New phenylpropanoid-conjugated pentacyclic triterpenoids from the whole plants of Leptopus lolonum with their antiproliferative activities on cancer cells

Article abstract of DOI:10.1016/j.bioorg.2021.104628

Most of Euphorbiaceae plants are considered as folk medicinal plants because of their various pharmacological effects. However, there are eight Leptopus genus plants which belong to Euphorbiaceae have never be investigated. Thus, four Leptopus genus plant

Full text of DOI:10.1016/j.bioorg.2021.104628

Bioorganic Chemistry 107 (2021) 104628
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
New phenylpropanoid-conjugated pentacyclic triterpenoids from the whole
plants of Leptopus lolonum with their antiproliferative activities on
cancer cells
,
,
,
,
,b
Shi-Zhou Qia b, Ting Liua b, Miao Wanga b, Xin-Xin Zhanga b, Yi-Ren Yanga, Wen-Hua Jinga
,
Li-Ping Longa b, Kai-Ru Songa b, Yue Jina b, Hui-Yuan Gaoa
,
,
,
,b,*
a School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China
b Key Laboratory of Structure-Based Drug Design and Discovery of Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of
China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Most of Euphorbiaceae plants are considered as folk medicinal plants because of their various pharmacological
effects. However, there are eight Leptopus genus plants which belong to Euphorbiaceae have never be investi-
gated. Thus, four Leptopus genus plants were collected to study their chemical constituents and pharmacological
activities. In the present work, the cytotoxicities of the extracts of four Leptopus genus plants were evaluated
before phytochemical experiments. And nine new phenylpropanoid-conjugated pentacyclic triterpenoids, along
with twenty-two known compounds were isolated from the whole plants of Leptopus lolonum. The structures of
these new compounds were unequivocally elucidated by HRESIMS and 1D/2D NMR data. All triterpenoids were
screened for their cytotoxicities against four cancer cell lines including HepG2, MCF-7, A549 and HeLa. Among
these isolates, the triterpenoid with a phenylpropanoid unit showed increasing cytotoxicity on cancer cells,
which suggested the importance of the phenylpropanoid moiety.
Leptopus lolonum
Euphorbiaceae
Pentacyclic triterpenoids
Phenylpropanoid
Cytotoxicity
1. Introduction
plants. All of the above indicates that understanding the medicinal ef-
ficacy and material basis of Leptopus genus plants is of great value for the
The family of Euphorbiaceae includes over 300 genus and 8000
species spread around the world, most of which are considered as me-
dicinal plants for their multiple pharmacological effects. Among these
genus, the Leptopus genus, also known as “Que-She-Mu” or “Hei-Gou-Ye”
in China, is a small perennial shrub that grows as a tree 0.5–4 m tall
which distributes in tropical and subtropical Asia [1]. There are almost
20 species plants belonging to Leptopus genus worldwide and 9 species
distribute in the southern regions of China [2]. In Chinese traditional
medicine, the leaves of Leptopus pachyphyllus are used to treat skin ulcers
and hemostasis, and the roots of Leptopus chinensis could be used to treat
diarrhea [1,3]. Few phytochemical and pharmacological investigations
on L. chinensis were reported describing the presence of bioactive com-
pounds such as triterpenoids and sterols, and some of these compounds
displayed potential anti-cancer activity [4]. However, the main chemi-
cal constituents and biological activities of L. chinensis were not fully
investigated. In addition, so far, there is no any study about the chemical
constituents and pharmacological activities of other Leptopus genus
development of medicinal plant resources. Thus, four Leptopus genus
plants including Leptopus lolonum, Leptopus chinensis, Leptopus clarkei,
Leptopus yunnanensis were collected and their cytotoxic effects were also
be evaluated. Additionally, the L. lolonum was selected to conduct
further phytochemical experiments, and nine new phenylpropanoid-
conjugated pentacyclic triterpenoids, along with twenty-two known
compounds were isolated. Biological assays were conducted using
human hepatocellular carcinoma cells (HepG2), human breast cancer
cells (MCF-7), human lung cancer cells (A549), and human cervi-
cal cancer cells (HeLa). Furthermore, the synthetic route of the
phenylpropanoid-conjugated triterpenoid was also be investigated.
Therefore, in this article, we describe the isolation, structural determi-
nation, synthetic route of this kind of triterpenoid, and evaluation of
cytotoxicities of these compounds and the extract of four Leptopus genus
plants.
* Corresponding author.
E-mail address: sypugaohy@163.com (H.-Y. Gao).
https://doi.org/10.1016/j.bioorg.2021.104628
Received 16 November 2020; Accepted 4 January 2021
Available online 7 January 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

S.-Z. Qi et al.
Bioorganic Chemistry 107 (2021) 104628
2. Materials and methods
g) was chromatographed over a silica gel column using a gradient of PE-
Acetone (from 1:0 to 0:1), and was separated into 8 fractions (Fr.11.1-
Fr.11.8). Fr.11.5 (455.3 mg) was separated by HPLC, using an gradient
solvent system 85%-95% MeOH in H2O yielded compounds 28 (4.8 mg,
tR = 41.3 min), 13 (17.0 mg, tR = 45.5 min), 21 (10.2 mg, tR = 64.2 min)
14 (9.0 mg, tR = 78.3 min) and 24 (6.4 mg, tR = 88.0 min). Fr.11.7
(305.3 mg) was separated by HPLC, using an isocratic solvent system
85% MeCN in H2O yielded compound 29 (3.1 mg, tR = 35.2 min).
2.1. General experimental procedures
Optical rotations were determined on a AUTOPOL IV automatic
polarimeter (Rudolph ResearchAnalytical, USA). IR spectra were ob-
tained with a Bruker IFS-55 Fourier transform infrared (FT-IR) spec-
trometer (Bruker, German). UV spectra were measured with Shimadzu
UV-2401A spectrophotometer (Shimadzu, Japan). NMR spectra were
obtained by Bruker AV-600 spectrometers (Bruker, German) using tet-
ramethyl silane as an internal standard. HRESIMS spectra were obtained
using a Bruker micro-TOF-Q mass spectrometer. Open-column chro-
matography was performed using silica gel (200–300 mesh, Qingdao
Marine Chemical Co., Ltd.). TLC was performed with precoated silica gel
GF254 glass plates (Qingdao Marine Chemical Co., Ltd). Preparative
HPLC was conducted on an Agela P1050 pump with Agela UV1000D UV
spectrophotometric detector at 210 and 254 nm using a YMC Pack ODS-
2.3.1. 3β-O-(trans-p-coumaroyl)-lupane-28-al-20-ol (1)
White amorphous powder; [α] + 38.5 (c 0.1, MeOH); UV (MeOH)
λ
max (log ε): 212 (2.19), 312 (2.70) nm; IR (KBr) νmax 3425, 2920, 2840,
1721, 1632, 1589, 1510 cmꢀ 1; 1H (600 MHz) and 13C NMR (150 MHz)
Table 1
The 1H NMR data of compounds 1–6 (600 MHz).
Position
1a
2b
3a
4b
5b
6b
A column column (5 μm, 20 × 250 mm).
1
1.69 m
0.84 m
1.68 o
1.51 m
4.60 dd
(10.8,
1.67 m
0.95 m
1.65 o
1.56 m
4.49 dd
(11.4,
1.67 m
0.95 m
1.65 m
1.49 m
4.60 dd
(10.8,
1.75 m
1.04 m
1.64 m
1.07 m
4.49 dd
(11.4,
1.74 m
1.00 m
1.62 o
1.57 m
4.62 dd
(11.4,
1.81 m
0.91 m
1.67 m
1.59 m
4.50 dd
(12.0,
2.2. Plant materials
2
3
Four Leptopus genus plants were purchased in august 2018 from
Yunan Yuancai Biotechnology Co., Ltd. (Kunming, Yunnan, China), and
identified by Associate Prof. Jiu-Zhi Yuan of Shenyang Pharmaceutical
University. Four voucher specimens [LL-18-08-14 (L. lolonum); LY-18-
08-14 (L. yunnanensis); LCH-18-08-27 (L. chinensis); LCL-18–08-27
(L. clarkei)] were deposited in the School of Traditional Chinese Medi-
cine, Shenyang Pharmaceutical University, Shenyang, China.
4.8)
4.8)
4.2)
5.4)
5.4)
4.8)
5
6
0.81 m
1.45 o
1.35 m
1.37 o
1.26 m
1.37 o
1.27 m
1.70 m
1.03 m
1.74 m
1.70 o
1.45 o
2.04 m
1.24 m
1.80 m
2.20 m
2.02 m
1.30 m
1.62 m
0.84 m
1.58 m
1.39 m
1.36 o
1.36 o
1.39 m
1.16 m
1.65 o
1.05 m
1.65 o
1.88 o
1.36 o
2.02 o
1.23 m
1.65 m
2.02 o
1.88 o
1.36 o
1.47 m
0.85 m
1.42 m
1.28 m
1.37 m
1.28s
0.84 m
1.56 o
1.30 m
1.48 o
1.58 m
1.49 m
1.35 m
1.56 o
1.17 m
1.74 m
1.68 o
1.16 m
1.90 m
1.23 m
1.71 m
2.04 m
1.97 m
0.85 m
1.62 o
1.39 o
1.39 o
1.24 m
1.46 o
1.35 m
1.61 m
0.76 m
1.60 m
1.39 o
1.39 o
1.25 m
1.48 m
1.14 m
1.66 o
7
9
11
1.39 m
2.3. Extraction and isolation
12
1.68 m
1.03 m
1.70 o
The dried whole plants of L. lolonum (18.0 kg) were repeatedly
extracted with 75% EtOH to give 1.96 kg crude extract. Then the crude
extract was suspended in H2O and partitioned with petroleum ether
(PE), CHCl3, EtOAc and n-BuOH to yield PE (124.5 g), CHCl3 (23.5 g),
EtOAc (48.6 g) and n-BuOH (165.1 g) fractions. The CHCl3 part was
subjected to silica gel CC eluted with a gradient of PE-EtOAc (from 100:1
to 0:1) and CH2Cl2-MeOH (from 20:1 to 0:1) to obtain 18 fractions.
Fraction 9 (1.8 g) was chromatographed over a silica gel column using a
gradient of PE-EtOAc (from 1:0 to 0:1), and was separated into 13
fractions (Fr.9.1-Fr.9.13). Furthermore, Fr.9.5 (102 mg) was purified by
HPLC, using a gradient solvent system 80%-95% MeOH in H2O over 80
min yielded compounds 6 (7.3 mg, tR = 33.2 min), 1 (15.2 mg, tR = 46.2
min), 4 (6.3 mg, tR = 59.4 min). Fr.9.7 (89.5 mg) was separated by
HPLC, using an isocratic solvent system 90% MeOH in H2O over 120 min
yielded compounds 2 (7.2 mg, tR = 35.5 min), 5 (15.9 mg, tR = 44.7
min), 17 (5.7 mg, tR = 56.4 min), 18 (8.2 mg, tR = 85.1 min). Fr.9.8
(85.0 mg) was purified by Sephadex LH-20 column chromatography
eluted with MeOH and then semipreparative HPLC using an isocratic
solvent system 85% MeOH to yield 10 (4.3 mg, tR = 45.1 min), 12 (3.9
mg, tR = 55.1 min) and 31 (3.8 mg, tR = 61.0 min). Fr.10 (1.7 g) was
subjected to the RP-18 column and eluted with MeOH-H2O (3:10 to
10:0) to afford 15 fractions (Fr.10.1-Fr.10.15). Fr.10.8 (115.3 mg) was
separated by HPLC, using a gradient solvent system 80%-95% MeOH in
13
15
1.74 m
1.83 o
1.92 m
1.38 m
1.85 m
1.03 m
1.66 o
2.04 m
1.92 m
1.28 m
1.82 o
1.29 m
0.83 s
0.88 s
0.85 s
1.00 s
0.94 s
1.70 o
1.46 o
1.41 m
1.90 m
1.25 m
1.71 m
1.88 m
1.87 m
1.29 m
1.64 m
16
1.78 m
0.88 m
1.63 m
1.80 m
1.46 o
1.36 m
1.63 m
1.14 m
0.80 s
0.82 s
0.76 s
0.90 s
0.91 s
18
19
21
22
1.66 m
23
24
25
26
27
28
0.86 s
0.88 s
0.90 s
0.90 s
0.99 s
9.60 s
0.81 s
0.86 s
0.82 s
0.83 s
0.96 s
9.59 s
0.89 s
0.91 s
0.99 s
0.88 s
1.07, s
4.34
0.82 s
0.88 s
0.85 s
1.02 s
0.95 s
3.57 dd
(10.2,
4.8)
d (10.8)
3.83
3.04 dd
(10.2,
4.8)
d (10.8)
29
30
32
2′
1.16 s
1.27 s
1.03 s
1.10 s
1.14 s
1.24 s
2.07 s
7.43
0.98 s
1.09 s
1.05 s
1.07 s
1.01 s
1.23 s
7.42
6.99
7.03
7.62
7.55
d (8.4)
6.83
d (1.8)
d (9.0)
6.83
d (1.8)
d (8.4)
6.74
d (8.4)
6.78
3′
5′
6′
H2O yielded compounds 30 (10.8 mg, tR = 38.7 min), 19 (7.8 mg, tR
=
d (8.4)
6.83
d (8.4)
6.83
d (8.4)
6.74
d (8.4)
6.78
44.6 min) and 20 (9.1 mg, tR = 58.9 min). Fr.10.9 (88.7 mg) was purified
6.75
6.99
via preparative HPLC using an isocratic solvent system of 80% MeCN in
d (8.4)
7.42
d (8.4)
6.98 dd
(8.4,1.8)
d (8.4)
7.43
d (8.4)
6.75 dd
(8.4,
d (8.4)
7.62
d (8.4)
7.55
H2O to yield compounds 9 (15.0 mg, tR = 40.2 min), 11 (3.9 mg, tR
=
d (8.4)
d (9.0)
d (8.4)
d (8.4)
46.7 min) and 3 (5.2 mg, tR = 49.5 min) and 15 (2.2 mg, tR = 53.0 min).
Fr.10.10 (106.9 mg) was purified via preparative HPLC using an iso-
cratic solvent system of 90% MeOH in H2O to yield compounds 7 (7.0
mg, tR = 30.8 min), 8 (2.9 mg, tR = 35.3 min), 23 (5.2 mg, tR = 51.1 min)
and 16 (3.2 mg, tR = 55.7 min). Fr.10.11 (272.4 mg) was purified by
HPLC, using an gradient solvent system 90%-95% MeOH in H2O to yield
compounds 22 (4.3 mg, tR = 53.2 min), 25 (5.2 mg, tR = 61.2 min), 26
(6.0 mg, tR = 82.4 min) and 27 (2.8 mg, tR = 88.0 min). Fraction 11 (2.2
1.8)
7′
8′
7.62
7.45
7.60
6.87
6.84
7.53
d (15.6)
6.29
d (15.6)
6.23
d (15.6)
6.29
d (12.6)
5.73
d (12.6)
5.77
d (15.6)
6.37
d (15.6)
d (15.6)
d (15.6)
d (12.6)
d (12.6)
d (15.6)
“o” represents the overlapped peak.
a
b
Measured in CDCl3.
Measured in DMSO‑d6.
2

S.-Z. Qi et al.
Bioorganic Chemistry 107 (2021) 104628
spectral data in CDCl3, see Table 1 and Table 2; HRESIMS: m/z 603.4037
[M ꢀ H]ꢀ (calcd for C39H56O5, 604.4128).
2.3.5. 3β-O-(cis-caffeoyl)-norlupane-17β,20-diol (5)
White amorphous powder; [ ] + 38.9 (c 0.1, MeOH); UV (MeOH)
α
λmax (log ε): 218 (2.89), 327 (2.58) nm; IR (KBr) νmax 3455, 2937, 2875,
2.3.2. 3β-O-(trans-caffeoyl)-lupane-28-al-20-ol (2)
1737, 1630, 1605, 1485 cmꢀ 1; 1H (600 MHz) and 13C NMR (150 MHz)
spectral data in DMSO‑d6, see Table 1 and Table 2; HRESIMS: m/z
591.4053 [M ꢀ H]ꢀ (calcd for C38H56O5, 592.4128).
White amorphous powder; [α] + 42.1 (c 0.1, MeOH); UV (MeOH)
λ
max (log ε): 218 (3.08), 330 (2.91) nm; IR (KBr) νmax 3468, 2904, 2861,
1752, 1634, 1602, 1521 cmꢀ 1; 1H (600 MHz) and 13C NMR (150 MHz)
spectral data in DMSO‑d6, see Table 1 and Table 2; HRESIMS: m/z
619.4032 [M ꢀ H]ꢀ (calcd for C39H56O6, 620.4077).
2.3.6. 3β-O-(trans-p-coumaroyl)-norlupane-17β-hydroperoxide-20-ol (6)
White amorphous powder; [α] + 27.5 (c 0.1, MeOH); UV (MeOH)
λ
max (log ε): 214 (3.26), 320 (2.86) nm; IR (KBr) νmax 3547, 2964, 2866,
2.3.3. 3β-O-(trans-p-coumaroyl)-lupane-28-O-acetyl-20-ol (3)
1725, 1633, 1608, 1464 cmꢀ 1; 1H (600 MHz) and 13C NMR (150 MHz)
spectral data in DMSO‑d6, see Table 1 and Table 2; HRESIMS: m/z
631.3970 [M + Na]+ (calcd for C38H56O6, 608.4077).
Light yellow amorphous powder; [α] + 45.9 (c 0.1, MeOH); UV
(MeOH) λmax (log ε): 215 (3.43), 317 (2.57) nm; IR (KBr) νmax 3389,
2940, 2880, 1724, 1640, 1604, 1567, 1502 cmꢀ 1; 1H (600 MHz) and 13
C
NMR (150 MHz) spectral data in CDCl3, see Table 1 and Table 2; HRE-
2.3.7. Ethyl 3β-O-(trans-caffeoyl)-lupane-28-oate (9)
SIMS: m/z 647.4297 [M ꢀ H]ꢀ (calcd for C41H60O6, 648.4390).
White amorphous powder; [α] + 43.7 (c 0.1, MeOH); UV (MeOH)
λmax (log ε): 213 (2.27), 317 (2.95) nm; IR (KBr) νmax 3397, 2940, 2883,
2.3.4. 3β-O-(cis-caffeoyl)-20-ol-betulin (4)
1733, 1629, 1593, 1514 cmꢀ 1; 1H (600 MHz) and 13C NMR (150 MHz)
spectral data in CDCl3, see Table 2; HRESIMS: m/z 645.4128 [M ꢀ H]ꢀ
(calcd for C41H58O6, 646.4233).
White amorphous powder; [α] + 57.5 (c 0.1, MeOH); UV (MeOH)
λ
max (log ε): 213 (3.15), 313 (2.84) nm; IR (KBr) νmax 3328, 2920, 2864,
1720, 1630, 1601, 1525 cmꢀ 1; 1H (600 MHz) and 13C NMR (150 MHz)
spectral data in DMSO‑d6, see Table 1 and Table 2; HRESIMS: m/z
621.4156 [M ꢀ H]ꢀ (calcd for C39H58O6, 622.4233).
2.3.8. 24-O-(cis-p-coumaroyl)-3β-hydroxyl-olean-12-en-28-oic acid (15)
White amorphous powder; [α] + 16.4 (c 0.1, MeOH); UV (MeOH)
λ
max (log ε): 204 (3.19), 312 (2.90) nm; IR (KBr) νmax 3360, 2913, 2887,
1723, 1634, 1601, 1501 cmꢀ 1; 1H (600 MHz) and 13C NMR (150 MHz)
spectral data in CDCl3, see Table 2; HRESIMS: m/z 617.3846 [M ꢀ H]ꢀ
(calcd for C39H54O6, 618.3920).
Table 2
The 13C NMR data of compounds 1–6 (150 MHz).
Position
2.3.9. 24-O-(cis-p-feruloyl)-3β-hydroxyl-olean-12-en-28-oic acid (16)
1a
2b
3a
4b
5b
6b
White amorphous powder; [α] + 14.3 (c 0.1, MeOH); UV (MeOH)
λ
max (log ε): 211 (3.04), 263 (2.87) nm; IR (KBr) νmax 3387, 2921, 2874,
1
38.6
38.1
38.6
38.3
38.4
38.4
1728, 1628, 1608, 1503 cmꢀ 1; 1H (600 MHz) and 13C NMR (150 MHz)
spectral data in CDCl3, see Table 2; HRESIMS: m/z 647.3932 [M ꢀ H]ꢀ
(calcd for C40H56O7, 648.4026).
2
24.0
81.0
38.2
55.5
18.4
34.8
41.5
50.5
37.2
21.5
28.4
38.4
38.4
29.4
29.7
61.8
47.2
49.6
73.8
29.3
33.2
28.2
16.9
16.5
16.4
14.7
207.2
25.1
31.4
23.5
79.8
37.6
54.5
17.8
34.0
40.8
49.6
36.6
20.9
27.3
37.9
43.1
28.8
29.0
60.7
46.3
48.9
71.2
28.3
32.9
27.7
16.5
15.8
16.0
14.4
207.7
26.1
30.9
24.0
81.0
38.3
55.0
18.4
34.6
41.7
50.3
36.8
21.5
27.4
37.2
43.6
29.2
30.5
48.1
49.0
49.8
73.7
28.5
34.2
28.2
16.9
16.4
16.4
15.2
63.1
24.9
31.9
171.9
21.3
127.7
130.1
116.0
157.6
116.0
130.1
144.0
116.7
167.4
24.0
80.3
37.0
55.0
18.3
34.4
41.5
50.1
36.4
21.4
27.4
38.1
43.4
28.2
28.4
49.0
48.7
49.0
71.9
30.1
33.5
27.9
17.1
16.3
16.4
15.5
58.9
25.4
32.2
23.8
80.4
37.9
55.3
18.2
34.0
41.2
50.7
36.5
21.6
27.2
37.1
42.3
28.2
31.1
78.7
49.7
54.0
70.8
23.4
40.5
28.1
16.9
16.8
15.8
14.5
24.0
80.3
37.1
55.1
18.3
34.6
41.3
50.3
35.3
21.6
27.5
38.1
42.8
28.2
29.2
90.6
49.3
48.3
71.8
29.4
32.9
28.0
17.1
16.7
16.4
14.6
3
4
5
6
7
2.4. Cell viability assay
8
9
The MTT assay was used to evaluate the cytotoxicities of triterpe-
noids and extracts of four Leptopus genus plants according to the pub-
lished method [5]. Four cancer cells of A549, HepG2, MCF-7 and HeLa
were cultured in DMEM medium supplemented with 10% fetal bovine
serum, 1 × penicillin–streptomycin in a humidified 37 ℃ incubator
supplied with 5% CO2, respectively. Briefly, cells were harvested with
trypsin, seeded into 96-well plates at 5000 cells/well and then incubated
for 12 h. The test samples which dissolved in DMSO were added to each
well, and cells were incubated for another 48 h. Control wells were
treated with 1% aqueous DMSO. And doxorubicin and ginsenoside Rg3
were used as two positive control. After 48 h incubation, 20 µL of MTT
(5 mg/mL) was added to each well and incubated for 4 h at 37 ℃ with
5% CO2. Next, the medium was aspirated and precipitated formazan
crystals dissolved in DMSO (150 µL/well). The absorbance of each well
was measured at 570 nm with a microplate reader (Thermo Scientific
Multiskan MK3, Shanghai, China).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
1′
29.1
31.3
27.0
31.0
2.5. General procedure for synthesizing phenylpropanoid-conjugated
triterpenoid
127.5
130.1
116.1
158.1
116.1
130.1
144.3
116.1
167.5
125.5
114.8
145.6
148.3
121.3
115.7
144.8
114.4
166.3
126.0
115.3
146.0
148.8
121.8
116.2
145.3
114.9
166.8
126.0
132.9
115.3
159.2
115.3
132.9
143.4
116.5
166.3
125.5
130.7
116.2
160.3
116.2
130.7
144.9
115.3
166.9
2′
Tanachatchairatan provided a method to synthesize this kind of tri-
terpenoid to study their antimycobacterial activities [6]. However,
during the course of hydrolyzing the acetyl group with alkaline solution,
the formed ester bond may also be hydrolyzed. In this study, the tert-
butyldimethylsilyl chloride (TBDMSCl) was used to protect PhOH and
tetrabutylammonium fluoride (TBAF) was used to remove this protec-
tive group. Briefly, 4-hydroxyl-cinnamic acid, caffeic acid or ferulic acid
and 8 eq Et3N were dissolved in anhydrous CH2Cl2 (15 mL). Then 4 eq
TBDMSCl in anhydrous CH2Cl2 (15 mL) was slowly added to the
3′
4′
5′
6′
7′
8′
9′
a
b
Measured in CDCl3.
Measured in DMSO‑d6.
3

S.-Z. Qi et al.
Bioorganic Chemistry 107 (2021) 104628
prepared cooled solution. The reaction mixture was stirred for 12 h at
room temperature, washed with 1 M HCl (2 × 30 mL) and water (30
mL), dried over MgSO4, filtered and concentrated in vacuo. And formed
brown oil was dissolved in MeOH (30 mL) and water (30 mL) before 1 eq
K2CO3 was added. This reaction mixture was stirred for another 4 h and
concentrated in vacuo to remove the MeOH. Then the EtOAc (2 × 50
mL) and concentrated HCl (4 mL, pH 0) were added and combined
organic phase was washed with brine (100 mL), dried over MgSO4,
filtered and concentrated in vacuo. The crude product was further pu-
rified by flash column chromatography (petroleum ether: EtOAc 5:1) to
afford product. Next, linking with tert-butyldimethylsilyl acid chlorides
were prepared by reacting the corresponding carboxylic acids with
oxalyl chloride at reflux for 2 h, and the remaining oxalyl chloride was
removed. The residue was dissolved in anhydrous CH2Cl2 (10 mL),
added to a solution of betulinic acid, and heated to reflux for 2 h at 60 ℃.
After the completion of the esterification and pure compound was ob-
tained, 2 eq dry TBAF (1.0 M in THF) was added to reaction mixture and
stirred for 4 h. Finally, solvent was removed and the crude product was
dissolved in CH2Cl2 (3 × 20 mL), washed with water (3 × 20 mL), dried
with anhydrous MgSO4 and solvent was removed in vacuo to afford
phenylpropanoid-conjugated triterpenoid, which was further purified
by flash chromatography on silica gel (PE: EtOAc, 5:1 to 3:1).
3. Results and discussion
3.1. Structure elucidation
Compound 1 was obtained as a white amorphous powder. The
Fig. 1. The structures of compounds H1, H2 and 1–31.
4

S.-Z. Qi et al.
Bioorganic Chemistry 107 (2021) 104628
molecular formula was assigned as C39H56O5 according to the HRESIMS
(m/z 603.4037 [M ꢀ H]ꢀ , calcd 604.4128) and 13C NMR data. The IR
spectrum showed typical absorption bands of hydroxy and carbonyl
groups at 3425 and 1721 cmꢀ 1, respectively. The 1H NMR spectrum of 1
(Table 1) exhibited resonances for seven singlet methyl protons at δH
1.27, 1.16, 0.99, 0.90, 0.90, 0.88, 0.86, an oxygenated methine proton at
δH 4.60 (1H, dd, J = 10.8, 4.8 Hz), two olefinic protons at δH 7.62 (1H, d,
J = 15.6 Hz) and 6.29 (1H, d, J = 15.6 Hz), a set of AA’XX’-type aro-
matic protons at δH 7.42 (2H, d, J = 8.4 Hz) and 6.83 (2H, d, J = 8.4 Hz),
8.4, 1.8 Hz), 6.75 (1H, d, J = 8.4 Hz) and 6.23 (1H, d, J = 15.6 Hz), and
δ 166.3, 148.3, 145.6, 144.8, 125.5, 121.3, 115.7, 114.8 and 114.4. The
C
location of trans-caffeoyl moiety was confirmed at C-3 via the HMBC
cross-peak of H-3/C-9′ (Fig. 2). The β-orientation of C-3 was also
deduced by the NOESY correlations of H-3α and H-23 (Fig. 3). Collec-
tively, the structure of compound 2 was confirmed and named 3β-O-
(trans-caffeoyl)-lupane-28-al-20-ol.
Compound 3 was isolated as a light yellow amorphous powder, and
the HRESIMS (m/z 647.4297 [M ꢀ H]ꢀ , calcd 648.4390) and 13C NMR
data suggested that it possesses a molecular formula of C41H60O6. The
lupane nature of 3 was supported by a comparison of its NMR data with
those of 1 and 2, which showed signals of protons and carbons for the
lupane triterpenoid and trans-coumaroyl moiety. However, the 1H and
13C NMR data of 3 (Tables 1 and 2) showed the presence of methyl
acetate group [δH 4.34 (1H, d, J = 10.8 Hz), 3.83 (1H, d, J = 10.8 Hz),
2.07 (3H, s); δ 171.9, 63.1 and 21.3] replacing the aldehyde group (δ
207.2) of 1. It Ccould be further confirmed by the key HMBC correlationCs
and an aldehydic proton at δ 9.60. The 13C NMR spectrum of 1
H
(Table 2) revealed 39 resonances including an aldehydic carbonyl car-
bon at δ 207.2, an ester group at δ 167.5, six aromatic and two olefinic
carbonsCat δC 116.1–158.1, an oxygCenated methine carbon at δC 81.0 and
an oxygenated quaternary carbon at δ 73.8. The comparison of its 1H
and 13C NMR data with those of luCpane-3β,20,28-triol caffeate [7]
indicated that 1 possesses the same skeleton, except for the presence of a
trans-coumaroyl moiety and an aldehyde group which observed in 1.
And the position of ester bond between the trans-coumaroyl moiety and
3,20-dihydroxyl betulinic aldehyde was determined at C-3 by the key
of δ 4.34/3.83 with δ 171.9 (C-31) and 48.1 (C-17), and of δ 2.07
H
H
with δC 171.9 (C-31) (FCig. 2). The structure of 3 was finally established
HMBC correlation of δH 4.60 (H-3) with δ 167.5 (C-9′) (Fig. 2). The 2D
as 3β-O-(trans-p-coumaroyl)-lupane-28-O-acetyl-20-ol.
C
structure of 1 was established with the aid of HSQC and HMBC spectra.
Compound 4 was purified as a white amorphous powder and the
molecular formula was assigned as C39H58O6 based on the HRESIMS ion
signal at m/z 621.4156 [M ꢀ H]ꢀ . The 1H and 13C NMR spectra (Tables 1
and 2) of 4 were very similar to those of 2, with the difference being the
replacement of trans-olefinic protons with a large coupling constant [δH
7.45 (1H, d, J = 15.6 Hz), 7.45 (1H, d, J = 15.6 Hz)] by cis-olefinic
protons with a smaller coupling constant [δH 6.87 (1H, d, J = 12.6 Hz),
5.73 (1H, d, J = 12.6 Hz)]. The position of cis-caffeoyl moiety was
determined to be C-3 by the HMBC correlations of H-3/C-9′ (Fig. 2).
Consequently, the structure of compound 4 was defined as shown and
named 3β-O-(cis-caffeoyl)-lupane-20,28-diol.
The β-orientation of C-3 was confirmed by the strong NOESY correla-
tions of H-3α and H-23 (Fig. 3). Thus, the structure of 1 was assigned as
3β-O-(trans-p-coumaroyl)-lupane-28-al-20-ol.
Compound 2 was isolated as a white amorphous powder and
assigned the molecular formula C39H56O6 according to the HRESIMS
(m/z 619.4032 [M ꢀ H]ꢀ , calcd 620.4077) and 13C NMR data. The 1H
and 13C NMR spectra (Tables 1 and 2) of 2 showed a close resemblance
to those of 1, suggesting that 2 was 3,20-dihydroxyl betulinic aldehyde
with a trans-caffeoyl moiety. And it could be deduced by the NMR data at
δ 7.45 (1H, d, J = 15.6 Hz), 6.99 (1H, d, J = 1.8 Hz), 6.98 (1H, dd, J =
H
HO
HO
HO
O
O
CHO
CHO
O
O
O
O
O
O
HO
HO
HO
HO
OH
1
2
3
HO
HO
HO
OH
OH
OOH
O
OH
OH
O
O
O
HO
O
O
4
5
6
COOH
COOH
O
HO
O
HO
O
O
O
O
O
O
HO
OH
H3CO
OH
OH
9
15
16
Fig. 2. Key HMBC correlations of compounds 1–6, 9, 15 and 16.
5

S.-Z. Qi et al.
Bioorganic Chemistry 107 (2021) 104628
9
9
3
27
5
3
5
27
23
23
1
2
3
9
5
27
23
9
3
27
5
23
9
15
Fig. 3. Key NOESY correlations of compounds 1, 2, 9 and 15.
The molecular formula of compound 5 was deduced as C38H56O5
based on HRESIMS ([M ꢀ H]ꢀ , m/z 591.4053), again corresponding to a
lupane skeleton. However, several noticeable differences were observed
in the NMR data (Tables 1 and 2) of 5 and 1. The most conspicuous
differences in their NMR signals were the replacement of cis-olefinic
group [δH (1.27, t, 7.2 Hz); δ 14.4], and the absence of an oxygenated
quaternary carbon at δ 71.2Cin 9. Combined with the HMBC spectrum
C
(Fig. 2) could confirmed the exist of ethyl methanoate which linked with
C-17. Thus, the structure of compound 9 was assigned as Ethyl 3β-O-
(trans-caffeoyl)-lupane-28-oate.
protons [δ 6.84 (1H, d, J = 12.6 Hz), 5.77 (1H, d, J = 12.6 Hz)] and the
Compound 15 was obtained as a white amorphous powder, and the
molecular formula C39H54O6 was calculated from the [M ꢀ H]ꢀ ion at
m/z 617.3846 in its HRESIMS. Examination of the 1H and 13C NMR data
(Table 3) of 15 suggested that this compound shares close structural
similarities with (23E)-feruloylhederagenin, except that the trans-fer-
uloyl moiety was replaced by a cis-p-coumaroyl moiety [10]. The long
H
presence of an oxygenated quaternary at δC 78.7 in 5. The structure of
28-norlupane skeleton was deduced by the HMBC (Fig. 2) and the
comparison of its NMR data with those of 28-norlup-20(29)-ene-3β,17β-
diol [8]. The β-orientation of C-17 can be deduced by the chemical shift
(δC 37.1) of C-13 (17β: about 38 ppm; 17α: about 43 ppm) according the
published study [8]. Thus, the structure of 5 was consequently estab-
lished as 3β-O-(cis-p-coumaroyl)-norlupane-17β,20-diol.
range HMBC correlations (Fig. 2) of 15 between H-24 (δ 4.63 and 4.25)
H
with δC 167.4 (C-9′) and several key NOESY correlations (H-23/H-3; H-
3/H-5) (Fig. 3) suggested that the trans-p-coumaroyl moiety attached to
C-24 and relative stereochemistry of C-3 was in β-oriented. Hence,
compound 15 was established as 24-O-(cis-p-coumaroyl)-3β-hydroxyl-
olean-12-en-28-oic acid.
Compound 6 has the molecular formula C38H56O6 as determined by
HRESIMS data ([M + Na]+, m/z 631.3970). The 1H and 13C NMR data
(Tables 1 and 2) of 6 suggested its close similarity to the known 28-
norlup-20(29)-en-3-hydroxy-17-hydroperoxide [9], with an additional
trans-p-coumaroyl moiety. The 13C NMR spectrum of 6 showed a qua-
ternary carbon signal at δC 90.6, assigned to C-17 based on HMBC data.
Similarly with those of 5, the relative configuration of C-17 in 7 could
also be determined by 13C NMR chemical shifts because of the presence
of γ-substituent effect. The β-configuration of C-17 in 6 was deduced by
the chemical shift value of its C-13 (δC 38.1) and C-19 (δC 48.3) [9]. The
location of this additional trans-p-coumaroyl group was confirmed at C-3
by the HMBC correlations of δH 4.50 with 166.9 (C-9′) (Fig. 2). There-
fore, the structure of 6 was determined as 3β-O-(trans-p-coumaroyl)-
norlupane-17β-hydroperoxide-20-ol.
Compound 16 showed an [M ꢀ H]ꢀ ion at m/z 647.3932 (calcd for
C40H56O7, 648.4026) in the HRESIMS spectrum, corresponding to the
molecular formula C40H56O7. The NMR data (Table 3) of 16 were very
similar with 15, suggested that compound 16 possesses the same skel-
eton as that of 15. And the major difference was the presence of a trans-
feruloyl group replacing the cis-p-coumaroyl group. The location of the
trans-feruloyl moiety was assigned to C-24 due to the key HMBC cor-
relations of δH 4.37/4.18 with δC 166.4 (C-9′) (Fig. 2). The β-orientation
of C-24 was deduced from the comparison of NMR data between 16 and
15 and the same biogenetic relationship. Thus, the structure of 16 was
established as 24-O-(cis-feruloyl)-3β-hydroxyl-olean-12-en-28-oic acid.
Structure elucidation of known compounds was carried out by a
combination of spectroscopic means and comparison with references.
The compounds were determined to be monogynol A (7) [11], 3β-O-
(trans-caffeoyl)-20-ol-betulin (8) [11], 3β-O-trans-coumaroylbetulinic
acid (10) [12], 3β-O-(cis-coumaroyl)-betulinic acid (11) [13], 3β-O-
Compound 9 was obtained as a white amorphous powder with the
identical molecular formula C41H58O6 from its HRESIMS showing a
deprotonated ion at m/z 645.4128 [M ꢀ H]ꢀ (calcd for C41H58O6,
646.4233). The 1H and 13C NMR data (Table 3) of 9 were found to be
closely comparable to those of 2, but there were slight shifts in the
presence of an oxygenated methine (δH 4.17; δC 59.9) and a methyl
6

S.-Z. Qi et al.
Bioorganic Chemistry 107 (2021) 104628
methylcholest-4-ene-6
α
-ol-3-one (22) [18], 24-ethylcholest-4-ene-6β-
Table 3
The 1H NMR and 13C NMR data of compounds 9, 15 and 16 in CDCl3 (1H: 600
ol-3-one (23) [18], 11β-hydroxy-ergost-4-en-3-one (24) [19], 7-oxo-
MHz, 13C: 150 MHz).
Position
β-sitosterol (25) [20], 7-oxocampesterol (26) [21], (3α, 24R)-3-
hydroxyergost-5-en-7-one (27) [21], Stigmasta-4,22-dien-6β-ol-3-one
(28) [22], 7-Ketostigmasterol (29) [23], ergosterol peroxide (30) [24],
9,11-dehydroergosterol peroxide (31) [24] (Fig. 1).
9
15
16
δH (J in Hz)
δC
38.3
δH (J in Hz)
δC
38.7
δH (J in Hz)
δC
38.5
1
2
3
1.70 m
1.01 m
1.68 o
1.58 m
1.66 m
0.99 m
1.77 o
0.98 m
3.2. The synthetic route of the phenylpropanoid-conjugated triterpenoid.
23.8
81.2
1.62 m
22.9
80.6
23.0
79.4
1.17 m
1.08 m
3.28 dd
(12.0, 4.2)
4.60 dd
3.44 dd
(12.0, 4.8)
Three common phenylpropionic acid were found to be randomly
attach to triterpenoid skeleton. In order to systematically study the
structure–activity relationships, further structural modification, and
pharmaceutical effect of this kind of compounds, an efficient synthetic
route has been researched as shown in Fig. 7, and compounds H1, H2
were obtained.
(10.8, 6.0)
4
5
6
38.1
55.4
18.2
42.5
56.4
18.9
42.2
56.1
18.9
0.82 m
1.51 m
1.40 o
1.37 m
1.00 m
1.42 m
1.34 m
1.32 m
1.22 m
0.88 m
1.57 o
1.33 m
1.42 m
1.25 m
7
34.3
33.3
33.0
8
40.8
50.5
37.1
20.9
39.4
47.6
37.3
23.8
39.2
47.7
36.9
23.5
3.3. The cytotoxicities of four Leptopus genus plants and the characteristic
triterpenoids of L. lolonum
9
1.31 m
1.57 m
1.88 m
5.30 brs
1.57 o
1.89 m
10
11
1.42 m
1.27 m
1.70 m
1.03 m
1.69 o
Cytotoxicities of four Leptopus genus plants toward four cancer cell
lines A549, HepG2, MCF-7 and HeLa were examined. Results (Fig. 4,
Table S1) showed that PE, CHCl3 fractions displayed significant cyto-
toxicities on four cancer cells, and CHCl3 fractions showed different
12
25.5
123.0
5.28 brt
(3.6)
122.5
13
14
15
38.4
42.4
29.6
143.7
41.2
27.2
143.5
41.1
27.1
1.40 o
1.15 m
2.22 m
1.39 o
1.70 o
1.09 m
1.70 o
1.76 o
1.08 m
2.00 m
1.63 m
level of cytotoxicities with IC50 values of 30.19 to 77.92 μg/mL. The very
weak cytotoxicities of n-BuOH fractions (Table S1) of four plants were
observed. And this phenomenon might be related with the attachment of
phenylpropanoid at C-3 and C-24 of triterpenoid skeleton leading to the
less triterpenoid saponins. Considering the timeliness of chemical
isolation and the plant’s Euphorbiaceae background, the constituents of
CHCl3 fraction of L. lolonum were first investigated. As a result, a ser-
ies of compounds including triterpenoids and sterols were isolated and
most triterpenoids were found to possess trans or cis p-coumaroyl, caf-
feoyl and feruloyl moieties at C-3 or C-24 of oleanene or lupane-type
triterpenoid skeleton. Moreover, the major feature of the isolated
lupane-type triterpenoids is that the double bond (C-20/29) are oxidized
and a hydroxyl group is attached to C-20. This is the first time to report
the main bioactive constituents of L. lolonum and the abundant presence
of 20-hydroxyl-lupane triterpenoids.
16
32.2
27.9
27.6
17
18
56.4
49.4
46.7
41.5
46.5
41.6
1.60 m
3.03 m
2.86 dd
(13.8, 4.2)
1.59 m
2.82 dd
(13.8, 4.8)
1.57 o
19
47.0
45.9
45.9
1.21 m
1.23 o
20
21
150.6
30.6
30.9
33.9
30.7
33.8
1.88 m
1.37 m
1.26 m
1.80 m
1.64 m
1.24 s
4.63
1.33 m
1.23 o
1.80 m
1.57 m
1.13 s
4.37
22
1.89 m
1.40 o
0.88 s
0.90 s
37.1
32.7
32.4
23
24
28.0
16.2
22.7
66.3
22.4
65.4
d (11.4)
4.25
d (11.4)
4.18
d (11.4)
1.06 s
0.72 s
1.14 s
d (11.4)
0.93 s
0.74 s
1.12 s
25
26
27
28
29
0.87 s
0.93 s
0.97 s
16.7
15.9
15.9
17.6
15.6
16.8
3.4. The cytotoxicities of compounds H1, H2 and 1–20.
14.7
26.2
25.8
176.4
109.6
184.4
32.8
181.6
32.9
Compounds H1, H2 and 1–20 were also evaluated their cytotoxic-
ities on four cancer cells of HepG2, MCF-7, A549 and HeLa (Fig. 5,
Table S2). And doxorubicin and ginsenoside Rg3 were used as the pos-
itive control. All the compounds except compounds 7, 19 and 20 showed
moderate cytotoxicities on cancer cells. A preliminary structure–activity
relationship (Fig. 6) suggested that triterpenoids with a phenyl-
propanoid moiety exhibited more potent effects than those without such
a unit, which was deduced from the results that compounds 10, 11, H1,
H2, 17 and 18 showed relatively high cytotoxicities, but not compounds
14 and 19. It is evident that the binding site of the phenylpropanoid
moiety may be not play an important role in efficacy by comparing the
effects of compounds 11 (at C-3), H2 (at C-3), 15 (at C-24) and 16 (at C-
24) with similar potency. And the comparison of IC50 values of com-
pounds 8 and 13 indicated that the hydroxyl group at C-20 might play an
negative effect on the cytotoxicity of triterpenoids. In addition, com-
pound 10, with a trans-coumaroyl moiety (A), showed more potent
cytotoxicity than 11, with cis-coumaroyl moiety (B). This showed that
trans-coumaroyl moiety might be responsible for the increasing bioac-
tivity of compound 10. Taken together, phenylpropanoids in triterpe-
noids are important for their cytotoxicities on cancer cells.
4.74 s
0.92 s
0.95 s
0.91 s
0.92 s
4.60 s
30
31
32
1′
1.69 s
19.4
59.9
23.8
23.5
4.17 m
1.27 t (7.2)
14.4
127.6
114.4
144.5
146.3
115.5
122.3
126.2
130.3
116.1
159.2
116.1
130.3
127.1
112.8
146.0
147.1
113.9
125.5
2′
7.11 d (1.8)
7.01 d (7.8)
6.66 d (8.4)
7.69 d (1.2)
3′
4′
5′
6.88 d (7.8)
7.01 dd
(7.8, 1.8)
7.56
6.66 d (8.4)
7.01 d (7.8)
6.88 d (8.4)
7.12 dd
(8.4, 1.2)
6.80
6′
7′
8′
144.5
116.3
167.6
7.46
145.2
114.5
167.4
144.5
116.4
d (15.6)
6.27
d (15.6)
5.91
d (13.2)
5.78
d (15.6)
d (16.2)
d (13.2)
9′
166.4
56.0
OCH3
3.92 s
“o” represents the overlapped peak.
coumaroylbetulin (12) [14], 3β-O-caffeoylbetulin (13) [14], betulinic
acid (14) [14], 3β-O-(trans-caffeoyl)-betulinic acid (H1) [15], 3β-O-
trans-feruloylbetulinic acid (H2) [14], 3β-(p-hydroxy-trans-cinnamoy-
loxy)olean-12-en-28-oic acid (17) [16], 3-O-caffeoyloleanolic acid (18)
[13], oleanolic acid (19) [13], Methyl olean-12-en-2β,3β-diol-28-oate
(20) [17], 24-methylcholest-4-ene-6β-ol-3-one (21) [18], 24-
3.5. The short discussion on phenylpropanoid-conjugated triterpenoids
To our knowledge, about 200 phenylpropanoid-conjugated penta-
cyclic triterpenoids have been reported until now. And they displayed a
7

S.-Z. Qi et al.
Bioorganic Chemistry 107 (2021) 104628
Fig. 4. The cytotoxicities of four Leptopus genus plants on four cancer cells. #: IC50 > 300 μg/mL.
Fig. 5. The cytotoxicities of compounds 1–6, 9, 10, 15 and 16. #: IC50 > 100 μM.
Fig. 6. The structure–activity relationships of phenylpropanoid-conjugated triterpenoid.
8

S.-Z. Qi et al.
Bioorganic Chemistry 107 (2021) 104628
O
O
O
Cl
C2Cl2O2
OH
Et3N, TBDMSCl
CH2Cl2, 0 rt, 18 h
OH
TBDMSO
CH2Cl2, reflux
HO
R
-
TBDMSO
R
R
Betulinic acid, Et3N
CH2Cl2, reflux, 60
COOH
COOH
TBAF, THF
rt, 4 h
O
O
O
O
TBDMSO
HO
R
R
Fig. 7. The synthetic route of the phenylpropanoid-conjugated triterpenoid.
variety of biological activities including anti-inflammatory, antioxidant,
anticancer, anti-hyperglycaemic, anti-ischemic stroke, antiosteoclasto-
genesis, anti-HIV as well as neuroprotective activity [25-28]. In most
published articles, the significant increasing pharmacological activities
are observed when phenylpropanoid moiety attached to triterpenoid
skeleton. In addition, it is worth noting that this triterpenoid exists in
some edible fruits such as apple, pear, actinidia arguta and jujube, and
play a key role in respective reported bioactivity [13,29-31]. These
findings showed that phenylpropanoid-conjugated triterpenoid might
has potential possibility of being lead compound because of the less drug
toxicity. Moreover, in the study of finding quality-marker ingredients of
Panax quinquefolius [32], metabolomics analysis revealed the presence
of phenylpropanoid-conjugated triterpenoids in the blood of mice. It
indicated that this kind of triterpenoid could enter the blood to exert
their effects and they have certain medicinal properties. Nevertheless,
there is not much research to report the detailed mechanisms of anti-
cancer in vitro and in vivo. Thus, more pharmacological investigations
about phenylpropanoid-conjugated triterpenoids should be carried out.
reported in this paper.
Acknowledgments
This study was supported by National Natural Science Foundation of
China (No. 31670359), Liaoning Revitalization Talents Program (No.
XLYC 1905019) and Natural Science Foundation of Liaoning Province
(No. 201602691).
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.104628.
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