Iridal-type triterpenoids with ichthyotoxic activity from Belamcanda chinensis

Article abstract of DOI:10.1021/np980271b

Ichthyotoxic activity-guided fractionation of the hexane and ether extracts of Belamcanda chinensis (Iridaceae) has resulted in the isolation of eleven iridal-type triterpenoids including six new compounds, 3-O- tetradecanoyl-16-O-acetylisoiridogermanal (4), 3-O-decanoyl-16-O- acetylisoiridogermanal (5), belachinal (7), anhydrobelachinal (9), epianhydrobelachinal (10), and isoanhydrobelachinal (11). Structures of the new iridals were determined by spectral and chemical methods. The absolute configuration of isoiridogermanal (1) at C-16 was determined to be R by the modified Mosher's method. Of these compounds, 16-O-acetylisoiridogermanal (3), 7, and spiroiridal (12) exhibited potent ichthyotoxic activity against killie-fish (Oryzias latipes).

Full text of DOI:10.1021/np980271b

J . Nat. Prod. 1999, 62, 89-93
89
Ir id a l-Typ e Tr iter p en oid s w ith Ich th yotoxic Activity fr om
Bela m ca n d a ch in en sis
Hideyuki Ito, Satomi Onoue, Yoko Miyake, and Takashi Yoshida*
Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700-8530, J apan
Received J une 24, 1998
Ichthyotoxic activity-guided fractionation of the hexane and ether extracts of Belamcanda chinensis
(Iridaceae) has resulted in the isolation of eleven iridal-type triterpenoids including six new compounds,
3-O-tetradecanoyl-16-O-acetylisoiridogermanal (4), 3-O-decanoyl-16-O-acetylisoiridogermanal (5), belachi-
nal (7), anhydrobelachinal (9), epianhydrobelachinal (10), and isoanhydrobelachinal (11). Structures of
the new iridals were determined by spectral and chemical methods. The absolute configuration of
isoiridogermanal (1) at C-16 was determined to be R by the modified Mosher’s method. Of these compounds,
16-O-acetylisoiridogermanal (3), 7, and spiroiridal (12) exhibited potent ichthyotoxic activity against killie-
fish (Oryzias latipes).
1
Naturally occurring ichthyotoxic substances also often
exhibit a variety of biological properties such as insecticide,
tumor-promoting, antitumor and antifungal activities, and/
or inhibitory effect on activation of Epstein-Barr virus
early antigen induced by TPA, etc.1-4 In a recent study of
Iris germanica L. (Iridaceae),5 we reported the character-
ization of several iridal-type triterpenoids including new
iridals, irisgermanicals A, B, and C, as ichthyotoxic com-
ponents. These active compounds included R-irigermanal,
iriflorental, and iripallidal, which were previously demon-
strated to have a potent in vivo antiulcerogenic effect on
indomethacin- or stress-induced ulceration in rats.6 These
findings prompted further investigation of the iridaceous
species Belamcanda chinensis (L.) DC, as part of a research
program aimed at finding novel biologically active natural
products using an ichthyotoxic assay as a primary indica-
tor. The dried rhizomes of B. chinensis have been used as
a traditional Chinese medicine for disorders of the throat
including coughing and pharyngitis.7 Although belamcan-
dal, a stimulant of the throat membrane, other related
iridals8,9 and many isoflavonoids10,11,12 have been isolated
from the plant, no ichthyotoxic principle has been reported.
In a bioassay-directed separation using killie-fish (Oryzias
latipes; Medaka), hexane and ether extracts of the B.
chinensis rhizomes afforded a series of iridals including six
new compounds. This paper deals with the isolation of
these compounds and their characterization by extensive
2D NMR and chemical methods, and the assessment of
their ichthyotoxic activity.
C46H78O6. The UV (255 nm) absorption and the H and 13C
NMR signals at δH 10.16 (1H, s), δC 189.7, 162.4, and 133.3
indicated the presence of a fully substituted R,â-unsatur-
ated aldehyde function. The 1H NMR spectrum also
exhibited signals due to three nonconjugated olefinic
protons, five vinyl methyl, and two tertiary methyl groups.
Two additional signals at δ 0.87 (t, J ) 7.5 Hz) and 2.01
(s) were ascribed to a terminal methyl of a fatty acid
residue and acetyl methyl, respectively. Acetyl and tet-
radecanoyl residues were suggested by significant fragment
ions at m/z 667 [(M - CH3COOH) + H]+ and 211
[CH3(CH2)12CO]+ in the ESIMS, and by NMR spectra
showing methylene proton and two ester carbonyl carbon
resonances. These spectral features, along with 13C NMR
data indicating 9 sp2 and 21 sp3 carbon signals for the
parent alcohol, implied that compound 4 was a diester of
isoiridogermanal (1). Comparison of the 1H NMR spectrum
of 4 with that of iristectorene B (2) (3-O-tetradecanoate of
1)14 indicated that 4 was the acetyl derivative of 2. This
conclusion was supported by a significant downfield shift
of the H-16 signal (δ 3.91 in 2 to δ 4.97 in the 1H NMR
spectrum of 4). Their structural relationships were sub-
stantiated by methanolysis of 4 yielding 1, 2, and 3, in
addition to methyl tetradecanoate (GLC). Compound 4 was
thus characterized as 3-O-tetradecanoyl-16-O-acetylisoiri-
dogermanal. This is the first isolation from natural source
14
although 4 was previously reported as derivative of 2.
Compound 5 showed a pseudo-molecular ion [M + NH4]+
at m/z 688 in the ESIMS and its molecular formula was
determined to be C42H70O6 by HRESIMS. Although insuf-
ficient 5 was available to measure the 13C NMR spectrum,
the 1H NMR was almost superimposable with that of 4
except for methylene protons in a fatty acid residue. Taking
the molecular weight, 56 (CH2 × 4) less than 4, into
consideration, compound 5 was assigned to 3-O-decanoyl-
16-O-acetylisoiridogermanal. Isoiridogermanal (1), the par-
ent alcohol of 2-5, was first isolated from Iris florentina
and I. pallida, and its stereostructure, except for the
configuration at C-16, was established13 as shown.
Resu lts a n d Discu ssion
Fresh rhizomes of B. chinensis were extracted with
hexane followed by MeOH. The concentrated MeOH extract
was partitioned with ether. The active hexane and ether
extracts were fractionated separately by an extensive series
of chromatographic procedures and afforded eleven iridals
(1-5, 7-12) including six new compounds (4, 5, 7, 9-11).
The known compounds were identified as isoiridogermanal
(1),13 iristectorene B (2),14 16-O-acetylisoiridogermanal (3),8
28-deacetylbelamcandal8 and (+)-(6R,10S,11S,14S,26R)-
26-hydroxy-15-methylidenespiroirid-16-enal (12),15 by com-
parisons of their spectral data with those in the literature.
The electrospray ionization (ESI) MS of 4 gave a [M +
NH4]+ peak at m/z 744, corresponding to molecular formula
The CD spectra for 1-5 are similar (positive Cotton
effect at ca. 250 nm), indicating that the absolute configu-
rations at C-10 and C-11 of 4 and 5 are the same as those
of 1. The absolute configuration of 1 at C-16 has been
examined by application of the modified Mosher’s method16
using methoxy-(trifluoromethyl)phenylacetic acid (MTPA)
esters as follows. Most of the protons of 1, except for some
* To whom correspondence should be addressed. Tel. and fax: +81-86-
251-7936. E-mail: yoshida@pheasant.pharm.okayama-u.ac.jp.
10.1021/np980271b CCC: $18.00
© 1999 American Chemical Society and American Society of Pharmacognosy
Published on Web 12/03/1998

90 J ournal of Natural Products, 1999, Vol. 62, No. 1
Ito et al.
Ta ble 1. 1H NMR Data (δ) of 7 and 9-11 (500 MHz in CDCl3)a
7
9
10
11
H-1
H-3
10.24 [10.16] s
3.64 m
3.58 m
1.30-1.25 m
2.10-2.30 m
3.64 [3.19] m
2.70 m
10.30 s
10.34 s
1.83 s
4.22 dd (5, 12)
3.55 dt (3, 12)
1.60-1.40 m
2.93 m
3.75 t (6)
2.58 m
4.21 dd (5, 12.5)
3.58 dt (3, 12.5)
1.60-1.38 m
2.95 m
3.82 d (10.5)
2.64 dt (5.5, 13.5)
2.53 brd (13.5)
1.80-1.60 m
2.10 m
5.16 q (8)
5.44 d (8)
6.11 d (15)
6.40 dd (11, 15)
5.86 d (11)
2.11 m
1.60-1.46 m
5.08 m
1.68 s
1.82 s
5.42 s
1.27 s
1.78 brs
1.78 brs
4.22 dd (5, 12.5)
3.55 dt (3, 12.5)
1.59-1.35 m
2.90 m
3.16 d (8.5)
3.23 brt (13.5)
2.57 brd (13.5)
1.82-1.39 m
2.04 m
4.89 q (8)
5.63 d (8.5)
6.18 d (15)
6.41 dd (11, 15)
5.87 d (11)
2.11 m
1.60-1.48 m
5.09 m
1.68 s
10.18 s
5.43 s
1.32 s
1.78 brs
1.78 brs
H-4
H-5
H-6
H-8
2.51 m
2.56 m
H-9
1.80-1.65 m
1.98 dd (8, 13)
4.89 [5.06] q (8)
5.58 [5.35] d (8)
6.16 [6.11] d (15.5)
6.40 [6.43] dd (11, 15.5)
5.88 d (11)
2.30-2.05 m
1.49-1.40 m
5.08 m
1.68 s
1.81 s
5.68 [5.52] s
1.30 s
1.78 brs
1.84-1.60 m
1.98 dd (8, 13)
4.89 q (8)
5.59 d (8)
6.16 d (15.5)
6.40 dd (11, 15.5)
5.87 d (11)
2.11 m
1.60-1.42 m
5.09 m
1.68 s
1.78 s
H-12
H-13
H-14
H-16
H-17
H-18
H-20
H-21
H-22
H-24
H-25
H-26
H-27
H-28
H-29
H-30
5.45 s
1.33 s
1.78 brs
1.78 brs
1.78 brs
1.60 s
1.60 s
1.60 s
1.60 s
a
J values (Hz) are in parentheses, and data of the second anomer are found in brackets.
methylene protons, were assigned based on the 1H-1H
COSY and TOCSY spectra and comparison with NMR data
of derivatives (2-5). The (R) and (S)-MTPA esters, 6a and
6b, were prepared by treatment of a trityl derivative (6) of
1 with (R)- and (S)-MTPA. The positive and negative ∆δ
(δS - δR) (Hz) values [-33 Hz (14-H), -73.5 Hz (28-CH3),
+15, +30 Hz (17-CH2), +53 Hz (18-H), +20.5 Hz (29-CH3)]
obtained for the (R)- and (S)-MTPA esters (6a and 6b) led
to assignment of the R configuration at C-16. Thus, the
full stereostructures of 1 and its mono- (2 and 3) and
diesters (4 and 5) are as shown.
F igu r e 1. Selective NOE’s of 7.
showed absorption maxima characteristic of the conjugated
1
triene chromophore. The H NMR spectrum of 7 exhibited
duplicate signals in a ratio of ca. 6:4 for each proton (partly
overlapped), as clearly represented by singlets at δ 10.24
(10.16) and 5.68 (5.52) which are attributable to an
aldehyde and a hemiacetal proton, respectively. Its 1H
(Table 1) and 13C NMR spectra resembled those of the
spirobicyclic-iridal [(6R,10S,11R)-26ú-hydroxy-(13R)-oxa-
spiroirid-16-enal] (8)5,17 that was isolated as a mixture of
two anomers from Iris pseudacorus and I. japonica. The
1
major differences between the H NMR spectra of 7 and 8
were an upfield shift (0.26 ppm) of H-13 and a downfield
shift (0.10 ppm) of H-14 in 7 relative to those in 8, implying
7 to be a 13-epimer of 8. The structural assignment for 7
was confirmed by the NOESY spectrum. Although the C-27
methyl signal in 8 was reported to show NOE correlations
with H-26 and H-14,5 the corresponding methyl signal in
7 displayed a definite cross-peak with H-13 signal, but not
with H-14 (Figure 1). Other NOE’s similar to those of 8
were also consistent with the proposed structure 7 having
the S-configuration at C-13.
Anhydrobelachinal (9) was obtained as a white amor-
phous powder, and the molecular formula was established
1
to be C30H44O4 by HRESIMS. The UV and H NMR (Table
The positive ion FABMS of 7 gave a [M + Na]+ peak at
m/z 509, corresponding to C30H46O5. The UV spectrum
1) of 9 revealed some features similar to 7 although the
1H NMR spectrum of 9 showed no duplication of signals.

Triterpenoids with Ichthyotoxic Activity from B. chinesis
J ournal of Natural Products, 1999, Vol. 62, No. 1 91
F igu r e 2. Selective NOE’s of 9.
1
In a comparison of the H NMR data for 9 with those of 7,
the C-3 methylene protons of the former resonated at δ 4.22
and 3.55, while those in the latter appeared as essentially
equivalent signals near δ 3.64. An upfield shift of the H-26
signal relative to that of 7 was also observed. Taking a
molecular weight of 18 mass units less than that of 7 into
consideration, these spectral features suggested an acetal
structure in 9. The presence of a seven-membered acetal
ring was substantiated by the 1H-13C long-range COSY
(J CH ) 8 Hz) spectrum showing a three-bond coupling
between H-3 and C-26. Additional evidence for the acetal
structure was provided by the NOESY spectrum of 9 which
displayed an NOE cross-peak between H-3 and H-26. The
C-27 methyl signal also showed definite NOE’s with H-26
and H-13 (Figure 2). Inspection of the MM2 molecular
model calculation of 9 indicated that these NOE interac-
tions were only possible if 9 had the relative configurations
at C-26, C-27 and C-13 shown in the formula. The absolute
stereochemistry of 9 was concluded to be the same as that
of 7 as evidenced by a close similarity of CD spectra
between 7 and 9.
during the purification or extraction process. The possibility
of their formation when exposed to Si gel was ruled out as
when a CH2Cl2 solution of 7 with Si gel was left at room
temperature for a week, 7 was recovered unchanged, with
no trace of 9, 10, or 11 detected by HPLC. Furthermore, a
lipid fraction obtained by supercritical fluid extraction
(SFE)18 [CO2 flow rate 12.0 L/min; 22 Mpa; 40 °C with
entrainer MeOH (1.0 mL/min)] of fresh B. chinensis
rhizomes was shown, by HPLC, to contain compounds
9-11, as well as 7 and other iridals. SFE using CO2 is
known as a quick and mild extraction method with
relatively little risk of structural alteration of the plant
ingredients,18 and was reported to be applicable to selective
extraction of iridals.20 From these observations, compounds
9-11 were considered natural products rather than arti-
facts.
Among iridals isolated in the present study, compounds
3, 7, and spiroiridal (12) were highly toxic to killie-fish with
median tolerance limit (TLm)1 values of 1.6-3.5 µg/mL after
24 h (Table 2). Although most monocyclic iridals are
nontoxic to fish,5 16-O-acetylisoiridogermanal (3) showed
moderate ichthyotoxic activity. Acetylation of 3 enhanced
the activity. Nevertheless, it appears that esterification of
the 3-OH of monocyclic and spiro-type iridals with a higher
fatty acid, markedly lowers toxicity. Ethers such as 9 and
10 are also much less toxic indicating that the free hydroxyl
group at C-3 is important for the toxicity. The TLm values
of the active iridals are comparable to that of buddledin B
which was isolated as an ichthyotoxic constituent from
Buddleja davidii roots20 and later found to be cytotoxic
against P-388 lymphocytic cells.
The molecular formulas of compounds 10 and 11 were
both indicated by their HRESIMS data to be C30H44O4,
which was the same as that of 9. The UV and 1H NMR
spectra of 10 and 11 (Table 1) were also very similar to
1
those of 9. Significant differences between H NMR spectra
of 10 and 9 were observed in the chemical shifts of H-13
and H-14 which were at lower and upper fields, respec-
tively, in 10 than the corresponding signals in 9. The
differences of these signals in 9 and 10 were comparable
with those observed for 7 and 8, the epimeric pairs at C-13.
Compound 10 was thus concluded to be a C-13 epimer of
9, which was consistent with the HMBC data.
Comparison of the 1H NMR data of 11 and 9 revealed
an upfield shift (0.59 ppm) of H-6 and a downfield shift
(0.65 ppm) for one of the C-8 methylene proton signals in
the former related to the corresponding signals in the
latter. This tendency was analogous to that reported for
irisgermanical B and iriflorental which are the geometrical
isomers at the R,â-unsaturated aldehyde moiety.5 Structure
11 was confirmed by the NOESY data that included NOE
correlations between the H-6 (δ 3.16) and vinyl methyl (δ
1.83) signals and also between the aldehyde (δ 10.18) and
H-8 (δ 3.23) protons. The other NOE’s similar to those of 9
led us to assign the same stereochemistry at the chiral
centers of the ring in 11 as that in 9.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. ESIMS were deter-
mined on a Micromass Auto Spec OA-Tof spectrometer in the
direct inlet mode using a solvent, 50% MeOH-0.1% AcONH4,
and FABMS on a VG 70SE spectrometer using 3-nitrobenzyl
alcohol as the matrix agent. 1H and 13C NMR spectra were
recorded on a Varian VXR 500 spectrometer, and the chemical
shifts are given in δ (ppm) referenced to TMS. Optical rotations
were measured on a J asco DIP-1000 polarimeter. Kieselgel 60
(Merck), Sephadex LH-20 (Pharmacia), and YMC ODS AQ120-
S50 (YMC) were used for column chromatography. Analytical
HPLC was performed with the following conditions: reversed-
phase; Inertsil ODS-2 (4.6 × 250 mm, GL Sciences), 40 °C,
solvent system of H2O-MeOH, normal phase; YMC-Pack
Structural relationships between belachinal (7) and its
anhydro analogues (9-11) were established by treatment
of 7 with p-toluenesulfonic acid in CH2Cl2 yielding 9, 10,
and 11 as well as some uncharacterized products.
The facile chemical conversion of 7 into anhydro deriva-
tives prompted us to examine the possibility that com-
pounds 9-11 might be artifacts produced from 7 or 8

92 J ournal of Natural Products, 1999, Vol. 62, No. 1
Ito et al.
Ta ble 2. Ichthyotoxic Activity of the Iridals Isolated from B.
chinensis
20), 37.0 (C-9), 36.6 (C-12), 34.3 (C-2′), 31.9 (C-12′), 31.5 (C-
17), 29.67 (CH2), 29.64 (CH2), 29.61 (C-7′), 29.46 (C-6′), 29.34
(C-11′), 29.28 (C-5′), 29.17 (C-4′), 28.6 (C-4), 26.7 (C-21), 26.6
(C-5), 26.3 (C-27), 25.7 (C-24), 25.0 (C-3′), 23.8 (C-8), 22.7 (C-
13′), 21.8 (C-13), 21.3 (16-COCH3), 17.9 (C-26), 17.7 (C-30), 16.2
(C-29), 14.1 (C-14′), 11.8 (C-28), 11.0 (C-25); ESIMS m/z 744
[M + NH4]+, 667 [M - CH3COOH+H]+, 649 [M - CH3COOH
- H2O + H]+, 211 [CH3 (CH2)12CO]+; HRESIMS m/z [M +
NH4]+ 744.6142 (calcd for C46H82NO6, 744.6142).
TLm
Compounds
(µg/mL)a
monocyclic Iridals
isoiridogermanal (1)
9.3
3.5
0.8
16-O-acetylisoiridogermanal (3)
3,16-di-O-acetylisoiridogermanal (3a )
monocyclic iridal esters
3-O-tetradecanoyl-16-O-acetylisoiridogermanal (4)
3-O-decanoyl-16-O-acetylisoiridogermanal (5)
iristectorene B (2)
spiroiridals
belachinal (7)
(6R,10S,11R)-26ú-hydroxy-(13R)-oxaspiroirid-16-enal (8) 5.5
anhydrobelachinal (9)
epianhydrobelachinal (10)
(+)-(6R,10S,11S,14S,26R)-26-hydroxy-15-
methylidenespiroirid-16-enal (12)
buddledin B
>10
>10
>30
Meth a n olysis of 4: A solution of 4 (3 mg) in 0.1% metha-
nolic NaOH (1 mL) was kept at room temperature and the
reaction mixture was monitored by reversed-phase HPLC
which showed formation of 2 and 3 at an early stage and then
of 1 as a final product. Identification of the final product 1
was confirmed after prep. HPLC by 1H NMR data. The
liberated fatty acid ester was identified as methyl tetrade-
canoate by GLC analysis.
3-O-Deca n oyl-16-O-a cetylisoir id oger m a n a l (5): Glassy
solid; [R]D +30.0° (c 0.39, CH2Cl2); UV (CH3CN) λmax (log ꢀ):
255 (4.19) nm; CD (MeOH) ∆ꢀ (nm) +3.2 (257); 1H NMR
(CDCl3, 500 MHz) δ 10.17 (1H, s, H-1), 5.26 (1H, t, J ) 7.5
Hz, H-14), 5.05 (1H, m, H-18), 5.05 (1H, m, H-22), 4.97 (1H,
brt, J ) 6.5 Hz, H-16), 4.01 (2H, t, J ) 6.5 Hz, H-3), 3.27 (1H,
brd, J ) 10.0 Hz, H-6), 2.56 (2H, m, H-8), 2.33/2.20 (each 1H,
m, H-17), 2.27 (2H, t, J ) 7.5 Hz, H-2′), 2.01 (3H, s, 16-COCH3),
1.84 (3H, s, H-25), 1.67 (3H, s, H-24), 1.59 (3H, s, H-29), 1.58
(3H, s, H-30), 1.52 (3H, s, H-28), 1.26 (16H, CH2, brs), 1.15
(3H, s, H-27), 1.07 (3H, s, H-26), 0.88 (3H, t, J ) 7.0 Hz, H-10′);
ESIMS m/z 688 [M + NH4]+, 610 [M - CH3COOH]+, 592 [M
- CH3COOH - H2O]+; HRESIMS m/z [M + NH4]+ 688.5492
(calcd for C42H74NO6, 688.5516).
2.8
>10
>10
1.6
1.2
a
TLm: median tolerance limit after 24 h.
SIL A-003 (4.6 × 250 mm, YMC), solvent system of n-hexane-
EtOH. In preparative HPLC, a YMC-Pack ODS-A324 column
(10 × 300 mm) was used in the reversed-phase mode and
Inertsil Sil (10 × 250 mm) was used in the normal phase mode,
with a UV detector (254 and 280 nm) or photodiode-array
detector.
P la n t Ma ter ia l. Rhizomes of B. chinensis cultivated at the
herbarium of Faculty of Pharmaceutical Sciences, Okayama
University, were collected in J anuary, 1995. A voucher speci-
men (OPH-I03) is kept at the same herbarium.
Tr ityla tion of 1. Isoiridogermanal (1) (11 mg) and triphenyl
methyl chloride (20 mg) were dissolved in 2 mL of dried
pyridine, and the mixture was allowed to stand for 24 h at
room temperature. Preparative TLC [Si gel, CHCl3-MeOH
(70:1)] of the crude tritylation product obtained after usual
workup afforded 4 mg of 3-O-tritylisoiridogermanal (6) as a
colorless oil: 1H NMR (CDCl3, 500 MHz) δ 10.12 (1H, s, H-1),
7.41-7.22 (15H, phenyl-H), 5.24 (1H, t, J ) 7.0 Hz, H-14), 5.08
(1H, m, H-18), 5.06 (1H, m, H-22), 3.92 (1H, t, J ) 7.0 Hz,
H-16), 3.24 (1H, brd, J ) 9.5 Hz, H-6), 3.03 (1H, m, H-3), 3.01
(1H, m, H-3), 2.55, 2.51 (each 1H, m, H-8), 1.82 (3H, s, H-25),
1.68 (3H, brs, H-24), 1.62 (3H, s, H-29), 1.60 (3H, s, H-30),
1.55 (3H, s, H-28), 1.13 (3H, s, H-26), 1.05 (3H, s, H-27).
(R)-MTP A ester 6a : To a solution of 3-O-tritylisoiridoger-
manal (6) (9.5 mg) in dry CH2Cl2 (1 mL) was added Et3N (0.6
µL), DMAP (7 mg), and (R)-MTPA chloride (0.6 µL) and the
mixture was stirred at room temperature for 15 h. The mixture
was then purified by preparative TLC [Si gel, CHCl3-MeOH
(100:1)] to give the (R)-MTPA ester 6a (12.9 mg) as colorless
oil: 1H NMR (CDCl3, 500 MHz) δ 10.12 (1H, s, H-1), 7.50-
7.22 (20H, phenyl-H), 5.39 (1H, t, J ) 7.0 Hz, H-14), 5.32 (1H,
m, H-18), 5.06 (1H, m, H-22), 4.92 (1H, t, J ) 7.0 Hz, H-16),
3.50 (3H, s, MTPA -OCH3), 3.22 (1H, brd, J ) 9.5 Hz, H-6),
3.04 (1H, m, H-3), 3.01 (1H, m, H-3), 2.40 (1H, m, H-17), 2.24
(1H, m, H-17), 2.00 (1H, m, H-21), 1.91 (1H, m, H-13), 1.82
(3H, s, H-25), 1.68 (3H, brs, H-24), 1.55 (3H, s, H-29), 1.54
(3H, s, H-28), 1.12 (3H, s, H-26), 1.05 (3H, s, H-27); ESIMS
m/z 950 [M + NH4]+.
(S)-MTP A ester 6b: Ester 6b was prepared in a way
similar to that for 6a . 6b: 1H NMR (CDCl3, 500 MHz) δ 10.10
(1H, s, H-1), 7.48-7.21 (20H, phenyl-H), 5.32 (1H, t, J ) 7.0
Hz, H-14), 5.28 (1H, m, H-18), 5.07 (1H, m, H-22), 5.02 (1H, t,
J ) 7.0 Hz, H-16), 3.53 (3H, s, MTPA -OCH3), 3.20 (1H, brd,
J ) 9.5 Hz, H-6), 3.04 (1H, m, H-3), 3.00 (1H, m, H-3), 2.46
(1H, m, H-17), 2.27 (1H, m, H-17), 2.03 (1H, m, H-21), 1.97
(1H, m, H-13), 1.82 (3H, s, H-25), 1.63 (3H, brs, H-24), 1.57
(3H, s, H-29), 1.39 (3H, s, H-28), 1.13 (3H, s, H-26), 1.04 (3H,
s, H-27); ESIMS m/z 950 [M + NH4]+.
Bela ch in a l (7): Glassy solid; [R]D +33.0° (c 1.0, CH2Cl2);
UV (EtOH) λmax (log ꢀ) 259 (sh) (4.51), 269 (4.54), 280 (4.58),
290 (4.47) nm; CD (CH3CN) ∆ꢀ (nm) +5.0 (248), -3.3 (280),
-1.3 (290), +0.1 (305); 1H NMR, Table 1; 13C NMR (CDCl3) δ
190.5 (190.1) (C-1), 161.3 (160.7) (C-7), 139.7 (140.1) (C-19),
135.7 (137.2) (C-15), 134.4 (134.3) (C-16), 133.0 (C-2), 131.8
E xt r a ct ion a n d Isola t ion . Fresh rhizomes (1 kg) of B.
chinensis were chopped and soaked in hexane (2 L) three times
for 24 h each time at room temperature to yield a hexane
extract (2.5 g). The residue was further extracted with MeOH
(2 L × 3). The concentrated solution was diluted with H2O
and extracted successively with ether and n-BuOH to give
Et2O (9.5 g), n-BuOH (13 g), and H2O (36.8 g) extracts.
Ichthyotoxic activity was exhibited by the hexane and Et2O
extracts with TLm values of 4.6 and 4.2 ppm, respectively. The
hexane extract was subjected to column chromatography over
Si gel (3.8 i.d. × 40 cm) using CHCl3 and CHCl3-acetone. The
eluate with CHCl3-acetone (8:2) (800 mg) was further purified
by repeated column chromatography over ODS gel (1.1 i.d. ×
26 cm) with MeOH-H2O (93:7) to give 2 (10 mg), 4 (30 mg),
and 5 (2 mg). The Et2O extract (6 g) was chromatographed
over Si gel (3.0 i.d. × 58 cm) with CHCl3-acetone (8:2 f 7:3
f 6:4 f 5:5) and acetone-MeOH (1:1) in a stepwise gradient
mode. The eluate with CHCl3-acetone (6:4) (316 mg) was
purified by a combination of column chromatography over
Sephadex LH-20 (1.2 i.d. × 30 cm) with CHCl3-MeOH and
preparative HPLC on a RP-18 column (MeOH-H2O, 17:3) to
yield 9 (14 mg) and 10 (17 mg). The CHCl3-acetone (8:2) eluate
was divided into three parts (each ca. 400 mg) and fractionated
separately by column chromatography over Sephadex LH-20
and preparative HPLC in a way similar to that described above
to give additional 9 (49 mg total) and 10 (55 mg), and 11 (18
mg), 12 (68 mg), 1 (209 mg), 7 (51 mg), 3 (76 mg), and 28-
deacetylbelamcandal (7 mg).
3-O-Tetradecanoyl-16-O-acetylisoiridogermanal(4): Glassy
solid; [R]D +25.0° (c 1.0, CH2Cl2); UV (MeOH) λmax (log ꢀ) 255
1
(4.15) nm; CD (MeOH) ∆ꢀ (nm) +1.4 (254); H NMR (CDCl3,
500 MHz) δ 10.16 (1H, s, H-1), 5.26 (1H, t, J ) 7.0 Hz, H-14),
5.06 (1H, m, H-18), 5.06 (1H, m, H-22), 4.97 (1H, t, J ) 7.0
Hz, H-16), 4.01 (2H, t, J ) 6.5 Hz, H-3), 3.27 (1H, brd, J ) 9.5
Hz, H-6), 2.55 (2H, m, H-8), 2.34, 2.20 (each 1H, m, H-17),
2.27 (2H, t, J ) 7.5 Hz, H-2′), 2.01 (3H, s, 16-COCH3), 1.83
(3H, s, H-25), 1.67 (3H, s, H-24), 1.59 (3H, s, H-29), 1.58 (3H,
s, H-30), 1.52 (3H, s, H-28), 1.24 (CH2, brs), 1.14 (3H, s, H-27),
1.06 (3H, s, H-26), 0.87 (3H, t, J ) 7.5 Hz, H-14′); 13C NMR
(CDCl3, 125 MHz) δ 189.7 (C-1), 173.9 (C-1′), 170.3 (16-
COCH3), 162.4 (C-7), 137.7 (C-19), 133.3 (C-15), 133.3 (C-2),
131.4 (C-23), 127.8 (C-14), 124.1 (C-22), 119.1 (C-18), 78.9 (C-
16), 74.9 (C-10), 64.2 (C-3), 44.7 (C-11), 43.2 (C-6), 39.7 (C-

Triterpenoids with Ichthyotoxic Activity from B. chinesis
J ournal of Natural Products, 1999, Vol. 62, No. 1 93
(C-23), 130.8 (C-14), 125.5 (125.7) (C-17), 125.2 (125.1) (C-18),
123.9 (124.0) (C-22), 99.6 (105.3) (C-26), 74.3 (74.2) (C-10), 73.0
(C-13), 62.0 (62.5) (C-3), 61.0 (C-11), 42.6 (46.0) (C-6), 40.2
(39.0) (C-20), 39.0 (39.1) (C-9), 32.0 (CH2), 30.9 (CH2), 28.1
(27.7) (C-27), 26.7 (C-21), 25.6 (C-24), 24.0 (23.9) (C-8), 17.8
(C-30), 16.9 (C-29), 12.7 (13.1) (C-28), 11.1 (C-25); FABMS m/z
509 [M + Na]+; ESIMS m/z 504 [M + NH4]+; HRESIMS m/z
[M + NH4]+ 504.3657 (calcd for C30H50NO5, 504.3689).
An h yd r obela ch in a l (9): Glassy solid; [R]D +62.5° (c 0.45,
CH2Cl2); UV (MeOH) λmax (log ꢀ) 257 sh (4.21), 268 (4.25), 282
(4.28), 291 (sh) (4.19) nm; CD (MeOH) ∆ꢀ (nm): +4.9 (249),
-0.6 (273), +0.3 (298); 1H NMR, see Table 1; 13C NMR (CDCl3)
δ 189.9 (C-1), 164.3 (C-7), 139.2 (C-19), 135.3 (C-15), 134.1 (C-
16), 133.7 (C-2), 131.7 (C-23), 129.1 (C-14), 125.3 (C-17), 125.0
(C-18), 124.0 (C-22), 110.9 (C-26), 77.9 (C-13), 73.3 (C-10), 70.7
(C-3), 61.2 (C-11), 43.8 (46.4) (C-6), 40.1 (C-20), 39.1 (C-9), 32.0
(CH2), 31.9 (CH2), 27.8 (C-27), 26.6 (C-21), 25.7 (C-24), 24.4
(C-8), 17.7 (C-30), 16.8 (C-29), 12.6 (C-28), 10.7 (C-25); EIMS
m/z 468 [M]+; ESIMS m/z 469 [M + H]+; HRESIMS m/z [M +
H]+ 469.3302 (calcd for C30H45O4, 469.3318).
E p ia n h yd r obela ch in a l (10): Glassy solid; [R]D +7.8° (c
0.64, CH2Cl2); UV (MeOH) λmax (log ꢀ) 259 (sh) (4.12), 270
(4.18), 281 (4.23), 290 (sh) (4.11) nm; CD (CH3CN) ∆ꢀ (nm)
+8.3 (252), -7.9 (280); 1H NMR, see Table 1; 13C NMR (CDCl3)
δ 190.1 (C-1), 165.0 (C-7), 139.7 (C-19), 137.1 (C-15), 134.2 (C-
16), 131.7 (C-2), 129.8 (C-23), 129.2 (C-14), 125.3 (C-17), 125.0
(C-18), 123.9 (C-22), 110.0 (C-26), 75.2 (C-13), 74.2 (C-10), 70.5
(C-3), 60.5 (C-11), 43.8 (45.0) (C-6), 40.1 (C-20), 38.8 (C-9), 32.5
(CH2), 31.6 (CH2), 28.3 (C-27), 26.6 (C-21), 25.7 (C-24), 24.4
(C-8), 17.7 (C-30), 16.9 (C-29), 13.2 (C-28), 10.8 (C-25); EIMS
m/z 468 [M]+; ESIMS m/z 469 [M + H]+; HRESIMS m/z [M +
H]+ 469.3313 (calcd for C30H45O4, 469.3318).
mL) at room temperature for 5 h. After disappearance of the
starting material by HPLC, the reaction mixture was concen-
trated and subjected to preparative HPLC (YMC-Pack ODS-
A324 (10 i.d. x 300 mm), H2O-MeOH 15:85, 40 °C) to yield 9
(0.8 mg), 10 (1.0 mg) and 11 (0.8 mg) which were identical
with naturally occurring compounds by HPLC and 1H NMR
spectral comparisons.
Ack n ow led gm en t. The NMR instrument used in this
study was the property of the SC NMR Laboratory of Okayama
University. This work was supported in part by a Grant-in-
Aid for Scientific Research (No. 09672143) from the Ministry
of Education, Science, Sports and Culture of J apan.
Refer en ces a n d Notes
(1) Okuda, T.; Yoshida, T.; Koike, S.; Toh, N. Phytochemistry 1975, 14,
509-515.
(2) Luyengi, L.; Lee, I.-S.; Mar, W.; Fong, H. H. S.; Pezzuto, J . M.;
Kinghorn, A. D. Phytochemistry 1994, 36, 1523-1526.
(3) Gomez, E.; Giron, O. C.; Cruz, A. A.; J oshi, B. S.; Chittawong, V.;
Miles, D. H. J Nat. Prod. 1989, 52, 649-651.
(4) Ito, H.; Muranaka, T.; Mori, K.; J in, K.; Yoshida, T. Chem. Pharm.
Bull. 1997, 45, 1720-1722.
(5) Miyake, Y.; Ito, H.; Yoshida, T. Can. J . Chem. 1997, 75, 734-741.
(6) Mutoh, Y.; Ichikawa, H.; Kitagawa, O.; Kumagai, K.; Watanabe, M.;
Ogawa, E.; Seiki, M.; Shirataki, Y.; Yokoe, I.; Komatsu, M. Yakugaku
Zasshi 1994, 114, 980-994.
(7) Chang, S. Dictionary of Chinese Crude Drugs: Shanghai Scientific
Technologic Publisher: Shanghai, 1977; p 1883.
(8) Abe, F.; Chen, R.-F.; Yamauchi, T. Phytochemistry 1991, 30, 3379-
3382.
(9) Seki, K.; Haga, K.; Kaneko, R. Phytochemistry 1995, 38, 703-709.
(10) Kutani, N.; Hayashi, K. Yakugaku Zasshi 1944, 64, 16.
(11) Morita, N.; Shimokooiyama, M.; Shimizu, M.; Arisawa, M. Chem.
Pharm. Bull. 1972, 20, 730-733.
(12) Fukuyama, Y.; Okino, J .; Kodama, M. Chem. Pharm. Bull. 1991, 39,
1877-1879.
Isoa n h yd r obela ch in a l (11): Glassy solid; [R]D +30.1° (c
0.5, CH2Cl2); UV (MeOH) λmax (log ꢀ) 261 (4.20), 270 (4.23),
280 (4.25), 289 (sh) (4.16) nm; 1H NMR, see Table 1; 13C NMR
(CDCl3) δ 10.8 (C-1), 164.9 (C-7), 139.3 (C-19), 135.2 (C-15),
134.6 (C-16), 133.8 (C-2), 131.7 (C-23), 128.9 (C-14), 125.2 (C-
17), 124.9 (C-18), 123.9 (C-22), 111.1 (C-26), 77.9 (C-13), 73.4
(C-10), 70.7 (C-3), 61.2 (C-11), 50.6 (43.2) (C-6), 40.1 (C-20),
39.8 (C-9), 39.7 (CH2), 31.9 (CH2), 32.8 (C-27), 26.6 (C-21), 25.7
(C-24), 17.7 (C-30), 16.8 (C-29), 12.6 (C-28), 190.8 (C-25); EIMS
m/z 468 [M]+; ESIMS m/z 469 [M + H]+; HRESIMS m/z [M +
H]+ 469.3338 (calcd for C30H45O4, 469.3318).
(13) Krick, W.; Marner, F.-J .; J aenicke, L. Z. Naturforsch. 1983, 38C, 179-
184.
(14) Seki, K.; Haga, K.; Kaneko, R. Phytochemistry 1994, 37, 807-815.
(15) Marner, F.-J .; Karimi-Nejad, Y.; J aenicke, L. Helv. Chim. Acta 1990,
73, 433-438.
(16) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J . Am. Chem.
Soc. 1991, 113, 4092-4096.
(17) Marner, F.-J .; Littek, A.; Arnold, R.; Seferiadis, K.; J aenicke, L. Helv.
Chim. Acta 1990, 73, 433-438.
(18) Ishii, H.; Tan, S.; Wang, J . P.; Chen, I.-S.; Ishikawa, T. Yakugaku
Zasshi 1991, 111, 376-385.
(19) Bicchi, C.; Rubiolo, P. Flavour Fragrance J . 1993, 8, 261-267.
(20) Yoshida, T.; Nobuhara, J .; Uchida, M.; Okuda, T. Chem. Pharm. Bull.
1978, 26, 2535-2542.
Ch em ica l Con ver sion of 7 in to 9-11. Compound 7 (5 mg)
was treated with p-toluenesulfonic acid (1.3 mg) in CHCl3 (2
NP980271B