Synthesis and biological evaluation of rigid polycyclic derivatives of the Diels-Alder adduct tricyclo[6.2.1.02,7]undeca-4,9-dien-3,6-dione

Article abstract of DOI:10.3390/12020271

Part of our research program concentrates on the discovery of new bioactive compounds prepared either by total synthesis or molecular transformation of compounds with bioactivity profiles. In this work we have focused our interest on chemical transformati

Full text of DOI:10.3390/12020271

Molecules 2007, 12, 271-282
molecules
ISSN 1420-3049
http://www.mdpi.org
Full Paper
Synthesis and Biological Evaluation of Rigid Polycyclic
Derivatives of the Diels-Alder Adduct
2,7
Tricyclo[6.2.1.0 ]undeca-4,9-dien-3,6-dione
1
1
1
1,*
Felicia Megumi Ito , Jacqueline Marques Petroni , Dênis Pires de Lima , Adilson Beatriz ,
2
3
3
Maria Rita Marques , Manoel Odorico de Moraes , Letícia Veras Costa-Lotufo , Raquel
3
3
3
Carvalho Montenegro , Hemerson Iury Ferreira Magalhães and Cláudia do Ó Pessoa
1
Department of Chemistry, Federal University of Mato Grosso do Sul, Campo Grande, MS, Brazil. E-
mails: megumi@nin.ufms.br, jacque_petroni@hotmail.com, dlima@nin.ufms.br
Department of Morphophysiology, Federal University of Mato Grosso do Sul, Campo Grande, MS,
Brazil. E-mail: mrmarx@nin.ufms.br
2
3
Department of Physiology and Pharmacology, Federal University of Ceará, Fortaleza, CE, Brazil.
E-mail: odorico@ufc.br, lvcosta@secrel.com.br, hemersoniury@yahoo.com.br, cpessoa@ufc.br
*
Author to whom correspondence should be addressed; E-mail: adilbeat@nin.ufms.br; Homepage:
http://www.dqi.ufms.br/~lp4/adilson.htm.
Received: 30 January 2007; in revised form: 14 February 2007 / Accepted: 16 February 2007 /
Published: 27 February 2007
Abstract: Part of our research program concentrates on the discovery of new bioactive
compounds prepared either by total synthesis or molecular transformation of compounds
with bioactivity profiles. In this work we have focused our interest on chemical
2
,7
transformations of the Diels-Alder adduct tricyclo[6.2.1.0 ]undeca-4,9-dien-3,6-dione in
order to obtain cage-like compounds and derivatives, followed by an evaluation of their
biological activity.
2
,7
Keywords: Tricyclo[6.2.1.0 ]undecane ring system, cage-like compounds, Diels-Alder
adducts, biological activity.

Molecules 2007, 12
272
Introduction
Since the discovery of the Diels-Alder reaction [1], it has become one of the most employed
strategies in organic synthesis [2]. This all began in 1928, when the cycloaddition between
2
,7
cyclopentadiene (1) and p-benzoquinone (2), forming mostly the endo adduct tricyclo[6.2.1.0 ]-
undeca-4,9-dien-3,6-dione (3), as shown in Scheme 1, was described.
2
,7
Scheme 1. Preparation of tricyclo [6.2.1.0 ]undeca-4,9-dien-3,6-dione.
H
O
H
O
+
O
1
2
O
endo-3
A unique aspect of adduct 3 is its high symmetry, which allows for facile selective reactions at one
or both carbonyl groups by means of classical and non-classical reagents, including microbial
transformations [3]. Another feature to be considered is its cage-like framework, which forces
functional groups into close spatial proximity, facilitating subsequent reactions, as exemplified, for
instance, by the synthesis of Cookson’s diketone (4) [4] (Scheme 2).
Scheme 2. Preparation of Cookson’s diketone (4).
UV
O
O
O
O
3
4
Although compound 3 has been extensively studied, its use in the medicinal chemistry field has not
been exploited very much. On the other hand, there is considerable interest nowadays in studying the
biological activity of cage-like rigid polycyclic molecules, which owing to their structural
peculiarities, may interact in a specific way with biological receptors and/or trap smaller chemical
species in their interior. It is known, for example, that the incorporation of a rigid carbogenic structure
in biologically active compounds often increases their physiological activity. One of their advantages
is a slowing down of metabolic degradation and consequent prolongation of the activity [5-7].

Molecules 2007, 12
Figure 1: Examples of bioactive cage-like polycyclic compounds.
273
H C
3
NH2
CH
NH2
H N
2
H N
2
C H
6
5
CH3
5
6
7
8
Considering the important antiviral activity of amantadine (5) and rimantadine (6), additional
studies related to cage-like polycyclic compounds led to the discovery of other compounds with
similar properties, such as the pentacycloundecanes 7 and 8 [6] (Figure 1).
Accordingly, as a part of a research program aimed at finding new synthetic compounds with
interesting bioactivity profiles, we decided to investigate the preparation of some rigid polycyclic
oxygenated compounds, taking advantage of the structural versatility of the Diels-Alder adduct 3. The
central idea in this work was to make accessible new structurally complex molecules which are both
easy to prepare and endowed with potential biological activity.
Results and Discussion
Chemistry
The reaction shown in Scheme 1 is widely known in literature [2, 8-11], however, from an
experimental point of view, a few drawbacks can be noted, such as the use of toxic solvents and the
need to perform the reaction at very low temperatures. These features prompted us to search for a more
efficient and higher yielding methodology to generate 3. As a result of these studies the cycloaddition
reaction was performed in a 5:1 mixture of hexane-ethyl acetate to both force the kinetic control of the
reaction and to solubilize the reagents. This procedure led to a 97% yield of the endo isomer after
1
recrystallization from hexane, as proven by its H-NMR spectrum.
Schemes 3 and 4 show some of the compounds chosen for chemical transformation and screening
for biological activity that were prepared from adduct 3. Compounds 9 and 10 (Scheme 3) were easily
obtained using described methods [3, 12]. As one may observe, the keto-alcohol 10 possesses
appropriate structural and chemical functionality for the insertion of new groups and building rigid
cage-like polycyclic molecules, in conformity with our initial plan.
2
,7
Scheme 3. Selective reductions of tricyclo[6.2.1.0 ]undeca-4,9-dien-3,6-dione (3).
O
O
O
Zn/AcOH
NaBH4
MeOH/H O
o
5
1
5-60 C
2 h
2
O
O
HO
5
h
9
10
3
(
85%)
(90%)
.

Molecules 2007, 12
274
The structures of compounds 3, 9 and 10 were confirmed by comparison of their spectroscopic data
with those reported in the literature [8, 12 and 3, respectively]. Next, and starting from compound 10,
five rigid polycyclic compounds were obtained, possessing various and diverse chemical
functionalities, as shown in Scheme 4.
Scheme 4. Syntheses of derivatives of keto-alcohol 10.
1
1
1
1
0
O
2
O
9
8
3
7
4
O
O
O
6
15
5
NaH, THF
reflux
MeO
12
O
MeO
MeO
MeO
O
OMe
Cl
Br
OMe
O
11
NBS
CH Cl
HO
+
2
2
Py/toluene
reflux, 24 h
o
0
C, 2 h
O
10
14
O
NaBH MeOH
4,
o
0
C
r.t, 1 h
O
13
Br
OH
NaH, THF
O
O
O
reflux
16
17
Gallate 12 was prepared by submitting 10 to simple esterification in refluxing toluene with the acid
chloride 11 in the presence of pyridine. The structure and stereochemistry of the resulting ester 12
were assigned based on NMR spectral analyses, including HSQC, HMBC, COSY and NOESY
1
experiments. The H-NMR spectrum showed a multiplet at 5.52 ppm, assigned to the hydrogen
attached to C6, which is probably deshielded by the ester carbonyl. It also showed a multiplet (4H) at
1
.95-2.45 ppm, assigned to the two methylene groups at C4 and C5. The two doublets at 1.26 and 1.37
ppm (J = 8.4 Hz) correspond to the geminal methylene protons at C11. The broad signal at 3.24 ppm
was ascribed to the hydrogen attached to C1 and the signals of the protons on C2, C7 and C8 were
superimposed at around 2.91-3.03 ppm. The olefinic hydrogens of C9 and C10 appear as double-
doublets at 5.93 and 6.2 ppm, respectively, while the aromatic ring singlets and methoxyls are
observed at 7.21 and 3.83 ppm. A comparison of the NMR data of substance 12 with that of a similar
tricyclic ketoester described by Marchand et al., which contains an endo-6-OC(O)Ar group were Ar =
3
,5-dinitrophenyl [3], also confirmed the structure proposed for compound 12. Compound 13 was
obtained along with 12 (Scheme 4), probably due to the mild acid conditions of reaction mixture
favoring protonation of double bond followed by ring closure via the hydroxyl group [13] (Scheme 5).
With the purpose of verifying this proposal, we checked the stability of compound 10. We have
observed that 10 is stable in pure ethyl acetate, however, it is rapidly converted into 13 when a small
amount of p-toluenesulfonic acid is added to the solution.

Molecules 2007, 12
Scheme 5. Mechanistic proposal for the formation of compound 13.
275
H
H
O
O
H+
-H+
13
HO
HO
The structure of compound 13 was confirmed on the basis of its spectral data. In contrast to 10, no
1
olefinic proton signals were observed in the H-NMR spectrum. As main signals a multiplet at 4.22
ppm, ascribed to the hydrogen attached to C9, and the triplet at 4.41 ppm assigned to the hydrogen at
1
3
C6 were observed. The C-NMR data were in agreement with the proposal structure. The appearance
of C10 as an additional secondary carbon (CH
2
) at 35.1 ppm was decisive for confirmation of the 10
+
→
13 transformation. Moreover, the mass spectrum registered the expected molecular ion (M ) at m/z
=
178.
The bromoether 14 was obtained in quantitative yield from alcohol 10 by bromination with NBS in
dichloromethane [14]. Treatment of tetracycle 14 with NaH in THF under reflux induced abstraction
of the acidic proton at C2 and subsequent intramolecular cyclopropanation leading to formation of
compound 15. Its structure was confirmed by spectroscopic studies comprising 2D experiments
1
(
COSY, HSQC, HMBC and NOESY). The H-NMR spectrum showed the hydrogen at C6 as a
multiplet at 4.35 ppm, whereas the C9 hydrogen was a double doublet at 4.55 ppm. The hydrogen of
the cyclopropane ring (C10-H) is in a current shielding region and can be seen at 1.69 ppm, coupled
with the C9-H (J = 5.0 Hz). The triplet at 2.51 ppm was attributed to the C8-H that is coupled with the
1
3
C9-H and C7-H (J = 2.8 Hz). The C-NMR spectrum showed signals in accordance with the chemical
transformation performed, mainly at 21.8 (C1), 33.1 (C2) and 35.3 (C10) ppm (see Table 1). The mass
+
spectrum showed the molecular ion peak (M ) at m/z = 176.
When we attempted to prepare the highly strained compound 17 by reduction of the carbonyl in 14
from the convex face, followed by displacement of the bromide, only the known bromohydrin 16 [15]
was formed (Scheme 4). Even forcing conditions, such as treatment of 16 with NaH in THF under
reflux, failed to yield 17.
o
o
Bromohydrin 16 (m.p. 136-138 C; Lit. [15], m.p. 133-134 C), was characterized by means of its
1
13
spectroscopic data ( H- and C-NMR, IR) and mass spectrum (cf. Experimental Part). Its mass
+
spectrum showed molecular ion peak at m/z 258/260 and a (M -18) peak at m/z 240/242, due to loss of
one water molecule from the hexane ring system.
1
3
Table 1 list the experimental C-NMR data of compounds 13, 14, 15 and 16, allowing a rapid
comparison of the chemical shifts of all carbons of these molecules and supporting the main
2
,7
transformations carried out on the 6,9-epoxytricyclo[6.2.1.0 ]undecane ring system (Figure 2).
Figure 2.
1
1
1
1
0
2
3
9
8
7
4
O
6
5

Molecules 2007, 12
276
1
3
a
Table 1. C-NMR (CDCl
3
) spectral data of the compounds 13, 14, 15 and 16
14 16 15
b
Position
13
C
δ
C
δ
C
δ
C
δ
1
2
3
4
5
6
7
8
9
39.3
51.2
214.0
37.4
26.5
74.4
43.5
49.1
79.7
35.1
37.2
42.2
49.8
211.8
35.0
25.9
75.3
46.9
48.6
88.4
55.1
34.9
42.9
39.5
69.7
26.7
26.1
76.1
44.2
49.1
88.7
56.5
35.4
21.8
33.1
209.4
38.3
30.4
74.1
42.0
46.3
82.2
35.3
30.3
1
1
0
1
a
Data acquired at 75 MHz.
b
Confirmed by bidimensional NMR techniques. Signal multiplicity of
carbons in all compounds was assigned by DEPT-135 experiments.
Biological Evaluation
. Assay for cytotoxic activity
Cytotoxic analyses by the MTT method are used in the screening program of the U.S. National
1
Cancer Institute (NCI), which tests more than 10,000 samples per year [16]. It is a fast, sensitive and
cheap methodology, described for the first time by Mosman [17] in 1983 and subsequently modified in
1
996 by Alley et al. [18]. This evaluation allows one to easily determine the cytotoxicity of a
particular compound, but it does not provide any insight into the mechanism of action [19].
The cytotoxic activity of compounds 3, 9, 10, 12, 14, 15, 16 and 18 are listed in Table 2, with the
respective IC50 values. Among the eight samples analyzed, compound 3 showed moderate albeit
unspecific cytotoxicity, with IC values of 11.34, 13.62 and 13.52 µg/mL towards the HL-60, HCT-8
5
0
and MDA-MB 435 cell lines, respectively. Similarly, the novel gallate 12 was cytotoxic towards the
HCT-8, SF-295 and MDA-MB 435 cell lines, with IC50 values of 6.68, 7.58 and 13.51 µg/mL,
respectively.
Figure 3: Methyl gallate.
O
OMe
MeO
OMe
OMe
18

Molecules 2007, 12
277
On the other hand, methyl gallate 18 (Figure 3) did not show any cytotoxic effects on the cell
growth of the tested cell lines, suggesting that the cage structure present in 12 may play an important
role in any observed increase of activity. Compound 9 was noticeably cytotoxic towards the MDA-MB
4
35 cell line with an IC of 10.4 µg/mL. On the other hand, the other samples did not show important
50
cytotoxic effects.
Table 2. Cytotoxicity of compounds 3, 9, 10, 12, 14, 15, 16 and 18, based on
inhibition of tumor cell growth.
a
Cells
b
Compound
IC50 [µg/mL, confidence interval]
HL-60
HCT-8
SF-295
MDA-MB 435
3
9
11.34
13.62
(5.52 – 33.63)
> 25
> 25
> 25
> 25
7.58
13.52
(
8.68 – 14,80)
> 25
(11.57 – 15.80)
10.47
4.80 – 22.05)
> 25
(
1
0
2
4
5
6
8
> 25
> 25
> 25
> 25
> 25
> 25
> 25
1
1
1
1
1
6.68
13.51
(
4.22 – 10.56)
> 25
(6.12 – 9.39)
> 25
(11.16 – 16.37)
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
a
2
Cells were plated in 96-well plates incubated with a 5% CO atmosphere, at 37ºC, for 72h, in presence of
concentrations of pure compounds (0.39-25 µg/mL). Each concentration was tested in triplicate and the
analyses were performed in duplicate.
b
The IC50 corresponds to inhibitory concentration for 50% of cellular growth (95% Confidence Interval).
2
. Assay of hemolytic activity in Swiss mouse (Mus musculus) erythrocytes
In order to verify whether the observed cytotoxic and antimitotic activities are related to membrane
disruption, compounds were tested for their ability to induce lysis of mouse erythrocytes. The
erythrocyte membrane is a dynamic structure that can dictate significant changes in its interaction with
drugs [20, 21]. The results revealed the absence of hemolytic activity for compounds 3, 12, 9, 16 and
1
8 at the highest tested concentration (200 µg/mL), suggesting that both cytotoxic and antimitotic
activities were not related to the lytic properties or membrane instability induced by these compounds,
which may be probably caused by a more specific pathway. The others compounds were not tested.

Molecules 2007, 12
278
Conclusions
From the results presented it may be concluded that a polycyclic framework could be a useful
scaffold for generating new lead compounds with biological activity and further synthetic studies are
warranted, aimed at the preparation of hybrid compounds similar, for instance, to compound 12, which
represents a kind of hybrid between a polycyclic and a trimethoxy residue found in some cytotoxic
molecules such as podophyllotoxin and colchicine. Another topic of potential interest would be
molecular modeling studies aimed at revealing possible structural modifications that could be made in
the prepared compounds so they would be active in the receptor site(s) of proteins involved in the
structure-cytotoxicity activity relationships, such as tubuline.
Experimental Section
General
1
13
H- and C-NMR spectra were obtained at 300 and 75 MHz respectively on a Bruker AVANCE
DPX-300 spectrometer. Chemical shifts were referenced to TMS and coupling constants are given in
Hertz. Infrared (IR) spectra were obtained on a Perkin Elmer equipment model 783. Mass spectra were
recorded on a GC/Mass Spectrometer model QP5000 (Shimadzu). Solvents were purified before use
and chemical reagents were commercially available and used without previous treatment.
p-Benzoquinone was prepared by oxidation of hydroquinone [22]. Column chromatography was
performed using Merck 60 Å (70-230 mesh) silica gel, eluting with a hexane-ethyl acetate gradient.
TLC analyses employed Merck (60F254/0.2 mm) sílica gel plates Melting points were recorded on
Köffler equipment and are reported without correction.
Chemistry
2
,7
Synthesis of rel-(1S,2S,7R,8R)-tricyclo[6.2.1.0 ]undeca-4,9-dien-3,6-dione (3)
Cyclopentadiene (14.6 g, 221 mmol) was added to a solution of p-benzoquinone (22 g, 203 mmol)
o
in hexane-ethyl acetate (5:1), cooled at 0 C. The resulting mixture was stirred for 4 hours. Afterwards,
the solvent was removed under vacuum and the product was recrystallized from hexane to give the title
o
1
compound as yellow crystals: 97% yield; m.p.: 71-75 C; H-NMR (CDCl
3
) δ (ppm): 1.46 (dt, J =
8
6
1
.8/1.3 Hz, 1H), 1.55 (dt, J = 8.8/1.8 Hz, 1H), 3.20 (m, 2H), 3.50 (m, 2H), 6.09 (t, J = 1.8 Hz, 2H),
1
3
.60 (s, 2H); C-NMR (CDCl
36.6 (CH).
3
2
) δ (ppm): 43.4 (CH), 46.3 (CH), 49.8 (CH ), 67.7 (CH), 131.3 (CH),
5
,10
Synthesis of rel-(2S,3S,4S,5S,6S,10S,1R,9R)-2-bromine-3,6-epoxytricyclo[6.2.1.0 ]undecan-9-ol
16)
(
NaBH (0.034g, 0.89 mmol) was added to an ice-bath-cooled solution of compound 14 [14] (0.153
4
g, 0.6 mmol) in MeOH (1.6 mL). The ice bath was then removed and the reaction mixture was stirred
for 1 hour at room temperature, before being quenched by addition of a drop of acetic acid. The
2 2
mixture was extracted with CH Cl (3 x 2 mL) and washed with distilled water. The organic layer was

Molecules 2007, 12
279
dried over anhydrous MgSO
4
, filtered and the solvent was removed under vacuum. The product was
purified by flash chromatography on silica gel column using hexane-ethyl acetate (2:1) as eluent to
1
afford a white amorphous solid: 72% yield; m.p.: 136-138°C; H-NMR (CDCl ) δ (ppm): 1.36-1.42
3
(
m, 1H), 1.50 (d, J = 11 Hz, 1H), 1.59-1.67 (m, 3H), 1.97 (ddd, J = 17.8/6.5/3.2 Hz, 1H), 2.13 (d, J =
1 Hz, 1H), 2.30 (m, 1H), 2.36 (m, 1H), 2.61 (broad signal, -OH), 2.90 (m, 1H), 3.95 (dd, J = 17.8/7.8
1
1
3
Hz, 1H), 4.15 (m, 1H), 4.22 (d, J = 2.0 Hz, 1H), 4.54 (d, J = 5 Hz, 1H); C- NMR (CDCl ) δ (ppm):
3
2
6.1 (CH
2 2 2
), 26.7 (CH ), 35.4 (CH ), 39.5 (CH), 42.9 (CH), 44.2 (CH), 49.1 (CH), 56.5 (CH), 69.7
-1
+ 81
(
(
CH), 76.1 (CH), 88.7 (CH); IR (KBr) ν (cm ): 3269, 2975-2869; MS m/z: 260 [(M + 2) , Br], 258
M , Br), 242 [(M – H O + 2) , Br], 240 [(M – H O) , Br].
+
79
+
81
+ 79
2
2
2
,7
Synthesis of rel-(1S,3S,7S,2R,8R)-6-oxo-3-(3,4,5-trimethoxyphenylcarbonyloxy) tricyclo[6.2.1.0 ]-
2
,7
undec-9-ene (12) and rel- (1S,2S,6S,8S,7R,9R)-6,9-epoxytricyclo[6.2.1.0 ]undecan-3-one (13)
Under a nitrogen atmosphere, thionyl chloride (0.270 mL; 3.6 mmol) was added to a solution of
3
,4,5-trimethoxybenzoic acid (0.385 g; 1.8 mmol) in CH Cl (5.5 mL). The mixture was refluxed for 4
2 2
hours. After evaporation of the solvent under reduced pressure the acid chloride 11 was dissolved in
anhydrous toluene (5.5 mL). To this solution it was added compound 10 (0.318 g, 1.8 mmol) and
pyridine (5.0 mL) and the mixture was refluxed during 24 hours. The reaction was monitored by TLC
and after completion it was diluted with dichloromethane and the resulting solution was washed with a
4 2 4
saturated solution of CuSO . The organic layer was dried over anhydrous Na SO and concentrated
under reduced pressure. The products were purified by silica gel column chromatography using
1
hexane-ethyl acetate (1:1) as eluent. Compound 12: white solid: 15% yield; m.p.: 42-46 °C; H-NMR
(CDCl
3
) δ (ppm): 1.26 (d, J = 8.4 Hz, 1H), 1.37 (d, J = 8.4 Hz, 1H), 1.92-2.05 (m, 2H), 2.16-2.29 (m,
2
1
4
H), 2.91-3.03 (m, 3H), 3.24 (broad signal, 1H), 3.83 (s, 9H), 5.52 (m, 1H), 5.93 (dd, J = 5.3/2.7 Hz,
1
3
H), 6.12 (dd, J = 5.3/2.9 Hz, 1H), 7,21 (s, 2H); C-NMR (CDCl
3
2 2
) δ (ppm): 25.6 (CH ), 35.6 (CH ),
4.4 (CH), 45.0 (CH), 45.4 (CH), 49.6 (CH ), 51.7 (CH), 56.1 (CH
2
3
), 60.8 (CH ), 70.0 (CH), 106.8
3
(
CH), 125.0 (C), 135.4 (CH), 135.8 (CH), 142.2 (C), 152.9 (C), 165.1 (C), 211.5 (C=O). Compound
1
1
1
2
1
3: colorless oil: 9 % yield; H-NMR (CDCl
3
) δ (ppm): 1.31 (d, J = 10.4, 1H), 1.35 (d, J = 3.2 Hz,
H), 1.44 (m, 1H), 1.50 (m, 1H), 1.82 (tdd, J = 13.0/13.0/5.4/1.3 Hz, 1H), 2.12 (m, 1H), 2.19 (m, 1H),
.26 (d, J = 5.4 Hz, 1H), 2.37-2.58 (m, 3H), 2.89 (t, J = 4.0 Hz, 1H), 4.22 (m, 1H), 4.40 (t, J = 6.3 Hz,
1
3
H); C-NMR (CDCl
3
) δ (ppm): 26.5 (CH
2
), 35.1 (CH
2
), 37.2 (CH
2
), 37.4 (CH
2
), 39.3 (CH), 43.5
+
(CH), 49.1 (CH), 51.2 (CH), 74.4 (CH), 79.7 (CH), 214.0 (C=O); MS m/z: 178 [M ].
2
,7 2,10
Synthesis of 6,9-epoxytetracyclo[6.2.1.0 .0 ]undecan-3-one (15)
A solution of compound 14 (0.60 g, 2.3 mmol) in THF (1.6 mL) was added to a mixture of 60%
NaH (0.11 g, 2.8 mmol) cooled in water-ice bath. The mixture was then stirred for 1 hour at room
temperature and 6 hours under reflux before extraction with dichloromethane (3 x 2 mL). The organic
4
layer was dried over MgSO , filtered and the solvent was removed on a rotavapor. The product was
purified by silica gel column chromatography using hexane-ethyl acetate (2:1) as eluent: 76% yield.
1
H-NMR (CDCl
3
) δ (ppm): 1.69 (ddd, J = 5.0/1.5/0.7 Hz, 1H), 1.73 (d, J = 11.3 Hz, 1H), 1.79 (d, J =
1
1.3 Hz, 1H), 2.03 (m, 1H), 2.30-2.44 (m, 5H), 2.51 (t, J = 2.8 Hz, 1H), 4.35 (m, 1H), 4.55 (dd, J =

Molecules 2007, 12
280
1
3
5
3
.0/2.8 Hz, 1H); C-NMR (CDCl
8.3 (CH
3
) δ (ppm): 21.8, (CH), 30.3 (CH
2
), 30.4 (CH
2
), 33.1 (C), 35.3 (CH),
+
2
), 41.8 (CH), 46.3 (CH), 74.1 (CH), 82.2 (CH), 209.4 (C=O); MS m/z: 176 [M ].
Biological Evaluation
1
. Assay for cytotoxic activity
The cytotoxicity of the compounds was tested against HL-60 (human leukemia), HCT-8 (human
colon), SF-295 (human central nervous system) and MDA-MB 435 (human breast cancer) cell lines
obtained from the National Cancer Institute, Bethesda, MD, USA. Cells were grown in RPMI-1640
medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 µg/mL streptomycin and
1
00 µg /mL penicillin and incubated at 37 °C with a 5% CO atmosphere. Cells were plated in 96-well
2
5
5
plates (10 cells/well for adherent cells or 0.5 x 10 cells/well for suspended cells in 100 µL of
medium). After 24 h, the compounds (0.39 to 25.0 µg/mL) dissolved in DMSO 1% were added to each
well and incubated for 72 h. Control group received the same amount of DMSO. Doxorubicin
®
(
Doxolem , Zodiac Produtos Farmacêuticos S/A, Brazil) was used as positive control. Tumor cell
growth was quantified by the ability of living cells to reduce the yellow dye 3-(4,5-dimethyl-2-
thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to a purple formazan product [17]. At the end
of the incubation, the plates were centrifuged and the medium was replaced by fresh medium (200 µL)
containing 0.5 mg/mL MTT. Three h later, the MTT formazan product was dissolved in DMSO (150
µL), and absorbance was measured using a multiplate reader (Spectra Count, Packard, Ontario,
Canada). The drug effect was quantified as the percentage of the absorbance of reduced dye at 550 nm
in relation to control wells.
2
. Swiss mouse (Mus musculus) erythrocyte hemolysis assay
The test was performed in 96-well plates following the method described by Costa-Lotufo et al.
[
2
23]. Each well received 100 µL of 0.85% NaCl solution containing 10mM CaCl . The first well was
the negative control that contained only the vehicle (DMSO 10%), and, in the second well, test
substance (100 µL) half was added. The compounds were tested at concentrations ranging from 3.9 to
2
00 µg/mL. The serial dilution continued until the 11th well. The last well received 0.1% Triton X-100
in 0.85% saline (20 µL) to produce 100% hemolysis (positive control). Then, each well received a 2%
suspension of mouse erythrocytes in 0.85% saline containing 10 mM CaCl (100 µL). After incubation
2
at room temperature for 30 min and centrifugation, the supernatant was removed and the hemoglobin
liberated was measured spectroscopically as absorbance at 540 nM.
Statistical Analysis
The IC50 values and their 95% confidence intervals (CI 95%) were obtained by nonlinear regression
using the GRAPHPAD program (Intuitive Software for Science, San Diego, CA, U.S.A.). For the
hemolytic assay, the differences were analyzed by ANOVA followed by Student Newman Keuls test
compared to negative control at a significance level of 5%.

Molecules 2007, 12
281
Acknowledgements
We are thankful to the Fundação de Apoio ao Desenvolvimento do Ensino, Ciência e Tecnologia do
Estado de Mato Grosso do Sul (FUNDECT) and the Financiadora de Estudos e Projetos (FINEP) for
financial support in the form of grants and fellowship awards. We also thank to Kardol Indústria
Química Ltda., for providing solvents and reagents and Professor Dr. Cláudio F. Tormena (Unicamp)
for access to the GC/Mass Spectrometer.
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Sample Availability: Samples of all compounds are available from authors.
2007 by MDPI (http://www.mdpi.org). Reproduction is permitted for noncommercial purposes.
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