Synthesis and structure-activity relationship study of novel cytotoxic carbamate and N-acylheterocyclic bearing derivatives of betulin and betulinic acid

Article abstract of DOI:10.1016/j.bmc.2010.04.085

Chemical transformation studies were conducted on betulin and betulinic acid, common plant-derived lupane-type triterpenes. The concise synthesis, via a stepwise approach, of betulin and betulinic acid carbamate and N-acylheterocyclic containing derivatives is described. All new compounds, as well as betulinic acid were tested in vitro for their cytotoxic activity. Most of the compounds have shown a better cytotoxic profile than betulinic acid, including the synthesized betulin derivatives. Compounds 25 and 32 were the most promising derivatives, being up to 12-fold more potent than betulinic acid against human PC-3 cell lines (IC₅₀ values of 1.1 and 1.8 μM, respectively).

Full text of DOI:10.1016/j.bmc.2010.04.085

Bioorganic & Medicinal Chemistry 18 (2010) 4385–4396
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and structure–activity relationship study of novel cytotoxic
carbamate and N-acylheterocyclic bearing derivatives
of betulin and betulinic acid
b
b,
Rita C. Santos a, Jorge A. R. Salvador a, , Silvia Marín , Marta Cascante
,
*
*
João N. Moreira c,d, Teresa C. P. Dinis d,e
a Laboratório de Química Farmacêutica, Faculdade de Farmácia, Universidade de Coimbra, Pólo das Ciências da Saúde, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal
b IBUB—Department of Biochemistry and Molecular Biology, Faculty of Biology, Institute of Biomedicine of University of Barcelona (IBUB) and IDIBAPS,
Unit Associated with CSIC, Diagonal 645, 08028 Barcelona, Spain
c Laboratório de Tecnologia Farmacêutica, Faculdade de Farmácia, Universidade de Coimbra, Pólo das Ciências da Saúde, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal
d Centro de Neurociências e Biologia Celular, Universidade de Coimbra, 3004-517 Coimbra, Portugal
e Laboratório de Bioquímica, Faculdade de Farmácia, Universidade de Coimbra, Pólo das Ciências da Saúde, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
Chemical transformation studies were conducted on betulin and betulinic acid, common plant-derived
lupane-type triterpenes. The concise synthesis, via a stepwise approach, of betulin and betulinic acid car-
bamate and N-acylheterocyclic containing derivatives is described. All new compounds, as well as betu-
linic acid were tested in vitro for their cytotoxic activity. Most of the compounds have shown a better
cytotoxic profile than betulinic acid, including the synthesized betulin derivatives. Compounds 25 and
32 were the most promising derivatives, being up to 12-fold more potent than betulinic acid against
Received 19 January 2010
Revised 19 March 2010
Accepted 25 April 2010
Available online 29 April 2010
Keywords:
human PC-3 cell lines (IC50 values of 1.1 and 1.8
l
M, respectively).
Ó 2010 Elsevier Ltd. All rights reserved.
Triterpenoids
Betulinic acid
20-Methylimidazole
Triazole
Cytotoxicity
1. Introduction
vanced modifications may be performed without loss of the
desired biological properties.5
Natural products have been used to treat human disease for
thousands of years and play an increasingly important role in drug
discovery and development. In fact, the majority of anticancer and
anti-infectious agents are of natural origin.1,2
The naturally occurring lupane-type triterpenoids betulin 1 and
betulinic acid 2 (Fig. 1) have been thoroughly investigated during
the past years for their promising medical properties,6–9 and par-
ticularly their chemopreventive and antitumor activities.10,11
Compound 2, derived from birch bark, exhibits a broad spec-
trum of anticancer activities and potential clinical value as an
anti-HIV, antibacterial and antimalarial agent.6,10 Discovery of 2
as a growth inhibitor of melanoma cell lines and confirmation of
its anticancer activity in mice bearing human melanoma xeno-
grafts12 led to considerable interest in 2 as an anticancer drug
Terpenes are a wide-spread group of natural compounds with
considerable practical significance which are synthesized in many
plants by rearrangement of squalene epoxide. Almost all terpenes
have biological activities in animals including man, and they also
play a meaningful role in human medicine.3 From this point of
view, the most important groups of terpenes are triterpenes, triter-
penes glycosides, and other triterpenoids. Pentacyclic triterpenoids
are a class of pharmacologically active and structurally rich natural
products with privileged motifs for further modifications and
structure–activity relationship (SAR) analyses.4 To date several re-
ports have been published demonstrating that either simple or ad-
* Corresponding authors. Tel.: +351 239488479; fax: +351 239827126 (J.A.R.S.);
tel.: +34 93 402 15 9; fax: +34 93 402 12 19 (M.C.).
E-mail addresses: salvador@ci.uc.pt (J.A.R. Salvador), martacascante@ub.edu
(M. Cascante).
Figure 1. Chemical structure of betulin 1 and betulinic acid 2.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.04.085

4386
R. C. Santos et al. / Bioorg. Med. Chem. 18 (2010) 4385–4396
candidate, and a significant number of scientific publications in
this field has been published. Triterpenoid 2 and its derivatives
were shown to be effective against a variety of carcinoma cell lines
derived from lung, ovarian, cervical, head and neck carcinomas, as
well as from lymphoma, neuroblastoma, medulloblastoma, glio-
blastoma, and other types of tumors.6,10,12–14 In addition to tumor
cell lines, 2 was also cytotoxic against primary cancer cells isolated
from tumor specimens obtained from neuroblastoma, glioblas-
toma, and leukemia.15,16 Also, 2 was cytotoxic in different models
of drug resistance, for example primary pediatric acute leukemia
samples that were refractory to standard chemotherapeutic
agents.15 By comparison, normal cells of different origin have been
reported to be much more resistant to 2 than cancer cells, which is
indicative of some selectivity for tumor cells.17,18 This feature
makes 2 unique in comparison to compounds that are currently
used in cancer therapy, such as taxol, camptothecin (CPT), elipti-
cine, etoposide, vinblastine or vincristine, among others, which
are very toxic and inhibit replication of both cancer and normal
cells. Reports have demonstrated that the cytotoxicity induced by
2 involves modulation of Bcl-2 family and cell cycle regulatory pro-
place at carbon C-28 and then at carbon C-3, as determined by
1H NMR. Thus, to obtain the C-3 lupane carbamate 7, compound
6 was previously prepared according the general procedure with
CDT, and was then selectively hydrolyzed at C-28 with silica gel
and purified by flash column chromatography (FCC) to afford 7,
in 63% yield (Scheme 1). In order to avoid the formation of
multiple derivatives, compound 1 was first acetylated at the C-
28 position with acetic anhydride (Ac2O) and imidazole, in CHCl3,
to give betulin 28-acetate 8 (Scheme 1), that was then treated
with CBMI or CDT in THF, at reflux, to afford compounds 9 and
10 in 82% and 88% yield, respectively after FCC (Scheme 1). The
synthetic pathway employed for the synthesis of 3-acetylbetulin
11 (Scheme 1) was inspired by the procedure developed by Tietze
et al.39 Initially, 1 was acetylated at the C-3 and C-28 positions
with acetic anhydride and 4-dimethylaminopyridine (DMAP) in
anhydrous THF to afford betulin 3,28-diacetate in quantitative
yield, without further purification. Thereafter, the 3,28-diacetate
was deacetylated at the C-28 position in the presence of 1 equiv
of methanolic potassium hydroxide, in THF, in 89% isolated
yield.
teins,19–22 regulation of the nuclear factor kappa B (NF-
j
B),23,24 as
The synthesis of derivative 15 (Scheme 1) began with the epox-
idation of the isopropenyl group of the commercially available 1
with m-chloroperbenzoic acid (m-CPBA), followed by acid cata-
lyzed epoxy-ring opening to afford the epimeric isomer (20R-alde-
hyde) 14 (39%) as the major product.40 This compound was then
treated with CBMI in THF, at reflux, to afford 15 in 75% yield, after
FCC (Scheme 1). The intermediate 16 (Scheme 2) was synthesized
by methoxylation of compound 1 in two steps, as previously de-
scribed.41 The reaction with CBMI and CDT afforded derivatives
17–20 in isolated yields ranging from 17% to 74% (Scheme 2).
The second series of derivatives, originated from compound 2,
was synthesized following the reaction sequence shown in
Schemes 3 and 4. Derivatives 23 and 24 (Scheme 3) were prepared
directly from the reaction of 2 with 5 equiv of CBMI in anhydrous
THF, in 77% and 22% yield, respectively, after FCC. However, only
derivative 25 (Scheme 3) was obtained after reaction with CDT,
in 82% isolated yield, under the same reaction conditions. This fact
could be explained by the similarity of the reaction rate of the hy-
droxyl and carboxyl groups with CDT. The methyl ester 21 (Scheme
3) was prepared in 83% yield from 2 using CH3I and K2CO3, in dry
DMF. Reaction of 21 with CBMI gave the 20-methylimidazole carba-
mate derivative 26, in quantitative yield (Scheme 3). For the
synthesis of compound 27 (Scheme 3), prior to the introduction
of the 20-methylimidazole moiety at C-28, betulonic acid 22 was
prepared by oxidation of 3b-hydroxyl group under acidic
conditions (CrO3 and sulfuric acid). Derivative 27 was obtained in
84% isolated yield using the same reaction protocol mentioned
above.
The new double bond between C1 and C2 was formed by dehy-
drogenation of 22 with 2,3-dichloro-5,6-dicyanobenzoquinone
(DDQ) in dioxane to afford compound 28 (Scheme 4).42 This com-
pound was then treated with CBMI to afford 29, in 87% isolated
yield. For reasons that are currently unclear, all attempts to pre-
pare the N-acyltriazole derivatives from intermediates 22 and 28
were unsuccessful.
For the synthesis of derivatives 31 and 32, intermediate 30
(Scheme 4) was prepared according to a previously published
procedure using oxygen in the presence of potassium tert-butoxide
(t-BuOK), in 72% yield.43 Further reaction with CBMI or CDT and
purification by FCC led to derivatives 31 and 32, in 83% and 77%
isolated yield, respectively.
well as inhibition of aminopeptidase N,25 and growth factor-in-
duced angiogenesis.26 DNA topoisomerases have been shown to
be important targets for several chemotherapeutic agents.27–30
Compound 2 and some of its derivatives have also been reported
as potent inhibitors of eukaryotic topoisomerases I and II.31–35
Since natural derivatives of 2 bear anticancer activity, the syn-
thesis of derivatives of this triterpenoic acid, which is commer-
cially available, could be of great value in discovering potential
bioactive semisynthetic compounds. In fact, many studies reported
that modifications of triterpenoids 1 and 2 at the C-3, C-20, and C-
28 positions can produce a number of potentially important deriv-
atives with potent anticancer activity.6,11,36–38
Recently, we focused our attention on the synthesis of lupane-
type imidazole carbamates and N-acylimidazole bearing deriva-
tives. Our results showed that addition of an imidazolyl moiety
at the C-3 and/or C-28 positions of 1 and 2 can result in more po-
tent in vitro anticancer agents than 2, with IC50 values between 0.8
and 26.1 lM, in human cancer cell lines of different tumor types.
These IC50 values were 2–45 times lower than those obtained with
compound 2. The promising results prompted us to extend our
study to 20-methylimidazole and triazole derivatives, in order to
establish meaningful SAR. Hence, we report here the synthesis of
22 novel 20-methylimidazole and triazole carbamates and N-acyl-
heterocyclic bearing derivatives of compounds 1 (3–7, 9, 10, 12,
13, 15, and 17–20) and 2 (23–27, 29, 31, and 32). The in vitro cyto-
toxic activity of the synthesized compounds was evaluated against
human hepatocellular carcinoma (HepG2), leukemia (Jurkat), cer-
vical adenocarcinoma (HeLa), colon adenocarcinoma (HT-29),
prostate adenocarcinoma (PC-3), and fibroblasts (BJ) cells. The
compounds were also screened for their ability to inhibit topoiso-
merase I.
2. Results and discussion
2.1. Chemistry
Similar transformations were used to prepare the 20-methylim-
idazole and triazole derivatives. The first series of derivatives was
synthesized according to the reaction sequence shown in Schemes
1 and 2. Compound 1 was the starting material used directly for
the synthesis of derivatives 3–6, through reaction with 1,10-car-
bonylbis(20-methylimidazole) (CBMI) and 1,10-carbonyl-di(1,2,4-
triazole) (CDT) as previously reported (Scheme 1).38 In the case
of the disubstituted derivatives, the first carbamoylation takes
The structures of all the newly synthesized compounds (3–7, 9,
10, 12, 13, 15, 17–20, 23–27, 29, 31, and 32) were assigned by 1D
and 2D NMR experiments (1H, 13C, DEPT135, COSY, HMQC, and
HMBC), IR and MS spectroscopy.

R. C. Santos et al. / Bioorg. Med. Chem. 18 (2010) 4385–4396
4387
Scheme 1. Synthesis of derivatives 3–7, 9, 10, 12, 13, and 15. Reagents and conditions: (a) CBMI or CDT, dry THF, N2, reflux; (b) CDT, dry THF, N2, reflux; (c) silica gel, rt; (d)
Ac2O, imidazole, CHCl3, reflux; (e) Ac2O, DMAP, THF, rt; (f) KOH, MeOH, THF, rt; (g) m-CPBA, CH2Cl2, 0–5 °C; (h) H2SO4 (2 M), 0–5 °C; (i) CBMI, dry THF, N2, reflux.
2.2. Antiproliferative activity
compounds with respect to their potential cytotoxicity against hu-
man cancers. The in vitro inhibition of cell viability mediated by
the different synthesized compounds was studied on HepG2 (hu-
man hepatocellular carcinoma; adherent epithelial cell line), Jurkat
(human acute T cell leukemia; suspended cell line), HeLa (human
2.2.1. Cytotoxicity in human cancer cell lines
Several cancer cell lines were cultured and used in experiments
in order to obtain the SAR information of the new lupane-type

4388
R. C. Santos et al. / Bioorg. Med. Chem. 18 (2010) 4385–4396
Scheme 2. Synthesis of derivatives 17–20. Reagents and conditions: (a) CBMI or CDT, dry THF, N2, reflux.
at C-3 and/or C-28 positions were found to be cytotoxic against hu-
man cancer cell lines.38,46–48 Moreover, the synthesized N-acylhet-
erocyclic derivatives induced more potent cytotoxicity in the
cancer cell lines than the carbamate derivatives which suggests
that the beneficial impact on the cytotoxic activity of the N-hetero-
cyclic moieties is highly dependent on the lupane-type triterpe-
noid used. Compound 2 derivatives (23–27, 29, 31, and 32)
(Table 2) were the most potent compounds showing cytotoxic
activity against the tested tumor cells. The N-acyltriazole deriva-
tives 25 and 32 (Table 2) were about 19- and 12-fold, more active
than 2 against PC-3 cells. Compounds 7, 17, 18, 23, 24, and 27, 29,
and 31 (Tables 1 and 2) also exhibited a better cytotoxic profile
than 2. These preliminary results allowed us to predict which
groups are required to obtain a good cytotoxic activity. We first
investigated the cytotoxicity of acetyl esters at the C-28 and C-3
positions of 20-methylimidazole (9 and 12) (Table 1) and triazole
(10 and 13) (Table 1) carbamate derivatives and no cytotoxic activ-
ity was found, even at concentrations up to 30 lM. These results
were in accordance with a previous work reporting loss of cytotox-
icity on acylated derivatives.49 We also investigated the influence
of combining substitution at C-3 and C-28 positions. For com-
pounds of Group I, the introduction of a carbamate moiety at C-3
in the C-28 substituted derivatives (3, 4, 17, and 18) (Table 1) affor-
ded compounds 5, 6, 19, and 20 (Table 1) with lower cytotoxic
activity against the tested cancer cell lines. In accordance with
observations from Kim et al.,36 this loss of toxic effect suggested
a size limitation at the C-3 position. For Group II compounds,
introduction of a carbonyl group at C-3 originated compound 27
(Table 2) with a significant cytotoxic activity (IC50 values of 4.2,
Scheme 3. Synthesis of derivatives 23–27. Reagents and conditions: (a) CH3I,
K2CO3, dry DMF, rt; (b) Jones reagent, acetone, 0 °C; (c) CBMI or CDT, dry THF, N2,
reflux.
cervix adenocarcinoma; adherent epithelial cell line), HT-29 (hu-
man colon adenocarcinoma; adherent epithelial cell line), and
PC-3 (human prostate adenocarcinoma; adherent epithelial cell
line) cancer cell lines. These cell lines were chosen because they
represent cancer types with great incidence in the human popula-
tion. All cell lines were exposed to 10 serial threefold dilutions of
each drug for 72 h. Cytotoxicity was evaluated using the MTT or
XTT assay. Compounds were subdivided into two groups: com-
pound 1 derivatives (3–7, 9, 10, 12, 13, 15, 17–20 Group I), and
compound 2 derivatives (23–27, 29, 31, and 32, Group II). Cytotox-
icity results are displayed in Tables 1 and 2 and expressed as the
concentration inhibiting 50% of cell growth (IC50). The parent tri-
terpenoid 2, currently in clinical trials,44,45 was used as a positive
4.7, 5.3, and 5.5 lM in HeLa, HT-29, Jurkat and HepG2 cells, respec-
tively). This result suggested that more than the size limitation of
the moiety used, there is great significance in the electronic density
at C-3 position, for the cytotoxic effect.
It has been shown in previous SAR studies that the free C-28
carboxylic acid function is important for the cytotoxicity of
2,6,10,11,36 as the corresponding reduced form 1 is devoid of such
activity.36 As shown in Table 2, the lower cytotoxic activity of the
3,28-N-acyl-20-methylimidazole derivative 24 (Table 2) (IC50
7.8–20.1
(Table 2) (IC50 6.8–12.5
(IC50 12.9–16.4 M) compared with compounds 23 (Table 2)
(IC50 5.7–7.5 M) and 31 (Table 2) (IC50 4.7–9.4 M), bearing a free
l
M), the 28-N-acyl-20-methylimidazole derivative 29
M) and the 28-methyl ester 26 (Table 2)
l
l
control (IC50 12.8–36.4
lM). Compounds with IC50 values >30
lM
l
l
were considered inactive.
carboxyl group at C-28 position, also suggested the importance of
the hydrogen bonding capability and/or acidity in the cytotoxic
effect. These observations are consistent with the reported
in vitro cytotoxic activities described by Kim et al.,36 and Chatterjee
et al.,50 Our previous SAR investigation also showed that the addi-
tion of an imidazolyl moiety at the C-28 position of 2 significantly
decreased cytotoxicity compared to its carboxylic acid derivative.38
However, this extended study revealed that there are some
As shown in Tables 1 and 2, the synthesized compounds exhib-
ited different degrees of cytotoxicity towards the tested cell lines.
Generally, the cytotoxicity results indicate that the introduction of
the 20-methylimidazolyl and triazolyl moieties has a positive im-
pact on the anticancer activity of the lupane-type triterpenoids.
These results are in good agreement with previous investigations
in which lupane-type derivatives bearing N-heterocyclic moieties

R. C. Santos et al. / Bioorg. Med. Chem. 18 (2010) 4385–4396
4389
Scheme 4. Synthesis of derivatives 29, 31 and 32. Reagents and conditions: (a) DDQ, dioxane, N2, reflux; (b) O2, t-BuOK, t-BuOH, 40 °C; (c) CBMI, dry THF, N2, reflux; (d) CBMI
or CDT, dry THF, N2, reflux.
Table 1
Cytotoxic activity of compound 1 derivatives 3–7, 9, 10, 12, 13, 15, and 17–20 (Group I), in six human cell lines, after 72 h of exposure
a
Compound
IC50
(l
M
SD)
HepG2
Jurkat
HeLa
HT-29
PC-3
BJ
2
3
4
36.4 1.5
8.1 0.4
ND
26.9 2.2
15.8 2.4
ND
26.0 2.1
11.0 1.7
ND
12.8 1.7
16.2 2.1
7.6 0.9
>30
21.5 2.0
ND
4.7 0.4
ND
ND
ND
ND
ND
5
>30
>30
>30
6
7
9
ND
7.3 1.2
>30
ND
12.2 2.1
>30
ND
6.9 1.3
>30
17.5 1.8
4.9 0.6
>30
7.9 1.0
2.1 0.2
ND
ND
39.4 2.5
ND
10
12
13
15
17
18
19
20
ND
>30
ND
14.3 2.4
14.4 1.3
9.1 1.8
28.2 2.5
ND
ND
>30
ND
21.3 4.1
24.8 4.7
9.5 2.7
>30
ND
>30
ND
16.6 1.5
8.9 2.1
24.3 2.7
13.4 0.6
ND
>30
>30
>30
15.8 1.7
10.4 1.6
5.8 0.3
>30
>30
ND
>30
ND
ND
2.6 0.2
ND
ND
ND
ND
ND
ND
46.6 3.5
ND
ND
12.3 0.8
6.4 0.4
ND
Exponentially growing cells were treated with the compounds at different concentrations for 72 h. Cell-growth inhibition was analyzed by the MTT (HepG2, HeLa, HT-29, PC-
3, and BJ) or XTT (Jurkat) assay. The assay was done using three replicates and repeated four times.
ND = not determined.
a
IC50 is the concentration of compound that inhibits 50% of cell growth.
exceptions to this general tendency. In fact, compound 25 (Table 2
and Fig. 2 C), a disubstituted derivative of 2, was found to exhibit a
compared to 2 (IC50 21.5 lM). In fact, compounds 25 and 32 exhib-
ited very similar cytotoxic behavior, as it can be observed in the
dose dependent effect curves for cell viability (Fig. 2 C and D), sug-
gesting that both compounds share the same mechanism of cell
death in the tested cell lines.
The selectivity was studied by incubating the compounds with a
non-tumoral cell line (BJ). All the tested compounds are more cyto-
toxic towards cancer cell lines than normal cells (Tables 1 and 2)
demonstrating a selective cytotoxic activity for malignant cells.
Compounds 7, 18, 25, and 32 showed IC50 values 15–18 times low-
er on PC-3 cells than on non-tumoral BJ cells (Tables 1 and 2).
In terms of SAR, these in vitro cytotoxic results suggest that
simple modifications of the parent structure either in 1 or 2 can
produce new potentially interesting derivatives. Also, derivatives
potent cytotoxic profile, with IC50 values of 1.9 and 1.1
HT-29 and PC-3 cancer cell lines, respectively.
lM against
Although colon cancer cells were reported to be almost com-
pletely refractory to treatment with 2, in our assays the highest
cytotoxic activity of this compound was observed in the HT-29
adenocarcinoma cell line. Nevertheless, analogues 7 (Table 1), 25,
27, and 32 (Table 2) showed better IC50 values (65 lM), in HT-29
cells than compound 2. PC-3 prostate cancer cells were more sen-
sitive to the tested compounds than the other cancer cell lines.
Compounds 7, 18 (Table 1 and Fig. 2 A and B), 25, and 32 (Table
2 and Fig. 2 C and D) showed remarkable activities against PC-3
cells, with IC50 values of 2.1, 2.6, 1.1, and 1.8 lM, respectively,

4390
R. C. Santos et al. / Bioorg. Med. Chem. 18 (2010) 4385–4396
Table 2
Cytotoxic activity of compound 2 derivatives 23–27, 29, 31, and 32 (Group II), in six human cell lines, after 72 h of exposure
a
Compound
IC50
(l
M
SD)
HepG2
Jurkat
HeLa
HT-29
PC-3
BJ
2
23
24
25
26
27
29
31
32
36.4 1.5
7.3 1.0
11.6 0.8
6.8 1.1
13.4 1.7
5.5 1.1
6.8 1.5
4.7 0.2
7.4 1.3
26.9 2.2
6.1 1.3
20.1 3.2
11.6 2.6
16.4 2.4
5.3 0.9
12.5 1.2
9.4 0.8
10.5 2.6
26.0 2.1
7.5 1.5
12.4 1.7
4.1 0.9
13.4 1.5
4.2 1.2
10.6 0.7
6.4 0.8
5.4 0.4
12.8 1.7
5.7 0.6
7.8 1.6
1.9 0.7
12.9 0.5
4.7 1.1
9.0 1.7
5.9 0.3
3.3 0.3
21.5 2.0
ND
ND
1.1 0.1
ND
ND
ND
ND
1.8 0.1
ND
41.3 2.3
ND
19.4 1.6
ND
48.4 3.9
ND
37.2 4.3
27.9 2.6
Exponentially growing cells were treated with the compounds at different concentrations for 72 h. Cell-growth inhibition was analyzed by the MTT (HepG2, HeLa, HT-29, PC-
3, and BJ) or XTT (Jurkat) assay. The assay was done using three replicates and repeated four times.
ND = not determined.
a
IC50 is the concentration of compound that inhibits 50% of cell growth.
Figure 2. Dose-dependent effect of triazole derivatives (A: 7, B: 18, C: 25, and D: 32) on cell viability of different cell lines (PC-3, HT-29). Data shown are mean (standard error
of at least three independent experiments).
of 2 were found to be generally more potent in vitro cytotoxic
agents than those of 1, with the exception of the triazole deriva-
tives 7 (Table 1 and Fig. 2 A) and 18 (Table 1 and Fig. 2 B). These
compounds displayed potent cytotoxic activity against prostate
carcinoma (PC-3) and colorectal adenocarcinoma (HT-29) human
cell lines. A brief and schematic representation of SAR for cytotox-
icity of compound 2 derivatives is presented in Figure 3.
to investigate the effect of our synthesized derivatives on topoiso-
merase I activity, using the topoisomerase I–DNA relaxation assay
and CPT as a positive control (Fig. 4).
Figure 4 and Table 3 show the catalytic inhibition of topoiso-
merase I by derivatives 3–7, and 15, 17–20, 23–27, 29, 31, and
32 at 100 lM, in comparison with 100 lM CPT (Fig. 4, lane 5 and
Table 3). In this preliminary study, compounds 23 (Fig. 4, lane 7
and Table 3) and 31 (Fig. 4, lane 9 and Table 3), with a carboxyl
group at C-28, showed better topoisomerase I inhibitory activity
than CPT, indicating that the free carboxylic group is important
for the inhibition of topoisomerase I catalytic activity. A relation-
ship between the topoisomerase I inhibitory effects and the cyto-
toxicity observed with the synthetic lupane-type triterpenoids
could not be clearly established in this study. Although compounds
2.2.2. Topoisomerase I inhibitory activity
DNA topoisomerase I is a critical cellular enzyme involved in tu-
mor growth, therefore, inhibition of topoisomerase constitutes a
useful strategy for the identification of potential antitumor agents.
Considering the promising activity of triterpenoids as inhibitors of
topoisomerase I,31 including 2 and its derivatives,32–34 we decided

R. C. Santos et al. / Bioorg. Med. Chem. 18 (2010) 4385–4396
4391
Figure 3. Graphical depiction of SAR for cell cytotoxicity based on the IC50 results for compound 2 derivatives.
25 and 32 displayed the best cytotoxicity profile, they were not the
Table 3
Catalytic inhibition of topoisomerase I by compound 1 (Group I) and 2 (Group II)
derivatives
most potent inhibitors of topoisomerase I, bearing instead an
inhibitory potential similar to CPT for this enzyme. This lack of cor-
relation between the topoisomerase I inhibitory effect and the
cytotoxicity of the different tested compounds suggested that
there could be multiple mechanisms responsible for the cytostatic
activity of these compounds, one of them being the inhibition of
the herein assayed enzyme.
Group
Compound
Topoisomerase I
inhibitiona (100
l
M)
I
3
4
5
0
1+
0
6
0
7
2+
2+
0
2+
0
15
17
18
19
20
23
24
25
26
27
29
31
32
CPT
3. Conclusions
In conclusion, the cytotoxic activity of semisynthetic lupane-
type carbamate and N-acylheterocyclic bearing derivatives has
been investigated and SAR has been established. The overall results
suggest that these new lupane-type derivatives can inhibit the
growth of various cancer cell lines at micromolar concentrations
and are promising new experimental anticancer agents. Moreover
the cytotoxicity of these compounds appears to be selective seeing
that non-tumoral BJ cells tolerated substantially higher doses of
these compounds than tumor cells. From the library of compounds
synthesized, 28-(1H-triazol-1-yl)-28-oxo-lup-20(29)-en-3b-yl-1H-
imidazole-1-carboxylate 25 and 2-hydroxy-28-(1H-triazol-1-yl)-
lup-1,20(29)-dien-3,28-dione 32 were the most potent towards
the tumor cells tested. Compounds 7, 15, 18, 23, 25, 31, and 32
showed topoisomerase I inhibitory activity comparable to or better
than CPT.
0
II
3+
1+
2+
0
0
0
3+
2+
2+
DNA relaxation assay was carried out as described in Section 4.
Topoisomerase inhibitory activity was scored as 3+ (strong), 2+ (moderate), 1+
(weak), and 0 (none), with the positive control drug CPT scoring 2+.
a
melting point B-540 apparatus and are uncorrected. IR spectra
were obtained using a JASCO FT/IR-420 spectrophotometer. NMR
spectra were recorder on a Bruker Digital NMR-Avance 300 appa-
ratus and on a Bruker Digital NMR-Avance 400 apparatus in CDCl3
with Me4Si as the internal standard. Elucidation of the chemical
structures was based on 1H, 13C, DEPT135, COSY, HMQC, and HMBC
NMR experiments. Chemical shifts values (d) are given in ppm and
the coupling constants (J) are presented in Hz. Mass spectra were
obtained using a Finnigan Polaris Q GC/MS Benchtop Ion Trap mass
spectrometer with a direct insertion probe.
Work is currently in progress in our laboratory in order to elu-
cidate the exact mechanism of the anticancer action of these lu-
pane-type derivatives and results will be reported in due course.
4. Experimental
4.1. Chemistry
4.1.1. General
Compounds 1 and 2, CBMI, CDT, AC2O, imidazole, DMAP, KOH,
m-CPBA, H2SO4, CH3I, K2CO3, DDQ, t-BuOK and tert-butyl alcohol
(t-BuOH) were purchased from Sigma–Aldrich Co. (St. Louis, Mis-
souri), whereas solvents were obtained from VWR (Portugal). For
thin layer chromatography (TLC) analysis Kieselgel 60HF254/Kiesel-
gel 60G was used and FCC was performed using Kieselgel 60 (230–
400 mesh, Merk). Melting points were determined using a BUCHI
4.1.2. 3b-Hydroxy-lup-20(29)-en-28-yl-20-methyl-1H-imidazole-
1-carboxylate (3) and lup-20(29)-en-3b,28-di-yl-(20-methyl-1H-
imidazole-1-carboxylate) (5)
To a solution of compound 1 (200 mg, 0.45 mmol) in anhydrous
THF (8 ml), CBMI (238 mg, 1.35 mmol) was added. After 9 h under
magnetic stirring at reflux temperature and N2 atmosphere, the
Figure 4. Inhibitory effects of the synthesized derivatives on the catalytic activity of topoisomerase I. Lane 1, relaxed DNA; lane 2, supercoiled plasmid DNA alone (250 ng);
lane 3, same as lane 2 + topoisomerase I (5 U); lane 4, same as lane 3 + 0.5% DMSO; lane 5, topoisomerase I (5 U) + 100 M CPT and DNA. Lanes 6–14, topoisomerase I
(5 U) + DNA and 100 M of the synthesized compounds: lane 6 (15); lane 7 (23); lane 8 (24); lane 9 (31); lane 10 (7); lane 11 (25), lane 12 (18); lane 13 (4); lane 14 (32).
l
l

4392
R. C. Santos et al. / Bioorg. Med. Chem. 18 (2010) 4385–4396
reaction was completed as verified by TLC control. The reaction
mixture was poured onto water (30 ml) and extracted with diethyl
ether (3 ꢀ 30 ml). The combined organic extract was then washed
with water (30 ml), and brine (30 ml), dried with anhydrous
Na2SO4, filtered and concentrated under reduced pressure to give
a yellowish solid. This solid was submitted to FCC with petroleum
ether 40–60 °C/ethyl acetate (4:1) and afforded compound 3
(143 mg, 57%): mp (benzene) 163–165 °C; IR (film) mmax 3313,
mixture was stirred at room temperature for 15 h. The solid was
filtered off and the filtrate was poured onto water (30 ml) and ex-
tracted with diethyl ether (3 ꢀ 30 ml). The organic phase was then
washed with water (30 ml), and brine (30 ml), dried with Na2SO4,
filtered, and evaporated to dryness. The crude product was purified
by FCC eluting with petroleum ether 40–60 °C/ethyl acetate (3:2)
to yield compound 7 (152 mg, 63%): mp (acetone/n-hexane) 221–
224 °C; IR (film) mmax 3406, 3070, 1764, 1642, 886 cmꢁ1
(CDCl3, 300 MHz) d 8.79 (s, 1H, H-50), 8.08 (s, 1H, H-30), 4.79 (dd,
J = 9.1, 7.5 Hz, 1H, H-3 ), 4.69 (br s, 1H, H-29a), 4.59 (br s, 1H, H-
;
1H NMR
3073, 1758, 1642, 884 cmꢁ1 1H NMR (CDCl3, 300 MHz) d 7.38 (d,
;
J = 1.7 Hz, 1H, H-50), 6.91 (d, J = 1.7 Hz, 1H, H-40), 4.72 (br s, 1H,
H-29a), 4.62–4.59 (m, 2H, H-29b and H-28a), 4.17 (d, J = 10.9 Hz,
1H, H-28b), 3.19 (dd, J = 10.8 Hz, J = 5.2 Hz, 1H, H-3a), 2.71 (s, 3H,
a
29b), 3.80 (d, J = 10.8 Hz, 1H, H-28a), 3.34 (d, J = 10.8 Hz, 1H, H-
28b), 2.40 (dt, J = 10.5 Hz, J = 5.8 Hz, 1H, H-19), 1.69 (s, 3H, H-30),
1.04 (s, 3H), 0.99 (s, 6H), 0.98 (s, 3H), 0.90 (s, 3H); 13C NMR (CDCl3,
75 MHz) d 153.4 (C30), 150.4 (C20), 147.2 (OCO), 145.3 (C50), 109.7
(C29), 87.8 (C3), 60.4 (C28); EI-MS m/z (% rel. intensity): 537 (6)
M+, 119 (49), 107 (46), 105 (60), 93 (55), 91 (100), 81 (46), 79
(53), 77 (51), 70 (38).
CH3-20), 2.47 (m, 1H, H-19), 1.70 (s, 3H, H-30), 1.06 (s, 3H), 1.01
(s, 3H), 0.97 (s, 3H), 0.83 (s, 3H), 0.77 (s, 3H); 13C NMR (CDCl3,
75 MHz) d 149.6 (C20), 149.5 (OCO), 147.9 (C20), 126.5 (C40),
118.1 (C50), 110.2 (C29), 78.8 (C3), 67.3 (C28); EI-MS m/z (% rel.
intensity): 550 (10) M+, 189 (26), 187 (26), 133 (25), 119 (37),
107 (28), 91 (30), 83 (100), 81 (27), 79 (26). And compound 5
(112 mg, 38%): mp (acetone/n-hexane) 127–129 °C; IR (film) mmax
4.1.5. 3b-Hydroxylup-20(29)-en-28-yl acetate (8)
3070, 1753, 1645, 880 cmꢁ1
1H, H-50), 7.34 (s, 1H, H-500), 6.86 (s, 1H, H-40), 6.85 (s, 1H, H-400),
4.73 (s, 1H, H-29a), 4.67–4.58 (s, 3H, H-3 , H-28a, H-29b), 4.15
;
1H NMR (CDCl3, 300 MHz) d 7.36 (s,
Details of the synthesis of this compound were reported previ-
ously.39 Compound 8 (323 mg, 84%): IR (film) mmax 3471, 3070,
a
1734, 1642, 1244, 1102, 880 cmꢁ1 1H NMR (CDCl3, 300 MHz) d
;
(d, J = 10.8 Hz, 1H, H-28b), 2.66 (s, 3H, CH3-20), 2.65 (s, 3H, CH3-
200), 2.49 (m, 1H, H-19), 1.71 (s, 3H, H-30), 1.08 (s, 3H), 1.02 (s,
3H), 0.96 (s, 3H), 0.95 (s, 3H), 0.90 (s, 3H); 13C NMR (CDCl3,
75 MHz) d 150.0 (C20), 149.6 (OCO), 149.5 (OCO), 147.9 (C20 and
C200), 127.9 and 127.8 (C40, C400), 118.0 (C50 and C500), 110.3 (C29),
85.9 (C3), 66.7 (C28); EI-MS m/z (% rel. intensity): 658 (2) M+,
127 (13), 119 (12), 105 (10), 95 (19); 93 (13), 91 (16), 83 (100),
81 (19), 79 (11).
4.69 (s, 1H, H-29a), 4.59 (s, 1H, H-29b), 4.24 (d, J = 10.9 Hz, 1H, H-
28a), 3.86 (d, J = 10.9 Hz, 1H, H-28b), 3.18 (dd, J = 11.0 Hz,
J = 4.7 Hz, 1H, H-3a), 2.45 (dt, J = 10.9 Hz, J = 5.8 Hz, 1H, H-19),
2.07 (s, 3H, OCOCH3), 1.68 (s, 3H, H-30), 1.03 (s, 3H), 0.97 (s, 3H),
0.96 (s, 3H), 0.82 (s, 3H), 0.76 (s, 3H); 13C NMR (CDCl3, 75 MHz) d
171.6 (OCOCH3), 150.2 (C20), 109.9 (C29), 78.9 (C3), 62.8 (C28);
EI-MS m/z (% rel. intensity): 484 (12) M+, 203 (54), 189 (100),
187 (71), 147 (46), 145 (49), 133 (67), 119 (61), 105 (69), 91 (56).
4.1.3. 3b-Hydroxy-lup-20(29)-en-28-yl-1H-triazole-1-carboxylate
(4) and lup-20(29)-en-3b,28-di-yl-(1H-triazole-1-carboxylate (6)
The method followed that described for compound 3 but using
compound 1 (200 mg, 0.45 mmol) and CDT (295 mg, 1.8 mmol) in
anhydrous THF (8 ml) at reflux for 8 h. The resulting yellowish so-
lid was purified by FCC using petroleum ether 40–60 °C/ethyl ace-
tate (3:2) to afford compound 4 (175 mg, 72%): mp (acetone/n-
hexane) 200–203 °C; IR (film) mmax 3389, 3070, 1791, 1762, 1642,
4.1.6. 28-Acetoxy-lup-20(29)-en-3b-yl-20-methyl-1H-imidazole-
1-carboxylate (9)
The method followed that described for compound 3 but using
compound 8 (242 mg, 0.5 mmol) and CBMI (176 mg, 1 mmol) in
anhydrous THF (10 ml) at reflux for 9 h. The resulting white solid
was purified by FCC using petroleum ether 40–60 °C/ethyl acetate
(4:1) to afford compound 9 (243 mg, 82%): mp (acetone/n-hexane)
173–175 °C; IR (film) mmax 3070, 1740, 1642, 882 cmꢁ1
(CDCl3, 400 MHz)
J = 1.5 Hz, 1H, H-40), 4.69–4.64 (m, 2H, H-3
;
1H NMR
7.36 (d, J = 1.5 Hz, 1H, H-50), 6.90 (d,
and H-29a), 4.60 (br
882 cmꢁ1
;
1H NMR (CDCl3, 300 MHz) d 8.83 (br s, 1H, H-50), 8.09
d
(br s, 1H, H-30), 4.74–4.72 (m, 2H, H-29a and H-28a), 4.63 (br s,
a
1H, H-29b), 4.30 (d, J = 10.8 Hz, 1H, H-28b), 3.19 (dd, J = 10.8 Hz,
J = 5.2 Hz, 1H, H-3a), 2.49 (dt, J = 10.7 Hz, J = 6.0 Hz, 1H, H-19),
s, 1H, H-29b), 4.26 (d, J = 11.0 Hz, 1H, H-28a), 3.85 (d, J = 11.0 Hz,
1H, H-28b), 2.69 (s, 3H, CH3-20), 2.45 (dt, J = 10.7 Hz, J = 5.7 Hz,
1H, H-19), 2.08 (s, 3H, OCOCH3), 1.69 (s, 3H, H-30), 1.05 (s, 3H),
0.99 (s, 3H), 0.96 (s, 3H), 0.95 (s, 3H), 0.89 (s, 3H); 13C NMR (CDCl3,
100 MHz) d 171.6 (OCOCH3), 150.0 (C20), 149.1 (OCO), 147.9 (C20),
126.8 (C40), 118.1 (C50), 109.9 (C29), 86.4 (C3), 62.7 (C28); EI-MS m/
z (% rel. intensity): 592 (6) M+, 467 (100), 407 (58), 107 (45), 105
(56), 95 (70), 91 (52), 83 (40), 81 (61), 67 (53).
1.70 (s, 3H, H-30), 1.06 (s, 3H), 1.01 (s, 3H), 0.97 (s, 3H), 0.84 (s,
13
3H), 0.77 (s, 3H); C NMR (CDCl3, 75 MHz) d 153.6 (C30), 149.5
(C20), 147.9 (OCO), 145.4 (C50), 110.3 (C29), 78.9 (C3), 68.4
(C28); EI-MS m/z (% rel. intensity): 537 (4) M+, 190 (74), 189
(100), 187 (89), 133 (72), 119 (98), 107 (75), 105 (82), 91 (93),
79 (89). And compound 6 (68 mg, 24%): mp (acetone/n-hexane)
157–159 °C; IR (film) mmax 3070, 1787, 1763, 1642, 882 cmꢁ1 1H
;
NMR (CDCl3, 400 MHz) d 8.83 (br s, 1H, H-50), 8.80 (br s, 1H, H-
4.1.7. 28-Acetoxy-lup-20(29)-en-3b-yl-1H-triazole-1-carboxylate
(10)
500) 8.09 (br s, 1H, H-30), 8.08 (br s, 1H, H-300), 4.82–4.73 (m, 3H,
H-3
a
, H-28a, H-29a), 4.64 (br s, 1H, H-29b), 4.30 (d, J = 10.8 Hz,
The method followed that described for compound 3 but using
compound 8 (242 mg, 0.5 mmol) and CDT (246 mg, 1.5 mmol) in
anhydrous THF (10 ml) at reflux for 8 h. The resulting white solid
was purified by FCC using petroleum ether 40–60 °C/ethyl acetate
(4:1) to afford compound 10 (256 mg, 88%): mp (acetone/n-hex-
1H, H-28b), 2.50 (m, 1H, H-19), 1.72 (s, 3H, H-30), 1.09 (s, 3H),
1.02 (s, 3H), 1.00 (s, 3H), 0.98 (s, 3H), 0.92 (s, 3H); 13C NMR (CDCl3,
100 MHz) d 153.6 and 153.5 (C30, C300), 149.4 (C20), 147.9 (OCO),
147.2 (OCO), 145.4 (C50), 145.3 (C500), 110.3 (C29), 87.7 (C3), 69.3
(C28); EI-MS m/z (% rel. intensity): 632 (4) M+, 189 (86), 187
(62), 133 (67), 119 (79), 107 (60), 105 (83), 95 (67), 93 (74), 91
(100).
ane) 221–224 °C; IR (film) mmax 3070, 1787, 1766, 1733 cmꢁ1 1H
;
NMR (CDCl3, 400 MHz) d 8.79 (s, 1H, H-50), 8.08 (s, 1H, H-30),
4.79 (dd, J = 9.0 Hz, J = 7.5 Hz, 1H, H-3 ), 4.69 (br s, 1H, H-29a),
a
4.60 (br s, 1H, H-29b), 4.26 (d, J = 10.9 Hz, 1H, H-28a), 3.85 (d,
J = 10.9 Hz, 1H, H-28b), 2.45 (dt, J = 10.9 Hz, J = 5.8 Hz, 1H, H-19),
2.07 (s, 3H, OCOCH3), 1.69 (s, 3H, H-30), 1.05 (s, 3H), 0.99 (s, 3H),
0.98 (s, 3H), 0.97 (s, 3H), 0.90 (s, 3H); 13C NMR (CDCl3, 100 MHz)
d 171.6 (OCOCH3), 153.5 (C30), 150.1 (C20), 147.3 (OCO), 145.3
(C50), 109.9 (C29), 87.9 (C3), 62.8 (C28); EI-MS m/z (% rel. inten-
4.1.4. 28-Hydroxy-lup-20(29)-en-3b-yl-1H-triazole-1-carboxylate
(7)
A solution of compound 1 (200 mg, 0.45 mmol) and CDT
(443 mg, 2.70 mmol) in anhydrous THF (8 ml) was refluxed for
10 h to afford compound 6. Silica gel (200 mg) was added and this

R. C. Santos et al. / Bioorg. Med. Chem. 18 (2010) 4385–4396
4393
sity): 579 (4) M+, 203 (51), 189 (84), 187 (69), 159 (47), 119 (56),
4.1.12. 3b-Hydroxy-(20R)-lupan-29-oxo-28-yl-20-methyl-1H-
107 (57), 105 (75), 91 (100), 79 (54).
imidazole-1-carboxylate (15)
The method followed that described for compounds 3 but
using compound 14 (194 mg, 0.42 mmol) and CBMI (148 mg,
0.84 mmol) in anhydrous THF (8 ml), at reflux for 6 h. The result-
ing white solid was purified by FCC eluting with petroleum ether
40–60 °C/ethyl acetate (3:2) to afford compound 15 (179 mg,
75%): mp (acetone/n-hexane) 132–134 °C; IR (film) mmax 3365,
4.1.8. 28-Hydroxylup-20(29)-en-3b-yl acetate (11)
Details of the synthesis of this compound were reported previ-
ously.39 Compound 11 (259 mg, 89%): IR (film) mmax 3440, 3070,
1729, 1642, 1246, 978, 882 cmꢁ1 1H NMR (CDCl3, 300 MHz) d
;
4.68 (s, 1H, H-29a), 4.58 (s, 1H, H-29b), 4.47 (dd, J = 10.3 Hz,
J = 5.7 Hz, 1H, H-3 ), 3.79 (d, J = 10.6 Hz, 1H, H-28a), 3.33 (d,
a
1759, 1716 cmꢁ1 1H NMR (CDCl3, 400 MHz) d 9.85 (d, J = 1.8 Hz,
;
J = 10.7 Hz, 1H, H-28b), 2.39 (dt, J = 10.7 Hz, J = 5.9 Hz, 1H, H-19),
2.04 (s, 3H, OCOCH3), 1.69 (s, 3H, H-30), 1.02 (s, 3H), 0.97 (s, 3H),
0.85 (s, 6H), 0.84 (s, 3H); 13C NMR (CDCl3, 75 MHz) d 171.1
(OCOCH3), 150.5 (C20), 109.7 (C29), 80.9 (C3), 60.5 (C28); EI-MS
m/z (% rel. intensity): 484 (9) M+, 203 (71), 189 (100), 187 (55),
107 (61), 105 (52), 95 (77), 91 (67), 81 (60), 79 (85).
1H, H-29), 7.35 (d, J = 1.7 Hz, 1H, H-50), 6.87 (d, J = 1.7 Hz, 1H,
H-40), 4.55 (d, J = 10.9 Hz, 1H, H-28a), 4.09 (d, J = 10.9 Hz, 1H,
H-28b), 3.21 (dd, J = 10.4 Hz, J = 4.6 Hz, 1H, H-3a), 2.66 (s, 3H,
CH3-20), 1.16 (d, J = 7.0 Hz, 3H, H-30), 1.07 (s, 3H), 0.98 (s, 6H),
0.85 (s, 3H), 0.77 (s, 3H); 13C NMR (CDCl3, 100 MHz) d 206.3
(C29), 149.2 (OCO), 144.3 (C20), 130.1 (C40), 118.1 (C50), 78.9
(C3), 66.0 (C28); EI-MS m/z (% rel. intensity): 566 (8) M+, 189
(67), 161 (72), 147 (72), 133 (83), 105 (80), 91 (93), 83 (68), 81
(100), 79 (62).
4.1.9. 3b-Acetoxy-lup-20(29)-en-28-yl-20-methyl-1H-imidazole-
1-carboxylate (12)
The method followed that described for compound 3 but using
compound 11 (242 mg, 0.5 mmol) and CBMI (176 mg, 1 mmol) in
anhydrous THF (10 ml) at reflux for 7 h. The resulting white solid
was purified by FCC using petroleum ether 40–60 °C/ethyl acetate
(4:2) to afford compound 12 (253 mg, 86%): mp (acetone/n-hex-
ane) 99–102 °C; IR (film) mmax 3073, 1757, 1731, 1642, 1245,
4.1.13. 30-Methoxylup-20-(29)-en-3b,28-diol (16)
Details of the synthesis of these compounds were reported pre-
viously.41 Compound 16 (1.4 g, 64%): IR (film) mmax 3347, 3073,
1645 cmꢁ1 1H NMR (CDCl3, 300 MHz) d 4.92 (s, 1H, H-29a), 4.91
;
(s, 1H, H-29b), 3.86 (br s, 2H, H-30), 3.78 (d, J = 10.5 Hz, 1H, H-
28a), 3.35 (s, 3H, OCH3), 3.31 (d, J = 10.5 Hz, 1H, H-28b), 3.18 (dd,
J = 10.8 Hz, J = 5.2 Hz, 1H, H-3a), 2.28 (dt, J = 10.8 Hz, J = 5.4 Hz,
1H, H-19) 1.02 (s, 3H), 0.98 (s, 3H), 0.97 (s, 3H), 0.82 (s, 3H), 0.76
(s, 3H); 13C NMR (CDCl3, 75 MHz) d 150.9 (C20), 109.0 (C29), 78.9
(C3), 74.8 (C30), 60.2 (C28), 58.3 (OCH3); EI-MS m/z (% rel. inten-
sity): 473 (25) M+, 201 (93), 189 (86), 187 (100), 145 (75), 131
(66), 121 (71), 119 (73), 95 (66), 81 (69).
882 cmꢁ1 1H NMR (CDCl3, 300 MHz) d 7.38 (d, J = 1.8 Hz, 1H, H-
;
50), 6.90 (d, J = 1.8 Hz, 1H, H-40), 4.72 (br s, 1H, H-29a), 4.62–4.58
(m, 2H, H-28a and H-29b), 4.47 (dd, J = 10.2 Hz, J = 5.8 Hz, 1H, H-
3a
), 4.16 (d, J = 10.8 Hz, 1H, H-28b), 2.70 (s, 3H, CH3-20), 2.48 (dt,
J = 10.7 Hz, J = 5.7 Hz, 1H, H-19), 2.05 (s, 3H, OCOCH3), 1.71 (s,
3H, H-30), 1.06 (s, 3H), 0.99 (s, 3H), 0.86 (s, 3H), 0.85 (s, 3H),
0.84 (s, 3H); 13C NMR (CDCl3, 75 MHz) d 170.9 (OCOCH3), 149.7
(C20), 149.5 (OCO), 147.9 (C20), 127.1 (C40), 118.1 (C50), 110.2
(C29), 80.8 (C3), 67.0 (C28); EI-MS m/z (% rel. intensity): 592 (14)
M+, 189 (20), 187 (16), 145 (15), 119 (22), 107 (19), 105 (25), 91
(28), 83 (100), 79 (16).
4.1.14. 3b-Hydroxy-30-methoxylup-20(29)-en-28-yl-20-methyl-
1H-imidazole-1-carboxylate (17) and 30-methoxylup-20(29)-
en-3b,28-di-yl-(20-methyl-1H-imidazole-1-carboxylate) (19)
The method followed that described for compound 3 but using
compound 16 (213 mg, 0.45 mmol) and CBMI (238 mg, 1.35 mmol)
in anhydrous THF (8 ml), at reflux for 7 h. The resulting white solid
was purified by FCC eluting with petroleum ether 40–60 °C/ethyl
acetate (2:1) to afford compound 17 (193 mg, 74%): mp (acetone/
4.1.10. 3b-Acetoxy-lup-20(29)-en-28-yl-1H-triazole-1-carboxylate
(13)
The method followed that described for compound 3 but using
compound 11 (242 mg, 0.5 mmol) and CDT (246 mg, 1.5 mmol) in
anhydrous THF (10 ml) at reflux for 6 h. The resulting white solid
was purified by FCC using petroleum ether 40–60 °C/ethyl acetate
(4:2) to afford compound 13 (213 mg, 74%): mp (acetone/n-hex-
ane) 111–114 °C; IR (film) mmax 3070, 1795, 1770, 1729, 1642,
n-hexane) 109–112 °C; IR (film) mmax 3389, 3070, 1759, 1645 cmꢁ1
;
1H NMR (CDCl3, 300 MHz) d 7.37 (d, J = 1.7 Hz, 1H, H-50), 6.88 (d,
J = 1.7 Hz, 1H, H-40), 4.96 (br s, 1H, H-29a), 4.94 (br s, 1H, H-29b),
4.58 (d, J = 10.4 Hz, 1H, H-28a), 4.14 (d, J = 10.4 Hz, 1H, H-28b),
3.88 (s, 2H, H-30), 3.36 (s, 3H, OCH3), 3.19 (dd, J = 10.8 Hz,
1247, 882 cmꢁ1 1H NMR (CDCl3, 300 MHz) d 8.82 (s, 1H, H-50),
;
8.09 (s, 1H, H-30), 4.75–4.72 (m, 2H, H-28a and H-29a), 4.63 (br s,
1H, H-29b), 4.47 (dd, J = 10.2 Hz, J = 5.8 Hz, 1H, H-3 ), 4.30 (d,
J = 5.1 Hz, 1H, H-3a
), 2.68 (s, 3H, CH3-20), 2.38 (dt, J = 11.1 Hz,
a
J = 5.3 Hz, 1H, H-19), 1.06 (s, 3H), 1.01 (s, 3H), 0.97 (s, 3H), 0.83
(s, 3H), 0.76 (s, 3H); 13C NMR (CDCl3, 75 MHz) d 150.4 (C20),
149.8 (OCO), 147.9 (C20), 127.4 (C40), 118.0 (C50), 109.6 (C29),
78.8 (C3), 74.9 (C30), 66.7 (C28), 58.3 (OCH3); EI-MS m/z (% rel.
intensity): 580 (9) M+, 189 (25), 187 (28), 119 (27), 107 (23), 105
(25), 91 (24), 83 (100), 81 (27), 79 (25). And compound 19
(65 mg, 21%): mp (acetone/n-hexane) 116–118 °C; IR (film) mmax
J = 10.7 Hz, 1H, H-28b), 2.49 (dt, J = 10.6 Hz, J = 6.0 Hz, 1H, H-19),
2.05 (s, 3H, OCOCH3), 1.71 (s, 3H, H-30), 1.06 (s, 3H), 1.00 (s, 3H),
0.86 (s, 3H), 0.85 (s, 3H), 0.84 (s, 3H); 13C NMR (CDCl3, 75 MHz) d
171.0 (OCOCH3), 153.6 (C30), 149.5 (C20), 147.9 (OCO), 145.4
(C50), 110.3 (C29), 80.8 (C3), 68.4 (C28); EI-MS m/z (% rel. inten-
sity): 579 (6) M+, 202 (58), 189 (88), 187 (78), 145 (59), 119 (78),
107 (62), 105 (77), 91 (100), 79 (58).
3070, 1754, 1642 cmꢁ1 1H NMR (CDCl3, 400 MHz) d 7.36 (br s,
;
1H, H-50), 7.34 (br s, 1H, H-500), 6.87 (br s, 1H, H-40), 6.86 (s, 1H,
4.1.11. 3b,28-Dihydroxy-(20R)-lupan-29-al (14)
H-400), 4.98 (s, 1H, H-29a), 4.95 (s, 1H, H-29b), 4.65 (dd,
Details of the synthesis of these compounds were reported pre-
J = 11.1 Hz, J = 4.8 Hz, 1H, H-3a), 4.58 (d, J = 10.8 Hz, 1H, H-28a),
viously.40,51 Compound 14 (354 mg, 39%): IR (film) mmax 3393,
4.14 (d, J = 10.8 Hz, 1H, H-28b), 3.89 (s, 2H, H-30), 3.37 (s, 3H,
OCH3), 2.66 (s, 3H, CH3-20), 2.65 (s, 3H, CH3-200), 2.39 (dt,
J = 11.3 Hz, J = 5.5 Hz, 1H, H-19), 1.08 (s, 3H), 1.03 (s, 3H), 0.95 (s,
6H), 0.90 (s, 3H); 13C NMR (CDCl3, 100 MHz) d 150.4 (C20), 149.9
(OCO), 149.5 (OCO), 147.9 (C20 and C200), 127.9 (400), 127.7 (C40),
118.0 (C50 and C500), 109.8 (C29), 85.9 (C3), 75.1 (C30), 66.5
(C28), 58.4 (OCH3); EI-MS m/z (% rel. intensity): 688 (11) M+, 187
(23), 185 (22), 145 (32), 119 (22), 105 (26), 95 (26), 91 (46), 83
(100), 81 (27).
1714 cmꢁ1 1H NMR (CDCl3, 300 MHz) d 9.86 (d, J = 2.0 Hz, 1H, H-
;
29), 3.77 (d, J = 10.8 Hz, 1H, H-28a), 3.26 (d, J = 10.8 Hz, 1H, H-
28b), 3.20 (dd, J = 10.9 Hz, J = 5.1 Hz, 1H, H-3 ) 2.60 (m, 1H, H-
a
20), 1.10 (d, J = 6.9 Hz, 3H, H-30), 1.03 (s, 3H), 0.98 (s, 3H), 0.95
(s, 3H), 0.84 (s, 3H), 0.77 (s, 3H); 13C NMR (CDCl3, 75 MHz) d
206.8 (CHO), 78.9 (C3), 60.2 (C28); EI-MS m/z (% rel. intensity):
458 (2) M+, 369 (100), 207 (43), 192 (51), 189 (72), 161 (67), 133
(31), 121 (31), 107 (36), 95 (33).

4394
R. C. Santos et al. / Bioorg. Med. Chem. 18 (2010) 4385–4396
4.1.15. 3b-Hydroxy-30-methoxylup-20(29)-en-28-yl-1H-triazole-
1-carboxylate (18) and 30-methoxylup-20(29)-en-3b,28-di-yl-(1H-
triazole-1-carboxylate) (20)
50), 6.88 (d, J = 1.6 Hz, 1H, H-40), 4.75 (br s, 1H, H-29a), 4.68–4.61
(m, 2H, H-3 and H-29b), 3.05 (dt, J = 10.7 Hz, J = 4.3 Hz, 1H, H-
a
19), 2.67 (s, 3H, CH3-20), 1.70 (s, 3H, H-30), 0.99 (s, 3H), 0.96 (s,
3H), 0.95 (s, 3H), 0.94 (s, 3H), 0.89 (s, 3H); 13C NMR (CDCl3,
75 MHz) d 180.6 (C28), 150.6 (C20), 149.3 (OCO), 147.9 (C20),
127.3 (C40), 118.0 (C50), 109.6 (C29), 86.2 (C3); EI-MS m/z (% rel.
intensity): 564 (10) M+, 439 (100), 393 (61), 203 (58), 123 (50),
121 (50), 109 (57), 95 (74), 83 (66), 81 (97). And compound 24
(90 mg, 22%): mp (acetone/n-hexane) 164–166 °C; IR (film) mmax
The method followed that described for compound 3 but using
compound 16 (213 mg, 0.45 mmol) and CDT (295 mg, 1.8 mmol) in
anhydrous THF (8 ml), at reflux for 7 h. The resulting white solid
was purified by FCC eluting with petroleum ether 40–60 °C/ethyl
acetate (1:1) to afford compound 18 (147 mg, 57%): mp (acetone/
n-hexane) 137–140 °C; IR (film) mmax 3414, 3070, 1782, 1766,
1645 cmꢁ1
;
1H NMR (CDCl3, 300 MHz) d 8.82 (br s, 1H, H-50),
3070, 1752, 1722, 1642 cmꢁ1
(br s, 1H, H-50), 7.34 (br s, 1H, H-500), 6.86 (br s, 2H, H-40 and
H400), 4.79 (br s, 1H, H-29a), 4.66–4.62 (m, 1H, H-3
, H-29b), 3.06
;
1H NMR (CDCl3, 400 MHz) d 7.39
8.09 (br s, 1H, H-30), 4.97 (br s, 1H, H-29a), 4.94 (br s, 1H, H-29b),
4.72 (d, J = 10.6 Hz, 1H, H-28a), 4.29 (d, J = 10.6 Hz, 1H, H-28b),
3.88 (s, 2H, H-30), 3.36 (s, 3H, OCH3), 3.19 (dd, J = 10.8 Hz,
a
(dt, J = 11.0 Hz, J = 4.3 Hz, 1H, H-19), 2.65 (s, 3H, CH3-20), 2.63 (s,
3H, CH3-200), 1.73 (s, 3H, H-30), 1.01 (s, 3H), 0.96 (s, 3H), 0.94 (s,
6H), 0.89 (s, 3H); 13C NMR (CDCl3, 100 MHz) d 175.2 (C28), 150.0
(C20), 149.5 (OCO), 148.9 and 147.9 (C20, C200), 127.7 and 127.1
(C40, C400), 118.0 (C50 and C500), 110.1 (C29), 85.9 (C3); EI-MS m/z
(% rel. intensity): 628 (2) M+, 519 (18), 127 (30), 119 (17), 105
(20), 95 (22), 93 (18), 91 (19), 83 (100), 81 (24).
J = 5.0 Hz, H-3a), 2.39 (dt, J = 11.2 Hz, J = 5.3 Hz, 1H, H-19), 1.06
(s, 3H), 1.01 (s, 3H), 0.97 (s, 3H), 0.83 (s, 3H), 0.76 (s, 3H); 13C
NMR (CDCl3, 75 MHz) d 153.6 (C30), 150.3 (C20), 147.9 (OCO),
145.4 (C50), 109.7 (C29), 78.9 (C3), 74.9 (C30), 68.1 (C28), 58.3
(OCH3); EI-MS m/z (% rel. intensity): 567 (13) M+, 201 (71), 189
(78), 187 (70), 145 (72), 131 (61), 119 (71), 105 (82), 91 (100),
79 (57). And compound 20 (52 mg, 17%): mp (acetone/n-hexane)
172–175 °C; IR (film) mmax 3070, 1782, 1763, 1646 cmꢁ1
;
1H NMR
4.1.19. 28-(1H-Triazol-1-yl)-28-oxo-lup-20(29)-en-3b-yl-1H-
triazole-1-carboxylate (25)
(CDCl3, 400 MHz) d 8.83 and 8.79 (both s, each 1H, H-50, H-500),
8.09 and 8.08 (both s, each 1H, H-30, H-300), 4.98 (br s, 1H, H-29a),
The method followed that described for compound 3 but using
compound 2 (297 mg, 0.65 mmol) and CDT (640 mg, 3.9 mmol) in
anhydrous THF (12 ml) at reflux for 7 h. The resulting yellowish
solid was purified by FCC using petroleum ether 40–60 °C/ethyl
acetate (3:2) to afford compound 25 (322 mg, 82%): mp (acetone/
n-hexane) 253–256 °C; IR (film) mmax 3070, 1787, 1762, 1734,
4.96 (br s, 1H, H-29b), 4.82–4.71 (m, 2H, H-3a and H-28a), 4.29
(d, J = 10.8 Hz, 1H, H-28b), 3.89 (s, 2H, H-30), 3.37 (s, 3H, OCH3),
2.40 (dt, J = 11.1 Hz, J = 5.4 Hz, 1H, H-19), 1.09 (s, 3H), 1.03 (s,
3H), 0.99 (s, 3H), 0.98 (s, 3H), 0.91 (s, 3H); 13C NMR (CDCl3,
100 MHz) d 153.6 and 153.5 (C30, C300), 150.3 (C20), 147.9 (OCO),
147.2 (OCO), 145.4 and 145.3 (C50, C500), 109.8 (C29), 87.7 (C3),
74.9 (C30), 68.1 (C28), 58.3 (OCH3); EI-MS m/z (% rel. intensity):
663 (15) M+, 201 (70), 119 (81), 107 (66), 105 (80), 95 (75), 91
(100), 81 (94), 79 (82), 67 (74).
1642 cmꢁ1 1H NMR (CDCl3, 300 MHz) d 8.93 (s, 1H, H-50), 8.79
;
(s, 1H, H-500), 8.07 (s, 1H, H-300), 8.00 (s, 1H, H-30), 4.81–4.78 (m,
2H, H-3 and H-29a), 4.66 (br s, 1H, H-29b), 2.96 (m, 1H, H-19),
a
1.73 (s, 3H, H-30), 1.02 (s, 3H), 0.99 (s, 3H), 0.97 (s, 3H), 0.95 (s,
3H), 0.92 (s, 3H); 13C NMR (CDCl3, 75 MHz) d 173.4 (C28), 153.6
(C300), 152.1 (C30), 149.9 (C20), 147.3 (OCO), 145.3 (C500), 145.1
(C50), 110.1 (C29), 87.8 (C3); EI-MS m/z (% rel. intensity): 602
(10) M+, 202 (70), 190 (86), 189 (93), 188 (100), 187 (65), 173
(72), 105 (62), 91 (85), 70 (84).
4.1.16. Methyl 3b-hydroxylup-20(29)-en-28-oate (21)
Details of the synthesis of these compounds were reported pre-
viously.38 Compound 21 (129 mg, 83%): IR (film) mmax 3320, 3070,
1720, 1643 cmꢁ1 1H NMR (CDCl3, 300 MHz) d 4.71 (br s, 1H, H-
;
29a), 4.58 (br s, 1H, H-29b), 3.67 (s, 3H, COOCH3), 3.18 (dd, 1H,
J = 10.9 Hz, J = 4.5 Hz, H-3 ) 2.43 (m, 1H, H-19), 1.69 (s, 3H, H-
a
4.1.20. Methyl 3b-(20-methyl-1H-imidazole-1-carbonyloxy)-lup-
20(29)-en-28-oate (26)
30), 0.96 (s, 3H), 0.94 (3H), 0.92 (s, 3H), 0.82 (s, 3H), 0.75 (s, 3H);
13C NMR (CDCl3, 75 MHz) d 177.2 (C28), 149.7 (C20), 110.1 (C29),
80.6 (C3); EI-MS m/z (% rel. intensity): 470 (25) M+, 286 (26), 253
(52), 247 (29), 203 (36), 192 (100), 189 (100), 175 (64), 119 (47),
105 (51).
The method followed that described for compound 3 but using
compound 21 (100 mg, 0.2 mmol) and CBMI (70 mg, 0.4 mmol) in
anhydrous THF (4 ml), at reflux for 9 h. The crude product was
purified by FCC eluting with petroleum ether 40–60 °C/ethyl ace-
tate (3:2) to yield compound 26 (115 mg, 93%): mp (acetone/n-
4.1.17. 3-Oxolup-20(29)-en-28-oic acid (22)
hexane) 205–207 °C; IR (film) mmax 3073, 1752, 1728, 1642 cmꢁ1
;
Details of the synthesis of this compound were reported previ-
1H NMR (CDCl3, 300 MHz) d 7.34 (br s, 1H, H-50), 6.85 (br s, 1H,
H-40), 4.74 (s, 1H, H-29a), 4.65 (dd, J = 11.2 Hz, J = 5.1 Hz, 1H, H-
ously.52 Compound 22 (685 mg, 67%): IR (film) mmax 3070, 1703,
1686, 1642 cmꢁ1
;
1H NMR (CDCl3, 300 MHz) d 4.77 (br s, 1H, H-
3a), 4.61 (s, 1H, H-29b), 3.67 (s, 3H, COOCH3), 3.00 (dt,
29a), 4.64 (br s, 1H, H-29b), 3.04 (dt, J = 10.7 Hz, J = 4.3 Hz, 1H, H-
19), 1.72 (s, 3H, H-30), 1.09 (s, 3H), 1.04 (s, 3H), 1.02 (s, 3H), 0.99
(s, 3H), 0.95 (s, 3H); 13C NMR (CDCl3, 75 MHz) d 218.2 (C3), 182.2
(C28), 150.3 (C20), 109.7 (C29); EI-MS m/z (% rel. intensity): 454
(19) M+, 408 (24), 393 (20), 248 (85), 189 (100), 175 (62), 133
(55), 119 (76), 105 (69), 79 (52).
J = 10.4 Hz, J = 3.8 Hz, 1H, H-19), 2.65 (s, 3H, CH3-20), 1.69 (s, 3H,
H-30), 0.98 (s, 3H), 0.95 (s, 3H), 0.94 (s, 3H), 0.93 (s, 3H), 0.88 (s,
3H); 13C NMR (CDCl3, 75 MHz) d 176.6 (C28), 150.5 (C20), 149.5
(OCO), 147.9 (C20), 127.7 (C40), 118.0 (C50), 109.7 (C29), 86.0 (C3);
EI-MS m/z (% rel. intensity): 578 (13) M+, 393 (100), 189 (68),
119 (56), 105 (64), 95 (71), 91 (75), 83 (87), 81 (70), 79 (52).
4.1.18. 3b-(20-Methyl-1H-imidazole-1-carbonyloxy)-lup-20(29)-
en-28-oic acid (23) and 28-(20-methyl-1H-imidazol-1-yl)-28-oxo-
lup-20(29)-en-3b-yl-20-methyl-1H-imidazole-1-carboxylate (24)
The method followed that described for compound 3 but using
compound 2 (297 mg, 0.65 mmol) and CBMI (573 mg, 3.25 mmol)
in anhydrous THF (12 ml) at reflux for 8 h. The resulting yellowish
solid was purified by FCC using petroleum ether 40–60 °C/ethyl
acetate (3:2) to afford compound 23 (283 mg, 77%): mp (acetone/
n-hexane) 170–174 °C; IR (film) mmax 3070, 1756, 1703, 1642,
4.1.21. 28-(20-Methyl-1H-imidazol-1-yl)-lup-20(29)-en-3,28-
dione (27)
The method followed that described for compound 3 but using
compound 22 (205 mg, 0.45 mmol) and CBMI (238 mg, 1.35 mmol)
in anhydrous THF (8 ml), at reflux for 8 h. The crude product was
purified by FCC eluting with petroleum ether 40–60 °C/ethyl ace-
tate (3:2) to yield compound 27 (197 mg, 84%): mp (benzene)
196–198 °C; IR (film) mmax 3073, 1721, 1703, 1642 cmꢁ1
(CDCl3, 400 MHz)
J = 1.5 Hz, 1H, H-40), 4.78 (br s, 1H, H-29a), 4.65 (br s, 1H, H-29b),
;
1H NMR
d
7.40 (d, J = 1.5 Hz, 1H, H-50), 6.86 (d,
883 cmꢁ1 1H NMR (CDCl3, 300 MHz) d 7.35 (d, J = 1.6 Hz, 1H, H-
;

R. C. Santos et al. / Bioorg. Med. Chem. 18 (2010) 4385–4396
4395
3.06 (dt, J = 11.1 Hz, J = 4.6 Hz, 1H, H-19), 2.63 (s, 3H, CH3-20), 1.72
4.1.26. 2-Hydroxy-28-(1H-triazol-1-yl)-lup-1,20(29)-dien-3,28-
dione (32)
(s, 3H, H-30), 1.06 (s, 3H), 1.02 (s, 3H), 1.00 (s, 3H), 0.98 (s, 3H), 0.94
(s, 3H); 13C NMR (CDCl3, 100 MHz) d 218.0 (C3), 175.2 (C28), 149.9
(C20), 148.9 (C20), 126.9 (C40), 117.9 (C50), 110.0 (C29); EI-MS m/z
(% rel. intensity): 518 (2) M+, 409 (100), 245 (54), 203 (50), 189
(76), 119 (50), 105 (58), 91 (71), 81 (72), 79 (49).
The method followed that described for compound 3 but using
compound 30 (300 mg, 0.70 mmol) and CDT (500 mg, 3.0 mmol) in
anhydrous THF (12 ml), at reflux for 10 h. The resulting white solid
was purified by FCC eluting with petroleum ether 40–60 °C/ethyl
acetate (1:4) to afford compound 32 (260 mg, 77%): mp (acetone/
n-hexane) 149–153 °C; IR (film) mmax 1644, 1667, 1723, 1762,
4.1.22. 3-Oxolup-1,20(29)-dien-28-oic acid (28)
3073, 3434 cmꢁ1 1H NMR (CDCl3, 400 MHz) d 8.93 (s, 1H, 30-H),
;
Details of the synthesis of these compounds were reported pre-
7.99 (s, 1H, 50-H), 6.43 (s, 1H, 1-H), 4.78 (s, 1H, H-29b), 4.67 (s,
1H, H-29a), 2.97 (dt, J = 10,4 Hz e J = 4,0 Hz, 1H, H-19), 1.71 (s,
3H, H-30), 0.99 (3H), 1.05 (6H), 1.14 (3H), 1.19 (3H); 13C NMR
(CDCl3, 100 MHz) d 201.1 (C3), 172.7 (C28), 152.1 (C50), 149.5
(C20), 145.3 (C30), 143.9 (C2), 128.7 (C1), 110.3 (C29); EI-MS m/z
(% rel. intensity): 519 (23) M+, 424 (100), 216 (89), 212 (62), 184
(59), 118 (55), 104 (43), 90 (85), 79 (43), 69 (39).
viously.38 Compound 28 (179 mg, 45%); IR (film)mmax 3070, 1730,
; d 7.11 (d,
1689, 1645 cmꢁ1 1H NMR (CDCl3, 400 MHz)
J = 10.3 Hz, 1H, H-1), 5.80 (d, J = 10.3 Hz, 1H, H-2), 4.76 (s, 1H, H-
29a), 4.63 (s, 1H, H-29b), 3.03 (m, 1H, H-19), 1.70 (s, 3H, H-30),
1.13 (s, 3H), 1.07 (s, 3H), 1.06 (s, 3H), 1.02 (s, 3H), 0.99 (s, 3H);
13C NMR (CDCl3, 100 MHz) d 205.9 (C3), 181.7 (C28), 160.1 (C1),
150.2 (C20), 125.1 (C2), 109.9 (C29); EI-MS m/z (% rel. intensity):
452 (17) M+, 213 (100), 150 (39), 137 (34), 95 (31), 91 (42), 81
(36), 79 (41), 77 (29), 67 (34).
4.2. Antiproliferative activity
4.2.1. General
4.1.23. 28-(20-Methyl-1H-imidazol-1-yl)-lup-1,20(29)-dien-3,28-
dione (29)
All cell lines were purchased from the American Type Culture
Collection (ATCC, Rockville, MD). RPMI 1640 medium, Dulbecco’s
Modified Eagle Medium (DMEM-D5796), Dulbecco’s Phosphate Buf-
fered Saline (DPBS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-
razolium bromide (MTT) powder and dimethylsulfoxide (DMSO)
were obtained from Sigma–Aldrich Co. (St. Louis, Missouri). Penicil-
The method followed that described for compounds 3 but using
compound 28 (204 mg, 0.45 mmol) and CBMI (238 mg, 1.35 mmol)
in anhydrous THF (8 ml), at reflux for 9 h. The crude product was
purified by FCC eluting with petroleum ether 40–60 °C/ethyl ace-
tate (3:1) to yield compound 29 (202 mg, 87%): mp (acetone/n-
hexane) 99–103 °C; IR (film) mmax 3073, 1760, 1721, 1668,
lin/Streptomycin (P/S) and L-glutamine were obtained from Gibco-
BRL (Grand Island, New York). Sodium pyruvate and trypsin/EDTA
(0.05%/0.02%) were obtained from Biological Industries (Kibbutz
Beit Haemek, Israel). Fetal Bovine Serum (FBS) were purchased from
PAA Laboratories (Pasching, Austria) and XTT kit was obtained from
Roche Applied Science (Mannheim, Germany). Topoisomerase I drug
screening kit was obtained from TopoGen, Inc. (Port Orange, FL).
HT-29, HeLa, PC-3 and BJ cells were routinely maintained in
DMEM supplemented with 10% heat-inactivated FBS and 1% P/S
1642 cmꢁ1 1H NMR (CDCl3, 400 MHz) d 7.42 (br s, 1H, H-50),
;
7.12 (d, J = 10.3 Hz, 1H, H-1), 6.90 (br s, 1H, H-40), 5.80 (d,
J = 10.3 Hz, 1H, H-2), 4.80 (s, 1H, H-29a), 4.67 (s, 1H, H-29b) 3.06
(dt, J = 10.8 Hz, J = 4.3 Hz, 1H, H-19), 2.67 (s, 3H, CH3-20), 1.73 (s,
3H, H-30), 1.12 (s, 3H), 1.07 (s, 6H), 1.01 (s, 6H); 13C NMR (CDCl3,
100 MHz) d 205.4 (C3), 175.0 (C28), 159.6 (C1), 149.6 (C20),
148.9 (C20), 126.4 (C40), 125.1 (C2), 118.0 (C50), 110.1 (C29); EI-
MS m/z (% rel. intensity): 516 (3) M+, 408 (37), 407 (100), 243
(56), 205 (37), 189 (42), 135 (43), 105 (37), 91 (44), 81 (36).
(10,000 units/10,000 lg/ml) solution. HepG2 cells were main-
tained in DMEM supplemented with 10% heat-inactivated FBS, 1%
P/S and 1 mM of sodium pyruvate. Jurkat cells were grown in RPMI
1640 medium supplemented with 10% heat-inactivated FBS, 1% P/S
4.1.24. 2-Hydroxy-3-oxolup-1,20(29)-dien-28-oic acid (30)
Details of the synthesis of these compounds were reported pre-
viously.43 Compound 30 (273 mg, 73%): mmax 3389, 3073, 1730,
and 2 mM -glutamine. All cell cultures were incubated at 37 °C in
L
an atmosphere of 5% CO2.
1698, 1669, 1645 cmꢁ1 1H NMR (CDCl3, 300 MHz) d 6.45 (s, 1H,
;
H-1), 4.75 (s, 1H, H-29a), 4.64 (s, 1H, H-29b), 3.02 (m, 1H, H-19),
1.70 (s, 3H, H-30), 1.20 (s, 3H), 1.13 (s, 3H), 1.10 (s, 3H), 1.00 (s,
3H), 0.98 (s, 3H); 13C NMR (CDCl3, 75 MHz) d 201.2 (C3), 182.4
(C28), 150.1 (C20), 143.9 (C2), 128.9 (C1), 109.9 (C29); EI-MS m/z
(% rel. intensity): 469 (11) M+, 321 (43), 213 (100), 189 (32), 150
(45), 136 (29), 91 (63), 80 (34), 75 (54), 69 (65).
4.2.2. Cell viability assay
The cell viability of HeLa, HepG2, HT-29, PC-3, and BJ cells in the
presence of the synthesized compounds was determined by the
MTT assay. Briefly, exponentially growing cells were plated in
96-well plates at a density of 1–8 ꢀ 103 cells/well, for 24 h before
treatment. The growth medium was replaced with one containing
either the test compounds dissolved in DMSO (final DMSO concen-
tration <0.1%) at different concentrations or only DMSO, in tripli-
cate wells, and cells were incubated for 72 h. After incubation
with the compounds, the medium was removed, and cell viability
was assessed by the MTT colorimetric assay. MTT solution (0.5 mg/
4.1.25. 2-(20-Methyl-1H-imidazole-1-carbonyloxy)-3-oxolup-
1,20(29)-dien-28-oic acid (31)
The method followed that described for compound 3 but using
compound 30 (210 mg, 0.45 mmol) and CBMI (396 mg, 2.25 mmol)
in anhydrous THF (8 ml), at reflux for 7 h. The resulting white solid
was purified by FCC eluting with petroleum ether 40–60 °C/ethyl
acetate (2:3) to afford compound 31 (215 mg, 83%): mp (acetone/
n-hexane) 141–143 °C; IR (film) mmax 3394, 3070, 1824, 1770,
ml, 100
again at 37 °C for 1 h. MTT solution was removed, formazan crys-
tals were dissolved in DMSO (100 l) and the absorbance was
ll) was added to each well and the plates were incubated
l
immediately read at 550 nm on an ELISA plate reader (Tecan Sun-
rise MR20-301, TECAN, Austria).
1687, 1645 cmꢁ1 1H NMR (CDCl3, 400 MHz) d 7.41 (d, J = 1.7 Hz,
;
1H, H-50), 6.99 (s, 1H, H-1), 6.92 (d, J = 1.7 Hz, 1H, H-40), 4.75 (s,
1H, H-29a), 4.62 (s, 1H, H-29b), 3.04 (dt, J = 10.9 Hz, J = 4.0 Hz, 1H,
H-19), 2.66 (s, 3H, CH3-20), 1.69 (s, 3H, H-30), 1.21 (s, 3H), 1.16
(s, 6H), 1.05 (s, 3H), 1.01 (s, 3H); 13C NMR (CDCl3, 100 MHz) d
196.9 (C3), 180.7 (C28), 150.2 (C20), 148.6 (OCO), 147.4 (C2),
145.7 (C20), 142.0 (C1), 127.6 (C40), 118.4 (C50), 109.8 (C29); EI-
MS m/z (% rel. intensity): 576 (3) M+, 215 (100), 213 (62), 107
(35), 105 (46), 93 (35), 91 (62), 81 (34), 79 (40), 67 (34).
For Jurkat cells, the cell viability was determined by the XTT as-
say. Briefly, exponentially growing cells were plated in 96-well
plates at a density of 5.5 ꢀ 103 cells/well, treated with different
concentrations of compounds or vehicle (medium with DMSO) in
triplicate and incubated for 72 h. After incubation the XTT labeling
mixture (100 ll) was added to each of the wells and after a 4 h
incubation period at 37 °C the absorbance was read at 450 nm on
an ELISA plate reader.

4396
R. C. Santos et al. / Bioorg. Med. Chem. 18 (2010) 4385–4396
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R.C.S. thanks Fundação para a Ciência e a Tecnologia for support-
ing this work (SFRH/BD/23770/2005). J.A.R.S. thanks Universidade
de Coimbra for financial support. M.C. thanks Spanish Government:
Ministerio de Ciencia e Innovación (SAF2008-00164); from Red
Temática de Investigación Cooperativa en Cáncer, Instituto de Salud
Carlos III (ISCIII-RTICC, RD06/0020/0046 and RD06/0020/1037) and
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.04.085.
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