Anti-tumor promoting effects of sesquiterpenes from Maytenus cuzcoina (celastraceae)

Article abstract of DOI:10.1016/S0968-0896(00)00107-3

Ten sesquiterpenoids (1-10), with a dihydro-β-agarofuran skeleton, were isolated from Maytenus cuzcoina (Celastraceae). Their structures were elucidated on the basis of spectral analysis, including homo- and heteronuclear correlations NMR experiments (COSY, ROESY, HMQC and HMBC), and chemical correlations. The compounds have been tested for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), as a test for potential cancer chemopreventive agents. Compounds 1-3, 6 and 7 showed strong inhibitory effects on EBV-EA induction (100% inhibition at 1000 mol ratio/TPA). Their structure-activity relationship is discussed. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(00)00107-3

Bioorganic & Medicinal Chemistry 8 (2000) 1773±1778
Anti-Tumor Promoting E€ects of Sesquiterpenes from
Maytenus cuzcoina (Celastraceae)
Antonio G. Gonzalez,a Benigna M. Tincusi,a Isabel L. Bazzocchi,a,*
Harukuni Tokuda,b Hoyoku Nishino,b Takao Konoshima,c
Ignacio A. Jimeneza and Angel G. Raveloa
a
Â
Â
Instituto Universitario de Bio-Organica ``Antonio Gonzalez'', Universidad de La Laguna, Avda.
Â
Â
Astrofõsico Francisco Sanchez 2, La Laguna, 38206 Tenerife, Canary Islands, Spain
bKyoto Prefectural University of Medicine, Kamigyo-Ku, Kyoto 602-0841, Japan
cKyoto Pharmaceutical University, Yamashina-Ku, Kyoto 607-8414, Japan
Received 27 January 2000; accepted 21 March 2000
AbstractÐTen sesquiterpenoids (1±10), with a dihydro-b-agarofuran skeleton, were isolated from Maytenus cuzcoina (Celastraceae).
Their structures were elucidated on the basis of spectral analysis, including homo- and heteronuclear correlations NMR experiments
(COSY, ROESY, HMQC and HMBC), and chemical correlations. The compounds have been tested for their inhibitory e€ects on
Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), as a test for potential
cancer chemopreventive agents. Compounds 1±3, 6 and 7 showed strong inhibitory e€ects on EBV-EA induction (100% inhibition at
1000 mol ratio/TPA). Their structure±activity relationship is discussed. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
insect-antifeedant and insecticidal activities.16 Recently,
anti-HIV17 and reversal multidrug-resistence18 sesqui-
terpenes of this type, have been reported. In addition,
dihydro-b-agarofuran sesquiterpenes with antitumor-
promoting activity have been described.19 21
Inhibition of the tumor promotion stage in the multistage
of chemical carcinogenesis have been regarded as the most
promising method for cancer chemoprevention.1 In the
search for cancer chemopreventive agents, the inhibitory
e€ects on Epstein-Barr virus early antigen (EBV-EA)
induction by the tumor promoter, 12-O-tetradecanoyl-
phorbol-13-acetate (TPA), have been conducting as a pri-
mary screening test.2 Recently, studies on antitumor
promoting activities of several natural products,3 6 using
this in vitro assay, which have correlated well with those in
some animal models,7,8 have been reported.
This paper reports the inhibitory e€ects produced by ten
dihydro-b-agarofuran sesquiterpenes (1±10) isolated from
the fruits of Maytenus cuzcoina (Celastraceae) on the
Epstein-Barr virus early antigen (EBV-EA) activation
induced by 12-O-tetradecanoylphorbol-13-acetate (TPA).
The structure of the new compounds (1±8) were deter-
mined on the basis of spectroscopic data, including
1H±13C heteronuclear correlation (HMQC), long-range
correlation with inverse detection (HMBC), and ROESY
NMR experiments. The known compounds 9 and 10 were
identi®ed as eumaytenol22 and euonymine23 by compar-
ison of their spectral with data reported in the literature.
Species of the family Celastraceae have a long history in
traditional medicine.9 As part of an intensive study of the
bioactive metabolites from species of this family, we had
previously reported quinone-methide10,11 and dimeric
triterpenes,12 showing antimicrobial and cytotoxic
activities. On the other hand, sesquiterpene esters, based
on the dihydro-b-agarofuran [5,11-epoxy-5b,10a-eudes-
man-4(14)-ene] skeleton, are chemotaxonomic indicators
of the family,13 and they have attracted a great deal of
interest because of their immunosuppressive,14 cytotoxic,15
Results and Discussion
Repeated chromatography of the n-hexane:Et2O (1:1)
extracts of the fruits of M. cuzcoina on Sephadex LH-20
and Si gel a€orded, in addition to the known compounds
eumaytenol22 and euonymine,23 the new compounds 1±
8 (Fig. 1).
*Corresponding author. Tel.: +34-922-318576; fax: +34-922-318571;
e-mail: ilopez@ull.es
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00107-3

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A. G. Gonzalez et al. / Bioorg. Med. Chem. 8 (2000) 1773±1778
1774
Table 2. 13C NMR (100 MHz) data (d, CDCl3) of 1±8a
Position
1
2
3
4
5
6
7
8
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
70.0 d 70.0 d 72.4 d 70.1 d 70.0 d 69.9 d 68.2 d 68.8 d
68.8 d 68.6 d 67.8 d 69.4 d 68.3 d 68.2 d 68.0 d 68.0 d
42.3 t 42.3 t 44.2 t 42.5 t 42.3 t 41.5 t 42.2 t 42.2 t
69.9 s 69.9 s 70.1 s 70.0 s 69.9 s 69.9 s 69.7 s 69.8 s
91.6 c 91.2 s 91.5 s 91.3 s 91.2 s 91.2 s 90.9 s 91.0 s
79.2 d 79.3 d 79.3 d 79.3 d 79.2 d 79.3 d 77.9 d 78.6 d
48.9 d 48.9 d 48.8 d 48.9 d 48.8 d 48.9 d 48.9 d 49.0 d
31.1 t 31.2 t 31.1 t 31.2 t 31.1 t 31.3 t 34.5 t 34.6 t
72.0 d 72.1 d 72.7 d 72.0 d 72.0 d 72.0 d 70.2 d 70.4 d
51.0 s 51.1 s 51.1 s 51.0 s 51.0 s 51.1 s 54.6 s 54.9 s
84.7 s 84.6 s 84.3 s 84.8 s 84.6 s 84.7 s 84.5 s 84.6 s
25.6 c 25.7 c 25.6 c 25.7 c 25.6 c 25.7 c 25.5 c 25.7 c
29.5 c 29.7 c 29.6 c 29.7 c 29.6 c 29.7 c 29.3 c 29.4 c
25.5 c 25.1 c 25.4 c 25.4 c 25.0 c 25.1 c 24.7 c 24.7 c
21.5 c 21.4 c 21.8 c 21.8 c 21.4 c 21.7 c 65.3 t 65.4 t
C-9
C-10
C-11
C-12
C-13
C-14
C-15
aData are based on DEPT and 1H±13C (HMQC and HMBC) experi-
ments.
1
singlet at d 3.06 in the H NMR spectrum, interchange-
able with D2O, con®rmed the presence of a tertiary
hydroxyl group. All these data indicated that compound
1 was a pentasubstituted dihydro-b-agarofuran sesqui-
1
terpene. In its H NMR spectrum (Table 1) also were
observed an ABX2 system with signals at d 5.71 (1H, m), d
5.49 (1H, d, J=3.5 Hz), d 2.12 and 2.17 (2H, m), assign-
able to protons H-2, H-1 and H-3, respectively, and sig-
nals at d 5.68 (1H, s) and d 4.95 (1H, d, J=6.8 Hz),
assigned to protons H-6 and H-9, respectively. A tertiary
methyl at d 1.57 binding to a quaternary carbon at d
69.9 in the 13C NMR spectrum (Table 2) and three
angular methyls, were also observed. The relative ste-
reochemistry of 1 was established on the basis of the
coupling constants and con®rmed by a ROESY experi-
ment (Fig. 2), showing NOE e€ects between H-1 and H-2
and between H-15 and H-6, H-9 and H-14. The regio-
substitution was established by an HMBC experiment,
showing three-bond correlation between the carboxyl
Figure 1.
Compound 1 showed the molecular formula C32H34O13
by HR-EI/MS. The IR spectrum showed absorption
bands for hydroxyl groups (3547 cm 1) and ester groups
(1721 cm 1). The presence of a band a 235 nm in its UV
spectrum, a fragment at m/z 112 (C4H3OCOOH) in the
EI/MS, and signals for nine aromatic protons between d
6.68 and 8.19 and twelve aromatic carbons in the 1H and
13C NMR spectra, respectively, suggested the presence of
three furoylate groups in the molecule; while a singlet at d
1
signal of the acetate group at d 169.7 and the H signal
at d 5.49 assigned to H-1. All these data established the
structure of 1 as 1a-acetoxy-2a,6b,9b-trifuroyloxy-4b-
hydroxy-dihydro-b-agarofuran.
1
1.75 (3H, s) in the H NMR spectrum and fragments at
60 m/z (CH3COOH) and 42 m/z (CH2ˆCO) in the EIMS
suggested the existence of an acetate group. When 1 was
treated with acetic anhydride in pyridine, the compound
was unaltered, data which together with the presence of a
The structure of compounds 2±4 were elucidated by spec-
tral methods, 1H and 13C NMR studies (Tables 1 and 2),
2D 1H±1H and 1H±13C correlations, ROESY experiment
Table 1. 1H NMR (400 MHz) data (d, CDCl3, J are given in Hz in parentheses) of 1±8
Position
H-1
1
2
3
4
5
6
7
8
5.49 d
(3.5)
5.39 d
(3.5)
5.33 d
(3.3)
5.53 d
(3.6)
5.42 d
(3.6)
5.43 d
(3.6)
5.53 d
(3.4)
5.63 d
(3.4)
H-2
H-6
H-7
5.71 m
5.68 s
2.36 t
(3.1)
5.49 m
5.63 s
2.32 t
(2.9)
4.35 m
5.63 s
2.33 t
(3.1)
5.80 m
5.71 s
2.36 t
(3.0)
5.55 m
5.65 s
2.34 t
(3.2)
5.55 m
5.65 s
2.34 t
(3.2)
5.50 m
6.18 s
2.34 t
5.54 m
6.29 s
2.38 t
(3.3)
(3.1)
H-8
2.17 m,
2.57 m
4.95 d
(6.8)
2.15 m,
2.51 m
4.91 d
(6.7)
2.05 m,
2.52 m
4.94 d
(6.8)
2.22 m,
2.58 m
4.95 d
(6.8)
2.18 m,
2.55 m
4.93 d
(6.8)
2.18 m,
2.52 m
4.93 d
(6.8)
2.33 m,
2.61 m
5.36 d
(7.2)
2.24 m,
2.69 m
5.50 d
(7.2)
H-9
H-15
1.59 s
1.52 s
1.58 s
1.64 s
1.54 s
1.54 s
4.40,4.96
dAB (12.9)
4.46, 4.99
dAB (13.0)

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A. G. Gonzalez et al. / Bioorg. Med. Chem. 8 (2000) 1773±1778
1775
A detailed study of the spectroscopic data of compound
6, which had the molecular formula C32H40O12 (HR-EI/
MS), showed it to be related to 5. The most notable
di€erences being the existence of one 2-methylbutyrate
with signals at d 0.88 t (3H, J=7.4 Hz), 1.12 d (3H,
J=7.0 Hz), 1.61 m (2H) and 2.34 m (1H) in the 1H
NMR spectrum, instead of the signals corresponding to
the propionate group in 5.
Compound 7, with the molecular formula C31H36O14
1
(HREIMS), in a study of its IR, UV, H and 13C NMR
data (Tables 1 and 2), and 2D experiments was shown
to be a dihydro-b-agarofuran sesquiterpene with three
acetate, two furoylate and one tertiary hydroxyl group,
located at 1a, 2a, 4b, 6b, 9b and 15. An HMBC experi-
ment established the regiosubstitution partners and the
relative stereochemistry was resolved by analysis of a
ROESY experiment (Fig. 2).
Figure 2. ROESY experiment of compounds 1±8.
(Fig. 2), and chemical correlations (Scheme 1). Thus,
acetylation and benzoylation of 3 gave two derivatives
(Scheme 1), the spectral data of which were identical to
those of 2 and 4, respectively.
The spectroscopic data of compound 8 and comparison
with these of 7 allowed to determine its structure. Com-
pounds 1±6 and 7±8 have the basic polyhydroxylated
skeletons of 2a,4b-dihydroxy-celorbicol24 and 3-deoxy-
maytol,25 respectively.
Compound 5 showed the molecular formula C30H36O12
by HREIMS. The EIMS contained fragmentation ions
attributable to losses of m/z 18, 60, 74 and 112, suggest-
ing the presence in the molecule of hydroxyl, acetate,
propionate and furoylate groups. This was con®rmed by
1
the H NMR spectrum, showing an acetate methyl at d
Antitumor-promoting activity
1.74 as singlet, a methyl at d 1.13 as triplet and a meth-
ylene at d 2.31 as quartet assigned to a propionate
group, and six aromatic protons between d 6.74 and
8.18, data that were in accordance with the 13C NMR
spectrum. When 5 was acetylated under standard con-
ditions, it a€orded the product unaltered, con®rming
the existence of a tertiary alcohol. The relative stereo-
chemistry of 5 was established by a ROESY experiment,
while an HMBC experiment, showing long-range corre-
lations between the carboxyl signals of the acetate group
at d 169.6 and the propionate group at d 173.1 with the
1H signals at d 5.42 (H-1) and d 5.55 (H-2), respectively,
con®rmed the regiosubstitution in the molecule.
Compounds 1±10 were tested for their inhibitory e€ects
on Epstein-Barr virus early antigen (EBV-EA), induced
by the tumor promoter, TPA, in Raji cells. Their inhi-
bitory e€ects on the activation of the early antigen and
the viability of Raji cells are shown in Table 3.
Compounds 1±3, 6 and 7 have strong anti-tumor pro-
moting activity, even at 10 mol ratio/TPA (100% inhibi-
tory activity at 1000 mol ratio/TPA, and more than 70%
and 25% even at 500 mol ratio/TPA and 100 mol ratio/
TPA, respectively), and preserved high viability of Raji
cells (more than 70% at 10 to 1000 mol ratio/TPA).
Scheme 1.
Table 3. Percentage of Epstein-Barr virus early antigen induction in the presence of compounds 1±10 with respect to a positive controlc
Concentration
(mol ratio/TPA)a
1
2
3
4
5
6
7
8
9
10
1000
0b
0
0
11.7
(60)
42.9
87.1
100
6.3
(70)
34.9
77.9
100
0
0
12.6
(60)
48.0
86.9
100
10.5
(70)
40.2
84.6
100
26.4
(70)
59.6
86.7
100
(70)
30.2
76.9
94.6
(70)
27.4
74.8
93.0
(70)
28.5
76.7
95.8
(70)
32.7
78.0
96.2
(70)
25.9
72.0
91.0
500
100
10
aValues in parentheses represent viability percentages of Raji cells; unless otherwise stated, the viability percentages of Raji cells were more than 80%.
bMol ratio/TPA (32 pmol=20 ng/mL), 1000 mol ratio=32 nmol, 500 mol ratio=16 nmol, 100 mol ratio=3.2 nmol, and 10 mol ratio=0.32 nmol.
cValues represent percentages of EBV-EA induction to the positive control values (100%) (n=3).

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A. G. Gonzalez et al. / Bioorg. Med. Chem. 8 (2000) 1773±1778
1776
Compounds 7 and 8 based on the same 3-deoxy-maytol
skeleton showed, however, a very di€erent inhibitory
activity. Thus, while 7 was the most active of the com-
pounds assayed (9.0% inhibition of induction at 10 mol
ratio/TPA), compound 8 showed an insigni®cant inhibi-
tory activity (87.4% at 1000 mol ratio/TPA). Compound
9 with a skeleton based on 4b-hydroxy-celorbicol,22
showed an inhibitory activity comparable to 8.
Plant material
M. cuzcoina (local name ``paltay-paltay'') was collected
at Huayllabamba-Urquillos, Province of Urubamba,
Cusco (Peru), in December 1993. A voucher (``cuz'' 02765
A.T. 1004 MO) is deposited in the herbarium of Vargas,
Department of Botany, in the National University of San
Antonio Abad de Cusco.
Compounds 2, 1, 3 and 6, with a basic skeleton of
2a,4b-dihydro-celorbicol, showed increasing inhibitory
activity in this order. These results clearly indicated that
the acetoxy and furoyloxy groups increase the inhibition
of Epstein-Barr virus activation for this type of com-
pounds. Furthermore, the substitution of the acetate
group in compound 2 (7.0% inhibition of induction at
10 mol ratio/ATP) for a furoyloxy in 1, an hydroxy in 3
or a methylbutyroyloxy group in 6, decrease progres-
sively the activity (5.4%, 4.2% and 3.8% inhibition of
induction at 10mol ratio/TPA, respectively). On the other
hand, when a benzoyloxy or a propionyloxy group are
present on C-2, as in 4 and 5, respectively, compounds
show less activity. It suggests that the substituent at C-2
plays an important role in the inhibitory activity. Such
strict structural requirements remind us of the speci®c
target(s) of sesquiterpenes being involved in the action
mechanisms of EBV activation,19 and thus deserve fur-
ther mechanistic studies.
Bioassays
Chemicals. The cell culture reagent and n-butyric acid
were purchased from Nacalai Tesque, Inc. (Kyoto,
Japan). 12-O-Tetradecanoylphorbol-13-acetate (TPA)
was obtained from Sigma Chemical Co. (St. Louis, MO).
In vitro EBV-EA induction e€ect. The EBV genome-
carrying lymphoblastoid cells, Raji cells, derived from
Barkitt's lymphoma, were cultivated in RPMI-1640
medium. The Raji cells were incubated for 48 h at 37 ꢀC
in a medium containing n-butyric acid (4 mmol), TPA
(32 pmol), and various amounts of test compounds.
Smears were made from the cells suspension, and the
EBV-EA-inducing cells were stained by means of an
indirect immuno¯uorescence technique. The details of
the in vitro assay on EBV-EA induction have been
reported previously.7,8
Extraction and isolation
It is interesting to note that compound 10, with a macro-
lide bridge showed the lowest inhibitory activity (73.6% at
1000 mol ratio/TPA). It seems that the macrolide bridge
leads to a decrease in inhibitory activities, as was pointed
out by Tokuda et al.,19 and suggests that the size of the
molecule could strongly a€ect the inhibitory activity.
The fruits (400 g) of M. cuzcoina were extracted with n-
hexane:Et2O (1:1) in a Soxhlet apparatus. Removal of
the solvent under vacuum gave 70 g of residue, which
was chromatographed on a Si-gel column, using mix-
tures of n-hexane-EtOAc of increasing polarity as sol-
vent. In this way, after several chromatographies on
Sephadex LH-20 and Si gel, the known compounds
eumaytenol22 and euonymine,23 and the new compounds
1 (26 mg), 2 (620 mg), 3 (393 mg), 4 (58 mg), 5 (61 mg), 6
(123 mg), 7 (54 mg), and 8 (68 mg), were obtained.
The inhibitory activities of these compounds are greater
than those of glycyrrhizin26 and retinoic acid,27 which are
known as typical antitumor promoters, and also stronger
than other dihydro-b-agarofuran sesquiterpenes pre-
viously reported.19 21 From these results, it was concluded
that these sesquiterpenes might be valuable chemopre-
ventive agents, and the fruits of Maytenus cuzcoina
would be e€ective resources of preventive agents.
1ꢀ-Acetoxy-2ꢀ,6ꢁ,9ꢁ-trtifuroyloxy-4ꢁ-hydroxy-dihydro-
ꢁ-agarofuran (1). Amorphous solid; [a]2d5 +34.10 (c 1.0,
CHCl3); UV lmax nm: 235, 221; IR nmax cm 1: 3547, 2928,
1721, 1576, 1508, 1309, 1232, 1161, 768; 1H NMR d: 1.54
(3H, s), 1.56 (3H, s), 1.57 (3H, s), 1.75 (3H, s), 2.12 (1H,
m), 2.17 (1H, m), 3.06 (1H, br s), 6.68 (1H, d, J= 2.0 Hz),
6.75 (1H, d, J=2.0 Hz), 6.84 (1H, d, J=2.0 Hz), 7.44 (3H,
m), 7.95 (1H, s), 8.04 (1H, s), 8.18 (1H, s), for other sig-
nals, see Table 1; 13C NMR d: 20.4 (q); 109.5 (d); 109.7 (d);
109.8 (d); 118.6 (s); 119.2 (2 Â s); 143.7 (d); 143.8 (2Âd);
147.5 (d); 148.5 (d); 149.0 (d); 161.8 (s); 161.9 (s); 162.2 (s);
169.7 (s), for other signals, see Table 2; EI/MS m/z% : 626
(M+, 1); 611 (1); 514 (1); 499 (3); 472 (1) 402 (11); 290 (4);
233 (3); 192 (11); 95 (100); 57 (3); HR-EI/MS: m/z
626.20728 (calcd for C32H34O13, 626.19994).
Experimental
General
IR spectra were recorded in CDCl3 on a Bruker IFS 55
spectrophotometer, and UV spectra were collected in
absolute EtOH on a JASCO V-560.1H and 13C NMR
spectra were recorded on a Bruker at 400 MHz and
100 MHz, respectively. Optical rotations were measured
on a Perkin-Elmer 241 automatic polarimeter, and [a]d
values are given in 10
ꢀcm2/g. CD spectra on a
1
JASCO J-600 spectropolarimeter. EI/MS and HR-EI/
MS were recorded on a Micromass Autospec spectro-
meter. TLC 1500/LS 25 Schleicher and Schuell foils
were used for thin layer chromatography, while silica
gel (0.2±0.63 mm) and Sephadex LH-20 were used for
column chromatography.
1ꢀ,2ꢀ-Diacetoxy-6ꢁ,9ꢁ-difuroyloxy-4ꢁ-hydroxy-dihy-
dro-ꢁ-agarofuran (2). Amorphous solid; [a]2d5 +20.10 (c
2.4, CHCl3); UV lmax nm :235, 221; IR nmax cm 1: 3550,
3029, 2957, 1747, 1712, 1577, 1366, 1309, 1243, 1160,
761; H NMR d: 1.49 (3H, s), 1.50 (3H, s), 1.51 (3H; s),
1.72 (3H, s), 1.98 (1H, m), 2.15 (1H, m), 2.02 (3H, s),
1

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A. G. Gonzalez et al. / Bioorg. Med. Chem. 8 (2000) 1773±1778
1777
2.30 (1H, m), 3.00 (1H, br s), 6.72 (1H, d, J=1.8 Hz),
6.82 (1H, d, J=1.8 Hz), 7.41 (1H, d, J=1.8 Hz), 7.43
(1H, d, J=1.8 Hz), 8.00 (1H, s), 8.16 (1H, s), for other
signals, see Table 1; 13C NMR d: 20.4 (q); 21.1 (q); 109.7
(d); 109.8 (d); 118.7 (s); 119.3 (s); 143.8 (d); 144.0 (d);
148.6 (d); 149.1 (d); 161.9 (s); 162.2 (s); 169.7 (2 Â s), for
other signals, see Table 2; EI/MS m/z%: 574 (M+, 1)
559 (1), 514 (1), 470 (2), 447 (5), 402 (13), 290 (5), 233
(5), 192 (14), 105 (17), 95 (100), 57 (2); HR-EI/MS: m/z
574.20554 (calcd for C29H34O12 574.20503).
235, 221; IR nmax cm 1: 3550, 2970, 2931, 1721, 1577,
1508, 1309, 1232, 1160, 874, 762; 1H NMR d: 1.13 (3H, t,
J=7.6 Hz), 1.51 (3H, s), 1.52 (3H, s), 1.53 (3H, s), 1.74
(3H, s), 1.99 (1H, m), 2.18 (1H, m), 2.31 (2H, m), 6.74
(1H, d, J=1.6 Hz), 6.84 (1H, d, J=1.8 Hz), 7.43 (1H, d,
J=1.6 Hz), 7.45 (1H, d, J=1.8 Hz), 8.03 (1H, s), 8.18
(1H, s), for other signals, see Table 1; 13C NMR d: 9.02
(q), 20.4 (q), 27.8 (t), 109.6 (d), 109.7 (d), 118.6 (s), 119.2
(s), 143.7 (d), 143.9 (d), 148.5 (d), 149.0 (d), 161.8 (s),
162.2 (s), 169.6 (s), 173.1 (s), for other signals, see Table
2; EI/MS m/z (%): 588 (M+, 1), 587 (3), 514 (2), 475 (5),
460 (0.4), 454 (0.4), 402 (27), 290 (5), 233 (6), 192 (21), 105
(4), 95 (100), 57 (8); HR-EI/MS: m/z 587.21143 (calcd for
C30H35O12 587.21285).
1ꢀ-Acetoxy-6ꢁ,9ꢁ-difuroyloxy-2ꢀ,4ꢁ-dihydroxy-dihy-
dro-ꢁ-agarofuran (3). Amorphous solid; [a]2d5 +11.20 (c
0.5, CHCl3); UV lmax nm: 236, 222; IR nmax cm 1: 3520,
3136, 2927, 2855, 1721, 1576, 1508, 1310, 1160, 1077,
874, 762; 1H NMR d: 1.50 (3H, s), 1.53 (3H, s), 1.56 (3H,
s), 1.84 (3H, s), 2.05 (1H, m), 2.18 (1H, m), 2.98 (1H, s),
6.74 (1H, d, J=1.8 Hz), 6.83 (1H, d, J=1.8 Hz), 7.42
(1H, d, J=1.8 Hz), 7.43 (1H, d, J=1.8 Hz), 8.02 (1H, s),
8.18 (1H, s), for other signals, see Table 1; 13C NMR d:
20.7 (q), 109.6 (d), 109.7 (d), 118.7 (s), 119.2 (s), 143.7 (d),
143.9 (d), 148.5 (d), 149.1 (d), 161.9 (s), 162.2 (s), 169.9 (s),
for other signals, see Table 2; EI/MS m/z (%): 518
(M+ 14, 1), 472 (5), 453 (5), 446 (6), 416 (32), 361 (29),
312 (45), 287 (66), 252 (99), 125 (97), 95 (100), 57 (10); HR-
EI/MS: m/z 518.17702 (calcd for C26H30O11 518.17881).
1ꢀ-Acetoxy-6ꢀ,9ꢁ-difuroyloxy-2ꢀ-(2)-methylbutyroy-
loxy-4ꢁ-hydroxy-dihydro-ꢁ-agarofuran (6). Pale yellow
amorphous solid; [a]2d5 +18.30 (c 0.8, CHCl3); UV lmax
nm 238, 222; IR nmax cm 1: 3548, 3136, 2966, 2931,
1720, 1577, 1366, 1308, 1160, 874, 762; 1H NMR d: 0.88
(3H, t, J=7.4 Hz), 1.12 (3H, d, J=7.0Hz), 1.50 (3H, s),
1.51 (3H, s), 1.53 (3H, s), 1.61 (2H, m), 1.73 (3H, s), 1.97
(1H, m), 2.18 (1H, m), 2.34 (1H, m), 3.00 (1H, s), 6.74 (1H,
d, J=1.7 Hz), 6.83 (1H, d, J=1.7Hz), 7.42 (1H, d,
J=1.7Hz), 7.44 (1H, d, J=1.7Hz), 8.02 (1H, s), 8.17 (1H,
s), for other signals, see Table 1; 13C NMR d: 11.5 (q),
16.7 (q), 20.4 (q), 26.5 (t), 42.6 (d), 109.7 (d), 109.8 (d),
118.7 (s), 119.3 (s), 143.8 (d), 144.0 (d), 148.6 (d), 149.1
(d), 161.9 (s), 162.3 (s), 169.6 (s), 175.6 (s), for other
signals, see Table 2; EI/MS m/z (%): 601 (M+ 15, 1),
514 (1), 489 (5), 402 (14), 290 (7), 233 (6), 215 (4), 192
(17), 95 (100), 85 (13), 57 (38); HR-EI/MS: m/z
601.22971 (calcd for C31H37O12 601.22850).
Acetylation of 3. Ac2O (4 drops) was added to compound
3 (5.0 mg) dissolved in pyridine (2 drops), and the mixture
left at room temperature for 16h. EtOH (3Â2 mL) was
added and carried almost to dryness in a rotavapor, and
this process was repeated with CHCl3 (3Â2.0 mL), and
puri®ed by preparative TLC with a mixture of n-hexane:
EtOAc (1:1), to give product 2 (5.0 mg).
1ꢀ,2ꢀ,15-Triacetoxy-6ꢁ,9ꢁ-difuroyloxy-4ꢁ-hydroxy-di-
hydro-ꢁ-agarofuran (7). Amorphous solid; [a]2d5: +23.90
Benzoylation of 3. Compound 3 (5.0 mg) was dissolved
in dry pyridine (0.5 mL) and benzoyl chloride (6 drops),
and some crystals of 4-(dimethylamino)-pyridine were
added under argon atmosphere. The mixture was heated
at 60 ꢀC for 15 h, poured over H2O extracted with
EtOAc, and puri®ed on preparative TLC with a mixture
of n-hexane:EtOAc (1:1) to give 4 (4.5 mg).
1
(c 0.8, CHCl3); UV lmax nm: 234, 220; IR nmax cm
:
1
3549, 2930, 1749, 1722, 1576, 1367, 1309, 1158, 762; H
NMR d: 1.49 (3H, s), 1.54 (3H, s), 1.57 (3H, s), 1.72 (3H,
s), 2.01 (1H, m), 2.11 (3H, s), 2.17 (1H, m), 2.30 (3H, s),
2.93 (1H, br s), 6.74 (1H, d, J=1.8 Hz), 6.81 (1H, d,
J=1.7Hz), 7.43 (1H, d, J=1.8 Hz), 7.44 (1H, d,
J=1.7Hz), 8.02 (1H, s), 8.14 (1H; s), for other signals, see
Table 1; 13C NMR d: 20.3 (q), 21.0 (q), 21.2 (q), 109.7
(2Âd), 128.5 (s), 129.0 (s), 143.8 (d), 143.9 (d), 148.7 (d),
148.9 (d), 161.4 (s), 162.3 (s), 169.3 (s), 169.6 (s), 171.4
(s), for other signals, see Table 2; EI/MS m/z (%): 617
(M+-15, 1), 572 (1), 505 (2), 460 (11), 275 (9), 192 (12),
105 (38), 95 (100), 57 (7); HR-EI/MS: m/z 617.18915
(calcd for C30H33O14 617.18703).
1ꢀ-Acetoxy-2ꢀ-benzoyloxy-6ꢁ,9ꢁ-difuroyloxy-4ꢁ-dihy-
dro - ꢁ - agarofuran (4). Pale yellow amorphous solid;
[a]2d5 +40.90 (c 0.8, CHCl3); UV lmax nm: 231, 217; IR
nmax cm 1: 3544, 2956, 2931, 1747, 1721, 1507, 1395,
1160, 761, 712; 1H NMR d: 1.53 (3H, s), 1.56 (3H, s), 1.61
(3H, s), 1.73 (3H, s), 2.22 (2H, m), 3.06 (1H, s), 6.74 (1H,
d, J=2.0 Hz), 6.83 (1H, d, J=1.8 Hz), 7.43 (3H, m), 7.56
(2H, m), 7.94 (1H, d, J=2.0 Hz), 7.96 (2H, m), 8.10
(1H, s), 8.18 (1H, s), for other signals, see Table 1; 13C
NMR d: 20.5 (q), 109.7 (2Âd), 118.7 (s), 119.2 (s), 128.6
(2Âd), 129.5 (2Âd), 129.9 (s), 132.2 (d), 143.8 (d), 144.0
(d), 148.6 (d), 149.1 (d), 161.9 (s), 162.3 (s), 165.7 (s),
169.8 (s), for other signals, see Table 2; EI/MS m/z (%):
621 (M+ 15, 1), 509 (3), 402 (14), 290 (4), 247 (2), 230
(3), 215 (3), 192 (16), 105 (68), 95 (100), 57 (1), HR-EI/
MS: m/z 621.19492 (calcd for C33H33O12 621.19720).
1ꢀ,2ꢀ,15-Triacetoxy-6ꢁ,9ꢁ-dibenzoyloxy-4ꢁ-hydroxy-
dihydro-ꢁ-agarofuran (8). Amorphous solid; [a]d25
+40.20 (c 0.7, CHCl3); UV lmax nm: 274, 232, 202; IR
1
n
max cm 1: 3552, 2928, 1750, 1716, 1366, 1275, 714; H
NMR d: 1.53 (3H; s), 1.57 (3H, s), 1.59 (3H, s), 1.71 (3H,
s), 2.04 (1H, m), 2.19 (1H, m), 2.10 (3H, s), 3.09 (1H, br s),
7.46 (4H, m), 7.58 (2H, m), 8.05 (2H, m), 8.19 (2H, m), for
other signals, see Table 1; 13C NMR d: 20.2 (q), 21.0 (q),
21.2 (q), 128.3 (2Âd), 128.6 (2Âd), 128.9 (s), 129.7 (s), 130.0
(4Âd), 133.3 (d), 133.5 (d), 165.1 (s), 165.9 (s), 169.1 (s),
169.5 (s), 170.5 (s), for other signals, see Table 2; EIMS
m/z (%): 637 (M+ 15, 2), 592 (1), 515 (4), 470 (15), 275
1ꢀ-Acetoxy-6ꢁ,9ꢁ-difuroyloxy-2ꢀ-propyonyloxy-4ꢁ-
hydroxy-dihydro-ꢁ-agarofuran (5). Pale yellow amor-
phous solid; [a]d25 +17.40 (c 1.8, CHCl3); UV lmax nm:

Â
A. G. Gonzalez et al. / Bioorg. Med. Chem. 8 (2000) 1773±1778
1778
(7), 202 (5), 122 (3), 105 (100), 57 (5); HREIMS: m/z
637.22868 (calcd for C34H37O12 637.22850).
11. Gonzalez, A. G.; Alvarenga, N. L.; Ravelo, A. G.; Baz-
zocchi, I. L.; Ferro, E. A.; Navarro, A. G.; Moujir, L. M.
Bioorg. Med. Chem. 1996, 4, 815.
12. Gonzalez, A. G.; Alvarenga, N. L.; Estevez-Braun, A.;
Ravelo, A. G.; Bazzocchi, I. L.; Moujir, L. M. Tetrahedron
1996, 52, 9597.
Acknowledgements
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568.
15. Kuo, Y.; King, M.; Chen, G.; Chen, H.; Chen, C.; Chen,
L.; Lee, K. J. Nat. Prod. 1994, 57, 263.
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This work has been supported by DGES (Projects
PB96-1039 and PB96-1033) and by a Grant-in-Aid for
Scienti®c Research from the Ministry of Education,
Science, Sports and Culture and the Ministry of Health
and Welfare of Japan.
17. Duan, H.; Takaishi, Y.; Bando, M.; Kido, M.; Imakura,
Y.; Lee, K. Tetrahedron Lett. 1999, 40, 2969.
18. Perez-Victoria, J. M.; Tincusi, B. M.; Jimenez, I. A.; Baz-
zocchi, I .L.; Gupta, M. P.; Castanys, S.; Gamarro, F.;
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