New 29-nor-cycloartanes with a 3,4-seco- and a novel 2,3-seco-structure from the leaves of Sinocalycanthus chinensis

Article abstract of DOI:10.1016/j.bmc.2011.03.055

Eight new triterpenoids, including sinocalycanchinensins A-E (1-5) with a 3,4-seco-29-nor-cycloartane skeleton, sinocalycanchinensin F (6) possessing a novel 2,3-seco-29-nor-cycloartane skeleton, and 29-nor-cycloartanes, sinocalycanchinensins G and H (7 and 8), have been isolated from the leaves of Sinocalycanthus chinensis. Their structures were elucidated on the basis of spectroscopic examinations. The cytotoxicities of the isolated new triterpenes against a panel of human cancer cell lines, including multi-drug resistant (MDR) cancer cell lines, were also evaluated. Compound 5 demonstrated enhanced cytotoxicity against MDR KB cells in the presence of colchicine, although all the compounds showed moderate or no cytotoxicities.

Full text of DOI:10.1016/j.bmc.2011.03.055

Bioorganic & Medicinal Chemistry 19 (2011) 2790–2796
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New 29-nor-cycloartanes with a 3,4-seco- and a novel 2,3-seco-structure
from the leaves of Sinocalycanthus chinensis
⇑
Yoshiki Kashiwada , Kazuya Nishimura, Shin-ichiro Kurimoto, Yoshihisa Takaishi
Graduate School of Pharmaceutical Sciences, University of Tokushima, 1-78 Shomachi, Tokushima 770-8505, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Eight new triterpenoids, including sinocalycanchinensins A–E (1–5) with a 3,4-seco-29-nor-cycloartane
skeleton, sinocalycanchinensin F (6) possessing a novel 2,3-seco-29-nor-cycloartane skeleton, and 29-
nor-cycloartanes, sinocalycanchinensins G and H (7 and 8), have been isolated from the leaves of Sinoc-
alycanthus chinensis. Their structures were elucidated on the basis of spectroscopic examinations. The
cytotoxicities of the isolated new triterpenes against a panel of human cancer cell lines, including
multi-drug resistant (MDR) cancer cell lines, were also evaluated. Compound 5 demonstrated enhanced
cytotoxicity against MDR KB cells in the presence of colchicine, although all the compounds showed mod-
erate or no cytotoxicities.
Received 28 February 2011
Revised 22 March 2011
Accepted 23 March 2011
Available online 29 March 2011
Keywords:
Sinocalycanthus chinensis
Calycanthaceae
3,4-seco-29-nor-Cycloartane
2,3-seco-29-nor-Cycloartane
MDR
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
tween EtOAc, BuOH, and water. The EtOAc-soluble portion was fur-
ther partitioned between hexane and 90% MeOH. Repeated
Sinocalycanthus chinensis Cheng et S.Y. Chang, the only repre-
sentative in the genus Sinocalycanthus in the family Calycantha-
ceae, is native to Zhejiang of China. This plant is cultivated as an
ornamental tree in China because of the attractive cream and yel-
low semi-double flowers, which appear on the terminal twigs from
May to June.1 The leaves of S. chinensis are used as a remedy for
cold, cough, and wheeziness,2 while the flowers and roots of this
plant have been used for the treatment of stomachache.3 However,
there have been few reports of the constituents of S. chinensis.
In our chemical studies of medicinal plants aimed at searching
for biologically active compounds, we have examined the leaves of
S. chinensis, which has resulted in the isolation of eight new triterp-
enoids, including five 3,4-seco-29-nor-cycloartanes, sinocalycan-
chromatographies of the 90% MeOH-soluble fraction over Sepha-
dex LH-20, MCI gel CHP20P, YMC ODS-A, and purification by HPLC
with Gel-Permeation Chromatography (GPC), ODS, and silica gel to
afford eight new triterpenes (1–8), designated sinocalycanchinen-
sins A–H (Fig. 1), together with 14 known compounds. The known
compounds were identified as 8
diol,5 b-eudesmol,6 cryptomeridiol,6 11-hydroxy-4
inane,7 b-sitosterol-3-O-b- -glucoside,8 kaempferol,9 quercetin,9
kaempferol-3-O-b- quercetin-3-O-b-
-glucoside,10 -glucoside,10
a
-acetoxy-elemol,4
8a
,11-elemo-
a
-methoxy-sel-
D
D
D
scopoletin,11 ferulic acid,12 p-coumaric acid,12 and isopropyl vanil-
late13 by comparison of their physical and spectral data with those
reported in the literature.
Sinocalycanchinensin A (1) was obtained as a white amorphous
powder. The molecular formula of 1 was determined as C29H44O4
by HRESIMS (m/z 479.3112 [M+Na]+). The 1H NMR spectrum
showed a characteristic pair of doublet signals [dH 0.32, 0.49 (each
1H, d, J = 4.0 Hz)], ascribable to the cyclopropane methylene sig-
nals of a cycloartane-type triterpene. It also revealed the presence
of a vinylic methyl group [dH 1.92 (3H, s)], two tertiary methyl
groups [dH 0.94 and 0.98 (each 3H, s)], a secondary methyl group
[dH 0.90 (3H, d, J = 6.0 Hz)], a vinyl group [dH 4.99 (1H, d,
J = 10.0 Hz); 5.05 (1H, d, J = 17.2 Hz); 5.67 (1H, ddd, J = 8.8, 10.0,
17.2 Hz)], and a tri-substituted olefinic group [dH 6.10 (1H, t,
J = 7.2 Hz)]. The 13C NMR spectrum showed the signals of 29 car-
bon atoms: two carboxyl carbons, four olefinic carbons, including
one quaternary, two methines, and a methylene, four methyl car-
bons, 11 methylene carbons, four methine carbons, and four qua-
ternary carbons. The HMBC correlations of H2-19 with C-1 and of
chinensins A–E (1–5),
a
novel 2,3-seco-29-nor-cycloartane,
sinocalycanchinensin F (6), and two 29-nor-cycloartanes, sinocaly-
canchinensins G and H (7 and 8), together with 14 known com-
pounds. The cytotoxicities of the isolated new triterpenes against
a panel of human cancer cell lines, including multi-drug resistant
cancer cell lines, were also evaluated. In this paper, we describe
the structure elucidation and cytotoxicities of these compounds.
2. Results and discussion
The leaves of S. chinensis were extracted with MeOH at room
temperature. The MeOH extract was partitioned successively be-
⇑
Corresponding author. Tel./fax: +81 88 633 7276.
E-mail address: kasiwada@ph.tokushima-u.ac.jp (Y. Kashiwada).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.03.055

Y. Kashiwada et al. / Bioorg. Med. Chem. 19 (2011) 2790–2796
2791
Figure 1. Structures of sinocalycanchinensins A–F (1–8).
H2-2 with the carboxyl carbon at dC 180.4, together with the con-
nectivity of C-1 and C-2 shown by the 1H–1H COSY spectrum, im-
plied the presence of a 3,4-seco-structure.14 In addition, the HMBC
correlations of the proton signals of the vinyl group with C-5 as
well as the olefinic proton [dH 5.67 (1H, ddd, J = 8.8, 10.0,
17.2 Hz)] with C-6 indicated the absence of Me-29. These spectral
data indicated the presence of a 29-nor-3,4-seco-cycloartane skel-
eton,15,16 which were further supported by the following HMBC
correlations: H2-1 with C-5, C-10 and C-19; H-8 with C-9 and C-
14; H2-11 with C-9 and C-19; Me-18 with C-12, C-13, and C-17;
Me-30 with C-13, C-14, and C-15. In contrast, the olefinic proton
signal at dH 6.10 displayed HMBC correlations with the carboxyl
carbon (dC 173.6) and the methyl carbon (dC 20.5) as well as C-22
(dC 35.8), indicating that the double bond is present at C-24 and
C-25, and the methyl group and the carboxylic acid group are at-
tached at C-25. The geometry of the C24(25) double bond was as-
signed as Z from the NOESY correlation of H-24 with the vinyl
methyl group. Thus, the planar structure of 1 was assigned. The
1. This result was further supported by the HRESIMS, which
showed an [M+Na]+ peak at m/z 493.3280, corresponding to a
molecular formula of C30H46O4 (m/z 493.3294). The location of
the methyl ester group was assigned to be C-3 from the HMBC cor-
relation of the OMe signal with the carboxyl carbon signal at dC
174.4, which also had the HMBC correlation from H2-1. The NOESY
correlations observed in 2 indicated with the presence of the same
configurations seen in 1. From this spectral examination, the struc-
ture of 2 was assigned as shown.
Sinocalycanchinensin C (3) was obtained as a colorless oil and
shown to possess a molecular formula of C49H82O4 by HRESIMS
(m/z 733.6187, [M+Na]+). The 1H and 13C NMR spectra of 3 were
closely correlated with those of 2. It also showed signals due to a
trisubstituted double bond [dH 5.33 (1H, t, J = 7.2 Hz); dC 118.2,
142.6], an oxymethylene [dH 4.58 (2H, d, J = 7.2 Hz); dC 61.3], a vi-
nyl methyl group [dH 1.69 (3H, s); dC 16.4], four secondary methyl
groups [dH 0.85, 0.86, 0.88 ꢀ 2 (each 3H, d, J = 7.2 Hz); dC 19.7, 19.8,
22.6, 22.7], nine sp3 methylene carbons [dC 24.5, 24.8, 25.1, 36.7,
37.3, 37.4 (2C), 39.4, 39.9], and three sp3 methine carbons [dC
28.0, 32.7, 32.8], but the methoxyl signal seen in 2 was absent. In
the HMBC spectrum of 3, the oxymethylene proton signal showed
a correlation with the C-3 carboxyl carbon and the olefinic carbons
[dC 118.2, 142.6], which in turn displayed correlations with the vi-
nyl methyl signal. In contrast, two of the secondary methyls
showed HMBC correlations with the same methine and methylene
carbons, whereas the other two displayed HMBC correlations with
two methylene carbons in each case. From this spectral observa-
tion, 3 was presumed to be a diterpene alcohol ester of 1. Alkaline
hydrolysis of 3 gave 2 and an alcohol, which was identified as
configuration of H-5 was assigned as
a from the NOESY correlation
of H-19 with H-4 as well as H-5 with Me-30. Further examination
of the NOESY spectrum indicated that the configurations in 1 were
consistent with those of the cycloartane-type skeleton from the
following key NOESY correlations: H-19/H-8, H-8/H-18, and H-
18/H-20 on the b-face; and Me-30/H-17, and H-17/Me-21 on the
a-face (Fig. 2). On the basis of the examination, the structure of 1
was characterized as shown.
The 1H and 13C NMR spectra of sinocalycanchinensin B (2) were
quite similar to those of 1 except for the observation of a methoxy
signal [dH 3.65 (3H, s); dC 51.5], suggesting 2 to be a methyl ester of

2792
Y. Kashiwada et al. / Bioorg. Med. Chem. 19 (2011) 2790–2796
Figure 2. Key HMBC (left) and NOESY (right) of sinocalycanchinensin A.
phytol17 by comparison of physical and spectral data with those of
the authentic sample. The geometry of the phytol moiety in 3 was
assigned as E from the NOESY correlation of H2-10 and Me-200.
Based on these examinations, the structure of 3 was characterized
as shown.
identical to 5. On the basis of these observations, 5 was character-
ized as shown.
Sinocalycanchinensin F (6) was obtained as a white amorphous
powder, and its molecular formula was assigned as C32H50O6 by
HRESIMS(m/z553.3504,[M+Na]+). The1Hand13CNMRspectrawere
similar to those of 1, showing signals of the cyclopropyl methylene
group [dH 0.39, 0.48 (each 1H, d, J = 4.4 Hz); dC 29.6], two tertiary
methyl groups [dH 0.94, 0.95 (each 3H, s); dC 18.2, 19.4], a secondary
methyl group [dH 0.90 (3H, d, J = 6.0 Hz); dC 18.1], a trisubstituted
double bond [dH 6.09 (1H, t, J = 7.2 Hz); dC 125.7, 147.1], and a car-
boxyl carbon (dC 172.4). Signals corresponding to the vinyl group
found in 1 were absent, while those for an oxymethylene group [dH
4.12, 4.22 (each 1H, m); dC 62.8], a methoxy group [dH 3.68 (3H, s),
dC 51.6], an acetyl group [dH 2.06 (3H, s); dC 21.1, 171.2], and a sec-
ondary methyl group [dH 1.08 (3H, d, J = 6.8 Hz), dC 11.1] were ob-
served. The oxymethylene was assigned to C-2 from the HMBC
correlation of these proton signals with C-1 and C-10. These proton
signals also exhibited an HMBC correlation with the acetyl carbonyl
carbon, indicating the location of the acetyl group at C-2. In contrast,
the secondary methyl signal showed HMBC correlations with C-5
and the carboxyl carbon at dC 177.0, which in turn coupled with
the methoxyl proton signal, suggesting the presence of a 1-carb-
omehoxy-ethyl group at C-5. The other HMBC correlations were
good agreement of the presence of 2,3-seco-29-nor-cycloartane
strucutre: H2-1 with C-10 and C-19; H-8 with C-14; H-5 with C-
10; H2-11 with C-8, C-9 and C-19; Me-18 with C-12, C-13, and C-
17; Me-30 with C-13, C-14, and C-15 (Fig. 4). The NOESY correlations
of Me-26 and H-24 indicated the geometry of the C24(25) double
bond to be Z. The sterochemistry of C-4 was assigned to be S⁄ from
the NOESY correlations of H-4 and H-2b as well as Me-28 and H-
6b. The following NOESY correlations indicated that the configura-
tions in 6 were identical with those in cycloartane-type triterpene:
H-4/H-19, H-19/H-8, H-8/H-18, and H-18/H-20 on the b-face, and
the NOESY correlations of H-5/Me-30, Me-30/H-17, and H-17/Me-
The 1H and 13C NMR spectra of sinocalycanchinensin D (4)
resembled those of 1, indicating that 4 was also a 29-nor-3,4-
seco-cycloartane-type triterpene. This was also supported by the
following HMBC correlations: H2-1 with C-5, C-10 and C-19; H2-2
with C-3; H-8 with C-9 and C-14; H2-11 with C-9 and C-19; Me-
18 with C-12, C-13, and C-17; Me-30 with C-13, C-14, and C-15;
Me-27 with C-24, C-25, and C-26. In addition, the existence of an
oxymethine [dH 4.48 (1H, dt, J = 3.6, 13.2 Hz); dC 80.6] was also
indicated, and this oxymethine was assigned to be C-22 from the
HMBC correlations of the carbon resonance for this oxymethine
with Me-21 and H-24. The presence of a d-lactone structure was
suggested from the 13C resonance of C-26 (dC 166.5). This was fur-
ther supported by HRESIMS, which gave an [M+Na]+ ion peak at m/
z 491.3152, corresponding to a molecular formula of C30H44O4. The
NOESY correlations of H-4/H-19, H-19/H-8, H-8/H-18, and H-18/H-
20 on the b-face, as well as the NOESY correlations of H-5/Me-30,
Me-30/H-17, and H-17/Me-21 on the
a-face, suggested that the
configurations in 5 were identical to those in the cycloartane-type
triterpenes (Fig. 3). The absolute configuration at C-22 of the lac-
tone moiety was elucidated as R based on the positive cotton effect
(De
+ 3.65 at 253 nm) in the CD spectrum of 4.18 On the basis of
this examination, the structure of 4 was elucidated as shown.
The 1H and 13C NMR spectra of sinocalycanchinensin E (5) were
quite similar to those of 4 except for the appearance of a methoxyl
signal (dH 3.64) and an upfield shift of the carboxyl carbon reso-
nance [dC 174.2 (ꢁ4.8 ppm)], suggesting that 5 was a methyl ester
of 4. The molecular formula of 5, confirmed as C30H44O4 by HRE-
SIMS, was also consistent with this observation. The location of
the methyl ester group was assigned to be C-3 from the HMBC cor-
relation of the OMe signal with the C-3 carboxyl carbon signal at dC
172.7. The structural confirmation was obtained by the treatment
of 4 with trimethylsilyldiazomethane, which yielded a product
21 on the a-face (Fig. 4). From the spectral evidence described above,
sinocalycanchinesin F (6) was elucidated as a 29-nor-2,3-seco-cycl-
oartane-type triterpene, as shown.
Figure 3. Key HMBC (left) and NOESY (right) of sinocalycanchinensin D (4).

Y. Kashiwada et al. / Bioorg. Med. Chem. 19 (2011) 2790–2796
2793
Figure 4. Key HMBC (left) and NOESY (right) of sinocalycanchinensin F.
Sinocalycanchinensins G (7) and H (8) were obtained as a white
amorphous powder, and both were shown to possess the same
(MDR) human cancer cell lines, including KB-C2 (colchicine-resis-
tant KB) and K562/Adr (doxorubicin-resistant K562) (Table 1).
Compound 8 showed weak cytotoxicities against all the tested cell
molecular formula,
C29H44O3, by HRESIMS (m/z 463.3175,
[M+Na]+ in 7; m/z 463.3188, [M+Na]+ in 8). The 1H and 13C NMR
spectra of 7 and 8 were quite similar to each other, and were clo-
sely correlated with those of 1. However, both of them showed the
absence of the vinyl group and one of the carboxylic acid groups,
but instead exhibited the presence of a secondary methyl group
and a carbonyl group. The HMBC correlations of the secondary
methyl signal with C-3 and the carbonyl carbon resonance in each
case indicated the assignment of the carbonyl group to be C-3 and
the location of the secondary methyl group to be C-4 in 7 and 8. In
contrast, the carbonyl resonance also had an HMBC correlation
with the methylene proton resonances assignable to H2-2, which
further coupled with C-10, thus indicating the presence of a 29-
nor-3-oxo-cycloartane structure in each case. Compounds 7 and
8 were considered to be geometric isomers based on the carbon
resonance for the vinyl methyls being different. The geometries
of the double bond in 7 and 8 were assigned as Z and E, respec-
tively, from the following NOESY correlations: Me-26/H-24 in 7;
Me-27/H2-23 in 8. The configuration of H-4 was also assigned as
b from the NOESY correlation of H-19 and H-4. The following
NOESY correlations were also consistent with the presence of the
cycloartane-type skeleton in 7 and 8: H-4/H-19, H-19/H-8, H-8/
H-18, and H-18/H-20 on the b-face, and the NOESY correlations
lines, with IC50 values ranging from 14.8 to 18.8 lg/mL, while the
cytotoxicities of compound 7 were slightly less potent than those
of 8. Compound 6 also showed similar cytotoxicities against the
tested cell lines except for MCF-7. In contrast, compound 3 was
not a cytoxic compound, having IC50 values >100 lg/mL against
all the tested cell lines. Compound 5 was also a weak cytotoxic
compound, but it showed significant enhanced cytotoxicity against
KB-C2 cells in the presence of colchicine with an IC50 value of
1.51 lg/mL. Since colchicine had no effect on the growth of KB-
C2 cells at this concentration level, it was suggested that com-
pound 5 might show some MDR-reversing effects. Overall, 3,4-
seco-cycloartanes were less cytotoxic than the others.
4. Experimental
4.1. General experimental procedures
Optical rotations were measured on a JASCO P-2200 polarime-
ter. NMR spectra were recorded on a Bruker AVANCE-400 instru-
ment (1H NMR: 400 MHz, 13C NMR:100 MHz) using TMS as an
internal standard. HRESIMS were obtained on a Waters LCT Pre-
mier. CD spectra were run on a CD-J600 spectropolarimeter (JAS-
CO). Column chromatography: silica gel 60 N (KANTO CHEMICAL,
of H-5/Me-30, Me-30/H-17, and H-17/Me-21 on the a-face. Based
on this evidence, the structures of 7 and 8 were concluded to be
as shown.
63–210 lm), Sephadex LH-20 (GE Healthcare Bioscience), ODS
(YMC ODS A), MCI gel CHP20P (MITSUBISHI CHEMICAL CORPORA-
TION), and Toyopearl HW-40C (TOSHO); HPLC: gel permeation
chromatography (Asahipak, GS-310 2G, MeOH), silica gel (KANTO
Eight new triterpenoids, including five 3,4-seco-29-nor-cycloar-
tanes, a novel 2,3-seco-29-nor-cycloartane, and two 29-nor-cycl-
oartanes, have been isolated from the leaves of S. chinensis.
Compound 5 represents the first example of a 29-nor-2,3-seco-
cycloartane triterpene. In contrast, 29-nor-3,4-seco-cycloartane tri-
terpenes have been reported only from the aerial part of Antirhea
acutata (Rubiaceae), and this is the second example of the isolation
of this type of triterpene. In addition, sinocalycanchinensin C (3) is
the first example of a phytol ester of triterpene. Phytol, a compo-
nent of chlorophylls, is known to be derived from a mevalonate-
independent pathway, while cycloartanes are a metabolite of the
mevalonate pathway.19 Sinocalycanchinensin C is therefore a bio-
synthetically interesting compound, as it is considered to be de-
rived from the products of both mevalonate and mevalonate-
independent pathways.
CHENICAL, Mightysil Si 60
TO CHEMICAL, Mightysil RP-18 GP
u
20 ꢀ 250 mm), octadecyl silane (KAN-
u
20 ꢀ 250 mm).
Table 1
Cytotoxicity (IC50 in lg/mL) of compounds 1–8 against human cancer cell lines and
a
multidrug-resistant human cancer cell lines with or without colchicine (for KB-C2)
in vitro
KB
KB-C2 KB-C2 (+col.b) MCF-7 K562 K562/Adr
1
2
3
4
5
6
7
8
30.7
20.9
>100 >100
31.7
62.2
16.6
19.9
18.6
30.4
22.2
33.4
18.9
>100
25.6
1.51
17.5
21.4
17.2
11.6
61.6
35.7
>100
39.2
>100
33.8
23.7
18.6
0.033
29.9
16.5
>100
16.7
22.4
13.2
14.8
14.8
1.49
38.9
17.6
26.4
49.2
21.7
21.6
18.8
7.9
7.0
20.5
19.5
7.3
19.4
8.5
3. Bioassay data
Daunorubicin 0.45
Compounds 1–8 were examined for their cytotoxic activity
against a panel of human cancer cell lines, namely, the KB (human
epidermoid carcinoma of nasopharynx), K562 (leukemia), and
MCF-7 (breast carcinoma) cell lines, as well as multidrug-resistant
a
Cell lines: KB (human epidermoid carcinoma of the nasopharynx), KB-C2
(multidrug-resistant KB), K562 (leukemia), and MCF-7 (breast carcinoma).
b
2.5 lM colchicine.

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Y. Kashiwada et al. / Bioorg. Med. Chem. 19 (2011) 2790–2796
4.2. Plant material
MeOH(3:2 ? 0:1)], YMC ODS-A [H2O–MeOH(2:3 ? 0:1)], and silica
gel [n-hexane-acetone (9:1)] to give 8 -acetoxy-elemol (79 mg).
a
The leaves of S. chinensis Cheng et S.Y. Chang, cultivated in the
Godzu-Branch Herbal Garden of the Niigata University of Phar-
macy and Applied Life Sciences (NUPALS), Agano city, Niigata, were
collected in September 2004. The plant was identified by Professor
Yasumasa Ikeshiro of the NUPALS, and a voucher specimen (NU-
PAL090104) has been deposited in the herbarium of the NUPALS.
Fraction 1.4 was further fractionated by MCI gel CHP20P chromatog-
raphy [H2O–MeOH (3:2 ? 0:1)] into 11 fractions (frs. 1.4.1–1.4.11).
Fraction 1.4.4 was repeatedly chromatographed over YMC ODS-A
[H2O–MeOH (3:7 ? 0:1)] and silica gel [n-hexane-acetone (4:1)]
to afford4 (12 mg). Repeatedchromatography of fraction 1.4.10over
YMC ODS-A [H2O–MeOH (3:7 ? 0:1)], silica gel [n-hexane-acetone
(9:1)], and GPC on HPLC (MeOH) gave 6 (12 mg). Repeated chroma-
tography of fr. 1.4.9 over YMC ODS-A [H2O–MeOH (3:7 ? 0:1)] and
silica gel [n-hexane-EtOAc (9:1)] furnished b-eudesmol (13 mg). Fr.
1.5 was fractionated by YMC ODS-A chromatography [H2O–MeOH
(3:2 ? 0:1)] into 17 fractions (frs. 1.5.1–1.5.17). Fr. 1.5.11 was fur-
ther chromatographed over Toyoperal HW-40C [H2O–MeOH
4.3. Extraction and isolation
The fresh leaves of S. chinensis (7.6 kg) were extracted with MeOH
(3 ꢀ 54 L) at room temperature. The MeOH extracts were concen-
trated under reduced pressure to give a residue (880 g) that was par-
titioned between EtOAc and H2O. After removal of the solvent of the
EtOAc layer, theextracts were furtherpartitioned between n-hexane
and 90%MeOH, giving an n-hexane-soluble fraction (79 g) and a 90%
MeOH-soluble fraction (77 g). The 90% MeOH-soluble fraction was
subjected to chromatography over Sephadex LH-20 with EtOH to
give four fractions (frs. 1–4). Fraction 1 (44.4 g) was rechromato-
graphed over Sephadex LH-20 [H2O–MeOH (2:3?0:1)] to afford a
further 10 fractions (frs. 1.1–1.10). Fr. 1.3 was rechromatographed
over MCI gel CHP20P [H2O–MeOH (3:2?0:1)] to furnish 11 fractions
(frs. 1.3.1–1.3.11). Fraction 1.3.5 was further fractionated by Toyo-
pearl HW-40C chromatography [H2O–MeOH (1:1 ? 0:1)] into eight
fractions (frs. 1.3.5.1–1.3.5.8). Scopoletin was crystallized from fr.
1.3.5.3(MeOH)to give a pure sample(50 mg). Fr. 1.3.5.5was purified
by GPC on HPLC to afford ferulic acid (49 mg). Silica gel chromatog-
raphy of fr. 1.3.5.7 with CHCl3–MeOH (20:1) gave p-coumaric acid
(29 mg). Fraction 1.3.7 was fractionated by YMC ODS-A [H2O–MeOH
(3:7 ? 0:1)] into further seven fractions (frs. 1.3.7.1–1.3.7.7). Silica
gel column chromatography of fr. 1.3.7.2 with n-hexane-EtOAc
(4:1) furnished isopropyl vanillate (6 mg). Frs. 1.3.7.4 was chro-
matographed over silica gel [n-hexane-acetone (9:1)] to afford
(2:3 ? 0:1)] to afford
a further five fractions (frs. 1.5.11.1–
1.5.11.5). Repeated chromatography of fr. 1.5.11.3 over Sephadex
LH-20 (acetone), silica gel [n-hexane-acetone (9:1)], and GPC on
HPLC furnished 6 (8 mg). Fr. 1.5.11.5 was applied on a silica gel col-
umn with n-hexane-acetone (9:1) to afford 1 (162 mg) and 5
(31 mg). Fr. 1.5.13 was repeatedly chromatographed over Sephadex
LH-20 (acetone), and silica gel [n-hexane-EtOAc (9:1)], and then
purified by silica gel HPLC [CHCl3–MeOH (50:1)] to give 8 (4 mg).
Fraction 1.6 was repeatedly chromatographed over YMC ODS-A
[H2O–MeOH (2:3 ? 0:1)] and silica gel [n-hexane-EtOAc (20:1)],
and then purified by Mightysil RP-18 on HPLC to give 2 (101 mg)
and 7 (61 mg). Fr. 1.8 was rechromatographed over MCI gel CHP20P
[H2O–MeOH (3:2 ? 0:1)] to furnish 10 fractions (frs. 1.8.1–1.8.10).
Fraction 1.8.8 was repeatedly chromatographed over YMC ODS-A
[H2O–MeOH (2:3 ? 0:1)], Toyopearl HW-40C [H2O–MeOH (9:1)],
and silica gel [n-hexane-EtOAc (20:1)] to give 3 (16 mg). Crystalliza-
tion of fr. 1.8.6 furnish b-sitosterol-3-O-b-D-glucoside (73 mg). Fr. 2
(18.6 g) was rechromatographed over Sephadex LH-20 (70% MeOH)
to give eight fractions (frs. 2.1–2.8). Crystallization of fr. 2.7 (MeOH)
afforded kaempferol (3.0 g). MCI gel CHP20P chromatography of fr.
2.4 with H2O–MeOH (7:3 ? 0:1) and then crystallization furnished
kaempferol-3-O-b-D-glucoside (577 mg). Fr. 3 (1.0 g) was fraction-
ated by Sephadex LH-20 [H2O–MeOH (2:3 ? 0:1)] into a further
8a
,11-elemodiol (134 mg) and cryptomeridiol (21 mg), while silica
gel column chromatography of fr. 1.3.7.6 with n-hexane-acetone
(9:1) yielded 11-hydroxy-4 -methoxy-selinane (20 mg). Fr. 1.3.8
a
was repeatedly chromatographed over Toyopearl HW-40C [H2O–
ten fractions (frs. 3.1–3.10). Quercetin was crystallized from fr. 3.9
Table 2
1H NMR data for compounds 1–8 in CDCl3
a
Position
4
1
2
3b
4
5
6
7
8
5.67 (ddd, 8.8,
10.0, 17.2)
0.98 (s)
5.66 (ddd, 8.8,
8.8, 17.6)
0.97 (s)
5.67 (ddd, 8.4, 10.0,
17.2)
0.98 (3H, s)
0.32 (d, 4.0)
5.66 (ddd, 8.8, 10.0,
17.6)
1.02 (s)
5.65 (ddd, 8.4, 10.0,
17.2)
1.01 (s)
2.92 (m)
2.24 (m)
2.22
(m)
1.02 (s)
0.41
(d, 4.0)
0.64
(d, 4.0)
0.94
(d, 6.8)
1.20
18
19
0.95 (s)
0.39 (d,
4.4)
0.64 (d,
4.4)
0.90 (d,
6.0)
1.14 (m)
1.01 (s)
0.41 (d,
4.0)
0.63 (d,
4.0)
0.92 (d,
6.0)
1.17 (m)
0.32 (d, 4.0))
0.31 (d, 4.4)
0.34 (d, 4.4)
0.31 (d, 4.4)
0.49 (d, 4.0
0.90 (d, 6.0)
1.17 (m)
0.47 (d, 4.4)
0.90 (d, 7.2)
1.15 (m)
0.47 (d, 4.0)
0.91 (d, 6.4)
1.17 (m)
0.50 (d, 4.4)
0.48 (d, 4.4)
21
22
0.98 (d, 6.4)
0.96 (d, 6.4)
4.48 (dt, 3.6, 13.2)
4.46 (dt, 3.6, 13.2)
(m)
1.55 (m)
1.55 (m)
1.56 (m)
1.55 (m)
1.56 (m)
1.58
(m)
24
6.10 (t, 7.2)
6.09 (t, 7.2)
6.10 (t, 6.8)
6.61 (brd, 6.4)
6.60 (brd, 6.4)
6.09 (t,
7.2)
6.11 (t,
6.8)
6.91
(t, 7.2)
26
27
28
1.92 (s)
—
4.99 (d, 10.0), 5.05 (d,
17.2)
0.94 (s)
1.92 (s)
—
4.98 (d, 10.0), 5.04 (d,
17.2)
0.93 (s)
1.93 (s)
—
4.98 (dd, 2.0, 10.0),
5.04 (dd, 2.0, 17.2)
0.93 (3H, s)
—
—
1.93 (s)
–
1.08 (d,
6.8)
0.94 (s)
3.68 (s)
2.06 (s)
1.93 (s)
–
1.00 (d,
6.8)
1.92 (s)
1.91 (s)
1.86 (s)
1.00
(d, 6.4)
0.92 (s)
4.99 (dd, 2.0, 10.0),
5.05 (dd, 2.0, 17.6)
0.93 (s)
4.97 (dd, 1.6, 10.0),
5.03 (dd, 1.6, 17.2)
0.92 (s)
30
OMe
OAc
0.92 (s)
3.65 (s)
3.64 (s)
a
d (ppm); 400 MHz.
b
For phytol moiety: 0.85 (3H, d, J = 7.2 Hz, Me-190), 0.86 (3H, d, J = 7.2 Hz, Me-180), 0.88 (6H, d, J = 7.2 Hz, Me-160 and Me-17’), 1.69 (3H, s, Me-200), 2.01 (2H, t, J = 7.6 H, H2-
40), 4.58 (2H, d, J = 7.2 Hz, H2-10), 5.33 (1H, t, J = 6.0 Hz, H-30).

Y. Kashiwada et al. / Bioorg. Med. Chem. 19 (2011) 2790–2796
2795
Table 3
NMR (400 MHz, CDCl3) d 0.85, 0.86 (each 3H, d, J = 6.6 Hz, Me-18
and Me-19), 0.88 (6H, d, J = 6.5 Hz, Me-16 and Me-17), 1.68 (3H,
s, Me-20), 2.00 (2H, br t, J = 6.8 Hz, H2-4), 4.16 (2H, d, J = 7.2 Hz,
H2-1), 5.42 (1H, tq, J = 6.8, 1.2 Hz, H-3); 13C NMR (100 MHz, CDCl3)
d 16.2 (C-20), 19.7 (C-19), 19.7 (C-18), 22.6 (C-16), 22.7 (C-17), 24.5
(C-9), 24.8 (C-13), 25.1 (C-5), 28.0 (C-15), 32.7 (C-11), 32.8 (C-7),
36.6 (C-6), 37.3 (C-), 37.4 (C-), 37.4 (C-), 39.4 (C-14), 39.9 (C-4),
59.4 (C-1), 123.1 (C-2), 140.3 (C-3), and sinocalycanchinensin B
(2) (2.4 mg).
13C NMR data for compounds 1–8 in CDCl3
a
Position
1
2
3b
28.8
31.9
4
5
6
7
8
1
2
3
4
5
6
7
8
28.6
31.8
28.8
31.7
28.6
31.6
28.8
31.7
31.3
62.8
32.9
40.9
32.9
40.9
180.4 174.4 174.0 179.0 174.2 177.0 213.7 213.7
142.8 142.9 142.9 142.7 142.7
41.4
38.8
22.8
24.9
48.2
21.9
25.6
27.6
33.0
45.1
48.9
35.6
28.1
52.2
18.2
29.6
36.0
18.1
35.8
26.9
50.0
46.1
25.9
25.2
47.1
25.0
29.3
27.2
32.8
45.4
48.8
35.4
28.1
52.2
17.9
27.0
36.0
18.1
35.8
26.9
50.0
46.1
25.9
25.2
47.1
24.9
29.3
27.2
32.8
45.5
48.8
35.4
28.1
52.2
17.9
26.9
36.0
18.1
34.8
25.9
42.3
29.0
24.5
48.0
23.7
27.6
27.2
33.0
45.1
48.9
35.7
28.1
52.1
18.3
28.1
36.0
18.1
35.8
26.8
42.2
29.0
24.6
48.1
23.7
27.7
27.1
33.0
45.1
48.9
35.8
28.1
52.2
18.3
28.1
36.0
18.1
35.8
26.9
42.2
29.1
24.6
48.2
23.7
27.7
27.2
33.1
45.1
48.9
35.8
28.1
52.3
18.4
28.2
36.0
18.1
35.9
27.0
42.3
28.9
24.6
48.2
23.5
27.7
27.1
32.9
45.6
48.5
35.8
27.0
48.0
18.1
28.1
39.1
13.0
80.6
23.6
42.2
28.9
24.5
48.1
23.5
27.7
27.0
32.9
45.6
48.5
35.8
27.0
48.1
18.1
28.0
39.1
13.0
80.5
23.5
9
4.8. Sinocalycanchinensin D (4)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
30
OMe
OAc
Amorphous powder; ½a D20
ꢂ
+97.0 (c 1.0, CHCl3); HRESIMS: m/z
477.2982 [M+Na]+ (Calcd for C29H42O4, 477.2981); 1H NMR data,
see Table 2; 13C NMR data, see Table 3; CD (MeOH; 2.0 ꢀ 10ꢁ4 M,
De) kmax CD: 253 (+3.65).
4.9. Sinocalycanchinensin E (5)
Colorless oil; ½a D20
ꢂ
+77.8 (c 2.4, CHCl3); HRESIMS: m/z 491.3152
[M+Na]+ (Calcd for C30H44O4, 491.3137); 1H NMR data, see Table 2;
13C NMR data, see Table 3; CD (MeOH; 2.0 ꢀ 10ꢁ4 M,
De) kmax CD:
253 (+3.63).
147.3 147.3 147.3 139.4 139.3 147.1 147.4 145.6
125.8 125.8 125.7 17.0 16.9 125.7 125.7 126.6
20.5 20.5 20.5 128.3 128.3 20.6 20.5 172.5
173.6 173.4 173.2 166.6 166.5 172.4 172.7
4.10. Methylation of 4
12.0
10.7
19.2
114.1 114.1 114.1 114.3 114.1
11.1
19.4
51.6
21.1
171.2
10.7
19.1
A solution of 4 (5 mg) in MeOH (2 mL) was treated with was
treated with trimethylsilyldiazomethane (TMSCHN2, 0.5 mL in
hexane) at room temperature for 1 h with stirring. After removal
of solvent by evaporation, the residue was purified by prep. TLC
(silica gel, CHCl3–MeOH, 97:3) to afford a product, which was
shown to be identical with 4 by spectral comparison.
19.4
19.4
51.5
19.5
19.6
19.5
51.4
a
d (ppm); 100 MHz.
b
For phytol moiety: 61.3 (C-10), 118.2 (C-20), 142.6 (C-30), 39.9 (C-40), 25.1 (C-50),
36.7 (C-60), 32.7 (C-70), 37.4 (C-80), 24.5 (C-90), 37.4 (C-100), 32.8 (C-110), 37.3 (C-
120), 24.8 (C-130), 39.4 (C-140), 28.0 (C-150), 22.6 (C-160), 22.7 (C-170), 19.8 (C-180),
19.7 (C-190),16.4 (C-200).
4.11. Sinocalycanchinensin F (6)
Colorless oil; ½a D20
ꢂ
+55.4 (c 0.4, CHCl3); HRESIMS: m/z 553.3504
(MeOH) to give a pure sample (214 mg). Fr. 3.4 was chromato-
graphed over MCI gel CHP20P [H2O–MeOH (7:3 ? 0:1)] to afford
[M+Na]+ (Calcd for C32H50O6, 553.3505); 1H NMR data, see
Table 2;13C NMR data, see Table 3.
quercetin-3-O-b-D-glucoside (19 mg).
4.12. Sinocalycanchinensin G (7)
4.4. Sinocalycanchinensin A (1)
White amorphous powder; ½a D20
ꢂ
+40.3 (c 5.2, CHCl3); HRESIMS:
Amorphous powder; ½a D20
ꢂ
+51.1 (c 6.2, CHCl3); HRESIMS: m/z
m/z 463.3175 [M+Na]+ (Calcd for C29H44O3, 463.3188); 1H NMR
479.3112 [M+Na]+ (Calcd for C29H44O4, 479.3137); 1H NMR data,
see Table 2; 13C NMR data, see Table 3.
data, see Table 2;13C NMR data, see Table 3.
4.13. Sinocalycanchinensin H (8)
4.5. Sinocalycanchinensin B (2)
White amorphous powder; ½a D20
ꢂ
+39.6 (c 0.4, CHCl3); HRESIMS:
Colorless oil; ½a D20
ꢂ
+57.9 (c 2.9, CHCl3); HRESIMS: m/z 493.3280,
m/z 463.3188 [M+Na]+ (Calcd for C32H50O6, 463.3188); 1H NMR
[M+Na]+ (Calcd for C30H46O4Na, 493.3294); 1H NMR data, see Ta-
ble 2; 13C NMR data, see Table 3.
data, see Table 3;13C NMR data, see Table 2.
4.14. Cell lines and cell culture
4.6. Sinocalycanchinensin C (3)
KB (human epidermoid carcinoma of the nasopharynx), MCF-
7 (breast carcinoma), K562 (leukemia), and K562/Adr (multi-
drug-resistant human erythromyelogenous leukemia) cells were
obtained from the Cell Resource Center for Biomedical Research
(Tohoku University). Multidrug-resistant human epidermoid car-
cinoma KB-C2 cells were kindly provided by Professor Shin-ichi
Akiyama (Kagoshima University, Japan). KB cells were cultured
in Dulbecco’s modified Eagles medium (DMEM) with 10% fetal
bovine serum (FBS). KB-C2 cells were maintained in DMEM med-
Colorless oil; ½a D20
ꢂ
+38.6 (c 1.6, CHCl3); HRESIMS: m/z 733.6187
[MꢁH]ꢁ (Calcd for C49H82O4, 733.6135); 1H NMR data, see Table 2;
13C NMR data, see Table 3.
4.7. Alkaline Hydrolysis of 3
A solution of 3 (6 mg) in 1% NaOMe–MeOH (2 mL) was heated
at 60 °C with stirring for 1 day. The reaction mixture was neutral-
ized with ion-exchange resin (Dowex 50WX8), filtered, and con-
centrated under reduced pressure. The residue was
chromatographed over silica gel [n-hexane-acetone (9:1)] to give
ium in the presence of 10% FBS and 5 lg/mL colchicine. MCF-7
and K562 cells were cultured in RPMI1640 supplemented with
10% FBS. K562/Adr (doxorubicin-resistant K562 cell line) cells
were cultured in RPMI1640 medium containing 10% FBS and
phytol (1.2 mg) as a colorless oil; ½a D20
ꢂ
+0.7 (c 0.12, CHCl3); 1H

2796
Y. Kashiwada et al. / Bioorg. Med. Chem. 19 (2011) 2790–2796
0.5 lM doxorubicin. All cells were incubated at 37 °C in a
References and notes
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.03.055.