Synthesis and evaluation of tumor cell growth inhibition of methyl 3-amino-6-[(hetero)arylethynyl]thieno[3,2-b]pyridine-2-carboxylates. Structure-activity relationships, effects on the cell cycle and apoptosis

Article abstract of DOI:10.1016/j.ejmech.2010.11.009

The methyl 3-amino-6-bromothieno[3,2-b]pyridine-2-carboxylate, recently reported by some of us, was reacted in Sonogashira couplings with several (hetero)arylacetylenes. The growth inhibitory activity of the novel methyl 3-amino-6-[(hetero)arylethynyl]thieno[3,2-b]pyridine-2-carboxylates obtained was evaluated on three human tumor cell lines (MCF-7, NCI-H460, A375-C5). The para-methoxyphenyl and the ortho- and para-aminophenyl derivatives were the most promising compounds, and their effects were further studied regarding alterations in the normal cell cycle distribution and induction of apoptosis in the NCI-H460 cell line. All three compounds altered cell cycle distribution and the ortho-aminophenyl derivative was further shown to induce apoptosis in the same cell line.

Full text of DOI:10.1016/j.ejmech.2010.11.009

European Journal of Medicinal Chemistry 46 (2011) 236e240
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and evaluation of tumor cell growth inhibition of methyl 3-amino-6-
[(hetero)arylethynyl]thieno[3,2-b]pyridine-2-carboxylates. Structureeactivity
relationships, effects on the cell cycle and apoptosis
,
a
b
b
Maria-João R.P. Queiroz a , Ricardo C. Calhelha , Luís A. Vale-Silva , Eugénia Pinto ,
*
Gabriela M. Almeida c, M. Helena Vasconcelos b
,
c
a Centro de Química, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal
b Serviço de Microbiologia/Centro de Química Medicinal da Universidade do Porto (CEQUIMED-UP), Faculdade de Farmácia da Universidade do Porto, Rua Aníbal Cunha 164,
4050-047 Porto, Portugal
c 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:
The methyl 3-amino-6-bromothieno[3,2-b]pyridine-2-carboxylate, recently reported by some of us, was
reacted in Sonogashira couplings with several (hetero)arylacetylenes. The growth inhibitory activity of
the novel methyl 3-amino-6-[(hetero)arylethynyl]thieno[3,2-b]pyridine-2-carboxylates obtained was
evaluated on three human tumor cell lines (MCF-7, NCI-H460, A375-C5). The para-methoxyphenyl and
the ortho- and para-aminophenyl derivatives were the most promising compounds, and their effects
were further studied regarding alterations in the normal cell cycle distribution and induction of
apoptosis in the NCI-H460 cell line. All three compounds altered cell cycle distribution and the ortho-
aminophenyl derivative was further shown to induce apoptosis in the same cell line.
Received 19 July 2010
Received in revised form
5 October 2010
Accepted 7 November 2010
Available online 8 December 2010
Keywords:
Thieno[3,2-b]pyridines
Sonogashira coupling
Antitumoral activity
Cell cycle
Ó 2010 Elsevier Masson SAS. All rights reserved.
Apoptosis
1. Introduction
For the most promising compounds the effects on the cell cycle
distribution and induction of apoptosis were studied, in order to
Recently several thieno[3,2-b]pyridines have shown important
biological activities, namely antitumor and antiangiogenesis or
dual activity, essentially by acting as inhibitors of tyrosine kinase
receptors I [1], II [2] III [3], or nonreceptors IV [4] which have
been implicated in the growth and progression of various human
cancers and, therefore, have been crucial in the development of
anticancer therapies. As we have recently prepared the methyl
3-amino-6-bromothieno[3,2-b]pyridine-2-carboxylate [5] it was
decided to use it in the Pd/Cu-catalyzed Sonogashira coupling [6]
with several terminal (hetero)arylacetylenes in the synthesis
of novel methyl 3-amino-6-[(hetero)arylethynyl]thieno[3,2-b]
pyridine-2-carboxylates. Their potential as antitumor agents
was evaluated through the in vitro growth inhibitory activity on
three selected human tumor cell lines, MCF-7, NCI-H460,
and A375-C5 and some structureeactivity relationships (SARs)
were established.
investigate their possible mechanisms of action.
2. Results and discussion
2.1. Synthesis
The 6-bromothieno[3,2-b]pyridine 1 [5] was used as the thieno
[3,2-b]pyridine component in the Pd/Cu catalyzed Sonogashira
reaction [5e7] with several (hetero)arylacetylenes to obtain the
novel methyl 3-amino-6-[(hetero)arylethynyl]thieno[3,2-b]pyri-
dine-2-carboxylates 2aem in high to excellent yields using the
conditions depicted in Scheme 1, already used by us in the
synthesis of arylethynyl benzo[b]thiophene derivatives [7].
2.2. Growth inhibition 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 types, namely breast adenocarcinoma
* 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.2010.11.009

M.-J.R.P. Queiroz et al. / European Journal of Medicinal Chemistry 46 (2011) 236e240
237
Table 1
N
N
Growth inhibitory activity of the 6-(hetero)arylethynylthieno[3,2-b]pyridines 2 on
the three human tumor cell lines in study.
Ar
HetAr
S
S
S
O
F
Compounds
GI50
(m
M)a
NH
O
Me or H
MCF-7
A375-C5
NCI-H460
N
N
H
N
H
2a
2b
2c
2d
2e
2f
2g
2h
2i
>150.0
>150.0
>150.0
H
>47.3b
>47.3b
>47.3b
I
II
33.4 ꢁ 4.7
0.46 ꢁ 0.05
4.1 ꢁ 0.7
10.8 ꢁ 1.2
2.6 ꢁ 1.4
20.6 ꢁ 1.1
>150
>75.0b
49.0 ꢁ 4.7
0.32 ꢁ 0.05
5.7 ꢁ 3.0
11.5 ꢁ 0.5
8.9 ꢁ 0.9
16.6 ꢁ 1.1
>150
>37.5b
N
N
N
3.1 ꢁ 2.1
11.6 ꢁ 0.6
4.8 ꢁ 0.4
17.7 ꢁ 0.1
>150
Ar
NEt
S
S
NC
O
F
F
HN
Cl
OMe
Cl
O
O
2j
2k
2l
29.0 ꢁ 1.2
37.4 ꢁ 4.1
>150
48.4 ꢁ 1.6
13.9 ꢁ 1.9
>150
24.0 ꢁ 2.0
34.9 ꢁ 5.4
>150
N
N
N
H
III
2m
27.7 ꢁ 2.7
29.6 ꢁ 0.2
24.8 ꢁ 0.5
IV
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
Due to the low solubility of the compound in DMSO the maximum concentra-
(MCF-7), melanoma (A375-C5), and non-small cell lung cancer
(NCI-H460), after a continuous exposure of 48 h. The results are
summarized in Table 1.
tion tested was lower than 150 mM. Doxorubicin was used as positive control (GI50:
MCF-7 ¼ 43.3 ꢁ 2.6 nM; A375-C5 ¼ 130.2 ꢁ 10.1 nM; NCI-H460 ¼ 35.6 ꢁ 1.6 nM).
From the analysis of Table 1 it can be concluded that the
p-methoxylated compound 2d presents the lowest GI50 values
tendency for selective activity excluding the melanoma cell line
(A375-C5), which is clear for compound 2d, is already detectable in
2c. The p-dimethylamino derivative 2h presents higher GI50 values
than the free amino compounds. Among the fluorinated
compounds 2i and 2j only the latter shows moderate GI50 values
(below 1.0
cell lines. The aminated compounds 2ee2g were also effective at
inhibiting cell growth, with GI50 values between 2.6 and 11.6 M for
mM), with marked selectivity for MCF-7 and NCI-H460
m
the three cell lines, the best being the o- and p-aminophenyl
compounds 2e and 2g.
The methoxylated compounds 2b and 2c were not very potent
at inhibiting cell growth. They are not very soluble, nevertheless for
compound 2c it was possible to determine the GI50 values for MCF-
(24.0e48.4 mM). Considering the fact that the unsubstituted phe-
nylacetylene derivative 2a did not show any activity at the highest
tested concentration, the cell growth inhibitory activity presented
by the compounds discussed above appears to be closely depen-
dent on the substitution pattern.
7 and NCI-H460 which were between 33.4 and 49.0 mM. The
The pyridine derivative 2k shows some selectivity against
A375-C5 cell line, with a GI50 value of 13.9 mM. The position of the
NH2
N
CeC bond in the pyridine ring appears to be determinant for the
cell growth inhibitory activity, since compound 2l was not active at
the highest concentration tested.
The thiophene derivative 2m presents moderate growth inhib-
itory activity against the three cell lines in study (GI50 values
CO2Me
S
Br
X
1
between 24.8 and 29.6 mM).
R1
R2
2.3. Cell cycle analysis and detection of apoptosis
Y
R3
i
i
S
The most active compounds were chosen to be further investi-
gated regarding their mechanism of action. The NCI-H460 cell line
was incubated with the GI50 concentrations of compounds 2d, 2e
and 2g for 48 h and their effects on the normal cell cycle distri-
bution and induction of apoptosis analyzed. The results show that
all three compounds interfere with the normal cell cycle distribu-
tion of this cell line. Compound 2d caused a small increase in the
percentage of cells in G0/G1 and a reduction in the percentage of
cells in the S-phase of the cell cycle (Table 2). A similar but more
marked effect was observed upon incubation with compound 2g.
Compound 2e induced an increase in the percentage of cells in
G2/M phases of the cell cycle accompanied by a decrease of cells in
S-phase (Table 2). From these results it appears that compounds 2d
and 2g induce a cell cycle arrest on phases G0/G1, whereas
compound 2e appears to cause a G2/M cell cycle arrest. These
compounds were also shown to induce apoptosis, as determined by
the annexin V-FICT/PI flow cytometry assay, particularly compound
2e (Table 2). This observation was confirmed by the presence of
a sub-G1 peak in the cell cycle profile analysis (indicative of DNA
degradation, characteristic of apoptosis) upon incubation with
compound 2e (data not shown).
NH2
NH2
N
N
CO2Me
CO2Me
R1
Y
S
S
R2
R3
S
X= Y= CH unless stated
2m, 70%
X
2a R1 = R2 = R3 = H, 70%
2b R1 = OMe, R2 = R3 = H, 80%
2c R2 = OMe, R1 = R3 = H, 75%
2d R3 = OMe, R1 = R2 = H, 70%
2e R1 = NH2, R2 = R3 = H, 70%
2f R2 = NH2, R1 = R3 = H, 60%
2g R3 = NH2, R1 = R2 = H, 75%
2h R3 = NMe2, R1 = R2 = H, 80%
2i R1 = F, R2 = R3 = H, 75%
2j R3 = F, R1 = R2 = H, 90%
2k R1 = R2 = R3 = H, X = N,70%
2l R1 = R2 = R3 = H, Y = N, 90%
Scheme 1. Synthesis of compounds 2ae2m. Conditions: PdCl2(PPh3)2 (5 mol%), CuI
(3 mol%), NEt3 (3 equiv.), DMF, 1.5 h, 100 ꢀC.

238
M.-J.R.P. Queiroz et al. / European Journal of Medicinal Chemistry 46 (2011) 236e240
Table 2
4.1.1. General procedure for the synthesis of compounds 2ae2m
Compound 1 (150 mg, 0.540 mmol), the (hetero)arylacetylene
(1.1 equiv.), PdCl2(PPh3)2 (5 mol%), CuI (3 mol%), and NEt3 (3 equiv.)
were added to dry DMF (2e3 mL) into a dry Schlenk tube, under
argon, 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 a solid which was submitted to column
chromatography using 50% ether/petroleumetheraseluent. The solid
obtained was washed with petroleum ether and fully characterized.
Cell cycle analysis (% of cells in phases G0/G1, S, G2/M) and % of NCI-H460 cells
undergoing apoptosis after a 48 h treatment with compounds 2d, 2e and 2g at their
GI50 concentrations.
G0/G1
(cells %)
S (cells %)
G2/M
(cells %)
Apoptosis
(cells %)
Control
DMSO
2d
2e
2g
57.1 ꢁ 1.6
55.1 ꢁ 1.0
59.7 ꢁ 3.2
54.7 ꢁ 2.7
68.0 ꢁ 0.7
28.2 ꢁ 1.4
28.8 ꢁ 1.2
22.0 ꢁ 1.7
23.1 ꢁ 2.8
18.6 ꢁ 0.8
14.7 ꢁ 0.9
16.1 ꢁ 0.9
18.3 ꢁ 1.9
24.6 ꢁ 2.4
13.4 ꢁ 0.6
11.7 ꢁ 1.6
12.2 ꢁ 1.2
18.7 ꢁ 2.8
25.3 ꢁ 1.8a
17.0 ꢁ 2.1
Untreated cells were used as the control and a solvent control (DMSO) was also
included. Results are the mean ꢁ standard error of three to six independent
experiments performed in duplicate.
a
Apoptosis induction by compound 2e was statistically significantly when
4.1.1.1. Methyl 3-amino-6-(2-phenylethynyl)thieno[3,2-b]pyridine-2-
compared to the control (P < 0.05).
carboxylate (2a). Yellow solid (117 mg, 70%), m.p. 175e177 ꢀC. 1H
NMR (CDCl3, 400 MHz)
d 3.93 (3H, s, OMe), 6.24 (2H, br s, NH2),
7.38e7.42 (3H, m, Ar-H), 7.56e7.60 (2H, m, Ar-H), 8.18 (1H, d,
According to these findings, the compounds are likely to present
different mechanisms of action. The effect of compound 2e in cell
growth inhibition seems to be due to the induction of both
apoptosis and cell cycle arrest in the G2/M phase (Table 2).
Compounds 2d and 2g did not cause such a marked induction of
apoptosis (Table 2) and thus their growth inhibitory effects seem to
be mainly due to cell cycle related events.
J ¼ 2 Hz, Ar-H), 8.73 (1H, d, J ¼ 2 Hz, Ar-H) ppm. 13C NMR (CDCl3,
100.6 MHz)
d 51.75 (OMe), 86.07 (C), 93.88 (C), 100.84 (2-C), 119.11
(2 ꢂ CH), 122.29 (10-C), 128.50 (2 ꢂ CH), 129.02 (C), 131.74 (C),
133.49 (CH), 133.72 (C),144.97 (3a-C),147.30 (C), 148.73 (CH), 165.17
(C]O) ppm. MS (EI) m/z 308 (Mþ, 100), 276 (66). HRMS Mþ calcd.
for C17H12N2O2S 308.0619; found 308.0624.
4.1.1.2. Methyl 3-amino-6-[2-(2-methoxyphenyl)ethynyl]thieno[3,2-
3. Conclusion
b]pyridine-2-carboxylate (2b). Yellow solid (146 mg, 80%), m.p.
193e195 ꢀC.1H NMR (CDCl3, 400 MHz)
d 3.92 (3H, s, OMe), 3.95 (3H,
Novel methyl 3-amino-6-[(hetero)arylethynyl]thieno[3,2-b]
pyridine-2-carboxylates were synthesized and tested for their in
vitro growth inhibitory activity on three human tumor cell lines. All
compounds were fully soluble at the GI50 concentration. Overall,
the para-methoxyphenyl and the ortho- and para-aminophenyl
derivatives are the most promising compounds, presenting very
low GI50 values and therefore their effects on cell cycle distribution
and induction of apoptosis were further investigated in the
NCI-H460 cell line. Compounds 2d, 2e and 2g had an effect on cell
cycle distribution, with a G0/G1 arrest being detected for the para-
methoxyphenyl and the para-aminophenyl derivatives and a G2/M
arrest for the ortho-aminophenyl derivative. Additionally, the
ortho-aminophenyl compound was found to significantly induce
apoptosis at the GI50 concentration in the tested cell line. Hence, at
least two mechanisms of action appear to be involved in the activity
of this compound, cell cycle related events and induction of
apoptosis. The obtained data suggest that the results are dependent
on the compound substitution pattern, nevertheless it is important
to bear in mind that the physical properties of the compounds,
particularly cell permeability, may be influencing their relative
activity.
s, OMe), 6.22 (2H br s, NH2), 6.93e6.98 (2H, m, Ar-H), 7.34e7.39 (1H,
m, Ar-H), 7.53 (1H, dd, J ¼ 7.6 and 2 Hz, Ar-H), 8.20(1H, d, J ¼ 2 Hz, Ar-
H), 8.75 (1H, d, J ¼ 2 Hz, Ar-H) ppm. 13C NMR (CDCl3, 100.6 MHz)
d
51.71 (OMe), 55.83 (OMe), 90.06 (C), 90.45 (C), 100.68 (2-C),110.74
(CH), 111.52 (C), 119.50 (C), 120.57 (CH), 130.57 (CH), 133.38 (CH),
133.65 (CH),133.67 (C),144.89 (C),147.38 (C),148.89 (CH),160.16 (C),
165.21 (C]O) ppm. MS (EI) m/z 338 (Mþ, 100), 306 (39). HRMS Mþ
calcd. for C18H14N2O3S 338.0725; found 338.0727.
4.1.1.3. Methyl 3-amino-6-[2-(3-methoxyphenyl)ethynyl]thieno[3,2-
b]piridine-2-carboxylate (2c). Yellow solid (137 mg, 75%), m.p.
177e179 ꢀC. 1H NMR (CDCl3, 400 MHz)
d 3.85 (3H, s, OMe), 3.93
(3H, s, OMe), 6.23 (2H, br s, NH2), 6.94e6.97 (1H, m, Ar-H),
7.09e7.11 (1H, m, Ar-H), 7.16e7.19 (1H, m, Ar-H), 7.30 (1H, app. t,
J ¼ 8 Hz, 50-H), 8.19 (1H, d, J ¼ 1.6 Hz, Ar-H), 8.73 (1H, d, J ¼ 1.6 Hz,
Ar-H) ppm. 13C NMR (CDCl3, 100.6 MHz)
d 51.74 (OMe), 55.33
(OMe), 85.89 (C), 93.78 (C), 100.84 (2-C), 115.65 (CH), 116.48 (CH),
119.03 (C), 123.26 (C), 124.29 (CH), 129.58 (50-CH), 133.49 (CH),
133.69 (C), 145.05 (C),147.32 (C), 148.78 (CH), 159.42 (C), 165.18 (C]
O) ppm. MS (EI) m/z 338 (Mþ, 100), 306 (53). HRMS Mþ calcd. for
C18H14N2O3S 338.0725; found 338.0723.
4. Experimental
4.1.1.4. Methyl 3-amino-6-[2-(4-methoxyphenyl)ethynyl]thieno[3,2-
b]pyridine-2-carboxylate (2d). Yellow solid (130 mg, 70%), m.p.
4.1. Synthesis
190e192 ꢀC. 1H NMR (CDCl3, 300 MHz):
d 3.85 (3H, s, OMe), 3.92
(3H, s, OMe), 6.25 (2H, br s, NH2), 6.92 (2H, d, J ¼ 8.4 Hz, 30 and 50-
Melting points (ꢀC) 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 II
at 400 and 100.6 MHz, respectively. Heteronuclear correlations,
1He13C, HMQC or HMSQ 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 chromathography
(TLC). Dry flash was performed on Macherey-Nagel silica gel
150e230 mesh. Petroleum ether refers to the boiling range
40e60 ꢀC. Ether refers to diethylether.
H), 7.52 (2H, d, J ¼ 8.4 Hz, 20 and 60-H), 8.16 (1H, d, J ¼ 2 Hz, Ar-H),
8.71 (1H, d, J ¼ 2 Hz, Ar-H) ppm. 13C NMR (CDCl3, 75.4 MHz):
d 51.74
(OMe), 55.34 (OMe), 84.90 (C), 94.17 (C), 100.63 (2-C), 114.16 (30and
50-CH), 114.28 (C), 119.52 (C), 133.25 (CH), 133.29 (20 and 60-CH),
133.78 (C), 144.56 (C),147.27 (C), 148.56 (CH), 160.20 (C), 165.18 (C]
O) ppm. MS (EI): m/z (%) 338 (Mþ, 100), 306 (39). HRMS Mþ
C18H14N2O3S: calcd. 338.0725; found 338.0724.
4.1.1.5. Methyl 3-amino-6-[2-(2-aminophenyl)ethynyl]thieno[3,2-b]
pyridine-2-carboxylate (2e). Yellow solid (122 mg, 70%), m.p.
168e170 ꢀC. 1H NMR (DMSO-d6, 400 MHz)
d
3.82 (3H, s, OMe), 5.68
(2H, br s, NH2), 6.52e6.57 (1H, m, Ar-H), 6.73 (1H, br d, J ¼ 9.2 Hz,

M.-J.R.P. Queiroz et al. / European Journal of Medicinal Chemistry 46 (2011) 236e240
239
Ar-H), 6.93 (2H, br s, 20-NH2), 7.08e7.14 (1H, m, Ar-H), 7.26 (1H, dd,
J ¼ 7.6 and 1.6 Hz, Ar-H), 8.63 (1H, d, J ¼ 1.6 Hz, Ar-H), 8.85 (1H, d,
115.88 (d, J ¼ 21 Hz, 30 and 50-CH), 118.43 (d, J ¼ 3 Hz, C), 118.92 (C),
133.42 (CH), 133.70 (d, J ¼ 10 Hz, 20 and 60-CH), 133.76 (C), 145.07
(C), 147.29 (C), 148.69 (CH), 162.92 (d, J ¼ 252 Hz, CeF), 165.17 (C]
O) ppm. Elemental Analysis calcd. for C17H11FN2O2S C, 62.57; H,
3.40; N, 8.58; S, 9.83%. Found C, 62.25; H, 3.53; N, 8.24; S, 9.99%.
J ¼ 1.6 Hz, Ar-H) ppm. 13C NMR (DMSO-d6, 100.6 MHz)
d 51.57
(OMe), 90.88 (C), 91.60 (C), 97.94 (2-C), 104.38 (C), 114.03 (CH),
115.69 (CH), 119.02 (C), 130.46 (CH), 131.89 (CH), 132.97 (C), 133.50
(CH), 144.57 (C), 147.77 (C), 148.51 (CH), 150.15 (C), 164.40 (C]O)
ppm. Elemental Analysis calcd for C17H13N3O2S C, 63.14; H, 4.05; N,
12.99; S, 9.92%. Found C, 63.05; H, 4.05; N, 12.59; S, 9.48%.
4.1.1.11. Methyl 3-amino-6-[2-(pyridin-2-yl)ethynyl]thieno[3,2-b]pyri-
dine-2-carboxylate (2k). Yellow solid (117 mg, 70%), m.p. 226e
228 ꢀC. 1H NMR (DMSO-d6, T ¼ 60 ꢀC, 300 MHz)
d 3.84 (3H, s, OMe),
6.81 (2H, br s, NH2), 7.42e7.47 (1H, m, Ar-H), 7.70 (1H, d, J ¼ 7.8 Hz,
4.1.1.6. Methyl 3-amino-6-[2-(3-aminophenyl)ethynyl]thieno[3,2-b]
pyridine-2-carboxylate (2f). Yellow solid (105 mg, 60%), m.p.
Ar-H), 7.85e7.91 (1H, m, Ar-H), 8.63e8.66 (2H m, Ar-H), 8.84 (1H, d,
203e205 ꢀC. 1H NMR (DMSO-d6, 400 MHz)
d
3.82 (3H, s, OMe), 5.30
J ¼ 2 Hz, Ar-H) ppm. 13C NMR (DMSO-d6, T ¼ 60 ꢀC, 75.4 MHz)
d 51.33
(2H, br s, NH2), 6.64 (1H, ddd, J ¼ 8.1, 2.4 and 1.2 Hz, Ar-H), 6.73 (1H,
dt, J ¼ 7.6 and 1.2 Hz, Ar-H), 6.77 (1H, m, Ar-H), 6.92 (2H, br s, NH2),
7.01 (1H, app. t, J ¼ 8 Hz, 50-H), 8.57 (1H, d, J ¼ 2 Hz, Ar-H), 8.76 (1H,
(OMe), 85.03 (C), 92.47 (C), 99.06 (2-C),117.04 (C),123.66 (CH),127.33
(CH), 132.83 (C), 134.32 (CH), 136.54 (CH), 141.52 (C), 145.33 (C),
147.22 (C), 148.43 (C), 150.01 (CH), 164.08 (C]O) ppm. MS (EI) m/z
309 (Mþ, 100), 277 (63). HRMS calcd. for C16H11N3O2S Mþ 309.0572;
found 309.0571.
d, J ¼ 2 Hz, Ar-H) ppm. 13C NMR (DMSO-d6, 100.6 MHz)
d 51.62
(OMe), 84.97 (C), 94.46 (C), 98.35 (20-C), 115.17 (40-CH), 116.14 (20-
CH), 118.42 (C), 118.94 (60-CH), 121.68 (C), 129.32 (50-CH), 133.10 (C),
133.92 (CH), 144.94 (C), 147.65 (C), 148.53 (CH), 148.91 (C), 164.40
(C]O) ppm. MS (EI) m/z 323 (Mþ, 100), 291 (56). HRMS calcd. for
C17H13N3O2S Mþ 323.0728; found 323.0729.
4.1.1.12. Methyl 3-amino-6-[2-(pyridin-3-yl)ethynyl]thieno[3,2-b]pyri-
dine-2-carboxylate(2l). Yellow solid (150 mg, 90%), m.p. 205e207 ꢀC.
1H NMR (CDCl3, 400 MHz)
d 3.93 (3H, s, OMe), 6.22 (2H, br s, NH2),
7.36e7.38 (1H, m, Ar-H), 7.44e7.50 (1H, m, Ar-H), 7.64e7.70 (1H, m,
Ar-H), 7.87 (1H, d, J ¼ 8 Hz, Ar-H), 8.21 (1H,d, J ¼ 2 Hz, Ar-H), 8.74 (1H,
4.1.1.7. Methyl 3-amino-6-[2-(4-aminophenyl)ethynyl]thieno[3,2-b]
pyridine-2-carboxylate (2g). Yellow solid (130 mg, 75%), m.p.
d, J ¼ 2 Hz, Ar-H) ppm. 13C NMR (CDCl3, 100.6 MHz):
d 51.78 (OMe),
211e213 ꢀC. 1H NMR (DMSO-d6, 300 MHz)
d
3.81 (3H, s, OMe), 5.70
89.52 (C), 90.08 (C), 101.18 (2-C), 118.18 (C), 119.78 (C), 123.29 (CH),
128.55 (C), 132.02 (CH), 133.61 (C), 133.65 (CH), 138.78 (CH), 145.54
(C), 147.27 (C), 148.74 (CH), 165.13 (C]O) ppm. MS (EI) m/z 309 (Mþ,
53), 277 (100). HRMS calcd. for C16H11N3O2S Mþ 309.0572; found
309.0578.
(2H, br s, NH2), 6.57 (2H, d, J ¼ 8.4 Hz, 30 and 50-CH), 6.91 (2H, br s,
NH2), 7.25 (2H, d, J ¼ 8.4 Hz, 20 and 60-CH), 8.46 (1H, d, J ¼ 2 Hz, Ar-
H), 8.70 (1H, d, J ¼ 2 Hz, Ar-H) ppm. 13C NMR (DMSO-d6, 75.4 MHz)
d
51.60 (OMe), 83.87 (C), 96.06 (C), 97.85 (2-C), 107.07 (C), 113.62 (30
and 50-CH),119.43 (C),132.92 (20 and 60-CH),132.97 (CH),133.22 (C),
144.33 (C), 147.77 (C), 148.29 (CH), 150.15 (C), 164.46 (C]O) ppm.
MS (EI) m/z 323 (Mþ, 100), 291 (51). HRMS calcd. for C17H13N3O2S
Mþ 323.0728; found 323.0727.
4.1.1.13. Methyl 3-amino-6-[2-(thien-3-yl)ethynyl)]thieno[3,2-b]pyri-
dine-2-carboxylate (2m). Yellow solid (119 mg, 70%), m.p. 165e
167 ꢀC. 1H NMR (CDCl3, 400 MHz)
d 3.93 (3H, s, OMe), 6.22 (2H, br s,
NH2), 7.24 (1H, dd, J ¼ 4.8 and 1.2 Hz, Ar-H), 7.35 (1H, dd, J ¼ 4.8 and
2.8 Hz, Ar-H), 7.62 (1H, dd, J ¼ 2.8 and 1.2 Hz, Ar-H), 8.16 (1H, d,
J ¼ 2 Hz, Ar-H), 8.71 (1H, d, J ¼ 2 Hz, Ar-H) ppm. 13C NMR (CDCl3,
4.1.1.8. Methyl 3-amino-6-{2-[4-(dimethylamino)phenyl]ethynyl}thie-
no[3,2-b]pyridine-2-carboxylate (2h). Green solid (152 mg, 80%), m.p.
191e193 ꢀC.1H NMR (DMSO-d6, 300 MHz)
d
2.96 (6H, s, 2 ꢂ Me), 3.81
75.4 MHz) d 51.74 (OMe), 85.72 (C), 89.05 (C),100.78 (2-C),119.09 (C),
(3H, s, OMe), 6.73 (2H, d, J ¼ 8.7 Hz, 30 and 50-CH), 6.92 (2H, br s, NH2),
121.40 (C), 125.76 (CH), 129.74 (2 ꢂ CH), 133.32 (CH), 133.70 (C),
144.97 (C), 147.32 (C), 148.68 (CH), 165.18 (C]O) ppm. MS (EI) m/z
314 (Mþ, 100), 282 (90). HRMS calcd. for C15H10N2O2S2 Mþ 314.0184;
found 314.0183.
7.40 (2H, d, J ¼ 8.7 Hz, 20 and 60-CH), 8.49 (1H, d, J ¼ 2 Hz, Ar-H), 8.72
(1H, d, J ¼ 2 Hz, Ar-H) ppm. 13C NMR (DMSO-d6, 75.4 MHz)
d 39.59
(2 ꢂ Me), 51.56 (OMe), 84.51 (C), 95.60 (C), 97.86 (2-C), 107.33 (10C),
111.79 (30 and 50-CH), 119.24 (C), 132.66 (20 and 60-CH), 133.05 (CH),
133.16 (C),144.36 (3a-C),147.72 (C),148.28 (CH), 150.44 (40-C),164.41
(C]O) ppm. MS (EI) m/z 351 (Mþ, 100), 319 (33). HRMS calcd. for
C19H17N3O2S Mþ 351.1041; found 351.1042.
4.2. Biological activity
4.2.1. Material and methods
4.2.1.1. Reagents. Fetal bovine serum (FBS), L-glutamine, phosphate
4.1.1.9. Methyl 3-amino-6-[2-(2-fluorophenyl)ethynyl]thieno[3,2-b]
pyridine-2-carboxylate (2i). Yellow solid (132 mg, 75%), m.p.
buffered saline (PBS) and trypsin were from Gibco Invitrogen Co.
(Scotland, UK). RPMI-1640 medium was from Cambrex (New Jersey,
USA). Acetic acid, dimethyl sulfoxide (DMSO), doxorubicin, peni-
cillin, streptomycin, ethylenediaminetetraacetic acid (EDTA), sul-
forhodamine B (SRB) and trypan blue were from SigmaChemical Co.
(Saint Louis, USA). Tricloroacetic acid (TCA) and Tris were sourced
from Merck (Darmstadt, Germany).
174e176 ꢀC. 1H NMR (CDCl3, 400 MHz)
d 3.93 (3H, s, OMe), 6.22
(2H, br s, NH2), 7.14e7.18 (2H, m, Ar-H), 7.35e7.41 (1H, m, Ar-H),
7.53e7.58 (1H, m, Ar-H), 8.21 (1H, d, J ¼ 2 Hz, Ar-H), 8.75 (1H, d,
J ¼ 2 Hz, Ar-H) ppm. 13C NMR (CDCl3, 100.6 MHz)
d 51.75 (OMe),
87.10 (C), 91.03 (C), 101.00 (2-C), 111.06 (d, J ¼ 16 Hz, C), 115.68 (d,
J ¼ 21 Hz, CH), 118.68 (C), 124,12 (d, J ¼ 3 Hz, CH), 130.78 (d,
J ¼ 8 Hz, CH), 133.53 (d, J ¼ 13 Hz, CH), 133.63 (CH), 145.29 (C),
147.31 (C), 148.77 (CH), 162.73 (d, J ¼ 251 Hz, CeF), 165.16 (C]O)
ppm. Elemental Analysis calcd. for C17H11FN2O2S C, 62.57; H, 3.40;
N, 8.58; S, 9.83%. Found C, 62.39; H, 3.43; N, 8.40; S, 9.43%.
4.2.1.2. Solutions of the compounds. Stock solutions of the tested
compounds were prepared in DMSO and kept at ꢃ70 ꢀC. 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).
4.1.1.10. Methyl 3-amino-6-[2-(4-fluorophenyl)ethynyl]thieno[3,2-b]
pyridine-2-carboxylate (2j). Yellow solid (159 mg, 90%) m.p.
167e169 ꢀC. 1H NMR (CDCl3, 400 MHz)
d 3.93 (3H, s, OMe), 6.23
(2H, br s, NH2), 7.07e7.11 (2H, m, Ar-H), 7.54e7.58 (2H, m, Ar-H),
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
8.17 (1H, d, J ¼ 2 Hz, Ar-H), 8.71 (1H, d, J ¼ 2 Hz, Ar-H) ppm. 13C NMR
(CDCl3, 100.6 MHz) d 51.76 (OMe), 85.83 (C), 92.75 (C), 100.89 (2-C),

240
M.-J.R.P. Queiroz et al. / European Journal of Medicinal Chemistry 46 (2011) 236e240
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). They were routinely main-
tained as adherent cell cultures in RPMI-1640 medium supple-
mented with 5% heat-inactivated FBS, 2 mM glutamine and
using an Epics XL-MCL Coulter flow cytometer plotting at least
20,000 events per sample. Cell cycle distribution and apoptosis data
analysis were subsequently performed using FlowJo 7.2 software
(Tree Star, Inc.). Experiments wereperformed in duplicate in three to
six independent occasions and the average and standard error of the
obtained results were presented. Statistical analysis was performed
for the induction of apoptosis results by the non-parametric Fried-
man’s test followed by Dunn’s Post-test using GraphPad Prism 5
software. P values <0.05 were considered as statistically significant.
antibiotics (penicillin 100 U/mL, streptomycin 100 m
g/mL), at 37 ꢀC
in a humidified atmosphere containing 5% CO2. Exponentially
growing cells were obtained by plating 1.5 ꢂ105 cells/mL for MCF-7
and 0.75 ꢂ105 cells/mL for A375-C5 and NCI-H460, followed by
a 24 h incubation.
Acknowledgments
4.2.1.4. Growth inhibition assay. The effects on the in vitro 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
to assess cell growth [8,9]. 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
To the Foundation for Science and Technology (FCT, Portugal)
through the financial support to the research centres, the national
NMR network (Bruker Avance III 400) REDE/1517/RMN/2005, the
research project PTDC/QUI-QUI/111060/2009 which is also financed
by COMPETE/QREN/EU. RCC and LAVS acknowledge FCT for their
PhD (SFRH/BD/29274/2006) and post-doctoral (SFRH/BPD/29112/
2006) grants, respectively. GMA is supported by FCT and the
European Social Fund. IPATIMUP is an Associate Laboratory of the
Portuguese Ministry of Science, Technology and Higher Education
and is partially supported by FCT.
150 mM (if possible). Following the exposure period adherent cells
were fixed in situ with TCA, 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). Doseeresponse 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 inhibi-
ted 50% of the net cell growth was calculated as described else-
where [9]. Doxorubicin was tested in the same manner, to be used
as a positive control.
References
[1] 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.
[2] 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.
[3] S. Raeppel, S. Claridge, O. Saavedra, F. Gaudette, L. Zhan, M. Mannion, N. Zhou,
F. Raeppel, M.-C. Granger, L. Isakovick, R. Deziel, H. Nguyen, N. Beaulieu,
C. Beaulieu, I. Dupont, M.-F. Robert, S. Lefebvre, M. Dubay, J. Rahil, J. Wang,
H. Ste-Croix, A.R. MacLeod, J. Besterman, A. Vaisburg, Bioorg. Med. Chem. Lett.
19 (2009) 1323e1328.
4.2.1.5. Flow cytometric assays for cell cycle distribution analysys and
apoptosis detection with annexin V-FITC/PI assay. NCI-H460 cells
were plated at 0.75 ꢂ105 cells/mL in 6-well plates and left incu-
bating for 24 h. Cells were then incubated with complete medium
only (control), medium with the compound’s solvent (DMSO) or
with compounds 2d, 2e and 2g at their respective GI50 concentra-
tions previously obtained by the SRB assay (0.32, 5.7 and 8.9
mM).
[4] D.H. Boschelli, B. Wu, A.C.B. Susa, H. Durutlic, F. Ye, Y. Raifeld, J.M. Golas,
F. Boschelli, J. Med. Chem. 47 (2004) 6666e6668.
Cells were harvested following 48 h incubation with the
a
[5] R.C. Calhelha, M.-J.R.P. Queiroz, Tetrahedron Lett. 51 (2010) 281e283.
[6] For a recent review on the Sonogashira reaction see: R. Chincilla, C. Nájera
Chem. Rev. 107 (2007) 874e922.
[7] M.-J.R.P. Queiroz, R.C. Calhelha, L.A. Vale-Silva, E. Pinto, M.S.-J. Nascimento,
Eur. J. Med. Chem. 44 (2009) 1893e1899.
[8] P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vistica,
J.T. Warren, H. Bokesch, S. Kenney, M.R. Boyd, J. Natl. Cancer Inst. 83 (1991)
757e776.
[9] A. Monks, D. Scudiero, P. Skehan, R. Shoemaker, K. Paull, 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.
compounds and further processed for either cell cycle analysis or
apoptosis detection. For cell cycle analysis, cells were fixed in 70%
ethanol and subsequently resuspended in PBS containing 0.1 mg/
mL RNase A and 5 mg/mL propidium iodide, prior to analysis.
Induced apoptosis was assayed by the Human Annexin V-FITC/PI
apoptosis Kit (Bender MedSystems, Vienna, Austria) according to
the manufacturer’s instructions [10].
Cellular DNA content (for cell cycle distribution analysis and
presence of sub-G1 peak, suggestive of apoptosis induction) and
measurement of phosphatidylserine externalization were analyzed
[10] G.M. Almeida, T.L. Duarte, P.B. Farmer, W.P. Steward, G.D.D. Jones, Int.
J. Cancer 122 (2008) 1810e1819.