Synthesis of novel 3-(aryl)benzothieno[2,3-c]pyran-1-ones from Sonogashira products and intramolecular cyclization: Antitumoral activity evaluation

Article abstract of DOI:10.1016/j.ejmech.2008.11.002

Several novel 3-(aryl)benzothieno[2,3-c]pyran-1-ones (tricyclic lactones) were prepared either by a tandem one-pot Sonogashira coupling and intramolecular cyclization, reacting the 3-bromobenzo[b]thiophene-2-carboxylic acid with arylacetylenes, or by Sono

Full text of DOI:10.1016/j.ejmech.2008.11.002

European Journal of Medicinal Chemistry 44 (2009) 1893–1899
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of novel 3-(aryl)benzothieno[2,3-c]pyran-1-ones from Sonogashira
products and intramolecular cyclization: Antitumoral activity evaluation
,
a
b
b
Maria-Joa˜o R.P. Queiroz a , Ricardo C. Calhelha , Luıs A. Vale-Silva , Eugenia Pinto ,
*
´
´
b
´
M. Sa˜o-Jose Nascimento
a Centro de Qu´ımica, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal
b
ˆ
´
´
´
Laborato´rio de Microbiologia, Centro de Estudos de Qu´ımica Organica, Fitoquımica e Farmacologia/Centro de Quımica Medicinal – Faculdade de Farmacia da Universidade do Porto
(CEQOFFUP/CEQUIMED), Rua An´ıbal Cunha 164, 4050-047 Porto, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
Several novel 3-(aryl)benzothieno[2,3-c]pyran-1-ones (tricyclic lactones) were prepared either by
Received 16 October 2008
Received in revised form
6 November 2008
Accepted 6 November 2008
Available online 13 November 2008
a
tandem one-pot Sonogashira coupling and intramolecular cyclization, reacting the 3-bromo-
benzo[b]thiophene-2-carboxylic acid with arylacetylenes, or by Sonogashira coupling of the methyl
3-bromobenzo[b]thiophene-2-carboxylate or the methyl 3-bromo-6-methoxybenzo[b]thiophene-2-
carboxylate with arylacetylenes followed by an electrophilic intramolecular cyclization using iodine or
TFA in two separate steps. The Sonogashira products and the tricyclic lactones obtained were evaluated
for their capacity to inhibit the in vitro growth of three human tumor cell lines, MCF-7 (breast adeno-
carcinoma), NCI-H460 (non-small cell lung cancer) and SF-268 (CNS cancer). Most of the compounds
Keywords:
Benzo[b]thiophenes
Sonogashira coupling
Intramolecular lactonization
Benzothieno[2,3-c]pyran-1-ones
Antitumor activity
showed a high growth inhibitory effect on all the tested cell lines, with GI50 values in the mM range.
A structure–activity relationship was established for the Sonogashira products and for the tricyclic
lactones, namely related to the presence and position of substituents (OMe and/or F) in the benzo-
thiophene moiety or in the phenyl ring.
Ó 2008 Elsevier Masson SAS. All rights reserved.
1. Introduction
cancer (SF-268), was evaluated. Both types of compounds have
shown ability to inhibit the growth of all the cell lines tested, with
a
-Pyrones [1] and isocoumarins [2] (Fig. 1) represent two
GI50 values in the mM range, and it was possible to establish some
important classes of naturally occurring lactones, which are
structural subunits in numerous natural products that exhibit
a wide range of biological activities, such as antimicrobial [3],
androgen-like [4], phytotoxic [5] and pheromonal [6]. In partic-
ular 3-substituted isocoumarins have shown promising pharma-
cological activities [7]. The angiogenesesis inhibitor NM-3 [8],
which belongs to this class of compounds, is presently undergoing
clinical trials. This molecule induces also mitotic phase arrest and
apoptosis of human multiple myeloma cells [9]. Promising cyto-
toxic activities of 4-alkynyl substituted 2-pyrones have also been
reported [10].
Herein we present the synthesis of the new benzothieno[2,3-
c]pyran-1-one skeleton by intramolecular cyclization of Sonoga-
shira coupling products obtained in the benzo[b]thiophene series.
The effect of both the alkynyl precursors and the cyclized products
(tricyclic lactones) on the in vitro growth of three tumor cell lines,
representative of different tumor types, namely breast adenocar-
cinoma (MCF-7), non-small cell lung cancer (NCI-H460), and CNS
structure–activity relationships.
2. Results and discussion
2.1. Chemistry
3-Arylbenzothieno[2,3-c]pyran-1-ones 1 were prepared first
through a tandem one-pot procedure of Sonogashira coupling fol-
lowed by a regeoselective 6-endo-dig intramolecular lactonization
from the commercial 3-bromobenzo[b]thiophene-2-carboxylic
acid and different arylacetylenes (Scheme 1). The corresponding
arylacetylene dimers, identified by GC–MS, were also obtained in
some amount, as the less polar products.
When 2 equiv. of the phenylacetylene were used, the benzo-
thienopyranone 1a was isolated in approximately the same yield,
but a bigger amount of the phenylacetylene dimer was formed.
In these reactions, the 4-alkynyl-3-arylbenzothieno[2,3-c]pyran-1-
one derivatives, which correspond to the major products in the
work of Pal et al. from 3-iodothiophene-2-carboxylic acid and
several acetylenes [11], were not obtained.
As the product yields were not high, we have performed the
Sonogashira coupling [12] on the methyl 3-bromobenzo
* Corresponding author. Tel.: þ351 253604378; fax: þ351 253604382.
E-mail address: mjrpq@quimica.uminho.pt (M.-J.R.P. Queiroz).
0223-5234/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.11.002

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R3
O
O
R1
O
O
R2
isocoumarin
α-pyrone
S
CO2Me
Fig. 1. General structures of
a
-pyrones and isocoumarins.
3b and 3d-g
i
R2
R3
Br
6
5
i
4
I
7
R1
3
O2
COOH
S
R1
8
S
9
R2
1
O
S
+
O
O
1a R1 = R2 = H, 40 %
R2
4a R1 = R2 = H, R3 = OMe, 50 %
4b R1 = R2 = H, R3 = F, 62 %
4c R1 = OMe, R2 = R3 = H, 70 %
4d R1 = OMe, R2 = F, R3 = H, 65 %
4e R1 = OMe, R2 = H, R3 = F, 67 %
1b R1 = H, R2 = OMe, 35 %
1c R1 = F, R2 = H, 38 %
R1
Scheme 1. Synthesis of the 3-arylbenzothieno[2,3-c]pyran-1-ones 1a–c. (i)
PdCl2(PPh3)2 (5 mol%), CuI (3 mol%), arylacetylene (1.1 equiv.), NEt3 (3 equiv.), dry DMF,
Ar, 100 ꢀC, 1.5 h.
Scheme 3. Synthesis of 3-aryl-4-iodobenzothieno[2,3-c]pyran-1-ones. (i) I2 (3 equiv.),
dry CH2Cl2, rt, 27 h, under Argon.
[b]thiophene-2-carboxylate 2a and on the methyl 3-bromo-6-
methoxybenzo[b]thiophene-2-carboxylate 2b, obtained from the
corresponding amino compounds previously synthesized by our
group [13a] and following a knownprocedure using t-butylnitrite (t-
BuONO) and CuBr2 [13b], with arylacetylenes. The Sonogashira
coupling products 3a–g were obtained in good yields, using condi-
tions similar to the ones of the one-pot procedure, but 1.5 equiv. of
the arylacetylene were needed to completely consume the bromi-
nated compound (Scheme 2). The corresponding dimers of the
arylacetylenes were also isolated as less polar products and were
identified by GC–MS.
derivatives, by a palladium-catalyzed hydrodehalogenation [15], or
may be further functionalized by C–C bond formation using metal-
catalyzed cross-couplings [14c].
Recently, Biehel et al. described the synthesis of isocoumarins in
less than 1 h, by a 6-endo-dig cyclization of 2-(2-arylethynyl)ary-
lesters using TFA as a Brønsted acid at rt [16]. The same conditions
were applied by us for the intramolecular cyclization of the Sono-
gashira products 3d, 3e and 3g and we were able to obtain the
corresponding tricyclic lactones 1d–f in excellent yields, but heat-
ing the reaction mixture at 50 ꢀC for 2 h (Scheme 4).
Compounds 3 were submitted to iodolactonization, using iodine
in dichloromethane at room temperature [14], and the corre-
Nevertheless, starting from compound 3b we have obtained 1b
in 90% yield, at rt for 30 min., which is in agreement with what
Biehel et al. reported for the cases in which methoxy groups were
present in the arylacetylene [16].
sponding 3-aryl-4-iodobenzothieno[2,3-c]pyran-1-ones
4 were
obtained regioselectively in good yields (Scheme 3).
The mechanism of iodolactonization should involve the elec-
trophilic attack of iodine on the triple bond, regioselectively
forming a vinyl cation. A subsequent intramolecular nucleophilic
attack of the methoxycarbonyl group, followed by elimination of
methyl iodide, yields the iodo cyclized products. Compounds 4 can
either be used to prepare the corresponding dehalogenated
2.2. Effect on the growth of human tumor cell lines
Almost all the compounds prepared in series 1, 3, and 4 were
evaluated for their capacity to inhibit the in vitro growth of three
human tumor cell lines, MCF-7 (breast adenocarcinoma), NCI-H460
(non-small cell lung cancer) and SF-268 (CNS cancer). The results
are summarized in Table 1 and discussed below.
R3
Br
R1
R1
CO2Me
3d-e and 3g
S
i
R2
S
2a R = H
2b R = OMe
i
CO2Me
R2
R1
+
3a R1 = R2 = R3 = H, 48 %
3b R1 = R2 = H, R3 = OMe, 45%
R3
O
S
3c R1 = R3 = H, R2 = F, 63 %
3d R1 = R2 = H, R3 = F, 60 %
3e R1 = OMe, R2 = R3 = H, 60 %
3f R1 = OMe, R2 = F, R3 = H, 55 %
3g R1 = OMe, R2 = H, R3 = F, 73 %
R2
O
1d R1 = H, R2 = F, 95 %
1e R1 = OMe, R2 = H, 90 %
1f R1 = OMe, R2 = F, 84 %
Scheme 2. Synthesis of the Sonogashira coupling products 3a–g. (i) PdCl2(PPh3)2
(5 mol%), arylacetylene (1.5 equiv.), CuI (3 mol%), NEt3 (3 equiv.), dry DMF, Ar, 100 ꢀC,
1.5 h.
Scheme 4. Synthesis of 3-arylbenzothieno[2,3-c]pyran-1-ones. (i) TFA (2 mL), 2 h,
50 ꢀC.

M.-J.R.P. Queiroz et al. / European Journal of Medicinal Chemistry 44 (2009) 1893–1899
1895
Table 1
Effect of the compounds on the growth of three human tumor cell lines.
Compounds
GI50
(
m
M)a
Compounds
GI50
MCF-7
(m
M)a
MCF-7
SF-268
NCI-H460
SF-268
NCI-H460
F
O
>112.5
>112.5
>112.5
60.6 ꢁ 0.4
76.1 ꢁ 4.2b
56.2 ꢁ 4.0
S
S
3d
1a
O
O
CO2Me
OMe
MeO
>75.0
>75.0
>75.0
79.6 ꢁ 0.1
11.4 ꢁ 2.7
12.6 ꢁ 0.4
54.9 ꢁ 2.0
42.3 ꢁ 3.1
10.3 ꢁ 2.3b
10.1 ꢁ 1.0
S
S
3e
1b
CO2Me
O
MeO
F
O
F
19.4 ꢁ 3.2
12.4 ꢁ 0.5
15.5 ꢁ 0.5
7.2 ꢁ 1.7
S
S
3f
CO2Me
O
1c
F
F
MeO
>25.0c
>25.0c
>25.0c
20.9 ꢁ 4.5b
O
S
3g
S
1d
CO2Me
O
OMe
I
MeO
O
O
>75.0
>75.0
>75.0
>150.0
>150.0
>150.0
S
S
4a
1e
O
O
F
F
I
MeO
108.7d
28.9d
117.5 ꢁ 1.0
58.9 ꢁ 5.3
25.2 ꢁ 4.9
11.9 ꢁ 0.6
>150.0
20.7 ꢁ 3.8
11.3 ꢁ 2.8
>150.0
35.8 ꢁ 5.9
10.9 ꢁ 0.1
>150.0
O
O
S
S
4b
1f
O
O
I
MeO
O
F
71.6 ꢁ 3.5
64.7 ꢁ 3.1
S
3a
S
4d
CO2Me
O
OMe
F
I
MeO
O
90.6 ꢁ 10.3
40.9 ꢁ 4.1
>150.0
103.4 ꢁ 9.1
30.2 ꢁ 2.5
S
S
3b
4e
CO2Me
O
25.7 ꢁ 5.5
F
S
3c
CO2Me
a
The lowest concentrations causing 50% of cell growth inhibition (GI50) after a continuous exposure of 48 h, expressed as means ꢁ SEM of three independent experiments
performed in duplicate.
b
Results from two independent experiments performed in duplicate.
Due to the compound’s low solubility in DMSO the maximum concentration tested was 25 mM.
Results from one experiment performed in duplicate. GI50: MCF-7 ¼42.8 ꢁ 8.2 nM; NCI-H460 ¼ 94.0 ꢁ 8.7 nM and SF-268 ¼ 94.0 ꢁ 7.0 nM. Doxorubicin was used as
c
d
positive control.

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Concerning the 3-arylbenzothieno[2,3-c]pyran-1-ones of series
1, the ortho-fluorinated compound in the phenyl ring relative to the
C–C bond 1c clearly shows the best results, presenting quite low
encourage further studies in order to elucidate the mechanisms of
action underlying the antitumor activity observed.
GI50 values (12–20
mM) for the three tumor cell lines. The presence
4. Experimental
of a fluorine atom in the para-position decreases the solubility of
1d, in comparison to 1c, and it didn’t show any activity at
4.1. Synthesis
a concentration of 25 mM. In the 7-methoxylated compounds, the
presence of a fluorine atom on the phenyl ring in compound 1f was
shown to be important for the activity in the SF-268 cell line,
presenting a GI50 of 28.9 mM.
Melting points (ꢀC) were determined on a SMP3 Stuart appa-
ratus and are uncorrected. 1H and 13C NMR spectra were recorded
on a Varian Unity Plus at 300 and 75.4 MHz, or on an Avance III at
400 and 100.6 MHz, respectively. Heteronuclear correlations, 1H,
13C, HMQC and HMBC were performed to attribute some signals.
MS (EI) and HRMS data were recorded by the mass spectrometry
service of the University of Vigo, Spain. Elemental analysis was
performed on a LECO CHNS 932 elemental analyser. The reactions
were monitored by thin layer chromatography (TLC). Column
chromatography was performed on Macherey-Nagel silica gel 230–
400 mesh. Petroleum ether refers to the boiling range 40–60 ꢀC.
Ether refers to diethylether. When solvent gradient was used, the
increase of polarity was made from neat petroleum ether to
mixtures of ether/petroleum ether, increasing the ether proportion
by 10% in each step until the isolation of the product.
Among the Sonogashira coupling products the best results were
obtained for the derivatives of the methoxylated benzo[b]thio-
phene moiety bearing a fluorine atom on the arylethynyl unit 3f
and 3g. The presence of the fluorine atom in the ortho-position in 3f
seems to bring some selectivity against the CNS cancer cell line SF-
268. Regarding derivatives 3a–d, the presence of an ortho-fluorine
atom on the arylethynyl moiety of 3c leads to an increase of the
growth inhibitory activity towards the three cell lines. Neverthe-
less, its activity was lower than the one of the methoxylated
analogue 3f. A similar tendency was also observed between 3d and
3g, confirming the benefit of the methoxy group.
Regarding the iodolactonization products 4, the presence of
a para-fluorine atom relative to the C–C bond on the phenyl ring in
compound 4b produced an obvious raise in the antitumor activity
in comparison with the para-methoxylated 4a. When comparing 4d
and 4e, two iodo cyclized derivatives with a 7-methoxy group, the
presence of an ortho-fluorine atom in 4d appears to be essential for
the activity, yielding some of the lowest GI50 values for all tumor
cell lines tested.
Therefore, as depicted in Table 1 the majority of the tested
compounds showed a growth inhibitory effect on the three tumor
cell lines tested. Four of them, however, must be highlighted for their
low GI50 values, namely 1c, 3f, 3g, and 4d. In this study every
main structural skeleton, namely the 3-arylbenzothieno[2,3-
c]pyran-1-ones (series 1), the Sonogashira coupling products, methyl
3-(arylethynyl)benzo[b]thiophene-2-carboxylates (series 3), and the
3-aryl-4-iodobenzothieno[2,3-c]pyran-1-ones (series 4), yielded at
leastone highlyactive compound. Nevertheless, the results appear to
indicate series 3 (the Sonogashira coupling products) as the most
favourable basic structure, since all its derivatives were found to be
consistently active, even if showing variable potencies.
4.1.1. General procedure for the one-pot Sonogashira coupling and
intramolecular lactonization
3-Bromobenzo[b]thiophene-2-carboxylic
acid
(150 mg,
0.583 mmol), the arylacetylene (1.1 equiv.), PdCl2(PPh3)2 (5 mol%), CuI
(3 mol%), and NEt3 (3 equiv.) were added into a dry Schlenk tube,
under argon and dry DMF (2–3 mL), and the mixture was heated
with stirring at 100 ꢀC for 1.5 h. After cooling, water (5 mL) and ethyl
acetate (5 mL) were added and the phases were separated. The
aqueous phase was then extracted with additional ethyl acetate
(3 ꢂ 5 mL). The organic phases were collected, dried (MgSO4),
filtered, and, finally, removal of the solvent under reduced pressure
gave an oil or an oily solid which was submitted to column chro-
matography using solvent gradient.
4.1.1.1. 3-(Phenyl)benzothieno[2,3-c]pyran-1-one
(1a). 3-Bromo-
benzo[b]thiophene-2-carboxylic acid (150 mg, 0.583 mmol), phe-
nylacetylene (0.07 mL, 0.641 mmol), heating for 1.5 h. Compound
1a was isolated, after column chromatography using 40% ether/
petroleum ether, as a white solid (65.0 mg, 40%) that gave white
crystals after crystallization from ether/petroleum ether, m.p. 212–
3. Conclusions
214 ꢀC. 1H NMR (CDCl3, 300 MHz):
5H, Ar-H), 7.93–7.98 (m, 3H, Ar-H), 8.10 (m, 1H, Ar-H) ppm. 13C NMR
(CDCl3, 75.4 MHz): 96.86 (4-CH), 122.47 (C), 123.59 (CH), 123.64
d 7.39 (s, 1H, 4-H), 7.46–7.64 (m,
We were able to prepare several novel 3-arylbenzothieno[2,3-
c]pyran-1-ones regioselectively in moderate yields, in a tandem one-
pot procedure from 3-bromobenzo[b]thiophene-2-carboxylic acid
and the appropriate arylacetylenes. We have also synthesized
3-aryl-4-iodobenzothieno[2,3-c]pyran-1-ones in good yields by
iodolactonization of the also new methyl 3-(arylethynyl)benzo[b]-
thiophene-2-carboxylates. The latter were obtained by Sonogashira
coupling of the methyl 3-bromobenzo[b]thiophene-2-carboxylate
or of methyl 3-bromo-6-methoxybenzo[b]thiophene-2-carboxylate
with several arylacetylenes. 3-Arylbenzothieno[2,3-c]pyran-1-ones
were also prepared in excellent yields, by treating the Sonogashira
products with TFA.
d
(CH), 125.40 (CH), 125.44 (2 ꢂ CH), 128.95 (2 ꢂ CH), 129.18 (CH),
130.34 (CH), 131.70 (C), 134.24 (C), 143.60 (C), 143.73 (C), 157.35 (C),
158.76 (C) ppm; MS (EI): m/z (%) 278 (Mþ, 100), 250 (Mþ ꢃ CO, 88),
221 (89). HRMS Mþ calct. for C17H10O2S 278.0402, found 278.0399.
4.1.1.2. 3-(4-Methoxyphenyl)benzothieno[2,3-c]pyran-1-one (1b). 3-
Bromobenzo[b]thiophene-2-carboxylic acid (150 mg, 0.583 mmol),
4-methoxyphenylacetylene (85.0 mg, 0.641 mmol), heating for
1.5 h. Compound 1b was isolated, after column chromatography
using 40% ether/petroleum ether, as a white solid (64 mg, 35%) that
gave white crystals after crystallization from ether/petroleum ether,
The synthesized compounds were found to have a very prom-
ising antitumor activity, with some derivatives presenting GI50
m.p. 202–204 ꢀC. 1H NMR: (CDCl3, 300 MHz):
d 3.89 (s, 3H, OMe),
values approaching 10
mM. The Sonogashira coupling products
7.00 (d, 2H, J ¼ 9 Hz, 30 and 50-H), 7.28 (s, 1H, 4-H), 7.51–7.64 (m, 2H,
appear to constitute the most favourable basic structure. Further-
more, the presence of a methoxy group on the benzothiophene
moiety and a fluorine atom on the arylethynyl unit is associated
with the best antitumor activity. However, a similar high activity
was found for two of the lactone compounds, both presenting an
ortho-fluorinated phenyl ring relative to the C–C bond. The data of
the present study bring new perspectives to these compounds and
Ar-H), 7.89 (d, 2H, J ¼ 9 Hz, 20 and 60-H), 7.95 (m, 1H, Ar-H), 8.09 (m,
1H, Ar-H) ppm. 13C NMR (CDCl3, 75.4 MHz):
d 55.43 (OMe), 95.48 (4-
CH), 114.35 (2 ꢂ CH), 121.40 (C), 123.59 (CH), 123.67 (CH), 124.28 (C),
125.29 (CH), 127.07 (2 ꢂ CH), 129.12 (CH), 134.27 (C), 143.64 (C),
144.16 (C),157.56 (C),158.95 (C),161.37 (C) ppm. MS (EI): m/z (%) 308
(Mþ, 100), 280 (Mþ ꢃ CO, 84), 265 (26), 237 (41). HRMS Mþ calct. for
C18H12O3S 308.0507, found 308.0514.

M.-J.R.P. Queiroz et al. / European Journal of Medicinal Chemistry 44 (2009) 1893–1899
1897
4.1.1.3. 3-(2-Fluorophenyl)benzothieno[2,3-c]pyran-1-one (1c). 3-
Bromobenzo[b]thiophene-2-carboxylic acid (150 mg, 0.583 mmol),
2-fluorophenylacetylene (0.07 mL, 0.641 mmol), heating for 1.5 h.
Compound 1c was isolated, after column chromatography using
30% ether/petroleum ether, as a white solid (66 mg, 38%) that gave
white crystals after crystallization from ether/petroleum ether, m.p.
(m, 2H, Ar-H), 7.68–7.73 (m, 2H, Ar-H), 7.84–7.87 (m, 1H, Ar-H),
8.13–8.16 (m, 1H, Ar-H) ppm. 13C NMR (CDCl3, 75.4 MHz):
52.49
d
(OMe), 82.70 (C), 98.62 (C), 122.62 (CH), 122.83 (C), 123.57 (C),
124.87 (CH), 125.29 (CH), 127.72 (CH), 128.44 (2 ꢂ CH), 128.96 (CH),
132.00 (2 ꢂ CH), 133.52 (C), 139.70 (C), 139.83 (C), 162.46 (C) ppm.
MS (EI): m/z (%) 292 (Mþ, 100), 277 (Mþ ꢃ Me, 65), 189 (26). HRMS
165–167 ꢀC. 1H NMR (CDCl3, 400 MHz):
d
7.20–7.26 (m, 1H, Ar-H),
M
þ calct. for C18H12O2S 292.0558, found 292.0555.
7.29–7.33 (m, 1H, Ar-H), 7.41–7.47 (m, 1H, Ar-H), 7.55–7.59 (m, 1H,
Ar-H), 7.61–7.66 (m, 1H, Ar-H), 7.67 (s, 1H, 4-H), 7.97–8.00 (m, 1H,
Ar-H), 8.07–8.14 (m, 2H, Ar-H) ppm. 13C NMR: (CDCl3, 100.6 MHz)
102.00 (CH, d, J ¼ 16 Hz), 116.54 (CH, d, J ¼ 23 Hz), 119.95 (C, d,
J ¼ 10 Hz), 123.13 (C), 123.59 (CH), 123.84 (CH), 124.77 (CH, d,
J ¼ 3 Hz), 125.49 (CH), 128.76 (CH), 129.25 (CH), 131.50 (CH, d,
J ¼ 9 Hz), 134.35 (C), 143.62 (C), 143.75 (C), 151.67 (C), 158.53 (C),
159.98 (CF, d, J ¼ 253 Hz) ppm. MS (EI): m/z (%) 296 (Mþ, 88), 268
(Mþ ꢃ CO, 91), 239 (100). HRMS Mþ calct. for C17H9FO2S 296.0307,
found 296.0309.
4.1.3.2. Methyl 3-[(4-methoxyphenyl)ethynyl] benzo[b]thiophene-2-
carboxylate (3b). Compound 2a (150 mg, 0.550 mmol), 4-methox-
yphenylacetylene (0.11 mL, 0.825 mmol), heating for 1.5 h.
Compound 3b was isolated after column chromatography, using
20% ether/petroleum ether, as a yellow solid (80.0 mg, 45%). Crys-
tallization from ether/petroleum ether gave yellow crystals, m.p.
106–108 ꢀC. 1H NMR (CDCl3, 400 MHz):
d 3.87 (s, 3H, OMe), 4.01 (s,
3H, OMe), 6.94 (d, 2H, J ¼ 8.8 Hz, 30 and 50-H), 7.48–7.55 (m, 2H, Ar-
H), 7.64 (d, 2H, J ¼ 8.8 Hz, 20 and 60-H), 7.83–7.86 (m,1H, Ar-H), 8.12–
8.15 (m,1H, Ar-H) ppm. 13C NMR (CDCl3,100.6 MHz):
d 52.45 (OMe),
4.1.2. General procedure [13b] for the synthesis of methyl 3-
bromobenzo[b]thiophene-2-carboxylates 2a and 2b
55.35 (OMe), 81.77 (C), 99.03 (C), 114.14 (30and 50-CH), 114.93 (C),
122.61 (CH), 124.06 (C), 124.93 (CH), 125.20 (CH), 127.69 (CH),
132.71 (C), 133.60 (20 and 60-CH), 139.74 (C), 139.87 (C), 160.25 (C),
162.58 (C) ppm. MS (EI): m/z (%) 322 (Mþ, 100), 307 (Mþ ꢃ Me, 77),
84 (51). HRMS Mþ calct. for C19H14O3S 322.0664, found 322.0659.
t-BuONO (1.5 equiv.) and CuBr2 (1.2 equiv.) were added to the
appropriate methyl 3-aminobenzo[b]thiophene-2-carboxylates
[13a] (5 mmol), in dry acetonitrile (10 mL), and the mixture was
heated at 65 ꢀC in a silicone bath for 30 min. After cooling, the
mixture was poured into 25 mL of HCl 2 M and stirred. An orange
solid was formed and then filtered and dried in the oven at 50 ꢀC.
4.1.3.3. Methyl
3-[(2-fluorophenyl)ethynyl]benzo[b]thiophene-2-
carboxylate (3c). Compound 2a (150 mg, 0.550 mmol), 2-fluo-
rophenylacetylene (0.09 mL, 0.825 mmol), heating for 1.5 h.
Compound 3c was isolated after column chromatography, using
20% ether/petroleum ether, as a white solid (101 mg, 63%). Crys-
tallization from ether/petroleum ether gave white crystals, m.p.
4.1.2.1. Methyl 3-bromobenzo[b]thiophene-2-carboxylate (2a).
Compound 2a (935 mg, 70%) was obtained from methyl 3-
aminobenzo[b]thiophene-2-carboxylate [13a] (1.00 g, 5 mmol),
following the general procedure described above. Crystalliza-
tion from ethyl acetate/petroleum ether gave orange crystals,
103–105 ꢀC. 1H NMR (CDCl3, 300 MHz):
d 4.02 (s, 3H, OMe), 7.14–
7.23 (m, 2H, Ar-H), 7.35–7.44 (m, 1H, Ar-H), 7.50–7.56 (m, 2H, Ar-H),
7.65–7.71 (m, 1H, Ar-H), 7.83–7.89 (m, 1H, Ar-H), 8.15–8.20 (m, 1H,
m.p. 67–69 ꢀC. 1H NMR (CDCl3, 400 MHz):
7.49–7.56 (m, 2H, Ar-H), 7.82–7.86 (m, 1H, Ar-H), 7.98–8.02 (m,
1H, Ar-H) ppm. 13C NMR (CDCl3, 100.6 MHz):
52.57 (OMe),
d 3.98 (s, 3H, OMe),
Ar-H) ppm. 13C NMR (CDCl3, 75.4 MHz):
d 52.55 (OMe), 87.67 (C),
d
91.70 (C), 111.59 (C, d, J ¼ 16 Hz), 115.63 (CH, d, J ¼ 21 Hz), 122.59
(CH), 123.02 (C), 124.11 (CH, d, J ¼ 4 Hz), 124.94 (CH), 125.46 (CH),
127.77 (CH), 130.75 (CH, d, J ¼ 8 Hz), 133.77 (CH), 134.08 (C), 139.67
(C), 139.81 (C), 162.46 (C), 162.83 (CF, d, J ¼ 252 Hz), ppm. MS (EI):
m/z (%) 310 (Mþ, 100), 295 (Mþ ꢃ Me, 40), 279 (35). HRMS Mþ calct.
for C18H11FO2S 310.0464, found 310.0464.
115.08 (C), 122.66 (CH), 125.35 (CH), 125.65 (CH), 127.20 (C),
128.17 (CH), 138.61 (C), 139.34 (C), 161.85 (C) ppm. C10H7BrO2S
requires C, 44.30; H, 2.60; S, 11.83%. Found C, 44.49; H, 2.55; S,
11.85%.
4.1.2.2. Methyl 3-bromo-6-methoxybenzo[b]thiophene-2-carboxylate
(2b). Compound 2b (1.05 g, 70%) was obtained from 3-amino-6-
methoxybenzo[b]thiophene-2-carboxylate [13a] (1.12 g, 0.500
mmol), following the general procedure described above. Crystal-
lization from ether/petroleum ether gave beige crystals, m.p. 165–
4.1.3.4. Methyl
3-[(4-fluorophenyl)ethynyl]benzo[b]thiophene-2-
carboxylate (3d). Compound 2a (150 mg, 0.550 mmol), 4-fluo-
rophenylacetylene (0.09 mL, 0.825 mmol), heating for 1.5 h.
Compound 3d was isolated after column chromatography, using
20% ether/petroleum ether, as a white solid (100 mg, 60%). Crys-
tallization from ether/petroleum ether gave white crystals, m.p.
167 ꢀC. 1H NMR (CDCl3, 300 MHz):
d 3.91 (s, 3H, OMe), 3.96 (s, 3H,
OMe), 7.10 (dd, 1H, J ¼ 9 and 2.1 Hz, 5-H), 7.23 (d, 1H, J ¼ 2.1 Hz, 7-
H), 7.84 (d, 1H, J ¼ 9 Hz, 4-H). 13C NMR (CDCl3, 75.4 MHz):
d
52.38
94–96 ꢀC. 1H NMR (CDCl3, 400 MHz):
d 4.01 (s, 3H, OMe), 7.08–7.15
(OMe), 55.71 (OMe) 104.01 (CH), 114.96 (C), 116.56 (CH), 124.30 (C),
126.18 (CH), 132.62 (C), 141.18 (C), 160.40 (C), 161.87 (C); MS (EI): m/
z (%) 302 (Mþ 81Br,100), 300 (Mþ 79Br, 96), 271 (Mþ 81Br ꢃ OMe, 82),
269 (Mþ 79Br ꢃ OMe, 81). HRMS Mþ calct. for C11H799BrO3S
299.9456, found 299.9462.
(m, 2H, Ar-H), 7.49–7.56 (m, 2H, Ar-H), 7.65–7.71 (m, 2H, Ar-H),
7.85–7.87 (m, 1H, Ar-H), 8.11–8.13 (m, 1H, Ar-H) ppm. 13C NMR
(CDCl3, 100.6 MHz):
d 52.51 (OMe), 82.48 (C), 97.50 (C), 115.83
(2 ꢂ CH, d, J ¼ 22 Hz), 118.99 (C), 122.67 (CH), 123.48 (C), 124.81
(CH), 125.33 (CH), 127.78 (CH), 133.53 (C), 133.97 (2 ꢂ CH, d,
J ¼ 8 Hz), 139.61 (C), 139.87 (C), 162.40 (C), 162.83 (CF, d, J ¼ 250 Hz)
ppm. MS (EI): m/z (%) 310 (Mþ, 100), 295 (Mþ ꢃ Me, 76), 207 (49).
HRMS Mþ calct. for C18H11FO2S 310.0464, found 310.0463.
4.1.3. General procedure for the Sonogashira coupling
Compounds 2a and 2b were submitted to a general procedure
similar to the one described in Section 4.1.1, obtaining the corre-
sponding Sonogashira coupling products 3a–e.
4.1.3.5. Methyl 6-methoxy-3-(phenylethynyl)benzo[b]thiophene-2-
carboxylate (3e). Compound 2b (150 mg, 0.500 mmol), phenyl-
acetylene (0.08 mL, 0.750 mmol), heating for 1.5 h. Compound 3e
was isolated after column chromatography, using 20% ether/
petroleum ether, as a white solid (97.0 mg, 60%). Crystallization
from ether/petroleum ether gave white crystals, m.p. 123–125 ꢀC.
4.1.3.1. Methyl 3-(phenylethynyl)benzo[b]thiophene-2-carboxylate
(3a). Compound 2a (150 mg, 0.550 mmol), phenylacetylene
(0.09 mL, 0.825 mmol), heating for 1.5 h. Compound 3a was iso-
lated after column chromatography, using 20% ether/petroleum
ether, as a beige solid (78 mg, 48%). Crystallization from ether/
petroleum ether gave beige crystals, m.p. 82–84 ꢀC. 1H NMR (CDCl3,
1H NMR (CDCl3, 300 MHz):
d 3.92 (s, 3H, OMe), 3.99 (s, 3H, OMe),
7.12 (dd, 1H, J ¼ 8.7 and 2.4 Hz, 5-H), 7.26 (d, 1H, J ¼ 2.4 Hz, 7-H),
300 MHz):
d
4.02 (s, 3H, OMe), 7.40–7.44 (m, 3H, Ar-H), 7.50–7.54
7.39–7.42 (m, 3H, Ar-H), 7.66–7.72 (m, 2H, Ar-H), 8.00 (d, 1H,

1898
M.-J.R.P. Queiroz et al. / European Journal of Medicinal Chemistry 44 (2009) 1893–1899
J ¼ 8.7 Hz, 4-H) ppm. 13C NMR (CDCl3, 75.4 MHz):
d
52.35 (OMe),
2H, Ar-H), 7.55–7.72 (m, 4H, Ar-H), 7.99–8.02 (m, 1H, Ar-H), 9.36–
9.40 (m, 1H, Ar-H) ppm. 13C NMR (CDCl3, 75.4 MHz):
65.56 (C),
55.66 (OMe), 82.85 (C), 98.30 (C), 104.11 (7-CH), 116.16 (5-CH),
122.85 (C), 123.40 (C), 125.68 (4-CH), 128.43 (2 ꢂ CH), 128.93 (CH),
130.91 (C), 132.00 (2 ꢂ CH), 133.73 (C), 141.71 (C), 160.04 (C), 162.52
(C) ppm. MS (EI): m/z (%) 322 (Mþ, 100), 307 (Mþ ꢃ Me, 59). HRMS
Mþ calct. for C19H14O3S 322.0664, found 322.0662.
d
115.51 (2 ꢂ CH, d, J ¼ 23 Hz), 123.62 (CH), 124.43 (CH), 125.11 (C),
126.35 (CH), 128.97 (CH), 131.20 (C, d, J ¼ 5 Hz), 132.38 (2 ꢂ CH, d,
J ¼ 9 Hz), 135.64 (C), 141.09 (C), 143.98 (C), 156.42 (C), 158.19 (C),
163.61 (CF, d, J ¼ 252 Hz) ppm. MS (EI): m/z (%), 422 (Mþ, 97), 394
(46), 267 (57), 239 (100). HRMS Mþ calct. for C17H8FIO2S 421.9274,
found 421.9279.
4.1.3.6. Methyl 6-methoxy-3-[(2-fuorophenyl)ethynyl]benzo[b]thio
phene-2-carboxylate (3f). Compound 2b (150 mg, 0.500 mmol),
2-fluoropenylacetylene (0.08 mL, 0.750 mmol), heating for 1.5 h.
Compound 3f was isolated after column chromatography, using
20% ether/petroleum ether, as a white solid (93.0 mg, 55%).
Crystallization from ether/petroleum ether gave white crystals,
4.1.4.3. 4-Iodo-3-(phenyl)-7-methoxybenzothieno[2,3-c]pyran-1-one
(4c). From compound 3e (100 mg, 0.300 mmol) and I2 (228 mg,
0.900 mmol), compound 4c was isolated as a white solid (94 mg,
70%), m.p. 274–276 ꢀC. 1H NMR (DMSO-d6, 400 MHz):
d 3.91 (s, 3H,
m.p. 146–148 ꢀC. 1H NMR (CDCl3, 400 MHz):
d
3.92 (s, 3H, OMe),
OMe), 7.32 (dd, 1H, J ¼ 9.2 and 2.4 Hz, 6-H), 7.53–7.56 (m, 3H, Ar-H),
4.00 (s, 3H, OMe), 7.12–7.22 (m, 3H, Ar-H), 7.27–7.28 (1H, signal
partially obscured by the signal of CHCl3, Ar-H), 7.36–7.42 (m, 1H,
Ar-H), 7.65–7.69 (m, 1H, Ar-H), 8.03 (d, 1H, J ¼ 8.8 Hz, 4-H) ppm. 13C
7.64–7.67 (m, 2H, Ar-H), 7.85 (d, 1H, J ¼ 2.4 Hz, 8-H), 9.19 (d, 1H,
J ¼ 9.2 Hz, 5-H) ppm. 13C NMR (DMSO-d6, 100.6 MHz):
d 55.86
(OMe), 67.21 (C), 106.24 (8-CH), 115.25 (6-CH), 121.62 (C), 126.76 (5-
CH),128.36 (2 ꢂ CH),128.85 (C), 129.97 (2 ꢂ CH),130.17 (CH),135.35
(C), 141.28 (C), 145.70 (C), 157.33 (C), 157.60 (C), 160.31 (C) ppm. MS
(EI): m/z (%) 434 (Mþ,100). HRMS Mþ calct. for C18H11IO3S 433.9474,
found 433.9482.
NMR (CDCl3, 100.6 MHz):
d 52.40 (OMe), 55.68 (OMe), 87.84 (C),
91.38 (C), 104.15 (CH), 111.64 (C, d, J ¼ 16 Hz), 115.63 (CH, d,
J ¼ 20 Hz),116.31 (CH),122.87 (C),124.10 (CH, d, J ¼ 4 Hz),125.77 (4-
CH), 130.71 (CH, d, J ¼ 8 Hz), 131.48 (C), 133.73 (C), 133.78 (CH),
141.71 (C), 160.10 (C), 162.53 (C), 162.84 (CF, d, J ¼ 241 Hz) ppm. MS
(EI): m/z (%) 340 (Mþ, 100). HRMS Mþ calct. for C19H13FO3S
340.0569, found 340.0571.
4.1.4.4. 4-Iodo-3-(2-fluorophenyl)-7-methoxybenzothieno[2,3-
c]pyran-1-one (4d). From compound 3f (100 mg, 0.300 mmol)
and I2 (228 mg, 0.900 mmol), compound 4d was isolated as a white
solid (87 mg, 65%), m.p. 228–230 ꢀC. 1H NMR (DMSO-d6, 400 MHz):
4.1.3.7. Methyl 6-methoxy-3-[(4-fuorophenyl)ethynyl]benzo[b]thio
phene-2-carboxylate (3g). Compound 2b (150 mg, 0.500 mmol), 4-
fluorophenylacetylene (0.09 mL, 0.825 mmol), heating for 1.5 h.
Compound 3g was isolated after column chromatography, using
20% ether/petroleum ether, as a white solid (123 mg, 73%). Crys-
tallization from ether/petroleum ether gave white crystals, m.p.
d
3.91 (s, 3H, OMe), 7.43 (dd,1H, J ¼ 9.3 and2.5 Hz, 6-H), 7.41–7.46 (m,
2H, Ar-H), 7.61–7.68 (m, 2H, Ar-H), 7.87 (d,1H, J ¼ 2.5 Hz, 8-H), 9.15 (d,
1H, J ¼ 9.3 Hz, 5-H) ppm. 13C NMR (DMSO-d6, 100.6 MHz):
d 55.86
(OMe), 70.57 (C), 106.24 (8-CH), 115.49 (6-CH), 116.06 (CH, d,
J ¼ 20.12 Hz), 122.16 (C), 123.46 (C, d, J ¼ 15.09 Hz), 124.85 (CH),
126.48 (5-CH), 128.56 (C), 132.03 (CH), 132.96 (CH, d, J ¼ 9.1 Hz),
140.69 (C), 145.73 (C), 152.57 (C), 157.40 (C), 158.84 (CF, d,
J ¼ 248.5 Hz), 160.40 (C) ppm. MS (EI): m/z (%) 452 (Mþ, 100), 424
(21), 297 (42). HRMS Mþ calct. for C18H10FIO3S 451.9379, found
451.9388.
160–162 ꢀC. 1H NMR (CDCl3, 400 MHz):
d 3.92 (s, 3H, OMe), 3.98 (s,
3H, OMe), 7.08–7.14 (m, 3H, Ar-H), 7.26 (d,1H, J ¼ 2.4 Hz, 7-H), 7.64–
7.68 (m, 2H, Ar-H), 7.97 (d, 1H, J ¼ 9.2 Hz, 4-H) ppm. 13C NMR
(CDCl3,100.6 MHz): d 52.35 (OMe), 55.67 (OMe), 82.63 (C), 97.17 (C),
104.14 (CH), 115.70 (CH), 116.06 (2 ꢂ CH, d, J ¼ 27 Hz), 119.00 (C, d,
J ¼ 3 Hz), 123.30 (C), 125.60 (CH), 130.91 (C), 133.63 (C), 133.96
(2 ꢂ CH, d, J ¼ 8 Hz), 141.74 (C), 160.09 (C), 162.45 (C), 162.94 (CF, d,
J ¼ 249 Hz) ppm. HRMS Mþ calct. for C19H13FO3S 340.0569, found
340.0569.
4.1.4.5. 4-Iodo-3-(4-fluorophenyl)-7-methoxybenzothieno[2,3-
c]pyran-1-one (4e). From compound 3g (100 mg, 0.300 mmol)
and I2 (228 mg, 0.900 mmol), compound 4e was isolated as a white
solid (91 mg, 67%), m.p. >300 ꢀC. 1H NMR (CDCl3, 400 MHz):
d 3.96
4.1.4. General procedure for the iodolactonization to compounds 4
Dry dichloromethane (2–3 mL), the Sonogahira product, and I2
(3 equiv.) were added into a dry Schlenk tube, under argon, and the
mixture was stirred at room temperature for 27 h. A precipitate
appeared during the reaction. Diethyl ether was added (5 mL) and
more solid was formed. The reaction mixture was filtered under
vacuum to retain the product, which was then washed with ether.
(s, 3H, OMe), 7.16–7.23 (m, 3H, Ar-H), 7.41 (d, 1H, J ¼ 2.4 Hz, 8-H),
7.65–7.69 (m, 2H, Ar-H), 9.24 (d, 1H, J ¼ 9.2 Hz, 5-H) ppm. 13C NMR
(CDCl3, 100.6 MHz): d 55.72 (OMe), 65.67 (C), 105.09 (8-CH), 115.04
(CH), 115.48 (2 ꢂ CH, d, J ¼ 22 Hz), 122.47 (C), 127.25 (5-CH), 129.34
(C), 131.28 (C, d, J ¼ 5 Hz), 132.36 (2 ꢂ CH, d, J ¼ 9 Hz), 141.34 (C),
146.57 (C), 156.56 (C), 158.13 (C), 160.66 (C), 163.62 (CF, d,
J ¼ 251 Hz) ppm. MS (EI): m/z (%) 452 (9). 97 (100), 83 (98), 81 (79),
HRMS Mþ calct. for C18H10FIO3S 451.9379, found 451.9366.
4.1.4.1. 4-Iodo-3-(4-methoxyphenyl)benzothieno[2,3-c]pyran-1-one
(4a). From compound 3b (100 mg, 0.300 mmol) and I2 (228 mg,
0.900 mmol), compound 4a was isolated as a white solid (66.0 mg,
4.1.5. General synthetic procedure for the lactonization of the
Sonogashira products 3d–g using TFA
50%), m.p. 238–240 ꢀC. 1H NMR (CDCl3, 400 MHz):
d
3.90 (s, 3H,
The Sonogashira products 3d, 3e, and 3g (0.300 mmol) were
stirred in TFA (3 mL), for 2 h at 50 ꢀC, in a water bath. After that, the
TFA was removed, under reduced pressure, and the greenish solid
obtained was washed with ether to give compounds 1d–f as light
yellow solids.
OMe), 7.01 (d, 2H, J ¼ 9 Hz, 30 and 50-H), 7.58–7.67 (m, 4H, Ar-H),
7.98–8.01 (m, 1H, Ar–H), 9.38–9.41 (m, 1H, Ar-H) ppm. 13C NMR
(CDCl3, 100.6 MHz):
d
55.40 (OMe), 64.75 (C), 113.56 (2 ꢂ CH, 30 and
50-CH), 123.58 (CH), 124.27 (CH), 124.64 (C), 126.47 (CH), 127.36 (C),
128.84 (CH), 131.83 (2 ꢂ CH, 20 and 60-CH), 135.74 (C), 141.50 (C),
143.98 (C), 157.49 (C), 158.51 (C), 161.03 (C) ppm. MS (EI): m/z (%)
434 (Mþ, 100), 406 (66), 264 (97), 208 (91). HRMS Mþ calct. for
C18H11IO3S 433.9474, found 433.9467.
4.1.5.1. 3-(4-Fluorophenyl)benzothieno[2,3-c]pyran-1-one (1d).
From compound 3d (100 mg, 0.300 mmol) compound 1d was iso-
lated as a light yellow solid (85 mg, 95%), m.p. 275–277 ꢀC. 1H NMR
(CDCl3, 400 MHz):
7.67 (m, 2H, Ar-H), 7.95–8.01 (m, 3H, Ar-H), 8.11–8.13 (m, 1H, Ar-H)
ppm. 13C NMR (CDCl3, 100.6 MHz):
96.72 (C), 96.72 (CH), 116.18
d 7.17–7.24 (m, 2H, Ar-H), 7.36 (s, 1H, 4-H), 7.55–
4.1.4.2. 4-Iodo-3-(4-fluorophenyl)benzothieno[2,3-c]pyran-1-one
(4b). From compound 3d (100 mg, 0.300 mmol) and I2 (228 mg,
0.900 mmol), compound 4b was isolated as a white solid (78 mg,
d
(2 ꢂ CH, d, J ¼ 22 Hz), 122.43 (C), 123.66 (CH), 123.70 (CH), 125.46
62%), m.p. 235–237 ꢀC. 1H NMR (CDCl3, 300 MHz):
d 7.17–7.24 (m,
(CH), 127.60 (2 ꢂ CH, d, J ¼ 9 Hz), 128.10 (C, d, J ¼ 3 Hz), 129.27 (CH),

M.-J.R.P. Queiroz et al. / European Journal of Medicinal Chemistry 44 (2009) 1893–1899
1899
134.26 (C), 143.72 (C), 156.54 (C), 158.63 (C), 163.99 (CF, d,
J ¼ 251 Hz) ppm. MS (EI): m/z (%) 296 (Mþ, 81), 268 (83), 267 (57),
239 (100). HRMS Mþ calct. for C17H9FO2S 296.0307, found
296.0306.
Institute (NCI, USA), the ‘‘In vitro Anticancer Drug Discovery
Screen’’, which uses the protein-binding dye sulforhodamine B to
assess cell growth [17,18]. Briefly, cells were plated in 96-well trays
at appropriate densities (1.5 ꢂ105cells/mL for MCF-7 and SF-268
and 0.75 ꢂ104 cells/mL for NCI-H460) to ensure exponential
growth throughout the experimental period and then allowed to
attach overnight. Attached cells were exposed for 48 h to five serial
concentrations of each compound, starting from a maximum
4.1.5.2. 3-(Phenyl)-7-methoxybenzothieno[2,3-c]pyran-1-one
(1e). From compound 3e (100 mg, 0.300 mmol) compound 1e was
isolated as a light yellow solid (83 mg, 90%), m.p. 215–217 ꢀC. 1H
NMR (CDCl3, 300 MHz):
d
3.93 (s, 3H, OMe), 7.15 (dd, 1H, J ¼ 9.0 and
concentration of 150 mM. Following this period, adherent cells were
2.4 Hz, 6-H), 7.31 (s, 1H, 4-H), 7.36 (d, 1H, J ¼ 2.1 Hz, 8-H), 7.45–7.54
fixed in situ, washed, and stained with SRB. The bound stain was
solubilized and the absorbance was measured at 492 nm in a plate
reader (EAR 400, STL-Labinstruments). A dose–response curve was
obtained for each test compound–cell line pair and the growth
inhibition 50% (GI50), corresponding to the concentration of
compound that inhibited 50% of the net cell growth, was calculated
as described elsewhere [18]. Doxorubicin, tested in the same
manner, was used as a positive control.
(m, 3H, Ar-H), 7.92–7.98 (m, 3H, Ar-H) ppm. 13C NMR (CDCl3,
75.4 MHz):
d 55.72 (OMe), 96.81 (CH), 105.25 (CH), 116.08 (CH),
120.16 (C), 124.41 (CH), 125.45 (2 ꢂ CH), 127.88 (C), 128.94 (2 ꢂ CH),
130.28 (CH), 131.81 (C), 143.77 (C), 145.87 (C), 155.42 (C), 158.74 (C),
161.05 (C) ppm. MS (EI): m/z (%) 308 (Mþ, 100), 280 (60), 237 (46).
HRMS Mþ calct. for C18H12O3S 308.0507, found 308.0504.
4.1.5.3. 3-(4-Fluorophenyl)-7-methoxybenzothieno[2,3-c]pyran-1-
one (1f). From compound 3g (100 mg, 0.300 mmol) compound 1f
was isolated as a light yellow solid (82 mg, 84%), m.p. 258–260 ꢀC.
Acknowledgments
1H NMR (CDCl3, 400 MHz):
d
3.94 (s, 3H, OMe), 7.14–7.21 (m, 3H, Ar-
H), 7.26 (s, 1H, 4-H), 7.38 (d, 1H, J ¼ 2.4 Hz, 8-H), 7.92–7.98 (m, 3H,
Ar-H) ppm. 13C NMR (CDCl3, 100.6 MHz):
55.76 (OMe), 96.63 (CH),
The authors thank the Foundation for the Science and Tech-
nology (FCT, Portugal) and FEDER for financial support to CQ-Univ.
Minho, through project POCI/QUI/59407/2004. R.C. Calhelha
acknowledges FCT for his Ph.D. grant (SFRH/BD/29274/2006). L.A.
Vale-Silva acknowledges FCT for his post-doctoral grant (SFRH/
BPD/29112/2006).
d
105.32 (CH), 116.13 (2 ꢂ CH, d, J ¼ 22 Hz), 116.17 (CH), 120.05 (C),
124.42 (CH), 127.56 (2 ꢂ CH, d, J ¼ 9 Hz), 127.86 (C), 128.15 (C, d,
J ¼ 4 Hz), 143.72 (C), 145.96 (C), 156.57 (C), 158.58 (C), 161.13 (C),
163.94 (CF, d, J ¼ 252 Hz) ppm. MS (EI): m/z (%) 326 (Mþ, 6), 183
(46), 81 (100), 83 (82). HRMS Mþ calct. for C18H11FO3S 326.0413,
found 326.0416.
References
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growth of the three human tumor cell lines in vitro were evaluated
according to the procedure adopted by the National Cancer