Cytotoxic dihydroagarofuranoid sesquiterpenes from the seeds of Celastrus orbiculatus

Article abstract of DOI:10.1021/np800052d

A chemical study on the seeds of Celastrus orbiculatus has led to the isolation of nine new (1-9) and 13 known dihydro-β-agarofuran derivatives. The identification and structural elucidation of the new compounds were based on spectroscopic data analysis, and the absolute configurations of compounds 1-6, 8-10, and 16, as well as derivatives 2a and 6a, were determined by CD studies or by chemical methods. All compounds isolated were evaluated for cytotoxic activity against HL-60 human leukemia cells.

Full text of DOI:10.1021/np800052d

J. Nat. Prod. 2008, 71, 1005–1010
1005
Cytotoxic Dihydroagarofuranoid Sesquiterpenes from the Seeds of Celastrus orbiculatus
Yingdong Zhu, Zehong Miao, Jian Ding, and Weimin Zhao*
Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203,
People’s Republic of China
ReceiVed January 23, 2008
A chemical study on the seeds of Celastrus orbiculatus has led to the isolation of nine new (1-9) and 13 known
dihydro-ꢀ-agarofuran derivatives. The identification and structural elucidation of the new compounds were based on
spectroscopic data analysis, and the absolute configurations of compounds 1-6, 8-10, and 16, as well as derivatives
2a and 6a, were determined by CD studies or by chemical methods. All compounds isolated were evaluated for cytotoxic
activity against HL-60 human leukemia cells.
Celastrus orbiculatus Thunb. (Celastraceae) is a perennial shrub
that has been used in Chinese folk medicine as a treatment for
rheumatoid arthritis and bacterial infections.1 The family Celas-
traceae is well known for producing various dihydro-ꢀ-agarofuran
derivatives, which have attracted much interest due to their broad
range of biological activities such as insecticidal,2 reversal of the
multidrug resistance (MDR) phenotype,3,4 cytotoxic,5 antitumor-
promoting,6 antitubercular,7 immunosuppressive,8 and anti-inflam-
matory effects.9 As part of an ongoing search for new bioactive
metabolites from plants used in traditional Chinese medicine, a
chemical investigation has been undertaken on the seeds of C.
orbiculatus. Herein, we report the isolation and structural elucidation
of nine new sesquiterpenes (1-9) and 13 known secondary
metabolites, along with their cytotoxic activity against HL-60 human
leukemia cells.
with petroleum ether and EtOAc, yielding petroleum ether and
EtOAc fractions. The two fractions were subjected to a series of
chromatographic steps to afford nine new dihydro-ꢀ-agarofuran
derivatives (1-9) and 13 known metabolites.
Compound 1, isolated as a white, amorphous powder, showed
an accurate [M + Na]+ ion at m/z 629.2727 in the HRESIMS,
corresponding to the molecular formula C35H42O9Na. It displayed
IR absorptions indicative of the presence of ester groups at 1743
and 1727 cm-1. The UV spectrum exhibited an absorption
maximum at 274 nm, which suggested the existence of aromatic
moieties.7 The H NMR spectrum of 1 (Table 1) indicated the
1
presence of signals due to one acetyl group at δ 2.12 (3H, s), two
benzoyl groups at δ 7.61 (2H, d, J ) 7.4 Hz), 6.92 (2H, t, J ) 7.4
Hz), 7.18 (1H, t, J ) 7.4 Hz), and 7.59 (2H, d, J ) 7.4 Hz), 7.10
(2H, t, J ) 7.4 Hz), and 7.32 (1H, t, J ) 7.4 Hz), respectively, and
also signals of a butyrate group at δ 0.88 (3H, t, J ) 7.6 Hz), 1.62
(2H, m), and 2.34 (2H, t, J ) 7.6 Hz), with those assignments
1
supported by H-1H COSY and HMBC correlations (Figure 1).
In addition, resonances belonging to acylated oxymethine protons
at δ 5.96 (1H, s), 5.67 (1H, d, J ) 4.6 Hz), 5.54 (1H, dd, J ) 11.2,
4.0 Hz), and 5.52 (1H, brs), two sets of typical methylene protons
at δ 1.80 and 1.78 (both 1H, m), and δ 2.20 and 1.48 (1H, m,
each), and four characteristic methyl groups appearing as a doublet
at δ 1.10 (3H, d, J ) 7.2 Hz) and three singlets at δ 1.44, 1.59,
and 1.61 (3H, s, each) were also observed. The 13C NMR spectrum
of 1 (Table 2) indicated 35 carbon signals separated by DEPT
experiments into four carbonyls at δ 172.3, 169.9, 165.5, and 164.8,
three quaternary sp3 carbons with two linked to an oxygen atom,
two quaternary sp2 carbons, 16 tertiary carbons comprising six sp3
carbons with four linked to an oxygen atom and 10 sp2 carbons,
four secondary sp3 carbons, and six methyl carbons. The complete
assignments of the protonated carbons were made from the HSQC
spectrum, while a detailed analysis of the 1H-1H COSY and HMBC
spectra of 1 led to the establishment of a tetrasubstituted dihydroa-
garofuran sesquiterpene for the structure of 1 (Figure 1). The
regiosubstitution of the ester functions was determined by HMBC
correlations of the carbonyl signals of the benzoate groups at δ
164.8 and 165.5 with signals at δ 5.67 (H-9) and 5.54 (H-1) and
of the carbonyl signal of the acetate group at δ 169.9 with the signal
at δ 5.96 (H-6), while the carbonyl signal of the butyrate group at
δ 172.3 correlated with the signal at δ 5.52 (H-8). The relative
configuration of 1 was established on the basis of a ROESY
experiment (Figure 2), in which NOE effects were found between
Me-14 and H-6 and Me-15, between H-2′′ (δ 1.62) of the C-8
butyrate group and H-6, between Me-12 and H-8 and H-9, and
between H-9 and H-1. The absolute configuration of 1 was
confirmed by the dibenzoate chirality method, an extension of the
circular dichroism exciton chirality method,10 which showed a
Davidoff-type split curve with a first Cotton effect at 239.7 nm
Results and Discussion
Powdered, air-dried seeds of C. orbiculatus (10.0 kg) were
extracted with 95% EtOH at room temperature (3 × 72 h). After
removal of solvent, the aqueous residue was partitioned in sequence
* Corresponding author. Tel & Fax: 86-21-50806052. E-mail: wmzhao@
mail.shcnc.ac.cn.
10.1021/np800052d CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 05/29/2008

1006 Journal of Natural Products, 2008, Vol. 71, No. 6
Zhu et al.
Table 1. 1H NMR Spectroscopic Data of 1-9 (400 MHz, CDCl3)a
position
1
1
2
3
4
5
6
7
8
9
5.54 dd
(11.2, 4.0)
1.80 m
1.78 m
1.48 m
2.20 m
2.26 m
5.96 s
2.50 d (4.2)
5.52 brs
5.67 d (4.6)
1.59 s
4.08 dd
4.08 dd
(11.0, 4.4)
1.61 m
1.47 m
1.39 m
2.00 m
2.16 m
6.16 s
2.52 d (4.5)
5.58 dd
(10.3, 5.7)
1.82 m
1.80 m
1.52 m
2.24 m
2.32 m
5.16 s
2.58 brs
5.78 brs
5.78 brs
1.65 s
5.36 dd
(11.0, 4.8)
1.68 m
1.64 m
1.42 m
2.12 m
2.19 m
6.15 s
2.45 d (4.4)
4.38 t (4.4)
5.50 d (4.4)
1.46 s
5.50 d (3.3)
5.49 d (3.5)
5.52 dd
(12.0, 4.3)
5.46 dd
(11.4, 3.9)
1.61 m
(12.0, 4.3)
1.62 m
1.50 m
1.42 m
2.12 m
2.28 m
4.41 s
2R
4.39 brd (2.7)
5.55 dd (3.5, 3.1) 1.96 m
1.48 m
2ꢀ
1.50 m
1.39 m
2.00 m
2.15 m
3R
1.82 m
2.26 m
2.25 m
5.39 s
2.00 m
2.10 m
1.89 m
2.20 m
2.45 m
5.49 s
3ꢀ
4
6
7
8
5.88 s
2.42 m 1.85 m
2.06 m
2.55 d (4.5)
5.50 dd (4.5, 5.0) 4.39 t (4.5)
5.63 d (5.0)
1.54 s
2.17 brs
2.43 brs
2.11 brs
2.36 m 2.08 m 2.18 m 2.04 m
4.78 d (6.9)
1.38 s
2.50 m 2.22 m 2.24 m 2.14 m
5.10 d (6.9)
1.47 s
9
12
5.58 d (4.5)
1.47 s
4.81 d (6.2)
1.45 s
4.98 d (6.4)
1.40 s
13
1.44 s
1.38 s
1.40 s
1.59 s
1.39 s
1.36 s
1.32 s
1.47 s
1.52 s
14
15
OAc-1
OAc-2
OAc-6
OAc-8
1.10 d (7.2)
1.61 s
1.01 d (7.5)
1.38 s
1.01 d (7.3)
1.39 s
1.31 d (7.3)
1.70 s
1.02 d (7.1)
1.56 s
1.24 d (7.3)
1.49 s
1.87 s
1.43 s
1.38 s
1.82 s
2.05 s
3.55 m 3.61 m 1.19 d (7.2)
1.15 s
1.62 s
1.32 s
1.60 s
2.12 s
2.07 s
2.08 s
2.10 s
2.15 s
2.07 s
a Data for additional ester groups are provided in the Experimental Section.
effect at 241.4 nm and a second positive effect at 221.7 nm. Therefore,
2 was established as (1S,4R,5S,6R,7R,8R,9S,10S)-6,8-diacetoxy-9-
benzoyloxy-1-hydroxydihydro-ꢀ-agarofuran.
Compounds 3 and 4 were assigned the molecular formulas
C24H32O7 and C36H38O8, respectively, as deduced from their
HRESIMS and NMR data. The NMR spectroscopic data (Tables 1
and 2) revealed that compounds 3 and 4 both possessed an identical
dihydro-ꢀ-agarofuran skeleton to that of 2. A difference in the 1H
NMR spectrum of 3 was due to an additional hydroxyl group at
C-8 instead of an acetate group in 2. Thus, 3 was determined as
the 8-deacetyl derivative of 2. Similarly, the 1H NMR and 13C NMR
spectroscopic data of 4 corresponded to those of 2 except that two
additional benzoate groups signals appeared in 4, and no acetate
group signals were present. An analysis of the NMR spectra of 4
revealed that 4 is the 1,8-dibenzoyloxy-6-hydroxy derivative of 2.
The relative configurations of compounds 3 and 4 were resolved
by analysis of the coupling constants and confirmed by ROESY
experiments.
Benzoylation of 3 yielded the known derivative 10. The absolute
configuration of 10 was determined by CD studies, with the curve
showing a first negative Cotton effect at 236.5 nm and a second
positive one at 221.9 nm. As a result, the structure and absolute
configuration of 3 were proposed as (1S,4R,5S,6R,7R,8R,9S,10S)-
6-acetoxy-9-benzoyloxy-1,8-dihydroxydihydro-ꢀ-agarofuran. The
CD spectrum of 4 showed a very close curve to that of 10,
supporting the structure and absolute configuration assignment as
(1S,4R,5S,6R,7R,8R,9S,10S)-1,8,9-tribenzoyloxy-6-hydroxydihydro-
ꢀ-agarofuran.
Compound 5 gave a molecular formula of C33H38O8, as deduced
from its HRESIMS and NMR data. Examination of the NMR spectra
(Tables 1 and 2) revealed that this compound was a trisubstituted
dihydro-ꢀ-agarofuran sesquiterpene with the presence of a free tertiary
hydroxyl and a cinnamyl group [δ 6.92 (2H, d, J ) 7.5 Hz), 7.16
(2H, t, J ) 7.5 Hz), and 7.25 (1H, t, J ) 7.5 Hz), and δ 7.22 (1H, d,
J ) 15.9 Hz) and 5.68 (1H, d, J ) 15.9 Hz)]. The HMBC experiment
established the regiosubstitution in the molecule of 5, and the relative
stereochemistry was resolved by analysis of coupling constants and a
ROESY experiment, which showed 5 to be the 1-cinnamyloxy
derivative of 3. The CD spectrum of 5 showed a split curve very similar
to that of 1, and its absolute configuration was accordingly established
as (1S,4R,5S,6R,7R,8R,9S,10S)-6-acetoxy-9-benzoyloxy-1-cinnamyl-
oxy-8-hydroxydihydro-ꢀ-agarofuran.
Figure 1. Main 1H-13C long-range correlation (1Hc13C) and
1H-1H correlation (s) signals in the HMBC and COSY spectra
of 1, 2, 6, and 8.
and a second one at 223.3 nm, due to the couplings of the two
benzoate chromophores at C-1R and C-9R. Thus, the structure and
absoluteconfigurationof1wereassignedas(1S,4R,5S,6R,7R,8R,9S,10S)-
6-acetoxy-1,9-dibenzoyloxy-8-butyryloxydihydro-ꢀ-agarofuran.
Compound 2, purified as a white, amorphous powder, gave the
molecular formula C26H34O8, as deduced from the HRESIMS and
NMR analysis. The NMR data (Tables 1 and 2) of 2 revealed 2 was
very similar to those of 1 except that one benzoate group at C-1 in 1
was displaced by one free hydroxyl group in 2, and one additional
acetate group at C-8 in 2 appeared, instead of one butyrate group in 1.
The HMBC experiment (Figure 1) established the regiosubstitution
in the molecule of 2, and the relative configuration was resolved by
analysis of a ROESY experiment (Figure 2). Thus compound 2 was
assigned as the 8-acetoxy-1-debenzoyl derivative of 1. To determine
the absolute configuration of 2, it was necessary to introduce another
chromophoric group. Benzoylation of 2 yielded the benzoate derivative,
2a, which was suitable for applying the dibenzoate chirality method.10
Its CD spectrum showed a split curve with a first negative Cotton
Compounds 6 and 7 were both assigned the molecular formula
C28H36O8 by HRESIMS. The 1H and 13C NMR data (Tables 1 and
2) of 6 and 7 indicated that these two compounds were triesterified
dihydro-ꢀ-agarofuran sesquiterpenes with the presence of free
hydroxy groups. The HMBC experiments (Figure 1) established

Cytotoxic Dihydroagarofuranoid Sesquiterpenes from C. orbiculatus
Journal of Natural Products, 2008, Vol. 71, No. 6 1007
Table 2. 13C NMR Spectroscopic Data of 1-9 (100 MHz, CDCl3)a
position
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
79.0 d
22.3 t
26.5 t
33.8 d
91.1 s
75.1 d
52.5 d
71.0 d
74.4 d
49.1 s
81.7 s
24.1 q
30.6 q
16.8 q
12.2 q
76.2 d
25.6 t
26.7 t
33.9 d
91.1 s
75.3 d
52.4 d
71.6 d
75.2 d
49.6 s
81.3 s
24.0 q
30.6 q
16.7 q
10.7 q
76.4 d
25.7 t
26.9 t
34.0 d
91.5 s
75.0 d
54.3 d
70.4 d
77.7 d
49.5 s
81.1 s
24.1 q
30.8 q
16.8 q
11.1 q
79.5 d 79.0 d
73.6 d
69.0 d
32.5 t
33.7 d
89.8 s
79.1 d
48.6 s
31.4 t
72.9 d
49.6 s
82.4 s
25.9 q
30.6 q
18.9 q
20.7 q
70.0 d
69.2 d
40.6 t
69.5 s
90.3 s
30.9 t
43.4 d
30.5 t
73.1 d
47.5 s
83.8 s
24.3 q
30.0 q
25.5 q
20.6 q
73.3 d
22.1 t
22.7 t
44.9 d
88.8 s
80.2 d
48.8 d
32.3 t
72.6 d
50.2 s
82.6 s
26.0 q
30.7 q
62.6 t
17.9 q
74.1 d
21.6 t
26.9 t
33.7 d
91.4 s
78.1 d
50.8 d
32.4 t
73.8 d
50.1 s
82.6 s
26.3 q
31.0 q
18.1 q
19.0 q
22.4 t
26.7 t
21.9 t
26.5 t
33.6 d 33.7 d
92.5 s 91.1 s
73.1 d 74.6 d
54.5 d 54.2 d
72.4 d 69.8 d
74.6 d 76.4 d
48.6 s 48.7 s
82.0 s 81.2 s
24.5 q 24.0 q
31.1 q 30.6 q
17.4 q 16.7 q
12.6 q 12.3 q
OAc-1
OAc-2
OAc-6
OAc-8
20.9 q 170.0 s 20.6 q 170.0 s 20.8 q 169.9 s 20.8 q 170.0 s
21.2 q 169.8 s
21.2 q 169.8 s 21.3 q 170.0 s
21.3 q 169.9 s 21.2 q 169.9 s 21.4 q 169.9 s
20.9 q 169.9 s
a Data for additional ester groups are provided in the Experimental Section.
Figure 2. Main NOE correlation signals (T) in the ROESY spectra of 1, 2, 6, and 8, and CD exciton coupling (dashed arrow) for 1.
the regiosubstitution in the molecules of 6 and 7, and their relative
stereochemistry was resolved by analysis of coupling constants and
ROESY experiments (Figure 2). The hydroxyl group in 6 was
located at C-2 based on the HMBC cross-peak between C-2 at δ
69.0 and H-4 at δ 2.25 and between H-2 at δ 4.39 and C-10 at δ
49.6. The hydroxy group in 7 was attached to C-4 on the basis of
the HMBC correlations between the hydroxyl proton at δ 2.76 (OH-
4) and carbons at δ 69.5 (C-4) and 25.5 (C-14). Accordingly, the
structure 7 was deduced as 1R,2R-diacetoxy-9ꢀ-cinnamyloxy-4ꢀ-
hydroxydihydro-ꢀ-agarofuran.
the benzoate derivative, 6a. The CD spectrum of 6a showed a broad
positive Cotton effect at 276.3 nm, while the second maximum
could not be observed due possibly to the strong positive absorption
overlaying background ellipticity.10 Thus, the structure and absolute
configuration of 6 were proposed as (1R,2S,4R,5S,6R,7R,9S,10R)-
1,6-diacetoxy-9-cinnamyloxy-2-hydroxydihydro-ꢀ-agarofuran.
Compounds 8 and 9 were assigned the molecular formulas C31H36O8
and C24H32O6, respectively, as deduced from their HRESIMS and
NMR data. The 1H and 13C NMR data (Tables 1 and 2) indicated that
compound 8 was a triesterified dihydro-ꢀ-agarofuran sesquiterpene with
one acetate, two benzoyl, and one secondary hydroxyl group, and 9
was a diesterified dihydro-ꢀ-agarofuran sesquiterpene with one acetate,
To determine the absolute configuration of 6, it was necessary
to introduce another chromophoric group. Benzoylation of 6 yielded

1008 Journal of Natural Products, 2008, Vol. 71, No. 6
Zhu et al.
DU-7 spectrometer. IR spectra were recorded using a Perkin-Elmer
577 spectrometer. LRESIMS were measured using a Finnigan LCQ-
Deca instrument, and HRESIMS data were obtained on a Mariner mass
spectrometer. NMR experiments were run on a Bruker AM 400
spectrometer with TMS as internal standard. Preparative HPLC was
carried out using a Varian SD-1 instrument equipped with a Merck
NW25 C18 column (12 µM, 20 mm × 250 mm) and ProStar 320 UV/
vis detector. Column chromatographic (CC) separations were performed
using silica gel H60 (300-400 mesh), zcx-II (100-200 mesh) (Qingdao
Haiyang Chemical Group Corporation, Qingdao, People’s Republic of
China), ODS (40-63 µM) (Merck), and Sephadex LH-20 (Pharmacia
Biotech AB, Uppsala, Sweden) as packing materials. HSGF254 silica
gel TLC plates (Yantai Chemical Industrial Institute, Yantai, People’s
Republic of China) and RP-18 WF254 TLC plates (Merck) were used
for analytical TLC.
Table 3. Cytotoxic Activity of Compounds 1, 5, and 11-16
against the HL-60 Human Leukemia Cell Line
compound
IC50 (µM)
1
5
11
12
13
14
15
5.3
8.3
6.8
2.8
6.8
3.3
7.2
1.9
0.2
16
etoposidea
a Etoposide was used as a positive control.
Plant Material. The seeds of C. orbiculatus were collected in a
suburb of Liaoyuan, Jilin Province, People’s Republic of China, in
January 2007, and identified by Professor Jingui Shen of Shanghai
Institute of Materia Medica, Chinese Academy of Sciences. A voucher
specimen (no. 20061202) is deposited at the Herbarium of Shanghai
Institute of Materia Medica, Chinese Academy of Sciences.
one benzoyl, and one tertiary hydroxyl group. The HMBC experiments
(Figure 1) established the regiosubstitution in the molecules of 8 and
9, and their relative stereochemistry was resolved by analysis of
coupling constants and ROESY experiments (Figure 2). The hydroxyl
group in 8 was sited at C-14 on the basis of the HMBC cross-peaks
between the carbon at δ 62.6 (C-14) and the proton at δ 2.45 (H-4)
and between protons at δ 3.61, 3.55 (H2-14) and the carbon at δ 22.7
(C-3). The hydroxyl group in 9 was established at C-6 on the basis of
the HMBC correlations between the carbon at δ 78.1 (C-6) and the
proton at δ 2.24, 2.14 (H-8) and between the proton at δ 4.41 (H-6)
and carbons at δ 82.6 (C-11) and 32.4 (C-8). The CD spectrum of 8
displayed a weak split curve with a first positive Cotton effect at 242.7
nm and a second negative one at 221.4 nm ascribable to the
homobenzoate interaction at C-6ꢀ and C-9ꢀ, providing its structure
and absolute configuration as (1S,4S,5S,6R,7R,9S,10S)-1-acetoxy-6,9-
dibenzoyloxy-14-hydroxydihydro-ꢀ-agarofuran. Cinnamylation of 9
gave the known compound 16, which was suitable for applying the
dibenzoate chirality method. The CD spectrum of 16 showed a split
curve with a first positive Cotton effect at 279.1 nm and a second
negative one at 235.0 nm. Thus, the structure and the absolute
configuration of 9 were accordingly deduced as (1S,4R,5S,6R,7R,9S,10S)-
1-acetoxy-9-benzoyloxy-6-hydroxydihydro-ꢀ-agarofuran.
In addition to the nine new dihydro-ꢀ-agarofuran derivatives
(1-9), 13 known metabolites were also isolated and characterized
by comparison with literature data as 6ꢀ-acetoxy-1R,8R,9R-
tribenzoyloxydihydro-ꢀ-agarofuran (10),11 celafolin C-1 (11),12 9R-
acetoxy-1ꢀ,6R-dibenzoyloxydihydro-ꢀ-agarofuran (12),13 1ꢀ-acetoxy-
9R-cinnamyloxydihydro-ꢀ-agarofuran (13),13 2R,9ꢀ-diacetoxy-1R-
cinnamyloxydihydro-ꢀ-agarofuran (14),14 6ꢀ-acetoxy-1R,8R-diben-
zoyloxy-9R-hydroxydihydro-ꢀ-agarofuran (15),15 celafolin A-1
(16),12 celafolin B-3,12 6ꢀ,9ꢀ-diacetoxy-1R-benzoyloxydihydro-ꢀ-
agarofuran,14 1R,6R,14-triacetoxy-9ꢀ-benzoyloxydihydro-ꢀ-agaro-
furan,16 triptogelin B-1,17 celafolin B-1,12 and 6ꢀ-acetoxy-8R,9R-
dibenzoyloxy-1R,2R-dihydroxydihydro-ꢀ-agarofuran.18
To test the potential anticancer activities of all the isolates
obtained, we used a standard in vitro cytotoxicity evaluation system,
the HL-60 cell line, to analyze their cytotoxic activities by MTT
assays. As a result, the new compounds 1 and 5 and known
sesquiterpene derivatives 11-16 were observed to exhibit cytotoxic
activities with IC50 values ranging from 1.9 to 8.3 µM (Table 3),
while the others exhibited less than 50% of cell growth inhibition
at a concentration of up to 10 µM. Preliminary analysis of the
structure-activity relationship from these natural sesquiterpenes
revealed that compounds with a hydroxy group at C-6, C-8, or C-9
(4, 5, 9, and 15) had a slightly decreased cytotoxicity, while
compounds with a free hydroxy group at C-1, C-2, or C-14 showed
no such activity, as deduced from 2, 6, 8, triptogelin B-1, celafolin
B-1, celafolin B-3, and 6ꢀ-acetoxy-8R,9R-dibenzoyloxy-1R,2R-
dihydroxydihydro-ꢀ-agarofuran.
Extraction and Isolation. Powdered and air-dried seeds of C.
orbiculatus (10.0 kg) were percolated with 95% EtOH at room
temperature (3 × 72 h). The solvents were evaporated in vacuo, and
the residue was suspended in H2O and then partitioned with petroleum
ether and EtOAc (2 L × 3 each), successively, yielding petroleum ether
(1.1 kg) and EtOAc (20.5 g) extracts. The petroleum ether-soluble
fraction (1.1 kg) was subjected to silica gel CC eluting with a gradient
of petroleum ether and acetone (100:1 to 0:1), and six fractions (F1-F6)
were obtained. F2 (135.2 g) was chromatographed on silica gel eluting
with petroleum ether-acetone (P-A) (40:1) to give 12 (100.1 mg)
and 13 (1.0 g). Then, F3 (120.1 g) was separated into four subfractions
(F31-F34) by CC eluting with P-A (40:1). F33 (50.5 g) and F34 (10.5
g) were subjected to CC over silica gel eluting with P-A (40:1),
followed by preparative HPLC using a gradient of MeOH-H2O (70%
to 100% over 80 min, 10 mL ·min-1) to afford 6ꢀ,9ꢀ-diacetoxy-1R-
benzoyloxydihydro-ꢀ-agarofuran (200.1 mg) and 16 (150.2 mg), and
11 (1.3 g), respectively. F4 (80.2 g) was purified by a combination of
silica gel CC eluting with P-A (40:1) and preparative HPLC, using a
gradient of MeOH-H2O (70% to 100% over 80 min, 10 mL ·min-1),
as well as by preparative TLC (CHCl3-acetone, 100:1), to yield 1 (41.2
mg), 10 (80.2 mg), 1R,6R,14-triacetoxy-9ꢀ-benzoyloxydihydro-ꢀ-
agarofuran (160.3 mg), and 14 (630.2 mg). F6 (60.2 g) was separated
by ODS CC eluting with a gradient of MeOH-H2O (1:1, 7:3, 9:1, and
1:0) to give three subfractions (F61-F63). F61 (1.5 g) was chromato-
graphed on silica gel eluting with P-A (10:1) and then purified by
preparative TLC (CHCl3-acetone, 20:1) to obtain 2 (26.9 mg). F62
(15.3 g) was subjected to silica gel CC eluting with CHCl3-acetone
(100:1), and four fractions (F621-F624) were obtained. F621 (3.2 g) was
separated by a combination of silica gel CC eluting with P-A (10:1)
and preparative HPLC using a gradient of MeOH-H2O (60% to 100%
over 80 min, 10 mL · min-1) and further by preparative TLC
(CHCl3-acetone, 40:1) to give triptogelin B-1 (144.2 mg). Compounds
4 (7.0 mg), 5 (113.2 mg), 7 (15.8 mg), 9 (3.0 mg), and 15 (49.5 mg)
were obtained from F623 (1.4 g) by using the same steps as described
for F621. F624 (1.0 g) was purified by preparative HPLC using a gradient
of MeOH-H2O (60% to 100% over 70 min, 10 mL ·min-1) followed
by CC eluting with P-A (8:1) and then passed through a Sephadex
LH-20 column with ethanol as eluent, to afford 6 (90.2 mg), celafolin
B-1 (30.1 mg), and celafolin B-3 (11.2 mg). The EtOAc extract (20.5
g) was subjected to silica gel CC eluting with P-A (10:1), and four
fractions (F1-F4) were obtained. Purification of F4 (9.6 g) by repeated
preparative HPLC using a gradient of MeOH-H2O (40% to 100% over
80 min, 10 mL ·min-1) and further by preparative TLC (CHCl3-MeOH,
50:1) resulted in the isolation of compounds 3 (10.2 mg), 8 (6.0 mg),
and 6ꢀ-acetoxy-8R,9R-dibenzoyloxy-1R,2R-dihydroxydihydro-ꢀ-aga-
rofuran (15.2 mg).
(1S,4R,5S,6R,7R,8R,9S,10S)-6-Acetoxy-1,9-dibenzoyloxy-8-bu-
20
tyryloxydihydro-ꢀ-agarofuran (1): white, amorphous powder; [R]D
-35.0 (c 0.24, CHCl3); UV (EtOH) λmax (log ꢀ) 227 (4.34), 274 (3.46)
nm; CD (MeOH) λext (∆ꢀ) 239.7 (-7.71), 223.3 (+14.56) nm; IR (KBr)
Experimental Section
ν
max 2968, 1743, 1727, 1452, 1382, 1280, 1226, 1112, 1093, 962, 707
General Experimental Procedures. Optical rotations were measured
with a Perkin-Elmer 241MC instrument. CD spectra were recorded on
a JASCO J-810 spectrometer. UV spectra were obtained on a Beckman
cm-1; 1H NMR (400 MHz, CDCl3) δ OBz-1 [7.61 (2H, d, J ) 7.4 Hz,
H-2′/6′), 6.92 (2H, t, J ) 7.4 Hz, H-3′/5′), and 7.18 (1H, t, J ) 7.4 Hz,

Cytotoxic Dihydroagarofuranoid Sesquiterpenes from C. orbiculatus
Journal of Natural Products, 2008, Vol. 71, No. 6 1009
H-4′)], OBut-8 [2.34 (2H, t, J ) 7.6 Hz, H-1′′), 1.62 (2H, m, H-2′′),
and 0.88 (3H, t, J ) 7.6 Hz, H-3′′)], and OBz-9 [7.59 (2H, d, J ) 7.4
Hz, H-2′′′/6′′′), 7.10 (2H, t, J ) 7.4 Hz, H-3′′′/5′′′), and 7.32 (1H, t, J
) 7.4 Hz, H-4′′′)], for other signals, see Table 1; 13C NMR (100 MHz,
CDCl3) δ OBz-1 [129.8 (s, C-1′), 129.2 (d, C-2′/6′), 127.5 (d, C-3′/5′),
132.2 (d, C-4′), and 165.5 (s, CO2-1)], OBut-8 [36.4 (t, C-1′′), 18.3 (t,
C-2′′), 13.7 (q, C-3′′), and 172.3 (s, CO2-8)], and OBz-9 [129.6 (s,
C-1′′′), 129.1 (d, C-2′′′/6′′′), 127.8 (d, C-3′′′/5′′′), 132.4 (d, C-4′′′), and
164.8 (s, CO2-9)], for other signals, see Table 2; ESIMS m/z 629 [M
+ Na]+; HRESIMS m/z 629.2727 [M + Na]+ (calcd for C35H42O9Na,
629.2727).
NMR (100 MHz, CDCl3) δ OBz-1 [129.9 (s, C-1′), 129.1 (d, C-2′/6′),
127.5 (d, C-3′/5′), 132.1 (d, C-4′), and 165.5 (s, CO2-1)], OBz-8 [130.0
(s, C-1′′), 129.6 (d, C-2′′/6′′), 128.5 (d, C-3′′/5′′), 133.2 (d, C-4′′), and
165.3 (s, CO2-8)], and OBz-9 [129.6 (s, C-1′′′), 129.1 (d, C-2′′′/6′′′),
127.7 (d, C-3′′′/5′′′), 132.2 (d, C-4′′′), and 164.8 (s, CO2-9)], for other
signals, see Table 2; ESIMS m/z 621 [M + Na]+; HRESIMS m/z
621.2455 [M + Na]+ (calcd for C36H38O8Na, 621.2464).
(1S,4R,5S,6R,7R,8R,9S,10S)-6-Acetoxy-9-benzoyloxy-1-cinnamyl-
oxy-8-hydroxydihydro-ꢀ-agarofuran (5): white, amorphous powder;
[R]D20 -12.0 (c 0.27, CHCl3); UV (EtOH) λmax (log ꢀ) 223 (4.40), 281
(4.32) nm; CD (MeOH) λext (∆ꢀ) 251.5 (-8.11), 227.8 (+26.96) nm;
IR (KBr) νmax 2931, 1718, 1637, 1450, 1328, 1282, 1251, 1091, 1027,
979, 711 cm-1;1H NMR (400 MHz, CDCl3) δ OCin-1 [6.92 (2H, d, J
) 7.5 Hz, H-2′/6′), 7.16 (2H, t, J ) 7.5 Hz, H-3′/5′), 7.25 (1H, t, J )
7.5 Hz, H-4′), 5.68 (1H, d, J ) 15.9 Hz, H-R), and 7.22 (1H, d, J )
15.9 Hz, H-ꢀ)], and OBz-9 [7.93 (2H, d, J ) 7.8 Hz, H-2′′/6′′), 7.19
(2H, t, J ) 7.8 Hz, H-3′′/5′′), and 7.27 (1H, t, J ) 7.8 Hz, H-4′′)], for
other signals, see Table 1; 13C NMR (100 MHz, CDCl3) δ OCin-1
[133.9 (s, C-1′), 127.7 (d, C-2′/6′), 128.2 (d, C-3′/5′), 129.7 (d, C-4′),
117.9 (d, C-R), 143.9 (d, C-ꢀ), and 166.0 (s, CO2-1)], and OBz-9 [129.8
(s, C-1′′), 129.5 (d, C-2′′/6′′), 128.2 (d, C-3′′/5′′), 132.6 (d, C-4′′), and
165.0 (s, CO2-9)], for other signals, see Table 2; ESIMS m/z 585 [M
+ Na]+; HRESIMS m/z 585.2460 [M + Na]+ (calcd for C33H38O8Na,
585.2464).
(1S,4R,5S,6R,7R,8R,9S,10S)-6,8-Diacetoxy-9-benzoyloxy-1-hy-
20
droxydihydro-ꢀ-agarofuran (2): white, amorphous powder; [R]D
-59.0 (c 0.14, CHCl3); UV (EtOH) λmax (log ꢀ) 229 (4.18), 273 (3.11)
nm; IR (KBr) νmax 3533, 2925, 1747, 1708, 1452, 1369, 1282, 1236,
1097, 1035, 962, 711 cm-1; H NMR (400 MHz, CDCl3) δ OBz-9
1
[7.98 (2H, d, J ) 7.7 Hz, H-2′/6′), 7.40 (2H, t, J ) 7.7 Hz, H-3′/5′),
and 7.55 (1H, t, J ) 7.7 Hz, H-4′)], for other signals, see Table 1; 13
C
NMR (100 MHz, CDCl3) δ OBz-9 [130.1 (s, C-1′), 129.5 (d, C-2′/6′),
128.4 (d, C-3′/5′), 133.0 (d, C-4′), and 165.8 (s, CO2-9)], for other
signals, see Table 2; ESIMS m/z 497 [M + Na]+; HRESIMS m/z
497.2128 [M + Na]+ (calcd for C26H34O8Na, 497.2151).
Benzoylation of 2. Compound 2 (5.0 mg) was dissolved in dry
pyridine (0.5 mL), and benzoyl chloride (6 drops) and a catalytic amount
of 4-(dimethylamino)pyridine were added. Then, the mixture was stirred
at room temperature for 48 h, poured over H2O, extracted with EtOAc,
and purified by preparative TLC with a solvent of petroleum
ether-EtOAc (5:1), to give compound 2a (4.0 mg, Rf 0.28).
(1R,2S,4R,5S,6R,7R,9S,10R)-1,6-Diacetoxy-9-cinnamyloxy-2-hy-
20
droxydihydro-ꢀ-agarofuran (6): white, amorphous powder; [R]D
+36.0 (c 0.24, CHCl3); UV (EtOH) λmax (log ꢀ) 279 (4.27) nm; IR
(KBr) νmax 3505, 2919, 1731, 1693, 1639, 1450, 1369, 1240, 1093,
1024, 979, 771 cm-1; H NMR (400 MHz, CDCl3) δ OCin-9 [7.55
1
(1S,4R,5S,6R,7R,8R,9S,10S)-6,8-Diacetoxy-1,9-dibenzoyloxydihy-
20
(2H, m, H-2′/6′), 7.38 (2H, m, H-3′/5′), 7.38 (1H, m, H-4′), 6.38 (1H,
d, J ) 16.0 Hz, H-R), and 7.70 (1H, d, J ) 16.0 Hz, H-ꢀ)], for other
signals, see Table 1; 13C NMR (100 MHz, CDCl3) δ OCin-9 [134.2 (s,
C-1′), 128.1 (d, C-2′/6′), 128.7 (d, C-3′/5′), 130.2 (d, C-4′), 117.9 (d,
C-R), 145.2 (d, C-ꢀ), and 165.9 (s, CO2-9)], for other signals, see Table
2; ESIMS m/z 523 [M + Na]+; HRESIMS m/z 523.2297 [M + Na]+
(calcd for C28H36O8Na, 523.2308).
Benzoylation of 6. Compound 6 (5.0 mg) was benzoylated under
the same conditions described above for compound 2, to give compound
6a (4.7 mg, Rf 0.31).
dro-ꢀ-agarofuran (2a): white, amorphous powder; [R]D -32.0 (c
0.10, CHCl3); CD (MeOH) λext (∆ꢀ) 241.4 (-13.58), 221.7 (+18.47)
1
nm; H NMR (400 MHz, CDCl3) δ 5.54 (1H, m, H-1), 1.80 (2H, m,
H-2), 2.20 (2H, m, H-3), 2.26 (1H, m, H-4), 5.95 (1H, s, H-6), 2.51
(1H, d, J ) 4.4 Hz, H-7), 5.52 (1H, m, H-8), 5.65 (1H, d, J ) 5.0 Hz,
H-9), 1.42 (3H, s, H-12), 1.58 (3H, s, H-13), 1.09 (3H, d, J ) 7.1 Hz,
H-14), 1.61 (3H, s, H-15), OAc-6 [2.12 (3H, s)], OAc-8 [2.10 (3H,
s)], OBz-1 [7.60 (2H, d, J ) 7.6 Hz, H-2′/6′), 6.91 (2H, t, J ) 7.6 Hz,
H-3′/5′), and 7.14 (1H, t, J ) 7.6 Hz, H-4′)], and OBz-9 [7.60 (2H, d,
J ) 7.6 Hz, H-2′′/6′′), 7.11 (2H, t, J ) 7.6 Hz, H-3′′/5′′), and 7.33
(1H, t, J ) 7.6 Hz, H-4′′)]; 13C NMR (100 MHz, CDCl3) δ 79.0 (d,
C-1), 22.3 (t, C-2), 26.6 (t, C-3), 33.9 (d, C-4), 91.1 (s, C-5), 75.1 (d,
C-6), 52.4 (d, C-7), 71.4 (d, C-8), 74.3 (d, C-9), 49.2 (s, C-10), 81.7
(s, C-11), 30.6 (q, C-12), 24.1 (q, C-13), 16.7 (q, C-14), 12.2 (q, C-15),
OAc-6 [21.3 (q), 169.9 (s, CO2-6)], OAc-8 [20.9 (q), 169.9 (s, CO2-
8)], OBz-1 [129.9 (s, C-1′), 129.2 (d, C-2′/6′), 127.5 (d, C-3′/5′), 132.1
(d, C-4′), and 165.5 (s, CO2-1)], and OBz-9 [129.6 (s, C-1′′), 129.1 (d,
C-2′′/6′′), 127.9 (d, C-3′′/5′′), 132.4 (d, C-4′′), and 164.8 (s, CO2-9)].
(1S,4R,5S,6R,7R,8R,9S,10S)-6-Acetoxy-9-benzoyloxy-1,8-dihy-
(1R,2S,4R,5S,6R,7R,9S,10R)-1,6-Diacetoxy-2-benzoyloxy-9-cin-
namyloxydihydro-ꢀ-agarofuran (6a): white, amorphous powder;
20
[R]D +31.0 (c 0.18, CHCl3); CD (MeOH) λext (∆ꢀ) 276.3 (+14.15)
1
nm; H NMR (400 MHz, CDCl3) δ 5.71 (1H, d, J ) 3.7 Hz, H-1),
5.85 (1H, brd, J ) 3.0 Hz, H-2), 1.95/2.40 (both 1H, m, H-3), 2.39
(1H, m, H-4), 5.44 (1H, s, H-6), 2.23 (1H, brs, H-7), 2.18/2.55 (each
1H, m, H-8), 4.78 (1H, d, J ) 7.1 Hz, H-9), 1.41 (3H, s, H-12), 1.42
(3H, s, H-13), 1.28 (3H, d, J ) 7.2 Hz, H-14), 1.58 (3H, s, H-15),
OAc-1 [1.80 (3H, s)], OAc-6 [2.12 (3H, s)], OBz-2 [7.98 (2H, d, J )
8.0 Hz, H-2′/6′), 7.45 (2H, t, J ) 8.0 Hz, H-3′/5′), and 7.56 (1H, t, J
) 8.0 Hz, H-4′)], and OCin-9 [7.55 (2H, m, H-2′′/6′′), 7.38 (2H, m,
H-3′′/5′′), 7.38 (1H, m, H-4′′), 6.38 (1H, d, J ) 16.0 Hz, H-R), 7.70
(1H, d, J ) 16.0 Hz, H-ꢀ)]; 13C NMR (100 MHz, CDCl3) δ 70.0 (d,
C-1), 70.5 (d, C-2), 30.9 (t, C-3), 33.0 (d, C-4), 88.9 (s, C-5), 78.4 (d,
C-6), 48.2 (d, C-7), 30.7 (t, C-8), 72.0 (d, C-9), 49.0 (s, C-10), 82.3 (s,
C-11), 30.0 (q, C-12), 25.2 (q, C-13), 18.2 (q, C-14), 19.9 (q, C-15),
OAc-1 [19.9 (q), 169.3 (s, CO2-1)], OAc-6 [20.6 (q), 169.2 (s, CO2-
6)], OBz-2 [129.8 (s, C-1′), 128.8 (d, C-2′/6′), 128.0 (d, C-3′/5′), 132.4
(d, C-4′), and 165.1 (s, CO2-2)], and OCin-9 [133.8 (s, C-1′′), 127.6
(d, C-2′′/6′′), 128.3 (d, C-3′′/5′′), 129.7 (d, C-4′′), 117.4 (d, C-R), 144.7
(d, C-ꢀ), and 165.4 (s, CO2-9)].
20
droxydihydro-ꢀ-agarofuran (3): white, amorphous powder; [R]D
-42.0 (c 0.19, CHCl3); UV (EtOH) λmax (log ꢀ) 229 (4.13), 273 (2.99)
nm; IR (KBr) νmax 3540, 2817, 1731, 1708, 1450, 1384, 1282, 1255,
1099, 1027, 962, 713 cm-1; H NMR (400 MHz, CDCl3) δ OBz-9
1
[8.03 (2H, d, J ) 7.9 Hz, H-2′/6′), 7.45 (2H, t, J ) 7.9 Hz, H-3′/5′),
and 7.56 (1H, t, J ) 7.9 Hz, H-4′)], for other signals, see Table 1; 13
C
NMR (100 MHz, CDCl3) δ OBz-9 [130.2 (s, C-1′), 129.7 (d, C-2′/6′),
128.6 (d, C-3′/5′), 133.3 (d, C-4′), and 166.1 (s, CO2-9)], for other
signals, see Table 2; ESIMS m/z 455 [M + Na]+; HRESIMS m/z
455.2042 [M + Na]+ (calcd for C24H32O7Na, 455.2046).
Benzoylation of 3. Compound 3 (5.0 mg) was benzoylated under
the same conditions described above for 2, to yield the known
compound 10 (3.7 mg, Rf 0.35), whose absolute configuration was
determined by CD study to be (1S,4R,5S,6R,7R,8R,9S,10S)-6-acetoxy-
1,8,9-tribenzoyloxydihydro-ꢀ-agarofuran.
1r,2r-Diacetoxy-9ꢀ-cinnamyloxy-4ꢀ-hydroxydihydro-ꢀ-agarofu-
ran (7): white, amorphous powder; [R]D20 +83.0 (c 0.06, CHCl3); UV
(EtOH) λmax (log ꢀ) 217 (4.19), 279 (4.32) nm; IR (KBr) νmax 3504,
2929, 1745, 1697, 1637, 1450, 1384, 1367, 1282, 1253, 1143, 1027,
771 cm-1; 1H NMR (400 MHz, CDCl3) δ OCin-9 [7.58 (2H, m, H-2′/
6′), 7.40 (2H, m, H-3′/5′), 7.40 (1H, m, H-4′), 6.38 (1H, d, J ) 15.9
Hz, H-R), and 7.71 (1H, d, J ) 15.9 Hz, H-ꢀ)], for other signals, see
Table 1; 13C NMR (100 MHz, CDCl3) δ OCin-9 [134.3 (s, C-1′), 128.3
(d, C-2′/6′), 128.8 (d, C-3′/5′), 130.4 (d, C-4′), 118.0 (d, C-R), 145.4
(d, C-ꢀ), and 166.1 (s, CO2-9)], for other signals, see Table 2; ESIMS
m/z 523 [M + Na]+; HRESIMS m/z 523.2317 [M + Na]+ (calcd for
C28H36O8Na, 523.2308).
(1S,4R,5S,6R,7R,8R,9S,10S)-1,8,9-Tribenzoyloxy-6-hydroxydihy-
20
dro-ꢀ-agarofuran (4): white, amorphous powder; [R]D -134.0 (c
0.19, CHCl3); UV (EtOH) λmax (log ꢀ) 227 (4.57), 273 (3.49) nm; CD
(MeOH) λext (∆ꢀ) 235.0 (-49.36), 220.9 (+21.26) nm; IR (KBr) νmax
1
2929, 1727, 1602, 1452, 1319, 1282, 1107, 1068, 956, 703 cm-1; H
NMR (400 MHz, CDCl3) δ OBz-1 [7.58 (2H, d, J ) 7.8 Hz, H-2′/6′),
6.87 (2H, t, J ) 7.8 Hz, H-3′/5′), and 7.14 (1H, t, J ) 7.8 Hz, H-4′)],
OBz-8 [8.00 (2H, d, J ) 8.1 Hz, H-2′′/6′′), 7.46 (2H, t, J ) 8.1 Hz,
H-3′′/5′′), and 7.58 (1H, t, J ) 8.1 Hz, H-4′′)], and OBz-9 [7.44 (2H,
d, J ) 7.7 Hz, H-2′′′/6′′′), 6.98 (2H, t, J ) 7.7 Hz, H-3′′′/5′′′), and
7.24 (1H, t, J ) 7.7 Hz, H-4′′′)], for other signals, see Table 1; 13C
(1S,4S,5S,6R,7R,9S,10S)-1-Acetoxy-6,9-dibenzoyloxy-14-hydroxy-
20
dihydro-ꢀ-agarofuran (8): white, amorphous powder; [R]D +20.0

1010 Journal of Natural Products, 2008, Vol. 71, No. 6
Zhu et al.
(c 0.03, CHCl3); UV (EtOH) λmax (log ꢀ) 230 (4.32), 275 (3.79) nm;
CD (MeOH) λext (∆ꢀ) 242.7 (+2.04), 221.4 (-1.38) nm; IR (KBr) νmax
2925, 1716, 1452, 1384, 1276, 1240, 1107, 1026, 713 cm-1; 1H NMR
(400 MHz, CDCl3) δ OBz-6 [8.12 (2H, d, J ) 7.5 Hz, H-2′/6′), 7.50
(2H, m, H-3′/5′), and 7.61 (1H, m, H-4′)], and OBz-9 [8.10 (2H, d, J
) 8.3, H-2′′/6′′), 7.46 (2H, m, H-3′′/5′′), and 7.58 (1H, m, H-4′′)], for
other signals, see Table 1; 13C NMR (100 MHz, CDCl3) δ OBz-6 [129.7
(s, C-1′), 129.7 (d, C-2′/6′), 128.8 (d, C-3′/5′), 133.5 (d, C-4′), and
165.8 (s, CO2-6)], OBz-9 [129.6 (s, C-1′′), 130.0 (d, C-2′′/6′′), 128.3
(d, C-3′′/5′′), 133.3 (d, C-4′′), and 165.5 (s, CO2-9)], for other signals,
see Table 2; ESIMS m/z 559 [M + Na]+; HRESIMS m/z 559.2300 [M
+ Na]+ (calcd for C31H36O8Na, 559.2308).
Acknowledgment. The authors are grateful for partial financial
support from the Science and Technology Commission of Shanghai
Municipality (06DZ22028).
References and Notes
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(1S,4R,5S,6R,7R,9S,10S)-1-Acetoxy-9-benzoyloxy-6-hydroxydihy-
dro-ꢀ-agarofuran (9): white, amorphous powder; [R]D20 +55.0 (c 0.18,
CHCl3); UV (EtOH) λmax (log ꢀ) 230 (3.99), 273 (2.98) nm; IR (KBr)
1
νmax 2925, 1715, 1450, 1384, 1276, 1107, 714 cm-1; H NMR (400
MHz, CDCl3) δ OBz-9 [8.10 (2H, d, J ) 7.5 Hz, H-2′/6′), 7.42 (2H,
t, J ) 7.5 Hz, H-3′/5′), and 7.58 (1H, t, J ) 7.5 Hz, H-4′)], for other
signals, see Table 1; 13C NMR (100 MHz, CDCl3) δ OBz-9 [129.8 (s,
C-1′), 130.1 (d, C-2′/6′), 128.3 (d, C-3′/5′), 133.2 (d, C-4′), and 165.7
(s, CO2-9)], for other signals, see Table 2; ESIMS m/z 439 [M + Na]+;
HRESIMS m/z 439.2085 [M + Na]+ (calcd for C24H32O6Na, 439.2097).
Cinnamoylation of 9. Compound 9 (2.5 mg) was dissolved in dry
pyridine (0.5 mL), and cinnamoyl chloride (3 drops) and a catalytic
amount of 4-(dimethylamino)pyridine were added. Then, the mixture
was stirred at rt for 48 h, poured into H2O, extracted with EtOAc, and
purified on preparative TLC developed with a solvent of petroleum
ether-EtOAc (5:1), to give compound 16 [1.5 mg, Rf 0.45; CD (MeOH)
λext (∆ꢀ) 279.1 (+5.22), 235.0 (-4.01) nm].
Cytotoxicity Assays. HL-60 human leukemia cells were plated into
96-well plates containing 90 µL of medium. Cells were treated in
triplicate with gradient concentrations of the tested compounds for 72 h.
Thereafter, 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide
(Sigma, St. Louis, MO) solution was added to each well. After 4 h of
incubation at 37 °C, 50 µL of extraction buffer (10% SDS, 5%
isobutanol, and 0.01 M hydrochloric acid) was added, the cells were
incubated overnight at 37 °C, and the absorbance was then measured
at 570 nm using a 96-well multiscanner (Molecular Devices, Missis-
sauga, Ontario, Canada). The concentrations giving 50% growth
inhibition (IC50) were calculated with the Logit method. The anticancer
drug etoposide was used as a positive control.
NP800052D