Chemical constituents from Aspidosperma illustre (Apocynaceae)

Article abstract of DOI:10.1590/S0103-50532010000800004

A new natural product oleanane-type triterpene, olean-12-ene-11α- methoxy-3β-acetate (10) was isolated from Aspidosperma illustre, together with β-amyrin (3), lupeol (4), β-amyrin acetate (5), lupeol acetate (6), olean-12-ene-28-hydroxy-3β-tetradecanoate (7), olean-12-ene-28- carboxy-3β-hexadecanoate (8), ursolic acid (9) triterpenes, and two monoterpenic indole alkaloids, β-yoimbine (1) and 1,2- dehydroaspidospermidine (2). These compounds were characterized on their spectral data basis, mainly one-(¹H,¹³C, APT) and two-dimensional (¹H-¹H-COSY,¹H- ¹H-NOESY, HMQC and HMBC) NMR, and mass spectra, involving also comparison with data from the literature.

Full text of DOI:10.1590/S0103-50532010000800004

J. Braz. Chem. Soc., Vol. 21, No. 8, 1434-1438, 2010.
Printed in Brazil - ©2010 Sociedade Brasileira de Química
0103 - 5053 $6.00+0.00
Chemical Constituents from Aspidosperma illustre (Apocynaceae)
Lara F. Barbosa, Leda Mathias, Raimundo Braz-Filhoand Ivo J. Curcino Vieira*
Laboratório de Ciências Químicas, Universidade Estadual do Norte Fluminense Darcy Ribeiro,
28013-602 Campos dos Goytacazes-RJ, Brazil
A partir de Aspidosperma illustre, foi obtido como produto natural um novo triterpeno da série
oleanano, o olean-12-en-11a-metóxi-3β-acetato (10), além dos triterpenos β-amyrina (3), lupeol (4),
acetato de β-amyrina (5), acetato de lupeol (6), olean-12-en-28-hidróxi-3β-tetradecanoato (7),
olean-12-en-28-carboxi-3β-hexadecanoato (8), ácido ursólico (9) e dois alcalóides indólicos
monoterpênicos, β-ioimbina (1) e 1,2-desidroaspidospermidina (2).As substâncias isoladas foram
1
identificadas através de métodos espectroscópicos, principalmente uni (RMN H, 13C, APT) e
bidimensionais (1H-1H-COSY, 1H-1H-NOESY, HMQC e HMBC) e de massas, envolvendo também
comparação com dados de literatura.
A new natural product oleanane-type triterpene, olean-12-ene-11a-methoxy-3β-acetate (10)
was isolated from Aspidosperma illustre, together with β-amyrin (3), lupeol (4), β-amyrin acetate
(5), lupeol acetate (6), olean-12-ene-28-hydroxy-3β-tetradecanoate (7), olean-12-ene-28-carboxy-
3β-hexadecanoate (8), ursolic acid (9) triterpenes, and two monoterpenic indole alkaloids,
β-yoimbine (1) and 1,2-dehydroaspidospermidine (2). These compounds were characterized on
their spectral data basis, mainly one- (1H, 13C, APT) and two-dimensional (1H-1H-COSY, 1H-1H-
NOESY, HMQC and HMBC) NMR, and mass spectra, involving also comparison with data from
the literature.
Keywords: Aspidosperma illustre, Apocynaceae, oleanane triterpenes, alkaloids
Introduction
olean-12-ene-11a-methoxy-3β-acetate (10), along
with seven known triterpenes, β-amyrin (3), lupeol (4),
β-amyrin acetate (5), lupeol acetate (6), olean-12-ene-
28-hydroxy-3β-tetradecanoate (7), olean-12-ene-28-
carboxy-3β-hexadecanoate (8), ursolic acid (9) and two
known monoterpenic indole alkaloids, β-yoimbine (1) and
1,2-dehidroaspidospermidine (2). The structures of known
and new compound are being mentioned for the first time
in this species and were established on the basis of spectral
data, mainly 1H and 13C (1D and 2D) NMR spectra, mass
spectrometry and by comparison with literature data.
The Aspidosperma (Apocynaceae) genus is endemic to
Americas and is found mainly in regions between Mexico
and Argentina.1 Aspidosperma genus continues to be
fascinating as an expressive source of indole alkaloids with
novel skeletons,2 which are interesting from a biosynthetic
perspective and reported biological properties.2,3 Species
of the Aspidosperma genus are applied broadly by popular
medicine as potential antimalarial agents, leishmaniasis
treatment, uterus and ovary inflammation, as contraceptive,
in diabetes, in stomach problems, against cancer, fever and
rheumatism.3
Results and Discussion
Aspidosperma illustre, commonly known as “Tambu-
Pequiá” in Atlantic forests in the North of Espírito Santo
State, appears as a tree of 5-20 m. This species is not
reported on studies of chemical composition described in
the literature.
The hexane and MeOH extracts of stem bark and leaves
of A. illustre were subjected to a classical chromatographic
methods to yield the a new oleanane-type triterpene,
olean-12-ene-11a-methoxy-3β-acetate (10), in addition
to known, β-amyrin (3),4 lupeol (4),4 β-amyrin acetate
(5),4 lupeol acetate (6),4 olean-12-ene-28-hydroxy-
3β-tetradecanoate (7),4 olean-12-ene-28-carboxy-3β-
hexadecanoate (8),5,6 ursolic acid (9),4 β-yoimbine (1)7-9
In the present paper, we describe the isolation and
characterization of a novel oleanane-type triterpene,
*e-mail: curcino@uenf.br

Vol. 21, No. 8, 2010
Barbosa et al.
1435
Table 1. 1H (400 MHz) and 13C (100 MHz) NMR data of triterpene (10),
in CDCl3 as solvent and TMS used as internal reference. Chemical shifts
(d, ppm) and coupling constants (J, Hz, in parenthesis)*
and 1,2-dehydroaspidospermidine (2),10 were identified
1
13
on the basis of H and C NMR spectral data, including
1
1H-1H-COSY, H-1H-NOESY, HSQC and HMBC NMR
experiments,11,12 which were also used to complete
unambiguous 1H and 13C chemical shift assignments of the
triterpenoid 10, the new natural product.
10
HMQC
HMBC
dC
dH
2JCH
3JCH
C
The pentacyclic triterpene (10) was obtained as an
amorphous solid. Comparative analysis of {1H}- andAPT-
13C NMR spectra (Table 1), involving the corroboration
4
38.0
43.1
38.0
149.7
41.7
32.3
31.1
171.0
-
-
-
-
-
-
-
-
3H-23; 3H-24
H-9; 3H-26
H-5; 3H-25
8
3H-27
1
1
1
of H NMR spectra (1D H NMR and 2D H-1H-
COSY), allowed to recognize the presence of 33 signals
corresponding to eight nonhydrogenated [(C)8: six sp3,
two sp2 (including one carbonyl groups at dC 171.0 and
one sp2 olefinic at dC 149.7], six methine [(CH)6: five sp3
(including two oxygenated linked to acetyl group at dC 80.8
and to methoxyl function at dC 75.7) and one sp2 olefinic at
dC 121.8], nine methylene [(CH2)9, all sp3] and ten methyl
[(CH3)10: including two oxygenated attributed to acetate (dC
21.4/dH 2.06, s) and methoxyl groups (dC 53.4/dH 3.21, s)]
carbon atoms, allowing to deduce the expanded molecular
formula (C)7(C=O)(CH)6(CH2)9 (CH3)9(OCH3) for 10.
The LREI-MS (70 eV) spectrum of 10 showed of
molecular peak [M+.] at m/z 498 Daltons, allowing in
conjugation with the 13C NMR spectral data to propose
molecular formula C33H54O3 (10), containing seven
degrees of unsaturation and consistent with the presence
of one acetyl group and one double bond in a pentacyclic
triterpenoid acetylated and methoxylated, compatible
with the structure triterpenic sustaining an acetate group
at carbon atom CH-3 [dC 80.8/dH 4.51 (dd, J 8.8 and
7.6 Hz)] in an β-amyrin skeleton (3, β-amyrin).4 In fact,
heteronuclear long-range coupling (3JCH) of this carbon
atom (dC 80.8) with H-5 (dH 0.87, value approximated
deduced through of HMQC spectrum) and both methyl
groups linked to quaternary carbon C-4 [dH 0.85 (3H-23)
and 0.88 (3H-24)], as shown in Table 1.
10
13
14
17
20
1’
CH
3
3H-27
3H-27
3H-28
H-9; 3H-26
3H-29; 3H-30
3H-2’
80.8
55.3
4.51
(dd, 8.8, 7.6)
H-5; 3H-23;
3H-24
5
0.87
1.72
3H-23; 3H-24;
3H-25
9
51.0
75.7
3H-25; 3H-26
MeO-11
11
3.88
(dd, 9.4, 3.5)
H-9
12
121.8
46.9
5.30
(d, 3.5)
3H-28
18
CH2
1
2.35 (m)
39.1
23.8
18.3
1.42, 1.22
1.72, 1.60
H-9; 3H-25
2
6
1.98 (t, 13.5),
1.69
H-5
7
33.2
26.6
26.7
46.5
34.7
37.0
3H-26
3H-27
15
16
3H-28
19
1.60, 1.30
3H-29; 3H-30
3H-29; 3H-30
3H-28
21
22
CH3
23
The 13C NMR spectrum of 10 revealed signals at dC
121.8 (CH-12) and at dC 149.7 (C-13), indicating the
presence of a trisubstituted double bond. In the HMQC
28.2
16.7
16.9
18.2
25.1
28.5
33.1
23.7
53.4
21.4
0.85(s)
0.88 (s)
1.07 (s)
1.00(s)
1.21 (s)
0.83 (s)
0.88 (s)
0.88 (s)
3.21 (s)
2.05 (s)
3H-24
3H-23
24
25
H-1b;H-9
1
spectrum a cross-peak correlation JCH was observed
26
between the CH-12 at dC 121.8 and the singlet signal at dH
5.30, which was assigned to the vinylic hydrogen.
27
28
The stereochemistry of the stereogenic carbons CH-3
and CH-11 of 10 was determined from the coupling
constants of relevant hydrogens and from the observed
1H-1H-NOESY. The values corresponding to vicinal
interaction (3JH,H) between the hydrogen atoms H-3 and H-2
suggested axial-axial interaction, since the H-3 signal (dH
4.51, dd, J 8.8 and 7.6 Hz) revealed axial-axial coupling
(J 8.8 Hz, Table 1); similarly the multiplicity observed in the
signal of the hydrogen H-11 (dH 3.88, dd, J 9.4 and 3.5 Hz)
29
3H-30
3H-29
30
MeO-11
Me-2’
*Number of hydrogens bound to carbon atoms deduced by comparative
analysis of {1H}- and DEPT-13C NMR spectra. Chemical shifts and
coupling constants (J) were obtained of 1D 1H NMR spectrum.
1H-1H-COSY and H-1H-NOESY experiments were also used to these
1
assignments. Superimposed 1H signals are described without multiplicity
and chemical shifts deduced by HMQC and HMBC spectra.

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Chemical Constituents from Aspidosperma illustre (Apocynaceae)
J. Braz. Chem. Soc.
and both H-12 (dH 5.30, d, J 3.5 Hz) and H-9 (dH 1.72)
allowed to recognize axial-axial interaction of H-11ax with
H-9ax, since the H-11 signal revealed axial-axial coupling
(J 9.4 Hz, Table 1) and H-11ax and H-12eq by = 3.5 Hz,
consistent with the relative configuration shown in 10a.
atoms CH-12 e C-13 in the mass spectrometer, revealing
the absence of peaks produce by reaction Retro-Diels-
Alder (RDA), as observed in other triterpenes with such
characteristics.13,14
Confirmation of structure 10 was done by comparison
with authentic sample of derivative monoacetate of
triterpene 11-a-methoxy-β-amyrin isolated fromMyroxylon
balsamum (Fabaceae).15
According to the literature, the triterpene 10 was also
obtained as an intermediate reaction by allylic oxidation
by N-bromosuccimide with β-amyrin acetate.16
Thus, the pentacyclic isolated from Aspidosperma
illustre was characterized as olean-12-ene-11a-methoxy-
3β-acetate (10), a new natural product.
1
Consistent with these observations, the H-1H-NOESY
spectrum of 10 showed cross-peaks assigned to dipolar
interaction (spatial proximity, vide 10a) of H-11 (dH 3.88)
with 3H-25 (dH 1.07) and 3H-26 (dH 1.00); hydrogen atoms
of the methyl group present in the 3β-O-acetyl (dH 2.05)
with both 3H-23 (dH 0.85) and 3H-24 (dH 0.88).
The oxidation at CH-11 of 10 is adequate to introduce
significant modification in fragmentation of this pentacyclic
triterpene containing double bond between the carbon
Figure 1. Structure of the compounds isolated from A. illustre.

Vol. 21, No. 8, 2010
Barbosa et al.
1437
The results of the extensive application of 1D and
2D NMR spectral techniques were also used to confirm
of hexane/ethyl acetate to afford five fractions. Fraction 1
(1.83 g) was rechromatographed over a silica gel column
with a gradient of hexane/ethyl acetate yielding of a mixture
5 and 6 (15.0 mg). Fraction 2 (1.0 g) and 5 (848.7 mg)
was rechromatographed over a silica gel column with a
gradient of hexane/ethyl acetate supplying of a mixture
3 and 4 (42.0 mg). Fraction 3 (3.26 g) presented as an
amorphous white solid identified as compound (3). Fraction
4 (682.4 mg) was rechromatographed over a silica gel
column with a gradient of hexane/ethyl acetate yielding
of a compound (10) (7.0 mg).
A portion of the methanol extract (10.0 g) was
chromatographed over silica gel column with a gradient
of CH2Cl2/MeOH supplying four fractions. Fraction 1
(589.0 mg) was rechromatographed over silica gel column
with a gradient of CH2Cl2/MeOH yielding of a compound
(7, 9.0 mg) and mixture of compounds (3) e (4) (53.0 mg).
Fraction 3 (360.0 mg) was rechromatographed over silica
gel column with a gradient of CH2Cl2/MeOH furnishing
two compounds (1, 319.0 mg) and (2, 10.0 mg).
Dried and powdered leaves (1.78 kg) were extracted
with methanol at room temperature, furnishing, after
solvent evaporation, 159.5 g of crude methanol extract.
A portion of the methanol extract (40.0 g) was
partitioned with CH2Cl2/H2O, supplying CH2Cl2 phase
(23.2 g). A portion of the CH2Cl2 phase (5.0 g) was
chromatographed over a silica gel column with a gradient
of hexane/ethyl acetate supplying seven fractions. The
fractions 4 (603.3 mg) and 5 (4.1 g) after successive
chromatography’s furnishing of compound (8) (138.4 mg).
The fraction seven (963.7 mg) was rechromatographed over
a silica gel column with a gradient of hexane/ethyl acetate
yielding of compound (9) (83.8 mg).
1
the structure and to establish the H and 13C resonance
assignments of 10 (Table 1).
Experimental
General procedures
Optic rotation measures were obtained on a
Perkin Elmer 343 digital polarimeter. Melting points
were obtained on a Microquímica MQRPF and are
uncorrected. FTIR spectra were recorded on a FTIR-8300
Shimadzu spectrometer using KBr disk. ESI-MS
(high resolution) mass spectra were obtained on a
MICROMASSUltrOTOF-Q (Brüker Daltonics, Billerica,
MA) mass spectrometer, using the negative ion mode of
analysis and EI-MS (low resolution) mass spectra were
obtained on Shimadzu QP5050A mass spectrometer.
Column chromatographic purifications were carried out
over silica gel (70-230 mesh). Silica gel 60F254 was used
in thin layer chromatography analysis.
1H and 13C NMR spectra were measured on a Jeol
Eclipse 400 spectrometer, operating at 400 (1H) and
100 (13C) MHz. CDCl3 was used as solvent and TMS as
internal reference. Chemical shifts are given in the d scale
(ppm) and coupling constants J in Hz. One dimensional
1
(1D) H and 13C NMR spectra were acquired under
standard conditions by using a direct detection 5 mm
1H/13C dual probe. Standard pulse sequences were used for
two dimensional spectra by using a multinuclear inverse
detection 5 mm probe with field gradient.
Plant materials
The water phase (23.2 g) was extracted with ethyl acetate
furnishing the ethyl acetate phase (5.35 g). The portion
of the ethyl acetate phase (2.0 g) was chromatographed
over silica gel column with a gradient of CH2Cl2/MeOH
supplying thirteen fractions. The fractions 3 (16.6 mg)
and 5 (12.7 mg) were submitted to a preparative TLC with
hexane/ethyl acetate supplied by compounds 7 (7.0 mg)
and 3 and 4 mixture (4.0 mg), respectively.
The stem barks and leaves of Aspidosperma illustre
(Vell.) Kuhlm. & Piraja were collected in November 2004
at Reserva Florestal de Linhares, Linhares, Espírito Santo
State, Brazil.A voucher specimen (CVRD 338) is deposited
at the Reserva Florestal Vale do Rio Doce Herbarium,
Linhares, Espírito Santo State.
Extraction and isolation
Transesterification reaction
Dried and powdered stem bark (3.08 kg) from A. illustre
(Vell.) Kuhlm. and Piraja were extracted with hexane and
methanol at room temperature, furnishing, after solvent
evaporation, 10.1 g and 46.6 g of crude hexane and
methanol extracts, respectively.
After purification, the triterpene 8 was submitted to
transesterification reaction with boron trifluorate methanol
(BF3-MeOH). In a flask were added 20 mg of triterpene
8 and 5 mL of BF3-MeOH 20%. The solution was heated
to 90 °C for 10 h. After was added 10 mL of H2O to the
reaction and the aqueous phase separated from the organic
phase by liquid-liquid extraction with hexane. The organic
The hexane extract (10.1 g) from stem bark was
chromatographed over silica gel column with a gradient

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Chemical Constituents from Aspidosperma illustre (Apocynaceae)
J. Braz. Chem. Soc.
phase was concentrated, dried with Na2SO4 and evaporated
for subsequent analysis of gas chromatography.
4. Ahmad,V. U.; Rahman, A.U.; Handbook of Natural Products
Data, Pentacyclic Triterpenoids, Vol. 2, Elsevier: New York,
1994.
Olean-12-ene-11a-methoxy-3b-acetate (10)
5. Carvalho, G. J.; Carvalho, M. G.; Ferreira, D. T.; Faria, T. J.;
Braz-Filho, R.; Quim. Nova 2001, 24, 24.
Amorphous solid, mp 148-152 °C; [a]D23 = + 8.06o
(CHCl3, c 0.062); LREI-MS (rel. int.) 498 (29, M+•), 483 (7,
M - Me•), 466 (21, M - MeOH), 451 (9, M - MeOH - Me•),
391 (8, M - MeOH - Me• - AcOH), 293 (32), 255 (58), 253
(20), 191 (41);1H and 13C NMR: see Table 1.
6. Mallavadhani, U. V.; Mahapatra, A.; Raja, S. S.; Manjula, C.;
J. Agric. Food Chem. 2003, 51, 1952.
7. Breitmaier, E.; Voelter, W.; Carbon-13 NMR Spectroscopy:
High-Resolution Methods and Applications in Organic
Chemistry and Biochemistry, 3rd ed., VCH: Weinheim, 1987.
8. Shamma, M.; Carbon-13 NMR Shift Assignments of Amines
and Alkaloids, Plenum Press: New York, 1979.
9. Malik, A.;Afza, N.; Siddiqui, S.; Heterocycles 1981, 16, 1727.
10. Hugel, G.; Royer, D.; Sigaut, F.; Levy, J.; J. Org. Chem. 1991,
56, 4631.
Supplementary Information
Supplementary data are available free of charge at
http://jbcs.sbq.org.br, as PDF file.
Acknowledgments
11. Claridge, T. D.W.; High-Resolution NMRTechniques in Organic
Chemistry; Pergamon: Amsterdam, 1999.
The authors thank Fundação de Amparo à Pesquisa do
Estado do Rio de Janeiro (FAPERJ-RJ-Brasil) for visitant
research fellowship and grants, to Conselho Nacional de
Desenvolvimento Científico (CNPq-Brasil) for research
fellowship. We are grateful to Prof. Norberto Peporine
Lopes (FCFRP-USP) for the high-resolution mass spectra.
12. Friebolin, H.; Basic One and Two-Dimensional NMR
Spectroscopy; 2nd ed., VCH: New York, 1993.
13. Barnes, R. A.; Pereira, A. L.; Scofield, T. C. V.; Braz-Filho, R.;
Pinto, A. C.; Chem. Pharm. Bull. 1984, 32, 3674.
14. Branco,A.; Pinto,A. C.; Braz-Filho, R.; An. Acad. Bras. Cienc.
2004, 76, 505.
15. Mathias, L.; Vieira, I. J. C.; Braz-Filho, R.; Rodrigues-Filho,
E.; J. Braz. Chem. Soc. 2000, 11, 195.
References
16. Finucane, B. W.; Thomson, J. B.; J. Chem. Soc. Perkin I 1972,
14, 1856.
1. Pereira, M. M.; Jácome, R. L. R. P.; Alcântara, A. F. C.; Alves,
R. B.; Raslan, D. S.; Quim. Nova 2007, 30, 1.
2. Oliveira, V. B.; Vieira, I. J. C.; Braz-Filho, R.; Mathias, L.;
Lopes, N. P.; Crotti, A. E. M.; Uchoa, D. E. A.; J. Braz. Chem.
Soc. 2009, 20, 753.
Received: September 13, 2009
Web Release Date: March 22, 2010
3. Oliveira, V. B.; Freitas, M. S. M.; Mathias, L.; Braz-Filho, R.;
Vieira, I. J. C.; Rev. Bras. Pl. Med. 2009, 11, 92.

J. Braz. Chem. Soc., Vol. 21, No. 8, S1-S9, 2010.
Printed in Brazil - ©2010 Sociedade Brasileira de Química
0103 - 5053 $6.00+0.00
Chemical Constituents from Aspidosperma illustre (Apocynaceae)
Lara F. Barbosa, Leda Mathias, R. Braz-Filhoand Ivo J. Curcino Vieira*
Laboratório de Ciências Químicas, Universidade Estadual do Norte Fluminense Darcy Ribeiro,
28013-602 Campos dos Goytacazes-RJ, Brazil
Figure S1. 1H NMR spectrum of triterpene 10 (400 MHz, CDCl3).
*e-mail: curcino@uenf.br

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Chemical Constituents from Aspidosperma illustre (Apocynaceae)
J. Braz. Chem. Soc.
Figure S2. 13C NMR spectrum of triterpene 10 (100 MHz, CDCl3).
Figure S3. Expansion of 13C NMR spectrum of triterpene 10 (100 MHz, CDCl3).

Vol. 21, No. 8, 2010
Barbosa et al.
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Figure S4. Expansion of 13C NMR spectrum of triterpene 10 (100 MHz, CDCl3).
Figure S5. 13C NMR-APT spectrum of triterpene 10 (100 MHz, CDCl3).

S4
Chemical Constituents from Aspidosperma illustre (Apocynaceae)
J. Braz. Chem. Soc.
Figure S6. Expansion 13C NMR-APT spectrum of triterpene 10 (100 MHz, CDCl3).
Figure S7. Expansion 13C NMR-APT spectrum of triterpene 10 (100 MHz, CDCl3).

Vol. 21, No. 8, 2010
Barbosa et al.
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Figure S8. HMQC spectrum of triterpene 10 (400 MHz, CDCl3).
Figure S9. Expansion of HMQC spectrum of triterpene 10 (400 MHz, CDCl3).

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Chemical Constituents from Aspidosperma illustre (Apocynaceae)
J. Braz. Chem. Soc.
Figure S10. HMBC spectrum of triterpene 10 (400 MHz, CDCl3).
Figure S11. Expansion of HMBC spectrum of triterpene 10 (400 MHz, CDCl3).

Vol. 21, No. 8, 2010
Barbosa et al.
S7
Figure S12. 1H-1H-COSY spectrum of triterpene 10 (400 MHz, CDCl3).
Figure S13. 1H-1H-NOESY spectrum of triterpene 10 (400 MHz, CDCl3).

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Chemical Constituents from Aspidosperma illustre (Apocynaceae)
J. Braz. Chem. Soc.
Figure S14. LREIMS spectrum of triterpene 10 (70 eV).
Figure S15. Proposed fragmentation mechanisms of triterpene 8 by MS/MS (HRESIMS) of the peaks at m/z 693.5829 ([M-H+]-) with production of
fragments 8a and 8b and EIMS (70 eV) to furnish 8c and 8d, only peaks classified as principals.

Vol. 21, No. 8, 2010
Barbosa et al.
S9
Figure S16. Proposed fragmentation mechanisms to justify principal peaks observed in the mass spectrum (LREIMS, 70 eV) of 10 (in parenthesis
percentage of relative abundance).