Chemical constituents of the roots of Euphorbia micractina

Article abstract of DOI:10.1021/np900305j

Nine minor new tirucallane (1-7) and euphane (8 and 9) triterpenoids including five hydroperoxides, together with 18 known compounds, have been isolated from an ethanolic extract of the roots of Euphorbia micractina. Their structures including absolute co

Full text of DOI:10.1021/np900305j

1620
J. Nat. Prod. 2009, 72, 1620–1626
Chemical Constituents of the Roots of Euphorbia micractina
Wendong Xu, Chenggen Zhu, Wei Cheng, Xiaona Fan, Xiaoguang Chen, Sen Yang, Ying Guo, Fei Ye, and Jiangong Shi*
Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College (Key Laboratory of BioactiVe
Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education), Beijing 100050, People’s Republic of China
ReceiVed May 19, 2009
Nine minor new tirucallane (1-7) and euphane (8 and 9) triterpenoids including five hydroperoxides, together with 18
known compounds, have been isolated from an ethanolic extract of the roots of Euphorbia micractina. Their structures
including absolute configurations were elucidated by spectroscopic and chemical analysis. In the in vitro assays, betulin
(10) showed a selective cytotoxic activity against A2780 ovarian cells with an IC50 value of 6.1 µM and inhibitory
activity against protein tyrosine phosphatase 1B (PTP1B) with an IC50 value of 15.3 µM. Jolkinol B (11) showed a
potent activity against HIV-1 replication with an IC50 value of 12.6 µM. However, compounds 1-9 and the other
known compounds were inactive in the three assays used.
Species of the genus Euphorbia (Euphorbiaceae) are sources of
various secondary metabolites with interesting chemical structures
and significant bioactivities.1 Several plants of this genus have long
been used for the treatment of various diseases such as edema,
ascites, warts, and cancer in the People’s Republic of China.2
Euphorbia micractina Boiss. is widely distributed at high altitudes
(2700-5000 m) in western mainland China, and its roots are used
in Chinese folk medicine for the treatment of tumors and warts.3
Some structurally interesting diterpenes were reported from this
plant.4-6 As part of a program to access chemical diversity of
Chinese traditional medicines and their biological effects, we carried
out an investigation of the roots of E. micractina. This paper
describes the isolation, structure elucidation, and selected in vitro
bioassays of nine minor, new triterpenoids including seven tiru-
callane (1-7) and two euphane derivatives (8 and 9), together with
18 known compounds, from an ethanolic extract of this plant
material. Among them, compounds 2-4, 7, and 8 are uncommon
triterpene hydroperoxides.
Results and Discussion
Compound 1, a white, amorphous solid, [R]20 -1.3 (c 0.08,
D
MeOH), showed IR absorptions for hydroxy (3333 cm-1) and
double-bond (1648 cm-1) functional groups. The EIMS of 1
exhibited a molecular ion peak at m/z 442 [M]+ •, and the HREIMS
at m/z 442.3799 [M]+ • indicated that 1 has a molecular formula of
C30H50O2 (calcd for C30H50O2, m/z 442.3811). The 1H NMR
spectrum of 1 in acetone-d6 showed signals due to an olefinic
methylene at δ 4.88 (br s, H-26a) and 4.74 (br s, H-26b), two
oxygen-bearing methines at δ 3.96 (dt, J ) 6.0 and 4.2 Hz, H-24)
and 3.17 (dt, J ) 9.6 and 5.4 Hz, H-3), and two exchangeable
hydroxy protons at δ 3.63 (d, J ) 4.2 Hz, HO-24) and 3.37 (d, J
) 5.4 Hz, HO-3). In addition, it displayed an olefinic methyl singlet
at δ 1.69 (H3-27), five aliphatic methyl singlets at δ 0.81 (H3-18),
0.98 (H3-19), 1.00 (H3-28), 0.80 (H3-29), and 0.91 (H3-30), and an
aliphatic methyl doublet at δ 0.94 (J ) 6.6 Hz, H3-21), together
with partially overlapped multiplets attributed to aliphatic meth-
ylenes and methines between δ 1.10 and 2.20. These data helped
classify 1 as a dihydroxylated triterpene containing a terminal
double-bond unit.7,8 This was supported by the 13C NMR and DEPT
spectra of 1, which showed 30 carbon signals consisting of seven
methyls, 11 methylenes (one sp2), five methines (two oxygen-
bearing), and seven quaternary carbons (three olefinic) (Table 2).
The structure of 1 was finalized by the analysis of its 2D NMR
data. The proton and protonated carbon signals in the NMR spectra
of 1 were assigned unambiguously (Tables 1 and 2) on the basis
of H-1H COSY and HMQC spectroscopic analysis. The H-1H
COSY spectrum showed homonuclear coupling correlations from
HO-3 through H-3 and H2-2 to H2-1, from H-5 through H2-6 to
H2-7, from H2-15 through H2-16, H-17, H-20, H2-22, and H2-23 to
H-24 (in which H-20 correlated with H3-21), from H2-11 to H2-12,
and from H2-26 to H3-27. These correlations indicated unambigu-
ously the nature of the partial structural units with vicinal coupling
protons in 1 (Supporting Information, Figure S1, units with thick
lines). In the HMBC spectrum of 1, two- and three-bond correlations
from H3-18 to C-12, C-13, C-14, and C-17, from H3-19 to C-1,
C-5, C-9, and C-10, from H3-21 to C-17, C-20, and C-22, from
1
1
* To whom correspondence should be addressed. Tel: 86-10-83154789.
Fax: 86-10-63017757. E-mail: shijg@imm.ac.cn.
10.1021/np900305j CCC: $40.75  2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 08/24/2009

Chemical Constituents of Euphorbia micractina
Journal of Natural Products, 2009, Vol. 72, No. 9 1621

1622 Journal of Natural Products, 2009, Vol. 72, No. 9
Xu et al.
Table 2. 13C NMR Spectroscopic Data (δ) of Compounds 1-9a
has a 24R configuration.10,11 Therefore, the structure of 1 was
determined as (-)-(24R)-tirucalla-8,25-diene-3ꢀ,24-diol.
position
1
2
3
4
5
6
7
8
9
Compound 2, a white, amorphous solid, [R]20 -1.9 (c 0.05,
D
1
2
3
4
5
6
7
8
36.1 35.3 35.3 35.3
28.8 28.4 28.4 28.3
78.5 77.7 77.7 77.7
39.7 39.5 39.5 39.5
52.0 49.2 49.2 49.2
19.7 36.3 36.3 36.3
35.2 36.6
28.3 28.5
77.7 78.6
39.5 39.7
49.2 49.3
36.2 24.4
34.6 35.3 35.2
28.2 28.4 28.3
77.6 77.7 77.7
39.3 39.5 39.5
49.1 49.2 49.2
36.2 36.3 36.3
MeOH), exhibited quasimolecular ion peaks at m/z 473 [M + H]+
and 495 [M + Na]+ in the positive ESIMS. The molecular formula
of 2 was indicated as C30H48O4 by the HRESIMS at m/z 495.3468
[M + Na]+ (calcd for C30H48O4Na, 495.3445). The IR spectrum of
2 suggested the presence of hydroxy (3313 cm-1) and R,ꢀ-
unsaturated carbonyl (1714 and 1633 cm-1) groups. The NMR
spectroscopic features of 2 in acetone-d6 were found to be similar
to those of 1. However, comparison of the NMR data of the
tetracyclic ring moieties of 1 and 2 showed that the resonance due
to a methylene (H2-7 and C-7) of 1 was replaced by the resonance
of a carbonyl carbon (δ 197.6) in 2. Meanwhile, the resonances of
the protons and carbons around the double bond between C-8 and
C-9 of 1 were changed significantly in 2 (Tables 1 and 2). This
suggested that 2 is a analogue of tirucalla-8,25-dien-3ꢀ-ol-7-one
and was confirmed by analysis of the 2D NMR data of 2, which
resulted in an unambiguous assignment of the 1D NMR data (Tables
1 and 2). In particular, HMBC correlations from H2-6 to C-5, C-7,
and C-10, from H3-19 to C-1, C-5, C-9, and C-10, and from both
H3-28 and H3-29 to C-3, C-4, and C-5 (Supporting Information,
Figure S1, arrows), in combination with their chemical shifts,
demonstrated unequivocally the presence of an 8-en-7-one unit and
a ꢀ-hydroxy group at C-3 in 2. The 1D and 2D NMR data analysis
indicated that the side-chain moiety of 2 is very similar to that of
1. However, the resonances due to H-24, H2-26, and HO-24 were
changed from δ 3.96 (dt, J ) 6.0, 4.2 Hz, H-24), 4.88 (s, H-26a),
4.74 (s, H-26b), and 3.63 (d, J ) 4.2 Hz, HO-24) in 1 to δ 4.20 (t,
J ) 7.0 Hz, H-24), 4.90 (2H, s, H2-26), and 10.41 (s, HOO-24) of
2, respectively. Moreover, the resonances of C-24, C-25, and C-26
of 2 were shifted by ∆δC +13.7, -3.7, and +3.3 ppm, respectively,
as compared with those of 1. These data, in combination with the
molecular formula, were consistent with the presence of a hydro-
peroxy group at C-24 for 2.12 The NOESY spectroscopic data
(Supporting Information) and the chemical shift of H3-21 (δH 0.94,
d, J ) 6.5 Hz)8 of 2 suggested that the relative configurations of
the tetracyclic ring moiety and C-20 in 2 are identical to those of
1. This inference was supported by the CD spectrum of 2, which
showed a positive Cotton effect at λ 339 nm (∆ε +0.97) for a n f
π* transition and a negative Cotton effect at λ 258 nm (∆ε -1.49)
for a π f π* transition. Molecular modeling using the MM2
program indicated that the cyclohexenone moiety of the ring system
possesses a twisted boat conformation for the lowest energy
conformational isomer (Supporting Information, Figure S2). On the
basis of the octant rule for cyclohexenones,13 the Cotton effects
observed indicated a configuration for the tetracyclic ring moiety
of 2, identical to that of 1. Reduction of 2 with triphenylphosphine
yielded 2a. After an unambiguous assignment of the 1H NMR data
28.4 197.6 197.6 197.7 197.6 119.5 199.8 197.6 NDb
134.2 139.2 139.2 139.1 139.1 142.1 150.1 139.2 NDb
135.3 165.6 165.6 165.7 165.7 146.2 155.3 165.6 NDb
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
38.0 40.0 40.0 40.0
22.1 24.1 24.1 24.1
31.6 30.7 30.7 30.6
44.8 45.4 45.3 45.3
50.7 48.4 48.5 48.4
30.5 32.3 32.4 32.4
28.6 29.2 29.2 29.2
51.0 49.5 49.5 49.3
15.8 15.9 15.9 15.9
20.6 18.8 18.8 18.8
37.2 36.9 37.1 37.4
19.2 19.0 19.1 19.1
32.9 32.9 32.9 40.1
39.8 36.9
24.1 115.9 201.9 24.2 24.1
38.8 40.0 40.0
30.5 39.1
45.3 44.7
48.4 50.3
32.4 31.9
29.2 28.6
49.2 51.8
15.9 16.6
18.7 21.2
37.5 36.8
19.1 18.9
51.8 30.7 30.7
45.9 45.4 45.4
48.4 48.5 48.4
32.6 32.3 32.3
28.3 29.3 29.3
49.6 48.9 49.2
18.5 16.1 16.1
18.1 18.8 18.7
37.1 36.6 36.8
18.7 19.3 19.4
40.0 32.72 39.7 31.9 32.7
32.6 28.0 28.4 128.7 124.6 32.71 128.3 28.2 32.1
76.4 90.1 89.9 137.2 141.6 76.0 137.4 89.8 76.3
149.3 145.6 146.0 81.5
70.1 149.6
81.5 146.0 NDb
25.0 113.5 110.3
25.0 17.1 17.7
27.9 27.8 27.8
15.6 15.6 15.6
24.2 24.7 24.7
110.5 113.8 113.4 25.0b 29.3 110.1
17.5 16.9 17.1 25.1b 29.3 17.9
28.6 27.8 27.8 27.8
16.2 15.6 15.6 15.6
24.7 24.6 24.7 24.6
27.8 28.3
15.6 15.9
24.6 23.6
a
13C NMR data (δ) were measured in acetone-d6 for 1-4 and 6-9 at
150 MHz. and for 5 at 125 MHz. The assignments were based on
DEPT, H-1H COSY, HSQC, and HMBC experiments. b ND means the
1
signal was not detected.
H3-27 to C-24, C-25, and C-26, from both H3-28 and H3-29 to C-3,
C-4, and C-5, and from H3-30 to C-8, C-13, C-14, and C-15
(Supporting Information, Figure S1, arrows), together with the
chemical shifts of these protons and carbons, suggested that 1 is a
stereoisomer of either tirucalla-8,25-diene-3,24-diol or eupha-8,25-
diene-3,24-diol.8 In a NOE difference experiment of 1, irradiation
of H-3 enhanced H-5 and H3-28, suggesting that the hydroxy group
at C-3 has an equatorial ꢀ-orientation. This was in good agreement
with the coupling constants between H-3 with H-2a (5.4 Hz) and
H-2b (9.6 Hz), indicating that H-3 has an axial R-orientation.
Irradiation of H3-19 enhanced H3-29 and H3-30, while irradiation
of H3-30 enhanced H-12a, H-17, and H3-19, indicating that these
protons are oriented on the same side of the ring system. In addition,
H-20 and H3-21 were enhanced by irradiation of H3-18, while H3-
18 and H-12b were enhanced by irradiation of H3-21. These NOE
enhancements, in combination with the chemical shift of H3-21,8
suggested that 1 is a C-24 epimeric isomer of tirucalla-8,25-diene-
3ꢀ,24-diol rather than of eupha-8,25-diene-3ꢀ,24-diol. The absolute
configuration at C-24 of 1 was determined by the modified Mosher’s
method.9 Compound 1 was treated with (R)-(+)- and (S)-(-)-R-
methoxy-R-(trifuromethyl)phenylacetic acid (MTPA) to yield the
corresponding two pairs of (S)- and (R)-MTPA monoesters, 1a/1c
and 1b/1d (Scheme 1). The chemical shifts of the two pairs of
diastereomers 1a/1c and 1b/1d were carefully assigned by analysis
of their 1H-1H COSY and NOE difference data. From the
MTPA determination rule,9 the positive and negative ∆δ
(δS-MTPA ester - δR-MTPA ester) values observed for protons were located
on the right and on the left side of the MTPA plane, respectively.
As shown in Scheme 1, for the pair of diastereomers 1a/1c the ∆δ
values for H2-1 and H2-2 are negative, whereas those for H3-28
and H3-29 are positive. This verified that 1 possesses a 3S
configuration, which is the same as that of the majority of natural
tirucalla-3ꢀ-ol derivatives. For the pair of diastereomers 1b/1d the
∆δ values for H2-26 and H3-27 are negative, whereas those for
H3-21, H-20, H2-22, and H2-23 are positive. This indicated that 1
1
of 2a (Experimental Section) by the H-1H COSY experiment,
1
comparison of the H NMR data of the side-chain moiety of 2a
and 1 suggested that the absolute configuration at C-24 of 2 and 1
is identical. Therefore, the structure of 2 was determined as (-)-
(24R),24-hydroperoxytirucalla-8,25-dien-3ꢀ-ol-7-one.
Compound 3, a white, amorphous solid, gave spectroscopic data
1
almost identical to those of 2. Comparison of the H-1H COSY
spectra of 2 and 3 (Supporting Information) indicated clearly that
H-24 of 3 was coupled with two protons having a large difference
in their chemical shifts [δ 1.65 (m, H-23a) and 1.33 (m, H-23b)],
whereas H-24 of 2 was coupled with two overlapped protons [δ
1.49 (m, H2-23)]. In addition, resonances for C-23, C-25, and C-26
in the 13C NMR spectrum of 3 were shifted, respectively, by ∆δC
+0.4, +0.4, and -0.4 ppm (Table 2), when compared with those
of 2. These data suggested that 3 is a C-24 epimer of 2, which was
supported by the NOESY and CD spectroscopic data of 3
(Supporting Information). Thus, the structure of 3 was determined
as (-)-(24S),24-hydroperoxytirucalla-8,25- dien-3ꢀ-ol-7-one.

Chemical Constituents of Euphorbia micractina
Journal of Natural Products, 2009, Vol. 72, No. 9 1623
1
Scheme 1. ∆δ (δS - δR) Values Obtained from the H NMR Spectra of the MTPA Esters 1a-1d
The IR and HRESIMS data of 4 (Experimental Section)
demonstrated that it is also an isomer of 2. Comparison of the H
Compound 6, a white, amorphous solid, [R]20 -23.1 (c 0.61,
D
MeOH), exhibited a molecular ion peak at m/z 440 [M]+• in the
1
NMR data between 4 and 2 indicated that the resonances assigned
to the oxymethine (H-24), the olefinic methylene (H2-26), and the
olefinic methyl (H3-27) of the side-chain moiety of 2 were replaced
by resonances attributable to two olefinic methines [δ 5.60 (m, H-23
and H-24)] and two aliphatic methyls attached to an oxygen-bearing
quaternary carbon [δ 1.26 (s, H3-26 and H3-27)] in 4. This suggested
that the terminal double bond between C-25 and C-26 and the
hydroperoxy group at C-24 in 2 were moved in 4 to C-23 and C-24
and C-25, respectively. This was supported by the replacement of
the carbon resonances attributed to the terminal double bond and
hydroperoxy-bearing methine of the side-chain moiety of 2 by
resonances assignable to two methines of a double bond [δ 128.7
(C-23) and 137.2 (C-24)]11,12 and a hydroperoxy-bearing quaternary
carbon [δ 81.5 (C-25)] in the 13C NMR spectrum of 4. The
deduction was further confirmed by 2D NMR experiments on 4,
in particular, the HMBC spectrum, in which two- and three-bond
correlations from H3-21 to C-17 and C-20, from H2-22 to C-20,
C-23, and C-24, from H-24 to C-22, C-23, and C-25, from H2-26
to C-24, C-25, and C-27, and from H3-27 to C-24, C-25, and C-26,
together with their chemical shifts, proved the presence of a 25-
hydroperoxy-23-ene unit in the side-chain moiety. Although the
EIMS. The molecular formula, C30H48O2, was indicated from the
HREIMS at m/z 440.3640 [M]+• (calcd for C30H48O2, 440.3654).
The IR and NMR spectroscopic features of 6 were similar to those
1
of 1. However, the H NMR spectrum of 6 in acetone-d6 showed
signals attributable to two additional olefinic methines for the
tetracyclic ring moiety at δ 5.37 (br s, H-7) and 5.24 (br s, H-11),
while the 13C NMR and DEPT spectra of 6 displayed signals
attributable to two trisubstituted double bonds for the tetracyclic
ring moiety at δ 146.2, 142.1, 119.5, and 115.9. In addition, the
resonances assignable to C-6, C-8, C-9, and C-12 of 6 were
deshielded by ∆δC +4.7, +7.9, +10.9, and +7.5 ppm, respectively,
as compared with those of 1, whereas the resonance due to C-5 of
6 was shielded by ∆δC -2.7 ppm. These data indicated that 6 is a
derivative of 1 possessing double bonds between both C-7 and C-8
and C-9 and C-11, respectively. This was confirmed by the 2D
NMR spectroscopic data analysis of 6. In particular, HMBC
correlations from H3-19 to C-1, C-5, C-9, and C-10, from H-7 to
C-5, C-6, C-9, and C-14, and from H-11 to C-8, C-12, and C-13,
in combination with chemical shifts of these protons and carbons,
proved the location of the double bonds in 6. Therefore, the structure
of 6 was determined as (-)-(24R)-tirucalla-7,9(11),25-triene-3ꢀ,24-
diol.
1
H-23 and H-24 signals were partially overlapped in the H NMR
spectrum of 4 in acetone-d6, the trans geometric configuration of
Compound 7, a white, amorphous solid, [R]20 +2.7 (c 0.04,
D
MeOH), showed IR absorptions for hydroxy (3310 cm-1) and R,ꢀ-
unsaturated carbonyl (1713 and 1668 cm-1) groups. Its molecular
formula, C30H46O5, was indicated from the HRESIMS at m/z
509.3272 [M + Na]+ (calcd for C30H46O5Na, 509.3237). The NMR
spectroscopic features of 7 were similar to those of 4. Comparison
of NMR data (Tables 1 and 2) between 7 and 4 revealed that they
have the same side chain and a difference in the tetracyclic ring
moiety. The resonances due to C-11 and H2-11 of 4 were replaced
by a carbonyl signal for 7 [δC 201.9]. In addition, resonances
attributable to C-7, C-8, and C-12 of 7 were deshielded by ∆δC
+2.1, +11.0, and +21.2 ppm, respectively, whereas resonances
assignable to C-9 and C-10 of 7 were shielded, in turn, by ∆δC
-10.4 and -1.2 ppm. This suggested that 7 is an 11-oxo derivative
of 4, which was confirmed by comparison of the NMR data between
7 and 11-oxo-kansenonol.8 Thus, the structure of 7 was assigned
as (+)-(23E),25-hydroperoxytirucalla-8,23-dien-3ꢀ-ol-7,11-dione.
The spectroscopic data of 8 (Tables 1 and 2 and Experimental
Section) were consistent with this compound being an isomer of
the 23-ene unit was indicated unequivocally by a vicinal coupling
1
constant of 16.2 Hz between H-23 and H-24 observed in the H
NMR spectrum of this compound in pyridine-d5.11 The similarity
of the CD (Experimental Section) and specific rotation data between
4 ([R]20 -3.2) and 2 ([R]20 -1.9) proposed that they have the
D
D
same stereochemistry.8 Therefore, the structure of 4 was determined
as (-)-(23E),25-hydroperoxytirucalla-8,23-dien-3ꢀ-ol-7-one.
Compound 5, a white, amorphous solid, [R]20 -2.5 (c 0.04,
D
MeOH), gave the molecular formula C30H48O3 (calcd for
C30H48O3Na, m/z 479.3496), as indicated from its positive HRES-
IMS at m/z 479.3524 [M + Na]+, one oxygen atom less than that
of 4. The differences in the 1H NMR spectroscopic features between
5 and 4 were that an exchangeable singlet ascribed to a hydroxy
proton of 5 at δ 3.37 (s, HO-25) replaced the signal assigned to
the hydroperoxy proton of 4 [δ 9.94 (s, HOO-25)], and the
overlapped signals due to H3-26 and H3-27 of 5 were shielded by
∆δH -0.04 and -0.03 ppm, respectively, as compared with those
of 4. In addition, comparison of the 13C NMR spectra of 5 and 4
showed that the resonance attributable to C-25 of 5 was shielded
significantly by ∆δC -11.4 ppm and that the resonances assignable
to C-26 and C-27 of 5 were deshielded by ∆δC +4.3 and +4.2
ppm, respectively. This suggested that 5 is (-)-tirucalla-8,23-diene-
3ꢀ,25-diol-7-one. Although the resonances for H-23 and H-24 of
5 were overlapped at δ 5.60 in actone-d6 and at δ 5.94 in pyridine-
d5, a 23E configuration was assigned for 5 on the basis of the vicinal
coupling constant of 15.6 Hz between H-23 and H-24 observed in
the 1H NMR spectrum of 5 in C6D6.14 The CD and specific rotation
data of 5 (Experimental Section) supported the same relative
configuration in the tetracyclic ring system of 5 as that of 2.
Therefore, the structure of 5 was determined as (-)-(23E)-tirucalla-
8,23-diene-3ꢀ,25-diol-7-one.
1
3. After the H and 13C NMR data of 8 were assigned unambigu-
ously by 2D NMR data analysis, a careful comparison of the data
with analogous values for 3 (Tables 1 and 2) revealed that for 8
the resonances for H-20, H-22a, and H-22b were deshielded by
∆δH +0.07, +0.16, and +0.08 ppm, respectively, whereas the
resonances for H3-21, and C-17, C-20, and C-22 were shielded, in
turn, by ∆δH -0.08 and ∆δC -0.6, -0.5, and -1.0 ppm. This
suggested that 8 is the C-21 epimer of 3, which was supported by
the NOESY data. For compound 8, NOE correlations very similar
to those of 3 were observed except for the appearance of NOEs
between H3-21 and H-16R and the absence of a NOE between H3-
18 and H3-21.8 The chemical shift of H3-21 (δH 0.86, d, J ) 6.0
Hz) and the positive optical rotation ([R]20D +6.2) were consistent

1624 Journal of Natural Products, 2009, Vol. 72, No. 9
Xu et al.
with 8 belonging to the euphane series of triterpenoids.8 Accord-
ingly, the structure of 8 was determined as (+)-(24S),24-hydrop-
eroxyeupha-8,25-dien-3ꢀ-ol-7-one.
7% H2SO4 in 95% EtOH followed by heating. Unless otherwise noted,
all chemicals were obtained from commercially available sources and
were used without further purification.
Plant Material. The roots of Euphorbia micractina were collected
at Zhang County, Gansu Province, People’s Republic of China, in
September 2002. The plant was identified by Associate Professor Lin
Ma (Institute of Materia Medica, Beijing). A voucher specimen (no.
02086) was deposited at the Herbarium of the Department of Medicinal
Plants, Institute of Materia Medica.
The IR and the NMR spectroscopic features of 9 resembled those
of 8. The HRESIMS at m/z 479.3539 [M + Na]+ of 9 indicated
that it has a molecular formula of C30H48O3 (calcd for C30H48O3Na,
479.3496), with one oxygen less than 8. Comparison of the 1H NMR
data between these two compounds (Tables 1 and 2) indicated that
the triplet for H-24 of 8 at δH 4.22 was replaced by a double triplet
of 9 at δH 3.96 (J ) 6.0 and 5.0 Hz), while the singlet for the
hydroperoxy proton of 8 at δH 10.41 was replaced by an exchange-
able doublet for a hydroxy proton of 9 at δH 3.65 (J ) 5.0 Hz). In
addition, as compared with those of 8, the 13C NMR resonances
for C-22 and C-23 of 9 were deshielded, in turn, by ∆δC +0.8 and
+3.9 ppm. Also, the resonances for C-24 and C-26 of 9 were
shielded by ∆δC -13.5 and -3.2 ppm, respectively. However, the
13C NMR resonances of the quaternary carbons C-7, C-8, C-9, and
C-25 were not clearly observed for 9 (Supporting Information) due
to a limitation of the sample amount available. The differences in
the above spectroscopic data, in combination with the similarity in
the CD and specific rotation data between 9 and 8, led to the
proposal of the presence of a hydroxy group at C-24 in 9, replacing
the hydroperoxy group of 8. Therefore, the structure of 9 was
assigned as (+)-(24S)-eupha-8,25-diene-3ꢀ,24-diol-7-one.
The known compounds isolated in the present investigation were
identified by comparison of spectroscopic data with those reported
in the literature as betulin (10),15 erythrodiol,16 uvaol,17 heliosco-
pinolide E,18 3-acetoxyhelioscopinolide B,19 helioscopinolide A,20
helioscopinolide B,21 jolkinol D,22 jolkinol B (11),22,23 latilagascene
F,23 20-O-acetylingenol-3-O-(2′E,4′Z)-decadienoate,24 glycerol mono-
palmitate,25 (Z)-12-octadecenic-R-glycerol monoester,26,27 p-hy-
droxyphenethyl anisate,28 scopoletin,29 E-cinnamic acid,30 1-tria-
contanol,31 and ꢀ-sitosterol.32
Extraction and Isolation. The air-dried root of E. micractina (11.2
kg) was powdered and extracted with 30 L of 95% EtOH at room
temperature for 3 × 48 h. The EtOH extract was evaporated under
reduced pressure to yield a dark brown residue (1092 g). The residue
was suspended in H2O (1500 mL) and then partitioned with EtOAc (4
× 1000 mL). After removing the solvent, the EtOAc extract (323.5 g)
was subjected to CC over silica gel, eluting with a gradient of increasing
acetone concentrations (0-100%) in petroleum ether, to afford 20
fractions (A1-A20) on the basis of TLC analysis. Fraction A9 (6.4 g)
was separated by normal-phase silica gel CC, eluting with a gradient
of increasing acetone concentrations (5-100%) in petroleum ether, to
give 10 subfractions (A9-1-A9-10). Subfraction A9-5 (1.8 g) was
further separated by flash column chromatography over reversed-phase
silica gel (C-18), eluting with a gradient of increasing EtOH concentra-
tions (0-100%) in H2O, to afford nine fractions (A9-5-1-A9-5-9).
Fraction A9-5-4 (35 mg) was chromatographed over Sephadex LH-
20, eluting with petroleum ether-CHCl3-MeOH (5:5:1), to give three
further fractions. These were separately purified by reversed-phase
semipreparative HPLC, using MeOH-H2O (70:30) or MeCN-H2O (70:
30) as mobile phase, to yield 2 (1.6 mg), 3 (0.9 mg), 4 (2.4 mg), 5 (0.8
mg), 7 (0.7 mg), 8 (1.4 mg), 9 (0.7 mg), and 10 (22.7 mg). Fraction
A9-5-6 (45 mg) was chromatographed over Sephadex LH-20, eluting
with petroleum ether-CHCl3-MeOH (5:5:1), and then purified repeat-
edly by reversed-phase semipreparative HPLC, using MeOH-H2O (85:
15) or MeCN-H2O (80:20) as mobile phase, to yield 6 (5.6 mg).
Fraction A10 (7.6 g) was separated by normal-phase silica gel CC,
eluting with a gradient of increasing acetone concentrations (5-100%)
in petroleum ether, to give nine subfractions (A10-1-A10-9). Sub-
fraction A10-4 (2.0 g) was further separated by flash column chroma-
tography over reversed-phase silica gel eluting with a gradient of
increasing EtOH concentrations (0-100%) in H2O to afford four
fractions (A10-4-1-A10-4-4). Fraction A10-4-2 (800 mg) was chro-
matographed over Sephadex LH-20, eluting with petroleum
ether-CHCl3-MeOH (5:5:1), to give seven fractions (A10-4-2-1-A10-
4-2-7). Fraction A10-4-2-4 was purified repeatedly by normal-phase
silica gel CC, using CHCl3-MeOH (50:1), to yield 1 (30.6 mg) and
11 (306.3 mg).
In the in vitro bioassays performed in this study, compound 10
showed a selective cytotoxic activity against the A2780 ovarian
cancer cell line with an IC50 value of 6.1 µM [the positive control
camptothecin (CPT) gave an IC50 value of 0.3 µM] and inhibitory
activity against protein tyrosine phosphatase 1B (PTP1B), with an
IC50 value of 15.3 µM. The positive control in this assay was
oleanolic acid (IC50, 4.2 µM). Compound 11 showed activity against
HIV-1 replication with an IC50 value of 12.6 µM [the positive
control zidovudine (AZT) gave an IC50 value of 0.05 µM]. However,
compounds 1-9 and the other known compounds were inactive in
the three assays.
(-)-(24R)-Tirucalla-8,25-diene-3ꢀ,24-diol (1): white, amorphous
solid; [R]20D -1.3 (c 0.08, MeOH); UV (MeOH) λmax (log ꢀ) 204 (3.41)
nm; CD (MeOH) 281 (∆ꢀ +0.04), 218 (∆ꢀ -0.72), 203 (∆ꢀ -0.20);
IR νmax 3333, 3071, 2938, 2874, 1648, 1466, 1452, 1373, 1335, 1296,
Experimental Section
1
1249, 1191, 1152, 1095, 1062, 1026, 893, 865, 649 cm-1; H NMR
General Experimental Procedures. Optical rotations were measured
on a PE model 343 polarimeter. UV spectra were measured on a Cary
300 spectrometer. CD spectra were recorded on a JASCO-815 CD
spectrometer. IR spectra were recorded on a Nicolet 5700 FT-IR
microscope instrument (FT-IR microscope transmission). 1D- and 2D-
NMR spectra were obtained at 500 or 600 MHz for 1H and 125 or 150
MHz for 13C, respectively, on INOVA 500 MHz or SYS 600 MHz
spectrometers, in acetone-d6, CDCl3, pyridine-d5, or C6D6, with solvent
peaks used as references. EIMS and HREIMS data were measured with
a Micromass Autospec-Ultima ETOF spectrometer. ESIMS data were
measured with a Q-Trap LC/MS/MS (Turbo Ionspray Source) spec-
trometer. HRESIMS data were measured using an AccuToFCS JMS-
T100CS spectrometer. Column chromatography (CC) was performed
with silica gel (200-300 mesh, Qingdao Marine Chemical Inc.,
Qingdao, People’s Republic of China) and Pharmadex LH-20 (Amer-
sham Biosciences, Inc., Shanghai, People’s Republic of China).
Preparative TLC separation was performed with high-performance silica
gel preparative TLC plates (HSGF254, glass precoated, Yantai Jiangyou
Silica Gel Development Co., Ltd., Yantai, People’s Republic of China).
HPLC separation was performed on an instrument consisting of a
Waters 600 controller, a Waters 600 pump, and a Waters 2487 dual λ
absorbance detector, with a Prevail (250 × 10 mm i.d.) column packed
with C18 (5 µm). TLC was carried out with glass precoated silica gel
GF254 plates. Spots were visualized under UV light or by spraying with
(acetone-d6, 600 MHz) data, see Table 1; 13C NMR (acetone-d6, 150
MHz) data, see Table 2; EIMS m/z 442 [M]+•; HREIMS m/z 442.3799
[M]+• (calcd for C30H50O2, 442.3811).
Preparation of (S)- and (R)-MTPA Esters of 1. 1-(3-Dimethyl-
aminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 75.0 mg),
DMAP (53.1 mg), and (S)-MTPA (61.3 mg) were added to a solution
of 1 (27.6 mg) in dried CH2Cl2 (3 mL). The mixture was stirred at
room temperature for 24 h. The reaction mixture was evaporated under
reduced pressure and then partitioned between CHCl3 and H2O. After
the solvent was removed, the CHCl3 extract was purified by preparative
TLC with petroleum ether-EtOAc (6:1), and then HPLC using
MeOH-H2O (99:1), to afford the (S)-MTPA esters 1a (6.5 mg; 16.5%)
and 1b (11.2 mg; 28.4%). By the same procedure, the (R)-MTPA esters
1c (1.4 mg; 8.2%) and 1d (5.1 mg; 29.8%) were prepared. 1a: colorless
1
gum; H NMR (CDCl3, 600 MHz) δ 7.54 (2H, m, H-2′ and H-6′ of
MTPA moiety), 7.40 (3H, m, H-3′, H-4′, and H-5′ of MTPA moiety),
3.53 (3H, s, MeO of MTPA moiety), 4.93 (1H, br s, H-26a), 4.84 (1H,
br s, H-26b), 4.72 (1H, dd, J ) 12.0, 4.2 Hz, H-3), 4.02 (1H, dd, J )
6.6 and 4.8 Hz, H-24), 1.81 (1H, m, H-2a), 1.73 (3H, s, H3-27), 1.64
(1H, m, H-2b), 1.49 (1H, m, H-1a), 1.31 (1H, m, H-1b), 1.24 (1H, m,
H-5), 0.96 (3H, s, H3-19), 0.924 (3H, s, H3-28), 0.920 (3H, d, J ) 5.4
Hz, H3-21), 0.86 (3H, s, H3-30), 0.83 (3H, s, H3-29), 0.76 (3H, s, H3-
1
18). 1b: colorless gum; H NMR (CDCl3, 600 MHz) δ 7.52 (2H, m,
H-2′ and H-6′ of MTPA moiety), 7.39 (3H, m, H-3′, H-4′, and H-5′ of

Chemical Constituents of Euphorbia micractina
Journal of Natural Products, 2009, Vol. 72, No. 9 1625
1
MTPA moiety), 3.57 (3H, s, MeO of MTPA moiety), 5.35 (1H, t, J )
7.2 Hz, H-24), 4.97 (1H, br s, H-26a), 4.93 (1H, br s, H-26b), 3.23
(1H, dd, J ) 12.0, 4.8 Hz, H-3), 1.76 (1H, m, H-23a), 1.62 (1H, m,
H-23b), 1.60 (3H, s, H3-27), 1.46 (1H, m, H-22a), 1.40 (1H, m, H-20),
1.00 (3H, s, H3-28), 0.97 (1H, m, H-22b), 0.95 (3H, s, H3-19), 0.91
(3H, d, J ) 6.0 Hz, H3-21), 0.86 (3H, s, H3-30), 0.80 (3H, s, H3-29),
0.75 (3H, s, H3-18). 1c: colorless gum; 1H NMR (CDCl3, 600 MHz) δ
7.56 (2H, m, H-2′ and H-6′ of MTPA moiety), 7.40 (3H, m, H-3′, H-4′,
and H-5′ of MTPA moiety), 3.57 (3H, s, MeO of MTPA moiety), 4.93
(1H, br s, H-26a), 4.84 (1H, br s, H-26b), 4.74 (1H, dd, J ) 11.4, 4.8
Hz, H-3), 4.02 (1H, dd, J ) 6.6 and 6.0 Hz, H-24), 1.88 (1H, m, H-2a),
1.76 (1H, m, H-2b), 1.73 (3H, s, H3-27), 1.51 (1H, m, H-1a), 1.33
(1H, m, H-1b), 1.24 (1H, m, H-5), 0.99 (3H, s, H3-19), 0.92 (3H, d, J
) 6.6 Hz, H3-21), 0.86 (3H, s, H3-30), 0.84 (3H, s, H3-28), 0.81 (3H,
1148, 1100, 1032, 977, 891, 844, 688, 618 cm-1; H NMR (acetone-
d6, 500 MHz) data, see Table 1; 13C NMR (acetone-d6, 125 MHz) data,
see Table 2; ESIMS m/z 457 [M + H]+, 479 [M + Na]+, and 495 [M
+ K]+; HRESIMS m/z 479.3524 [M + Na]+ (calcd for C30H48O3Na,
479.3496).
(-)-(24R)-Tirucalla-7,9(11),25-triene-3ꢀ,24-diol (6): white, amor-
phous solid; [R]20 -23.1 (c 0.61, MeOH); UV (MeOH) λmax (log ꢀ)
D
205 (3.19), 238 (1.90) nm; CD (MeOH) 236 (∆ꢀ -2.12); IR νmax 3391,
3041, 2958, 2926, 2872, 1714, 1655, 1613, 1510, 1459, 1372, 1292,
1243, 1184, 1153, 1110, 1072, 1037, 984, 903, 813 cm-1; H NMR
1
(acetone-d6, 600 MHz) data, see Table 1; 13C NMR (acetone-d6, 150
MHz) data, see Table 2; EIMS m/z 440 [M]+•; HREIMS m/z 440.3640
[M]+• (calcd for C30H48O2, 440.3654).
(+)-(23E),25-Hydroperoxytirucalla-8,23-dien-3ꢀ-ol-7,11-dione (7):
1
white, amorphous solid; [R]20 +2.7 (c 0.04, MeOH); UV (MeOH)
s, H3-29), 0.76 (3H, s, H3-18). 1d: colorless gum; H NMR (CDCl3,
D
600 MHz) δ 7.51 (2H, m, H-2′ and H-6′ of MTPA moiety), 7.39 (3H,
m, H-3′, H-4′, and H-5′ of MTPA moiety), 3.54 (3H, s, MeO of MTPA
moiety), 5.39 (1H, t, J ) 7.2 Hz, H-24), 5.06 (1H, br s, H-26a), 4.98
(1H, br s, H-26b), 3.24 (1H, dd, J ) 12.0, 4.8 Hz, H-3), 1.72 (3H, s,
H3-27), 1.69 (1H, m, H-23a), 1.56 (1H, m, H-23b), 1.36 (1H, m, H-22a),
1.36 (1H, m, H-20), 1.00 (3H, s, H3-28), 0.95 (3H, s, H3-19), 0.89
(1H, m, H-22b), 0.87 (3H, d, J ) 6.6 Hz, H3-21), 0.85 (3H, s, H3-30),
0.80 (3H, s, H3-29), 0.73 (3H, s, H3-18).
(-)-(24R),24-Hydroperoxytirucalla-8,25-dien-3ꢀ-ol-7-one (2): white,
amorphous solid; [R]20D -1.9 (c 0.05, MeOH); UV (MeOH) λmax (log
ꢀ) 202 (3.68), 254 (1.87) nm; CD (MeOH) 339 (∆ꢀ +0.97), 258 (∆ꢀ
-1.49), 237 (∆ꢀ -0.61), 221 (∆ꢀ -1.17), 207 (∆ꢀ -0.53); IR νmax
3313, 2939, 1714, 1633, 1588, 1457, 1403, 1373, 1331, 1267, 1190,
1099, 1033, 980, 916, 863, 803, 689, 607 cm-1; 1H NMR (acetone-d6,
500 MHz) data, see Table 1; 13C NMR (acetone-d6, 150 MHz) data,
see Table 2; ESIMS m/z 473 [M + H]+ and 495 [M + Na]+; HRESIMS
m/z 495.3468 [M + Na]+ (calcd for C30H48O4Na, 495.3445).
λmax (log ꢀ) 204 (3.33), 219 (1.23), 271 (1.36) nm; CD (MeOH) 274
(∆ꢀ +3.03), 233 (∆ꢀ -2.96), 214 (∆ꢀ -1.43), 202 (∆ꢀ -1.91); IR
νmax 3310, 3160, 2970, 2930, 1713, 1668, 1458, 1407, 1377, 1230,
1184, 1034, 983, 918, 734, 608 cm-1; 1H NMR (acetone-d6, 500 MHz)
data, see Table 1; 13C NMR (acetone-d6, 150 MHz) data, see Table 2;
ESIMS m/z 509 [M + Na]+ and 521 [M + Cl]-; HRESIMS m/z
509.3272 [M + Na]+ (calcd for C30H46O5Na, 509.3237).
(+)-(24S),24-Hydroperoxyeupha-8,25-dien-3ꢀ-ol-7-one (8): white,
amorphous solid; [R]20D +6.2 (c 0.08, MeOH); UV (MeOH) λmax (log
ꢀ) 203 (3.52), 254 (2.11) nm; CD (MeOH) 338 (∆ꢀ +0.49), 259 (∆ꢀ
-0.59), 239 (∆ꢀ -0.34), 221 (∆ꢀ -0.68), 207 (∆ꢀ -0.42), 197 (∆ꢀ
-0.77); IR νmax 3315, 2932, 1715, 1656, 1586, 1457, 1412, 1373, 1333,
1267, 1184, 1098, 1033, 985, 917, 894, 861, 694, 617 cm-1; 1H NMR
(acetone-d6, 500 MHz) data, see Table 1; 13C NMR (acetone-d6, 150
MHz) data, see Table 2; ESIMS m/z 473 [M + H]+ and 495 [M +
Na]+; HRESIMS m/z 495.3475 [M + Na]+ (calcd for C30H48O4Na,
495.3445).
Reduction of 2. A solution of 2 (0.6 mg) in dried CH2Cl2 (1.0 mL)
was treated with triphenylphosphine (6.5 mg) at room temperature for
24 h. The reaction mixture was evaporated under reduced pressure and
then purified by preparative TLC with CHCl3-EtOAc (10:1) as
(+)-(24S)-Eupha-8,25-diene-3ꢀ,24-diol-7-one (9): white, amor-
phous solid; [R]20 +5.1 (c 0.04, MeOH); UV (MeOH) λmax (log ꢀ)
D
203 (3.39), 254 (1.77) nm; CD (MeOH) 337 (∆ꢀ +0.50), 255 (∆ꢀ
-0.58), 239 (∆ꢀ -0.42), 220 (∆ꢀ -0.78); IR νmax 3320, 2955, 2920,
1713, 1647, 1588, 1457, 1377, 1331, 1303, 1257, 1167, 1076, 1035,
1
developing solvent, to afford 2a (0.5 mg): H NMR (acetone-d6, 600
1
998, 974, 897, 841, 808, 616 cm-1; H NMR (acetone-d6, 500 MHz)
MHz) δ 4.87 (1H, br s, H-26a), 4.73 (1H, br s, H-26b), 3.95 (1H, dt,
J ) 5.4, 4.2 Hz, H-24), 3.63 (1H, d, J ) 4.2 Hz, OH-24), 3.57 (1H, d,
J ) 4.8 Hz, OH-3), 3.24 (1H, dt, J ) 8.4, 4.8 Hz, H-3), 2.43 (1H, dt,
J ) 21.0, 9.0 Hz, H-11a), 2.32 (1H, ddd, J ) 21.0, 7.8, 1.0 Hz, H-11b),
2.30 (2H, d, J ) 9.0 Hz, H2-6), 2.09 (1H, ddd, J ) 13.0, 10.0, 2.5 Hz,
H-15a), 1.93 (1H, m, H-16a), 1.86 (1H, dt, J ) 12.6, 3.6 Hz, H-1a),
1.76-1.82 (2H, m, H2-12), 1.71 (1H, t, J ) 9.0 Hz, H-5), 1.69 (2H,
m, H2-2), 1.68 (3H, s, H3-27), 1.61 (1H, m, H-23a), 1.57 (1H, m,
H-22a), 1.48 (1H, m, H-17), 1.48 (1H, m, H-15b), 1.47 (1H, m, H-1b),
1.42 (1H, m, H-20), 1.40 (1H, m, H-23b), 1.34 (1H, m, H-16b), 1.07
(3H, s, H3-19), 0.98 (1H, m, H-22b), 0.97 (3H, s, H3-28), 0.95 (3H, s,
H3-30), 0.94 (3H, d, J ) 6.6 Hz, H3-21), 0.87 (3H, s, H3-29), 0.76
(3H, s, H3-18).
data, see Table 1; 13C NMR (acetone-d6, 150 MHz) data, see Table 2;
ESIMS m/z 457 [M + H]+, 479 [M + Na]+ and 495 [M + K]+;
HRESIMS m/z 479.3539 [M + Na]+ (calcd for C30H48O3Na, 479.3496).
Cells, Culture Conditions, and Cell Proliferation Assay. See ref
33. Camptothecin (CPT) was used as the positive control.
PTP1B Inhibition Assay. See ref 34. Oleanolic acid was used as
the positive control.
Anti-HIV Activity Assay. A cell-based VSVG/HIV-1 pseudotyping
system was used for evaluating the anti-HIV replication activity of the
compounds as described previously.35 Briefly, vesicular stomatitis virus
glycoprotein (VSV-G) plasmid was cotransfected with env-deficient
HIV-1 vector, pNL4-3.luc.R-E-,36 into 293 cells by using a modified
Ca3(PO4)2 method.37 Sixteen hours post-transfection, plates were washed
by PBS, and fresh DMEM with 10% FBS was added into the plates.
Forty-eight hours post-transfection, supernatant, which contained
VSVG/HIV-1 virions, was harvested and filtered through a 0.45 µm
filter. VSVG/HIV-1 pseudotyped virions were quantified in terms of
their p24 concentrations detected by ELISA (ZeptoMetrix, cat. no.
0801111), then diluted to 0.2 ng p24/mL, and used directly or stored
at -80 °C.
For the infection assay, 293T cells were plated on 24-well plates at
a density of 6 × 104 cells per well, one day prior to infection.
Compounds were incubated with target cells for 15 min prior to adding
VSVG/HIV-1. The same amount of solvent alone was used as blank
control. Forty-eight hours post-infection, cells were lysed in 50 µL of
Cell Lysis Reagent (Promega). Luciferase activity of the cell lysate
was measured by a FB15 luminometer (Berthold Detection System),
according to the manufacture’s instructions. In this assay, the positive
control was zidovudine (AZT).
(-)-(24S),24-Hydroperoxytirucalla-8,25-dien-3ꢀ-ol-7-one (3): white,
amorphous solid; [R]20D -4.7 (c 0.08, MeOH); UV (MeOH) λmax (log
ꢀ) 200 (3.54), 254 (1.91) nm; CD (MeOH) 339 (∆ꢀ +0.57), 258 (∆ꢀ
-0.86), 238 (∆ꢀ -0.37), 220 (∆ꢀ -0.78); IR νmax 3316, 2936, 2623,
2498, 1711, 1646, 1589, 1452, 1373, 1332, 1268, 1222, 1188, 1097,
1035, 1014, 985, 916, 863, 807, 690, 617, 605 cm-1; 1H NMR (acetone-
d6, 500 MHz) data, see Table 1; 13C NMR (acetone-d6, 150 MHz) data,
see Table 2; ESIMS m/z 473 [M + H]+ and 967 [2 M + Na]+;
HRESIMS m/z 495.3463 [M + Na]+ (calcd for C30H48O4Na, 495.3445).
(-)-(23E),25-Hydroperoxytirucalla-8,23-dien-3ꢀ-ol-7-one (4): white,
amorphous solid; [R]20D -3.2 (c 0.16, MeOH); UV (MeOH) λmax (log
ꢀ) 202 (2.99), 254 (1.87) nm; CD (MeOH) 339 (∆ꢀ +0.97), 259 (∆ꢀ
-1.58), 238 (∆ꢀ -0.76), 220 (∆ꢀ -1.39), 207 (∆ꢀ -0.94), 199 (∆ꢀ
-1.28); IR νmax 3313, 3160, 2939, 2606, 2498, 2360, 2140, 2045, 1938,
1742, 1661, 1647, 1587, 1476, 1445, 1400, 1372, 1331, 1261, 1173,
1096, 1080, 1035, 979, 917, 852, 807, 687, 610 cm-1 1H NMR
;
(acetone-d6, 500 MHz) data, see Table 1; 13C NMR (acetone-d6, 150
MHz) data, see Table 2; ESIMS m/z 473 [M + H]+ and 495 [M +
Na]+; HRESIMS m/z 495.3478 [M + Na]+ (calcd for C30H48O4Na,
495.3445).
Acknowledgment. Financial support from the National Natural
Sciences Foundation of China (NNSFC; grant no. 30825044), the
Program for Changjiang Scholars and Innovative Research Team in
University (PCSIRT, grant no. IRT0514), and the National “973”
Program of China (grant nos. 2004CB13518906 and 2006CB504701)
is acknowledged.
(-)-(23E)-Tirucalla-8,23-diene-3ꢀ,25-diol-7-one (5): white, amor-
phous solid; [R]20 -2.5 (c 0.04, MeOH); UV (MeOH) λmax (log ꢀ)
D
202 (3.13), 254 (2.01) nm; CD (MeOH) 340 (∆ꢀ +0.79), 258 (∆ꢀ
-1.35), 236 (∆ꢀ -0.66), 221 (∆ꢀ -1.03), 206 (∆ꢀ -0.37); IR νmax
3335, 2962, 2932, 1642, 1584, 1454, 1414, 1370, 1333, 1268, 1185,

1626 Journal of Natural Products, 2009, Vol. 72, No. 9
Xu et al.
Supporting Information Available: Figure S1, main 1H-1H COSY
(thick lines) and HMBC (arrows) correlations of 1, 2, and 6 and main
HMBC (arrows) correlations of the side chain of 4. Figure S2,
conformation and octant projection diagram of 2. CD, HRMS, 1H and
13C NMR, 1H-1H COSY, HMQC, HMBC, and NOE spectra of
compounds 1-4, 6, and 8. CD, HRMS, 1H and 13C NMR of 5, 7, and
9. 1H NMR and 1H-1H COSY of 1a-1d and 2a. This material is
available free of charge via the Internet at http://pubs.acs.org.
(18) He, S. L.; Chen, R. Y. Chin. Tradit. Herbal Drugs 2005, 36, 662–
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(19) Talapatra, S. K.; Pal, P.; Das, G.; Biswas, K.; Porel, A.; Talapatra, B.
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