Activity of lupane triterpenoids from Maytenus species as inhibitors of nitric oxide and prostaglandin E2

Article abstract of DOI:10.1016/j.bmc.2005.10.063

In the present study, we report that three new lupane triterpenes (1-3), in addition to 16 known ones (4-19), were isolated from the root bark of Maytenus cuzcoina and the leaves of Maytenus chiapensis. Their structures were elucidated by spectral analysis, including homonuclear and heteronuclear correlation NMR experiments (COSY, ROESY, HSQC, and HMBC). The natural compounds and derivatives 6a, 6b, 9a, and 9b have been tested for potential anti-inflammatory activity, and several compounds including 3-epicalenduladiol (2), 11α-hydroxy- glochidone (3), rigidenol (6), acetoxy-rigidenol (6a), 11α-acetoxy-30- chloro-3-oxo-lup-20(29)-ene (6b), betulin (9), 28-acetoxy-betulin (9a), epibetulin (12), epibetulinic acid (13), and betulonic acid (16) exhibited potent inhibitory effects on NO and prostaglandin E₂ production in mouse macrophages (RAW 264.7) stimulated with bacterial endotoxin. The structure-activity relationship is discussed in detail.

Full text of DOI:10.1016/j.bmc.2005.10.063

Bioorganic & Medicinal Chemistry 14 (2006) 1573–1579
Activity of lupane triterpenoids from Maytenus species as
inhibitors of nitric oxide and prostaglandin E2
a
a
a
b
´
´
Carolina P. Reyes, Marvin J. Nu´nez, Ignacio A. Jimenez, Jeroˆme Busserolles,
˜
b
a,
*
´
Marıa J. Alcaraz and Isabel L. Bazzocchi
aInstituto Universitario de Bio-Orga´nica Antonio Gonza´lez, Universidad de La Laguna and Instituto Canario de Investigacio´n
del Ca´ncer, Avda. Astrofı´sico Francisco Sa´nchez 2, La Laguna, 38206 Tenerife, Canary Islands, Spain
bDepartamento de Farmacologı´a, Universidad de Valencia, Facultad de Farmacia, Av. Vicent Andre´s Estelle´s s/n, Valencia, Spain
Received 8 April 2005; accepted 5 October 2005
Available online 5 December 2005
Abstract—In the present study, we report that three new lupane triterpenes (1–3), in addition to 16 known ones (4–19), were isolated
from the root bark of Maytenus cuzcoina and the leaves of Maytenus chiapensis. Their structures were elucidated by spectral anal-
ysis, including homonuclear and heteronuclear correlation NMR experiments (COSY, ROESY, HSQC, and HMBC). The natural
compounds and derivatives 6a, 6b, 9a, and 9b have been tested for potential anti-inflammatory activity, and several compounds
including 3-epicalenduladiol (2), 11a-hydroxy-glochidone (3), rigidenol (6), acetoxy-rigidenol (6a), 11a-acetoxy-30-chloro-3-oxo-
lup-20(29)-ene (6b), betulin (9), 28-acetoxy-betulin (9a), epibetulin (12), epibetulinic acid (13), and betulonic acid (16) exhibited
potent inhibitory effects on NO and prostaglandin E2 production in mouse macrophages (RAW 264.7) stimulated with bacterial
endotoxin. The structure–activity relationship is discussed in detail.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
Stimulation of macrophages with bacterial lipopolysac-
charide (LPS) or cytokines causes the expression of
inducible nitric oxide synthase (iNOS) and cyclooxygen-
ase-2 (COX-2) with the consequent generation of large
quantities of NO and prostaglandins (PGs).10 A variety
of agents induce iNOS producing high concentrations of
NO which contribute to the antimicrobial and antitu-
mor action of macrophages.11 Nevertheless, a high pro-
duction of NO contributes to tissue injury as occurs in
chronic inflammatory diseases.12 Interaction of NO with
superoxide anion produces the reactive species peroxyni-
trite which can lead to energy depletion and modifica-
tion of proteins, lipids, carbohydrates, and nucleic
acids. Thus, NO and related species have been implicat-
ed in atherosclerosis, arthritis, endotoxemia, and other
pathologies.13 COX activity catalyzes the rate-limiting
step in prostanoid synthesis. The inducible COX-2
isozyme plays an important role in inflammation, pain,
fever, and cancer,14 whereas the pathogenesis of gas-
trointestinal damage by non-steroidal anti-inflammato-
ry drugs has been mainly related to the inhibition of
the constitutive isoform COX-1.15 COX-2 is overex-
pressed in several human cancers and possesses proan-
giogenic as well as procarcinogenic effects.16 Thus,
inhibitors of NO and PG production in macrophages
Species of the family Celastraceae have a long history in
the traditional medicine.1 Over the last 30 years or so, a
large number of secondary metabolites exhibiting a wide
range of bioactivity have been extracted from species
of the genus Maytenus, including a large number of
dihydro-b-agarofuran sesquiterpenoids,2 phenolic and
quinone–methide triterpenes,3, and a diverse array of
triterpenoids.
Triterpenoids from the Celastraceae belonged to the
lupane, oleanane, friedelane, taraxerane, glutinane,
ursane, dammarane, and baccharane series.4 Lupane
triterpenoids are pentacyclic compounds with 30 carbon
atoms, biosynthetically derived from the cyclization of
squalene, and a vast class of natural products whose
structural diversity includes a wide array of functional
groups.5 Compounds of this class are reported to be bio-
active with cytotoxic,6 antitumor-promoting,7 antiviral,8
and anti-inflammatory activities.9
*
Corresponding author. Tel.: +34 922 318594; fax: +34 922 318571;
e-mail: ilopez@ull.es
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.10.063

1574
C. P. Reyes et al. / Bioorg. Med. Chem. 14 (2006) 1573–1579
are potential anti-inflammatory or cancer chemopre-
ventive drugs.
28-diacetoxy-betulin (9b)22 by comparison of their spec-
tral data with values reported in the literature.
As part of an intensive investigation on the biological
activities of natural products from plants of the family
Celastraceae, we have isolated 19 lupane triterpenes
(1–19) (Fig. 1) from the root bark of Maytenus cuzcoina
and the leaves of Maytenus chiapensis. The natural
compounds and derivatives 6a, 6b, 9a, and 9b were
investigated for their pharmacological activity as
inhibitors of NO and PGE2 production in macrophages.
The structures of the new compounds (1–3) and the
derivative of rigidenol, 11a-acetoxy-30-chloro-3-oxo-
lup-20(29)-ene (6b), were determined on the basis of
spectroscopic data, including 1H–13C heteronuclear
correlation (HSQC), long-range correlation with inverse
detection (HMBC), and ROESY experiments. The
known compounds (4–19, 6a, 9a, and 9b) were identified
as 28-hydroxy-3,20-dioxo-29-norlupane (4),17 nepeticin
(5),18 rigidenol (6),18 glochidone (7),19 lupeol (8),19 betulin
(9),17 betulinic aldehyde (10),20 betulin 3-caffeate (11),21
epibetulin (12),17 epibetulinic acid (13),20 lupenone
(14),19 betulone (15),17 betulonic acid (16),17 3-epiglochi-
diol (17),19 glochidiol (18),19 glochidonol (19),19 acet-
oxy-rigidenol (6a),18 28-acetoxy-betulin (9a),22 and 3,
2. Results and discussion
Repeated chromatography of the n-hexane/Et2O (1:1)
extract of the root bark of M. cuzcoina and the CH2Cl2
extract of the leaves of M. chiapensis on silica gel and
Sephadex LH-20 yielded three new lupane triterpenoids
(1–3).
Compound 1 was assigned the molecular formula
C30H50O2 by HREIMS. The IR spectrum suggested
that it contained hydroxyl groups (3413 cmꢀ1) and a
terminal double bond (2926, 1731, and 883 cmꢀ1).
1
The H NMR spectrum (Table 1) showed six methyl
groups [dH 0.77, 0.85, 0.94, 0.97, 1.03, 1.05 (each
3H, s)], an isopropenyl group [dH 1.68 (3H, s), 4.58
(1H, br s), and 4.71 (1H, br s)], and two oxymethine
protons geminal to hydroxyl groups [dH 3.36 (1H, t,
J = 7.3 Hz) and 3.92 (1H, dt, J = 5.3, 10.7 Hz)], and
a typical lupene Hb-19 proton signal dH 2.35 (1H,
dt, J = 4.9, 10.8 Hz). All these data indicate that
compound 1 is a two-substituted lupane triterpene.
29
30
20
19
22
HO
25
R
12
18
14 16
28
1
9
OH
R2
R1
3
27
5
7
HO
O
24
23
1 R1= H, R2= OH
5 R1= OH, R2= H
2
3 R= OH
7 R= H
O
R2
R1
R2
CH2OH
R1
O
O
4
8
R1= OH, R2= CH3
6
R1= OH, R2= CH3
9
R1= OH, R2= CH2OH
6a R1= OAc, R2= CH3
10 R1= OH, R2= CHO
6b R1= OAc, R2= CH2Cl
11 R1= OCaf, R2= CH2OH
9a R1= OH, R2= CH2OAc
9b R1= OAc, R2= CH2OAc
HO
R
R
R2
R1
HO
O
14 R= CH3
15 R= CH2OH
16 R= COOH
12 R= CH2OH
13 R= COOH
17 R1= OH, R2= H
18 R1= H, R2= OH
19 R1= R2= O
O
O
OCaf :
OH
OH
Figure 1. Chemical structures of compounds 1–19 isolated from Maytenus chiapensis, Maytenus cuzcoina, and the derivatives 6a, 6b, 9a, and 9b.

C. P. Reyes et al. / Bioorg. Med. Chem. 14 (2006) 1573–1579
Table 1. 1H NMR (400 MHz) data (d, CDCl3, J are given hertz in parentheses) of 1–4
1575
H
1
2
3
4a
1
2.30, 1.47
1.22 dd (12.4, 17.2)
1.38 dd (6.4, 12.4)
1.53 dd (6.0, 12.4)
1.93 dt (6.0, 18.8)
3.38 t (2.6)
1.20
8.30 d (10.4)
1.00, 1.52
2
1.97, 1.48
5.71 d (10.4)
1.35, 2.36
3
5
3.36 t (7.3)
1.20
1.28
1.55
1.10b
1.25
1.45
1.16
1.17
1.37
1.40
0.96
6
1.41b
1.35,1.90
1.44
1.75
7
1.32
1.47
1.41b
1.40b
9
11
12
13
15
3.92 dt (5.3, 10.7)
1.18, 1.99
1.77 t (10.9)
1.02, 1.56
1.20, 1.43
1.05, 1.69
1.65
4.85 dt (4.8, 10.7)
1.98
1.90 t (10.9)
1.24, 1.40b
1.31 d (5.0)
1.56 dd (8.0, 13.6)
3.60 dd (4.7, 11.2)
1.40mb
16
18
19
21
22
23
24
25
26
27
28
29
1.45, 2.28
1.42
1.45b, 1.56
1.45b
1.10b, 1.98
2.25 t (11.7)
2.31 m
1.90
2.35 dt (4.9, 10.8)
1.34, 1.92
1.25, 1.40
0.94 s
2.50 dt (5.6,10.4)
1.40b, 1.98
1.28, 1.65 t (10.4)
0.93 s
2.40 dt (4.9, 10.8)
1.40b, 2.01
1.31, 1.40b
1.12 sb
1.23, 2.10
1.07 s
0.99 s
0.85 s
1.05 s
0.82 s
0.84 s
1.12 sb
1.26 s
1.11 s
0.73 s
0.89 s
1.03 s
0.97 s
0.77 s
1.04 s
1.00 s
0.98 s
0.80 s
0.93 s
2.98, 3.55 dAB (10.6)
1.84 s
0.79 s
4.59 br s
4.58 br s
4.71 br s
1.68 s
4.61 br s
4.73 br s
1.70 s
4.68 br s
1.68 s
30
a C6D6.
b Overlapping signals.
Table 2. 13C NMR (100 MHz) data (d, CDCl3) of 1–4a
Its 13C NMR spectrum (Table 2) revealed 30 carbon
signals, which were assigned by DEPT experiments
as seven methyls, nine methylenes, five methines, five
quaternary carbons, two alcoholic methines, and two
olefinic carbons. The two sp2 carbons observed at dC
150.2 (s) and 109.8 (t) in the 13C NMR spectrum con-
firmed the D20,29-functionality of a lupane skeleton.
The positions of the two hydroxyl groups were deter-
mined through spectral data. Thus, the multiplicity
and 1H NMR shift of the oximethine group at dH
3.36 agree with those of the biogenetic hydroxyl group
at the C-3 position and were confirmed by an HMBC
experiment in which the protons of the geminal meth-
yl groups (dH 0.94, 0.85) at C-4 showed 2J correla-
tions with C-4 (dC 37.8) and 3J interactions with
C-3 (dC 75.9) and C-5 (dC 48.9). An HMBC experi-
ment established the regiosubstitution of the second
hydroxyl group at C-11 since 2J correlations of the
oxymethine proton signal (dH 3.92) to C-9 (dC 55.6)
and C-12 (dC 37.6), and 3J correlations to C-13 (dC
37.1) and C-10 (dC 39.1), were observed.
C
1
2
3
4
1
35.4 t
25.6 t
75.9 d
37.8 s
48.9 d
18.3 t
35.1 t
42.7 s
55.6 d
39.1 s
70.6 d
37.6 t
37.1 d
42.7 s
27.3 t
35.4 t
43.0 s
47.7 d
47.7 d
150.2 s
29.8 t
39.8 t
22.3 q
28.7 q
16.2 q
17.2 q
14.6 q
18.0 q
109.8 t
19.3 q
33.3 t
25.2 t
76.2 d
37.5 s
49.0 d
18.2 t
34.1 t
41.1 s
49.7 d
37.2 s
20.7 t
24.7 t
37.2 d
44.2 s
36.8 t
77.0 d
48.6 s
47.7 d
47.6 d
150.0 s
29.9 t
37.7 t
28.2 q
22.1 q
15.9 q
16.0 q
16.3 q
11.7 q
109.8 t
19.3 q
123.6 d
165.1 d
205.3 s
45.0 s
52.9 d
18.9 t
34.5 t
42.9 s
48.9 d
40.6 s
70.6 d
37.3 t
37.1 d
42.8 s
27.3 t
35.3 t
43.0 s
47.5 d
47.7 d
150.2 s
29.7 t
39,8 t
21.4 q
28.2 q
19.9 q
17.4 q
14.4 q
18.0 q
113.5 t
19.3 q
39.3 t
33.5 t
214.9 s
46.9 s
54.6 d
19.6 t
33.8 t
40.7 s
49.5 d
36.6 s
21.2 t
27.6 t
36.3 d
42.5 s
27.3 t
34.1 t
47.8 s
49.0 d
51.7 d
209.4 s
27.1 t
29.0 t
26.6 q
21.0 q
14.5 q
15.6 q
15.7 q
60.1 t
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
The relative stereochemistry of the oxymethine proton
at the C-3 position was established as b, on the basis
of the coupling constants and confirmed by ROESY
experiment, showing NOE effects with the Me-23 and
Me-24 resonating at dH 0.94 and 0.85, respectively,
which indicated an equatorial position for H-3 on the
b-face and an axial orientation for the hydroxyl group
on the a-face. Similarly, the relative stereochemistry of
the hydroxyl group at C-11 was assigned as a on the
28.8 q
a Data are based on DEPT, HSQC, and HMBC experiments.

1576
C. P. Reyes et al. / Bioorg. Med. Chem. 14 (2006) 1573–1579
basis of the coupling constants and confirmed by a
ROESY experiment, showing a correlation between
the oxymethine proton at d 3.92 and the protons of
the methyl group at C-26 (dH 1.03). All of these data
and comparison with those found in the literature for
nepeticin (5)18 established the structure of 1 as 3-
epinepeticin.
The isolated lupane triterpenes 1–19 and the derivatives
(6a, 6b, 9a, and 9b) were tested for their inhibitory activ-
ity on the production of NO (measured as nitrite) and
PGE2 in mouse macrophages (RAW 264.7), as a preli-
minary screening assay system to find new anti-inflam-
matory compounds. Stimulation of RAW 264.7
macrophages with LPS (1 lg/mL) for 18 h induced
iNOS and COX-2. Non-stimulated cells generated
175.3 18.5 ng/mL nitrite and 4.4 0.9 ng/mL PGE2.
Control cells were incubated with vehicle and LPS for
18 h, and generated 1628.3 86.8 ng/mL nitrite and
31.1 3.1 ng/mL PGE2. The compounds were tested in
this system at concentrations not significantly affecting
cell viability (Table 3). As expected, the reference drugs
1400 W (iNOS inhibitor) and NS 398 (COX-2 inhibitor)
potently decreased the production of nitrite and PGE2,
respectively. Several lupane triterpenes inhibited both
NO and PGE2 generation, with some differences in
potency and IC50 values in the micromolar range (Table
3). The most effective compounds were 2, 3, 6, 9, 12, 13,
and 16. In addition, 8, 11, and 18 preferentially inhibited
the generation of PGE2.
The structure of compound 2 with the same molecular
weight as 1 was elucidated by spectral methods (Ta-
bles 1 and 2), showing it was related to 1, the differ-
ence being the presence of a secondary hydroxyl
group at C-16 (dH 3.60, dd, J = 4.7, 11.2 Hz) instead
of C-11 (dH 3. 92) as in 1. This difference was well
2
established by an HMBC experiment showing J cor-
relations of the oxymethine proton signal at dH 3.60
to C-17 (dC 48.6) and 3J interactions of the carbons
C-14 (dC 44.2), C-18 (dC 47.7), and C-28 (dC 11.7).
The relative stereochemistry of the oxymethine proton
at C-16 was assigned as a since NOESY correlations
with the signals corresponding to Me-27 and H-18,
resonating at d 1.00 and 1.40, respectively, were ob-
served. These data and those given in the literature
for calenduladiol23 revealed that
2
was 3-
The preliminary structure–activity relationship of these
natural lupane triterpenes revealed the following trends.
The stereochemistry of the hydroxyl group at C-3 can
modify the activity (1 and 17 vs 5 and 18, respectively).
Interestingly, the esterification by caffeic acid of the C-3
hydroxyl group in compound 9 suppresses the activity
on NO (11). It can also be observed that the presence
of oxygenated groups at C-28 has important conse-
quences on the activity. In this respect, the substitution
of a C-28 methyl (8, 14) by a hydroxymethyl group (9,
15) increases the potency mainly on NO, whereas the
presence of a C-28 carboxyl group (13, 16) increases
the potency on NO and PGE2 production and also the
cytotoxicity. In contrast, the introduction of either a
C-28 (10) or C-20 (4) carbonyl group is detrimental.
On the other hand, the presence of the a-hydroxyl group
at C-11 (3 vs 7) results in a higher inhibitory activity,
especially for NO. The acetylation of 9 at C-28 (9a)
increases the potency and reduces the cytotoxicity of this
compound, although the double acetylation at C-28 and
C-3 strongly reduces the activity (9b). Also, the acetyla-
tion of 6 at C-11 (6a) or the chlorination at C-30 (6b)
increases the potency of the compound.
epicalenduladiol.
Compound 3 with the molecular formula C30H46O2 was
shown in the study of its IR, UV, H, and 13C NMR
1
data (Tables 1 and 2) and 2D 1H–1H (COSY and
1
ROESY) and H–13C (HSQC and HMBC) experiments
to be a lupane triterpene with an unsaturated a,b-ketone
and one secondary hydroxyl group, whose positions
were established by an HMBC experiment. These data
and comparison with those found in the literature for
glochidone (7)19 established the structure of 3 as 11a-
hydroxyglochidone.
The detailed 1H and 13C NMR (Tables 1 and 2)
assignments of the one known lupane triterpene
28-hydroxy-3,20-dioxo-29-norlupane (4),17 which have
not been previously reported, were achieved by 1D
and 2D techniques including DEPT, HMBC, HSQC,
COSY, and ROESY.
The betulin (9) analogues 9a and 9b were prepared by
acetylation reaction,22 where the reactivity of the
hydroxyl group on C-28 is much higher than that
on C-3, the C-28 acetoxy could be obtained in a high-
er yield (77.5%) keeping the temperature reaction at
0 ꢁC.
In summary, our data indicate that several lupane triter-
penes are effective in vitro inhibitors of NO and PGE2
production by macrophages. Compounds able to reduce
the excessive production of these mediators have a po-
tential for the prevention and treatment of different
inflammatory pathologies.
We prepared by standard acetylation reaction the acet-
oxy-rigidenol (6a). We want to introduce a double bond
at C-1 by the treatment of 6a with phenylselenyl chloride
in ethyl acetate and the sequential addition of pyridine
and m-chloroperbenzoic acid.24 However, instead
of the unsaturated a,b-ketone desired we obtained a
11a-acetoxy-30-chloro-3-oxo-lup-20(29)-ene (6b) by the
chlorination of the isopropenyl group at C-19. This
was verified with the studies of the spectroscopic data
of 6b, including 1H–13C heteronuclear correlation
(HSQC), long-range correlation with inverse detection
(HMBC), and ROESY experiments.
3. Experimental
3.1. General
Optical rotations were measured on a Perkin Elmer 241
automatic polarimeter and the [aD] are given in
10ꢀ1 deg cm2 gꢀ1. IR (film) spectra were recorded on a
Bruker IFS 55 spectrophotometer, and UV spectra were

C. P. Reyes et al. / Bioorg. Med. Chem. 14 (2006) 1573–1579
Table 3. Effect of compounds 1–19 on NO and PGE2 production
1577
Compound
Viabilitya (10 lM)
NO
PGE2
% inhibitiona
IC50 (lM)
% inhibitiona
IC50 (lM)
b
b
1
100.0 0.0
91.4 1.5
88.4 1.9
94.9 3.3
96.8 3.1
100.0 0.0
90.9 2.3
98.4 1.2
100.0 0.0
98.8 0.7
69.9 1.2c
84.4 1.2
90.1 1.6
100.0 0.0
98.2 1.0
91.0 5.3
67.1 4.1c
92.7 3.1
100.0 0.0
72.1 2.9c
96.1 2.2
100.0 0.0
99.0 1.0
99.0 0.2
98.2 0.4
22.0 3.6c
38.7 3.8c
53.1 4.2d
19.4 2.8c
4.0 4.0
42.7 1.7d
67.7 4.8d
69.4 4.3d
0.0 0.0
N.D.
15.6
8.2
20.2 0.2c
52.1 2.9d
53.6 5.9d
0.0 0.0
27.1 4.2c
44.6 4.7d
59.6 3.9d
78.8 5.3d
33.2 0.6c
42.1 2.1d
47.7 4.3d
69.5 5.1d
32.5 6.6c
0.0 0.0
52.1 2.6d
49.3 2.7d
68.7 5.7d
35.2 4.9c
34.5 12.7c
58.4 3.9d
0.0 0.0
N.D.
6.9
5.4
2
3
4
N.D.
N.D.
12.9
5.3
N.D.
N.D.
20.7
3.9
5
6
6a
6b
7
4.7
0.3
N.D.
N.D.
5.0
N.D.
18.0
12.9
1.7
8
8.0 2.5
9
9a
50.1 3.0d
55.9 5.0d
27.4 4.4c
19.5 4.0
9.3 3.2
4.7
9b
10
N.D.
N.D.
N.D.
12.8
0.7
N.D.
N.D.
10.8
9.6
11
12
44.9 5.7d
89.1 4.4d
7.5 3.3
27.3 2.3c
69.2 5.1d
26.5 2.8c
25.8 0.5c
14.3 1.7
71.8 6.4d
N.D.
13
14
0.6
N.D.
N.D.
0.3
N.D.
N.D.
2.7
15
16
17
18
N.D.
N.D.
N.D.
0.4
N.D.
15.5
N.D.
N.D.
2.5 nM
47.2 5.1d
0.0 0.0
N.D.
19
1400 W
NS 398
N.D.
100.0 0.9d
N.D., not determined.
a Data show means SEM (n = 6). Compounds were assayed at 10 lM. Percentages of inhibition for NO and PGE2 production were obtained at
5 lM for 9, 13, and 16.
b Values represent the concentration required to produce 50% inhibition of the response, along with the 95% confidence limits.
c P < 0.05.
d P < 0.01 with respect to the control group.
collected in absolute EtOH on a JASCO V-560 spectro-
photometer. 1H and 13C NMR spectra were recorded on
a Bruker AMX-500, a Bruker Avance 400, or a Bruker
Avance 300 spectrometer. EIMS and HREIMS were
recorded on a Micromass Autospec spectrometer.
Purification was performed using silica gel (particle size
(n-hexane/CHCl3/MeOH, 2:1:1), to afford 64 fractions.
In this way, after several chromatographies on Sephadex
LH-20 (n-hexane/CHCl3/MeOH, 2:1:1), silica gel
(CH2Cl2–Et2O of increasing polarity), and preparative
HPTLC developed with n-hexane/Et2O (4:6) gave rise
to the new compounds 1 (20.5 mg) and 3 (12.3 mg), in
addition to the known compounds nepeticin (5,
220.0 mg), rigidenol (6, 200.0 mg), glochidone (7,
75.0 mg), lupeol (8, 10.0 mg), betulin (9, 16.0 mg), betu-
linic aldehyde (10, 7.0 mg), epibetulin (12, 30.0 mg),
epibetulinic acid (13, 28.0 mg), lupenone (14, 7.0 mg),
betulone (15, 10.0 mg), betulonic acid (16, 3.0 mg), 3-
epiglochidiol (17, 350.0 mg), glochidiol (18, 40.0 mg),
and glochidonol (19, 125.0 mg).
40–63 lM, Merck and HPTLC-Platten-Sil 20 UV254
Panreac) and Sephadex LH-20 (Pharmacia).
,
3.2. Plant material
Maytenus cuzcoina Loesener was collected at Hua-
yllabamba-Urquillos, Province of Urubamba, Cusco
(Peru), in December 1998, and was identified by Prof.
Alfredo Tupayachi; a voucher specimen (ÔcuzÕ 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. M. chiapensis Lundell
was collected at the Parque Nacional El Imposible, El
Salvador, in August 1999 and was identified by Prof.
Edi Montalvo; a voucher specimen (ISB-88) is on file
The leaves of M. chiapensis (2.1 kg) were extracted with
EtOH in a Soxhlet apparatus. Evaporation of the
solvent under reduced pressure provided 400.2 g crude ex-
tract, which was partitioned into a CH2Cl2/H2O (1:1, v/v)
solution. Removal of the CH2Cl2 from the organic-solu-
ble extract under reduced pressure yielded 77.0 g residue,
which was chromatographed on a silica gel column using
increasing polarity mixtures of n-hexane–EtOAc as an
eluant to afford 54 fractions. Fractions 15–35 (8.0 g) were
subjected to column chromatography over Sephadex LH-
20 (n-hexane/CHCl3/MeOH, 2:1:1) and silica gel
(CH2Cl2–acetone of increasing polarity). Preparative
HPTLC developed with CH2Cl2/EtO2 (9:1) was used to
´
in the Jardin Botanico La Laguna, El Salvador.
3.3. Extraction and isolation
The root bark of M. cuzcoina (900.0 g) was extracted
with n-hexane–Et2O in a Soxhlet apparatus. The extract
(25.5 g) was chromatographed on Sephadex LH-20

1578
C. P. Reyes et al. / Bioorg. Med. Chem. 14 (2006) 1573–1579
purify the new compound 2 (30.0 mg), in addition to the
known compounds 28-hydroxy-3,20-dioxo-29-norlupane
(4, 32.0 mg), glochidone (7, 24.0 mg), lupeol (8, 10.0 mg),
betulin (9, 45.0 mg), betulin 3-caffeate (11, 45.0 mg), lupe-
none (14, 50.0 mg), betulone (15, 60.0 mg), 3-epiglochidi-
ol (17, 60.0 mg), and glochidonol (19, 15.0 mg).
3.5. Chlorination of acetoxyrigidenol
A solution of 6a (150.0 mg, 0.3 mM) and phenylselenyl
chloride (98%) (76.0 mg, 0.4 mM) in EtOAc (65.0 mL)
was stirred at room temperature for 3 h. To the stirred
mixture, saturated aqueous NaHCO3 solution was add-
ed. After most of the aqueous layer was removed, pyri-
dine (0.3 mL, 0.8 mM) and m-chloroperbenzoic acid
(50–60%) (284.0 mg, 0.8 mM) were added to the organic
layer. The mixture was washed with 5% aqueous NaOH
solution (three times), saturated aqueous NH4Cl solu-
tion (three times), and saturated NaCl solution (three
times); dried over anhydrous MgSO4; and filtered. The
mixture was evaporated to dryness and the residue was
purified by flash chromatography on silica gel (eluting
40–70% ethyl acetate in n-hexane) to yield 11a-acet-
20
3.3.1. Compound 1. Lacquer; ½aꢁ 13.6 (c 0.5, CHCl3);
D
IR mmax cmꢀ1: 3413, 2926, 2855, 1731, 1458, 1381,
1070, 990, 883, 757; 1H NMR d: see Table 1; 13C
NMR d: see Table 2; EI/MS m/z %: 442 (M+, 1), 424
(18), 406 (100), 391 (64), 255 (38), 216 (17), 201 (38),
189 (26), 175 (19), 159 (21), 121 (35), 95 (43), 81 (34);
HR-EI/MS: m/z 442.3790 (calcd for C30H50O2
442.3811).
20
3.3.2. Compound 2. Lacquer; ½aꢁ +4.1 (c 0.87, CHCl3);
oxy-30-chloro-3-oxo-lup-20(29)-ene
(6b)
(55.3 mg,
D
20
IR mmax cmꢀ1: 3430, 3010, 2946, 2844, 1645, 1460, 1385,
32%) as an amorphous solid; ½aꢁ +5.5 (c 0.12, CHCl3);
D
1
1035, 900; H NMR d: see Table 1; 13C NMR d: see
IR mmax cmꢀ1: 2957, 2870, 1728, 1712, 1643, 1456, 1381,
1246, 1019, 971, 910, 756, 580; 1H NMR d: 0.80 (3H, s),
0.90 (3H, s), 1.01 (3H, s), 1.04 (3H, s), 1.09 (3H, s), 1.09
(3H, s), 1.11 (3H, s), 1.85 (1H, m), 1.78 (1H, m), 1.96
(3H, s), 2.38 (1H, m), 4.05 (2H, s), 5.02 (1H, s), 5.08
(1H, s), 5.09 (1H, m); 13C NMR d: 14.3 (q), 16.7 (q),
17.5 (q), 17.9 (q), 19.5 (t), 20.3 (q), 21.9 (q), 27.3 (t),
27.7 (q), 32.2 (t), 33.7 (t), 34.0 (t), 34.1 (t), 35.2 (t),
36.6 (d), 37.8 (s), 39.6 (t), 42.1 (t), 42.5 (s), 42.7 (s),
43.1 (s), 43.3 (d), 47.5 (s), 48. 1 (t), 49.3 (d), 52.3 (d),
54.1 (d), 72.8 (d), 112.7 (t), 150.5 (s), 169.9 (s), 218.3
(s); EI/MS m/z %: 456 (M+ꢀ60, 100.0), 441 (24.1), 422
(11.5), 407 (3.4), 311 (16.7), 250 (13.9), 237 (17.4), 107
(12.4), 81.1 (8.5); HR-EI/MS: m/z 456.3179 (calcd for
C30H45OCl 456.3159).
Table 2; EI/MS m/z %: 442 (M+, 9), 424 (10), 406
(100), 363 (17), 201 (37), 189 (67), 147 (42), 134 (79),
107 (54), 95 (45), 69 (29), 55 (34); HR-EI/MS: m/z
442.3760 (calcd for C30H50O2 442.3811).
20
3.3.3. Compound 3. Lacquer; ½aꢁ +23.8 (c 0.6, CHCl3);
D
UV kmax nm: 229; IR mmax cmꢀ1: 3447, 2925, 2854,
1732, 1659, 1459, 1382, 1261, 1075, 937, 883, 803,
1
697; H NMR d: see Table 1; 13C NMR d: see Table
2; EI/MS m/z %:438 (M+, 16), 420 (100), 405 (21), 339
(7), 329 (38), 283 (13), 255 (9), 229 (15), 216 (11), 201
(15), 189 (25), 175 (20), 150 (31), 121 (41), 95 (47), 69
(57); HR-EI/MS: m/z 438.3494 (calcd for C30H46O2
438.3498).
3.4. Acetylation of rigidenol
3.6. Acetylation of betulin
To solution of rigidenol (6) (160.3 mg, 0.4 mM) and cat-
alytic amount of 4-dimethylaminopyridine (DMAP) in
dichloromethane (8 mL) were added triethylamine
(0.2 mL, 1.4 mM) and acetic anhydride (0.1 mL,
1.0 mM). After being stirred for 30 min at room temper-
ature, until TLC showed complete conversion, the mix-
ture was quenched with EtOH and stirred for 30 min.
The mixture was evaporated to dryness and the residue
was purified by flash chromatography on silica gel (elut-
ing 20–40% ethyl acetate in n-hexane) to yield acetoxy-
To a solution of betulin (9) (442.0 mg, 1.0 mM) in pyri-
dine (4.0 mL) was added acetic anhydride (0.1 mL,
1.0 mM) at 0 ꢁC. The reaction mixture was stirred for
4 h at 0 ꢁC, until TLC showed complete conversion.
The mixture was evaporated to dryness and the residue
was purified by flash chromatography on silica gel (elut-
ing 5–50% ethyl acetate in n-hexane) to give 28-acetoxy-
betulin (9a) (375.2 mg, 77.5%) and 3,28-diacetoxy-betu-
lin (9b) (40.3 mg, 7.6%).
rigidenol (6a) (169.4 mg, 87.9%) as an amorphous solid;
3.7. Pharmacological activity
20
D
½aꢁ +31.2 (c 0.25, CHCl3); IR mmax cmꢀ1: 2959, 2926,
2854, 1728, 1712, 1643, 1456, 1379, 1243, 1018, 801;
1H NMR d: 0.74 (3H, s), 0.85 (3H, s), 0.99 (3H, s),
1.00 (3H, s), 1.04 (3H, s), 1.06 (3H, s), 1.48 (3H, s),
1.91 (3H, s), 2.25 (1H, m), 2.34 (1H, m), 2.48 (1H, m),
4.56 (1H, br s); 4.70 (1H, br s), 5.08 (1H, dt, J = 5.2,
11.1 Hz); 13C NMR d: 14.2 (q), 16.6 (q), 17.5 (q), 18.0
(q), 19.4 (q), 19.5 (t), 20.3 (q), 21.9 (q), 27.3 (t), 27.7
(q), 29.7 (t), 32.3 (t), 33.8 (t), 34.1 (t), 35.3 (t), 36.7 (d),
37.7 (s), 39.8 (t), 42.1 (t), 42.5 (s), 42.6 (s), 43.0 (s);
47.3 (d), 47.4 (s), 47.7 (d), 52.2 (d), 54.0 (d), 72.7 (d),
109.9 (t), 149.8 (s), 169.8 (s), 218.2 (s); EI/MS m/z %:
482 (M+, 0.2), 440 (0.2), 422 (100.0), 407 (30.4), 379
(5.6), 339 (6.9), 311 (10.7), 216 (16.7), 203 (22.0), 147
(9.1), 121 (8.3), 81 (5.5); HR-EI/MS: m/z 482.3762 (calcd
for C32H50O3 482.3760).
3.7.1. Nitrite and PGE2 production by stimulated macro-
phages. The mouse macrophage RAW 264.7 cell line
(American Type Culture Collection, Manassas, VA,
USA) was cultured in DMEM supplemented with 10% fe-
tal bovine serum, 2 mM L-glutamine, and 1% penicillin/
streptomycin. Cell viability was assessed by the mitochon-
drial-dependent reduction of 3-(4,5-dimethylthiazol-2-
yl)-2,5 diphenyltetrazolium bromide (MTT) to formazan.
After incubation with test compounds for 18 h, cells were
incubated with MTT (200 lg/mL) for 60 min. The medi-
um was then removed and the cells were solubilized in
DMSO (100 lL) to quantitate formazan at 550 nm.25
Macrophages (4 · 105/well) were incubated with
Escherichia coli [serotype 0111:B4] LPS (1 lg/mL) at
37 ꢁC for 18 h in the presence of test compounds or vehicle

C. P. Reyes et al. / Bioorg. Med. Chem. 14 (2006) 1573–1579
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Acknowledgments
We are indebted to the DGES (BQU2003-09558-CO2-
01) and ICIC (09/2004) projects and E. C. Marie
Curie HPMD-CT-2001-00102 for financial assistance.
We thank the Servicio de Parques Nacionales y Vida
´
Silvestre, Direccion de Recursos Renovables del
Ministerio de Agricultura y Ganaderıa (MAG), and
Fundacion Ecologica de El Salvador (SALVANATU-
RA), for supplying the species M. chiapensis, and
Prof. Alfredo Tupayachi for kindly providing the spe-
cies M. cuzcoina. M.J.N. thanks the AECI and ICIC,
and C.P.R. to the Gobierno de Canarias for a
fellowship.
´
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