Semi-synthesis of acylated triterpenes from olive-oil industry wastes for the development of anticancer and anti-HIV agents

Article abstract of DOI:10.1016/j.ejmech.2013.12.049

A broad set of potential bioactive conjugate compounds has been semi-synthesized through solution- and solid-phase organic procedures, coupling two natural pentacyclic triterpene acids, oleanolic (OA) and maslinic acids (MA), at the hydroxyl groups of the A-ring of the triterpene skeleton, with 10 different acyl groups. These acyl OA and MA derivatives have been tested for their anti-proliferative (against the b16f10 murine melanoma cancer cells) and antiviral (as inhibitors of the HIV-1-protease) effects. Several derivatives have shown high levels of early and total apoptosis (up to 90%). Most of the compounds that exhibited anti-proliferative effects also generated ROS, probably involving the activation of an intrinsic apoptotic route. The only four compounds that did not cause the release of ROS could be related to the participation of a probable extrinsic activation of the apoptosis mechanism. A great number of these acyl OA and MA derivatives have proved to be potent inhibitors of the HIV-1-protease, the most active inhibitors having IC ₅₀ values between 0.31 and 15.6 μM, these values being between 4 and 186 times lower than their non-acylated precursors. The potent activities exhibited in the apoptosis-activation processes and in the inhibition of the HIV-1-protease by some OA and MA acylated derivatives imply that these compounds could be used as new, safe, and effective anticancer and/or antiviral drugs.

Full text of DOI:10.1016/j.ejmech.2013.12.049

European Journal of Medicinal Chemistry 74 (2014) 278e301
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Semi-synthesis of acylated triterpenes from olive-oil industry wastes
for the development of anticancer and anti-HIV agents
,
a
a
,
b
Andres Parra a , Samuel Martin-Fonseca , Francisco Rivas
, Fernando J. Reyes-Zurita ,
*
**
Marta Medina-O’Donnell a, Antonio Martinez a, Andres Garcia-Granados a,
***
Jose A. Lupiañez b , Fernando Albericio
,
c,d
a Departamento de Quimica Organica, Facultad de Ciencias, Universidad de Granada, E-18071 Granada, Spain
b Departamento de Bioquimica y Biologia Molecular I, Facultad de Ciencias, Universidad de Granada, E-18071 Granada, Spain
c Institute for Research in Biomedicine and CIBER BBN, Baldiri Reixac 10, 08028 Barcelona, Spain
d School of Chemistry and Physics, University of KwaZulu-Natal, 4001 Durban, South Africa
a r t i c l e i n f o
a b s t r a c t
Article history:
A broad set of potential bioactive conjugate compounds has been semi-synthesized through solution-
and solid-phase organic procedures, coupling two natural pentacyclic triterpene acids, oleanolic (OA) and
maslinic acids (MA), at the hydroxyl groups of the A-ring of the triterpene skeleton, with 10 different acyl
groups. These acyl OA and MA derivatives have been tested for their anti-proliferative (against the b16f10
murine melanoma cancer cells) and antiviral (as inhibitors of the HIV-1-protease) effects. Several de-
rivatives have shown high levels of early and total apoptosis (up to 90%). Most of the compounds that
exhibited anti-proliferative effects also generated ROS, probably involving the activation of an intrinsic
apoptotic route. The only four compounds that did not cause the release of ROS could be related to the
participation of a probable extrinsic activation of the apoptosis mechanism. A great number of these acyl
OA and MA derivatives have proved to be potent inhibitors of the HIV-1-protease, the most active in-
Received 26 September 2013
Received in revised form
20 December 2013
Accepted 23 December 2013
Available online 8 January 2014
Keywords:
Triterpene
Oleanolic acid
Maslinic acid
Apoptosis
hibitors having IC50 values between 0.31 and 15.6 mM, these values being between 4 and 186 times lower
Anti-HIV
Green chemistry
than their non-acylated precursors. The potent activities exhibited in the apoptosis-activation processes
and in the inhibition of the HIV-1-protease by some OA and MA acylated derivatives imply that these
compounds could be used as new, safe, and effective anticancer and/or antiviral drugs.
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
hydroxyolean-12-en-28-oic acid, OA, 1) and maslinic acid (2a,3b-
dihydroxyolean-12-en-28-oic acid, MA, 2) (Fig. 1), two natural
pentacyclic triterpenoids widely found in nature [1e3]. A method
to obtain large quantities of these triterpene acids from olive-mill
wastes has been reported by our research group [4]. Both acids,
and several closely related triterpene compounds, exhibit a wide
range of biological activities, and some may be used in medicine
[5e9]. These compounds could be useful to semi-synthesize other
biologically or chemically valuable products [10,11]. Moreover,
there is firm evidence supporting the beneficial effects of
consuming olive oil, and OA and MA are two bioactive compounds
contained in its residual waste [12].
The olive-oil industry produces a large volume of solid and
liquid wastes, which require complex disposal problems and po-
tential environmental pollution. In fact, olive-oil waste has histor-
ically been one of the biggest problems associated with this
industry. Nevertheless, these residues are promising sources of
diverse substrates, such as, phenolic compounds, oligosaccharides,
manitol and two triterpenic acids (oleanolic and maslinic acids),
which can be converted into value-added products. Thus, our
starting vegetal material was the residue of both liquid and solid
wastes from the two-phase industrial olive-oil extraction.
The isolated triterpene compounds were oleanolic acid (3
b
-
Triterpenoids are natural products widely distributed among
diverse organisms, including bacteria, fungi, yeasts, plants, and
mammals. Nevertheless, the majority of triterpenes are found in
plants, especially as glycoside derivatives (saponins). Pentacyclic
triterpenes are a group of compounds which are broadly distrib-
uted throughout the plant kingdom and which can be classified
into three major types based on their structural skeleton: oleanane,
* Corresponding author. Tel.: þ34 958 240480; fax: þ34 958 248437.
** Corresponding author. Tel./fax: þ34 958 240479.
*** Corresponding author. Tel.: þ34 958 243089.
E-mail addresses: aparra@ugr.es (A. Parra), frivas@ugr.es (F. Rivas), jlcara@ugr.es
(J.A. Lupiañez).
0223-5234/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.12.049

A. Parra et al. / European Journal of Medicinal Chemistry 74 (2014) 278e301
279
potential and the release of ROS (reactive oxygen species) [41,42].
This apoptotic route involves the mitochondria-dependent activa-
tion of initiator caspases, which in turn activates downstream ex-
ecutive caspases, such as caspase-3. The extrinsic death-receptor
pathway is triggered by death ligands through the formation of the
death-inducing signalling complex that results in caspase-8 and
caspase-10 activation, triggering downstream apoptotic signalling
[43,44]. An initial release of ROS accompanied of apoptosis induc-
tion could indicate the activation of the intrinsic apoptotic pathway,
whereas that apoptotic induction without the release of ROS would
suggest the activation of the extrinsic apoptotic pathway. The
evaluation of the levels of ROS, at the cytotoxic and apoptotic
concentrations, can help to determine the possible molecular
mechanism involved in the apoptotic response found against the
different compounds tested.
Fig. 1. Structures of compounds 1e4.
A major aim in the field of diversity-oriented synthesis is the
effective production of small-molecule libraries to undertake new
biological probes and to discover new therapeutic leader com-
pounds [45]. Terpenoids have long served as important small-
molecule drugs, leading to drug discoveries [46,47]. In fact,
terpenoid cores are prevalent among privileged scaffolds able to
bind multiple biological targets, and a variety of terpenoid-based
libraries have been reported [48,49]. Combinatorial chemistry
represents the possibility of synthesizing and testing a large
number of compounds for their bioactivity simultaneously, instead
of one by one [50,51]. On the other hand, the SPOS (solid-phase
organic synthesis) is a powerful tool in the synthesis of small
molecules to discover new remarkable pharmacological com-
pounds. In this synthetic technique, the derivatives are synthe-
sized by using an excess of reagents to drive reactions to
completion, being isolated easily by simple filtration and removed
from the support material [49]. The SPOS has a principal advan-
tage during synthesis in solution, which facilitates the purification
process, allowing automation of the whole synthesis. However,
SPOS also presents some major disadvantages; for example, it can
be problematic to adapt some conventional solution reactions to a
solid-phase process. There are only few articles related to the
solid-phase of triterpenoids by attaching a solid support to the C-
28 position [52e54]. This methodology would not only serve to
protect the carboxyl group, but also could simplify the entire
synthesis procedure.
In the present paper, we have carried out the semi-synthesis of a
number of oleanolic and maslinic acid derivatives, starting from
these natural compounds isolated from olive-oil wastes. This
preparation has been made using solution-phase organic synthesis
processes, which have been compared with the corresponding
solid-phase organic synthesis ones. Previously, we have also tested
the degree of incorporation of OA and MA to the solid support
employed. Finally, these acyl OA and MA derivatives were also
tested in a screening mode, for different potential bioactivities such
as anticancer and anti-HIV properties. The main purpose of this
study is to determine the bioactive potential of these acyl OA and
MA derivatives as anti-proliferative and pro-apoptotic compounds
as well as anti-virals on HIV-serine protease. With respect to the
anticancer properties, we firstly determined the cytotoxic potential
of the different derivatives against the murine melanoma cell line
b16f10, and we secondly investigated whether the cytotoxicity of
the most effective compounds was caused by the induction of
apoptosis. We also determined the levels of ROS, proposing a
plausible molecular mechanism for its apoptotic effects. Finally, we
have performed HIV-protease inhibition assays, being the action of
the acyl derivatives of the triterpene acids, much more potent than
that of the natural precursor compounds. Hence, these derivatives
may represent an effective therapeutic strategy for treating cancer
or HIV infection.
ursane, and lupine [13,14]. Biologically, these are the most impor-
tant triterpenoid structures for their anti-inflammatory, anti-HIV,
hepatoprotective, analgesic, antimicrobial, and virostatic properties
[15e23]. Although some natural compounds of the terpenoid
family have emerged as promising anti-HIV substances, impor-
tantly, their chemical modification may allow improvement of both
their potency and selectivity in this assay [24e26]. The potential
anti-HIV properties of diverse compounds can be tested using an
FRET (fluorescence resonance energy transfer) HIV protease-
inhibitor assay. This assay is considered an effective means for
drug screening [27,28]. The continuous quantification of HIV-pro-
tease activity uses an FRET peptide, in which the fluorescence is
quenched by a quencher group until the peptide is cleaved into two
separate fragments.
Apoptosis or programmed cell death is a well-ordered and
tightly regulated physiological process of cell self-destruction,
with specific morphological and biochemical changes in the nu-
cleus and cytoplasm, which plays a crucial role in the development
of cancer. Apoptotic death is known to involve a cascade of pro-
teolytic events driven mainly by a family of cysteine proteases,
called caspases. Defects in apoptotic pathways contribute funda-
mentally to a range of disease states. Agents that suppress the
proliferation of malignant cells, by inducing apoptosis, may
represent a useful mechanistic approach to both chemoprevention
and chemotherapy of cancer. The discovery of small-molecule
drugs capable of inducing apoptosis in cancer cells is a funda-
mental and a highly attractive goal. Pro-apoptotic agents would be
expected to have beneficial effects compared to existing cancer
therapeutics [29e31]. Recently, our research group has demon-
strated that some MA derivatives induce noteworthy apoptosis in
b16f10 murine melanoma cells, and therefore they may be
considered valuable molecules for their use as anti-proliferative
agents [32]. MA also shows anti-tumour effects in Caco-2 and
HT29 colon-cancer cell lines [33,34], and in the astrocytoma cell
line [35], via mitochondrial or intrinsic apoptotic pathway [36,37].
In addition, our group has demonstrated for the first time, that the
MA can modify certain cytoskeleton proteins of HT29, which is one
of the bases of their anti-proliferative activity against colon-cancer
cells [38], as well as the effectiveness of MA as a potential che-
mopreventive against colorectal cancer when used against the
development of intestinal tumours in the ApcMin/þ mice model
[39]. MA also displays cytotoxic effects in different tumour-cell
lines: non-small lung cells, ovary, melanoma, central nervous
system, and colon [40].
During apoptotic activation, two major pathways have been
described: the mitochondrial or intrinsic pathway, and the death-
receptor or extrinsic pathway. The intrinsic pathway involves
mitochondrial disruption, changing the mitochondrial-membrane

280
A. Parra et al. / European Journal of Medicinal Chemistry 74 (2014) 278e301
Table 1
2. Results and discussion
Solution-phase acetylation of OA.
2.1. Chemistry
Time (h)
Temperature
Compound
OA (1)
3-Acetyl-OA (5)
Oleanolic acid (1, 3
and maslinic acid (2, 2
b
-hydroxyolean-12-en-28-oic acid, OA) [1]
Yield (%)
Rt (min)
Yield (%)
Rt (min)
13.89
a
,3 -dihydroxyolean-12-en-28-oic acid,
b
0.5
1
12
24
Reflux
Reflux
rt
8.9
5.6
18.4
5.3
9.72
91.1
94.4
81.6
94.7
MA) [2] are natural compounds belonging to the pentacyclic tri-
terpene family and are widely distributed in the plant kingdom. In
our case, these compounds were isolated from the olive-pressing
residues by extraction processes with different solvents [4]. These
triterpenoid acids were benzylated by treatment with benzyl
rt
chloride to obtain the corresponding derivatives
3
(28-
Table 2
benzyloleanolic acid, OA-Bn) and 4 (28-benzylmaslinic acid, MA-
Bn) (Fig. 1). Both compounds were easily recognizable from their
spectroscopic properties, since in their 1H and 13C NMR spectra
appeared the signals of the benzyl methylene group (dH 5.05, 2H,
AB system, J ¼ 12.0 Hz, and dC 66.15, respectively in both com-
pounds), which matched those found in the literature [32,55].
Solution-phase acetylation of MA.
Time Temp. Compound
(h)
MA (2)
2-Acetyl-MA 3-Acetyl-MA 2,3-Diacetyl-
(6) (7) MA (8)
Yield (%) Rt (min) Yield Rt (min) Yield Rt (min) Yield Rt (min)
(%)
(%)
(%)
0.5 Reflux 10.3
4.69
20.7 6.93
2.2
30.8
14.1
30.8
9.2 8.56
59.8 10.12
89.9
40.5
80.8
3.8
2.1.1. Solution-phase acylations of triterpenes 1e4
1
Reflux 6.2
1.7
19.0
0.8
20.1
30.2
To compare the acylation process, in different phases and con-
ditions, of OA (1) and MA (2), we proceeded to test their acetylation
in solution-phase at diverse times and under different temperature
conditions. The acetylation reactions of OA and MA were carried
out with acetic anhydride in pyridine, consistently using an excess
of 4:1 of the acylating agent with respect to the hydroxyl group
(Scheme 1). The composition of the different acetylation mixtures
was studied using HPLC (Tables 1 and 2).
In this acetylation process in the solution-phase, OA was acet-
ylated to form the 3-acetyl derivative 5 [56] in good yields (80e
95%), even at rt (Table 1). In the acetylation reaction of MA (2), three
derivatives were formed: 2-acetyl-MA (6), 3-acetyl-MA (7), and
2,3-diacetyl-MA (8) [57]. This acylation process was conducted at
several reaction times and temperatures, improving the yield of the
double acetylated derivative (8) with the increase in the tempera-
ture and reaction time (80% at 24 h at rt, and 90% at 1 h and reflux)
(Table 2).
Once the reaction conditions (24 h at rt) for the acetylations of
OA and MA were optimized, we carried out the semi-syntheses in
solution-phase of several derivatives of compounds 1e4, by acyla-
tion of the hydroxyl groups of the A-ring with 10 acid anhydrides
(Fig. 2), to later check their biological properties. The acyl groups
were selected with a wide structural variety in order to obtain
acylated derivatives of OA and MA with diverse physical and
chemical characteristics. Thus, acyl groups IeV have an increasing
number of carbon atoms, and therefore the polarity decreases
12
24
12
24
rt
rt
0
9.7
4.3
45.3
10.5
ꢀC
ꢀC
0
44.6
14.7
progressively from the acetyl to the lauroyl group. On the other
hand, acyl groups VI and VII have aromatic rings, and phthaloyl
group (VII) also another carboxylic group at the end of the carbon
chain. Finally, acyl groups VIIIeX, derived from succinic and glutaric
anhydrides, also have an additional carboxylic group that conferred
them with singular polarity and solubility properties.
The above-mentioned acylated derivatives were formed by
treating the compounds 1e4 with the corresponding acid anhy-
dride in pyridine for 24 h at rt, in a relationship of 4:1 (anhy-
dride:hydroxyl group). Thus, we obtained 57 acylated derivatives
(9e65), given in Figs. 3 and 4.
The main spectroscopic differences between the acylated com-
pounds (9e27) and the corresponding substrates (OA (1), and OA-
Bn (3)) were the important deshieldings of the signals of the
geminal protons to the hydroxyl group at C-3 (1H spectra), and the
notable variations in the chemical shifts of C-3 (13C spectra). Thus,
the signal of the geminal proton of H-3 for OA and OA-Bn was
situated at dH 3.19 and 3.18, respectively, whereas the same signal
was deshielded up to dH 4.40e4.50 for the derivatives with the acyl
group IeV and VIIIeX, and up to dH 4.70e4.80 for the acyl groups VI
and VII (aromatic ring). In the same way, the chemical shift of C-3
Scheme 1. Acetylation tests of OA and MA in the solution-phase.

A. Parra et al. / European Journal of Medicinal Chemistry 74 (2014) 278e301
281
Fig. 2. Structures of the acyl groups used for the acylations of OA and MA.
Fig. 3. Structures of compounds 9e27 from the acylation of OA.
was situated at dC 78.69 for OA and dC 79.23 for OA-Bn, moving to dC
80e83 when this carbon was acylated.
In the acylation processes of MA (2) and MA-Bn (4), using the
optimized reaction conditions of 24 h at rt, the major 2-acyl
derivatives and 2,3-diacyl derivatives were formed, probably due
to the increased steric hindrance of the hydroxyl group at C-3 by
the presence of the geminal methyl groups on C-4. For this reason,
the minor 3-acyl derivatives were not considered for this study. The
Fig. 4. Structures of compounds 28e65 from the acylation of MA.

282
A. Parra et al. / European Journal of Medicinal Chemistry 74 (2014) 278e301
Scheme 2. Control and quantification of load of OA and MA to the CTC resin.
main 1H NMR chemical shift differences between the acylated
compounds (28e65) and the corresponding substrates (MA (2), and
MA-Bn (4)) were the position of the signals of H-2 and H-3, situated
at dH 3.45 and 2.84 for MA, and at dH 3.59 and 2.89 for MA-Bn,
respectively. However, in the corresponding 2-acyl derivatives, the
H-2 signals were situated between dH 4.90e5.20, and for the 2,3-
diacyl derivatives, the H-3 signals were deshielded up to dH 4.70e
4.80 for the derivatives with the acyl group IeV and VIIIeX, and up
to dH 5.50e5.80 for the acyl groups VI and VII (aromatic ring).
Additionally, the 13C NMR chemical shifts of the signals of C-2 and
C-3 were situated at dC 68.24 and 83.17 for MA and dC 68.98 and
83.90 for MA-Bn, respectively, whereas the C-2 signals were
deshielded up to dC 73e74 for the 2-acyl derivatives, and up to dC
69e71 for the 2,3-diacyl derivatives. The C-3 signals were shielded
down to dC 80e82 for both monoacylated and diacylated
derivatives.
phase processes. The 2-chlorotrityl chloride polymer resin (Barlos
resin, PL Cl-Trt-Cl resin, CTC resin) was chosen as the solid support,
since this resin allows the release of the compounds by treatment
under mild acidic conditions that do not affect the triterpene
skeleton. First, it became necessary to study and to quantify the
incorporation of OA and MA into this resin. Therefore, a sequence of
chemical reactions was carried out using amino acids with an Fmoc
(fluorenylmethyloxycarbonyl) group incorporated, since this group
absorbs at a wavelength (301 nm) that does not interfere with that
of the other compounds. In this way, the above-mentioned tri-
terpene acids were attached to the resin through its 28-carboxyl
group, and later, a treatment was made with Fmoc-Leu-OH and
MeOH to cap the resin. Afterwards, Fmoc-Gly-OH was attached to
the hydroxyl groups of the A-ring, and finally, the resin was cleaved
with TFA in DCM, producing free Fmoc-Leu-OH, and OA and MA,
which incorporated Fmoc-Gly-OH in their hydroxyl groups (66e
69). Additionally, to determine the better yield of triterpene acid
loaded to the solid support, we made three tests with different
proportions of OA and MA (0.5, 1.0, and 3.0 equiv with respect to
the resin) (Scheme 2).
2.1.2. Solid-phase acylations of OA and MA
Several of these acylated derivatives (acetic, hexanoic, benzoic,
and succinic) of OA and MA, were also semi-synthesized by solid-

A. Parra et al. / European Journal of Medicinal Chemistry 74 (2014) 278e301
283
The incorporation of OA to the CTC resin was almost quantitative
with 3 equiv of this triterpene acid. The maximum incorporation of
MA into the resin was about a 92%, also with 3 equiv of this com-
pound. This percentage was distributed between the C-2 derivative
(68.1%), the C-2/C-3 derivative (17.2%), and the C-3 derivative (6.8%)
(Table 3). This distribution of derivatives was reasonable, given that
the C-3 hydroxyl group has more steric hindrance due to the
presence of the angular methyl groups on C-4. This quantification
was achieved by HPLC at 301 nm of absorbance, and the Fmoc-Gly-
derivatives of OA and MA, used as reference compounds, were
prepared via the solution-phase. Thus, the reaction of OA (1) with
Fmoc-Gly-OH in DCM with DIPCDI and DMAP at reflux for 12 h,
gave 3-Fmoc-Gly-OA (66) (Table 3 and Scheme 2). The 1H NMR
spectrum of this compound (66) presented the following deshiel-
ded signals: dH 4.01 (2H, d, J ¼ 3.5 Hz, 2H-20 glycine methylene),
4.26 (1H, t, J ¼ 6.8 Hz, H-300 proton of the cyclopentane ring of the
Fmoc group), 4.43 (2H, d, J ¼ 6.8 Hz, H-200 methylene of the Fmoc
group), 4.62 (1H, dd, J1 ¼ J2 ¼ 7.5 Hz, H-3), and 5.31 (1H, dd,
J1 ¼ J2 ¼ 3.3 Hz, H-12). In its 13C NMR spectrum the signals of the
carbonyl and methylene groups of the glycine (dC 170.1 and 43.2),
those of the carbonyl and methylene groups of the Fmoc group (dC
156.5 and 67.4), and those of C-3 and C-28 (dC 82.8 and 184.0), were
detected.
the nearly complete benzoylation process of OA (94.8%) required
4 h of reaction time, and the acylation with succinic anhydride was
much more difficult and slow, reaching a yield of only 53.7% after
48 h of reaction (Table 4).
The solid-phase acylations of MA (2) with the same four acid
anhydrides provided more complex mixtures of compounds
because of the presence of the two hydroxyl groups in the A-ring of
the substrate (2). Nevertheless, the acylation reactions with the
more apolar and aliphatic acid anhydrides (acetic and hexanoic)
only gave the doubly acylated compounds (8 and 33, respectively)
in 2 h. The benzoylation process took place more slowly, requiring
24 h for a yield of 94.9% of the doubly benzoyl derivative (37). In this
acylation reaction, we identified the 2-benzoyl derivative (36) but
did not detect the possible 3-benzoyl derivative. Finally, the solid-
phase succinylation of MA yielded 81.1% of the diacylated deriva-
tive (41) after 48 h of reaction. This yield was higher than the one
reached in the formation of the succinyl derivative of OA (15). The
succinylation reaction of MA probably first formed the 2-succinyl
derivative (40), which seems to help the entry at the more hin-
dered C-3 hydroxyl group, and therefore the doubly acylated
compound (41) gave very high yield. In this case, we also detected
very low proportions of what may be a possible 3-succinyl deriv-
ative of MA (Table 5).
The reaction of MA (2) with Fmoc-Gly-OH in DCM with DIPCDI
and DMAP at rt for 24 h, gave 2-Fmoc-Gly-MA (67), 3-Fmoc-Gly-MA
(68), and 2,3-diFmoc-Gly-MA (69) (Table 3 and Scheme 2). The
NMR spectra of compounds 67 and 68 differed only in the signals of
H-2/C-2 and H-3/C-3. In the 1H NMR spectrum of 67, the signal of H-
2 appeared at dH 5.06 and that of H-3 at dH 3.19, whereas for 68, the
signals of these protons were detected at dH 3.80 and 4.60,
respectively. In the 13C NMR spectrum of 67, the signal of C-2
appeared at dC 74.9 and that of C-3 at dC 80.7, whereas for 68, the
signals of these carbons were detected at dC 67.3 and 86.6,
respectively. The doubly substituted derivative 2,3-diFmoc-Gly-MA
(69) had a 1H NMR spectrum in which the signal of H-2 appeared at
dH 5.34, and that of H-3 at dH 4.80. In the 13C NMR spectrum of 69,
the signal of C-2 was placed at dC 71.0 and that of C-3 at dC 82.1.
The acid anhydrides selected to carry out the acylation reactions
in solid-phase were: acetic, hexanoic, benzoic and succinic. First,
OA and MA were incorporated into the resin under the above-
described optimized conditions (3 equiv of the triterpene acid).
After this, the four selected acid anhydrides were added to the
corresponding syringe in a molar relationship of 4:1 (anhy-
dride:hydroxyl group), in the presence of DMAP and Et3N. Finally,
after 24 h of orbital stirring at rt, the resin was cleaved with TFA/
DCM, and the residue was analyzed by HPLC (Scheme 3).
In these acylation studies, we found that the acylated derivatives
of OA and MA that were achieved from a cyclic acid anhydride
(phtalic, succinic, glutaric, and 3,3-dimethylglutaric) were com-
pounds of very difficult chromatographic isolation and also of
complicated spectroscopic identification, due to the presence of
two or three free carboxylic groups in the molecule. Therefore, to
avoid these experimental problems, we selected the succinyl de-
rivatives to study the protection of the free carboxylic groups,
through a benzylation reaction. Accordingly, when OA (incorpo-
rated to the resin) was succinylated in solid-phase, cleaved from the
resin, and benzylated with benzyl chloride, the corresponding
benzyl oleanolate (3, 40%), and benzyl 3-(benzylsuccinyl) ole-
anolate (70, 55%) were formed (Scheme 4). The NMR spectra of 70
presented the characteristic signals of a doubly benzylated com-
pound, 10 aromatic protons at dH 7.36e7.35, two AB systems at dH
5.13 and 5.07, 12 aromatic carbons between 136 and 128 ppm, and
two methylene groups at dC 66.5 and 65.9 (see the Experimental
Section). In a similar process, MA was also succinylated and ben-
zylated to give: benzyl maslinate (4, 6%), benzyl 2-(benzylsuccinyl)
maslinate (71, 8%), benzyl 3-(benzylsuccinyl) maslinate (72, 6%),
and benzyl 2,3-di(benzylsuccinyl) maslinate (73, 74%) (Scheme 4).
The benzylsuccinyl derivatives of MA (71e73) were also easily
chromatographically separated, and identified from their NMR
properties. Hence, with these benzylation reactions the proportions
of succinylated derivatives achieved from the solid-phase succi-
nylation of OA and MA could be determined more accurately.
The solid-phase acylation reactions of OA (1) with the acetic and
hexanoic anhydrides were almost completed in 30 min. However,
2.2. Cytotoxicity
Table 3
Tests of incorporation of the triterpenic acids to the CTC resin.
We determined the bioactive potential of these semi-
synthesized oleanolic and maslinic acid derivatives (1e65), as
anti-proliferative and pro-apoptotic compounds, and as anti-virals
on HIV-serine protease (Fig. 5).
Cytotoxicity effects have been previously described in a wide
variety of pentacyclic triterpenes, involving a mechanism that
implied MAPK (mitogen-activated protein kinases), death receptor,
and mitochondrial disruption [19,33]. Also, the cytotoxic effects of
3-O-acyl derivatives of several pentacyclic triterpenes have been
described [58,59].
Test incorporation OA to the resin
Equivalents of OA respect to the resin
0.5 equiv
1 equiv
3 equiv
Fmoc-Leu-OH (Rt ¼ 11.59 min)
32.2%
67.8%
13.9%
86.1%
1.1%
98.9%
Fmoc-Gly-OA (66) (Rt ¼ 34.74 min)
Test incorporation MA to the resin
Equivalents of MA respect to
the resin
0.5 equiv
1 equiv
3 equiv
Fmoc-Leu-OH (Rt ¼ 11.61 min)
39.4%
55.8%
1.9%
17.8%
67.3%
3.8%
7.9%
68.1%
6.8%
2-(Fmoc-Gly)-MA (67) (Rt ¼ 31.84 min)
3-(Fmoc-Gly)-MA (68) (Rt ¼ 33.42 min)
2,3-di(Fmoc-Gly)-MA (69) (Rt ¼ 35.82 min)
We examined the effects of the semi-synthesized acylated MA
and OA derivatives on the proliferation of b16f10 cell line using the
2.9%
11.1%
17.2%
MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

284
A. Parra et al. / European Journal of Medicinal Chemistry 74 (2014) 278e301
Scheme 3. Solid-phase acylation reactions of OA and MA.
bromide) assay. Thus, these murine melanoma cells were treated
with increasing doses (range 0e80 g/mL) of each derivative, and
C-3 (R2) in the acyl MA derivatives appears to inhibit the cytotox-
icity of these compounds. The exception observed with the diacetyl
derivative 8 may be due to the small size of this acyl group (Table 6
and Chart 1). Chart 1 shows a comparison of the growth-inhibitory
effects of the acyl OA and MA derivatives on b16f10 murine mela-
noma cells, describing the ratio between the IC50 values of the
triterpene precursors (OA, MA, OA-Bn and MA-Bn) and the IC50
values of their cytotoxic acyl derivatives (last column of Table 6).
Several cytometry studies, described below, were made to
determine the apoptotic properties of the compounds tested (1e
65). The in vitro IC50 growth-inhibitory concentrations of the new
semi-synthesized compounds were, in many cases, similar of those
of their precursors (1e4), but sufficiently low to investigate their
apoptotic properties.
m
their viability was determined by formazan dye uptake, and
expressed as a percentage of untreated control cells. The results of
these assays showed that only some 24% of the compounds tested
(1e65) displayed cytotoxic effects on this cell line. The numbers
and structures of these cytotoxic compounds and their IC50 values
are given in Table 6. Comparing the IC50 data for OA (106.4
mM) and
MA (36.2 M), we deduce that the hydroxyl group at C-2 enhances
m
the cytotoxicity of this compound (2). Furthermore, considering the
IC50 data for OA and MA, and those of their corresponding 28-
benzyl derivatives (3 and 4), we find that benzylation reduces the
IC50 concentrations by about 50% (Table 6).
The acyl OA derivatives with cytotoxic effects were only com-
pounds 5, 9, and 13, and only the acetyl derivative (5,
IC50 ¼ 64.7
cursor (1). Nevertheless, the acyl 28-benzyl OA derivatives (24e27)
with cytotoxic effects (IC50 from 14.2 to 29.9 M), showed an IC50
underneath its precursor (3). In terms of structureeactivity rela-
tionship (SAR), the better cytotoxic results for the acyl OA de-
rivatives were achieved when the acyl group at C-3 (R2) was a polar
dicarboxylic fragment, such as phthaloyl, succinyl, glutaryl, or 3,3-
dimethylglutaryl (compounds 24e27), and a benzyl group was
placed at the carboxylic group (R3) (Table 6 and Chart 1).
m
M) showed a lower IC50 concentration than its pre-
2.3. Apoptosis
m
Different types of pentacyclic triterpenoids have shown anti-
cancer properties ranging from inhibition of tumour-cell prolifer-
ation, induction of apoptosis, suppression of angiogenesis, and
metastasis. These triterpenoids have the ability to suppress various
key steps of initiation, progression, and promotion of tumours, and
therefore have the potential for use in both chemoprevention and
therapy of various cancers with several common molecular targets
[33,60]. Recently, the anticancer properties of a 3-O-acyl derivative
of a pentacyclic triterpene have been reported [61].
The acyl MA derivatives (8, 30, and 32) exhibited relatively low
cytotoxicity (IC50 from 34.6 to 106.6
mM), although in the same
range as found in its precursor (2). Similarly, several acyl 28-benzyl
MA derivatives (46, 58, 62, and 64) showed low IC50 (from 28.3 to
73.2 mM), but above of its precursor (4). The acyl MA derivatives
that showed cytotoxicity had only one acyl group at C-2 (R1),
whereas the hydroxyl group at C-3 (R2) was free, except the diacetyl
derivative 8. The presence of double acyl groups at C-2 (R1) and at
The apoptotic determination assays were conducted through
double staining with An-V (annexin V) conjugated FITC (fluorescein
isothiocyanate) and PI (propidium iodide). Early events in the
apoptotic processes are loss of plasma-membrane asymmetry
accompanied by translocation of PS (phosphatidylserine) from the
inner to the outer membrane leaflet, thereby exposing PS to the

A. Parra et al. / European Journal of Medicinal Chemistry 74 (2014) 278e301
285
Table 4
Solid-phase acylation of OA.
annexin V/propidium iodide flow-cytometry for three of the com-
pounds that display higher apoptotic cell populations (24, 25, and
46).
Time (h)
Acyl group
Compound
OA
Though the structureeactivity relationship of these OA and MA
derivatives was not absolutely well defined, it seems that the
presence of a benzyl group blocking the carboxylic group at C-28
and a polar dicarboxylic acyl substituent at C-2 (R1) or C-3 (R2),
enhanced the apoptotic properties of these derivatives, compared
with the corresponding precursors. The potent activities on the
apoptosis-activation process reached by these derivatives suggest
that they could be used as anticancer drugs.
3-Acyl-OA
Yield (%)
Yield (%)
Rt (min)
Rt (min)
0.5
0.5
2
4
2
4
8
24
48
Acetyl
Hexanoyl
Benzoyl
1.5
1.1
23.5
3.2
94.4
90.1
79.6
59.8
43.6
9.5
98.3
96.4
75.1
94.8
2.5
14.1
22.4
25.0
Succinyl
9.0
6.8
18.2
36.9
53.7
2.4. ROS production
Mitochondria constitute a key target in synthetic and natural
triterpenoids [7]. Pentacyclic triterpene compounds have proved to
be able to activate the intrinsic apoptotic pathway, mediated with
mitochondrial disruption [36,37], and the extrinsic apoptotic
pathway [33], mediated by an enhanced receptor [62]. The over
production of ROS in OA and MA treatment on several cancer cell
lines has also been reported [34,35,63]. Changes in the
mitochondrial-membrane potential can be examined by moni-
toring the cell fluorescence after double staining with Rh123
(rhodamine 123) and PI (propidium iodide). Rh123 is a membrane-
permeable fluorescent cationic dye that is selectively taken up by
mitochondria directly proportional to the MMP (mitochondrial-
membrane permeabilization) [64]. Most studies on the apoptosis
molecular mechanism show that when the intrinsic apoptotic
mechanism is activated, a rapid production of the ROS is generated
by the mitochondrial disruption, although this also can be triggered
in the final steps of extrinsic apoptotic activation if the bit protein is
activated by the initiator caspases 8 or 10 [65,66].
We analyzed the ROS production to elucidate the possible
mechanism involved in the apoptotic responses in the b16f10
mouse melanoma cells to the acyl OA and MA derivatives, at the
same times and concentrations of the cytotoxic and apoptosis re-
sponses. These studies were performed to determine whether ROS
generation occurs simultaneously or not with apoptosis. If so, the
mitochondrial disruption would be implicated in the apoptotic
response, but if not the apoptosis could be caused by the extrinsic
apoptotic-route activation. Changes in the MMP were analyzed by
flow-cytometry after staining with Rh123.
external environment. The phospholipid-binding protein annexin V
has a high affinity for PS and binds to cells fluorescently labelled
with FITC. The percentages of apoptosis were determined with
annexin VeFITC/PI flow-cytometric analysis. Apoptosis in b16f10
cells was assessed 72 h after treatment with the different previ-
ously cytotoxic OA and MA derivatives at the IC50 concentrations
before administered (Table 6), and at the IC50 ꢁ 2 concentrations
(twice IC50 concentrations. ꢁ2 values corresponding to 50%
viability). FACS (flow-activated cell sorter) analysis using annexin
VeFITC staining and PI accumulation was used to differentiate early
apoptotic cells (An-Vþ and PIꢂ) from late apoptotic (An-Vþ and PIþ),
necrotic (An-Vꢂ and PIþ) or normal cells (An-Vꢂ and PIꢂ).
All the cytotoxic compounds showed apoptotic effects on the
treated cells with a high percentage of total apoptosis between 51
and 95% at IC50 concentration, except product 62 (25.2% of total
apoptosis), and between 60 and 98% at IC50 ꢁ 2 concentrations
(Table 7). In addition, the percentages of the necrotic population for
all these acyl OA and MA derivatives were irrelevant. Several
compounds (9, 24, 25, 27, 30, and 46) showed a total apoptosis
above 70%, at its IC50 concentrations; three of these were the acyl
28-benzyl OA derivatives (24, 25, and 27) with a polar dicarboxylic
substituent at C-3 (R2), which displayed the most cytotoxic prop-
erties. Ten compounds (8, 24e27, 30, 32, 46, 58, and 64) showed a
percentage of total apoptosis above 80%, at its IC50 ꢁ 2 concentra-
tions, higher percentages corresponding to compounds with a
benzyl group blocking the carboxylic group of the triterpene skel-
eton, and a polar dicarboxylic acyl substituent at C-2 (R1) or C-3
(R2). In addition, seven of the compounds tested (9, 24, 27, 30, 32,
46, and 58) showed a higher percentage of early apoptosis than late
apoptosis, at its IC50 concentrations (between 42 and 66% of total
cell population), whereas nine compounds (9, 24e27, 30, 32, 46,
and 62) did (between 44 and 74% of total cells) at its IC50 ꢁ 2
concentrations (Table 7 and Chart 2). Fig. 6 presents the diagrams of
The results at the IC50 concentration, showed strong Rh123
fluorescence intensity in the response in many of the compounds
tested, except for compounds 24, 25, 26, and 58 (Chart 3). From
these results, it can be interpreted that compounds 5, 8, 9,13, 27, 30,
32, 46, 62, and 64 caused mitochondrial disruption in the activation
of apoptosis, whereas in the compounds 24, 25, 26, and 58, the
Table 5
Solid-phase acylation of MA.
Time (h)
Acyl group
Compound
MA
2-Acyl-MA
Yield (%)
3-Acyl-MA
Yield (%)
2,3-Diacyl-MA
Yield (%)
Yield (%)
Rt (min)
Rt (min)
Rt (min)
Rt (min)
2
2
2
4
8
24
2
4
8
Acetyl
Hexanoyl
Benzoyl
1.2
0.8
2.2
1.8
1.1
4.7
0.0
0.0
47.3
32.9
23.7
3.4
41.2
31.5
20.1
14.5
7.1
e
0.0
0.0
0.0
0.0
0.0
0.0
1.7
2.4
3.8
4.2
4.8
e
e
e
96.4
97.1
49.4
64.3
73.9
94.9
23.4
37.1
56.3
69.2
81.1
10.0
24.9
19.8
e
10.1
0.6
Succinyl
32.5
27.6
18.4
10.2
6.3
3.7
1.3
1.5
24
48

286
A. Parra et al. / European Journal of Medicinal Chemistry 74 (2014) 278e301
Scheme 4. Solid-phase succinylation reactions and consecutive solution-phase benzylation reactions of OA and MA.
induction of apoptosis was not concomitant with the ROS genera-
tion. The results found at the IC50 ꢁ 2 concentration were very
similar to those found at the IC50 concentration, and showed the
generation or not of ROS for the same compounds. These results
could be related with an extrinsic activation of the apoptosis
mechanism for compounds 24, 25, 26, and 58, and the others by the
intrinsic apoptotic-route activation. In any case, further molecular
studies in this sense will be necessary to confirm this assertion.
Fig. 7 shows the diagram of Rh123/PI flow-cytometry, for the
compounds with minor (24 and 25) and with major (32 and 62)
Rh123 staining. The Rh123/PI labelling of b16f10 cells was carried
out as described in the experimental section. This diagram in-
dicates that, for compounds 24 and 25 (negative ROS production),
the major cell population was distributed into the Q1 and Q3 left
quadrants, whereas for compounds 32 and 62 it was contained into
the Q2 and Q4 right quadrants.
2.5. Anti-HIV-1-protease activity
Pentacyclic triterpenes have been found to exhibit anti-HIV
activities. The modes of action of the anti-HIV triterpenes have
been reported to be associated with virus entry, reverse tran-
scription, virus assembly, and maturation [67]. In many cases, the
anti-HIV maturation activity has been associated with the presence
of an acyl group at C-3 of the pentacyclic triterpene skeleton
[24,26,68e70], whereas a side chain at C-28 has been related with
the anti-HIV-1 entry activity [53,71].
The determination of the concentration of the products that
account for the inhibition of 50% activity of HIV-1-protease or the
IC50 concentration using the FRET (fluorescence resonance energy
transfer) technique with a fluorescence resonance energy transfer
peptide. The different compounds were considered to be inhibitors
of the HIV-1-protease when their IC50 values were within the
Finally, we can conclude that, although the ROS production
structureeactivity relationship was not absolutely clear, the com-
pounds in which the ROS production was totally or partially
negative had a polar dicarboxylic substituent at C-2 (R1), and a
benzyl group blocked the carboxyl group at C-28 of the triterpene
skeleton.
concentration range (0e1200 mg/mL). The results from this test for
all the acyl OA and MA derivatives (1e65) showed strong inhibitory
capacity of the HIV-1-protease by the 38% of the compounds
(Table 8 and Chart 4). From the set of the acyl OA derivatives
assayed, only five compounds (5, 14, 16, 17, and 24) presented anti-
HIV activity. Most of the active acyl OA derivatives had a polar
Fig. 5. General structures of the tested acyl OA and MA derivatives.

A. Parra et al. / European Journal of Medicinal Chemistry 74 (2014) 278e301
287
Table 6
Growth-inhibitory effects of acyl OA and MA derivatives on b16f10 murine melanoma cells.
Comp. #
R1
R2
R3
IC50 concentration (
m
M)a
IC50 of precursor/IC50 of compound #b
OA (1)
5
9
13
e
e
e
e
H
H
H
H
H
106.4 ꢃ 3.7
64.7 ꢃ 2.8
1.0
1.6
0.7
0.7
Acetyl
Propanoyl
Benzoyl
161.1 ꢃ 9.8
162.1 ꢃ 18.4
OA-Bn (3)
e
e
e
e
e
H
Bn
Bn
Bn
Bn
Bn
52.2 ꢃ 0.9
26.8 ꢃ 0.9
23.2 ꢃ 0.3
29.9 ꢃ 0.9
14.2 ꢃ 1.0
1.0
1.9
2.3
1.7
3.7
24
25
26
27
Phthaloyl
Succinyl
Glutaryl
3,3-Dimethylglutaryl
MA (2)
8
30
32
H
H
Acetyl
H
H
H
H
H
H
36.2 ꢃ 3.8
44.1 ꢃ 3.8
106.6 ꢃ 11.3
34.6 ꢃ 2.1
1.0
0.8
0.3
1.0
Acetyl
Butanoyl
Hexanoyl
MA-Bn (4)
H
Acetyl
Phthaloyl
Glutaryl
H
H
H
H
H
Bn
Bn
Bn
Bn
Bn
19.0 ꢃ 0.2
39.6 ꢃ 0.8
73.2 ꢃ 2.4
63.2 ꢃ 2.1
28.3 ꢃ 1.1
1.0
0.5
0.3
0.3
0.7
46
58
62
64
3,3-Dimethylglutaryl
a
The IC50 values were calculated considering control untreated cells as 100% of viability. Cell-growth inhibition was analyzed by the MTT assay, as described in the
Experimental section. All assays were made four times using six replicates. Values, means ꢃ S.E.M.
b
The last column represents the ratio between the IC50 of each precursor (OA, OA-Bn, MA or MA-Bn) and the IC50 of the related acyl derivatives.
dicarboxylic substituent at C-3 (R2), and exhibited an excellent
inhibitory capacity, with IC50 values between 0.31 and 0.70 M,
3. Conclusions
m
which are between 82 and 186 times lower than its precursor (OA)
(see Table 8 and Chart 4).
The comparative study of the solution- and solid-phase semi-
synthesis of the OA and MA derivatives showed that the solid-
phase procedures were shorter, easier, cleaner, and with better
yields than the corresponding solution processes, when using an
aliphatic or an aromatic acyl group. In this solid-phase process, we
determined a high degree of incorporation (above 90%) of OA and
MA to the CTC resin, when coupling with 3 equiv of these acids.
The acyl OA and MA derivatives were tested for their anti-
proliferative effects. Some of these derivatives (24%) displayed
cytotoxic effects (low IC50) as growth inhibitors against the b16f10
murine melanoma cells. These cytotoxic compounds showed
apoptotic effects on the treated cells, with high levels of early and
total apoptosis, reaching in several cases, values of apoptosis pop-
ulation up to 90% of the total cells. The study of the structureeac-
tivity relationship indicated that the more cytotoxic and pro-
There were 18 acyl MA derivatives that inhibited the HIV-1-
protease (Table 8). Only compounds 32, 50, 53, and 55, showed
lower activities than their precursors, and all of them had an
aliphatic substituent at C-2 (R1) or at C-2 and C-3 (R2), such as
butanoyl, hexanoyl or lauroyl group. The most active acyl MA de-
rivatives had polar dicarboxylic substituents, such as phthaloyl,
succinyl or glutaryl group, and exhibited a good inhibitory capacity,
with IC50 values between 0.8 and 15.6 mM, which were between 4
and 78 times lower than its precursor (MA) (see Table 8 and Chart
4). Chart 4 shows a comparative of the inhibitory effects of the acyl
OA and MA derivatives on the HIV-1-protease, describing the ratio
between the IC50 values of the triterpene precursors (OA and MA)
and the IC50 values of their acyl derivatives (last column of Table 8).
Chart 1. Comparison of the growth-inhibitory effects of the acyl OA and MA derivatives on b16f10 murine melanoma cells, describing the ratio between the IC50 values of the
triterpene precursors (1e4) and the IC50 values of their cytotoxic acyl derivatives. DMG ¼ 3,3-dimethylglutaryl, Glu ¼ glutaryl, Succ ¼ succinyl, Phta ¼ phthaloyl, Bz ¼ benzoyl,
Laur ¼ lauroyl, Hex ¼ hexanoyl, But ¼ butanoyl, Prop ¼ propanoyl, Ac ¼ acetyl, Precursors ¼ OA (1), MA (2), OA-Bn (3), and MA-Bn (4).

288
A. Parra et al. / European Journal of Medicinal Chemistry 74 (2014) 278e301
Table 7
derivatives had a polar dicarboxylic substituent at C-2 or at C-2 and
C-3 of the triterpene skeleton.
Flow-cytometry analysis of annexin VeFITC staining and PI accumulation after
exposure of b16f10 murine melanoma cells to acyl OA and MA derivatives.
The potent activities on the apoptosis-activation process and on
the inhibition of the HIV-1-protease reached by some of the tested
OA and MA acylated derivatives suggest that they could be used in
the future as new, safe, and effective anticancer and/or antiviral
drugs.
Comp. #
Conc.a
Populationb
Normal
Early
Late
Total
Necrosis
apoptosis apoptosis apoptosis
Control
0
77.9 ꢃ 1.5 3.9 ꢃ 0.4 9.8 ꢃ 2.2 13.7 ꢃ 2.7 4.0 ꢃ 0.6
44.9 ꢃ 5.1 16.4 ꢃ 2.3 28.2 ꢃ 2.0 44.6 ꢃ 4.3 10.1 ꢃ 1.0
OA (1)
IC50
4. Experimental
IC50 ꢁ 2 49.8 ꢃ 6.4 29.1 ꢃ 6.0 12.4 ꢃ 1.6 41.5 ꢃ 7.5 8.7 ꢃ 1.4
5
IC50
IC50 ꢁ 2 46.3 ꢃ 6.8 14.6 ꢃ 0.6 33.4 ꢃ 3.5 48.0 ꢃ 4.1 5.7 ꢃ 2.8
IC50
24.3 ꢃ 6.8 47.0 ꢃ 0.0 27.4 ꢃ 7.0 74.4 ꢃ 7.0 1.3 ꢃ 0.1
IC50 ꢁ 2 37.2 ꢃ 8.9 44.3 ꢃ 4.7 16.5 ꢃ 4.3 60.8 ꢃ 9.1 2.0 ꢃ 0.1
IC50
44.6 ꢃ 1.9 14.0 ꢃ 1.1 37.6 ꢃ 0.5 51.6 ꢃ 1.6 3.8 ꢃ 0.4
IC50 ꢁ 2 31.6 ꢃ 0.2 9.5 ꢃ 0.4 50.5 ꢃ 1.2 60.0 ꢃ 1.6 8.5 ꢃ 1.8
39.5 ꢃ 6.5 14.4 ꢃ 2.4 40.3 ꢃ 5.8 54.7 ꢃ 7.2 5.8 ꢃ 1.4
4.1. General experimental chemical procedures
9
Measurements of NMR spectra (300.13 MHz 1H and 75.47 MHz
13C) were made in CDCl3, CD3OD, DMSO-d6 or Pyridine-d5 (which
also provided the lock signal) using a VARIAN Inova unity (300 MHz
1H NMR). The 13C chemical shifts were assigned with the aid of
distortionless enhancement by polarization transfer (DEPT) using a
flip angle of 135ꢀ. IR spectra were recorded on a MATTSON SAT-
ELLITE FTIR spectrometer. Optical rotations were measured on a
PerkineElmer 241 polarimeter at 25 ꢀC. Purity of new compounds
was determined by a WATERS ACQUITY UPLC system (ultra-per-
formance liquid chromatography) coupled with a WATERS SYNAPT
G2 HRMS spectrometer (high-resolution mass spectra) with ESI
(electrospray ionization). Melting points (mp) were determined
using a Kofler (Reichter) apparatus and were uncorrected. All re-
action solvents and chromatography solvents were distilled prior to
use. Commercially available reagents were used without further
purification. Merck silica-gel 60 aluminium sheets (ref. 1.16835)
were used for TLC, and spots were rendered visible by spraying
with H2SO4eAcOH, followed by heating to 120 ꢀC, and also visu-
alized under UV at 254 nm. Merck silica-gel 60 (0.040e0.063 mm,
ref. 1.09385) was used for flash chromatography. CH2Cl2 (Fisher, ref.
D/1852/17), CHCl3 (Fisher, ref. C/4960/17), or n-hexane (Merck, ref.
1.04374), with increasing amounts of Me2CO (Fisher, ref. A/0600/
17), MeOH (Fisher, ref. M/4000/17), or AcOEt (Fisher, ref. E/0900/
17), were used as eluents (all the solvents had an analytical reagent-
grade purity). RP-HPLC analyses were made with a WATERS C18
13
OA-Bn (3) IC50
IC50 ꢁ 2 54.3 ꢃ 5.4 23.0 ꢃ 5.1 18.6 ꢃ 2.1 41.6 ꢃ 6.5 4.1 ꢃ 1.5
IC50
3.9 ꢃ 0.3 64.3 ꢃ 5.6 30.3 ꢃ 5.2 94.6 ꢃ 0.5 1.5 ꢃ 0.2
IC50 ꢁ 2 3.7 ꢃ 0.5 66.9 ꢃ 2.8 28.5 ꢃ 3.3 95.4 ꢃ 0.5 1.0 ꢃ 0.0
IC50
16.9 ꢃ 2.1 16.6 ꢃ 2.8 55.5 ꢃ 8.2 72.1 ꢃ 5.5 11.1 ꢃ 3.4
IC50 ꢁ 2 7.7 ꢃ 2.5 74.0 ꢃ 0.8 17.6 ꢃ 3.3 91.6 ꢃ 2.4 0.7 ꢃ 0.0
IC50
25.8 ꢃ 4.5 20.6 ꢃ 1.9 37.9 ꢃ 1.9 58.4 ꢃ 0.1 15.9 ꢃ 4.4
IC50 ꢁ 2 8.1 ꢃ 0.8 65.8 ꢃ 0.1 24.6 ꢃ 1.1 90.4 ꢃ 1.0 1.6 ꢃ 0.1
IC50
8.3 ꢃ 2.0 44.0 ꢃ 4.4 42.6 ꢃ 7.7 86.5 ꢃ 3.3 5.3 ꢃ 1.3
IC50 ꢁ 2 4.9 ꢃ 0.6 52.8 ꢃ 5.7 36.1 ꢃ 5.6 88.8 ꢃ 0.1 6.4 ꢃ 0.7
67.9 ꢃ 3.5 15.6 ꢃ 1.2 12.2 ꢃ 1.7 27.8 ꢃ 0.8 4.3 ꢃ 2.6
24
25
26
27
MA (2)
IC50
IC50 ꢁ 2 8.3 ꢃ 1.8 68.8 ꢃ 3.9 21.0 ꢃ 1.9 89.8 ꢃ 2.5 2.0 ꢃ 0.7
IC50
31.4 ꢃ 1.3 8.7 ꢃ 0.0 52.9 ꢃ 3.0 61.6 ꢃ 3.0 7.1 ꢃ 1.7
IC50 ꢁ 2 9.7 ꢃ 5.6 29.6 ꢃ 7.6 59.3 ꢃ 6.5 88.9 ꢃ 6.0 1.5 ꢃ 0.5
IC50
21.0 ꢃ 8.2 55.6 ꢃ 5.5 22.1 ꢃ 2.0 77.7 ꢃ 8.5 1.2 ꢃ 0.3
IC50 ꢁ 2 10.2 ꢃ 1.6 53.0 ꢃ 2.9 30.0 ꢃ 5.7 83.0 ꢃ 2.8 6.8 ꢃ 1.2
IC50
34.8 ꢃ 1.7 42.4 ꢃ 3.3 19.0 ꢃ 0.4 61.4 ꢃ 3.6 3.9 ꢃ 1.9
IC50 ꢁ 2 18.9 ꢃ 0.5 62.7 ꢃ 0.9 17.5 ꢃ 0.5 80.2 ꢃ 0.5 1.1 ꢃ 0.0
10.9 ꢃ 1.7 20.8 ꢃ 2.8 54.2 ꢃ 3.5 74.9 ꢃ 2.5 11.7 ꢃ 0.5
8
30
32
MA-Bn (4) IC50
IC50 ꢁ 2 11.3 ꢃ 4.2 86.2 ꢃ 5.0 2.2 ꢃ 1.0 88.4 ꢃ 4.1 0.1 ꢃ 0.1
IC50
12.9 ꢃ 0.9 66.0 ꢃ 0.9 20.0 ꢃ 0.3 86.0 ꢃ 0.6 1.2 ꢃ 0.3
IC50 ꢁ 2 1.1 ꢃ 0.1 71.3 ꢃ 2.2 27.1 ꢃ 2.1 98.4 ꢃ 0.1 0.6 ꢃ 0.0
IC50
32.8 ꢃ 4.7 42.2 ꢃ 6.3 22.5 ꢃ 1.4 64.7 ꢃ 4.9 2.5 ꢃ 0.2
IC50 ꢁ 2 2.9 ꢃ 0.7 43.6 ꢃ 3.7 46.2 ꢃ 1.4 89.7 ꢃ 2.3 7.5 ꢃ 1.7
IC50
73.3 ꢃ 1.7 10.6 ꢃ 1.7 14.6 ꢃ 0.1 25.2 ꢃ 1.6 1.6 ꢃ 0.1
IC50 ꢁ 2 20.2 ꢃ 4.3 59.1 ꢃ 9.1 19.8 ꢃ 1.2 78.9 ꢃ 7.8 0.9 ꢃ 0.6
IC50
35.4 ꢃ 4.5 16.5 ꢃ 0.8 46.3 ꢃ 4.2 62.8 ꢃ 5.0 1.8 ꢃ 0.5
IC50 ꢁ 2 2.2 ꢃ 0.3 26.9 ꢃ 3.5 65.3 ꢃ 3.3 92.2 ꢃ 0.2 5.6 ꢃ 0.5
5.0 ꢃ 0.6 84.2 ꢃ 5.4 10.6 ꢃ 4.7 94.7 ꢃ 0.7 0.3 ꢃ 0.1
46
58
62
64
reverse-phase column (0.4
mm
diameter of particle;
150 mm ꢁ 3.9 mm) with a flow rate of 1 mL/min, on a WATERS
Liquid Chromatograph (HPLC), model ALLIANCE 2690 (Waters,
USA). Wavelengths of 210 and 301 nm were selected for the purity
analyses. The analyses were performed using a linear gradient of
30e0% of B in 30 min and maintained for 5 min without B, where A
was CH3CN containing 0.1% TFA and B was H2O containing 0.1% TFA.
CTC resin (1.27 mmol/g), Fmoc-amino acid derivatives, DIEA,
DIPCDI, and DMF were purchased from different sources.
a
Cell lines were treated at concentrations equal to its IC50 or IC50 ꢁ 2 values.
Values are expressed as means ꢃ S.E.M. of three experiments in duplicate.
b
Normal cells were annexin Vꢂ PIꢂ early apoptotic cells were annexin Vþ PIꢂ
whereas late apoptotic cells were annexin Vþ PIþ total apoptosis (early apoptosis
plus late apoptosis) were annexin Vþ and necrotic cells were annexin Vꢂ PIþ.
apoptotic compounds had a benzyl group on the carboxylic group
at C-28, and polar dicarboxylic fragments on the hydroxyl groups of
the A-ring of the triterpene skeleton.
4.2. Isolation of OA (1) and MA (2)
OA and MA were isolated from solid olive-oil-production
wastes, which were extracted successively in a Soxhlet with hex-
ane and EtOAc. Hexane extracts were a mixture of OA and MA
(80:20), whereas this relationship was (20:80) for the EtOAc ex-
tracts. Both products were purified from these mixtures by column
chromatography over silica gel, eluting with a CHCl3/MeOH or
CH2Cl2/acetone mixtures of increasing polarity [1,2,72].
Most of the compounds that exhibited anti-proliferative effects
also triggered ROS generation. Therefore, the apoptosis would
probably be caused by the participation of an intrinsic apoptotic-
route activation, and the mitochondrial disruption would be
involved in the apoptotic response. Only four compounds (24, 25,
26, and 58) did not release ROS, and these results could be related
with the participation of a plausible extrinsic activation of the
apoptosis mechanism. Further molecular studies are necessary to
test these conclusions, but both mechanisms on the apoptosis in-
duction by triterpenes compounds have been described in the
literature.
4.3. Synthesis of benzyl OA and MA derivatives
4.3.1. Benzyl oleanolate (3)
BnCl (418 mL) was added in a relationship of 2:1 to a solution of
A great number of acyl OA and MA derivatives (38%) were
demonstrated to be outstanding inhibitors of the anti-HIV-1-
protease, with IC50 values between 4 and 186 times lower than
their non-acylated precursors. The most active acyl OA and MA
OA (1, 912 mg, 2 mmol) in DMF (8 mL) with K2CO3 (0.61 g). The
reaction was stirred for 4 h at 55 ꢀC. The mixture was diluted with
water and extracted with DCM, and the organic layer dried with
anhydrous Na2SO4. The solvent was removed under reduced

A. Parra et al. / European Journal of Medicinal Chemistry 74 (2014) 278e301
289
Chart 2. Flow-cytometry analysis of annexin VeFITC staining and PI accumulation after exposure of b16f10 murine melanoma cells to acyl OA and MA derivatives for 72 h with
respect to untreated control cells. Cell lines were treated at concentrations equal to its IC50 or IC50 ꢁ 2 values. Early apoptotic cells (yellow bars) were annexin Vþ PIꢂ, whereas late
apoptotic cells (green bars) were annexin Vþ PIþ, total apoptosis (blue bars) were annexin Vþ, and necrotic cells (red bars) were annexin Vꢂ PIþ. (For interpretation of the references
to colour in this figure legend, the reader is referred to the web version of this article.)
pressure, and the residue was purified by column chromatography
using DCM/acetone (10:1) to give 3 as a white solid (710 mg, 83%)
[55].
acetone (10:1) to give 6, 7, and 8 as white solids (percentages in
Table 2) [57].
4.5. General method for acylation of 1e4 in solution-phase
4.3.2. Benzyl maslinate (4)
BnCl (418
m
L) was added in a relationship of 2:1 to a solution of
The corresponding acid anhydrides (IeX) were added respec-
tively, in a relationship of 4:1 (anhydride:hydroxyl group), to a
solution of compounds (1e4) in pyridine. The reactions were stir-
red for 24 h at rt. In all cases, cold water was added to the mixture
and afterwards 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 chro-
matography using DCM/acetone or hexane/ethyl acetate.
MA (2, 944 mg, 2 mmol) in DMF (8 mL) with K2CO3 (0.61 g). The
reaction was stirred for 4 h at 55 ꢀC. The mixture was diluted with
water and extracted with DCM, and the organic layer dried with
anhydrous Na2SO4. The solvent was removed under reduced pres-
sure, and the residue was purified by column chromatography us-
ing DCM/acetone (10:1) to give 4 as a white solid (690 mg, 81%)
[32].
4.4. Acetylations of OA and MA
4.5.1. Acylation of OA with propanoic anhydride
Propanoic anhydride (113 mL) was added slowly to a solution of
4.4.1. Acetylations of OA with Ac2O
OA (100 mg, 0.219 mmol) in pyridine (2 mL). The mixture was
treated as described in the general method for acylation to give 9 as
Ac2O (1 mL) was added slowly to a solution of OA (1, 1368 mg,
3 mmol) in pyridine (10 mL). The reaction was performed under
different conditions (see Table 1). Cold water was added to the
different mixtures, and afterwards these were extracted with DCM,
and the organic layers were dried with anhydrous Na2SO4. In all
cases, 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 (percentages in Table 1)
[56].
a white solid (89 mg, 83%), mp 245e247 ꢀC; [
a]
þ 71 (c 1 in
D
CHCl3:MeOH, 2:1); IR nmax(KBr)/cmꢂ1 3388, 3015, 2829 and 1720;
dH (CDCl3) 5.26 (dd, 1H, J1 ¼ J2 ¼ 3.5 Hz), 4.48 (dd, 1H, J1 ¼ 5.5,
J2 ¼ 10.5 Hz), 2.80 (dd, 1H, J1 ¼ 4.3, J2 ¼ 13.9 Hz), 2.30 (q, 2H,
J ¼ 6.5 Hz), 1.11, 0.92, 0.91, 0.88, 0.84, 0.83, 0.73 (s, 3H each); dC
(CDCl3) 183.8, 174.5, 143.8, 122.8, 80.9, 55.5, 47.8, 46.8, 46.1, 41.8,
41.2, 39.5, 38.3 (2C), 37.2, 34.0, 33.3, 32.8, 32.7, 30.9, 29.9, 28.8, 28.3,
27.9, 26.1, 23.8 (2C), 23.1, 18.4, 17.3, 16.9, 15.6, 9.6; ESI-HRMS m/z
calcd for C33H53O4 [M þ 1] 513.3944, found 513.3948.
4.4.2. Acetylations of MA with Ac2O
Ac2O (2 mL) was added slowly to a solution of MA (2, 1416 mg,
3 mmol) in pyridine (10 mL). The reaction was performed under
different conditions (see Table 2). Cold water was added to the
different mixtures, and afterwards these were extracted with DCM,
and the organic layers were dried with anhydrous Na2SO4. In all
cases, the solvent was removed under reduced pressure and the
residue was purified by column chromatography using DCM/
4.5.2. Acylation of OA with butanoic anhydride
Butanoic anhydride (143 mL) was added slowly to a solution of
OA (100 mg, 0.219 mmol) in pyridine (2 mL). The mixture was
treated as described in the general method to give 10 as a white
solid (96 mg, 87%), mp 215e217 ꢀC; [
a]
D þ 79 (c 1 in CHCl3:MeOH,
2:1); IR nmax(KBr)/cmꢂ1 3379, 2989, 2815 and 1699; dH (CDCl3) 5.26
(dd, 1H, J1 ¼ J2 ¼ 3.4 Hz), 4.48 (dd, 1H, J1 ¼ 5.3, J2 ¼ 10.5 Hz), 2.80

290
A. Parra et al. / European Journal of Medicinal Chemistry 74 (2014) 278e301
Fig. 6. Diagrams of annexin V/propidium iodide flow-cytometry for 24, 25, and 46. Q1 quadrant (necrotic cells). Q2 quadrant (late apoptotic cells). Q3 quadrant (normal cells). Q4
quadrant (early apoptotic cells). Left diagrams IC50 concentrations. Right diagrams IC50 ꢁ 2 concentrations.
(dd, 1H, J1 ¼ 4.3, J2 ¼ 13.9 Hz), 2.30 (t, 2H, J ¼ 6.5 Hz), 1.11, 0.92, 0.91,
0.88, 0.84, 0.83, 0.73 (s, 3H each); dC (CDCl3) 184.0, 173.7, 143.8,
122.8, 80.8, 55.5, 47.8, 46.8, 46.1, 41.8, 41.2, 39.5, 38.3 (2C), 37.2, 37.0,
34.0, 33.3, 32.8, 32.7, 30.9, 29.9, 28.3, 27.9, 26.1, 23.8, 23.6, 23.1, 18.9,
18.4, 17.3, 16.9, 16.5, 13.9; ESI-HRMS m/z calcd for C34H55O4 [M þ 1]
527.4100, found 527.4101.
dH (CDCl3) 5.26 (dd, 1H, J1 ¼ J2 ¼ 3.5 Hz), 4.80 (dd, 1H, J1 ¼ 5.4,
J2 ¼ 10.4 Hz), 2.80 (dd, 1H, J1 ¼ 4.3, J2 ¼ 13.9 Hz), 2.27 (t, 2H,
J ¼ 6.5 Hz), 1.11, 0.92, 0.91, 0.89, 0.86, 0.84, 0.83 (s, 3H each); dC
(CDCl3) 183.2, 174.0, 143.8, 122.8, 80.8, 55.6, 47.8, 46.8, 46.1, 41.9,
41.3, 39.6, 38.3, 38.0, 37.9, 35.1, 34.0, 33.3, 32.8, 32.7, 32.2, 30.9, 29.8
(4C), 29.7, 29.5, 28.3, 27.9, 26.2, 25.4, 23.7 (2C), 23.6, 23.2, 22.9, 18.4,
17.3, 17.0, 15.6, 14.3; ESI-HRMS m/z calcd for C42H71O4 [M þ 1]
639.5361, found 639.5352.
4.5.3. Acylation of OA with hexanoic anhydride
Hexanoic anhydride (203 mL) was added slowly to a solution of
OA (100 mg, 0.219 mmol) in pyridine (2 mL). The mixture was
treated as described in the general method to give 11 as a white
4.5.5. Acylation of OA with benzoic anhydride
Benzoic anhydride (166 mL) was added slowly to a solution of OA
solid (104 mg, 90%), mp 159e161; [
a
]
þ 69 (c 1 in CHCl3:MeOH,
(100 mg, 0.219 mmol) in pyridine (2 mL). The mixture was treated
as described in the general method for acylation to give 13 as a
D
2:1); IR nmax(KBr)/cmꢂ1 3383, 2979, 2832 and 1679; dH (CDCl3) 5.26
(dd, 1H, J1 ¼ J2 ¼ 3.5 Hz), 4.48 (dd, 1H, J1 ¼ 5.5, J2 ¼ 10.5 Hz), 2.80
(dd, 1H, J1 ¼ 4.5, J2 ¼ 13.9 Hz), 2.27 (t, 2H, J ¼ 6.6 Hz), 1.11, 0.92, 0.91,
0.89, 0.84, 0.83, 0.74 (s, 3H each); dC (CDCl3) 183.8, 173.9, 143.8,
122.8, 80.8, 55.6, 47.8, 46.8, 46.1, 41.9, 41.3, 39.6, 38.3, 37.9, 37.2, 35.1,
34.1, 33.3, 32.8, 32.7, 31.6, 30.9, 29.9, 28.3, 27.9, 26.1, 25.1, 23.8, 23.7,
23.2, 22.5, 18.4, 17.3, 17.0, 15.6, 14.1; ESI-HRMS m/z calcd for
white solid (81 mg, 69%), mp 153e155 ꢀC; [
a
]
þ 36 (c 1 in
D
CHCl3:MeOH, 2:1); IR nmax(KBr)/cmꢂ1 3390, 3001, 2840 and 1689;
dH (CDCl3) 7.42 (m, 5H), 5.28 (dd, 1H, J1 ¼ J2 ¼ 3.5 Hz), 4.70 (dd, 1H,
J1 ¼ 5.5, J2 ¼ 10.5 Hz), 2.80 (dd, 1H, J1 ¼ 4.3, J2 ¼ 13.8 Hz), 1.14, 1.00,
0.98, 0.93, 0.90, 0.83, 0.77 (s, 3H each); dC (CDCl3) 183.1,166.5,143.8,
132.9 (2C), 131.2 (2C), 129.8, 128.5, 122.8, 81.8, 55.6, 47.8, 46.8, 46.1,
41.9, 41.3, 39.6, 38.4 (2C), 37.3, 34.1, 32.9, 32.7 (2C), 30.9, 29.9, 28.5,
27.9, 26.2, 23.8, 23.7, 23.2, 18.5, 17.3, 17.2, 15.7; ESI-HRMS m/z calcd
for C37H53O4 [M þ 1] 561.3937, found 561.3944.
C
36H59O4 [M þ 1] 555.4413, found 555.4421.
4.5.4. Acylation of OA with lauric anhydride
Lauric anhydride (336 mg, 0.876 mmol) was added slowly to a
solution of OA (100 mg, 0.219 mmol) in pyridine (2 mL). The
mixture was treated as described in the general method for acyl-
4.5.6. Acylation of OA with phthalic anhydride
Phthalic anhydride (130 mg, 0.876 mmol) was added slowly to a
solution of OA (100 mg, 0.219 mmol) in pyridine (2 mL). The
mixture was treated as described in the general method for
ation to give 12 as a colourless oil (97 mg, 73%), [
a
]
þ 37 (c 1 in
D
CHCl3:MeOH, 2:1); IR nmax(film)/cmꢂ1 3379, 2963, 2840 and 1681;

A. Parra et al. / European Journal of Medicinal Chemistry 74 (2014) 278e301
291
Chart 3. Flow-cytometry analysis of rhodamine 123 and PI staining after exposure of b16f10 murine melanoma cells to acyl OA and MA derivatives for 72 h with respect to the
control untreated cells. Cell lines were treated at concentrations equal to its IC50 or IC50 ꢁ 2 values. ROS-negative cells (yellow bars) were rhodamine 123ꢂ with PIþ or PIꢂ. ROS-
positive cells (green bars) were rhodamine 123þ with PIþ or PIꢂ. Values are expressed as means ꢃ S.E.M. of three experiments in duplicate. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)
acylation to give 14 as a white solid (123 mg, 93%), mp 166e168 ꢀC;
mixture was treated as described in the general method for acyl-
[
a]
D þ 26 (c 1 in CHCl3:MeOH, 2:1); IR nmax(KBr)/cmꢂ1 3380, 3053,
ation to give 16 as a white solid (73 mg, 59%), mp 190e192 ꢀC;
2855 and 1699; dH (CDCl3) 7.80e7.50 (m, 4H), 5.26 (dd, 1H,
J1 ¼ J2 ¼ 3.6 Hz), 4.71 (dd, 1H, J1 ¼ 5.5, J2 ¼ 10.3 Hz), 2.81 (dd, 1H,
J1 ¼ 4.3, J2 ¼ 13.9 Hz), 1.12, 0.91, 0.91, 0.89, 0.89, 0.83, 0.73 (s, 3H
each); dC (CDCl3) 185.0, 172.8, 168.0, 143.8, 133.8, 132.2, 130.9, 130.3,
130.0, 129.2, 122.8, 83.4, 55.6, 47.7, 46.7, 46.1, 41.8, 41.1, 39.5, 38.2
(2C), 38.1, 34.0, 32.8, 33.3, 32.7, 30.9, 29.7, 28.4, 27.9, 26.2, 23.8, 23.6,
23.0, 18.4, 17.5, 17.0, 15.7; ESI-HRMS m/z calcd for C38H53O6 [M þ 1]
605.3850, found 605.3842.
[a
]
D þ 49 (c 1 in CHCl3:MeOH, 2:1); IR nmax(KBr)/cmꢂ1 3379, 3018,
2847 and 1700; dH (CDCl3) 5.25 (dd, 1H, J1 ¼ J2 ¼ 3.5 Hz), 4.49 (dd,
1H, J1 ¼ 5.6, J2 ¼ 10.6 Hz), 2.80 (dd, 1H, J1 ¼ 4.3, J2 ¼ 13.9 Hz), 2.45e
2.35 (m, 6H), 1.10, 0.92, 0.91, 0.88, 0.85, 0.82, 0.71 (s, 3H each); dC
(CDCl3) 185.0, 179.6, 172.7, 143.8, 122.8, 81.3, 55.5, 47.7, 46.8, 46.0,
41.7, 41.1, 39.5, 38.2, 38.0, 37.2, 34.0, 33.3 (2C), 33.0, 32.7, 32.6, 30.9,
29.7, 28.4, 27.9, 26.2, 23.8, 23.6, 23.1, 20.4, 18.4, 17.2, 17.1, 15.6; ESI-
HRMS m/z calcd for C35H55O6 [M þ 1] 571.3999, found 571.4005.
4.5.7. Acylation of OA with succinic anhydride
4.5.9. Acylation of OA with 3,3-dimethylglutaric anhydride
Succinic anhydride (88 mg, 0.876 mmol) was added slowly to a
solution of OA (100 mg, 0.219 mmol) in pyridine (2 mL). The
mixture was treated as described in the general method for acyl-
ation to give 15 as a white solid (57 mg, 47%), mp 190e192 ꢀC;
3,3-Dimethylglutaric anhydride (124 mg, 0.876 mmol) was added
slowly to a solution of OA (100 mg, 0.219 mmol) in pyridine (2 mL).
The mixture was treated as described in the general method for
acylation to give 17 as a white solid (81 mg, 62%), mp 220e222 ꢀC;
[
a]
D þ 49 (c 1 in CHCl3:MeOH, 2:1); IR nmax(KBr)/cmꢂ1 3394, 3018,
[a]
þ 9 (c 1 in CHCl3:MeOH, 2:1); IR nmax(KBr)/cmꢂ1 3399, 3022,
D
2854 and 1712; dH (CDCl3) 5.25 (dd, 1H, J1 ¼ J2 ¼ 3.5 Hz), 4.51 (dd,
1H, J1 ¼ 5.5, J2 ¼ 10.5 Hz), 2.80 (dd, 1H, J1 ¼ 4.4, J2 ¼ 13.8 Hz), 2.70e
2.60 (m, 4H), 1.10, 0.92, 0.90, 0.88, 0.83, 0.81, 0.72 (s, 3H each); dC
(CDCl3) 184.9, 178.6, 171.8, 143.8, 122.8, 81.7, 55.7, 47.7, 46.8, 46.0,
41.7, 41.1, 39.5, 38.3, 38.2, 37.2, 34.0, 33.3, 32.7, 32.6, 30.9, 29.7, 29.6,
29.4, 28.3, 27.9, 26.2, 23.8, 23.6, 23.0, 18.5, 17.2, 17.0, 15.7; ESI-HRMS
m/z calcd for C34H53O6 [M þ 1] 557.3844, found 557.3842.
2845 and 1704; dH (CDCl3) 5.25 (dd,1H, J1 ¼ J2 ¼ 3.5 Hz), 4.50 (dd,1H,
J1 ¼5.5, J2 ¼10.5 Hz), 2.81 (dd,1H, J1 ¼4.3, J2 ¼13.7 Hz), 2.70e2.25(m,
4H), 1.13, 1.10, 1.07, 0.93, 0.91, 0.88, 0.85, 0.80, 0.70 (s, 3H each); dC
(CDCl3) 184.9,178.0,172.1,143.7,122.9, 81.2, 55.4, 47.5, 46.8, 46.1, 45.4,
44.4, 41.6, 41.0, 39.5, 38.0 (2C), 37.3, 34.0, 34.0, 32.7 (2C), 32.6, 30.9,
29.9, 28.8, 28.6 (2C), 28.0, 26.2, 23.8, 23.6, 22.9, 18.5, 17.6, 17.3, 15.8;
ESI-HRMS m/z calcd for C37H59O6 [M þ 1] 599.4312, found 599.4301.
4.5.8. Acylation of OA with glutaric anhydride
4.5.10. Acylation of OA-Bn with Ac2O
Glutaric anhydride (100 mg, 0.876 mmol) was added slowly to a
solution of OA (100 mg, 0.219 mmol) in pyridine (2 mL). The
Ac2O (69
mL) was added slowly to a solution of OA-Bn (100 mg,
0.183 mmol) in pyridine (2 mL). The mixture was treated as in the

292
A. Parra et al. / European Journal of Medicinal Chemistry 74 (2014) 278e301
Fig. 7. Diagram of rhodamine 123/propidium iodide flow-cytometry, for the compounds with minor (24 and 25) and major (32 and 62) Rh123 staining. The right quadrants of each
diagram (Q2 and Q4) represent cells positive stained with Rh123.
general method for acylation above to give 18 as a white solid
4.46 (dd, 1H, J1 ¼ 5.3, J2 ¼ 10.4 Hz), 2.98 (dd, 1H, J1 ¼ 4.3,
J2 ¼ 13.9 Hz), 2.02 (s, 3H), 1.10, 0.91, 0.90, 0.88, 0.84, 0.83, 0.59 (s, 3H
each); dC (CDCl3) 183.8, 171.2, 143.9, 136.7, 128.6, 128.2 (2C), 128.1
(2C), 122.6, 81.1, 66.1, 55.5, 47.8, 47.0, 46.1, 41.9, 41.6, 39.5, 38.4, 37.9,
(95 mg, 89%), mp 173e175 ꢀC; [
a]
D þ 47 (c 1 in CHCl3:MeOH, 2:1);
IR nmax(KBr)/cmꢂ1 3376, 3001, 2865 and 1710; dH (CDCl3) 7.31 (m,
5H), 5.26 (dd, 1H, J1 ¼ J2 ¼ 3.4 Hz), 5.05 (AB system, 2H, J ¼ 12.0 Hz),
Table 8
HIV-1-protease inhibition effects of OA and MA derivatives.
Comp. #
R1
R2
R3
IC50
(
m
M) (% HIV-1-protease activity inhibition)a
IC50 OA/IC50 comp. #b
OA (1)
5
14
16
17
e
e
e
e
e
e
H
Acetyl
Phthaloyl
Glutaryl
3,3-Dimethylglutaryl
Phthaloyl
H
H
H
H
H
Bn
57.7 ꢃ 3.9
3.4 ꢃ 0.4
1.0
17.0
186.1
103.0
82.4
144.3
0.31 ꢃ 0.03
0.56 ꢃ 0.16
0.70 ꢃ 0.07
0.4 ꢃ 0.1
24
IC50 MA/IC50 comp. #b
MA (2)
31
32
38
39
40
41
42
43
44
45
50
53
55
58
59
60
61
63
H
H
H
H
H
H
H
H
H
H
62.7 ꢃ 4.9
31.2 ꢃ 5.9
124.4 ꢃ 5.8
14.2 ꢃ 2.1
2.3 ꢃ 0.3
3.5 ꢃ 0.5
1.9 ꢃ 0.4
3.2 ꢃ 0.3
1.4 ꢃ 0.3
14.3 ꢃ 1.8
27.5 ꢃ 3.3
226 ꢃ 24
150 ꢃ 8
1.0
2.0
0.5
4.4
27.3
17.9
33.0
19.6
44.8
4.4
2.3
0.3
0.4
0.9
78.4
33.0
4.0
12.1
16.1
Butanoyl
Hexanoyl
Phthaloyl
Phthaloyl
Succinyl
Succinyl
Glutaryl
Glutaryl
3,3-Dimethylglutaryl
3,3-Dimethylglutaryl
Butanoyl
Hexanoyl
Lauroyl
Phthaloyl
Phthaloyl
Succinyl
Butanoyl
H
H
Phthaloyl
H
Succinyl
H
Glutaryl
H
3,3-Dimethylglutaryl
H
Hexanoyl
Lauroyl
H
Phthaloyl
H
H
H
H
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
73 ꢃ 4
0.8 ꢃ 0.1
1.9 ꢃ 0.5
15.6 ꢃ 2.9
5.2 ꢃ 0.7
3.9 ꢃ 0.3
Succinyl
Glutaryl
Succinyl
Glutaryl
a
The IC50 values were calculated based on: Vinh ¼ V0$IC50/([I] þ IC50). The percentage of protease-activity inhibition was calculated according to: % inhibition ¼ [1 ꢂ (Vinh
V0)]$100. All assays were performed three times in triplicates.
/
b
The last column represents the ratio between the IC50 of each precursor (OA or MA) and the IC50 of the related acyl derivatives.

A. Parra et al. / European Journal of Medicinal Chemistry 74 (2014) 278e301
293
system, 2H, J ¼ 12.0 Hz), 4.47 (dd,1H, J1 ¼5.5, J2 ¼ 10.6 Hz), 2.89 (dd,
1H, J1 ¼4.4, J2 ¼ 13.9 Hz), 2.27 (t, 2H, J ¼ 6.5 Hz),1.11, 0.90, 0.89, 0.88,
0.87, 0.83, 0.59 (s, 3H each); dC (CDCl3) 177.6, 173.9, 143.9, 136.7,
128.6, 128.2 (2C), 128.1 (2C), 122.7, 80.8, 66.1, 55.5, 47.8, 47.0, 46.1,
41.9, 41.6, 39.6, 38.4, 38.0, 37.1, 35.0, 34.1, 33.3, 32.9, 32.6, 31.6, 30.9,
28.3, 28.1, 27.9, 26.1, 23.9, 23.8, 23.6, 23.3, 22.5, 18.4, 17.1, 17.0, 15.5,
14.1; ESI-HRMS m/z calcd for C43H65O4 [M þ 1] 645.4883, found
645.4861.
4.5.14. Acylation of OA-Bn with lauric anhydride
Lauric anhydride (280 mg, 0.732 mmol) was added slowly to a
solution of OA-Bn (100 mg, 0.183 mmol) in pyridine (2 mL). The
mixture was treated as described in the general method for acyl-
ation to give 22 as a colourless oil (126 mg, 91%), [
a
]
þ 24 (c 1 in
D
CHCl3:MeOH, 2:1); IR nmax(film)/cmꢂ1 3368, 2978, 2861 and 1712;
dH (CDCl3) 7.33 (m, 5H), 5.27 (dd, 1H, dd, J1 ¼ J2 ¼ 3.6 Hz), 5.06 (AB
system, 2H, J ¼ 12.0 Hz), 4.47 (dd,1H, J1 ¼5.6, J2 ¼ 10.5 Hz), 2.90 (dd,
1H, J1 ¼ 4.3, J2 ¼ 13.9 Hz), 2.30 (t, 2H, J ¼ 6.6 Hz), 1.11, 0.90, 0.89,
0.88, 0.86, 0.84, 0.59 (s, 3H each); dC (CDCl3) 177.6, 173.8, 143.9,
136.6, 128.6, 128.2 (2C), 128.1 (2C), 122.6, 80.7, 66.1, 55.5, 47.7, 46.9,
46.1, 41.9, 41.6, 39.5, 38.3, 37.9, 37.1, 35.0, 34.1, 33.3, 32.9, 32.6, 32.1,
30.9, 29.8 (3C), 29.5 (2C), 29.4, 28.3 (2C), 27.8, 26.0, 23.8 (2C), 23.6,
23.3, 22.9, 18.4, 17.1, 17.0, 15.5, 14.1; ESI-HRMS m/z calcd for
Chart 4. Comparative of the inhibitory effects of the acyl OA and MA derivatives on the
HIV-1-protease, describing the ratio between the IC50 values of the triterpene pre-
cursors (OA and MA) and the IC50 values of their acyl derivatives. DMG
¼
3,3-
dimethylglutaryl, Glu ¼ glutaryl, Succ ¼ succinyl, Phta ¼ phthaloyl, Bz ¼ benzoyl,
Laur ¼ lauroyl, Hex ¼ hexanoyl, But ¼ butanoyl, Prop ¼ propanoyl, Ac ¼ acetyl,
precursors ¼ OA (1) and MA (4).
C
49H77O4 [M þ 1] 729.5822, found 729.5826.
37.1, 34.1, 33.3, 32.9 (2C), 30.9, 28.3, 27.8 (2C), 26.0, 23.9, 23.6, 23.3,
21.5, 18.4, 17.1, 16.9, 15.6; ESI-HRMS m/z calcd for C39H57O4 [M þ 1]
589.4257, found 589.4252.
4.5.15. Acylation of OA-Bn with benzoic anhydride
Benzoic anhydride (138 L) was added slowly to a solution of
m
OA-Bn (100 mg, 0.183 mmol) in pyridine (2 mL). The mixture was
treated as described in the general method for acylation to give 23
4.5.11. Acylation of OA-Bn with propanoic anhydride
Propanoic anhydride (94 mL) was added slowly to a solution of
as a colourless oil (90 mg, 73%), [
a
]
D þ 31 (c 1 in CHCl3:MeOH, 2:1);
IR nmax(film)/cmꢂ1 3380, 3015, 2849 and 1699; dH (CDCl3) 7.98e7.33
(m, 5H), 7.15 (m, 5H), 5.12 (dd, 1H, J1 ¼ J2 ¼ 3.5 Hz), 5.06 (AB system,
2H, J ¼ 12.0 Hz), 4.55 (dd, 1H, J1 ¼ 5.3, J2 ¼ 10.5 Hz), 2.73 (dd, 1H,
J1 ¼ 4.2, J2 ¼ 13.7 Hz), 0.96, 0.83, 0.83, 0.87, 0.93, 0.72, 0.45 (s, 3H
each); dC (CDCl3) 177.6, 166.5, 143.8, 134.7, 132.9 (2C), 130.7 (2C),
129.7, 129.1, 128.6, 128.2 (2C), 128.1 (2C), 122.6, 81.8, 66.1, 55.6, 47.8,
47.0, 46.1, 41.9, 41.6, 40.0, 38.3, 37.2 (2C), 34.1, 32.9, 33.3, 32.6, 30.9,
28.4, 27.8, 26.1, 23.8 (2C), 23.6, 23.3, 18.4, 17.2, 17.1, 15.6; ESI-HRMS
m/z calcd for C44H59O4 [M þ 1] 651.4413, found 651.4404.
OA-Bn (100 mg, 0.183 mmol) in pyridine (2 mL). The mixture was
treated as described in the general method for acylation to give 19
as a white solid (97 mg, 85%), mp 153e155 ꢀC; [
a]
þ 47 (c 1 in
D
CHCl3:MeOH, 2:1); IR nmax(KBr)/cmꢂ1 3391, 2989, 2855 and 1690;
dH (CDCl3) 7.31 (m, 5H), 5.26 (dd, 1H, J1 ¼ J2 ¼ 3.5 Hz), 5.05 (AB
system, 2H, J ¼ 12.0 Hz), 4.47 (dd,1H, J1 ¼5.5, J2 ¼ 10.5 Hz), 2.89 (dd,
1H, J1 ¼ 4.2, J2 ¼ 13.7 Hz), 2.30 (q, 2H, J ¼ 6.5 Hz), 0.11, 0.92, 0.91,
0.89, 0.88, 0.83, 0.59 (s, 3H each); dC (CDCl3) 177.7, 174.5, 143.9,
136.7, 128.6, 128.2 (2C), 128.1 (2C), 122.7, 80.8, 66.1, 55.5, 47.8, 47.0,
46.1, 41.9, 41.6, 40.0, 38.4, 38.0, 37.2, 34.1, 33.3, 32.9, 32.6, 30.9, 28.3
(2C), 27.9, 26.1, 23.9, 23.6, 23.3, 18.8, 18.4, 17.1, 16.9, 15.5, 13.9; ESI-
HRMS m/z calcd for C40H59O4 [M þ 1] 603.4413, found 603.4439.
4.5.16. Acylation of OA-Bn with phthalic anhydride
Phthalic anhydride (108 mg, 0.732 mmol) was added slowly to a
solution of OA-Bn (100 mg, 0.183 mmol) in pyridine (2 mL). The
mixture was treated as described in the general method for acyl-
4.5.12. Acylation of OA-Bn with butanoic anhydride
Butanoic anhydride (119 mL) was added slowly to a solution of
OA-Bn (100 mg, 0.183 mmol) in pyridine (2 mL). The mixture was
treated as described in the general method for acylation to give 20
ation to give 24 as a colourless oil (107 mg, 82%), [
a
]
D þ 48 (c 1 in
CHCl3:MeOH, 2:1); IR nmax(film)/cmꢂ1 3376, 3060, 2842 and 1711;
dH (CDCl3) 8.02e7.56 (m, 4H), 7.33 (m, 5H), 5.27 (dd, 1H,
J1 ¼ J2 ¼ 3.5 Hz), 5.05 (AB system, 2H, J ¼ 12.0 Hz), 4.77 (dd, 1H,
J1 ¼ 5.5, J2 ¼ 10.5 Hz), 2.89 (dd, 1H, J1 ¼ 4.3, J2 ¼ 13.9 Hz), 1.12, 0.94,
0.90, 0.89, 0.89, 0.85, 0.59 (s, 3H each); dC (CDCl3) 177.7, 171.0, 168.1,
143.8, 136.7, 133.3, 132.1, 131.9, 131.1, 130.4, 129.4, 128.6, 128.2 (2C),
128.1 (2C), 122.6, 83.1, 66.1, 55.7, 47.8, 47.0, 46.1, 42.0, 41.6, 39.6,
38.4, 38.2, 37.2, 34.1, 32.9, 33.3, 32.6, 30.9, 28.4, 27.9, 27.8, 26.1, 23.9,
23.7, 23.2, 18.4, 17.1, 17.0, 15.2; ESI-HRMS m/z calcd for C45H59O6
[M þ 1] 695.4331 found 695.4312.
as a white solid (108 mg, 93%), mp 108e110 ꢀC; [
a]
þ 40 (c 1 in
D
CHCl3:MeOH, 2:1); IR nmax(KBr)/cmꢂ1 3387, 2982, 2843 and 1699;
dH (CDCl3) 7.31 (m, 5H), 5.26 (dd, 1H, J1 ¼ J2 ¼ 3.5 Hz), 5.05 (AB
system, 2H, J ¼ 12.0 Hz), 4.48 (dd,1H, J1 ¼5.5, J2 ¼ 10.5 Hz), 2.89 (dd,
1H, J1 ¼ 4.3, J2 ¼ 13.8 Hz), 2.25 (t, 2H, J ¼ 6.4 Hz), 1.11, 0.90, 0.89,
0.88, 0.84, 0.83, 0.59 (s, 3H each); dC (CDCl3) 177.6, 173.6, 143.9,
136.6, 128.5, 128.2 (2C), 128.1 (2C), 122.6, 80.7, 66.1, 55.5, 47.7, 46.9,
46.0, 41.9, 41.6, 39.5, 38.3 (2C), 36.9, 34.1, 33.3, 32.9, 32.6, 30.9, 28.8,
28.2, 27.8, 26.0, 23.8 (2C), 23.6, 23.3, 18.8, 18.4, 17.1, 16.9, 15.5, 13.9;
ESI-HRMS m/z calcd for C41H61O4 [M þ 1] 617.4584, found 617.4570.
4.5.17. Acylation of OA-Bn with succinic anhydride
Succinic anhydride (73 mg, 0.732 mmol) was added slowly to a
solution of OA-Bn (100 mg, 0.183 mmol) in pyridine (2 mL). The
mixture was treated as described in the general method for acyl-
4.5.13. Acylation of OA-Bn with hexanoic anhydride
Hexanoic anhydride (169 mL) was added slowly to a solution of
OA-Bn (100 mg, 0.183 mmol) in pyridine (2 mL). The mixture was
treated as described in the general method for acylation to give 21
ation to give 25 as a colourless oil (62 mg, 51%), [
a
]
þ 38 (c 1 in
D
CHCl3:MeOH, 2:1); IR nmax(film)/cmꢂ1 3377, 3020, 2859 and 1720;
dH (CDCl3) 7.31 (m, 5H), 5.26 (dd, 1H, J1 ¼ J2 ¼ 3.4 Hz), 5.05 (AB
system, 2H, J ¼ 12.0 Hz), 4.48 (dd,1H, J1 ¼5.4, J2 ¼ 10.6 Hz), 2.89 (dd,
1H, J1 ¼ 4.3, J2 ¼ 13.8 Hz), 2.70e2.40 (m, 4H), 1.10, 0.90, 0.88, 0.88,
as a white solid (111 mg, 90%), mp 73e75 ꢀC; [
a
]
þ 49 (c 1 in
D
CHCl3:MeOH, 2:1); IR nmax(KBr)/cmꢂ1 3360, 2989, 2845 and 1708;
dH (CDCl3) 7.32 (m, 5H), 5.27 (dd, 1H, J1 ¼ J2 ¼ 3.5 Hz), 5.05 (AB

294
A. Parra et al. / European Journal of Medicinal Chemistry 74 (2014) 278e301
0.83, 0.83, 0.59 (s, 3H each); dC (CDCl3) 178.4, 177.7, 172.9, 143.8,
136.6, 128.6, 128.2 (2C),128.1 (2C), 122.6, 81.7, 66.1, 55.5, 47.7, 46.9,
46.0, 41.9, 41.6, 39.5, 38.3, 37.9, 37.1, 34.0, 33.3, 32.8, 32.6, 30.9, 29.6,
29.5, 29.3, 28.2, 27.8, 26.0, 23.9, 23.6, 23.2, 18.4, 17.1, 16.9, 15.5; ESI-
HRMS m/z calcd for C41H59O6 [M þ 1] 647.4332, found 647.4312.
treated as described in the general method for acylation to give 30
as a white solid (18 mg, 15%), mp 138e140 ꢀC; [
a
]
þ 52 (c 1 in
D
CHCl3:MeOH, 2:1); IR nmax(KBr)/cmꢂ1 3353, 2980, 2839 and 1719;
dH (CDCl3) 5.23 (dd, 1H, J1 ¼ J2 ¼ 3.5 Hz), 4.92 (ddd, 1H, J1 ¼ 4.3,
J2 ¼ 10.0, J3 ¼ 11.5 Hz), 3.17 (d, 1H, J ¼ 10.0 Hz), 2.78 (dd, 1H, J1 ¼ 3.5,
J2 ¼ 14.0 Hz), 2.28 (t, 2H, J ¼ 6.5 Hz), 1.08, 1.01, 0.92, 0.89, 0.86, 0.81,
0.70 (s, 3H each); dC (CDCl3) 184.3, 174.5, 143.9, 122.5, 81.1, 73.2,
55.3, 47.8, 46.7, 46.1, 43.9, 41.8, 41.1, 40.0, 39.5, 38.5, 36.7, 34.0, 33.3,
32.6 (2C), 30.9, 28.7, 27.8, 26.1, 23.8, 23.7, 23.0, 18.7, 18.4, 17.3, 16.8,
16.5, 13.8; ESI-HRMS m/z calcd for C34H54O5Na [M þ Na] 565.3869,
found 565.3862; and 31 as a white solid (109 mg, 80%), mp 142e
4.5.18. Acylation of OA-Bn with glutaric anhydride
Glutaric anhydride (84 mg, 0.732 mmol) was added slowly to a
solution of OA-Bn (100 mg, 0.183 mmol) in pyridine (2 mL). The
mixture was treated as described in the general method for acyl-
ation to give 26 as a colourless oil (84 mg, 67%), [
a
]
þ 26 (c 1 in
D
CHCl3:MeOH, 2:1); IR nmax(film)/cmꢂ1 3378, 3033, 2867 and 1715;
dH (CDCl3) 7.31 (m, 5H), 5.26 (dd, 1H, J1 ¼ J2 ¼ 3.5 Hz), 5.05 (AB
system, 2H, J ¼ 12.0 Hz), 4.48 (dd,1H, J1 ¼5.5, J2 ¼ 10.5 Hz), 2.89 (dd,
1H, J1 ¼ 4.3, J2 ¼ 13.9 Hz), 2.42e2.34 (m, 6H), 1.10, 0.90, 0.88, 0.88,
0.83, 0.83, 0.59 (s, 3H each); dC (CDCl3) 178.4, 177.7, 172.9, 143.9,
136.6, 128.6, 128.2 (2C), 128.1 (2C), 122.6, 81.3, 66.1, 55.5, 47.7, 47.0,
46.1, 41.9, 41.6, 39.5, 38.3, 37.9, 37.1, 34.1, 33.9 (2C), 33.3, 32.9, 32.6,
30.9, 28.3, 27.8, 27.8, 26.0, 23.9, 23.6, 23.3, 20.3, 18.4, 17.1, 17.0, 15.6;
ESI-HRMS m/z calcd for C42H61O6 [M þ 1] 661.4486, found
661.4468.
144 ꢀC; [
a]
D þ 42 (c 1 in CHCl3:MeOH, 2:1); IR nmax(KBr)/cmꢂ1 3365,
3000, 2848 and 1780; dH (CDCl3) 5.28 (dd, 1H, J1 ¼ J2 ¼ 3.5 Hz), 5.12
(ddd, 1H, J1 ¼ 4.3, J2 ¼ 10.5, J3 ¼ 11.5 Hz), 4.80 (d, 1H, J ¼ 10.5 Hz),
2.83 (dd, 1H, J1 ¼ 4.5, J2 ¼ 13.8 Hz), 2.30 and 2.22 (t, 2H each,
J ¼ 6.5 Hz), 1.14, 1.08, 0.94, 0.93, 0.91, 0.91, 0.70 (s, 3H each); dC
(CDCl3) 184.6, 173.5 (2C), 143.9, 122.5, 80.3, 70.1, 55.1, 47.8, 46.8,
46.0, 44.1, 41.8, 41.1, 39.7, 39.6, 38.4, 36.7 (2C), 34.0, 33.3, 32.7 (2C),
30.9, 28.7, 27.9, 26.1, 23.8, 23.7, 23.0, 18.8, 18.6, 18.4, 17.9, 17.4, 16.6,
14.0, 13.9; ESI-HRMS m/z calcd for C38H59O6 [M ꢂ 1] 611.4312,
found 611.4305.
4.5.19. Acylation of OA-Bn with 3,3-dimethylglutaric anhydride
3,3-Dimethylglutaric anhydride (105 mg, 0.732 mmol) was
added slowly to a solution of OA-Bn (100 mg, 0.183 mmol) in
pyridine (2 mL). The mixture was treated as described in the gen-
eral method for acylation to give 27 as a colourless oil (84 mg, 65%);
4.5.22. Acylation of MA with hexanoic anhydride
Hexanoic anhydride (389 mL) was added slowly to a solution of
MA (100 mg, 0.211 mmol) in pyridine (2 mL). The mixture was
treated as described in the general method for acylation to give 32
as a white solid (22 mg, 19%), mp 124e126 ꢀC; [
a]
þ 40 (c 1 in
D
[
a]
D þ 20 (c 1 in CHCl3:MeOH, 2:1); IR nmax(film)/cmꢂ1 3390, 3021,
CHCl3:MeOH, 2:1); IR nmax(KBr)/cmꢂ1 3405, 3053, 2849 and 1690;
dH (CDCl3) 5.23 (dd, 1H, J1 ¼ J2 ¼ 3.3 Hz), 4.93 (ddd, 1H, J1 ¼ 4.3,
J2 ¼ 10.3, J3 ¼ 11.3 Hz), 3.16 (d, 1H, J ¼ 10.3 Hz), 2.78 (dd, 1H, J1 ¼ 4.5,
J2 ¼ 14.0 Hz), 2.28 (t, 2H, J ¼ 6.6 Hz), 1.09, 1.01, 0.99, 0.88, 0.86, 0.82,
0.71 (s, 3H each); dC (CDCl3) 184.5, 174.7, 143.8, 122.5, 81.0, 73.1,
55.3, 47.7, 46.7, 46.0, 43.9, 41.8, 41.1, 39.9, 39.5, 38.4, 34.7, 34.0, 33.2,
32.6 (2C), 31.4, 30.8, 28.7, 27.8, 26.1, 24.6, 23.7 (2C), 23.0, 22.5, 18.4,
17.2, 16.8, 16.5, 14.1; ESI-HRMS m/z calcd for C36H57O5 [Mâ ꢂ 1]
569.4206, found 569.4209; and 33 as a colourless oil (101 mg, 70%),
2865 and 1698; dH (CDCl3) 7.31 (m, 5H), 5.25 (dd, 1H,
J1 ¼ J2 ¼ 3.6 Hz), 5.05 (AB system, 2H, J ¼ 12.0 Hz), 4.50 (dd, 1H,
J1 ¼ 5.4 Hz, J2 ¼ 10.3 Hz), 2.81 (dd, 1H, J1 ¼ 4.4, J2 ¼ 13.8 Hz), 2.70e
2.25 (m, 4H), 1.13, 1.10, 0.93, 0.91, 0.88, 0.85, 0.80, 0.70, 0.59 (s, 3H
each); dC (CDCl3) 177.7, 176.4, 172.5, 143.9, 136.7, 128.6, 128.2 (2C),
128.1 (2C), 122.6, 81.6, 66.1, 55.6, 47.8, 47.0, 46.1, 45.8, 45.4, 41.9,
41.6, 39.5, 38.4, 37.8, 37.1, 34.1, 33.3, 32.9, 32.6 (2C), 30.9, 28.3, 28.2,
28.1, 27.8, 27.8, 26.1, 23.9, 23.6, 23.3, 18.4, 17.1, 17.0, 15.5; ESI-HRMS
m/z calcd for C44H65O6 [M þ 1] 689.4767, found 689.4781.
[a]
D e 21 (c 1 in CHCl3:MeOH, 2:1); IR nmax(film)/cmꢂ1 3340, 3013,
2839 and 1701; dH (CDCl3) 5.19 (dd, 1H, J1 ¼ J2 ¼ 3.5 Hz), 5.02 (ddd,
1H, J1 ¼ 4.4, J2 ¼ 10.5, J3 ¼ 11.2 Hz), 4.69 (d, 1H, J ¼ 10.5 Hz), 2.75 (dd,
1H, J1 ¼ 4.3, J2 ¼ 13.5 Hz), 2.22 and 2.13 (t, 2H each, J ¼ 6.5 Hz), 1.13,
1.11, 1.05, 0.98, 083, 0.81, 0.67 (s, 3H each); dC (CDCl3) 183.0, 173.7,
173.6, 143.9, 122.5, 80.3, 70.1, 55.1, 47.8, 46.7, 46.1, 44.1, 41.9, 41.2,
39.7, 39.6, 38.4, 34.8 (2C), 34.0, 33.3, 32.7 (2C), 31.7, 31.5, 30.9, 28.7,
27.9, 26.1, 25.1, 24.9, 23.8, 23.1, 22.9, 22.6 (2C), 18.4, 17.9, 17.4, 16.6,
14.1, 14.1; ESI-HRMS m/z calcd for C42H68O6Na [M þ Na] 691.4914,
found 691.4913.
4.5.20. Acylation of MA with propanoic anhydride
Propanoic anhydride (217 mL) was added slowly to a solution of
MA (100 mg, 0.211 mmol) in pyridine (2 mL). The mixture was
treated as described in the general method for acylation to give 28
as a white solid (22 mg, 20%), mp 155e157 ꢀC; [
a
]
D
þ 49 (c 1 in
CHCl3:MeOH, 2:1); IR nmax(KBr)/cmꢂ1 3376, 3015, 2855 and 1711; dH
(CDCl3) 5.26 (dd, 1H, J1 ¼ J2 ¼ 3.5 Hz), 4.92 (ddd, 1H, J1 ¼ 4.8,
J2 ¼ 10.5, J3 ¼ 11.2 Hz), 3.17 (d, 1H, J ¼ 10.5 Hz), 2.80 (dd, 1H, J1 ¼ 4.3,
J2 ¼ 13.9 Hz), 2.30 (q, 2H, J ¼ 6.5 Hz), 1.10, 1.08, 1.01, 0.99, 0.88, 0.81,
0.70 (s, 3H each); dC (CDCl3) 184.2, 175.3, 143.9, 122.4, 81.0, 73.2,
55.3, 47.7, 46.7, 46.0, 43.8, 41.8, 41.0, 40.0, 39.5, 38.6, 34.0, 33.2, 32.6,
32.6, 30.8, 28.7, 28.1, 27.8, 26.1, 23.8, 23.7, 23.0, 18.4, 17.3, 16.8, 16.5,
9.3; ESI-HRMS m/z calcd for C33H53O5 [M þ 1] 529.3893, found
529.3886; and 29 as a white solid (89 mg, 73%), mp 224e226 ꢀC;
4.5.23. Acylation of MA with lauric anhydride
Lauric anhydride (645 mg, 1.680 mmol) was added slowly to a
solution of MA (100 mg, 0.211 mmol) in pyridine (2 mL). The
mixture was treated as described in the general method for acyl-
ation to give 34 as a white solid (37 mg, 27%), mp 30e32 ꢀC;
[
a]
D þ 32 (c 1 in CHCl3:MeOH, 2:1); IR nmax(KBr)/cmꢂ1 3299, 3017,
[a
]
D þ 20 (c 1 in CHCl3:MeOH, 2:1); IR nmax(KBr)/cmꢂ1 3384, 2956,
2796 and 1699; dH (CDCl3) 5.28 (dd, 1H, J1 ¼ J2 ¼ 3.4 Hz), 5.14 (ddd,
1H, J1 ¼ 4.3, J2 ¼ 10.1, J3 ¼ 11.2 Hz), 4.80 (d, 1H, J ¼ 10.1 Hz), 2.84 (dd,
1H, J1 ¼ 4.5, J2 ¼ 12.0 Hz), 2.32 and 2.26 (q, 2H each, J ¼ 6.5 Hz), 1.08,
1.01, 0.99, 0.92, 0.91, 0.91, 0.76 (s, 3H each); dC (CDCl3) 184.6, 173.5
(2C), 143.9, 122.5, 80.5, 70.1, 55.1, 47.8, 46.8, 46.0, 44.1, 41.8, 41.1,
39.7, 39.6, 38.4, 34.0, 33.3, 32.6 (2C), 30.9, 28.7, 28.1 (2C), 27.9, 26.1,
23.8, 23.7, 23.0, 18.4, 17.9, 17.4, 16.6, 9.6, 9.4; ESI-HRMS m/z calcd for
2827 and 1689; dH (CDCl3) 5.24 (dd, 1H, J1 ¼ J2 ¼ 3.4 Hz), 4.94 (ddd,
1H, J1 ¼ 4.3, J2 ¼ 10.3, J3 ¼ 11.3 Hz), 3.18 (d, 1H, J ¼ 10.3 Hz), 2.79 (dd,
1H, J1 ¼ 4.5, J2 ¼ 13.8 Hz), 2.31 (t, 2H, J ¼ 6.6 Hz), 1.10, 1.03, 1.01, 0.90,
0.88, 0.83, 0.73 (s, 3H each); dC (CDCl3) 184.8, 174.8, 143.8, 122.5,
81.1, 73.1, 55.4, 47.8, 46.8, 46.1, 43.9, 41.9, 41.1, 40.0, 39.6, 38.4, 34.8,
34.3, 33.3, 32.6 (2C), 32.1, 30.9, 29.7 (3C), 29.3 (2C), 29.1, 28.7, 27.8,
26.1, 24.8, 23.8, 23.7, 23.1, 22.8, 18.5, 16.9, 16.8, 16.5, 14.2; ESI-HRMS
m/z calcd for C42H71O5 [M þ 1] 655.5302, found 655.5316; and 35 as
C
36H57O6 [M þ 1] 585.4155, found 585.4144.
a colourless oil (100 mg, 57%), [
a
]
D þ 45 (c 1 in CHCl3:MeOH, 2:1); IR
4.5.21. Acylation of MA with butanoic anhydride
nmax(film)/cmꢂ1 3390, 3000, 2880 and 1770; dH (CDCl3) 5.18 (dd, 1H,
J1 ¼ J2 ¼ 3.3 Hz), 5.01 (ddd, 1H, J1 ¼ 4.3, J2 ¼ 10.5, J3 ¼ 11.3 Hz), 4.68
(d, 1H, J ¼ 10.5 Hz), 2.73 (dd, 1H, J1 ¼ 4.3, J2 ¼ 13.8 Hz), 2.21 and 2.13
Butanoic anhydride (276 L) was added slowly to a solution of
m
MA (100 mg, 0.211 mmol) in pyridine (2 mL). The mixture was

A. Parra et al. / European Journal of Medicinal Chemistry 74 (2014) 278e301
295
(t, 2H each, J ¼ 6.5 Hz), 1.13, 1.04, 1.05, 0.97, 0.80, 0.77, 0.65 (s, 3H
each); dC (CDCl3) 184.5, 173.7, 173.6, 143.9, 122.5, 80.3, 70.0, 55.1,
47.8, 46.8, 46.0, 44.1, 41.8, 41.1, 39.7, 39.6, 38.4, 34.9, 34.8, 34.0, 33.3,
32.7 (2C), 32.2 (2C), 30.9, 30.0 (4C), 29.9 (4C), 29.8, 29.6 (2C), 29.3,
28.7, 27.9, 26.1, 25.4, 25.2, 23.8, 23.7, 23.1, 22.9 (2C), 18.4, 17.9, 17.3,
16.6, 14.4, 14.1; ESI-HRMS m/z calcd for C54H93O6 [M þ 1] 837.6972,
found 837.6967.
J3 ¼ 11.5 Hz), 5.47 (dd, 1H, J1 ¼ J2 ¼ 3.6 Hz), 3.55 (d, 1H, J ¼ 10.8 Hz),
3.30 (dd, 1H, J1 ¼ 4.6, J2 ¼ 14.0 Hz), 2.80e2.60 (m, 4H), 1.30, 1.26,
1.08, 1.05, 1.03, 1.00, 0.98 (s, 3H each); dC (Py-d5) 180.6, 175.6, 173.4,
145.4, 122.8, 80.2, 74.3, 56.1, 48.5, 47.2, 47.1, 44.9, 42.8, 42.5, 40.9,
40.3, 39.1, 34.8, 33.8, 33.7, 33.6, 31.5, 30.9, 30.6, 29.7, 28.8, 26.7, 24.4,
24.3, 24.2, 19.3, 18.1, 17.9, 17.0; ESI-HRMS m/z calcd for C34H52O7Na
[M þ Na] 595.3611, found 595.3612; and 41 as a white solid (45 mg,
33%), mp 113e115 ꢀC; [ e 6 (c 1 in MeOH); IR nmax(KBr)/cmꢂ1
a]
D
4.5.24. Acylation of MA with benzoic anhydride
3346, 3021, 2803 and 1688; dH (Py-d5) 5.50 (ddd, 1H, J1 ¼ 4.6,
J2 ¼ 10.9, J3 ¼ 11.4 Hz), 5.44 (dd, 1H, J1 ¼ J2 ¼ 3.5 Hz), 5.15 (d, 1H,
J ¼ 10.9 Hz), 3.30 (dd, 1H, J1 ¼ 4.6, J2 ¼ 14.0 Hz), 2.80e2.55 (m, 8H),
1.29, 1.25, 1.04, 1.01, 0.98, 0.96, 0.95 (s, 3H each); dC (Py-d5) 180.9,
176.0,175.7,173.5,173.4,145.7,122.5, 81.2, 70.7, 55.5, 48.2, 47.2, 47.0,
44.6, 42.7, 42.4, 40.5 (2C), 39.2, 34.7, 33.8, 33.6, 33.3, 31.8, 31.0, 30.9,
30.6, 30.5, 29.0, 28.7, 26.7, 24.3, 24.2, 24.1, 18.9, 18.4, 17.7, 16.9; ESI-
HRMS m/z calcd for C38H57O10 [M þ 1] 673.3947, found 673.3952.
Benzoic anhydride (318 mL) was added slowly to a solution of
MA (100 mg, 0.211 mmol) in pyridine (2 mL). The mixture was
treated as described in the general method for acylation to give 36
as a white solid (46 mg, 37%), mp 95e97 ꢀC; [
a]
þ 28 (c 1 in
D
CHCl3:MeOH, 2:1); IR nmax(KBr)/cmꢂ1 3420, 3030, 2822 and 1717;
dH (CDCl3) 8.10e7.45 (m, 5H), 5.26 (dd, 1H, J1 ¼ J2 ¼ 3.4 Hz), 5.20
(ddd, 1H, J1 ¼ 4.3, J2 ¼ 10.2, J3 ¼ 11.3 Hz), 3.39 (d, 1H, J ¼ 10.2 Hz),
2.83 (dd, 1H, J1 ¼ 4.5, J2 ¼ 13.7 Hz), 1.13, 1.07, 1.07, 0.92, 0.90, 0.89,
0.77 (s, 3H each); dC (CDCl3) 184.8, 172.2, 143.8, 133.9 (2C), 130.4
(2C), 129.8, 128.7, 122.6, 81.2, 74.2, 55.4, 47.8, 46.8, 46.1, 44.0, 41.9,
41.2, 40.1, 39.6, 38.7, 34.0, 33.3, 32.7, 32.6, 30.9, 28.8, 27.9, 26.2, 23.8,
23.7, 23.1, 18.5, 17.1, 16.9, 16.6; ESI-HRMS m/z calcd for C37H51O5
[M ꢂ 1] 575.3737, found 575.3745; and 37 as a white solid (64 mg,
4.5.27. Acylation of MA with glutaric anhydride
Glutaric anhydride (193 mg, 1.680 mmol) was added slowly to a
solution of MA (100 mg, 0.211 mmol) in pyridine (2 mL). The
mixture was treated as described in the general method for acyl-
ation to give 42 as a colourless oil (38 mg, 31%), [
a
]
þ 17 (c 1 in
D
45%), mp 70e72 ꢀC; [
a]
þ 28 (c 1 in CHCl3:MeOH, 2:1); IR
CHCl3:MeOH, 2:1); IR nmax(film)/cmꢂ1 3395, 2979, 2833 and 1725;
dH (DMSO-d6) 5.27 (dd, 1H, J1 ¼ J2 ¼ 3.5 Hz), 5.00 (ddd, 1H, J1 ¼ 4.6,
J2 ¼ 10.7, J3 ¼ 11.3 Hz), 3.20 (d, 1H, J ¼ 10.7 Hz), 2.90 (dd, 1H, J1 ¼ 4.3,
J2 ¼ 13.8 Hz), 2.35e2.15 (m, 4H), 1.20, 1.17, 1.09, 1.07, 0.97, 0.89, 0.85
(s, 3H each); dC (DMSO-d6) 181.9,175.7,173.7, 145.5,122.5, 81.1, 73.8,
56.2, 49.1, 47.7, 47.4, 44.6, 43.1, 42.9, 41.1, 40.7, 39.6, 35.1, 34.0 (2C),
33.7, 32.2, 31.8, 30.9, 29.7, 28.8, 26.6, 24.7, 24.2 (2C), 21.6, 19.7, 18.4,
17.8,17.1; ESI-HRMS m/z calcd for C35H55O7 [M þ 1] 587.3948, found
D
nmax(KBr)/cmꢂ1 3402, 2983, 2842 and 1700; dH (CDCl3) 7.95e7.35
(m, 10 H), 5.28 (dd, 1H, J1 ¼ J2 ¼ 3.4 Hz), 5.20 (ddd, 1H, J1 ¼ 4.5,
J2 ¼ 10.3, J3 ¼ 11.5 Hz), 5.23 (d, 1H, J ¼ 10.3 Hz), 2.84 (dd, 1H, J1 ¼ 4.3,
J2 ¼ 14.0 Hz), 1.20, 1.16, 1.11, 1.03, 0.93, 0.91, 0.80 (s, 3H each); dC
(CDCl3) 184.0, 166.7, 166.5, 143.9, 133.9 (4C), 129.8 (4C), 129.8 (2C),
128.5 (2C), 122.5, 81.2, 71.3, 55.4, 47.9, 46.8, 46.0, 44.2, 41.9, 41.2,
40.1, 39.7, 38.7, 34.1, 33.3, 32.7 (2C), 30.9, 28.8, 27.9, 26.2, 23.8, 23.7,
23.1, 18.5, 18.1, 17.4, 16.8; ESI-HRMS m/z calcd for C44H57O6 [M þ 1]
681.4155, found 681.4155.
587.3957; and 43 as a colourless oil (53 mg, 35%), [a]D e 14 (c 1 in
MeOH); IR nmax(film)/cmꢂ1 3396, 3001, 2843 and 1710; dH (Py-d5)
5.49 (dd, 1H, J1 ¼ J2 ¼ 3.4 Hz), 5.45 (ddd, 1H, J1 ¼ 4.4, J2 ¼ 10.7,
J3 ¼ 11.5 Hz), 5.14 (d, 1H, J ¼ 10.7 Hz), 3.20 (dd, 1H, J1 ¼ 4.3,
J2 ¼ 14.0 Hz), 2.35e2.10 (m, 8H), 1.32, 1.09, 1.03, 1.01, 0.98, 0.98, 0.95
(s, 3H each); dC (Py-d5) 180.7, 176.3, 176.2, 173.7, 173.5, 145.6, 122.7,
81.2, 70.8, 55.6, 48.0, 47.3, 46.7, 44.4, 42.9, 42.2, 41.4, 40.3, 39.1, 34.5,
34.4 (2C), 34.2, 34.1, 33.6, 33.5, 33.1, 31.6, 28.9, 28.5, 26.5, 24.1, 24.0,
23.9, 21.5, 21.4, 18.8, 18.2, 17.6, 16.7; ESI-HRMS m/z calcd for
4.5.25. Acylation of MA with phthalic anhydride
Phthalic anhydride (250 mg, 1.680 mmol) was added slowly to a
solution of MA (100 mg, 0.211 mmol) in pyridine (2 mL). The
mixture was treated as described in the general method for acyl-
ation to give 38 as a white solid (33 mg, 25%), mp 190e192 ꢀC;
[
a]
þ 11 (c 1 in CHCl3:MeOH, 2:1); IR nmax(KBr)/cmꢂ1 3375, 3015,
D
2822 and 1722; dH (CDCl3) 8.30e7.30 (m, 4H), 5.74 (ddd, 1H, J1 ¼4.5,
J2 ¼ 10.6, J3 ¼ 11.5 Hz), 5.40 (dd, 1H, J1 ¼ J2 ¼ 3.5 Hz), 3.33 (d, 1H,
J ¼ 10.6 Hz), 2.70 (dd, 1H, J1 ¼ 4.5, J2 ¼ 13.8 Hz), 1.29, 1.25, 1.11, 1.07,
1.06, 1.02, 0.97 (s, 3H each); dC (CDCl3) 180.2, 169.9, 168.3, 145.1,
133.8, 132.9, 132.3 (2C), 128.4, 128.1, 122.1, 82.3, 74.8, 55.5, 47.9, 46.8
(2C), 43.9, 42.1, 41.4, 40.4, 39.9, 38.6, 34.4, 33.5, 33.3, 33.1, 31.2, 28.9,
27.4, 26.4, 24.1, 23.9 (2C), 18.6, 18.3, 17.4, 16.5; ESI-HRMS m/z calcd
for C38H51O7 [M ꢂ 1] 619.3635, found 619.3649; and to give 39 as a
C40H60O10Na [M þ Na] 723.4084, found 723.4073.
4.5.28. Acylation of MA with 3,3-dimethylglutaric anhydride
3,3-Dimethylglutaric anhydride (239 mg, 1.680 mmol) was
added slowly to a solution of MA (100 mg, 0.211 mmol) in pyridine
(2 mL). The mixture was treated as described in the general method
for acylation to give 44 as a colourless oil (45 mg, 33%), [
a
]
D þ 44 (c
1 in CHCl3:MeOH, 2:1); IR nmax(film)/cmꢂ1 3299, 2987, 2801 and
1740; dH (DMSO-d6) 5.27 (dd, 1H, J1 ¼ J2 ¼ 3.4 Hz), 4.97 (ddd, 1H,
J1 ¼ 4.3, J2 ¼ 10.5, J3 ¼ 11.5 Hz), 3.19 (d, 1H, J ¼ 10.5 Hz), 2.88 (dd, 1H,
J1 ¼ 4.3, J2 ¼ 14.0 Hz), 2.45e2.35 (m, 4H), 1.19, 1.17, 1.16, 1.14, 1.01,
0.96, 0.93, 0.88, 0.84 (s, 3H each); dC (DMSO-d6) 181.8, 177.0, 174.9,
145.4, 122.5, 81.1, 73.9, 56.7, 49.1, 47.7, 47.4, 45.4, 45.2, 45.1, 43.1,
42.8, 41.1, 40.7, 39.6, 35.1, 34.7, 34.0, 33.9, 33.8, 31.8, 30.8, 29.7, 29.4,
29.0, 26.6, 24.7, 24.2 (2C), 19.7, 17.8, 17.6, 17.1; ESI-HRMS m/z calcd
for C37H59O7 [M þ 1] 615.4261, found 615.4252; and 45 as a col-
colourless oil (65 mg, 40%), [
a
]
D þ 38 (c 1 in CHCl3:MeOH, 2:1); IR
nmax(film)/cmꢂ1 3391, 2999, 2843 and 1740; dH (CDCl3) 8.30e7,30
(m, 8H), 5.80 (ddd, 1H, J1 ¼ 4.3, J2 ¼ 10.3, J3 ¼ 11.3 Hz), 5.60 (d, 1H,
J ¼ 10.3 Hz), 5.42 (dd, 1H, J1 ¼ J2 ¼ 3.5 Hz), 2.69 (dd, 1H, J1 ¼ 4.4,
J2 ¼ 13.9 Hz), 1.30, 1.28, 1.10, 1.08, 1.06, 1.03, 0.98 (s, 3H each); dC
(CDCl3) 180.3, 170.7, 169.9, 168.7, 168.3, 145.2, 133.8 (2C), 132.9 (2C),
132.3 (4C), 128.4 (2C), 128.1 (2C), 122.2, 82.4, 72.2, 55.5, 48.0, 46.8
(2C), 43.9, 42.1, 41.4, 40.3, 39.9, 38.6, 34.4, 33.5, 33.3, 33.1, 31.2, 28.9,
27.4, 26.4, 24.1, 23.9 (2C), 18.6, 18.3, 17.4, 16.5; ESI-HRMS m/z calcd
for C46H57O10 [M þ 1] 769.3952, found 769.3954.
ourless oil (59 mg, 36%), [a]
D e 13 (c 1 in MeOH); IR nmax(film)/cmꢂ1
3380, 3024, 2841 and 1724; dH (Py-d5) 5.49 (ddd, 1H, J1 ¼ 4.4,
J2 ¼ 10.6, J3 ¼ 11.3 Hz), 5.46 (dd, 1H, J1 ¼ J2 ¼ 3.5 Hz), 5.21 (d, 1H,
J ¼ 10.6 Hz), 3.60 (dd, 1H, J1 ¼ 4.5, J2 ¼ 13.9 Hz), 2.90e2.65 (m, 8H),
1.43, 1.41, 1.39 and 1.37 (s, 3H), 1.07, 1.05, 1.04, 1.03, 0.99, 0.99, 0.98
(s, 3H each); dC (Py-d5) 180.7, 175.3 (2C), 172.5, 172.4, 145.5, 122.6,
80.7, 70.8, 55.6, 48.0, 47.7, 46.7, 46.2 (2C), 45.2 (2C), 44.6, 42.8, 42.1,
40.4, 40.3, 39.0, 34.7 (2C), 34.5, 33.5, 33.4, 33.1, 31.6, 29.0, 28.6 (2C),
28.2 (2C), 28.1, 26.4, 24.4, 23.9, 23.4, 19.1, 18.3, 17.5, 16.7; ESI-HRMS
m/z calcd for C44H68O10Na [M þ Na] 779.4710, found 779.4712.
4.5.26. Acylation of MA with succinic anhydride
Succinic anhydride (169 mg, 1.680 mmol) was added slowly to a
solution of MA (100 mg, 0.211 mmol) in pyridine (2 mL). The
mixture was treated as described in the general method for acyl-
ation to give 40 as a white solid (40 mg, 30%), mp 121e123 ꢀC;
[
a
]
D þ 11 (c 1 in CHCl3:MeOH, 2:1); IR nmax(KBr)/cmꢂ1 3365, 3022,
2839 and 1711; dH (Py-d5) 5.53 (ddd, 1H, J1 ¼ 4.6, J2 ¼ 10.8,

296
A. Parra et al. / European Journal of Medicinal Chemistry 74 (2014) 278e301
4.5.29. Acylation of MA-Bn with Ac2O
18.4, 17.1, 16.9, 16.6, 13.9; ESI-HRMS m/z calcd for C41H61O5 [M þ 1]
Ac2O (134
mL) was added slowly to a solution of MA-Bn (100 mg,
633.4519, found 633.4523; and 51 as a white solid (100 mg, 79%),
0.177 mmol) in pyridine (2 mL). The mixture was treated as
mp 47e49 ꢀC; [
a
]
D þ 5 (c 1 in CHCl3:MeOH, 2:1); IR nmax(KBr)/cmꢂ1
described in the general method for acylation to give 46 as a white
3412, 3020, 2862 and 1730; dH (CDCl3) 7.30 (m, 5H), 5.23 (dd, 1H,
J1 ¼ J2 ¼ 3.5 Hz), 5.06 (ddd, 1H, J1 ¼ 4.6, J2 ¼ 10.3, J3 ¼ 11.2 Hz), 5.03
(AB system, 2H, J ¼ 12.0 Hz), 4.74 (d, 1H, J ¼ 10.3 Hz), 2.87 (dd, 1H,
J1 ¼4.5, J2 ¼ 13.9 Hz), 2.24 and 2.15 (t, 2H each, J ¼ 6.5 Hz),1.08, 0.99,
0.94, 0.91, 0.90, 0.86, 0.85 (s, 3H each); dC (CDCl3) 177.4, 173.3, 173.2,
143.9,136.5,128.5 (2C),128.1, 128.0 (2C),122.3, 80.2, 69.9, 66.1, 55.0,
47.6, 46.8, 46.0, 44.1, 41.8, 41.5, 39.6, 39.5, 38.3, 36.6 (2C), 34.0, 33.2,
32.7, 32.5, 30.8, 28.6, 27.7, 26.0, 23.8, 23.6, 23.2, 18.7, 18.5, 18.4, 17.8,
17.0, 16.5, 13.9, 13.8; ESI-HRMS m/z calcd for C45H67O6 [M þ 1]
703.4938, found 703.4946.
solid (17 mg, 15%), mp 85e87 ꢀC; [
a
]
þ 14 (c 1 in CHCl3:MeOH,
D
2:1); IR nmax(KBr)/cmꢂ1 3396, 3015, 2845 and 1701; dH (CDCl3) 7.31
(m, 5H), 5.25 (dd, 1H, J1 ¼ J2 ¼ 3.4 Hz), 5.05 (AB system, 2H,
J ¼ 12.0 Hz), 4.93 (ddd, 1H, J1 ¼ 4.3, J2 ¼ 10.3, J3 ¼ 11.3 Hz), 3.16 (d,
1H, J ¼ 10.3 Hz), 2.88 (dd, 1H, J1 ¼ 4.8, J2 ¼ 13.8 Hz), 2.04, 1.09, 1.03,
0.97, 0.90, 0.88, 0.84, 0.57 (s, 3H each); dC (CDCl3) 177.6, 171.8, 144.0,
136.3, 128.6 (5C), 122.4, 81.1, 73.5, 66.2, 55.4, 47.8, 46.9, 46.1, 43.9,
41.9, 41.6, 40.0, 39.6, 38.5, 34.1, 33.3, 32.8, 32.6, 30.9, 28.8, 27.8, 26.1,
23.9, 23.7, 23.2, 21.6, 18.5, 17.1, 16.9, 16.6; ESI-HRMS m/z calcd for
C
39H56O5Na [M þ Na] 627.4025, found 627.4017; and 47 as a white
solid (94 mg, 75%), mp 78e80 ꢀC; [
a]
þ 54 (c 1 in CHCl3:MeOH,
4.5.32. Acylation of MA-Bn with hexanoic anhydride
D
2:1); IR nmax(KBr)/cmꢂ1 3353, 2995, 2808 and 1685; dH (CDCl3) 7.30
(m, 5H), 5.25 (dd, 1H, J1 ¼ J2 ¼ 3.5 Hz), 5.08 (ddd, 1H, J1 ¼ 4.3,
J2 ¼ 10.5, J3 ¼ 11.5 Hz), 5.03 (AB system, 2H, J ¼ 12.0 Hz), 4.70 (d, 1H,
J ¼ 10.5 Hz), 2.88 (dd, 1H, J1 ¼ 4.3, J2 ¼ 13.8 Hz), 2.01, 1.94, 1.08, 0.99,
0.89, 0.87, 0.87, 0.86, 0.55 (s, 3H each); dC (CDCl3) 177.5, 170.9, 170.6,
143.9,136.6,128.5 (2C),128.2,128.0 (2C),122.3, 80.8, 70.2, 66.1, 55.1,
47.7, 46.9, 46.0, 44.1, 41.9, 41.5, 39.5 (2C), 38.3, 34.0, 33.3, 32.7, 32.5,
30.9, 28.6, 27.7, 26.0, 23.8, 23.6, 23.2, 21.3, 21.0, 18.4, 17.9, 17.8, 16.6;
ESI-HRMS m/z calcd for C41H59O6 [M þ 1] 647.4312, found 647.4320.
Hexanoic anhydride (328 mL) was added slowly to a solution of
MA-Bn (100 mg, 0.177 mmol) in pyridine (2 mL). The mixture was
treated as described in the general method for acylation to give 52
as a white solid (18 mg, 15%), mp 40e42 ꢀC; [
a]
e 6 (c 1 in
D
CHCl3:MeOH, 2:1); IR nmax(KBr)/cmꢂ1 3375, 3012, 2841 and 1753;
dH (CDCl3) 7.31 (m, 5H), 5.25 (dd, 1H, J1 ¼ J2 ¼ 3.4 Hz), 5.02 (AB
system, 2H, J ¼ 12.0 Hz), 4.93 (ddd,1H, J1 ¼4.5, J2 ¼ 9.9, J3 ¼ 11.5 Hz),
3.16 (d, 1H, J ¼ 9.9 Hz), 2.78 (dd, 1H, J1 ¼ 4.3, J2 ¼ 14.0 Hz), 2.31 (t,
2H, J ¼ 6.6 Hz), 1.09, 1.02, 0.98, 0.90, 0.87, 0.87, 0.84 (s, 3H each); dC
(CDCl3) 177.5, 174.6, 144.0, 136.6, 128.6 (2C), 128.2, 128.1 (2C), 122.4,
81.1, 73.2, 66.1, 55.4, 47.8, 46.9, 46.1, 43.9, 41.9, 41.6, 40.0, 39.6, 38.5,
34.8, 34.1, 33.3, 32.7, 32.6, 31.5, 30.9, 28.8, 27.8, 26.1, 24.9, 23.8, 23.7
(2C), 22.5, 18.5, 17.1, 16.9, 16.5, 14.1; ESI-HRMS m/z calcd for
4.5.30. Acylation of MA-Bn with propanoic anhydride
Propanoic anhydride (182 mL) was added slowly to a solution of
MA-Bn (100 mg, 0.177 mmol) in pyridine (2 mL). The mixture was
treated as described in the general method for acylation to give 48
C
43H65O5 [M þ 1] 661.4832, found 661.4844; and 53 as a colourless
as a white solid (24 mg, 22%), mp 46e48 ꢀC; [
a
]
þ 24 (c 1 in
oil (100 mg, 76%), [ D e 24 (c 1 in CHCl3:MeOH, 2:1); IR nmax(film)/
a]
D
CHCl3:MeOH, 2:1); IR nmax(KBr)/cmꢂ1 3401, 2989, 2823 and 1745;
dH (CDCl3) 7.31 (m, 5H), 5.25 (dd, 1H, J1 ¼ J2 ¼ 3.3 Hz), 5.05 (AB
system, 2H, J ¼ 12.0 Hz), 4.93 (ddd, 1H, J1 ¼ 4.3, J2 ¼ 10.2,
J3 ¼ 11.3 Hz), 3.18 (d, 1H, J ¼ 10.2 Hz), 2.89 (dd, 1H, J1 ¼ 4.6,
J2 ¼ 14.0 Hz), 2.33 (2H, q, J ¼ 6.5 Hz), 1.12, 1.09, 1.03, 0.98, 0.90, 0.87,
0.84 (s, 3H each); dC (CDCl3) 177.6, 175.3, 144.0, 136.6, 128.6 (2C),
128.2, 128.1 (2C), 122.4, 81.1, 73.3, 66.2, 55.4, 47.8, 46.9, 46.1, 43.9,
42.0, 41.6, 40.0, 39.6, 38.5, 34.1, 33.3, 32.8, 32.6, 30.9, 28.8, 28.1, 27.8,
26.1, 23.9, 23.7, 23.3, 18.5, 17.1, 16.9, 16.5, 9.4; ESI-HRMS m/z calcd
for C40H59O5 [M þ 1] 619.4363, found 619.4368; and 49 as a white
cmꢂ1 3432, 3022, 2849 and 1740; dH (CDCl3) 7.29 (m, 5H), 5.24 (dd,
1H, J1 ¼ J2 ¼ 3.5 Hz), 5.04 (ddd, 1H, J1 ¼ 4.8, J2 ¼ 10.3, J3 ¼ 11.3 Hz),
5.02 (AB system, 2H, J ¼ 12.0 Hz), 4.74 (d, 1H, J ¼ 10.3 Hz), 2.87 (dd,
1H, J1 ¼ 4.5, J2 ¼ 13.6 Hz), 2.26 and 2.16 (t, 2H each, J ¼ 6.5 Hz), 1.13,
1.08, 0.99, 0.89, 0.87, 0.87, 0.85 (s, 3H each); dC (CDCl3) 177.4, 173.5,
173.4, 143.9, 136.5, 128.5 (2C), 128.2, 128.0 (2C), 122.3, 80.2, 69.4,
66.1, 55.1, 47.7, 46.8, 46.0, 44.1, 41.9, 41.5, 39.6, 39.5, 38.3, 34.7, 34.6,
34.0, 33.2, 32.7, 32.5, 31.5, 31.4, 30.8, 28.6, 27.7, 26.0, 24.9, 24.7, 23.8,
23.6, 23.2, 22.4 (2C), 18.4, 17.8, 17.0, 16.5, 14.0 (2C); ESI-HRMS m/z
calcd for C49H75O6 [M þ 1] 759.5564, found 759.5560.
solid (81 mg, 67%), mp 34e36 ꢀC; [
a]
D þ 7 (c 1 in CHCl3:MeOH, 2:1);
IR nmax(KBr)/cmꢂ1 3400, 3042, 2835 and 1704; dH (CDCl3) 7.30 (m,
5H), 5.32 (dd, 1H, J1 ¼ J2 ¼ 3.5 Hz), 5.10 (ddd, 1H, J1 ¼ 4.3, J2 ¼ 9.5,
J3 ¼ 11.5 Hz), 5.05 (AB system, 2H, J ¼ 12.0 Hz), 4.74 (d, 1H,
J ¼ 9.5 Hz), 2.88 (dd, 1H, J1 ¼ 4.5, J2 ¼ 13.7 Hz), 2.28 and 2.22 (t, 2H
each, J ¼ 6.5 Hz), 1.09, 1.03, 1.01, 0.92, 0.90, 0.88, 0.86 (s, 3H each); dC
(CDCl3) 177.6, 174.3, 173.2, 144.0, 136.6, 128.6 (2C), 128.2 (2C), 128.1,
122.4, 80.5, 70.1, 66.2, 55.1, 47.7, 47.0, 46.1, 44.2, 42.0, 41.6, 39.7, 39.6,
38.4, 34.0, 33.3, 32.6 (2C), 30.9, 28.7, 28.1 (2C), 27.8, 26.1, 23.9, 23.7,
23.3, 18.5, 17.9, 17.1, 16.6, 9.6, 9.4; ESI-HRMS m/z calcd for C43H63O6
[M þ 1] 675.4625, found 675.4626.
4.5.33. Acylation of MA-Bn with lauric anhydride
Lauric anhydride (544 mg, 1.422 mmol) was added slowly to a
solution of MA-Bn (100 mg, 0.177 mmol) in pyridine (2 mL). The
mixture was treated as described in the general method for acyl-
ation to give 54 as a colourless oil (44 mg, 33%), [
a
]
þ 25 (c 1 in
D
CHCl3:MeOH, 2:1); IR nmax(film)/cmꢂ1 3388, 3012, 2815 and 1700;
dH (CDCl3) 7.31 (m, 5H), 5.25 (dd, 1H, J1 ¼ J2 ¼ 3.4 Hz), 5.02 (AB
system, 2H, J ¼ 12.0 Hz), 4.93 (ddd, 1H, J1 ¼ 4.3, J2 ¼ 10.6,
J3 ¼ 11.5 Hz), 3.16 (d, 1H, J ¼ 10.6 Hz), 2.88 (dd, 1H, J1 ¼ 4.5,
J2 ¼ 13.6 Hz), 2.29 (t, 2H, J ¼ 6.6 Hz), 1.10, 1.03, 0.98, 0.90, 0.88, 0.86,
0.84 (s, 3H each); dC (CDCl3) 177.6, 174.7, 144.0, 136.6, 128.6 (2C),
128.2, 128.1 (2C), 122.4, 81.1, 73.3, 66.2, 55.4, 47.8, 46.9, 46.1, 43.9,
42.0, 41.6, 40.0, 39.6, 38.5, 34.9, 34.1, 33.3, 32.7, 32.6, 32.1, 30.9, 29.8
(3C), 29.7 (2C), 29.4, 28.8, 27.8, 26.1, 25.3, 23.9, 23.7, 23.3, 22.9, 18.5,
17.1, 16.9, 16.6, 14.3; ESI-HRMS m/z calcd for C49H77O5 [M þ 1]
745.5771, found 745.5758; and 55 as a colourless oil (102 mg, 61%),
4.5.31. Acylation of MA-Bn with butanoic anhydride
Butanoic anhydride (232 mL) was added slowly to a solution of
MA-Bn (100 mg, 0.177 mmol) in pyridine (2 mL). The mixture was
treated as in the general method for acylation above to give 50 as a
white solid (12 mg, 10%), mp 33e35 ꢀC; [
a
]
D
þ 53 (c 1 in
CHCl3:MeOH, 2:1); IR nmax(KBr)/cmꢂ1 3376, 3015, 2842 and 1710;
dH (CDCl3) 7.31 (m, 5H), 5.25 (dd, 1H, J1 ¼ J2 ¼ 3.5 Hz), 5.05 (AB
system, 2H, J ¼ 12.0 Hz), 4.92 (ddd,1H, J1 ¼4.4, J2 ¼ 9.8, J3 ¼ 11.3 Hz),
3.16 (d, 1H, J ¼ 9.8 Hz), 2.88 (dd, 1H, J1 ¼ 4.5, J2 ¼ 14.0 Hz), 2.27 (t,
2H, J ¼ 6.5 Hz), 1.10, 1.03, 0.93, 0.89, 0.89, 0.87, 0.84 (s, 3H each); dC
(CDCl3) 177.5, 174.5, 144.0, 136.6, 128.6 (2C), 128.2 (2C), 128.1, 122.4,
81.1, 73.3, 66.2, 55.4, 47.8, 46.9, 46.1, 44.0, 41.6, 41.5, 40.0, 39.5, 38.5,
36.8, 34.1, 33.4, 32.8, 32.6, 30.9, 28.8, 27.8, 26.1, 23.9, 23.8, 23.3, 18.8,
[a
]
þ 1 (c 1 in CHCl3:MeOH, 2:1); IR nmax(film)/cmꢂ1 3419, 2999,
D
2850 and 1708; dH (CDCl3) 7.31 (m, 5H), 5.25 (dd, 1H,
J1 ¼ J2 ¼ 3.3 Hz), 5.06 (ddd, 1H, J1 ¼ 4.3, J2 ¼ 10.5, J3 ¼ 11.2 Hz), 5.05
(AB system, 2H, J ¼ 12.0 Hz), 4.75 (d, 1H, J ¼ 10.5 Hz), 2.88 (dd, 1H,
J1 ¼ 4.3, J2 ¼ 13.7 Hz), 2.21 and 2.12 (q, 2H each, J ¼ 6.5 Hz), 1.09,
1.00, 0.90, 0.87, 0.87, 0.86, 0.83 (s, 3H each); dC (CDCl3) 177.6, 173.7,
173.6, 144.0, 136.6, 128.6 (2C), 128.2, 128.1 (2C), 122.4, 80.4, 70.1,
66.2, 55.1, 47.7, 46.9, 46.1, 44.2, 41.9, 41.6, 39.6 (2C), 38.3, 34.9, 34.8,

A. Parra et al. / European Journal of Medicinal Chemistry 74 (2014) 278e301
297
34.0, 33.3, 32.7 (2C), 32.2 (2C), 30.0, 29.9 (4C), 29.8 (4C), 29.6 (3C),
30.9, 28.7, 27.8, 26.1, 24.9 (2C), 23.9, 23.7, 23.3, 22.9, 22.2, 18.5, 17.9,
17.1, 16.6, 14.3 (2C); ESI-HRMS m/z calcd for C61H99O6 [M þ 1]
927.7442, found 927.7434.
acylation to give 60 as a colourless oil (34 mg, 27%), [
a
]
D þ 13 (c 1 in
CHCl3:MeOH, 2:1); IR nmax(film)/cmꢂ1 3421, 2983, 2845 and 1722;
dH (CDCl3) 7.30 (m, 5H), 5.24 (dd, 1H, J1 ¼ J2 ¼ 3.4 Hz), 5.05 (ddd, 1H,
J1 ¼ 4.5, J2 ¼ 9.9, J3 ¼ 11.2 Hz), 5.04 (AB system, 2H, J ¼ 12.0 Hz), 3.20
(d, 1H, J ¼ 9.9 Hz), 2.70e0.50 (m, 4H), 2.88 (dd, 1H, J1 ¼ 4.4,
J2 ¼ 13.9 Hz), 1.14, 1.09, 1.01, 0.95, 0.89, 0.87, 0.78 (s, 3H each); dC
(CDCl3) 177.6 (2C), 172.8, 143.9, 136.6, 128.6 (2C), 128.2 (2C), 128.1,
122.4, 80.9, 73.7, 66.2, 55.3, 47.7, 46.9, 46.1, 43.8, 41.9, 41.6, 39.8,
39.5, 38.5, 34.1, 33.3, 32.7, 32.5, 30.9, 29.9, 29.8, 28.8, 27.8, 26.1, 23.8,
23.4, 23.3, 18.4, 17.0, 16.9, 16.5; ESI-HRMS m/z calcd for C41H59O7
[M þ 1] 663.4261, found 663.4272; and 61 as a colourless oil
4.5.34. Acylation of MA-Bn with benzoic anhydride
Benzoic anhydride (268 mL) was added slowly to a solution of
MA-Bn (100 mg, 0.177 mmol) in pyridine (2 mL). The mixture was
treated as described in the general method for acylation to give 56
as a white solid (44 mg, 35%), mp 120e122 ꢀC; [
a
]
D
þ 14 (c 1 in
CHCl3:MeOH, 2:1); IR nmax(KBr)/cmꢂ1 3401, 3022, 2848 and 1712;
dH (CDCl3) 7.45e8.10 (m, 5H), 7.32 (m, 5H), 5.25 (dd, 1H,
J1 ¼ J2 ¼ 3.5 Hz), 5.20 (ddd, 1H, J1 ¼ 4.7, J2 ¼ 10.2, J3 ¼ 11.1 Hz), 5.05
(AB system, 2H, J ¼ 12.0 Hz), 3.35 (d, 1H, J ¼ 10.2 Hz), 2.88 (dd, 1H,
J1 ¼ 4.5, J2 ¼ 13.8 Hz), 1.11, 1.08, 1.05, 0.92, 0.90, 0.89, 0.88 (s, 3H
each); dC (CDCl3) 177.6, 167.2, 143.9, 136.6, 133.9, 130.4, 129.8 (2C),
128.7 (2C), 128.6 (2C), 128.2 (2C), 128.1, 122.4, 81.1, 74.3, 66.1, 55.4,
47.8, 46.9, 46.1, 44.0, 41.9, 41.6, 40.1, 39.5, 38.6, 34.1, 33.3, 32.8, 32.6,
30.9, 28.8, 27.8, 26.1, 23.8, 23.7, 23.2, 18.5, 17.1, 16.9, 16.6; ESI-HRMS
m/z calcd for C44H58O5Na [M þ Na] 689.4182, found 689.4175; and
(65 mg, 43%), [
a
]
þ 10 (c 1 in CHCl3:MeOH, 2:1); IR nmax(film)/
D
cmꢂ1 3379, 2984, 2855 and 1700; dH (CDCl3) 7.30 (m, 5H), 5.25 (dd,
1H, J1 ¼ J2 ¼ 3.5 Hz), 5.10 (ddd, 1H, J1 ¼ 4.4, J2 ¼ 10.3, J3 ¼ 11.3 Hz),
5.04 (AB system, 2H, J ¼ 12.0 Hz), 4.75 (d, 1H, J ¼ 10.3 Hz), 2.89 (dd,
1H, J1 ¼ 4.6, J2 ¼ 13.7 Hz), 2.70e2.55 (m, 8H), 1.09, 0.99, 0.90, 0.88,
0.88, 0.86, 0.56 (s, 3H each); dC (CDCl3) 178.6 (2C), 177.6, 172.1, 171.9,
144.0,136.6,128.7 (2C),128.3 (2C),128.2,122.3, 81.2, 70.7, 66.2, 55.1,
47.8, 46.9, 46.1, 44.2, 41.9, 41.6, 39.7, 39.6, 38.4, 34.1, 33.4, 32.7, 32.6,
30.9, 29.3 (2C), 29.1 (2C), 28.7, 27.8, 26.1, 23.9, 23.7, 23.3, 18.5, 18.0,
17.1, 16.6; ESI-HRMS m/z calcd for C45H62O10Na [M þ Na] 785.4241,
found 785.4246.
57 as a white solid (70 mg, 51%), mp 75e77 ꢀC; [
a] e 18 (c 1 in
D
CHCl3:MeOH, 2:1); IR nmax(KBr)/cmꢂ1 3379, 2991, 2845 and 1729;
dH (CDCl3) 7.95e7.35 (m, 10H), 7.31 (m, 5H), 5.53 (ddd, 1H, J1 ¼ 4.6,
J2 ¼ 10.4, J3 ¼ 11.5 Hz), 5.30 (dd, 1H, J1 ¼ J2 ¼ 3.5 Hz), 5.27 (d, 1H,
J ¼ 10.4 Hz), 5.13 (AB system, 2H, J ¼ 12.0 Hz), 2.97 (dd, 1H, J1 ¼ 4.5,
J2 ¼ 13.7 Hz), 1.21, 1.20, 1.14, 1.06, 0.96, 0.94, 0.68 (s, 3H each); dC
(CDCl3) 177.6, 166.7, 166.5, 144.0, 136.7, 133.1, 133.0, 130.8, 129.8
(2C), 129.8 (2C), 129.1 (2C), 128.5 (4C), 128.3 (2C), 128.2 (2C), 122.4,
81.3, 71.3, 66.2, 55.4, 47.9, 47.0, 46.1, 44.2, 42.0, 41.7, 40.1, 39.7, 38.6,
34.2, 33.4, 32.8, 32.6, 31.0, 28.8, 27.9, 26.2, 23.9, 23.8, 23.3, 18.6, 18.1,
17.2, 16.8; ESI-HRMS m/z calcd for C51H63O6 [M þ 1] 771.4625,
found 771.4601.
4.5.37. Acylation of MA-Bn with glutaric anhydride
Glutaric anhydride (162 mg, 1.422 mmol) was added slowly to a
solution of MA-Bn (100 mg, 0.177 mmol) in pyridine (2 mL). The
mixture was treated as described in the general method for acyl-
ation to give 62 as a colourless oil (27 mg, 21%), [
a
]
þ 19 (c 1 in
D
CHCl3:MeOH, 2:1); IR nmax(film)/cmꢂ1 3401, 3015, 2871 and 1699;
dH (CDCl3) 7.31 (m, 5H), 5.24 (dd, 1H, J1 ¼ J2 ¼ 3.5 Hz), 4.98 (AB
system, 2H, J ¼ 12.0 Hz), 4.96 (ddd, 1H, J1 ¼ 4.4, J2 ¼ 10.2,
J3 ¼ 11.5 Hz), 3.18 (d, 1H, J ¼ 10.2 Hz), 2.87 (dd, 1H, J1 ¼ 4.8,
J2 ¼ 13.8 Hz), 2.45e2.30 (m, 4H), 1.09, 1.02, 0.97, 0.89, 0.83, 0.78,
0.57 (s, 3H each); dC (CDCl3) 177.9, 177.6, 173.7, 144.0, 136.6, 128.7
(2C), 128.3 (2C), 128.2, 122.3, 81.2, 70.7, 66.2, 55.1, 47.8, 46.9, 46.1,
44.2, 41.9, 41.6, 39.7, 39.6, 38.4, 34.1, 33.4, 32.7, 32.6, 30.9, 29.3, 29.1,
28.7, 27.8, 26.1, 23.9, 23.7, 23.3, 20.2, 18.5, 18.0, 17.1, 16.6; ESI-HRMS
m/z calcd for C42H60O7Na [M þ Na] 699.4237, found 699.4261; and
4.5.35. Acylation of MA-Bn with phthalic anhydride
Phthalic anhydride (210 mg, 1.422 mmol) was added slowly to a
solution of MA-Bn (100 mg, 0.177 mmol) in pyridine (2 mL). The
mixture was treated as described in the general method for acyl-
ation to give 58 as a colourless oil (36 mg, 27%), [
a
]
þ 45 (c 1 in
D
CHCl3:MeOH, 2:1); IR nmax(film)/cmꢂ1 3427, 3030, 2862 and 1731;
dH (CDCl3) 8.30e7.30 (m, 4H), 7.32 (m, 5H), 5.64 (ddd, 1H, J1 ¼ 4.4,
J2 ¼ 10.5, J3 ¼ 11.3 Hz), 5.38 (dd, 1H, J1 ¼ J2 ¼ 3.5 Hz), 5.08 (AB
system, 2H, J ¼ 12.0 Hz), 3.33 (d, 1H, J ¼ 10.5 Hz), 2.72 (dd, 1H,
J1 ¼4.7, J2 ¼ 14.0 Hz),1.28,1.26,1.10,1.08,1.05,1.01, 0.94 (s, 3H each);
dC (CDCl3) 180.7, 169.4, 168.3, 145.1, 136.7, 133.8, 132.9, 132.3, 129.9,
128.5 (2C), 128.4, 128.2 (4C), 122.2, 82.3, 74.9, 66.2, 55.5, 47.8, 46.9,
46.8, 43.8, 42.2, 41.4, 40.4, 39.9, 38.6, 34.4, 33.4, 33.2, 33.1, 31.2, 28.9,
27.4, 26.3, 24.2, 24.0, 23.9, 18.6, 18.3, 17.4, 16.5; ESI-HRMS m/z calcd
for C45H58O7Na [M þ Na] 733.4080, found 733.4068; and 59 as a
63 as a colourless oil (75 mg, 51%), [
a
]
þ 1 (c 1 in CHCl3:MeOH,
D
2:1); IR nmax(film)/cmꢂ1 3378, 3021, 2861 and 1725; dH (CDCl3) 7.32
(m, 5H), 5.25 (dd, 1H, J1 ¼ J2 ¼ 3.5 Hz), 5.10 (ddd, 1H, J1 ¼ 4.5,
J2 ¼ 10.5, J3 ¼ 11.3 Hz), 5.04 (AB system, 2H, J ¼ 12.0 Hz), 4.75 (d, 1H,
J ¼ 10.5 Hz), 2.88 (dd, 1H, J1 ¼ 4.3, J2 ¼ 13.8 Hz), 2.50e2.10 (m, 8H),
1.09, 1.02, 0.99, 0.88, 0.87, 0.87, 0.56 (s, 3H each); dC (CDCl3) d 178.6
(2C), 177.5, 172.9 (2C), 143.9, 136.6, 128.6 (2C), 128.2 (2C), 128.1,
122.3, 80.4, 70.2, 66.1, 55.1, 47.7, 46.9, 46.1, 44.0, 41.9, 41.5, 39.6, 39.5,
38.4, 34.1, 33.4 (2C), 33.3, 33.1 (2C), 32.7, 32.5, 30.9, 28.6, 27.8, 26.1,
23.8, 23.7, 23.2, 20.3, 20.2, 18.4, 17.9, 17.0, 16.6; ESI-HRMS m/z calcd
for C47H66O10Na [M þ Na] 813.4554, found 813.4523.
colourless oil (89 mg, 55%), [
a
]
þ 1 (c 1 in CHCl3:MeOH, 2:1); IR
D
nmax(film)/cmꢂ1 3366, 3019, 2868 and 1701; dH (CDCl3) 8.30e7.30
(m, 8H), 7.32 (m, 5H), 5.78 (ddd, 1H, J1 ¼ 4.5, J2 ¼ 10.3, J3 ¼ 11.5 Hz),
5.62 (d, 1H, J ¼ 10.3 Hz), 5.40 (dd, 1H, J1 ¼ J2 ¼ 3.5 Hz), 5.05 (AB
system, 2H, J ¼ 12.0 Hz), 2.70 (dd, 1H, J1 ¼ 4.7, J2 ¼ 13.7 Hz), 1.20,
1.16, 1.11, 1.09, 1.07, 1.04, 0.98 (s, 3H each); dC (CDCl3) 180.1, 170.6,
169.9, 168.6, 168.2, 145.1, 136.7, 133.8 (2C), 132.9 (2C), 132.3 (2C),
129.8 (2C), 128.5 (2C), 128.4 (2C), 128.2 (2C), 128.1 (2C), 128.0, 122.2,
82.4, 72.1, 66.3, 55.5, 48.0, 46.9, 46.8, 43.9, 42.2, 41.4, 40.2, 39.9,
38.6, 34.4, 33.5, 33.4, 33.2, 31.1, 28.9, 27.5, 26.4, 24.1, 23.9 (2C), 18.6,
18.3, 17.4, 16.5; ESI-HRMS m/z calcd for C53H63O10 [M þ 1] 859.4421,
found 859.4420.
4.5.38. Acylation of MA-Bn with 3,3-dimethylglutaric anhydride
3,3-Dimethylglutaric anhydride (202 mg, 1.422 mmol) was
added slowly to a solution of MA-Bn (100 mg, 0.177 mmol) in
pyridine (2 mL). The mixture was treated as described in the gen-
eral method for acylation to give 64 as a colourless oil (33 mg, 25%),
[a
]
D þ 25 (c 1 in CHCl3:MeOH, 2:1); IR nmax(film)/cmꢂ1 3383, 3010,
2829 and 1715; dH (CDCl3) 7.31 (m, 5H), 5.25 (dd, 1H,
J1 ¼ J2 ¼ 3.4 Hz), 5.04 (AB system, 2H, J ¼ 12.0 Hz), 4.94 (ddd, 1H,
J1 ¼ 4.5, J2 ¼ 10.5, J3 ¼ 11.3 Hz), 3.19 (d, 1H, J ¼ 10.5 Hz), 2.87 (dd, 1H,
J1 ¼ 4.3, J2 ¼ 13.7 Hz), 2.45e2.35 (m, 4H), 1.14, 1.12, 1.10, 1.02, 0.98,
0.89, 0.87, 0.84, 0.56 (s, 3H each); dC (CDCl3) 177.6 (2C) 172.8, 144.0,
136.6, 128.6 (2C), 128.2 (2C), 128.1, 122.4, 80.9, 73.5, 66.2, 55.3, 47.8,
46.9, 46.1, 45.4, 45.1, 43.9, 42.0, 41.6, 39.9, 39.6, 38.5, 34.1, 33.3, 33.0,
32.8, 32.6, 30.9, 28.8, 28.6, 28.5, 27.8, 26.1, 23.9, 23.7, 23.2, 18.5, 17.1,
16.9, 16.5; ESI-HRMS m/z calcd for C44H64O7Na [M þ Na] 727.4550,
4.5.36. Acylation of MA-Bn with succinic anhydride
Succinic anhydride (142 mg, 1.422 mmol) was added slowly to a
solution of MA-Bn (100 mg, 0.177 mmol) in pyridine (2 mL). The
mixture was treated as described in the general method for

298
A. Parra et al. / European Journal of Medicinal Chemistry 74 (2014) 278e301
found 727.4552; and 65 as a colourless oil (71 mg, 47%), [
a
]
D þ 10 (c
min. A wavelength of 301 nm was selected for the purity analysis,
using a linear gradient of 30e0% of B in 30 min and maintained for
5 min without B, where A was CH3CN containing 0.1% TFA and B was
H2O containing 0.1% TFA.
1 in CHCl3:MeOH, 2:1); IR nmax(film)/cmꢂ1 3401, 3027, 2839 and
1732; dH (CDCl3) 7.31 (m, 5H), 5.25 (dd, 1H, J1 ¼ J2 ¼ 3.5 Hz), 5.06
(ddd, 1H, J1 ¼ 4.5, J2 ¼ 10.3, J3 ¼ 11.3 Hz), 5.04 (AB system, 2H,
J ¼ 12.0 Hz), 4.77 (d, 1H, J ¼ 10.3 Hz), 2.88 (dd, 1H, J1 ¼ 4.3,
J2 ¼ 13.8 Hz), 2.55e2.25 (m, 8H), 1.12, 1.12, 1.09, 1.00, 0.89, 0.87, 0.87,
0.84, 0.56 (s, 3H each); dC (CDCl3) 177.6, 177.1 (2C), 172.1, 171.9, 144.1,
136.6, 128.6 (2C), 128.2 (3C), 122.3, 80.4, 70.5, 66.2, 55.1, 47.7, 46.9,
46.1, 45.5 (2C), 45.1 (2C), 44.2, 42.0, 41.6, 39.6 (2C), 38.4, 34.1, 33.3,
32.6, 32.5, 31.1 (2C), 30.9, 28.8, 28.2 (2C), 28.0 (2C), 27.8, 26.1, 23.9,
23.7, 23.5, 18.5, 18.0, 17.1, 16.6; ESI-HRMS m/z calcd for C51H73O10
[M ꢂ 1] 845.5204, found 845.5194.
4.6.1. Reaction of OA with Fmoc-Gly
A solution of Fmoc-Gly with DIPCDI and DMAP in DCM/DMF was
added to a solution of 1 in DCM, as described in the above step 4 of
the general procedure for the quantization of the incorporation of
OA to the CTC resin. The reaction was stirred for 2 h at reflux. Then,
cold water was added to the mixture and afterwards 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 66 as a white solid, with the percentages given in Table 3, mp
4.6. General procedure for the quantization of the incorporation of
OA and MA to the CTC resin
115e117 ꢀC; [
a
]
D þ 27 (c 1 in CHCl3); IR nmax(KBr)/cmꢂ1 3620, 2941,
2395 and 1699; dH (CDCl3) 7.79 (d, 2H, J ¼ 6.5 Hz), 7.63 (d, 2H,
J ¼ 6.5 Hz), 7.45e7.29 (m, 4H), 5.31 (dd, 1H, J1 ¼ J2 ¼ 3.3 Hz), 4.62
(dd, 1H, J1 ¼ J2 ¼ 7.5 Hz), 4.43 (d, 1H, J ¼ 6.8 Hz), 4.26 (t, 1H,
J ¼ 6.8 Hz), 4.01 (d, 2H, J ¼ 3.5 Hz), 1.07, 1.01, 0.89, 0.87, 0.85, 0.84,
0.59 (s, 3H each); dC (CDCl3) 184.0, 170.1, 156.5, 144.0 (2C), 143.8,
141.5 (2C), 127.9 (2C), 127.3 (2C), 125.3 (2C), 122.6, 120.2 (2C), 82.8,
67.4, 55.5, 47.8, 47.3, 46.7, 46.1, 43.2, 41.8, 41.1, 39.6, 38.2, 38.1, 37.2,
34.0, 33.3, 32.7, 32.6, 30.9, 28.3, 27.9, 26.1, 23.8 (2C), 23.7, 23.1, 18.4,
17.3, 16.8, 15.6; ESI-HRMS m/z calcd for C47H62NO6 [M þ 1]
736.4577, found 736.4570.
Step 1. Portions of 50 mg of CTC resin (loading 1.27 mmol/g)
were placed in six polypropylene syringes (10 mL) fitted with
polyethylene filter disks. The resin was washed with DMF
(2 mL ꢁ 3) and DCM (2 mL ꢁ 3), swelled with DCM (2 mL, 20 min)
and drained. Each syringe was treated with the corresponding so-
lution of OA (1) or MA (2) and DIEA indicated below and placed in
orbital agitation for 48 h. Afterwards, the resin was filtered, washed
with DMF (2 mL ꢁ 3) and DCM (2 mL ꢁ 3), and then drained.
OA (1)
DIEA
DCM
4.6.2. Reaction of MA with Fmoc-Gly
Test A (syringe 1)
Test B (syringe 2)
Test C (syringe 3)
14.5 mg, 0.5 equiv
29.0 mg, 1.0 equiv
87.0 mg, 3.0 equiv
110
220
660
m
m
m
L, 10 equiv
L, 20 equiv
L, 60 equiv
1 mL
1 mL
1 mL
A solution of Fmoc-Gly with DIPCDI and DMAP in DCM/DMF was
added to a solution of 2 in DCM, as described in the above step 4 of
the general procedure for the quantization of the incorporation of
MA to the CTC resin. The reaction was stirred for 12 h at rt. Then,
cold water was added to the mixture and afterwards 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 (20:1) to
give as white solids, with the percentages given in Table 3: 67, mp
MA (2)
DIEA
DCM
Test A (syringe 4)
Test B (syringe 5)
Test C (syringe 6)
15.0 mg, 0.5 equiv
29.9 mg, 1.0 equiv
89.9 mg, 3.0 equiv
110
220
660
m
m
m
L, 10 equiv
L, 20 equiv
L, 60 equiv
1 mL
1 mL
1 mL
170e172 ꢀC; [
a]
D þ 6 (c 1 in CHCl3); IR nmax(KBr)/cmꢂ1 3636, 2930,
Step 2. The six syringes with the resin were now treated with the
corresponding six solutions of Fmoc-Leu-OH (112 mg, 5 equiv each)
2389 and 1700; dH (CDCl3) 7.76 (d, 2H, J ¼ 6.4 Hz), 7.60 (d, 2H,
J ¼ 6.4 Hz), 7.43e7.29 (m, 4H), 5.26 (dd, 1H, J1 ¼ J2 ¼ 3.3 Hz), 5.06
(ddd,1H, J1 ¼4.5, J2 ¼ 10.5, J3 ¼ 11.3 Hz), 4.42 (d, 2H, J ¼ 6.4 Hz), 4.24
(t, 1H, J ¼ 6.4 Hz), 4.02e3.92 (m, 4H) 3.19 (d, 1H, J ¼ 10.5 Hz), 2.80
(dd, 1H, J1 ¼ 3.4, J2 ¼ 10.5 Hz), 1.12, 1.04, 1.03, 0.93, 0.92, 0.86 0.75 (s,
3H each); dC (CDCl3) 182.5, 170.3, 157.3, 143.8, 143.8 (2C), 141.5 (2C),
127.9 (2C), 127.3 (2C), 125.7 (2C), 122.4, 120.2 (2C), 80.7, 74.9, 67.5,
55.3, 47.7, 47.2, 46.7, 46.0, 43.7, 43.3, 41.2, 39.9, 39.5, 38.6, 34.0, 33.2,
32.6 (2C), 30.8, 29.9, 28.7, 27.8, 26.1, 23.7, 23.6, 23.1 18.4, 17.2, 16.8,
16.5; ESI-HRMS m/z calcd for C47H62NO7 [M þ 1] 752.4526, found
and DIEA (110
stirring for 2 h.
mL, 10 equiv each) in DCM, and placed in orbital
Step 3. For the capping of the resin, 0.5 mL of MeOH were added
to each syringe and maintained with orbital stirring for 30 min. The
resin was filtered, washed with DMF (2 mL ꢁ 3) and DCM (2 mL ꢁ 3)
and then drained.
Step 4. The solutions of Fmoc-Gly, DIPCDI, DMAP in DCM/DMF
specified below were added to the different syringes and placed in
orbital stirring for 1 h. The syringes were filtered, rinsed with DCM
and treated twice more with the same solutions. After the three
treatments, the resin was filtered, washed with DMF (2 mL ꢁ 3) and
DCM (2 mL ꢁ 3), and then drained.
752.4529; 68, mp 143e145 ꢀC; [
a
]
D þ 3 (c 1 in CHCl3); IR nmax(KBr)/
cmꢂ1 3619, 2922, 2388 and 1705; dH (CDCl3) 7.76 (d, 2H, 6.5 Hz),
7.59 (d, 2H, J ¼ 6.5 Hz), 7.42e7.28 (m, 4H), 5.29 (dd, 1H,
J1 ¼ J2 ¼ 3.5 Hz), 4.60 (d, 1H, J ¼ 12.5 Hz), 4.42 (d, 2H, J ¼ 12.5 Hz),
4.24 (t, 1H, J ¼ 6.5), 4.05e4.00 (m, 4H) 3.80 (ddd, 1H, J1 ¼ 3.5,
J2 ¼ 10.5, J3 ¼ 12.5 Hz), 2.83 (dd, 1H, J1 ¼ 3.5, J2 ¼ 10.5 Hz), 1.14, 0.98,
0.93, 0.91, 0.90, 0.85, 0.76 (s, 3H each); dC (CDCl3) 185.32, 170.8,
157.3, 143.9, 143.8 (2C), 141.5 (2C), 127.9 (2C), 127.2 (2C), 125.2 (2C),
122.5, 120.2 (2C), 86.6, 67.5, 67.3, 55.3, 47.7, 47.3, 46.6, 46.0, 43.4,
41.9, 41.3, 39.5, 39.4, 38.4, 34.0, 33.2, 32.7, 32.6, 30.8, 29.9, 28.8, 27.8,
26.1, 23.7, 23.6, 23.1 18.4, 17.8, 17.3, 16.7; ESI-HRMS m/z calcd for
Fmoc-Gly-OH
DIPCDI
98 L, 10 equiv
DMAP
Syringes 1, 2 and 3
Syringes 4, 5 and 6
188 mg, 10 equiv
377 mg, 20 equiv
m
7 mg, 1 equiv
15 mg, 2 equiv
196 L, 20 equiv
m
Step 5. The cleavage of the OA or MA derivatives from the resin
was performed by adding to each syringe a solution of TFA (1%) in
DCM (2 min ꢁ 3).
C
47H62NO7 [M þ 1] 752.4526, found 752.4525; and 69, mp 121e
123 ꢀC; [ D e 1 (c 1 in CHCl3); IR nmax(KBr)/cmꢂ1 3615, 2927, 2390
a]
The resulting mixtures of Fmoc-Gly-OA or Fmoc-Gly-MA and
Fmoc-Leu-OH were then analyzed by HPLC. RP-HPLC analyses were
and 1710; dH (CDCl3) 7.79 (d, 4H, J ¼ 6.5 Hz), 7.63 (d, 4H, J ¼ 6.5 Hz),
7.45e7.29 (m, 8H), 5.34 (ddd, 1H, J1 ¼ 4.5, J2 ¼ 10.5, J3 ¼ 11.3 Hz),
5.23 (dd, 1H, J1 ¼ J2 ¼ 3.5 Hz), 4.80 (d, 1H, J ¼ 10.5 Hz), 4.18 (t, 2H,
J ¼ 6.6 Hz), 3.95e3.87 (m, 8H) 2.80 (dd, 1H, J1 ¼ 3.5, J2 ¼ 13.4 Hz),
carried out with a WATERS C18 reverse-phase column (0.4
diameter of particle; 150 mm ꢁ 3.9 mm) with a flow rate of 1 mL/
mm

A. Parra et al. / European Journal of Medicinal Chemistry 74 (2014) 278e301
299
1.11, 1.04, 0.85, 0.81, 0.81, 0.75 0.53 (s, 3H each); dC (CDCl3) 183.7,
170.3, 169.9, 157.0 (2C), 143.9 (4C), 143.8, 141.4 (4C), 127.9 (4C), 127.3
(4C), 125.7 (4C),122.2,120.1 (4C), 82.1, 71.0, 67.5 (2C), 55.0, 47.7, 47.2
(2C), 46.7, 46.0, 43.9, 43.1, 42.9, 41.8, 41.1, 39.6, 39.5, 38.5, 34.0 (2C),
32.6, 32.5, 30.9, 28.6, 27.8, 26.1, 23.8, 23.6, 23.0 18.3, 17.8, 17.3, 16.6;
ESI-HRMS m/z calcd for C64H75N2O10 [M þ 1] 1031.5422, found
1031.5428.
was stirred for 4 h at 55 ꢀC. Then, the reaction mixture was diluted
with water and 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 DCM/acetone (10:1) to give benzyl maslinate (4, 32 mg, 5.7%);
benzyl 2-(benzylsuccinyl) maslinate (71, 57 mg, 7.6%) as a colour-
less oil; [
a
]
D þ 6 (c 1 in CHCl3); IR nmax(film)/cmꢂ1 3301, 3012, 2915
and 1783; dH (CDCl3) 7.36e7.34 (m, 10H), 5.27 (dd, 1H,
J1 ¼ J2 ¼ 3.4 Hz), 5.14 and 5.08 (2 AB systems, 2H each, J ¼ 12.0 Hz),
5.00 (ddd, 1H, J1 ¼ 3.0, J2 ¼ 6.0, J3 ¼ 10.5 Hz), 3.14 (d, 1H, J ¼ 6.0 Hz),
2.91 (dd, 1H, J1 ¼ 3.5, J2 ¼ 12.0 Hz), 2.75e2.60 (m, 4H), 1.12, 1.05,
0.99, 0.92, 0.90, 0.86, 0.59 (s, 3H each); dC (CDCl3) 177.5, 172.7, 172.5,
143.9, 136.5, 135.8, 128.7, 128.5 (2C), 128.4 (2C), 128.3 (2C), 128.1
(2C), 128.0, 122.3, 80.7, 73.9, 66.8, 66.1, 55.2, 47.6, 46.8, 46.0, 43.6,
41.8, 41.5, 39.7, 39.4, 38.4, 34.0, 33.2, 32.7, 32.5, 30.8, 29.7, 29.6, 28.7,
27.7, 26.0, 23.8, 23.6, 23.1, 18.3, 17.0, 16.8, 16.4; ESI-HRMS m/z calcd
for C48H65O7 [M þ 1] 753.4730, found 753.4714; benzyl 3-(ben-
4.7. General method for solid-phase acylation
Two portions of CTC resin (250 mg, 1.27 mmol/g) were placed in
two polypropylene syringes (20 mL) fitted with polyethylene filter
disks. The resin was washed with DMF (2 mL ꢁ 3) and DCM
(2 mL ꢁ 3), swelled with DCM (2 mL, 20 min) and drained. OA (1)
and MA (2) were incorporated into the resin as described in the
above general procedure, and placed in orbital stirring for 24 h.
Afterwards the resin was recoupled with the same solutions for
another 24 h. For the capping of the resin, 0.5 mL of MeOH were
added to each syringe and maintained with orbital stirring for
30 min. The resin was filtered, washed with DMF (2 mL ꢁ 3) and
DCM (2 mL ꢁ 3), and then drained.
zylsuccinyl) maslinate (72, 43 mg, 5.5%) as a colourless oil; [
a
]
D þ 3
(c 1 in CHCl3); IR nmax(film)/cmꢂ1 3343, 3076, 2915 and 1783; dH
(CDCl3) 7.35e7.34 (m, 10H), 5.29 (dd, 1H, J1 ¼ J2 ¼ 3.5 Hz), 5.13 and
5.07 (2 AB systems, 2H each, J ¼ 12.5 Hz), 4.57 (d, 1H, J ¼ 6.5 Hz),
3.75 (ddd, 1H, J1 ¼ 3.5, J2 ¼ 6.5, J3 ¼ 10.5 Hz), 2.91 (dd, 1H, J1 ¼ 3.5,
J2 ¼ 12.0 Hz), 2.80e2.65 (m, 4H), 1.13, 0.95, 0.92, 0.90, 0.87, 0.84,
0.60 (s, 3H each); dC (CDCl3) 177.5, 173.0, 172.9, 143.8, 136.5, 135.7,
128.7,128.6 (2C),128.5 (2C),128.3 (2C),128.1 (2C),128.0,122.4, 85.6,
67.2, 66.9, 66.0, 55.2, 47.5, 46.9, 46.8, 45.8, 41.8, 41.5, 39.5, 39.3, 38.2,
34.0, 33.2, 32.5, 32.4, 30.8, 29.5, 29.4, 28.5, 27.6, 25.9, 23.6, 23.4,
23.0, 18.3, 17.7, 16.9, 16.6; ESI-HRMS m/z calcd for C48H65O7 [M þ 1]
753.4730, found 753.4722; and benzyl 2,3-di(benzylsuccinyl)
These two syringes were each split into four portions (1/4 each
one) to obtain four syringes of resin-OA and other four syringes of
resin-MA, which were now respectively acylated with acetic, hex-
anoic, benzoic, and succinic anhydrides at a molar relationship of
4:1 (anhydride:OH group), and placed in orbital agitation for 24 h at
rt. These acylation reactions were conducted in the presence of
DMAP (0.5 equiv), Et3N (0.5 equiv) and DCM/DMF (1 mL/1 mL) as
solvent. The resin was filtered, washed with DMF (2 mL ꢁ 3) and
DCM (2 mL ꢁ 3), and then drained. The cleavage of the acyled de-
rivatives from the resin was carried out by addition of each syringe
of a solution of DCM with TFA (1%) (2 min ꢁ 3).
maslinate (73, 721 mg, 76.5%) as a colourless oil; [
a
]
þ 57 (c 1 in
D
CHCl3); IR nmax(film)/cmꢂ1 3343, 3076, 2915 and 1783; dH (CDCl3)
7.38e7.31 (m, 15H), 5.29 (dd, 1H, J1 ¼ J2 ¼ 3.5 Hz), 5.14 (ddd, 1H,
J1 ¼ 3.0, J2 ¼ 6.0, J3 ¼ 10.5 Hz), 5.12 and 5.04 (m, 6H each), 4.79 (d,
1H, J ¼ 6.0 Hz), 2.93 (dd, 1H, J1 ¼ 3.5, J2 ¼ 12.0 Hz), 2.67e2.57 (m,
8H), 1.14, 1.03, 1.02, 0.94, 0.92, 0.89, 0.60 (s, 3H each); dC (CDCl3)
177.4, 172.2, 172.1, 172.0, 171.9, 143.7, 136.5, 135.8 (2C), 128.7, 128.5
(4C), 128.4 (4C), 128.3 (4C), 128.1, 128.0, 122.1, 80.7, 70.3, 66.5, 66.4,
66.0, 54.9, 47.5, 46.7, 45.8, 43.8, 41.7, 41.4, 39.5, 39.3, 38.1, 33.9, 33.2,
32.5, 32.4, 30.8, 29.3, 29.2, 29.1, 29.0, 28.4, 27.6, 25.9, 23.7, 23.5, 23.1,
18.2, 17.7, 16.8, 16.4; ESI-HRMS m/z calcd for C59H75O10 [M þ 1]
943.5360, found 943.5335.
4.7.1. Solid-phase succinylation and consecutive solution-phase
benzylation of OA
OA (150 mg, 0.33 mmol) was succinylated in solid-phase ac-
cording with the general procedure described above. Then, the
solvent was evaporated under reduced pressure and 110 mg of
residue were obtained. BnCl (83 mL) was added in a relationship 2:1
to a solution of this residue in DMF (5 mL) with K2CO3 (0.12 g). The
reaction was stirred for 4 h at 55 ꢀC. Then, the reaction mixture was
diluted with water and 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 chro-
matography using DCM/acetone (10:1) to give benzyl oleanolate (3,
72 mg, 40.1%), and benzyl 3-(benzylsuccinyl) oleanolate (70,
4.8. Biological experimental procedures
4.8.1. Drugs
The different compounds used in cell treatment were dissolved
before use at 10 mg/mL in 50% DMSO and 50% PBS. A stock solution
was frozen and stored at e 20 ꢀC. Prior to the experiments, this
solution was diluted in cell-culture medium. Apoptosis and
mitochondrial-membrane potential were measured at the IC50
(concentration causing 50% reduction in growth compared to the
control after 72 h of treatment) and the IC50 ꢁ 2 (twice IC50
concentration. ꢁ2 value corresponding to 50% viability)
concentrations.
133 mg, 54.9%) as a colourless oil; [
a
]
þ 77 (c 1 in CHCl3); IR
D
nmax(film)/cmꢂ1 3293, 2985, 2815 and 1699; dH (CDCl3) 7.36e7.35
(m, 10H), 5.31 (dd, 1H, J1 ¼ J2 ¼ 3.4 Hz), 5.13 and 5.07 (2 AB systems,
2H each, J ¼ 12.0 Hz), 4.53 (dd, 1H, J1 ¼ 5.5, J2 ¼ 10.6 Hz), 2.93 (dd,
1H, J1 ¼ 3.5, J2 ¼ 12.0 Hz), 2.75e2.60 (m, 4H), 1.15, 0.94, 0.92, 0.91,
0.87, 0.85, 0.63 (s, 3H each); dC (CDCl3) 178.4, 172.1, 171.8, 143.7,
136.5, 135.8, 128.6, 128.4 (2C), 128.3 (2C), 128.2 (2C), 128.0 (2C),
127.9, 122.4, 81.3, 66.5, 65.9, 55.3, 47.6, 46.8, 45.9, 41.7, 41.4, 39.3,
37.7, 36.9, 38.1, 33.9, 33.1, 32.9, 32.4, 30.7, 29.6, 29.3, 29.2, 28.1, 27.7,
25.9, 23.7, 23.5, 23.1, 18.2, 16.9, 16.8, 15.4; ESI-HRMS m/z calcd for
4.8.2. Cell culture
C
48H63O6 [M ꢂ 1] 735.4625, found 735.4623.
Murine melanoma cell line b16f10 (ECACC CRL-6475, provided
by the cell bank of the University of Granada, Spain) was cultured in
DMEM supplemented with 2 mM glutamine, 10% heat-inactivated
FCS, 10,000 units/mL of penicillin and 10 mg/mL of streptomycin.
Subconfluent monolayer cells were used in all experiments.
4.7.2. Solid-phase succinylation and consecutive solution-phase
benzylation of MA
MA (472 mg, 1 mmol) was succinylated in solid-phase according
with the general procedure described above. Then, the solvent was
evaporated under reduced pressure and 390 mg of residue were
4.8.3. Cell-proliferation activity assay
obtained. BnCl (364
tion of this residue in DMF (8 mL) with K2CO3 (0.53 g). The reaction
m
L) was added in a relationship 2:1 to a solu-
The effect of treating each product upon proliferation in b16f10
murine melanoma cells was measured using the MTT assay (Sigma,

300
A. Parra et al. / European Journal of Medicinal Chemistry 74 (2014) 278e301
MO, USA), which is based on the ability of live cells to cleave the
tetrazolium ring, thus producing formazan, which absorbs at
570 nm.
Cell viability was determined by measuring the absorbance of
MTT dye staining of living cells [33]. For this assay, 11$103 b16f10
cells were grown on a 96-well plate and incubated with the
calculated according to:
%
inhibition
¼
[1
ꢂ
(Vinh/V0)]$100
(Vinh ¼ enzymatic activity in the presence of the inhibitor;
V0 ¼ enzymatic activity of the control). The assays were performed
with a solution containing HIV-1-protease (10 hM), the fluorogenic
substrate (10
EDTA, 0.5 mM DTT, 1 M NaCl, 2.5% glycerol), and an increasing
concentrations of inhibitor from 0.17 g/mL to 1.7 mg/mL, in a total
volume of 140 L. The fluorescence was analyzed using an LS 50B
mM) in an assay buffer (50 mM sodium acetate, 1 mM
different products (0e80
m
g/mL). After 72 h, 100
mL of MTT solution
m
(0.5 mg/mL) was added to each well. After 2 h of incubation the
cells were washed twice with PBS and the formazan was resus-
m
fluorescence spectrometer at excitation/emission
420 nm.
¼
320 nm/
pended in 200
mL DMSO. Relative cell viability was measured by
absorbance at 550 nm on an ELISA plate reader (Tecan Sunrise
MR20-301, TECAN, Austria).
Acknowledgements
4.8.4. Annexin VeFICT/propidium iodide flow-cytometric analysis
The extension apoptosis was analyzed with flow-cytometry by
using a FACScan flow-cytometer (fluorescence-activated cell sorter)
(Coulter Corporation, Hialeah, FL, USA). In brief, 11$104 b16f10 cells
were plated in 12-well plates with 2 mL of medium following
treatment with the compounds for 72 h, at the IC50 concentrations,
calculated previously, and at twice the IC50 concentrations
(IC50 ꢁ 2). The cells were collected and resuspended in binding
buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2).
This work was financially supported by grants from the Minis-
terio de Ciencia e Innovación (CTQ2009e13898) and the Consejería
de Innovación, Ciencia y Empresa of the Junta de Andalucía (FQMe
0139). We thank David Nesbitt for reviewing the English in the
manuscript.
References
[1] A. Garcia-Granados, A. Martinez, J.N. Moliz, A. Parra, F. Rivas, 3
yolean-12-en-28-oic acid (oleanolic acid), Molecules 3 (1998) M87.
[2] A. Garcia-Granados, A. Martinez, J.N. Moliz, A. Parra, F. Rivas, 2
b
-hydrox-
Annexin VeFITC conjugate (1
mg/mL) was added and incubated for
a
,3 -dihy-
b
30 min at rt in darkness. Just before FACS analysis, cells were
droxyolean-12-en-28-oic acid (maslinic acid), Molecules 3 (1998) M88.
[3] J. Pollier, A. Goossens, Oleanolic acid, Phytochemistry 77 (2012) 10e15.
[4] A. Garcia-Granados, Process for the industrial recovery of oleanolic and
maslinic acids contained in the olive milling byproducts, PCT. Int. Appl. WO
9804331, 1998.
stained with 20 mL of 1 mg/mL PI solution. In each experiment,
approximately 20$103 cells were analyzed and the experiment was
duplicated three times.
[5] J.M. Lou-Bonafonte, C. Arnal, M.A. Navarro, J. Osada, Efficacy of bioactive
compounds from extra virgin olive oil to modulate atherosclerosis develop-
ment, Mol. Nutr. Food Res. 56 (2012) 1043e1057.
[6] I. Sogno, N. Vannini, G. Lorusso, R. Cammarota, D.M. Noonan, L. Generoso,
M.B. Sporn, A. Albini, Anti-angiogenic activity of a novel class of chemo-
preventive compounds: oleanic acid terpenoids, Recent Results Cancer Res.
181 (2009) 209e212.
4.8.5. Flow-cytometry analysis of the mitochondrial-membrane
potential
Oxidative damage was studied by flow-cytometry analysis of the
ROS levels, using dihydrorhodamine 123 oxidized to the highly
fluorescent product rhodamine. The formation of rhodamine can be
monitored by fluorescence spectroscopy using excitation and
emission wavelengths of 500 and 536 nm, respectively. The intra-
cellular measurement of the reactive oxygen species was made by
cytometry determination of rhodamine 123. In the same way as in
the apoptosis assays, 11$104 b16f10 cells were plated in 12-well
plates, and treated with the compounds that exhibited cytotoxic
activity for 72 h, at the IC50 and IC50 ꢁ 2 concentrations. After
treatment, the medium was removed and a fresh medium with
[7] A. Petronelli, G. Pannitteri, U. Testa, Triterpenoids as new promising anti-
cancer drugs, Anti-Cancer Drugs 20 (2009) 880e892.
[8] N. Sultana, A. Ata, Oleanolic acid and related derivatives as medicinally
important compounds, J. Enzym. Inhib. Med. Chem. 23 (2008) 739e756.
[9] F. Mengoni, M. Lichtner, L. Battinelli, M. Marzi, C.M. Mastroianni, V. Vullo,
G. Mazzanti, In vitro anti-HIV activity of oleanolic acid on infected human
mononuclear cells, Planta Med. 68 (2002) 111e114.
[10] M.B. Sporn, K.T. Liby, M.M. Yore, L. Fu, J.M. Lopchuk, G.W. Gribble, New syn-
thetic triterpenoids: potent agents for prevention and treatment of tissue
injury caused by inflammatory and oxidative stress, J. Nat. Prod. 74 (2011)
537e545.
[11] K.I. Wolska, A.M. Grudniak, B. Fiecek, A. Kraczkiewicz-Dowjat, A. Kurek,
Antibacterial activity of oleanolic and ursolic acids and their derivatives, Cent.
Eur. J. Biol. 5 (2010) 543e553.
[12] V.R. Preedy, R.R. Watson, Olives and Olive Oil in Health and Disease Preven-
tion, Elsevier, London, 2010.
DHR, at a final concentration of 5
incubation, the medium was removed and the cells were washed
and resuspended in PBS with 5 g/mL. The intensity of fluorescence
mg/mL, was added. After 30 min of
m
from Rh123 and PI was determined using a FACScan flow-cytom-
eter (fluorescence-activated cell sorter) (Coulter Corporation, Hia-
leah, FL, USA).
[13] R.A. Hill, J.D. Connolly, Triterpenoids, Nat. Prod. Rep. 29 (2012) 780e818 and
references therein.
[14] H. Sheng, H. Sun, Synthesis, biology and clinical significance of pentacyclic tri-
terpenes: a multi-target approach to prevention and treatment of metabolic and
vascular diseases, Nat. Prod. Rep. 28 (2011) 543e593 and references therein.
[15] N. Sultana, Z.S. Saify, Naturally occurring and synthetic agents as potential
anti-inflammatory and immunomodulants, Anti-Inflamm. Anti-Allergy Agents
Med. Chem. 11 (2012) 3e19.
4.8.6. Anti-HIV protease activity
The HIV activity was determined using recombinant HIV pro-
tease from E. coli source (PQITLWQRPL VTIKIGGQLK EALLDTGADD
TVLEEMNLPG
RWKPKMIGGI
GGFIKVRQYD
QILIEICGHK
[16] A. Bishayee, S. Ahmed, N. Brankov, M. Perloff, Triterpenoids as potential
agents for the chemoprevention and therapy of breast cancer, Front. Biosci. 16
(2011) 980e996.
[17] G. Kuttan, P. Pratheeshkumar, K.A. Manu, R. Kuttan, Inhibition of tumor pro-
gression by naturally occurring terpenoids, Pharm. Biol. 49 (2011) 995e1007.
[18] V.R. Yadav, S. Prasad, B. Sung, R. Kannappan, B.B. Aggarwal, Targeting in-
flammatory pathways by triterpenoids for prevention and treatment of can-
cer, Toxins 2 (2010) 2428e2466.
[19] J.A.R. Salvador, Pentacyclic Triterpenes as Promising Agents in Cancer, Nova
Science Publishers, New York, 2010.
[20] J. Rios, Effects of triterpenes on the immune system, J. Ethnopharmacol. 128
(2010) 1e14.
[21] J. Feng, W. Chen, Y. Zhao, X. Ju, Anti-tumor activity of oleanolic, ursolic and
glycyrrhetinic acid, Open Nat. Prod. J. 2 (2009) 48e52.
[22] M.N. Laszczyk, Pentacyclic triterpenes of the lupane, oleanane and ursane
group as tools in cancer therapy, Planta Med. 75 (2009) 1549e1560.
[23] P. Dzubak, M. Hajduch, D. Vydra, A. Hustova, M. Kvasnica, D. Biedermann,
L. Markova, M. Urban, J. Sarek, Pharmacological activities of natural
AIGTVLVGPT PVNIIGRNLL TQIGCTLNF) from BioVendor GmbH
(Heidelberg, Germany). The proteolytic activity of HIV-1-protease
was measured using the FRET (fluorescence resonance energy
transfer). The synthetic peptide (Abz-Ala-Arg-Val-Nle-Tyr(NO2)-
Glu-Ala-Nle-NH2) from Sigma (St. Louis, MO, USA) corresponding to
the p-24-p17 cleavage site in the natural gag precursor was used as
substrate. In the FRET HIV-1 PR substrate, the fluorescence was
quenched by an appropriate fluorescence quencher until this
peptide was cleaved into two separate fragments by HIV-1-prote-
ase at the cleavage site; when the peptide was cleaved, the fluo-
rescence was monitored.
The decrease in the percentage of the fluorescence activity was
used to determine the percentage of the activity inhibition, and was

A. Parra et al. / European Journal of Medicinal Chemistry 74 (2014) 278e301
301
triterpenoids and their therapeutic implications, Nat. Prod. Rep. 23 (2006)
394e411 and references therein.
[50] T. Kodadek, The rise, fall and reinvention of combinatorial chemistry, Chem.
Commun. 47 (2011) 9757e9763.
[24] B.K. Cassels, M. Asencio, Anti-HIV activity of natural triterpenoids and hemi-
synthetic derivatives 2004e2009, Phytochem. Rev. 10 (2011) 545e564.
[25] C.R. Dorr, S. Yemets, O. Kolomitsyna, P. Krasutsky, L.M. Mansky, Triterpene
derivatives that inhibit human immunodeficiency virus type 1 replication,
Bioorg. Med. Chem. Lett. 21 (2011) 542e545.
[26] R.Y. Kuo, K. Qian, S.L. Morris-Natschke, K.H. Lee, Plant-derived triterpenoids
and analogues as antitumor and anti-HIV agents, Nat. Prod. Rep. 26 (2009)
1321e1344.
[27] H. Gehringer, K. Von der Helm, S. Seelmeir, B. Weissbrich, J. Eberle,
H. Nitschko, Development and evaluation of a phenotypic assay monitoring
resistance formation to protease inhibitors in HIV-1-infected patients, J. Virol.
Methods 109 (2003) 143e152.
[28] S.M. Rodems, B.D. Hamman, C. Lin, J. Zhao, S. Shah, D. Heidary, L. Makings,
J.H. Stack, B.A. Pollok, A FRET-based assay platform for ultra-high density drug
screening of protein kinases and phosphatases, Assay Drug Dev. Technol. 1
(2002) 9e19.
[29] N.I. Ziedan, H. Kadri, A.D. Westwell, The development of pro-apoptotic cancer
therapeutics, Mini Rev. Med. Chem. 8 (2008) 711e718.
[30] L. Cipak, L. Grausova, E. Miadokova, L. Novotny, P. Rauko, Dual activity of
triterpenoids: apoptotic versus antidifferentiation effects, Arch. Toxicol. 80
(2006) 429e435.
[31] J.M.R. Patlolla, C.V. Rao, Triterpenoids for cancer prevention and treatment:
current status and future prospects, Curr. Pharm. Biotechnol. 13 (2012) 147e
155.
[32] A. Parra, F. Rivas, S. Martin-Fonseca, A. Garcia-Granados, A. Martinez, Maslinic
acid derivatives induce significant apoptosis in b16f10 murine melanoma
cells, Eur. J. Med. Chem. 46 (2011) 5991e6001.
[51] A. Ganesan, Natural products as a hunting ground for combinatorial chemis-
try, Curr. Opin. Biotechnol. 15 (2004) 584e590.
[52] P.E. Lopez, A. Isidro-Llobet, C. Gracia, L.J. Cruz, A. Garcia-Granados, A. Parra,
M. Alvarez, Use of p-nitrobenzyloxycarbonyl (pNZ) as a permanent protecting
group in the synthesis of Kahalalide F analogs, Tetrahedron Lett. 46 (2005)
7737e7741.
[53] A. Parra, F. Rivas, P.E. Lopez, A. Garcia-Granados, A. Martinez, F. Albericio,
N. Marquez, Solution- and solid-phase synthesis and anti-HIV activity of
maslinic acid derivatives containing amino acids and peptides, Bioorg. Med.
Chem. 17 (2009) 1139e1145.
[54] S. Wang, W. Fang, Design and synthesis of a dual linker for solid phase syn-
thesis of oleanolic acid derivatives, Molecules 16 (2011) 4748e4763.
[55] R. Weis, W. Seebacher, Complete assignment of 1H and 13C NMR spectra of
new pentacyclic triterpene acid benzyl esters, Magn. Reson. Chem. 40 (2002)
455e457.
[56] A. Garcia-Granados, J. Dueñas, J.N. Moliz, A. Parra, F.L. Perez, J.A. Dobado,
J. Molina, Semi-synthesis of triterpene A-ring derivatives from oleanolic and
maslinic acids. Theoretical and experimental 13C chemical shifts, J. Chem. Res.
(M) (2000) 326e339.
[57] A. Garcia-Granados, J. Dueñas, E. Melguizo, J.N. Moliz, A. Parra, F.L. Perez,
J.A. Dobado, J. Molina, Semi-synthesis of triterpene A-ring derivatives from
oleanolic and maslinic acids. Part II. Theoretical and experimental 13C
chemical shifts, J. Chem. Res. (M) (2000) 653e670.
[58] J. Hao, J. Liu, X. Wen, H. Sun, Synthesis and cytotoxicity evaluation of oleanolic
acid derivatives, Bioorg. Med. Chem. Lett. 23 (2013) 2074e2077.
[59] F.B.H. Ahmad, M.G. Moghaddam, M. Basri, M.B. Abdul Rahman, Anticancer
activity of 3-O-acylated betulinic acid derivatives obtained by enzymatic
synthesis, Biosci. Biotechnol. Biochem. 74 (2010) 1025e1029.
[60] M.K. Shanmugam, A.H. Nguyen, A.P. Kumar, B.K.H. Tan, G. Sethi, Targeted
inhibition of tumor proliferation, survival, and metastasis by pentacyclic tri-
terpenoids: potential role in prevention and therapy of cancer, Cancer Lett.
320 (2012) 158e170.
[33] F.J. Reyes, J.J. Centelles, J.A. Lupiañez, M. Cascante, (2a,3b)-2,3-
dihydroxyolean-12-en-28-oic acid, a new natural triterpene from Olea euro-
paea, induces caspase dependent apoptosis selectively in colon adenocarci-
noma cells, FEBS Lett. 580 (2006) 6302e6310.
[34] M.E. Juan, J.M. Planas, V. Ruiz-Gutierrez, H. Daniel, U. Wenzel, Anti-
proliferative and apoptosis-inducing effects of maslinic and oleanolic acids,
two pentacyclic triterpenes from olives, on HT-29 colon cancer cells, Br. J.
Nutr. 100 (2008) 36e43.
[35] R. Martin, J. Carvalho-Tavares, E. Ibeas, M. Hernandez, V. Ruiz-Gutierrez,
M.L. Nieto, Acidic triterpenes compromise growth and survival of astrocytoma
cell lines by regulating reactive oxygen species accumulation, Cancer Res. 67
(2007) 3741e3751.
[36] F.J. Reyes-Zurita, E.E. Rufino-Palomares, J.A. Lupiañez, M. Cascante, Maslinic
acid, a natural triterpene from Olea europaea L., induces apoptosis in HT29
human colon-cancer cells via the mitochondrial apoptotic pathway, Cancer
Lett. 273 (2009) 44e54.
[37] F.J. Reyes-Zurita, G. Pachon-Pena, D. Lizarraga, E.E. Rufino-Palomares,
M. Cascante, J.A. Lupiañez, The natural triterpene maslinic acid induces
apoptosis in HT29 colon cancer cells by a JNK-p53-dependent mechanism,
BMC Cancer 11 (2011) 154.
[61] Y. Qurishi, A. Hamid, P.R. Sharma, Z.A. Wani, D.M. Mondhe, S.K. Singh,
M.A. Zargar, S.S. Andotra, B.A. Shah, S.C. Taneja, A.K. Saxena, PARP cleavage
and perturbance in mitochondrial membrane potential by 3-
-boswellic acid results in cancer cell death and tumor regression in murine
models, Future Oncol. 8 (2012) 867e881.
a-propionyloxy-
b
[62] I. Murtaza, M. Saleem, V.M. Adhami, B.B. Hafeez, H. Mukhtar, Suppression of
cFLIP by lupeol, a dietary triterpene, is sufficient to overcome resistance to
TRAIL-mediated apoptosis in chemoresistant human pancreatic cancer cells,
Cancer Res. 69 (2009) 1156e1165.
[63] J. Wei, M. Liu, H. Liu, H. Wang, F. Wang, Y. Zhang, L. Hana, X. Lina, Oleanolic
acid arrests cell cycle and induces apoptosis via ROS-mediated mitochondrial
depolarization and lysosomal membrane permeabilization in human
pancreatic cancer cells, J. Appl. Toxicol. 33 (2013) 756e765.
[64] R.K. Emaus, R. Grunwald, J.J. Lemasters, Rhodamine 123 as a probe of trans-
membrane potential in isolated rat-liver mitochondria: spectral and meta-
bolic properties, Biochim. Biophys. Acta 850 (1986) 436e448.
[38] E.E. Rufino-Palomares, F.J. Reyes-Zurita, L. Garcia-Salguero, K. Mokhtari,
P.P. Medina, J.A. Lupiañez, J. Peragon, Maslinic acid,
tumoural agent, interferes with cytoskeleton protein expression in HT29
human colon-cancer cells, J. Proteomics 83 (2013) 15e25.
a
triterpenic anti-
[65] H. Li, H. Zhu, C.J. Xu, J. Yuan, Cleavage of BID by caspase 8 mediates the
mitochondrial damage in the Fas pathway of apoptosis, Cell 94 (1998) 491e
501.
[39] S. Sanchez-Tena, F.J. Reyes-Zurita, S. Diaz-Moralli, M.P. Vinardell, M. Reed,
F. Garcia-Garcia, J. Dopazo, J.A. Lupiañez, U. Günther, M. Cascante, Maslinic
acid-enriched diet decreases intestinal tumorigenesis in ApcMin/þ mice
through transcriptomic and metabolomic reprogramming, PLoS One 8 (2013)
e59392.
[66] X. Luo, I. Budihardjo, H. Zou, C. Slaughter, X. Wang, Bid, a Bcl2 interacting
protein, mediates cytochrome c release from mitochondria in response to
activation of cell surface death receptors, Cell 94 (1998) 481e490.
[67] L. Huang, C.H. Chen, The molecular targets of anti-HIV-1 triterpenes, an up-
date, Med. Chem. Rev. Online 2 (2005) 423e427.
[40] Y.K. Kim, S.K. Yoon, S.Y. Ryu, Cytotoxic triterpenes from stem bark of Phys-
ocarpus intermedius, Planta Med. 66 (2000) 485e486.
[41] X. Wang, The expanding role of mitochondria in apoptosis, Genes Dev. 15
(2001) 2922e2933.
[68] K. Qian, R.Y. Kuo, C.H. Chen, L. Huang, S.L. Morris-Natschke, K.H. Lee, Anti-
AIDS agents 81. Design, synthesis, and structureeactivity relationship study of
betulinic acid and moronic acid derivatives as potent HIV maturation in-
hibitors, J. Med. Chem. 53 (2010) 3133e3141.
[42] S.Y. Sun, N. Hail Jr., R. Lotan, Apoptosis as a novel target for cancer chemo-
prevention, J. Natl. Cancer Inst. 96 (2004) 662e672.
[43] O. Micheau, J. Tschopp, Induction of TNF receptor I-mediated apoptosis via
two sequential signaling complexes, Cell 114 (2003) 181e190.
[44] A. Ashkenazi, Targeting the extrinsic apoptosis pathway in cancer, Cytokine
Growth Factor Rev. 19 (2008) 325e331.
[45] S.T. Derek, Diversity-oriented synthesis: exploring the intersections between
chemistry and biology, Nat. Chem. Biol. 1 (2005) 74e84.
[46] D.J. Newman, G.M. Cragg, Natural products as sources of new drugs over the
30 years from 1981 to 2010, J. Nat. Prod. 75 (2012) 311e335.
[47] A. Ganesan, The impact of natural products upon modern drug discovery,
Curr. Opin. Chem. Biol. 12 (2008) 306e317.
[69] Y.M. Zhu, J.K. Shen, H.K. Wang, L.M. Cosentino, K.H. Lee, Synthesis and anti-
HIV activity of oleanolic acid derivatives, Bioorg. Med. Chem. Lett. 11 (2001)
3115e3118.
[70] K. Qian, K. Nakagawa-Goto, D. Yu, S.L. Morris-Natschke, T.J. Nitz, N. Kilgore,
G.P. Allaway, K.H. Lee, Anti-AIDS agents 73: structureeactivity relationship
study and asymmetric synthesis of 3-O-monomethylsuccinyl-betulinic acid
derivatives, Bioorg. Med. Chem. Lett. 17 (2007) 6553e6557.
[71] K. Qian, D. Yu, C.H. Chen, L. Huang, S.L. Morris-Natschke, T.J. Nitz, K. Salzwedel,
M. Reddick, G.P. Allaway, K.H. Lee, Anti-AIDS agents. 78. Design, synthesis,
metabolic stability assessment, and antiviral evaluation of novel betulinic acid
derivatives as potent anti-human immunodeficiency virus (HIV) agents,
J. Med. Chem. 52 (2009) 3248e3258.
[48] M.E. Welsch, S.A. Snyder, B.R. Stockwell, Privileged scaffolds for library design
and drug discovery, Curr. Opin. Chem. Biol. 14 (2010) 347e361.
[49] V.L. Eifer-Lima, C.S. Graebin, F.D.T. Uchoa, P.D. Duarte, A.G. Corrêa, Highlights
in the solid-phase organic synthesis of natural products and analogues, J. Braz.
Chem. Soc. 21 (2010) 1401e1423.
[72] A. Martinez, F. Rivas, A. Perojil, A. Parra, A. Garcia-Granados, A. Fernandez-
Vivas, Biotransformation of oleanolic and maslinic acids by Rhizomucor miehei,
Phytochemistry 94 (2013) 229e237.