Cytotoxic terpenoids from Juglans sinensis leaves and twigs

Article abstract of DOI:10.1016/j.bmcl.2012.01.010

Bioassay-guided fractionation of an 80% MeOH extract of Juglan sinensis leaves and twigs has resulted in the isolation of three new triterpenes (1-3) and two new sesquiterpenes (4-5) along with two known sesquiterpenes (6-7). The new compounds were determined to be 3β, 11α, 19α, 24, 30-pentahydroxy-20β, 28-epoxy-28β-methoxy-ursane (1), 1α, 3β-dihydroxy-olean-18-ene (2), 2α, 3α, 23-trihydroxy-urs-12-en- 28-oic acid 28-O-β-d-glucopyranoside (3), (4S, 5S, 7R, 8R, 14R)-8, 11-dihydroxy-2, 4-cyclo-eudesmane (4), 15-hydroxy-α-eudesmol-11-O-β- d-glucopyranoside (5), by spectroscopic analysis. The cytotoxicity of compounds (1-7) against four cancer cell lines such as B16F10, Hep-2, MCF-7 and U87-MG was evaluated. Compounds 1, 2, 6 and 7 showed potent cytotoxicity against all of four cancer cell lines, respectively.

Full text of DOI:10.1016/j.bmcl.2012.01.010

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2079–2083
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Cytotoxic terpenoids from Juglans sinensis leaves and twigs
Heejung Yang a, Hyun-Jong Cho a, So Hee Sim b, Young Keun Chung b, Dae-Duk Kim a, Sang Hyun Sung a,
Jinwoong Kim a, Young Choong Kim a,
⇑
a College of Pharmacy and Research Institute of Pharmaceutical Science, Seoul National University, Daehak-Dong, Gwanak-Gu, Seoul 151-742, Republic of Korea
b Department of Chemistry, Seoul National University, Daehak-Dong, Gwanak-Gu, Seoul 151-742, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Bioassay-guided fractionation of an 80% MeOH extract of Juglan sinensis leaves and twigs has resulted in
the isolation of three new triterpenes (1–3) and two new sesquiterpenes (4–5) along with two known ses-
Received 22 November 2011
Revised 3 January 2012
Accepted 4 January 2012
Available online 13 January 2012
quiterpenes (6–7). The new compounds were determined to be 3b, 11
28-epoxy-28b-methoxy-ursane (1), 1 , 3b-dihydroxy-olean-18-ene (2), 2
en-28-oic acid 28-O-b- -glucopyranoside (3), (4S, 5S, 7R, 8R, 14R)-8, 11-dihydroxy-2, 4-cyclo-eudesmane
(4), 15-hydroxy- -eudesmol-11-O-b- -glucopyranoside (5), by spectroscopic analysis. The cytotoxicity of
a
, 19a
, 24, 30-pentahydroxy-20b,
a
a
, 3a, 23-trihydroxy-urs-12-
D
a
D
Keywords:
compounds (1–7) against four cancer cell lines such as B16F10, Hep-2, MCF-7 and U87-MG was evaluated.
Compounds 1, 2, 6 and 7 showed potent cytotoxicity against all of four cancer cell lines, respectively.
Ó 2012 Elsevier Ltd. All rights reserved.
Juglans sinensis
Triterpenes
Sesquiterpenes
Oleanane
Ursane
Eudesmane
Juglans sinensis Dode (Juglandaceae) is a walnut tree native to
Korea, Japan and China. The edible seed of J. sinensis (walnut) has
been used for the treatment of ascaris, cold, hemorrhoids, burn
and athlete’s foot in Korean traditional medicine.1 It was reported
that the extract of the root bark and fruits of J. sinensis, respec-
tively, showed the anti-proliferative effect against human cancer
cell lines such as Caco-2 colon cancer cells, HepG-2 liver cancer
cells and MDA-MB-231 human breast cancer cells.2–4 On the
background of these findings, we attempted to isolate cytotoxic
constituents of J. sinensis. We previously reported that oleanane-,
ursane- and lupane-type triterpenes obtained from the leaves
and twigs of J. sinensis showed anti-proliferative effects on HSC-
T6 cells, immortalized rat hepatic stellate cells via apoptosis.5 We
also reported that four diarylheptanoids isolated from J. sinensis
significantly attenuated the toxicity induced by the glutamate in
HT22 cells through scavenging reactive oxygen species.6 In the
present study, we could additionally isolate three new triterpenes
(1–3) and two new sesquiterpenes (4–5) along with two known
sesquiterpenes (6–7) (Fig. 1).7–11 The two known compounds were
identified as (4S, 5S, 7R, 8S, 14R)-8, 11-dihydroxy-2, 4-cyclo-eudes-
mane (6) and 11-hydroxy-2, 4-cycloeudesman-8-one (7), respec-
tively.12 The 1D and 2D NMR spectroscopies, ESI-MS analysis and
GC after acid hydrolysis of compounds 3 and 5 were applied for
the elucidation of their structures. The isolated seven compounds
were evaluated for cytotoxicity against four cancer cell lines of
B16F10 (mouse melanoma), Hep-2 (human larynx carcinoma),
MCF-7 (human breast cancer), and U87-MG (human glioblastoma).
Compound 1 is whitish amorphous powder and was obtained as
½
a 2D5
ꢀ
ꢁ22.6 (c 0.80, MeOH). A molecular formula of compound 1
was assigned as C31H54O8 on the basis of its HRFABMS (positive
mode, m/z 559.3596 [M+Na]+, calcd for 559.3611). The 1H and
13C NMR, and HSQC spectral data displayed signals for an acetal
group at dH/dC 4.35 (1H, s, H-28)/109.4 (C-28), a methoxy group
at dH/dC 3.38 (3H, s, OCH3)/54.5 (OCH3), two oxygenated methylene
groups at dH/dC 4.59 and 3.74 (2H, d, J = 10.8 Hz, H-24a and H-24b)/
64.7 (C-24), and 4.35 and 4.06 (2H, d, J = 11.1 Hz, H-30a and
H-30b)/65.7 (C-30), two oxygenated methine groups at dH/dC 4.28
(1H, td, J = 8.0, 10.9 Hz, H-11)/69.5 (C-11) and 3.70 (1H, dd,
J = 4.4, 11.7 Hz, H-3)/80.1 (C-3), and five tertiary methyl groups
at dH/dC 1.80 (3H, s, H-29)/18.8 (C-29), 1.56 (3H, s, H-23)/23.9 (C-
23), 1.26 (3H, s, H-25)/17.9 (C-25), 1.09 (3H, s, H-26)/17.2 (C-26)
and 1.03 (3H, s, H-27)/15.6 (C-27). The connectivity of H-1/H-2/
H-3, H-5/H-6/H-7, H-9/H-11/H-12/H-13/H-18, H-15/H-16 and
H-21/H-22 was confirmed through the 1H–1H COSY analysis
(Fig. 2). In the HMBC spectrum, the locations of two oxygenated
methine carbons were determined to be at C-3 and C-11, respec-
tively, through the cross peaks of dH/dC 3.70 (H-3)/64.7 (C-24),
43.9 (C-4) and 23.9 (C-23), and 4.28 (H-11)/57.3 (C-10). By the cor-
related signal from dH 3.38 (OCH3) to dC 109.4 (C-28), it was sug-
gested that a methoxy group was attached to an acetoxy carbon.
Also, the signals correlating the acetal proton at dH 4.35 (H-28)
with the carbons at dC 77.6 (C-20), 54.5 (OCH3), 49.7 (C-18), 31.4
(C-16), and 35.7 (C-22) indicated that C-28 attached to a methoxy
⇑
Corresponding author. Tel.: +82 2 880 7842; fax: +82 2 888 2933.
E-mail address: youngkim@snu.ac.kr (Y.C. Kim).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.010

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H. Yang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2079–2083
OH
O
30
OH
20
29
19
21
22
18
HO
13
14
12
26
28
17
16
11
OH
O
25
9
OCH3
15
1
4
HO
HO
2
3
10
8
O
5
7
27
OH
6
HO
HO
O
24
23
HO
HO
OH
OH
OH
2
3
1
14
R
2
1
9
10
8
7
5
13
H
O
6
3
OH
4
11
12
HO
H
OH
O
HO
15
HO
OH
β
4 R = -OH
5
6
α
R = -OH
7 R = O
Figure 1. The structures of compounds 1–7.
OH
OH
O
HO
O
OH
OCH3
HO
HO
O
OH
O
HO
HO
HO
HO
OH
OH
OH
2
1
3
OH
H
O
OH
H
OH
HO
O
HO
HO
OH
5
4
Figure 2. The key 2D correlations for compounds 1–5. Arrow; HMBC correlation, bold line; 1H–1H COSY correlation.
group was connected with C-20 through an oxygen atom. A ter-
tiary methyl proton at dH 1.80 (H-29), and two oxygenated meth-
ylene protons at dH 4.35 (H-30a) and 4.05 (H-30b) had the cross
peaks with dC 77.6 (C-20), 76.2 (C-19) and 49.7 (C-18), and with
dC 77.6, 76.2 and 23.4 (C-21), respectively. These results suggested
that a tertiary methyl group and a hydroxyl methylene carbon
were located at C-19 attached to a hydroxyl group and at C-20,
respectively (Fig. 2). Taken into account all of the above described
spectroscopic data, compound 1 was considered as ursane-type tri-
terpene with multiple hydroxyl moieties and no olefinic moiety.
The relative configurations of H-3, C-23 and C-24 were deter-
was determined as 3b, 11a, 19a, 24, 30-pentahydroxy-20b, 28-
epoxy-28b-methoxy-ursane.
Compound 2 was whitish amorphous powder, ½a D25
ꢀ
+10.3 (c
0.50, pyridine), and the molecular formula (C30H50O2) was estab-
lished by the positive HRFABMS (m/z 442.3789 [M]+, calcd for
442.3811). The signals in the 1H and 13C NMR and HSQC experi-
ments indicated that compound 2 was a D18 oleanane-type
triterpene by the chemical shifts of two olefinic carbons at dC
143.3 (C-18) and 129.8 (C-19) (Kagawa et al., 1998), and it also in-
cluded two oxygenated methine carbons at dC 72.8 (C-3) and 71.9
(C-1) (Table 1). In the HMBC spectrum, the positions of two hydro-
xyl methine carbons were determined to be at C-1 and C-3 by the
signals from dH 1.01 (3H, s, H-25) to dC 71.9 (C-1), from dH 4.31 (1H,
dd, J = 4.7, 12.0 Hz, H-3) to dC 28.7 (C-23) and 16.6 (C-24) (Fig. 2).
Their configurations of two hydroxyl groups attached at C-1 and
mined as
a, a and b, respectively, based on the observed NOESY
correlations from H-23 to H-3 and H-5, and from H-24 to H-2b
(dH 2.17) and H-25 (dH 1.26) in consistent with the upfield chemical
shift of C-23 (dC 23.9) in the 13C NMR spectrum (Nishimura et al.,
1999). The signals of H-11 and H-29 also showed the positive
NOE effects on H-25 and H-26 (dH 1.09), and on H-13 (dH 2.35)
and OCH3 (dH 4.35), respectively. Thus, all the orientations of
H-11, C-29 and OCH3 were determined as b-orientation (Fig. 3).
From the obtained spectral data and above analysis, compound 1
C-3 were confirmed as a- and b-oriented, respectively, by the split-
ting patterns of H-1 (s) and H-3 (dd), and the chemical shifts of C-1
(dC 71.9) and C-3 (dC 72.8) compared to the previously reported
values.13,14 In NOESY spectrum, the relative stereochemistry was
reconfirmed by the correlations peaks from dH 3.97 (H-1) to dH

H. Yang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2079–2083
2081
CH3
H3CO
O
H
CH3
CH3
H
CH3
CH3
H
H
OH
H
H
3
HO
CH
H
CH3
HO
CH3
H
H
CH3
H
HO
H
HO
HO
H
H3C OH
H
H
HO
OH
OH
H
H
H
H
H
CH3
H3C
H
H
1
2
3
H
H
H
CH3
H
H
H
H
H
H
OH
OH
CH3
CH
3
CH3
H
H
H3C
CH3
HO
H3C
H
H
β
O- -D-glu
H
4
5
Figure 3. The NOESY spectral data of compounds 1–5. Dashed arrow; NOESY correlation.
2.58 (H-9) and 1.01 (H-25) and from dH 4.31 (H-3) to dH 1.67 (H-5)
and 1.29 (H-23) (Fig. 3). With above explanations, compound 2 was
cross peaks from H-1/H-2/H-3, H-5/H-6/H-7/H-8/H-9 established
the connectivity of the protonated carbons (Fig. 2). In the HMBC
experiment, the correlation peaks of dH/dC 1.25 (H-12 and H-13)/
75.2 (C-11) and 54.4 (C-7) indicated that the isopropyl moiety
was attached at C-7. Additionally, the HMBC analysis confirmed
that the locations of two methyl groups and a hydroxyl moiety
were at C-4, C-10 and C-8 in turn by the basis of the correlation
peaks from dH 1.04 (H-15) to dC 55.4 (C-5), 33.5 (C-3) and 26.2
(C-4), from dH 0.93 (H-14) to dC 55.4 (C-5), 52.6 (C-10) and 44.6
(C-1), and from dH 4.00 (H-8) to dC 52.6 (C-10) and 24.1 (C-6)
(Fig. 2). On the basis of these spectroscopic data, the planar
structure of compound 4 was identified with that of (4S, 5S, 7R,
8S, 14R)-8, 11-dihydroxy-2, 4-cycloeudesmane (6), the absolute
stereochemistry of which was determined by X-ray crystallogra-
phy and modified Mosher’s method (See the Supplementary data).7
The proposed relative stereochemical assignments at H-5, C-7, H-8,
C-14 and C-15 were established by the NOESY spectrum from dH
0.96 (1H, dd, J = 2.1, 13.4 Hz, H-5) to dH 0.35 (H-3b) and 1.37 (1H,
td, J = 4.0, 10.6 Hz, H-7), from dH 4.00 (H-8) to dH 1.85 (1H, dd,
J = 5.0, 11.4 Hz, H-9a) and 0.93 (H-14), and from dH 1.04 (H-15)
to dH 1.70 (1H, m, H-6a) (Fig. 3). The characteristic cross peak be-
tween dH 4.00 (H-8) and 0.93 (H-14) indicated that the orientation
of OH-8 was b-side different from that of compound 6. The abso-
lute stereochemistry of hydroxyl group at C-8 was determined as
R using the application of modified Mosher’s method (Fig. 4). With
above observed spectral data, the structure of compound 4 was
confirmed as (4S, 5S, 7R, 8R, 14R)-8, 11-dihydroxy-2, 4-cyclo-
eudesmane.
Compound 5 was isolated as brownish oil exhibiting C21H36O7
by HRFABMS m/z 423.2376 [M+Na]+ (calcd for 423.2359). In the
1H NMR analysis, it was observed the characteristic signals indicat-
ing an olefinic proton at dH 5.75 (1H, br s, H-3), two oxygenated
methylene protons at dH 4.41(1H, d, J = 12.5 Hz, H-15a) and 4.19
(1H, d, J = 12.5 Hz, H-15b), along with an anomeric proton at dH
4.98 (1H, d, J = 7.7 Hz, H-10). The 13C NMR spectrum showed signals
displaying the presence of two olefinic carbons at dC 140.3 (C-4)
and 122.4 (C-3), an oxygenated quaternary carbon at dC 80.0
(C-11), an oxygenated methylene carbon at dC 64.8 (C-15) and an
anomeric carbon at dC 98.7 (C-10) (Table 2). With above observed
1D NMR spectrum, compound 5 was a eudesmane sesquiterpene
having a sugar moiety. The 1H–1H COSY spectrum showed the con-
nectivity of protonated carbons (H-1/H-2/H-3, H-5/H-6/H-7/H-8/
determined as 1a, 3b-dihydroxy-olean-18-ene.
Compound 3 was isolated as whitish amorphous powder, ½a D25
ꢀ
+39.6 (c 1.32, pyridine) and showed the ion peak at m/z 651.4130
[M+H]+ (calcd for 651.4108) in positive mode HRFABMS indicating
its molecular formula (C36H58O10). The characteristic signals at dH/
dC 5.41 (1H, br s, H-12)/126.0 (C-12) and dC 138.5 (C-13) and dH/dC
6.23 (1H, d, J = 8.2 Hz, H-10)/95.7 (C-10) in 1D NMR and HSQC spec-
tral data indicated that compound 3 was a
D
12 ursane-type bearing
a sugar residue.15 In HMBC experiment, it was postulated that the
positions of three hydroxyl groups were located at C-2, C-3 and C-
23, respectively, from dH 4.25 (1H, t, J = 8.8 Hz, H-2) to dC 78.8 (C-3),
from dH 4.10 (1H, d, J = 1.8 Hz, H-3) to dC 66.2 (C-2) and 71.1 (C-23)
(Fig. 2). The sugar residue was determined to locate at C-28 by the
correlation peak from dH 6.23 (H-10) to dC 176.2 (C-28). With these
spectroscopies data, the structure of compound 3 was expected
similar to 2a, 3a, 23-trihydroxy-urs-12-en-28-oic acid, except for
the presence of a sugar moiety attached to C-28.11 The different
NOE effects showed that the signal of H-3 had indisputable
cross-peak with H-2 correlating with those of H-24 and H-25
(Fig. 3). This result indicated that all the hydroxyl moieties at C-2
and C-3 of compound 3 were oriented as the
tively. The sugar was identified as -glucose by GC analysis after
acid hydrolysis. The amplitude of the coupling constant at dH
6.23 (J = 8.2 Hz) suggested that a -glucose was oriented as b-side.
The aglycone of 3 was confirmed as 2 , 3 , 23-trihydroxy-urs-12-
a-position, respec-
D
D
a
a
en-28-oic acid by comparing with the previously reported litera-
ture.16 On the basis of the above description, compound 3 was
confirmed as 2
b- -glucopyranoside.
Compound 4 was obtained as colorless amorphous crystal. The
a, 3a, 23-trihydroxy-urs-12-en-28-oic acid 28-O-
D
molecular formula C15H26O2 was established by the positive
HRFABMS (m/z 239.2000, calcd for [M+H]+ 239.2011). The 1H and
13C NMR, and HSQC spectral data suggested the presence of an oxy-
genated methine carbon at dH 4.00 (1H, dd, J = 5.0, 10.6 Hz, H-8)/dC
69.6 (C-8), four methyl carbons at dH 1.25 (6H, s, H-12 and H-13)/dC
30.1 (C-12) and 24.0 (C-13), 1.04 (3H, s, H-15)/dC 18.7 (C-15) and
0.93 (3H, s, H-14)/dC 20.2 (C-14) (Table 1). In the 1H NMR spectrum,
the signals at dH 0.81 (1H, m, H-3a), 0.81 (1H, m, H-1b) and 0.35
(1H, t, J = 3.9 Hz, H-3b) indicated that the structure of compound
4 included a cyclopropane ring.17 In the 1H–1H COSY analysis, the

2082
H. Yang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2079–2083
Table 1
1H and 13C NMR spectroscopic data for compounds 2–3 in pyridine-d5
Position
1a
2b
3a
dH (J in Hz)
dC
41.8
dH (J in Hz)
dC
dH (J in Hz)
dC
1
2
3.36, m
1.49, m
2.17, m
1.99, m
3.97, s
71.9, d
1.91, m
1.76, m
4.25, t (8.8)
42.8, t
29.0
2.38, m
2.26, m
36.1, t
66.2, d
3
4
5
6
3.70, dd (4.4, 11.7)
80.1
43.9
57.0
18.9
4.31, dd (4.7, 12.0)
48.5, d
39.8, t
48.5, d
18.7, t
4.10, d (1.8)
2.02, m
78.8, d
41.8, s
43.4, d
18.3, t
1.06, m
1.72, m
1.43, m
1.42–1.30, m
1.67, m
1.67, m
1.45, m
1.46, m
1.53, m
1.34, m
1.68, m
1.34, m
7
35.8
34.7, t
33.2, t
8
9
10
11
43.8
57.3
39.5
69.5
40.8, s
41.6, d
41.8, s
26.4, t
40.3, s
48.0, d
38.3, s
23.7, t
1.78, m
2.58, m
1.87, m
2.03, m
5.41, br s
4.28, td (8.0, 10.9)
1.44, m
1.34, m
1.81, m
12
2.86, m
1.72, m
2.35, m
41.7
27.9, t
126.0, d
13
14
15
35.3
41.2
28.2
2.33, m
38.8, d
44.1, s
27.9, t
138.5, s
42.5, s
28.6, t
1.92, m
1.01, m
1.34, m
1.80, m
1.12, m
1.43, m
2.40, td (4.7, 13.8)
1.09, m
2.01, m
16
31.4
37.7, t
24.6, t
1.90, m
17
18
19
20
21
34.4
49.7
76.2
77.6
23.4
34.6, s
143.3, s
129.8, d
32.5, s
33.6, t
48.3,s
1.81, m
2.48, d (11.3)
1.34, m
0.84, m
1.34, m
1.23, m
1.85, m
1.68, m
3.87, m
3.71, d (10.8)
0.83, s
53.3, d
39.2, d
39.1, d
30.8, t
4.87, br s
2.69, m
2.06, m
1.42–1.30, m
1.45, m
1.31, m
1.33, m
22
23
24
35.7
23.9
64.7
38.1, t
28.7, q
16.2, q
36.7, t
71.1, t
17.7, q
1.56, s
1.29, s
1.11, s
4.59, d (10.8)
3.74, d (10.8)
1.26, s
1.09, s
1.03, s
4.35, m
1.80, s
4.35, d (11.1)
4.06, d (11.1)
3.38, s
25
26
27
28
29
30
17.7
17.2
15.6
109.4
18.8
65.7
1.01, s
1.16, s
0.92, s
1.06, s
0.98, s
0.97, s
17.8, q
16.4, q
15.1, q
25.4, q
31.5, q
29.3, q
1.16, s
1.02, s
1.07, s
17.7, q
17.3, q
23.7, q
176.2, s
17.2, q
21.2, q
0.87, d (6.4)
0.84, d (6.4)
OCH3
54.5
b-
10
20
30
40
50
60
D-Glucose
6.23, d (8.2)
4.17, m
4.25, m
4.32, m
3.99, m
95.7, d
74.0, d
78.9, d
71.2, d
79.1, d
62.3, t
4.39, m
Multiplicity of 13C NMR data was determined by DEPT experiments (s = quaternary, d = methine, t = methylene, q = methyl).
a
1H and 13C data were measured at 500 and 125 MHz in pyridine-d5, respectively.
b
1H and 13C data were measured at 400 and 100 MHz in pyridine-d5, respectively.
dH 4.41 (H-15a) and 4.19 (H-15b) to dC 140.5 (C-4), 122.4 (C-3) and
44.8 (C-5), from dH 1.44 (3H, s, H-12) and 1.34 (3H, s, H-13) to dC
80.0 (C-11) and 48.4 (C-7), and from dH 0.81 (3H, s, H-14) to dC
40.6 (C-9), 38.0 (C-1) and 32.4 (C-10) displayed the positions of
0.00
+0. 0 2
+0. 0 2
+0.05
+0.12
OH
0.00
-0.04
-0.04
two olefinic carbons
(D
3), an oxygenated methylene carbon
+0 .0 2
-0.21
0.00
+0.01
0.00
-0.05
(C-15), an quaternary oxygenated carbon (C-11) and three methyl
carbons (C-12, C-13 and C-14) (Fig. 2). Also, the location of a sugar
moiety was at C-11 by the cross peak from dH 4.98 (H-10) to dC 80.0
(C-11). The doublet (J = 7.7 Hz) of an anomeric proton indicated
H
OH
-0.12
-0.01
4a
4b
R = (S)-MTPAester
R = (R)-MTPAester
that
afforded
tral data, the aglycone of compound 5 was the derivative of
-eudesmol substituted with a hydroxyl group at C-15 and a -glu-
cose at C-11.18,19 The stereochemistry of H-5, C-11 and C-14 was
confirmed as , b and b-configuration, respectively, by the cross
D
-glucose was oriented as b-configuration. Acid hydrolysis
D-glucose as the sugar residue. On the basis of these spec-
Figure 4.
Dd (dS–dR) Values obtained from MTPA esters for compound 4.
a
D
H-9). In the HMBC experiment, the key correlation peaks from dH
5.75 (1H, br s, H-3) to dC 64.8 (C-15), 44.8 (C-5) and 38.0 (C-1), from
a

H. Yang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2079–2083
2083
Table 2
cell lines (Table 3). Interestingly, compound 6 having OH-8
a
signif-
1H and 13C NMR spectroscopic data for compounds 4–5
icantly inhibited cell growth of the cancer cells, whereas com-
pound 4 which has identical planar structure of compound 6
having OH-8b showed little or no cytotoxicity.
Position
4a
5b
dH (J in Hz)
dC
dH (J in Hz)
dC
1
2
3
1.70, m
0.81, m
1.12, m
44.6, t
1.30, m
38.0, t
Acknowledgment
26.6, d
33.5, t
2.11, m
1.98, d (17.9)
5.75, br s
23.2, t
This research was supported by a Grant (2011K000290) from
Brain Research Center of the 21st Century Frontier Research Pro-
gram funded by the Ministry of Science and Technology, Korea.
0.81, m
0.35, t (3.9)
122.4, d
4
5
6
26.2, s
55.4, d
24.1, t
140.3, s
44.8, d
23.8, t
0.96, dd (2.1, 13.4)
1.70, m
1.07, m
2.29, d (12.4)
2.66, d (12.4)
1.22, m
Supplementary data
7
8
1.37, td (4.0, 10.6)
4.00, td (5.0, 10.6)
54.4, d
69.6, d
1.82, m
1.73, d (8.6)
1.42, m
1.43, m
1.17, m
48.4, d
23.0, t
Supplementary data (original spectral data of 1–5, detailed
descriptions of experimental and bioassay protocols) associated
with this article can be found, in the online version, at
doi:10.1016/j.bmcl.2012.01.010.
9
1.85, dd (5.0, 11.4)
1.15, t (11.4)
46.2, t
40.6, t
10
11
12
13
14
15
52.6, s
75.2, s
30.1, q
24.0, q
20.2, q
18.7, q
32.4, s
80.0, s
25.4, q
23.9, q
15.9, q
64.8, t
References and notes
1.25, s
1.25, s
0.93, s
1.04, s
1.44, s
1.34, s
0.81, s
4.41, d (12.5)
4.19, d (12.5)
1. Park, J. H.; Lee, J. K. The Encyclopedia of Medicinal Plants; Shin-Il Books: Seoul,
2000.
2. Hasan, T. N.; B, L. G.; Shafi, G.; Al-Hazzani, A. A.; Alshatwi, A. A. Asian Pac. J.
Cancer Prev. 2011, 12, 525.
3. Negi, A. S.; Luqman, S.; Srivastava, S.; Krishna, V.; Gupta, N.; Darokar, M. P.
Pharm. Biol. 2011, 49, 669.
4. Carvalho, M.; Ferreira, P. J.; Mendes, V. S.; Silva, R.; Pereira, J. A.; Jeronimo, C.;
Silva, B. M. Food Chem. Toxicol. 2010, 48, 441.
b-
1’
2’
3’
4’
5’
6’
D-Glucose
4.98, d (7.7)
3.92, t (8.1)
4.20, t (9.0)
4.10, t (9.3)
3.85, m
98.7, d
75.4, d
78.8, d
71.9, d
78.1, d
63.0, t
5. Yang, H.; Jeong, E. J.; Kim, J.; Sung, S. H.; Kim, Y. C. J. Nat. Prod. 2011, 74, 751.
6. Yang, H.; Sung, S. H.; Kim, J.; Kim, Y. C. Planta Med. 2011, 77, 841.
7. 3b, 11a, 19a, 24, 30-Pentahydroxy-20b, 28-epoxy-28b-methoxy-ursane (1):
4.44, td (2.3, 12.0)
4.25, dd (5.7, 11.8)
whitish amorphous powder; ½a D25
ꢀ : ꢁ22.6 (c 0.8, MeOH); IR (KBr) vmax: 3375,
2928, 1636, 1456, 1378, 1194, 1101, 1039 cmꢁ1; FABMS (positive mode): m/z
559 [M+Na]+; HRFABMS (positive mode): m/z 559.3596 [M+Na]+ (calcd m/z
559.3611).
Multiplicity of 13C NMR data was determined by DEPT experiments (s = quaternary,
d = methine, t = methylene, q = methyl).
8.
9.
1
a
, 3b-Dihydroxy-olean-18-ene (2): whitish amorphous powder; ½a D25
ꢀ
: +10.3 (c
1H and 13C NMR data were measured at 400 and 100 MHz in CDCl3,
a
0.50, pyridine); IR (KBr)
vmax: 3336, 2938, 2865, 1649, 1452, 1376, 1052,
respectively.
1032 cmꢁ1; FABMS (positive mode): m/z 442 [M]+; HRFABMS (positive mode):
1H and 13C NMR data were measured at 500 and 125 MHz in pyridine-d5,
b
m/z 442.3789 [M]+ (calcd for 442.3811).
respectively.
2
a, 3
a
, 23-Trihydroxy-urs-12-en-28-oic acid 28-O-b-
D
-glucopyranoside (3):
whitish amorphous powder; ½a D25
ꢀ
: +39.6 (c 1.32, pyridine); IR (KBr) vmax: 3388,
2921, 1749, 1647, 1455, 1375, 1142, 1070, 1025 cmꢁ1; FABMS (positive mode):
m/z 673 [M+Na]+; HRFABMS (positive mode): m/z 651.4130 [M+H]+ (calcd for
651.4108).
Table 3
The cytotoxic activities of compounds 1–7 in vitro
10. (4S, 5S, 7R, 8R, 14R)-8, 11-Dihydroxy-2, 4-cyclo-eudesmane (4): colorless
amorphous crystal; ½a D25
ꢀ : ꢁ0.6 (c 0.40, MeOH); IR (MeOH) mmax: 3334, 2940,
Cell linesa (IC50
lM)
2858, 1716, 1651, 1455, 1375, 1176, 1150, 1058, 1015, 979, 907, 788 cmꢁ1
;
Compound
FABMS (positive mode): m/z 239 [M+H]+; HRFABMS (positive mode): m/z
239.2000 [M+H]+ (calcd for 239.2011); (S)-MTPA ester (4a): 1H NMR (CDCl3,
500 MHz) d 5.21 (1H, td, J = 10.9, 4.8 Hz), 2.17 (1H, m, H-9a), 1.90 (1H, dt,
J = 7.9, 4.0 Hz, H-6a), 1.75 (1H, m, H-1a), 1.71 (1H, m, H-7), 1.24 (1H, m, H-9b),
1.15 (2H, m, H-2 and H-6b), 1.06 (6H, br s, H-12 and H-15), 1.03 (3H, br s, H-
14), 1.01 (1H, m, H-5), 0.95 (3H, br s, H-13), 0.85 (1H, dd, J = 8.7, 4.2 Hz, H-3a),
0.83 (1H, dd, J = 10.0, 3.2 Hz, H-1b), 0.37 (1H, t, J = 3.9 Hz, H-3b); (R)-MTPA
ester (4b): 1H NMR (CDCl3, 500 MHz) d 5.25 (1H, td, J = 10.9, 4.8 Hz), 2.12 (1H,
dd, J = 11.5, 4.8 Hz, H-9a), 1.90 (1H, dt, J = 12.9, 4.1 Hz, H-6a), 1.75 (1H, m, H-7),
1.73 (1H, m, H-1a), 1.20 (1H, m, H-6b), 1.18 (3H, br s, H-12), 1.16 (3H, br s, H-
13), 1.15 (1H, m, H-2), 1.12 (1H, m, H-9b), 1.07 (3H, br s, H-15), 1.03 (3H, br s,
H-14), 0.99 (1H, dd, J = 13.3, 3.3 Hz, H-5), 0.85 (1H, dd, J = 11.4, 7.1 Hz, H-3a),
0.81 (1H, dd, J = 12.4, 3.3 Hz, H-1b), 0.36 (1H, t, J = 4.0 Hz, H-3b).
B16F10
Hep-2
MCF-7
U87-MG
1
2
3
4
5
6
7
1.53 0.30
0.30 0.01
>10
>10
>10
5.52 1.62
5.40 1.51
>10
>10
>10
1.25 0.36
3.86 0.16
>10
>10
>10
3.29 0.13
1.31 0.22
>10
>10
>10
>10
0.30 0.02
4.22 1.38
5.25 0.61
1.81 0.51
4.11 0.20
1.13 0.39
1.16 0.55
Cell viability was measured by MTS assay. Cancer cells were treated with com-
pounds 1–7 for 24 h. Results are expressed as the mean S.D. of four independent
experiments.
Cancer cell lines; B16F10 (mouse melanoma), Hep-2 (human larynx carcinoma),
MCF-7 (human breast cancer), and U87-MG (human glioblastoma).
11. 15-Hydroxy-
ꢁ381.3 (c 0.1, MeOH); IR (MeOH)
1076, 1036 cmꢁ1
a
-eudesmol-11-O-b-
D
-glucopyranoside (5): brownish oil; ½a D25
ꢀ
:
a
m
max: 3388, 2924, 1648, 1455, 1375, 1264,
;
FABMS (positive mode): m/z 423 [M+Na]+; HRFABMS
(positive mode): m/z 423.2376 [M+Na]+ (calcd for 423.2359).
12. Maeda, H.; Kakoki, N.; Ayabe, M.; Koga, Y.; Oribe, T.; Matsuo, Y.; Tanaka, T.;
Kouno, I. Phytochemistry 2011, 72, 796.
peaks from dH 2.29 (1H, m, H-5) to dH 1.82 (1H, m, H-7) and 1.17
(1H, m, H-9b), and from dH 0.81 (3H, s, H-14) to dH 2.11 (1H, m,
H-2a), 1.42 (1H, m, H-8b) and 1.22 (1H, m, H-6b) in the
NOESY experiment (Fig. 3). Consequently, the structure of com-
pound 5 was concluded as 15-hydroxy-a-eudesmol-11-O-b-D-
glucopyranoside.
13. Gonzalez, A. G.; Fraga, B. M.; Gonzalez, P.; Hernandez, M. G.; Ravelo, A. G.
Phytochemistry 1919, 1981, 20.
14. Katerere, D. R.; Gray, A. I.; Nash, R. J.; Waigh, R. D. Phytochemistry 2003, 63, 81.
15. Seebacher, W.; Simic, N.; Weis, R.; Saf, R.; Kunert, O. Magn. Reson. Chem. 2003,
41, 636.
16. Lee, I. K.; Kim do, H.; Lee, S. Y.; Kim, K. R.; Choi, S. U.; Hong, J. K.; Lee, J. H.; Park,
Y. H.; Lee, K. R. Arch. Pharmacol. Res. 2008, 31, 1578.
In the study, the cytotoxicity of compounds 1–7 was evaluated
using four cancer cell lines such as B16F10 (mouse melanoma),
Hep-2 (human larynx carcinoma), MCF-7 (human breast cancer),
and U87-MG (human glioblastoma). Compounds 1, 2, 6 and 7
exhibited potent cytotoxic activities against all of the four cancer
17. Bardon, A.; Mitre, G. B.; Kamiya, N.; Toyota, M.; Asakawa, Y. Phytochemistry
2002, 59, 205.
18. Jakupovic, J.; Lehmann, L.; Bohlmann, F.; King, R. M.; Robinson, H.
Phytochemistry 1988, 27, 3831.
19. Raharivelomanana, P.; Bianchini, J. P.; Cambon, A.; Azzaro, M.; Faure, R. Magn.
Reson. Chem. 1995, 33, 233.