Synthesis and cytotoxic activity of α-santonin derivatives

Article abstract of DOI:10.1016/j.ejmech.2009.03.036

Ten α-santonin derivatives were synthesized in moderate to high yields. Four derivatives namely 10α-acetoxy-3-oxo-1,7αH,6,11βH-guai-4-en-6,12-olide (2), isofotosantonic acid (3), 10α-hydroxy-3-oxo-1,7αH,6,11βH-guai-4-en-6,12-olide (4), and lumisantonin (5

Full text of DOI:10.1016/j.ejmech.2009.03.036

European Journal of Medicinal Chemistry 44 (2009) 3739–3745
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and cytotoxic activity of
a
-santonin derivatives
a
a
,
a
a
*
Francisco F.P. Arantes , Luiz C.A. Barbosa , Elson S. Alvarenga , Ant oˆ nio J. Demuner ,
b
b
b
b
b
Daniel P. Bezerra , Jos e´ R.O. Ferreira , Let ı´ cia V. Costa-Lotufo , Cl a´ udia Pessoa , Manoel O. Moraes
a
Department of Chemistry, Federal University of Viçosa, Av. P.H. Rolfs, S/N, CEP 36570-000 Viçosa, MG, Brazil
Department of Physiology and Pharmacology, Federal University of Cear a´ , Rua Coronel Nunes de Melo 1127, CEP 60431-970 Fortaleza, CE, Brazil
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 31 July 2008
Received in revised form
Ten
acetoxy-3-oxo-1,7
,7 H,6,11 H-guai-4-en-6,12-olide (4), and lumisantonin (5), were prepared by different photochemical
reactions using -santonin (1) as starting material. These transformations were carried out in either
anhydrous acetic acid, acetic acid/water (1:1 v/v) or acetonitrile, using different types of reactors and
ultraviolet light sources. Treatment of -santonin (1) with lithium diisopropyl amide (LDA) followed by
capture of the organolithium with phenyl selenium chloride produced the compound 3-oxo-7 H,6 H,11-
phenylselenyl)-eudesma-1,4-dien-6,12-olide (6). Subsequent treatment of compound 6 with hydrogen
peroxide gave 3-oxo-7 H,6 H-eudesma-1,4,11-trien-6,12-olide (7). Photochemical reaction of compound
7 led to the formation of 11,13-dehydrolumisantonin (8) and 10 -acetoxy-3-oxo-1,7 H,6 H-guai-4,
11-dien-6,12-olide (9). Sodium borohydride reduction of compounds 2 and 4 afforded the derivatives
-acetoxy-3 -hydroxy-1,7 H,6,11 H-guai-4-en-6,12-olide (10) and ,10 -hydroxy-1,7 H,6,11 H-
guai-4-en-6,12-olide (11).
The cytotoxicity of the synthesized compounds were evaluated against the cancer cell lines HL-60
a
-santonin derivatives were synthesized in moderate to high yields. Four derivatives namely 10
a-
a
H,6,11 H-guai-4-en-6,12-olide (2), isofotosantonic acid (3), 10 -hydroxy-3-oxo-
b
a
1
a
b
2
March 2009
a
Accepted 26 March 2009
Available online 5 April 2009
a
a
b
Keywords:
(
a
-Santonin derivatives
a
b
Photochemistry
Sesquiterpene lactones
Cytotoxicity
a
a
b
10
a
b
a
b
3
b
a
a
b
(
(
leukemia), SF-295 (central nervous system), HCT-8 (colon), MDA-MB-435 (melanoma), UACC-257
melanoma), A549 (lung), OVACAR-8 (ovarian), A704 (renal), and PC3 (prostate). The compounds with
higher activity, possessing IC50 values in the range of 0.36–14.5
mM, showed as common structural
feature the presence of an -methylidene- -butyrolactone moiety in their structures. The biological
a
g
assays conducted with normal cells (PBMC) revealed that the compounds are selective against cancer cell
lines. The modified lactones seem to be interesting lead structures towards anticancer drug
development.
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
known for their various biological activities such as anti-inflam-
matory, phytotoxic, antimicrobial, antiprotozoal, and, cytotoxicity
According to the World Health Organization (WHO), cancer is an
important health problem that claims the lives of more than seven
million people worldwide on an annual basis [1]. As a consequence,
search for new anticancer drugs is highly demanding nowadays.
However, cytotoxic agents have very little or no specificity, which
leads to systemic toxicity, causing undesirable side effects. There-
fore, the development of innovative and efficacious tumor-specific
drug delivery protocols or systems is urgently needed [2,3]. Within
this framework, the natural products continue to be a rich source of
new promising substances [4]. Sesquiterpene lactones (SQLs) are
a class of naturally occurring plant terpenoids of Asteraceae family,
against different tumor cell lines [5–9].
These different activities have been linked mainly to the
a-methylene-g-lactone functionality, which is prone to react with
suitable nucleophiles, e.g., sulfhydryl groups of cysteine, in a Michael
addition fashion. These reactions are nonspecific, leading to the
inhibition of a large number of enzymes or factors involved in key
biological processes [10–15]. It is well known, however, that the
a-methylene-g-lactone moiety is not an absolute requirement for
cytotoxicity. It has been demonstrated that thiols do undergo
‘‘Michael-type’’ additions not only to the exocyclic methylene of
sesquiterpene lactones but also to the
pentenone moiety [12].
a,b-unsaturated cyclo-
The precise mechanism by which SQLs inhibit the cell growth
remains unclear. Several experimental data indicate that cytotoxic
activity of these compounds is strongly related to their inhibitory
*
Corresponding author. Tel.: þ55 31 3899 3068; fax: þ55 31 3899 3065.
E-mail address: lcab@ufv.br (L.C.A. Barbosa).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.03.036

3740
F.F.P. Arantes et al. / European Journal of Medicinal Chemistry 44 (2009) 3739–3745
1
effect on many thiol-containing enzymes, involved in the synthesis
and processing of proteins, RNA and DNA [16–18]. In addition,
sesquiterpene lactones exert their cytotoxic effects by triggering
apoptosis in many types of cell lines [19,20].
It was recently found that a sesquiterpene lactone, parthenolide,
can selectively kill primitive leukemia cells without affecting
normal stem and progenitor hematopoietic cells [21]. These data
indicate that SQLs and related compounds may represent a prom-
ising class of antileukemic agents.
characterized by IR, H and 13C NMR spectrometry as well as mass
spectrometry.
To prepare compound 2, we utilized a reaction previously
described in the literature [28–32]. Thus, irradiation of
(1) with high pressure mercury lamp, using anhydrous acetic acid
as solvent in borosilicate reactor, afforded 10 -acetoxy-3-oxo-
1,7 H,6,11 H-guai-4-en-6,12-olide (2) in 26% yield. The infrared
spectrum of compound 2 showed a strong absorption at 1729 cm
due to C]O stretching of the acetyl group. The signals at 170.35
2.00 in the C and H NMR spectra respectively, confirmed
a-santonin
a
a
b
ꢀ1
d
C
1
3
1
In our continuous effort to discover novel cytotoxic compounds
and d
H
[22–26], we describe herein the preparation of several derivatives
the presence of the acetyl group.
from -santonin (1), a sesquiterpene lactone isolated from Arte-
misia santonica [27]. The evaluation of the cytotoxic effects of these
a
Using acetic acid/water mixture (1:1 v/v), keeping the
other conditions unchanged, isofotosantonic acid (3) and the
lactones against tumor and normal cells is also described.
10a-hydroxy-3-oxo-1,7aH,6,11bH-guai-4-en-6,12-olide (4) were
obtained in 44% and 32% yields, respectively. It should be pointed
out that Barton and co-workers [28] carried out a similar reaction
irradiating compound 1 in a mixture of acetic/water (9:11 v/v),
from ꢀ5 C to þ5 C, for 90 min. In this case, compounds 3 and 4
were obtained in 16% and 18% yields, respectively. In addition,
Greene and Edgar [33] reported the isolation of compound 4
exclusively, in 31% yield, running the reaction in a mixture of
2
. Results and discussion
ꢁ
ꢁ
2.1. Synthesis
The sesquiterpene lactones 2–11 were synthesized by a series of
reactions as depicted in Scheme 1. The compounds were fully
O
(
8)
hv
O
Hg-BP/RQ
O
O
anhydrous AcOH
OAc
O
(7)
O
O
H2O2 30%
O
(
9)
O
SePh
O
(6)
O
O
PhSeCl
LDA
HOOC
(3)
O
OAc
OAc
O
hv
hv
Hg-AP/RB
NaBH4
HO
Hg-AP/RB
O
OH
anhydrous AcOH
AcOH/H O 1:1
O
anhydrous MeOH
2
O
O
O
O
(1)
O
(
10)
(
2)
O
O
O
hv
(4)
Hg-BP/RQ (a, b)
O
MeCN
NaBH4
anhydrous MeOH
OH
O
HO
O
(5)
O
O
(11)
O
Scheme 1. Synthesis of lactone derivatives of a-santonin (1).

F.F.P. Arantes et al. / European Journal of Medicinal Chemistry 44 (2009) 3739–3745
3741
acetic acid/water (7:8 v/v) in a quartz reactor under refluxing
3. Conclusion
conditions for 6.5 h.
The photochemical reaction of
a
-santonin (1) in a quartz
The readily available
a-santonin (1) was used as starting mate-
reactor, using low pressure mercury lamp as source of ultraviolet
radiation and acetonitrile as solvent, gave lumisantonin (5) in 83%
yield.
rial for the preparation of ten sesquiterpene lactone derivatives
which were evaluated against cancer cells. Compounds 7–9, pos-
sessing the
a-methylidene-g-butyrolactone group in their struc-
Compounds 2 and 4 were further submitted to reduction
ture, displayed significant cytotoxic activities. Thus, the results
obtained reinforce the fact that the presence of such structural
motif has a significant role in the mechanism by which these
compounds exert their biological activities.
reactions with sodium borohydride, affording 10
a
-acetoxy-3
b
-
-
hydroxy-1,7
hydroxy-1,7
a
a
H,6,11
H,6,11
b
b
H-guai-4-en-6,12-olide (10) and the 3 ,10a
b
H-guai-4-en-6,12-olide (11) in 86% and 72%
yields, respectively. The stereochemistry of the resulting alcohols
was proposed on the assumption of a preferred attack of the
hydride from the less hindered side of the carbonyl group [31]. The
infrared spectra of compounds 10 and 11 showed a broad band at
The biological assays revealed that compounds 7–9 are selective
against normal cell lines (PBMC), an important feature towards the
development of new drugs against cancer. These SQLs may repre-
sent a promising class of anticancer agents.
ꢀ1
3
400 cm
observed around
around 78.00 in the C NMR spectrum, along with other signals,
associated with the O–H stretching. The multiplets
1
d
H
4.50 in the H NMR spectrum and the signals
4. Experimental part
13
d
C
helped to confirm the identity of the synthesized alcohols 10
and 11.
4.1. Synthesis
The 3-oxo-7
obtained in 72% yield after reaction of
selenium chloride (PhSeCl), in the presence of lithium diisopropyl
a
H,6
b
H-eudesma-1,4,11-trien-6,12-olide (7) was
All reactions were carried out under a protective atmosphere of
dry nitrogen. Methanol, acetic acid, diisopropylamine, tetrahydro-
furan and acetonitrile were purified as described by Perrin and
a-santonin (1), with phenyl
amide (LDA), followed by treatment of the selenyde 6 with
Armarego [34]. Commercial a-santonin (1) was purchased from
1
hydrogen peroxide (H
2
O
2
). In the H NMR spectrum of compound 7
Aldrich (Milwaukee, WI, USA) and utilized without further purifi-
cation. Infrared spectra were recorded on a Perkin Elmer Paragon
1000 FTIR spectrophotometer, using potassium bromide (1% w/w)
H H
the presence of a pair of doublets at d 5.56 and d 6.24, corre-
sponding to hydrogens of the exocyclic double bond, assisted in
confirming the formation of this substance. Subsequently, lactone 7
was submitted to a photochemical reaction in a quartz reactor,
using anhydrous acetic acid as solvent, yielding compounds 11,13-
ꢀ1
disk scanning from 500 to 4000 cm . Flash column chromatog-
raphy was performed using Crosfield Sorbil C60 silica gel (32–
63 mm). Analytical thin layer chromatography analyses were
dehydrolumisantonin (8) and 10
,11-dien-6,12-olide (9) in 18% and 4.5% yields, respectively. The
rational for the preparation of these derivatives 7–9 was the fact
that many sesquiterpene with -methylidene- -butyrolactone
a
-acetoxy-3-oxo-1,7
a
H,6
b
H-guai-
conducted on precoated silica gel plates. Melting points were
determined on an electrothermal digital apparatus model MQAPF-
301 (Microquimica, Brazil), without correction. The H and C NMR
spectra were recorded on Brucker AVANCE DRX 400 spectrometer
4
1
13
a
g
moiety in their structure have displayed cytotoxicity against several
tumor cell lines [10–14].
3
at 400 and 100 MHz respectively using CDCl as solvent and TMS as
internal standard. Low resolution mass spectra were obtained on
SHIMADZU GCMS-QP5050A instrument by direct injection using
ꢁ
the following temperature program: 40 C/min until temperature
ꢁ
ꢁ
ꢁ
2.2. Cytotoxicity assay
reaches 60 C; then 80 C/min until temperature reaches 300 C;
ꢁ
detector temp: 280 C. Values are reported as a ratio of mass to
The cytotoxicity of the sesquiterpene lactones 2–11 was evalu-
charge (m/z) in Daltons and relative intensities are quoted as
ated against HL-60 (leukemia), SF-295 (central nervous system),
HCT-8 (colon), MDA-MB-435 (melanoma), UACC-257 (melanoma),
A549 (lung), OVACAR-8 (ovarian), A704 (renal), and PC3 (prostate)
human cancer cell lines, using a previously described MTT assay
a percentage value. HRMS data were recorded under conditions of
chemical ionization (CI) on
lution ¼ 10,000 FWHM) in CI mode using NH
a Fisons Autospec-oaTof (reso-
þ
3
as the ionization
gas.
[20]. Doxorubicin was used as positive control. Table 1 summarizes
the IC50 data ( M) for cytotoxic activity. The results indicated that
m
4.1.1. 10a-Acetoxy-3-oxo-1,7aH,6,11bH-guai-4-en-6,12-olide (2)
compounds 7–9 exhibited relatively high cytotoxicity against all
A solution of
a-santonin (0.50 g, 2.0 mmol) in anhydrous acetic
tumor cell lines tested, with IC50 values in the range of 0.36–
acid (120 mL) was degassed with nitrogen for 30 min. The solution
was then irradiated under high pressure mercury lamp (125 W) for
23 h in a pyrex vessel. The solvent was evaporated under reduced
pressure to give a yellow oil which was dissolved in hot methanol
14.5 mM. Cytotoxic effectiveness of these compounds was compa-
rable to the well-known sesquiterpene lactones, parthenolide and
helenalin [16,19]. Compound 5 exhibited low cytotoxic activity with
ꢁ
IC50 values in the range of 49.84–91.46
investigated were not able to significantly inhibit cell growth under
assay conditions (IC50 > 100 M).
mM. The other compounds
and left in a freezer at ꢀ5 C for 2 h. The white crystals formed were
filtered under reduced pressure and washed with cold methanol to
yield 24.3 mg (0.08 mmol) of lactone (2). The filtrate was purified
by flash column chromatography eluted with hexane/ethyl acetate
(3:2 v/v) yielding a further 139.7 mg (0.46 mmol) of compound 2.
The yield of this reaction was 26% (164 mg; 0.54 mmol).
m
The cytotoxicity of compounds 7–9 was also evaluated against
normal cells (PBMC). The results presented in Table 1 show that the
effects of these lactones are less pronounced in normal cells.
Although the precise mechanism of action of sesquiterpene
lactones as inhibitors of cell growth is still unclear, several exper-
imental data indicate that cytotoxic activity of the more active
compounds herein investigated can be related to the presence of
ꢁ
ꢀ1
mp ¼ 176.6–177.8 C; IR (KBr,
n
max/cm ): 2976, 2934, 2876, 1783,
1
1729, 1707, 1645, 1459, 1371, 1246, 1178, 1045, 982, 969; H NMR
(400 MHz, CDCl
1.44–1.51 (m, 1H, H8 ), 1.91 (s, 3H, H15), 2.00 (s, 3H, CH
2.11 (m, 1H, H8), 2.15–2.25 (m, 2H, H7 and H9 ), 2.33 (dq, 1H,
3
):
d
1.09 (s, 3H, H14), 1.29 (d, 3H, J13,11 ¼6.7, H13),
0
3
CO), 2.06–
0
the
a
-methylidene-
g-butyrolactone moiety in their structure. It is
possible that the
addition reaction with bionucleophiles, especially thiol groups of
cysteine [14].
a
-santonin derivatives can react via Michael
J
11,7 ¼ 12.2, J11,13 ¼ 6.7, H11), 2.41 (dd, 1H, J2,2
0
¼ 19.4, J2,1 ¼ 2.1, H2),
0
2.50 (dd, 1H, J
2
0
,2 ¼ 19.4, J
2
0
,1 ¼6.2, H2 ), 2.62 (td, 1H, J9,9
0
¼ 13.6,
1
3
J9,8 ¼ J9,8
0
¼ 4.1, H9), 4.16 (m, 1H, H1), 4.80 (d, 1H, J6,7 ¼ 10.6, H6);
C

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F.F.P. Arantes et al. / European Journal of Medicinal Chemistry 44 (2009) 3739–3745
Table 1
Cytotoxic activity (IC50
[
mM]) of lactones derivatives of
a
-santonin.
Cell lines
Lactones
Doxorubicin
1
2
3
4
5
6
7
8
9
10
11
Tumor cells
HL-60
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
80.16
>100
>100
>100
>100
>100
>100
>100
>100
>100
1.14
0.23–2.77
2.70
0.67–1.90
2.92
0.98–4.86
6.74
2.30
1.87–2.84
2.15
1.90–2.40
1.96
1.64–2.29
4.57
3.26–5.32
4.87
4.32–5.12
14.50
14.01–14.87
3.88
3.12–4.21
3.61
1.60
1.09–2.35
3.06
2.52–3.70
0.36
0.16–0.79
8.84
5.62–10.23
7.76
5.56–8.23
10.23
9.53–11.56
8.67
8.01–8.98
9.24
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.04
0.03–0.05
0.48
0.34–0.72
0.02
0.02–0.03
0.96
0.68–1.32
0.60
0.51–0.70
0.72
0.54–1.54
0.51
0.26–0.96
2.00
65.68–91.31
SF-295
HCT-8
68.78
5
3.37–78.66
59.19
6.54–65.25
91.46
2.23–99.6
4
MDA-MB-435
UACC-257
A549
8
5.56–7.21
6.05
>100
5.26–6.56
69.35
13.43
12.56–13.98
6.94
6.53–7.26
10.14
9.23–11.23
13.04
5
5.26–74.23
49.84
5.55–56.28
85.43
9.23–95.23
66.45
9.29–75.37
OVACAR8
A704
4
6
3.01–3.89
5.07
4.85–5.79
8.56–10.23
14.41
14.02–14.97
1.46–2.72
1.32
0.62–1.56
PC3
5
12.56–13.78
Normal cells
PBMC
Nd
Nd
Nd
Nd
Nd
Nd
3.23
13.36
13.45
Nd
Nd
1.77
1.64–5.33
5.74–29.10
5.92–29.60
0.92–3.13
Doxorubicin was used as positive control. Data are presented as IC50 values and 95% confidence interval from two independent experiments, performed two times. Nd: not
determined.
0
NMR (100 MHz, CDCl
3
):
CO), 25.37 (C8), 36.83 (C2), 37.95 (C9), 41.38 (C11), 47.27 (C1),
8.28 (C7), 81.25 (C6), 85.55 (C10), 143.30 (C4), 160.82 (C5), 170.35
d
9.49 (C15), 12.46 (C13), 20.04 (C14), 22.29
(s, 3H, H14), 1.29 (d, 3H, J13,11 ¼6.9, H13), 1.42–1.47 (m, 1H, H8 ),
0
(
CH
3
1.75–1.85 (m, 1H, H9 ), 1.90 (s, 3H, H15), 2.00–2.25 (m, 3H, H7, H8,
4
H9), 2.32 (dq, 1H, J11,7 ¼ 12.2, J11,13 ¼ 6.9, H11), 2.54 (dd, 1H,
þ
ꢂ
0
(
CH
3
CO), 177.03 (C12), 207.00 (C3); MS, m/z (%): 306 [M ], (0.4),
J
2
0
,2 ¼ 19.7, J
2
0
,1 ¼5.9, H2 ), 2.60 (dd, 1H, J2,2
0
¼19.7, J2,1 ¼3.0, H2),
13
2
46 (100), 231 (51), 203 (15), 190 (25), 173 (64), 145 (34), 121 (18),
3.23 (m, 1H, H1), 4.82 (d, 1H, J6,7 ¼ 11.0, H6); C NMR (100 MHz,
CDCl ): : 9.45 (C15), 12.50 (C13), 21.32 (C14), 25.89 (C8), 37.17 (C2),
41.44 (C11), 45.38 (C9), 48.51 (C7), 50.51 (C1), 74.46 (C10), 81.49
C6), 143.12 (C4), 161.30 (C5), 177.15 (C12), 207.71 (C3); MS, m/z (%):
1
(
05 (33), 91 (61), 77 (47), 55 (95), 53 (40). HRMS (CI) C17
H
23
O
5
3
d
þ
MH ) requires 307.1540, found 307.1542.
(
þ
ꢂ
4
1
.1.2. Isofotosantonic acid (3) and the 10
,7 H,6,11 H-guai-4-en-6,12-olide (4)
A solution of -santonin (2.0 g, 8.13 mmol) in a mixture of water
80 mL) and acetic acid (80 mL) in a pyrex vessel was degassed by
a
-hydroxy-3-oxo-
264, [M ], (82), 221 (12), 206 (22), 193 (55), 169 (31), 149 (31), 133
(44), 105 (38), 91 (51), 77 (48), 55 (100), 53 (50). HRMS (CI)
a
b
þ
a
C
15
H
21
O
4
(MH ) requires 265.1434, found 265.1439.
(
a flow of nitrogen for 30 min. The reaction mixture was then irra-
diated with a high pressure mercury lamp (125 W) for 27 h. The
solvent was evaporated under reduced pressure (60 C) to give
4.1.3. Photochemical synthesis of lumisantonin (5)
A solution of -santonin (2.0 g, 8.13 mmol) in acetonitrile
a
ꢁ
(120 mL) in a quartz tube was degassed by a flow of nitrogen for
30 min. The reaction mixture was then irradiated under four low
pressure mercury lamps (4 ꢃ15 W) for 2 h. The solvent was evap-
a yellow oil which was purified by flash column chromatography
(
3
hexane/ethyl acetate 1:2 v/v) yielding (3) as a white solid (0.95 g,
.6 mmol) in 44% yield and compound 4, also as a white solid
ꢁ
orated under reduced pressure (40 C) to give a yellow solid which
(
0.687 g, 2.6 mmol) in 32% yield.
was recrystallized in a mixture of acetone and hexane (1:2 v/v) to
ꢁ
ꢀ1
Data for 3. mp ¼ 145.8–147.0 C; IR (KBr,
n
max/cm ): 2979, 2931,
give 5 as yellow crystals (0.416 g, 1.69 mmol) in 83% yield.
ꢁ
ꢀ1
2
600–3400, 1782, 1709, 1654, 1455,1373, 1240, 1182, 1137, 1007, 875,
mp ¼ 145.8–147.3 C; IR (KBr,
n
max/cm ): 2933, 2876, 1782, 1699,
1
1
7
1
(
2
43, 622; H NMR (400 MHz, CDCl
.35 (dddd, 1H, J7a,7e y J7a,8a y J7a,6 y 10.8, J7a,8e ¼ 4.1, H7a), 1.63
s, 3H, H14), 1.77 (s, 3H, H15), 1.78–1.88 (m, 2H, H6 and H8a), 2.00–
.06 (m, 1H, H7e), 2.34 (dq, 1H, J11,6 ¼ 12.6, J11,13 ¼ 6.9, H11), 2.85
3
):
d
1.23 (d, 1H, J13,11 ¼6.9, H13),
1570, 1456, 1252, 1027, 998, 836; H NMR (400 MHz, CDCl
3
):
d
1.11
(s, 3H, H14), 1.12–1.21 (m, 1H, H8a), 1.22 (s, 3H, H15), 1.25 (d, 1H,
13,11 ¼6.9, H13), 1.57–1.69 (m, 1H, H7), 1.79–1.98 (m, 3H, H8e, H9e,
H9a), 2.30 (dq, 1H, J11,7 ¼ 12.3, J11,13 ¼ 6.9, H11), 3.81 (d, 1H,
6,7 ¼ 10.8, H6), 6.01 (d, 1H, J3,4 ¼ 5.7, H3), 7.59 (d, 1H, J4,3 ¼ 5.7, H4);
J
(
ddd, 1H, J8e,8a ¼ 13.8, J8e,7a ¼ 4.1, J8e,7e ¼ 2.3, H8e), 2.98 (ddd, 1H,
J
1
3
J
J
J
2,2
0
¼ 17.4, J2,3 ¼ 7.8, J2,5 ¼1.2, H2), 3.03 (ddd, 1H, J
2
0
,2 ¼17.4,
3
C NMR (100 MHz, CDCl ): d 7.68 (C15), 12.76 (C13), 17.45 (C14),
0
0
¼ 6.5, J
0
¼ 2.0, H2 ), 4.12 (dc, 1H, J5,6 ¼ 10.8, H5), 5.67 (ddd, 1H,
22.77 (C8), 29.88 (C9), 40.66 (C5), 41.48 (C11), 42.97 (C1), 48.97
(C7), 50.38 (C10), 77.89 (C6), 131.68 (C3), 157.99 (C4), 178.88 (C12),
2
3
2
5
1
3
3,2 ¼ 7.8, J3,2
0
¼ 6.5, J3,5 ¼1.4, H3). C NMR (100 MHz, CDCl
12.44 (C13), 19.97 (C15), 22.14 (C14), 27.43 (C7), 30.39 (C8), 33.74
C2), 42.22 (C11), 54.20 (C6), 83.36 (C5), 110.40 (C3), 127.56 (C9),
30.84 (C10),140.21 (C4),177.50 (C1),178.60 (C12); MS, m/z (%): 264
3
):
þ
ꢂ
d
206.91 (C2); MS, m/z (%): 246 [M ], (35), 173 (49), 145 (24), 135
þ
(
1
(60), 107 (38), 91 (81), 77 (52), 55 (100). HRMS (CI) C15
requires 247.1329, found 247.1333.
19
H O
3
(MH )
þ
ꢂ
[M
], (32), 246 (26), 218 (15), 191 (90), 175 (23), 145 (32), 131(47),
1
05 (33), 91 (61), 77 (38), 55 (100), 53 (35). HRMS (CI) C15
H
21
O
4
4.1.4. 3-Oxo-7aH,6bH,11-(phenylselenyl)-eudesma-1,4-dien-6,
12-olide (6)
þ
(
MH ) requires 265.1434, found 265.1437.
ꢁ
ꢀ1
Data for 4. mp ¼ 162.4–163.2 C; IR (KBr,
n
max/cm ): 3449,
To
a
mixture of anhydrous diisopropylamine (1.9 mL,
3
062, 2971, 2928, 2857, 1777, 1699, 1641, 1458, 1311, 1209, 1177,
13.5 mmol) and anhydrous THF (10 mL) in a two necked round
1
ꢁ
1100, 1055, 991, 735, 707, 629; H NMR (400 MHz, CDCl
3
):
d
0.97
bottomed flask under nitrogen atmosphere at ꢀ78 C, was added

F.F.P. Arantes et al. / European Journal of Medicinal Chemistry 44 (2009) 3739–3745
3743
n-BuLi (14.2 mL, 13.6 mmol). The mixture was stirred for 30 min
and -santonin (3.0 g, 12.2 mmol) in anhydrous THF (35 mL) was
a white solid (0.035 g, 0.12 mmol) in 4.5% yield and compound 9 as
a
a yellow solid (0.12 g; 0.49 mmol) in 18%.
ꢁ
ꢀ1
n
max/cm ): 2929,
added. Phenyl selenium chloride (2.57 g, 13.4 mmol) in anhydrous
THF was added to the reaction mixture after 30 min and stirred for
Data for 8. mp ¼ 153.1–154.2 C; IR (KBr,
2876, 1776, 1698, 1568, 1381, 1258, 1134, 1039, 1073, 976, 838, 689;
ꢁ
1
a further 20 min at ꢀ78 C. Distilled water (30 mL) was added to
H NMR (400 MHz, CDCl
H15), 1.30–1.40 (dddd, 1H,
8a,9e ¼ 2.6, H8a), 1.88–2.00 (m, 2H, H9a, H9e), 2.14–2.21 (m, 1H,
3
) d (J/Hz): 1.17 (s, 3H, H14), 1.25 (s, 3H,
ꢁ
the mixture at 25 C; the resulting mixture was transferred to
J8a,8e y J8a,9a y J8a,7 y J6,7 ¼ 11.2,
a separatory funnel, and extracted with DCM (3 ꢃ 30 mL). The
combined organic layers were washed with brine (30 mL), dried
with anhydrous magnesium sulphate, filtered, and concentrated
under reduced pressure. The residue was purified by flash column
chromatography eluted with hexane/ethyl acetate (3:2 v/v), and
recrystallized with hexane/ethyl acetate (2:1 v/v) to afford
compound 6 as white crystals (1.9 g; 4.72 mmol; 39%). mp ¼ 196.9–
J
H8e), 2.56–2.65 (m, 1H, H7), 3.82 (d, 1H, J6,7 ¼ 11.2, H6), 5.50 (d, 1H,
J
13,13
0
¼ 3.1, H13), 6.06 (d, 1H, J3,4 ¼ 5.8, H3), 6.19 (d, 1H, J13
0
,13 ¼ 3.1,
0
13
H13 ), 7.65 (d, 1H, J4,3 ¼ 5.8, H4); C NMR (100 MHz, CDCl
3
) d: 7.50
(C15), 16.92 (C14), 20.99 (C8), 28.62 (C9), 40.48 (C5), 43.23 (C1),
45.49 (C7), 50.13 (C10), 77.55 (C6), 119.24 (C13), 131.49 (C3), 138.36
þ
ꢂ
(C11), 157.10 (C4), 170.00 (C12), 206.40 (C2); MS, m/z (%): 244 [M ],
ꢁ
ꢀ1
1
1
97.4 C; IR (KBr,
n
max/cm ): 3072, 2943, 2915, 2870, 1769, 1665,
(30), 229 (11), 215 (10), 201 (15), 173 (19), 145 (17), 105 (20), 84
1
þ
636, 1615, 1475, 1453, 1443, 1273, 1199, 1033, 742, 693; H NMR
(100), 77(32), 53 (72), 51 (98). HRMS (CI) C15
H
17
O
3
(MH ) requires
(
400 MHz, CDCl
3
):
d
1.33 (s, 3H, H14), 1.53 (m, 1H, H9e), 1.61 (s, 3H,
245.1172, found 245.1180.
ꢁ
ꢀ1
H13), 1.92–2.00 (m, 4H, H7, H8e, H8a, H9a), 2.1 (s, 3H, H15), 5.22 (d,
1
H1), 7.35 (t, 2H, J
H4 ), 7.65 (d, 2H, J
CDCl
4
Data for 9. mp ¼ 139.7–141.0 C; IR (KBr,
n
max/cm ): 2955, 2916,
H, J6,7 ¼ 9.6, H6), 6.25 (d, 1H, J2,1 ¼9.9, H2), 6,68 (d, 1H, J1,2 ¼ 9.9,
2848, 1771, 1727, 1705, 1645, 1472, 1370, 1245,1103, 1058, 1018, 997,
0
0
1
0
0
¼ J
0
0
¼ 7.3, H3 and H5 ), 7.45 (t, 1H, J
0
0
¼ 7.3,
963, 891; H NMR (400 MHz, CDCl
):
d
1.10 (s, 3H, H14), 1.46–1.56
3
,2
3
0
,4
4
,3
3
0
0
0
13
0
0
¼ 7.3, H2 and H6 ); C NMR (100 MHz,
(m,1H, H8 ), 1.95 (s, 3H, H15), 2.01 (s, 3H, CH
CO), 2.22–2.32 (m, 2H,
2
,3
3
0
):
d
11.01 (C15), 20.6 (C8), 22.34 (C13), 25.08 (C14), 37.55 (C9),
H8, H9 ), 2.43 (dd, 1H, J2,2
0
¼ 19.3, J2,1 ¼ 2.6, H2), 2.54 (dd, 1H,
¼ 13.4, J9,8 ¼ J9,8 ¼ 4.4,
¼ 1.4, H7), 4.20 (dd, 1H,
¼ 6.2, J1,2 ¼ 2.6, H1), 4.81 (d, 1H, J6,7 ¼ 10.9, H6), 5.61 (d, 1H,
3
0
0
1.34 (C10), 48.86 (C7), 57.58 (C11), 79.32 (C6), 123.94 (C1 ), 126.04
J
2
0
,2 ¼ 19.3, J
2
0
,1 ¼6.2, H2 ), 2.68 (td, 1H, J9,9
0
0
0
0
0
0
0
(
C2), 129.24 (C4, C2 , C6 ),130.03 (C4 ), 138.22 (C3 , C5 ), 150.92 (C5),
H9), 3.14 (tc, 1H, J7,6 ¼ 10.9, J7,8 ¼ J7,8
0
þ
ꢂ
1
2
9
4
54.64 (C1), 174.72 (C12), 186.15 (C3). MS, m/z (%): 402 [M ], (1),
44 (100), 229 (48), 216 (65), 201 (52), 188 (22), 157 (15), 105 (20),
1 (50), 77 (63), 55 (39). HRMS (CI) C21
03.0807, found 403.0811.
J
J
1,2
13,13
0
0
0
13
0
¼ 3.2, H13), 6.33 (d, 1H, J13 13 ¼ 3.2, H13 ); C NMR (100 MHz,
): 9.55 (C15), 20.03 (C14), 22.29 (CH CO), 24.48 (C8), 36.90
(C2), 37.54 (C9), 44.47 (C7), 47.32 (C1), 81.66 (C6), 85.49 (C10),
þ
23
H O
3
Se (MH ) requires
CDCl
3
d
3
1
20.83 (C13), 137.58 (C11), 143.21 (C4), 160.30 (C5), 168.80 (C12),
þ
ꢂ
4
.1.5. 3-Oxo-7
a
H,6bH-eudesma-1,4,11-trien-6,12-olide (7)
170.35 (CH
3
CO), 206.81 (C3); MS, m/z (%): 304 [M ], (0.3), 262 (35),
To a solution of compound 6 (1.7 mL, 4.2 mmol) in anhydrous
244 (100), 229 (17), 201 (20), 187 (48), 159 (22), 91 (36), 67 (33), 53
þ
THF (10 mL), and acetic acid (0.65 mL) in a two necked round
(55). 51 (24). HRMS (CI), C17
21
H O
5
(MH ) requires 305.1384, found
ꢁ
bottomed flask at 0 C was added hydrogen peroxide 30% (3.0 mL).
305.1379.
ꢁ
The mixture was stirred for 2 h at 0 C, and the reaction was
ꢀ
1
quenched with aqueous sodium bicarbonate (2 mol L , 10 mL).
The mixture was transferred into a separatory funnel, and extracted
with diethyl ether (3 ꢃ 30 mL). The combined organic layers was
washed with brine (30 mL), dried with anhydrous magnesium
sulphate, filtered, and concentrated under reduced pressure. The
residue was purified by flash column chromatography eluted with
hexane/ethyl acetate (2:1 v/v) to afford compound 7 as a yellow
4.1.7. 10
a-Acetoxy-3b-hydroxy-1,7aH,6,11bH-guai-4-en-6,
12-olide (10)
A mixture of sodium borohydride (0,056 g; 1.47 mmol), lactone
2 (0.225 g; 0.73 mmol), and anhydrous methanol (50 mL) in
a round bottomed flask was stirred for 2 h. The reaction was
quenched with saturated ammonium chloride aqueous solution
(30 mL), filtered and the solid was washed with methanol. The
ꢁ
ꢁ
solid (0.75 g; 3.1 mmol; 72%). mp ¼ 148.7–149.6 C; IR (KBr,
filtrate was concentrated under reduced pressure (40 C) and the
ꢀ1
n
max/cm ): 3044, 2937, 2870, 1777, 1663, 1635, 1615, 1458, 1254,
aqueous residue transferred to a separatory funnel which was
extracted with ethyl acetate (3 ꢃ 40 mL). The combined organic
layers were washed with brine (40 mL), dried with anhydrous
magnesium sulphate, filtered and concentrated under reduced
pressure. The yellow solid was purified by flash column chroma-
tography eluted with hexane/ethyl acetate (1:1 v/v) to give
compound 10 as a white solid (195 mg, 0.63 mmol) in 86% yield.
1
1
041, 985, 963, 907, 834; H NMR (400 MHz, CDCl
3
): d 1.32 (s, 3H,
H14), 1.56–1.63 (m, 2H, H8e, H9a), 1.79 (dddd, 1H, J8a,8e y -
J
J
8a,9a y J8a,7 y 11.6, J8a,9e ¼ 3.5, H8a), 1.94 (dc, 1H, J9e,8a ¼ 3.5,
9e,9a ¼ 13.4, H9e), 2.17 (s, 3H, H15), 2.71 (tc, 1H, J7,6 y J7,8a ¼ 11.6,
H7), 4.77 (d, 1H, J6,7 ¼ 11.6, H6), 5.56 (d, 1H, J13,13
0
¼ 2.9, H13), 6.24
0
(
J
2
(
(
(
5
2
d, 1H, J13
0
,13 ¼ 2.9, H13 ), 6.27 (d, 1H, J2,1 ¼9.9, H2), 6.70 (d, 1H,
1
3
ꢁ
ꢀ1
1,2 ¼ 9.9, H1); C NMR (100 MHz, CDCl
5.19 (C14), 37.68 (C9), 41.31 (C10), 50.30 (C7), 81.42 (C6), 119.61
C13), 126.02 (C2), 129.05 (C4), 137.56 (C11), 150.61 (C5), 154.62
3
):
d
10.81 (C15), 21.68 (C8),
mp ¼ 114.9–116.4 C; IR (KBr,
n
max/cm ): 3436, 2973, 2935, 2875,
1
1771, 1725, 1456, 1369, 1251, 1180, 850, 736; H NMR (400 MHz,
CDCl
3
):
d
(J/Hz): 1.20 (s, 3H, H14), 1.23 (d, 3H, J13,11 ¼6.5, H13), 1.36–
þ
ꢂ
0
C1), 169.04 (C12), 186.15 (C3); MS, m/z (%): 244 [M ], (45), 216
1.40 (m, 1H, H8 ), 1.57 (ddd, 1H, J2,2
(s broad, 1H, OH), 1.89 (s, 3H, H15), 1.98 (s, 3H, CH
(m, 4H, H7, H8, H9 , H11), 2.36–2.48 (m, 2H, H2 and H9), 3.75–3.78
(m, 1H, H1), 4.54–4.59 (m, 1H, H3), 4.67 (dc, 1H, J6,7 ¼ 10.9, H6);
NMR (100 MHz, CDCl ): 12.81 (C13), 12.91 (C15), 20.56 (C14),
22.80 (CH CO), 25.61(C8), 35.05 (C2), 38.35 (C9), 41.84 (C11), 49.34
(C7), 51.66 (C1), 78.16 (C3), 81.92 (C6), 86.87 (C10), 131.59 (C5),
0
¼14.0, J2,1 y J2,3 y 6.4, H2), 1.73
14), 201 (27), 173 (30), 145 (36), 105 (29), 91 (95), 77 (76), 65 (87),
3
CO), 1.99–2.25
þ
0
0
3 (100). HRMS (CI) C15
45.1179.
H
17
O
3
(MH ) requires 245.1172, found
1
3
C
3
d
4
.1.6. Photochemical synthesis of 11,13-dehydrolumisantonin (8)
3
and 10
a
-acetoxy-3-oxo-1,7 H,6 H-guai-4,11-dien-6,12-olide (9)
a
b
þ
ꢂ
A solution of compound 7 (0.65 g, 2.66 mmol) in anhydrous
acetic acid (120 mL) in a quartz tube was degassed by a flow of
nitrogen for 30 min. The reaction mixture was then irradiated
under four low pressure mercury lamps (4 ꢃ15 W) for 4 h. The
144.50 (C4), 170.65 (CH CO), 178.48 (C12); MS, m/z (%): 308 [M ],
3
248 (67), 233 (100), 174 (49), 159 (34), 91 (44), 55 (74). HRMS (CI),
þ
C
17
25
H O
5
(MH ) requires 309.1697, found 309.1695.
ꢁ
solvent was evaporated under reduced pressure (50 C) to give
4.1.8. 3b,10a-Hydroxy-1,7aH,6,11bH-guai-4-en-6,12-olide (11)
a yellow oil which was purified by flash column chromatography
A mixture of sodium borohydride (0.05 g; 1.47 mmol), lactone 4
eluted with hexane/ethyl acetate (3:2 v/v) to afford compound 8 as
(0.12 g; 0.45 mmol), and anhydrous methanol (30 mL) in a round

3744
F.F.P. Arantes et al. / European Journal of Medicinal Chemistry 44 (2009) 3739–3745
5
bottomed flask was stirred for 3 h. The reaction was quenched and
elaborated as described for the preparation of compound 10,
affording compound 11 as a white solid (86 mg, 0.32 mmol) in 72%
in 96-well plates (3 ꢃ 10 cells/well in 100
m
l of medium). After
ꢀ1
24 h, the compounds (0.09–25
mg mL ) dissolved in DMSO were
added to each well (using the HTS – high-throughput screening-
biomek 3000-Beckman Coulter, Inc. Fullerton, Calif o´ rnia, EUA) and
incubated for 72 h. Doxorubicin was used as positive control.
ꢁ
ꢀ1
yield; mp ¼ 168.5–169.5 C; IR (KBr,
n
max/cm ): 3389, 2973, 2929,
2
860, 1759, 1668, 1455, 1380, 1234, 1179, 1103, 985, 865, 735, 695;
1
H NMR (400 MHz, CDCl
3
):
d
: 1.06 (s, 3H, H14), 1.23 (d, 3H,
Twenty four hours before the end of the incubation, 10
mL of stock
0
ꢀ1
J
13,11 ¼7.0, H13), 1.30–1.45 (m, 1H, H8 ), 1.60–1.69 (m, 4H, OH, OH,
solution (0.312 mg mL ) of the Alamar Blue (resazurin – Sigma
Aldrich Co. – St. Louis, MO/USA) were added to each well. The
absorbance was measured using a multiplate reader (DTX 880
Multimode Detector, Beckman Coulter, Inc. Fullerton, Calif o´ rnia,
USA). The drug effect was quantified as the percentage of control
absorbance at 570 nm and 595 nm.
0
H2, H9 ), 1.89 (s, 1H, H15), 1.93–1.99 (m, 3H, H7, H8, H9), 2.20 (dq,
1
H,
J
11,7 ¼ 12.2,
J
11,13 ¼ 7.0, H11), 2.50 (dt, 1H,
J
2
0
,2 ¼16.0,
0
J
2
0
,3 ¼ J
2
0
,1 ¼8.0, H2 ), 2.90–2.95 (m, 1H, H1), 4.55 (m, 1H, H3), 4.70
13
(
1
(
1
2
dc, 1H, J6,7 ¼ 10.9, H6); C NMR (100 MHz, CDCl
2.99 (C13), 21.55 (C14), 25.67 (C8), 34.74 (C2), 41.49 (C11), 44.83
C9), 49.14 (C7), 54.66 (C1), 74.56 (C10), 77.62 (C3), 81.84 (C6),
3
): d 12.45 (C15),
þ
ꢂ
31.80 (C4), 143.98 (C5), 178.20 (C12); MS, m/z (%): 266 [M ], (0,1),
4.3. Statistical analysis
48 (55), 233 (8), 215 (17), 205 (12), 190 (83), 177 (30), 108 (54), 91
þ
(
44), 79 (57), 55 (100), 53 (38). HRMS (CI), C15
H
23
O
4
(MH ) requires
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).
2
67.1591, found 267.1587.
(
4
4
.2. Cytotoxic assays
Acknowledgements
.2.1. Tumor cell assay
The cytotoxic effects of the synthesized compounds were eval-
We are grateful to the following Brazilian agencies: Conselho
Nacional de Desenvolvimento Cient ı´ fico e Tecnol o´ gico (CNPq) for
research fellowships (LCAB, AJD), Fundaç a˜ o de Amparo a` Pesquisa
de Minas Gerais (FAPEMIG), Coordenaç a˜ o de Aperfeiçoamento de
Pessoal de N ı´ vel Superior (CAPES) e FINEP for financial support. We
are also grateful to Akshat Rathi (Oxford University – UK) for
suggestions and corrections made on the manuscript.
uated against HL-60 (leukemia), SF-295 (central nervous system),
HCT-8 (colon), MDA-MB-435 (melanoma), UACC-257 (melanoma),
A549 (lung), OVACAR-8 (ovarian), A704 (renal), and PC3 (prostate)
human cancer cell lines, all obtained from the National Cancer
Institute, Bethesda, MD, USA. The cells were grown in RPMI-1640
medium supplemented with 10% fetal bovine serum, 2 mM gluta-
mine, 100
bated at 37 C with a 5% CO
m
g/mL streptomycin and 100 U/mL penicillin, and incu-
ꢁ
2
atmosphere.
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cells to reduce the yellow dye 3-(4,5-dimethyl-2-thiazolyl)-2,5-
diphenyl-2H-tetrazolium bromide (MTT) to a purple formazan
product [35]. For all experiments, the cells were seeded in 96-well
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(
[
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(
150
mL) containing 0.5 mg/mL MTT. After 3 h, the formazan
19–26.
product was dissolved in 150
mL DMSO and the absorbance was
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7