New diterpenoids from the roots of Euphorbia ebracteolata Hayata

Article abstract of DOI:10.1080/14786410903388017

Three new diterpenoids, ingenol-5,20-O,O-isopropylidene-3-palmitate (1), ingenol-5,20-O,O-isopropylidene-3-myristinate (2) and 3,19-dihydroxy-1(10),15- rosadien-2-one (3), were isolated from the roots of Euphorbia ebracteolata Hayata. Their structures wer

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New diterpenoids from the roots of
Euphorbia ebracteolata Hayata
a
a
a
a
Bin Deng , Shu-Zhen Mu , Jian-Xin Zhang & Xiao-Jiang Hao
a
Key Laboratory of Chemistry for Natural Products of Guizhou
Province and Chinese Academy of Sciences , Guiyang, P.R. China
Published online: 09 Sep 2010.
To cite this article: Bin Deng , Shu-Zhen Mu , Jian-Xin Zhang & Xiao-Jiang Hao (2010) New
diterpenoids from the roots of Euphorbia ebracteolata Hayata, Natural Product Research: Formerly
Natural Product Letters, 24:16, 1503-1509, DOI: 10.1080/14786410903388017
To link to this article: http://dx.doi.org/10.1080/14786410903388017
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Natural Product Research
Vol. 24, No. 16, 10 October 2010, 1503–1509
New diterpenoids from the roots of Euphorbia ebracteolata Hayata
Bin Deng, Shu-Zhen Mu, Jian-Xin Zhang and Xiao-Jiang Hao*
Key Laboratory of Chemistry for Natural Products of Guizhou Province and Chinese
Academy of Sciences, Guiyang, P.R. China
(
Received 5 October 2009; final version received 26 October 2009)
Three new diterpenoids, ingenol-5ꢀ,20-O,O-isopropylidene-3ꢀ-palmitate
(1), ingenol-5ꢀ,20-O,O-isopropylidene-3ꢀ-myristinate (2) and 3ꢀ,19-
dihydroxy-1(10),15-rosadien-2-one (3), were isolated from the roots of
Euphorbia ebracteolata Hayata. Their structures were deduced by spectro-
scopic means and analytic techniques.
Keywords: Euphorbia; Euphorbia ebracteolata Hayata; diterpenoids
1
. Introduction
Euphorbia ebracteolata Hayata is a type of perennial herb widely distributed in
China, Korea and Japan (Fu, Yu, & Zhu, 2006). The root of E. ebracteolata is a
traditional Chinese medicine to treat pulmonary tuberculosis, psoriasis, neuropathic
dermatitis and chronic bronchitis (Xie, 1996). Previous phytochemical studies have
demonstrated the presence of some terpenoids, acetophenone derivatives and
flavonoids in this plant (Yan & Zhao, 2008). Our further research has led to the
isolation of seven diterpenoids from the roots of E. ebracteolata (Deng, Mu, Huang,
Song, & Hao, 2009). As a continuation of our investigations into the chemical
constituents of this plant, three new diterpenoids, ingenol-5ꢀ,20-O,O-isopropylidene-
3
ꢀ-palmitate (1), ingenol-5ꢀ,20-O,O-isopropylidene-3ꢀ-myristinate (2) and 3ꢀ,19-
dihydroxy-1(10),15-rosadien-2-one (3), were obtained from this plant. In this article,
we report the isolation and structural elucidation of three new diterpenoids (1–3,
Figure 1).
2
. Results and discussion
Compound 1 was obtained as yellow oil. Its molecular formula was determined
þ
as C H O by (þ)-HR-ESI-MS (m/z 649.4455, [M þ Na] , Calcd 649.4444). IR
3
9
62
6
ꢀ
1
absorptions at 3525, 3435 and 1728 cm
and carbonyl groups. All of the 39 carbons observed in the C-NMR and DEPT
spectra could be classified into two trisubstituted olefins, two carbonyls, four sp
3
quaternaries, six sp methines and 16 sp methylenes, together with seven methyls, as
indicated the presence of hydroxyl
1
3
3
3
shown in Table 1. Among them, two carbonyls (ꢁ 207.5 and 174.4) were assigned as
C
*Corresponding author. Email: haoxj@mail.kib.ac.cn
ISSN 1478–6419 print/ISSN 1029–2349 online
ß 2010 Taylor & Francis
DOI: 10.1080/14786410903388017
http://www.informaworld.com

1
504
B. Deng et al.
1
7
H
17
1
5
12
15
1
3
1
2
16
1
6
1
8
O
11
10
13
14
H
1
4
20
1
1
1
14
8
O
2
9
6
2
9
8
1
9
10
3
H
7
5
4
7
3
5
6
HO
H
19
18
RO HO
20
O
HO
1″
O
2
″
3
″
1
2
R=CO(CH2)14CH3
R=CO(CH2)12CH3
3
Figure 1. Structures of compounds 1–3.
1
13
3
Table 1. H-NMR (400 MHz) and C-NMR (100 MHz) data of compounds 1–2 in CDCl .
1
2
Position
ꢁ
C
ꢁ
H
[J (Hz)]
ꢁ
C
H
ꢁ [J (Hz)]
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
132.3
136.4
81.7
84.1
73.9
135.7
122.2
43.6
207.5
72.2
37.7
31.1
23.4
22.9
24.1
28.5
15.6
17.4
16.7
6.04 (s)
5.54 (s)
4.00 (s)
5.78 (s)
132.2
136.4
81.7
84.1
73.8
135.7
122.2
43.6
207.5
72.1
37.7
31.0
23.4
22.9
24.2
28.5
15.6
17.3
15.7
6.04 (s)
5.54 (s)
3.99 (s)
5.78 (s)
4.14 (m)
4.14 (m)
1
1
1
1
1
1
1
1
1
1
2
2.56 (m)
2.56 (m)
2.18 (m), 1.79 (m)
0.72 (dd, 8.4, 14.8)
0.86 (dd, 6.4, 13.2)
2.18 (m), 1.76 (m)
0.72 (dd, 8.4, 14.8)
0.86 (dd, 6.4, 13.2)
1.05 (s)
1.09 (s)
0.99 (d, 7.2)
1.76 (s)
4.23 (m)
1.05 (s)
1.09 (s)
0.99 (d, 6.8)
1.76 (s)
4.23 (m)
0
64.3
64.3
0
1
174.4
34.7
25.2
29.7–29.4
29.3
29.2
31.9
22.7
14.1
174.8
34.7
25.1
29.6–29.1
31.9
22.7
0
0
0
2
3
4
2.37 (m)
1.66 (m)
1.26 (s)
1.26 (s)
1.26 (s)
1.24 (m)
1.26 (m)
0.86 (m)
2.39 (m)
1.68 (m)
1.26 (s)
1.24 (m)
1.26 (m)
0.86 (m)
0
–11
0
1
1
1
1
1
2
3
4
5
0
0
0
0
0
0
0
14.1
6
0
0
0
1
100.3
20.7
26.8
100.3
20.7
26.8
2
1.41 (s)
1.46 (s)
1.41 (s)
1.46 (s)
3

Natural Product Research
1505
a ketone carbonyl and an ester carbonyl, respectively. One methylene (ꢁ 64.3,
C
ꢁH 4.23), one methine (ꢁ 73.9, ꢁ 4.00) and two quaternary carbons (ꢁ 84.1 and
C
H
C
1
00.3) were ascribed to be bearing oxygen atoms. The H-NMR spectrum of 1
1
revealed a broad singlet at ꢁ 1.26 (20H, s), which indicated the presence of a
H
long-chain fatty acid moiety. The chemical shifts at ꢁ 29.3–29.7 and 174.4 further
C
confirmed the above deduction.
Comparison of the NMR data of 1 with those of the known compound,
ingenol-3-myristinate, from Euphorbia wallichii (Li & Suo, 2005) suggested that 1
possessed an ingenane-type skeleton. The main difference between them was the
presence of three extra carbons at ꢁ 100.3 (s), 26.8 (q), and 20.7 (q) in 1 and their
C
1
1
molecular formulae. Further analysis of 1D-NMR, H– H COSY and HMBC
spectra, as shown in Figure 2, indicated that one acetonyl unit was connected to the
C-5 and C-20 via two oxygen atoms, respectively, on the basis of HMBC correlations
0
0
00
00
of H-20 (ꢁ 4.23, 2H, m), H-2 (ꢁ 1.41, 3H, s) and H-3 (ꢁ 1.46, 3H, s) to C-1 , and
H
H
H
00
00
of H-5 (ꢁ 4.00, 1H, s) to C-20, C-2 and C-3 (Figure 2). The location of the
H
long-chain fatty acid moiety at C-3 was determined by the HMBC cross-peaks from
0
H-3 (ꢁ 5.54, 1H, s) and C-1 (ꢁ 174.4). To further confirm the long-chain fatty acid
H
C
moiety, the basic hydrolysis of compound 1 was carried to obtain the free long-chain
fatty acid, which was determined as palmitic acid by a main peak in its GC-MS
spectrum. Thus, the planar structure of 1 was deduced as ingenol-5,20-O,O-
isopropylidene-3-palmitate.
The relative configuration of 1 was established by the analysis of its ROESY
spectrum, as shown in Figure 2. Correlations of H-1–H-3, H -18–H -19, H-3–H-5
3
3
0
0
00
and H-5–H -2 indicated that H-1, H-3, H-5 and H -2 were in ꢂ-orientation, while
3
3
0
0
correlations of H-8–H-7, H-7–H-11, H-11–H-17, H-17–H -20 and H -20–H -3
3
2
2
0
0
indicated that H-7, H-8, H -17, H -20 and H -3 were in ꢀ-orientation, which
3
2
3
consisted of with the structural characters of ingenane-type diterpenoids. Thus, the
structure of 1 was deduced as ingenol-5ꢀ, 20-O,O-isopropylidene-3ꢀ-palmitate.
1
13
Compound 2 was obtained as yellow oil. The H-NMR, C-NMR and DEPT
data of 2 (Table 1) were almost the same as those of 1, indicating that their structures
should be very similar. However, the positive ESI-MS and negative ESI-MS spectra
1
7
17
15
H
H
1
3
13
1
2
12
16
1
5
1
6
1
8
O
18
2
O
1
4
H
14
H
1
1
1
1
11
10
9
8
9
H
2
8
1
9
10
7
19
4
7
3
H
O
5
O
3
6
4
6
5
HO
HO
O
20
C
O
20
C
1′
3
′
3′
1
′
1
4
′
O
14
′
O
1
2
′
2′
(
H C)
1
″
O
(H C)
10
″
O
2
10
2
1
6
′
16′
1
5
′
15′
2
″
2″
3
″
3″
1
1
HMBC
H– H COSY
ROESY
1
Figure 2. Key H– H COSY, HMBC and ROESY correlations of 1.
1

1
506
B. Deng et al.
1 13
Table 2. H-NMR (400 MHz) and C-NMR (100 MHz) data of compound 3 in CDCl
3
.
Position
ꢁC
ꢁH [J (Hz)]
6.13 (d, 2.4)
4.13 (s)
Position
ꢁC
ꢁH [J (Hz)]
1
2
3
4
5
6
7
8
9
119.8
199.1
80.5
46.9
44.0
17.7
24.9
30.6
39.1
11
12
13
14
15
16
17
18
19
34.1
39.5
36.1
32.3
150.3
109.3
22.1
20.5
63.3
1.78 (m), 1.83 (m)
1.20 (m), 1.24 (m)
1.35 (m), 1.38 (m)
5.79 (dd, 10.8, 17.6)
4.88 (m), 4.92 (m)
0.98 (s)
1.35 (s)
3.51 (d, 11.2)
2.69 (m)
1.88 (m), 1.94 (m)
1.42 (m), 1.45 (m)
1.78 (m)
3.64 (d, 11.2)
1.04 (s)
10
177.3
20
19.1
þ
ꢀ
of 2 showed ion peaks at m/z 621 {[M þ Na] } and m/z 597 {[M ꢀ H] }, respectively,
and its molecular formula was determined to be C H O by (þ)-HR-ESI-MS (m/z
3
7
58
6
þ
6
21.4120 [M þ Na] , Calcd 621.4131), which is smaller than that of 1 by two CH
2
units. Moreover, further analysis using LC-MS spectra of 1 and 2 also showed their
different retention times and molecular weights. Combining the above evidence, the
difference between 1 and 2 should be a different fatty acid chain at C-3. Compared
with 1, the absence of two CH units in 2 implied the presence of a myristic acid unit
2
at C-3 of 2, instead of the palmitic one in 1. Thus, the structure of 2 was deduced to
be ingenol-5ꢀ,20-O,O-isopropylidene-3ꢀ-myristinate.
Compound 3 was obtained as white solid powder with the molecular formula
þ
C H O , derived from its (þ)-HR-ESI-MS at m/z 341.2094 [M þ Na]
2
0
30
3
þ
C H O Na , Calcd 341.2092) and C-NMR. The IR absorption bands at 3425
20 30 3
13
(
ꢀ1
1
and 1673 cm indicated the presence of hydroxyl and carbonyl groups. The H- and
1
C-NMR data (Table 2) of 3 showed 20 carbon signals, including one carbonyl
group (ꢁ 199.1), one trisubstituted [ꢁ 6.13 (1H, d, 2.4 Hz); ꢁ 119.8 (d), 177.3 (s)]
and one monosubstituted olefin [ꢁ 5.79 (1H, dd, 10.8, 17.6 Hz), ꢁ 4.88 (2H, m);
3
C
H
C
H
H
ꢁC 150.3 (d), 109.3 (t)], three tertiary methyls [ꢁ 0.98, 1.35 and 1.04 (each CH , s);
H
3
ꢁC 22.1, 20.5 and 19.1], one-oxygened methylene [ꢁ 3.51 (1H, d), 3.64 (1H, d);
H
ꢁC 63.3] and one oxygened methine [ꢁ 4.13 (1H, s); ꢁ 80.5].
H
C
Considering the structural characteristics of diterpenoids isolated from the genus
Euphorbia, all of the spectral data of 3 implied that its structure was a rosane-type
diterpenoid, similar to the known compound 18-hydroxyhugorosenone from
Hugonia casteneifolia (Ladislaus, Reiner, Mayunga, Stephan, & Hans, 1998).
1
1
Detailed analysis of the 2D-NMR (including HMQC, H– H COSY and HMBC)
of suggested that possessed the same planar structure as
8-hydroxyhugorosenone, but the differences of chemical shift values between 3
3
3
1
and 18-hydroxyhugorosenone in ring A (especially, C-3, C-4, C-5 and C-19)
indicated that they should possess different stereochemistry, as shown in Figure 3.
The relative stereochemistry of 3 was then elucidated by ROESY spectrum, as
shown in Figure 3. Correlations of H-3–H-5, H-5–H-8 and H-5–H -18 indicated that
3
H-5, H-8 and H -18 were in ꢂ-orientation, while the oxygened methylene group
3

Natural Product Research
1507
1
7
17
12
15
12
11
11
15
2
0
13
1
6
16
2
0
1
3
1
4
1
1
4
O
O
2
10
14
10
9
9
6
8
2
3
8
H
H
H
A
5
5
7
3
7
HO
HO
4
6
H
H
19
18
19 18
HO
HO
1
1
HMBC
H– H COSY
ROESY
1
Figure 3. Key H– H COSY, HMBC and ROESY correlations of 3.
1
took ꢀ-configuration, which was opposite to 18-hydroxyhugorosenone. Thus, the
structure of 3 was deduced to be 3ꢀ,19-dihydroxy-1(10),15-rosadien-2-one.
3
3
. Experimental
.1. General experimental procedures
Optical rotations were measured on a Jasco-20C digital polarimeter. IR (KBr)
spectra were recorded on a Bruker Tensor 27 FT-IR spectrophotometer. UV spectra
were recorded on a Shimadzu UV-2401PC spectrophotometer. 1D- and 2D-NMR
spectra were obtained on an INOVA-400 MHz NMR spectrometer in CDCl using
3
TMS as the internal standard. ESI-MS and LC-MS were carried out on a
HP 1100MSD (ESI) apparatus. HR-ESI-MS was measured an API Qstar Pulsar 1
spectrometer. GC-MS was carried out on a HP 6890-5975C (EI) apparatus.
Semi-preparative HPLC was performed on an Agilent 1100 apparatus. Column
chromatography was performed on silica gel (200–300 mesh and H-60, Qingdao
Marine Chemical Company, Qingdao, China) and Sephadex LH-20 (40–70 mm,
Amersham Pharmacia Biotech AB, Sweden). Solvents used for extraction and
isolation were distilled prior to use.
3
.2. Plant material
The roots of E. ebracteolata were collected from Changchun, Jilin Province, China in
February 2006. The sample was identified by Prof. L. Gao from the Natural Drug
Resources Laboratory of the Yunnan Institute of Materia Medica.
3
.3. Extraction and isolation
Air-dried and powdered roots of E. ebracteolata (20 kg) were extracted three times
with 95% ethanol (3 ꢁ 30 L, each for 5 days) at room temperature and concentrated
in vacuo to give a crude extract. The extract was then dissolved in H O and
2

1
508
B. Deng et al.
partitioned successively with petroleum ether (PE) and CHCl . The PE fraction
3
(735 g) was chromatographed on a silica gel column (200–300 mesh) eluted with a
PE : acetone gradient (100 : 0–0 : 100) to obtain fractions 1–6. Fraction 2 (65 g) was
submitted to a silica gel CC eluted with a PE : EtOAc gradient (1 : 0–0 : 1) to give four
subfractions, 2.1–2.4. Fraction 2.2 (7 g) was submitted to repeated chromatography
over silica gel H and preparative TLC to afford compound 3 (6 mg). Fraction 4 (18 g)
was submitted to a silica gel CC eluted with a CHCl : acetone gradient (1 : 0–0 : 1) to
3
give five subfractions, 4.1–4.5. Fraction 4.1 (8 g) was submitted to Sephadex LH-20
TM
(
(
1
MeOH) and semi-preparative HPLC using Kromail
1
100-5-C18 ODS column
ꢂ
ꢀ
10 mm ꢁ 250 mm) with 95% MeOH at 2 mL min under 30 C to afford compounds
(11 mg) and 2 (14 mg).
3
.3.1. Ingenol-5ꢀ,20-O,O-isopropylidene-3ꢀ-palmitate (1)
2
4
Yellow oil. ½ꢂꢃ ¼ þ8.97 (c 0.13, CHCl ); UV ꢃ
(CHCl ): 239 nm; IR ꢄ
1
(KBr):
525, 3435, 2954, 2924, 1728, 1462, 1380, 1161 cm ; for H-NMR and C-NMR
D
3
max
3
max
13
ꢀ1
3
þ
data, see Table 1; positive ESI-MS m/z: 649 [M þ Na] ; negative ESIMS m/z:
ꢀ
þ
þ
6
6
25 [M ꢀ H] ; (þ)-HR-ESI-MS m/z 649.4455 [M þ Na] (C H O Na , Calcd
3
9
62
6
49.4444).
3
.3.2. Ingenol-5ꢀ,20-O,O-isopropylidene-3ꢀ-myristinate (2)
1
D
6
Yellow oil. ½ꢂꢃ ¼ 0.00 (c 0.47, CHCl ); UV ꢃ
(CHCl ): 239 nm; IR ꢄ
3 max
3
max
ꢀ
1
KBr): 3428, 3068, 2924, 1726, 1463, 1380, 1079 cm ; for H-NMR and C-NMR
1
13
(
þ
data, see Table 1; positive ESIMS m/z: 621 [M þ Na] ; negative ESI-MS m/z: 597
ꢀ
þ
þ
[
M ꢀ H] ; (þ)-HR-ESI-MS m/z 621.4120 [M þ Na] (C H O Na , Calcd
3
7
58
6
6
21.4131).
3
.3.3. 3ꢀ,19-dihydroxy-1(10),15-rosadien-2-one (3)
2
D
4
White solid powder. ½ꢂꢃ ¼ þ64.71 (c 0.17, CHCl ); UV ꢃ
(CHCl ): 252 nm;
3
3
max
ꢀ1
1
(KBr): 3425, 2918, 2850, 1673, 1467, 1379, 1102, 1050 cm ; for H-NMR
IR ꢄ
max
1
3
þ
and C-NMR data, see Table 1; (þ) ESI-MS m/z: 341 [M þ Na] ; negative ESI-MS
ꢀ
þ
þ
m/z: 317 [M ꢀ H] ; (þ)-HR-ESI-MS m/z 341.2094 [M þ Na] (C H O Na , Calcd
2
0
30
3
3
41.2092).
3
.4. Basic hydrolysis and GC-MS analysis of 1
Compound 1 (3 mg) was dissolved in 5 mL MeOH, and then 0.025 g NaOH was
added. The mixture was stirred at room temperature for 3 h. After acidification with
5
washed by H O, dried by Na SO and evaporated under reduced pressure. The crude
% HCl, the mixture was extracted with CHCl . The extraction of CHCl was
3 3
2
2
4
residue was subjected to a silica gel CC to obtain an ingenol derivative and fatty acid.
Then the fatty acid was analysed by GC-MS.

Natural Product Research
1509
3
.5. LC-MS analysis of the mixture of 1 and 2
Compounds 1 (2 mg) and 2 (2 mg) were mixed together, and then analysed by an
TM
HP 1100MSD (ESI) using a Kromail
with 100% MeOH at 1 mL min under 30 C.
100-5-C18 ODS column (4.6 mm ꢁ 250 mm)
ꢀ1
ꢂ
Acknowledgements
This work was financial supported by the Specialized Foundation for Talent Guizhou
Province (No. 2008114) and the Specialized Foundation for Modernization of Traditional
Chinese Medicine of Guizhou province (No. 20065041).
References
Deng, B., Mu, S.Z., Huang, L.J., Song, Z.Q., & Hao, X.J. (2009). Studies on diterpenoids
from the roots of Euphorbia ebracteolata Hayata. China Journal of Chinese Materia
Medica, 34(6), 789–791.
Fu, G.M., Yu, B.Y., & Zhu, D.N. (2006). Two novel phloroglucinol derivatives from
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