Three novel quassinoids, javanicolides A and B, and javanicoside A, from seeds of Brucea javanica

Article abstract of DOI:10.1016/j.tet.2003.10.048

Two novel quassinoids, javanicolides A and B, and one novel quassinoid glucoside, javanicoside A were isolated from the seeds of Brucea javanica Merr. (Simaroubaceae), along with four known quassinoids, yadanziolides A and D, and bruceins D and E, and two known quassinoid glucosides, yadanziosides D and L. Their structures were elucidated by the analysis of spectral data and chemical evidence.

Full text of DOI:10.1016/j.tet.2003.10.048

Tetrahedron 59 (2003) 9985–9989
Three novel quassinoids, javanicolides A and B, and
javanicoside A, from seeds of Brucea javanica
*
Ik Hwi Kim, Ryoko Suzuki, Yukio Hitotsuyanagi and Koichi Takeya
School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
Received 8 August 2003; accepted 9 October 2003
Abstract—Two novel quassinoids, javanicolides A and B, and one novel quassinoid glucoside, javanicoside A were isolated from the seeds
of Brucea javanica Merr. (Simaroubaceae), along with four known quassinoids, yadanziolides A and D, and bruceins D and E, and two
known quassinoid glucosides, yadanziosides D and L. Their structures were elucidated by the analysis of spectral data and chemical
evidence.
q 2003 Elsevier Ltd. All rights reserved.
1. Introduction
novel quassinoids, javanicolides A (1) and B (2), and known
quassinoids, bruceins D (5) and E, and yadanziolides A and
D (4). The seventh fraction of the preparative reversed-
phase HPLC gave javanicoside A (3) and yadanziosides D
and L. Identification of those known compounds was made
by the comparison of their spectral data with those in the
literature.
Brucea javanica Merr. is a shrub which is distributed from
southeast Asia to northern Australia. Its seeds, having been
used for the treatment of dysentery, malaria and cancer,1,2
are also known as a rich source of quassinoids.3–6 In the
present study, from seeds of this plant we isolated three
novel quassinoids 1, 2 and 3 possessing unique structural
features along with six known quassinoids, yadanziolides A
and D (4),3,7 bruceins D (5) and E,8 and yadanziosides D and
L.4,9 The three new quassinoids are javanicolide A (1), a
C19-quassinoid characterized by an isomeric 1,2-seco-1-
nor-6(5!10)abeo-picrasane skeleton, javanicolide B (2), a
C20-quassinoid characterized by a hemiacetal linkage at
C-20, and javanicoside A (3), a C20-quassinoid glucoside
characterized by an A/B cis ring juncture. This paper
describes their isolation and structure elucidation (Fig. 1).
Javanicolide A (1) was obtained as an amorphous powder.
Its molecular formula was determined to be C19H24O9 by
the [MþNa]þ ion peak at m/z 419.1297 (calcd for
C19H24O9Na 419.1318) in the HRESIMS. Its IR spectrum
showed the presence of hydroxyl groups (3408 cm21), d-
and conjugated g-lactones (1737 cm21) and an olefinic
(1640 cm21) bond. The 1H NMR spectrum showed the
presence of two tertiary methyls (d 1.35 and 1.77), one
olefinic methyl (d 1.92), a pair of protons giving AB-quartet
signals (d 4.46 and 4.84, J¼6.9 Hz) and one olefinic proton
(d 5.91) (Table 1). Analysis of the 13C NMR and HMBC
spectra suggested the presence of a b-methyl-a,b-unsatu-
rated-g-lactone group attached to ring B (Table 2). These
observations and general similarity of the 1H and 13C NMR
spectra of 1 and 4 suggested that 1 possessed the 1,2-seco-1-
nor-6(5!10)abeo-picrasane skeleton as 4. A long-range
coupling observed between H-12 and C-16 in the HMBC
spectrum indicated that a lactone linkage exists between
C-12 and C-16 in 1. The stereochemistry of 1 was
determined by the analysis of NOESY spectrum (Fig. 2).
The NOESY correlations between H3-19 and Ha-20, and
Ha-6 and H-9 indicated a trans B/C ring juncture, that
between H3-19 and OH-11 revealed that the configuration
of C-11 hydroxyl group to be b, and the correlations
between H-7 and Hb-20, and H-15 and H3-21 indicated
that the hydroxyl groups at C-7 and C-15 were both
of a-configuration. The stereochemistry at C-5 was
2. Results and discussion
By Diaion HP-20 resin column chromatography of the
n-BuOH-soluble portion from a hot MeOH extract of the
seeds of B. javanica, a quassinoid-rich H2O/MeOH (1:1)
eluate was obtained. By preparative reversed-phase HPLC
of this eluate using MeOH/H2O (26:74 and 1:0) afforded
nine fractions. When left standing, the first fraction
produced crude crystals, which upon subsequent reversed-
phase HPLC gave known quassinoids, bruceins D (5) and
E. Reversed-phase HPLC of the mother liquid afforded two
Keywords: Brucea javanica; quassinoid; javanicolides A and B;
javanicoside A; Simaroubaceae.
*
Corresponding author. Tel.: þ81-426-76-3007; fax: þ81-426-77-1436;
e-mail: takeyak@ps.toyaku.ac.jp
0040–4020/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2003.10.048

9986
I. H. Kim et al. / Tetrahedron 59 (2003) 9985–9989
Figure 1.
determined by the observation of correlations between the
ring A and ring B protons. Correlations were observed
between H-5 and Ha-6, H-5 and H-9, and H3-18 and Ha-6.
We did not particularly investigate the absolute configur-
ations at 5 and 10, but on the basis of these observations and
biosynthetic considerations, we concluded that compound 1
possessed 5S, 10S configurations as in yadanziolide D (4).
Accordingly, javanicolide A (1) was shown to have the
structure given in Figure 1.
2 was confirmed by the analysis of NOESY spectrum
(Fig. 3). Correlations were observed between H-1, H-5 and
H-9, between H-9 and H-15, Hb-6 and H3-19, H3-19 and
H-20 and H-7 and OH-20. On the basis of these observations
and biosynthetic considerations, the stereochemistry at C-20
was assigned to S and the stereochemistry at the rest of the
stereocenters to be the same as those in 5. From these
observations, javanicolide B (2) was determined to have the
structure shown in Figure 1. This is the first example of
quassinoids having a hemiacetal linkage at C-20.
Several quassinoids having the 1,2-seco-1-nor-
6(5!10)abeo-picrasane skeleton are reported in several
Simaroubaceous plants.7,10–15 However, all the reported
compounds having this skeleton possess a lactone linkage
between C-7 and C-16, whereas javanicolide A (1) has a
lactone unit formed between C-12 and C-16. Javanicolide A
(1) is the first example of this type of quassinoids.
Javanicoside A (3) was obtained as an amorphous powder.
Its molecular formula was determined to be C32H42O16 as it
gave the [MþNa]þ ion peak at m/z 705.2313 (calcd for
1
C32H42O16Na 705.2371) in the HRESIMS. Its H NMR
spectrum showed the presence of a tertiary methyl (d 1.80),
three olefinic methyls (d 1.59, 2.11 and 2.50) and a
carbomethoxy (d 3.64) group (Table 1). The IR spectrum
showed the presence of hydroxyl (3347 cm21), d-lactone
and ester (1746 cm21), and a,b-unsaturated ketone
(1681 cm21) groups. The ESIMS showed a fragment ion
peak [MH2C6H10O5]þ at m/z 521, which suggested that 3
was a glycoside. Its 13C NMR and HMBC spectra
demonstrated the presence of a b-D-glucose unit (d 104.9,
78.1, 77.9, 75.6, 71.1 and 62.4), a ketone group at C-2 (d
193.8), and a senecioyl group (d 165.8, 158.5, 116.0, 26.9
and 20.1) (Table 2). An HMBC correlation between H-100
and C-3 revealed that the sugar moiety was linked to the C-3
oxygen atom. The sugar component was identified as
D-glucose by acid hydrolysis of 3 followed by the HPLC
analysis of the hydrolysate using an aminopropyl-bonded
silica gel column and an optical rotation detector. The
relatively large J value (7.7 Hz) of the anomeric proton of
the glucosyl moiety indicated a b anomeric orientation.
In the ROESY spectrum, H-20, H3-40 and H3-50 were
Javanicolide B (2) was obtained as an amorphous powder.
Its molecular formula was determined to be C20H26O10 by
the [MþNa]þ ion peak at m/z 449.1438 (calcd for
C20H26O10Na 449.1424) in the HRESIMS, which was
equivalent to that of brucein D (5) (C20H26O9) plus one
oxygen atom. Comparison of the NMR spectra with those of
5 revealed that 2 had A, B, C and D rings of the same
structure as in 5 and that, accordingly, the structural
differences between these two quassinoids resided only in
the E ring. The most distinguished features observed in the
NMR spectra were that C-20 of 2 gave a methine carbon
resonance (d 98.1) (Table 2), and the proton attached to
C-20 gave a broad doublet signal of one proton at very low
field of d 6.64 (Table 1), suggesting that two oxygen atoms
were connected to C-20. Thus, 2 was determined to be an
analogue of 5 in which one of the C-20 methylene protons of
5 was replaced by a hydroxyl group. The stereochemistry of

I. H. Kim et al. / Tetrahedron 59 (2003) 9985–9989
Table 1. H NMR spectral data for compounds 1–3 in C5D5Na
9987
1
Table 2. 13C NMR spectral data for compounds 1–3 in C5D5N
Position
1b
2c
3c,d
Position
1a
2b
3b
1 a
b
3
5
6 a
b
4.35 (s)
6.12 (s)
3.13 (d, 13.5)
3.33 (d, 15.5)
2.57 (d, 15.5)
1
2
3
4
5
6
7
8
82.7
198.2
125.0
163.4
43.4
27.6
77.6
52.9
46.0
48.7
75.4
81.1
85.9
82.4
70.5
174.8
22.1
11.6
98.1
20.1
51.0
193.8
146.6
147.7
48.1
27.6
83.1
46.2
31.9
39.7
73.3
76.3
82.7
48.8
68.4
167.0
16.7
26.5
73.3
170.8
52.3
165.8
116.0
158.5
26.9
20.1
104.9
75.6
78.1
71.1
77.9
62.4
172.6
119.9
167.9
94.0
46.0
73.1
63.1
48.7
47.1
71.9
87.7
83.9
82.9
72.0
172.3
16.2
19.5
73.4
17.2
5.91 (s)
5.25 (s)
2.49 (–e)
2.26 (dd, 13.8, 3.1) 2.39 (dt, 14.7, 2.8) 2.46 (–e)
1.79 (dd, 13.8, 4.9) 2.03 (ddd, 14.7,
13.5, 2.2)
2.14 (–e)
7
9
4.60 (br m)
3.01 (d, 4.0)
5.14 (m)
5.87 (s-like)
2.96 (d, 5.2)
5.37 (m)
4.96 (–e)
3.01 (d, 3.7)
4.91 (br s)
5.13 (br s)
4.01 (br d, 11)
6.48 (br s)
2.50 (s)
1.80 (s)
5.04 (d, 7.3)
3.85 (d, 7.3)
9
11
12
14
15
18
19
20 a
b
10
11
12
13
14
15
16
18
19
20
21
OMe
10
4.71 (d, 2.0)
4.63 (d, 4.0)
4.99 (s)
1.92 (s)
1.35 (s)
4.84 (d, 6.9)
4.46 (d, 6.9)
1.77 (s)
6.05 (s)
1.74 (s)
1.49 (s)
6.64 (br d, 6)
21
OMe
20
2.18 (s)
3.64 (s)
5.68 (s)
1.59 (s)
2.11 (s)
5.43 (d, 7.7)
4.20 (dd, 8.7, 7.7)
4.30 (dd, 9.2, 8.7)
4.37 (t, 9.2)
4.18 (m)
400
500
100
2
20
30
300
400
500
600
400
500
100
2
4.46 (dd, 11.7, 4.0)
4.57 (dd, 11.7, 2.7)
300
400
500
600
1-OH
7-OH
6.80 (s)
7.02 (br s)
6.94 (d, 6.2)
11-OH
12-OH
14-OH
15-OH
20-OH
5.07 (d, 5.8)
7.48 (d, 4.0)
7.14 (s)
6.64 (br s)
8.23 (br s)
a
7.52 (s)
f
–
Measured at 100 MHz.
Measured at 125 MHz.
f
b
–
7.68 (br d, 6)
a
Multiplicity and J values in Hz are given in parentheses.
Measured at 400 MHz.
Measured at 500 MHz.
The hydroxyl protons of the sugar moiety were not assigned due to
broadening and overlapping of the signals.
b
c
d
e
f
Multiplicity not determined due to overlapping of the signals.
Signals not identified because of extreme broadening.
demonstrated to be correlated with 21-OCH3 (Fig. 4).
Therefore, the senecioyl group was determined to link to the
oxygen atom at C-15, as those ROESY correlations were
possible only when the senecioyloxy group was present at
C-15 with b-configuration. The HMBC spectrum, however,
was not able to give information about the location of the
senecioyl group, as the H-15 resonance was extremely
broad. The analysis of ROESY spectrum afforded further
information about the stereochemistry of 3 (Fig. 4).
Correlations observed between Hb-1 and H-5, H-5 and
H3-19, Hb-6 and H3-19 suggested that these protons and the
methyl group involved were all of b-configuration, and that
the A/B rings were cis fused, whereas those between H-7
and H-14, H-14 and Hb-20, H3-19 and Ha-20 suggested that
the methyleneoxy bridge between C-8 and C-13 was of
b-configuration. Accordingly, javanicoside A (3) was
determined to have the structure shown in Figure 1. Quite
a few natural C20-quassinoids have been reported, but when
they have a hydrogen atom at 5, the A/B ring juncture is
always trans. This is the first example of the C20-
quassinoids having an A/B cis ring juncture with a hydrogen
at 5.
Figure 2. Selected NOESY correlations for 1.
Figure 3. Selected NOESY correlations for 2.

9988
I. H. Kim et al. / Tetrahedron 59 (2003) 9985–9989
Figure 4. Selected ROESY correlations for 3.
Javanicolides A (1) and B (2), and javanicoside A (3)
showed a weak cytotoxic activity on P-388 murine leukemia
cells with IC50 values of 17, 8.0, and 60 mg/mL,
respectively.
Diaion HP-20 (110 g) column and eluted sequentially with
H2O/MeOH mixtures (1:0, 1:1, 1:4 and 0:1) and finally with
acetone to give five fractions. The 1:1 H2O/MeOH eluate
(9.7 g) was further separated by reversed-phase HPLC
using MeOH/H2O (26:74 and 1:0) to afford nine fractions
(Frs. 1–9). Those fractions were evaporated to dryness.
MeOH was added to Fr. 1 (2.3 g). When the solution was
left standing, it produced precipitate of 451.8 mg of crude
crystals, which was separated and further subjected to
reversed-phase HPLC using MeOH/H2O (17:83) to afford
bruceins D (5) (95.0 mg) and E (253.8 mg). From the
mother liquid (1.8 g) by repeated reversed-phase HPLC
using either MeOH/H2O or MeCN/H2O mixture afforded
compounds 1 (4.3 mg) and 2 (5.1 mg), bruceins D (5)
(28.9 mg) and E (287.4 mg), and yadanzioldides A
(197.2 mg) and D (4) (7.7 mg). Fr. 7 (349.4 mg) gave
compound 3 (27.0 mg), yadanziosides D (121.8 mg) and L
(95.4 mg) when separated by using reversed-phase HPLC
using MeCN/H2O (15:85).
3. Experimental
3.1. General
Optical rotations were measured on a Jasco DIP-360 digital
polarimeter, UV spectra on a Hitachi U-2001 spectro-
photometer and IR spectra on a Perkin–Elmer 1710
spectrometer. NMR spectra were measured on Bruker
1
DRX-500 and DPX-400 spectrometers. The H chemical
shifts in C5D5N were referenced to the residual C5D4HN
resonance at 7.21 ppm, and the 13C chemical shifts to the
solvent resonance at 135.5 ppm. Mass spectra were obtained
on a Micromass LCT spectrometer. Preparative HPLC was
performed on a Tosoh CCPP-D system equipped with a
Jasco 875-UV detector (at 220 nm) and a reversed-phase
column, Inertsil PREP-ODS (10 mm, 20 mm i.d.£250 mm)
or Mightysil RP 18 GP (5 mm, 20 mm i.d.£250 mm)
(MeOH/H2O or MeCN/H2O, flow rate 10 mL/min).
Analytic HPLC was performed on a Tosoh CCPM system
equipped with a Tosoh CCP PX-8010 controller, a Tosoh
RI-8010 detector, a Shodex OR-2 optical rotation detector
and a CAPCELL PAK column, NH2 UG80 (5 mm, 4.6 mm
i.d.£250 mm) (MeCN/H2O (85:15), flow rate 1 mL/min).
3.3.1. Javanicolide
A
(1). Amorphous powder;
[a]D¼252.38 (c 0.09, MeOH); UV (MeOH) lmax nm
(log 1): 220 (4.08), 279 (3.19); IR (film) nmax cm21: 3408,
2950, 1737, 1640. 1H and 13C NMR: refer to Tables 1 and 2;
HRESIMS m/z 419.1297 [MþNa]þ, calcd for C19H24O9Na
419.1318.
3.3.2. Javanicolide
B
(2). Amorphous powder;
[a]D¼þ63.78 (c 0.10, MeOH); UV (MeOH) lmax nm
(log 1): 239 (4.17); IR (film) nmax cm21: 3364, 2946, 1734,
1663. 1H and 13C NMR: refer to Tables 1 and 2; HRESIMS
m/z 449.1438 [MþNa]þ, calcd for C20H26O10Na 449.1424.
3.2. Plant material
The seeds of Brucea javanica Merr. were purchased in
China in 2000, and identified by Dr Koichi Takeya,
Professor of Medicinal Plant Chemistry of Tokyo
University of Pharmacy and Life Science. A voucher
specimen was deposited in the herbarium of this university.
3.3.3. Javanicoside
A
(3). Amorphous powder;
[a]D¼241.38 (c 0.54, MeOH); UV (MeOH) lmax nm
(log 1): 224 (4.33); IR (film) nmax cm21: 3347, 2945, 1746,
1681. 1H and 13C NMR: refer to Tables 1 and 2; HRESIMS
m/z 705.2313 [MþNa]þ, calcd for C32H42O16Na 705.2371.
3.3. Extraction and isolation
3.3.4. Acid hydrolysis of 3. A solution of 3 (5.3 mg) in
0.1 M H2SO4 (1 mL) was heated at 908C for 30 min under
an Ar atmosphere. After cooling, H2O (5 mL) was added to
the mixture, and the whole was extracted with CHCl3
(3£5 mL). The combined CHCl3 layers were washed with
brine, dried over Na2SO4, and evaporated to give the
aglycon (2.1 mg). The H2O layer was passed through a short
Amberlite IRA-400 column and evaporated to dryness to
give a sugar fraction (1.7 mg). The sugar fraction was
dissolved in MeOH/H2O (2:8) and after passing through a
Dried and ground seeds of B. javanica (20 kg) were
extracted with hot MeOH (4£18 L). The solvent was
removed in vacuo to give a residue (1 kg), which was
suspended in H2O (2 L). Then the suspension was extracted
with n-hexane (2£1 L), CHCl3 (2£1 L), and n-BuOH
(2£1 L), successively, and the solvent removed in vacuo
to afford n-hexane-soluble (439 g), CHCl3-soluble (105 g),
and n-BuOH-soluble (363 g) portions, respectively. A part
(40 g) of the n-BuOH-soluble portion was placed on a

I. H. Kim et al. / Tetrahedron 59 (2003) 9985–9989
9989
5. Sakaki, T.; Yoshimura, S.; Ishibashi, M.; Tsuyuki, T.;
Takahashi, T.; Honda, T.; Nakanish, T. Bull. Chem. Soc. Jpn
1985, 58, 2680–2686.
Sep-Pak C18 cartridge, it was analyzed by HPLC using
MeCN/H2O (85:15). The sugar component was identified as
D-glucose by the comparison of the HPLC retention time, tR
11.80 (D-glucose tR 11.55) and the sign (positive) of optical
rotations.
6. Ohnishi, S.; Fukamiya, N.; Okano, M. J. Nat. Prod. 1995, 58,
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8. Lee, K. H.; Imakura, Y.; Sumida, Y.; Wu, R. Y.; Hall, I. H.
J. Org. Chem. 1979, 44, 2180–2185.
Data for aglycon. 1H NMR (500 MHz, C5D5N) d 5.73 (1H,
s, H-20), 5.09 (1H, br s, H-12), 5.09 (1H, d, J¼7.3 Hz,
Ha-20), 4.98 (1H, s, H-7), 4.88 (1H, br s, H-11), 3.99 (1H, br
s, H-14), 3.90 (1H, d, J¼7.3 Hz, Hb-20), 3.66 (3H, s, OMe),
3.38 (1H, d, J¼16.0 Hz, Ha-1), 3.10 (1H, br s, H-9), 2.55
(1H, m, Ha-6), 2.53 (1H, d, J¼16.0 Hz, Hb-1), 2.45 (1H, m,
H-5), 2.34 (3H, s, H3-18), 2.17 (1H, m, Hb-6), 2.11 (3H, s,
H3-50), 1.85 (3H, s, H3-19), 1.60 (3H, s, H3-40); HRESIMS
m/z 521.2018 [MþH]þ, calcd for C26H33O11 521.2023.
9. Yoshimura, S.; Sasaki, T.; Ishibashi, M.; Tsuyuki, T.;
Takahashi, T.; Honda, T. Bull. Chem. Soc. Jpn 1985, 58,
2673–2679.
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Takahashi, T.; Itai, A.; Iitaka, Y.; Matsushita, K. Chem. Lett.
1981, 1797–1798.
11. Itokawa, H.; Qin, X. R.; Morita, H.; Takeya, K. J. Nat. Prod.
1993, 56, 1766–1771.
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