Daphnane-type diterpenoid glucosides and further constituents of euphorbia pilosa

Article abstract of DOI:10.1002/cbdv.201300154

Phytochemical investigation of whole plants of Euphorbia pilosa led to the isolation and identification of two new daphnane-diterpenoid glucosides, euphopilosides A and B (1 and 2, resp.), and a new ent-abietane, euphopilolide (3), together with eight known compounds. Compounds 1 and 2 are the first daphnane-type diterpenoid glycosides. Their structures were elucidated by a combination of 1D- and 2D-NMR, and MS analyses, and acid hydrolysis. Compounds 1-9 were evaluated for their in vitro cytotoxicities against five human tumor cell lines, HL-60, SMMC-7721, A-549, MCF-7, and SW-480. Compound 7 showed moderate inhibitory activity against all five cell lines.

Full text of DOI:10.1002/cbdv.201300154

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CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
Daphnane-Type Diterpenoid Glucosides and Further Constituents of
Euphorbia pilosa
by Xu-Dong Zhanga)b), Wei Ni a), Huan Yana), Gen-Tao Lia), Hui-Min Zhongb), Yan Li a),
and Hai-Yang Liu*a)
a) State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of
Botany, Chinese Academy of Sciences, Kunming 650201, P. R. China
(phone: þ86-871-65223246; fax: þ86-871-65223245; e-mail: haiyangliu@mail.kib.ac.cn)
b) College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology,
Qingdao 266042, P. R. China
Phytochemical investigation of whole plants of Euphorbia pilosa led to the isolation and
identification of two new daphnane-diterpenoid glucosides, euphopilosides A and B (1 and 2, resp.),
and a new ent-abietane, euphopilolide (3), together with eight known compounds. Compounds 1 and 2
are the first daphnane-type diterpenoid glycosides. Their structures were elucidated by a combination of
1D- and 2D-NMR, and MS analyses, and acid hydrolysis. Compounds 1–9 were evaluated for their in
vitro cytotoxicities against five human tumor cell lines, HL-60, SMMC-7721, A-549, MCF-7, and SW-480.
Compound 7 showed moderate inhibitory activity against all five cell lines.
Introduction. – The genus Euphorbia is the largest in the spurge family, comprising
more than 2000 species [1]. Some species of Euphorbia have been used as medicinal
plants for the treatment of skin diseases, gonorrhea, migraine, and intestinal parasites,
and as wart cures [2]. More than 550 compounds including sesquiterpenoids,
diterpenoids, triterpenoids, steroids, cerebrosides and glycerols, phenolics, flavonoids,
and miscellaneous compounds have been reported from the Euphorbia species [3].
Diterpenoids (more than 446 compounds) are the characteristic compounds of the
genus with many different core frameworks such as jatrophanes, lathyranes, tiglianes,
daphnanes, ingenanes, and myrsinols, and they possess many biological features such as
antiproliferative activity, cytotoxicity, effects on the cell division, and DNA-damaging,
PEP-inhibitory, and anti-HIV-1 activities [3].
Euphorbia pilosa L. is a perennial, erect herb and grows in subalpine meadow,
grassland, or forest edge [4]. Its chemical composition has not been reported. As part of
our ongoing efforts to discover new bioactive constituents from Euphorbia species [5],
the AcOEt-soluble extract of E. pilosa was investigated, resulting in the isolation of two
new daphnane-type diterpenoid glucosides, named euphopilosides A and B (1 and 2,
resp.), and a new ent-abietane, named euphopilolide (3), together with eight known
compounds, langduin A (4) [6], phorbol (5) [7], jokiolide A (6) [6], jokiolide B (7) [6],
ent-atis-16-ene-3,14-dione (8) [8], 13b-hydroxy-ent-abiet-8(14)-en-7-one (9) [9],
annphenone (10) [10], and obtusifoliol (11) [11]. Componds 1 and 2 are the first
daphnane-type diterpenoid glycosides. Herein, we report the isolation and structure
elucidation of the new compounds. In addition, compounds 1–9 were evaluated for
their cytotoxicities against five human tumor cell lines.
ꢀ 2014 Verlag Helvetica Chimica Acta AG, Zꢁrich

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
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Results and Discussion. – Euphopiloside A (1), isolated as a white amorphous
powder, displayed a [MþNa]þ ion peak at m/z 533.2370 (calc. 533.2362) in the HR-
ESI-MS, which, combined with 13C-NMR data (Table 1), provided the molecular
formula C26H38O10, requiring eight degrees of unsaturation. The IR spectrum indicated
the presence of OH (3426 cmꢀ1) and (1698 cmꢀ1) C¼O groups, and C¼C bond
(1629 cmꢀ1). The 1H-NMR spectrum of 1 (Table 1) exhibited signals for four Me group
(d(H) 1.02 (d, J¼6.9), 1.23 (d, J¼6.9), 1.27 (d, J¼6.8), and 1.67 (s)), two trisubstituted
olefinic H-atom (d(H) 5.92 (d, J¼5.4) and 7.75 (br. s)), and an anomeric H-atom (d(H)
4.90 (d, J¼7.8 )) . The 13C-NMR and DEPT spectra (Table 1) revealed the presence of
two C¼O groups (d(C) 213.0 and 208.9), two trisubstituted C¼C bonds (d(C) 131.0,
135.2, 139.2, and 157.9), four Me, three CH2 (one oxygenated one), and five CH groups,
and two oxygenated quaternary C-atoms (d(C) 74.6 and 76.7), as well as of a
glucopyranosyl moiety (d(C) 104.4, 75.1, 78.7, 71.9, 78.6, and 63.0). Acid hydrolysis of 1
yielded langduin A (4) and d-glucose as sugar residue, which was identified by GC
analysis of its trimethylsilated l-cysteine adduct. The b-configuration of glucopranosyl
was determined by the coupling constant (3J(1,2)>7) of the anomeric H-atom.
Comparison of the NMR data of 1 (Table 1) with those of langduin A (4) [6] confirmed
that compound 1 was a glucoside derivative of 4. This conclusion was verified by 1H,1H-
COSY, HMBC, HSQC, and ROESY experiments (Fig.). The HMBC between the
signals at d(H) 4.90 (HꢀC(1’)) and d(C) 75.6 (C(20)) implied that the glucopyranosyl

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CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
Table 1. 1H- and 13C-NMR Data for Compounds 1 and 2 (d in ppm, J in Hz). Atom numbering as
indicated in the Formulae.
Position
1a)
2b)
d(H)
d(C)
d(H)
7.55 (s)
d(C)
1
2
3
4
5
7.75 (br. s)
157.9
135.2
208.9
74.6
160.0
135.2
209.2
72.6
3.16 (br. s, 2 H)
38.8
2.63 (d, J¼19.0),
2.34 (d, J¼19.0)
38.4
6
7
8
139.2
131.0
46.0
76.7
58.7
37.8
48.0
133.6
133.8
53.3
84.7
51.2
5.92 (d, J¼5.4)
4.33–4.35 (m)
5.48 (br. s)
2.77 (br. s)
9
10
11
12
3.66 (br. s)
3.25–3.29 (m)
3.43 (br. s)
2.30–2.32 (m)
42.6
36.7
2.71 (dd, J¼7.9, 14.6),
2.20 (dd, J¼9.0, 11.9),
2.54 (dd, J¼7.5, 14.6)
1.26 (dd, J¼9.0, 13.9)
13
14
15
16
17
18
19
20
213.0
55.9
29.2
22.1
17.9
18.9
10.5
75.6
110.9
57.3
32.0
21.8
19.6
20.7
2.89 (dd, J¼2.7, 11.9)
2.11–2.15 (m)
1.23 (d, J¼6.9)
1.02 (d, J¼6.9)
1.27 (d, J¼6.8)
1.67 (s)
1.59–1.61 (m)
1.57–1.59 (m)
1.00 (d, J¼5.5)
0.98 (d, J¼5.5)
0.92 (d, J¼6.7)
1.75 (s)
4.17 (d, J¼11.2),
4.08 (d, J¼11.2)
3.44 (s)
4.27 (d, J¼7.8)
3.15–3.17 (m)
3.19–3.21 (m)
3.26–3.29 (m)
3.30–3.35 (m)
3.83 (d, J¼11.9),
3.65 (dd, J¼5.3, 11.9)
8.81
76.0
4.58 (d, J¼11.7),
4.29 (d, J¼11.7)
MeO
1’
2’
3’
4’
50.5
101.2
73.6
70.0
73.6
76.5
61.0
4.90 (d, J¼7.8)
3.97–4.00 (m)
3.90–3.93 (m)
4.16–4.18 (m)
4.18–4.20 (m)
4.55–4.58 (m),
4.33 (dd, J¼6.0, 12.0)
104.4
75.1
78.7
71.9
78.6
63.0
5’
6’
a) Recorded in C5D5N, at 500 and 125 MHz for 1H and 13C, respectively. b) Recorded in CD3OD, at 400
and 100 MHz for 1H and 13C, respectively.
was attached at C(20) of langduin A (4). The downfield shift exhibited by C(20) (d(C)
75.6) compared to that of langduin A (4) (þ6.4) corroborated this deduction. Based on
these results, the structure of compound 1 was elucidated as 4b,9a,20-trihydroxy-13,15-
secotiglia-1,6-diene-3,13-dione 20-O-b-d-glucopyranoside. This is the first daphnane-
type diterpenoid glycoside.
Euphopiloside B (2) had a molecular formula C27H40O10 deduced from HR-ESI-MS
(m/z 547.2522 ([MþNa]þ ; calc. 547.2519)) and 13C-NMR data (Table 1). Comparison
of the 1H- and 13C-NMR data indicated that 2 differed from 1 by the presence of a ketal
(d(C) 110.9) and a MeO group (d(H) 3.44; d(C) 50.5) instead of a C¼O group (d(C)
213.0) moiety at C(13) in 1. The HMBC of the C(13) signal (d(C) 110.9) with those of

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
763
1
Figure. Key H,1H-COSY (—) and HMB (H!C) correlations of 1–3
CH2(12) (d(H) 1.26 (dd, J¼9.0, 13.9) and 2.20 (dd, J¼9.0, 11.9)), HꢀC(14) (d(H)
1.59–1.61 (m)), HꢀC(15) (d(H) 1.57–1.59 (m)), and MeO (d(H) 3.44) indicated the
location of the ketal group at C(13) (Fig.). ROESY Correlation of the MeO signal
(d(H) 3.44) with those of HꢀC(8) (d(H) 2.77 (br. s)), HꢀC(11) (d(H) 2.30–2.32 (m)),
HꢀC(15) (d(H) 1.57–1.59 (m)), Me(16) (d(H) 1.00 (d, J¼5.5)), and Me(17) (d(H)
0.98 (d, J¼5.5)) evidenced that the MeO group at C(12) was b-oriented. Therefore, the
structure of 2 was established as 9a,13a-epoxy-4b,20-dihydroxy-13b-methoxy-13,15-
secotiglia-1,6-diene-3-one 20-O-b-d-glucopyranoside.
Euphopilolide (3) gave rise to [MþNa]þ ion peak in the HR-ESI-MS at m/z
339.1932, corresponding to the molecular formula C20H28O3. The UV and IR spectra
displayed absorptions due to an a,b-unsaturated-g-lactone (moiety 222 nm, and 1754
1
and 1683 cmꢀ1, resp.). The H-NMR spectrum (Table 2) exhibited signals for three
tertiary Me groups (d(H) 0.88, 0.92, and 1.05), one olefinic Me group (d(H) 1.95), two
CHꢀO groups (d(H) 3.75 and 4.97–5.01). The 13C-NMR spectrum of 3 (Table 2)
displayed signals of a C¼O group (d(C) 173.9), one tetrasubstituted C¼C bond (d(C)
Table 2. 1H- and 13C-NMR (in CDCl3, at 400 and 125 MHz, resp.) Data for Compound 3 (d in ppm,
J in Hz)
Position
1
d(H)
d(C)
Position
11
d(H)
d(C)
1.45–1.47 (m),
41.3
2.27 (dd, J¼5.5, 13.3),
1.35–1.37 (m)
4.97–5.01 (m)
23.7
1.18 (dd, J¼6.1, 12.5)
1.52–1.56 (m, 2 H)
1.92–1.94 (m),
2
3
18.5
40.8
12
13
75.6
155.8
1.06–1.09 (m)
4
5
6
33.2
54.0
20.9
14
15
16
3.75 (s)
56.0
128.5
173.9
1.01–1.03 (m)
1.74 (dd, J¼2.9, 11.3),
1.41–1.43 (m)
7
1.96–1.99 (m),
34.7
17
1.95 (s)
8.7
1.63 (d, J¼13.8)
8
9
10
61.0
49.1
39.2
18
19
20
0.92 (s)
0.88 (s)
1.05 (s)
34.0
22.0
19.2
1.94–1.96 (m)

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CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
128.5 and 155.8), four Me, six CH2, and four CH groups (two oxygenated ones at d(C)
56.0 and 75.6), and three quaternary C-atoms (one oxygenated one at d(C) 61.0). These
data are very similar to those of jolkinolide A (6) except for the presence of one more
CHꢀO (d(C) 75.6) and CH2 group (d(C) 23.7) instead of a trisubstituted C¼C bond
between C(11) and C(12). This was supported by the correlations from the signal of
CH2(11) (d(H) 1.35–1.37 (m) and 2.27 (dd, J¼5.5, 13.3)) to that of HꢀC(12) (d(H)
4.97–5.01 (m)) in the 1H,1H-COSY spectrum (Fig.) and the long-range correlations of
the signal of C(12) with HꢀC(9) (d(H) 1.94–1.96 (m)), CH2(11) (d(H) 1.35–1.37 (m)
and 2.27 (dd, J¼5.5, 13.3)), those of HꢀC(14) (d(H) 3.75 (s)), and Me(17) (d(H) 1.95
(s)) in the HMBC spectrum (Fig.). The HꢀC(12) was a-oriented on the basis of
ROESY correlation of HꢀC(12) (d(H) 4.97–5.01 (m)) with Me(20) (d(H) 1.05 (s)).
Thus, the structure of compound 3 was elucidated as 8b,14b-epoxy-ent-abiet-13(15)-en-
16,12b-olide.
Euphopilosides A and B (1 and 2, resp.) are the first daphnane-type diterpenoid
glycosides. In 2011, we reported the first tigliane-type glycosides, fischerosides A–C,
isolated from Euphorbia fischeriana [5]. Interestingly, they had the same glucosylation
position, i.e., C(20). Compounds 1–9 were evaluated for their cytotoxic activities
against five human cancer cell lines (HL-60, SMMC-7721, A-549, MCF-7, and SW-480)
using the MTT (¼ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide)
assay, and cisplatin was used as the positive control. Only jokiolide B (7) showed
cytotoxic activities against the five cell lines with IC50 values of 4.18ꢁ0.18, 6.06ꢁ0.27,
4.12ꢁ0.34, 17.64ꢁ0.98, and 11.40ꢁ0.08 mm, respectively.
Experiment Part
General. TLC: SiO2 plates; detection by UV254 lamp and 10% H2SO4 in EtOH, followed by heating.
Column chromatography (CC): silica gel (SiO2, 200–300 mesh; Qingdao Marine Chemical Inc., P. R.
China), RP-18 (40–63 mm, Merck), MCI GEL CHP20P (75–150 mm, Mitsubishi Chemical Co.), and
Sephadex LH-20 (25–100 mm, GE Healthcare). GC: HP5890 Gas chromatograph; H2 flame ionization
detector. Optical rotations: JASCO P-1020 digital polarimeter. UV Spectra: Shimadzu UV-2401PC
spectrophotometer, MeOH solns.; lmax (log e) in nm. IR Spectra: Bruker Tensor-27 infrared
spectrophotometer, KBr pellets; ˜n in cmꢀ1. NMR Spectra: Bruker AM-400 and DRX-500 instruments;
d in ppm rel. to Me4Si as internal standard, J in Hz. ESI- and HR-ESI-MS: API QSTAR time-of-flight
(TOF) spectrometer.
Plant Material. Whole plants of E. pilosa were collected in June 2010 in Panshi, Jilin Province, P. R.
China, and identified by Dr. En-De Liu of Kunming Institute of Botany. A voucher specimen
(No. HY00011) was deposited with the State Key Laboratory of Phytochemistry and Plant Resources in
West China.
Extraction and Isolation. The dried and powered material (10 kg) was extracted with 95% EtOH
(25 lꢂ3) under reflux for a total of 6 h to give a crude extract. After removal of the solvent in vacuo, the
residue was suspended in H2O (10 l) and successively extracted with AcOEt (4ꢂ10 l). The solvent was
removed, and the AcOEt extract (1.17 kg) was subjected to CC (SiO2; petroleum ether (PE)/AcOEt
1:0!0 :1) to give four fractions, Fr. A–D. Fr. B (68 g) was subjected to MPLC (SiO2; PE/acetone
(500 :1!50 :1) to give Subfr. B1 and B2. And then Fr. B2 was subjeced to MPLC (RP-18 gel MeOH/H2O
40%!70%) to give two combined fractions, B21 –B22. Fr. B21 was purified by CC (SiO2 PE/aectone 25 :1)
to afford 3 (5.1 mg), 6 (5.1 mg), 7 (3.1 mg), and 8 (2.7 mg). Compounds 4 (1.6 mg), 5 (3.6 mg), 9
(2.0 mg), and 11 (500 mg) were obtained after crystallization from PE/aectone from the Fr. B22. Fr. D
(86 g) was separated by CC (MCI GEL CHP20P; MeOH/H2O 40%!70%) to give Fr. D1 and D2. Fr. D1
was submitted to CC (SiO2; CHCl3/MeOH 40 :1!25 :1) and purified by prep. TLC (CHCl3/MeOH

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
765
20 :1) to give 1 (53 mg) and 2 (6.1 mg). Fr. D2 was subjected to CC (SiO2; CHCl3/MeOH 25 :1!10 :1) to
afford 10 (3.9 mg).
Euphopiloside A (¼ [(3aR,6aS,7S,10R,10aR,10bS)-3a,10a-3,3a,4,6a,7,8,9,10,10a,10b-decahydrodi-
hydroxy-2,10-dimethyl-3,8-dioxo-7-(propan-2-yl)benzo[e]azulen-5-yl]methyl b-d-Glucopyranoside; 1).
White amorphous powder. [a]1D4 ¼ þ38.5 (c¼0.14, MeOH). UV (MeOH): 202 (4.02). IR (KBr): 3426,
2959, 2926, 1698, 1629, 1460, 1077, 1044. 1H- and 13C-NMR: Table 1. ESI-MS (pos.): 533 [MþNa]þ . HR-
ESI-MS: 533.2370 ([MþNa]þ , C26H38NaO1þ0 ; calc. 533.2362).
Euphopiloside B (¼ [(3aR,6aS,7S,8S,10R,10aR,10bR)-3a,4,6a,7,8,9,10,10b-octahydro-3a-hydroxy-8-
methoxy-2,10-dimethyl-3-oxo-7-(propan-2-yl)-3H-8,10a-epoxybenzo[e]azulen-5-yl]methyl b-d-Gluco-
pyranoside; 2). White amorphous powder. [a]1D3 ¼ þ12.3 (c¼0.13, MeOH). UV (MeOH): 235 (3.71),
201 (3.96). IR (KBr): 3426, 2957, 2925, 1703, 1629, 1461, 1077. 1H- and 13C-NMR: Table 1. ESI-MS (pos.):
547 [MþNa]þ . HR-ESI-MS: 547.2522 ([MþNa]þ , C27H40NaO1þ0 ; calc. 547.2519).
Euphopilolide (¼(1aS,3aS,7aR,7bS,8aR,11bR)-3,3a,4,5,6,7,7a,7b,8,8a-Decahydro-4,4,7a,11-tetra-
methyl-2H-oxireno[1,10a]phenanthro[3,2-b]furan-10(11bH)-one; 3). White needle. [a]1D3 ¼ þ51.4 (c¼
0.09, CHCl3). UV (MeOH): 222 (4.12). IR (KBr): 3486, 2951, 2928, 1754, 1683, 1456, 1091, 1022. 1H- and
13C-NMR: Table 2. ESI-MS (pos.): 339 [MþNa]þ . HR-ESI-MS: 339.1932 ([MþNa]þ , C20H28NaOþ3 ;
calc. 339.1936).
Acid Hydrolysis, and GC and HPLC Analyses. Compounds 1 and 2 (2 mg) were separately
hydrolyzed with 4m CF3COOH/dioxane 1:1 (2 ml) for 4 h at 908. After extraction with AcOEt (3ꢂ
5 ml), the aq. layer was evaporated to dryness. The dried residue was dissolved in 0.5 ml of anh. pyridine
and treated with l-cysteine methyl ester hydrochloride (1.5 mg), and the mixture was stirred at 608 for
1 h. 1-(Trimethylsilyl)-1H-imidazole (0.5 ml) was added to the mixtures, which was stirred at 608 for
another 30 min. The mixture (4 ml) was analyzed by GC. d-Glucose (tR 19.01 min) was detected in the
hydrolysate of 1 and 2, and its identity was confirmed by co-injection with a derivatized authentic sample.
The AcOEt layer of compound 1 was evaporated to dryness. The residue showed the same peak at
15.23 min as that of langduin A (4) by HPLC analysis (conditions: Zorbax SB-C18, 5 mm, 46ꢂ150 mm;
solvent, MeCN/H2O 10!50% (0!40 min); flow rate, 1.0 ml/min; detection, UVabsorbance at 203 nm)
under the same conditions.
Cytotoxicity Assay. The cytotoxicity assay was performed according to the MTT method [12] by using
of the following five human cancer cell lines: human myeloid leukemia HL-60, hepatocellular carcinoma
SMMC-7721, lung cancer A-549, breast cancer MCF-7, and colon cancer SW-480. Each experiment was
repeated three times. Cisplatin was used as the positive control antitumor drug and exhibited IC50 values
for the cell lines of 3.29ꢁ0.18, 9.26ꢁ0.27, 9.98ꢁ0.34, 15.92ꢁ0.98, and 14.43ꢁ0.08 mm, resp.
This work was supported by National Natural Science Funding of China (31170333 and 81072543),
the Young Academic and Technical Leader Raising Foundation of Yunnan Province (2008PY066),
Academy-Locality Cooperation Project of Chinese Academy of Sciences (XBKM-2011-004), and funds
(P2010-ZZ02) from the State Key Laboratory of Phytochemistry and Plant Resources in West China.
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Received May 10, 2013