Synthesis of new heteroaryl and heteroannulated indoles from dehydrophenylalanines: Antitumor evaluation

Article abstract of DOI:10.1016/j.bmc.2008.04.004

A 3-(dibenzothien-4-yl)indole and a phenylbenzothienoindole or a 3-(dibenzofur-4-yl)indole and a phenylbenzofuroindole were prepared by a metal-assisted C-N intramolecular cyclization of the methyl esters of N-Boc-(E) or (Z)-β-dibenzothien-4-yl or β-dibenzofur-4-yl dehydrophenylalanines. The latter were obtained by Suzuki cross-coupling of the methyl esters of N-Boc-(E) or (Z)-β-bromodehydrophenylalanines with dibenzothien-4-yl or dibenzofur-4-yl boronic acids, in high yields. The intramolecular cyclization from E or Z pure Suzuki-coupling products gave the corresponding heteroaryl and heteroannulated indoles, in different ratios, by either direct cyclization or cyclization after isomerisation. Three of the cyclized compounds, the two heteroarylindoles and the phenylbenzothienoindole, 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 methyl 3-(dibenzothien-4-yl)indole-2-carboxylate was the most potent compound with GI₅₀ values ranging from 11 to 17 μM.

Full text of DOI:10.1016/j.bmc.2008.04.004

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 5584–5589
Synthesis of new heteroaryl and heteroannulated indoles
from dehydrophenylalanines: Antitumor evaluation
Maria-Joao R. P. Queiroz,a,* Ana S. Abreu,a M. Solange D. Carvalho,a
˜
a
b
b
´
Paula M. T. Ferreira, Nair Nazareth and M. Sao-Jose Nascimento
˜
aCentro de Quı´mica, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal
ˆ
Laborato´rio de Microbiologia, Faculdade de Farma´cia, Centro de Estudos de Quı´mica Organica,
Fitoquı´mica e Farmacologia da Universidade do Porto, (CEQOFFUP), 4050-047 Porto, Portugal
b
Received 6 December 2007; accepted 1 April 2008
Available online 6 April 2008
Abstract—A 3-(dibenzothien-4-yl)indole and a phenylbenzothienoindole or a 3-(dibenzofur-4-yl)indole and a phenylbenzofuroin-
dole were prepared by a metal-assisted C–N intramolecular cyclization of the methyl esters of N-Boc-(E) or (Z)-b-dibenzothien-
4-yl or b-dibenzofur-4-yl dehydrophenylalanines. The latter were obtained by Suzuki cross-coupling of the methyl esters of
N-Boc-(E) or (Z)-b-bromodehydrophenylalanines with dibenzothien-4-yl or dibenzofur-4-yl boronic acids, in high yields. The
intramolecular cyclization from E or Z pure Suzuki-coupling products gave the corresponding heteroaryl and heteroannulated
indoles, in different ratios, by either direct cyclization or cyclization after isomerisation. Three of the cyclized compounds, the
two heteroarylindoles and the phenylbenzothienoindole, 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 methyl 3-(dibenzothien-4-yl)indole-2-carboxylate was the most potent compound with GI50 values ranging from 11 to 17 lM.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
dium catalysis in the synthesis of heterocycles including
indoles.4
Many planar heteroaromatic derivatives have shown
anti-proliferative activity in vitro and some of them
are important anticancer drugs.1 Some of these types
of compounds have the indole nucleus which is a struc-
tural component of a large number of biologically active
natural and unnatural compounds. Recently the high
anti-proliferative activity of 2-phenylindole-3-carbalde-
hydes in two human breast cancer cell lines (MDA-
MB 231 and MCF-7) was reported and the authors were
able to show that the tubulin is the primary target of
these agents inhibiting its polymerization.2,3
In recent years we have been interested in the synthesis
of different heteroaromatic systems from brominated
dehydroamino acids following a strategy of Suzuki
cross-coupling and metal-assisted intramolecular C–N
cyclization, developed by us.5,6 Thus we were able to
obtain benzo[b]thienylthienoindoles and benzo[b]thi-
enylbenzothienopyrroles,5b 3-arylindoles bearing elec-
tron-donating or electron-withdrawing groups5c and a
(dibenzothien-4-yl)benzothienoindole or a (dibenzofur-
4-yl)benzofuroindole5d from the methyl ester of
N-(tert-butoxycarbonyl)-b,b-dibromodehydroalanine.5a
We have also prepared benzo[b]thienylindoles and
phenylthienoindoles from the methyl ester of N-(tert-
butoxycarbonyl)-(Z)-b-bromodehydrophenylalanine and
bromobenzo[b]thiophenes using a one-pot borylation
of the latter and Suzuki-coupling reaction (BSC), fol-
lowed by the same type of intramolecular cyclization.6
The synthesis of indoles has been extensively reported
and developed in a variety of methods. In the last 40
years palladium-catalyzed reactions have achieved an
important place in organic chemistry and a great deal
of studies have been directed toward the use of palla-
Keywords: Dehydrophenylalanines; Suzuki-coupling; Metal-assisted
C–N intramolecular cyclization; Heteroarylindoles; Heteroannulated
indoles; Antitumor evaluation.
In the present work we describe the synthesis of a
3-(dibenzothien-4-yl)indole and a phenylbenzothienoin-
dole or a 3-(dibenzofur-4-yl)indole and a phenylbenzo-
furoindole by a metal-assisted intramolecular C–N
*
Corresponding author. Tel.: +351 253604378; fax: +351 253678983;
e-mail: mjrpq@quimica.uminho.pt
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.04.004

M.-J. R. P. Queiroz et al. / Bioorg. Med. Chem. 16 (2008) 5584–5589
5585
cyclization of E or Z pure stereoisomers of b-dibenzoth-
B(OH)2
H
N
H
N
COOMe
COOMe
Br
X
ien-4-yl or b-dibenzofur-4-yl dehydrophenylalanines,
respectively. The latter were obtained in high yields by
Suzuki cross-couplings of b-bromodehydrophenylala-
nines7 with dibenzothien-4-yl or dibenzofur-4-yl boronic
acids.
Boc
Boc
i
1-
E
X
Three of the cyclized products obtained, the two hetero-
arylindoles and the phenylbenzothienoindole, were evalu-
ated for their capacity to inhibit the in vitro growth of
three human tumor cell lines, MCF-7 (breast adenocarci-
noma), NCI-H460 (non-small cell lung cancer), and
SF-268 (CNS cancer). The methyl 3-(dibenzothien-4-
yl)indole-2-carboxylate showed to be the most potent
compound whereas the phenylbenzothienoindole exhib-
ited only a moderate inhibitory effect against the human
tumor cell lines tested. The activities of the heteroarylin-
doles studied showed that the oxygen isostere, the methyl
2-
3-
E
E
X=S, 97%
X=O, 95%
B(OH)2
X
H
N
H
COOMe
N
COOMe
Boc
Boc
Br
1-
i
Z
X
3-(dibenzofur-4-yl)indole-2-carboxylate, presents
weaker growth inhibitory effect although in NCI-H460
cell line the results for these two compounds are similar.
a
2-
3-
Z
X=S, 85%
X=O, 95%
Z
Scheme 1. Synthesis of the Suzuki cross-coupling products 2-E, 2-Z,
3-E, and 3-Z.
2. Results and discussion
2.1. Synthesis
H
The methyl esters of N-Boc-(E) or (Z)-b-hetero-
aryldehydrophenylalanines were prepared in high to
excellent yields by Suzuki cross-coupling of pure E or
Z stereoisomers of b-bromodehydrophenylalanine 1-E
or 1-Z, already described by us,7 with dibenzothien-4-
yl or dibenzofur-4-yl boronic acids, using similar condi-
tions to the ones applied by us in bis-Suzuki cross-cou-
plings.5b The Suzuki-coupling products 2-E, 2-Z, 3-E,
and 3-Z (Scheme 1) were obtained with maintenance
of the starting material stereochemistry, which was con-
firmed by NOE difference experiments. The saturation
of the a-NH enhanced the signals of the phenyl protons
on the E-isomers while the saturation of the OCH3 pro-
tons of the ester group enhanced the signals of the phe-
nyl protons on the Z-isomers.
N
COOMe
Boc
X
X= S or O
2 or 3-E or Z
50 mol% Pd(OAc)2,
3 equiv. Cu(OAc)2,
DMF
NH
NH
COOMe
COOMe
+
X
X
The intramolecular metal-assisted C–N cyclization of
the Suzuki-coupling products 2-E, 2-Z, 3-E, and 3-Z
gave in both cases two new heterocyclic compounds,
the 3-(dibenzothien-4-yl)indole 4 and the phenylbenz-
othienoindole 5 or the 3-(dibenzofur-4-yl)indole 6 and
the phenylbenzofuroindole 7 in different ratios depend-
ing on the starting stereoisomer (Scheme 2 and Table
1). These compounds result either from direct cycliza-
tion or cyclization after isomerization of theSuzuki-cou-
pling products.
4 X = S
6 X = O
5 X = S
7 X = O
Scheme 2. Synthesis of heteroaryl and heteroannulated indoles 4–7
from the pure stereoisormers 2-E, 2-Z, 3-E, and 3-Z.
Table 1. Heteroaryl and heteroannulated indoles 4–7 prepared via
Scheme 2
The heteroarylindoles 4 and 6 are always the major
products relative to the heteroannulated indoles 5 and
7, respectively. When the starting material is 2-Z com-
pound 4 is only 1.5–2 in excess relative to the phenyl-
benzothienoindole 5, but in the other cases and
specially in the reactions of 3-E and 3-Z the excess of
the 3-(dibenzofur-4-yl)indoles is much higher (Table
1). The cyclized products were separated by column
chromatography.
Starting
material
Cyclized products
(ratios, yields)
Time and temperature
2-E
4/5 (3:1, 41%/14%)
3 h 30 min, 130 ꢁC +
3 h 30 min, 160 ꢁC
5 h, 130 ꢁC + 2 h, 160 ꢁC
12 h, 160 ꢁC
5 h, 160 ꢁC
3 h, 160 ꢁC
2-Z
2-Z
3-E
3-Z
4/5 (1.5:1, 37%/26%)
4/5 (2:1, 20%/10%)
6/7 (6:1, 30%/5%)
6/7 (5:1, 49%/10%)

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M.-J. R. P. Queiroz et al. / Bioorg. Med. Chem. 16 (2008) 5584–5589
We have already postulated a possible mechanism for
the intramolecular Pd(II) C–N cyclization, involving
the formation of a palladacycle. After extrusion of
Pd(0) it is thought that Cu(OAc)2 reoxidizes it to Pd(II),
avoiding the use of a stoichiometric amount of
Pd(OAc)2. As acetic acid is formed, the Boc group is re-
moved.6 The Pd(II) may also be involved in the isomeri-
sation of the Suzuki-coupling products.
3. Conclusions
Suzuki cross-coupling products were obtained in high to
excellent yields, from E or Z b-bromodehydrophenylala-
nines and dibenzothien-4-yl or dibenzofur-4-yl boronic
acids, maintaining the stereochemistry of the starting
materials. The metal-assisted (Pd/Cu) C–N intramolecu-
lar cyclization of the Suzuki products gave new hetero-
aryl and heteroannulated indoles in different ratios, by
either direct cyclization or cyclization after
isomerization.
2.2. Effect on the growth of human tumor cell lines
The effect of compounds 4–6 was evaluated on the in vi-
tro growth of three human tumor cell lines representing
different tumor types, namely, breast adenocarcinoma
(MCF-7), non-small cell lung cancer (NCI-H460) and
CNS cancer (SF-268), after a continuous exposure of
48 h. The results are summarized in Table 2.
The inhibitory activity of the new heteroarylindoles 4
and 6 and of the phenylbenzothienoindole 5, on the
growth of human tumor cell lines, MCF-7 (breast ade-
nocarcinoma), NCI-H460 (non-small cell lung cancer)
and SF-268 (CNS cancer), was studied. The results
showed that the methyl 3-(dibenzothien-4-yl)indole-2-
carboxylate (4) has an interesting growth inhibitory
activity in all the tumor cell lines tested, giving to this
skeleton new perspectives for the development of new
antitumor agents.
All the compounds were able to inhibit the growth of the
human tumor cell lines in a dose-dependent manner
(data not shown).
The 3-(dibenzothien-4-yl)indole 4 showed the better re-
sults, exhibiting an equivalent potency in all the three tu-
mor cell lines, while compound 5 showed only a
moderated growth inhibitory effect. Comparing the
activities of the heteroarylindoles 4 and 6 it is observed
that the oxygen isostere 6 presents a weaker growth
inhibitory effect although the results in NCI-H460 cell
line are comparable.
4. Experimental
4.1. Synthesis
Melting points (ꢁC) were determined in a Gallenkamp
1
apparatus and are uncorrected. H and 13C NMR spec-
Table 2. Effect of Compounds 4–6 on the growth of three human tumor cell lines
Compound
GI50 (lM)
MCF-7
NCI-H460
SF-268
NH
S
11.0 0.6
COOMe
12.7 1.5
17.0 1.2
3-(Dibenzothien-4-yl)indole 4
NH
COOMe
72.7 17.5
40.3 12.8
50.0 9.1
S
Phenylbenzothienoindole 5
NH
26.5a
18.0 5.0
35.0a
O
COOMe
3-(Dibenzofur-4-yl)indole 6
Results are given in concentrations that were able to cause 50% of cell growth inhibition (GI50) after a continuous exposure of 48 h and show
means SEM of three-independent experiments performed in duplicate.
a Results fromtwo-independent experiments performed in duplicate. Doxorubicin was used as positive control, GI50: MCF-7 = 42.8 8.2 nM; NCI-
H460 = 94.0 8.7 nM, and SF-268 = 94.0 7.0 nM.

M.-J. R. P. Queiroz et al. / Bioorg. Med. Chem. 16 (2008) 5584–5589
5587
tra were recorded on a Varian Unity Plus at 300 and
75.4 MHz, respectively, unless stated. HMQC and
HMBC were used to attribute some signals. NOE differ-
ence experiments were also performed. MS (EI) or
(FAB) and HRMS data were recorded by the mass spec-
trometry service of the University of Vigo, Spain. Ele-
mental analysis was performed on a LECO CHNS 932
elemental analyzer.
matography using solvent gradient from pure petro-
leum ether to 30% diethyl ether/petroleum ether,
compound 2-Z was obtained as a white solid, after
some washes with petroleum ether, (217 mg, 85%),
1
mp 187–189 ꢁC; H NMR (300 MHz, CDCl3): d 1.40
(9H, s, CH3Boc), 3.63 (3H, s, OCH3), 5.93 (1H, broad
s, NH), 7.20–7.33 (6H, m, ArH), 7.45–7.53 (3H, m,
ArH), 7.77–7.80 (1H, m, ArH), 8.15–8.20 (2H, m,
ArH) ppm. 13C NMR (75.4 MHz, CDCl3): d 28.06
(C(CH3)3), 52.21 (OCH3), 81.47 (OC(CH3)3), 121.52
(CH), 121.69 (CH), 122.68 (CH), 124.46 (CH), 124.98
(CH), 127.08 (CH), 127.41 (C), 128.11 (CH), 128.20
(2· CH), 128.47 (CH), 128.87 (2· CH), 132.95 (C),
135.36 (C), 136.44 (C), 138.32 (C), 139.67 (C), 152.61
(C@O), 166.18 (C@O) ppm. MS: (FAB) m/z (%) 461
(M++2, 8), 460 (M++1, 27), 459 (M+, 42), 404 (31),
359 (M+ ꢀBoc, 100). Elemental analysis Calcd (%)
C27H25NO4S C, 70.57; H, 5.48; N, 3.05; S, 6.98, found
C, 70.40; H, 5.46; N, 3.10; S, 7.08.
PdCl2(dppf)ÆCH2Cl2 (1:1) corresponds dichloro[1,10-
bis(diphenylphosphane) ferrocene]palladium(II) com-
plex with dichloromethane (1:1) to and were purchased
from Sigma–Aldrich.
The reactions were monitored by thin-layer chromatog-
raphy (TLC). Column chromatography was performed
on Macherey-Nagel silica gel 230–400 mesh. Petroleum
ether refers to the boiling range 40–60 ꢁC. When solvent
gradient was used, the increase of polarity was made
from neat petroleum ether to mixtures of ether/petro-
leum ether, increasing 10% of ether each time until the
isolation of the product.
4.1.1.3. Methyl ester of N-(tert-butoxycarbonyl)-(E)-
b-(dibenzofur-4-yl)dehydrophenylalanine (3-E). From
Boc-(E)-DPhe-(b-Br)-OMe 1-E (205 mg, 0.575 mmol)
according to the general procedure and column chroma-
tography using solvent gradient from pure petroleum
ether to 40% diethyl ether/petroleum ether compound
3-E was obtained as a white solid (241 mg, 95%), mp
4.1.1. General procedure for the synthesis of compounds
2-E, 2-Z, 3-E, and 3-Z. To a solution of the pure stereo-
isomer E or Z of the methyl ester of N-Boc-b-bro-
modehydrophenyalanine [Boc-DPhe-(b-Br)-OMe]7 (200
mg, 0.561 mmol) in THF/H2O (10:1), the dibenzoth-
ien-4-yl or the dibenzofur-4-yl boronic acids (1.5 equiv),
Cs2CO3 (1.4 equiv), and PdCl2(dppf)ÆCH2Cl2 (1:1)
(15 mol%) were added. The reaction was heated for
1 h 30 min at 70–80 ꢁC. The THF was removed under
reduced pressure and the residue in ethyl acetate
(25 mL) was washed with water and brine (2· 10 mL).
The organic layer was dried with MgSO4, filtered and re-
moval of the solvent gave an oil which was submitted to
column chromatography.
1
110–112 ꢁC; H NMR (300 MHz, CDCl3): d 1.49 (9H,
s, CH3Boc), 3.43 (3H, s, OCH3), 6.21 (1H, broad s,
NH), 7.17–7.19 (1H, m, ArH) 7.26–7.51 (9H, m, ArH),
7.88–7.91 (1H, m, ArH), 7.93–7.96 (1H, m, ArH) ppm.
13C NMR (75.4 MHz, CDCl3): d 28.13 (C(CH3)3),
51.93 (OCH3), 81.30 (OC(CH3)3), 111.68 (CH), 120.38
(CH), 120.63 (CH), 122.57 (CH), 122.76 (CH), 124.04
(C), 124.14 (C), 124.20 (C), 127.21 (CH), 127.61 (C),
128.33 (CH), 128.46 (CH), 128.75 (2· CH), 129.60 (2·
CH), 138.17 (C), 152.94 (C), 153.79 (C), 155.97 (C),
165.89 (C@O) ppm. MS: (FAB) m/z (%) 444 (M++1,
98), 443 (M+, 25), 388 (M+ ꢀC(CH 3)3, 100), 343
(M+ꢀBoc, 96). HRMS: [M++H] Calcd for C27H26NO5
444.1811, found 444.1817.
4.1.1.1. Methyl ester of N-(tert-butoxycarbonyl)-(E)-
b-(dibenzothien-4-yl)dehydrophenylalanine (2-E). From
Boc-(E)-DPhe-(b-Br)-OMe 1-E (170 mg, 0.477 mmol)
according to the general procedure and column chroma-
tography using solvent gradient from pure petroleum
ether to 30% diethyl ether/petroleum ether compound
2-E was obtained as a white solid (213 mg, 97%), mp
4.1.1.4. Methyl ester of N-(tert-butoxycarbonyl)-(Z)-
b-(dibenzofur-4-yl)dehydrophenylalanine (3-Z). From
Boc-(E)-DPhe-(b-Br)-OMe 1-Z (205 mg, 0.575 mmol)
according to the general procedure and column chroma-
tography using solvent gradient from pure petroleum
ether to 40% diethyl ether/petroleum ether, compound
3-Z was obtained as a white solid (241 mg, 95%), mp
1
151–153 ꢁC; H NMR (300 MHz, CDCl3): d 1.48 (9H,
s, CH3Boc), 3.40 (3H, s, OCH3), 6.30 (1H, broad s,
NH), 7.34–7.49 (9H, m, ArH), 7.71–7.74 (1H, m, ArH),
8.09–8.15 (2H, m, ArH) ppm. 13C NMR (75.4 MHz,
CDCl3): d 28.13 (C(CH3)3), 52.03 (OCH3), 81.49
(OC(CH3)3), 121.08 (CH), 121.56 (CH), 122.64 (CH),
124.22 (CH), 124.29 (CH), 126.67 (CH), 127.55 (CH),
128.17 (C), 128.63 (CH), 128.85 (2· CH), 129.73 (2·
CH), 134.46 (C), 135.23 (C), 136.04 (C), 136.29 (C),
139.76 (C), 139.80 (C), 152.67 (C@O), 165.87 (C@O)
ppm. MS: (FAB) m/z (%) 460 (M+ +1, 30), 459 (M+,
38), 404 (M+ ꢀ55, 40), 359 (M+ ꢀBoc, 100). HRMS:
1
158–159 ꢁC; H NMR (300 MHz, CDCl3): d 1.38 (9H,
s, CH3Boc), 3.61 (3H, s, OCH3), 5.98 (1H, broad s,
NH), 7.13–7.15 (1H, m, ArH), 7.21–7.57 (9H, m,
ArH), 7.95–8.00 (2H, m, ArH) ppm. 13C NMR
(75.4 MHz, CDCl3): d 28.08 (C(CH3)3), 52.13 (OCH3),
81.00 (OC(CH3)3), 111.96 (CH), 120.65 (CH), 120.98
(CH), 122.55 (C), 122.94 (CH), 123.11 (CH), 123.86
(C), 124.99 (C), 127.50 (CH), 127.67 (C), 127.88 (CH),
128.07 (2· CH), 129.13 (CH), 129.15 (2· CH), 139.27
(C), 152.74 (C), 152.86 (C), 156.16 (C), 166.24 (C@O)
ppm. MS: (FAB) m/z (%) 444 (M+ +1, 20), 443 (M+,
30), 388 (M+ ꢀC(CH3)3, 21), 343 (M+ ꢀBoc, 100).
HRMS: [M++H] Calcd for C27H26NO5 444.1811, found
444.1801.
+
[M+H] Calcd for C27H26NO4S 460.1583, found 460.1576.
4.1.1.2. Methyl ester of N-(tert-butoxycarbonyl)-(Z)-
b-(dibenzothien-4-yl)dehydrophenylalanine (2-Z). From
Boc-(Z)-DPhe-(b-Br)-OMe 1-Z (200 mg, 0.561 mmol)
according to the general procedure and column chro-

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M.-J. R. P. Queiroz et al. / Bioorg. Med. Chem. 16 (2008) 5584–5589
4.1.2. General procedure for the synthesis of heteraryl and
heteroannulated indoles. To a solution of 2-E, 2-Z, 3-E
or 3- Z in DMF (0.1 M), Pd(OAc)2 (50 mol%) and
Cu(OAc)2.H2O (3 equiv) were added and the mixture
was heated at 130–160ꢁC (Table 1), monitoring the
reaction by TLC. Ethyl acetate (30 mL) was then
added and the organic layer washed with water and
brine (2· 15 mL), dried with MgSO4, filtered and
the solvent was evaporated at reduce pressure to give
an oil that was submitted to column chromatography.
lesser polar product) was obtained as a white solid
(36.0 mg, 30%), mp 226–228 ꢁC, H NMR (300 MHz,
1
CDCl3): d 3.68 (3H, s, OCH3), 7.15–7.20 (1H, m,
ArH), 7.34–7.55 (6H, m, ArH), 7.58–7.66 (2H, m,
ArH), 8.03 (2H, dd, J = 7.8 and 1.2 Hz ArH), 9.34
(1H, broad s, NH) ppm. 13C NMR (75.4 MHz; CDCl3):
d 51.86 (OCH3), 111.65 (CH), 111.89 (CH), 117.62 (C),
118.39 (C), 119.88 (CH), 120.63 (CH), 121.02 (CH),
121.85 (CH), 122.43 (CH), 122.63 (CH), 124.04 (C),
124.27 (C), 124.43 (C), 125.86 (CH), 127.04 (CH),
127.82 (C), 129.25 (CH), 135.92 (C), 154.36 (C),
156.08 (C), 162.54 (C@O) ppm. MS: (EI) m/z (%) 341
(M+, 28), 309 (M+ꢀOCH3, 100) 281 (M+ꢀCOOCH3,
19), 280 (34). HRMS: M+ Calcd for C22H15NO3
341.1052, found 341.1059. Compound 7 was isolated
as a beige solid (6.00 mg, 5%) mp 247–249 ꢁC, 1H
NMR (400 MHz, CDCl3): d 3.85 (3H, s, OCH3), 7.31–
7.37 (2H, m, ArH), 7.43 (1H, d, J = 8.8 Hz ArH),
7.47–7.56 (4H, m, ArH), 7.76–7.78 (2H, m, ArH),
7.91–7.93 (2H, m, ArH), 9.34 (1H, broad s, NH) ppm.
13C NMR (100.6 MHz; CDCl3): d 51.88 (OCH3),
107.48 (CH), 111.83 (CH), 114.48 (C), 116.53 (C),
118.46 (CH), 119.31 (CH), 122.38 (C), 122.74 (CH),
122.91 (C), 124.77 (C), 125.09 (CH), 127.40 (2· CH),
127.51 (CH), 131.07 (2· CH), 133.18 (C), 136.21 (C),
150.56 (C), 155.42 (C), 162.10 (C@O) ppm. MS: (EI)
m/z (%) 341 (M+, 20), 309 (M+ꢀOCH3, 100) 281 (M+
ꢀCOOCH3, 17), 280 (32). HRMS: M+ Calcd for
C22H15NO3 341.1052, found 341.1051.
4.1.2.1. Methyl 3-(dibenzothien-4-yl)-1H-indole-2-car-
boxylate (4) and methyl 1-phenyl-3H-benzothieno[2,
3-e]indole-2-carboxylate (5). From compound 2- E
(138.0 mg, 0.300 mmol) according to the general proce-
dure, heating for 3 h 30 min at 130 ꢁC + 3 h 30 min at
160 ꢁC and column chromatography using solvent gra-
dient from pure petroleum ether to 10% ethyl acetate/
petroleum ether, compound 4 (the lesser polar product)
was obtained as a yellow solid (44.0 mg, 41%), mp 230–
1
232 ꢁC, H NMR (300 MHz, CDCl3): d 3.70 (3H, s,
OCH3), 7.12–7.17 (1H, m, ArH), 7.38–7.63 (7H, m,
ArH), 7.75–7.78 (1H, m, ArH), 8.21–8.25 (2H, m,
ArH), 9.21 (1H, broad s, NH) ppm. 13C NMR
(75.4 MHz; CDCl3): d 51.95 (OCH3), 111.90 (CH),
120.67 (CH), 120.91 (CH), 121.67 (CH), 121.77 (C),
121.98 (CH), 122.68 (CH), 123.55 (C), 124.22 (CH),
124.32 (CH), 126.05 (CH), 126.59 (CH), 127.30 (C),
128.76 (CH), 129.05 (C), 135.62 (C), 135.79 (C),
135.93 (C), 139.61 (C), 141.02 (C), 162.21 (C@O) ppm.
MS: (EI) m/z (%) 359 (M++2, 3), 358 (M++1, 11), 357
(M+, 75), 325 (M+ꢀOCH3, 100) 296 (M+ꢀCOOCH 3,
70). HRMS:Calcd for M+ C22H15NO2S 357.0824, found
357.0820. Compound 5 was isolated as a yellow solid
Compounds 6 (53.0 mg, 49%) and 7 (11.0 mg, 10%) were
also obtained from compound 3-Z (140.0 mg,
0.316 mmol) heating for 3 h at 160 ꢁC in a 5:1 ratio.
1
(15.0 mg, 14%), mp 246–248 ꢁC, H NMR (300 MHz,
4.2. Biological activity
CDCl3): d 3.81 (3H, s, OCH3), 7.33–7.39 (1H, m,
ArH), 7.42–7.48 (1H, m, ArH), 7.53–7.60 (6H, m,
ArH), 7.73–7.76 (1H, m, ArH), 8.12–8.15 (2H, m,
ArH), 9.24 (1H, broad s, NH) ppm. 13C NMR
(75.4 MHz; CDCl3): d 51.87 (OCH3), 109.48 (CH),
119.72 (CH), 120.60 (CH), 122.44 (C), 122.60 (CH),
122.93 (C), 124.30 (CH), 125.22 (CH), 127.97 (2· CH),
128.02 (CH), 129.66 (C), 129.71 (C), 130.83 (2· CH),
132.98 (C), 133.24 (C), 134.40 (C), 135.63 (C), 138.68
(C), 162.07 (C@O) ppm. MS: (EI) m/z (%) 359 (M++2,
2), 358 (M++1, 7), 357 (M+, 40), 325 (M+ꢀOCH3, 100)
296 (M+ꢀCOOCH3, 62). HRMS: M+ Calcd for
C22H15NO2S 357.0824, found 357.0828.
4.2.1. Material and methods
4.2.1.1. Reagents. Fetal bovine serum (FBS) and
L-glutamine, were from Gibco Invitrogen Co. (Scotland,
UK). RPMI-1640 medium was from Cambrex (New Jer-
sey, USA). Dimethyl sulfoxide (DMSO), doxorubicin,
penicillin, streptomycin and sulforhodamine B (SRB)
were from SigmaChemical Co. (Saint Louis, USA).
4.2.1.2. Samples. Stock solutions of compounds 4–6
were prepared in DMSO and kept at ꢀ20 ꢁC. Appropri-
ate dilutions of the compounds were freshly prepared
just prior the assays. Final concentrations of DMSO
did not interfere with the cell growth.
Compounds 4 (63.0 mg, 37%) and 5 (44.0 mg, 26%) were
also obtained from compound 2-Z (220.0 mg,
0.479 mmol) after heating for 5 h at 130 ꢁC + 2 h at
160 ꢁC, in a 1.5:1 ratio. The same reaction was done
heating for 12 h at 160 ꢁC and compounds 4 and 5 were
obtained in a 2:1 ratio (Table 1).
4.2.1.3. Cell cultures. Three human tumor cell lines,
MCF-7 (breast adenocarcinoma), NCI-H460 (non-small
cell lung cancer), and SF-268 (CNS cancer) were used.
MCF-7 was obtained from the European Collection of
Cell Cultures (ECACC, Salisbury, UK) and NCI-H460
and SF-268 were kindly provided by the National Can-
cer Institute (NCI, Bethesda, USA). They grow as
monolayer and routinely maintained in RPMI-1640
medium supplemented with 5% heat inactivated FBS,
2 mM glutamine and antibiotics (penicillin 100 U/mL,
streptomycin 100 lg/mL), at 37 ꢁC in a humidified atmo-
sphere containing 5% CO2. Exponentially growing cells
were obtained by plating 1.5 · 105 cells/mL for MCF-7
4.1.2.2. Methyl 3-(dibenzofur-4-yl)-1H-indole-2-car-
boxylate (6) and methyl 1-phenyl-3H-benzofuro[2,3-
e]indole-2-carboxylate (7) . From compound 3- E
(155.0 mg, 0.350 mmol) according to the general proce-
dure, heating for 5 h at 160 ꢁC and column chromatog-
raphy using solvent gradient from pure petroleum ether
to 10% ethyl acetate/petroleum ether, compound 6 (the

M.-J. R. P. Queiroz et al. / Bioorg. Med. Chem. 16 (2008) 5584–5589
5589
and SF-268 and 0.75 · 104 cells/mL for NCI-H460, fol-
lowed by 24 h of incubation. The effect of the vehicle
solvent (DMSO) on the growth of these cell lines was
evaluated in all the experiments by exposing untreated
control cells to the maximum concentration (0.5%) of
DMSO used in each assay.
authors thank National Cancer Institute, Bethesda,
MD (USA), for kindly providing the tumor cell lines.
References and notes
1. Demeunynck, M.; Bailly, C.; Wilson, W. D. Small Molecule
DNA and RNA Binders; Wiley-VHC Verlag GmbH & Co.
KGaA: Weinheim, 2003.
4.2.1.4. Tumor cell growth assay. The effects of 4– 6
on the in vitro growth of human tumor cell lines were
evaluated according to the procedure adopted by the
National Cancer Institute (NCI, USA) in the ‘In vitro
Anticancer Drug Discovery Screen’ that uses the pro-
tein-binding dye sulforhodamine B to assess cell
growth.8,9 Briefly, exponentially, cells growing in 96-well
plates were then exposed for 48 h to five serial concen-
trations of each compound, starting from a maximum
concentration of 150 lM. Following this exposure peri-
od adherent cells were fixed, washed, and stained. The
bound stain was solubilized and the absorbance was
measured at 492 nm in a plate reader (Bio-Tek Instru-
ments Inc., Powerwave XS, Wincoski, USA). For each
test compound and cell line, a dose–response curve
was obtained and the growth inhibition of 50% (GI50),
corresponding to the concentration of the compounds
that inhibited 50% of the net cell growth, was calculated
as described elsewhere.9 Doxorubicin was used as a po-
sitive control and tested in the same manner.
´
2. Kaufmann, D.; Pojarova, M.; Vogel, S.; Liebl, R.; Gastpar,
R.; Gross, D.; Nishino, T.; Pfaller, T.; von Angerer, E.
Bioorg. Med. Chem. 2007, 15, 5122–5136.
3. For a recent review on indoles as potent inhibitors of
tubulin polymerization see: Brancale, A.; Silvestri, R. Med.
Res. Rev. 2007, 27, 209–238.
4. Cacchi, S.; Fabrizi, G. Chem. Rev. 2005, 105, 2873–2920.
5. (a) Abreu, A. S.; Silva, N. O.; Ferreira, P. M. T.; Queiroz,
M-J. R. P. M.-J. R. P. Tetrahedron Lett. 2003, 44, 3377–
3379; (b) Abreu, A. S.; Silva, N. O.; Ferreira, P. M. T.;
Queiroz, M-J. R. P. M.-J. R. P.; Venanzi, M. Eur. J. Org.
Chem. 2003, 4792–4796; (c) Queiroz, M-J. R. P. M.-J. R. P.;
Abreu, A. S.; Castanheira, E. M. S.; Ferreira, P. M. T.
Tetrahedron 2007, 63, 2215–2222; (d) Queiroz, M-J. R. P.
M.-J. R. P.; Castanheira, E. M. S.; Carvalho, M. S. D.;
Abreu, A. S.; Ferreira, P. M. T.; Karadeniz, H.; Erdem, A.
Tetrahedron 2008, 64, 382–391.
6. Abreu, A. S.; Ferreira, P. M. T.; Queiroz, M-J. R. P. M.-J.
R. P.; Ferreira, I. C. F. R.; Calhelha, R. C.; Estevinho, L.
M. Eur. J. Org. Chem. 2005, 2951–2957.
7. Abreu, A. S.; Ferreira, P. M. T.; Monteiro, L. S.; Queiroz,
M.-J. R. P.; Ferreira, I. C. F. R.; Calhelha, R. C.;
Estevinho, L. M. Tetrahedron 2004, 60, 11821–11828.
8. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMa-
hon, J.; Vistica, D.; Warren, J. T.; Bokesch, H.; Kenny, S.;
Boyd, M. R. J. Natl. Cancer Inst. 1990, 82, 1107–1112.
9. Monks, A.; Scudiero, D.; Skehan, P.; Shoemaker, R.; Paul,
K.; Vistica, D.; Hose, C.; Langley, J.; Cronise, P.; Vaigro-
Wolff, A.; Gray-Goodrich, M.; Campbell, H.; Mayo, J.;
Boyd, J. M. J. Natl. Cancer Inst. 1991, 83, 757–776.
Acknowledgments
Foundation for the Science and Technology (Portugal)
and FEDER for financial support through Centro de
Quımica of Univ. Minho and Project POCI/QUI/
59407/2004. A. S. Abreu acknowledges FCT for a
post-doc. Grant No. SFRH/BPD/24548/2005. The
´