Sesquiterpenes from maytenus jelskii as potential cancer chemopreventive agents

Article abstract of DOI:10.1021/np900476a

Seven new (1-4 and 7-9) sesquiterpenes with a dihydro-β-agarofuran skeleton, along with four known compounds (5, 6, 10, and 11), have been isolated from the leaves of Maytenus jelskii. The structures of the new compounds were elucidated by means of spectroscopic data analysis, including ID and 2D NMR techniques, and their absolute configurations were determined by circular dichroism 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-tetradecanoylphorbol13-acetate (TPA). Compound 10 was found to be an effective antitumor-promoting agent and also showed a potent chemopreventive effect in an in vivo two-stage carcinogenesis model.

Full text of DOI:10.1021/np900476a

J. Nat. Prod. 2010, 73, 127–132
127
Sesquiterpenes from Maytenus jelskii as Potential Cancer Chemopreventive Agents
Nayra R. Perestelo,† Ignacio A. Jime´nez,† Harukuni Tokuda,‡ Hirotaka Hayashi,‡ and Isabel L. Bazzocchi*,†
Instituto UniVersitario de Bio-Orga´nica “Antonio Gonza´lez”, UniVersidad de La Laguna, AVenida Astrof´ısico Francisco Sa´nchez 2, La Laguna,
38206 Tenerife, Canary Islands, Spain, and Department of Complementary and AlternatiVe Medicine, Graduate School of Medical Science,
Kanazawa UniVersity, 13-1 Takara-machi, Kanazawa 920-8640, Japan
ReceiVed August 3, 2009
Seven new (1-4 and 7-9) sesquiterpenes with a dihydro-ꢀ-agarofuran skeleton, along with four known compounds (5,
6, 10, and 11), have been isolated from the leaves of Maytenus jelskii. The structures of the new compounds were
elucidated by means of spectroscopic data analysis, including 1D and 2D NMR techniques, and their absolute
configurations were determined by circular dichroism 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). Compound 10 was found to be an effective antitumor-promoting agent and also showed a potent
chemopreventive effect in an in vivo two-stage carcinogenesis model.
Species of the Celastraceae family have been used for centuries
throughout South America and mainland China as insect repellents
and insecticides in traditional agriculture and for the treatment of
a plethora of medical aliments from stomach complaints and fever
to rheumatoid arthritis and cancer.1,2 Many of the medicinal
properties associated with these species have now been attributed
to a large family of highly oxygenated sesquiterpenoids, based on
the tricyclic dihydroagarofuran skeleton, which are chemotaxonomic
markers for the family.3,4 Due to their structural diversity and range
of biological activities, this intriguing class of natural products are
considered as a “privileged structures”.5
Cancer chemoprevention is defined as the use of specific natural
and synthetic agents to reverse or suppress carcinogenesis and to
prevent the development of invasive cancer.6 In particular, the
inhibition of the promotion stage of carcinogenesis is expected to
be an efficient approach for cancer prevention. Natural products
from plants and their synthetic derivatives play an important role
in developing innovative agents to inhibit the onset of cancer. In
liquid chromatography (VLC), medium-pressure liquid chroma-
fact, several classes of natural products are known to be potential
chemopreventive agents.7,8
Results and Discussion
The EtOH extract of the leaves of M. jelskii was partitioned into
a CH2Cl2-H2O (1:1, v/v) solution. The CH2Cl2 fraction was
subjected to multiple chromatographic steps, involving vacuum-
tography (MPLC), and preparative TLC on silica gel and Sephadex
LH-20, to yield 11 sesquiterpene esters (1-11). The structures of
the new compounds, 1-4 and 7-9, were deduced as described
below.
As part of our search for bioactive metabolites from Celastraceae
species, we have previously reported sesquiterpenes as antitumor
promoters from Celastraceae species.9-11 In continuing research
toward the discovery of naturally occurring cancer chemopreventive
agents, we report herein on the isolation and structure elucidation
of seven new sesquiterpenes (1-4 and 7-9), in addition to four
known compounds (5, 6, 10, and 11), from Maytenus jelskii
Zahlbruchner. This species is a small shrub that grows in the forests
of Bolivia, Peru, and Ecuador and whose chemical investigation
has not been previously reported. The structures of the new
compounds were determined by application of 1D and 2D NMR
techniques, including COSY, HSQC, HMBC, and ROESY experi-
ments. Absolute configurations were determined by CD studies and
chemical correlations. The compounds have been tested for their
antitumor-promoting effects on EBV-EA activation in Raji cells
induced by the tumor promoter TPA (12-O-tetradecanoylphorbol-
13-acetate) as a test for potential cancer chemopreventive effects.
Compound 10, the most active analogue in this in vitro assay, was
further evaluated in an in vivo two-stage carcinogenesis model.
Compound 1 was obtained as a colorless lacquer and showed
the molecular formula C35H40O10 by analysis of its HREIMS (m/z
620.2639, calcd 620.2621). The IR absorption bands at 3524, 1746,
1731, and 1714 cm-1 indicated hydroxy and ester functions, and
the UV spectrum revealed the occurrence of aromatic ring absorp-
tions at 223 and 274 nm. The mass spectrum exhibited peaks
attributable to the presence of methyl (M+ - 15, m/z 605, CH3),
benzoate (M+ - 15 - 122, m/z 483, C6H5COOH), cinnamate (M+
- 15 - 148, m/z 457, C8H7COOH), and acetate (M+ - 15 - 148
- 60, m/z 397, CH3COOH) groups. This was confirmed by the 1H
and 13C NMR spectra (Table 1), which included signals of 10
aromatic and two olefinic protons (δH 6.40-7.75), 12 aromatic and
two olefinic carbons (δC 118.2-144.7), and two carboxyl groups
at δC 164.9 and 165.9, assigned to a benzoyl and a cinnamoyl group.
Signals of two acetyl groups [δH 1.77 (s), δC 20.9 (q), 170.2 (s)
and δH 2.28 (s), δC 20.3 (q), 170.4 (s)] were also observed. When
1 was treated with acetic anhydride in pyridine, compound 1 was
unaltered; this fact together with the presence of a singlet at δH
3.38, interchangeable with D2O, confirmed the presence of a tertiary
hydroxy group. In its 1H NMR spectrum (Table 1) an ABX system
* To whom correspondence should be addressed. Tel: +34-922-318594.
Fax: +34- 922-318571. E-mail: ilopez@ull.es.
† Universidad de La Laguna.
was observed, with signals at δ 5.26 (JAB ) 2.8 Hz), 5.29 (JBA
)
2.8 Hz, JBX ) 11.0 Hz), and 6.24 (JXB ) 11.0 Hz), assignable to
‡ Kanazawa University.
protons H-1ax, H-2ax, and H-3eq, respectively, and two methine
10.1021/np900476a  2010 American Chemical Society and American Society of Pharmacognosy
Published on Web 02/10/2010

128 Journal of Natural Products, 2010, Vol. 73, No. 2
Perestelo et al.
Table 1. 1H-13C NMR (δ, CDCl3, J in Hz in parentheses) Data of Compounds 1-4
1
2
3
4
a
a
a
a
position
δH
δC
δH
δC
δH
δC
δH
δC
1
2
3
4
5
6
6.24 d (11.0)
5.29 dd (2.8, 11.0)
5.26 d (2.8)
67.8 d
68.6 d
75.3 d
69.7 s
89.5 s
32.5 t
6.22 d (11.3)
5.31 dd (2.8, 11.3)
3.84 dd (2.8, 11.0)
68.1 d
70.7 d
77.0 d
70.9 s
92.0 s
32.1 t
6.19 d (10.9)
5.27 dd (3.2, 10.9)
5.25 d (3.2)
68.1 d
68.7 d
75.6 d
69.9 s
89.7 s
32.9 t
6.17 d (11.2)
5.31 dd (2.8, 11.2)
3.85 dd (2.8, 10.9)
68.3 d
70.7 d
77.2 d
70.8 s
92.1 s
32.1 t
1.86 d (12.2)
2.40 dd (3.8, 12.2)
2.04 m
2.12 m
4.82 d (5.0)
1.85 d (12.9)
2.52 m
2.03 m
2.12 m
4.82 d (5.2)
1.86 m (12.2)
2.40 m
2.04 m
2.14 m
4.79 d (5.1)
1.86 d (12.2)
2.53 m
7
8
9
10
11
12
13
14
15
42.4 d
30.3 t
42.2 d
31.0 t
73.5 d
48.1 s
85.3 s
29.8 q
24.2 q
23.8 q
20.2 q
42.6 d
30.7 t
72.5 d
47.9 s
84.2 s
29.8 q
24.2 q
24.6 q
19.7 q
2.02b m
42.2 d
30.9 t
72.9 d
48.1 s
85.4 s
29.8 q
24.1 q
23.8 q
20.2 q
2.02b m
73.0 d
47.5 s
83.7 s
29.8 q
24.0b q
24.0b q
19.7 q
4.84 d (5.4)
1.31 s
1.35 s
1.46 s
1.42 s
1.48 s
1.31 s
1.47 s
1.46 s
1.50 s
1.36 s
1.47 s
1.43 s
1.48 s
1.46b s
1.46b s
1.46b s
a Based on DEPT, HSQC, and HMBC experiments. b Overlapping signals.
Figure 1. ROE effects (solid line) and CD exciton coupling (dashed line) for compounds 1 (left) and 9 (right).
protons at δ 4.82 (d, J ) 5.0 Hz, H-9) and 2.04 (m, H-7). The
geometry of the disubstituted double bond of the cinnamate moiety
was determined as trans by the large vicinal coupling constant value
of the olefinic proton signals at δ 6.40 (d, J ) 16.0 Hz) and 7.48
(overlapping signal). A methyl group at δ 1.46 attached to a carbon
at δ 69.7 bearing a hydroxy group (Me-14), and three methyls at
δ 1.31 (Me-12) and 1.46 (Me-13 and Me-15) were confirmed from
the 13C NMR spectrum. All these data indicated that 1 is a polyester
sesquiterpene with a 1,2,3,4,9-pentasubstituted dihydro-ꢀ-agarofuran
skeleton.
configuration of 1 was established as (1R,2S,3S,4S,5R,7R,9S,10R)-
2,3-diacetoxy-1-benzoyloxy-9-cinnamoyloxy-4-hydroxydihydro-ꢀ-
agarofuran.
The HREIMS of compound 2 gave a molecular ion at m/z
578.2539, corresponding to the molecular formula C33H38O9. Its
1H and 13C NMR data (Table 1) were closely related to those of 1,
except for the absence of the signals assigned to the acetyl group
at C-3 and the shift of the signal corresponding to the H-3 proton
1
from δH 5.26 in 1 to δH 3.84 in 2. Detailed assignments of the H
and 13C NMR data and the relative configuration were determined
on the basis of COSY, HSQC, and HMBC experiments. The
absolute configuration of 2 was established by chemical correlation.
Thus, acetylation of 2 yielded a compound for which the spectro-
scopic data were identical to those of 1.
The regiosubstitution of 1 was determined by a HMBC experi-
ment, showing three-bond correlations between the carboxyl signals
of the acetate groups at δC 170.2 and 170.4 and the resonances at
δH 5.29 (H-2) and 5.26 (H-3), respectively. The carboxyl signal of
the cinnamate group at δC 165.9 was correlated with the signal at
δH 4.82 (H-9), whereas the carboxyl signal of the benzoate group
at δC 164.9 was linked to the resonance at δH 6.24 (H-1). The
relative configuration of 1 was established on the basis of the
coupling constants and confirmed by a ROESY experiment.
Therefore, the J1,2 (11.0 Hz) and J2,3 (2.8 Hz) values indicated a
trans and cis relationship between H-1/H-2 and H-2/H-3, respec-
tively. The ROESY experiment showed ROE effects of H-2 to Me-
14 and Me-15, H-3 to Me-14, and H-9 to Me-15 (Figure 1).
Compounds 3 and 4 were closely related to 1 and 2, respectively.
Consequently, the stereostructure elucidation of these compounds
was greatly aided by the comparison of their spectroscopic data.
Even so, a complete set of 2D NMR spectra (COSY, HSQC, and
HMBC) was acquired for each metabolite in order to gain the
1
complete and unambiguous assignments of the H and 13C NMR
resonances, as listed in Table 1. The same molecular formula and
relative stereochemistry, previously assigned to 1 and 2 were also
confirmed for 3 and 4, respectively, by HREIMS and 2D NMR
ROESY spectroscopy. Their NMR spectra (Table 1) revealed the
absence of the trans-cinnamate double-bond signals and the
presence of signals assignable to a cis-cinnamate double bond [δH
6.03 d, 6.92 d (J ) 12.5 Hz) for 3; δH 6.05 d, 6.96 d (J ) 12.6 Hz)
for 4], suggesting an isomerization of the double bond at the
cinnamate group. Since compounds 3 and 4 have the same basic
polyhydroxy dihydro-ꢀ-agarofuran sesquiterpenoid core as 1, their
absolute configurations were proposed to be the same based on
biogenetic grounds.
The absolute configuration of 1 was resolved by the dibenzoate
chirality method, an extension of the circular dichroism exciton
chirality procedure.12 The angle (73.0°) between the two chro-
mophores (benzoate and cinnamate) was calculated by molecular
mechanics calculations using the PC model.13 Therefore, compound
1 was considered suitable for a CD study, showing a Davidoff-
type split curve with a first positive Cotton effect at 269.8 nm (∆ε
+16.8) and a second negative effect at 227.4 nm (∆ε -15.2), due
to the couplings of the benzoate and cinnamate groups at C-1R
and C-9ꢀ, respectively (Figure 1). Accordingly, the absolute
The structure and absolute configuration of compound 5 was
elucidated on the basis of spectroscopic data analysis, including

Sesquiterpenes from Maytenus jelskii
Journal of Natural Products, 2010, Vol. 73, No. 2 129
Table 2. 1H-13C NMR (δ, CDCl3, J in Hz in parentheses) Data of Compounds 7-9
7
8
9
a
a
a
position
δH
δC
δH
δC
δH
δC
1
2
3
4
5
6
7
8
6.24 d (11.3)
5.36 dd (3.1, 11.3)
5.24 d (3.1)
67.7 d
68.1 d
75.0 d
71.3 s
91.0 s
79.7 d
49.1 d
31.0 t
72.4 d
49.8 s
84.9 s
30.2 q
26.4 q
24.0 q
20.4 q
6.18 d (11.4)
5.33 dd (2.3, 11.4)
3.79 d (2.3)
67.8 d
70.2 d
76.8 d
72.7b s
92.7 s
80.0 d
48.9 d
31.4 t
72.7b d
50.4 s
86.4 s
29.8 q
26.3 q
23.7 q
20.8 q
6.29 d (11.3)
5.34 dd (2.6, 11.3)
3.81 d (2.6)
67.7 d
70.3 d
77.9 d
71.4 s
92.5 s
80.7 d
48.1 d
31.6 t
72.9 d
51.7 s
86.3 s
30.0 q
25.9 q
24.0 q
20.8 q
4.51 s
4.51 d (5.5)
2.19b m
5.76 s
2.34 t (2.5)
2.25 m
2.20b m
2.20b m
4.78 d (6.4)
2.19b m
9
4.76 d (6.4)
5.11 d (6.5)
10
11
12
13
14
15
1.60 s
1.55 s
1.82 s
1.56 s
1.61 s
1.54 s
1.77 s
1.59 s
1.60 s
1.53b s
1.53b s
1.67 s
a Based on DEPT, HSQC, and HMBC experiments. b Overlapping signals.
1D and 2D NMR experiments (COSY, ROESY, HSQC, and
HMBC). This compound, described previously as a semisynthetic
substance,14 has been isolated from a natural source for the first
time.
Table 3. Relative Ratio of Epstein-Barr Virus Early Antigen
Activation in the Presence of Compounds 1, 2, and 5-11a
concentration (mol ratio/TPA)b
compound
1000
500
100
10
Compound 7 gave the molecular formula C35H40O11 (HREIMS),
and its IR spectrum indicated the presence of ester and hydroxy
groups. The 1H and 13C NMR spectra (Table 2) combined with the
mass data were used to establish its structure as that of a dihydro-
ꢀ-agarofuran sesquiterpene polyester containing two acetates, one
1
2
5
6
7
8
9
10
11.8 (60)c
10.9 (60)
8.9 (60)
9.3 (60)
10.1 (60)
5.3 (60)
13.7 (60)
0.0 (60)
7.5 (60)
8.6 (70)
53.2
52.0
39.6
50.1
51.3
35.2
54.2
33.2
36.6
33.3
86.3
86.2
87.4
84.3
85.2
85.2
87.1
77.5
87.6
82.1
100
100
100
100
100
100
100
98.6
100
100
1
benzoate, one cinnamate, and two hydroxy groups. The H NMR
data of 7 were similar to that of 1, except for the presence of an
oxymethine proton at δH 4.51 replacing methylene resonances at
δH 1.86 (d, J ) 12.2 Hz) and 2.40 (dd, J ) 3.8, 12.2 Hz), suggesting
that this compound possesses an additional hydroxy group. The
11
ꢀ-carotene
a Values represent percentages relative to the positive control value
(100%) (n ) 3). b TPA concentration was 20 ng/mL (32 pmol/mL).
c Values in parentheses represent viability percentages of Raji cells.
1
position of the hydroxy group at C-6 was confirmed by H-1H
COSY and HMBC correlations. The regiosubstitution of 7 was
established by a HMBC experiment, showing three-bond correla-
tions between H-2 (δH 5.36) and H-3 (δH 5.24) with the carboxylic
carbons of the two acetate groups at δC 170.1 and 170.3,
respectively, and couplings of H-1 (δH 6.24) with the carboxylic
carbon of the benzoate (δC 164.8) and H-9 (δH 4.78) with the
carboxylic carbon of the cinnamate (δC 165.6) group. The ROESY
experiment enabled the relative configuration of the hydroxy and
ester groups to be determined as 1R, 2ꢀ, 3ꢀ, 4ꢀ, 6ꢀ, and 9ꢀ. The
structure of compound 8 was elucidated by spectroscopic methods,
including HREIMS, IR, UV, and NMR data (Table 2) and
(1R,2S,3S,4S,5S,6R,7R,9S,10R)-2-acetoxy-1,6,9-tribenzoyloxy-3,4-
dihydroxydihydro-ꢀ-agarofuran by CD studies (Figure 1), showing
a curve with a first Cotton effect at 237.0 (∆ε ) +18.2) and a
second one at 219.8 (∆ε ) -5.1).
The known sesquiterpenes 5,14 6,14 10,15 and 1116 were identified
from their spectroscopic data upon comparison with values reported
in the literature. The isolated compounds have the basic polyhy-
droxy dihydro-ꢀ-agarofuran sesquiterpenoid core of 6-deoxyma-
gellanol15 for 1-4 and magellanol15 for 5-11.
1
comparison with those of 7. Thus, the H NMR spectrum of 8
The antitumor-promoting effect of dihydro-ꢀ-agarofuran sesquit-
erpenes9,10 previously reported prompted us to examine the purified
constituents from M. jelskii as potential chemopreventive agents.
Thus, the inhibitory effects of compounds 1, 2, and 5-11 on EBV-
EA activation induced by the tumor promoter TPA in Raji cells,
conducted as a primary screening test in the search for cancer-
chemopreventive agents,17 were assessed (Table 3). Compounds 3
and 4 could not be assessed due to the small amounts obtained.
The evaluated compounds preserved a high viability of Raji cells
(60% at 1000 mol ratio/TPA), indicating that their cytotoxicity
seems to be moderate against these cells. All the compounds tested
showed some degree of inhibitory effect on EBV activation.
Compound 8, with an inhibition value of 94.7% at a 103 mol ratio/
TPA, was also more active than ꢀ-carotene, which has been
intensively studied in cancer chemoprevention using animal mod-
els.8 Moreover, compound 10 exhibited a remarkable biological
effect, proving to be more potent than the control at all the
concentrations assessed.
indicated that it is the 3-deacetyl derivative of 7, since the signal
corresponding to the H-3 proton shifted from δ 5.24 to δ 3.79, and
the signals for the acetyl group at δH 2.32 (s), δC 20.9, and δC
1
170.3 were not observed. Detailed assignments of the H and 13C
NMR signals and the regiosubstitution and relative configuration
were based on 2D NMR experiments. The absolute configurations
of 7 and 8 were determined by chemical correlations. Thus, when
compounds 7 and 8 were acetylated, the previously described
compound 5 was obtained, for which the absolute configuration
has been established by CD studies.14
Compound 9 was obtained as a colorless lacquer. Its molecular
formula was established as C38H40O11 by HREIMS and its 1H and
13C NMR data (Table 2). These values indicated that compound 9
was a 1,2,3,4,6,9-hexasubstituted dihydro-ꢀ-agarofuran sesquiter-
pene. The regiosubstitution was established by the long-range
1H-13C HMBC couplings observed between the H-1 (δH 6.29),
H-6 (δH 5.76), and H-9 (δH 5.11) proton resonances and the
carboxyl signals of the benzoate groups at δC 164.6, 166.1, and
165.3, respectively, whereas H-2 (δH 5.34) was coupled to the
carboxyl signal of the acetate group (δC 170.5). The ROESY
experiment enabled the relative configuration as 1R, 2ꢀ, 3ꢀ, 4ꢀ,
6ꢀ, and 9ꢀ. Its absolute configuration was established as
On the basis of the results of the in vitro assay, we investigated
the effect of compound 10 on mouse skin papillomas in a two-
stage carcinogenesis test, using 7,12-dimethylbenz[a]anthracene
(DMBA) as initiator and TPA as promoter. This model is used

130 Journal of Natural Products, 2010, Vol. 73, No. 2
Perestelo et al.
Experimental Section
General Experimental Procedures. Optical rotations were measured
on a Perkin-Elmer 241 automatic polarimeter in CHCl3 at 25 °C, and
the [R]D values are given in 10-1 deg cm2 g-1. CD spectra were recorded
in MeCN on a JASCO J-600 spectropolarimeter. UV spectra were
obtained in absolute EtOH on a JASCO V-560 instrument. IR (film)
spectra were measured in CHCl3 on a Bruker IFS 55 spectrophotometer.
1H and 13C NMR spectra were performed on a Bruker Avance 400 at
400 and 100 MHz, respectively, and chemical shifts are shown in δ
(ppm) with TMS as an internal reference. EIMS and HREIMS were
recorded on a Micromass Autospec spectrometer. Silica gel 60 (particle
size 15-40 and 63-200 µm) for column chromatography, silica gel
60 F254 for TLC, and nanosilica gel 60 F25 for preparative high-
performance TLC were purchased from Macherey-Nagel, and Sephadex
LH-20 was obtained from Pharmacia Biotech. Compounds used for
CD were purified by high-performance TLC and eluted with a mixture
of n-hexane-EtOAc (6:4).
Plant Material. Maytenus jelskii was collected in the Urubamba
Province, Cuzco, Peru´, in December 2006. A voucher specimen (CUZ
29845) was identified by Professor Alfredo Tupayachi Herrera and
deposited at the Herbarium of Missouri Botanical Garden, St. Louis,
MO.
Extraction and Isolation. The dried leaves (790 g) of M. jelskii
were sliced into chips and extracted with EtOH in a Soxhlet apparatus
for 48 h. Evaporation of the solvent under reduced pressure provided
171.1 g of crude extract, which was partitioned into a CH2Cl2-H2O
(1:1, v/v) solution. The CH2Cl2 (61.2 g) fraction was submitted to VLC
on a silica gel column, using mixtures of n-hexane-EtOAc of increasing
polarity as eluents, to afford nine fractions (I-IX). Fraction VII was
chromatographed on Sephadex LH-20, using as eluent n-hexane-
CHCl3-MeOH (2:1:1), to provide five fractions (A-E). Three fractions
(B-D) were purified further by silica gel flash column chromatography,
eluted in a step gradient manner with CH2Cl2-Me2CO (from 10:1 to
7:3), and further purified by preparative TLC with n-hexane-1,4-
dioxane (6:4), n-hexane-Et2O (2:8), and CH2Cl2-Me2CO (8:2) to give
the new compounds 1 (7.6 mg), 2 (6.5 mg) 3 (1.3 mg), 4 (1.4 mg), 7
(9.2 mg), 8 (10.8 mg), and 9 (21.8 mg), in addition to the known
compounds 5, 6, 10, and 11.
Figure 2. Inhibition of TPA-induced tumor promotion by multiple
application of 10. All mice were initiated with DMBA (390 nmol)
and promoted with 1.7 nmol of TPA, given twice weekly starting
1 week after initiation. (A) Percentage of mice bearing papillomas.
(B) Average number of papillomas per mouse. (b) Control (TPA
alone); (O) TPA + 85 nmol of 10. After 20 weeks of promotion,
the group treated with 10 was significantly different from the
positive control (p < 0.05) on papillomas per mouse.
(1R,2S,3S,4S,5R,7R,9S,10R)-2,3-Diacetoxy-1-benzoyloxy-9-trans-
cinnamoyloxy-4-hydroxydihydro-ꢀ-agarofuran (1): colorless lacquer;
[R]25 +42.4 (c 3.7, CHCl3); CD λext (MeCN) 269.8 (∆ε ) +16.8),
D
241.4 (∆ε ) 0), 227.4 (∆ε ) -15.2) nm; UV λmax (EtOH) (log ε) 274
(4.3), 223 (4.4) nm; IR νmax (film) 3524, 2960, 2926, 2854, 1746, 1731,
since mouse skin proves to be a unique target to differentiate
between the effect of a test agent as either an anti-initiator or an
anti-promoter under appropriate experimental conditions,18 in which
retinoic acid, a well-known inhibitor of tumor promotion, is used
as a positive control.19 As shown in Figure 2A, papilloma-bearing
mice in the positive control group treated with DMBA (390 nmol)
and TPA (1.7 nmol, twice/week) were noted as early as week 6,
with the percentage of papilloma-bearing mice increasing rapidly,
reaching 100% at week 10. In contrast, the percentage of papilloma-
bearing mice in the group treated with 10 (85 nmol) along with
DMBA/TPA was 20% at week 10 and 53.3% at week 15, increasing
to 90% at week 20. These tumor-inhibitory effects were also seen
as a reduction in the number of papillomas in the compound 10-
treatment group. Thus, as demonstrated in Figure 2B, mice treated
with compound 10 developed 3.9 papillomas/mice by week 20,
while the positive control group reached 8.6 papillomas/mice at
the end of the experiment. These results showed that when 10 was
applied before each TPA treatment, the formation of papillomas
on mouse skin was remarkably delayed and the number of
papillomas was significantly reduced.
A preliminary structure-activity relationship study, based on the
results of the in vitro assays, showed the following trends. The
replacement of an acetate by a benzoate group on C-6 decreased
the activity (22.5% in 10 vs 12.9% in 9). In addition, the results
indicated that a substituent at C-9 on the core skeleton is relevant
for the activity, and the presence of a cinnamate group has a
detrimental effect on activity with respect to the presence of a
benzoate group (6 vs 10, 15.7% and 22.5% of inhibition). In general,
we can conclude that the most active compounds from this series
have a magellanol-type basic polyhydroxylated core.
1
1714, 1636, 1224, 1140, 1024, 756, 712 cm-1; H NMR (CDCl3) δ
1.77 (3H, s, OAc-2), 2.28 (3H, s, OAc-3), 3.38 (1H, s, OH-4), 6.40
(1H, d, J ) 16.0 Hz, OCin), OBz, OCin [7.30 (2H, m), 7.43 (4H, m),
7.48 (3H, m), 7.75 (2H, d, J ) 7.2 Hz)], for other signals, see Table
1; 13C NMR (CDCl3) δ 20.3 (q, OAc-3), 20.9 (q, OAc-2), 118.2 (d,
OCin), OBz, OCin [127.9 (2 × d), 128.0 (2 × d), 128.6 (2 × d), 129.1
(2 × d), 129.7 (s), 129.9 (d), 132.7 (d), 134.4 (s)], 144.7 (d, OCin),
164.9 (s, OBz-1), 165.9 (s, OCin-9), 170.2 (s, OAc-2), 170.4 (s, OAc-
3), for other signals, see Table 1; EIMS m/z 620 [M]+ (3), 605 (14),
579 (1), 483 (1), 457 (21), 397 (4), 248 (11), 131 (100), 105 (89);
HREIMS m/z 620.2639 [M]+ (calcd for C35 H40 O10, 620.2621).
(1R,2S,3S,4S,5R,7R,9S,10R)-2-Acetoxy-1-benzoyloxy-9-trans-cin-
namoyloxy-3,4-dihydroxydihydro-ꢀ-agarofuran (2): colorless lac-
quer; [R]25D +112.7 (c 1.6, CHCl3); UV λmax (EtOH) (log ε) 278 (4.8),
223 (4.9) nm; IR νmax (film) 3474, 2931, 1740, 1733, 1716, 1639, 1277,
1241, 1169, 757, 714 cm-1; 1H NMR (CDCl3) δ 1.88 (3H, s, OAc-2),
3.38 (1H, s, OH-4), 3.55 (1H, d, J ) 11.0 Hz, OH-3), 6.49 (1H, d, J
) 16.0 Hz, OCin), OBz, OCin [7.31 (2H, m), 7.45 (5H, m), 7.58 (2H,
m), 7.78 (2H, d, J ) 7.2 Hz)], for other signals, see Table 1;13C NMR
(CDCl3) δ 20.8 (q, OAc-2), 118.6 (d, OCin), OBz, OCin [127.7 (4 ×
d), 128.8 (2 × d), 129.3 (2 × d), 129.8 (s), 130.2 (d), 132.9 (d), 134.6
(s)], 144.8 (d, OCin), 165.0 (s, OBz-1), 166.1 (s, OCin-9), 170.6 (s,
OAc-2), for other signals, see Table 1; EIMS m/z 578 [M+] (22), 563
(4), 545 (4), 503 (1), 456 (1), 430 (4), 396 (2), 265 (4), 248 (6), 131
(100), 105 (97); HREIMS m/z 578.2539 [M+] (calcd for C33H38O9,
578.2516).
(1R,2S,3S,4S,5R,7R,9S,10R)-2,3-Diacetoxy-1-benzoyloxy-9-cis-cin-
namoyloxy-4-hydroxydihydro-ꢀ-garofuran (3): colorless lacquer;
[R]25 +20.4 (c 0.3, CHCl3); UV λmax (EtOH) (log ε) 276 (3.1), 224
D
(3.4) nm; IR νmax (film) 3521, 2928, 2857, 1735, 1635, 1454, 1372,
1
1279, 1144, 1027, 761, 716 cm-1; H NMR (CDCl3) δ 1.78 (3H, s,

Sesquiterpenes from Maytenus jelskii
Journal of Natural Products, 2010, Vol. 73, No. 2 131
OAc-2), 2.25 (3H, s, OAc-3), 3.39 (1H, s, OH-4), 6.03 (1H, d, J )
12.5 Hz, OCin), 6.92 (1H, d, J ) 12.5 Hz, OCin), OBz, OCin [7.13
(2H, d, J ) 7.7 Hz), 7.31 (4H, m), 7.48 (2H, m), 7.83 (2H, d, J ) 7.7
Hz)], for other signals, see Table 1; 13C NMR (CDCl3) δ 20.7 (q, OAc-
2), 21.4 (q, OAc-3), 119.9 (d, OCin), OBz, OCin [128.1 (2 × d), 128.9
(2 × d), 129.8 (2 × d), 130.3 (2 × d), 131.5 (s), 131.9 (d), 133.1 (d),
134.7 (s)], 144.8 (d, OCin), 164.7 (s, OCin-9), 165.4 (s, OBz-1), 170.6
(s, OAc-2), 171.0 (s, OAc-3), for other signals, see Table 1; EIMS m/z
620 [M+] (4), 605 (11), 515 (1), 470 (4), 457 (27), 454 (5), 396 (7),
370 (1), 307 (3), 265 (4), 248 (13), 173 (4), 131 (93), 105 (100);
HREIMS m/z 620.2625 [M+] (calcd for C35 H40 O10, 620.2621).
Acetylation of 2. A mixture of acetic anhydride (2 drops), triethyl-
amine (4 drops), compound 2 (3 mg), and 4-(dimethylamino)pyridine
in dichloromethane (2 mL) was stirred at room temperature for 16 h.
The reaction was quenched by the addition of ethanol (0.5 mL),
followed by stirring for 30 min at room temperature. The mixture was
evaporated to dryness, and the residue was purified by preparative TLC,
using n-hexane-EtOAc (1:1), to give compound 1 (2.6 mg).
OBz [7.28 (2H, m), 7.44 (5H, m), 7.55 (4H, m), 7.96 (2H, d, J ) 7.2
Hz), 8.25 (2H, d, J ) 7.1 Hz)], for other signals, see Table 2; 13C
NMR (CDCl3) δ 23.9 (q, OAc-2), 3 × OBz [128.0 (2 × d), 128.2 (2
× d), 128.7 (2 × d), 129.2 (2 × d), 129.4 (s), 129.5 (s), 129.6 (s),
130.2 (2 × d), 130.3 (2 × d), 132.9 (d), 133.1 (d), 133.5 (d)], 164.6 (s,
OBz-1), 165.3 (s, OBz-9), 166.1 (s, OBz-6), 170.5 (s, OAc-2), for other
signals, see Table 2; EIMS m/z 657 [M - CH3]+ (1), 639 (2), 612 (2),
532 (1), 490 (2), 264 (1), 105 (100); HREIMS m/z 657.2321 [M -
CH3]+ (calcd for C37H37O11, 657.2336).
In Vitro Epstein-Barr Virus Early Antigen Induction Assay.
EBV genome-carrying lymphoblastoid cells (Raji cells, derived from
Burkitt’s lymphoma) were cultivated in 10% fetal bovine serum (FBS)
in RPMI-1640 medium (Nissui). Spontaneous activation of EBV-EA
in the subline Raji cells was less than 0.1%. The inhibition of EBV-
EA activation was assayed using Raji cells (virus nonproducer type)
as described previously.20 The indicator cells were incubated for 48 h
at 37 °C in 1 mL of a medium containing n-butyric acid (4 mmol),
TPA (32 pmol) in dimethylsulfoxide (DMSO) as inducer, and various
amounts of test compounds in 5 µL of DMSO. Smears were made
from the cell suspensions, and the activated cells that were stained by
EBV-EA positive serum from nasopharyngeal carcinoma patients were
detected by an indirect immunofluorescence technique. In each assay,
at least 500 cells were counted, and the number of stained cells (positive
cells) present was recorded. Triplicate assays were performed for each
compound. The average EBV-EA induction of the test compounds was
expressed as a relative ratio to the control experiment (100%), which
was carried out with only n-butyric acid (4 mmol) plus TPA (32 pmol).
EBV-EA induction was around 35%. The viability of treated Raji cells
was assayed by the Trypan Blue staining method.
In Vivo Two-Stage Mouse Skin Carcinogenesis Test. The animals
(specific pathogen-free female ICR mice, 6 weeks old) were divided
into three experimental groups of 15 mice each. The back of each mouse
was shaved with surgical clippers, and each mouse was topically treated
with DMBA (100 µg, 390 nmol) in acetone (0.1 mL) as an initiation
treatment. One week after initiation with DMBA, papilloma formation
was promoted by the application of TPA (1 µg, 1.7 nmol) in acetone
(0.1 mL) twice a week. Groups II and III received a topical application
of compound 10 (85 nmol) in acetone (0.1 mL) 1 h before each
promotion treatment, respectively. The incidence of papillomas was
observed weekly for 20 weeks, and the percentage of mice bearing
papillomas and the average number of papillomas per mouse were
recorded. In our experiment, only typical papillomas larger than about
1 mm in diameter were counted in each case. Details of the in vivo
two-stage carcinogenesis test have been reported previously.19
(1R,2S,3S,4S,5R,7R,9S,10R)-2-Acetoxy-1-benzoyloxy-9-cis-cin-
namoyloxy-3,4-dihydroxydihydro-ꢀ-agarofuran (4): colorless lac-
quer; [R]25D +20.7 (c 0.2, CHCl3); UV λmax (EtOH) (log ε) 275 (3.7),
224 (4.0) nm; IR νmax (film) 3515, 2927, 2857, 1732, 1632, 1604, 1451,
1
1384, 1278, 1165, 765, 715 cm-1; H NMR (CDCl3) δ 1.92 (3H, s,
OAc-2), 3.38 (1H, s, OH-4), 3.53 (1H, d, J ) 10.9 Hz, OH-3), 6.05
(1H, d, J ) 12.6 Hz, OCin), 6.96 (1H, d, J ) 12.6 Hz, OCin), OBz,
OCin [7.16 (2H, d, J ) 7,7 Hz), 7.33 (4H, m), 7.50 (2H, m), 7.87 (2H,
d, J ) 8.3 Hz), for other signals, see Table 1; 13C NMR (CDCl3) δ
20.9 (q, OAc-2), 119.8 (d, OCin), OBz, OCin [127.7 (2 × d), 128.4 (2
× d), 128.9 (d), 129.5 (2 × d), 129.9 (2 × d), 130.2 (s), 133.0 (d),
134.5 (s)], 144.6 (d, OCin), 164.6 (s, OCin-9), 165.1 (s, OBz-1), 170.6
(s, OAc-2), for other signals, see Table 1; EIMS m/z 578 [M+] (15),
552 (10), 545 (4), 430 (6), 370 (2), 322 (6), 279 (2), 248 (7), 149 (25),
131 (22), 105 (100), 57 (27); HREIMS m/z 578.2498 [M+] (calcd for
C33H38O9, 578.2516).
(1R,2S,3S,4S,5S,6R,7R,9S,10R)-2,3-Diacetoxy-1-benzoyloxy-9-
trans-cinnamoyloxy-4,6-dihydroxydihydro-ꢀ-agarofuran (7): color-
less lacquer; [R]25 +111.5 (c 0.9, CHCl3); UV λmax (EtOH) (log ε)
D
280 (4.3), 224 (4.4) nm; IR νmax (film) 3501, 3474, 2963, 2931, 1749,
1733, 1716, 1637, 1260, 1114, 1019, 759, 715 cm-1; 1H NMR (CDCl3)
δ 1.81 (3H, s, OAc-2), 2.32 (3H, s, OAc-3), 3.99 (1H, br s, OH-4),
4.54 (1H, br s, OH-6), 6.40 (1H, d, J ) 16.0 Hz, OCin), OBz, OCin
[7.29 (2H, m), 7.45 (5H, m), 7.55 (2H, m), 7.74 (2H, d, J ) 7.2 Hz)],
for other signals, see Table 2; 13C NMR (CDCl3) δ 20.3 (q, OAc-2),
20.9 (q, OAc-3), 117.9 (d, OCin-9), OBz, OCin [128.0 (2 × d), 128.1
(2 × d), 128.6 (2 × d), 128.8 (s), 129.1 (2 × d), 129.5 (s), 130.0 (d),
132.8 (d), 134.3 (s)], 145.0 (d, OCin-9), 164.8 (s, OBz-1), 165.6 (s,
OCin-9), 170.1 (s, OAc-2), 170.3 (s, OAc-3), for other signals, see
Table 2; EIMS m/z 636 [M+] (1), 621 (3), 603 (19), 561 (2), 488 (2),
481 (1), 473 (7), 428 (1), 356 (2), 306 (1), 264 (3), 131, (100), 105
(81); HREIMS m/z 636.2597 [M+] (calcd for C35H40O11, 636.2571).
(1R,2S,3S,4S,5S,6R,7R,9S,10R)-2-Acetoxy-1-benzoyloxy-9-trans-
cinnamoyloxy-3,4,6-trihydroxydihydro-ꢀ-agarofuran (8): colorless
Acknowledgment. This work has been supported by the Spanish
Grant CTQ2006-13376/BQU. One of the authors (N.R.P) thanks the
Gobierno Auto´nomo de Canarias for a fellowship. We thank Prof. Jesu´s
T. Va´zquez for carrying out the CD experiments. Thanks are also due
to Alfredo Tupayachi for collecting the plant material.
1
Supporting Information Available: H and 13C NMR spectra for
compounds 1-4 and 7-9. This material is available free of charge via
the Internet at http://pubs.acs.org.
lacquer; [R]25 +96.5 (c 0.6, CHCl3); UV λmax (EtOH) (log ε) 280
D
(4.5), 223 (4.6) nm; IR νmax (film) 3447, 2925, 2854, 1733, 1637, 1273,
1163, 1111, 979, 756, 713 cm-1; 1H NMR (CDCl3) δ 1.91 (3H, s, OAc-
2), 4.20 (1H, s, OH-4), 5.15 (1H, d, J ) 5.5 Hz, OH-6), 6.45 (1H, d,
J ) 16.0 Hz, OCin), OBz, OCin [7.30 (2H, m), 7.41 (5H, m), 7.54
References and Notes
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(2H, m), 7.75 (2H, d, J ) 7.1 Hz)], for other signals, see Table 2; 13
C
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Acetylation of 7 and 8. When compounds 7 (3.8 mg) and 8 (3.2
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(1R,2S,3S,4S,5S,6R,7R,9S,10R)-2-Acetoxy-1,6,9-tribenzoyloxy-3,4-
dihydroxydihydro-ꢀ-agarofuran (9): colorless lacquer; [R]25D +57.0
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219.8 (∆ε ) -5.1) nm; UV λmax (EtOH) (log ε) 223 (4.3) nm; IR νmax
(film) 3526, 2926, 2855, 1732, 1715, 1277, 1111, 1025, 757, 710 cm-1
;
1H NMR (CDCl3) δ 1.89 (3H, s, OAc-2), 3.91 (1H, s, OH-4), 3 ×

132 Journal of Natural Products, 2010, Vol. 73, No. 2
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NP900476A