (d, each, 1H, Bn-CH , J = 12.4 Hz), 4.58 (dd, 1H, H-3, J =
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58 11.6 Hz, 4.8 Hz), 3.76 (m, 1H, Pro-CH), 3.10 (m, 1H, Pro-CHH′NH),
59 2.92 (m, 1H, Pro-CHH′NH), 2.81 (dt, 1H, H-1, J = 14.0 Hz, 3.2 Hz),
60 2.43(m, 1H, Pro-CHH′(CH)), 2.33 (s, 1H, H-9), 2.15 (m, 1H,
61 Pro-CHH′(CH)), 2.04-1.38, 1.32-1.18, 1.07-0.95, 0.81-0.78 (21H,
62 methylene and methine of triterpenoid structure and proline), 1.34 (s, 3H,
63 H-27), 1.15 (brs, 6H, H-29 and H-25), 1.10 (s, 3H, H-26), 0.89 (s, 3H,
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64 H-23), 0.87 (s, 3H, H-24), 0.73 (s, 3H, H-28). C NMR (100MHz,
65 CDCl ): δ (ppm) 200.1 (C-11), 176.3 (C-30), 175.2 (Pro-COO), 169.2
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66 (C-13), 136.3 (C ), 128.7 (C ), 128.7 (C ), 128.6 (C-12), 128.5 (C ),
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67 128.4 (C ), 128.4 (C ), 81.2 (C-3), 66.3 (Bn-CH ), 61.8 (C-9), 60.3
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68 (Pro-CH), 55.1 (C-5), 48.3 (C-18), 47.0 (Pro-CH ), 45.5 (C-8), 44.1
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69 (C-20), 43.3 (C-14), 41.2 (C-19), 38.9 (C-1), 38.4 (C-4), 37.8 (C-22),
70 37.0 (C-10), 32.8 (C-7), 31.9 (C-17), 31.3 (C-21), 30.5 (Pro-CH ), 28.5
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72 (Pro-CH ), 23.7 (C-2), 23.4 (C-27), 18.8 (C-26), 17.5 (C-6), 16.9 (C-24),
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73 16.5 (C-25). HRMS (ESI) m/z: 658.4461 [M+H] , calcd. for C H NO
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60
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74 658.4471.
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76 4.1.4.5.Benzyl 3β-(L-sarcosine)-11-oxo-olean-12-en-30-oate(Compound
77 13). According to Method B, compound 13 was obtained as white powder;
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78 yield: 68.4%; m.p.: 182.9 ℃ , [a] =+172 (c 0.3 mg/mL, MeOH); H NMR
D
79 (400MHz, CDCl ): δ (ppm) 7.42-7.29 (m, 5H, H-Ar), 5.53 (s, 1H, H-12),
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80 5.18, 5.09 (d, each, 1H, Bn-CH , J = 12.0 Hz), 4.62 (dd, 1H, H-3, J =
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81 11.6 Hz, 4.4 Hz), 3.37 (brs, 2H, Sar-CH ), 2.81 (dt, 1H, H-1, J = 13.6 Hz,
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82 3.2 Hz), 2.45 (s, 3H, Sar-CH ), 2.33 (s, 1H, H-9), 2.04-1.92, 1.86-1.38,
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83 1.32-1.18, 1.06-0.94, 0.81-0.78 (19H, methylene and methine of
84 triterpenoid structure and sarcosine), 1.34 (s, 3H, H-27), 1.16 (brs, 6H,
85 H-29 and H-25), 1.10 (s, 3H, H-26), 0.88 (brs, 6H, H-23 and H-24), 0.73
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86 (s, 3H, H-28). C NMR (100MHz, CDCl ): δ (ppm) 200.1 (C-11), 176.3
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87 (C-30), 172.2 (Sar-COO), 169.2 (C-13), 136.3 (C ), 128.8 (C ), 128.8
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88 (C ), 128.6 (C-12), 128.5 (C ), 128.4 (C ), 128.4 (C ), 81.4 (C-3), 66.4
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89 (Bn-CH ), 61.8 (C-9), 55.1 (C-5), 52.9 (Sar-CH NH), 48.4 (C-18), 45.5
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90 (C-8), 44.1 (C-20), 43.3 (C-14), 41.2 (C-19), 38.9 (C-1), 38.3 (C-4), 37.8
91 (C-22), 37.0 (C-10), 36.2 (Sar-CH ), 32.8 (C-7), 31.9 (C-17), 31.3 (C-21),
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92 28.6 (C-29), 28.4 (C-23), 28.3 (C-28), 26.6 (C-16), 26.5 (C-15), 23.8
93 (C-2), 23.4 (C-27), 18.8 (C-26), 17.5 (C-6), 16.9 (C-24), 16.5 (C-25).

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94 HRMS (ESI) m/z: 632.4287 [M+H] , calcd. for C H NO 632.4315.
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96 4.1.4.6.Benzyl 3β-(L-leucine)-11-oxo-olean-12-en-30-oate(Compound
97 14). According to Method B, compound 14 was obtained as white powder;
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98 yield: 65.4%; m.p.: 186.1 ℃ , [a] =+172 (c 0.3 mg/mL, MeOH); H NMR
D
99 (400MHz, CDCl ): δ (ppm) 7.40-7.30 (m, 5H, H-Ar), 5.54 (s, 1H, H-12),
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00 5.18, 5.10 (d, each, 1H, Bn-CH , J = 12.4 Hz), 4.56 (dd, 1H, H-3, J =
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01 11.6 Hz, 4.8 Hz), 3.47 (m, 1H, Leu-CHNH ), 2.82 (dt, 1H, H-1, J = 13.6
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02 Hz, 3.6 Hz), 2.34 (s, 1H, H-9), 2.04-1.92, 1.83-1.38, 1.32-1.18, 1.08-0.98,
03 0.81-0.78 (22H, methylene and methine of triterpenoid structure and
04 leucine),
05 1.34 (s, 3H, H-27), 1.16 (brs, 6H, H-29 and H-25), 1.10 (s, 3H, H-26),
06 0.96-0.93 (br, 6H, Leu-CH ), 0.89-0.88 (m, 6H, H-23 and H-24), 0.73 (s,
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07 3H, H-28). C NMR (100MHz, CDCl ): δ (ppm) 200.1 (C-11), 176.3
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08 (C-30), 176.2 (Leu-COO), 169.2 (C-13), 136.3 (C ), 128.8 (C ), 128.8
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09 (C ), 128.6 (C-12), 128.5 (C ), 128.4 (C ), 128.4 (C ), 81.3 (C-3), 66.4
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10 (Bn-CH ), 61.8 (C-9), 55.2 (C-5), 53.3 (Leu-CHNH ), 48.4 (C-18), 45.5
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11 (C-8), 44.1 (C-20), 43.3 (C-14), 42.3 (Leu-CH ), 41.2 (C-19), 38.9 (C-1),
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12 38.3 (C-4), 37.8 (C-22), 37.0 (C-10), 32.8 (C-7), 31.9 (C-17), 31.3 (C-21),
13 28.6 (C-29), 28.4 (C-23), 28.3 (C-28), 26.6 (C-16), 26.5 (C-15), 25.0
14 (Leu-CH), 23.7 (C-2), 23.4 (C-27), 23.2 (Leu-CH ), 18.8 (C-26), 17.5
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15 (C-6), 17.0 (C-24), 16.5 (C-25). HRMS (ESI) m/z: 674.4768 [M+H] ,