Novel oleanolic vinyl boronates: Synthesis and antitumor activity

Article abstract of DOI:10.1016/j.ejmech.2013.01.040

A series of novel oleanane-type pentacyclic triterpenoids bearing a boronate ester moiety at C3 have been synthesized by palladium-catalyzed cross-coupling of bis(pinacolato)diboron with vinyl triflates, in the presence of base, and these compounds were fully characterized by 1D and 2D NMR techniques. Evaluation of their antiproliferative effects on a panel of hematological-based and solid tumor cell lines identified three active oleanolic vinyl boronates that inhibited the growth of leukemia (Jurkat, K562), Burkitt's lymphoma (Jijoye), cervix (Hela), colon (SW480), and ovary (SKOV-3) cancer cells without concomitant inhibition of non-tumoral human fibroblasts. Their mechanisms of action were investigated on the leukemia Jurkat cell line. The results show that the incorporation of boron in the oleanolic acid core combined with the presence of amide bonds afforded compounds with desirable biological effects such as apoptosis induction and inhibition of proteasomal activity on tumor cells, which makes them potential templates for further development in the anticancer drug setting.

Full text of DOI:10.1016/j.ejmech.2013.01.040

European Journal of Medicinal Chemistry 63 (2013) 46e56
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Novel oleanolic vinyl boronates: Synthesis and antitumor activity
,
b
,
,
c
,
c,
Vânia M. Moreira a 1, Jorge A.R. Salvador b , Sérgio Simões d, Federica Destro e,
*
Riccardo Gavioli e
a Centre of Chemistry, Faculty of Sciences and Technology, University of Coimbra, Portugal
b Laboratory of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Coimbra, Portugal
c Center for Neuroscience and Cell Biology, Coimbra, Portugal
d Laboratory of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra, Portugal
e Department of Biochemistry and Molecular Biology, University of Ferrara, Ferrara, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel oleanane-type pentacyclic triterpenoids bearing a boronate ester moiety at C3 have
been synthesized by palladium-catalyzed cross-coupling of bis(pinacolato)diboron with vinyl triflates, in
the presence of base, and these compounds were fully characterized by 1D and 2D NMR techniques.
Evaluation of their antiproliferative effects on a panel of hematological-based and solid tumor cell lines
identified three active oleanolic vinyl boronates that inhibited the growth of leukemia (Jurkat, K562),
Burkitt’s lymphoma (Jijoye), cervix (Hela), colon (SW480), and ovary (SKOV-3) cancer cells without
concomitant inhibition of non-tumoral human fibroblasts. Their mechanisms of action were investigated
on the leukemia Jurkat cell line. The results show that the incorporation of boron in the oleanolic acid
core combined with the presence of amide bonds afforded compounds with desirable biological effects
such as apoptosis induction and inhibition of proteasomal activity on tumor cells, which makes them
potential templates for further development in the anticancer drug setting.
Received 6 December 2012
Received in revised form
29 January 2013
Accepted 31 January 2013
Available online 9 February 2013
Keywords:
Triterpenoids
Oleanolic
Boronates
Apoptosis
Proteasome
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
[17], gastro and hepatoprotective [18], and antiviral [19]. Chemical
modifications of OA to produce derivatives have been successful in
Pentacyclic triterpenoids are secondary plant metabolites found
in fruit peel, leaves and stem bark [1e3]. Their potential interest in
cancer has now been well demonstrated by the successful clinical
utilities of oleanolic acid (OA), glycyrrhetic acid, asiaticoside, and
carbenoxolone as market drugs [4]. OA bears an impressive myriad
of reported biological activities [5e7] including antibacterial [8],
antiparasitic [9], antiosteoporotic [10], antifertility [11], anti-
hypertensive [12], antihyperlipidemic [13], antidiabetic [14],
immunomodulatory [15], anti-inflammatory [16], antinociceptive
improving not only its activity but also its pharmacokinetic prop-
erties. Although quite many OA derivatives have now been reported
in the literature, none have been synthesized bearing boron. Boron
chemistry remained relatively unexploited in the medicinal
chemistry arena until the FDA approved the boronic acid protea-
some inhibitor bortezomib (VelcadeÒ) for the treatment of multiple
myeloma and non-Hodgkin lymphoma, in 2003 [20]. Since then,
some promising compounds have been developed and tested
among which is PT-100 (Val-boroPro, Talabostat) that targets
dipeptidyl peptidases, such as fibroblast activation protein. In 2004,
a phase II clinical trial of PT-100 was launched for the treatment of
advanced non-small cell lung cancer [21]. However, it has been
placed on hold in 2007 due to efficacy issues. The compound has
also been recently studied on Phase I and II clinical trials in other
cancers including melanoma and brain tumors.
The physical, chemical and biological properties of boron offer
medicinal chemists an excellent opportunity to explore and pio-
neer new areas of drug discovery [22]. Boron is non-toxic, has Lewis
acid behavior due to its empty p-orbital, and bears electrophilic
character. Its empty p-orbital can be occupied by a lone pair of
electrons, allowing it to form dative bonds with biological nucle-
ophiles of enzyme residues (such as serine) and hydroxyl groups
Abbreviations: AA, amino acid; B2pin2, bis(pinacolato)diboron; ChT-L, chymo-
tryptic-like; PdCl2(PPh3)2, dichlorobis(triphenylphosphine)palladium(II); DMEM,
Dulbecco’s modified Eagle medium; DMF, dimethylformamide; DMSO, dime-
thylsulfoxide; DTBMP, di-t-butyl-methylpyridine; FCC, flash column chromatog-
raphy; HMBC, heteronuclear multiple-bond correlation; HMQC, heteronuclear
multiple quantum coherence; IR, infra-red; NMR, nuclear magnetic resonance;
NOESY, nuclear Overhauser effect spectroscopy; PBS, phosphate saline buffer; PI,
propidium iodide; Tf2O, triflic anhydride; THF, tetrahydrofuran; T-L, tryptic-like;
SAR, structureeactivity relationship.
* Corresponding author. Laboratory of Pharmaceutical Chemistry, Faculty of
Pharmacy, University of Coimbra, Portugal. Tel.: þ351 239 488 400; fax: þ351 239
488 503.
E-mail address: salvador@ci.uc.pt (J.A.R. Salvador).
Present address: Division of Pharmaceutical Chemistry, Faculty of Pharmacy,
1
University of Helsinki, Finland.
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.01.040

V.M. Moreira et al. / European Journal of Medicinal Chemistry 63 (2013) 46e56
47
from carbohydrates and nucleic acids. Thus, several boron-
containing compounds, including analogs of natural products
methyl ester [31] to afford compound 2 which was further reacted
with triflic anhydride (Tf2O) in the presence of base to give the vinyl
triflate intermediate 3, in 83% yield after purification by flash col-
umn chromatography (FCC) [32,33]. This compound was coupled
with bis(pinacolato)diboron using bis(triphenylphosphine)palla-
dium(II) chloride (PdCl2(PPh3)2) as catalyst to afford the oleanolic
vinyl boronate 4, in 89% yield.
Deprotection of the carboxylate ester was then performed using
lithium iodide in refluxing DMF to give compound 5 [34]. Reaction
of compound 5 with oxalyl chloride gave the acyl chloride inter-
mediate 6 [35] which was reacted either with ammonia or with an
amino acid methyl ester hydrochloride to afford compounds 7, and
8 and 10, respectively [36]. Deprotection of the carboxyl group of
the amino acid side chain was finally performed by alkaline
hydrolysis and compounds 9 and 11 were obtained in 98% yield. The
synthesis of the oleanolic vinyl boronates 12e14 started from
compound 4 (Scheme 2).
such as
b-lactamic antibiotics and curcumin, have been reported to
be inhibitors of serine and aspartic proteases, metalloproteases,
g-
glutamyl transpeptidase, arginase, surfactin synthetase C-terminal
thioesterase, cysteine proteases, tyrosine kinases, as well as to be
threonine-based inhibitors [22]. In continuation of our efforts to
design new triterpenoids as anticancer agents [2,23e29], we have
successfully prepared a series of novel oleanane-type pentacyclic
triterpenoids bearing a boronate ester moiety. We have screened
the compounds for cytotoxic activity against
a panel of
hematological-based and solid tumor cell lines and against non-
tumoral human fibroblasts. We have also studied their effects on
cell cycle, ability to induce apoptosis and to inhibit proteasomal
activity in leukemia Jurkat cells.
2. Results and discussion
Thus, oxidation of compound 4 with the KMnO4/FeSO4$nH2O
system [37] gave the 12-oxo derivative 12, in 92% yield, which by
reaction with bromine and hydrobromic acid in refluxing acetoni-
trile afforded the enone 13 [38]. The oxime 14 was prepared by
reaction of compound 4 with hydroxylamine hydrochloride in
pyridine, at reflux [39]. Reaction of oleanonic acid 15 with oxalyl
chloride afforded the acyl chloride derivative intermediate 16 [35]
which was reacted with ammonia to give the amide 17 (Scheme 3).
2.1. Chemistry
The synthesis of the oleanolic vinyl boronates 4e11 started from
oleanolic acid 1 (Scheme 1) and exploited the palladium-catalyzed
cross-coupling reaction of bis(pinacolato)diboron (B2pin2) with 1-
alkenyl (vinyl) triflates in the presence of di-t-butyl-methylpyr-
idine (DTBMP) [30]. The 28-carboxyl group was first protected as the
Scheme 1. Synthesis of the oleanolic boronates 4e11. Reagents and conditions: a. Jones reagent; b. CH3I, K2CO3, DMF, r.t.; c. Tf2O, DTBMP, CH2Cl2, reflux; d. B2pin2, PdCl2(PPh3)2,
Na2CO3, THF, reflux; e. LiI, DMF, reflux; f. (COCl)2, CHCl3, r.t.; g. NH3 (aq), THF, 0 ꢀC; h. AA methyl ester hydrochloride, NEt3, CH2Cl2, r.t.; i. NaOH (4 N), THF:MeOH, r.t.

48
V.M. Moreira et al. / European Journal of Medicinal Chemistry 63 (2013) 46e56
Scheme 2. Synthesis of the oleanolic boronates 12e14. Reagents and conditions: a. KMnO4/FeSO4$nH2O, t-BuOH, H2O, CH2Cl2, r.t.; b. HBr/Br2, CH3CN, reflux; c. NH2OH.HCl, pyr,
reflux.
Reaction of 17 with triflic anhydride in the presence of base affor-
ded the oleanolic vinyl triflate 18 bearing a nitrile group at C-28
[40]. Reaction of compound 18 with bis(pinacolato)diboron in the
presence of bis(triphenylphosphine)palladium(II) chloride afforded
compound 19, in 91% yield.
Elucidation of the final structures of the synthesized compounds
was performed by infra-red (IR), 1D and 2D nuclear magnetic res-
onance (NMR) spectroscopy. The specific IR absorptions for the
asymmetric and symmetric SO2-vibrations of the oleanolic vinyl
triflates 3 and 18 were observed around 1400e1430 and 1200e
1250 cmꢁ1 as well as the respective CeF absorptions at 1190e
1130 cmꢁ1. The 2-H signal was seen as a double doublet at 5.56
and 5.57 ppm, on the 1H NMR spectra of compounds 3 and 18,
respectively. In addition, the 13C NMR signal for the CF3 carbon was
observed as a pair of peaks at 116.8 and 120 ppm with the coupling
constant of 319 Hz [41,42]. For the oleanolic vinyl boronates 4e14
and 19, the 2-H multiplet was consistently found at 6.35e
6.43 ppm on the 1H NMR spectra. The pinacolborane ring methyl
group protons were observed as a singlet at 1.24e1.25 ppm on the
1H NMR spectra of the compounds [30]. The corresponding methyl
carbon signals were found at 24.6 and 24.8 ppm whereas the sig-
nals for the two quaternary carbons of the pinacolborane ring were
found at 82.6e82.8 ppm. No signal for the quaternary carbon
attached to the boron atom was ever observed on the 13C NMR
spectra of the oleanolic vinyl boronates 4e14 and 19. COSY analysis
of compound 10 showed the expected correlations between the
proton signals of the amino acid side chain. Thus, correlations were
found for the amide proton signal at 6.60 ppm and the signal at
4.45 ppm belonging to the single proton of the adjacent carbon.
This signal at 4.45 ppm also correlated with the signal at 1.37 ppm
belonging to the methyl protons of the same carbon. Analysis of the
heteronuclear multiple quantum coherence (HMQC) spectroscopy
Scheme 3. Synthesis of the oleanolic boronate 19. Reagents and conditions: a. (COCl)2, CHCl3, r.t.; b. NH3 (aq), THF, 0 ꢀC; c. Tf2O, DTBMP, CH2Cl2, reflux; d. B2pin2, PdCl2(PPh3)2,
Na2CO3, THF, reflux.

V.M. Moreira et al. / European Journal of Medicinal Chemistry 63 (2013) 46e56
49
structureeactivity relationships (SAR) information. Only the ole-
anolic vinyl boronates bearing an amide bond at the C28 position 7,
9, and 11 were active. All other modifications at C28 such as the
methyl ester, the nitrile or even the free carboxyl group, or at ring C
such as the 12-oxo group, the enone and the oxime moieties,
combined with the vinylic boronate structure of ring A failed to
provide compounds with cytotoxic activity. In addition, for com-
pounds 9 and 11, bearing an amino acid residue at C28, activity was
only observed when the terminal carboxyl group was unprotected.
Thus, the corresponding methyl ester amino acid derivatives 8 and
10 were inactive against the cell lines tested. We next examined the
effects of the active compounds on apoptosis. We found that
Fig. 1. NOESY correlations of the amide proton of the side chain of compound 10.
treatment of Jurkat cells with 20 mM of compounds 7, 9 and 11
spectra allowed the assignment of C12 at 123.5 ppm and C2 at
138.5 ppm as well as confirmation of the methyl carbon signals of
the pinacolborane ring which were found at 24.6 and 24.7 ppm. The
correlation observed on the heteronuclear multiple-bond correla-
tion (HMBC) spectroscopy spectrum of compound 10 between the
resulted in activation of the executioner caspases 3 and 7 (Fig. 2A).
Accordingly, expansion of a sub-G0 fraction on the cell cycle
profile after propidium iodide (PI) staining which was consistent
with the increase in the percentage of cells in late apoptosis/
necrosis observed after double staining occurred (Fig. 3A and B,
respectively). Treatment of Jurkat cells with 20 mM of compound 7
overnight did not, however, produce significant changes in the cell
cycle (data not shown).
To try to further shed some light into the possible mechanisms
of action of compounds 7, 9 and 11, we decided to investigate their
effects on proteasomal activity [43]. Although the vast majority of
proteasome inhibitors are small peptides (either boron-containing
or not) [44e47], a number of natural products including tyropeptin
A, epoxomicin, lactacystin, salinosporamide A, belactosins, argyrin
A, and withaferin A, chalcones [48] as well as some pentacyclic
triterpenoids [49e56] have also been reported to inhibit the pro-
teasome. A first screening using proteasomes semi-purified from
Jurkat cells pellets revealed that compound 9 inhibited the
chymotrypsin-like (ChT-L) proteasomal activity at both 50 and
18
the assignment of this signal to C28. The signal of the 18
b
-H signal at 2.63 ppm and the 13C signal at 177.5 ppm allowed
b-H also
displayed the expected correlations with the signals of C12 and C13
(143.6 ppm). The other quaternary 13C signal at 173.6 ppm was
assigned to the carboxylic group carbon of the methyl ester side
chain. Consistently, this signal displayed correlations with all the
proton signals of the amino acid side chain. The nuclear Overhauser
effect spectroscopy (NOESY) spectrum of compound 10 displayed
the selected correlations shown in Fig. 1 for the amide proton of the
amino acid side chain. Similar correlations were seen after 2D NMR
analysis of compound 11.
2.2. Biology
100
activity was only inhibited at 100
50%. Compound 11 was an effective inhibitor of both proteasomal
activities only at 100 M, whereas compound 7 was not active at
the concentrations tested. Thus, although an amide bond at C28
common to all the compounds was important for the cytotoxic
activity of the oleanolic vinyl boronates, in order to have protea-
some inhibition the presence of the amino acid moiety seemed
mandatory.
The effects on compound 9 and 11 on the proteasomes of intact
Jurkat cells were also examined (Fig. 4). The natural product
epoxomicin, a potent inhibitor of the ChT-L activity of the protea-
some [57] was used as a positive control. After pre-incubation of
m
M, in 36 and 41%, respectively (Table 2). The trypsin-like (T-L)
The ability of the novel synthesized oleanolic vinyl boronates 4e
14 and 19 and of oleanolic acid 1 to inhibit the proliferation of a
panel of tumor cell lines comprising both hematological-based
(Jurkat, K562, and Jijoye) and solid (Hela, SKOV-3, and SW480)
tumor cells was evaluated by MTT assay (Table 1). Compounds 7, 9,
and 11 were active. Thus, compounds 9 and 11 inhibited the pro-
liferation of the tumor cell lines tested with IC50 values ranging
mM by this compound, in about
m
from 13.4 to 28
this inhibition of proliferation was selective for malignant cells
seeing that the compounds were inactive (IC50 > 50 M) on non-
mM and 19.4 to 31.2 mM, respectively. In addition,
m
tumoral human fibroblasts. A very interesting cytotoxicity profile
was observed for compound 7. The compound preferentially
inhibited the proliferation of hematological-based cell lines with
intact Jurkat cells with compounds 9 and 11 at 50 mM, inhibition of
IC50 values of 21.6, 8.3, and 8.4
respectively, when compared to solid tumor cell lines. Thus, an IC50
value of 34.7 M could be determined for Hela cells alone with
compound 7 (Table 1). Moreover, its effects on cell proliferation
were also selective as the compound was inactive against the
human fibroblasts.
mM for Jurkat, K562, and Jijoye cells,
the ChT-L and T-L activities of the proteasome occurred (Fig. 4A and
B). Impairment of the ChT-L activity was more pronounced after
treatment with compound 9 than with compound 11 (25 and 37% of
control, respectively), and the effect of the compounds on the T-L
activity was similar. Additional confirmation of proteasomal
inhibition was observed by accumulation of ubiquitinated proteins
after incubation of Jurkat cells with compounds 9 and 11 (Fig. 2B).
m
This cytotoxicity screening allowed us to not only identify the
active vs. inactive compounds but also provided important
Table 1
Cell viability and proliferation assay for the oleanolic boronates 7, 9, and 11.
Compound
Cell line (IC50
Jurkat
, m
M)a,b
K562
Jijoye
Hela
SKOV-3
SW480
Human
fibroblasts
7
9
11
21.6 ꢂ 2.6
15.5 ꢂ 2.4
21.4 ꢂ 2.9
8.3 ꢂ 1.8
18.2 ꢂ 0.4
20.1 ꢂ 0.2
8.4 ꢂ 2.9
13.4 ꢂ 2.6
19.4 ꢂ 0.4
34.7 ꢂ 2.1
28.0 ꢂ 0.9
30.5 ꢂ 1.1
> 50
> 50
>50
>50
>50
15.4 ꢂ 2.1
17.9 ꢂ 1.8
31.2 ꢂ 2.4
28.7 ꢂ 2.1
a
IC50 is the concentration that inhibits 50% of cellular growth. IC50 values were determined by MTT assay, after 48 h of treatment. The reported values are expressed as a
mean ꢂ SD of three independent experiments.
b
IC50 values for compounds 4, 5, 8, 10, 12e14, and 19 (>50 mM).

50
V.M. Moreira et al. / European Journal of Medicinal Chemistry 63 (2013) 46e56
Fig. 2. A. Caspase 3/7 activation after overnight treatment of Jurkat cells with compounds 7, 9, 11 (20
experiments is shown. One-way ANOVA with a Dunnett post-test was used to analyze the data: *p < 0.01 vs. control. B. Effect of compounds 9 and 11 on the levels of poly-
ubiquitinated proteins in Jurkat cells. Jurkat cells were exposed to the compounds (50 M). Lysates were prepared and assayed for ubiquitinated protein levels by western blot.
Epoxomicin (0.1 M) was used as positive control. A representative example of three independent experiments is shown.
mM), and epoxomicin (0.1 mM). A representative example of three independent
m
m
These results show that compounds 9 and 11 are apoptosis
inducers and inhibit the proteasomal activity of Jurkat cells pellet
and are in addition cell-permeable bearing a similar effect on the
proteasomes of intact Jurkat cells. Nonetheless, the fact that this
proteasomal inhibition is somewhat weaker than that of cell via-
bility and proliferation suggests that additional targets may be
important for the activity of these compounds.
Finnigan PolarisQGC/MS Benchtop Ion Trap mass spectrometer.
Melting points were recorded on a BUCHI Melting point B-540
apparatus and are uncorrected. The purity (>95%) of the tested
compounds was determined by elemental analysis performed at
the Centro de Apoio Científico e Tecnolóxico á Investigación
(C.A.C.T.I.), Universidade de Vigo, Campos Lagoas e Marcosende, 15,
36310 Vigo.
3. Conclusions
4.2. Methyl 3-oxoolean-12-en-28-oate (2)
We herein report the synthesis and structural elucidation of a
series of novel oleanane-type pentacyclic triterpenoids bearing a
boronate ester moiety, and the study of their antitumoral effects.
We have found that the oleanolic vinyl boronates bearing an amide
bond at C28 7, 9, and 11 could inhibit the proliferation of the solid-
and hematological-based tumor cell lines tested and that this
inhibition was selective toward tumor cell lines. In addition, the
compounds induced caspase-dependent apoptosis of leukemia
Jurkat cells. Further insights on their possible mechanisms of action
have been provided. Compounds 9 and 11, bearing an amino acid
moiety at C28, were inhibitors of the proteasomal activity of both
Jurkat cells pellets and intact Jurkat cells. To the best of our
knowledge, our results show for the first time the potential of
boron-containing oleanane-type derivatives as templates that can
be further modified in the pursuit of anticancer agents. Efforts are
currently underway to optimize these compounds and further
precise their mechanisms of action.
Compound 2 was prepared according to the literature staring
from OA (1) [31].
4.3. Methyl oleana-2,12-dien-28-oate 3-trifluoromethanesulfonate
(3)
To a solution of compound 2 (1.0 g, 2.14 mmol) in dichloro-
methane (20 mL) stirred at room temperature under N2 atmos-
phere Tf2O (438
mL, 2.6 mmol) was added followed by DTBMP
(616 mg, 3 mmol). The solution darkened and temperature was
then raised to reflux. After 4 h 30 the reaction was complete and
water (75 mL) and ethyl acetate (150 mL) were added with stirring.
The aqueous phase was extracted twice with ethyl acetate
(2 ꢃ 100 mL) and washed with 10% aqueous HCl (2 ꢃ 50 mL), 10%
aqueous NaHCO3 (2 ꢃ 50 mL), 10% aqueous Na2SO3 (2 ꢃ 50 mL),
water (50 mL) and brine (50 mL), dried over anhydrous Na2SO4,
filtered, and the solvent was removed under reduced pressure to
afford a yellow oil which turned into a yellow solid with drying
(1.20 g). The solid was purified by FCC using ethyl acetate:petro-
leum ether 1:10 to afford compound 3 (1.06 g; 83%). Mp (ethyl
acetate:petroleum ether 40e60): 98e100 ꢀC. IR 1727, 1412, 1244,
4. Experimental
4.1. Chemistry
All reagents were obtained from SigmaeAldrich Co. For thin
layer chromatography (TLC) analysis, Kieselgel 60HF254/Kieselgel
60G was used. IR spectra were obtained using a JASCO FT/IR-420
spectrophotometer (FTIR-ATR). NMR spectra were obtained using
a Brucker Digital NMRdAvance 400 apparatus spectrometer, in
CDCl3 with Me4Si as the internal standard. NMR data was obtained
at the Nuclear Magnetic Resonance Laboratory of the Coimbra
Chemistry Centre (www.nmrccc.uc.pt), Universidade de Coimbra,
supported in part by grant REEQ/481/QUI/2006 from FCT, POCI-
2010 and FEDER, Portugal. Mass spectra were recorded on a
1209, 1142 cmꢁ1. 1H NMR (CDCl3)
3H), 0.99 (s, 3H),1.03 (s, 3H),1.13 (s, 6H), 2.87 (m,1H,18
d
0.77 (s, 3H), 0.90 (s, 3H), 0.93 (s,
-H), 3.62 (s,
b
3H, 28-OCH3), 5.31 (m, 1H, 12-H), 5.56 (dd, 1H, 2-H, J1 ¼1.77 Hz and
J2 ¼ 6.60 Hz); 13C NMR
113.7 (C2), 116.8 and 120 (J ¼ 319 Hz, CF3),
d
122 (C12), 143.8 (C13), 155.4 (C3), 178.2 (C28). EI-MS m/z (%) 600 (3)
Mþ, 262 (23), 248 (20), 203 (100), 189 (66), 133 (43), 119 (23), 105
(17). Anal. Calcd. for C32H47F3O5S: C, 63.97; H; 7.89. Found: C, 63.57;
H, 7.84.

V.M. Moreira et al. / European Journal of Medicinal Chemistry 63 (2013) 46e56
51
Fig. 3. Effect of compounds 9 and 11 on the cell cycle. FACS analysis by single (PI alone, A) staining and double (PI and annexin V, B) staining of Jurkat cells after overnight treatment
with DMSO or compounds 9 and 11 at 20 M. A representative example of three independent experiments is shown.
m
4.4. Methyl 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
oleana-2,12-dien-28-oate (4)
(200 mL) and water (50 mL) were added. The aqueous phase was
further extracted with ethyl acetate (2 ꢃ 100 mL). The organic
phase was then filtered through a Celite pad, washed with 10%
aqueous HCl (50 mL), 10% aqueous NaHCO3 (50 mL), 10% aqueous
Na2SO3 (50 mL), water (50 mL) and brine (50 mL), dried over
anhydrous Na2SO4, filtered, and the solvent was removed under
reduced pressure to afford compound 4 (516 mg, 89%) as a yellow
solid. Mp (THF:methanol): 208e210 ꢀC. IR 1726, 1622 cmꢁ1. 1H NMR
To a mixture of compound 3 (600 mg, 1 mmol) and PdCl2(PPh3)2
(50 mg, 0.07 mmol), THF (12 mL) was added at room temperature
followed by B2pin2 (380 mg, 1.5 mmol). A 0.8 M solution of Na2CO3
(4.8 mL; 3.84 mmol) was then charged dropwise into the reaction
flask. The solution darkened and temperature was then raised to
reflux. After 1 h the reaction was complete. The reaction mixture
was concentrated under reduced pressure and ethyl acetate
(CDCl3)
d 0.76 (s, 3H), 0.89 (s, 6H), 0.92 (s, 3H), 1.03 (s, 3H), 1.08 (s,
3H), 1.13 (s, 3H), 1.24 (s, 12H, pinacol-[C(CH3)2]2), 2.87 (m, 1H, 18
b
-

52
V.M. Moreira et al. / European Journal of Medicinal Chemistry 63 (2013) 46e56
Table 2
Proteasomal activity inhibition screening with the oleanolic boronates 7, 9, and 11.
Compound
Proteasomal activity (%)a,b
ChT-L activity
T-L activity
25
25
m
M
50
m
M
100
m
M
m
M
50
m
M
100 mM
7
9
11
95.8 ꢂ 3.8
83.6 ꢂ 2.7
91.7 ꢂ 2.8
92.4 ꢂ 3.4
64 ꢂ 2.1
85.6 ꢂ 2.7
59.3 ꢂ 2.1
47.2 ꢂ 2.9
81.6 ꢂ 1.1
85.3 ꢂ 1.2
85.1 ꢂ 1.8
77.9 ꢂ 1.9
84.1 ꢂ 2.8
70.7 ꢂ 2.1
72.5 ꢂ 2.9
51.4 ꢂ 2.1
50.8 ꢂ 2.3
84.3 ꢂ 2.2
a
Semi-purified proteasomes from Jurkat cells pellets. Values are expressed in % of control (DMSO) and represent residual proteasome activity after treatment with the
compounds. The reported values are expressed as a mean ꢂ SD of three independent experiments.
b
After treatment with MG132 (10
m
M), used as a positive control, ꢄ20% of both ChT-L and T-L activities remained.
H), 3.62 (s, 3H, 28-OCH3), 5.31 (m, 1H, 12-H), 6.37 (m, 1H, 2-H); 13
C
solid was washed with n-hexane (3 ꢃ 1 mL). The product was used
without further purification for the following steps. Compound 6:
NMR
d 82.6 (pinacol-[C(CH3)2]2), 122.6 (C12), 138.7 (C2), 143.6
(C13), 178.3 (C28); EI-MS m/z (%) 578 (3) Mþ, 315 (23), 262 (44), 203
(100), 189 (19), 173 (16), 133 (24), 119 (13). Anal. Calcd. for
C37H59BO4: C, 76.80; H, 10.28. Found: C, 77.07; H, 10.68.
90 mg; 97%. Mp 287e289 ꢀC. IR 1785, 1367 cmꢁ1. 1H NMR (CDCl3)
d
0.81 (s, 3H), 0.90 (s, 3H), 0.91 (s, 3H), 0.92 (s, 3H),1.03 (s, 3H),1.08 (s,
3H), 1.15 (s, 3H), 1.25 (s, 12H, pinacol-[C(CH3)2]2), 2.83 (m, 1H, 18
H), 5.36 (m, 1H, 12-H), 6.38 (m, 1H, 2-H); 13C NMR
82.7 (pinacol-
b
-
d
[C(CH3)2]2),124 (C12),138.6 (C2),141.8 (C13),179.8 (C28). EI-MS m/z
(%) 582 (2) Mþ, 518 (20), 214 (15), 201 (95), 189 (100), 144 (63), 118
(69), 90 (55). Anal. Calcd. for C36H56BClO3: C, 74.15; H, 9.68. Found:
C, 74.36; H, 9.96.
4.5. 3-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-oleana-2,12-
dien-28-oic acid (5)
To a mixture of compound 4 (200 mg, 0.35 mmol) and lithium
iodide (816 mg, 6.1 mmol), dry DMF (4 mL) was added under N2
atmosphere and the mixture was heated to reflux. After 52 h the
reaction was complete and water (25 mL) and ethyl acetate (75 mL)
were added with stirring. The aqueous phase was extracted twice
with ethyl acetate (2 ꢃ 50 mL) and washed with 10% aqueous HCl
(25 mL), water (25 mL) and brine (25 mL), dried over anhydrous
Na2SO4, filtered, and the solvent was removed under reduced
pressure to afford a yellow solid (215 mg). The compound was
purified by FCC using ethyl acetate:petroleum ether 40e60 (1: 10)
to afford compound 5 (120 mg; 62%). Mp (acetone:n-hexane): 319e
4.7. 3-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-oleana-2,12-
dien-28-amide (7)
A solution of compound 6 (400 mg, 0.68 mmol) in THF (20.8 mL)
was added dropwise to ammonia (0.4 mL) cooled in an ice bath,
under magnetic stirring. After 3 h the reaction was complete. The
reaction mixture was evaporated to dryness and ethyl acetate
(125 mL) and water (25 mL) were added. The aqueous phase was
further extracted with ethyl acetate (2 ꢃ 50 mL) and the resulting
organic phase was washed with 10% aqueous HCl (25 mL), 10%
aqueous NaHCO3 (25 mL), water (25 mL) and brine (25 mL), dried
over anhydrous Na2SO4, filtered, and the solvent was removed
under reduced pressure to afford a brown solid (355 mg; 92%)
which was purified by FCC using ethyl acetate:petroleum ether 40e
60 4:1 to 2:1 as eluent, to afford compound 7 (220 mg; 62%). Mp
(ethyl acetate:petroleum ether 40e60): 294e296 ꢀC. IR 3160, 1670,
321 ꢀC. IR 3377, 1697, 1622 cmꢁ1. 1H NMR (CDCl3)
(s, 6H), 0.92 (s, 3H), 1.00 (s, 3H), 1.07 (s, 3H), 1.13 (s, 3H), 1.25 (s, 12H,
pinacol-[C(CH3)2]2), 2.81 (m, 1H, 18 -H), 5.30 (m, 1H, 12-H), 6.38
(m,1H, 2-H); 13C NMR
d 0.78 (s, 3H), 0.89
b
d
82.6 (pinacol-[C(CH3)2]2),122.8 (C12),138.8
(C2), 143.4 (C13), 184.2 (C28). EI-MS m/z (%) 565 (2) Mþ, 316 (18),
248 (64), 203 (100), 187 (21), 133 (50), 119 (30), 91 (19). Anal. Calcd.
for C36H57BO4: C, 76.58; H, 10.17. Found: C, 76.51; H, 10.57.
1362 cmꢁ1. 1H NMR (CDCl3)
0.91 (s, 3H), 1.03 (s, 3H), 1.08 (s, 3H), 1.18 (s, 3H), 1.25 (s, 12H,
pinacol-[C(CH3)2]2), 2.52 (m, 1H, 18 -H), 5.40 (m, 1H, 12-H), 5.70 (br
s, 1H), 5.97 (br s, 1H), 6.37 (m, 1H, 2-H); 13C NMR
82.7 (pinacol-
d 0.86 (s, 3H), 0.90 (s, 3H), 0.90 (s, 3H),
4.6. 3-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-oleana-2,12-
dien-28-oyl chloride (6)
b
d
Oxalyl chloride (0.36 mL; 4.16 mmol) was added dropwise to a
solution of compound 5 (90 mg; 0.16 mmol) in chloroform (2 mL),
at room temperature, and the resulting mixture was stirred for
30 min. The solvent was evaporated to dryness and the resulting
[C(CH3)2]2), 123.2 (C12), 138.4 (C2), 144.6 (C13), 181.8 (C28). EI-MS
m/z (%) 563 (4) Mþ, 259 (18), 246 (20), 202 (100), 188 (27), 132
(48), 118 (26), 104 (20). Anal. Calcd. for C36H58BNO3: C, 76.71; H,
10.37; N, 2.48. Found: C, 77.01; H, 10.83; N, 2.59.
Fig. 4. Inhibition of the ChT-L (A) and T-L (B) proteasomal activities after pre-treatment of Jurkat cells with compounds 9 and 11 (50 mM), or epoxomicin (0.1 mM), for 30 min,
followed by collection of the pellet and semi-purification of the proteasome. A representative example of three independent experiments is shown (AFU ¼ arbitrary fluorescence
units).

V.M. Moreira et al. / European Journal of Medicinal Chemistry 63 (2013) 46e56
53
4.8. Methyl N-[3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
oleana-2,12-dien-28-oyl]glycinate (8)
FCC using ethyl acetate:petroleum ether 40e60 from 6:1 to 3:1 to
afford compound 10 (400 mg; 67%). Mp (ethyl acetate:petroleum
ether 40e60): 209e211 ꢀC. IR 1741, 1663, 1366 cmꢁ1 1H NMR
.
To a solution of compound 6 (600 mg; 1.02 mmol) and glycine
methyl ester hydrochloride (174 mg; 1.38 mmol) in dichloro-
methane (6.5 mL), triethylamine (0.3 mL; 2.22 mmol) was added,
and the mixture was stirred at room temperature, for 6 h. Addi-
tional glycine methyl ester hydrochloride (58 mg; 0.46 mmol),
triethylamine (0.1 mL; 0.74 mmol), and dichloromethane (0.5 mL)
were then added and after 2 h the reaction was complete. The
reaction mixture was evaporated to dryness and ethyl acetate
(150 mL) and water (30 mL) were added. The aqueous phase was
further extracted with ethyl acetate (2 ꢃ 75 mL) and the resulting
organic phase was washed with 10% aqueous HCl (30 mL), 10%
aqueous NaHCO3 (30 mL), water (30 mL) and brine (30 mL), dried
over anhydrous Na2SO4, filtered, and the solvent was removed
under reduced pressure to afford a brown solid (595 mg; 91%)
which was purified by FCC using ethyl acetate:petroleum ether 40e
60 from 6:1 to 3:1 to afford compound 8 (385 mg; 65%). Mp (ethyl
acetate:petroleum ether 40e60): 149e151 ꢀC. IR 1752, 1646,
(CDCl3)
3H), 1.07 (s, 3H), 1.16 (s, 3H), 1.24 (s, 12H, pinacol-[C(CH3)2]2), 1.37
(d, 3H, eNCH(CH3)e), 2.63 (m, 1H, 18 -H), 3.73 (s, 3H, OCH3), 4.45
(m, 1H, eNCH(CH3)e), 5.46 (m, 1H, 12-H), 6.37 (m, 1H, 2-H), 6.60
(m, 1H, NH); 13C NMR
82.7 (pinacol-[C(CH3)2]2), 123.5 (C12), 138.5
(C2), 143.6 (C13), 173.6 (COOCH3), 177.5 (C28). EI-MS m/z (%) 649
(20) Mþ, 189 (27), 158 (33), 144 (45), 132 (100), 118 (95), 104 (80), 90
(67). Anal. Calcd. for C40H64BNO5: C, 73.94; H, 9.93; N, 2.16. Found:
C, 74.19; H, 10.25; N, 2.22.
d 0.72 (s, 3H), 0.87 (s, 3H), 0.90 (s, 3H), 0.91 (s, 3H), 1.01 (s,
b
d
4.11. N-[3-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-oleana-
2,12-dien-28-oyl]alanine (11)
The method followed that of compound 9 using compound 10
(150 mg; 0.23 mmol), methanol (1.1 mL), THF (1.5 mL), and NaOH
4 N (0.6 mL; 2.4 mmol), at room temperature, for 1 h, to afford
compound 11 (144 mg; 98%). Mp (ethyl acetate:petroleum ether
1362 cmꢁ1. 1H NMR (CDCl3)
0.91 (s, 3H), 1.01 (s, 3H), 1.07 (s, 3H), 1.16 (s, 3H), 1.24 (s, 12H,
pinacol-[C(CH3)2]2), 2.61 (m, 1H, 18 -H), 3.78 (s, 3H, OCH3), 3.82
and 4.13 (m, 1H and m, 1H, eNCH2e), 5.48 (m, 1H, 12-H), 6.37 (m,
1H, 2-H), 6.55 (m, 1H, NH); 13C NMR
82.7 (pinacol-[C(CH3)2]2),
d
0.73 (s, 3H), 0.88 (s, 3H), 0.90 (s, 3H),
40e60): 244e246 ꢀC. IR 1733, 1622, 1367 cmꢁ1. 1H NMR (CDCl3)
d
0.73 (s, 3H), 0.87 (s, 3H), 0.91 (s, 6H), 1.01 (s, 3H), 1.07 (s, 3H), 1.16 (s,
3H), 1.25 (s, 12H, pinacol-[C(CH3)2]2), 1.43 (d, 3H, eNCH(CH3)e),
b
2.56 (m, 1H, 18
b
-H), 4.43 (m, 1H, eNCH(CH3)e), 5.48 (m, 1H, 12-
82.7 (pinacol-
d
H), 6.37 (m, 1H, 2-H), 6.71 (m, 1H, NH); 13C NMR
d
123.6 (C12), 138.5 (C2), 144.1 (C13), 170.5 (COOCH3), 178.4 (C28). EI-
MS m/z (%) 635 (30) Mþ, 162 (26), 158 (35), 144 (47), 132 (100), 118
(93), 104 (67), 100 (53). Anal. Calcd. for C39H62BNO5: C, 73.68; H,
9.83; N, 2.20. Found: C, 73.70; H, 10.23; N, 2.30.
[C(CH3)2]2), 124 (C12), 138.5 (C2), 143.7 (C13), 175.5 (COOH), 179.7
(C28). EI-MS m/z (%) 635(6) Mþ, 214(20), 202 (65), 189 (100), 172
(60), 118 (60), 90 (60), 82 (35). Anal. Calcd. for C39H62BNO5: C,
73.68; H, 9.83; N, 2.20. Found: C, 73.96; H, 10.23; N, 2.11.
4.9. N-[3-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-oleana-
4.12. Methyl 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-12-
2,12-dien-28-oyl] glycine (9)
oxoolean-2-en-28-oate (12)
To a solution of compound 8 (150 mg; 0.24 mmol) in methanol
(1.1 mL) and THF (1.5 mL), NaOH 4 N (0.6 mL; 2.4 mmol) was added
and the reaction mixture was stirred, at room temperature, for 1 h.
The reaction mixture was evaporated to dryness and ethyl acetate
(100 mL) and water (25 mL) were added. The aqueous phase was
further extracted with ethyl acetate (2 ꢃ 50 mL) and the resulting
organic phase was washed with 10% aqueous HCl (25 mL), 10%
aqueous NaHCO3 (25 mL), water (25 mL) and brine (25 mL), dried
over anhydrous Na2SO4, filtered, and the solvent was removed
under reduced pressure to afford compound 9 (144 mg; 98%). Mp
(ethyl acetate:petroleum ether 40e60): 268e270 ꢀC. IR 1733, 1651,
To a solution of compound 4 (100 mg; 0.17 mmol) in dichloro-
methane (2 mL) a mixture of KMnO4 (361 mg; 2.28 mmol) and
FeSO4$nH2O (180 mg) previously grounded in a mortar was added,
at room temperature, under strong magnetic stirring, followed by t-
butanol (0.09 mL) and water (18 mL). After 6 h the reaction was
complete. The reaction mixture was diluted with ethyl ether
(100 mL), filtered through a Celite pad with further washing with
ethyl ether (2 ꢃ 75 mL). The resulting organic phase was washed
with 10% aqueous NaHCO3 (30 mL), water (30 mL), and brine
(30 mL), dried over anhydrous Na2SO4, filtered, and the solvent was
removed under reduced pressure to afford compound 12 (9ꢁ51mg,
1366 cmꢁ1. 1H NMR (CDCl3)
1.02 (s, 3H),1.08 (s, 3H),1.17 (s, 3H),1.25 (s,12H, pinacol-[C(CH3)2]2),
2.57 (m, 1H, 18 -H), 3.89 and 4.09 (m, 1H and m, 1H, eNCH2e), 5.49
(m, 1H, 12-H), 6.37 (m, 1H, 2-H), 6.77 (m, 1H, NH); 13C NMR
82.7
d
0.74 (s, 3H), 0.88 (s, 3H), 0.90 (s, 6H),
92%). Mp (acetonitrile): 179e181 ꢀC. IR 1727, 1622, 1367 cm 1H
.
NMR (CDCl3)
0.98 (s, 3H), 1.03 (s, 3H), 1.08 (s, 3H), 1.25 (s, 12H, pinacol-
[C(CH3)2]2), 2.63 (m, 13 -H), 2.79 (m, 1H, 18 -H), 3.68 (s, 3H, 28-
OCH3), 6.35 (m, 1H, 2-H); 13C NMR
82.7 (pinacol-[C(CH3)2]2),
d 0.83 (s, 3H), 0.90 (s, 3H), 0.94 (s, 3H), 0.98 (s, 3H),
b
d
b
b
(pinacol-[C(CH3)2]2), 124 (C12), 138.5 (C2), 143.8 (C13), 172.2
(COOH), 179.8 (C28). EI-MS m/z (%) 621 (6) Mþ, 162 (20), 158 (24),
144 (30), 132 (100), 118 (70), 100 (50), 94 (35). Anal. Calcd. for
C38H60BNO5: C, 73.41; H, 9.73; N, 2.25. Found: C, 73.19; H, 10.07; N,
2.29.
d
138 (C2), 178.4 (C28), 212 (C12). EI-MS m/z (%) 594 (5) Mþ, 277 (70),
217 (100), 202 (64), 174 (54),132 (38), 118 (44),104 (37). Anal. Calcd.
for C37H59BO5: C, 74.73; H, 10.00. Found: C, 74.61; H, 10.40.
4.13. Methyl 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-12-
4.10. Methyl N-[3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
oxooleana-2,9(11)-dien-28-oate (13)
oleana-2,12-dien-28-oyl]alaninate (10)
To a solution of compound 12 (150 mg; 0.25 mmol) in aceto-
The method followed that of compound 8 using compound 6
nitrile (7.3 mL), 48% aqueous HBr (12.2 mL) was added followed by
(600 mg; 1.02 mmol),
(192 mg; 1.38 mmol), dichloromethane (6 mL) and triethylamine
(0.3 mL; 2.22 mmol), at room temperature, for 6 h, after which
L
-alanine methyl ester hydrochloride
bromine (1 M solution in acetonitrile, 0.35 mL; 0.35 mmol) and the
mixture was heated to reflux. After 1 h, additional bromine (0.1 mL;
0.1 mmol) was added and after 1.5 h the reaction was complete. The
reaction mixture was evaporated to dryness and ethyl acetate
(75 mL) and water (20 mL) were added. The aqueous phase was
further extracted with ethyl acetate (2 ꢃ 50 mL) and the resulting
organic phase was washed with 10% aqueous NaHCO3 (25 mL), 10%
additional
L-alanine methyl ester hydrochloride (64 mg;
0.46 mmol), dichloromethane (0.5 mL) and triethylamine (0.1 mL;
0.74 mmol) were added, and after 2 h the reaction was complete
and afforded a brown solid (600 mg; 90%) which was purified by

54
V.M. Moreira et al. / European Journal of Medicinal Chemistry 63 (2013) 46e56
aqueous Na2SO3 (25 mL), and water (25 mL), dried over anhydrous
Na2SO4, filtered, and the solvent was removed under reduced
pressure to afford compound 13 as a yellowish solid (140 mg; 94%).
Mp (acetonitrile): 224e226 ꢀC. IR 1719, 1657, 1628, 1371 cmꢁ1. 1H
reaction was complete and worked up to afford compound 18
(386 mg, 91%). Mp (ethyl acetate:petroleum ether 40e60): 169e
170 ꢀC. IR 2227, 1407, 1244, 1207, 1140 cmꢁ1 1H NMR (CDCl3)
. d
0.91 (s, 3H), 0.95 (s, 3H), 1.03 (s, 3H), 1.05 (s, 3H), 1.08 (s, 3H), 1.15 (s,
NMR (CDCl3)
(s, 3H), 1.24 (s, 3H), 1.25 (s, 12H, pinacol-[C(CH3)2]2), 2.87 (m, 1H,
13 -H), 3.02 (m, 1H, 18 -H), 3.69 (s, 3H, 28-OCH3), 5.78 (m, 1H, 11-
H), 6.43 (m, 1H, 2-H), 13C NMR
82.8 (pinacol-[C(CH3)2]2), 123.9
(C11), 137.8 (C2), 178.3 and 178.3 (C10 and C28), 200.4 (C12). EI-MS
m/z (%) 592 (15) Mþ, 278 (11), 200 (10), 186 (20), 174 (100), 132 (67),
104 (93), 90 (65). Anal. Calcd. for C37H57BO5: C, 74.98; H, 9.69.
Found: C, 74.85; H, 10.09.
d
0.89 (s, 3H), 0.99 (s, 6H), 1.08 (s, 3H), 1.12 (s, 3H), 1.16
6H), 2.59 (m, 1H, 18b-H), 5.41 (m, 1H, 12-H), 5.57 (dd, 1H, 2-H,
J1 ¼ 1.76 Hz and J2 ¼ 6.60 Hz); 13C NMR
d 113.5 (C2), 116.8 and 120
b
b
(J ¼ 319 Hz, CF3), 124.3 (C12), 125.5 (CN), 141.6 (C13), 155.4 (C3); EI-
MS m/z (%) 418 (80) Mþ ꢁ OTf, 390 (32), 213 (40), 186 (85), 172
(100), 156 (70), 144 (65), 130 (55). Anal. Calcd. for C31H44F3NO3S: C,
65.58; H, 7.81; N, 2.47. Found: C, 65.69; H, 8.11; N, 2.52.
d
4.18. 3-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-oleana-2,12-
diene-28-nitrile (19)
4.14. Methyl 12-hydroxyimino-3-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)-olean-2-en-28-oate (14)
The method followed that of compound 4 using compound 18
(100 mg; 0.18 mmol), PdCl2(PPh3)2 (9 mg, 0.012 mmol), THF (2 mL),
B2pin2 (67 mg, 0.26 mmol), and a 0.8 M solution of Na2CO3
(0.84 mL; 0.68 mmol), at reflux. After 1.5 h additional PdCl2(PPh3)2
(4.5 mg, 0.006 mmol), B2pin2 (34 mg, 0.13 mmol), and THF (0.5 mL)
were added and after 2 h the reaction was complete and worked up
to afford compound 19 (87 mg, 91%). Mp (THF:methanol): 292e
To a solution of compound 12 (80 mg; 0.13 mmol) in dry pyr-
idine (1.3 mL), hydroxylamine hydrochloride (60 mg; 0.86 mmol)
was added. The mixture was warmed to reflux under N2 atmos-
phere and after 5 h the reaction was complete. Ethyl acetate (50 mL)
and 5% aqueous HCl (20 mL) were added and the aqueous phase
was further extracted with ethyl acetate (2 ꢃ 50 mL). The resulting
organic phase was washed with 5% aqueous HCl (25 mL), 10%
aqueous NaHCO3 (25 mL), water (25 mL), and brine (25 mL), dried
over anhydrous Na2SO4, filtered, and the solvent was removed
under reduced pressure to afford compound 14 (74 mg, 90%). Mp
294 ꢀC. IR 2227, 1367 cmꢁ1. 1H NMR (CDCl3)
3H), 0.94 (s, 3H), 1.04 (s, 3H), 1.06 (s, 3H), 1.09 (s, 3H), 1.14 (s, 3H),
1.25 (s, 12H, pinacol-[C(CH3)2]2), 2.57 (m, 1H, 18 -H), 5.39 (m, 1H,
12-H), 6.37 (m, 1H, 2-H); 13C NMR
82.7 (pinacol-[C(CH3)2]2), 124.9
d 0.90 (s, 3H), 0.93 (s,
b
d
(C12), 125.6 (CN),138.5 (C2), 141.4 (C13). EI-MS m/z (%) 545 (22) Mþ,
315 (15), 228 (100), 213 (80), 172 (70), 146 (55), 92 (60), 90 (74).
Anal. Calcd. for C36H56BNO2: C, 79.24; H, 10.34; N, 2.57. Found: C,
79.02; H, 10.03; N, 2.74.
(acetonitrile): 177e179 ꢀC. IR 1717, 1628, 1362 cmꢁ1
(CDCl3) 0.84 (s, 3H), 0.85 (s, 3H), 0.88 (s, 3H), 0.93 (s, 3H), 0.93 (s,
3H), 1.02 (s, 3H), 1.07 (s, 3H), 1.25 (s, 12H, pinacol-[C(CH3)2]2), 2.57
(m, 1H, 13 -H), 2.85 (m, 1H, 18 -H), 3.06 (m, 1H), 3.68 (s, 3H, 28-
OCH3), 6.37 (m, 1H, 2-H); 13C NMR
82.7 (pinacol-[C(CH3)2]2),
.
1H NMR
d
b
b
d
4.19. Biology
138.3 (C2), 160.9 (C12), 178.7 (C28). EI-MS m/z (%) 609 (5) Mþ, 174
(80), 132 (84), 118 (100), 106 (93), 104 (85), 100 (75), 90 (65). Anal.
Calcd. for C37H60BNO5: C, 72.89; H, 9.92; N, 2.30. Found: C, 73.03; H,
10.32; N, 2.23.
Human Jurkat, K562, Jijoye, Hela, SW480, SKOV-3 cells and
fibroblasts were obtained from the American Type Culture Collec-
tion (USA). Jurkat, K562, Jijoye, SW480 and SKOV-3 cells were
routinely grown in RPMI 1640 supplemented with 10% fetal bovine
4.15. 2-Oxoolean-12-en-28-oyl chloride (16)
serum,100 IU/mL penicillin, and 100 mg/mL streptomycin at 5% CO2.
Hela cells and human fibroblasts were routinely grown in Dulbec-
co’s modified Eagle medium (DMEM) supplemented with 10% fetal
bovine serum, 100 IU/mL penicillin, and 100 mg/mL streptomycin at
5% CO2. PBMC were obtained from healthy individuals after Ficoll-
Hypaque density separation. MG132 and epoxomycin were pur-
chased from Enzo Life Sciences AG, Lausen, Switzerland.
The method followed that of compound 6 using oleanonic acid
15 (100 mg; 0.22 mmol), oxalyl chloride (0.5 mL; 5.72 mmol), and
chloroform (1.5 mL), at room temperature. After 4 h the reaction
was complete and worked up to afford compound 16 (102 mg;
98%). IR 1786, 1703 cmꢁ1. 1H NMR (CDCl3)
d
0.83 (s, 3H), 0.91 (s, 6H),
1.04 (s, 3H), 1.05 (s, 3H), 1.08 (s, 3H), 1.15 (s, 3H), 2.84 (m, 1H, 18 -H),
b
5.34 (m, 1H, 12-H); 13C NMR
d
123.5 (C12), 142.0 (C13), 179.7 (COCl),
4.20. Cell viability and proliferation assays
217.6 (C3). EI-MS m/z (%) 472 (4) Mþ, 409 (6), 265 (12), 204 (24), 201
(100), 187 (15), 131 (15), 90 (16).
Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) assay. For the MTT assay
cells were seeded at a concentration of 25 ꢃ 103 cells/well on 96-
well plates and treated with the compounds at the specified con-
centrations. After the indicated periods of time, the cells were
incubated with a 5 mg/mL solution of MTT in phosphate saline
buffer (PBS) for 2 h. Following this incubation lysis buffer (20% w/v
SDS in 1:1 DMF:H2O at pH 4.7) was added to the cells and after 4 h
the plates were read at 570 nm (Sunrise, TECAN, Austria GmbH).
4.16. 2-Oxoolean-12-en-28-amide (17)
The method followed that of compound 7 using compound 16
(500 mg, 1.1 mmol), THF (20 mL), and ammonia (0.5 mL), cooled in
an ice bath. After 1.5 h the reaction was complete and worked up to
afford compound 17 (476 mg; 99%). IR 3459, 1700, 1669 cmꢁ1 1H
.
NMR (CDCl3)
d
0.87 (s, 3H), 0.89 (s, 6H), 1.03 (s, 6H), 1.07 (s, 3H), 1.16
122.5
(s, 3H), 5.37 (m, 1H, 12-H), 5.91 (br s, 2H, CONH2); 13C NMR
d
(C12), 144.8 (C13), 181.3 (CONH2), 217.4 (C3). EI-MS m/z (%) 453 (15)
Mþ, 233 (69), 202 (100), 190 (56), 186 (40), 132 (84), 118 (43), 104
(39).
4.21. Proteasome inhibition assays e cell pellets [43]
Jurkat cells (5 ꢃ 108) or PBMC (80 ꢃ 106) were washed with cold
PBS and resuspended in buffer (400 mL) containing 50 mM TriseHCl
4.17. 28-Cyanooleana-2,12-diene 3-trifluoromethanesulfonate (18)
(pH 7.4), 5 mM MgCl2, 1 mM DTT (SigmaeAldrich, Co), 2 mM ATP,
and 250 mM sucrose. An amount equivalent to half the volume of
cell suspension was added in glass beads and the mixture was
vortexed for 2 min at 4 ꢀC. Beads and cell debris were removed by
7 min centrifugation at 1000 g, followed by 20 min of centrifugation
The method followed that of compound 3 using compound 17
(340 mg, 0.75 mmol), dichloromethane (7 mL), Tf2O (0.23 mL,
1.4 mmol) and DTBMP (310 mg, 1.51 mmol), at reflux. After 1.5 h the

V.M. Moreira et al. / European Journal of Medicinal Chemistry 63 (2013) 46e56
55
at 10,000 g, at 4 ꢀC. The lysates were cleared by ultracentrifugation
for 1 h at 100,000 g followed by another ultracentrifugation for 5 h
at the same rpm, at 4 ꢀC. Proteasome-containing pellets were
cells were seeded at a concentration of 25 ꢃ 103 cells/well on 96-
well plates and treated with the compounds at the specified con-
centrations. After overnight treatment, the cells were incubated
resuspended in homogenization buffer (200
mL) [50 mM TriseHCl
with 100 ml/well of substrate solution and then the luminescence
(pH 7.4), 100 mM KCl, 15% glycerol]. Protein concentration was
determined using the BCA protocol (Pierce, Rockford, IL). The flu-
orogenic substrates Suc-LLVY-AMC and Boc-LRR-AMC (Enzo Life
Sciences AG, Lausen, Switzerland) were used to measure the ChT-L
and T-L activities of the proteasome, respectively. Semipurified
proteasomes were pretreated with vehicle (DMSO) or the com-
pounds for 30 min in a buffer containing 50 mM TriseHCl (pH 7.4),
was immediately read by Wallac EnVision 2104 Multilabel Reader
(PerkinElmer, Monza, Italy). Samples were run in triplicate.
Acknowledgments
Jorge A.R. Salvador thanks Universidade de Coimbra for financial
support. Vânia M. Moreira thanks Fundação para a Ciência e a
Tecnologia for financial support (SFRH/BPD/45037/2008). The
authors would like to acknowledge Alessandro Canella at the
Department of Biochemistry and Molecular Biology, University of
Ferrara, Italy and Teresa Gagliano, Federico Tagliati, and Degli
Uberti EC at the Section of Endocrinology, Department of Bio-
medical Sciences and Advanced Therapies, University of Ferrara,
Italy
5 mM MgCl2, 500 m
M EDTA, 1 mM DTT, and 2 mM ATP, at 37 ꢀC, and
after this time the different peptide substrates were added to the
mixture. Fluorescence (Spectrafluor plus, Tecan, Salzburg, Austria)
was read at 360 nm excitation wavelength and 465 nm emission
wavelength for 2 h, at 37 ꢀC.
4.22. Proteasome inhibition assays e Jurkat cells [43]
Jurkat cells (1.6 ꢃ 106 cells/well) were plated on 24-well plates
and treated with the compounds at the specified concentrations or
with the vehicle for 30 min after which the pellets were collected,
washed with PBS and the lysates were prepared and assayed
according to the procedure reported above.
Appendix A. Supplementary data
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.ejmech.2013.01.040.
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