Synthesis and anticancer activities of glycyrrhetinic acid derivatives

Article abstract of DOI:10.3390/molecules21020199

A total of forty novel glycyrrhetinic acid (GA) derivatives were designed and synthesized. The cytotoxic activity of the novel compounds was tested against two human breast cancer cell lines (MCF-7, MDA-MB-231) in vitro by the MTT method. The evaluation results revealed that, in comparison with GA, compound 42 shows the most promising anticancer activity (IC₅₀ 1.88 ± 0.20 and 1.37 ± 0.18 μM for MCF-7 and MDA-MB-231, respectively) and merits further exploration as a new anticancer agent.

Full text of DOI:10.3390/molecules21020199

molecules
Article
Synthesis and Anticancer Activities of Glycyrrhetinic
Acid Derivatives
Yang Li 1,2, Ling Feng , Zhi-Fang Song , Hai-Bei Li * and Qi-Yong Huai *
1
1
1,
1,
1
Marine College, Shandong University, Weihai 264209, China; lychemlh@163.com (Y.L.);
fenglingjpf@163.com (L.F.); zhifangdl@163.com (Z.-F.S.)
Zhong Yuan Academy of Biological Medicine, Liaocheng People’s Hospital/Affiliated Liaocheng Hospital,
2
Taishan Medical University, Liaocheng 252000, China
*
Correspondence: lihaibei@sdu.edu.cn (H.-B.L.); huaiqy01@163.com (Q.-Y.H.);
Tel.: +86-631-568-8303 (H.-B. L. & Q.-Y.H.)
Academic Editors: Jean Jacques Vanden Eynde and Annie Mayence
Received: 7 December 2015 ; Accepted: 2 February 2016 ; Published: 6 February 2016
Abstract: A total of forty novel glycyrrhetinic acid (GA) derivatives were designed and synthesized.
The cytotoxic activity of the novel compounds was tested against two human breast cancer cell
lines (MCF-7, MDA-MB-231) in vitro by the MTT method. The evaluation results revealed that, in
comparison with GA, compound 42 shows the most promising anticancer activity (IC50 1.88
˘
0.20
and 1.37 0.18 M for MCF-7 and MDA-MB-231, respectively) and merits further exploration as
˘
µ
a new anticancer agent.
Keywords: glycyrrhetinic acid (GA); derivatives; anticancer; MCF-7; MDA-MB-231
1
. Introduction
Breast cancer is one of the most common diseases amongst women throughout the world.
About 521,900 women lost their lives because of it in 2012, 197,600 of which were from developed
countries and 324,300 from developing countries [ ]. In 2016, 249,260 new breast cancer cases and
0,890 breast cancer deaths are projected to occur in the United States [ ]. A current hot research topic is
1
4
2
how to develop novel therapeutics with improved selectivity and higher anticancer activity by chemical
modifications of natural sources. Up to now, a large number of chemotherapeutic agents derived from
natural products and used for the treatment of cancer, have shown satisfactory therapeutic effects, like
vinblastine, vincristine, the camptothecin derivatives, e.g., topotecan, irinotecan and etoposide, were
derived from epipodophyllotoxin and paclitaxel [3].
Terpenoids are the largest group of natural compounds found in plants. Among terpenoids,
a large number of triterpenoids exhibit cytotoxicity against a variety of tumor cells as well as anticancer
efficacy in preclinical animal models [
transformation, survival, invasion, angiogenesis, metastasis, chemoresistance and radioresistance [
As a kind of triterpenoid, glycyrrhetinic acid (GA) has many valuable pharmacological properties,
such as antiviral [ ], anti-allergic [10], anti-inflammatory [11 12], anti-ulcer [13] and anticancer
activity [14 15]. It has been reported that GA exhibited selective toxicity to varieties of tumor cells,
making it an ideal lead compound for anticancer treatment [16 17]. Ferulic acid as a natural product that
also has apparent biological activities like antibacterial [18], anti-inflammatory [19 20] and anticancer
4–
6]. Those triterpenoids regulate tumor cell proliferation,
7].
8,
9
,
,
,
,
properties [21]. To improve the cytotoxicity of GA, many researchers have tried to enhance its potency
by various derivatizations. Some studies have shown that the addition of lipophilic fragments to
antitumor molecules could increase their anticancer activity [22
which were conjugated with amino acids provide improved bioacitivity [26
on the pro-drug principle and the previously reported therapeutic potential of GA, we designed
–
25]. In addition, natural products
–28]. In this study, based
Molecules 2016, 21, 199; doi:10.3390/molecules21020199
www.mdpi.com/journal/molecules

Molecules 2016, 21, 199
2 of 20
and synthesized a series of novel GA derivatives in which the 30-carboxyl group was coupled with
lipophilic fragments (ferulic acid analogs) and the 3-hydroxyl group was coupled with amino acids
(
L-methionine or L-selenomethionine) to improve the anticancer potency of GA.
In totally, forty derivatives of GA were successfully synthesized and and their structures
1
13
characterized by H-NMR, C-NMR, MS and elemental analysis. Their in vitro anticancer activities were
then tested, using a MTT assay, against two human breast cancer cell lines (MCF-7 and MDA-MB-231)
and one normal human retinal pigment epithelial cell line (hTERT-RPE1 cells). Most of the derivatives
exhibited much stronger inhibitory activity than GA against those two breast cancer cell lines (but
lower than the positive control doxorubicin) and relatively lower inhibitory activity against normal cells.
More importantly, one derivative, compound 42 (see Section 2 below), showed significantly stronger
cytotoxicity against both MCF-7 cells and MDA-MB-231 cells than GA itself. Our data suggested
that coupling lipophilic fragments (especially ferulic acid methyl ester) and amino acids (especially
L-selenomethionine) to GA is a promising approach to generate highly active anticancer compounds.
Further SAR development is in progress to discover more potential lead antitumor drugs.
2
. Results and Discussion
2
.1. Chemistry
The synthesis of new GA derivatives 17–56 was carried out according to the steps shown in
Scheme 1.
O
O
O
O
R'
R''
b
R''
OH
a
O
OH
OH
O
HO
HO
NH2
HN
R
R
1
-4
5-12
O
1
3-14
15-16
1
3
5
7
9
4-OH, R=3-OCH3;
3-OH, R=4-OCH3;
2 4-OH, R=3-H;
4 3-OH, R=4-H;
6 4-OH, R=3-OCH3, R'=CH2CH3;
8 4-OH, R=3-H, R'=CH2CH3;
10 3-OH, R=4-OCH3, R'=CH2CH3;
12 3-OH, R=4-H, R'=CH2CH3.
1
1
3,15 R''=S;
4,16 R''=Se
4-OH, R=3-OCH3, R'=CH3;
4-OH, R=3-H, R'=CH3;
3-OH, R=4-OCH3, R'=CH3;
1
1 3-OH, R=4-H, R'=CH3;
O
O
R'
O
COOH
C
O
R
17 4-OGA, R=3-OCH , R'=CH ;
3 2 3
18 4-OGA, R=3-OCH , R'=CH CH ;
O
c
O
3
3
H
H
19 4-OGA, R=3-H, R'=CH3;
20 4-OGA, R=3-H, R'=CH2CH3;
2
2
1 3-OGA, R=4-OCH3, R'=CH3;
3 3-OGA, R=4-H, R'=CH3;
22 3-OGA, R=4-OCH3, R'=CH2CH3;
24 3-OGA, R=4-H, R'=CH2CH3.
H
H
HO
HO
H
H
GA
17-24
O
O
R'
R'
O
C
O
O
C
O
O
O
d
e
R
R
17-20
O
O
H
H
O
O
O
H
H
R''
R''
O
O
O
H
H
HN
25-32
NH2
.HCl
41-48
2
2
2
3
5,41 R=OCH3, R'=CH3, R''=S;
7,43 R=OCH3, R'=CH2CH3, R''=S;
9,45 R=H, R'=CH3, R''=S;
26,42 R=OCH3, R'=CH3, R''=Se;
28,44 R=OCH3, R'=CH2CH3, R''=Se;
30,46 R=H, R'=CH3, R''=Se;
1,47 R=H, R'=CH2CH3, R''=S;
32,48 R=H, R'=CH2CH3, R''=Se
O
R
O
R
C
C
O
O
d
e
2
1-24
O
O
H
H
O
O
O
H
H
R''
R''
O
O
O
O
O
O
O
H
H
HN
NH2
.HCl
R'
R'
49-56
33-40
3
3,49 R=OCH3, R'=CH3, R''=S;
5,51 R=OCH3, R'=CH2CH3, R''=S;
7,53 R=H, R'=CH3, R''=S;
34,50 R=OCH3, R'=CH3, R''=Se;
36,52 R=OCH3, R'=CH2CH3, R''=Se;
38,54 R=H, R'=CH3, R''=Se;
3
3
3
9,55 R=H, R'=CH2CH3, R''=S;
40,56 R=H, R'=CH2CH3, R''=Se
Reagents and conditions: (a) CH
c) ferulic acid analogs, EDCI, DMAP, CH
DMAP, CH Cl , rt; (e) HCl(g) in CH Cl , rt.
3
OH or CH
2
CH
3
OH, H
2
SO
4
, reflux; (b) 1,4-dioxane-H
2
O, NaHCO
3
, (Boc)
2
O, rt;
(
2
Cl
2
, rt; (d) Boc-L-methionine or Boc-L-selenomethionine, EDCI,
2
2
2
2
Scheme 1. Preparation of glycyrrhetinic acid derivatives 17–56.

Molecules 2016, 21, 199
3 of 20
All these derivatives are new compounds which were not previously reported. The lipophilic
fragments 12 were obtained by the treatment of with methanol or ethanol catalyzed by
concentrated sulfuric acid. To avoid the formation of byproducts, we used the t-butyloxycarbonyl
Boc-) group to protect amino acids and thus obtained compounds 15 16, which were used in the next
step without further purification.
Compounds 17 24 were obtained through the formation of an ester bond between compounds
12 and GA after stirring for 12 h at room temperature in dry dichloromethane (DCM) catalyzed by
-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and 4-dimethylamino-pyridine
DMAP). We applied the same method to introduce the N-Boc group to protect amino acids at position
C-3 and obtained compounds 25 40. To get compounds 41 56, deprotection was performed by treating
the compounds with dry HCl gas in DCM.
5
–
1–4
(
–
–
5
–
1
(
–
–
2
.2. Anticancer Activities
The anticancer activities of compounds 17–24, 41–56 against MCF-7, MDA-MB-231 and hTERT-RPE1
cells were determined in vitro by an MTT assay. Doxorubicin was included in the experiments as positive
control, and the IC50 of GA is also presented to compare the anticancer activities. The data was calculated
and presented as IC50 values in Table 1. All the derivatives 17
in which their IC50 values ranged from 1.88 0.20 to 24.45
.37 ˘ 0.18 to 23.15 1.07
GA are 75.66 ˘ 1.52 and 84.70
the highest cytotoxicity with the lowest IC values of 1.88 ˘ 0.20 µM on MCF-7 cells and 1.37 ˘ 0.18 µM
–24
,
41–56 showed significant cytotoxicities,
˘
˘
1.36 M on MCF-7 cells and from
µ
1
˘
µ
M on MDA-MB-231 cells, while the IC values of the parent compound
50
˘
1.73 µM, respectively. Among the derivatives, compound 42 showed
5
0
on MDA-MB-231 cells, which were 40.24 and 61.82 times better than GA.
Table 1. IC50 values (in µM) of test compounds against MCF-7, MDA-MB-231 and hTERT-RPE1 cell lines.
IC50(µM) a
MDA-MB-231 hTERT-RPE1
IC50(µM) a
Compd.
GA
Compd.
MCF-7
MCF-7
MDA-MB-231 hTERT-RPE1
75.66˘ 1.52
13.64˘0.93
22.46 ˘ 1.26
20.29 ˘ 1.47
24.45 ˘ 1.36
8.54 ˘ 0.67
19.27 ˘ 1.01
14.90 ˘ 0.75
19.30 ˘ 0.98
6.00 ˘ 0.43
1.88 ˘ 0.20
8.62 ˘ 0.23
8.45 ˘ 0.32
84.70˘ 1.73
5.03 ˘ 0.82
8.14 ˘ 0.76
14.38 ˘ 0.52
14.46 ˘ 0.58
7.31 ˘ 0.16
9.41 ˘ 1.03
20.84 ˘ 1.20
23.15 ˘ 1.07
3.52 ˘ 0.61
1.37 ˘ 0.18
5.36 ˘ 0.44
3.49 ˘ 0.61
63.41 ˘ 1.07
17.32 ˘ 1.21
22.80 ˘ 0.97
29.63 ˘ 1.16
28.41 ˘ 0.87
18.59 ˘ 0.54
21.11 ˘ 0.73
24.09 ˘ 0.88
22.88 ˘ 0.68
10.36 ˘ 0.80
4.93 ˘ 0.36
16.28 ˘ 0.51
12.33 ˘ 0.46
45
46
47
48
49
50
51
52
53
54
55
56
7.24 ˘ 0.30
6.02 ˘ 0.35
2.65 ˘ 0.12
2.42 ˘ 0.23
8.70 ˘ 0.14
5.12 ˘ 0.58
8.81 ˘ 0.20
6.93˘ 0.63
7.20 ˘ 0.55
3.48 ˘ 0.82
5.79 ˘ 0.85
2.57 ˘ 0.40
0.49 ˘ 0.09
6.43 ˘ 0.84
6.27 ˘ 0.24
2.31 ˘ 0.65
1.86 ˘ 0.29
4.56 ˘ 0.36
3.13 ˘ 0.45
4.67 ˘ 0.74
3.27 ˘ 0.66
3.54 ˘ 0.46
2.46 ˘ 0.77
1.92 ˘ 0.91
1.53 ˘ 0.25
0.53 ˘ 0.14
8.48 ˘ 0.73
6.33 ˘ 0.19
5.65 ˘ 1.02
7.08 ˘ 0.73
8.92 ˘ 0.55
9.79 ˘ 0.37
12.52 ˘ 0.40
10.06 ˘ 0.28
8.49 ˘ 0.51
4.55 ˘ 0.44
6.63 ˘ 0.22
3.70 ˘ 0.29
2.76 ˘ 0.11
17
18
19
20
21
22
23
24
41
42
43
44
b
ADR
a
All values are given as means
˘
standard deviation. IC50 is the drug concentration effective in inhibiting 50%
b
of the cell growth measured by MTT method. The drug doxorubicin (ADR) was used as positive control in
this study.
In addition, it was observed that the introduction of different lipophilic fragments on the
0-carboxyl group or different amino acids on the 3-hydroxy group of GA results indifferent
3
cytotoxicities. When we combined ferulic acid methyl ester analogs at C-30, the cytotoxicity was
higher than with the corresponding ethyl ester analogs (the IC50 values: 17 < 18, 19 < 20, 21 < 22, 23 < 24)
.
It is also found that the lipophilic fragments with a methoxy group have higher anticancer activity (the
IC50 values: 17
position C-3 (compounds 42
compounds 41 43 45 47 49
<
19
,
18
<
20
,
,
,
21
44
51
<
23
46 48
53, and 55). Almost all the new synthetic GA derivatives exhibited
,
22
<
,
24). Meanwhile the introduction of L-selenomethionine at
52, 54, and 56), leads to lower IC50 values than L-methionine
,
,
,
50
(
,
,
,
,
,
much stronger inhibitory activity against MDA-MB-231 cells and relatively lower inhibitory activity
against MCF-7 cells than GA itself.

Molecules 2016, 21, 199
4 of 20
3
. Experimental Section
3
.1. General Information
All the chemicals and reagents were commercially available and used without further purification.
Routine thin-layer chromatography (TLC) was performed on silica gel plates (silica gel GF254 from
Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), and visualization was performed using UV.
1
13
H-NMR and C-NMR spectra were recorded on a Bruker-400 instrument (Bruker, Billerica, MA,
USA) at room temperature with TMS as an internal standard and CDCl or DMSO-d as solvents.
3
6
Chemical shifts are expressed in
δ (ppm) and coupling constants (J) in Hz. Mass spectra were recorded
with a MSQ Plus mass spectrometer (Thermo Scientific, Waltham, MA, USA). Melting points were
measured by an SGWX-4 micro melting point apparatus (Shanghai Precision & Scientific Instrument
Co. Ltd., Shanghai, China) and are uncorrected.
3
.2. General Method for Synthesizing Compounds 5–12
Ferulic acid (1) or trans-4-hydroxycinnamic acid (2) or isoferulic acid (3) or trans-3-hydroxycinnamic
acid (
mixture was concentrated in vacuo and the residue was dissolved in ethyl acetate. The organic layer
was washed with a 5% aqueous NaHCO solution and water. After drying over anhydrous Na SO ,
4) and five drops of H SO (95%) were refluxed in methanol or ethanol for 12 h. The reaction
2 4
3
2
4
the ethyl acetate was removed in vacuo. The residue was purified by column chromatography on silica
gel using ethyl acetate/petroleum ether mixtures as eluents to afford compounds 5–12.
3
.3. General Method for Synthesizing Compounds 15–16
The appropriate amino acid (13 or 14, 1 equiv.) and sodium bicarbonate (3 equiv.) was dissolved
in a 1:1 mixture of water and 1,4-dioxane. Di-tert-butyl dicarbonate (1.2 equiv.) was added and the
mixture was stirred at room temperature for 12 h. The 1,4-dioxane was removed under reduced
pressure and the mixture was extracted with ethyl acetate. Then the solution was acidified using
1
M hydrochloric acid solution and extracted three times with ethyl acetate. The combined organic
layers were washed with brine, dried over sodium sulfate, filtered, and the solvent was evaporated.
The crude protected amino acids 15–16 were used without any further purification.
3
.4. General Method for Synthesizing Compouds 17–24
GA (485 mg, 1 mmol) was dissolved in dry DCM (30 mL) and stirred at room temperature for
5
min. Then EDCI (230 mg, 1.2 mmol), DMAP (24 mg, 0.2 mmol) and compounds 5–12 (1 mmol)
were added to the solution, and then the reaction mixture was stirred at room temperature for 12 h.
The organic layer was washed with 1 M HCl solution and concentrated in vacuo. The residue was
purified by column chromatography on silica gel with ethyl acetate/petroleum as eluent to yield pure
compounds 17–24.
Ferulic acid methyl ester 3
β-hydroxy-11-oxo-olean-12-en-30-oate (17). Compound 17 was obtained from GA
˝
1
and ferulic acid methyl ester as a white solid (548.5 mg, 83%); m.p. 229–231 C; H-NMR (CDCl ):
δ
3
0
1
2
.72–0.74 (m, 1H, H-5), 0.83 (s, 3H, H-28), 0.90 (s, 3H, H-24), 0.96–1.00 (m, 1H, H-1’), 1.03 (s, 3H, H-23),
.08–1.11 (m, 1H, H-15’), 1.16 (s, 3H, H-26), 1.17 (s, 3H, H-25), 1.23–1.25 (m, 1H, H-16’), 1.27–1.30 (m,
H, H-22’ and 21’), 1.37 (s, 3H, H-29), 1.42 (s, 3H, H-27), 1.44–1.45 (m, 1H, H-22), 1.46–1.48 (m, 1H,
H-7’), 1.49–1.51 (m, 1H, H-6’), 1.61–1.63 (m, 1H, H-6), 1.65 (dd, J = 13.2, 4.0 Hz, 1H, H-19’), 1.67–1.68
m, 1H, H-2’), 1.70–1.73 (m, 1H, H-7), 1.77–1.79 (m, 1H, H-2), 1.89 (ddd, J = 14.4, 14.4, 5.2 Hz, 1H, H-16),
(
2
2
3
.02–2.04 (m, 1H, H-21), 2.08–2.10 (m, 1H, H-15), 2.12 (dd, J = 13.2, 4.0 Hz, 1H, H-18), 2.37 (s, 1H, H-9),
.43–2.45 (m, 1H, H-19), 2.80 (ddd, J = 13.2, 3.2, 3.2 Hz, 1H, H-1), 3.25 (dd, J = 10.8, 5.6 Hz, 1H, H-3),
.84 (s, 3H, COOCH ), 3.89 (s, 3H, OCH ), 5.73 (s, 1H, H-12), 6.42 (d, Jtrans = 12.8 Hz, 1H, H-β), 7.02 (d,
3
3
J = 6.8 Hz, 1H, Bn-H-5), 7.13 (s, 1H, Bn-H-3), 7.15 (d, J = 6.8 Hz, 1H, Bn-H-6), 7.68 (d, Jtrans = 12.8 Hz
,
13
1H, H-
α
); C-NMR (CDCl3):
δ
200.10, 174.43, 168.44, 166.88, 151.40, 149.95, 147.92, 128.43, 123.12,

Molecules 2016, 21, 199
5 of 20
121.17, 118.34, 113.48, 109.11, 78.66, 61.63, 55.68, 54.84, 51.64, 48.00, 45.33, 44.37, 43.11, 41.21, 38.04,
3
8.02, 37.42, 37.06, 32.75, 31.81, 31.13, 28.47, 28.05, 28.04, 27.28, 26.40, 26.35, 23.38, 18.59, 17.44, 16.30,
+
15.57; ESI-MS: m/z = 661.26 [M + H] . Anal. Calcd. for C H O (660.40): C, 74.51; H, 8.54%. Found:
41
56
7
C, 74.48; H, 8.56%.
Ferulic acid ethyl ester 3β-hydroxy-11-oxo-olean-12-en-30-oate (18). Compound 18 was obtained from GA
˝
1
and ferulic acid ethyl ester as a white solid (573.7 mg, 85%); m.p. 224–226 C; H-NMR (CDCl ):
δ
3
0
1
2
.72–0.74 (m, 1H, H-5), 0.83 (s, 3H, H-28), 0.90 (s, 3H, H-24), 0.96–1.00 (m, 1H, H-1’), 1.03 (s, 3H, H-23),
.08–1.11 (m, 1H, H-15’), 1.16 (s, 3H, H-26), 1.17 (s, 3H, H-25), 1.23–1.25 (m, 1H, H-16’), 1.27–1.30 (m,
H, H-22’ and H-21’), 1.37 (t, J = 5.6 Hz, 3H, CH ), 1.38 (s, 3H, H-29), 1.42 (s, 3H, H-27), 1.43–1.46 (m,
3
1
4
H, H-22), 1.47–1.48 (m, 1H, H-7’), 1.49–1.51 (m, 1H, H-6’), 1.62–1.64 (m, 1H, H-6), 1.65 (dd, J = 13.2,
.0 Hz, 1H, H-19’), 1.67–1.68 (m, 1H, H-2’), 1.70–1.72 (m, 1H, H-7), 1.76–1.78 (m, 1H, H-2), 1.89 (ddd,
J = 14.0, 14.0, 4.8 Hz, 1H, H-16), 2.03–2.05 (m, 1H, H-21), 2.08–2.10 (m, 1H, H-15), 2.12 (dd, J = 12.8,
3
3
.6 Hz, 1H, H-18), 2.37 (s, 1H, H-9), 2.43–2.45 (m, 1H, H-19), 2.80 (ddd, J = 13.6, 3.6, 3.6 Hz, 1H, H-1),
.25 (dd, J = 11.2, 5.2 Hz, 1H, H-3), 3.88 (s, 3H, OCH ), 4.30 (q, J = 5.6 Hz, 2H, CH2CH ), 5.73 (s, 1H,
3
3
H-12), 6.41 (d, Jtrans = 12.8 Hz, 1H, H-β), 7.02 (d, J = 6.8 Hz, 1H, Bn-H-5), 7.13 (s, 1H, Bn-H-3), 7.15 (d,
13
J = 6.8 Hz, 1H, Bn-H-6), 7.67 (d, Jtrans = 12.8 Hz, 1H, H-
α
); C-NMR (CDCl3):
δ 200.02, 174.30, 168.32,
1
5
2
66.80, 151.34, 149.95, 147.90, 128.45, 123.01, 121.12, 118.24, 113.47, 109.10, 78.63, 61.75, 60.63, 55.70,
4.87, 47.98, 45.32, 44.36, 43.13, 41.14, 38.00, 37.94, 37.56, 36.93, 32.65, 31.91, 31.15, 28.48, 28.06, 28.04,
+
7.25, 26.45, 26.38, 23.33, 18.63, 17.39, 16.32, 15.52, 14.28; ESI-MS: m/z = 675.23 [M + H] . Anal. Calcd.
for C H O (674.42): C, 74.74; H, 8.66%. Found: C, 74.70; H, 8.69%.
42
58
7
trans-4-Hydroxycinnamic acid methyl ester 3
β-hydroxy-11-oxo-olean-12-en-30-oate (19). Compound 19 was
obtained from GA and trans-4-hydroxycinnamic acid methyl ester as a white solid (530 mg, 84%);
˝
1
m.p. 202–205 C; H-NMR (CDCl ):
δ
0.69–0.72 (m, 1H, H-5), 0.81 (s, 3H, H-28), 0.86 (s, 3H, H-24),
3
0.94–0.98 (m, 1H, H-1’), 1.01 (s, 3H, H-23), 1.06–1.09 (m, 1H, H-15’), 1.14 (s, 3H, H-26), 1.14 (s, 3H,
H-25), 1.19–1.21 (m, 1H, H-16’), 1.24–1.26 (m, 2H, H-22’ and H-21’), 1.35 (s, 3H, H-29), 1.40 (s, 3H,
H-27), 1.43–1.45 (m, 1H, H-22), 1.47–1.48 (m, 1H, H-7’), 1.50–1.53 (m, 1H, H-6’), 1.60–1.62 (m, 1H, H-6),
1
1
.64 (dd, J = 13.2, 4.0 Hz, 1H, H-19’), 1.67–1.69 (m, 1H, H-2’), 1.72–1.74 (m, 1H, H-7), 1.75–1.78 (m,
H, H-2), 1.87 (ddd, J = 14.8, 14.8, 5.6 Hz, 1H, H-16), 2.04–2.06 (m, 1H, H-21), 2.11–2.13 (m, 1H, H-15),
2
2
.24 (dd, J = 13.6, 4.4 Hz, 1H, H-18), 2.35 (s, 1H, H-9), 2.43–2.46 (m, 1H, H-19), 2.78 (ddd, J = 12.8, 2.8,
.8 Hz, 1H, H-1), 3.23 (dd, J = 10.4, 6.0 Hz, 1H, H-3), 3.81 (s, 3H, COOCH ), 5.67 (s, 1H, H-12), 6.41 (d,
3
Jtrans = 16.0 Hz, 1H, H-
.68 (d, Jtrans = 16.0 Hz, 1H, H-
32.27, 129.25, 128.70, 122.12, 118.41, 78.74, 61.82, 54.94, 51.86, 48.43, 45.36, 44.39, 43.21, 41.11, 39.16,
β), 7.08 (d, J = 8.8 Hz, 2H, Bn-H-2 and 6), 7.55 (d, J = 8.8 Hz, 2H, Bn-H-3 and 5),
13
7
α); C-NMR (CDCl3): δ 200.03, 174.86, 168.65, 166.93, 162.26, 145.61,
1
3
9.09, 37.75, 37.10, 32.69, 31.88, 31.13, 28.54, 28.09, 28.06, 27.32, 26.41, 26.38, 23.49, 18.65, 17.49, 16.47,
+
15.62; ESI-MS: m/z = 631.21 [M + H] . Anal. Calcd. for C H O (630.39): C, 76.16; H, 8.63%. Found:
40
54
6
C, 76.13; H, 8.67%.
trans-4-Hydroxycinnamic acid ethyl ester 3β-hydroxy-11-oxo-olean-12-en-30-oate (20). Compound 20 was
obtained from GA and trans-4-hydroxycinnamic acid ethyl ester as a white solid (548.1 mg, 85%); m.p.
˝
95–196 C; H-NMR (CDCl ): δ 0.69–0.72 (m, 1H, H-5), 0.81 (s, 3H, H-28), 0.86 (s, 3H, H-24), 0.94–0.98
3
1
1
(
(
(
m, 1H, H-1’), 1.01 (s, 3H, H-23), 1.06–1.09 (m, 1H, H-15’), 1.14 (s, 3H, H-26), 1.14 (s, 3H, H-25), 1.19–1.21
m, 1H, H-16’), 1.24–1.26 (m, 2H, H-22’ and H-21’), 1.34 (t, J = 7.2 Hz, 3H, CH ), 1.35 (s, 3H, H-29), 1.40
3
s, 3H, H-27), 1.44–1.45 (m, 1H, H-22), 1.46–1.48 (m, 1H, H-7’), 1.49–1.53 (m, 1H, H-6’), 1.60–1.62 (m,
1
(
H, H-6), 1.63 (dd, J = 13.2, 4.0 Hz, 1H, H-19’), 1.67–1.69 (m, 1H, H-2’), 1.72–1.74 (m, 1H, H-7), 1.75–1.78
m, 1H, H-2), 1.86 (ddd, J = 14.4, 14.4, 5.2 Hz, 1H, H-16), 2.04–2.06 (m, 1H, H-21), 2.12–2.14 (m, 1H,
H-15), 2.24 (dd, J = 13.2, 4.0 Hz, 1H, H-18), 2.35 (s, 1H, H-9), 2.43–2.45 (m, 1H, H-19), 2.78 (ddd, J = 13.2,
3
.2, 3.2 Hz, 1H, H-1), 3.23 (dd, J = 10.8, 5.6 Hz, 1H, H-3), 4.27 (q, J = 7.2 Hz, 2H, CH CH ), 5.68 (s, 1H,
2 3
H-12), 6.40 (d, Jtrans = 16.0 Hz, 1H, H-β), 7.08 (d, J = 8.8 Hz, 2H, Bn-H-2 and 6), 7.55 (d, J = 8.8 Hz, 2H,
13
Bn-H-3 and 5), 7.67 (d, Jtrans = 16.0 Hz, 1H, H-
α); C-NMR (CDCl3): δ 200.11, 174.78, 168.70, 166.82,
1
62.12, 145.40, 132.12, 129.14, 128.61, 121.97, 118.33, 78.64, 61.76, 60.61, 54.86, 48.43, 45.33, 44.31, 43.16,

Molecules 2016, 21, 199
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4
1.00, 39.07, 39.03, 37.67, 37.01, 32.69, 31.88, 31.06, 28.54, 28.04, 28.02, 27.21, 26.39, 26.34, 23.38, 18.61,
+
1
7.42, 16.30, 15.54, 14.27; ESI-MS: m/z = 645.31 [M + H] . Anal. Calcd. for C H O (644.41): C, 76.36;
41 56 6
H, 8.75%. Found: C, 76.31; H, 8.79%.
Isoferulic acid methyl ester 3
GA and isoferulic acid methyl ester as a white solid (575 mg, 87%); m.p. 215–217 C; H-NMR (CDCl ):
β-hydroxy-11-oxo-olean-12-en-30-oate (21). Compound 21 was obtained from
˝
1
3
δ
0.70–0.72 (m, 1H, H-5), 0.81 (s, 3H, H-28), 0.89 (s, 3H, H-24), 0.94–0.98 (m, 1H, H-1’), 1.01 (s, 3H,
H-23), 1.06–1.09 (m, 1H, H-15’), 1.14 (s, 3H, H-26), 1.15 (s, 3H, H-25), 1.19–1.21 (m, 1H, H-16’), 1.24–1.26
m, 2H, H-22’ and 21’), 1.36 (s, 3H, H-29), 1.40 (s, 3H, H-27), 1.44 (m, 1H, H-22), 1.47 (m, 1H, H-7’),
.49 (m, 1H, H-6’), 1.62 (m, 1H, H-6), 1.64 (dd, 1H, J = 13.2, 4.0 Hz, H-19’), 1.67 (m, 1H, H-2’), 1.70
(
1
(
(
m, 1H, H-7), 1.75 (m, 1H, H-2), 1.88 (ddd, 1H, J = 14.4, 14.4, 5.2 Hz, H-16), 2.06 (m, 1H, H-21), 2.10
m, 1H, H-15), 2.13 (dd, 1H, J = 13.2, 4.0 Hz, H-18), 2.35 (s, 1H, H-9), 2.42 (m, 1H, H-19), 2.78 (ddd,
1
H, J = 13.2, 3.2, 3.2 Hz, H-1), 3.23 (dd, 1H, J = 10.8, 5.6 Hz, H-3), 3.79 (s, 3H, COOCH ), 3.86 (s,
3
3
H, OCH ), 5.70 (s, 1H, H-12), 6.30 (d, 1H, Jtrans = 16.0 Hz, H-
β), 6.96 (d, 1H, J = 8.8 Hz, Bn-H-3),
3
7
.17 (d, 1H, J = 2.0 Hz, Bn-H-6), 7.36 (dd, 1H, J = 8.8, 2.0 Hz, Bn-H-4), 7.61 (d, 1H, Jtrans = 16.0 Hz,
13
H-α); C-NMR (CDCl3): δ 200.23, 174.38, 169.22, 167.43, 152.86, 143.66, 139.91, 128.42, 127.56, 127.40,
121.68, 116.24, 112.25, 78.63, 61.76, 55.81, 54.85, 51.62, 47.95, 45.32, 44.34, 43.12, 41.16, 39.07, 39.06, 37.42,
3
7.00, 32.69, 31.81, 31.15, 28.53, 28.29, 28.04, 27.20, 26.45, 26.36, 23.34, 18.63, 17.42, 16.31, 15.55; ESI-MS:
+
m/z = 661.28 [M + H] . Anal. Calcd. for C H O (660.40): C, 74.51; H, 8.54%. Found: C, 74.46; H, 8.56%.
4
1
56
7
Isoferulic acid ethyl ester 3
β-hydroxy-11-oxo-olean-12-en-30-oate (22). Compound 22 was obtained from
˝
1
GA and isoferulic acid ethyl ester as a white solid (567 mg, 84%); m.p. 210–213 C; H-NMR (CDCl ):
3
δ
0.69–0.72 (m, 1H, H-5), 0.81 (s, 3H, H-28 ), 0.88 (s, 3H, H-24), 0.94–0.98 (m, 1H, H-1’), 1.01 (s, 3H,
H-23), 1.05–1.09 (m, 1H, H-15’), 1.14 (s, 3H, H-26), 1.15 (s, 3H, H-25), 1.19–1.21 (m, 1H, H-16’), 1.24–1.26
m, 2H, H-22’ and 21’), 1.33 (t, 3H, J = 7.2 Hz, CH ), 1.36 (s, 3H, H-29), 1.40 (s, 3H, H-27), 1.44 (m, 1H,
(
3
H-22), 1.47 (m, 1H, H-7’), 1.50 (m, 1H, H-6’), 1.62 (m, 1H, H-6), 1.64 (dd, 1H, J = 13.2, 4.0 Hz, H-19’),
.67 (m, 1H, H-2’), 1.70 (m, 1H, H-7), 1.75 (m, 1H, H-2), 1.87 (ddd, 1H, J = 14.0, 14.0, 4.8 Hz, H-16),
.06 (m, 1H, H-21), 2.10 (m, 1H, H-15), 2.13 (dd, 1H, J = 12.8, 3.6 Hz, H-18), 2.35 (s, 1H, H-9), 2.42 (m,
H, H-19), 2.78 (ddd, 1H, J = 13.6, 3.6, 3.6 Hz, H-1), 3.23 (dd, 1H, J = 11.2, 5.2 Hz, H-3), 3.86 (s, 3H,
OCH ), 4.25 (q, 2H, J = 7.2 Hz, CH CH ), 5.70 (s, 1H, H-12), 6.30 (d, 1H, Jtrans = 16.0 Hz, H- ), 6.96 (d,
H, J = 8.8 Hz, Bn-H-3), 7.18 (d, 1H, J = 2.0 Hz, Bn-H-6), 7.36 (dd, 1H, J = 8.8, 2.0 Hz, Bn-H-4), 7.60 (d,
1
2
1
β
3
2
3
1
1
1
1
3
H, Jtrans = 16.0 Hz, H-
α); C-NMR (CDCl3): δ 200.23, 174.38, 169.22, 167.43, 152.86, 143.66, 139.91,
28.42, 127.56, 127.40, 121.68, 116.24, 112.25, 78.63, 61.76, 55.81, 54.85, 51.62, 47.95, 45.32, 44.34, 43.12,
4
1.16, 39.07, 39.06, 37.42, 37.00, 32.69, 31.81, 31.15, 28.53, 28.29, 28.04, 27.20, 26.45, 26.36, 23.34, 18.63,
+
17.42, 16.31, 15.55; ESI-MS: m/z = 675.26 [M + H] . Anal. Calcd. for C H O (674.42): C, 74.74; H,
42
58
7
8
.66%. Found: C, 74.70; H, 8.69%.
trans-3-Hydroxycinnamic acid methyl ester 3
β-hydroxy-11-oxo-olean-12-en-30-oate (23). Compound 23 was
obtained from GA and trans-3-hydroxycinnamic acid methyl ester as a white solid (511 mg, 81%); m.p.
˝
88–190 C; H-NMR (CDCl ): δ 0.72–0.75 (m, 1H, H-5), 0.83 (s, 3H, H-28), 0.90 (s, 3H, H-24), 0.97–1.00
3
1
1
(
(
m, 1H, H-1’), 1.03 (s, 3H, H-23), 1.08–1.12 (m, 1H, H-15’), 1.16 (s, 3H, H-26), 1.17 (s, 3H, H-25), 1.20–1.23
m, 1H, H-16’), 1.27–1.28 (m, 2H, H-22’ and 21’), 1.38 (s, 3H, H-29), 1.42 (s, 3H, H-27), 1.44 (m, 1H, H-22),
1
1
.47 (m, 1H, H-7’), 1.50 (m, 1H, H-6’), 1.62 (m, 1H, H-6), 1.66 (dd, 1H, J = 13.2, 4.0 Hz, H-19’), 1.69 (m,
H, H-2’), 1.74 (m, 1H, H-7), 1.77 (m, 1H, H-2), 1.89 (ddd, 1H, J = 14.8, 14.8, 5.6 Hz, H-16), 2.08 (m, 1H,
H-21), 2.16 (m, 1H, H-15), 2.28 (dd, 1H, J = 13.6, 4.4 Hz, H-18), 2.37 (s, 1H, H-9), 2.43 (m, 1H, H-19), 2.81
ddd, 1H, J = 12.8, 2.8, 2.8 Hz, H-1), 3.25 (dd, 1H, J = 10.4, 6.0 Hz, H-3), 3.84 (s, 3H, COOCH ), 5.70 (s,
(
3
1
7
(
1
3
H, H-12), 6.46 (d, 1H, Jtrans = 16.0 Hz, H-
.42 (s, 1H, Bn-H-4), 7.44 (t, 1H, J = 7.6 Hz, Bn-H-5), 7.70 (d, 1H, Jtrans = 16.0 Hz, H-
CDCl3): 200.07, 174.89, 168.68, 167.04, 151.12, 145.65, 135.96, 129.89, 128.61, 125.61, 123.24, 120.69,
18.92, 78.65, 61.77, 54.86, 51.74, 48.36, 45.33, 44.28, 43.15, 40.97, 39.07, 39.04, 37.74, 37.02, 32.69, 31.88,
1.08, 28.58, 28.07, 28.05, 27.22, 26.39, 26.34, 23.39, 18.62, 17.42, 16.30, 15.54; ESI-MS: m/z = 631.23 [M + H]+
β), 7.09 (dt, 1H, J = 6.8, 2.4 Hz, Bn-H-6), 7.21 (s, 1H, Bn-H-2),
13
α); C-NMR
δ
.
Anal. Calcd. for C H O (630.39): C, 76.16; H, 8.63%. Found: C, 76.11; H, 8.67%.
40
54
6

Molecules 2016, 21, 199
7 of 20
trans-3-Hydroxycinnamic acid ethyl ester 3
β
-hydroxy-11-oxo-olean-12-en-30-oate (24). Compound 24 was
obtained from GA and trans-3-hydroxycinnamic acid ethyl ester as a white solid (561 mg, 87%); m.p.
˝
73–175 C; H-NMR (CDCl ): δ 0.72–0.75 (m, 1H, H-5), 0.83 (s, 3H, H-28), 0.90 (s, 3H, H-24), 0.97–1.00
3
1
1
(
m, 1H, H-1’), 1.03 (s, 3H, H-23), 1.08–1.12 (m, 1H, H-15’), 1.16 (s, 3H, H-26), 1.17 (s, 3H, H-25), 1.20–1.23
(
m, 1H, H-16’), 1.27–1.28 (m, 2H, H-22’ and 21’), 1.36 (t, 3H, J = 7.2 Hz, CH ), 1.38 (s, 3H, H-29), 1.42 (s,
3
3H, H-27), 1.44 (m, 1H, H-22), 1.47 (m, 1H, H-7’), 1.50 (m, 1H, H-6’), 1.62 (m, 1H, H-6), 1.66 (dd, 1H,
J = 13.2, 4.0 Hz, H-19’), 1.69 (m, 1H, H-2’), 1.74 (m, 1H, H-7), 1.77 (m, 1H, H-2), 1.89 (ddd, 1H, J = 14.4,
1
1
4.4, 5.2 Hz, H-16), 2.08 (m, 1H, H-21), 2.16 (m, 1H, H-15), 2.28 (dd, 1H, J = 13.2, 4.0 Hz, H-18), 2.37 (s,
H, H-9), 2.43 (m, 1H, H-19), 2.81 (ddd, 1H, J = 13.2, 3.2, 3.2 Hz, H-1), 3.25 (dd, 1H, J = 10.8, 5.6 Hz,
H-3), 4.29 (q, 2H, J = 7.2 Hz, CH2CH ), 5.70 (s, 1H, H-12), 6.46 (d, 1H, Jtrans = 16.0 Hz, H-
β), 7.08 (dt,
3
1
7
H, J = 6.8, 2.4 Hz, Bn-H-6), 7.22 (s, 1H, Bn-H-2), 7.42 (s, 1H, Bn-H-4), 7.44 (t, 1H, J = 7.6 Hz, Bn-H-5),
.68 (d, 1H, Jtrans = 16.0 Hz, H-α); C-NMR (CDCl3): δ 200.15, 174.76, 168.73, 166.96, 151.00, 145.48,
13
1
35.85, 129.73, 128.50, 125.49, 123.02, 120.57, 118.80, 78.56, 61.69, 60.32, 54.72, 48.21, 45.23, 44.20, 43.02,
4
0.86, 38.94, 38.90, 37.62, 36.95, 32.56, 31.73, 30.98, 28.40, 27.97, 27.93, 27.12, 26.25, 26.21, 23.23, 18.52,
+
1
7.30, 16.23, 15.46, 14.30; ESI-MS: m/z = 667.29 [M + Na] . Anal. Calcd. for C H O (644.41): C, 76.36;
41
56
6
H, 8.75%. Found: C, 76.29; H, 8.78%.
3
.5. General Method for Synthesizing Compouds 25–40
The protected amino acid (N-Boc-L-methionine or N-Boc-L-selenomethionine, 0.5 mmol) was
˝
dissolved in dry DCM and stirred at 0 C for 5 min. Then EDCI (115 mg, 0.6 mmol) and DMAP (12 mg,
˝
.1 mmol) were added to the solution, and the mixture was stirred at 0 C for 1 h. The appropriate GA
0
˝
derivative 17
–
24 (0.5 mmol) was then added to the mixture. After stirring at 0 C for another 1 h, the
reaction mixture was then stirred at room temperature for 12 h. The organic layer was washed with
M HCl solution and concentrated in vacuo. The residue was purified by column chromatography on
silica gel with ethyl acetate/petroleum to yield pure compound 25–40.
1
Ferulic acid methyl ester 3 -(Boc-L-methionine)-11-oxo-olean-12-en-30-oate (25). Obtained from 17 and
β
˝
1
N-Boc-L-methionine as a colourless powder (357 mg, 80%); m.p. 120–123 C; H-NMR (CDCl ):
δ
3
0
1
.82–0.84 (m, 1H, H-5), 0.89 (s, 3H, H-28), 0.91 (s, 3H, H-24), 0.92 (s, 3H, H-23), 1.07–1.11 (m, 1H, H-15’),
.14–1.16 (m, 1H, H-1’), 1.17 (s, 3H, H-26), 1.19 (s, 3H, H-25), 1.22–1.25 (m, 1H, H-16’), 1.27–1.31 (m, 2H,
H-22’ and 21’), 1.38 (s, 3H, H-29), 1.41 (s, 3H, H-27), 1.47 (s, 9H, Boc-CH ), 1.49–1.51 (m, 1H, H-22),
3
1
.53–1.55 (m, 1H, H-7’), 1.57–1.59 (m, 1H, H-6’), 1.62–1.64 (m, 1H, H-6), 1.67 (dd, J = 13.6, 4.4 Hz, 1H,
H-19’), 1.70–1.72 (m, 1H, H-2’), 1.73–1.74 (m, 1H, H-7), 1.77–1.80 (m, 1H, H-2), 1.89 (ddd, J = 14.8,
4.8, 5.6 Hz, 1H, H-16), 2.03–2.05 (m, 1H, H-21), 2.09–2.11 (m, 1H, H-15), 2.12 (s, 3H, SCH ), 2.15 (dd,
1
3
J = 13.6, 4.4 Hz, 1H, H-18), 2.38 (s, 1H, H-9), 2.43–2.45 (m, 1H, H-19), 2.57–2.60 (m, 2H, SCH ), 2.83
2
(
ddd, J = 13.6, 2.8, 2.8 Hz, 1H, H-1), 3.83 (s, 3H, COOCH ), 3.88 (s, 3H, OCH ), 4.80 (dd, J = 11.2, 5.2 Hz,
3 3
1
H, H-3), 5.14 (t, J = 8.4 Hz, 1H, COOCH ), 5.73 (s, 1H, H-12), 6.42 (d, Jtrans = 16.0 Hz, 1H, H-β), 7.02 (d,
3
J = 8.0 Hz, 1H, Bn-H-5), 7.13 (s, 1H, Bn-H-3), 7.15 (d, J = 8.0 Hz, 1H, Bn-H-6), 7.68 (d, Jtrans = 16.0 Hz,
13
1H, H-
α); C-NMR (CDCl3): δ 199.82, 174.40, 171.90, 169.23, 166.85, 154.87, 151.38, 149.94, 145.89,
1
28.40, 123.02, 121.15, 118.31, 113.49, 109.10, 81.93, 79.69, 61.54, 55.69, 54.94, 52.73, 51.67, 48.00, 45.33,
4
2
4.47, 43.11, 41.18, 38.06, 38.02, 37.40, 36.86, 32.64, 32.10, 31.81, 31.15, 29.63, 28.47, 28.26, 28.09, 28.04,
6.40, 26.34, 23.48, 23.28, 18.59, 17.32, 16.74, 16.30, 15.36; ESI-MS: m/z = 892.25 [M + H] . Anal. Calcd.
+
for C H NO S (891.50): C, 68.66; H, 8.25; N, 1.57; S, 3.59%. Found: C, 68.60; H, 8.28; N, 1.54; S, 3.60%.
5
1
73
10
Ferulic acid methyl ester 3
β-(Boc-L-selenomethionine)-11-oxo-olean-12-en-30-oate (26). Obtained from 17
˝
1
and N-Boc-L-selenomethionine as a colourless powder (389 mg, 83%); m.p. 125–127 C; H-NMR
(
(
1
CDCl3):
m, 1H, H-15’), 1.14–1.16 (m, 1H, H-1’), 1.18 (s, 3H, H-26), 1.19 (s, 3H, H-25), 1.23–1.25 (m, 1H, H-16’),
.27–1.30 (m, 2H, H-22’ and 21’), 1.38 (s, 3H, H-29), 1.42 (s, 3H, H-27), 1.46 (s, 9H, Boc-CH ), 1.49–1.52
δ 0.82–0.85 (m, 1H, H-5), 0.90 (s, 3H, H-28), 0.91 (s, 3H, H-24), 0.92 (s, 3H, H-23), 1.07–1.11
3
(m, 1H, H-22), 1.54–1.56 (m, 1H, H-7’), 1.58–1.60 (m, 1H, H-6’), 1.62–1.65 (m, 1H, H-6), 1.67 (dd,
J = 13.6, 4.4 Hz, 1H, H-19’), 1.70–1.72 (m, 1H, H-2’), 1.73–1.76 (m, 1H, H-7), 1.77–1.79 (m, 1H, H-2),

Molecules 2016, 21, 199
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1.88 (ddd, J = 14.8, 14.8, 5.6 Hz, 1H, H-16), 1.89-1.92 (m, 1H, H-21), 1.98–2.00 (m, 1H, H-15), 2.02 (s, 3H,
SeCH ), 2.12 (dd, J = 13.6, 4.4 Hz, 1H, H-18), 2.39 (s, 1H, H-9), 2.44–2.47 (m, 1H, H-19), 2.58–2.61 (m, 2H,
3
SeCH ), 2.83 (ddd, J = 13.6, 2.8, 2.8 Hz, 1H, H-1), 3.84 (s, 3H, COOCH ), 3.89 (s, 3H, OCH ), 4.59 (dd,
2
3
3
J = 11.2, 5.2 Hz, 1H, H-3), 5.12 (t, J = 8.4 Hz, 1H, CHCOO), 5.73 (s, 1H, H-12), 6.42 (d, Jtrans = 16.0 Hz
H, H-
d, Jtrans = 16.0 Hz, 1H, H-
,
1
(
β), 7.02 (d, J = 8.0 Hz, 1H, Bn-H-5), 7.13 (s, 1H, Bn-H-3), 7.15 (d, J = 8.0 Hz, 1H, Bn-H-6), 7.68
13
α
); C-NMR (CDCl3):
δ
199.82, 174.40, 171.82, 169.17, 166.85, 154.90, 151.40,
1
49.94, 145.89, 128.40, 123.02, 121.15, 118.32, 113.49, 109.10, 82.12, 79.72, 61.54, 55.70, 54.97, 52.71, 51.67,
4
2
8.02, 45.34, 44.47, 43.15, 41.20, 38.10, 38.06, 37.43, 36.86, 32.65, 32.07, 31.84, 31.15, 29.64, 28.50, 28.27,
8.12, 28.02, 26.41, 26.35, 23.45, 23.30, 18.60, 17.32, 16.75, 16.30, 15.37; ESI-MS: m/z = 962.18 [M + Na] .
+
Anal. Calcd. for C H NO Se (939.44): C, 65.23; H, 7.84; N, 1.49%. Found: C, 65.18; H, 7.89; N, 1.46%.
51
73
10
Ferulic acid ethyl ester 3
β
-(Boc-L-methionine)-11-oxo-olean-12-en-30-oate (27). Obtained from 18 and
˝
1
N-Boc-L-methionine as a colourless powder (357.8 mg, 79%); m.p. 111–113 C; H-NMR (CDCl ):
δ
3
0
1
.80–0.83 (m, 1H, H-5), 0.88 (s, 3H, H-28), 0.89 (s, 3H, H-24), 0.90 (s, 3H, H-23), 1.05–1.08 (m, 1H, H-15’),
.11–1.13 (m, 1H, H-1’), 1.15 (s, 3H, H-26), 1.17 (s, 3H, H-25), 1.20–1.23 (m, 1H, H-16’), 1.26–1.28 (m, 2H,
H-22’ and 21’), 1.34 (t, J = 7.2 Hz, 3H, CH ), 1.35 (s, 3H, H-29), 1.39 (s, 3H, H-27), 1.45 (s, 9H, Boc-CH ),
3
3
1
(
1
.48–1.50 (m, 1H, H-22), 1.51–1.54 (m, 1H, H-7’), 1.58–1.60 (m, 1H, H-6’), 1.61–1.63 (m, 1H, H-6), 1.68
dd, J = 13.6, 4.4 Hz, 1H, H-19’), 1.71–1.73 (m, 1H, H-2’), 1.75–1.77 (m, 1H, H-7), 1.78–1.81 (m, 1H, H-2),
.89 (ddd, J = 14.4, 14.4, 5.2 Hz, 1H, H-16), 2.04–2.06 (m, 1H, H-21), 2.08–2.09 (m, 1H, H-15), 2.10 (s, 3H,
SCH ), 2.15 (dd, J = 13.2, 4.0 Hz, 1H, H-18), 2.37–2.39 (m, 1H, H-9), 2.41–2.44 (m, 1H, H-19), 2.55–2.57
3
(
m, 2H, SCH ), 2.81 (ddd, J = 14.0, 3.2, 3.2 Hz, 1H, H-1), 3.86 (s, 3H, OCH ), 4.27 (q, J = 7.2 Hz, 2H,
2
3
C
H2CH ), 4.57 (dd, J = 11.6, 4.8 Hz, 1H, H-3), 5.12 (t, J = 8.4 Hz, 1H, CHCOO), 5.71 (s, 1H, H-12), 6.39
3
(
1
1
d, Jtrans = 16.0 Hz, 1H, H-
H, Bn-H-6), 7.65 (d, Jtrans = 16.0 Hz, 1H, H-α); C-NMR (CDCl3): δ 199.92, 174.28, 171.90, 169.32,
66.77, 155.10, 151.33, 149.92, 145.86, 128.38, 123.00, 121.10, 118.30, 113.49, 109.10, 82.03, 79.80, 61.60,
β
), 7.00 (d, J = 7.6 Hz, 1H, Bn-H-5), 7.11 (s, 1H, Bn-H-3), 7.12 (d, J = 7.6 Hz,
13
6
3
0.50, 55.67, 54.94, 52.71, 48.00, 45.32, 44.36, 43.13, 41.16, 38.05, 38.04, 37.36, 36.83, 32.61, 32.12, 31.81,
1.16, 29.63, 28.47, 28.26, 28.12, 28.04, 26.45, 26.34, 23.50, 23.28, 18.62, 17.30, 16.74, 16.32, 15.37, 14.26;
+
ESI-MS: m/z = 928.27 [M + Na] . Anal. Calcd. for C H NO S (905.51): C, 68.92; H, 8.34; N, 1.55; S,
52
75
10
3
.54%. Found: C, 68.86; H, 8.37; N, 1.51; S, 3.57%.
Ferulic acid ethyl ester 3β-(Boc-L-selenomethionine)-11-oxo-olean-12-en-30-oate (28). Obtained from 18 and
˝
1
N-Boc-L-selenomethionine as a colourless powder (390 mg, 82%); m.p. 118–120 C; H-NMR (CDCl ):
3
δ
0.82–0.85 (m, 1H, H-5), 0.90 (s, 3H, H-28), 0.91 (s, 3H, H-24), 0.92 (s, 3H, H-23), 1.07–1.10 (m, 1H,
H-15’), 1.11–1.13 (m, 1H, H-1’), 1.17 (s, 3H, H-26), 1.19 (s, 3H, H-25), 1.22–1.26 (m, 1H, H-16’), 1.27–1.30
m, 2H, H-22’ and 21’), 1.37 (t, J = 7.2 Hz, 3H, CH ), 1.38 (s, 3H, H-29), 1.42 (s, 3H, H-27), 1.47 (s, 9H,
(
3
Boc-CH ), 1.49–1.51 (m, 1H, H-22), 1.53–1.56 (m, 1H, H-7’), 1.58–1.60 (m, 1H, H-6’), 1.63–1.65 (m, 1H,
3
H-6), 1.67 (dd, J = 13.6, 4.4 Hz, 1H, H-19’), 1.73–1.75 (m, 1H, H-2’), 1.76–1.78 (m, 1H, H-7), 1.80–1.83
(m, 1H, H-2), 1.89 (ddd, J = 14.4, 14.4, 5.2 Hz, 1H, H-16), 1.97–1.99 (m, 1H, H-21), 2.00–2.01 (m, 1H,
H-15), 2.02 (s, 3H, SeCH ), 2.12 (dd, J = 13.2, 4.0 Hz, 1H, H-18), 2.39 (s, 1H, H-9), 2.43–2.46 (m, 1H,
3
H-19), 2.59–2.61 (m, 2H, SeCH ), 2.83 (ddd, J = 14.0, 3.2, 3.2 Hz, 1H, H-1), 3.88 (s, 3H, OCH ), 4.29 (q,
2
3
J = 7.2 Hz, 2H, CH CH ), 4.59 (dd, J = 11.6, 4.8 Hz, 1H, H-3), 5.13 (t, J = 8.4 Hz, 1H, CHCOO), 5.73
2
3
(
7
1
s, 1H, H-12), 6.41 (d, Jtrans = 16.0 Hz, 1H, H-
.14 (d, J = 8.0 Hz, 1H, Bn-H-6), 7.67 (d, Jtrans = 16.0 Hz, 1H, H-
71.83, 169.24, 166.77, 155.12, 151.36, 149.95, 145.86, 128.38, 123.03, 121.10, 118.30, 113.49, 109.10, 82.09,
β), 7.02 (d, J = 8.0 Hz, 1H, Bn-H-5), 7.13 (s, 1H, Bn-H-3),
13
α); C-NMR (CDCl3): δ 199.92, 174.28,
7
3
9.83, 61.60, 60.50, 55.68, 54.95, 52.68, 48.02, 45.32, 44.36, 43.14, 41.18, 38.08, 38.04, 37.37, 36.83, 32.62,
2.10, 31.81, 31.16, 29.63, 28.49, 28.27, 28.15, 28.03, 26.46, 26.36, 23.47, 23.29, 18.64, 17.30, 16.76, 16.32,
+
15.37, 14.26; ESI-MS: m/z = 978.12 [M + Na] . Anal. Calcd. for C H NO Se (953.11): C, 65.53; H,
52
75
10
7
.93; N, 1.47%. Found: C, 65.46; H, 7.98; N, 1.44%.
trans-4-Hydroxycinnamic acid methyl ester 3β-(Boc-L-methionine)-11-oxo-olean-12-en-30-oate (29). Obtained from
˝
1
1
9
and N-Boc-L-methionine as a colourless powder (362 mg, 84%); m.p. 107–109 C; H-NMR (CDCl ):
3
δ
0.82-0.85 (m, 1H, H-5), 0.88 (s, 3H, H-28), 0.91 (s, 3H, H-24), 0.92 (s, 3H, H-23), 1.08-1.11 (m, 1H,

Molecules 2016, 21, 199
9 of 20
H-15’), 1.13–1.15 (m, 1H, H-1’), 1.16 (s, 3H, H-26), 1.18 (s, 3H, H-25), 1.23–1.25 (m, 1H, H-16’), 1.27–1.31
m, 2H, H-22’ and 21’), 1.37 (s, 3H, H-29), 1.42 (s, 3H, H-27), 1.47 (s, 9H, Boc-CH ), 1.48–1.50 (m, 1H,
(
3
H-22), 1.51–1.53 (m, 1H, H-7’), 1.60–1.62 (m, 1H, H-6’), 1.64–1.66 (m, 1H, H-6), 1.67 (dd, J = 13.6, 4.4 Hz,
1
1
H, H-19’), 1.74–1.76 (m, 1H, H-2’), 1.77–1.78 (m, 1H, H-7), 1.80–1.83 (m, 1H, H-2), 1.89 (ddd, J = 15.2,
5.2, 6.0 Hz, 1H, H-16), 2.06–2.08 (m, 1H, H-21), 2.10–2.11 (m, 1H, H-15), 2.12-2.15 (s, 3H, SCH ), 2.26
3
(
dd, J = 14.0, 4.8 Hz, 1H, H-18), 2.39 (s, 1H, H-9), 2.43–2.46 (m, 1H, H-19), 2.57–2.60 (m, 2H, SCH ), 2.83
2
(
ddd, J = 13.2, 2.4, 2.4 Hz, 1H, H-1), 3.83 (s, 3H, COOCH ), 4.59 (dd, J = 10.8, 5.6 Hz, 1H, H-3), 5.14 (t,
3
J = 9.2 Hz, 1H, CHCOO), 5.70 (s, 1H, H-12), 6.43 (d, Jtrans = 16.0 Hz, 1H, H-
β), 7.10 (d, J = 8.4 Hz, 2H,
13
Bn-H-2 and 6), 7.57 (d, J = 8.4 Hz, 2H, Bn-H-3 and 5), 7.70 (d, Jtrans = 16.0 Hz, 1H, H-
α
); C-NMR
199.80, 174.83, 172.02, 168.76, 166.90, 162.24, 155.22, 145.41, 132.17, 129.13, 128.58, 121.92,
18.41, 81.98, 79.85, 61.73, 54.94, 52.88, 51.83, 48.43, 45.36, 44.39, 43.24, 41.10, 38.16, 38.12, 37.78, 37.15,
(
1
CDCl3): δ
3
2.66, 32.23, 31.88, 31.13, 29.87, 28.54, 28.18, 28.16, 28.06, 26.43, 26.40, 23.52, 23.39, 18.65, 17.33, 16.74,
+
1
6.42, 15.42; ESI-MS: m/z = 884.22 [M + Na] . Anal. Calcd. for C H NO S (861.48): C, 69.65; H, 8.30;
50
71
9
N, 1.62; S, 3.72%. Found: C, 69.59; H, 8.33; N, 1.58; S, 3.75%.
trans-4-Hydroxycinnamic acid methyl ester 3
β-(Boc-L-selenomethionine)-11-oxo-olean-12-en-30-oate (30).
Obtained from 19 and N-Boc-L-selenomethionine as a colourless powder (363 mg, 80%); m.p.
˝
1
1
15–118 C; H-NMR (400 MHz, CDCl ):
δ
0.82–0.85 (m, 1H, H-5), 0.88 (s, 3H, H-28), 0.91 (s, 3H,
H-24), 0.92 (s, 3H, H-23), 1.08–1.11 (m, 1H, H-15’), 1.13–1.15 (m, 1H, H-1’), 1.17 (s, 3H, H-26), 1.19 (s,
H, H-25), 1.22–1.26 (m, 1H, H-16’), 1.28–1.31 (m, 2H, H-22’ and 21’), 1.38 (s, 3H, H-29), 1.42 (s, 3H,
H-27), 1.47 (s, 9H, Boc-CH ), 1.49–1.51 (m, 1H, H-22), 1.53–1.55 (m, 1H, H-7’), 1.60–1.62 (m, 1H, H-6’),
3
3
3
1
1
.64–1.66 (m, 1H, H-6), 1.68 (dd, J = 13.6, 4.4 Hz, 1H, H-19’), 1.74–1.76 (m, 1H, H-2’), 1.77–1.79 (m,
H, H-7), 1.81–1.84 (m, 1H, H-2), 1.90 (ddd, J = 15.2, 15.2, 6.0 Hz, 1H, H-16), 1.97–1.99 (m, 1H, H-21),
2
2
.00–2.01 (m, 1H, H-15), 2.02 (s, 3H, SeCH ), 2.26 (dd, J = 14.0, 4.8 Hz, 1H, H-18), 2.39 (s, 1H, H-9),
3
.43–2.46 (m, 1H, H-19), 2.58–2.60 (m, 2H, SeCH ), 2.84 (ddd, J = 13.2, 2.4, 2.4 Hz, 1H, H-1), 3.83 (s, 3H,
2
COOCH ), 4.59 (dd, J = 10.8, 5.6 Hz, 1H, H-3), 5.13 (t, J = 9.2 Hz, 1H, CHCOO), 5.70 (s, 1H, H-12), 6.43
3
(
5
1
d, Jtrans = 16.0 Hz, 1H, H-
), 7.71 (d, Jtrans = 16.0 Hz, 1H, H-
55.25, 145.41, 132.16, 129.13, 128.58, 121.92, 118.44, 82.07, 79.88, 61.73, 54.98, 52.85, 51.83, 48.46, 45.36,
β), 7.10 (d, J = 8.4 Hz, 2H, Bn-H-2 and 6), 7.57 (d, J = 8.4 Hz, 2H, Bn-H-3 and
13
α); C-NMR (CDCl3): δ 199.80, 174.83, 171.90, 168.67, 166.90, 162.25,
4
2
4.39, 43.27, 41.13, 38.20, 38.12, 37.80, 37.15, 32.62, 32.20, 31.90, 31.15, 29.87, 28.57, 28.20, 28.18, 28.04,
6.44, 26.42, 23.49, 23.40, 18.67, 17.33, 16.75, 16.42, 15.42; ESI-MS: m/z = 932.16 [M + Na] . Anal. Calcd.
+
for C H NO Se (909.43): C, 66.06; H, 7.87; N, 1.54%. Found: C, 66.00; H, 7.91; N, 1.51%.
50
71
9
trans-4-Hydroxycinnamic acid ethyl ester 3β-(Boc-L-methionine)-11-oxo-olean-12-en-30-oate (31). Obtained from
˝
0 and N-Boc-L-methionine as a colourless powder (359 mg, 82%); m.p. 102–104 C; H-NMR (CDCl ):
3
1
2
δ
0.82–0.85 (m, 1H, H-5), 0.88 (s, 3H, H-28), 0.91 (s, 3H, H-24), 0.92 (s, 3H, H-23), 1.08–1.11 (m, 1H,
H-15’), 1.13–1.15 (m, 1H, H-1’), 1.16 (s, 3H, H-26), 1.18 (s, 3H, H-25), 1.23–1.26 (m, 1H, H-16’), 1.28–1.30
m, 2H, H-22’ and 21’), 1.36 (t, J = 7.2 Hz, 3H, CH ), 1.37 (s, 3H, H-29), 1.42 (s, 3H, H-27), 1.47 (s,
(
3
9
H, Boc-CH ), 1.49–1.50 (m, 1H, H-22), 1.51–1.54 (m, 1H, H-7’), 1.61–1.63 (m, 1H, H-6’), 1.64–1.66
3
(
1
(
m, 1H, H-6), 1.67 (dd, J = 13.6, 4.4 Hz, 1H, H-19’), 1.73–1.75 (m, 1H, H-2’), 1.77–1.79 (m, 1H, H-7),
.80–1.83 (m, 1H, H-2), 1.88 (ddd, J = 14.8, 14.8, 5.6 Hz, 1H, H-16), 2.07–2.09 (m, 1H, H-21), 2.10–2.11
m, 1H, H-15), 2.12 (s, 3H, SCH ), 2.27 (dd, J = 13.6, 4.4 Hz, 1H, H-18), 2.39 (s, 1H, H-9), 2.43–2.46 (m,
3
1
H, H-19), 2.57–2.59 (m, 2H, SCH ), 2.83 (ddd, J = 13.6, 2.8, 2.8 Hz, 1H, H-1), 4.29 (q, J = 7.2 Hz, 2H,
2
CH CH ), 4.59 (dd, J = 11.2, 5.2 Hz, 1H, H-3), 5.14 (t, J = 9.2 Hz, 1H, CHCOO), 5.70 (s, 1H, H-12), 6.42
2
3
(
5
1
d, Jtrans = 16.0 Hz, 1H, H-
), 7.69 (d, Jtrans = 16.0 Hz, 1H, H-
55.21, 145.37, 132.13, 129.14, 128.56, 121.95, 118.38, 82.00, 79.83, 61.62, 60.49, 54.94, 52.85, 48.44, 45.33,
β
), 7.10 (d, J = 8.4 Hz, 2H, Bn-H-2 and 6), 7.57 (d, J = 8.4 Hz, 2H, Bn-H-3 and
α
); 13C-NMR (CDCl3):
δ
199.83, 174.76, 171.93, 168.81, 166.79, 162.11,
4
4.31, 43.16, 40.99, 38.07, 38.04, 37.67, 36.84, 32.60, 32.25, 31.88, 31.06, 29.84, 28.53, 28.26, 28.12, 28.01,
+
26.39, 26.33, 23.47, 23.33, 18.61, 17.28, 16.75, 16.33, 15.41, 14.26; ESI-MS: m/z = 898.25 [M + Na] . Anal. Calcd.
for C H NO S (875.50): C, 69.91; H, 8.40; N, 1.60; S, 3.66%. Found: C, 69.84; H, 8.43; N, 1.55; S, 3.73%.
51
73
9

Molecules 2016, 21, 199
10 of 20
trans-4-Hydroxycinnamic acid ethyl ester 3
β
-(Boc-L-selenomethionine)-11-oxo-olean-12-en-30-oate (32).
Obtained from 20 and N-Boc-L-selenomethionine as a colourless powder (383 mg, 83%); m.p.
˝
1
1
10–112 C; H-NMR (CDCl ):
δ
0.82–0.85 (m, 1H, H-5), 0.89 (s, 3H, H-28), 0.91 (s, 3H, H-24), 0.92
3
(s, 3H, H-23), 1.08–1.11 (m, 1H, H-15’), 1.13–1.15 (m, 1H, H-1’), 1.17 (s, 3H, H-26), 1.19 (s, 3H, H-25),
1
.23–1.26 (m, 1H, H-16’), 1.28–1.30 (m, 2H, H-22’ and 21’), 1.37 (t, J = 7.2 Hz, 3H, CH ), 1.38 (s, 3H, H-29),
3
1
.42 (s, 3H, H-27), 1.47 (s, 9H, Boc-CH ), 1.48–1.50 (m, 1H, H-22), 1.51–1.53 (m, 1H, H-7’), 1.60–1.62
3
(m, 1H, H-6’), 1.64–1.66 (m, 1H, H-6), 1.67 (dd, J = 13.6, 4.4 Hz, 1H, H-19’), 1.73–1.75 (m, 1H, H-2’),
1
1
1
.77–1.79 (m, 1H, H-7), 1.81–1.84 (m, 1H, H-2), 1.89 (ddd, J = 14.8, 14.8, 5.6 Hz, 1H, H-16), 1.92–1.94 (m,
H, H-21), 1.97–1.99 (m, 1H, H-15), 2.02 (s, 3H, SeCH ), 2.26 (dd, J = 13.6, 4.4 Hz, 1H, H-18), 2.39 (s,
3
H, H-9), 2.46–2.49 (m, 1H, H-19), 2.57–2.60 (m, 2H, SeCH ), 2.84 (ddd, J = 13.6, 2.8, 2.8 Hz, 1H, H-1),
2
4
.29 (q, J = 7.2 Hz, 2H, CH2CH ), 4.59 (dd, J = 11.2, 5.2 Hz, 1H, H-3), 5.13 (t, J = 9.2 Hz, 1H, CHCOO),
3
5
.70 (s, 1H, H-12), 6.42 (d, Jtrans = 16.0 Hz, 1H, H-β), 7.10 (d, J = 8.4 Hz, 2H, Bn-H-2 and 6), 7.57 (d,
13
J = 8.4 Hz, 2H, Bn-H-3 and 5), 7.70 (d, Jtrans = 16.0 Hz, 1H, H-
α
); C-NMR (CDCl3):
δ 199.83, 174.75,
1
6
2
71.84, 168.78, 166.79, 162.13, 155.25, 145.37, 132.16, 129.14, 128.56, 121.94, 118.38, 82.09, 79.87, 61.62,
0.47, 54.96, 52.80, 48.49, 45.33, 44.36, 43.15, 41.03, 38.05, 38.04, 37.65, 36.78, 32.60, 32.23, 31.88, 31.00,
9.82, 28.59, 28.30, 28.15, 27.96, 26.41, 26.36, 23.42, 23.35, 18.60, 17.28, 16.77, 16.33, 15.40, 14.28; ESI-MS:
+
m/z = 946.23 [M + Na] . Anal. Calcd. for C H NO Se (923.45): C, 66.36; H, 7.97; N, 1.52%. Found: C,
51
73
9
6
6.33; H, 8.02; N, 1.48%.
Isoferulic acid methyl ester 3
N-Boc-L-methionine as a colourless powder (361 mg, 81%); m.p. 117–119 C; H-NMR (CDCl ):
β
-(Boc-L-methionine)-11-oxo-olean-12-en-30-oate (33). Obtained from 21 and
˝
1
δ
3
0
1
.82–0.85 (m, 1H, H-5), 0.89 (s, 3H, H-28), 0.90 (s, 3H, H-24), 0.92 (s, 3H, H-23), 1.07–1.11 (m, 1H, H-15’),
.14–1.16 (m, 1H, H-1’), 1.17 (s, 3H, H-26), 1.18 (s, 3H, H-25), 1.22–1.26 (m, 1H, H-16’), 1.28–1.29 (m, 2H,
H-22’ and 21’), 1.38 (s, 3H, H-29), 1.42 (s, 3H, H-27), 1.46 (s, 9H, Boc-CH ), 1.49 (m, 1H, H-22), 1.53 (m,
3
1
1
H, H-7’), 1.58 (m, 1H, H-6’), 1.62 (m, 1H, H-6), 1.67 (dd, 1H, J = 13.6, 4.4 Hz, H-19’), 1.73 (m, 1H, H-2’),
.77 (m, 1H, H-7), 1.80 (m, 1H, H-2), 1.88 (ddd, 1H, J = 14.8, 14.8, 5.2 Hz, H-16), 2.06 (m, 1H, H-21), 2.10
(
m, 1H, H-15), 2.12 (s, 3H, SCH ), 2.15 (dd, 1H, J = 13.2, 4.4 Hz, H-18), 2.39 (s, 1H, H-9), 2.44 (m, 1H,
3
H-19), 2.57 (m, 2H, SCH ), 2.83 (ddd, 1H, J = 13.2, 2.8, 2.8 Hz, H-1), 3.81 (s, 3H, COOCH ), 3.88 (s, 3H,
2
3
OCH ), 4.59 (dd, 1H, J = 11.2, 5.2 Hz, H-3), 5.14 (t, 1H, J = 8.4 Hz, CHCOO), 5.72 (s, 1H, H-12), 6.32 (d,
3
1
1
1
H, Jtrans = 16.0 Hz, H-
H, J = 8.4, 2.0 Hz, Bn-H-4), 7.63 (d, 1H, Jtrans = 16.0 Hz, H-
71.83, 169.03, 166.96, 154.82, 152.83, 143.64, 138.11, 128.42, 127.43, 127.38, 121.70, 116.21, 112.26, 81.90,
β), 6.98 (d, 1H, J = 8.4 Hz, Bn-H-3), 7.19 (d, 1H, J = 2.0 Hz, Bn-H-6), 7.38 (dd,
13
α
); C-NMR (CDCl3): δ 199.95, 174.34,
7
3
9.66, 61.56, 55.71, 54.87, 52.69, 51.53, 47.93, 45.29, 44.36, 43.03, 41.02, 38.95, 38.85, 37.84, 36.90, 32.63,
2.12, 31.83, 31.12, 29.60, 28.47, 28.33, 28.07, 28.05, 26.35, 26.28, 23.50, 23.13, 18.57, 17.30, 16.73, 16.25,
+
1
1
5.37; ESI-MS: m/z = 914.28 [M + Na] . Anal. Calcd. for C H NO S (891.50): C, 68.66; H, 8.25; N,
.57; S, 3.59%. Found: C, 68.60; H, 8.31; N, 1.53; S, 3.63%.
51
73
10
Isoferulic acid methyl ester 3
and N-Boc-L-selenomethionine as a colourless powder (375 mg, 80%); m.p. 124–125 C; H-NMR
CDCl3): 0.82–0.85 (m, 1H, H-5), 0.89 (s, 3H, H-28), 0.90 (s, 3H, H-24), 0.92 (s, 3H, H-23), 1.07–1.11
m, 1H, H-15’), 1.14–1.16 (m, 1H, H-1’), 1.18 (s, 3H, H-26), 1.19 (s, 3H, H-25), 1.22–1.26 (m, 1H, H-16’),
β
-(Boc-L-selenomethionine)-11-oxo-olean-12-en-30-oate (34). Obtained from
˝
1
2
1
(
(
δ
1
1
1
.28–1.29 (m, 2H, H-22’ and 21’), 1.38 (s, 3H, H-29), 1.42 (s, 3H, H-27), 1.47 (s, 9H, Boc-CH ), 1.49 (m,
H, H-22), 1.52 (m, 1H, H-7’), 1.60 (m, 1H, H-6’), 1.62 (m, 1H, H-6), 1.67 (dd, 1H, J = 13.6, 4.4 Hz, H-19’),
.73 (m, 1H, H-2’), 1.77 (m, 1H, H-7), 1.80 (m, 1H, H-2), 1.88 (ddd, 1H, J = 14.8, 14.8, 5.2 Hz, H-16), 1.93
3
(
m, 1H, H-21), 2.00 (m, 1H, H-15), 2.02 (s, 3H, SeCH ), 2.13 (dd, 1H, J = 13.2, 4.4 Hz, H-18), 2.39 (s, 1H,
3
H-9), 2.44 (m, 1H, H-19), 2.58 (m, 2H, SeCH ), 2.83 (ddd, 1H, J = 13.2, 2.8, 2.8 Hz, H-1), 3.81 (s, 3H,
2
COOCH ), 3.88 (s, 3H, OCH ), 4.59 (dd, 1H, J = 11.2, 5.2 Hz, H-3), 5.12 (t, 1H, J = 8.4 Hz, CHCOO), 5.73
3
3
(
s, 1H, H-12), 6.32 (d, 1H, Jtrans = 16.0 Hz, H-
β), 6.98 (d, 1H, J = 8.4 Hz, Bn-H-3), 7.19 (d, 1H, J = 2.0 Hz,
13
Bn-H-6), 7.38 (dd, 1H, J = 8.4, 2.0 Hz, Bn-H-4), 7.63 (d, 1H, Jtrans = 16.0 Hz, H-
1
1
α
); C-NMR (CDCl3):
99.95, 174.34, 171.75, 169.00, 166.97, 154.85, 152.85, 143.64, 138.11, 128.42, 127.43, 127.38, 121.70, 116.21,
12.26, 81.90, 79.70, 61.56, 55.73, 54.88, 52.67, 51.53, 47.96, 45.31, 44.36, 43.06, 41.05, 39.02, 38.85, 37.86,
δ

Molecules 2016, 21, 199
11 of 20
3
6.90, 32.65, 32.04, 31.85, 31.13, 29.58, 28.52, 28.33, 28.10, 28.03, 26.37, 26.30, 23.47, 23.10, 18.59, 17.30,
+
1
6.74, 16.26, 15.39; ESI-MS: m/z = 962.12 [M + Na] . Anal. Calcd. for C H NO Se (939.44): C, 65.23;
51 73 10
H, 7.84; N, 1.49%. Found: C, 65.15; H, 7.89; N, 1.43%.
Isoferulic acid ethyl ester 3
N-Boc-L-methionine as a colourless powder (376 mg, 83%); m.p. 111–114 C; H-NMR (CDCl ):
β-(Boc-L-methionine)-11-oxo-olean-12-en-30-oate (35). Obtained from 22 and
˝
1
δ
3
0
1
2
.80–0.83 (m, 1H, H-5), 0.88 (s, 3H, H-28), 0.89 (s, 3H, H-24), 0.90 (s, 3H, H-23), 1.05–1.09 (m, 1H, H-15’),
.14–1.16 (m, 1H, H-1’), 1.16 (s, 3H, H-26), 1.17 (s, 3H, H-25), 1.20–1.24 (m, 1H, H-16’), 1.26–1.28 (m,
H, H-22’ and 21’), 1.33 (t, 3H, J = 7.2 Hz, CH ), 1.36 (s, 3H, H-29), 1.40 (s, 3H, H-27), 1.45 (s, 9H,
3
Boc-CH ), 1.48 (m, 1H, H-22), 1.51 (m, 1H, H-7’), 1.58 (m, 1H, H-6’), 1.61 (m, 1H, H-6), 1.64 (dd, 1H,
3
J = 13.6, 4.4 Hz, H-19’), 1.72 (m, 1H, H-2’), 1.75 (m, 1H, H-7), 1.78 (m, 1H, H-2), 1.88 (ddd, 1H, J = 14.4
,
1
4
3
5
4.4, 5.2 Hz, H-16), 2.05 (m, 1H, H-21), 2.10 (m, 1H, H-15), 2.10 (s, 3H, SCH ), 2.14 (dd, 1H, J = 13.2,
3
.0 Hz, H-18), 2.37 (s, 1H, H-9), 2.43 (m, 1H, H-19), 2.55 (m, 2H, SCH ), 2.81 (ddd, 1H, J = 14.0, 3.2,
2
.2 Hz, H-1), 3.86 (s, 3H, OCH ), 4.25 (q, 2H, J = 7.2 Hz, CH CH ), 4.57 (dd, 1H, J = 11.6, 4.8 Hz, H-3),
3
2
3
.12 (t, 1H, J = 8.4 Hz, CHCOO), 5.71 (s, 1H, H-12), 6.30 (d, 1H, Jtrans = 16.0 Hz, H-β), 6.96 (d, 1H,
J = 8.4 Hz, Bn-H-3), 7.18 (d, 1H, J = 2.0 Hz, Bn-H-6), 7.36 (dd, 1H, J = 8.4, 2.0 Hz, Bn-H-4), 7.60 (d, 1H,
13
Jtrans = 16.0 Hz, H-
α
); C-NMR (CDCl3):
δ 200.13, 174.23, 171.83, 169.13, 166.88, 155.04, 152.80, 143.61,
1
4
2
38.08, 128.40, 127.40, 127.38, 121.65, 116.20, 112.26, 81.93, 79.78, 61.65, 60.65, 55.70, 54.87, 52.70, 47.93,
5.27, 44.27, 43.05, 41.05, 38.96, 38.85, 37.80, 36.87, 32.60, 32.15, 31.83, 31.14, 29.61, 28.47, 28.33, 28.10,
+
8.05, 26.37, 26.30, 23.52, 23.13, 18.59, 17.29, 16.73, 16.28, 15.38, 14.23; ESI-MS: m/z = 928.24 [M + Na] .
Anal. Calcd. for C H NO S (905.51): C, 68.92; H, 8.34; N, 1.55; S, 3.54%. Found: C, 68.87; H, 8.39;
52
75
10
N, 1.51; S, 3.56%.
Isoferulic acid methyl ester 3
and N-Boc-L-selenomethionine as a colourless powder (405 mg, 85%); m.p. 119–121 C; H-NMR
CDCl3): 0.80–0.83 (m, 1H, H-5), 0.88 (s, 3H, H-28), 0.89 (s, 3H, H-24), 0.90 (s, 3H, H-23), 1.05–1.09
m, 1H, H-15’), 1.12–1.14 (m, 1H, H-1’), 1.16 (s, 3H, H-26), 1.17 (s, 3H, H-25), 1.20–1.24 (m, 1H, H-16’),
β
-(Boc-L-selenomethionine)-11-oxo-olean-12-en-30-oate (36). Obtained from
˝
1
2
2
(
(
δ
1
1
.26–1.28 (m, 2H, H-22’ and 21’), 1.33 (t, 3H, J = 7.2 Hz, CH ), 1.36 (s, 3H, H-29), 1.40 (s, 3H, H-27),
3
.45 (s, 9H, Boc-CH ), 1.48 (m, 1H, H-22), 1.51 (m, 1H, H-7’), 1.59 (m, 1H, H-6’), 1.62 (m, 1H, H-6), 1.64
3
(
dd, 1H, J = 13.6, 4.4 Hz, H-19’), 1.71 (m, 1H, H-2’), 1.75 (m, 1H, H-7), 1.78 (m, 1H, H-2), 1.88 (ddd, 1H,
J = 14.4, 14.4, 5.2 Hz, H-16), 1.96 (m, 1H, H-21), 1.98(m, 1H, H-15), 2.00 (s, 3H, SeCH ), 2.10 (dd, 1H,
3
J = 13.2, 4.0 Hz, H-18), 2.37 (s, 1H, H-9), 2.43 (m, 1H, H-19), 2.55 (m, 2H, SeCH ), 2.81 (ddd, 1H, J = 14.0,
2
3
.2, 3.2 Hz, H-1), 3.86 (s, 3H, OCH ), 4.25 (q, 2H, J = 7.2 Hz, CH CH ), 4.57 (dd, 1H, J = 11.6, 4.8 Hz,
3 2 3
H-3), 5.10 (t, 1H, J = 8.4 Hz, CHCOO), 5.71 (s, 1H, H-12), 6.30 (d, 1H, Jtrans = 16.0 Hz, H-
β), 6.96 (d, 1H,
J = 8.8 Hz, Bn-H-3), 7.18 (d, 1H, J = 2.0 Hz, Bn-H-6), 7.36 (dd, 1H, J = 8.8, 2.0 Hz, Bn-H-4), 7.60 (d, 1H,
13
J
trans = 16.0 Hz, H-
α
); C-NMR (CDCl3):
δ 200.14 174.23, 171.77, 169.05, 166.88, 155.06, 152.83, 143.62,
1
4
2
38.08, 128.40, 127.40, 127.38, 121.65, 116.20, 112.26, 81.93, 79.80, 61.65, 60.65, 55.72, 54.89, 52.66, 47.94,
5.27, 44.27, 43.06, 41.08, 38.96, 38.86, 37.82, 36.87, 32.61, 32.12, 31.83, 31.14, 29.61, 28.48, 28.31, 28.12,
+
8.04, 26.39, 26.30, 23.49, 23.15, 18.61, 17.29, 16.75, 16.28, 15.38, 14.23; ESI-MS: m/z = 976.15 [M + Na] .
Anal. Calcd. for C H NO Se (953.11): C, 65.53; H, 7.93; N, 1.47%. Found: C, 65.48; H, 8.00; N, 1.43%.
52
75
10
trans-3-Hydroxycinnamic acid methyl ester 3β-(Boc-L-methionine)-11-oxo-olean-12-en-30-oate (37).
˝
Obtained from 23 and N-Boc-L-methionine as a colourless powder (349 mg, 81%); m.p. 109–111 C;
1
H-NMR (CDCl3):
.08–1.12 (m, 1H, H-15’), 1.13–1.15 (m, 1H, H-1’), 1.17(s, 3H, H-26), 1.19 (s, 3H, H-25), 1.23–1.25 (m, 1H,
H-16’), 1.26–1.28 (m, 2H, H-22’ and 21’), 1.39 (s, 3H, H-29), 1.42 (s, 3H, H-27), 1.47 (s, 9H, Boc-CH3),
.50 (m, 1H, H-22), 1.53 (m, 1H, H-7’), 1.61 (m, 1H, H-6’), 1.64 (m, 1H, H-6), 1.67 (dd, 1H, J = 13.6,
δ 0.82–0.85 (m, 1H, H-5), 0.90 (s, 3H, H-28), 0.91 (s, 3H, H-24), 0.92 (s, 3H, H-23),
1
1
4
6
.4 Hz, H-19’), 1.74 (m, 1H, H-2’), 1.77 (m, 1H, H-7), 1.80 (m, 1H, H-2), 1.89 (ddd, 1H, J = 15.2, 15.2,
.0 Hz, H-16), 2.07 (m, 1H, H-21), 2.10 (m, 1H, H-15), 2.13 (s, 3H, SCH ), 2.28 (dd, 1H, J = 14.0, 4.8 Hz,
3
H-18), 2.39 (s, 1H, H-9), 2.46 (m, 1H, H-19), 2.57 (m, 2H, SCH ), 2.84 (ddd, 1H, J = 13.2, 2.4, 2.4 Hz, H-1),
2
3
.83 (s, 3H, COOCH ), 4.59 (dd, 1H, J = 10.8, 5.6 Hz, H-3), 5.14 (t, 1H, J = 9.2 Hz, CHCOO), 5.71 (s, 1H,
3
H-12), 6.46 (d, 1H, Jtrans = 16.0 Hz, H-β), 7.09 (dt, 1H, J = 6.8, 2.4 Hz, Bn-H-6), 7.21 (s, 1H, Bn-H-2), 7.42

Molecules 2016, 21, 199
12 of 20
(
1
1
s, 1H, Bn-H-4), 7.44 (t, 1H, J = 7.6 Hz, Bn-H-5), 7.70 (d, 1H, Jtrans = 16.0 Hz, H-
α
); 13C-NMR (CDCl3):
99.80, 174.87, 171.91, 168.82, 167.02, 155.24, 151.11, 143.61, 135.95, 129.88, 128.57, 125.50, 123.22, 120.67,
18.89, 81.99, 79.82, 61.61, 54.92, 52.85, 51.76, 48.36, 45.32, 44.28, 43.15, 40.94, 38.57, 38.07, 37.72, 36.84,
δ
3
2.59, 32.23, 31.88, 31.07, 29.84, 28.57, 28.26, 28.12, 28.07, 26.37, 26.32, 23.53, 23.33, 18.61, 17.28, 16.75,
+
1
6.31, 15.41; ESI-MS: m/z = 884.27 [M + Na] . Anal. Calcd. for C H NO S (861.48): C, 69.65; H, 8.30;
50
71
9
N, 1.62; S, 3.72%. Found: C, 69.61; H, 8.35; N, 1.57; S, 3.78%.
trans-3-Hydroxycinnamic acid methyl ester 3
β-(Boc-L-selenomethionine)-11-oxo-olean-12-en-30-oate (38).
Obtained from 23 and N-Boc-L-selenomethionine as a colourless powder (377 mg, 83%); m.p.
˝
1
1
(
1
9
12–115 C; H-NMR (CDCl ):
δ
0.82–0.85 (m, 1H, H-5), 0.90 (s, 3H, H-28), 0.91 (s, 3H, H-24), 0.92
s, 3H, H-23), 1.08–1.11 (m, 1H, H-15’), 1.13–1.15 (m, 1H, H-1’), 1.17(s, 3H, H-26), 1.19 (s, 3H, H-25),
.23–1.25 (m, 1H, H-16’), 1.26–1.28 (m, 2H, H-22’ and 21’), 1.39 (s, 3H, H-29), 1.42 (s, 3H, H-27), 1.47 (s,
H, Boc-CH ), 1.50 (m, 1H, H-22), 1.53 (m, 1H, H-7’), 1.61 (m, 1H, H-6’), 1.65 (m, 1H, H-6), 1.67 (dd,
3
3
1H, J = 13.6, 4.4 Hz, H-19’), 1.74 (m, 1H, H-2’), 1.77 (m, 1H, H-7), 1.80 (m, 1H, H-2), 1.89 (ddd, 1H,
J = 15.2, 15.2, 6.0 Hz, H-16), 1.97 (m, 1H, H-21), 2.01 (m, 1H, H-15), 2.02 (s, 3H, SeCH ), 2.16 (dd, 1H,
3
J = 14.0, 4.8 Hz, H-18), 2.39 (s, 1H, H-9), 2.46 (m, 1H, H-19), 2.58 (m, 2H, SeCH ), 2.84 (ddd, 1H, J = 13.2
,
2
2
.4, 2.4 Hz, H-1), 3.83 (s, 3H, COOCH ), 4.59 (dd, 1H, J = 10.8, 5.6 Hz, H-3), 5.13 (t, 1H, J = 9.2 Hz
,
3
CHCOO), 5.71 (s, 1H, H-12), 6.46 (d, 1H, Jtrans = 16.0 Hz, H-
β
), 7.09 (dt, 1H, J = 6.8, 2.4 Hz, Bn-H-6), 7.21
);
199.80, 174.87, 171.83, 168.78, 167.02, 155.26, 151.12, 143.61, 135.96, 129.88, 128.57,
25.50, 123.22, 120.67, 118.90, 82.06, 79.84, 61.61, 54.93, 52.83, 51.76, 48.37, 45.32, 44.28, 43.16, 40.95,
(
s, 1H, Bn-H-2), 7.42 (s, 1H, Bn-H-4), 7.44 (t, 1H, J = 7.6 Hz, Bn-H-5), 7.70 (d, 1H, Jtrans = 16.0 Hz, H-
α
1
3
C-NMR (CDCl3):
δ
1
3
2
8.59, 38.07, 37.73, 36.84, 32.60, 32.21, 31.88, 31.07, 29.84, 28.58, 28.27, 28.14, 28.06, 26.38, 26.33, 23.49,
+
3.34, 18.62, 17.28, 16.76, 16.31, 15.41; ESI-MS: m/z = 932.22 [M + Na] . Anal. Calcd. for C H NO Se
50
71
9
(909.43): C, 66.06; H, 7.87; N, 1.54%. Found: C, 66.01; H, 7.91; N, 1.52%.
trans-3-Hydroxycinnamic acid ethyl ester
3
β
-(Boc-L-methionine)-11-oxo-olean-12-en-30-oate
(
39).
˝
Obtained from 24 and N-Boc-L-methionine as a colourless powder (341 mg, 78%); m.p. 107–108 C;
1
H-NMR (CDCl3):
.08–1.12 (m, 1H, H-15’), 1.13–1.15 (m, 1H, H-1’), 1.17(s, 3H, H-26), 1.19 (s, 3H, H-25), 1.23–1.25 (m, 1H,
H-16’), 1.26–1.28 (m, 2H, H-22’ and 21’), 1.36 (t, 3H, J = 7.2 Hz, CH ), 1.38 (s, 3H, H-29), 1.42 (s, 3H,
δ 0.82–0.85 (m, 1H, H-5), 0.90 (s, 3H, H-28), 0.91 (s, 3H, H-24), 0.92 (s, 3H, H-23),
1
3
H-27), 1.47 (s, 9H, Boc-CH ), 1.50 (m, 1H, H-22), 1.53 (m, 1H, H-7’), 1.61 (m, 1H, H-6’), 1.64 (m, 1H,
3
H-6), 1.67 (dd, 1H, J = 13.6, 4.4 Hz, H-19’), 1.74 (m, 1H, H-2’), 1.77 (m, 1H, H-7), 1.80 (m, 1H, H-2), 1.89
(
(
ddd, 1H, J = 14.8, 14.8, 5.6 Hz, H-16), 2.07 (m, 1H, H-21), 2.10 (m, 1H, H-15), 2.13 (s, 3H, SCH ), 2.28
3
dd, 1H, J = 13.6, 4.4 Hz, H-18), 2.39 (s, 1H, H-9), 2.46 (m, 1H, H-19), 2.56 (m, 2H, SCH ), 2.84 (ddd, 1H,
2
J = 13.6, 2.8, 2.8 Hz, H-1), 4.29 (q, 2H, J = 7.2 Hz, CH CH ), 4.59 (dd, 1H, J = 11.2, 5.2 Hz, H-3), 5.15 (t,
2
3
1H, J = 9.2 Hz, CHCOO), 5.71 (s, 1H, H-12), 6.46 (d, 1H, Jtrans = 16.0 Hz, H-β), 7.08 (dt, 1H, J = 6.8,
2
.4 Hz, Bn-H-6), 7.22 (s, 1H, Bn-H-2), 7.42 (s, 1H, Bn-H-4), 7.44 (t, 1H, J = 7.6 Hz, Bn-H-5), 7.69 (d, 1H,
13
J
trans = 16.0 Hz, H-
α
); C-NMR (CDCl3):
128.45, 125.38, 123.00, 120.54, 118.78
4.10, 43.16, 40.85, 38.58, 38.06, 37.69, 36.82, 32.57, 32.26, 31.85, 31.09, 29.85, 28.57, 28.26, 28.14, 28.07,
δ
199.92, 174.72, 171.91, 168.98, 166.93, 155.64, 150.97, 143.48,
1
35.84, 129.72
,
,
82.10, 79.97, 61.73, 60.45, 54.92, 52.83, 48.36, 45.32,
4
+
2
6.45, 26.32, 23.54, 23.33, 18.63, 17.27, 16.75, 16.32, 15.41, 14.30; ESI-MS: m/z = 898.30 [M + Na] . Anal. Calcd.
for C H NO S (875.50): C, 69.91; H, 8.40; N, 1.60; S, 3.66%. Found: C, 69.87; H, 8.46; N, 1.54; S, 3.70%.
51
73
9
trans-3-Hydroxycinnamic acid ethyl ester 3
β-(Boc-L-selenomethionine)-11-oxo-olean-12-en-30-oate (40).
Obtained from 24 and N-Boc-L-selenomethionine as a colourless powder (364 mg, 79%); m.p.
˝
1
1
(
1
09–112 C; H-NMR (CDCl ):
δ
0.82–0.85 (m, 1H, H-5), 0.90 (s, 3H, H-28), 0.91 (s, 3H, H-24), 0.92
s, 3H, H-23), 1.08–1.11 (m, 1H, H-15’), 1.13–1.15 (m, 1H, H-1’), 1.17(s, 3H, H-26), 1.19 (s, 3H, H-25),
.23–1.25 (m, 1H, H-16’), 1.26–1.28 (m, 2H, H-22’ and 21’), 1.36 (t, 3H, J = 7.2 Hz, CH ), 1.38 (s, 3H,
3
3
H-29), 1.42 (s, 3H, H-27), 1.47 (s, 9H, Boc-CH ), 1.50 (m, 1H, H-22), 1.53 (m, 1H, H-7’), 1.61 (m, 1H, H-6’),
3
1
1
.64 (m, 1H, H-6), 1.70 (dd, 1H, J = 13.6, 4.4 Hz, H-19’), 1.74 (m, 1H, H-2’), 1.77 (m, 1H, H-7), 1.80 (m,
H, H-2), 1.90 (ddd, 1H, J = 14.8, 14.8, 5.6 Hz, H-16), 1.98 (m, 1H, H-21), 2.01 (m, 1H, H-15), 2.02 (s, 3H,
SeCH ), 2.29 (dd, 1H, J = 13.6, 4.4 Hz, H-18), 2.39 (s, 1H, H-9), 2.46 (m, 1H, H-19), 2.58 (m, 2H, SeCH ),
3
2

Molecules 2016, 21, 199
13 of 20
2
.84 (ddd, 1H, J = 13.6, 2.8, 2.8 Hz, H-1), 4.29 (q, 2H, J = 7.2 Hz, CH CH ), 4.59 (dd, 1H, J = 11.2, 5.2 Hz,
2 3
H-3), 5.13 (t, 1H, J = 9.2 Hz, CHCOO), 5.71 (s, 1H, H-12), 6.46 (d, 1H, Jtrans = 16.0 Hz, H-
β), 7.08 (dt, 1H,
J = 6.8, 2.4 Hz, Bn-H-6), 7.22 (s, 1H, Bn-H-2), 7.42 (s, 1H, Bn-H-4), 7.44 (t, 1H, J = 7.6 Hz, Bn-H-5), 7.68
13
(
1
4
2
d, 1H, Jtrans = 16.0 Hz, H-
43.48, 135.86, 129.72 128.45, 125.36, 123.03, 120.51, 118.78
5.32, 44.13, 43.19, 40.87, 38.57, 38.09, 37.68, 36.79, 32.58, 32.26, 31.84, 31.03, 29.85, 28.60, 28.29, 28.15,
8.03, 26.46, 26.36, 23.51, 23.30, 18.62, 17.27, 16.78, 16.32, 15.41, 14.31; ESI-MS: m/z = 946.26 [M + Na]+
α
); C-NMR (CDCl3):
δ
199.92, 174.72, 171.82, 168.93, 166.93, 155.67, 150.98,
,
,
82.17, 80.00, 61.73, 60.44, 54.95, 52.82, 48.38,
.
Anal. Calcd. for C H NO Se (923.45): C, 66.36; H, 7.97; N, 1.52%. Found: C, 66.33; H, 8.01; N, 1.47%.
51
73
9
3
.6. General Method for Synthesizing Compouds 41–56
The Boc-protected compounds 25 40 (0.25 mmol) were dissolved in dry DCM. After saturation
–
with dry hydrogen chloride gas for 10 min, stirring at room temperature was then continued for 24 h.
After completion of the reaction (as monitored by TLC), the resulting precipitate was filtered and
washed with ethyl acetate until no parent substance can be detected; analytical samples were obtained
by re-crystallization to obtain pure compounds 41–56.
Ferulic acid methyl ester 3β-(l-methionine)-11-oxo-olean-12-en-30-oate hydrochloride (41). Obtained from 25
˝
1
as a white solid (155 mg, 81%); m.p. 203–205 C; H-NMR (DMSO-d ):
0
δ
0.71–0.74 (m, 1H, H-5),
.83 (s, 3H, H-28), 0.84 (s, 3H, H-24), 0.85 (s, 3H, H-23), 0.90–0.93 (m, 1H, H-15’), 1.00–1.03 (m, 1H,
H-1’), 1.07 (s, 3H, H-26), 1.08 (s, 3H, H-25), 1.12–1.14 (m, 1H, H-16’), 1.20–1.24 (m, 2H, H-22’ and 21’),
.32 (s, 3H, H-29), 1.41 (s, 3H, H-27), 1.43–1.45 (m, 1H, H-22), 1.48–1.50 (m, 1H, H-7’), 1.51–1.52 (m, 1H,
H-6’), 1.53–1.55 (m, 1H, H-6), 1.62 (dd, J = 13.2, 4.0 Hz, 1H, H-19’), 1.67–1.69 (m, 1H, H-2’), 1.71–1.73 (m,
6
1
1
1
H, H-7), 1.75–1.78 (m, 1H, H-2), 1.86 (ddd, J = 14.8, 14.8, 6.0 Hz, 1H, H-16), 1.91–1.93 (m, 1H, H-21),
.95–1.97 (m, 1H, H-15), 2.04 (s, 3H, SCH ), 2.28–2.31 (m, 1H, H-18), 2.43 (s, 1H, H-9), 2.54–2.56 (m, 1H,
3
H-19), 2.57–2.60 (m, 2H, SCH ), 2.64 (ddd, J = 13.2, 3.2, 3.2 Hz, 1H, H-1), 3.74 (s, 3H, COOCH ), 3.83 (s,
2
3
3
7
7
H, OCH ), 4.47 (dd, J = 11.2, 5.2 Hz, 1H, H-3), 5.46 (s, 1H, H-12), 6.72 (d, Jtrans = 16.0 Hz, 1H, H-
β
),
3
.11 (d, J = 8.0 Hz, 1H, Bn-H-5), 7.33 (dd, J = 8.0, 1.6 Hz, 1H, Bn-H-3), 7.55 (d, J = 1.6 Hz, 1H, Bn-H-6),
13
.67 (d, Jtrans = 16.0 Hz, 1H, H-
α); C-NMR (DMSO-d6): δ 199.91, 174.52, 173.90, 169.23, 166.87, 151.38,
1
49.96, 145.89, 128.40, 123.02, 121.15, 118.31, 113.51, 109.11, 81.45, 61.59, 55.69, 54.98, 52.84, 51.69, 48.03,
4
2
5.37, 44.50, 43.11, 41.28, 38.10, 38.04, 37.40, 36.86, 32.65, 32.12, 31.83, 31.25, 29.69, 28.52, 28.29, 28.10,
6.40, 26.38, 23.58, 23.30, 18.69, 17.42, 16.74, 16.33, 15.40; ESI-MS: m/z = 792.22 [M + H] . Anal. Calcd.
+
for C H ClNO S (791.44): C, 66.68; H, 8.03; N, 1.69; S, 3.87%. Found: C, 66.63; H, 8.07; N, 1.66; S, 3.90%.
4
6
66
8
Ferulic acid methyl ester 3β-(l-selenomethionine)-11-oxo-olean-12-en-30-oate hydrochloride (42). Obtained from
˝
1
2
(
1
3
6
as a white solid (156 mg, 77%); m.p. 210–213 C; H-NMR (DMSO-d ):
δ
0.71–0.74 (m, 1H, H-5), 0.83
s, 3H, H-28), 0.84 (s, 3H, H-24), 0.85 (s, 3H, H-23), 0.91–0.94 (m, 1H, H-15’), 1.00–1.04 (m, 1H, H-1’),
.07 (s, 3H, H-26), 1.08 (s, 3H, H-25), 1.12–1.15 (m, 1H, H-16’), 1.21–1.24 (m, 2H, H-22’ and 21’), 1.32 (s,
H, H-29), 1.41 (s, 3H, H-27), 1.43–1.45 (m, 1H, H-22), 1.49–1.51 (m, 1H, H-7’), 1.52–1.53 (m, 1H, H-6’),
6
1
1
1
.54–1.57 (m, 1H, H-6), 1.63 (dd, J = 13.2, 4.0 Hz, 1H, H-19’), 1.67–1.69 (m, 1H, H-2’), 1.71–1.73 (m,
H, H-7), 1.76–1.78 (m, 1H, H-2), 1.86 (ddd, J = 14.8, 14.8, 6.0 Hz, 1H, H-16), 1.88–1.90 (m, 1H, H-21),
.92–1.93 (m, 1H, H-15), 1.94 (s, 3H, SeCH ), 2.28–2.31 (m, 1H, H-18), 2.43 (s, 1H, H-9), 2.54–2.56 (m, 1H,
3
H-19), 2.57–2.59 (m, 2H, SeCH ), 2.64 (ddd, J = 13.2, 3.2, 3.2 Hz, 1H, H-1), 3.74 (s, 3H, COOCH ), 3.84
2
3
(
s, 3H, OCH ), 4.48 (dd, J = 11.2, 5.2 Hz, 1H, H-3), 5.46 (s, 1H, H-12), 6.72 (d, Jtrans = 16.0 Hz, 1H, H-
β),
3
7
7
.11 (d, J = 8.0 Hz, 1H, Bn-H-5), 7.33 (dd, J = 8.0, 1.6 Hz, 1H, Bn-H-3), 7.55 (d, J = 1.6 Hz, 1H, Bn-H-6),
.67 (d, Jtrans = 16.0 Hz, 1H, H-α); C-NMR (DMSO-d6): δ 199.92, 174.47, 173.82, 169.27, 166.85, 151.40,
13
1
49.94, 145.90, 128.42, 123.02, 121.16, 118.32, 113.49, 109.10, 82.42, 61.54, 55.72, 55.00, 52.81, 51.67, 48.02,
45.34, 44.54, 43.15, 41.30, 38.06, 38.00, 37.43, 36.88, 32.65, 32.17, 31.84, 31.28, 29.74, 28.50, 28.27, 28.12,
2
6.43, 26.36, 23.55, 23.32, 18.71, 17.43, 16.75, 16.34, 15.38; ESI-MS: m/z = 840.26 [M + H]+. Anal. Calcd.
for C H ClNO Se (839.39): C, 63.11; H, 7.60; N, 1.60%. Found: C, 63.03; H, 7.65; N, 1.54%.
46
66
8
Ferulic acid ethyl ester 3
as a white solid (142 mg, 73%); m.p. 191–194 C; H-NMR (DMSO-d ):
β-(l-methionine)-11-oxo-olean-12-en-30-oate hydrochloride (43). Obtained from 27
˝
1
δ
0.74–0.78 (m, 1H, H-5), 0.83
6

Molecules 2016, 21, 199
14 of 20
(s, 3H, H-28), 0.84 (s, 3H, H-24), 0.85 (s, 3H, H-23), 0.90–0.93 (m, 1H, H-15’), 1.00–1.02 (m, 1H, H-1’),
1
.07 (s, 3H, H-26), 1.08 (s, 3H, H-25), 1.12–1.14 (m, 1H, H-16), 1.17–1.23 (m, 2H, H-22’ and 21’), 1.27 (t,
J = 7.2 Hz, 3H, CH ), 1.31 (s, 3H, H-29), 1.40 (s, 3H, H-27), 1.45–1.47 (m, 1H, H-22), 1.48–1.50 (m, 1H,
3
H-7’), 1.51–1.52 (m, 1H, H-6’), 1.54–1.57 (m, 1H, H-6), 1.63 (dd, J = 13.2, 4.0 Hz, 1H, H-19’), 1.67–1.69
(
1
m, 1H, H-2’), 1.70–1.73 (m, 1H, H-7), 1.79–1.81 (m, 1H, H-2), 1.86 (ddd, J = 14.4, 14.4, 5.6 Hz, 1H, H-16),
.92-1.93 (m, 1H, H-21), 1.94–1.96 (m, 1H, H-15), 2.03 (s, 3H, SCH ), 2.28–2.31 (m, 1H, H-18), 2.42 (s, 1H,
3
H-9), 2.54–2.55 (m, 1H, H-19), 2.56–2.59 (m, 2H, SCH ), 2.63 (ddd, J = 13.6, 3.6, 3.6 Hz, 1H, H-1), 3.83 (s,
2
3
6
7
H, OCH ), 4.20 (q, J = 7.2 Hz, 2H, CH CH ), 4.47 (dd, J = 11.6, 4.8 Hz, 1H, H-3), 5.46 (s, 1H, H-12),
3
2
3
.72 (d, Jtrans = 16.0 Hz, 1H, H-β), 7.10 (d, J = 8.0 Hz, 1H, Bn-H-5), 7.31 (dd, J = 8.0, 1.6 Hz, 1H, Bn-H-3),
13
.55 (d, J = 1.6 Hz, 1H, Bn-H-6), 7.64 (d, Jtrans = 16.0 Hz, 1H, H-
α); C-NMR (DMSO-d6): δ 200.02,
174.37, 174.03, 169.45, 166.78, 151.33, 149.94, 145.86, 128.38, 123.03, 121.10, 118.30, 113.47, 109.12, 81.43,
6
3
1.60, 60.50, 55.67, 54.94, 53.52, 48.02, 45.33, 44.36, 43.15, 41.26, 38.07, 38.05, 37.36, 36.83, 32.63, 32.14,
1.79, 31.21, 29.72, 28.47, 28.37, 28.15, 26.45, 26.34, 23.63, 23.28, 18.75, 17.47, 16.72, 16.39, 15.46, 14.26;
+
ESI-MS: m/z = 828.30 [M + Na] . Anal. Calcd. for C H ClNO S (805.46): C, 67.00; H, 8.13; N, 1.66; S,
47
68
8
3
.81%. Found: C, 66.95; H, 8.20; N, 1.61; S, 3.85%.
Ferulic acid ethyl ester 3β-(l-selenomethionine)-11-oxo-olean-12-en-30-oate hydrochloride (44). Obtained from 28
˝
1
as a white solid (160 mg, 78%); m.p. 205–207 C; H-NMR (DMSO-d ):
δ
0.70–0.74 (m, 1H, H-5), 0.83
6
(s, 3H, H-28), 0.84 (s, 3H, H-24), 0.85 (s, 3H, H-23), 0.91–0.93 (m, 1H, H-15’), 1.00–1.03 (m, 1H, H-1’),
1.07 (s, 3H, H-26), 1.08 (s, 3H, H-25), 1.12–1.14 (m, 1H, H-16’), 1.20–1.24 (m, 2H, H-22’ and 21’), 1.27 (t,
J = 7.2 Hz, 3H, CH ), 1.32 (s, 3H, H-29), 1.41 (s, 3H, H-27), 1.46–1.48 (m, 1H, H-22), 1.49–1.50 (m, 1H,
3
H-7’), 1.51–1.52 (m, 1H, H-6’), 1.54–1.56 (m, 1H, H-6), 1.65 (dd, J = 13.2, 4.0 Hz, 1H, H-19’), 1.68-1.69 (m,
1
1
H, H-2’), 1.70–1.73 (m, 1H, H-7), 1.79–1.82 (m, 1H, H-2), 1.86 (ddd, J = 14.4, 14.4, 5.6 Hz, 1H, H-16),
.89–1.90 (m, 1H, H-21), 1.91–1.92 (m, 1H, H-15), 1.93 (s, 3H, SeCH ), 2.28–2.31 (m, 1H, H-18), 2.43
3
(
s, 1H, H-9), 2.54–2.56 (m, 1H, H-19), 2.57–2.60 (m, 2H, SeCH ), 2.63 (ddd, J = 13.6, 3.6, 3.6 Hz, 1H,
2
H-1), 3.83 (s, 3H, OCH ), 4.20 (q, J = 7.2 Hz, 2H, CH CH ), 4.47 (dd, J = 11.6, 4.8 Hz, 1H, H-3), 5.46 (s,
3
2
3
1
1
H, H-12), 6.72 (d, Jtrans = 16.0 Hz, 1H, H-β), 7.11 (d, J = 8.0 Hz, 1H, Bn-H-5), 7.32 (dd, J = 8.0, 1.6 Hz,
13
H, Bn-H-3), 7.56 (d, J = 1.6 Hz, 1H, Bn-H-6), 7.65 (d, Jtrans = 16.0 Hz, 1H, H-
200.06, 174.38, 173.98, 169.39, 166.87, 151.36, 149.95, 145.88, 128.42, 123.05, 121.13, 118.30, 113.51,
09.11, 81.49, 61.60, 60.50, 55.68, 54.97, 53.49, 48.04, 45.32, 44.37, 43.14, 41.28, 38.08, 38.04, 37.39, 36.84,
α); C-NMR (DMSO-d6):
δ
1
3
2.62, 32.13, 31.80, 31.26, 29.73, 28.49, 28.39, 28.13, 26.46, 26.37, 23.59, 23.29, 18.76, 17.50, 16.76, 16.42,
+
15.47, 14.27; ESI-MS: m/z = 854.18 [M + H] . Anal. Calcd. for C H ClNO Se (853.40): C, 63.47; H,
47
68
8
7
.71; N, 1.57%. Found: C, 63.44; H, 7.75; N, 1.53%.
trans-4-Hydroxycinnamic acid methyl ester 3
β
-(l-methionine)-11-oxo-olean-12-en-30-oate hydrochloride (45).
˝
1
Obtained from 29 as a white solid (143 mg, 76%); m.p. 205–207 C; H-NMR (DMSO-d ):
δ
0.71–0.74
m, 1H, H-5), 0.83 (s, 3H, H-28), 0.84 (s, 3H, H-24), 0.85 (s, 3H, H-23), 0.91–0.93 (m, 1H, H-15’), 1.00–1.04
m, 1H, H-1’), 1.07 (s, 3H, H-26), 1.08 (s, 3H, H-25), 1.17–1.20 (m, 1H, H-16’), 1.24–1.27 (m, 2H, H-22’
6
(
(
and 21’), 1.33 (s, 3H, H-29), 1.41 (s, 3H, H-27), 1.45–1.48 (m, 1H, H-22), 1.49–1.50 (m, 1H, H-7’), 1.52-1.53
m, 1H, H-6’), 1.54–1.57 (m, 1H, H-6), 1.64 (dd, J = 13.2, 4.0 Hz, 1H, H-19’), 1.68–1.70 (m, 1H, H-2’),
.71–1.73 (m, 1H, H-7), 1.79–1.82 (m, 1H, H-2), 1.87 (ddd, J = 15.2, 15.2, 6.4 Hz, 1H, H-16), 1.93–1.95
m, 1H, H-21), 1.97–2.00 (m, 1H, H-15), 2.04 (s, 3H, SCH ), 2.15–2.17 (m, 1H, H-18), 2.43 (s, 1H, H-9),
(
1
(
3
2
.53–2.55 (m, 1H, H-19), 2.56–2.59 (m, 2H, SCH ), 2.64 (ddd, J = 12.8, 2.8, 2.8 Hz, 1H, H-1), 3.74 (s, 3H,
2
COOCH ), 4.47 (dd, J = 10.8, 5.6 Hz, 1H, H-3), 5.46 (s, 1H, H-12), 6.65 (d, Jtrans = 16.0 Hz, 1H, H-
β),
3
7
2
.17 (d, J = 8.8 Hz. 2H, Bn-H-2 and 6), 7.69 (d, J = 8.8 Hz, 2H, Bn-H-3 and 5), 7.81 (d, Jtrans = 16.0 Hz,
H, H-α); C-NMR (DMSO-d6): δ 199.80, 174.83, 174.01, 168.76, 166.98, 162.27, 145.45, 132.17, 129.13,
13
128.58, 121.98, 118.45, 81.48, 61.73, 54.94, 53.68, 51.83, 48.43, 45.36, 44.39, 43.24, 41.10, 38.16, 38.12, 37.78,
3
7.15, 32.66, 32.43, 31.88, 31.13, 29.98, 28.54, 28.18, 28.14, 26.57, 26.40, 23.63, 23.39, 18.65, 17.33, 16.74,
+
16.42, 15.54; ESI-MS: m/z = 784.23 [M + Na] . Anal. Calcd. for C H ClNO S (761.43): C, 67.69; H,
45
64
7
8
.08; N, 1.75; S, 4.02%. Found: C, 67.65; H, 8.10; N, 1.69; S, 4.10%.

Molecules 2016, 21, 199
15 of 20
trans-4-Hydroxycinnamic acid methyl ester 3
β
-(l-selenomethionine)-11-oxo-olean-12-en-30-oate hydrochloride
˝
1
(46). Obtained from 30 as a white solid (156 mg, 80%); m.p. 218–221 C; H-NMR (DMSO-d ):
6
δ
0.71–0.74 (m, 1H, H-5), 0.83 (s, 3H, H-28), 0.84 (s, 3H, H-24), 0.85 (s, 3H, H-23), 0.90–0.93 (m, 1H,
H-15’), 1.00–1.03 (m, 1H, H-1’), 1.06 (s, 3H, H-26), 1.07 (s, 3H, H-25), 1.16–1.20 (m, 1H, H-16’), 1.23–1.27
m, 2H, H-22’ and 21’), 1.33 (s, 3H, H-29), 1.41 (s, 3H, H-27), 1.44–1.46 (m, 1H, H-22), 1.48–1.50 (m, 1H,
H-7’), 1.52–1.55 (m, 1H, H-6’), 1.56–1.59 (m, 1H, H-6), 1.65 (dd, J = 13.2, 4.0 Hz, 1H, H-19’), 1.68–1.69
m, 1H, H-2’), 1.71–1.74 (m, 1H, H-7), 1.78–1.81 (m, 1H, H-2), 1.86 (ddd, J = 15.2, 15.2, 6.4 Hz, 1H, H-16),
.88–1.90 (m, 1H, H-21), 1.91–1.92 (m, 1H, H-15), 1.93 (s, 3H, SeCH ), 2.15–2.18 (m, 1H, H-18), 2.43 (s, 1H,
(
(
1
3
H-9), 2.53–2.55 (m, 1H, H-19), 2.58–2.61 (m, 2H, SeCH ), 2.64 (ddd, J = 12.8, 2.8, 2.8 Hz, 1H, H-1), 3.73 (s,
2
3
7
2
H, COOCH ), 4.47 (dd, J = 10.8, 5.6 Hz, 1H, H-3), 5.45 (s, 1H, H-12), 6.64 (d, Jtrans = 16.0 Hz, 1H, H-β),
3
.17 (d, J = 8.8 Hz, 2H, Bn-H-2 and 6), 7.69 (d, J = 8.8Hz, 2H, Bn-H-3 and 5), 7.81 (d, Jtrans = 16.0 Hz
,
13
H, H-α
); C-NMR (DMSO-d6):
δ
199.83, 174.83, 173.95, 168.67, 167.03, 162.25, 145.44, 132.20, 129.13,
128.58, 121.97, 118.48, 81.52, 61.73, 54.96, 53.65, 51.83, 48.44, 45.36, 44.35, 43.27, 41.13, 38.18, 38.14, 37.80,
3
7.15, 32.62, 32.40, 31.93, 31.15, 30.00, 28.59, 28.20, 28.18, 26.59, 26.42, 23.65, 23.40, 18.70, 17.29, 16.72,
+
16.48, 15.53; ESI-MS: m/z = 810.14 [M + H] . Anal. Calcd. for C H ClNO Se (809.38): C, 63.93; H,
45
64
7
7
.63; N, 1.66%. Found: C, 63.86; H, 7.68; N, 1.61%.
trans-4-Hydroxycinnamic acid ethyl ester 3
β
-(l-methionine)-11-oxo-olean-12-en-30-oatehydrochloride (47).
˝
1
Obtained from 31 as a white solid (138 mg, 74%); m.p. 202–204 C; H-NMR (DMSO-d ):
δ
0.71–0.74
m, 1H, H-5), 0.83 (s, 3H, H-28), 0.84 (s, 3H, H-24), 0.85 (s, 3H, H-23), 0.91–0.94 (m, 1H, H-15’), 1.00–1.04
m, 1H, H-1’), 1.07 (s, 3H, H-26), 1.08 (s, 3H, H-25), 1.13–1.14 (m, 1H, H-16’), 1.18–1.22 (m, 2H, H-22’
6
(
(
and 21’), 1.27 (t, J = 7.2 Hz, 3H, CH ), 1.33 (s, 3H, H-29), 1.41 (s, 3H, H-27), 1.45–1.47 (m, 1H, H-22),
3
1
.49–1.51 (m, 1H, H-7’), 1.52–1.55 (m, 1H, H-6’), 1.57–1.60 (m, 1H, H-6), 1.64 (dd, J = 13.2, 4.0 Hz, 1H,
H-19’), 1.67–1.68 (m, 1H, H-2’), 1.71–1.74 (m, 1H, H-7), 1.79–1.82 (m, 1H, H-2), 1.87 (ddd, J = 14.8, 14.8,
.0 Hz, 1H, H-16), 1.93–1.95 (m, 1H, H-21), 1.97–1.99 (m, 1H, H-15), 2.04 (s, 3H, SCH ), 2.15–2.17 (m,
6
3
1
H, H-18), 2.43 (s, 1H, H-9), 2.52–2.54 (m, 1H, H-19), 2.56–2.58 (m, 2H, SCH ), 2.64 (ddd, J = 13.2, 3.2,
2
3
6
.2 Hz, 1H, H-1), 4.20 (q, J = 7.2 Hz, 2H, CH CH ), 4.47 (dd, J = 11.2, 5.2 Hz, 1H, H-3), 5.46 (s, 1H, H-12),
2 3
.63 (d, Jtrans = 16.0 Hz, 1H, H-β), 7.17 (d, J = 8.8 Hz, 2H, Bn-H-2 and 6), 7.67 (d, J = 8.8 Hz, 2H, Bn-H-3
13
and 5), 7.81 (d, Jtrans = 16.0Hz, 2H, H-
α
); C-NMR (DMSO-d6):
δ 199.94, 174.85, 174.05, 168.92, 166.91,
1
4
2
62.23, 145.47, 132.17, 129.36, 128.58, 122.02, 118.40, 81.40, 61.62, 60.63, 54.94, 53.68, 48.44, 45.47, 44.40,
3.21, 41.06, 38.05, 38.01, 37.63, 36.90, 32.60, 32.36, 31.88, 31.06, 29.96, 28.52, 28.30, 28.25, 26.46, 26.43,
+
3.60, 23.36, 18.74, 17.40, 16.75, 16.43, 15.54, 14.46; ESI-MS: m/z = 776.28 [M + H] . Anal. Calcd. for
C H ClNO S (775.45): C, 68.00; H, 8.19; N, 1.72; S, 3.95%. Found: C, 67.96; H, 8.21; N, 1.70; S, 3.97%.
46
66
7
trans-4-Hydroxycinnamic acid ethyl ester 3β-(l-selenomethionine)-11-oxo-olean-12-en-30-oate hydrochloride (48)
.
˝
1
Obtained from 32 as a white solid (145 mg, 73%); m.p. 208–211 C; H-NMR (DMSO-d ):
δ
0.71–0.74
6
(
(
m, 1H, H-5), 0.83 (s, 3H, H-28), 0.84 (s, 3H, H-24), 0.85 (s, 3H, H-23), 0.91–0.94 (m, 1H, H-15’), 1.00–1.04
m, 1H, H-1’), 1.07 (s, 3H, H-26), 1.08 (s, 3H, H-25), 1.13–1.14 (m, 1H, H-16’), 1.18–1.22 (m, 2H, H-22’
and 21’), 1.27 (t, J = 7.2 Hz, 3H, CH ), 1.33 (s, 3H, H-29), 1.41 (s, 3H, H-27), 1.45–1.47 (m, 1H, H-22),
3
1
.49–1.51 (m, 1H, H-7’), 1.52–1.55 (m, 1H, H-6’), 1.57–1.59 (m, 1H, H-6), 1.65 (dd, J = 13.2, 4.0 Hz, 1H,
H-19’), 1.68–1.70 (m, 1H, H-2’), 1.71–1.73 (m, 1H, H-7), 1.78–1.80 (m, 1H, H-2), 1.87 (ddd, J = 14.8, 14.8,
.0 Hz, 1H, H-16), 1.89–1.91 (m, 1H, H-21), 1.92–1.93 (m, 1H, H-15), 1.94 (s, 3H, SeCH ), 2.15–2.18 (m,
6
3
1
H, H-18), 2.43 (s, 1H, H-9), 2.52–2.54 (m, 1H, H-19), 2.56–2.59 (m, 2H, SeCH ), 2.64 (ddd, J = 13.2, 3.2,
2
3
.2 Hz, 1H, H-1), 4.20 (q, J = 7.2 Hz, 2H, CH2CH ), 4.47 (dd, J = 11.2, 5.2 Hz, 1H, H-3), 5.46 (s, 1H,
3
H-12), 6.63 (d, Jtrans = 16.0 Hz, 1H, H-
H, Bn-H-3 and 5), 7.81 (d, Jtrans = 16.0 Hz, 2H, H-
68.98, 166.88, 162.25, 145.49, 132.19, 129.28, 128.56, 122.06, 118.45, 81.49, 61.62, 60.59, 54.96, 53.64, 48.49,
β), 7.17 (d, J = 8.8 Hz, 2H, Bn-H-2 and 6), 7.67 (d, J = 8.8 Hz,
13
2
α); C-NMR (DMSO-d6): δ 199.92, 174.88, 173.98,
1
4
5.43, 44.36, 43.26, 41.03, 38.05, 38.04, 37.65, 36.89, 32.60, 32.33, 31.88, 31.10, 29.97, 28.59, 28.32, 28.26,
+
26.44, 26.37, 23.59, 23.35, 18.75, 17.38, 16.77, 16.46, 15.54, 14.48; ESI-MS: m/z = 846.32 [M + Na] . Anal. Calcd.
for C H ClNO Se (823.39): C, 64.29; H, 7.74; N, 1.63%. Found: C, 64.25; H, 7.77; N, 1.59%.
46
66
7

Molecules 2016, 21, 199
16 of 20
Isoferulic acid methyl ester 3β-(l-methionine)-11-oxo-olean-12-en-30-oate hydrochloride (49). Obtained from
˝
1
3
(
1
3
3
as a white solid (143 mg, 75%); m.p. 199–201 C; H-NMR (DMSO-d ):
δ
0.71–0.74 (m, 1H, H-5), 0.84
s, 3H, H-28), 0.85 (s, 3H, H-24), 0.85 (s, 3H, H-23), 0.91–0.94 (m, 1H, H-15’), 1.00–1.03 (m, 1H, H-1’),
.08 (s, 3H, H-26), 1.08 (s, 3H, H-25), 1.12–1.14 (m, 1H, H-16’), 1.20–1.24 (m, 2H, H-22’ and 21’), 1.33 (s,
H, H-29), 1.41 (s, 3H, H-27), 1.46 (m, 1H, H-22), 1.50 (m, 1H, H-7’), 1.52 (m, 1H, H-6’), 1.55 (m, 1H,
H-6), 1.64 (dd, 1H, J = 13.2, 4.0 Hz, H-19’), 1.68 (m, 1H, H-2’), 1.71 (m, 1H, H-7), 1.80 (m, 1H, H-2), 1.87
ddd, 1H, J = 14.8, 14.8, 5.6 Hz, H-16), 1.92 (m, 1H, H-21), 1.95 (m, 1H, H-15), 2.04 (s, 3H, SCH ), 2.28 (m,
6
(
3
1
H, H-18), 2.43 (s, 1H, H-9), 2.54 (m, 1H, H-19), 2.57 (m, 2H, SCH ), 2.64 (ddd, 1H, J = 12.8, 3.2, 3.2 Hz,
2
H-1), 3.72 (s, 3H, COOCH ), 3.82 (s, 3H, OCH ), 4.47 (dd, 1H, J = 11.2, 5.2 Hz, H-3), 5.48 (s, 1H, H-12),
3
3
6
.60 (d, 1H, Jtrans = 16.0 Hz, H-
β
), 7.18 (d, 1H, J = 8.4 Hz, Bn-H-3), 7.57 (d, 1H, J = 2.0 Hz, Bn-H-6), 7.62
13
(dd, 1H, J = 8.4, 2.0 Hz, Bn-H-4), 7.63 (d, 1H, Jtrans = 16.0 Hz, H-
α
); C-NMR (DMSO-d6):
δ 200.03,
1
6
3
74.45, 172.81, 169.03, 166.97, 152.83, 143.64, 138.11, 128.42, 127.43, 127.38, 121.70, 116.21, 112.29, 81.49,
1.60, 55.71, 54.89, 52.80, 51.55, 47.91, 45.32, 44.39, 43.03, 41.13, 38.95, 38.89, 37.84, 36.90, 32.63, 32.15,
1.84, 31.21, 29.63, 28.51, 28.35, 28.12, 26.35, 26.29, 23.59, 23.15, 18.67, 17.40, 16.73, 16.27, 15.41; ESI-MS:
+
m/z = 814.21 [M + Na] . Anal. Calcd. for C H ClNO S (791.44): C, 66.68; H, 8.03; N, 1.69; S, 3.87%.
46
66
8
Found: C, 66.62; H, 8.08; N, 1.66; S, 3.91%.
Isoferulic acid methyl ester 3 -(l-selenomethionine)-11-oxo-olean-12-en-30-oate hydrochloride (50).
Obtained from 34 as a white solid (158 mg, 78%); m.p. 201–203 C; H-NMR (DMSO-d ):
β
˝
1
δ
0.71–0.74
m, 1H, H-5), 0.84 (s, 3H, H-28), 0.85 (s, 3H, H-24), 0.85 (s, 3H, H-23), 0.91–0.94 (m, 1H, H-15’), 1.00–1.03
m, 1H, H-1’), 1.08 (s, 3H, H-26), 1.08 (s, 3H, H-25), 1.13–1.15 (m, 1H, H-16’), 1.20–1.24 (m, 2H, H-22’
6
(
(
and 21’), 1.33 (s, 3H, H-29), 1.41 (s, 3H, H-27), 1.46 (m, 1H, H-22), 1.50 (m, 1H, H-7’), 1.52 (m, 1H, H-6’),
1
1
.55 (m, 1H, H-6), 1.64 (dd, 1H, J = 13.2, 4.0 Hz, H-19’), 1.68 (m, 1H, H-2’), 1.71 (m, 1H, H-7), 1.80 (m,
H, H-2), 1.86 (ddd, 1H, J = 14.8, 14.8, 5.6 Hz, H-16), 1.89 (m, 1H, H-21), 1.92 (m, 1H, H-15), 1.93 (s,
3
H, SeCH ), 2.28 (m, 1H, H-18), 2.43 (s, 1H, H-9), 2.55 (m, 1H, H-19), 2.58 (m, 2H, SeCH ), 2.64 (ddd,
3 2
1
H, J = 12.8, 3.2, 3.2 Hz, H-1), 3.72 (s, 3H, COOCH ), 3.82 (s, 3H, OCH ), 4.47 (dd, 1H, J = 11.2, 5.2 Hz,
3
3
H-3), 5.48 (s, 1H, H-12), 6.60 (d, 1H, Jtrans = 16.0 Hz, H-
β
), 7.18 (d, 1H, J = 8.4 Hz, Bn-H-3), 7.57 (d, 1H,
13
J = 2.0 Hz, Bn-H-6), 7.62 (dd, 1H, J = 8.4, 2.0 Hz, Bn-H-4), 7.63 (d, 1H, Jtrans = 16.0 Hz, H-
α
); C-NMR
200.05, 174.43, 172.75, 169.11, 166.97, 152.85, 143.64, 138.13, 128.43, 127.43, 127.38, 121.70,
16.21, 112.26, 81.42, 61.56, 55.76, 54.91, 52.76, 51.53, 47.96, 45.31, 44.45, 43.06, 41.14, 39.00, 38.85, 37.86,
(
1
DMSO-d6): δ
3
6.91, 32.65, 32.15, 31.85, 31.26, 29.66, 28.52, 28.33, 28.13, 26.38, 26.30, 23.54, 23.14, 18.70, 17.33, 16.74,
+
16.30, 15.40; ESI-MS: m/z = 840.28 [M + H] . Anal. Calcd. for C H ClNO Se (839.39): C, 63.11; H,
46
66
8
7
.60; N, 1.60%. Found: C, 63.05; H, 7.64; N, 1.53%.
Isoferulic acid ethyl ester 3
as a white solid (144 mg, 74%); m.p. 192–195 C; H-NMR (DMSO-d ):
β
-(l-methionine)-11-oxo-olean-12-en-30-oate hydrochloride (51). Obtained from 35
˝
1
δ
0.71–0.74 (m, 1H, H-5), 0.84
6
(s, 3H, H-28), 0.85 (s, 3H, H-24), 0.85 (s, 3H, H-23), 0.91–0.94 (m, 1H, H-15’), 1.00–1.04 (m, 1H, H-1’),
1.08 (s, 3H, H-26), 1.08 (s, 3H, H-25), 1.13–1.15 (m, 1H, H-16’), 1.18–1.21 (m, 2H, H-22’ and 21’), 1.26
(
t, 3H, J = 7.2 Hz, CH ), 1.33 (s, 3H, H-29), 1.41 (s, 3H, H-27), 1.46 (m, 1H, H-22), 1.50 (m, 1H, H-7’),
3
1
1
1
.52 (m, 1H, H-6’), 1.54 (m, 1H, H-6), 1.64 (dd, 1H, J = 13.2, 4.0 Hz, H-19’), 1.68 (m, 1H, H-2’), 1.71 (m,
H, H-7), 1.80 (m, 1H, H-2), 1.86 (ddd, 1H, J = 14.4, 14.4, 5.6 Hz, H-16), 1.89 (m, 1H, H-21), 1.92 (m,
H, H-15), 2.04 (s, 3H, SCH ), 2.29 (m, 1H, H-18), 2.43 (s, 1H, H-9), 2.53 (m, 1H, H-19), 2.56 (m, 2H,
3
SCH ), 2.64 (ddd, 1H, J = 13.6, 3.6, 3.6 Hz, H-1), 3.82 (s, 3H, OCH ), 4.18 (q, 2H, J = 7.2 Hz, CH2CH3),
2
3
4.47 (dd, 1H, J = 11.6, 4.8 Hz, H-3), 5.48 (s, 1H, H-12), 6.59 (d, 1H, Jtrans = 16.0 Hz, H-
β), 7.18 (d, 1H,
J = 8.4 Hz, Bn-H-3), 7.59 (d, 1H, J = 2.0 Hz, Bn-H-6), 7.61 (dd, 1H, J = 8.4, 2.0 Hz, Bn-H-4), 7.62 (d, 1H,
13
Jtrans = 16.0 Hz, H-
α
); C-NMR (DMSO-d6):
δ 200.21, 174.46, 172.85, 169.25, 166.90, 152.80, 143.61,
1
4
2
38.08, 128.40, 127.41, 127.38, 121.65, 116.17, 112.26, 81.35, 61.65, 60.65, 55.70, 54.87, 53.47, 47.96, 45.27,
4.27, 43.08, 41.17, 38.99, 38.85, 37.80, 36.87, 32.61, 32.18, 31.80, 31.20, 29.70, 28.47, 28.42, 28.17, 26.37,
+
6.30, 23.64, 23.13, 18.72, 17.38, 16.70, 16.34, 15.43, 14.23; ESI-MS: m/z = 828.33 [M + Na] . Anal. Calcd.
for C H ClNO S (805.46): C, 67.00; H, 8.13; N, 1.66; S, 3.81%. Found: C, 66.96; H, 8.15; N, 1.61; S, 3.84%.
4
7
68
8

Molecules 2016, 21, 199
17 of 20
Isoferulic acid ethyl ester 3β-(l-selenomethionine)-11-oxo-olean-12-en-30-oate hydrochloride (52). Obtained from
˝
1
3
6
as a white solid (154 mg, 75%); m.p. 196–198 C; H-NMR (DMSO-d ):
δ
0.71–0.75 (m, 1H, H-5),
6
0.84 (s, 3H, H-28), 0.85 (s, 3H, H-24), 0.85 (s, 3H, H-23), 0.91–0.93 (m, 1H, H-15’), 1.00–1.04 (m, 1H,
H-1’), 1.08 (s, 3H, H-26), 1.08 (s, 3H, H-25), 1.13–1.15 (m, 1H, H-16’), 1.18–1.21 (m, 2H, H-22’ and 21’),
1
1
1
1
.26 (t, 3H, J = 7.2 Hz, CH ), 1.33 (s, 3H, H-29), 1.41 (s, 3H, H-27), 1.46 (m, 1H, H-22), 1.50 (m, 1H, H-7’),
.52 (m, 1H, H-6’), 1.54 (m, 1H, H-6), 1.64 (dd, 1H, J = 13.2, 4.0 Hz, H-19’), 1.68 (m, 1H, H-2’), 1.70 (m,
H, H-7), 1.80 (m, 1H, H-2), 1.86 (ddd, 1H, J = 14.4, 14.4, 5.6 Hz, H-16), 1.89 (m, 1H, H-21), 1.92 (m,
3
H, H-15), 1.94 (s, 3H, SeCH ), 2.30 (m, 1H, H-18), 2.43 (s, 1H, H-9), 2.55 (m, 1H, H-19), 2.58 (m, 2H,
3
SeCH ), 2.64 (ddd, 1H, J = 13.6, 3.6, 3.6 Hz, H-1), 3.82 (s, 3H, OCH ), 4.18 (q, 2H, J = 7.2 Hz, CH2CH3),
2
3
4.47 (dd, 1H, J = 11.6, 4.8 Hz, H-3), 5.48 (s, 1H, H-12), 6.59 (d, 1H, Jtrans = 16.0 Hz, H-
β), 7.18 (d, 1H,
J = 8.4 Hz, Bn-H-3), 7.59 (d, 1H, J = 2.0 Hz, Bn-H-6), 7.61 (dd, 1H, J = 8.4, 2.0 Hz, Bn-H-4), 7.62 (d, 1H,
13
J
trans = 16.0 Hz, H-
α
); C-NMR (DMSO-d6):
δ 200.24, 174.41, 172.87, 169.20, 166.96, 152.83, 143.62,
1
4
2
38.08, 128.43, 127.41, 127.36, 121.65, 116.20, 112.25, 81.33, 61.65, 60.65, 55.72, 54.91, 53.45, 47.94, 45.27,
4.27, 43.06, 41.18, 38.96, 38.86, 37.82, 36.88, 32.61, 32.15, 31.83, 31.23, 29.68, 28.48, 28.45, 28.15, 26.39,
+
6.30, 23.60, 23.15, 18.74, 17.42, 16.75, 16.37, 15.47, 14.25; ESI-MS: m/z = 854.24 [M + H] . Anal. Calcd.
for C H ClNO Se (853.40): C, 63.47; H, 7.71; N, 1.57%. Found: C, 63.44; H, 7.74; N, 1.52%.
47
68
8
trans-3-Hydroxycinnamic acid methyl ester 3
β-(l-methionine)-11-oxo-olean-12-en-30-oate hydrochloride (53).
˝
1
Obtained from 37 as a white solid (145 mg, 79%); m.p. 190–192 C; H-NMR (DMSO-d ):
δ
0.72–0.74
m, 1H, H-5), 0.84 (s, 3H, H-28), 0.85 (s, 3H, H-24), 0.85 (s, 3H, H-23), 0.91–0.93 (m, 1H, H-15’), 1.00–1.04
m, 1H, H-1’), 1.06 (s, 3H, H-26), 1.07 (s, 3H, H-25), 1.12–1.14 (m, 1H, H-16’), 1.17–1.20 (m, 2H, H-22’
6
(
(
and 21’), 1.34 (s, 3H, H-29), 1.41 (s, 3H, H-27), 1.45 (m, 1H, H-22), 1.49 (m, 1H, H-7’), 1.52 (m, 1H, H-6’),
1
1
.54 (m, 1H, H-6), 1.65 (dd, 1H, J = 13.2, 4.0 Hz, H-19’), 1.68 (m, 1H, H-2’), 1.70 (m, 1H, H-7), 1.79 (m,
H, H-2), 1.86 (ddd, 1H, J = 15.2, 15.2, 6.4 Hz, H-16), 1.94 (m, 1H, H-21), 1.97 (m, 1H, H-15), 2.03 (s,
3
H, SCH ), 2.18 (m, 1H, H-18), 2.43 (s, 1H, H-9), 2.52 (m, 1H, H-19), 2.54 (m, 2H, SCH ), 2.63 (ddd, 1H,
3 2
J = 12.8, 2.8, 2.8 Hz, H-1), 3.74 (s, 3H, COOCH ), 4.47 (dd, 1H, J = 10.8, 5.6 Hz, H-3), 5.48 (s, 1H, H-12),
3
6
7
.74 (d, 1H, Jtrans = 16.0 Hz, H-
.54 (s, 1H, Bn-H-2), 7.65 (d, 1H, J = 7.6 Hz, Bn-H-4), 7.71 (d, 1H, Jtrans = 16.0 Hz, H-
199.80, 174.87, 173.94, 168.82, 167.07, 151.16, 143.69, 135.91, 129.83, 128.57, 125.59, 123.26,
20.65, 118.94, 81.50, 61.61, 54.92, 53.63, 51.76, 48.36, 45.32, 44.28, 43.15, 40.94, 38.57, 38.07, 37.72, 36.84,
β), 7.15 (dd, 1H, J = 8.0, 2.0 Hz, Bn-H-6), 7.48 (t, 1H, J = 8.0 Hz, Bn-H-5),
13
α
); C-NMR
(DMSO-d6): δ
1
3
2.59, 32.41, 31.88, 31.07, 29.95, 28.57, 28.26, 28.16, 26.49, 26.32, 23.61, 23.33, 18.61, 17.28, 16.75, 16.31,
+
1
1
5.54; ESI-MS: m/z = 762.27 [M + H] . Anal. Calcd. for C H ClNO S (761.43): C, 67.69; H, 8.08; N,
.75; S, 4.02%. Found: C, 67.64; H, 8.13; N, 1.72; S, 4.10%.
45
64
7
trans-3-Hydroxycinnamic acid methyl ester 3β-(l-selenomethionine)-11-oxo-olean-12-en-30-oate hydro-chloride (54).
˝
1
Obtained from 38 as a white solid (148 mg, 76%); m.p. 194–196 C; H-NMR (DMSO-d ):
(
(
δ
0.72–0.74
m, 1H, H-5), 0.84 (s, 3H, H-28), 0.85 (s, 3H, H-24), 0.85 (s, 3H, H-23), 0.90–0.93 (m, 1H, H-15’), 1.00–1.03
m, 1H, H-1’), 1.06 (s, 3H, H-26), 1.07 (s, 3H, H-25), 1.12–1.14 (m, 1H, H-16’), 1.17–1.20 (m, 2H, H-22’
6
and 21’), 1.34 (s, 3H, H-29), 1.41 (s, 3H, H-27), 1.44 (m, 1H, H-22), 1.49 (m, 1H, H-7’), 1.52 (m, 1H, H-6’),
1
1
.54 (m, 1H, H-6), 1.65 (dd, 1H, J = 13.2, 4.0 Hz, H-19’), 1.68 (m, 1H, H-2’), 1.70 (m, 1H, H-7), 1.75 (m,
H, H-2), 1.86 (ddd, 1H, J = 15.2, 15.2, 6.4 Hz, H-16), 1.89 (m, 1H, H-21), 1.91 (m, 1H, H-15), 1.93 (s, 3H,
SeCH ), 2.18 (m, 1H, H-18), 2.43 (s, 1H, H-9), 2.55 (m, 1H, H-19), 2.60 (m, 2H, SeCH ), 2.63 (ddd, 1H,
3
2
J = 12.8, 2.8, 2.8 Hz, H-1), 3.74 (s, 3H, COOCH ), 4.47 (dd, 1H, J = 10.8, 5.6 Hz, H-3), 5.49 (s, 1H, H-12),
3
6
7
.74 (d, 1H, Jtrans = 16.0 Hz, H-
.54 (s, 1H, Bn-H-2), 7.65 (d, 1H, J = 7.6 Hz, Bn-H-4), 7.71 (d, 1H, Jtrans = 16.0 Hz, H-
199.82, 174.87, 173.89, 168.78, 167.09, 151.12, 143.63, 136.03, 129.88, 128.55, 125.53, 123.25,
20.67, 118.97, 81.56, 61.61, 54.93, 53.60, 51.76, 48.37, 45.32, 44.26, 43.16, 40.95, 38.59, 38.07, 37.76, 36.81,
β), 7.15 (dd, 1H, J = 8.0, 2.0 Hz, Bn-H-6), 7.48 (t, 1H, J = 8.0 Hz, Bn-H-5),
13
α
); C-NMR
(DMSO-d6): δ
1
3
2.63, 32.43, 31.88, 31.13, 29.94, 28.58, 28.27, 28.18, 26.45, 26.33, 23.66, 23.34, 18.68, 17.25, 16.79, 16.34,
+
1
5.57; ESI-MS: m/z = 832.18 [M + Na] . Anal. Calcd. for C H ClNO Se (809.38): C, 63.93; H, 7.63; N,
45
64
7
1
.66%. Found: C, 63.90; H, 7.66; N, 1.63%.

Molecules 2016, 21, 199
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trans-3-Hydroxycinnamic acid ethyl ester 3
Obtained from 39 as a white solid (136 mg, 73%); m.p. 183–185 C; H-NMR (DMSO-d ):
β
-(l-methionine)-11-oxo-olean-12-en-30-oate hydrochloride(55).
˝
1
δ
0.72–0.74 (m,
6
1
1
1
H, H-5), 0.84 (s, 3H, H-28), 0.85 (s, 3H, H-24), 0.85 (s, 3H, H-23), 0.91–0.93 (m, 1H, H-15’), 1.00–1.04 (m,
H, H-1’), 1.07 (s, 3H, H-26), 1.08 (s, 3H, H-25), 1.12–1.14 (m, 1H, H-16’), 1.17–1.20 (m, 2H, H-22’ and 21’),
.27 (t, 3H, J = 7.2 Hz, CH3), 1.34 (s, 3H, H-29), 1.42 (s, 3H, H-27), 1.45 (m, 1H, H-22), 1.49 (m, 1H, H-7’),
1
1
.52 (m, 1H, H-6’), 1.55 (m, 1H, H-6), 1.65 (dd, 1H, J = 13.2, 4.0 Hz, H-19’), 1.68 (m, 1H, H-2’), 1.70 (m,
H, H-7), 1.79 (m, 1H, H-2), 1.86 (ddd, 1H, J = 14.8, 14.8, 6.0 Hz, H-16), 1.95 (m, 1H, H-21), 1.98 (m, 1H,
H-15), 2.04 (s, 3H, SCH ), 2.19 (m, 1H, H-18), 2.43 (s, 1H, H-9), 2.52 (m, 1H, H-19), 2.56 (m, 2H, SCH ),
3
2
2
.64 (ddd, 1H, J = 13.2, 3.2, 3.2 Hz, H-1), 4.20 (q, 2H, J = 7.2 Hz, CH2CH ), 4.48 (dd, 1H, J = 11.2, 5.2 Hz,
3
H-3), 5.49 (s, 1H, H-12), 6.74 (d, 1H, Jtrans = 16.0 Hz, H-β), 7.15 (dd, 1H, J = 8.0, 2.0 Hz, Bn-H-6), 7.48
(
t, 1H, J = 8.0 Hz, Bn-H-5), 7.56 (s, 1H, Bn-H-2), 7.64 (d, 1H, J = 7.6 Hz, Bn-H-4), 7.70 (d, 1H, Jrans = 16.0 Hz
,
13
H-α); C-NMR (DMSO-d6): δ 200.03, 174.81, 172.05, 169.08, 167.02, 151.03, 143.58, 135.87, 129.79,
128.49, 125.58, 123.17, 120.60, 118.79, 81.48, 61.73, 60.61, 54.92, 53.65, 48.36, 45.43, 44.21, 43.16, 40.97,
3
8.58, 38.12, 37.66, 36.87, 32.55, 32.36, 31.85, 31.17, 29.98, 28.57, 28.35, 28.19, 26.41, 26.42, 23.79, 23.33,
+
18.76, 17.37, 16.75, 16.48, 15.58, 14.51; ESI-MS: m/z = 776.34 [M + H] . Anal. Calcd. for C H ClNO S
46
66
7
(775.45): C, 68.00; H, 8.19; N, 1.72; S, 3.95%. Found: C, 67.97; H, 8.22; N, 1.68; S, 4.01%.
trans-3-Hydroxycinnamic acid ethyl ester 3β-(l-selenomethionine)-11-oxo-olean-12-en-30-oate hydrochloride (56).
˝
1
Obtained from 40 as a white solid (149 mg, 75%); m.p. 191–194 C; H-NMR (DMSO-d ):
(
(
δ
0.72–0.75
m, 1H, H-5), 0.84 (s, 3H, H-28), 0.85 (s, 3H, H-24), 0.85 (s, 3H, H-23), 0.91–0.93 (m, 1H, H-15’), 1.01–1.04
m, 1H, H-1’), 1.07 (s, 3H, H-26), 1.08 (s, 3H, H-25), 1.13–1.15 (m, 1H, H-16’), 1.17–1.21 (m, 2H, H-22’
6
and 21’), 1.27 (t, 3H, J = 7.2 Hz, CH ), 1.35 (s, 3H, H-29), 1.42 (s, 3H, H-27), 1.45 (m, 1H, H-22), 1.48
3
(m, 1H, H-7’), 1.52 (m, 1H, H-6’), 1.57 (m, 1H, H-6), 1.65 (dd, 1H, J = 13.2, 4.0 Hz, H-19’), 1.68 (m,
1
1
1
H, H-2’), 1.71 (m, 1H, H-7), 1.76 (m, 1H, H-2), 1.86 (ddd, 1H, J = 14.8, 14.8, 6.0 Hz, H-16), 1.89 (m,
H, H-21), 1.91 (m, 1H, H-15), 1.94 (s, 3H, SeCH ), 2.19 (m, 1H, H-18), 2.44 (s, 1H, H-9), 2.54 (m,
3
H, H-19), 2.59 (m, 2H, SeCH ), 2.64 (ddd, 1H, J = 13.2, 3.2, 3.2 Hz, H-1), 4.20 (q, 2H, J = 7.2 Hz,
2
C
H2CH ), 4.48 (dd, 1H, J = 11.2, 5.2 Hz, H-3), 5.49 (s, 1H, H-12), 6.74 (d, 1H, Jtrans = 16.0 Hz, H-
β),
3
7
.15 (dd, 1H, J = 8.0, 2.0 Hz, Bn-H-6), 7.48 (t, 1H, J = 8.0 Hz, Bn-H-5), 7.56 (s, 1H, Bn-H-2), 7.65 (d, 1H,
13
J = 8.0 Hz), 7.69 (d, 1H, Jtrans = 16.0 Hz, H-
α
); C-NMR (DMSO-d6):
δ
200.02, 174.82, 171.96, 169.03,
81.44, 61.75, 60.58, 54.95,
3.62, 48.38, 45.38, 44.23, 43.19, 40.98, 38.57, 38.09, 37.65, 36.89, 32.58, 32.33, 31.84, 31.13, 29.96, 28.63,
8.40, 28.13, 26.46, 26.47, 23.65, 23.30, 18.72, 17.39, 16.78, 16.45, 15.60, 14.53; ESI-MS: m/z = 824.24 [M + H]+
Anal. Calcd. for C H ClNO Se (823.39): C, 64.29; H, 7.74; N, 1.63%. Found: C, 64.25; H, 7.77; N, 1.58%.
167.05, 151.08, 143.60, 135.91, 129.72, 128.54, 125.49, 123.13, 120.62, 118.78,
5
2
.
4
6
66
7
3
.7. Anticancer Assays
The cytotoxicities of the compounds were evaluated by the MTT assay, which is based on the
conversion of MTT to formazan crystals by mitochondrial dehydrogenases. The three lines of cells
(
0
MCF-7, MDA-MB-231 and hTERT-RPE1) were cultured in 96 well plates for 24 h with a density of
4
.8
ˆ
10 cells/well, afterwards treated them with varying concentrations of GA, doxorubicin or the
˝
derivatives for 24 h at 37 C. The medium was incubated with 20
µL of 5 mg/mL MTT solution for
3
h in a humidified incubator containing 5% CO . The purple coloured formazan crystals formed in
2
the wells were dissolved in DMSO and their absorbances were measured at 570 nm with a microplate
reader. Cell viability was expressed as a percentage of the value in control cultures.
4
. Conclusions
In summary, forty novel glycyrrhetinic acid derivates were designed, synthesized and evaluated
for their inhibitory activities as novel anticancer agents against two human breast cancer cell lines
MCF-7 and MDA-MB-231) and one normal human retinal pigment epithelial cell line (hTERT-RPE1).
(
Most of the prepared compounds displayed moderate cytotoxic activity against both cancer cell
lines and relatively lower inhibitory activity against the normal cell line. In particular, compound 42

Molecules 2016, 21, 199
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exhibited good antiproliferative activity, with IC50 values of 1.88
˘
0.20 and 1.37
˘
0.18
µ
M. The results
demonstrated that compound 42 could be a potential antitumor agent that deserves further research.
Our SAR study disclosed that incorporation of a lipophilic fragment or amino acid groups into
an anticancer natural product could increase the activity, compared with the parent compound.
Further studies in this area are in progress in our laboratory and will be reported in the future.
Acknowledgments: This work was supported by the National Natural Sciences Foundation of China (Grant No.
2
1403127) and Natural Science Foundation of Shandong Province of China (Grant No. ZR2014BQ015).
Author Contributions: Qi-Yong Huai and Yang Li designed the research. Synthesis of the compounds was
performed by Yang Li and Hai-Bei Li. The anticancer activity was finished by Ling Feng. Zhi-Fang Song contributed
in the spectral analysis of the compounds. Yang Li and Hai-Bei Li wrote the manuscript. All authors read and
approved the final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 17–56 are available from the authors.
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2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons by Attribution
CC-BY) license (http://creativecommons.org/licenses/by/4.0/).
(