Synthesis, cytotoxicity and antiplasmodial activity of novel ent-kaurane derivatives

Article abstract of DOI:10.1016/j.ejmech.2012.12.010

This paper reports on the syntheses and spectrometric characterisation of eleven novel ent-kaurane diterpenoids, including a complete set of ¹H, ¹³C NMR and crystallographic data for two novel ent-kaurane diepoxides. Moreover, the antineoplastic cytotoxicity for kaurenoic acid and the majority of ent-kaurane derivatives were assessed in vitro against a panel of fourteen cancer cell lines, of which allylic alcohols were shown to be the most active compounds. The good in vitro antimalarial activity and the higher selectivity index values observed for some ent-kaurane epoxides against the chloroquine-resistant W2 clone of Plasmodium falciparum indicate that this class of natural products may provide new hits for the development of antimalarial drugs.

Full text of DOI:10.1016/j.ejmech.2012.12.010

European Journal of Medicinal Chemistry 62 (2013) 168e176
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, cytotoxicity and antiplasmodial activity of novel
ent-kaurane derivatives
,
Ronan Batista a b, Pablo A. García c, María Angeles Castro c, José M. Miguel del Corral c,
,
Nivaldo L. Speziali d, Fernando de P. Varotti e f, Renata C. de Paula e,
Luis F. García-Fernández g, Andrés Francesch g, Arturo San Feliciano c,
,
Alaíde B. de Oliveira e
*
a Departamento de Estudos Básicos e Instrumentais, Universidade Estadual do Sudoeste da Bahia, BR 415, Km 03, s/nꢀ, 45.700-000 Itapetinga,
Bahia, Brazil
b Departamento de Química Orgânica, Instituto de Química, Universidade Federal da Bahia, Rua Barão de Geremoabo s/n, Ondina,
41950-350 Salvador, Bahia, Brazil
c Departamento de Química Farmacéutica, Facultad de Farmacia e CIETUS, Universidad de Salamanca, Campus Unamuno, 37007 Salamanca, Spain
d Departamento de Física, ICEx, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6.627, 31.270-901 Belo Horizonte, MG, Brazil
e Departamento de Produtos Farmacêuticos, Faculdade de Farmácia, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6.627,
31.270-901 Belo Horizonte, MG, Brazil
f Universidade Federal de São João Del-Rei, Campus Centro-Oeste, Centro de Ciências da Saúde, R. Sebastião Gonçalves Coelho, 400,
35501-296 Divinópolis, MG, Brazil
g PharmaMar SA, P. I. La Mina, 28770 Colmenar Viejo, Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
This paper reports on the syntheses and spectrometric characterisation of eleven novel ent-kaurane
diterpenoids, including a complete set of 1H, 13C NMR and crystallographic data for two novel ent-
kaurane diepoxides. Moreover, the antineoplastic cytotoxicity for kaurenoic acid and the majority of ent-
kaurane derivatives were assessed in vitro against a panel of fourteen cancer cell lines, of which allylic
alcohols were shown to be the most active compounds. The good in vitro antimalarial activity and the
higher selectivity index values observed for some ent-kaurane epoxides against the chloroquine-resistant
W2 clone of Plasmodium falciparum indicate that this class of natural products may provide new hits for
the development of antimalarial drugs.
Received 6 August 2012
Received in revised form
6 December 2012
Accepted 7 December 2012
Available online 20 December 2012
Keywords:
Wedelia paludosa D.C.
ent-Kaurane derivatives
In vitro antimalarial activity
Plasmodium falciparum
Antineoplastic activity
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
high risk for malaria. This at-risk population is represented mostly
by people living in the Brazilian Amazonia region. Among the four
Malaria is among the leading causes of death from a single
infectious agent in the developing world. An estimated 3.3 billion
people were at risk of malaria in 2010, of which 2.2 billion were at
low risk. The 1.2 billion at high risk were living in the WHO African
(47%) and South East Asian Regions (37%). Malaria episodes and
deaths in 2010 were estimated to be 216 million and 655,000,
respectively. The cases and deaths were mostly in the WHO African
region. Globally, malaria deaths rates were still high, approximately
86%, among children. In Brazil, 2% of the population (4,480,000) is at
etiological agents causing malaria in humans, Plasmodium falcipa-
rum is the most virulent and is responsible for nearly one million
deaths per year. However, in Brazil, Plasmodium vivax is the major
species [1]. As the burden of this disease is worsening mainly
because of the increasing resistance of P. falciparum to the widely
available antimalarial drugs [2], together with the lack of highly
effective vaccines and inadequate control of mosquito vectors,
malaria continues to be a global challenge. Thus, there is an urgent
need for new, more affordable and accessible antimalarial agents
with original modes of action [3,4]. Within this context, new anti-
malarial lead compounds may emerge from tropical plant sources, as
natural products have been playing a dominant role in the discovery
of leads in the development of drugs to treat human diseases [3,5].
* Corresponding author. Tel.: þ55 31 3409 6972; fax: þ55 31 3441 5575.
E-mail addresses: alaide.braga@pq.cnpq.br, alaidebraga@terra.com.br
(A.B. de Oliveira).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.12.010

R. Batista et al. / European Journal of Medicinal Chemistry 62 (2013) 168e176
169
Kaurane diterpenes have been identified from numerous
medicinal plants that have been used for the treatment of inflam-
mation, cancer, zoonosis and infection, as well as other disorders
and ailments [6e10]. Kaurenoic, iso-kaurenoic and grandiflorenic
acids [ent-kaur-16-en-19-oic (1), ent-kaur-15-en-19-oic (2) and ent-
kaur-9:11,16-dien-19-oic (3) acids, respectively] have been abun-
dantly found in Wedelia paludosa D.C. (Asteraceae) [8,9,11]. Kaur-
enoic (1) and grandiflorenic (3) acids have been shown to be
trypanocides [8,12], cytotoxic [10,13] and potent stimulators of
uterine contraction [6], whereas iso-kaurenoic acid (2) has been
found to display potent antifungal activity against the soil-borne
plant pathogenic fungi Pythium ultimum and Rhizoctonia solani [11].
Given the wide array of biological activities already described
for ent-kaurane diterpenes [7,14], along with the promising anti-
malarial activities observed for three zoapatlin diterpene lactones
structurally related to a rearranged kaurane skeleton [15,16], it was
assumed that kaurenoic acid (1) and its related ent-kaurane diter-
penoids and derivatives might also possess promising antimalarial
properties, even though the literature only reports a weak anti-
plasmodial activity for kaurenoic acid via the parasite lactate
dehydrogenase assay (pLDH) [17].
Thisworkdescribes the invitro antiplasmodial activity of kaurenoic
(1) and grandiflorenic (3) acids, both isolated from W. paludosa D.C.
Furthermore, hydroxyl and other oxygen substituents in the skeletons
of kaurane diterpenoids may improve their biological effects [7,18]. In
addition, the presence of epoxide groups is known to increase the
capacity of molecules to inhibit proteases by covalent reactions [19].
Accordingly, this paper also reports the synthesis and characterisation
of a number of oxidised/epoxidised ent-kaurane derivatives obtained
from the naturally occurring ent-kaurenes of W. paludosa D.C. and
describes the results of in vitro evaluation of the antineoplastic and
antimalarial activities for most of these compounds.
C21H32O4 by HRFABMS, but with slightly different specific rotation
values [8, ꢁ9.5ꢀ (CHCl3, c ¼ 0.9); 9, ꢁ7.2ꢀ (CHCl3, c ¼ 0.5)] and very
distinct melting points (8, 168e170 ꢀC; 9, 96e99 ꢀC). The structure
of both compounds was determined by means of bidimensional
spectroscopic experiments (COSY, ROESY, HMBC and HMQC). The
correlations observed allowed the complete assignment of the NMR
data (Table 1) and indicated that both derivatives possess the same
kaurane skeleton and the same atomic connectivities, the difference
being due to the relative configuration of their epoxide groups at C-
9/C-11 and/or C-16/C-17 positions. Fortunately, compounds 8 and 9
were obtained as colourless crystals after recrystallisation in hexane
and their structures were confirmed unequivocally by X-Ray crys-
tallography. The x-ray diffraction analysis of a single crystal of 8 and
9 (Fig.1) proved the ent-
11 for both compounds, whereas the epoxide group at C-16/C-17
was oriented at the ent- configuration on compound 8 and at the
ent- configuration on compound 9. The three epoxides (7, 8 and 9)
b orientation of the epoxide group at C-9/C-
b
a
were the results of the total epoxidation of the two major compo-
nents 4 and 6 of the starting mixture.
When grandiflorenic acid (3) alone was treated with MCPBA,
only the exocyclic double bond was epoxidised, as deduced from
the 1H and 13C NMR data of the reaction product. Column chro-
matography over silica gel afforded the two novel epoxides 10a and
10b in a 1:10 ratio. Comparison of their NMR data with that of 8 and
9 allowed the assignment of the ent-16
a configuration for the
minor isomer 10a and ent-16 for 10b, which is in agreement with
b
those expected from the stereoselective epoxidation of the C-16/C-
17 double bond taking place at the less hindered face (exo side).
To obtain new derivatives with hydroxyl groups at different
positions of the kaurenoic skeleton, a mixture of 4, 5 and 6 was
submitted to different oxidation reactions, such as hydroboration-
oxidation [23], allylic oxidation [24,25] by SeO2 and photo-
oxidation with Bengal Rose [26]. The first approach was to employ
9-borabicyclo[3.3.1]nonane (9-BBN) as a hydroboration agent to
favour the synthesis of less hindered ent-kaurane alcohols, taking
into account that the selectivity obtained with 9-BBN is generally
higher than that previously realised with diborane or disiamylborane
[27]. Unfortunately, no reactionwas observed when 9-BBN was used.
When the mixture of esters was treated with diborane generated in
situ, the alcohols 11e15 were obtained, which were separated by
column chromatography (CC). After 1 h of the hydroboration reac-
tion, alcohols 11, 12 and 13a/b were obtained, and partial hydroxyl-
ation of ester 6 was observed. However, when the reaction time was
extended to 2 h, complete hydroboration of all the double bonds was
observed, and the isolated products were 11, 12, 14 and 15.
Alcohol 11 came from the hydroboration of ester 5. The absolute
configuration of C-15 and C-16 for compound 11 was deduced from
their spectroscopic data and confirmed unequivocally by X-Ray
crystallography [18].
2. Results and discussion
2.1. Chemistry
The naturally occurring diterpenes kaurenoic (1), iso-kaurenoic
(2) and grandiflorenic (3) acids, obtained from W. paludosa [8,9,11],
were used as the starting materials for the chemical trans-
formations presented in this paper. The three acids are difficult to
separate by standard chromatographic techniques, although small
amounts of 1 and 3 can be isolated [8,11]. The transformations
performed in this work included the syntheses of 11 novel ent-
kaurane derivatives (8, 9, 10a, 10b, 13a, 13b, 14, 15, 21, 22 and 23)
and four known compounds (7, 11, 12 and 24) whose structures are
depicted in Scheme 1. Physical and spectrometric data for alcohol
11 are described here for the first time, although its crystallographic
data have been published before by our group [18]. The syntheses
and complete spectrometric characterisation of substances 7, 12, 16,
17, 18, 19 and 20 are available in the literature [12,20,21].
The first aim of the study was to synthesise ent-kaurane epox-
ides starting from the naturally occurring kaurenes. A mixture of
kaurenoic (1), iso-kaurenoic (2) and grandiflorenic (3) acids, ob-
tained previously from W. paludosa D.C. in a 14:8:17 ratio [11], were
esterified with an ethereal solution of diazomethane to afford
a mixture of esters 4, 5 and 6 in the same ratio. Then, the mixture of
esters was epoxidised [22], affording the known expected methyl
Alcohol 12 was identical to the compound obtained by hydro-
boration [12] of 4, and it was transformed into the more oxidised
derivatives 17e20 as previously described [20]. Also the new
derivative 21 was obtained from the reaction of aldehyde 17 with
propylenediamine.
Derivatives 13, 14 and 15 came from ester 6. The 1H and 13C NMR
data for alcohol 13 indicated that the double bond at C-9/C-11 was
present in the molecule and it was a 3:1 mixture of the two isomers
that could not be separated by chromatographic techniques.
However, the comparison of the C-16/C-17 NMR data of 13a and
13b with the C-16/C-17 NMR data available for related ent-kaurane
ent-kauran-16
with literature data [20,21], and two novel ent-kaurane diepoxides,
methyl ent-kauran-9 :11,16 :17-diepoxy-19-oate (8) and methyl
ent-kauran-9 :11,16 :17-diepoxy-19-oate (9).
b,17-epoxy-19-oate (7), identified by comparison
b
b
alcohols [28] allowed us to attribute the ent-16a configuration for
b
a
13a and ent-16 for 13b.
b
Diepoxides 8 and 9, obtained as colourless crystals, were shown
to be isomers via presentation of very similar IR, 13C and 1H NMR
spectra, in addition to presenting the same molecular formula
Compounds 14 and 15 were also shown to be isomers via the
presentation of the same molecular formula C21H34O4 but very
distinct melting points (14, 159e162 ꢀC; 15, 88e91 ꢀC). No olefinic

170
R. Batista et al. / European Journal of Medicinal Chemistry 62 (2013) 168e176
12
20
14
17
1
9
3
5
15
7
+
+
COOH
COOH
( 14 : 8 : 17 )
2
1
3
COOH
18
19
a
(100%)
+
+
CO2CH3
4
CO2CH3
5
6
CO2CH3
( 14 : 8 : 17 )
d
b
NO REACTION
c
(78%)
(17%)
(8%)
O
O
O
O
O
O
O
b
+
COOH
COOH
3
COOH
10-a
10-b
CO2CH3
CO2CH3
CO2CH3
9
7
8
(4%)
(36%)
(72 / 77%)
(86 / 80%)
(10%)
(15%)
(36%)
HO
HO
e
OH
OH
OH
OH
H
(43 / 100%)
H
H
H
H
OH
CO2CH3
12
CO2CH3
CO2CH3
13 a/b
(3:1)
15
11
14
CO2CH3
CO2CH3
e
(1:1)
(50%)
(17%)
(3%)
(12%)
COOH
H
CHO
H
H
OH
O
f
CHO
(70%)
17
16
CO2CH3 19
CO2CH3
CO2CH3 20
CO2CH3
18
CO2CH3
g
(77%)
OH
HN
OH
h
OH
+
+
N
H
OH
H
CO2CH3
4
CO2CH3
22
CO2CH3 23 (36%)
CO2CH3 24
(11%)
(10%)
21
CO2CH3
i
(70%)
Scheme 1. Synthesis of the ent-kaurane diterpenoids 4e24. Reagents and conditions: (a) CH2N2, Et2O, 4 h; (b) MCPBA, NaHCO3, CH2Cl2, 30 min.; (c) NaBH4, BF3.Et2O, THF, 1 or 2 h;
NaOH, H2O2, 50 ꢀC, 1 or 2vh; (d) 9-BBN, THF, 3 h; H2O2, NaOH, 50 ꢀC, 1 h; (e) Ref. [20]; (f) HCl, AcOH, 80 ꢀC, 48 h; (g) H2N(CH2)3NH2, CH2Cl2, MgSO4, 6 h; (h) Bengal Rose, i-PrOH, h
16 days; NaBH4, MeOH; and (i) SeO2, t-BuOOH, CH2Cl2, 45 min.
n,
carbon/hydrogen signals were found on their 13C/1H NMR spectra,
indicating the hydroxylation of both double bonds. The deshielded
bond of 3. Ent-16
derivatives on the basis of comparison of 13C NMR data with liter-
ature data [28]. NOE experiments confirmed the ent-11 -hydroxy
group in 14 and the ent-11 -hydroxy group in 15.
a configuration was assigned to both ent-kaurane
C-9 signals observed for 14 (
d
59.4) and 15 (
d
65.4) confirmed the
b
expected anti-Markovnikov hydroxylation on the C-9/C-11 double
a

R. Batista et al. / European Journal of Medicinal Chemistry 62 (2013) 168e176
171
Table 1
Complete assignments of 1H (400 MHz) and 13C (100 MHz) NMR data for the ent-
kaurane diepoxides 8 and 9 (CDCl3).
Position
8
9
dC, type
dH (J in Hz)
dC, type
dH (J in Hz)
1
2
3
39.5, CH2
1.46, m
39.5, CH2
1.91, m
1.25, m
1.87, m
1.49, m
2.21, d (12.0)
1.01, dt (4.2; 13.2)
1.36, dd (4.2; 12.0)
1.93, dd (4.4; 10.6)
1.71, d (4.1)
2.23, d (13.4)
1.03, dt (4.2; 13.4)
19.0, CH2
37.9, CH2
19.1, CH2
37.8, CH2
4
5
6
44.3, C
48.7, CH
19.8, CH2
44.2, C
48.7, CH
19.8, CH2
1.76, dd (4.4; 12.2)
1.41, d (5.2)
1.46, m
1.77, dd (3.9; 12.3)
1.49, m
1.43, m
7
38.9, CH2
1.56, m
39.2, CH2
1.91, m
1.91, dd (2.4; 11.5)
1.49, m
8
43.4, C
42.3, C
9
69.9, C
69.4, C
10
11
12
38.1, C
53.7, CH
29.1, CH2
38.1, C
54.2, CH
26.8, CH2
3.07, dd (0.9; 4.0)
2.04, dd (6.0; 15.4)
1.66, m
1.46, m
1.95, dd (4.4; 10.6)
1.65, m
3.10, dd (1.0; 4.0)
2.00, m
1.84, dd (5.5; 15.1)
1.69, bt (9.0)
1.95, m
13
14
38.9, CH
31.8, CH2
36.3, CH
32.1, CH2
1.88, m
15
45.6, CH2
2.50, dd (2.4; 14.5)
1.75, d (14.7)
46.2, CH2
2.25, dd (3.5; 14.4)
1.95, dd (4.1; 14.4)
16
17
69.0, C
50.6, CH2
66.1, C
54.8, CH2
2.84, d (4.5)
2.79, d (4.5)
1.21, s
2.83, d (5.3)
2.78, d (5.3)
1.19, s
18
19
20
1’
28.6, CH3
177.8, C
15.0, CH3
51.3, CH3
28.6, CH3
177.9, C
14.8, CH3
51.2, CH3
0.68, s
3.64, s
0.66, s
3.63, s
Additionally, allylic oxidation of methyl ester 4 by SeO2 afforded
22, while compounds 22, 23 and 24 were obtained from the photo-
oxidation of this ester using Bengal Rose as a photosensitising agent.
The configuration of 22e24 was deduced by comparison of their
NMR data with those of similar ent-kaurane derivatives [24,29e33].
Fig. 1. Molecular plot of the kaurane diepoxides 8 and 9 presenting the relative
configuration and the labelling scheme of the non-H atoms (displacement ellipsoids at
50% probability level).
As for the cytotoxicities of aldehydes 16 and 17, the ent-16
configuration on 17 appears to favour a slightly higher activity
against K-562 (GI50 ¼ 8.18 M) and LoVo (GI50 ¼ 8.69 M) cells than
the ent-16 configuration (16).
Finally, it is noteworthy to emphasise that the most potent
cytotoxicities depicted in Table 2 were displayed by the allylic
derivatives 22 and 23, which were active against IGROV (22,
a
2.2. Biological evaluation
m
m
2.2.1. Antineoplastic cytotoxicity
b
Most of the compounds prepared had their antineoplastic
cytotoxicity evaluated in vitro against a panel of 14 human tumour
cell lines representing diverse organs and tissues, including pros-
tate, ovary, breast, melanoma, lung, leukaemia, pancreas, colon and
cervical cells. In general, the compound derivatives presented low
cytotoxicity, and only those cell lines in which at least one diter-
penoid was active are depicted in Table 2 (IGROV, IGROV-ET, K-562,
PANC-1, HT-29, LoVo, LoVo-DOX and HELA cell lines). Against the
other cell lines tested (DU-145, SK-BR-3, SK-MEL-28, A-549, HT-29-
KF and HELA-APL), none of the ent-kaurene diterpenoids 1e24
demonstrated cytotoxicity at the maximum concentration tested.
These cell lines are not shown in Table 2.
It is notable that ent-kaurenoic acid (1) and most of the evaluated
ent-kaurane derivatives were inactive against all cell lines assayed
displaying GI50 values above 8.56
found in our assays, it is interesting to observe that compound 4 was
slightly more cytotoxic against IGROV cells (GI50 ¼ 8.44 M) when
compared to that of 1 (GI50 > 9.92 M) and this effect might be
Table 2
Cytotoxicity data (GI50, mM) for some ent-kaurane compounds.
Compound IGROV IGROV-ET K-562 PANC-1 HT-29 LoVo LoVo-DOX HELA
1
4
5
7
8
9
10b
11
12
13a/13b
14
15
16
17
>9.92 >9.92
8.44 >9.48
>9.92 >9.92 >9.92 >9.92 >9.92
>9.48 >9.48 >9.48 >9.48 >9.48
>9.48 >9.48 >9.48 >9.48 >9.48
>9.02 >9.02 >9.02 >9.02 >9.02
>8.66 >8.66 >8.66 >8.66 >8.66
>8.66 >8.66 >8.66 >8.66 >8.66
>9.48 >9.48 >9.48 >9.48 >9.48
>9.02 >9.02 >9.02 >9.02 >9.02
>9.02 >9.02 >9.02 >9.02 >9.02
>9.02 >9.02 >9.02 >9.02 >9.02
>8.56 >8.56 >8.56 >8.56 >8.56
>8.56 >8.56 >8.56 >8.56 >8.56
>9.02 >9.02 >9.02 >9.02 >9.02
8.18 >9.02 >9.02 8.69 >9.02
>9.92
>9.48
>9.48
>9.02
>8.66
>8.66
>9.48
>9.02
>9.02
>9.02
>8.56
>8.56
>9.02
>9.02
7.04
>9.48 >9.48
>9.02 >9.02
>8.66 >8.66
>8.66 >8.66
>9.48 >9.48
8.34 >9.02
mM. In spite of the low cytotoxicity
>9.02
7.23
>9.02 >9.02
>8.56 >8.56
>8.56 >8.56
>9.02 >9.02
>9.02 >9.02
7.52 >9.02
m
m
related to the methyl ester function on 4. A hydroxyl group, either at
C-15 or C-17 positions, also seems to increase cytotoxicity against
IGROV or IGROV-ET cell lines, but this augmentation is not observed
when C-16 or C-11 are hydroxylated or when there is a double bond
between C-9/C-11, as can be concluded by comparing the cytotox-
icities of 11, 12, 22 and 23 with those of 13a/13b, 14, 15 and 24.
22
>9.02 >9.02
7.40 8.21 >9.02
23
8.90 >9.02
>8.56 >8.56
>9.02 8.45 >9.02 0.71 4.28
>8.56 >8.56 >8.56 >8.56 >8.56
>9.02
>8.56
0.17
24
DOX HCla
0.11
6.17
0.17 0.24
0.15 0.24 2.21
a
Doxorubicin hydrochloride.

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R. Batista et al. / European Journal of Medicinal Chemistry 62 (2013) 168e176
GI50 ¼ 7.52
m
M; 23, GI50 ¼ 8.90
M), LoVo (22, GI50 ¼ 8.21
M), LoVo-DOX (23, GI50 ¼ 4.28 M) and HELA (22,
M) cell lines. These results might be interpreted in two
m
M), PANC-1 (23, GI50 ¼ 8.45
m
M),
was also observed when trypanocidal activity was evaluated for
these diterpenes against Trypanosoma cruzi, the causative agent of
Chagas’ disease (American Trypanosomiasis) [12]. In addition,
when the antiplasmodial activities of epoxides 7, 8 and 9 are
compared, one can conclude that an epoxide group at C-16/C17
HT-29 (22, GI50 ¼ 7.40
m
m
M; 23,
GI50 ¼ 0.71
GI50 ¼ 7.04
m
m
m
ways: the cytotoxicity of such allylic ent-kaurane alcohols could be
related to alkylation of bionucleophiles, such as DNA, proteins, by
derived phosphates/pyrophosphates or by Michael’s addition
decreases the activity when it is oriented at the ent-
(7 and 8, IC50 ¼ 9.6 ꢂ 1.8 and 10.4 ꢂ 2.0 M, respectively). This is
opposite to that observed for 9 (IC50 ¼ 6.9 ꢂ 2.0 M), which bears
an epoxide group at the ent- configuration. Finally, the perhy-
b configuration
m
reactions involving donors (protein, DNA, etc.) and
carbonyl moieties derived from oxidation of the allylic alcohol of
these ent-kaurane derivatives in neoplastic cells.
As mentioned before, the GI50 values found in our assays indi-
cated that our compounds are non-cytotoxic, however, these values
could be in same range of some compounds reported in the liter-
ature, such as some naturally occurring ent-kaurane diterpenes,
including kaurenoic (1) and grandiflorenic (3) acids, that have been
shown to exhibit moderate cytotoxicity against several lines of
cultured human neoplastic cells, with a majority of their GI50 values
a
,b
-unsaturated
m
a
dropyrimidine derivative 21 was the most potent antiplasmodial
compound obtained in this work, and its potent activity suggests
that the two secondary amino groups linked to C-17 position may
largely contribute to the augmentation of this effect on ent-kaurane
diterpenes.
To investigate the potential of ent-kaurane diterpenoids as lead
compounds for the development of novel antimalarial drugs, and
considering the availability of each diterpenoid, the cytotoxicities
of the compounds 1, 4, 5, 7, 8 and 9 were determined against HepG2
cells, allowing the calculation of their respective selectivity indexes
(SI). These results are also presented in Table 3. ent-Kaurenoic acid
(1) displayed a reasonable value of SI (9.1), and the lower SI value
observed for its methyl ester 4 (3.9) indicated that methylation of
the carboxylic acid group of 1 resulted in a more active derivative
with a lower selectivity against the P. falciparum W2 clone. On the
other hand, the incorporation of epoxide groups along the ent-
kaurane skeleton substantially increased the selectivity of this class
of diterpenes against the P. falciparum W2 clone, as can be deduced
from the SI values obtained for the monoepoxide 7 (SI ¼ 11.5) and
for the diepoxides 8 (SI ¼ 119.1) and 9 (SI ¼ 117.4). None of the ent-
kaurane diterpenoids 1e23 presented an antimalarial activity or
a selectivity performance equivalent to chloroquine, but these
results are very important for the structure-antimalarial activity
relationships studies of ent-kaurane diterpenoids and must be
considered in the context of the quest for novel derivatives with
better and more selective antiplasmodial activities.
As far as the authors are aware, this is the first report on the
promising selective antimalarial activity of ent-kaurane diterpe-
noids. The present discovery points to this class of diterpenes as
lead compounds for the development of novel antimalarial drugs
from some naturally occurring ent-kaurenes such as 1 and 3, which
are abundantly found in a few number of plant species, notably in
W. paludosa D.C. (Asteraceae) and Xylopia frutescens (Annonaceae)
[9], among others. The ent-kaurenes 1 and 3 could then be used as
starting material for chemical and/or microbiological semi-
synthetic routes aimed at obtaining more potent and selective
derivatives.
One concern related to the development of new antimalarials is
the development of resistant parasites. Indeed, this has happened
with atovaquone that was recently introduced in clinics. The rapid
resistance observed for atovaquone may be associated with its
action in the parasite electron transport chain [38] that targets the
cytochrome bc(1) complex, a key enzyme that catalyses transfer of
electrons maintaining the membrane potential of mitochondria.
Currently, atovaquone is the only drug in clinical use targeting the
P. falciparum bc(1) complex [39]. However, when antimalarial drugs
do not target enzymes, such as the blood schizonticides chloro-
quine, mefloquine, halophantrine and artesunate whose site of
action is within the digestive vesicle by complexation with hemin
and preventing its polymeryzation to the non-toxic pigment
hemozoin [40], the arising of resistance seems to happen much less
rapidly [41]. We have shown that one of our semi-synthetic kaur-
ane derivatives causes inhibition of the SERCA pump [42]. In this
case, a prolonged increase in the levels of cytoplasmic Ca2þ is ex-
pected what is considered to be an important mode of action
for antimalarial drugs, once Ca2þ is involved in a wide range
in the 10e30 mM range [7,12,34e36]. Furthermore, some ent-
kaurane derivatives were found to induce apoptosis via the acti-
vation of caspases-8 and -9 [37]. Within this context, diterpenes 22
and 23 may represent interesting models for the development of
more potent cytotoxic derivatives.
2.2.2. Antimalarial assay
Most of the compounds included in this work were assessed for
their in vitro antimalarial activity against the chloroquine-resistant
W2 clone of P. falciparum. These results are shown in Table 3.
According to the criteria recently established [3], the ent-kaur-
ane compounds 4, 5, 7, 8, 9, 21 and 22 disclosed good anti-
plasmodial activity against the chloroquine-resistant W2 strain of
P. falciparum, with IC50 values ranging from 5.4 to 10.4
diterpenes 1 and 3, as well as derivatives 11 and 13a/13b, were
moderately active, with IC50 values from 21.1 to 34.7 M.
Compounds 14, 15, 18, 20, 23 and 24 presented very low activity,
with IC50 values above 142.7 M (Table 3).
Interestingly, methyl kaurenoate (4, IC50 ¼ 8.2 ꢂ 4.1
methyl iso-kaurenoate (5, IC50 ¼ 8.8 ꢂ 1.6 M) displayed a substan-
tially higher activity than kaurenoic (1, IC50 ¼ 21.1 ꢂ3.3 M) and
M) acids, indicating that ester-
mM, while
m
m
mM) and
m
m
grandiflorenic (3, IC50 ¼ 23.3 ꢂ1.7
m
ification of the carboxylic acid group at C-19 improves the anti-
plasmodial activity of ent-kaurane diterpene acids. A similar effect
Table 3
Inhibitory concentrations (IC50) of some ent-kaurane diterpenoids against the
Plasmodium falciparum chloroquine-resistant clone W2 and their cytotoxicities
(CC50) against HepG2 cells.
Compound
W2 (IC50
,
m
M)a
HepG2 (CC50
192.4 ꢂ 25.5
N.D.
, m
M)a
SIb
9.1
1
3
4
5
21.1 ꢂ 3.3
23.3 ꢂ 1.7
8.2 ꢂ 4.1
8.8 ꢂ 1.6
9.6 ꢂ 1.8
10.4 ꢂ 2.9
6.9 ꢂ 2.0
34.7 ꢂ 4.5
33.1 ꢂ 6.0
>142.7
e
32.2 ꢂ 6.6
44.9 ꢂ 0.3
110.7 ꢂ 0.9
3.9
5.1
11.5
119.1
117.4
e
7
8
1238.5 ꢂ 200.6
810.2 ꢂ 105.9
N.D.
9c
11
13a/13b
14
15
18
20
21
22
N.D.
N.D.
e
e
>142.7
N.D.
e
>157.0
N.D.
e
>143.5
N.D.
e
5.4 ꢂ 1.5
8.4 ꢂ 2.4
>150.4
N.D.
N.D.
N.D.
e
e
23
24
e
>142.7
N.D.
e
Chloroquine
0.78 ꢂ 0.03
504.1 ꢂ 30.2
646.3
a
Values are averages ꢂ standard deviation (n ¼ 3).
Selectivity Index ¼ CC50 (HepG2) /IC50 (W2).
b
c
Caused haemolysis at the concentration of 139
m
M; N.D.¼ not determined.

R. Batista et al. / European Journal of Medicinal Chemistry 62 (2013) 168e176
173
of important process in malarial parasites, including cellular divi-
sion [43].
Finally, it is important to stress that acute toxicity and in vivo
efficacy assays need to be undertaken with ent-kaurane compounds
to confirm them as potential candidates for the development of
antimalarial drugs.
there were no peaks greater than 0.34 Å. Crystallographic data
(excluding structure factors) for the structures in this paper have
been deposited with the Cambridge Crystallographic Data Centre as
supplementary publication numbers CCDC 749702 and 749703.
Copies of the data can be obtained, free of charge, upon application
to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [fax: þ44 (0)1223-
336033 or e-mail: deposit@ccdc.cam.ac.uk].
3. Conclusion
4.3. Chemistry
This paper highlights once again the importance of naturally
occurring compounds as synthones in the preparation of semi-
synthetic series to be screened for biological activities. A total of
eleven novel ent-kaurane derivatives were obtained and the
complete set of 1H, 13C NMR and crystallographic data for two novel
diepoxidised ent-kaurane derivatives (8 and 9) was established. The
allylic alcohols 22 and 23 were shown to be the most potent anti-
neoplastic compounds, while diepoxide 9 and the perhydropyr-
imidinyl derivative 21 displayed the highest antimalarial activities.
Epoxidation along the ent-kaurane skeleton led to more selective
derivatives against the blood-stage P. falciparum chloroquine-
resistant W2 clone. These results indicate that ent-kaurane deriv-
atives are a promising source of new hits for antimalarial drug
research.
4.3.1. Isolation and methylation of diterpene acids
The mixture of diterpenes 1 þ 2 þ 3 (1.14 g, proportion 14:8:17,
3.77 mmol of total diterpenes) obtained previously from
W. paludosa D.C. [11] was methylated by the usual procedure with
an ethereal solution of diazomethane (100 mL), giving the respec-
tive mixture of methyl esters 4 þ 5 þ 6 in quantitative yield
(1.20 mg, proportion 14:8:17, 3.77 mmol of total esters). Small
amounts of 3 (52 mg, 0.17 mmol) and 5 (123 mg, 139 mmol) were
previously isolated from these mixtures [11]. Kaurenoic acid (1)
was also obtained as a pure compound from W. paludosa D.C.
ethanol extract [8], and its methyl ester derivative (4) was quanti-
tatively obtained under diazomethane methylation conditions.
4.3.2. Epoxidation
4. Experimental section
A portion of the methyl esters mixture (4 þ 5 þ 6, 506 mg;
1.60 mmol total) was treated with MCPBA (55%, 567 mg;
1.83 mmol) in dry CH2Cl2 (30 mL), and the system was stirred at
room temperature, in the presence of NaHCO3 excess (1 g). After
30 min, the CH2Cl2 solution was washed with aq. satd. Na2S2O3,
water and brine, and dried on Na2SO4. The solvent was evaporated
and the mixture of products (540 mg) was submitted to Flash
Column Chromatography (FCC) over silica gel, eluting with n-
hexaneeEtOAc (95:5), 20 mL per fraction, to yield compounds 7
(frs. 19e24, 217 mg, 78%), 9 (frs. 31e34, 14 mg, 8%) and 8 (frs. 36e
39, 31 mg, 17%).
Diterpene 2 (52 mg; 0.17 mmol) was submitted to the same
procedure above, yielding a mixture of products (32 mg), which
was, in turn, chromatographed (FCC) over silica gel, eluting with n-
hexaneeEtOAc (95:5), 20 mL per fraction, to afford 10a (fr. 22,
2 mg, <0.01 mmol, 2%) and 10b (frs. 24e29, 20 mg, 0.06 mmol,
36%) epoxides.
4.1. General procedures
Melting points were taken with a Microquıímica apparatus APF-
301 and uncorrected. Optical rotations were measured with a Per-
kin-Elmer 241 digital polarimeter. IR spectra were obtained on
a Shimadzu IR-400 spectrophotometer in diamond film. NMR
spectra were recorded either at 200 MHz for 1H and 50 MHz for 13
C
on a Bruker AC 200 or at 400 MHz for 1H and 100 MHz for 13C on
a Bruker Advance DRX 400, in deuterochloroform or deuter-
omethanol, with added TMS as an internal reference. One-
dimensional (1D) 1H and 13C NMR spectra were acquired under
standard conditions (5 mm DUAL-1H/13C direct detection). For all
two-dimensional NMR experiments, the 1H spectral width was set
to 5000 Hz and the 13C spectral width was set to 21.4 for HMQC and
to 30 KHz for HMBC, respectively. Chemical shift values are
expressed in ppm and coupling constants (J) in Hz. Column chro-
matography (CC) and flash column chromatography (FCC) were
performed with Merck silica gel 60 (0.063e0.200 and 0.040e
0.063 mm, respectively). HRMS were run in a VG TS-250 spec-
trometer working at 70 eV. TLC was carried out on Merck silica gel
60 F254 (0.25 mm thick). The solvents and reagents were purified
by standard procedures as necessary.
4.3.2.1. Methyl ent-9b:11,16b-diepoxy-kauran-19-oate (8). Mp 168e
170 ꢀC;
a]
D ꢁ9.5ꢀ, CHCl3, c 0.9. IR(film) nmax/cmꢁ1: 2934, 2872,1723,
1458, 1433, 1384, 1226, 1148, 980. 1H NMR and 13C NMR data,
Table 1. HRMS (FAB-POSI, M þ 1) Calcd. 347.2222; Found 347.2201.
4.3.2.2. Methyl ent-9b:11,16a-diepoxy-kauran-19-oate (9). Mp 96e
99 ꢀC;
a
]
ꢁ7.2ꢀ, CHCl3, c 0.5. IR(film) nmax/cmꢁ1: 2928, 2854,
D
4.2. Crystallographic analysis
1724, 1458, 1434, 1383, 1227, 1148. 1H NMR and 13C NMR data,
Table 1. HRMS (FAB-POSI, M þ 1) Calcd. 347.2222; Found 347.2253.
Data collection was performed on a Kappa-CCD-Enraf-Nonius
diffractometer using CRYSOM, which was also employed for unit
cell determination [44]. X-RAY80 was used for data reduction [45].
The structure was solved by direct methods using the SHELXL97
program [46]. SHELXTL/PC was employed for molecular graphics
[47]. The refinement of F2 against all reflections was conducted until
all atomic-parameter shifts were smaller than their standard devi-
ations. Most of the H atoms could be observed in Fourier difference
maps; nevertheless, their positions were calculated and refined
using a riding model approximation, with distance restraints
CdH ¼ 0.96e0.98 Å and with Uiso(H) ¼ 1.5Ueq (parent atom) for
methyl H atoms and 1.2Ueq (parent atom) for the remaining H atoms.
As the data set contained no Friedel pairs, the absolute configuration
was assumed from the synthesis. In the final difference Fourier map,
4.3.2.3. ent-16a
-Epoxy-kauran-19-oic acid (10a). Mp 120e123 ꢀC;
[
a
]
þ32.4ꢀ, CHCl3, c 0.11. IR(film) nmax/cmꢁ1: 3400e3100, 3035,
D
2928, 2858, 1691, 1651, 1464, 1377, 1264, 1232, 1181, 1162, 977, 966,
796. 1H NMR (CDCl3, 200 MHz) (ppm)
(d, 1H, C17-Ha, J ¼ 5.3), 2.72 (d, 1H, C17-Hb, J ¼ 5.3), 1.21 (s, 3H, C4-
CH3), 1.01 (s, 3H, C10-CH3). 13C NMR (CDCl3, 50 MHz) (ppm)
: 40.7
d: 5.36 (br s, 1H, C11-H), 2.86
d
(C1), 20.1 (C2), 38.2 (C3), 44.7 (C4), 46.4 (C5), 18.3 (C6), 30.1 (C7), 41.9
(C8), 155.7 (C9), 38.7 (C10), 115.6 (C11), 30.4 (C12), 38.1 (C13), 43.9
(C14), 49.0 (C15), 67.1 (C16), 54.4 (C17), 28.2 (C18), 182.7 (C19), 23.6
(C20). HRMS (FAB-POSI, M þ 1) Calcd. 317.2117; Found 317.2149.
4.3.2.4. ent-16b
-Epoxy-kauran-19-oic acid (10b). Mp 158e161 ꢀC;
a]
þ6.6ꢀ, CHCl3, c 0.85. IR(film) nmax/cmꢁ1: 3400e3100, 2946,
D

174
R. Batista et al. / European Journal of Medicinal Chemistry 62 (2013) 168e176
1709,1651, 1460,1434,1374,1254,1216,1171,1151, 976, 815. 1H NMR
(CDCl3, 200 MHz) (ppm) : 5.24 (br s, 1H, C11-H), 2.85 (d, 1H, C17-Ha,
J ¼ 4.8), 2.80 (d, 1H, C17-Hb, J ¼ 4.8), 1.24 (s, 3H, C4-CH3), 1.03 (s, 3H,
C10-CH3). 13C NMR (CDCl3, 50 MHz) (ppm)
: 40.7 (C1), 20.1 (C2),
38.2 (C3), 44.7 (C4), 46.7 (C5), 18.4 (C6), 30.0 (C7), 42.9 (C8), 156.6
(C9), 38.9 (C10), 114.2 (C11), 32.9 (C12), 40.7 (C13), 43.2 (C14), 50.0
(C15), 69.3 (C16), 50.9 (C17), 28.2 (C18), 183.9 (C19), 23.6 (C20). HRMS
(FAB-POSI, M þ 1) Calcd. 317.2117; Found 317.2148.
solid; [
a
]
þ41.7ꢀ, CHCl3, c 0.77. IR(film) nmax/cmꢁ1: 3364, 3060,
D
d
2928, 2867, 1723, 1651, 1463, 1221, 1147, 980. 1H NMR (CDCl3,
200 MHz) (ppm) d: 5.22/5.20 (br s, 1H, C11-H), 2.35 (m, 1H, C16-H),
d
3.58 (d, 2H, C17-Ha, J ¼ 7.9), 3.45 (m, 2H, C17-Hb), 1.15 (s, 3H, C4-
CH3), 0.90/0.89 (s, 3H, C10-CH3), 3.63 (s, 3H, C19-OCH3). 13C NMR
(CDCl3, 50 MHz) (ppm) d: 41.2 (C1), 20.2 (C2), 38.3 (C3), 45.0 (C4),
46.6 (C5), 18.5 (C6), 30.0/29.7 (C7), 44.8 (C8), 158.1/156.4 (C9), 38.6
(C10), 114.9/114.7 (C11), 30.0/37.2 (C12), 35.0/35.9 (C13), 44.8/42.6
(C14), 45.6/47.0 (C15), 45.1/48.1 (C16), 65.8/67.5 (C17), 28.0 (C18), 178.0
(C19), 23.3 (C20), 51.2 (C21). HRMS (FAB-POSI, M þ 1) Calcd.
333.2430; Found 333.2443.
4.3.3. Hydroboration-oxidation
4.3.3.1. Method A. A portion of the mixture of methyl esters
(4 þ 5 þ 6, 200 mg; 0.64 mmol) in anhydrous THF (2 mL) was added
to
a
solution of 9-borabicyclo[3.3.1]nonane (9-BBN; 155 mg,
4.3.3.6. Methyl ent-11
b,17-dihydroxy-9-epi-16a-kauran-19-oate
1.27 mmol) in dry THF (2 mL), and the system was stirred at room
temperature under inert atmosphere (N2) for 3 h. After this time,
the solution was cooled (0 ꢀC), and EtOH (5 mL), NaOH aq. 20%
(5 mL) and H2O2 30% v/v (3 mL) were added and then stirred at
50 ꢀC for 1 h before being washed with brine and dried on Na2SO4.
The solvent was evaporated, and only the mixture of esters
(4 þ 5 þ 6) was observed on TLC. No product was observed for this
reaction also under 1H NMR analysis.
(14). Mp 159e162 ꢀC; [
a
]
ꢁ63.5ꢀ, CHCl3, c 0.95. IR(film) nmax
/
D
cmꢁ1: 3363, 2986, 2932, 2856, 1722, 1464, 1450, 1384, 1371, 1334,
1233, 1154, 1096, 1049, 1032, 1021, 998. 1H NMR (CDCl3, 200 MHz)
(ppm)
C17-2H, J ¼ 4.0, 8.3), 1.17 (s, 3H, C4-CH3), 0.90 (s, 3H, C10-CH3), 3.62
(s, 3H, C19-OCH3). 13C NMR (CDCl3, 50 MHz) (ppm)
: 38.8 (C1),
d: 4.00 (m, 1H, C11-H), 2.38 (m, 1H, C16-H), 3.67 (dd, 2H,
d
19.4 (C2), 38.2 (C3), 44.1 (C4), 51.3 (C5), 20.2 (C6), 37.9 (C7), 42.5
(C8), 59.4 (C9), 37.3 (C10), 68.8 (C11), 36.1 (C12), 35.7 (C13), 37.9
(C14), 40.2 (C15), 46.4 (C16), 63.6 (C17), 28.9 (C18), 178.4 (C19), 22.7
(C20), 51.2 (C21). HRMS (FAB-POSI, M þ 1) Calcd. 351.2535; Found
351.2491.
4.3.3.2. Method B. A portion of the mixture of methyl esters
(4 þ 5 þ 6, 504 mg; 1.60 mmol) in anhydrous THF (20 mL) was
treated with NaBH4 (610 mg, 16.12 mmol) and BF3$Et2O (2.0 mL,
15.92 mmol). The system was stirred at room temperature under
inert atmosphere (Ar) for 1 h. After this time, the solution was
cooled (0 ꢀC), and then EtOH (10 mL), NaOH aq. 20% (10 mL) and
H2O2 30% v/v (5 mL) were added, before the solution was stirred at
50 ꢀC for 1 h and washed with brine and dried on Na2SO4. The
solvent was evaporated, and the mixture of products (556 mg) was
submitted to FCC over silica gel, eluting with n-hexaneeEtOAc
(9:1), 20 mL per fraction, to give alcohols 11 (frs. 2e25, 64 mg,
0.19 mmol, 72%),13a D 13b (3:1, frs. 27e29, 26 mg, 0.08 mmol,15%)
and 12 (frs. 32e41, 230 mg, 0.69 mmol, 86%).
4.3.3.7. Methyl ent-11a,17-dihydroxy-16a-kauran-19-oate (15).
Mp 88e91 ꢀC; [ ]D ꢁ51.3ꢀ, CHCl3, c 0.90. IR(film) nmax/cmꢁ1: 3363,
2925, 2872, 2854, 1724, 1465, 1446, 1376, 1325, 1235, 1191, 1156,
1095, 1030, 997, 975, 733. 1H NMR (CDCl3, 200 MHz) (ppm)
d: 3.86
(m, 3H, C11-H, C17-2H), 2.15 (m, 1H, C16-H), 1.17 (s, 3H, C4-CH3), 0.70
(s, 3H, C10-CH3), 3.63 (s, 3H, C19-OCH3). 13C NMR (CDCl3, 50 MHz)
(ppm) d: 40.2 (C1), 19.0 (C2), 37.9 (C3), 43.8 (C4), 56.7 (C5), 21.8 (C6),
39.9 (C7), 42.8 (C8), 65.4 (C9), 38.3 (C10), 66.8 (C11), 35.4 (C12), 37.7
(C13), 39.9 (C14), 42.1 (C15), 42.7 (C16), 63.0 (C17), 28.7 (C18), 177.9
(C19), 14.8 (C20), 51.1 (C21). HRMS (FAB-POSI, M þ 1) Calcd. 351.2535;
Found 351.2533.
4.3.3.3. Method C. A portion of the mixture of methyl esters
(4 þ 5 þ 6, 660 mg; 2.09 mmol) in anhydrous THF (25 mL) was
treated with NaBH4 (802 mg, 21.20 mmol) and BF3$Et2O (5.0 mL,
39.80 mmol). The system was stirred at room temperature under an
inert atmosphere (Ar) for 2 h. After this time, the solution was
cooled (0 ꢀC), and then EtOH (10 mL), NaOH aq. 20% (10 mL) and
H2O2 30% v/v (7 mL) were added, before being stirred at 50 ꢀC for
2 h and washed with brine and dried on Na2SO4. The solvent was
evaporated, and the mixture of products (894 mg) was submitted
to FCC over silica gel, eluting with n-hexaneeEtOAc (9:1), 20 mL per
fraction, to yield alcohols 11 (frs. 19e21, 81 mg, 0.24 mmol, 77%), 12
(frs. 25e31, 278 mg, 0.83 mmol, 80%), 14 (frs. 42e44, 25 mg,
0.07 mmol, 10%) and 15 (frs. 45e46, 87 mg, 0.25 mmol, 36%).
4.3.4. Synthesis of oxidised compounds from either epoxide 7 or
alcohol 12
The synthesis of aldehydes 16 and 17 (1:1) was previously per-
formed starting from epoxide 7, as well as the syntheses of
compounds 17, 18, 19 and 20 from alcohol 12 [20].
4.3.5. Synthesis of the perhydropyrimidinyl derivative 21
A solution of aldehyde 17 (50 mg, 0.15 mmol) in anhydrous
CH2Cl2 (3 mL) was added (dropwise) to 1,3-propanediamine
(375 mL, 4.5 mmol) containing MgSO4 (50 mg, 0.42 mmol), and
the system was stirred at room temperature under an inert argon
atmosphere for 6 h. Next, the solution was filtered and concen-
trated exhaustively under reduced pressure to give 21 (45 mg,
0.12 mmol) at 77% yield.
4.3.3.4. Methyl ent-15b-hydroxy-16a-kauran-19-oate (11). Mp
139e141 ꢀC; [
a]
ꢁ62.6ꢀ, CHCl3, c 1.00. IR(film) nmax/cmꢁ1: 3421,
D
2923, 2851, 1726, 1464, 1375, 1232, 1216, 1193, 1158, 1097, 1078,
4.3.5.1. Methyl ent-16a-(hexahydropyrimidin-2-yl)-17-nor-kauran-
1056, 1032, 997, 807, 737. 1H NMR (CDCl3, 200 MHz) (ppm)
d: 2.17
19-oate (21). Amorphous solid; [
a
]
D ꢁ58.9ꢀ, CHCl3, c 2.30. IR(film)
(d, 1H, C13-H, J ¼ 14.2), 3.23 (d, 1H, C15-H, J ¼ 4.4), 1.78 (m, 1H, C16
-
-
:
nmax/cmꢁ1: 3315, 2933, 2852, 1724, 1461, 1449, 1384, 1347, 1324,
H), 1.12 (d, 3H, C16-CH3, J ¼ 7.3), 1.18 (s, 3H, C4-CH3), 0.84 (s, 3H, C10
1234, 1192, 1150, 1024, 980, 774, 732. 1H NMR (CDCl3, 200 MHz)
CH3), 3.65 (s, 3H, C19-OCH3). 13C NMR (CDCl3, 50 MHz) (ppm)
d
(ppm)
d
: 3.22 (br s, 1H, C16-H), 3.46 (d, 1H, C17-H, J ¼ 9.6), 1.24 (s,
40.7 (C1), 19.1 (C2), 38.1 (C3), 43.8 (C4), 57.1 (C5), 21.4 (C6), 38.0 (C7),
47.6 (C8), 54.5 (C9), 39.6 (C10), 18.5 (C11), 24.9 (C12), 38.2 (C13), 35.8
(C14), 88.3 (C15), 47.2 (C16), 13.6 (C17), 28.7 (C18), 178.1 (C19), 15.5
(C20), 51.2 (C21). HRMS (FAB-POSI, M þ 1) Calcd. 335.2586; Found
335.2555.
3H, C4-CH3), 0.95 (s, 3H, C10-CH3), 3.63 (s, 3H, C19-OCH3), 2.80 (m,
4H, NC1 -2H, NC3 -2H), 3.14 (m, 2H, eNHe). 13C NMR (CDCl3,
50 MHz) (ppm) : 40.7 (C1), 19.1 (C2), 38.0 (C3), 43.8 (C4), 56.9
0
0
d
(C5), 22.1 (C6), 42.0 (C7), 44.1 (C8), 56.5 (C9), 39.4 (C10), 19.6 (C11),
26.2 (C12), 36.7 (C13), 40.3 (C14), 43.6 (C15), 47.0 (C16), 73.2 (C17),
28.6 (C18), 178.1 (C19), 15.4 (C20), 51.1 (C21), 46.0 (C1’), 27.8 (C2’),
45.9 (C3’). HRMS (FAB-POSI, M þ 1) Calcd. 389.3168; Found
389.3193.
4.3.3.5. Methyl ent-17-hydroxy-16
a-kaur-9(11)-en-19-oate/ent-17-
hydroxy-16 -kaur-9(11)-en-19-oate (13a/13b, 3:1). Amorphous
b

R. Batista et al. / European Journal of Medicinal Chemistry 62 (2013) 168e176
175
4.3.6. Synthesis of the alcohols 22e24 from the ester 4
HELA and HELA-APL, cervix epithelioid carcinoma. A conventional
colorimetric assay [48] was set up to estimate GI50 values, the drug
concentration that causes 50% cell growth inhibition after 72 h of
continuous exposure to the test molecules.
4.3.6.1. Method D. The methyl ester 4 (101 mg, 0.32 mmol) in dry
CH2Cl2 (5 mL) was treated with SeO2 (30 mg, 0.27 mmol) and t-
BuOOH in CH2Cl2 (5 mL, 7.94 mmol). After stirring for 45 min at
room temperature under an inert argon atmosphere, the solution
was concentrated, and the yellowish mixture of products was
submitted to FCC over silica gel, eluting with n-hexaneeEtOAc
(95:5), 10 mL per fraction, to yield alcohol 22 (frs. 10e11, 74 mg,
0.22 mmol, 70%).
Cells were seeded into 16-mm wells (multidishes, NUNC 42001)
at concentrations of 2 ꢃ104 cells/well, in 1-mL aliquots of
MEM10FCS medium containing the compound to be evaluated at
the concentrations tested. In each case, a set of control wells was
incubated in the absence of sample and counted daily to ensure the
exponential growth of cells. After 3 days at 37 ꢀC, under a 10% CO2,
98% humidity atmosphere, the cells were stained with crystal
violet, observed through an inverted microscope and the degree of
inhibition was determined by comparison with the controls. All
calculations represent the average of duplicated wells. For Jurkat-
77 cell-line assays, the cells were seeded into 25-mm wells (mul-
tidishes, NUNC 150318) at concentrations of 13 ꢃ 104 cells/mL, in
RPMI 1604-SBF10% medium containing the compound to be eval-
uated at the concentrations tested. In each case, a set of control
wells was incubated in the absence of sample and counted daily to
ensure the exponential growth of cells. After 4 days at 37 ꢀC, under
a 5% CO2, 90% humidity atmosphere, the cells were stained with
trypan blue, observed through an inverted microscope and the
degree of inhibition was determined by comparison with the
controls. All calculations represent the average of triplicate wells.
4.3.6.2. Method E. The methyl ester 4 (120 mg, 0.38 mmol) in i-
PrOH (50 mL) was treated with Bengal Rose (20 mg), and the
mixture was stirred for 16 days at room temperature and exposed
to sunlight. The solvent was removed under vacuum. The residue
was dissolved in MeOH (25 mL), and the solution was stirred for 1 h
at room temperature after being treated with NaBH4 (294 mg,
3.42 mmol). The solution was then concentrated, and the mixture
of products was dissolved in Et2O, washed with brine and dried on
Na2SO4. The organic solution was concentrated under reduced
pressure, and the mixture of products (108 mg) was submitted to
FCC over silica gel, eluting with n-hexaneeEtOAc (85:15), 20 mL per
fraction, to afford alcohols 22 (frs. 26e28, 10 mg, 0.03 mmol, 10%)
and 23 (frs. 30e34, 38 mg, 0.11 mmol, 36%). Final fractions 44e46,
eluted with EtOAc, yielded alcohol 24 (12 mg, 0.03 mmol, 11%).
4.3.6.3. Methyl ent-15
b
-hydroxy-kaur-16-en-19-oate (22). Mp 62e
4.4.2. Antimalarial assay
65 ꢀC; [
a]
ꢁ95.2ꢀ, CHCl3, c 0.50. IR(film) nmax/cmꢁ1: 3419, 2925,
Infected red blood cells were maintained in continuous culture
with human erythrocytes (blood group: 0 negative) in RPMI
medium supplemented with 10% human plasma (complete
medium), as previously described [49]. The synchronisation of the
parasites was achieved by sorbitol treatment [50], and parasitemia
was determined microscopically with Giemsa-stained smears.
The antimalarial effects of ent-kaurane compounds and controls
were measured with the [3H]-hypoxanthine incorporation assay
performed as described previously [51] with minor modifications
[52]. Briefly, ring-stage parasites in sorbitol-synchronised blood
cultures were added to 96-well culture plates at 1% parasitemia and
1% haematocrit and then incubated with the test drugs alone. The
compounds were diluted in complete medium without hypoxan-
thine, from 10 mg/mL stock solutions in DMSO at a final concen-
tration of 0.002% (vol/vol) and stored at ꢁ20 ꢀC. After a 24-h
D
2851, 1726, 1652, 1463, 1376, 1233, 1192, 1154, 1093, 1070, 1054,
1019, 1001, 897, 772. 1H NMR (CDCl3, 200 MHz) (ppm)
d: 2.74 (br s,
1H, C13-H), 3.80 (s, 1H, C15-H), 5.21 (s, 1H, C17-Ha), 5.08 (s, 1H, C17
Hb), 1.19 (s, 3H, C4-CH3), 0.84 (s, 3H, C10-CH3), 3.65 (s, 3H, C19
-
-
OCH3). 13C NMR (CDCl3, 50 MHz) (ppm)
d: 40.7 (C1), 19.1 (C2), 38.0
(C3), 43.8 (C4), 57.0 (C5), 21.0 (C6), 36.2 (C7), 47.7 (C8), 53.3 (C9), 39.6
(C10), 18.3 (C11), 32.6 (C12), 42.3 (C13), 35.2 (C14), 82.7 (C15), 160.3
(C16), 108.3 (C17), 28.7 (C18), 178.1 (C19), 15.6 (C20), 51.1 (C21). HRMS
(FAB-POSI, M þ 1) Calcd. 333.2430; Found 333.2472.
4.3.6.4. Methyl ent-17-hydroxy-kaur-15-en-19-oate (23). Mp 80e
82 ꢀC; [
a]
ꢁ51.1ꢀ, CHCl3, c 1.35. IR(film) nmax/cmꢁ1: 3436, 2930,
D
2848, 1727, 1674, 1469, 1446, 1377, 1367, 1326, 1233, 1192, 1158, 1131,
1090, 1024, 952. 1H NMR (CDCl3, 200 MHz) (ppm)
d: 2.54 (br s, 1H,
C13-H), 5.36 (s, 1H, C15-H), 4.19 (s, 2H, C17-2H), 1.17 (s, 3H, C4-CH3),
incubation period, 25
thine (0.5 Ci/well) was added per well, followed by another 18-h
m
L of medium containing [3H]-hypoxan-
0.85 (s, 3H, C10-CH3), 3.64 (s, 3H, C19-OCH3). 13C NMR (CDCl3, 50
m
MHz) (ppm)
d
: 40.7 (C1), 19.1 (C2), 38.0 (C3), 43.8 (C4), 56.7 (C5), 20.8
incubation at 37 ꢀC. The plates were frozen (ꢁ70 ꢀC for 24 h) and
thawed, and the cells were harvested (Tomtec 96-Harvester) on
glass fibre filters (Wallac, Turku, Finland) and then placed into
sample bags (Wallac) and immersed in scintillation fluid (Opti-
phase super mix; Wallac). Radioactive emission was counted with
a 1450 Microbeta reader (Wallac). The inhibition of parasite growth
was evaluated from the levels of [3H]-hypoxanthine incorporation;
i.e., the IC50 values were evaluated by comparing the incorporation
in drug-free control cultures and estimated by linear interpolation
[51] using curve-fitting software (Microcal Origin software 5.0). All
experiments were performed three times, and each sample was
tested in triplicate.
(C6), 39.2 (C7), 48.8 (C8), 47.5 (C9), 39.6 (C10), 18.8 (C11), 25.4 (C12),
41.0 (C13), 43.8 (C14), 135.4 (C15), 146.1 (C16), 61.2 (C17), 28.7 (C18),
178.0 (C19), 15.2 (C20), 51.1 (C21). HRMS (FAB-POSI, M þ 1) Calcd.
333.2430; Found 333.2397.
4.3.6.5. Methyl ent-16a,17-dihydroxy-kauran-19-oate (24). Mp 65e
67 ꢀC;
a]
ꢁ45.4ꢀ, CHCl3, c 0.90. IR(film) nmax/cmꢁ1: 3363, 2925,
D
2872, 2854, 1724, 1465, 1446, 1376, 1325, 1235, 1191, 1156, 1095,
1030, 997, 975, 733. 1H NMR and 13C NMR data were very similar to
those described in the literature [28]. HRMS (FAB-POSI, M þ 1)
Calcd. 351.2535; Found 351.2533.
4.4. Biological evaluation
4.4.3. Cytotoxicity assay against HepG2 cells
Hep G2A16 hepatoma cells were stored at 37 ꢀC, 5% CO2 in 75-
cm3 sterile culture flasks (CorningÒ) in RPMI1640 culture
medium (SigmaÒ) supplemented with 5% FBS, gentamicin (40 mg/
mL), with medium changes twice a week. The assays were per-
formed as described previously [53] with slight modifications.
Briefly, the cells were maintained with weekly passages and grown
to confluence (ATCC). The monolayers were trypsinised (0.05%
trypsin/0.5 mM EDTA), washed, counted, diluted in complete
4.4.1. Antineoplastic cytotoxicity assay
The cell lines assayed were DU-145, prostate carcinoma; IGROV
and IGROV-ET, ovarian; SK-BR-3, breast adenocarcinoma; SK-MEL-
28, malignant melanoma; A-549, lung carcinoma NSCL; K-562,
chronic myelogenous leukaemia; PANC-1, pancreatic epithelioid
carcinoma; HT-29, HT-29-KF and LoVo colon adenocarcinoma, and
LoVo-Dox, colon adenocarcinoma resistant to doxorubicin; and

176
R. Batista et al. / European Journal of Medicinal Chemistry 62 (2013) 168e176
medium, distributed in 96-well microliter plates (4 ꢃ104 cells/
well), then incubated for another 18 h at 37 ꢀC. The test compounds
and control drugs (DMSO, chloroquine diphosphate), assayed in
triplicate, were diluted to a final concentration of 0.01% DMSO in
culture medium to yield four drug concentrations obtained in serial
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the supernatant was removed and 18
in PBS) was added to each well, followed by 1 h, 30 min of incu-
bation at 37 ꢀC. Then, DMSO (180
L) was added to each well and
m
g/mL. After a 24-h incubation at 37 ꢀC,
L of MTT solution (6 mg/mL
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