Efficient synthesis of 6-(hetero)arylthieno[3,2-b]pyridines by Suzuki-Miyaura coupling. Evaluation of growth inhibition on human tumor cell lines, SARs and effects on the cell cycle

Article abstract of DOI:10.1016/j.ejmech.2010.09.014

A wide variety of new bi(hetero)aryl derivatives of the thieno[3,2-b]pyridine skeleton was obtained in high to excellent yields (65-91%) by Suzuki-Miyaura cross-coupling of the methyl 3-amino-6-bromothieno[3,2-b] pyridine-2-carboxylate, recently reported

Full text of DOI:10.1016/j.ejmech.2010.09.014

European Journal of Medicinal Chemistry 45 (2010) 5628e5634
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Efficient synthesis of 6-(hetero)arylthieno[3,2-b]pyridines by SuzukieMiyaura
coupling. Evaluation of growth inhibition on human tumor cell lines, SARs
and effects on the cell cycle
,
a
b
,
,
,d
Maria-João R.P. Queiroz a , Ricardo C. Calhelha , Luís A. Vale-Silva c, Eugénia Pinto b c, Raquel T. Lima b
,
*
,
d
M. Helena Vasconcelos b
a Centro de Química, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal
b Laboratório de Microbiologia, Faculdade de Farmácia da Universidade do Porto, Rua Aníbal Cunha 164, 4050-047 Porto, Portugal
c CEQUIMED-UP, Centro de Química Medicinal da Universidade do Porto, Rua Aníbal Cunha 164, 4050-047 Porto, Portugal
d Cancer Biology Group, IPATIMUP e Institute of Molecular Pathology and Immunology of the University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
A wide variety of new bi(hetero)aryl derivatives of the thieno[3,2-b]pyridine skeleton was obtained in
high to excellent yields (65e91%) by SuzukieMiyaura cross-coupling of the methyl 3-amino-6-bromo-
thieno[3,2-b]pyridine-2-carboxylate, recently reported by us, with aryl or heteroaryl pinacolboranes or
potassium trifluoroborates.
The coupling products obtained were evaluated for their growth inhibitory effect on three human
tumor cell lines, representing different tumor models, MCF-7 (breast adenocarcinoma), A375-C5
(melanoma) and NCI-H460 (non-small cell lung cancer). Some of the compounds showed an interesting
Received 14 January 2010
Received in revised form
2 September 2010
Accepted 8 September 2010
Available online 17 September 2010
Keywords:
activity against the tested cell lines, with GI50 values in the
some structureeactivity relationships (SARs). Several compounds presented GI50 values below 15
particularly a bithiophene and an o-aniline thienopyridine derivative. The first presented selectivity for
MCF-7 and NCI-H460 cell lines, with very low GI50 values (0.7e1.0 M), while the latter was active
against the three cell lines tested in this study, also presenting very low GI50 values (2.5e4.2 M). The
m
M range, and it was possible to establish
SuzukieMiyaura coupling
(Hetero)aryl potassium trifluoroborates
(Hetero)arylpinacolboranes
(Hetero)arylthieno[3,2-b]pyridines
Tumor cell growth inhibition
Cell cycle analysis
m
M,
m
m
effect of these two compounds on cell cycle progression was analyzed in the NCI-H460 cell line. Results
showed that both compounds interfered with the normal cell cycle distribution.
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
with several (hetero)arylboronated compounds. The potential of
the bi(hetero)aryl compounds obtained as antitumor agents was
Recently the thienopyridine skeleton has shown interesting
biological activities. (3-Amino-6-thien-2-yl-thieno[2,3-b]pyridin-
2-yl)arylmethanones showed selectivity against a tumorogenic cell
line, in very low EC50 values [1]. Diarylamine derivatives of thieno
[3,2-b]pyridines [2], substituted thieno[3,2-c]pyridine ureas [3] and
evaluated through the study of their in vitro growth inhibitory
effect on three human tumor cell lines, representing different
tumor models, MCF-7 (breast adenocarcinoma), NCI-H460 (non-
small cell lung cancer), and A375-C5 (melanoma), and it was
possible to establish some structureeactivity relationships. For the
most active compounds, a study of their effects on normal cell cycle
distribution was also performed in the NCI-H460 cell line.
diarylether
derivatives
of
thieno[3,2-b]pyridine
phenyl-
acetylthioureas [4] are inhibitors of the vascular endothelial growth
factor receptor (VEGFR-2) which mediates the biological function of
the vascular endothelial growth factor (VEGF), related to angio-
genesis and metastasis.
2. Results and discussion
Herein we report the synthesis of a wide variety of 6-(hetero)
arylthieno[3,2-b]pyridines by SuzukieMiyaura cross-coupling of
the methyl 3-amino-6-bromothieno[3,2-b]pyridine-2-carboxylate
2.1. Chemistry
The 6-bromothieno[3,2-b]pyridine 1 recently reported by us [5],
was used as the brominated component in the palladium-catalyzed
SuzukieMiyaura cross-coupling [6], with several (hetero)aryl
pinacolboronates [7] or potassium trifluoroborates [8]. These boron
compounds are easier to handle than the boronic acids due to their
* Corresponding author. Tel.: þ351 253604378; fax: þ351 253604382.
E-mail address: mjrpq@quimica.uminho.pt (M.-J.R.P. Queiroz).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.09.014

M.-J.R.P. Queiroz et al. / European Journal of Medicinal Chemistry 45 (2010) 5628e5634
Table 1 (continued)
5629
insensitivity to air and moisture. Furthermore the corresponding
GI50 (m
M)a
boronic acids did not react with compound 1 under the conditions
used.
Boronated
component
Biheteroaryl (h)
Bpin
N
MCF-7
A375-C5
NCI-H460
2l, 81%
10.7 ꢀ 1.8 10.1 ꢀ 2.7 7.7 ꢀ 1.1
= (Het)Ar-Bpin
O
NH2
B
NH2
Bpin
N
+
O
N
(Het)Ar
or
i
CO2Me
2m, 80%
2n, 82%
2o, 80%
> 150.0
> 150.0
> 150.0
CO2Me
S
Br
N
(Het)ArBF3K
S
(Het)Ar
1
Bpin
Bpin
2a-v
i) PdCl2(dppf).CH2Cl2 (1:1) (2 mol%), K2CO3 (6 equiv.), DME/H2O (3:1), 1-3 h, 90 ºC.
23.0 ꢀ 2.5 21.2 ꢀ 4.4 20.3 ꢀ 3.0
N
Me
Table 1
> 75.0b
> 75.0b
> 75.0b
Boronated coupling components, 6-(hetero)arylthieno[3,2-b]pyridines 2a-v product
yields and GI50 values presented by compounds 2 in three human tumor cell lines.
NC
Boronated
Biheteroaryl (
h) GI50
(m
M)a
component
BF3K
MCF-7
A375-C5
NCI-H460
BF3K
Bpin
H3C
O
2p, 90%
> 150.0
> 150.0
> 150.0
2a, 80%
2b, 70%
12.7 ꢀ 2.0 12.1 ꢀ 1.7 12.4 ꢀ 2.2
S
S
BF3K
BF3K
Bpin
2q, 75%
2r, 70%
> 150.0
> 150.0
> 150.0
> 150.0
> 150.0
> 150.0
26.9 ꢀ 2.7 > 150.0
18.0 ꢀ 5.0
F3C
Bpin
MeO
MeO
S
> 75.0b
0.7 ꢀ 0.03
> 120.0b
S
2c, 72%
2d, 76%
1.0 ꢀ 0.1
> 120.0b
Bpin
2s, 70%
56.6 ꢀ 3.2 82.0 ꢀ 3.7 31.8 ꢀ 5.2
OMe
Bpin
> 120.0b
O
2t, 84%
2u, 75%
2v, 65%
4.2 ꢀ 0.1
> 150.0
2.5 ꢀ 0.2
> 150.0
3.8 ꢀ 1.1
> 150.0
NH2
Bpin
Bpin
Bpin
H2N
H2N
N
N
2e, 72%
> 150.0
> 150.0
> 150.0
Me
Bpin
107.8 ꢀ 2.7 80.8 ꢀ 11.1 37.1 ꢀ 4.4
2f, 88%
2g, 90%
2h, 82%
> 112.3b
> 37.5b
NDc
> 112.3b
> 37.5b
NDc
> 112.3b
> 37.5b
NDc
N
Bpin
*This compound was obtained as an intermediate in the step 1 of Scheme 1.
a
The lowest concentrations causing 50% of cell growth inhibition (GI50) after
MeO
F
N
a continuous exposure of 48 h, expressed as means ꢀ SD of three independent
experiments performed in duplicate.
Bpin
b
Due to limited solubility of the compound in DMSO, the maximum concentra-
tion tested was lower than 150
m
M.
The compound precipitated when a solution in DMSO was added to the cell
culture medium. Doxorubicin was used as positive control (GI50 MCF-
c
N
:
OMe
7 ¼ 43.3 ꢀ 2.6 nM; A375-C5 ¼ 130.2 ꢀ 10.1 nM; NCI-H460 ¼ 35.6 ꢀ 1.6 nM).
Bpin
N
2i, 88%
> 75.0b
> 75.0b
> 75.0b
The 6-(hetero)arylthieno[3,2-b]pyridines 2aev were obtained
in high to excellent yields (65e91%) using the reaction conditions
depicted in Table 1. We have already successfully applied these
conditions to the synthesis of dehydroamino acid derivatives by
SuzukieMiyaura cross-couplings of
and -bromodehydrophenylalanine derivatives with (hetero)aryl
boronated compounds [9,10]. Thus the conditions are rather
general due to the different types of brominated substrates that can
be used in the SuzukieMiyaura coupling.
MeO
N
BF3K
Bpin
2j, 88%
2k, 91%
50.9 ꢀ 2.3 95.6 ꢀ 12.8 43.5 ꢀ 1.7
b,b-dibromodehydroalanine
b
9.8 ꢀ 1.7
9.1 ꢀ 1.7
9.5 ꢀ 1.7
N
For the synthesis of compound 2b neither the 2-thienyl pina-
colborane ester nor the corresponding potassium trifluoroborate
(continued on next page)

5630
M.-J.R.P. Queiroz et al. / European Journal of Medicinal Chemistry 45 (2010) 5628e5634
salt are commercially available so it was decided to perform a one
pot two steps, borylation with pinacolborane followed by Suzuki
observed for the quinoline derivative 2m (linked by the 6-position
of the quinoline), for which no activity was found at the maximum
tested concentration. The position of the CeC linkage in the
nitrogenated system of these compounds seems to be important for
the antitumoral activity. The N-methylindole 2n (linked by the
5-position of the indole) was active against the three cell lines,
coupling (BSC) with the brominated thieno[3,2-b]pyridine
1
(Scheme 1). Due to the electron-rich character of the thiophene
ring, compound 2b was only obtained using tri-t-butylphospho-
nium tetrafluoroborate (t-Bu3PHBF4) [11] as the ligand, in 70% yield
(Scheme 1). To our knowledge it is the first time that this ligand was
used in a BSC reaction.
presenting moderately low GI50 values (slightly over 20 mM).
The p-substituted phenyl derivatives, either with electron-
donating or electron-withdrawing groups, 2oer, were not active at
the concentrations tested. The dimethoxy derivative 2s, in turn,
showed activity against the MCF-7 and NCI-H460 cell lines, with
O
GI50 values from 30 to 60
cells (GI50 over 80 M). Among the aryl derivatives tested, the
2-aminophenyl derivative 2t presented the lowest GI50 values
(below 5 M) against all the tumor cell lines tested, while the
mM, but were less active against A375-C5
B
H
m
NH2
CO2Me
O
1.
N
Pd(OAc)2
m
S
Ligand, NEt3,
3-aminophenyl derivative 2u was not active at the tested concen-
trations. The 4-aminophenyl derivative 2v showed only moderately
Br
S
S
Dioxane, 100 ºC, 2h
low GI50 values against NCI-H460 cell lines (around 37 mM).
NH2
2b
In addition, analysis of the growth inhibition curves allowed to
conclude that all the active compounds inhibited the growth of the
human tumor cell lines in a dose-dependent manner (data not
shown).
N
2.
Br
CO2Me
S
1
Ba(OH)2.8H2O
100 ºC, 2h
Ligand = tri-t-butylphosphonium tetrafluoroborate
2.3. Cell cycle analysis
The most active compounds, 2c and 2t, were selected to be
further studied regarding their influence on the cell cycle distri-
bution of the NCI-H460 cells. With this purpose, cells were incu-
bated for 48 h with the previously determined GI50 concentrations
of the compounds (from Table 1) and also with twice the GI50
concentration. The reason for using both these concentrations was
to increase the possibility of detecting the effect on the cell cycle,
given the fact that the GI50 was determined with an assay that
detects alterations in protein content, not in cellular viability. Flow
cytometry results showed that both compounds interfere with the
normal cell cycle distribution of this cell line. When analyzing the
effect of the highest concentration tested (twice the GI50),
compound 2c caused 15% increase in the percentage of cells in G1
and 15% reduction in the percentage of cells in the S-phase of the
cell cycle (Fig. 1). Upon incubation with compound 2t (with twice
the GI50) there was a reduction in the percentage of cells in the S-
phase of about 10% and an increase of cells in the G2/M phases of
about 12% (Fig. 1). Therefore, compound 2c seems to induce a cell
Scheme 1. Synthesis of compound 2b by a BSC reaction.
2.2. Effect on the growth of human tumor cell lines
The in vitro growth inhibitory activity of all the compounds
prepared was evaluated using three human tumor cell lines, rep-
resenting different tumor models, namely breast adenocarcinoma
(MCF-7), melanoma (A375-C5), and non-small cell lung cancer
(NCI-H460). For all cell lines, the results were obtained after 48 h of
continuous exposure to the compounds. The results are summa-
rized in Table 1.
From the analysis of Table 1 it is possible to establish some
structureeactivity relationships (SARs). Comparing the thienopyr-
idines 2ae2e, bearing five-member rings, compound 2a (with
a thiophene ring linked at the 3-position) shows a low GI50 (around
12 mM) for all the tumor cell lines tested, while compound 2b (with
a thiophene ring linked at the 2-position) shows a moderately low
GI50 only for MCF-7 and NCI-H460 cell lines. Compound 2c (with
a 2,20-bithiophene system) presents the lowest GI50 values selec-
tively for MCF-7 and NCI-H460 cell lines (1.0 and 0.7 mM, respec-
tively). In turn, the thienopyridines bearing a furan linked at the
3-position, 2d, and a N-methylpyrazole linked at the 4-position, 2e,
did not show any activity at the tested concentrations.
Assays with the pyridine and pyrimidine derivatives 2fe2i were
technically difficult to perform due to problems related to solubility
in DMSO or, in the case of 2h, related to precipitation when the
DMSO solution was added to the aqueous cell culture medium.
These compounds did not show any activity at the tested
concentrations.
In the series of the thienopyridines substituted with naphtha-
lene, quinolines and an isoquinoline (2je2m), interesting results
were obtained. The naphthalene derivative 2j presents GI50 values
approaching 50 mM for MCF-7 and NCI-H460 cell lines, but a lower
activity against A375-C5 cells was registered. The quinoline deriv-
ative 2k (linked by the 3-position of the quinoline to the thieno-
pyridine moiety) and the isoquinoline derivative 2l (linked by the
4-position) both present low GI50 values against all the cell lines
Fig. 1. Cell cycle analysis of NCI-H460 cells treated for 48 h with compounds 2c or 2t at
their GI50 or 2 ꢁ GI50 concentrations (0.7 or 1.4
mM and 3.8 or 7.6 mM, respectively).
Untreated cells were used as the control and a solvent control was also included
(DMSO). Results are the mean ꢀ SD of three independent experiments performed in
duplicate.
studied (below 10 mM). This was in striking contrast to what was

M.-J.R.P. Queiroz et al. / European Journal of Medicinal Chemistry 45 (2010) 5628e5634
5631
cycle arrest on phases G0/G1, whereas compound 2t appears to
cause a G2/M cell cycle arrest. According to these findings, the
compounds tested probably present different mechanisms of
action.
thiophenetrifluoroborate (123 mg, 0.648 mmol) and heating for 3 h
gave compound 2a as a yellow solid (123 mg, 80%), m.p. 159e161 ꢂC,
after some washes with petroleum ether. 1H NMR (CDCl3, 400 MHz):
d
3.93 (3H, s, OMe), 6.23 (2H, broad s, NH2), 7.44e7.51 (2H, m, AreH),
7.61e7.62 (1H, m, AreH), 8.19 (1H, d, J ¼ 2.0 Hz, AreH), 8.88 (1H, d,
3. Conclusions
J ¼ 2.0 Hz, AreH) ppm. 13C NMR (CDCl3, 100.6 MHz):
d 51.66 (OMe),
99.74 (C),122.14 (CH), 126.02 (CH), 127.28 (CH), 127.92 (CH),130.62 (C),
134.63 (C), 138.43 (C), 145.30 (CH), 147.55 (C), 165.33 (C]O) ppm.
Anal. calcd. for C13H10N2O2S2: C, 53.77; H, 3.47; N, 9.65; S, 22.10%;
found: C, 53.73; H, 3.51; N, 9.56; S, 22.50%.
We were able to synthesize a large variety of new methyl 6-
(hetero)aryl-3-aminothieno[3,2-b]pyridine-2-carboxylates in high
to excellent yields (65e91%) by SuzukieMiyaura cross-couplings of
the methyl 6-bromo-3-aminothieno[3,2-b]pyridine-2-carboxylate
with (hetero)aryl pinacolborane esters or potassium tri-
fluoroborates using mild and general conditions. The growth
inhibitory effect of the coupling products was evaluated against
three human tumor cell lines and some SARs were established. The
most active compounds were shown to be: 2c (a 2,20-bithiophene
derivative) with selectivity against MCF-7 and NCI-H460 cell lines
4.1.1.2. Methyl 3-amino-6-(thien-2-yl)thieno[3,2-b]pyridine-2-carbox-
ylate (2b). A dried Schlenk tube was charged under argon with dry
dioxane (3 mL), 2-bromothiophene (150 mg, 0.1 mL, 0.900 mmol), Pd
(OAc)2 (5 mol%), tri-t-butylphosphonium tetrafluoroborate (20 mol
%), pinacolborane (3 equiv.) NEt3 (4 equiv.). The mixture was left
stirring for 2 h at 100 ꢂC. After cooling compound 1 (1 equiv.) and
BaOH.8H2O (3 equiv.) were added. The mixture was left stirring for
more 2 h at 100 ꢂC giving compound 2b as a yellow solid (102 mg,
70%), after the dry flash referred in the general procedure, m.p.
231e233 ꢂC, after some washes with petroleum ether. 1H NMR
presenting very low GI50 values (1 and 0.7
the o-aminophenyl derivative 2t, which displays GI50 values below
M for the three cell lines studied. Other compounds presented
mM, respectively), and
5
m
very good activity, such as the quinoline and isoquinoline deriva-
tives, 2k and 2l respectively, with the CeC bond in the nitrogenated
(DMSO-d6, 300 MHz): d 3.82 (3H, s, OMe), 6.90 (2H, br s, NH2),
ring and with GI50 values between 6 and 12
derivative 2a, with GI50 values between 10 and 14
m
M, and the thiophene
7.20e7.24 (1H, m, AreH), 7.69e7.72 (1H, m, AreH), 7.77e7.79 (1H, m,
AreH), 8.65 (1H, d, J ¼ 2.0 Hz, AreH), 8.99 (1H, d, J ¼ 2 Hz, AreH)
mM. The influ-
ence of the most active compounds, 2c and 2t, on the cell cycle
distribution was evaluated in the NCI-H460 cell line. Compound 2c
caused a cell cycle arrest in the G0/G1 phases, while compound 2t
caused a G2/M arrest, thus pointing out to different cellular
mechanisms of action. Based on the current results it is worthwhile
to pursue the lead 2c for further optimization.
ppm. 13C NMR (DMSO-d6, 75.4 MHz):
d 51.54 (OMe), 97.36 (C), 126.00
(CH), 127.38 (CH), 127.69 (CH), 128.88 (CH), 128.99 (C), 134.03 (C),
139.30 (C), 144.14 (CH), 145.06 (C), 147.88 (C), 164.50 (C]O) ppm. MS
(EI): m/z (%) 290 (Mþ, 100), 258 (80), 230 (27). HRMS Mþ calcd. for
C13H10N2O2S2 290.0184, found 290.0194.
4.1.1.3. Methyl 3-amino-6-(2,20-bithenyl-3-yl)thieno[3,2-b]pyridine-
2-carboxylate (2c). Compound 1 (150 mg, 0.500 mmol), 2,20-
bithiophene-5-boronic acid pinacol ester (190 mg, 0.648 mmol)
and heating for 2 h gave compound 2c as a yellow solid (144 mg,
72%), m.p. 218e219 ꢂC, after some washes with petroleum ether. 1H
4. Experimental
4.1. Synthesis
Melting points (ꢂC) were determined in a SMP3 Stuart apparatus
and are uncorrected. 1H and 13C NMR spectra were recorded on
a Varian Unity Plus at 300 and 75.4 MHz, and on a Bruker Avance at
400 and 100.6 MHz, respectively. Heteronuclear correlations,
1He13C, HMQC or HSQC 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).
Dry flash was performed on MachereyeNagel silica gel 150e230
mesh. Petroleum ether refers to the boiling range 40e60 ꢂC. Ether
refers to diethylether. DME refers to 1,2-dimethoxyethane. The
catalyst PdCl2(dppf).CH2Cl2 (1:1) refers to 1,10-bis(diphenylphos-
phino)ferrocene dichloropalladium (II), complex with dichloro-
methane (1:1) and was purchased from Aldrich.
NMR (CDCl3, 400 MHz): d 3.93 (3H, s, OMe), 6.29 (2H, broad s, NH2),
7.05e7.08 (1H, m, AreH), 7.22 (1H, d, J ¼ 4.0 Hz, AreH), 7.25e7.29
(2H, m, AreH), 7.38 (1H, d, J ¼ 4.0 Hz, AreH), 8.19 (1H, d, J ¼ 2.0 Hz,
AreH), 8.87 (1H, d, J ¼ 2.0 Hz, AreH) ppm. 13C NMR (CDCl3,
100.6 MHz):
d 51.74 (OMe), 100.28 (C), 124.34 (CH), 124.90 (CH),
125.17 (CH),125.74 (CH),127.22 (CH),128.03 (CH),129.20 (C),134.87
(C), 136.65 (C), 138.21 (C), 138.93 (C), 143.53 (CH), 144.65 (C), 147.15
(C), 165.16 (C]O) ppm. Anal. calcd. for C17H12N2O2S3: C, 54.82; H,
3.25; N, 7.52; S, 25.83%; found: C, 54.44; H, 3.23; N, 7.31; S, 26.23%.
4.1.1.4. Methyl 3-amino-6-(fur-3-yl)thieno[3,2-b]pyridine-2-carbox-
ylate (2d). Compound 1 (150 mg, 0.500 mmol), 3-furanboronic acid
pinacol ester (126 mg, 0.648 mmol) and heating for 2 h gave
compound 2d as a yellow solid (112 mg, 76%), m.p.170e171 ꢂC, after
some washes with petroleum ether. 1H NMR (CDCl3, 400 MHz):
d
3.92 (3H, s, OMe), 6.24 (2H, broad s, NH2), 6.77e6.78 (1H, m,
4.1.1. General procedure for the SuzukieMyaura coupling (except
for 2b)
AreH), 7.56e7.57 (1H, m, AreH), 7.87e7.88 (1H, m, AreH), 8.09 (1H,
d, J ¼ 2.0 Hz, AreH), 8.76 (1H, d, J ¼ 2.0 Hz, AreH) ppm. 13C NMR
In a round flask, DME (3 mL) and water (1 mL), the 3-amino-6-
bromothieno[3,2-b]pyridine-2-carboxylate (1) (hetero)aryl boronic
acid pinacol ester or potassium trifluoroborate (1.2 equiv.),
PdCl2(dppf).CH2Cl2 (1:1) (2 mol%), K2CO3 (6 equiv.) were added and
the mixture was heated with stirring at 90 ꢂC for some hours
(following the reaction by TLC). After cooling, the solvents were
evaporated under reduced pressure giving an oily solid which was
submitted to a dry flash column chromatography (silica 150e230
mesh) using ethyl acetate.
(CDCl3, 100.6 MHz): d 51.66 (OMe), 99.60 (C), 108.55 (CH), 123.12
(C), 127.40 (CH), 127.55 (C), 134.70 (C), 139.43 (CH), 144.41 (CH),
144.55 (C), 145.08 (C), 147.47 (C), 165.29 (C]O) ppm. Anal. calcd. for
C13H10N2O3S: C, 57.00; H, 3.67; N, 10.21; S, 11.69%; found: C, 57.10;
H, 3.89; N, 10.12; S, 12.10%.
4.1.1.5. Methyl 3-amino-6-(1-methyl-1H-pyrazol-4-yl)thieno[3,2-b]
pyridine-2-carboxylate (2e). Compound 1 (150 mg, 0.500 mmol), 1-
methylpyrazole-4-boronic acid pinacol ester (135 mg, 0.648 mmol)
and heating for 2 h gave compound 2e as a yellow solid (112 mg,
72%), m.p. 231e233 ꢂC, after some washes with petroleum ether. 1H
4.1.1.1. Methyl 3-amino-6-(thien-3-yl)thieno[3,2-b]pyridine-2-carbox-
ylate (2a). Compound 1 (150 mg, 0.540 mmol), potassium 3-
NMR (CDCl3, 400 MHz): d 3.92 (3H, s, OMe), 4.00 (3H, s, NMe), 6.22

5632
M.-J.R.P. Queiroz et al. / European Journal of Medicinal Chemistry 45 (2010) 5628e5634
(2H, broad s, NH2), 7.50 (1H, s, AreH), 7.86 (1H, s, AreH), 8.08 (1H,
d, J ¼ 2.0 Hz, AreH), 8.75 (1H, d, J ¼ 2.0 Hz, AreH) ppm. 13C NMR
(C) ppm. Anal. Calcd for C15H14N4O4S: C, 52.02; H, 4.07; N, 16.18; S,
9.26%; found: C, 52.32; H, 4.01; N, 15.98; S, 9.65%.
(CDCl3, 100.6 MHz):
d 39.30 (NMe), 51.64 (OMe), 99.28 (C), 119.51
(C), 126.69 (CH), 127.47 (CH), 127.84 (C), 134.83 (C), 137.02 (CH),
144.47 (CH), 144.72 (C), 147.59 (C), 165.36 (C]O) ppm. MS (EI): m/z
(%) 288 (Mþ, 100), 256 (69), 228 (35). HRMS Mþ calcd. for
C13H12N4O2S 288.0681, found 288.0682.
4.1.1.10. Methyl 3-amino-6-(naphthalen-2-yl)thieno[3,2-b]pyridine-
2-carboxylate (2j). Compound 1 (150 mg, 0.500 mmol), potassium
2-naphthalenetrifluoroborate (152 mg, 0.648 mmol) and heating
for 2 h gave compound 2j as a yellow solid (159 mg, 88%), m.p.
161e163 ꢂC, after some washes with petroleum ether. 1H NMR
4.1.1.6. Methyl 3-amino-6-(pyrid-3-yl)thieno[3,2-b]pyridine-2-carbox-
ylate (2f). Compound 1 (150 mg, 0.540 mmol), 3-pyridineboronic
acid pinacol ester (133 mg, 0.648 mmol) and heating for 3 h gave
compound 2f as a yellow solid (135 mg, 88%), m.p. 210e212 ꢂC, after
(CDCl3, 400 MHz):
d 3.94 (3H, s, OMe), 6.34 (2H, broad s, NH2),
7.53e7.58 (2H, m, AreH), 7.76 (1H, dd, J ¼ 8.6 and 2.0 Hz, AreH),
7.89e7.95 (2H, m, AreH), 7.99 (1H, d, J ¼ 8.6 Hz, AreH), 8.11 (1H, d,
J ¼ 2.0 Hz, AreH), 8.35 (1H, d, J ¼ 2.0 Hz, AreH), 8.98 (1H, d,
some washes with petroleum ether.1H NMR (CDCl3, 400 MHz):
d 3.94
J ¼ 2.0 Hz, AreH) ppm. 13C NMR (CDCl3, 100.6 MHz):
d 51.71 (OMe),
(3H, s, OMe), 6.26 (2H, broad s, NH2), 7.44e7.48 (1H, m, AreH),
7.94e7.97 (1H, m, AreH), 8.23 (1H, d, J ¼ 2.0 Hz, AreH), 8.69e8.71
(1H, m, AreH), 8.84 (1H, d, J ¼ 2.0 Hz, AreH), 8.92 (1H, d, J ¼ 2.0 Hz,
100.17 (C), 124.98 (CH), 126.66 (CH), 126.75 (CH), 126.80 (CH),
127.73 (CH), 128.29 (CH), 129.12 (CH), 129.60 (CH) 133.01 (C), 133.55
(C), 134.49 (C), 134.79 (C), 135.79 (C), 145.04 (C), 145.60 (CH), 147.30
(C),165.27 (C]O) ppm. MS (EI): m/z (%) 334 (Mþ,100), 302 (65), 274
(31). HRMS Mþ calcd. for C19H14N2O2S 334.0776, found 334.0777.
AreH) ppm. 13C NMR (CDCl3, 100.6 MHz):
d 51.76 (OMe), 100.44 (C),
123.87 (CH), 129.26 (CH), 132.50 (C), 133.31 (C), 134.59 (C), 134.71
(CH), 145.45 (CH), 146.24 (C), 147.35 (C), 148.40 (CH), 149.58 (CH),
165.24 (C]O) ppm. MS (EI): m/z (%) 285 (Mþ,100), 253 (71), 225 (34).
HRMS Mþ calcd. for C14H11N3O2S: 285.0572, found 285.0571.
4.1.1.11. Methyl 3-amino-6-(quinolin-3-yl)thieno[3,2-b]pyridine-2-
carboxylate (2k). Compound 1 (150 mg, 0.500 mmol), quinoline-3-
boronic acid pinacol ester (165 mg, 0.648 mmol) and heating for
1 h, gave compound 2k as a yellow solid (164 mg, 91%), m.p.
222e224 ꢂC, after some washes with petroleum ether. 1H NMR
4.1.1.7. Methyl 3-amino-6-(6-methoxypyrid-3-yl)thieno[3,2-b]pyri-
dine-2-carboxylate (2g). Compound 1 (150 mg, 0.500 mmol),
2-methoxypyridine-5-boronic acid pinacol ester (150 mg,
0.648 mmol) and heating for 1 h gave compound 2g as a yellow
solid (154 mg, 90%), m.p. 224e226 ꢂC, after some washes with
(CDCl3, 400 MHz):
d 3.93 (3H, s, OMe), 6.27 (2H, broad s, NH2),
7.63e7.66 (1H, m, AreH), 7.77e7.81 (1H, m, AreH) 7.93 (1H, d,
J ¼ 8.0 Hz, AreH), 8.19 (1H, d, J ¼ 8.0 Hz, AreH), 8.35 (1H, d,
J ¼ 2.0 Hz, AreH), 8.40 (1H, d, J ¼ 2.0 Hz, AreH), 8.96 (1H, d,
J ¼ 2.0 Hz, AreH), 9.22 (1H, broad s, AreH) ppm. 13C NMR (CDCl3,
petroleum ether. 1H NMR (CDCl3, 400 MHz):
d 3.93 (3H, s, OMe),
4.02 (3H, s, 60-OMe), 6.34 (2H, broad s, NH2), 6.90 (1H, d, J ¼ 9.0 Hz,
50-H), 7.84 (1H, dd, J ¼ 9.0 and 2.4 Hz, 40-H), 8.19 (1H, d, J ¼ 2.0 Hz,
AreH), 8.46 (1H, dd, J ¼ 2.4 and 0.4 Hz, 20-H), 8.79 (1H, d, J ¼ 2.0 Hz,
100.6 MHz):
d 51.74 (OMe), 100.47 (C), 127.52 (CH), 127.77 (C),
128.08 (CH), 129.34 (CH), 129,36 (CH), 130.22 (CH), 130.34 (C),
132.53 (C), 134.10 (CH), 134.63 (C), 145.66 (CH), 146.15 (C), 147.37
(C), 147.67 (C), 149.16 (CH), 165.23 (C]O) ppm. MS (EI): m/z (%) 335
(Mþ, 100), 303 (75), 275 (31). HRMS Mþ calcd. for C18H13N3O2S
335.0728, found 335.0732.
AreH) ppm. 13C NMR (CDCl3, 100.6 MHz):
d
51.77 (OMe), 53.80 (60-
OMe), 100.31 (C), 111.54 (50-CH), 126.19 (C), 129.11 (CH), 132.77 (C),
134.91 (C), 137.48 (40-CH), 144.44 (CH), 144.79 (C), 145.47 (20-CH),
147.01 (C), 164.41 (CeO), 165.17 (C]O) ppm. Anal. Calcd for
C15H13N3O3S: C, 57.13; H, 4.16; N, 13.33; S, 10.17%; found: C, 56.73;
H, 3.82; N, 13.10; S, 9.96%.
4.1.1.12. Methyl 3-amino-6-(isoquinolin-4-yl)thieno[3,2-b]pyridine-
2-carboxylate (2l). Compound 1 (150 mg, 0.500 mmol), isoquino-
line-4-boronic acid pinacol ester (165 mg, 0.648 mmol) and heating
for 1 h, gave compound 2l as a yellow solid (155 mg, 81%), m.p.
213e215 ꢂC, after some washes with petroleum ether. 1H NMR
4.1.1.8. Methyl 3-amino-6-(2-fluoropyridin-5-yl)thieno[3,2-b]pyri-
dine-2-carboxylate (2h). Compound 1 (150 mg, 0.500 mmol),
2-fluoropyridine-5-boronic acid pinacol ester (143 mg, 0.648 mmol)
and heating for 1 h gave compound 2h as a yellow solid (135 mg,
82%), m.p. 232e234 ꢂC, after some washes with petroleum ether. 1H
(CDCl3, 400 MHz):
d 3.95 (3H, s, OMe), 6.31 (2H, broad s, NH2),
7.73e7.80 (1H, m, AreH), 7.87 (1H, d, J ¼ 8.0 Hz, AreH) 8.14 (1H, d,
J ¼ 8.0 Hz, AreH), 8.21 (1H, d, J ¼ 1.6 Hz, AreH), 8.56 (1H, broad s,
AreH), 8.77 (1H, d, J ¼ 1.6 Hz, AreH), 9.39 (1H, broad s, AreH) ppm.
NMR (CDCl3, 400 MHz): d 3.94 (3H, s, OMe), 6.26 (2H, broad s, NH2),
7.11 (1H, m, AreH), 8.01 (1H, m, AreH), 8.20 (1H, d, J ¼ 2.0 Hz, AreH),
13C NMR (CDCl3, 100.6 MHz):
d 51.78 (OMe), 100.47 (C), 124.12 (CH),
8.51e8.52 (1H, m, AreH), 8.80 (1H, d, J ¼ 2.0 Hz, AreH) ppm.13C NMR
128.04 (CH), 128.34 (C), 128.48 (CH), 129.95 (C), 131.47 (C), 131.84
(CH), 132.12 (CH), 134.30 (C), 134.34 (C), 142.22 (CH), 146.30 (C),
147.42 (C), 147.56 (CH), 152.43 (CH), 165.28 (C]O) ppm. Anal. Calcd
for C18H13N3O2S: C, 64.46; H, 3.91; N, 12.53; S, 9.56%; found: C,
64.17; H, 3.90; N, 12.29; S, 9.32%.
(CDCl3, 100.6 MHz):
d 51.80 (OMe), 100.66 (C), 110.12 (CH, d,
J ¼ 37.0 Hz), 129.34 (CH), 130.42 (C), 134.64 (C), 140.02 (CH, d,
J ¼ 9.0 Hz),145.08 (CH), 146.13 (C),146.28 (CH, d, J ¼ 16.0 Hz),147.20
(C), 163.72 (CF, d, J ¼ 240.0 Hz), 165.18 (C]O) ppm. Anal. Calcd. for
C14H10FN3O2S: C, 55.44; H, 3.32; N,13.85; S,10.57%; found: C, 55.60;
H, 3.22; N, 13.72; S, 10.81%.
4.1.1.13. Methyl 3-amino-6-(quinolin-6-yl)thieno[3,2-b]pyridine-2-
carboxylate (2m). Compound 1 (150 mg, 0.500 mmol), quinoline-6-
boronic acid pinacol ester (165 mg, 0.648 mmol) and heating for
2 h, gave compound 2m as a yellow solid (145 mg, 80%), m.p.
174e175 ꢂC, after some washes with petroleum ether. 1H NMR
4.1.1.9. Methyl 3-amino-6-(2,4-dimethoxypyrimid-5-yl)thieno[3,2-b]
pyridine-2-carboxylate (2i). Compound 1 (150 mg, 0.500 mmol),
2,4-dimethoxypyrimidine-5-boronic acid pinacol ester (147 mg,
0.648 mmol) and heating for 1 h gave compound 2i as a yellow
solid (161 mg, 88%), m.p. 239e241 ꢂC, after some washes with
(CDCl3, 300 MHz):
d 3.94 (3H, s, OMe), 6.27 (2H, broad s, NH2),
petroleum ether. 1H NMR (CDCl3, 400 MHz):
d 3.93 (3H, s, OMe),
7.48e7.52 (1H, m, AreH), 8.02e8.10 (2H, m, AreH), 8.26e8.35 (3H,
m, AreH), 8.97e8.99 (2H, m, AreH) ppm. 13C NMR (CDCl3,
4.07 (6H, s, 2 ꢁ OMe), 6.24 (2H, broad s, NH2), 8.18 (1H, d, J ¼ 2 Hz,
Ar-H), 8.36 (1H, s, 60-H) 8.73 (1H, d, J ¼ 2.0 Hz, Ar-H) ppm. 13C NMR
75.4 MHz):
d 51.74 (OMe), 100.21 (C), 121.90 (CH), 126.37 (CH),
128.47 (C), 128.83 (CH), 129.40 (CH), 130.55 (CH), 134.59 (C), 134.94
(C), 135.65 (C), 136.40 (CH), 145.89 (C), 145.94 (CH), 147.45 (C),
147.83 (C), 151.00 (CH), 165.29 (C]O) ppm. MS (EI): m/z (%) 335
(CDCl3, 100.6 MHz):
d 51.69 (CO2Me), 54.38 (OMe), 55.09 (OMe),
100.11 (C), 112.68 (C), 128.31 (C), 130.75 (CH), 134.14 (C), 145.57 (C),
146.61 (60-CH), 147.39 (C), 157.93 (CH), 165.21 (C), 165.30 (C), 168.25

M.-J.R.P. Queiroz et al. / European Journal of Medicinal Chemistry 45 (2010) 5628e5634
5633
(Mþ, 100), 303 (71) 275 (28). HRMS Mþ calcd. for C18H13N3O2S
335.0728, found 335.0727.
potassium 4- methoxyphenyltrifluoroborate (139 mg, 0.648 mmol),
heating for 2 h gave compound 2r as a yellow solid (115 mg, 70%),
m.p. 174e176 ꢂC, after some washes with petroleum ether. 1H NMR
4.1.1.14. Methyl
3-amino-6-(1-methyl-1H-indol-5-yl)thieno[3,2-b]
(CDCl3, 400 MHz): d 3.89 (3H, s, OMe), 3.93 (3H, s, OMe), 6.35 (2H,
pyridine-2-carboxylate (2n). Compound 1 (150 mg, 0.500 mmol),
1-methylindole-5-boronic acid pinacol ester (167 mg, 0.648 mmol)
and heating for 2 h, gave compound 2n as a yellow solid (153 mg,
82%), m.p. 197e198 ꢂC, after some washes with petroleum ether. 1H
broad s, NH2), 7.05 (2H, d, J ¼ 8.8 Hz, 30 and 50-H), 7.59 (2H, d,
J ¼ 8.8 Hz, 20 and 60-H), 8.21 (1H, d, J ¼ 2 Hz, AreH), 8.83 (1H, d,
J ¼ 2 Hz, AreH) ppm. 13C NMR (CDCl3, 100.6 MHz):
d 51.71 (OMe),
55.43 (OMe), 99.89 (C), 114.79 (30 e 50-CH), 128.57 (20 e 60-CH),
128.93 (CH), 129.48 (C), 132.46 (C), 134.90 (C), 135.61 (C), 144.33 (C),
144.95 (CH), 147.21 (C), 147.96 (C), 160.18 (C), 165.27 (C]O) ppm.
MS (EI): m/z (%) 314 (Mþ, 100), 282 (61), 254 (29); HRMS Mþ calcd.
for C16H14N2O3S 314.0727, found 314.0725.
NMR (CDCl3, 400 MHz): d 3.86 (3H, s, NMe), 3.94 (3H, s, OMe), 6.26
(2H, broad s, NH2), 6.58e6.59 (1H, m, AreH), 7.14 (1H, d, J ¼ 2.8 Hz,
AreH), 7.44e7.53 (2H, m, AreH), 7.90e7.91 (1H, m, AreH), 8.25 (1H,
d, J ¼ 2.0 Hz, AreH), 8.93 (1H, d, J ¼ 2.0 Hz, AreH) ppm. 13C NMR
(CDCl3, 100.6 MHz):
d 33.00 (NMe), 51.62 (OMe), 99.26 (C), 101.58
(CH), 110.01 (CH), 120.02 (CH), 121.24 (CH), 128.79 (CH), 129.16 (C),
130.04 (C), 134.70 (C), 136.71 (C), 137.40 (C), 144.80 (C), 146.40 (CH),
147.74 (C), 165.47 (C]O) ppm. Anal. Calcd for C18H15N3O2S: C,
64.10; H, 4.48; N, 12.45; S, 9.50%; found: C, 63.70; H, 4.67; N, 12.10;
S, 9.46%.
4.1.1.19. Methyl 3-amino-6-(3,5-dimethoxyphenyl)thieno[3,2-b]pyri-
dine-2-carboxylate (2s). Compound 1 (150 mg, 0.500 mmol) and
3,5- dimethoxyphenylboronic acid pinacol ester (171 mg,
0.648 mmol), heating for 2 h gave compound 2s as a yellow solid
(120 mg, 70%), m.p. 182e184 ꢂC, after some washes with petroleum
ether. 1H NMR (CDCl3, 300 MHz):
d
3.88 (6H, s, 2 ꢁ OMe), 3.93 (3H,
4.1.1.15. Methyl 3-amino-6-(4-cyanopheny)thieno[3,2-b]pyridine-2-
carboxylate (2o). Compound 1 (150 mg, 0.500 mmol), 4-cyano-
phenylboronic acid pinacol ester (148 mg, 0.648 mmol) and heating
for 1 h, gave compound 2o as a yellow solid (133 mg, 80%), m.p.
285e286 ꢂC, after some washes with petroleum ether. 1H NMR
s, OMe), 6.28 (2H, broad s, NH2), 6.55 (1H, t, J ¼ 2.1 Hz, 40-H), 7.76
(2H, d, J ¼ 2.1 Hz, 20 and 60-H), 8.20 (1H, d, J ¼ 2.0 Hz, AreH), 8.83
(1H, d, J ¼ 2.0 Hz, AreH) ppm. 13C NMR (CDCl3, 75.4 MHz):
d 51.71
(OMe), 55.51 (2 ꢁ OMe), 100.02 (C), 100.21 (40-CH), 105.73 (20 e 60-
CH), 129.30 (CH), 134.52 (C), 135.85 (C), 139.43 (C), 145.52 (C),
145.68 (CH), 147.38 (C), 161.38 (2 ꢁ C), 165.30 (C]O) ppm. MS (EI):
m/z (%) 344 (Mþ, 100), 312 (63), 284 (25); HRMS Mþ calcd. for
C17H16N2O4S 344.0831, found 344.0829.
(CDCl3, 400 MHz): d 3.95 (3H, s, OMe), 6.33 (2H, broad s, NH2), 7.80
(2H, d, J ¼ 8.4 Hz, 2 ꢁ AreH), 7.83 (2H, d, J ¼ 8.4 Hz, 2 ꢁ AreH), 8.28
(1H, d, J ¼ 2.0 Hz, HetAreH), 8.85 (1H, d, J ¼ 2.0 Hz, HetAreH) ppm.
13C NMR (CDCl3, 100.6 MHz):
d 51.85 (OMe), 101.14 (C), 112.38 (C),
118.38 (C), 128.10 (2 ꢁ CH), 129.97 (CH), 133.04 (2 ꢁ CH), 133.77 (C),
134.73 (C), 141.78 (C), 144.90 (CH), 146.99 (C), 165.09 (C]O) ppm.
MS (EI): m/z (%) 309 (Mþ, 100), 277 (99) 249 (33), HRMS Mþ calcd.
for C16H11N3O2S 309.0572, found 309.0580.
4.1.1.20. Methyl 3-amino-6-(2-aminophenyl)thieno[3,2-b]pyridine-
2-carboxylate (2t). Compound 1 (150 mg, 0.500 mmol) and 2-
aminophenylboronic acid pinacol ester (142 mg, 0.648 mmol)
heating for 2 h gave compound 2t as a green solid (130 mg, 84%),
m.p. 154e156 ꢂC, after some washes with petroleum ether. 1H NMR
4.1.1.16. Methyl 6-(4-acetylphenyl)-3-aminothieno[3,2-b]pyridine-2-
carboxylate (2p). Compound 1 (150 mg, 0.500 mmol), potassium
4-acetylphenyltrifluoroborate (146 mg, 0.648 mmol), and heating
for 2 h, gave compound 2p as a yellow solid (158 mg, 90%), m.p.
214e216 ꢂC, after some washes with petroleum ether. 1H NMR
(CDCl3, 400 MHz): d
3.92 (5H, appt s, OMe and 20-NH2), 6.25 (2H,
broad s, NH2), 6.84 (1H, dd, J ¼ 8.0 and 1.2 Hz, 30-H), 6.92 (1H, appt
dt, J ¼ 7.6 and 1.2 Hz, 50-H), 7.18 (1H, dd, J ¼ 7.6 and 1.6 Hz, 60-H),
7.22e7.27 (1H, m, 40-H), 8.18 (1H, d, J ¼ 2.0 Hz, AreH), 8.72 (1H, d,
J ¼ 2.0 Hz, AreH) ppm. 13C NMR (CDCl3, 100.6 MHz):
d 51.68 (OMe),
(CDCl3, 400 MHz):
d
2.67 (3H, s, Me), 3.94 (3H, s, OMe), 6.35 (2H,
99.93 (C), 116.58 (30-CH), 119.73 (C), 123.82 (10-C), 129.70 (40-CH),
130.74 (60-CH), 131.39 (CH), 134.10 (C), 134.41 (C), 142.90 (C), 145.92
(C), 147.25 (CH), 147.40 (C), 165.31 (C]O) ppm. Anal. Calcd. for
C15H13N3O2S: C, 60.18; H, 4.38; N, 14.04; S, 10.71%; found C, 60.30;
H, 4.34; N, 13.78; S, 10.49%.
broad s, NH2), 7.75 (2H, d, J ¼ 7.2 Hz, 20 and 60-H), 8.10 (2H, d,
J ¼ 7.2 Hz, 30 and 50-H), 8.30 (1H, broad s, HetAreH), 8.88 (1H, broad
s, HetAreH) ppm. 13C NMR (CDCl3, 100.6 MHz):
d 26.70 (Me), 51.79
(OMe), 100.79 (C), 127.60 (20 and 60-CH), 129.26 (30and 50-CH),
129.88 (CH), 134.50 (C), 134.73 (C), 136.81 (C), 141.65 (C), 145.13
(CH), 145.50 (C), 147.04 (C), 165.12 (C]O), 197.38 (C]O) ppm. Anal.
Calcd for C17H14N2O3S: C, 62.56; H, 4.32; N, 8.58; S, 9.82%; found C,
62.39; H, 4.20; N, 8.53; S, 9.71.
4.1.1.21. Methyl 3-amino-6-(3-aminophenyl)thieno[3,2-b]pyridine-2-
carboxylate (2u). Compound 1 (150 mg, 0.500 mmol) and 3-ami-
nophenylboronic acid pinacol ester (142 mg, 0.648 mmol) heating
for 2 h gave compound 2u as a yellow solid (105 mg, 75%), m.p.
198e200 ꢂC, after some washes with petroleum ether. 1H NMR
4.1.1.17. Methyl 3-amino-6-[4-(trifluoromethyl) phenyl]thieno[3,2-b]
pyridine-2-carboxylate (2q). Compound 1 (150 mg, 0.500 mmol),
potassium 4-(trifluoromethyl)phenyltrifluoroborate (163 mg,
0.648 mmol) and heating for 2 h, gave compound 2q as a yellow
solid (132 mg, 75%), m.p. 162e164 ꢂC, after some washes with
(DMSO-d6, 400 MHz):
d
3.82 (3H, s, OMe), 5.24 (2H, br s, 30-NH2),
6.65 (1H, dd, J ¼ 8.0 and 1.2 Hz, AreH), 6.90e6.96 (4H, m, AreH and
3-NH2), 7.16 (1H, apparent t, J ¼ 7.6 Hz, 50-H), 8.52 (1H, d, J ¼ 1.6 Hz,
AreH), 8.85 (1H, d, J ¼ 1.6 Hz, AreH) ppm. 13C NMR (DMSO-d6,
petroleum ether. 1H NMR (CDCl3, 400 MHz):
d
3.94 (3H, s, OMe),
100.6 MHz): d 51.49 (OMe), 97.11 (C), 112.37 (CH), 114.09 (CH),
6.28 (2H, broad s, NH2), 7.74e7.80 (4H, m, AreH), 8.24 (1H, d,
114.79 (CH), 128.88 (CH), 129.73 (50-CH), 133.88 (C), 135.83 (C),
137.26 (C), 145.14 (C), 145.41 (CH), 147.92 (C), 149.36 (C), 164.56 (C]
O) ppm. MS (EI): m/z (%) 299 (Mþ, 100), 267 (70), 239 (35); HRMS
J ¼ 2.0 Hz, AreH), 8.85 (1H, d, J ¼ 2.0 Hz, AreH) ppm. 13C NMR
(CDCl3, 100.6 MHz):
d 51.76 (OMe), 100.59 (C), 124.00 (CF3, q,
J ¼ 273.0 Hz) 126.19 (2 ꢁ CH, q, J ¼ 4.0 Hz), 127.80 (2 ꢁ CH), 129.53
(CH), 130.56 (C, q, J ¼ 33.0 Hz) 134.31 (C), 134.58 (C), 140.90 (C),
145.44 (CH), 146.03 (C), 147.24 (C), 165.20 (C]O), ppm. MS (EI): m/z
(%) 352 (Mþ, 100), 320 (88), 292 (34), HRMS Mþ calcd. for
C16H11F3N2O2S 352.0493, found 352.0493.
M
þ calcd. for C15H13N3O2S 299.0728, found 299.0732.
4.1.1.22. Methyl 3-amino-6-(4-aminophenyl)thieno[3,2-b]pyridine-
2-carboxylate (2v). Compound (150 mg, 0.500 mmol) and
1
4-aminophenylboronic acid pinacol ester (142 mg, 0.648 mmol)
heating for 2 h gave compound 2v as a yellow solid (94 mg, 65%),
m.p. 179e181 ꢂC, after some washes with petroleum ether. 1H NMR
4.1.1.18. Methyl 3-amino-6-(4-methoxyphenyl)thieno[3,2-b]pyridine-
2-carboxylate (2r). Compound
1
(150 mg, 0.500 mmol) and
(DMSO-d6, 400 MHz): d
3.81 (3H, s, OMe), 5.44 (2H, br s, 40-NH2),

5634
M.-J.R.P. Queiroz et al. / European Journal of Medicinal Chemistry 45 (2010) 5628e5634
6.68 (2H, d, J ¼ 8.8 Hz, 30 and 50-H), 6.86 (2H, br s, 3-NH2), 7.54 (2H,
d, J ¼ 8.8 Hz, 20 and 60-H), 8.47 (1H, d, J ¼ 2.0 Hz, AreH), 8.89 (1H, d,
mL in 6-well plates and left incubating for 24 h. Cells were then
incubated with complete medium only (blank), medium with the
compound’s solvent (control DMSO) or with compounds 2c or 2t at
their respective GI50 or 2 ꢁ GI50 concentrations (previously deter-
mined by the SRB assay). Following 48 h incubation, cells were har-
vested, fixed in 70% ethanol and subsequently resuspended in PBS
J ¼ 2.0 Hz, AreH) ppm. 13C NMR (DMSO-d6, 100.6 MHz):
d 51.41
(OMe), 96.23 (C), 114.28 (30and 50-CH), 123.18 (C), 126.72 (CH),
127.90 (20 and 60-CH), 134.12 (C), 135.56 (C), 143.88 (C), 144.74 (CH),
148.12 (C), 149.51 (C), 164.61 (C]O) ppm. MS (EI): m/z (%) 299 (Mþ,
100), 267 (64), 239 (33); HRMS Mþ calcd. for C15H13N3O2S
299.0728, found 299.0740.
containing0.1 mg/mLRNase A and 5
analysis [14].
mg/mLpropidiumiodide, prior to
Cellular DNA content, for cell cycle distribution analysis, and
presence of sub-G1 peaks (suggestive of apoptosis induction) were
analyzed by flow cytometry using an Epics XL-MCL Coulter flow
cytometer plotting 20,000 events per sample. The percentage of
cells in the G1/G0, S, and G2/M phases of the cell cycle was
subsequently determined using FlowJo 7.2 software (Tree Star, Inc.).
Three independent experiments were performed in duplicate
and results are presented as the mean ꢀ SD of the results obtained.
4.2. In vitro antitumor evaluation
4.2.1. Material and methods
4.2.1.1. Reagents. Fetal bovine serum (FBS), L-glutamine, phosphate
buffered saline (PBS) and trypsin were purchased from Gibco
Invitrogen Co. (Scotland, UK). RPMI-1640 medium was purchased
from Cambrex (New Jersey, USA). Acetic acid, dimethyl sulfoxide
(DMSO), doxorubicin, penicillin, streptomycin, ethylene diamine
tetraacetic acid (EDTA), sulforhodamine B (SRB) and trypan blue
were purchased from SigmaChemical Co. (Saint Louis, USA). Tri-
chloroacetic acid (TCA) and Tris were sourced from Merck (Darm-
stadt, Germany).
Acknowledgments
Thanks are due to the Foundation for Science and Technology
(FCT, Portugal) and FEDER (European Communitarian Fund) for
financial support through the research centres and project PTDC/
QUI/81238/2006. The Bruker 400 NMR spectrometer was
purchased in the framework of the National Programme for
Scientific Re-equipment and is part of the national NMR network,
contract REDE/1517/RMN/2005, with funds from POCI 2010. R. C.
Calhelha and R. T. Lima acknowledge FCT for their PhD grants
(SFRH/BD/29274/2006 and SFRH/BD/21759/2005, respectively) and
L. Vale-Silva acknowledges FCT for his post-doctoral (SFRH/BPD/
29112/2006) grant. IPATIMUP is an Associate Laboratory of the
Portuguese Ministry of Science, Technology and Higher Education
and is partially supported by FCT.
4.2.1.2. Solutions of the compounds. Stocks solutions of the tested
compounds were prepared in DMSO and kept at ꢃ 70 ꢂC. Appro-
priate dilutions were freshly prepared in the test medium just prior
to the assays. The effect of the vehicle solvent (DMSO) on the
growth of the cell lines was evaluated by exposing untreated
control cells to the maximum concentration of DMSO used in the
assays (0.25%). No influence was found (data not shown).
4.2.1.3. Cell cultures. Three human tumor cell lines, MCF-7 (breast
adenocarcinoma), NCI-H460 (non-small cell lung cancer) and
A375-C5 (melanoma) were used. MCF-7 and A375-C5 were
obtained from the European Collection of Cell Cultures (ECACC,
Salisbury, UK), and NCI-H460 was kindly provided by the National
Cancer Institute (NCI, Bethesda, USA). Cell lines were routinely
maintained as adherent cell cultures in RPMI-1640 medium sup-
plemented with 5% heat-inactivated FBS, 2 mM glutamine and
References
antibiotics (penicillin 100 U/mL, streptomycin 100 m
g/mL), at 37 ꢂC
[1] I. Hayakawa, R. Shioya, T. Agatsuma, H. Furukawa, Y. Sugano, Bioorg. Med.
Chem. Lett. 14 (2004) 3411e3414.
in a humidified atmosphere containing 5% CO2. Exponentially
growing cells were obtained by plating 1.5 ꢁ 105cells/mL for MCF-7
and 0.75 ꢁ 105 cells/mL for A375-C5 and NCI-H460, followed by
a 24 h incubation.
[2] M.J. Munchhof, J.S. Beebe, J.M. Casavant, B.A. Cooper, J.L. Doty, R.C. Hidgon,
S.M. Hillerman, C.I. Doderstrom, E.A. Knauth, M.A. Marx, A.M.K. Rossi,
S.B. Sobolov, J. Sun, Bioorg. Med. Chem. Lett. 14 (2004) 21e24.
[3] H.R. Heyman, R.R. Frey, P.F. Bousquet, G.A. Cunha, M.D. Moskey, A.A. Ahmed,
N.B. Soni, P.A. Marcotte, L.J. Pease, K.B. Glaser, M. Yates, J.J. Bouska, D.H. Albert,
C.L. Black-Schaefer, P.J. Dandliker, K.D. Stewart, P. Rafferty, S.K. Davidsen,
M.R. Michaelides, M.L. Curtin, Bioorg. Med. Chem. Lett. 17 (2007) 1246e1249.
[4] S. Claridge, F. Raeppel, M.-C. Granger, N. Bernstein, O. Saavedra, L. Zhan,
D. Llewellyn, A. Wahhab, R. Deziel, J. Rahil, N. Beaulieu, H. Nguyen, I. Dupont,
A. Barsalou, C. Beaulieu, I. Chute, S. Gravel, M.-F. Robert, S. Lefebvre, M. Dubay,
R. Pascal, J. Gillespie, Z. Jin, J. Wang, J.M. Besterman, A.R. MacLeod, A. Vaisburg,
Bioorg. Med. Chem. Lett. 18 (2008) 2793e2798.
[5] R.C. Calhelha, M.-J.R.P. Queiroz, Tetrahedron Lett. 51 (2010) 281e283.
[6] For a recent review on the Suzuki reaction see: F. Alonso, I.P. Belestskaya,
M. Yus, Tetrahedron 64 (2008) 3047e3101.
[7] M. Murata, T. Oyama, S. Watanabe, Y. Masuda, J. Org. Chem. 65 (2000)
164e168.
4.2.1.4. Growth inhibition assay. The in vitro effects on the growth
of human tumor cell lines were evaluated according to the proce-
dure adopted by the NCI (USA) in their “In vitro Anticancer Drug
Discovery Screen”, using the protein-binding dye sulforhodamine B
(SRB) to assess cell growth [12,13]. Briefly, exponentially growing
cells were exposed for 48 h, in 96-well microtiter plates, to five
serial dilutions of each test compound, starting from a maximum
concentration of 150 mM (when possible). Following this exposure
period, adherent cells were 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 (Bio-tek Instruments Inc.,
Powerwave XS, Wincoski, USA). Dose-response curves were
obtained for each test compound and cell line, 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 [13]. Doxorubicin was tested following the
same protocol and used as a positive control.
[8] For a recent review on organotrifluoroborates see: H.A. Stefani, R. Cella,
A.S. Vieira, Tetrahedron 63 (2007) 3623e3658.
[9] M.-J.R.P. Queiroz, A.S. Abreu, R.C. Calhelha, M.S.D. Carvalho, P.M.T. Ferreira,
Tetrahedron 64 (2008) 382e391.
[10] M.-J.R.P. Queiroz, A.S. Abreu, M.S.D. Carvalho, N. Nazareth, M.S.-J. Nascimento,
Bioorg. Med. Chem. 16 (2008) 5584e5589.
[11] M.R. Netherton, G.C. Fu Org, Lett 3 (2001) 4295e4298.
[12] P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vistica,
J.T. Warren, H. Bokesch, S. Kenny, M.R. Boyd, J. Natl. Cancer Inst. 82 (1990)
1107e1112.
[13] A. Monks, D. Scudiero, P. Skehan, R. Shoemaker, K. Paul, D. Vistica, C. Hose,
J. Langley, P. Cronise, A. Vaigro-Wolff, M. Gray-Goodrich, H. Campbell, J. Mayo,
M. Boyd, J. Natl. Cancer Inst. 83 (1991) 757e776.
[14] G.M. Almeida, T.L. Duarte, P.B. Farmer, W.P. Steward, G.D.D. Jones, Int. J.
Cancer 122 (2009) 1810e1819.
4.2.1.5. Cell cycle analysis and detection of sub-G1 peak. For cell
cycle distribution analysis, H460 cells were plated at 0.75 ꢁ 105cells/