Maslinic acid derivatives induce significant apoptosis in b16f10 murine melanoma cells

Article abstract of DOI:10.1016/j.ejmech.2011.10.011

Maslinic acid (2α,3β-dihydroxyolean-12-en-28-oic acid), a natural dihydroxylated pentacyclic triterpene acid isolated from olive-pressing residues, has been investigated together with some of its derivatives regarding the induction of apoptosis in B16F10 melanoma cells. Some of the compounds tested are described in this work, but others come from previous studies. Ten of these derivatives induce over 80% of apoptosis, clearly promoting cell death in B16F10 melanoma. By contrast, the induction cell death through necrosis was very slightly significant with these compounds. These results indicate that maslinic acid derivatives are promising chemopreventive and chemotherapeutic agents.

Full text of DOI:10.1016/j.ejmech.2011.10.011

European Journal of Medicinal Chemistry 46 (2011) 5991e6001
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Maslinic acid derivatives induce significant apoptosis in b16f10 murine
melanoma cells
*
*
Andres Parra , Francisco Rivas , Samuel Martin-Fonseca, Andres Garcia-Granados, Antonio Martinez
Departamento de Quimica Organica, Facultad de Ciencias, Universidad de Granada, Fuentenueva, s/n, 18071-Granada, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 July 2011
Received in revised form
29 September 2011
Accepted 4 October 2011
Available online 12 October 2011
Maslinic acid (2a,3b-dihydroxyolean-12-en-28-oic acid), a natural dihydroxylated pentacyclic triterpene
acid isolated from olive-pressing residues, has been investigated together with some of its derivatives
regarding the induction of apoptosis in B16F10 melanoma cells. Some of the compounds tested are
described in this work, but others come from previous studies. Ten of these derivatives induce over 80%
of apoptosis, clearly promoting cell death in B16F10 melanoma. By contrast, the induction cell death
through necrosis was very slightly significant with these compounds. These results indicate that maslinic
acid derivatives are promising chemopreventive and chemotherapeutic agents.
Keywords:
Maslinic acid derivatives
Synthesis
Ó 2011 Elsevier Masson SAS. All rights reserved.
Cytotoxicity
Anticancer activity
Apoptosis
1. Introduction
Oleanolic acid (OA, 1, 3
b
-hydroxyolean-12-en-28-oic acid) and
maslinic acid (MA, 2, 2
a
,3b-dihydroxyolean-12-en-28-oic acid)
Natural products have a noteworthy role in drug discovery, as
almost half of all new drugs are natural compounds or directly
derived therefrom [1e4]. Triterpenoids are compounds derived
from squalene and widely present in plants used in traditional
medicine [5e9]. Tetra- and pentacyclic triterpenoids display anti-
inflammatory, anti-microbial, anti-HIV, anti-oxidant, hepato-
protective, and analgesic effects [10e13]. However, the disadvan-
tage of using many of these triterpenoids is the toxicity due to their
haemolytic and cytostatic properties. In addition, several natural
triterpene compounds and some derivatives induce apoptosis in
a wide variety of cancer cells, such as breast carcinoma, melanoma,
hepatoma, prostate carcinoma and leukaemia [14e17]. Apoptosis is
a programmed process of cell death, and inappropriately regulated
apoptosis is involved in diverse disease states, such as neuro-
degenerative disease and cancer. Agents that suppress the prolif-
eration of malignant cells, and even cause apoptosis, have the
potential to both prevent and treat cancer. The identification of new
cytotoxic agents that enhance or restore the capability of malignant
cancer cells to undergo apoptosis may be crucial for more effective
anticancer therapies.
(Fig. 1) are two pentacyclic triterpenes present in high concentra-
tions in olive-pomace oil, being the main components of the
protective wax-like coating of the olive skin [18,19]. A method to
obtain large amounts of both compounds from the olive-pressing
residues has been reported by our group [20]. Maslinic acid (MA,
2) has been found to reduce cell proliferation and promote cell
apoptosis in various types of human cancer [21e28]. Moreover, in
the last decade, our research group has published several papers on
the reactivity and some biological activities of OA and MA [29e35].
In the present paper, we have semi-synthesized a series of
MA derivatives (3e21), which have been tested for their
apoptosis-inducing abilities on B16F10 melanoma cells. This apo-
ptotic effect was also investigated for a set of triterpene com-
pounds previously developed in our laboratory (22e40). Some of
these MA derivatives exhibit strongly apoptotic effects without
any cytotoxic consequence.
2. Results and discussion
2.1. Chemistry
Starting with natural maslinic acid (MA, 2), we prepared several
derivatives to be tested as potential apoptotic agents. Thus,
compound 2 was transformed into its corresponding sodium salt
* Corresponding authors.
E-mail addresses: aparra@ugr.es (A. Parra), frivas@ugr.es (F. Rivas).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.10.011

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A. Parra et al. / European Journal of Medicinal Chemistry 46 (2011) 5991e6001
Several triterpenoids containing a nitrogen atom on C-28 have
shown significant biological activities [37,38]. On this basis, we
prepared a new series of MA derivatives with a nitrogen atom on C-
28, starting from this natural product (Scheme 2). Thus, MA (2) was
acetylated to obtain the diacetyl derivative 8 [39], which was
transformed into the corresponding amide derivative 9 by treat-
ment, firstly with thionyl chloride/DCM and then with MeOH/NH3.
The diacetyl amide 9 was deacetylated to form compound 10. The
13C NMR spectra of these amide derivatives (9 and 10) differed
mainly from that of 8 in the signal of C-28, because it is more
shielded in a carboxylic acid group (d 184.5 for 8) than in an amide
group ( 182.2 for 9 and 181.5 for 10). The amide derivative 9 was
d
also converted into the nitrile derivative 11 by a treatment with
thionyl chloride in DCM, which was then deacetylated to form
compound 12. In the 13C NMR spectra of both nitrile derivatives (11
and 12), the C-28 signals are the most relevant (d 125.7 for 11 and
125.8 for 12). Finally, the amine derivative 13 was prepared by the
treatment of 11 with LiAlH4 in THF. The NMR signals, for 13, of the
methylene group at C-28 (
d 3.23 and 3.57) and the signal of this
Fig. 1. Compounds 1 and 2.
carbon atom ( 69.9) confirmed the presence of an amine group.
d
Scheme 3 shows the third set of MA (2) derivatives that were
also tested against B16F10 melanoma cells. Thus, we treated MA (2)
with PCC/DCM to obtain a partially oxidized product (14, 40%), and
a previously described conjugated dicarbonyl product (15, 40%)
[39], which appeared in its ketoenolic form. Triterpenoids with the
same conjugated dicarbonylic system on the A-ring have proved to
be cytotoxic against several cancer-cell lines [40]. The NMR data of
(3) by several procedures (see Experimental). This derivative 3
presented NMR data very similar to that of MA (2), differing only in
its water solubility. Moreover, MA (2) was treated with LiAlH4 in
THF to form 2a-hydroxyerythrodiol (4) [36]. The subsequent reac-
tion of this triol (4) with 2,2-dimethoxypropane produced the ketal
derivative 5, in which both hydroxyl groups of the A-ring were
blocked. The tosyl derivative 6 was formed by treatment with tosyl
chloride in pyridine, and converted into the desired compound 7,
which has a eN3 group on C-28, through an azidation reaction
(Scheme 1).
14 (a singlet signal of 3-H at
d 3.92 and a signal of a carbonyl group
on C-2 at 211.2) confirmed the presence of a carbonyl group on C-
d
2, as a result of the oxidation of the hydroxyl group on this carbon of
MA (2). In addition, to test the influence that the configuration of
the hydroxyl group at C-2 exerted on the apoptotic activity,
compound 14 was reduced with NaBH4 to form the 2-epi-maslinic
acid (augustic acid, 16). The narrow 1H NMR signal of the geminal
Structures of these compounds 5e7 can be easily deduced from
their 1H and 13C NMR data. The NMR signals for compound 5 are
compatible with the presence of an acetonide group between C-2
and C-3 (d d 27.1, 27.4 and
1.38 and 1.40 in its 1H NMR spectrum and
proton of the hydroxyl group at C-2 (d 4.08, ddd, J 3.1, 3.1 and 4.4),
108.4 in its 13C NMR spectrum). Compound 6 had the same NMR
signals as compound 5 had, except for the corresponding signals of
for 16, confirmed the configuration in this carbon atom. Also, 14 and
15 were reduced with LiAlH4 in THF and, in both reactions, triol 17
was achieved, with a hydroxymethylene group placed at C-28 [41].
To test the influence, on the induction of apoptosis in cancer
cells, of a free carboxyl group at C-28 or its benzyl ester, a series of
derivatives (18e21) were synthesised. Several benzyl derivatives of
the tosyl group on C-28 (d
2.42, 7.30 and 7.74 in its 1H NMR spec-
trum). Finally, the 1H and 13C NMR spectra of 7 revealed the absence
of acetonide and tosyl groups as well as the presence of a eN3 group
on C-28 (d 69.9).
Scheme 1. (i) 1) NaOH, 2) NaCl 4%. (ii) LiAlH4/THF. (iii) 2,2-dimethoxypropane. (iv) TsCl/Py. (v) 1) NaN3/DCM, 2) p-toluenesulfonic acid.

A. Parra et al. / European Journal of Medicinal Chemistry 46 (2011) 5991e6001
5993
Scheme 2. (i) Ac2O/Py. (ii) 1) SOCl2/DCM, 2) MeOH/NH3 (1:1). (iii) KOH/MeOH (1:10). (iv) SOCl2/DCM. (v) LiAlH4/THF.
triterpenoids, previously described, exhibit more potent activity
than did their parent compounds [42]. Therefore, MA (2) was
treated with benzyl chloride and DMF to form 28-benzyl maslinic
acid (18). Oxidation of 18 with PCC/DCM led to two oxidized
compounds 19 and 20, 28-benzyl derivatives of 14 and 15,
respectively. The reduction of 19 with LiAlH4 in THF gave
compound 21 (28-benzyl augustic acid). Finally, deprotection of the
benzyl group at C-28 of 21, was carried out by hydrogenation with
H2/Pd/DCM to get 16. We easily identified all the compounds of this
series (18e21) by comparing their corresponding 1H and 13C NMR
spectra with those of the analogues with a free carboxyl group at C-
28 (2, 14e16) (Scheme 3).
Scheme 3. (i) PCC/DCM. (ii) NaBH4/i-PrOH. (iii) LiAlH4/THF. (iv) BnCl/K2CO3/DMF. (v) H2/Pd/DCM.

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A. Parra et al. / European Journal of Medicinal Chemistry 46 (2011) 5991e6001
Several compounds (22e40) [43e47], previously developed by
our research group, have also been tested as apoptotic agents. Their
structures are shown in Fig. 2.
in most cases), and there were 10 compounds that exhibited total
apoptosis (early and late apoptosis) over 80% (3, 10e12, 14, 15,
18e21). In most of these compounds, the early apoptosis repre-
sented over 90% of total apoptosis. Moreover, the compounds
described in previous articles (22e40), with a great structural
variation between them, showed smaller apoptotic effects, and
only 25, 27, 29, and 35 had a significant biological activity between
30 and 50% (see Fig. 3).
2.2. Investigation of apoptosis
Apoptosis is a regulated process of cell death that occurs during
embryonic development as well as maintenance of tissue homeo-
stasis. Annexin V labelled with FITC can identify and quantify
apoptotic cells on a single-cell basis by flow cytometry. Staining
cells with propidium iodide and Annexin V-FITC enables the
distinction of live, apoptotic, dead and late apoptotic, or necrotic
cells [48]. The percentages detected in this test for each cell type, at
different concentrations and periods of time, provide information
on the mechanism involved in the cell death.
The compounds with the best results of total apoptosis of the
above test, at 30
18e21), were selected to carry out new analyses for 24 h and 48 h at
lower concentrations (1, 10, and 20 M; Figs. 4 and 5). At
a concentration of 20 M for 24 h, compounds 3, 10e12, 18e19
mM of concentration for 24 h (3, 10e12, 14, 15,
m
m
showed a total apoptosis over 70%, whereas for 48 h, almost all the
compounds showed this percentage of apoptosis at least. At the
lowest concentration (1
mM), the apoptotic activities of certain
B16-F10 murine melanoma cell lines have been treated with
several MA or OA derivatives (1e40) to test their cytotoxic or
apoptotic effects. The results of the flow cytometry for these
melanoma cells, which have been treated with a 30 mM solution of
these compounds (1e40) in DMSO for 24 h, are shown in Fig. 3. All
compounds, for 24 h, were strongly significant: sodium maslinate
(3, 56.67%), 2,3-diacetoxy-28-cyanide MA (11, 68.62%), 28-cyanide
MA (12, 78.75%), and 28-benzoyl MA (18, 87.50%) (Fig. 6). In the
compounds with the highest apoptosis activity (11, 12 and 18), at
lower concentrations (1 and 10
mM), this apoptosis percentage
the compounds tested, presented very low necrosis (less than 10%
Fig. 2. Compounds 22e40.

A. Parra et al. / European Journal of Medicinal Chemistry 46 (2011) 5991e6001
5995
Fig. 3. % of apoptosis (total and early apoptosis) and necrosis at 30 mM of concentration for 24 h.
greatly decreased for 48 h compared to 24 h, possibly due to the fact
that the cells that were in apoptosis at 24 h were not present at
48 h, as a consequence of the very process of cell death. Although
the structure and activity relationships of these pentacyclic tri-
terpene derivatives are far from clear, it seems that the eCOONa,
eCONH2, eCN, and eCOOBn groups at C-28 enhanced the apoptotic
ability of these derivatives as compared to triterpenic acids
(eCOOH at C-28). Moreover, the necrosis percentage detected with
these MA derivatives was insignificant.
4. Experimental
4.1. General
Measurements of NMR spectra (300.13 MHz 1H and 75.47 MHz
13C) were made in CDCl3 or CD3OD (which also provided the lock
signal) using a BRUKER AM-300 spectrometer. The assignments of
13C chemical shifts were made with the aid of distortionless
enhancement by polarization transfer (DEPT) using a flip angle of
135ꢀ. Bruker’s programs were used for COSY (45ꢀ) and C/H and C/C
correlation. IR spectra were recorded on a MATTSON SATELLITE
FTIR spectrometer. High-resolution mass spectra were made in
a MICROMASS AUTOSPEC-Q spectrometer (EBE geometry). Mps
were determined using a Kofler (Reichter) apparatus and are
uncorrected. Optical rotations were measured on a PerkineElmer
241 polarimeter at 25 ꢀC. All reaction solvents and chromatography
solvents were distilled prior to use. Commercially available
reagents were used without further purification. TLC was carried
3. Conclusions
In conclusion, we have demonstrated that a number of maslinic
acid derivatives provide new insight into the anti-carcinogenic
action to inhibit B16F10 melanoma. Given that apoptosis induc-
tion is arguably the most important process to remove cells that
have lost growth control, these maslinic acid derivatives may be
considered valuable molecules for use as antiproliferative agents.
Fig. 4. Total apoptosis vs. concentration at 24 h. Data are presented as mean ꢁ SD from two independent experiments.

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A. Parra et al. / European Journal of Medicinal Chemistry 46 (2011) 5991e6001
Fig. 5. Total apoptosis vs. concentration at 48 h. Data are presented as mean ꢁ SD from two independent experiments.
out on commercially available TLC aluminium sheets, and spots
were rendered visible by spraying with H2SO4eAcOH, followed by
heating to 120 ꢀC, and also visualized under UV at 254 nm. Silica gel
acetonide group), 1.40 (3 H, s, Me acetonide group), 2.71 (1 H, dd,
J ¼ 4.3 and 13.9, H-18), 3.01 (1 H, d, J ¼ 9.1, H-3), 3.19 (1 H, d, J ¼ 12.5,
HB-28), 3.52 (1 H, d, J ¼ 12.5, HA-28), 3.66 (1 H, ddd, J ¼ 4.4, 9.1 and
11.2, H-2) and 5.17 (1 H, dd, J ¼ 3.5 and 3.5, H-12); dC (CDCl3) 16.4
(Me), 17.3 (Me), 17.5 (Me), 18.0 (C-6), 22.2 (C-16), 23.8 (Me), 24.0
(C-11), 25.8 (C-15), 26,2 (Me), 27.1 (Me acetonide group), 27.4 (Me),
27.4 (Me acetonide group), 28.7 (C-20), 31.3 (C-22), 32.9 (C-7), 33.4
(Me), 34.3 (C-21), 37.2 (C-10), 37.6 (C-4), 40.2 (C-8), 40.4 (C-14), 42.0
(C-17), 42.2 (C-1), 42.5 (C-18), 46.6 (C-19), 48.1 (C-9), 56.3 (C-5),
69.9 (C-28), 72.7 (C-2), 88.8 (C-3), 108.4 (C acetonide group), 122.3
(C-12) and 144.6 (C-13); HR-LSIMS (m/z) 497.4077 (C33H54O3þ
requires 497.4073).
(40e60 mm) was used for flash chromatography.
4.2. Synthesis
4.2.1. Sodium maslinate (3)
MA (2, 200 mg, 0.42 mmol) was added slowly to a stirred
solution of NaOH (263 mg) in hot water (20 mL) until its complete
dissolution. To facilitate this process, a solution of NaCl (4%) was
added. Then, the mixture was cooled and the solid was filtered,
washed with a saturated NaCl solution, and with water, to give 3 as
a white solid (210 mg, 99%); mp 267e269ꢀ; [
a]
þ54 (c 1 in
4.2.4. 2,3-Acetonide-2a,3b-dihydroxy-28-tosylolean-12-ene (6)
D
CHCl3:MeOH, 2:1); IR nmax(KBr)/cmꢂ1 3396, 3001, 2843 and 1710;
dH (CDCl3) 0.69 (3 H, s, Me), 0.72 (3 H, s, Me), 0.83 (3 H, s, Me), 0.86
(3 H, s, Me), 0.88 (3 H, s, Me), 0.90 (3 H, s, Me), 1.04 (3 H, s, Me), 2.73
(1 H, dd, J ¼ 4.3 and 13.9, H-18), 2.87 (1 H, d, J ¼ 9.1, H-3), 3.59 (1 H,
ddd, J ¼ 4.4, 9.1 and 11.2, H-2) and 5.03 (1 H, dd, J ¼ 3.5 and 3.5, H-
12); dC (CDCl3) 16.3 (Me),17.2 (Me),17.5 (Me),18.2 (C-6), 23.0 (C-16),
23.4 (C-11), 23.7 (Me), 25.7 (Me), 27.6 (C-15), 28.8 (Me), 30.7 (C-20),
32.4 (C-7), 32.5 (C-22), 33.1 (Me), 33.8 (C-21), 37.7 (C-10), 38.8 (C-
4), 38.9 (C-8), 41.5 (C-14), 41.7 (C-18), 45.6 (C-17), 46.9 (C-19), 46.9
(C-1), 47.4 (C-9), 54.9 (C-5), 67.1 (C-2), 82.2 (C-3), 119.6 (C-12), 146.1
(C-13) and 181.6 (C-28); HR-LSIMS (m/z) 481.3655 (C30H50O3Naþ
requires 481.3658).
Tosyl chloride (300 mg, 0.46 mmol) was added to a solution of 5
(500 mg, 0.8 mmol) in anhydrous pyridine (10 mL). The reaction
was stirred for 24 h at room temp. The mixture was extracted with
DCM and the organic layer dried with anhydrous Na2SO4. The
solvent was removed under reduced pressure and the residue was
purified by column chromatography using hexane/ethyl acetate
(8:1) to give 6 as a white solid in quantitative yield (663 mg); mp
101e103ꢀ; [
a
]
þ27 (c 1, CHCl3); IR nmax(KBr)/cmꢂ1 3379, 2895,
D
2845 and 1700; dH (CDCl3) 0.81 (3 H, s, Me), 0.84 (3 H, s, Me), 0.86
(3 H, s, Me), 0.86 (3 H, s, Me), 0.97 (3 H, s, Me), 1.02 (3 H, s, M), 1.08
(3 H, s, Me), 1.38 (3 H s, Me acetonide group), 1.39 (3 H, s, Me
acetonide group), 2.42 (3 H, s, Me tosyl group), 2.71 (1 H, dd, J ¼ 4.3
and 13.9, H-18), 3.00 (1 H, d, J ¼ 9.1, H-3), 3.50 (1 H, d, J ¼ 12.5,
HA-28), 3.66 (1 H, ddd, J ¼ 4.4, 9.1 and 11.2, H-2), 3.87 (1 H, d,
J ¼ 12.5, HB-28), 5.05 (1 H, dd, J ¼ 3.5 and 3.5, H-12), 7.30 (2 H, d,
J ¼ 9.0, H tosyl group) and 7.74 (2 H, d, J ¼ 9.0, H tosyl group); dC
(CDCl3) 16.4 (Me), 16.7 (Me), 17.4 (Me), 17.9 (C-6), 20.6 (Me tosyl
group), 21.8 (Me), 22.8 (C-16), 23.8 (Me), 23.9 (C-11), 25.7 (C-15),
26,2 (Me), 27.3 (Me acetonide group), 28.7 (Me acetonide group),
31.0 (C-20), 31.8 (C-22), 32.7 (C-7), 33.2 (Me), 33.9 (C-21), 35.5 (C-
10), 37.5 (C-4), 40.0 (C-8), 40.2 (C-14), 41.7 (C-17), 42.1 (C-18), 42.2
(C-1), 46.3 (C-19), 47.9 (C-9), 56.2 (C-5), 72.6 (C-2), 76.8 (C-28), 88.8
(C-3), 108.4 (C acetonide group), 123.1 (C-12), 128.2 (C tosyl group),
129.9 (C tosyl group), 133.3 (C tosyl group), 143.3 (C tosyl group)
and 144.6 (C-13); HR-LSIMS (m/z) 651.4157 (C40H60O5Sþ requires
651.4161).
4.2.2. Reduction of MA (2) with LiAlH4
LiAlH4 (10 mL, 1 M) was added to a solution of MA (2, 1 g,
2.1 mmol) in anhydrous THF (30 mL). The reaction was stirred for
2 h at reflux. MeOH was added to destroy the excess reagent, and
the solvent was removed under reduced pressure. The residue was
purified by column chromatography using DCM/acetone (4:1) to
give 4 as a white solid (0.932 g, 93%) [36].
4.2.3. 2,3-Acetonide-2a,3b,28-trihydroxyolean-12-ene (5)
A solution of 4 (250 mg, 0.55 mmol) in anhydrous 2,2-
dimethoxypropane (10 mL) was stirred for 12 h at room temp.
The solvent was removed under reduced pressure and the residue
was purified by column chromatography using DCM/acetone (10:1)
to give 5 as a white solid (273 mg, 99%); mp 52e54ꢀ; [
a
]
D þ 23 (c 1,
CHCl3); IR nmax(KBr)/cmꢂ1 3359, 2967, 2899 and 1680; dH (CDCl3)
0.85 (3 H, s, Me), 0.86 (3 H, s, Me), 0.86 (3 H, s, Me), 0.93 (3 H, s, Me),
1.01 (3 H, s, Me), 1.03 (3 H, s, Me), 1.14 (3 H, s, Me), 1.38 (3 H, s, Me
4.2.5. 2a,3b-Dihydroxyolean-12-en-28-azide (7)
NaN3 (90 mg) was added to a solution of 6 (300 mg, 0.48 mmol)
in DCM (25 mL). The reaction was stirred for 4 h at room temp. The

A. Parra et al. / European Journal of Medicinal Chemistry 46 (2011) 5991e6001
5997
Fig. 6. Flow cytometry for control/DMSO and compounds 11, 12, and 18 at a concentration of 1 mM for 24 h. Low left quadrant (LL, black): live cells. Low right quadrant (LR, green):
early apoptotic cells. Upper right quadrant (UR, orange): late apoptotic and dead cells. Upper left quadrant (UL, fuchsia): necrotic cells. (For interpretation of the references to colour
in this figure legend, the reader is referred to the web version of this article.)
mixture was extracted with DCM, p-toluenesulfonic acid (20 mg)
was added, and the reaction was stirred for 30 min at room temp.
The mixture was extracted with DCM, and the organic layer was
dried with anhydrous Na2SO4. The solvent was removed under
reduced pressure and the residue purified by column chromatog-
raphy using DCM/acetone (2:1) to give 7 as a white solid (77 mg,
42.6 (C-18), 46.7 (C-1), 46.8 (C-19), 47.7 (C-9), 55.5 (C-5), 69.2 (C-2),
69.9 (C-28), 84.1 (C-3), 122.4 (C-12) and 144.5 (C-13); HR-LSIMS
(m/z) 482.3820 (C30H49O2Nþ3 requires 482.3825).
4.2.6. Acetylation of MA (2) with Ac2O
Ac2O (2 mL) was added slowly to a solution of MA (2, 1.4 g,
0.294 mol) in pyridine (10 mL). The reaction was stirred for 2 h at
reflux. Cold water was added to the mixture and, after this was
extracted with DCM, the organic layer was dried with anhydrous
Na2SO4. The solvent was removed under reduced pressure and the
residue purified by column chromatography using DCM/acetone
(10:1) to give 8 as a white solid (924 mg, 70%) [39].
93%); mp 89e91ꢀ; [
a
]
D
þ62 (c 1, CHCl3:MeOH, 2:1); IR nmax(KBr)/
cmꢂ1 3392, 2950, 2883 and 1664; dH (CDCl3) 0.85 (3 H, s, Me), 0.87
(3 H, s, Me), 0.93 (3 H, s, Me), 0.95 (3 H, s, Me), 0.99 (3 H, s, Me), 1.03
(3 H, s, Me), 1.16 (3 H, s, Me), 2.71 (1 H, dd, J ¼ 4.3 and 13.9, H-18),
3.13 (1H, d, J ¼ 9.0, H-3), 3.34 (1 H, d, J ¼ 9.0, HA-28), 3.68 (1 H, d,
J ¼ 9.0, HB-28), 3.80 (1 H, ddd, J ¼ 4.4, 9.0 and 11.2, H-2) and 5.19
(1 H, dd, J ¼ 3.5 and 3.5, H-12); dC (CDCl3) 17.0 (Me), 17.1 (Me), 17.8
(Me), 18.6 (C-6), 22.2 (C-16), 22.2 (C-11), 25.7 (Me), 25.8 (C-15), 26.2
(Me), 28.8 (Me), 29.5 (C-20), 31.3 (C-22), 32.7 (C-7), 33.4 (Me), 34.3
(C-21), 37.2 (C-10), 38.4 (C-4), 39.4 (C-8), 40.1 (C-14), 42.0 (C-17),
4.2.7. 2a,3b-Diacetoxyolean-12-en-28-amide (9)
SOCl2 (2 mL) was added to a solution of 8 (500 mg, 0.87 mmol)
in anhydrous DCM (20 mL). The reaction was stirred for 2 h at

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A. Parra et al. / European Journal of Medicinal Chemistry 46 (2011) 5991e6001
reflux. The solvent was removed under reduced pressure and the
residue dissolved in MeOH. Then, a solution of MeOH/NH3 (1:1)
was added dropwise up to the cessation of the bubbling. The
mixture was stirred overnight at room temp. The mixture was
extracted with DCM and the organic layer dried with anhydrous
Na2SO4. The solvent was removed under reduced pressure, and the
residue purified by column chromatography using hexane/ethyl
acetate (2:1) to give 9 as a white solid (109 mg, 55%); mp 267e269ꢀ;
(C-1), 46.8 (C-17), 47.7 (C-9), 55.1 (C-5), 70.2 (C-2), 80.8 (C-3), 124.4
(C-12), 125.7 (C-28), 141.9 (C-13), 170.7 (C]O acetyl group) and
171.0 (C]O acetyl group); HR-LSIMS (m/z) 560.3710 (C34H51O4N-
Naþ requires 560.3716).
4.2.10. 2a,3b-Dihydroxyolean-12-en-28-nitrile (12)
A solution of KOH/MeOH (1:10, 5 mL) was added to a solution of
11 (100 mg, 0.18 mmol) in anhydrous THF (5 mL). Proceeding as
described above for compound 10, compound 12 was obtained as
[
a]
þ54 (c 1, CHCl3:MeOH, 2:1); IR nmax(KBr)/cmꢂ1 3399, 2912,
D
2855 and 1708; dH (CDCl3) 0.84 (3 H, s, Me), 0.85 (3 H, s, Me), 0.91
(3 H, s, Me), 0.93 (3 H, s, Me), 1.07 (3 H, s, Me), 1.10 (3 H, s, Me), 1.18
(3 H, s, Me), 1.94 (3 H, s, Me acetyl group), 2.02 (3 H, s, Me acetyl
group), 2.49 (1 H, dd, J ¼ 4.3 and 13.9, H-18), 4.71 (1H, d, J ¼ 9.1, H-
3), 5.04 (1 H, ddd, J ¼ 4.4, 9.1 and 11.2, H-2) and 5.90 (1 H, dd, J ¼ 3.5
and 3.5, H-12); dC (CDCl3) 17.8 (Me), 17.1 (Me), 17.6 (Me), 18.3 (C-6),
21.1 (Me acetyl group), 21.3 (Me acetyl group), 23.5 (Me), 23.9 (C-
11), 23.9 (C-16), 25.9 (Me), 27.4 (C-15), 28.6 (Me), 30.9 (C-20), 32.4
(C-22), 32.6 (C-7), 33.2 (Me), 34.2 (C-21), 38.3 (C-10), 39.4 (C-4),
39.5 (C-8), 42.2 (C-14), 42.6 (C-18), 44.1 (C-19), 46.5 (C-17), 46.8 (C-
1), 47.64 (C-9), 55.0 (C-5), 70.2 (C-2), 80.2 (C-3), 122.7 (C-12), 145.0
(C-13), 170.7 (C]O acetyl group), 171.0 (C]O acetyl group) and
182.2 (C-28); HR-LSIMS (m/z) 556.3998 (C34H54O5Nþ requires
556.4002).
a white solid (77 mg, 90%); mp 105e107ꢀ; [
a
]
þ40 (c 1,
D
CHCl3:MeOH, 2:1); IR nmax(KBr)/cmꢂ1 2955, 2383, 1755 and 1270;
dH (CDCl3) 0.82 (3 H, s, Me), 0.89 (3 H, s, Me), 0.92 (3 H, s, Me), 1.00
(3 H, s, Me), 1.01 (3 H, s, Me), 1.02 (3 H, s, Me), 1.12 (3 H, s, Me), 2.56
(1 H, dd, J ¼ 4.3 and 13.9, H-18), 2.99 (1H, d, J ¼ 9.1, H-3), 3.69 (1 H,
ddd, J ¼ 4.4, 9.1 and 11.2, H-2) and 5.35 (1 H, dd, J ¼ 3.5 and 3.5, H-
12); dC (CDCl3) 16.9 (Me), 17.0 (Me), 17.5 (Me), 18.6 (C-6), 23.6 (Me),
23.7 (C-16), 24.3 (C-11), 25.8 (Me), 28.2 (C-15), 28.9 (Me), 30.8 (C-
20), 32.5 (C-22), 33.1 (C-7), 33.2 (Me), 33.2 (C-21), 38.2 (C-10), 38.5
(C-8), 39.4 (C-4), 39.8 (C-14), 42.1 (C-17), 44.1 (C-18), 45.0 (C-19),
46.7 (C-1), 47.8 (C-9), 55.5 (C-5), 69.1 (C-2), 84.1 (C-3), 124.7 (C-12),
125.8 (C-28) and 141.9 (C-13); HR-LSIMS (m/z) 476.3513
(C30H47O2NNaþ requires 476.3504).
4.2.11. 2a,3b-Dihydroxyolean-12-en-28-amine (13)
4.2.8. 2
a
,3
b
-Dihydroxyolean-12-en-28-amide (10)
LiAlH4 (1 mL, 1 M) was added to a solution of 11 (100 mg,
0.2 mmol) in anhydrous THF (3 mL). The reaction was stirred for 2 h
at reflux. The mixture was extracted with DCM and the organic
layer dried with anhydrous Na2SO4. The solvent was removed
under reduced pressure and the residue purified by column chro-
matography using DCM/acetone (2:1) to give 13 as a white solid
A solution of KOH/MeOH (1:10, 5 mL) was added to a solution of
9 (100 mg, 0.18 mmol) in anhydrous THF (5 mL). The reaction was
stirred for 30 min at room temp. Then, cold water was added
dropwise up to the cessation of the bubbling. The mixture was
extracted with DCM. The organic layer was dried with anhydrous
Na2SO4. The solvent was removed under reduced pressure and the
residue was purified by column chromatography using DCM/
acetone (2:1) to give 10 as a white solid (76 mg, 90%); mp
(75 mg, 80%); mp 135e137ꢀ; [
a
]
D
þ63 (c 1, CHCl3:MeOH, 2:1); IR
nmax(KBr)/cmꢂ1 2933, 2342, 1761 and 1245; dH (CDCl3) 0.85 (3 H, s,
Me), 0.90 (3 H, s, Me), 0.91 (3 H, s, Me), 0.96 (3 H, s, Me), 1.03 (3 H, s,
Me),1.06 (3 H, s, Me), 1.19 (3 H, s, Me), 2.58 (1 H, dd, J ¼ 4.3 and 13.9,
H-18), 3.03 (1H, d, J ¼ 9.1, H-3), 3.23 (1 H, d, J ¼ 12.0, HA-28), 3.57
(1 H, d, J ¼ 12.0, HB-28), 3.71 (1 H, ddd, J ¼ 4.4, 9.1 and 11.2, H-2) and
5.22 (1 H, dd, J ¼ 3.5 and 3.5, H-12); dC (CDCl3) 17.0 (Me), 17.0 (Me),
17.1 (Me), 18.6 (C-6), 22.2 (C-16), 23.8 (C-11), 23.9 (C-15), 23.9 (Me),
25.7 (Me), 28.8 (Me), 29.5 (C-20), 31.3 (C-22), 32.7 (C-7), 33.4 (Me),
34.3 (C-21), 37.2 (C-10), 38.4 (C-8), 39.4 (C-4), 40.1 (C-14), 42.0 (C-
17), 42.6 (C-18), 46.7 (C-1), 46.7 (C-19), 47.8 (C-9), 55.5 (C-5), 69.8
(C-2), 69.9 (C-28), 84.1 (C-3), 122.4 (C-12) and 144.5 (C-13); HR-
LSIMS (m/z) 456.3913 (C30H51O2Nþ requires 456.3920).
160e162ꢀ; [
a]
þ60 (c 1, CHCl3:MeOH, 2:1); IR nmax(KBr)/cmꢂ1
D
3401, 2940, 2890 and 1698; dH (CDCl3) 0.86 (3 H, s, Me), 0.86 (3 H, s,
Me), 0.95 (3 H, s, Me), 0.95 (3 H, s, Me), 1.02 (3 H, s, Me), 1.06 (3 H, s,
Me), 1.20 (3 H, s, Me), 2.56 (1 H, dd, J ¼ 4.3 and 13.9, H-18), 3.03 (1H,
d, J ¼ 9.1, H-3), 3.71 (1 H, ddd, J ¼ 4.4, 9.1 and 11.2, H-2) and 5.90
(1 H, dd, J ¼ 3.5 and 3.5, H-12); dC (CDCl3) 16.9 (Me), 17.0 (Me), 17.3
(Me), 18.5 (C-6), 23.8 (Me), 23.8 (C-16), 24.0 (C-11), 26.0 (Me), 27.5
(C-15), 27.8 (Me), 30.9 (C-20), 32.6 (C-22), 32.7 (C-7), 33.2 (Me),
34.3 (C-21), 38.5 (C-10), 39.4 (C-4), 39.5 (C-8), 42.4 (C-14), 42.8 (C-
18), 46.6 (C-19), 46.7 (C-17), 46.9 (C-1), 47.8 (C-9), 55.5 (C-5), 69.1
(C-2), 84.1 (C-3), 122.9 (C-12), 145.2 (C-13) and 181.5 (C-28); HR-
LSIMS (m/z) 494.3592 (C30H49O3Naþ requires 494.3610).
4.2.12. Oxidation of MA (2) with PCC
PCC (90 mg, 5.4 mmol) was added to a solution of 2 (200 mg,
0.36 mmol) in dry DCM (10 mL). The mixture was stirred for 2 h at
room temp. The mixture was extracted with a solution of hexane/
diethyl ether (1:1) and the organic layer dried with anhydrous
Na2SO4. The solvent was removed under reduced pressure and the
residue was purified by column chromatography using hexane/
ethyl acetate (2:1) to give 14 as a white solid (67 mg, 40%); mp
4.2.9. 2a,3b-Diacetoxyolean-12-en-28-nitrile (11)
SOCl2 (2 mL) was added to a solution of 9 (200 mg, 0.36 mmol)
in anhydrous DCM (10 mL). The reaction was stirred for 3 h at
room temp. Then, cold water was added and the mixture was
extracted with DCM. The organic layer was dried with anhydrous
Na2SO4. The solvent was removed under reduced pressure and the
residue was purified by column chromatography using DCM/
acetone (10:1) to give 11 as a white solid (83 mg, 43%); mp
164e166ꢀ; [
a
]
þ85 (c 1, CHCl3:MeOH, 2:1); IR nmax(KBr)/cmꢂ1
D
3400, 2953, 2868 and 1639; dH (CDCl3) 0.71 (3 H, s, Me), 0.78 (3 H,
s, Me), 0.90 (3 H, s, Me), 0.94 (3 H, s, Me), 0.96 (3 H, s, Me), 1.22
(3 H, s, Me), 1.22 (3 H, s, Me), 2.87 (1 H, dd, J ¼ 4.3 and 13.9, H-18),
3.92 (1 H, s, H-3) and 5.32 (1 H, dd, J ¼ 3.5 and 3.5, H-12); dC
(CDCl3) 16.4 (Me), 16.8 (Me), 16.9 (Me), 18.7 (C-6), 23.1 (C-16), 23.6
(C-11), 23.8 (Me), 26.1 (Me), 27.6 (C-15), 29.6 (Me), 30.9 (C-20),
32.6 (C-7), 32.6 (C-22), 33.2 (Me), 34.0 (C-21), 39.9 (C-8), 41.2 (C-
18), 42.0 (C-14), 43.9 (C-10), 46.0 (C-4), 46.1 (C-19), 46.7 (C-17),
47.9 (C-9), 53.3 (C-1), 54.7 (C-5), 83.1 (C-3), 122.1 (C-12), 144.0 (C-
13), 183.2 (C-28) and 211.2 (C-2); HR-LSIMS (m/z) 469.3390
(C30H46Oþ4 requires 469.3396); and 15, also as a white solid (67 mg,
40%) [39].
144e146ꢀ; [
a
]
þ34 (c 1, CHCl3:MeOH, 2:1); IR nmax(KBr)/cmꢂ1
D
2929, 2360, 1741 and 1250; dH (CDCl3) 0.89 (3 H, s, Me), 0.90 (3 H, s,
Me), 0.92 (3 H, s, Me), 1.01 (3 H, s, Me), 1.07 (3 H, s, Me), 1.11 (3 H, s,
Me), 1.23 (3 H, s, Me), 1.95 (3 H, s, Me acetyl group), 2.03 (3 H, s, Me
acetyl group), 2.56 (1 H, dd, J ¼ 4.3 and 13.9, H-18), 4.73 (1H, d,
J ¼ 9.1, H-3), 5.07 (1 H, ddd, J ¼ 4.4, 9.1 and 11.2, H-2) and 5.34 (1 H,
dd, J ¼ 3.5 and 3.5, H-12); dC (CDCl3) 16.7 (Me), 17.4 (Me), 17.9 (Me),
18.4 (C-6), 21.1 (Me acetyl group), 21.3 (Me acetyl group), 23.6
(Me), 23.7 (C-16), 24.2 (C-11), 25.7 (Me), 28.2 (C-15), 28.7 (Me),
30.8 (C-20), 32.4 (C-22), 32.6 (C-7), 33.2 (Me), 34.2 (C-21), 38.2 (C-
10), 38.3 (C-4), 39.6 (C-8), 39.7 (C-14), 42.6 (C-18), 44.1 (C-19), 44.2

A. Parra et al. / European Journal of Medicinal Chemistry 46 (2011) 5991e6001
5999
4.2.13. Augustic acid (2
b
,3
b
-dihydroxyolean-12-en-28-oic acid) (16)
8), 41.6 (C-18), 42.0 (C-14), 43.9 (C-10), 45.9 (C-4), 46.1 (C-19), 46.9
(C-17), 46.8 (C-9), 53.3 (C-1), 54.7 (C-5), 66.17 (C benzyl group), 83.2
(C-3), 121.9 (C-12), 128.1 (C benzyl group), 128.2 (C benzyl group),
128.6 (C benzyl group), 136.6 (C benzyl group), 144.1 (C-13), 177.5
(C-28) and 211.2 (C-2); HR-LSIMS (m/z) 561.3943 (C37H53O4þ
requires 561.3944); and also 20 as a white solid (77 mg, 40%); mp
NaBH4 (40 mg) was added to a solution of 14 (100 mg,
0.21 mmol) in i-PrOH (5 mL). The reaction was stirred for 2 h at
room temp. MeOH was added to destroy the excess reagent, and the
solvent removed under reduced pressure. The residue was purified
by column chromatography using DCM/acetone (4:1) to give 16 as
a white solid (95.2 mg, 95%); mp 265e267ꢀ; [
a
]
þ83 (c 1,
199e201ꢀ; [
a
]
þ47 (c 1, CHCl3:MeOH, 2:1); IR nmax(KBr)/cmꢂ1
D
D
CHCl3:MeOH, 2:1); IR nmax(KBr)/cmꢂ1 3380, 2912, 2890 and 1676;
dH (CD3OD) 0.62 (3 H, s, Me), 0.69 (3 H, s, Me), 0.76 (3 H, s, Me), 0.94
(3 H, s, Me), 0.99 (3 H, s, Me), 1.04 (3 H, s, Me), 1.19 (3 H, s, Me), 2.99
(1 H, dd, J ¼ 4.3 and 13.9, H-18), 3.11 (1 H, d, J ¼ 3.1, H-3), 4.08 (1 H,
ddd, J ¼ 3.1, 3.1 and 4.4, H-2) and 5.19 (1 H, dd, J ¼ 3.5 and 3.5, H-
12); dC (CD3OD) 16.7 (Me), 17.6 (Me), 18.3 (Me), 18.8 (C-6), 23.2 (C-
16), 23.9 (Me), 24.1 (C-11), 26.4 (Me), 28.7 (C-15), 30.4 (Me), 30.7 (C-
20), 33.4 (Me), 33.5 (C-22), 33.7 (C-7), 34.4 (C-21), 37.8 (C-8), 39.2
(C-4), 40.3 (C-10), 42.2 (C-18), 42.8 (C-14), 45.0 (C-1), 46.5 (C-17),
46.6 (C-19), 48.7 (C-9), 56.4 (C-5), 71.9 (C-2), 78.8 (C-3), 123.1 (C-
12), 145.3 (C-13) and 180.6 (C-28); HR-LSIMS (m/z) 495.3455
(C30H48O4Naþ requires 495.3450).
3391, 2925, 2897 and 1700; dH (CDCl3) 0.57 (3 H, s, Me), 0.81 (3 H, s,
Me), 0.84 (3 H, s, Me), 1.02 (3 H, s, Me), 1.03 (3 H, s, Me), 1.10 (3 H, s,
Me), 1.12 (3 H, s, Me), 2.94 (1 H, dd, J ¼ 3.1 and 15.0, H-18), 5.10 (2 H,
AB system, J ¼ 12.0, H benzyl group), 5.25 (1 H, dd, J ¼ 3.5 and 3.5,
H-12), 6.24 (1 H s, H-1) and 7.38 (5 H, m, H benzyl group); dC (CDCl3)
17.5 (Me), 18.9 (C-6), 19.8 (Me), 22.0 (Me), 23.3 (C-16), 23,7 (11),
23.8 (Me), 26.0 (Me), 27.7 (C-15), 27.9 (Me), 30.9 (C-20), 32.6 (C-22),
32.8 (C-7), 33.3 (Me), 33.6 (C-8), 34.2 (C-21), 40.2 (C-14), 41.7 (C-
18), 42.3 (C-10), 43.2 (C-9), 44.1 (C-4), 46.0 (C-19), 47.0 (C-17), 54.1
(C-5), 66.3 (C benzyl group), 122.3 (C-12), 128.2 (C benzyl group),
128.2 (C benzyl group), 128.5 (C-1), 128.6 (C benzyl group), 136.5 (C
benzyl group), 143.9 (C-2), 144.3 (C-13), 177.6 (C-28) and 201.3 (C-
3); HR-LSIMS (m/z) 559.3765 (C37H51Oþ4 requires 559.3787).
4.2.14. 2b,3b,28-Trihydroxyolean-12-ene (17)
LiAlH4 (1 mL, 1 M) was added to a solution of 14 (100 mg,
0.21 mmol) in anhydrous THF (5 mL). Proceeding as described
above for the reduction of MA (2), compound 17 was obtained as
a white solid (93.2 mg, 93%). The compound 17 also was syn-
thesised from 15 under the same reaction conditions (92.1 mg, 91%)
[41].
4.2.17. Benzyl augustate (21)
LiAlH4 (1 mL, 1 M) was added to a solution of 19 (100 mg,
0.18 mmol) in anhydrous THF (10 mL). Proceeding as described
above for the reduction of MA (2), compound 21 was obtained as
a white solid (90.1 mg, 91%); mp 221e223ꢀ; [
a
]
D
þ64 (c 1,
CHCl3:MeOH, 2:1); IR nmax(KBr)/cmꢂ1 3371, 2940, 2891 and 1668;
dH (CDCl3) 0.60 (3 H, s, Me), 0.87 (3 H, s, Me), 0.90 (3 H, s, Me), 0.98
(3 H, s, Me), 0.98 (3 H, s, Me), 1.10 (3 H, s, Me), 1.18 (3 H, s, Me), 2.83
(1 H, dd, J ¼ 3.1 and 12.0, H-18), 3.10 (1 H, d, J ¼ 3.0, H-3), 3.98 (1 H,
ddd, J ¼ 3.0, 4.4 and 4.4, H-2), 5.00 (2 H, AB system, J ¼ 12.0, H
benzyl group), 5.28 (1 H, dd, J ¼ 3.5 and 3.5, H-12) and 7.48 (5 H, m,
H benzyl group); dC (CDCl3) 16.5 (Me), 17.1 (Me), 17.5 (Me), 18.3 (C-
6), 23.3 (C-16), 23.7 (C-11), 23.8 (Me), 26.1 (Me), 27.7 (C-15), 29.9
(Me), 30.9 (C-20), 32.6 (C-22), 32.9 (C-7), 33.3 (Me), 34.08 (C-21),
36.9 (C-8), 38.3 (C-4), 39.6 (C-10), 41.6 (C-18), 42.0 (C-14), 44.3 (C-
1), 46.1 (C-19), 47.0 (C-17), 48.3 (C-9), 55.43 (C-5), 66.1 (C benzyl
group), 71.3 (C-2), 78.7 (C-3), 122.8 (C-12), 128.1 (C benzyl group),
128.2 (C benzyl group), 128.6 (C benzyl group), 136.6 (C benzyl
group), 143.9 (C-13) and 177.6 (C-28); HR-LSIMS (m/z) 561.3937
(C37H53Oþ4 requires 561.3937).
4.2.15. Benzyl maslinate (18)
BnCl was added to a solution of MA (2, 1 g, 2 mmol) in DMF
(8 mL) with K2CO3 (0.61 g). The reaction was stirred for 4 h at 55 ꢀC.
The mixture was washed with water and extracted with DCM, and
the organic layer dried with anhydrous Na2SO4. The solvent was
removed under reduced pressure, the residue was purified by
column chromatography using DCM/acetone (10:1) to give 18 as
a white solid (690 mg, 81%); mp 226e228ꢀ; [
a
]
þ12 (c 1,
D
CHCl3:MeOH, 2:1); IR nmax(KBr)/cmꢂ1 3396, 2925, 2843 and 1704;
dH (CDCl3) 0.50 (3 H, s, Me), 0.72 (3 H, s, Me), 0.80 (3 H, s, Me), 0.82
(3 H, s, Me), 0.85 (3 H, s, Me), 0.92 (3 H, s, Me), 1.03 (3 H, s, Me), 2.82
(1 H, dd, J ¼ 3.5 and 12.2, H-18), 2.89 (1 H, d, J ¼ 9.1, H-3), 3.59 (1 H,
ddd, J ¼ 9.1 and 12.1, H-2), 4.97 (2 H, AB system, J ¼ 12.0, H benzyl
group), 5.21 (1 H, dd, J ¼ 3.5 and 3.5, H-12) and 7.48 (5 H, m, H
benzyl group); dC (CDCl3) 16.9 (Me), 17.2 (Me), 17.3 (Me), 18.7 (C-6),
23.3 (C-16), 23.8 (C-11), 24.0 (Me), 27.9 (C-15), 29.02 (Me), 30.9 (C-
20), 32.7 (C-22), 32.9 (C-7), 33.5 (Me), 34.0 (C-21), 38.4 (C-4), 39.2
(C-10), 39.5 (C-8), 41.5 (C-14), 41.9 (C-18), 46.0 (C-17), 46.2 (C-1),
46.8 (C-19), 47.9 (C-9), 55.6 (C-5), 66.3 (benzyl-C), 69.2 (C-2), 89.0
(C-3), 122.5 (C-12), 128.0 (C benzyl group), 128.1 (C benzyl group),
128.5 (C benzyl group), 136.5 (C benzyl group), 143.9 (C-13) and
177.2 (C-28); HR-LSIMS (m/z) 585.3910 (C37H54O4Naþ requires
585.3920).
4.2.18. Hydrogenation of 21
Palladium was added in catalytic amount to a solution of 21
(200 mg, 0.39 mmol) in anhydrous DCM (20 mL). The reaction was
stirred for 2 h at 4 atm hydrogen pressure. Then, the mixture was
filtered and the solvent removed under reduced pressure. The
residue was purified by column chromatography using DCM/
acetone (2:1) to give 16 as a white solid (165 mg, 90%).
4.3. Apoptosis tests
4.2.16. Oxidation of 18 with PCC
B16-F10 murine melanoma cell lines (ATCC Nꢀ: CRL-6475) were
provided by the Scientific Instrumentation Centre of University of
Granada. B16-F10 murine melanoma cells were grown in DMEM
PCC (90 mg, 5.4 mmol) was added to a solution of 18 (200 mg,
0.36 mmol) in DCM (10 mL). Proceeding as described above for the
oxidation of MA (2), compound 19 was obtained as a white solid
medium containing L-glutamine (4 mM), sodium bicarbonate
(77 mg, 40%); mp 107e109ꢀ; [
a
]
þ52 (c 1, CHCl3:MeOH, 2:1); IR
(1.5 g/L), sodium pyruvate (1 mM), glucose (4.5 g/L), and foetal
bovine serum (10%). Confluents B16-F10 melanoma cells in DMEM
medium with foetal bovine serum, 10%, were trypsinised and put
on 6-well culture dishes (300,000 cells per well). After 24 h, the
cells were treated with the compounds 1e40 (by duplicate with
compounds 3, 10e12, 14, 15, 18e21) in DMSO at different
concentrations for 24 or 48 h. As a control, B16F10 cells were
incubated in the presence of the vehicle (DMSO) or in medium
alone.
D
nmax(KBr)/cmꢂ1 3366, 2963, 2832 and 1720; dH (CDCl3) 0.50 (3 H, s,
Me), 0.61 (3 H, s, Me), 0.76 (3 H, s, Me), 0.82 (3 H, s, Me), 0.84 (3 H, s,
Me), 1.09 (3 H, s, Me), 1.10 (3 H, s, Me), 3.09 (1 H, dd, J ¼ 3.1 and 12.2,
H-18), 4.04 (1 H, s, H-3), 5.21 (2 H, AB system, J ¼ 12.0, H benzyl
group), 5.21 (1 H, dd, J ¼ 3.5 and 3.5, H-12) and 7.48 (5 H, m, H
benzyl group); dC (CDCl3) 16.4 (Me), 16.7 (Me), 16.8 (Me), 18.7 (C-6),
23.2 (C-16), 23.5 (C-11), 23.8 (Me), 26.0 (Me), 27.8 (C-15), 29.6 (Me),
30.9 (C-20), 32.5 (C-22), 32.5 (C-7), 33.3 (Me), 34.1 (C-21), 39.9 (C-

6000
A. Parra et al. / European Journal of Medicinal Chemistry 46 (2011) 5991e6001
[21] D.M. Wu, D. Zhao, D.Z. Li, D.Y. Xu, W.F. Chu, X.F. Wang, Maslinic acid induces
Afterwards, the cells were detached with EDTA-Trypsin (0.5 mg/
mL and 0.2 mg/mL, respectively), washed twice with temperate
apoptosis in salivary gland adenoid cystic carcinoma cells by Ca2þ-evoked p38
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phosphate-buffered saline (PBS), and resuspended in 1ꢃ Annexin-
binding buffer at a concentration 1 ꢃ 106 cells/mL. 5
mL of the
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Annexin V-FITC and 5 mL of propidium iodide were added to 100 mL
of each sample. The cells were incubated at room temperature for
15 min in darkness. After this incubation period, 400
mL of 1ꢃ
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Annexin-binding buffer were added and the samples analysed by
flow cytometry within 1 h.
expression and inhibition of NF-kB and AP-1 activation in Raji cells, Planta
Samples were analysed on a Becton Dickinson FACSVantage flow
cytometer with a filter BP530 ꢁ 30 nm in FL1 and a filter
BP585 ꢁ 70 nm in FL2 to collect the fluorescein and propidium
iodide signals, respectively. The acquisition process was at 500
cells/sec up to completing 10,000 cells with CELLQuest Software
(Becton Dickinson).
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Acknowledgments
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Acidic triterpenes compromise growth and survival of astrocytoma cell lines
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This work was supported by a grant from the Dirección General
de Programas y Transferencia de Conocimiento and by a project
from Ministerio de Ciencia e Innovación. We thank David Nesbitt
for reviewing the English of the manuscript.
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