Cholestane rhamnosides from the bulbs of Ornithogalum saundersiae

Article abstract of DOI:10.1016/S0031-9422(99)00240-X

Phytochemical examination of the bulbs of Ornithogalum saundersiae yielded six cholestane rhamnosides, two of which had previously been isolated from the same plant material. However, detailed spectroscopic analysis of the aglycone led us to revise the configuration of the C-11 hydroxyl group of the latter two and reassign their structures as (22S)cholest-5-ene- 3β,11α,16β,22-tetrol 16-O-α-L-rhamnopyranoside and (22S)-cholesta-5,24- diene-3β,11α,16β,22-tetrol 16-O-α-L-rhamnopyranoside, respectively. The other four are new naturally occurring constituents and their structures were determined to be (22S)-cholest-5-ene-3β,11α,16β,22-tetrol 16-O-(2,3-di-O- acetyl-α-L-rhamnopyranoside), (22S)-cholest-5-ene-3β,11α,16β,22-tetrol 16-O-{2-O-acetyl-3-O-(3,4,5-trimethoxybenzoyl)-α-L-rhamnopyranoside}, (22S)-cholest-5-ene-3β,11α,16β,22-tetrol 16-0-{2-O-acetyl-3-O-(p- methoxybenzoyl)-α-L-rhamnopyranoside} and (22S)-cholesta-5,24-diene- 3,11*16β,22-tetrol 16-O-(2,3-di-O-acetyl-α-L-rhamnopyranoside), respectively. The isolated compounds were evaluated for their cytostatic activity against leukemia HL-60 cells.

Full text of DOI:10.1016/S0031-9422(99)00240-X

Phytochemistry 52 (1999) 445±452
Cholestane rhamnosides from the bulbs of Ornithogalum
saundersiae
Minpei Kuroda, Yoshihiro Mimaki*, Yutaka Sashida
School of Pharmacy, Tokyo University of Pharmacy and Life Science, Horinouchi 1432-1, Hachioji, Tokyo 192-0392, Japan
Received 9 October 1998; received in revised form 2 March 1999
Abstract
Phytochemical examination of the bulbs of Ornithogalum saundersiae yielded six cholestane rhamnosides, two of which
had previously been isolated from the same plant material. However, detailed spectroscopic analysis of the aglycone led
us to revise the con®guration of the C-11 hydroxyl group of the latter two and reassign their structures as (22S)-
cholest-5-ene-3b,11a,16b,22-tetrol 16-O-a-L-rhamnopyranoside and (22S)-cholesta-5,24-diene-3b,11a,16b,22-tetrol 16-O-a-L-
rhamnopyranoside, respectively. The other four are new naturally occurring constituents and their structures were determined to
be (22S )-cholest-5-ene-3b,11a,16b,22-tetrol 16-O-(2,3-di-O-acetyl-a-L-rhamnopyranoside), (22S)-cholest-5-ene-3b,11a,16b,22-
tetrol 16-O-{2-O-acetyl-3-O-(3,4,5-trimethoxybenzoyl)-a-L-rhamnopyranoside}, (22S )-cholest-5-ene-3b,11a,16b,22-tetrol 16-O-{2-
O-acetyl-3-O-( p-methoxybenzoyl)-a-L-rhamnopyranoside} and (22S)-cholesta-5,24-diene-3b,11a,16b,22-tetrol 16-O-(2,3-di-O-
acetyl-a-L-rhamnopyranoside), respectively. The isolated compounds were evaluated for their cytostatic activity against leukemia
HL-60 cells. # 1999 Elsevier Science Ltd. All rights reserved.
Keywords: Ornithogalum saundersiae; Liliaceae; Bulbs; Cholestane rhamnosides; Cytostatic activity; HL-60 cells
1. Introduction
the isolation of six cholestane glycosides (1±6). We
now report the structural revision of 1 and 5, which
had previously been isolated from this plant (Kubo,
Mimaki, Sashida, Nikaido & Ohmoto, 1992a) and
structural determination of four new cholestane rham-
nosides (2±4 and 6) on the basis of spectroscopic
analysis, including two-dimensional NMR techniques
and the results of hydrolysis. The activity of 1±6
against leukemia HL-60 cells was also investigated.
During our systematic studies on the chemical con-
stituents of plants of the family Liliaceae and their bio-
logical activities, we have disclosed that the bulbs of
Ornithogalum saundersiae contain abundant cholestane
glycosides (Kubo et al., 1992b; Kuroda, Mimaki &
Sashida, 1999; Kuroda, Mimaki, Sashida, Nikaido &
Ohmoto, 1993; Kuroda et al., 1995, 1997; Mimaki et
al., 1996a,b,c, 1997), some of which are unique in
structures having a rearranged cholestane skeleton
(Kuroda et al., 1993, 1997, submitted for publication;
Mimaki et al., 1996a,c) and exhibit potent cytostatic
activity against malignant tumor cells (Kuroda et al.,
1997, 1999; Mimaki et al., 1996a,b,c, 1997). Further
analysis of the bulbs of O. saundersiae has resulted in
2. Results and discussion
Compounds 1±6 were puri®ed as described in
Section 3.
The physical properties and spectral data of 1 and 5
completely agreed with those of (22S)-cholest-5-ene-
3b,11b,16b,22-tetrol 16-O-a-L-rhamnopyranoside and
(22S )-cholesta-5,24-diene-3b,11b,16b,22-tetrol 16-O-a-
L-rhamnopyranoside, which had previously been iso-
lated from the same plant source (Kubo et al., 1992a).
* Corresponding author. Tel.: +81-426-76-4577; Fax: +81-426-76-
4579.
E-mail address: mimakiy@ps.toyaku.ac.jp (Y. Mimaki)
0031-9422/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(99)00240-X

446
M. Kuroda et al. / Phytochemistry 52 (1999) 445±452
Table 1
1H and 13C NMR spectral data for compound 1a in C5D5N
Position
1H
J (Hz)
13C
40.0
1 ax
1 eq
2 ax
2 eq
3
1.57
3.36 ddd
2.03
13.9, 3.3, 3.3
32.9
2.19 br dd
3.97 m
2.77
12.7, 2.1
21.1a
71.7
44.2
4 ax
4 eq
5
2.75
±
142.9
121.0
32.4
6
5.56 br d
1.74
2.07
5.4
7 a
7 b
8
1.72
1.42 dd
±
32.1
57.3
38.9
68.2
52.4
9
10
10.7, 10.7
11
4.43 ddd
1.67
10.7, 10.7, 4.7
12.0, 4.7
12 ax
12 eq
13
2.70 dd
±
1.19
43.4
54.6
37.2
14
15 a
15 b
16
2.37 ddd
1.58
12.7, 7.7, 7.7
4.84 ddd
1.80 dd
1.31 s
1.47 s
2.64
7.7, 7.2, 4.5
11.1, 7.2
71.8
58.1
14.7
19.3
36.0
15.1
75.1
31.9
17
18
19
20
21
22
1.26 d
4.20 br d
1.83
7.1
8.8
23 a
23 b
24 a
24 b
25
However, detailed analysis of the aglycone using exten-
sive spectroscopic methods led us to revise the con-
®guration of the C-11 hydroxyl group. Acid hydrolysis
of 1 with 1 M HCl in dioxane±H2O gave an aglycone
(1a) and L-rhamnose. The complete assignments of the
1H and 13C NMR signals of 1a were carried out by
the concerted use of the 1H-1H COSY, HMQC and
HMBC spectra as shown in . This NMR information
showed the presence of the C-3, C-11, C-16 and C-22
hydroxyl groups on a cholestane skeleton. The con-
®guration of the C-11 hydroxyl group was previously
assigned as an 11b(ax) by the conventional comparison
1.71
1.76
36.7
1.39
1.63
28.4
22.8
23.0
26
27
0.93 d
0.92 d
6.5
6.6
a W1/2
.
orientation in 1a were ascertained by the H and 13C
NMR data (Table 1), including NOE information
(Fig. 1), and by the application of the modi®ed
1
1
Mosher's method (Ohtani, Kusumi, Kashman
&
of the H NMR data with those of cholesterol. In the
high-resolution 1H NMR spectrum (500 MHz) of 1a
measured in pyridine-d5, to which was added the
vapor of HCl to remove the signals due to the
exchangeable protons, the signal due to H-11 was
observed as a ddd multiplicity at d 4.43 (3JH-11,H-
Kakisawa, 1991) (Fig. 2). Furthermore, we inquired
into the stereochemistry at C-20, which was conven-
tionally assigned as S in the previous report. The
stereochemistry was examined by using molecular
3
modeling, JH,H value and NOESY data. A combi-
3
3
nation of molecular mechanics (MM) and molecular
dynamics (MD) calculations in the MM2 force ®eld as
implemented in MacroModel 6.0 was performed on
two possible compounds with C-20S and C-20R con-
®gurations. The H17±C17±C20±H20 torsion angle was
almost identical between the minimum energy confor-
mers of C-20S and C-20R models; 176.48 for C-20S
and 172.48 for C-20R (Fig. 3). The experimental J
value (11.1 Hz) of H-17/H-20 almost corresponded to
that (20S: 12.8 Hz; 20R: 12.6 Hz) calculated through
9=10.7 Hz, JH-11,H-12ax=10.7 Hz and JH-11,H-
12eq=4.7 Hz). This unequivocally indicated an a(eq)-
orientation of the C-11 hydroxyl group, which was
strongly supported by NOE correlations from H-11 to
both Me-18 and Me-19. The signi®cant down®eld shift
of the H-1b(eq) proton [d 3.36 (ddd, J = 13.9, 3.3, 3.3
Hz)] was considered to be caused by the interaction
with the 11a-hydroxyl group. The 3b, 16b and 22S
con®gurations, and the usual steroidal ring and side
chain junctions, B/C trans and C/D trans, and 17b-

M. Kuroda et al. / Phytochemistry 52 (1999) 445±452
447
Fig. 1. NOE correlations of 1a.
Fig. 3. The lowest energy conformer of 1a.
the application of the given dihedral angle to the
advanced Karplus-type equation proposed by
Haasnoot, De Leeuw and Altona (1980). Since it was
indicated by the above data that H-20 lay toward Me-
18 and that the C-17/C-20 bond was frozen (Nes et al.,
1998), NOE correlations observed between Me-18 and
H-20, and between H-12b(eq) and Me-21 made it poss-
ible to assign the C-20 con®guration as S. The evi-
dence presented above led us to revise the structure of
1a to (22S )-cholest-5-ene-3b,11a,16b,22-tetrol and,
consequently, 1 to (22S)-cholest-5-ene-3b,11a,16b,22-
tetrol 16-O-a-L-rhamnopyranoside. Compound 5 was
correlated to 1 by hydrogenation (Kubo et al., 1992a)
and has also been reassigned as (22S )-cholesta-5,24-
3H, s)] and 13C NMR [d 170.4 and 170.1 (C.O), and
20.8 and 20.7 (Me)] spectra. Treatment of 2 with 3%
NaOMe in MeOH gave 1. Therefore, 2 was a diacetate
1
of 1. In the H NMR spectrum of 2, the signals assign-
able to H-2 and H-3 of the rhamnose moiety were
moved down®eld by 1.17 and 1.48 ppm in comparison
with those of 1 and observed at d 5.64 (dd, J = 3.3,
1.3 Hz) and 5.78 (dd, J = 9.9, 3.3 Hz), respectively.
Thus, the acetyl groups were linked to the rhamnose
C-2 and C-3 positions and the structure was formu-
lated as (22S )-cholest-5-ene-3b,11a,16b,22-tetrol 16-O-
(2,3-di-O-acetyl-a-L-rhamnopyranoside).
The H and 13C NMR signals arising from the agly-
cone and rhamnosyl moieties of compound
1
diene-3b,11a,16b,22-tetrol
side.
16-O-a-L-rhamnopyrano-
3
Compound 2, isolated as an amorphous solid, [a]D
28.08 (MeOH) showed in the HRFABMS (positive
mode) an accurate [M+Na]+ ion at m/z 687.4048 in
accordance with the empirical molecular formula
C37H60O10, also deduced on the basis of 13C NMR
(C45H68O13) were almost identical to those of 2. The
existence of a 3,4,5-trimethoxybenzoyl group as well as
an acetyl group in 3 was indicated by the IR (nmax
1
1745 and 1720 cm 1), UV [lmax 266 (log e 4.02)], H
NMR [d 7.58 (2H, s), 3.89 (3H, s) and 3.73 (3H Â 2,
s); d 2.18 (3H, s)] and 13C NMR [d 165.9 (C.O), 153.6
(C Â 2), 143.0 (C), 125.9 (C), 107.7 (CH Â 2), 60.6
(OMe) and 56.0 (OMe  2); 170.0 (C.O) and 20.8
(Me)] spectra. Alkaline methanolysis of 3 with 3%
NaOMe in MeOH furnished methyl 3,4,5-trimethoxy-
benzoate and 1. The down®eld shifts by O-acylation
could be detected at H-2 and H-3 of the rhamnosyl
moiety, leading to the two possible structures with
regard to the linkage positions of the two acyl groups,
that is, 2-O-acetyl-3-O-(3,4,5-trimethoxybenzoyl)-a-L-
1
data. The H NMR spectrum of 2 showed signals for
®ve steroid methyl protons at d 1.41 (3H, s), 1.22 (3H,
d, J = 6.9 Hz), 1.08 (3H, d, J = 6.5 Hz), 1.02 (3H, d,
J = 6.5 Hz) and 0.99 (3H, s), an ole®nic proton at d
5.50 (1H, br d, J = 5.2 Hz) and an anomeric proton
at d 5.02 (1H, d, J = 1.3 Hz). The above 1H NMR
features and 13C NMR spectral data were essentially
analogous to those of 1. The presence of two acetyl
groups in the molecule was shown by the IR (nmax
1
1745 and 1725 cm 1), H NMR [d 2.10 and 2.00 (each
rhamnosyl
or
2-O-(3,4,5-trimethoxybenzoyl)-3-O-
acetyl-a-L-rhamnosyl. This was solved by analysis of
the HMBC spectrum. A carbonyl carbon signal at d
170.0 showed long-range correlations with the proton
signals due to the acetyl methyl group and H-2 of
rhamnosyl moiety at d 5.82 (dd, J = 3.3, 1.4 Hz).
Another carbonyl carbon at d 165.9 was correlated to
the aromatic protons and H-3 proton of the rhamnosyl
at d 6.09 (dd, J = 9.7, 3.3 Hz). From the evidence pre-
sented above, the acetyl and 3,4,5-trimethoxybenzoyl
groups were shown to be located at C-2 and C-3 of
the rhamnosyl moiety, respectively, and the structure
Fig. 2. Chemical shift di€erences between (S)-MPTA ester (1c) of 1a
and (R)-MPTA ester (1b) of 1a.
of
3
was elucidated as (22S )-cholest-5-ene-

448
M. Kuroda et al. / Phytochemistry 52 (1999) 445±452
3b,11a,16b,22-tetrol
16-O-{2-O-acetyl-3-O-(3,4,5-tri-
3. Experimental
methoxybenzoyl)-a-L-rhamnopyranoside}.
The 1H and 13C NMR spectra of compound 4
(C43H64O11) were completely superimposable on those
of 3, except for the aromatic region signals due to the
substituted benzoyl moiety. The aromatic acid linked
to C-3 of the rhamnosyl residue was suggested to be p-
methoxybenzoic acid by the UV [lmax 260 (log e 3.68)],
1H NMR [d 8.21 and 6.96 (each 2H, d, J = 8.7 Hz),
and 3.66 (3H, s)] and 13C NMR [d 165.9 (C.O), 163.8
(C), 132.0 (C Â 2), 123.5 (C), 114.1 (CH Â 2) and 55.4
(OMe)] spectra. Alkaline hydrolysis of 4 gave p-meth-
oxybenzoic acid and 1. The structure of 4 was shown
to be (22S)-cholest-5-ene-3b,11a,16b,22-tetrol 16-O-{2-
O-acetyl-3-O-( p-methoxybenzoyl)-a-L-rhamnopyrano-
side}.
3.1. General
As for preceding paper, except, in addition, NMR
1
experiments used Bruker DPX-400 (400 MHz for H
NMR), Bruker DRX-500 (500 MHz for 1H NMR
spectrometers). HPLC also employed a Capcell Pak
C18 column (Shiseido, Japan, 4.6 mm i.d. Â 250 mm,
ODS, 5 mm) for analytical HPLC. Cell culture and
assay for cytostatic activity: microplate reader, Inter
Med Immuno-Mini NJ-2300 (Japan); 96-well ¯at-bot-
tom plate, Iwaki Glass (Japan); HL-60 cells, ICN
Biomedicals (USA); RPMI 1640 medium, Gibco BRL
(USA); MTT, Sigma (USA).
3.2. Plant material
Spectral properties of compound 6 (C37H58O10) were
closely related to those of 2 and indicative of it being
a cholestane rhamnoside with two acetyl groups. The
1H NMR spectrum of 6 showed two three-proton sing-
let signals at d 1.79 and 1.74 assignable to methyl
groups on a double bond, which were present as three-
proton doublet signals at d 1.08 (J = 6.5 Hz) and 1.02
(J = 6.5 Hz) attributable to the Me-26 and Me-27
groups in 2. All other signals were almost superimpo-
sable between these two compounds. Alkaline metha-
nolysis of 6 gave 5. The above-mentioned data
indicated that 6 was the corresponding 24-ene deriva-
tive of 2, and that the structure was (22S )-cholesta-
5,24-diene-3b,11a,16b,22-tetrol 16-O-(2,3-di-O-acetyl-
a-L-rhamnopyranoside).
The cytostatic activity of 1 6 on human promyelo-
cytic leukemia HL-60 cells was evaluated as in the pre-
ceding paper (Kuroda et al., 1999). Compounds 1, 2,
3, 4 and 6 exhibited potent cytostatic activity with the
GI50 values of 0.19, 6.9, 1.8, 0.022 and 0.80 mM, re-
spectively. Subsequent evaluation of 1, the main con-
stituent of the plant, in the National Cancer Institute
60 cell line assay (Monks et al., 1991) showed that the
mean concentrations required to achieve GI50, TGI
and LC50 levels against the panel of cells tested were
1.5, 20 and 69 mM, respectively. Compound 1 dis-
played a speci®c activity towards the leukemia cell
lines (mean GI50 0.052 mM; mean TGI 0.23 mM; mean
LC50 27 mM). Furthermore, some cell lines were sensi-
tive to it. These cell lines included the colon cancer
KM12 (GI50 0.41 mM; TGI 2.5 mM; LC50 14 mM),
CNS cancer SF-539 (GI50 0.015 mM; TGI 0.24 mM;
LC50 4.6 mM), melanoma SK-MEL-28 (GI50 0.22 mM;
TGI 2.6 mM; LC50 8.5 mM) and breast cancer MCF7
(GI50 0.022 mM; TGI 0.37 mM; LC50 10 mM) (Table
3). The acute toxicity assay on 1 has been done and
the maximum tolerated dose in mice was determined
to be 400 mg/kg. The in vivo antitumor testing for 1 is
in progress.
See preceding paper Kuroda et al., 1999.
3.3. Extraction and isolation
As for preceding paper, fraction I was subjected to
ODS silica gel CC eluting with MeOH±H2O (17:3) and
divided into ®ve additional fractions (Ia±Ie). Fraction
Ia was subjected to silica gel CC eluting with CHCl3±
MeOH (10:1) and preparative HPLC with MeCN±
H2O (1:1) to yield 2 (111 mg) and 6 (91.5 mg).
Fraction Ib was submitted to a silica gel column elut-
ing with CHCl3±MeOH (9:1) and preparative HPLC
with MeCN±H2O (11:9) to furnish 3 (81.7 mg) and 4
(11.5 mg). Fraction III was subjected to CC on ODS
silica gel eluting with MeOH±H2O (4:1) to give four
additional fractions (IIIa±IIId). Fraction IIIa was
chromatographed on silica gel eluting with CHCl3±
MeOH±H2O (50:10:1) and ODS silica gel with
MeOH±H2O (7:3) to a€ord a mixture of 1 and 5,
which was then separated by preparative HPLC using
MeCN±H2O (2:3) to yield 1 (520 mg) and 5 (485 mg).
3.4. Compound 1
Amorphous solid. [a ]2D6 39.08 (MeOH: c 0.50).
Positive-ion HRFABMS m/z 603.3891 [M+Na]+
1
(C33H56O8Na requires 603.3873). IR nmax (KBr) cm
:
3420 (OH), 2945 (CH). 1H NMR (C5D5N): d 5.51
(1H, br d, J = 4.9 Hz, H-6), 5.26 (1H, br s, H-1'),
4.47 (1H, br d, J = 3.3 Hz, H-2'), 4.30 (1H, dd,
J = 9.3, 3.3 Hz, H-3'), 1.68 (3H, d, J = 6.0 Hz, Me-
6'), 1.41 (3H, s, Me-19), 1.22 (3H, d, J = 6.9 Hz, Me-
21), 1.04 (3H, s, Me-18), 0.85 (3H Â 2, d, J = 6.5 Hz,
Me-26 and Me-27). 13C NMR: Table 2.
3.5. Acid hydrolysis of 1
A solution of 1 (15 mg) in 1 M HCl (dioxane±H2O,

M. Kuroda et al. / Phytochemistry 52 (1999) 445±452
449
Table 2
13C NMR spectral data for compounds 1±6 in C5H5N
3.6. Compound 1a
Amorphous solid. [a ]2D6 44.08 (MeOH: c 0.30).
EIMS m/z 434 [M]+. IR nmax (KBr) cm 1: 3370 (OH),
C
1
2
3
4
5
6
1
1
40.1
32.3
71.7
44.2
40.0
32.2
71.7
44.1
40.0
32.2
71.7
44.1
40.0
32.2
71.7
44.2
40.0
32.3
71.7
44.1
40.0
32.2
71.7
44.1
2930 (CH). H NMR and 13C NMR spectra: Table 1.
2
3
3.7. (R ),(S )-MTPA derivatives of 1a
4
5
142.9
120.9
32.9
31.9
57.2
38.9
68.2
51.9
43.0
54.6
35.6
82.4
57.9
14.4
19.3
36.0
11.9
73.2
34.4
36.8
28.7
22.9
22.8
104.9
72.4
72.7
74.0
71.0
18.4
142.9
120.7
32.9
31.7
57.0
38.8
68.1
51.8
42.8
54.4
35.4
83.3
57.7
14.2
19.2
36.1
12.0
72.7
34.5
36.6
29.0
22.8
22.9
101.2
71.3
72.9
71.0
70.9
18.1
170.1
20.7
170.4
20.8
143.0
120.8
32.9
31.8
57.1
38.8
68.1
51.8
42.9
54.4
35.4
83.6
57.7
14.3
19.3
36.2
12.0
72.7
34.6
36.6
29.0
22.9
22.9
101.2
71.5
73.8
71.3
71.0
18.2
170.0
20.8
142.9
120.8
32.9
31.7
57.1
38.8
68.1
51.8
42.9
54.4
35.4
83.3
57.7
14.3
19.3
36.2
12.1
72.7
34.6
36.6
29.0
22.9
23.0
101.3
71.5
73.2
71.2
71.0
18.2
170.0
20.7
142.9
120.8
32.9
31.8
57.2
38.8
68.2
51.9
43.0
54.5
35.6
82.3
57.8
14.4
19.3
35.1
11.8
73.1
35.3
123.0
132.4
25.9
18.1
104.9
72.0
72.6
74.0
70.9
18.4
142.9
120.7
32.9
31.8
57.0
38.8
68.1
51.8
42.9
54.4
35.7
83.2
57.7
14.3
19.3
35.4
12.0
72.0
35.4
123.5
132.2
26.0
18.1
101.2
71.5
72.9
71.0
71.0
18.1
170.2
20.8
Compound 1a (1.5 mg) was dissolved in dry CH2Cl2
(1.5 ml), to which were added (R )-MTPA (16.5 mg),
4-(dimethylamino)pyridine (15.9 mg) and 1-ethly-3-(3-
dimethylaminopropyl)carbodiimideÁHCl (41.2 mg), and
the solution was allowed to stand at room temperature
for 24 h under an Ar atmosphere. The reaction mix-
ture was diluted with H2O and extracted with Et2O.
The Et2O phase was chromatographed on silica gel
eluting with hexane±EtOAc (6:1) to give the (R )-
MTPA ester (1b) (0.8 mg) of 1a. Following this pro-
cedure, 1a (1.5 mg) was converted to the (S )-MTPA
ester (1c) (2 mg) of 1a. 1H NMR of 1b (CDCl3): d
5.47 (1H, d, J = 5.7 Hz, H-6), 5.39 (1H, t-like,
J = 7.0 Hz, H-22), 4.90 (1H, m, H-3), 4.36 (1H, m, H-
16), 4.00 (1H, m, H-11), 3.57 (3H  2, s, OMe  2),
2.13 (3H, m, H-20), 1.14 (3H, s, H-19), 0.93 (3H, d,
J = 6.9 Hz, Me-21), 0.87 (3H, d, J = 6.3 Hz, Me-26
or Me-27), 0.86 (3H, s, Me-18), 0.85 (3H, d, J = 6.5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
1
Hz, Me-26 or Me-27). H NMR of 1c (CDCl3): d 5.49
1'
2'
3'
4'
5'
(1H, d, J = 5.6 Hz, H-6), 5.41 (1H, t-like, J = 6.8 Hz,
H-22), 4.89 (1H, m, H-3), 4.42 (1H, m, H-16), 4.01
(1H, m, H-11), 3.57 (3H, s, OMe), 3.51 (3H, s, OMe),
2.16 (3H, m, H-20), 1.16 (3H, s, Me-19), 0.97 (3H, d,
J = 7.0 Hz, Me-21), 0.90 (3H, s, Me-18), 0.85 (3H, d,
J = 6.6Hz, Me-26 or Me-27), 0.83 (3H, d, J = 6.6 Hz,
Me-26 or Me-27).
6'
Ac
170.5
20.9
10
20
30
40
50
60
70
OMe
125.9
107.7
153.6
143.0
153.6
107.7
165.9
60.6
123.5
132.0
114.1
163.8
114.1
132.0
165.9
55.4
3.8. Conformational analysis 1a
See preceding paper (Kuroda et al., 1999) with the
following exception of equilibration time period: 100
ps; and temperature bath set at 300 K.
3.9. Compound 2
56.0
(Â2)
Amorphous solid. [a ]3D0 28.08 (MeOH: c 0.10).
Negative-ion FABMS m/z 663 [M±H] , 621 [M±Ac] ,
579 [M±Ac  2] . Positive-ion HRFABMS m/z
687.4048 [M+Na]+ (C35H58O9Na requires 687.4084).
IR nmax (KBr) cm 1: 3400 (OH), 2930 and 2865 (CH),
1745 and 1725 (C.O), 1465, 1365, 1240, 1220, 1125,
1:1, 5 ml) was heated at 1008C for 1 h under an Ar at-
mosphere. After cooling, the reaction mixture was neu-
tralized by passage through an Amberlite IRA-93ZU
(Organo, Japan) column and chromatographed on
silica gel eluting with CHCl3±MeOH (19:1; 1:1) to give
an aglycone (9 mg) and L-rhamnose (2 mg). L-
Rhamnose was identi®ed by HPLC analysis following
their conversion to the 1-[(S )-N-acetyl-a-methylbenzy-
lamino]-1-deoxyalditol acetate derivatives (Kuroda et
al., 1997; Oshima, Yamauchi & Kumanotani, 1982). Rt
(min): 20.61.
1
1045, 1015, 985, 955, 900, 805. H NMR (C5D5N): d
5.78 (1H, dd, J = 9.9, 3.3 Hz, H-3'), 5.64 (1H, dd,
J = 3.3, 1.3 Hz, H-2'), 5.50 (1H, br d, J = 5.2 Hz, H-
6), 5.02 (1H, d, J = 1.3 Hz, H-1'), 2.10 and 2.00 (each
3H, s, Ac  2), 1.70 (3H, d, J = 6.2 Hz, Me-6'), 1.41
(3H, s, Me-19), 1.22 (3H, d, J = 6.9 Hz, Me-21), 1.08
(3H, d, J = 6.5 Hz, Me-26 or Me-27), 1.02 (3H, d,

450
M. Kuroda et al. / Phytochemistry 52 (1999) 445±452
J = 6.5 Hz, Me-26 or Me-27), 0.99 (3H, s, Me-18).
13C NMR: Table 2.
3.14. Alkaline hydrolysis of 4
Compound 4 (3 mg) was treated with 4% KOH in
EtOH (2 ml) at room temperature for 1 h. The reac-
tion mixture was neutralized by passage through an
Amberlite IR-120B column and then chromatographed
on silica gel eluting with CHCl3±MeOH (9:1; 4:1) to
yield 1 (1 mg) and p-methoxybenzoic acid (0.5 mg).
3.10. Alkaline methanolysis of 2
Compound 2 (5 mg) was treated with 3% NaOMe
in MeOH (2 ml) at room temperature for 1 h. The
reaction mixture was neutralized by passage through
an Amberlite IR-120B (Organo, Japan) column and
then chromatographed on silica gel eluting with
CHCl3±MeOH (4:1) to yield 1 (3.2 mg).
3.15. Compound 5
Amorphous solid. [a ]2D8 42.08 (MeOH: c 0.54).
Positive-ion HRFABMS m/z 601.3735 [M+Na]+
3.11. Compound 3
1
(C33H54O8Na requires 601.3716). IR nmax (KBr) cm
:
1
Amorphous solid. [a]2D7 +6.08 (MeOH: c 0.10).
Negative-ion FABMS m/z 815 [M±H] . Positive-ion
HRFABMS m/z 817.4719 [M+H]+ (C45H69O13
requires 817.4738). IR nmax (KBr) cm 1: 3440 (OH),
2930 and 2870 (CH), 1745 and 1720 (C.O), 1585 and
1500 (aromatic ring), 1460, 1415, 1330, 1225, 1170,
1125, 1045, 1000, 955, 860, 810. UV lmax (MeOH) nm
3420 (OH), 2975 and 2940 (CH). H NMR (C5D5N): d
5.53 (1H, t, J = 7.0 Hz, H-24), 5.50 (1H, br d, J = 5.0
Hz, H-6), 5.23 (1H, br s, H-1'), 4.48 (1H, br d,
J = 2.9 Hz, H-2'), 4.30 (1H, dd, J = 9.8, 2.9 Hz, H-
3'), 1.69 (3H, s, Me-27), 1.68 (3H, d, J = 6.0 Hz, Me-
6'), 1.67 (3H, s, Me-26), 1.43 (3H, s, Me-19), 1.26 (3H,
d, J = 6.8 Hz, Me-21), 1.03 (3H, s, Me-18). 13C
NMR: Table 2.
1
(log e): 266 (4.02). H NMR (C5D5N): d 7.58 (2H, s,
H-20 and H-60), 6.09 (1H, dd, J = 9.7, 3.3 Hz, H-3'),
5.82 (1H, dd, J = 3.3, 1.4 Hz, H-2'), 5.52 (1H, br d,
J = 5.2 Hz, H-6), 5.11 (1H, d, J = 1.4 Hz, H-1'), 3.89
(3H, s, OMe), 3.73 (3H  2, s, OMe  2), 2.18 (3H, s,
Ac), 1.77 (3H, d, J = 6.0 Hz, Me-6'), 1.43 (3H, s, Me-
19), 1.28 (3H, d, J = 6.9 Hz, Me-21), 1.03 (3H, s, Me-
18), 1.02 (3H, d, J = 6.4 Hz, Me-26 or Me-27), 0.97
(3H, d, J = 6.4 Hz, Me-26 or Me-27). 13C NMR:
Table 2.
3.16. Compound 6
Amorphous solid. [a ]3D0 24.08 (MeOH: c 0.10).
Negative-ion FABMS m/z 661 [M±H] , 619 [M±Ac] ,
577 [M±Ac  2] . Positive-ion HRFABMS m/z
663.4103 [M+H]+ (C37H59O10 663.4108 requires
685.3928). IR nmax (KBr) cm 1: 3430 (OH), 2970, 2930
and 2865 (CH), 1745 and 1725 (C.O), 1445, 1375,
1
1240, 1130, 1065, 1050, 1020, 985, 960, 810. H NMR
(C5D5N): d 5.79 (1H, dd, J = 10.1, 2.8 Hz, H-3'), 5.64
(1H, br d, J = 2.8 Hz, H-2'), 5.50 (1H, br d, J = 5.1
Hz, H-6), 5.04 (1H, br s, H-1'), 2.10 (3H, s, Ac), 1.99
(3H, s, Ac), 1.79 (3H, s, Me-27), 1.74 (3H, s, Me-26),
1.70 (3H, d, J = 5.7 Hz, Me-6'), 1.41 (3H, s, Me-19),
1.25 (3H, d, J = 6.6 Hz, Me-21), 0.98 (3H, s, Me-18).
13C NMR: Table 2.
3.12. Alkaline methanolysis of 3
Compound 3 (10 mg) was hydrolysed as for 2 above
to yield 1 (6.5 mg) and methyl 3,4,5-trimethoxybenzo-
ate (1 mg).
3.13. Compound 4
Amorphous solid. [a]3D0 12.08 (MeOH: c 0.10).
Positive-ion HRFABMS m/z 779.4350 [M+Na]+
(C43H64O11Na requires 779.4346). IR nmax (KBr)
cm 1: 3440 (OH), 2950 and 2880 (CH), 1750 and 1725
(C.O), 1590 and 1490 (aromatic ring), 1460, 1415,
1325, 1250, 1230, 1215, 1120, 1050, 950, 860, 800. UV
lmax (MeOH) nm (log e): 260 (3.68). 1H NMR
(C5D5N): d 8.21 (2H, d, J = 8.7 Hz, H-20 and H-60),
6.96 (2H, d, J = 8.7 Hz, H-30 and H-50), 6.09 (1H,
dd, J = 9.4, 3.1 Hz, H-3'), 5.81 (1H, br d, J = 3.1 Hz,
H-2'), 5.53 (1H, br d, J = 4.5 Hz, H-6), 5.10 (1H, br
s, H-1'), 3.66 (3H, s, OMe), 2.15 (3H, s, Ac), 1.78 (3H,
d, J = 5.8 Hz, Me-6'), 1.44 (3H, s, Me-19), 1.27 (3H,
d, J = 6.6 Hz, Me-21), 1.03 (3H, s, Me-18), 1.02 (3H,
d, J = 6.2 Hz, Me-26 or Me-27), 0.98 (3H, d, J = 6.2
Hz, Me-26 or Me-27). 13C NMR: Table 2.
3.17. Alkaline methanolysis of 6
Compound 6 (5 mg) was hydrolysed as for 2 above
to yield 5 (2.5 mg).
3.18. Cell culture and assay for cytostatic activity
See preceding paper (Kuroda et al., 1999).
Acknowledgements
We wish to thank the Developmental Therapeutics
Program, National Cancer Institute, USA (Bethesda,
MD), for performing the cytostatic and cytotoxic
screening studies. We are also grateful to Dr. Y. Shida

M. Kuroda et al. / Phytochemistry 52 (1999) 445±452
451
Table 3
The GI50, TGI and LC50 values of compound 1 against the NCI 60 cell linesa
Panel/cell line
GI50 (mM)
TGI (mM)
LC50 (mM)
Leukemia
K-562
0.12
0.43
0.19
0.051
0.25
35
13
11
47
MOLT-4
RPMI-8226
SR
0.028
0.016
0.042
Non-small cell lung cancer
A 549/ATCC
HOP-62
1.5
0.32
27
±
±
±
±
±
±
±
±
NCI-H23
NCI-H522
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
5.3
1.3
b
±
4.6
±
±
±
0.18
±
±
±
±
±
HT29
KM12
SW-620
1.8
±
±
0.41
0.14
2.5
4.0
14
±
CNS cancer
SF-268
SF-295
1.2
±
±
0.021
0.015
±
0.10
0.24
±
±
SF-539
SNB-19
U251
4.6
±
0.010
±
±
Melanoma
MALME-3M
M14
2.5
1.2
±
5.5
±
±
71
±
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-62
Ovarian cancer
OVCAR-3
OVCAR-5
OVCAR-8
SK-OV-3
Renal cancer
786-0
7.2
0.22
0.74
0.50
2.6
34
3.3
8.5
±
±
±
±
±
±
±
±
±
±
±
6.2
±
±
0.11
0.46
±
±
12
±
±
±
±
±
±
±
±
A498
ACHN
CAKI-1
0.63
0.025
±
41
±
RXF-393
SN12C
UO-31
±
87
3.1
Prostate cancer
PC-3
DU-145
0.34
±
±
±
±
±
Breast cancer
MCF7
0.022
87
1.0
0.37
10
±
MCF7/ADR-RES
MDA-MB-231/ATCC
MDA-MB-435
MDA-N
±
±
±
±
±
±
0.98
1.2
±
±
T-47D
±
±
Mean value
1.5
20
69
a The LC50 is the concentration at which only 50% of the cells are viable, the GI50 value is the concentration that yields 50% growth and the
total growth inhibition (TGI) is the concentration at which no growth was observed.
b The value is more than 100 mM.

452
M. Kuroda et al. / Phytochemistry 52 (1999) 445±452
Mimaki, Y., Kuroda, M., Kameyama, A., Sashida, Y., Hirano, T.,
Oka, K., Dobashi, A., Koike, K., & Nikaido, T. (1996a). Bioorg.
Med. Chem. Lett., 6, 2635.
and Mr. H. Fukaya, Tokyo University of Pharmacy
and Life Science, for the measurements of the mass
spectra and elemental analysis.
Mimaki, Y., Kuroda, M., Kameyama, A., Sashida, Y., Hirano, T.,
Oka, K., Koike, K., & Nikaido, T. (1996b). Biosci. Biotech.
Biochem., 60, 1049.
Mimaki, Y., Kuroda, M., Kameyama, A., Sashida, Y., Hirano, T.,
Oka, K., Maekawa, R., Wada, T., Sugita, K., & Beutler, J. A.
(1997). Bioorg. Med. Chem. Lett., 7, 633.
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