Bioactivity-guided isolation of cytotoxic constituents of Brucea javanica collected in Vietnam

Article abstract of DOI:10.1016/j.bmc.2008.10.076

Five new triterpenoids (1-5), together with two known quassinoids, bruceantin (6) and bruceine A (7), and a known flavonolignan, (-)-hydnocarpin (8), were isolated from the chloroform-soluble subfraction of a methanol extract of the combined twigs, leaves

Full text of DOI:10.1016/j.bmc.2008.10.076

Bioorganic & Medicinal Chemistry 17 (2009) 2219–2224
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Bioactivity-guided isolation of cytotoxic constituents of Brucea javanica
collected in Vietnam
Li Pan a, Young-Won Chin a, Hee-Byung Chai a, Tran Ngoc Ninh b, Djaja Djendoel Soejarto c,
A. Douglas Kinghorn a,
*
a Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, 500 West 12th Avenue, Columbus, OH 43210, USA
b Institute of Ecology and Biological Resources, Vietnamese Academy of Science and Technology, Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam
c Program for Collaborative Research in the Pharmaceutical Sciences, Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, 833 South Wood Street,
University of Illinois at Chicago, Chicago, IL 60612, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 April 2008
Revised 4 June 2008
Accepted 31 October 2008
Available online 5 November 2008
Five new triterpenoids (1–5), together with two known quassinoids, bruceantin (6) and bruceine A (7),
and a known flavonolignan, (ꢀ)-hydnocarpin (8), were isolated from the chloroform-soluble subfraction
of a methanol extract of the combined twigs, leaves, and inflorescence of Brucea javanica collected in Viet-
nam. The structures of the new compounds 1–5 were established on the basis of spectroscopic methods.
All isolates were evaluated for cytotoxicity against a small panel of human cancer cell lines. Quassinoids 6
and 7 were found to be highly active against these cell lines. (ꢀ)-Hydnocarpin (8) showed a potentiating
effect when combined with both 6 and 7, during cytotoxicity testing using the MCF-7 human breast can-
cer cell line.
Keywords:
Brucea javanica
Simaroubaceae
Apotirucallane triterpenoids
Protolimonoid triterpenoid
Quassinoids
Ó 2008 Elsevier Ltd. All rights reserved.
Flavonolignan
Cytotoxicity
Potentiating effect
1. Introduction
using the MCF-7 human breast cancer cell line to monitor fraction-
ation. The chloroform-soluble partition of a methanol extract from
Brucea javanica (L.) Merr. (Simaroubaceae) is an evergreen
shrub distributed from southeast Asia to northern Australia. In
southeast Asia, all parts of B. javanica have been employed as an
antimalarial treatment, and the seeds of this plant have also been
used for the alleviation of dysentery and skin conditions such as
warts and corns.1–5 Previous investigations on the chemical con-
stituents of B. javanica have focused mainly on the fruits and seeds,
and have found this plant part to be a rich source of quassinoids,
the mixed B. javanica leaves, twigs, and inflorescence collected in
Vietnam was chosen for phytochemical study and biological eval-
uation due to its inhibitory effect on this cell line. In the present
work, activity-guided fractionation of the chloroform-soluble ex-
tract led to the isolation of five new triterpenoids (1–5), two
known quassinoids (6, 7), and a known flavonolignan (8). Herein,
the isolation and structure elucidation of compounds 1–5, as well
as the biological evaluation of all isolates obtained, are described.
6–12
which are the bitter-tasting principles of many species in the
family Simaroubaceae. Quassinoids have been documented from
B. javanica with a wide spectrum of biological effects, such as hav-
ing potential antibabesial, anti-HIV, antimalarial, antitubercular,
antitumor, cancer chemopreventive, and cytotoxic activities.13–20
Research on other plant parts of B. javanica is limited, but apotiru-
callane triterpenoids and b-carboline alkaloids occur as major con-
stituents besides the quassinoids.3,4
2. Results and discussion
2.1. Structure elucidation of new compounds
Bruceajavanone A (1) was obtained as a white amorphous resin.
The molecular formula was determined as C38H56O8 based on the
sodiated molecular ion peak at m/z 663.3873 [M+Na]+ (calcd
663.3873) in the HRESIMS. The IR spectrum showed absorptions
for hydroxyl (3458 cmꢀ1) and carbonyl (1730, 1671 cmꢀ1) groups.
As part of ongoing investigation of natural anticancer agents
from tropical plants, an initial screening procedure was conducted
The UV maximum at 228 nm (log
an
,b-unsaturated ketone group. In the 1H NMR spectrum, seven
tertiary methyl groups were observed at the high-field region from
e 4.12) suggested the presence of
a
* Corresponding author. Tel.: +1 614 247 8094; fax: +1 614 247 8642.
E-mail address: kinghorn.4@osu.edu (A. D. Kinghorn).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.10.076

2220
L. Pan et al. / Bioorg. Med. Chem. 17 (2009) 2219–2224
dH 0.95–1.35 ppm (see Table 1), and signals for two olefinic protons
at dH 7.08 (1H, d, J = 10.4 Hz, H-1) and 5.73 (1H, d, J = 10.4 Hz, H-2)
were consistent with the presence of an endocyclic double bond
conjugated to a ketone group, while another olefinic proton reso-
nating at dH 5.34 (s, H-15) was ascribed to a trisubstituted double
bond. Moreover, an acetyl group as well as a hexanoate group was
recognized from characteristic signals in the 1H and 13C NMR spec-
tra (see Tables 1 and 2). After subtraction of these two ester groups,
there were 30 carbon signals remaining in the 13C NMR spectrum,
which were sorted by DEPT into seven methyls, four methylenes,
four methines, four quaternary carbons, six oxygenated carbons
(including five secondary and a tertiary), a carbonyl group, a tri-
substituted double bond, and a 1,2-substituted double bond. These
characteristic NMR data suggested that compound 1 is an apotiru-
callane-type triterpenoid, with a structure similar to that of 21-O-
acetyl-toosendantriol.21 Compounds based on this triterpenoid
skeleton have been obtained previously from the stems and bark
of B. javanica as well as other species of the plant families Melia-
ceae and Rutaceae.4,21–24
22. The a-positions of OH-7 and the C-11 hexanoate group
were deduced from the NOE cross peaks of H-7/H-30, H-11/H-
30, and H-11/Hb-22. The b orientation of OAc-21 was elucidated
from the NOE effects of H-21 with H-20 as well as H-18. The
a,b-unsaturated ketone in the A ring obeyed the octant rule,
which made it possible to confirm the absolute configuration
of 1 using a circular dichroism (CD) spectroscopic measure-
ment.4,25 The CD curve of this compound showed a negative
band at 346 nm (
D
e
= ꢀ1.84) for a n–p* transition, which was
in agreement with the predicted negative Cotton effect for the
A ring based on the assignments of 5R and 10R. Thus, the
chemical structure of compound 1 was determined as shown,
and was accorded the trivial name, bruceajavanone A.
The HRESIMS of compound 2 gave a sodiated molecular ion
peak at m/z 705.3978 [M+Na]+, consistent with a molecular for-
mula of C40H58O9. The 1H NMR spectrum of compound 2 was very
similar to that of bruceajavanone A (1), except for an obvious
downfield shift of H-7 from dH 4.02 (br s, 1H) to dH 5.26 (br s,
1H) (Table 1). Moreover, a second acetate methyl group signal
H
O
H
H
O
O
26
24
O
O
O
23
O
25
O
21
22
3'
R3O
20
AcO
MeO
H
2'
27
18
H
H
H
12
1' O
OH
H
H
4'
O
R2O
H
16
17
5'
OH
11 30 13
19
H
H
1
9
14
2
15
8
10
H
6
5
7
3
4
O
O
O
OR1
OAc
28 H
29
H
4
5
1
2
3
R1 = H,
R1 = Ac,
R1 = Ac,
R2 = Hexanoyl,
R2 = Hexanoyl,
R2 = Tigloyl,
R3 = Ac
R3 = Ac
R3 = Ac
OMe
3''
OR
2''
1''
4''
5''
5'
2'
O
4'
3'
7''
OH
O
6'
6''
8
COOCH3
OR
HO
H
RO
O
OR
9
1'
8''
O
7
6
2
3
9''
O
4
10
5
H
H
OR
O
HO
O
O
H
8
R = H
6
7
R = (E)-3,4-Dimethyl-2-pentenoyl
R = Isopentanoyl
8a R = Ac
The presence of a five-membered hemiacetyl ring in 1 was
confirmed by the HMBC correlations from the H-21 methine
proton (dH 6.20, d, J = 4.1 Hz) to C-20 (dC 44.2), C-22 (dC 31.4),
as well as C-23 (dC 79.8). HMBC correlations from the high-field
oxymethine proton (dH 2.67, d, J = 7.6 Hz, H-24) as well as two
methyl protons (dH 1.32, 1.29, s, H-26 and H-27, each 3H) to C-
25 (dC 57.1) indicated the occurrence of an epoxide moiety with
two methyl substituents. The key 1H–1H COSY correlations of
H-23 (dH 3.90, ddd, J = 9.9, 7.3, 7.3 Hz) with H-24, and H-20
(dH 2.35, m) with H-17 (dH 1.96, m), established the connectiv-
ities of these different moieties. The acetyl and hexanoate
groups mentioned above were assigned to C-21 and C-11,
respectively, based on the key HMBC correlations between H-
21 with the carbonyl of the acetyl group (dC 169.7), and be-
tween H-11 (dH 5.45, dd, J = 9.5, 7.5 Hz) with the carbonyl of
the hexanoate group (dC 172.9). A free hydroxy group was lo-
cated at C-7, with HMBC correlations of H-7 (dH 4.02, br s) with
C-5, C-8, and C-30 observed. The NOE effects between H3-18
with H-20 and H-17 with H3-30 indicated that the five-mem-
was observed at dH 1.97 (s, 3H) besides that resonating at dH 2.07
(s, 3H). These observations suggested that the free hydroxy group
at C-7 in compound 1 is acetylated in 2. Corresponding evidence
in the 13C NMR spectrum was found from the resonances for a car-
bonyl group at dC 170.0 and the methyl signal at dC 20.5, along with
a downfield shift of C-7 by 2.8 ppm relative to 1 (Table 2). This
analysis was confirmed by a key HMBC correlation between H-7
and the carbonyl of the acetyl group at dC 170.0. The absolute con-
figuration of compound 2 was consistent with that of bruceajava-
none A (1) based on the analysis of NOESY spectrum as well as
the CD curve. Thus, the chemical structure of compound 2 (bru-
ceajavanone A 7-acetate) was determined as shown.
The molecular formula of 3 was defined as C39H54O9 from the
sodiated molecular ion peak at m/z 689.3651 [M+Na]+ in the HRE-
SIMS. Comparison of the 1D and 2D NMR data of 3 with those of 2
revealed that the only difference between these two compounds
was at the C-11 substituent, with the hexanoate group in 2 re-
placed by a tigloyl group in 3. The signals of the latter appeared
at dH 6.85 (1H, qd, J = 7.1, 1.0 Hz, H-30), 1.84 (3H, br d, J = 7.1 Hz,
H-40) and 1.88 (1H, br s, H-50) in the 1H NMR spectrum as well as
at dC 169.8 (s, C-10), 128.9 (s, C-20), 138.2 (d, C-30), 14.6 (q, C-40)
and 12.1 (q, C-50) in the 13C NMR spectrum. A key HMBC correla-
bered hemiacetal ring is located in an
a-position at C-17, and
the C-24, C-25 epoxide group was found to be b-oriented based
on the NOE correlations of H-23 with H -22 and H-24 with Hb-
a

L. Pan et al. / Bioorg. Med. Chem. 17 (2009) 2219–2224
2221
Table 1
1H NMR chemical shifts of compounds 1–5a.
Position
1
1
2
3
4
5
7.08, d (10.4)
5.73, d (10.4)
7.14, d (10.4)
5.76, d (10.4)
7.07, d (10.4)
5.72, d (10.4)
3. 66, br s
2.40, m
3.02, m
3.85, br s
2.39, m
2.95, m
2a
2b
5
2.48, m
2.24, m
2.27, m
2.38, m
6
7
8
9
11
12
12b
15
16
17
18
19
20
21
22
22b
23
24
26
1.89–1.92, m
4.02, br s
1.82–1.90, m
5.26, br s
1.83, m, 1.94, m
5.28, br s
1.81, m, 2.02, m
5.25, br s
5.42, br s
2.23, mb
2.20, m
3.14, m
5.35, m
1.02, m
2.40, mb
1.65, m
2.53, d (9.5)
5.45, dd (9.5, 7.5)
1.59, br d (14.8)
1.82, m
5.34, br s
2.26–2.30, m
1.96, m
1.11, s
1.24, s
2.35, m
6.20, d (4.1)
2.10, m
2.53, d (9.5)
5.43, dd (9.5, 7.5)
1.55, br d (14.8)
1.83, m
5.34, br s
2.12–2.18, m
1.95, m
1.10, s
1.28, s
2.32, m
6.20, d (4.1)
2.06, m
2.57, d (9.5)
5.49 dd (9.5, 7.5)
1.60, br d (15.0)
1.82, m
5.34, br s
2.17–2.20, m
1.92, m
1.10, s
1.28, s
2.32, m
6.17, d (4.1)
2.05, m
2.84, d (9.1)
5.56, m
1.85, m
a
1.85, m
5.36, br s
2.16–2.19, m
1.70, m
1.13, s
1.23, s
2.30, m
4.63, d (3.8)
1.99, m
1.42, m, 2.00, m
1.97, m
1.05, s
1.09, s
2.15, m
6.10, d (4.1)
2.10, m
1.68, m
3.91, m
2.67, d (7.6)
1.32, s
a
1.68, m
1.68, m
1.69, m
1.40, m
3.90, ddd (9.9, 7.3, 7.3)
2.67, d (7.6)
1.32, s
3.90, ddd (9.8, 7.2, 7.2)
2.67, d (7.5)
1.29, sb
3.90, ddd (9.8, 7.2, 7.2)
2.66, d (7.5)
1.32, s
3.90, ddd (11.6, 7.5, 4.7)
2.66, d (7.5)
1.32, s
27
1.29, s
1.27, sb
1.27, s
1.27, s
1.26, s
28
1.17, s
1.08, s
1.08, s
1.08, s
1.08, s
29
0.96, s
1.05, s
1.05, s
1.05, s
0.97, s
30
1.18, s
2.30, m
1.22, s
2.32, m
1.68, m
1.32, m
1.34, m
0.91, t (6.9)
1.97, s
1.24, s
1.24, s
0.88, s
20
30
1.64, m
6.85, qd (7.1, 1.0)
1.84, br d (7.1)
1.88, br s
6.82, qd (7.1, 1.0)
1.81, br d (7.1)
1.87, br s
6.82, qd (7.1, 1.0)
1.81, br d (7.1)
1.87, br s
40
1.34, mb
50
1.35, mb
60
0.91, t (6.9)
OAc-7
OAc-21
OMe-21
2.00, s
2.07, s
1.99, s
3.23, s
1.99, s
2.06, s
2.07, s
2.07, s
a
Measured at 400 MHz; obtained in CDCl3 with TMS as internal standard; J values (Hz) are given in parentheses. Assignments are based on 1H-1H COSY, HSQC, and HMBC
spectroscopic data.
b
Assignments may be interchangeable in the same column.
tion of H-11 (dH 5.49 m) with the carbonyl carbon of the tigloyl
group at dC 169.8 (C-10) was observed. Analysis of the NOESY spec-
trum and the CD curve indicated the absolute configuration of
compound 3 to be identical with those of compounds 1 and 2.
Therefore, the structure of compound 3 (bruceajavanone B) was
assigned as shown.
served between H-1 and H-2, which implied that H-1 adopted an
equatorial b orientation. This was confirmed by NOE effects be-
tween H-1 with H-19 and Hb-2. The
a orientation of the meth-
oxy group at C-21 was deduced from the NOE effects between
H-21 with H-17 and Hb-22. This assignment explained the nota-
ble downfield shift of around 4.5 ppm for C-17 (d 57.0), due to
Bruceajavanone C (4) was obtained as white amorphous resin,
with the molecular composition determined as C38H56O9 based
on the sodiated molecular ion peak at m/z 679.3823 [M+Na]+
(calcd for C38H56O9Na, 679.3822) in the HRESIMS. The 1D and
2D NMR spectra of 4 were similar in appearance to those of
1–3. However, in the 1H NMR spectrum of 4, two olefinic pro-
tons belonging to the double bond of the A ring present in 1–
3 were missing, while proton signals of a methylene group adja-
cent to a ketone group were recognized at dH 2.40 and 3.02
the cis-
c
substitution effect on H-17 in 1-3 compared with a
trans-
c
effect in 4. In the CD spectrum of 4, the n–p* transition
of the ketone group on A ring was blue-shifted to 292 nm due to
the lack of any conjugation effect, but was still a broad negative
band, suggesting that the absolute stereochemistry of A ring in
compound 4 was consistent with those of compounds 1–3. Thus,
the structure of compound 4 (bruceajavanone C) was elucidated
as shown.
The molecular formula of 5 was defined as C37H54O8 from the
sodiated molecular ion peak at m/z 649.3716 [M+Na]+ in the HRE-
SIMS. Close scrutiny of the NMR data revealed that the skeleton of
5 exhibited subtle differences from those of 1–4. Accordingly, a tri-
substituted double bond was located in the B ring at C-5/C-6 in-
stead of in the D ring at C-14/C15 like in compounds 1–4, based
on HMBC correlations from the olefinic proton (dH 5.42, br s, H-
6) to C-8, and from H-9 (dH 3.14, m) to C-5 (dC 141.6). A tertiary
methyl proton signal appeared at dH 0.88 (s, H3-30) and exhibited
HMBC correlations with C-13, C-14 as well as C-15. In the 1H-1H
COSY spectrum, the H-9 proton signal showed correlations not
only with H-11 (dH 5.35, m), but also with a methine proton at
dH 2.20 (H-8). This suggested that the tertiary methyl group of
C-30 at C-8 in 1–4 was migrated to C-14 in 5. NOE effects between
(each 1H, H-2a
, and H-2b). In the 13C NMR spectrum, a down-
field shift around 10 ppm for C-3 (dC 214.2) was observed due
to the lack of conjugation with a double bond. Moreover, an oxy-
methine proton signal appeared at dH 3.66 (br s, H-1) and
showed COSY correlations with H2-2. This suggested that the A
ring of 4 is saturated, with a hydroxyl group substituted at C-
1. HMBC correlations from H-1 to C-3 and C-19, as well as H2-
2 to C-3 confirmed this inference. Another difference between
4 and 3 was that a methoxyl group instead of an acetyl group
was located at C-21 in 4, which was confirmed by the HMBC
correlation from the methoxyl protons at dH 3.23 (s, 3H) to C-
21 (dC 110.0). The orientation of the hydroxy group on C-1 in
4 was assigned as
a from the small coupling constant value ob-

2222
L. Pan et al. / Bioorg. Med. Chem. 17 (2009) 2219–2224
Table 2
Cotton effect at 279 nm. These data were very similar to CD data
13C NMR chemical shifts of compounds 1–5a,b
.
published for trans-(2S,3S)-3-methyl-2-phenyl-1,4-benzodioxane,
which was derivatized by a stereoselective procedure to afford a
product with established absolute configuration.33Thus, the abso-
lute configuration of 8 was assigned as 700S and 800S, respectively.
This is first time (ꢀ)-hydnocarpin (8) has been isolated from B.
javanica, and the absolute configuration has been clarified in this
investigation.
Position
1
2
3
4
5
1
2
3
4
5
6
7
8
158.4, d
124.1, d
204.3, s
44.4, s
43.8, d
24.0, t
71.3, d
44.3, s
42.5, d
40.7, s
69.7, d
42.4, t
45.6, s
160.4, s
119.9, d
35.2, t
52.5, d
20.1, q
20.0, q
44.2, d
96.3, d
31.4, t
79.8, d
66.6, d
57.1, s
24.9, q
19.3, q
26.5, q
21.5, q
29.6, q
172.9, s
35.2, t
24.6, t
31.3, t
22.5, t
14.1, q
158.7, d
124.3, d
204.0, s
44.4, s
45.7, d
23.7, t
74.1, d
42.6, s
44.0, d
40.7, s
69.9, d
43.0, t
45.8, s
158.2, s
119.1, d
35.5, t
52.8, d
20.4, q
19.5, q
44.5, d
96.5, d
31.6, t
80.0, d
66.8, d
57.4, s
25.1, q
20.4, q
26.7, q
21.6, q
29.6, q
173.1, s
35.3, t
24.8, t
31.5, t
22.5, t
14.1, q
170.0, s
21.5, q
170.2, s
21.6, q
158.4, d
123.8, d
203.8, s
44.2, s
45.5, d
23.5, t
73.8, d
42.4, s
44.1, d
40.5, s
69.6, d
42.6, t
45.6, s
158.2, s
118.7, d
35.1, t
52.2, d
20.0, q
19.9, q
44.4, d
96.5, d
31.3, t
79.8, d
66.6, d
57.1, s
24.9, q
19.3, q
26.2, q
21.0, q
29.6, q
169.8, s
128.9, s
138.2, d
14.6, q
12.1, q
74.5, d
41.3, t
214.2, s
47.5, s
40.8, d
23.7, t
73.9, d
42.1, s
40.9, d
42.7, s
71.5, d
41.8, t
46.0, s
158.9, s
118.3, d
34.7, t
57.0, d
19.2, q
16.7, q
46.4, d
110.0, d
34.8, t
77.7, d
65.3, d
57.3, s
25.1, q
19.6, q
24.8, q
21.5, q
29.9, q
166.8, s
129.2, s
138.7, d
14.9, q
12.4, q
75.5, d
41.0, t
214.5, s
47.8, s
141.6, s
120.8, d
24.6, t
45.3, d
45.6, d
42.7, s
70.7, d
41.6, t
46.0, s
51.0, s
34.3, t
25.1, t
45.7, d
22.1, q
13.9, q
46.4, d
96.6, d
32.2, t
80.0, d
67.0, d
57.3, s
25.1, q
19.6, q
24.4, q
27.5, q
21.8, q
166.8, s
129.2, s
138.7, d
14.9, q
12.4, q
2.3. Biological activity evaluation
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
10
All pure compounds obtained in the present investigation were
evaluated their cytotoxic activity against three human cancer cell
lines (Lu1, LNCaP, and MCF-7) (Table 3). The five new compounds
(1–5) were considered inactive (ED50 >10 lM). The two quassi-
noids, bruceantin (6) and bruceine A (7), as shown in Table 3, dem-
onstrated significant inhibitory activities for all the three human
cancer cell lines, and for bruceantin (6), the cytotoxicities were ex-
tremely potent. Bruceantin (6) was formerly of interest for devel-
opment as an anticancer agent by the US National Cancer
Institute, but was eventually dropped because of a lack of demon-
strated efficacy in human clinical trials.34 Recently, it has been sug-
gested that this compound may have value in treating human
hematological malignancies.35
(ꢀ)-Hydnocarpin (8) did not show any discernible cytotoxicity
against the cancer cell lines itself (ED50 >10 lM). However, the
cytotoxicities against the MCF-7 cell line of bruceantin (6) and bru-
ceine A (7), were dramatically increased by 7.4-fold and 10-fold,
respectively, in the presence of (ꢀ)-hydnocarpin (8) at a concentra-
20
tion of 20 lg/mL (43 lM) (see Table 3). Hydnocarpin and its ana-
30
logs have been reported to possess
a bacterial multidrug
40
resistance pump-inhibiting effect,32,36,37 but its effect in potentiat-
ing the cytotoxic potency of potential anticancer agents does not
seem to have been reported to date.
50
60
OAc-7
170.0, s
21.5, q
169.7, s
21.4, q
170.4, s
21.5, q
OAc-21
169.7, s
21.4, q
170.4, s
21.5, s
3. Experimental
OMe-21
55.7, q
a
3.1. General experimental procedures
Measured at 100 MHz; obtained in CDCl3 with TMS as internal standard.
Assignments are based on HSQC, and HMBC NMR spectra.
b
Multiplicity obtained from the DEPT spectra.
Optical rotations were measured on a Perkin-Elmer 241 auto-
matic polarimeter. CD spectra were conducted on a JASCO J-810
spectropolarimeter. IR spectra were obtained on an ATI Mattson
Genesis Series FT-IR spectrometer. UV spectra were run on a Per-
kin-Elmer Lambda 10 UV/vis spectrometer. NMR spectroscopic data
were recorded at room temperature on a Bruker Avance DRX-400
spectrometer with tetramethylsilane (TMS) as an internal standard.
H-18 with H-9, as well as between H-30 with H-8 and H-17 indi-
cated a trans fusion of the C/D rings. Therefore, compound 5 was
found to be a derivative based on a protolimonoid carbon skeleton
rather than an apotirucallane skeleton.26,27 Investigation of the 2D
NMR data including the HMBC and NOESY spectra, enabled the
assignments of all substituent groups and the relative configura-
tion of 5, which was comparable with compounds 1–4. The CD
spectrum of 5 exhibited a negative Cotton effect at 289 nm, very
similar to that of compound 4. Thus, the structure of 5 was deter-
mined as shown, and has been named bruceajavaninone A.
HRESIMS and ESIMS were obtained on
a 3-Tesla Finnigan
Table 3
Cytotoxicity evaluation of compounds 6 and 7a.
Compound
Cell lineb
LNCaP
2.2. Identification of known compounds
Lu1
MCF-7
6
12
11
2.0
0.27
15
1.5
0.70
29
The structures of compounds 6 and 7 were identified as bruce-
antin (6) and bruceine A (7), respectively, by comparing their phys-
ical and spectroscopic data with reported values.28–30 These two
known quassinoids have been isolated previously as active princi-
ples from B. javanica.2,20,29 The structure of compound 8 was deter-
mined as the known flavonolignan, (ꢀ)-hydnocarpin,31,32 with the
1H and 13C NMR data fully assigned based on HSQC and HMBC
spectroscopic data. In order to determine the absolute configura-
tion of 8, (ꢀ)-hydnocarpin 5,7,400,900-tetraacetate (8a) was pro-
duced using standard acetylation conditions with (Ac)2O/
pyridine, to yield a methanol-soluble derivative. The CD spectrum
of 8a showed a positive Cotton effect at 223 nm and a negative
6c
NTd
79
NTd
96
7
7c
NTd
2.3
29
NTd
4.7
29
Paclitaxele
Camptothecine
a
Compounds 1–5 and 8 were inactive (ED50 >10
Key to cell lines used: Lu1, human lung cancer; LNCaP, hormone-dependent
l
M).
b
human prostate cancer; MCF-7, human breast cancer. Results are expressed as ED50
values (nM).
c
(ꢀ)-Hydnocarpin (8) was added at a concentration of 20
lg/mL (43 lM).
d
The potentiation effect of (ꢀ)-hydnocarpin (8) was not tested in these two cell
lines, for compounds 6 and 7.
e
Used as positive control substances.

L. Pan et al. / Bioorg. Med. Chem. 17 (2009) 2219–2224
2223
FTMS-2000 Fourier transform mass spectrometer. Column
chromatography was performed with Sephadex LH-20 (Supelco,
Bellefonte, PA), 65–250 or 230–400 mesh Si gel (Sorbent Technolo-
gies, Atlanta, GA). Analytical thin-layer chromatography was
conducted on precoated 250 lm thickness Partisil Si gel 60F254
glass plates (Whatman, Clifton, NJ), and preparative thin-layer
solvent (Rf = 0.70, 20 mg). F30304 was subjected to preparative
m), developed with a solvent system of
CH2Cl2ꢀhexanesꢀacetone (6:2:1), to give a mixture of 3 and 4
(Rf = 0.65, 25 mg). This mixture was purified by HPLC with
MeCNꢀH2O (60:40) as solvent system to afford 3 (7 mg) and 4
(11 mg), respectively. F30305 was purified by preparative TLC
TLC (20 ꢁ 20 cm, 1000
l
chromatography was performed on precoated 20 cm ꢁ 20 cm,
(20 ꢁ 20 cm, 500 m), with CH2Cl2ꢀhexanesꢀacetone (6:2:1) as
l
500
l
m or 1000
l
m thickness Partisil Si gel 60F254 glass plates
developing solvent, to yield 5 (Rf = 0.50, 8 mg). F308 was subjected
to passage over a Si gel column (2ꢁ 45 cm, 230–400 mesh) using a
solvent system of CHCl3ꢀacetone (10:1 to pure acetone), to yield
six fractions (F30801–F30806). F30803 was purified by HPLC, with
MeCNꢀH2O (35:65) as eluant, to afford 6 (17 mg) and 7 (15 mg),
respectively. F30805 was chromatographed on a Sephadex LH-20
column (2ꢁ 120 cm) using MeOH as solvent to afford 8 (15 mg).
(Whatman, Clifton, NJ). A 150 mm ꢁ 19 mm i.d., 5
PrepC18 column (Waters, Milford, MA) and a 10 mm ꢁ 19 mm i.d.,
m Sunfire guard column were used for preparative HPLC, along
lm Sunfire
5
l
with a Waters system composed of a 600 controller, a 717 Plus
autosampler, and a 2487 dual wavelength absorbance detector.
3.2. Plant material
3.5. Bruceajavanone A (1)
The combined leaves, twigs, and inflorescence (4.3 kg) were col-
lected at Hon Ba Mountain (12° 09.42800N, 109° 02.62400E., altitude
150 m), Khanh Hoa Province, Vietnam by Tran Ngoc Ninh and Djaja
Djendoel Soejarto in December, 2006, who also identified this
plant. A voucher specimen (Collection No. Soejarto and Ninh
13944) has been deposited in the John G. Searle Herbarium of
the Field Museum of Natural History, Chicago, Illinois.
White amorphous resin; ½a D23
ꢀ4.6 (c 0.2, MeOH); UV (MeOH)
ꢂ
kmax (loge) 228 (4.12) nm; IR mmax (film) 3458, 2958, 2930, 1730,
1671, 1457, 1379, 1228, 1093, 887 cmꢀ1; CD (c 7.8 ꢁ 10ꢀ5 M,
MeOH) kmax
(
D
e
) 232 (+11.93), 346 (ꢀ1.84) nm; HRESIMS obsd
m/z 663.3873 [M+Na]+, calcd for C38H56O8Na, 663.3873; 1 H and
13C NMR data, see Tables 1 and 2, respectively.
3.3. Cytotoxicity evaluation procedures
3.6. Bruceajavanone A 7-acetate (2)
The cytotoxic activity of extracts, chromatographic subfractions
of chloroform-soluble extract and all pure compounds were evalu-
ated against a panel of human cancer cell lines (Table 3), according
to established protocols.38
White amorphous resin; ½a D23
ꢂ
+24.1 (c 0.1, MeOH); UV (MeOH)
kmax (loge) 228 (4.16) nm; IR mmax (film) 2958, 2933, 1730, 1671,
1457, 1377, 1244, 1228, 1093, 890 cmꢀ1; CD (c 7.3 ꢁ 10ꢀ5 M,
MeOH) kmax
(
D
e
) 232 (+12.62), 345 (ꢀ1.82) nm; HRESIMS obsd
m/z 705.3978 [M+Na]+, calcd for C40H58O9 Na, 705.3979; 1H and
3.4. Extraction and isolation
13C NMR data, see Tables 1 and 2, respectively.
The dried and milled material contained leaves, twigs and inflo-
rescence (4.3 kg) of B. javanica was extracted using MeOH (3ꢁ 8 L,
each for 2 days) at rt. The solvent was removed under reduced pres-
sure to give 900 g of a thick brown syrup, which was suspended in a
3 L mixture of methanolꢀwater (9:1) solution and was then parti-
tioned with hexanes (3ꢁ 2 L). To the defatted residue was added
10% water (300 mL) and this was then partitioned with chloroform
(3ꢁ 2 L). Next, the chloroform partition was washed with water that
contained 1% NaCl (3ꢁ 2 L) to give 82 g of a partially detannified
chloroform-soluble extract. The chloroform-soluble extract was
found to be active against the MCF-7 cell line, with an ED50 value
3.7. Bruceajavanone B (3)
White amorphous resin; ½a D23
ꢂ
+34.3 (c 0.1, MeOH); UV (MeOH)
kmax (loge) 224 (4.77) nm; IR mmax (film) 2928, 2857, 1740, 1705,
1671, 1458, 1377, 1248, 1244, 1132, 1082, 1031, 953, 817 cmꢀ1
;
CD (c 7.5 ꢁ 10ꢀ5 M, MeOH) kmax
(D
e
) 222 (+28.24), 346 (ꢀ1.93)
nm; HRESIMS obsd m/z 689.3651 [M+Na]+, calcd for C39H54O9Na,
689.3666; 1H and 13C NMR data, see Tables 1 and 2, respectively.
3.8. Bruceajavanone C (4)
of <0.16
lg/mL. This was subjected to separation over a Si gel
White amorphous resin; ½a D23
ꢀ121.1 (c 0.2, MeOH); UV (MeOH)
ꢂ
column (9ꢁ 45 cm, 65–250 mesh, 1000 g), eluted with a solvent sys-
tem of CH2Cl2ꢀacetone with a gradient polarity (20:1 to 1:1, then
pure acetone) to yield 11 fractions, denoted as F1–F11. All these
fractions were evaluated against the MCF-7 cell line, and F3 was
kmax (loge) 221 (4.11) nm; IR mmax (film) 3591, 2965, 2933, 1728
(br), 1708, 1647, 1458, 1380, 1248, 1125, 1031, 879 cmꢀ1; CD
(c 7.6 ꢁ 10ꢀ5 M , MeOH) kmax
(
D
e) 219 (+27.09), 246 (ꢀ3.67), 292
(ꢀ2.00) nm; HRESIMS obsd m/z 679.3823 [M+Na]+, calcd for
C38H56O9Na, 679.3822; 1H and 13C NMR data, see Tables 1 and 2,
respectively.
found to be most active, with an ED50 value of <0.16 lg/mL.
Fraction F3 (20 g) was chromatographed on a silica gel column
(4ꢁ 40 cm, 230–400 mesh, 500 g) and eluted in a gradient manner
with hexanes-acetone (10:1 to pure acetone), to give eight subfrac-
tions (F301–F308). Subfraction F303 (2 g) was further subjected to
passage over a Sephadex LH-20 column (2ꢁ 120 cm), eluted with
CHCl3 first, to remove chlorophylls, and then with MeOH. Next,
subfraction F303 was applied to a silica gel column (1ꢁ 40 cm,
230–400 mesh) with a gradient solvent system of CHCl3ꢀacetone
(30:1 to pure acetone) to yield 12 subfractions (F30301–F30312).
Subfraction F30302 was purified by preparative TLC (20 ꢁ 20 cm,
3.9. Bruceajavaninone A (5)
White amorphous resin; ½a D23
ꢀ43.3 (c 0.2, MeOH); UV (MeOH)
ꢂ
kmax (loge
) 221 (4.09) nm; IR mmax (film) cmꢀ1 3596, 2990, 2927,
1738, 1711 (br), 1708, 1644, 1456, 1379, 1255, 1227, 1097, 953,
884 cmꢀ1; CD (c 8.0 ꢁ 10ꢀ5 M , MeOH) kmax
(
D
e) 246 (ꢀ0.63),
287 (ꢀ1.24) nm; HRESIMS obsd m/z 649.3716 [M+Na]+, calcd for
C37H54O8Na, 649.3716; 1H and 13C NMR data, see Tables 1 and 2,
respectively.
500
l
m), developed with
a
solvent system of CH2Cl2ꢀhex-
anesꢀacetone (6:1:0.4, Rf = 0.65), to afford 2 (15 mg). Repeated
chromatography of subfraction F30303 using Si gel columns with
a gradient solvent system of CHCl3ꢀacetone afforded impure 1,
which was finally purified by preparative TLC (20 ꢁ 20 cm,
3.10. (ꢀ)-Hydnocarpin (8)
Pale yellow amorphous powder; ½a D23
ꢂ
ꢀ32.0 (c 0.5, DMSO); 1H
500
l
m), using CH2Cl2ꢀhexanesꢀacetone (6:2:1) as developing
NMR (400 MHz, DMSO-d6, TMS) d 3.37 (1H, m, H-900b), 3.58 (1H,

2224
L. Pan et al. / Bioorg. Med. Chem. 17 (2009) 2219–2224
m, H-900a), 3.78 (3H, s, OMe-300), 4.27 (1H, m, H-800), 4.99 (1H, t,
J = 5.0 Hz, OH-900), 5.01 (1H, d, J = 8.0 Hz, H-700), 6.20 (1H, d,
J = 1.9 Hz, H-6), 6.52 (1H, d, J = 1.9 Hz, H-8), 6.81 (1H, d,
J = 8.0 Hz, H-500), 6.87 (1H, dd, J = 8.0, 1.5 Hz, H-600), 6.89 (1H, s,
H-3), 7.03 (1H, d, J = 1.5 Hz, H-200), 7.08 (1H, d, J = 8.5 Hz, H-50),
7.60 (1H, dd, J = 8.5, 1.9 Hz, H-60), 7.66 (1H, d, J = 1.9 Hz, H-20),
9.20 (1H, s, OH-400), 10.88 (1H, s, OH-7), 12.90 (1H, s, OH-5); 13C
NMR (100 MHz, DMSO-d6, TMS) d 55.7 (OMe-300), 60.07 (C- 900),
76.4 (C-700), 78.0 (C-800), 94.1 (C-8), 98.9 (C-6), 103.8 (C-3), 103.9
(C-10), 111.7 (C-200), 114.8 (C-20), 115.3 (C-500), 117.5 (C-50), 119.9
(C-60), 120.6 (C-600), 123.7 (C-10), 126.9 (C-100), 143.7 (C-30), 147.1
(C-40), 147.2 (C-400), 147.6 (C-300), 157.3 (C-9), 161.4 (C-5), 162.9
(C-7), 164.2 (C-2), 181.8 (C-4).
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3.11. (ꢀ)-Hydnocarpin 5,7,400,900-tetraacetate (8a)
½
a 2D3
ꢂ
ꢀ21.2 (c 0.05, MeOH); CD (c 6.5 ꢁ 10ꢀ5 M, MeOH) kmax
(De)
223 (+8.52), 279 (ꢀ0.50) nm; 1H NMR (400 MHz, CDCl3, TMS) d
2.10 (3H, s, OAc), 2.35 (3H, s, OAc), 2.37 (3H, s, OAc), 2.46 (3H, s,
OAc), 3.88 (3H, s, OMe-300), 4.04 (1H, dd, J = 12.0, 4.5 Hz, H-900b),
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The investigation was supported by grants U19-CA52956 and
P01-CA125066-01A1, funded by the National Cancer Institute,
NIH, Bethesda, Maryland. We acknowledge Mr. J. Fowble, College
of Pharmacy, The Ohio State University, OSU, for facilitating the
running of the 400 MHz NMR spectra. We thank the Institute of
Ecology and Biological Resources and authorities of Khanh Hoa
Province for permission in regard to the collection of the plant
material used for this study. We are also grateful to Mr. P.H.
Eichenseer and Ms. J.C. Hach of the OSU Campus Instrument Center
for the mass spectrometric data.
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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.2008.10.076.
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