Rubrosides A-H, new bioactive tetramic acid glycosides from the marine sponge Siliquariaspongia japonica

Article abstract of DOI:10.1021/jo981995v

Eight new tetramic acid glycosides named rubrosides A-H have been isolated from the marine sponge Siliquariaspongia japonica. Their structures were elucidated on the basis of spectral data as tetramic acid glycosides containing polyenes terminating in a 4-chloro-2-methyltetrahydrofuran ring. The absolute stereochemistry of the furan functionality in the two major metabolites, rubrosides D and F, was determined by the NMR method using chiral anisotropic reagents for tetrahydro-2-furoic acid derived by RuO₄ oxidation. The absolute stereochemistry of tetramic acid and of the sugar moieties in all rubrosides was deduced by chiral GC analysis of chemical degradation products. The rubrosides induced numerous large intracellular vacuoles in 3Y1 rat fibroblasts at concentrations of 0.5-1.0 μg/mL, and rubrosides A, C, D, and E were cytotoxic against P388 murine leukemia cells with IC₅₀ values of 0.046-0.21 μg/mL. Most rubrosides show antifungal activity against Aspergillus fumigatus and Candida albicans.

Full text of DOI:10.1021/jo981995v

J . Org. Chem. 1999, 64, 2331-2339
2331
Ru br osid es A-H, New Bioa ctive Tetr a m ic Acid Glycosid es fr om
th e Ma r in e Sp on ge Siliqu a r ia spon gia ja pon ica 1
Noriko U. Sata,† Shun-ichi Wada,‡ Shigeki Matsunaga,† Shugo Watabe,‡
Rob W. M. van Soest,§ and Nobuhiro Fusetani*,†
Laboratory of Aquatic Natural Products Chemistry and Laboratory of Aquatic Molecular Biology and
Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo,
Bunkyo-ku, Tokyo 113-8657, J apan, and Institute for Systematics and Ecology, The University of
Amsterdam, P.O. Box 94766, 1090 GT Amsterdam, Netherlands
Received October 2, 1998
Eight new tetramic acid glycosides named rubrosides A-H have been isolated from the marine
sponge Siliquariaspongia japonica. Their structures were elucidated on the basis of spectral data
as tetramic acid glycosides containing polyenes terminating in a 4-chloro-2-methyltetrahydrofuran
ring. The absolute stereochemistry of the furan functionality in the two major metabolites, rubrosides
D and F, was determined by the NMR method using chiral anisotropic reagents for tetrahydro-2-
furoic acid derived by RuO4 oxidation. The absolute stereochemistry of tetramic acid and of the
sugar moieties in all rubrosides was deduced by chiral GC analysis of chemical degradation products.
The rubrosides induced numerous large intracellular vacuoles in 3Y1 rat fibroblasts at concentra-
tions of 0.5-1.0 µg/mL, and rubrosides A, C, D, and E were cytotoxic against P388 murine leukemia
cells with IC50 values of 0.046-0.21 µg/mL. Most rubrosides show antifungal activity against
Aspergillus fumigatus and Candida albicans.
Marine sponges of the order Lithistida have proved to
be a rich source of metabolites with unusual structural
features as well as interesting biological activities, many
of which are reminiscent of microbial metabolites,2 e.g.,
discodermins,3 theonellamides,4 swinholides,5 discoder-
mide,6 and aurantosides.7 Faulkner and co-workers showed
that swinholides are in fact of bacterial origin.8 Recently,
we have initiated a program to discover marine natural
products that influence cell functions using 3Y1 rat
fibroblast cells,9 which proved to be useful for detection
of active compounds such as inhibitors of actin polym-
erization, spindle toxins, cell cycle arrestants, and others.
In the screening of a number of extracts, we encountered
a vermilion sponge, Siliquariaspongia japonica, collected
off Hachijo-jima Island, whose methanolic extract exhib-
ited significant activity in the 3Y1 rat fibroblast assay.
Bioassay-guided isolation afforded eight active metabo-
lites named rubrosides A-H, which proved to be new
tetramic acid glycosides containing an unusual 4-chloro-
2-methyl-tetrahydrofuran ring. This paper describes the
isolation, structure elucidation, and bioactivities of these
compounds.
The EtOH extract of the sponge (400 g wet weight) was
partitioned between ether and water. The aqueous phase
was extracted with n-BuOH, and the ether phase10 was
partitioned between MeOH/H2O (9:1) and n-hexane. The
n-BuOH and the aqueous MeOH phases were combined
and subjected to flash chromatography on ODS followed
by reversed-phase HPLC to give rubrosides A (1, 9.1 mg,
2.3 × 10-3 %), B (2, 5.1 mg, 1.3 × 10-3 %), C (3, 7.0 mg,
1.8 × 10-3 %), D (4, 126.3 mg, 3.2 × 10-2 %), E (5, 8.6
mg, 2.2 × 10-3 %), F (6, 108.1 mg, 2.7 × 10-2 %), G (7,
5.5 mg, 1.4 × 10-3 %), and H (8, 4.5 mg, 1.1 × 10-3 %) as
dark red solids (Chart 1).
* To whom correspondence should be addressed. Phone: +81-3-
3812-2111 (ext 5299). Fax: +81-3-5684-0622. E-mail: anobu@
hongo.ecc.u-tokyo.ac.jp.
† Laboratory of Aquatic Natural Products Chemistry.
‡ Laboratory of Aquatic Molecular Biology and Biotechnology.
§ Institute for Systematics and Ecology.
(1) Bioactive Marine Metabolites. Part 87. Part 86: Nakao, Y.;
Masuda, A.; Matsunaga, S.; Fusetani, N. J . Am. Chem. Soc., in press.
(2) Bewley, C. A.; Faulkner, D. J . Angew. Chem. Int. Ed. 1998, 37,
2162-2178.
(3) (a) Matsunaga, S.; Fusetani, N.; Konosu, S. Tetrahedron Lett.
1984, 25, 5165-5168. (b) Matsunaga, S.; Fusetani, N.; Konosu, S.
Tetrahedron. Lett. 1985, 26, 855-856. (c) Matsunaga, S.; Fusetani, N.;
Konosu, S. J . Nat. Prod. 1985, 48, 236-241. (d) Ryu, G.; Matsunaga,
S.; Fusetani, N. Tetrahedron Lett. 1994, 35, 8251-8254. (e) Ryu, G.;
Matsunaga, S.; Fusetani, N. Tetrahedron 1994, 50, 13409-13416.
(4) (a) Matsunaga, S.; Fusetani, N.; Hashimoto, K.; Walchli, M. J .
Am. Chem. Soc. 1989, 111, 2582-2588. (b) Matsunaga, S.; Fusetani,
N. J . Org. Chem. 1995, 60, 1177-1181.
(5) (a) Carmely, S.; Kashman, Y. Tetrahedton Lett. 1985, 26, 511-
514. (b) Kobayashi, M.; Tanaka, J .; Katori, T.; Matsuura, M.; Kitagawa,
I. Tetrahedron Lett. 1989, 30, 2963-2966. (c) Kobayashi, M.; Tanaka,
J .; Katori, T.; Kitagawa, I. Chem. Pharm. Bull. 1990, 38, 2960-2966.
(d) Tsukamoto, S.; Ishibashi, M.; Sasaki, T.; Kobayashi, J . J . Chem.
Soc., Perkin Trans. 1 1991, 3185-3188. (e) Tsukamoto, S.; Ishibashi,
M.; Sasaki, T.; Kobayashi, J . J . Chem. Soc., Perkin Trans. 1 1993, 1273.
(f) Dumdei, E. J .; Blunt, J . W.; Munro, M. H. G.; Pannell, L. K. J . Org.
Chem. 1997, 62, 2636-2639.
The most abundant rubroside D (4) exhibited UV
absorptions at 251 and 432 nm in MeOH, which were
shifted to 359 and 480 nm in 0.01 N HCl and to 251, 432,
and 453 nm in 0.01 N NaOH, reminiscent of the auran-
tosides.7 The FABMS revealed (M - H)- ions of equal
intensity at m/z 939 and 941, indicative of the presence
(6) Gunasekera, S. P.; Gunasekera, M.; McCarthy, P. J . Org. Chem.
1991, 56, 4830-4833.
(7) (a) Matsunaga, S.; Fusetani, N.; Kato, Y. J . Am. Chem. Soc. 1991,
113, 9690-9692. (b) Schmidt, E. W.; Harper, M. K.; Faulkner, D. J . J .
Nat. Prod. 1997, 60, 779-782. The structures of aurantosides have
been revised as shown (manuscript submitted).
(9) (a) Kimura, G.; Itagaki, A.; Summers, J . Int. J . Cancer 1975,
15, 694-706. (b) Watabe, S.; Wada, S.; Saito, S.; Matsunaga, S.;
Fusetani, N.; Ozaki, H.; Karaki, H. Cell Struct. Funct. 1996, 21, 199-
212.
(10) The organic phase was subjected to a series of solvent partition-
ing between n-hexane/MeOH-H2O (9:1) and CH2Cl2/MeOH-H2O
(6:4). The CH2Cl2 and 60% aqueous MeOH phases exhibited the activity
to induce morphological changes in cultured 3Y1 rat fibroblasts.
(8) Bewley, C. A.; Holland, N. D.; Faulkner, D. J . Experientia 1996,
52, 716-722.
10.1021/jo981995v CCC: $18.00 © 1999 American Chemical Society
Published on Web 03/11/1999

2332 J . Org. Chem., Vol. 64, No. 7, 1999
Sata et al.
Ch a r t 1
of either one bromine or two or three chlorine atoms. In
fact, the molecular formula of C44H56Cl2N2O16 was de-
termined by HRFABMS. We faced a problem with
E geometry (Table 1). Similarly, COSY analysis starting
1
acquiring NMR data of high quality. Although H NMR
signals were well dispersed in CD3OD, they were uni-
formly broad. Measurement in DMSO-d6 did not solve
this problem, which was finally overcome by using
pyridine-d5 as a solvent. Because of slow formation of the
pyridinium salt, the 1H NMR spectrum experienced a
transition state, appearing as a mixture of two com-
pounds and finally converging to a spectrum of a single
compound.11 To determine coupling constants, it was
1
absolutely necessary to measure the H NMR spectrum
in pyridine-d5.
Interpretation of the COSY spectrum starting from a
deshielded olefinic signal at δ 7.76 (d, J ) 15.0 Hz, H8),
together with analysis of coupling constants, led to partial
structure a , in which all double bonds except for ∆16 had
from a doublet at δ 6.52 (d, J ) 14.3 Hz, H18) resulted
in partial structure b, which consisted of a triene con-
nected to a disubstituted tetrahydrofuran ring. Coupling
constants indicated Z geometry of the ∆20 double bond
and E geometry for other two. Placement of a chlorine
atom at C-25 was consistent with 1H and 13C chemical
(11) At higher temperatures, 1H NMR signals became sharper and
dispersed. It is interesting that signals of tetramic acid and xylose
moieties changed over 4 d at 318 K. Similar results were also observed
for other rubrosides.

Rubrosides A-H
J . Org. Chem., Vol. 64, No. 7, 1999 2333
Ta ble 1. NMR Da ta for Ru br osid e D (4) in CD3OD a n d C5D5N
CD3OD
1H mult
C5D5N
1H mult
13C mult
J (Hz)
13C mult
J (Hz)
HMBC (C no.)
1
2
3
4
5R
5â
6
7
8
174.8 s
102.5 s
195.0 s
65.4 d
38.2 t
174.0 s
101.7 s
196.1 s
60.3 d
39.0 t
4.32 br
2.65 m
2.80 dd
5.32 dd
3.22 dd
3.37 dd
3.4, 6.7
6.7, 15.9
3.4, 15.9
6, 3
4, 6
4, 6, 3
4.9, 12.2
174.3 s
176.2 s
122.0 d
146.4 d
133.3 d
145.3 d
135.6 d
140.4 d
137.4 d
132.9 d
131.1 d
134.9 s
132.7 d
128.8 d
132.1 d
130.6 d
129.5 d
133.1 d
84.1 d
172.9 s
174.2 s
123.6 d
143.7 d
133.0 d
142.5 d
135.2 d
136.8 d
138.4 d
131.7 d
130.5 d
133.8 s
132.0 d
128.1 d
129.8 d
131.7 d
128.6 d
133.3 d
83.0 d
7.20 d
14.0
12.2, 14.0
7.76 d
15.0
7, 10
9
7.62 dd
6.60 m
6.88 m
6.58 m
6.73 dd
6.60 m
6.88 m
6.58 m
7.64 dd
6.42 dd
6.71 dd
6.46 dd
6.57 dd
6.61 dd
6.95 dd
6.59 d
11.3, 15.0
15.0, 11.3
11.6, 15.0
13.7, 11.6
10.9, 13.7
13.7, 10.9
11.0, 13.7
11.0
7, 8, 10, 11
8
9, 13, 12
11, 14
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26R
26â
27
28
NH2
11.3, 15.0
17, 14
17
6.53 d
14.0
10.7, 14.0
6.52 d
14.3
17, 20
20, 21, 17
22, 21
19
24, 20, 21
25, 24, 21
25
7.17 dd
6.20 m
6.18 m
6.88 m
5.92 dd
4.50 m
4.51 m
1.92 dd
2.78 dd
4.14 m
1.38 d
7.37 dd
6.22 dd
6.27 dd
7.14 dd
6.17 dd
4.49 dd
4.56 ddd
1.91 ddd
2.62 ddd
4.05 ddd
1.36 d
11.0, 14.3
10.7, 11.0
10.7, 10.7
15.3, 10.7
6.4, 15.3
4.0, 6.4
3.0, 4.0, 6.7
6.7, 7.6, 14.0
3.0, 6.4, 14.0
6.1, 6.4, 7.6
6.1
6.1, 15.0
63.3 d
44.9 t
63.1 d
44.3 t
27
6.4, 13.6
7.3, 13.6
25, 27, 24
27, 24
75.6 d
22.2 q
74.4 d
22.1 q
6.1
27, 26
7.80 brs
8.40 brs
6.10 d
4.68 dd
4.18 m
4.24 m
3.64 t
1′
2′
3′
4′
5′R
5â
1′′
2′′
3′′
86.1 d
81.3 d
79.2 d
70.5 d
69.2 t
80.9 d
81.6 d
69.9 d
79.7 d
69.4 t
9.2
8.9, 9.2
4, 5′, 2′, 1
1′, 1′′
2′, 5′
4.50 br
3.47 t
3.61 m
3.21 t
8.9
11.0
12.0
3′, 4′, 1′
3.86 m
5.02 brs
3.79 m
3.78 m
3.91 m
3.57 m
3.70 m
5.08 d
3.64 m
3.89 m
3.74 m
1.31 d
3.34 s
4.24 m
5.75 d
103.9 d
71.6 d
70.8 d
76.0 d
61.5 t
104.2 d
71.5 d
70.1 d
76.9 d
61.8 t
3.4
5.0, 9.8
3.1, 9.8
5′′, 3′′, 2′
3′′
2′′, 4′′
1′′′, 2′′
1′′, 4′′, 3′′
4′′
4′′, 3′′′
OMe, 3′′′
2′′′, 4′′′
3′′′, 1′′′
4′′′
4.43 dd
4.38 dd
4.20 m
4.02 m
4.24 m
5.29 d
3.91 dd
4.41 t
4.12 t
4′′
5′′R
5′′â
1′′′
2′′′
3′′′
4′′′
5′′′
OMe
98.9 d
87.3 d
79.7 d
79.5 d
20.8 q
58.3 q
4.3
6.1
99.7 d
87.2 d
79.1 d
79.1 d
20.9 d
57.6 q
4.5
4.5, 7.8
7.8
6.4
6.4
1.44 d
3.35 s
2′′′
shifts of δ 4.56 and 63.1, respectively. The presence of a
tetrahydrofuran ring was implied by a NOESY cross-
peak between H-24 and H-27 and supported by coupling
constants for H-24 to H-27, which were observed between
3.0 and 7.6 Hz.12 Units a and b were connected through
C-17/C-18 on the basis of the HMBC cross-peaks, H-18/
C-17 and H-16/C-17 (Table 1). We were not able to use
NOESY or ROESY data to assign stereochemistry of the
∆16-olefin because signals for H-16 and H-18 were
overlapped with those for H-13 and H-14, respectively;
the four signals were congested in a range of ap-
proximately 0.1 ppm. Therefore, Z geometry for this olefin
was inferred from: (1) chemical shifts of H-13 through
H-15 similar to those in aurantosides, especially the
significant downfield shift of H-15 due to the proximate
chlorine atom, and (2) the absence of a ROESY cross-
peak between H-15 and H-18. Instead, a distinct ROESY
cross-peak was observed between H-19 and H-22.
There was a pair of mutually coupled exchangeable
broad singlets at δ 7.80 and 8.40 in the 1H NMR spectrum
that exhibited NOESY cross-peaks with the methylene
protons in an isolated CHCH2 unit (C-4, C-5), thereby
suggesting the presence of a primary amide, which was
confirmed by HMBC data (H-4/C-6, H-5R/C-6, and H-5â/
C-6). Four broad nonprotonated 13C NMR signals at δ
196.1, 174.0, 174.2, and 101.713 were reminiscent of the
tetramic acid portion of the aurantosides, which was also
supported by acid/base shifts of UV absorptions and by
HMBC cross-peaks H-4, H-5â, and C-3 at δ 196.1 (unit
(13) (a) Shigemori, H.; Bae, M.-A.; Yazawa, K.; Sasaki, T.; Koba-
yashi, J . J . Org. Chem. 1992, 57, 4317-4320. (b) Kanazawa, S.;
Fusetani, N.; Matsunaga, S. Tetrahedron Lett. 1993, 34, 1065-1068.
(c) Ohta, S.; Ohta, E.; Ikegami, S. J . Org. Chem. 1997, 62, 6452-6453.
(d) Lee, V. J .; Rinehart, K. L., J r. J . Antibiot. 1980, 33, 408-415. (e)
Stickings, C. E. Biochem. J . 1959, 72, 332-340. (f) Phillips, N. J .;
Goodwin, J . T.; Fraiman, A.; Cole, R. J .; Lynn, D. G. J . Am. Chem.
Soc. 1989, 111, 8223-8231. (g) Sakuda, S.; Ono, M.; Furihata, K.;
Nakayama, J .; Suzuki, A.; Isogai, A. J . Am. Chem. Soc. 1996, 118,
7855-7856. (h) Nowak, A.; Steffan, B. Liebigs Ann. Chem. 1997, 1817-
1821.
(12) Stevens, J . D.; Fetcher, H. G., J r. J . Org. Chem. 1968, 33, 1799-
1805.

2334 J . Org. Chem., Vol. 64, No. 7, 1999
Sata et al.
F igu r e 1. Comparison of NOESY data of rubrosides D and F
in C5D5N.
c); connectivities of units b and c could be established
by HMBC cross-peaks H-8/C-7 and H-9/C-7 (Table 1).
The remaining portion consisted of three sugars, each
of which gave rise to an isolated spin system starting
from an anomeric proton. Interpretation of the HOHAHA
and COSY spectra together with HMQC data led to
assignment of a xylopyranose unit with an axial anomeric
proton (sugar I), an arabinopyranose unit with an
equatorial anomeric proton (sugar II), and a 5-deoxy-
pentofuranose unit (sugar III) possessing a methoxy
group on C-2′′′ as revealed by HMBC correlations (OCH3/
C-2′′′ and H-2′′′/OCH3). The sequence of the three sugars
was established by HMBC cross-peaks observed between
H-1′′′ (δ 5.29) and C-4′′ (76.9) and H-1′′ (5.75) and C-2′
(81.6). A chemical shift of δ 80.9 for C-1′ and an HMBC
cross-peak between H-1′ (δ 6.10) and a carbon at δ 174.0,
which was a part of tetramic acid, indicated that the
xylopyranose unit was linked to the tetramic acid unit
through a nitrogen atom. At this stage, we realized that
the gross structure of the glycosylated tetramic acid
moiety in rubroside D was identical with the correspond-
ing portion in aurantoside A. This was supported by NMR
data of the relevant portions, which were almost super-
imposable on those of aurantoside A (10), thus suggesting
their identical stereochemistry.
F igu r e 2. ∆δ values obtained for tetrahydro-2-furoic acid and
3-chloro-5-methyl-tetrahydro-2-furoic acid PGME esters.
∆δ ) δS(-) - δR(+)
.
models. Unexpectedly, the ∆δ values of the (R)-PGME
amide were all positive except for the R-H, which gave a
negative value, whereas the reverse was true for (S)-
PGME amide (Figure 2). It is likely that the conformation
of the chiral auxiliary was different from the reported
model as a result of the formation of a hydrogen bond
between the amide hydrogen and the ether oxygen. If the
pattern of distribution of ∆δ values was reproducible in
our system, it should be possible to assign the absolute
stereochemistry. Rubroside D (4) was oxidized with
ruthenium tetroxide17 to give 3-chloro-5-methyl-tetra-
hydro-2-furoic acid (17), which was converted to the
PGME amides 17a and 17b. The ∆δ values were positive
for all the protons except for the R-H (Figure 2). There-
fore, the stereochemistry of the methine carbon attached
to the carboxylic group was assigned as S. Incidentally,
the formation of a hydrogen bond between the amide NH
and the chlorine atom was ruled out by comparing the
∆δ values of the PGME amides.
The absolute stereochemistry of both xylose and ara-
binose were determined to be D by chiral GC analysis of
the acid hydrolysate.18 The relative stereochemistry of
sugar III was assigned on the basis of NOESY correla-
tions H-1′′′/H-3′′′, H-1′′′/H-4′′′, and H-3′′′/CH3-5′′′ to be
identical with 5-deoxyarabinoside in aurantoside A,
Treatment of 4 with H2 on Pd-C in MeOH afforded a
hexadecahydrorubroside D (9) in good yield. The 1H NMR
spectrum exhibited a broad signal at δ 1.23 for a long
methylene chain from H-8 to H-24, and the FABMS
showed a pseudomolecular ion at m/z 919 (M - H)-,
corresponding to C44H72ClN2O16.
1
which was supported by almost superimposable H and
13C NMR values for the relevant portions. Because
attempts to determine the absolute stereochemistry by
the Mosher method19 were not successful, we decided to
isolate sugar III. Rubroside D was subjected to metha-
nolysis followed by ODS flash chromatography to furnish
the methyl glycoside of sugar III. Distribution of the
positive and negative ∆δ values of the MTPA esters was
in agreement with 3′′′ R stereochemistry (Figure 3). It is
in agreement with the stereochemistry of des-O-methyl
sugar III in aurantoside B,7 which had been assigned by
the CD exciton chirality method.20
Stereochemistry of C-4 was determined as S by the
same method applied to aurantosides.7 Lemieux oxida-
tion14 followed by acid hydrolysis of rubroside D yielded
L-Asp as analyzed by chiral GC analysis. The relative
stereochemistry of the terminal tetrahydrofuran ring was
established on the basis of NOESY correlations, H-24/
H-27, H-25/H-27, H-26R/H-27, H-25/H-26R, and CH3-28/
H-26â (Figure 1), which disclosed that H-24, H-25, and
H-27 were on the same face of the five-membered ring.
To determine its absolute stereochemistry, we decided
to apply the NMR method using the chiral anisotropic
reagents (R)- and (S)-phenylglycine methyl esters
(PGME)15 developed by Kusumi and co-worker16 to a
furoic acid derived by cleavage of the C-22-C-23 double
bond. First, we examined the validity of this method by
using (R)- and (S)-tetrahydro-2-furoic acids as suitable
Rubroside F (6) had a molecular formula of C42H53
-
Cl3N2O16, which differs from 4 by the elements of C2H3-
(17) Carlsen, P, H.; Katsuki, T.; Martin, V. S.; Sharpless, K. B. J .
Org. Chem. 1981, 46, 3936-3938.
(18) Kˆınig, W. A.; Benecke, I.; Bretting, H. Angew. Chem., Int. Ed.
Eng. 1981, 20, 693-694.
(14) (a) Lemieux, R. U. Anal. Chem. 1954, 26, 920-921. (b) Lemieux,
R. U.; J ohnson, W. S. J . Org. Chem. 1956, 21, 478-479.
(15) Rachele, J . R. J . Org. Chem. 1963, 28, 2898.
(19) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J . Am.
Chem. Soc. 1991, 113, 4092-4096.
(20) Nakanishi, K.; Kuroyanagi, M.; Nambu, H.; Oltz, E. M.; Takeda,
R.; Verdine, G.; Zask, A. Pure Appl. Chem. 1984, 56, 1031-1048.
(16) Nagai, Y.; Kusumi, T. Tetrahedron Lett. 1995, 36, 1853-1856.

Rubrosides A-H
J . Org. Chem., Vol. 64, No. 7, 1999 2335
Ta ble 2. 1H NMR Da ta for Ru br osid es A (1), B (2), a n d G (7)
1a 2b
7a
4
4.31 br
2.66 br
2.80 m
7.22 br
7.61 br
6.62 m
6.90 m
6.57 m
4.20 br
2.52
2.60
4.29 br
2.80 m
5R
5â
8
7.19 br
7.55 br
6.88 m
7.04 m
6.67 m
6.70 m
6.72 m
6.80 m
6.70 m
7.25 d (15.2)
7.63 dd (11.9, 15.2)
6.63 m
6.73 dd (11.0, 16.0)
6.57 m
6.58 m
6.59 m
6.90 m
6.61 m
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24R
24â
25
26R
26â
27
28
NH2
6.70 dd (11.0, 13.8)
6.63 m
6.88 m
6.59 m
6.53 d (14.0)
6.69 d (15.0)
7.05 dd (10.1, 15.0)
6.23 m
6.51 d (14.4)
7.00 dd (11.5, 14.0)
6.40 dd (10.4, 11.5)
5.72 dd (7.6, 10.4)
4.88 br
4.54 ddd (2.4, 2.7, 6.4)
1.94 ddd (2.4, 6.1, 14.3)
2.84 ddd (6.4, 8.2, 4.3)
4.15 ddq (6.1, 6.1, 8.2)
1.37 d (6.1)
7.17 dd (10.7, 14.4)
6.20 dd (11.0, 10.7)
6.17 dd (10.7, 11.0)
6.88 m
5.92 dd (6.4, 15.0)
4.50 m
6.21 m
6.86 dd (10.4, 15.2)
5.89 dd (6.6, 15.2)
4.52 dd (4.0, 6.6)
4.69 ddd (3.1, 7.0, 4.0)
1.84 ddd (3.1, 6.1, 14.3)
2.77 ddd (7.0, 7.6, 14.3)
4.08 m
1.32 d (6.1)
6.99 brs
4.52 m
1.92 ddd (2.4, 6.1, 14.0)
2.77 ddd (6.4, 7.9, 14.0)
4.14 dq (6.4, 7.9)
1.38 d (6.4)
7.44 brs
1′
4.77 br
2′
4.02 br
3′
4′
5′R
5′â
1′′
3.47 t (8.9)
3.61 m
3.21 dd (7.6, 14.4)
3.87 m
5.02 br
3.78 m
3.30 m
3.40 m
3.10 t (10.7)
3.73 m
4.83 brs
3.61 m
3.32 m
3.54 ddd (5.5, 9.5, 9.7)
3.23 dd (9.7, 11.2)
3.87 dd (5.5, 11.2)
2′′
3′′
3.78 m
3.60 m
4′′
3.90 m
3.80 m
5′′R
5′′â
1′′′
2′′′
3′′′
4′′′
5′′′
OMe
3.57 dd (2.7, 12.5)
3.70
5.08 d (4.0)
3.67 m
3.87 m
3.75 m
3.41 m
3.53 m
4.81 d (4.6)
3.71 m
3.61 m
3.61 m
1.21 d (5.8)
1.31 d (6.1)
3.34 s
a
b
CD3OD at 300 K. DMSO-d6 at 300 K.
Chiral GC analysis of the methanolysis products
revealed D configuration for both xylose and arabinose,
and analysis of the Lemieux oxidation/acid hydrolysis
product implied 4S stereochemistry for the tetramic acid
portion. The relative stereochemistry of the terminal
tetrahydrofuran ring was assigned on the basis of
NOESY cross-peaks H-22/H-24â, H-22/CH3-26, H-23/H-
24R, H-23/H-25, H-24R/H-25, and H-24â/CH3-26 (Figure
1), which indicated that H-23 and H-25 were on the same
face of the tetrahydrofuran ring and H-22 and CH3-26
were on the opposite face. Absolute stereochemistry at
C-22 was determined in the same manner as in the case
of 4. Oxidation of 6 with RuO4 yielded 18, which was
converted to the PGME amides 18a and 18b. The ∆δ
values were negative for all of the protons except for the
R-hydrogen (Figure 2), thus revealing that the tetra-
hydrofuran ring in 6 was epimeric to that of 4 at the
carbon attached to the polyene chain.
F igu r e 3. ∆δ values obtained for 5-deoxy-1,2-dimethoxy-
arabinose MTPA ester. ∆δ ) δS(-) - δR(+)
.
Cl. One double bond was missing, and an additional
chlorine atom was present. NMR data suggested that 6
duplicated partial structures including relative stereo-
chemistry of the tetramic acid, sugar, and polyene
portions up to C-17 of 4 (Tables 3 and 4). Six contiguous
olefinic protons (H-18 through H-23) in 4 were replaced
by three mutually coupled olefinic signals. The COSY
spectrum revealed the proton signals attributable to the
terminal tetrahydrofuran ring, but their chemical shifts
and coupling constants were significantly different from
those in 4. HMBC data indicated that C-17 and the
tetrahydrofuran ring were connected via a 1-chlorobuta-
diene unit; a nonprotonated carbon (C-21) at δ 134.3 was
linked to a methine proton at δ 4.81 (H-22) on the
tetrahydrofuran ring.
Rubroside A (1) was smaller by a C2H2 unit than
rubroside D (4). Comparison of COSY, HMQC, and
HMBC data showed that they had the identical structure
except that the ∆22-olefin in 4 was missing in 1. Z
geometry for the ∆20-olefin was deduced from a coupling

2336 J . Org. Chem., Vol. 64, No. 7, 1999
Ta ble 3. 1H NMR Da ta for Ru br osid es C (3), E (5), F (6), a n d H (8)
Sata et al.
3a
5b
6c
8d
4
4.31 br
2.64 m
2.79 dd (4.0, 15.9)
7.21 d (15.0)
7.61 dd (11.3, 15.0)
6.62 m
6.90 m
6.55 m
6.72 m
6.61 m
5.48 br
3.30 br
3.44 dd (3.5, 16.5)
7.89 br
7.60 br
6.45 m
6.66 m
6.46 m
6.56 m
6.56 m
5.35 dd (2.7, 6.5)
3.24 dd (6.5, 15.6)
3.41 dd (2.7, 15.6)
7.89 d (15.3)e
4.78 br
3.16 br
3.45 brd (11.9)
8.12
7.65 dd (11.9, 14.2)
6.43 m
6.61 m
6.43 m
6.54 m
6.54 m
6.86 m
5R
5â
8
9
7.65 dd (11.3, 15.3)e
6.41 dd (14.7, 11.3)e
6.68 dd (11.3, 14.7)e
6.47 dd (13.8, 11.3)e
6.59 dd (9.2, 13.8)e
6.59 dd (13.8, 9.2)e
6.91 dd (11.1, 13.8)e
6.67 d (11.1)e
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24R
24â
25
26
27
28
NH2
6.88 m
6.57 d (11.3)
6.91 m
6.67 d (11.2)
6.63 d (11.2)
6.50 d (14.6)
6.70 m
6.48 dd (10.7, 15.3)
5.86 dd (6.7, 15.3)
4.42 t (6.4)
4.12 q (7.0)
1.82 ddd (7.6, 7.9, 12.8)
2.68 m
6.70 d (15.0)
7.18 dd (10.8, 15.0)
6.83 d (10.8)
6.70 d (14.7)e
6.68 d (15.0)
7.16 dd (11.0, 15.0)
6.80 d (11.0)
7.17 dd (10.9, 14.7)e
6.83 d (10.9)e
4.82 d (6.2)
4.81 d (5.9)
4.82 d (5.8)
4.58 ddd (6.2, 6.9, 7.3)
1.86 ddd (7.3, 8.1, 12.7)
2.57 ddd (6.2, 6.9, 12.7)
4.30 m
4.57 ddd (5.9, 6.4, 8.3)
1.85 ddd (8.3, 8.3, 12.7)
2.56 ddd (6.4, 6.4, 12.7)
4.30 ddq (6.3, 6.4, 8.3)
1.25 d (6.3)
4.57 ddd (6.9, 7.3, 5.8)
1.86 ddd (7.3, 8.1, 12.7)
2.57 ddd (6.9, 6.5, 12.7)
4.31 m
4.27 m
1.26 d (6.4)
1.26 d (6.2)
1.27 d (6.2)
7.60 brs
7.89 brs
7.75 brs
8.31 brs
6.13 d (8.3)
4.72 br
7.56 brs
7.87 brs
5.92
1′
2′
3.74 m
4.55
3′
4′
3.46 t (9.2)
3.61 m
4.30 m
4.15 m
4.17 m
4.22 m
4.17 t (9.2)
4.21 m
5′R
5′â
1′′
2′′
3′′
3.21 t (11.0)
3.87 m
5.02 brd (2.8)
3.77 m
3.77 m
3.91 m
3.75 dd (10.4, 11.6)
4.29 m
5.76 br
4.41 m
4.41 m
3.63 t (10.3)
4.22 m
5.79 d (3.9)
4.44 dd (3.9, 10.0)
4.38 dd (3.4, 10.0)
4.18 m
3.77 t (10.4)
4.30 m
4′′
4.22 m
5′′R
5′′â
1′′′
2′′′
3′′′
4′′′
5′′′
OMe
3.56 dd (2.7, 12.5)
3.68
5.08 d (4.6)
3.64 m
3.89 m
3.72 m
3.98 brd (11.5)
4.23 m
5.28 d (4.6)
4.47 m
4.43 m
4.18 t (6.5)
1.49 d (6.5)
4.05 dd (2.4, 12.7)
4.24 m
5.27 d (4.4)
3.91 dd (4.4, 7.8)
4.41 m
4.12 dd (6.4, 6.8)
1.44 d (6.4)
3.32 s
1.28 d (5.5)
3.33 s
a
b
d
CD3OD at 300 K. C5D5N at 300 K. c C5D5N at 313 K. C5D5N at 308 K. e Coupling constants were determined at 333 K.
constant of 10.4 Hz, whereas the geometry of other olefins
was assigned as E. Relative stereochemistry of the
terminal tetrahydrofuran ring was identical with that of
4 by comparison of the NMR data of the relevant protons.
Rubroside G (7) had a molecular formula of C33H38-
Cl2N2O9, which was significantly smaller than those of
1
the other congeners. It exhibited a H NMR spectrum less
crowded in the region between δ 3.00 and 5.00, whereas
signals in other regions were superimposable on those
of rubroside D. Interpretation of 2D NMR data revealed
the presence of a xylose moiety with an axial anomeric
proton and the absence of the other two sugars. There-
fore, rubroside G is a truncated derivative of rubroside
D.
The HRFAB mass spectrum of rubroside B (2) led to
the molecular formula of C43H54Cl2N2O16, which was one
CH2 unit smaller than that of 4. Absence of an O-methyl
signal in the 1H and 13C NMR spectra and an upfield shift
of C-2′′′(δ 77.5) were consistent with the replacement of
the 2′′′ OMe group in 4 by a hydroxyl group.
Rubroside H (8) showed features similar to those of
rubroside G; the two terminal sugars were missing, and
the remainder was superimposable on that of rubroside
F (6). Thus, 8 is a truncated derivative of rubroside F.
Rubroside C (3) had the same molecular formula as
rubroside A (1). They were different in the terminal
portion; 3 had a tetrahydrofuran ring identical with that
of 6 and (E)-∆20-olefin was assigned by a coupling
constant of 15.3 Hz. The rest of the molecule was
identical, as revealed by NMR data.
The rubrosides not only induced morphological changes
in 3Y1 rat fibroblasts but also were cytotoxic against
P388 murine leukemia cells and antifungal as depicted
in Table 5. Rubrosides induced numerous large vacuoles
and/or spindle-shaped cells in 3Y1 rat fibroblasts, which
may have resulted from disruption of membrane traf-
ficking and/or reduction of the adhesive ability of cells.
Interestingly, hexadecahydrorubroside D (9) was inactive
Rubroside E (5) was smaller by a CH2 unit than
rubroside F (6). Their NMR data were superimposable
except for the absence of the O-methyl signal and an
upfield shift of C-2′′′ in 5, thereby demonstrating that
rubroside E is the 2′′′-des-O-methyl derivative of rubro-
side F.

Rubrosides A-H
J . Org. Chem., Vol. 64, No. 7, 1999 2337
Ta ble 4. 13C NMR Da ta for Ru br osid es A (1), B (2), C (3),
E (5), F (6), G (7), a n d H (8)
in CD3OD; δC 39.5, δH 2.49 in DMSO-d6; δC 123.5, δH 7.19 in
C5D5N). Standard pulse sequences were employed for the 2D
NMR experiments.23 NOESY spectra were measured with a
mixing time (tm) of 500 ms. FAB mass spectra were obtained
1a
2b
3a
5c
6a
7c
8d
1
2
173.8 s 176.5 s
102.1 s
on
a J EOL SX102 spectrometer. Optical rotations were
measured on a J ASCO DIP-371 digital polarimeter. IR spectra
were recorded on a J ASCO FT/IR-5300 Fourier transform
infrared spectrometer.
3
195.6 s 195.0 s
4
5
60.0 d
39.0 t
59.7 d
38.8 t
59.8 d
38.5 t
59.5 d
39.0 t
38.2 t
36.5 t
38.0 t
Collection , Extr a ction , a n d Isola tion . The vermilion
sponge S. japonica (family Theonellidae, order Lithistida) was
collected at a depth of 15 m off Hachijo-jima Island, 300 km
south of Tokyo. The main skeleton was an interlocked mass
of small tetracrepid desmas of 150 µm diameter which were
caltropslike in having the cladi more or less equal in length
and shape. These desmas have characteristic conical spines.
At the surface, there was a thin discontinuous layer of
discotriaenes with diameter 100-140 mm, with rounded or
slightly irregular outline and irregular margins, that had very
short rhabds. Microrhabds formed a thick cover at the surface
and were dispersed in the interior. They were of three sorts:
small thin centrotylote, ca. 18 µm × 0.5 µm; long profusely
spined oxeotes, ca. 35 µm × 4 µm; and thick, almost smooth
spindles, ca. 25 µm × 6 µm. A voucher specimen (ZMAPOR.
13013) was deposited at the Zoological Museum of the Uni-
versity of Amsterdam, The Netherlands. The frozen sponge
(400 g wet wt) was sequentially extracted with EtOH (3 × 1
L). The EtOH extract was concentrated and partitioned
between H2O (500 mL) and Et2O (3 × 500 mL). The Et2O phase
was partitioned between n-hexane and MeOH/H2O (9:1). The
latter phase was partitioned between H2O and n-BuOH. The
active n-BuOH and 90% MeOH soluble portions were combined
to yield a residue (2.5 g), which was flash chromatographed
on ODS with aqueous MeOH. The 90% MeOH eluate was
fractionated by MPLC on ODS with CH3CN/H2O (55:45)
containing 0.05% TFA to yield nine fractions. The second
fraction was separated by C18 reversed-phase HPLC with CH3-
CN/H2O (55:45) containing 0.05% TFA, followed by MeOH/
H2O (85:15) containing 0.05% TFA to afford rubroside A (1,
9.1 mg, 2.3 × 10-3), rubroside B (2, 5.1 mg, 1.3 × 10-3), and
rubroside C (3, 7.0 mg, 1.8 × 10-3). The third fraction was
purified by C18 reversed-phase HPLC with CH3CN/H2O (55:
45) containing 0.05% TFA, followed by MeOH/H2O (85:15)
containing 0.05% TFA to furnish rubroside D (4, 126.3 mg,
3.2 × 10-2) and rubroside E (5, 8.6 mg, 2.2 × 10-3). The fourth
fraction was purified by C18 reversed-phase HPLC with CH3-
CN/H2O (55:45) containing 0.05% TFA and then with MeOH/
H2O (90:10) containing 0.05% TFA to afford rubroside F (6,
108.1 mg, 2.7 × 10-2). The fifth fraction was purified with C18
reversed-phase HPLC with CH3CN/H2O (55:45) containing
0.05% TFA, followed by MeOH/H2O (90:10) containing 0.05%
TFA and then CH3CN/H2O (60:40) containing 0.05% TFA to
yield rubroside G (7, 5.5 mg, 1.4 × 10-3). The sixth fraction
was purified with CH3CN/H2O (55:45) containing 0.05% TFA,
followed by MeOH/H2O (90:10) containing 0.05% TFA to obtain
rubroside H (8, 4.5 mg, 1.1 × 10-3 % wet weight).
6
7
8
9
170.5 s
172.4 s 173.0 s
174.8 s
124.2 d
122.2 d 120.6 d 121.8 d
146.3 d 144.1 d 146.6 d 142.7 d 142.5 d 140.5 d 141.1 d
133.4 d 132.5 d 134.2 d 133.8 d 132.6 d 134.8 d 134.0 d
145.1 d 143.7 d 145.2 d 141.6 d 141.4 d 140.0 d 140.2 d
135.7 d 134.9 d 135.5 d 135.6 d 134.1 d 134.1 d 135.8 d
140.2 d 138.7 d 140.2 d 137.8 d 137.4 d 137.0 d 136.8 d
137.7 d 136.8 d 137.3 d 137.8 d 137.4 d 136.8 d 137.7 d
132.7 d 130.3 d 132.1 d 130.9 d 130.7 d 130.6 d 130.2 d
131.5 d 131.1 d 131.0 d 132.0 d 131.7 d 130.4 d 131.8 d
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
NH2
1′
134.5 s 132.6 s
133.2 s 133.0 s 133.5 s
134.0 d 131.5 d 132.6 d 134.3 d 133.2 d 132.0 d 134.4 d
128.7 d 127.1 d 132.5 d 126.4 d 126.0 d 127.9 d 126.0 d
132.5 d 129.0 d 134.2 d 127.4 d 127.0 d 130.0 d 127.2 d
130.6 d 131.1 d 134.2 d
79.7 d 127.8 d
63.5 d 132.9 d
134.3 s 131.5 d 134.5 s
88.4 d 128.6 d
58.9 d 133.2 d
87.0 d
61.5 d
44.5 t
75.5 d
22.0 q
89.0 d
59.5 d
43.5 t
76.5 d
22.0 q
89.0 d
59.3 d
43.0 t
76.0 d
22.0 q
45.1 t
75.7 d
22.2 q
82.3 d
63.0 d
43.5 t
73.5 d
22.0 q
43.2 t
74.0 d
21.2 q
83.0 d
63.0 d
44.2 t
74.3 d
22.3 q
80.3 d
81.2 d
79.3 d
69.8 d
69.0 t
84.9 d
71.8 d
79.7 d
71.0 d
69.6 t
84.6 d
71.9 d
79.5 d
70.9 d
69.5 t
2′
3′
4′
5′
81.3 d
79.3 d
70.5 d
69.2 t
85.9 d
77.0 d
69.0 d
67.5 t
81.0 d
79.0 d
70.1 d
69.0 t
70.0 d
79.4 d
69.4 t
1′′
2′′
3′′
4′′
5′′
1′′′
2′′′
3′′′
4′′′
5′′′
OMe
103.8 d 102.0 d 104.0 d 104.5 d 103.5 d
71.6 d
70.8 d
76.0 d
61.5 t
98.9 d
87.3 d
79.7 d
79.5 d
20.8 q
58.3 q
69.8 d
68.5 d
73.5 d
60.0 t
98.0 d
77.5 d
77.8 d
79.7 d
20.5 q
71.5 d
70.3 d
76.0 d
61.5 t
70.8 d
70.2 d
76.2 d
61.2 d
71.0 d
69.4 d
76.3 d
61.2 t
99.0 d
86.7 d
78.7 d
78.7 d
20.6 q
57.1 q
98.5 d 102.0 d
87.0 d
79.8 d
79.5 d
20.5 q
58.0 q
77.8 d
74.9 d
79.0 d
21.5 d
CD3OD at 300 K. DMSO-d6 at 300 K. c C5D5N at 300 K.
C5D5N at 308 K.
a
b
d
in all tests, and aurantosides A (10) and B (11) were less
active in 3Y1 rat fibroblast and cytotoxic tests. Rubro-
sides are closely related to aurantosides in having the
common tetramic acid core with a C2 side chain. The
appending trisaccharide moiety and the polyene up to
C-17 were also identical. Aurantosides terminate in
olefins, whereas rubrosides terminate in a tetrahydro-
furan ring. The presence of a terminal tetrahydrofuran
ring at the end of a polyene conjugated with tetramic acid
is reminiscent of erythroskyrin and lipomycin, which
were isolated from Penicillium islandicum21 and Strep-
tomyces aureofaciens,22 respectively. Another interesting
structural feature of the rubrosides is the presence of
Z-olefins in the polyene unit.
R u br osid e A (1). Red amorphous solid. [R]24 -1824°
D
(c 0.001, MeOH); UV (MeOH) λmax 246 (ꢀ 29 100), 418 (110 400),
430 (110 200) nm; HRFABMS (matrix: glycerol) m/z 912.2859
1
(M - H)- (C42H5435Cl2N2O16 ∆ +0.9 mmu); H and 13C NMR
data, see Tables 2 and 4.
R u br osid e B (2). Red amorphous solid. [R]24 -1460°
D
(c 0.001, MeOH); UV (MeOH) λmax 250 (ꢀ 18 900), 432 (71 300),
457 (69 700) nm; HRFABMS (matrix: glycerol) m/z 924.2806
1
(M - H)- (C43H5435Cl2N2O16 ∆ -4.5 mmu); H and 13C NMR
data, see Tables 2 and 4.
R u br osid e C (3). Red amorphous solid. [R]24 -1088°
D
Exp er im en ta l Section
(c 0.001, MeOH); UV (MeOH) λmax 240 (ꢀ 18 600), 450 (60 600)
nm; HRFABMS (matrix: glycerol) m/z 912.2794 (M - H)-
(C42H5435Cl2N2O16 ∆ -5.7 mmu); 1H and 13C NMR data,
seeTables 3 and 4.
Gen er a l Exp er im en ta l P r oced u r es. The NMR spectra
were recorded on either a J EOL R-500 or R-600 spectrometer.
Chemical shifts were referenced to the solvent (δC 49.0, δH 3.30
(23) (a) Summers, M. F.; Marzilli, L. G.; Bax, A. J . Am. Chem. Soc.
1986, 108, 4285-4294. (b) Bax, A.; Subramanians, S. J . Magn. Reson.
1986, 67, 565-569. (c) Bax, A.; Azolos, A.; Dinya, Z.; Sudo, K. J . Am.
Chem. Soc. 1986, 108, 8056-8063.
(21) Beutler, J . A.; Hilton, B. D.; Clark, P. J . Nat. Prod. 1988, 51,
562-566.
(22) Schabacher, K.; Zeeck, A. Tetrahedron Lett. 1973, 2691-2694.

2338 J . Org. Chem., Vol. 64, No. 7, 1999
Ta ble 5. Biologica l Activities of Ru br osid es
Sata et al.
cytotoxicity
antifungal activityb
C. albicans A. fumigatus
morphological
change in 3Y1 cellsa
P388 cells (IC50 µg/mL)
rubroside A (1)
rubroside B (2)
rubroside C (3)
rubroside D (4)
rubroside E (5)
rubroside F (6)
rubroside G (7)
rubroside H (8)
vacuole,spindles
vacuole
vacuole,spindles
vacuole
vacuole,spindles
vacuole
vacuole
no change
no change
no change
no change
0.05
>0.5
0.2
0.2
0.2
11.2
17.8
9.5
11.0
10.6
inactive
10.0
inactive
inactive
inactive
18.0
inactive
inactive
inactive
inactive
inactive
inactive
inactive
inactive
11.3
0.2
>0.5
>0.5
>0.5
>0.5
>0.5
hexadecahydroruboside D (9)
aurontoside A (10)
auroantoside B (11)
11.8
17.2
a
b
Morphological change at 0.1 µg/mL after 24 h. Inhibitory zone (mm) at 2 µg/disk (8 mm).
R u br osid e D (4). Red amorphous solid. [R]24 -1024°
broside D, 15.833, 17.112, 19.025, and 20.545 min; products
from rubroside F, 15.650, 16.795, 19.385, and 20.420 min.
Because the retention times fluctuated, identity of the peaks
was examined by co-injection with the standards. Absolute
configuration of Xyl and Ara from 4 and 6 were both assigned
as D.
D
(c 0.001, MeOH); UV (MeOH) λmax 251 (ꢀ 16 400), 432 (109 100)
nm, UV (0.01 N HCl in MeOH) λmax 359 (23 600), 480 (101 800)
nm, UV (0.01 N NaOH in MeOH) λmax 251 (20 000), 432
(123 600), 453 (112 700) nm; IR (film) 3360 (br), 3190, 2960,
2920, 1660, 1610, 1570, 1520, 1440, 1400, 1135, 1050 (br),
1000, 950, 765, 750 cm-1; HRFABMS (matrix: glycerol) m/z
Lem ieu x Oxid a tion , Acid Hyd r olysis, a n d Ch ir a l GC
An a lysis of Ru br osid es D (4) a n d F (6). To a solution of
rubroside D (ca. 1.0 mg) in H2O (0.1 mL) were added KMnO4
(0.25 mL of 10 mg/mL solution in H2O) and NaIO4 (0.3 mL of
10 mg/mL solution in H2O), and the mixture was stirred at
room temperature for 10 min. The reaction mixture was
centrifuged for 10 min and filtered. The supernatant was
evaporated to afford a residue which was dissolved in 6 N HCl
(1.0 mL), and the mixture was heated at 105 °C for 2 h. After
evaporation of the solvent, the residue was chromatographed
on ODS with H2O and MeOH. The H2O fraction was dissolved
in 10% HCl in MeOH (0.5 mL) and heated at 100 °C for 2 h.
After removal of the solvent in a stream of N2, CH2Cl2 (0.2
mL) and trifluoroacetic anhydride (0.2 mL) were added to the
residue, and the mixture heated at 100 °C for 5 min. The
solvents were removed in a stream of N2, and the residue was
redissolved in CH2Cl2 (0.1 mL). A 2 µL portion was subjected
to GC analysis on a Chirasil-L-Val capillary column (25 m ×
0.25 mm, i.d.): detection, FID; initial temperature 80 °C for 5
min, final temperature 200 °C for 10 min, temperature raised
at 4 °C min-1. Rubroside F was treated in the same way.
Retention times: standard L-Asp (12.600 min), D-Asp (12.940
min); product from rubroside D (12.805 min); product from
rubroside F (12.805 min). Because the retention times fluctu-
ated, identity of the peaks was examined by co-injection with
the standards. Absolute stereochemistry of Asp from 4 and 6
were both assigned as L.
1
937.2883 (M - H)- (C44H5635Cl2N2O16 ∆ -3.7 mmu); H and
13C NMR data, see Table 1.
R u br osid e E (5). Red amorphous solid. [R]24 -1424°
D
(c 0.001, MeOH); UV (MeOH) λmax 243 (ꢀ 17 000), 450 (61 700)
nm; HRFABMS (matrix: glycerol) m/z 932.2304 (M - H)-
(C41H5135Cl3N2O16 ∆ 0.0 mmu); 1H and 13C NMR data, see
Tables 3 and 4.
R u br osid e
F
(6). Red amorphous solid. [R]24 -490°
D
(c 0.001, MeOH); UV (MeOH) λmax 250 (ꢀ 9100), 425 (80 000)
nm, UV (0.01 N HCl in MeOH) λmax 348 (14 500), 468 (70 900)
nm, UV (0.01 N NaOH in MeOH) λmax 250 (10 900), 425
(83 600), 450 (76 400) nm; IR (film) 3350 (br), 3180, 2910, 2840,
1660, 1610, 1575, 1550, 1520, 1440, 1400, 1135, 1070, 1040,
1000, 950, 750 cm-1; HRFABMS (matrix: glycerol) m/z:
1
945.2413 (M - H)- (C42H5335Cl3N2O16 ∆ +3.0 mmu); H and
13C NMR data, see Tables 3 and 4.
R u br osid e G (7). Red amorphous solid. [R]24 -1212°
D
(c 0.001, MeOH); UV (MeOH) λmax 248 (ꢀ 32 400), 435 (154 400),
460 (175 500) nm; HRFABMS (matrix: glycerol) m/z 676.1985
(M - H)- (C33H3835Cl2N2O9 ∆ +3.1 mmu); 1H and 13C NMR
data, see Tables 2 and 4.
R u br osid e H (8). Red amorphous solid. [R]24 -1308°
D
(c 0.001, MeOH); UV (MeOH) λmax 245 (ꢀ 13 700), 440 (44 700)
nm; HRFABMS (matrix: glycerol) m/z 684.1372 (M - H)-
(C31H3535Cl3N2O9 ∆ -3.6 mmu); 1H and 13C NMR data, see
Tables 3 and 4.
Hexa d eca h yd r or u br osid e
D
(9). Colorless oil. [R]24
Acid Meth a n olysis of Ru br osid e D. Rubroside D (8.0 mg,
8.5 × 10-3 mmol) in 10% HCl/MeOH (1.0 mL) was heated at
90 °C for 1 h. After evaporation of the solvent in a stream of
nitrogen, the residue was purified by flash chromatography
on ODS with H2O, 10% MeOH, and 20% MeOH. The 10%
MeOH eluate furnished sugar III (0.8 mg, yield 58%): 1H NMR
(CDCl3) δ 1.31 (3H, d, J ) 6.4 Hz, H-5), 3.37 (3H, s, 2-OMe),
3.40 (3H, s, 1-OMe), 3.63 (1H, d, J ) 2.8, H-2), 4.05 (1H, dq,
J ) 6.7, 4.6, H-4), 4.88 (1H, brs, H-1), 3.70 (1H, dd, J ) 3.1,
4.3, H-3)
D
+920° (c 0.001, MeOH); UV(MeOH) λmax 243 (ꢀ 20 300), 280
(20 300) nm; HRFABMS (matrix: glycerol) m/z 919.4500 (M
- H)- (C44H7235ClN2O16 ∆ -7.0 mmu); 1H NMR (C5D5N at 318
K) δ 4.47 (1H, m, H-25), 3.98 (1H, m, H-27), 3.75 (1H, dt, J )
3.7, 6.5 Hz, H-24), 3.41 (1H, br, H-5â), 3.40 (2H, m, H-8), 3.30
(1H, br, H-5R), 2.58 (1H, ddd, J ) 7.3, 7.3, 14.7, H-26â), 1.87
(1H, m, H-26R), 1.85 (2H, m, H-23), 1.75 (2H, m, H-9), 1.44
(4H, m, H-10 and H-22), 1.34 (1H, d, J ) 6.4, H-28), 1.23 (22H,
brs, H-11 to H-21).
Meth a n olysis a n d Ch ir a l GC An a lysis of Ru br osid es
D (4) a n d F (6). Rubroside D (1.5 mg) in 10% HCl-MeOH
(1.0 mL) was heated at 100 °C for 2 h. After evaporation of
the solvent, the residue was chromatographed on ODS with
H2O and MeOH. The H2O fraction was evaporated and reacted
with trifluoroacetic anhydride (0.2 mL) in CH2Cl2 (0.2 mL) at
100 °C for 5 min. The reaction mixture was dried in a stream
of N2 and redissolved in CH2Cl2 (0.1 mL), and a 2 µL portion
was subjected to GC analysis on a Chirasil-L-Val capillary
column (25 m × 0.25 mm, i.d.): detection, FID; initial
temperature 50 °C for 6 min, final temperature 160 °C for 1
min, temperature raised at 4 °C min-1. Rubroside F was
treated in the same way. Retention times: standard L-Xyl
(16.785, 20.283 min), D-Xyl (16.427, 19.963 min), L-Ara (17.365,
20.325 min), D-Ara (17.385, 20.740 min); products from ru-
(S)-(-)-MTP A Ester of Su ga r III. To a strirred solution
of sugar III (0.4 mg, 4.3 × 10-3 mmol) in dry pyridine (200
µL) was added (R)-(-)-MTPACl (7 µL), and the mixture was
stirred at room temperature. After 1 h, additional (R)-(-)-
MTPACl (7 µL) was added, and the mixture was stirred for 2
h at room temperature. The reaction mixture was purified by
column chromatography on silica gel (hexane/EtOAc, 9:1) to
furnish (S)-(-)-MTPA ester: 1H NMR (CDCl3) δ 7.686 (2H,
m, Ph), 7.508 (3H, m, Ph), 5.238 (1H, dd, J ) 4.6, 6.4 Hz, H-3),
4.875 (1H, d, J ) 4.3, H-1), 4.040 (1H, dq, J ) 4.6, 6.1, H-4),
3.907 (1H, dd, J ) 4.3, 6.4, H-2), 3.541 (3H, s, OMe), 3.428
(3H, s, 1-OMe), 3.250 (3H, s, 2-OMe), 1.495 (3H, d, J ) 6.1,
H-5).
(R)-(+)-MTP A Ester of Su ga r III. Sugar III (0.4 mg, 4.3
× 10-3 mmol) in dry pyridine (200 µL) and (S)-(+)-MTPACl (7

Rubrosides A-H
J . Org. Chem., Vol. 64, No. 7, 1999 2339
µL) were stirred at room temperature for 2 h. The reaction
mixture was purified by column chromatography on silica gel
(hexane/EtOAc, 9:1) to afford (R)-(+)-MTPA ester: 1H NMR
(CDCl3) δ 7.686 (2H, m, Ph), 7.508 (3H, m, Ph), 5.254 (1H, dd,
J ) 4.9, 6.7 Hz, H-3), 4.879 (1H, d, J ) 4.6, H-1), 3.952 (1H,
dd, J ) 4.6, 6.7, H-2), 3.908 (1H, dq, J ) 4.9, 6.4, H-4), 3.528
(3H, s, OMe), 3.431 (3H, s, 1-OMe), 3.387 (3H, s, 2-OMe), 1.477
(3H, d, J ) 6.4, H-5).
yield and 18, ∼1.0 mg, ∼60% yield). 1H NMR of acid 17:
(CDCl3-CD3OD) 4.58 (1H, ddd, J ) 3.0, 4.5, 6.5 Hz, H-3), 4.48
(1H, d, J ) 4.5, H-2), 4.17 (1H, ddq, J ) 6.0, 6.0, 7.5, H-5),
2.68 (1H, ddd, J ) 6.5, 7.5, 15.0, H-4), 1.97 (1H, ddd, J ) 3.0,
1
6.0, 15.0, H-4), 1.41 (1H, d, J ) 6.0, H-6). H NMR of acid 18:
(CDCl3-CD3OD) δ 4.52 (1H, brs, H-2), 4.51 (1H, dd, J ) 4.6,
6.1 Hz, H-3), 4.33 (1H, ddq, J ) 6.1, 6.7, 7.0, H-5), 2.59 (1H,
ddd, J ) 6.1, 6.7, 13.7, H-4), 1.84 (1H, ddd, J ) 4.6, 7.0, 13.7,
H-4), 1.34 (1H, d, J ) 6.1, H-6).
P GME Am id es of (R)- a n d (S)-Tet r a h yd r o-2-fu r oic
Acid s. L-(+)-R-phenylglycine (0.15 g, 1.0 mmol) and D-(-)-R-
phenylglycine (0.15 g, 1.0 mmol) were each separately dis-
solved in 2,2-dimethoxypropane (10 mL). To each solution, was
added concentrated HCl (1 mL), and the mixture was stirred
at room tempetrature for 14 h. After removal of solvent, the
residue was recrystallized from MeOH and Et2O to yield L-(+)-
R-PGME (171.6 mg, 85% yield) and D-(-)-R-PGME (141.4 mg,
70% yield). To a stirred solution of a mixture of (R)-tetrahydro-
2-furoic acid (2.6 mg, 0.022 mmol) and L-(+)-R-PGME (5.0 mg,
0.025 mmol) in DMF (1.0 mL) were successively added PyBOP
(13.0 mg, 0.025 mmol), HOBT (3.4 mg, 0.025 mmol), and
N-methylmorphorine (7.4 mg, 0.073 mmol) at 0 °C. The
mixture was stirred at room temperature for 18 h, and to the
reaction mixture were added benzene (1.5 mL) and ethyl
acetate (3.0 mL). The mixture was successively washed with
1 N HCl (3.0 mL), saturated sodium bicarbonate solution (3.0
mL), and brine (3.0 mL). The organic phase was dried over
Na2SO4, filtered, and concentrated to give a residue which was
chromatographed on silica gel with hexane/EtOAc (2:1, v/v)
to afford (R)-tetrahydro-2-furoyl-L-(+)-R-PGME amide (15a , 5.4
mg, 82% yield) as a colorless oil. Similarly, (R)-tetrahydro-2-
furoyl-D-(-)-R-PGME amide (15b, 5.6 mg, 85% yield) using
D-(-)-R-PGME, and (S)-tetrahydro-2-furoyl-L-(+)-R-PGME amide
(16a , 4.5 mg, 68% yield) and (S)-tetrahydro-2-furoyl-D-(-)-R-
PGME amide (16b, 4.7 mg, 72%yield) were obtained from (S)-
tetrahydro-2-furoic acid, respectively. 1H NMR of 15a : (CDCl3)
δ 7.496 (1H, br, -NHCH(Ph)COOMe), 7.338 (5H, s, -NHCH-
(Ph)COOMe), 5.560 (1H, d, J ) 8.0 Hz, -NHCH(Ph)COOMe),
4.345 (1H, dd, J ) 5.5, 8.5, H-2), 4.022 (1H, ddd, J ) 7.0, 7.5,
14.5, H-5), 3.883 (1H, ddd, J ) 7.0, 7.5, 14.5, H-5), 3.712 (3H,
s, -NHCH(Ph)COOMe), 2.253 (1H, m, H-3), 2.120 (1H, m, H3),
1.920 (1H, m, H-4), 1.913 (1H, m, H-4). 1H NMR of 15b:
(CDCl3) δ 7.586 (1H, br, -NHCH(Ph)COOMe), 7.335 (5H, s,
-NHCH(Ph)COOMe), 5.547 (1H, d, J ) 8.0 Hz, -NHCH(Ph)-
COOMe), 4.384 (1H, dd, J ) 6.0, 8.0, H-2), 3.932 (1H, ddd, J
) 7.0, 8.0, 14.8, H-5), 3.870 (1H, ddd, J ) 7.0, 8.0, 14.8, H-5),
3.712 (3H, s, -NHCH(Ph)COOMe), 2.229 (1H, m, H-3), 1.978
(1H, m, H-3), 1.873 (1H, m, H-4), 1.789 (1H, m, H-4). 1H NMR
of 16a : (CDCl3) δ 7.589 (1H, br, -NHCH(Ph)COOMe), 7.336
(5H, s, -NHCH(Ph)COOMe), 5.545 (1H, d, J ) 8.0 Hz,
-NHCH(Ph)COOMe), 4.385 (1H, dd, J ) 5.8, 8.2, H-2), 3.933
(1H, ddd, J ) 6.1, 8.2, 14.2, H-5), 3.871 (1H, ddd, J ) 7.0, 8.2,
14.2, H-5), 3.712 (3H, s, -NHCH(Ph)COOMe), 2.229 (1H, m,
H-3), 1.985 (1H, m, H-3), 1.873 (1H, m, H-4), 1.789 (1H, m,
P GME Am id es of 17 a n d 18. To a stirred solution of a
mixture of 17 (0.5 mg, 3.0 µmol) and L-(+)-PGME (2.5 mg, 12.0
µmol) in DMF (0.1 mL) were successively added PyBOP (6.5
mg, 12.5 µmol), HOBT (1.7 mg, 12.5 µmol), and N-methylmor-
pholine (3.7 mg, 36.6 µmol) at 0 °C. After the mixture was
stirred at room temperature for 20 h, benzene (1.0 mL) and
ethyl acetate (2.0 mL) were added. The resulting diluted
solution was successively washed with 1 N HCl (2.0 mL),
saturated sodium bicarbonate solution (2.0 mL), and brine (2.0
mL). The organic phase was dried over Na2SO4 and filtered,
and the filtrate was concentrated to furnish a residue which
was chromatographed on silica gel with hexane/EtOAc (2:1,
v/v) to afford L-(+)-R-PGME amide (17a ). Similarly, D-(-)-R-
PGME amide (17b) was prepared, and L-(+)-R-PGME amide
(18a ) and D-(-)-R-PGME amide (18b) were obtained from
3-chloro-5-methyl-tetrahydro-2-furoic acid (18). 1H NMR of
17a : (CDCl3) δ 7.311 (5H, s, -CH(Ph)COOMe), 5.636 (1H, d,
J ) 7.0 Hz, -NHCH(Ph)COOMe), 4.647 (1H, m, H-3), 4.386
(1H, d, J ) 4.0, H-2), 4.278 (1H, m, H-5), 3.677 (3H, s, -CH-
(Ph)COOMe), 2.686 (1H, dm, H-4), 2.055 (1H, m, H-4), 1.498
(3H, d, J ) 6.0, H-6). 1H NMR of 17b: (CDCl3) δ 7.311 (5H, s,
-CH(Ph)COOMe), 5.631 (1H, d, J ) 7.0, -NHCH(Ph)-
COOMe), 4.625 (1H, m, H-3), 4.416 (1H, d, J ) 4.0, H-2), 4.277
(1H, m, H-5), 3.735 (3H, s, -CH(Ph)COOMe), 2.677 (1H, m,
H-4), 2.000 (1H, m, H-4), 1.454 (3H, d, J ) 6.0, H-6). 1H NMR
of 18a : (CDCl3) δ 7.446 (5H, s, -CH(Ph)COOMe), 5.515 (1H,
d, J ) 7.0 Hz, -NHCH(Ph)COOMe), 4.607 (1H, d, J ) 3.0,
H-2), 4.553 (1H, m, H-3), 4.225 (1H, m, H-5), 3.397 (3H, s,
-CH(Ph)COOMe), 2.450 (1H, m, H-4), 1.958 (1H, m, H-4),
1
1.405 (3H, d, J ) 6.0, H-6). H NMR of 18b: (CDCl3) δ 7.440
(5H, s, -CH(Ph)COOMe), 5.531 (1H, d, J ) 7.0, -NHCH(Ph)-
COOMe), 4.684 (1H, m, H-3), 4.569 (1H, d, J ) 3.0, H-2), 4.404
(1H, m, H-5), 3.472 (3H, s, -CH(Ph)COOMe), 2.616 (1H, m,
H-4), 1.963 (1H, m, H-4), 1.433 (3H, d, J ) 6.0, H-6).
F ibr obla st Assa y. 3Y1 rat fibroblasts were cultured as
previously described.9 To each well of a 24-well microplate were
dispensed 1.0 × 104 cells suspended in 500 µL of the culture
medium. After incubation for 24 h, 5 µL of a test solution
dissolved in DMSO or MeOH was added to each well and
morphology of the cells was observed under an Olympus IX-
70 phase contrast microscope at 1 h, 5 h, 24 h, and 5 d after
addition of samples.
Ack n ow led gm en t. We are grateful to Professor P.
J . Scheuer, University of Hawaii, for reading the
manuscript. Thanks are also due to Professor T. Kusumi,
Tokushma Unversity, for his advice in application of the
PGME method, to Dr. K. Furihata, University of Tokyo,
for assistance with NMR measurements, and to Mr. K.
Suzuki, Yamanouchi Pharmaceutical Co. Ltd., for an-
tifungal assays. This work was partly supported by
Grant-in-Aids for Scientific Research from the Ministry
of Education, Science, Culture, and Sports of J apan. A
J SPS Research Fellowship for Young Scientists to
N.U.S. is also acknowledged.
1
H-4). H NMR of 16b: (CDCl3) δ 7.495 (1H, br, -NHCH(Ph)-
COOMe), 7.339 (5H, s, -NHCH(Ph)COOMe), 5.560 (1H, d, J
) 8.0 Hz, -NHCH(Ph)COOMe), 4.346 (1H, dd, J ) 5.5, 8.6,
H-2), 4.023 (1H, ddd, J ) 6.4, 7.9, 14.8, H-5), 3.884 (1H, ddd,
J ) 7.0, 8.2, 14.8, H-5), 3.713 (3H, s, -NHCH(Ph)COOMe),
2.253 (1H, m, H-3), 2.114 (1H, m, H-3), 1.921 (1H, m, H-4),
1.913 (1H, m, H-4).
Oxid a tion of Ru br osid es D (4) a n d F (6). Each sample
[8.4 mg (9.0 µmol) for rubroside D and 4.6 mg (5.0 µmol) for
rubroside F] in CCl4 (320 µL), MeCN (320 µL), and H2O (400
µL) was added to a 25 mM solution of ruthenium trichloride
hydrate (80 µL) and 20 mg of sodium metaperiodate, and the
mixture was stirred vigorously for 1.5 h at room temperature.
The reaction mixture was diluted with H2O (1 mL) and 1 N
HCl (1 mL) and extracted with ethyl acetate (3 × 4 mL). The
organic phase was dried over anhydrous MgSO4, and the
solvent was evaporated. The residue was purified on a short
silica gel column with CHCl3/MeOH/H2O (6:4:1) to yield
3-chloro-5-methyl-tetrahydro-2-furoic acids (17, 1.0 mg, 68%
Su p p or tin g In for m a tion Ava ila ble: Copies of 1H NMR,
13C NMR, COSY, HMQC, HMBC, and mass spectral data for
rubrosides A-H. This material is available free of charge via
the Internet at http://pubs.acs.org.
J O981995V