New di(hetero)arylethers and di(hetero)arylamines in the thieno[3,2-b]pyridine series: Synthesis, growth inhibitory activity on human tumor cell lines and non-tumor cells, effects on cell cycle and on programmed cell death

Article abstract of DOI:10.1016/j.ejmech.2013.09.023

New fluorinated and methoxylated di(hetero)arylethers and di(hetero)arylamines were prepared functionalizing the 7-position of the thieno[3,2-b]pyridine, using copper (C-O) or palladium (C-N) catalyzed couplings, respectively, of the 7-bromothieno[3,2-b]pyridine, also prepared, with ortho, meta and para fluoro or methoxy phenols and anilines. The compounds obtained were evaluated for their growth inhibitory activity on the human tumor cell lines MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer), HCT15 (colon carcinoma), HepG2 (hepatocellular carcinoma) and HeLa (cervical carcinoma). The most active compounds, a di(hetero)arylether with a methoxy group in the meta position relative to the ether function and two di(hetero)arylamines with a methoxy group either in the ortho or in the meta position relative to the NH, were further tested at their GI₅₀ concentrations on NCI-H460 cells causing pronounced alterations in the cell cycle profile and a strong and significant increase in the programmed death of these cells. The fluorinated and the other methoxylated compounds did not show important activity, presenting high GI₅₀ values in all the cell lines tested. Furthermore, the hepatotoxicity of the compounds was assessed using porcine liver primary cells (PLP2), established by some of us. Results showed that one of the most active compounds was not toxic to the non-tumor cells at their GI₅₀ concentrations showing to be the most promising as antitumoral.

Full text of DOI:10.1016/j.ejmech.2013.09.023

European Journal of Medicinal Chemistry 69 (2013) 855e862
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
New di(hetero)arylethers and di(hetero)arylamines in the thieno
3,2-b]pyridine series: Synthesis, growth inhibitory activity on human
[
tumor cell lines and non-tumor cells, effects on cell cycle and on
programmed cell death
a
,
a
a
,
b
a
*
Maria-João R.P. Queiroz , Daniela Peixoto , Ricardo C. Calhelha , Pedro Soares ,
c
c
,
d
a
b
Tiago dos Santos , Raquel T. Lima , Joana F. Campos , Rui M.V. Abreu ,
Isabel C.F.R. Ferreira , M. Helena Vasconcelos
b
c, e
a
Centro de Química, Escola de Ciências, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal
b
c
Centro de Investigação Montanha, ESA, Polytechnic Institute of Bragança, Campus de Sta Apolónia, Apartado 1172, 5301-855 Bragança, Portugal
Cancer Drug Resistance Group, Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), Porto, Portugal
Centre of Medicinal Chemistry of the University of Porto (CEQUIMED-UP), Portugal
d
e
Laboratory of Microbiology, Department of Biological Sciences, Faculty of Pharmacy of the University of Porto (FFUP), Porto, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 31 May 2013
Received in revised form
New fluorinated and methoxylated di(hetero)arylethers and di(hetero)arylamines were prepared func-
tionalizing the 7-position of the thieno[3,2-b]pyridine, using copper (CeO) or palladium (CeN) catalyzed
couplings, respectively, of the 7-bromothieno[3,2-b]pyridine, also prepared, with ortho, meta and para
fluoro or methoxy phenols and anilines. The compounds obtained were evaluated for their growth
inhibitory activity on the human tumor cell lines MCF-7 (breast adenocarcinoma), NCI-H460 (non-small
cell lung cancer), HCT15 (colon carcinoma), HepG2 (hepatocellular carcinoma) and HeLa (cervical car-
cinoma). The most active compounds, a di(hetero)arylether with a methoxy group in the meta position
relative to the ether function and two di(hetero)arylamines with a methoxy group either in the ortho or
in the meta position relative to the NH, were further tested at their GI50 concentrations on NCI-H460 cells
causing pronounced alterations in the cell cycle profile and a strong and significant increase in the
programmed death of these cells. The fluorinated and the other methoxylated compounds did not show
important activity, presenting high GI50 values in all the cell lines tested. Furthermore, the hepatotoxicity
of the compounds was assessed using porcine liver primary cells (PLP2), established by some of us.
Results showed that one of the most active compounds was not toxic to the non-tumor cells at their GI50
concentrations showing to be the most promising as antitumoral.
2
August 2013
Accepted 9 September 2013
Available online 21 September 2013
Keywords:
Di(hetero)arylethers
Di(hetero)arylamines
Thieno[3,2-b]pyridines
Metal-catalyzed couplings
Tumor and nontumor cells growth
inhibition
Cell cycle analysis and cell death
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
3-amino-6-[(hetero)arylamino]thieno[3,2-b]thienopyridine-2-car
boxylates [2]. In the 3-amino-6-[(hetero)aryl]thieno[3,2-b]pyri-
The thienopyridine skeleton has been reported as having
interesting biological activities namely antitumoral [1e7] and
antiangiogenic [8e12]. Sugano et al. reported that the (3-amino-6-
dine-2-carboxylate series, compounds III and IV were shown to be
the most promising compounds against MCF-7 (breast adenocar-
cinoma), A375-C5 (melanoma) and NCI-H460 (non-small cell lung
cancer) cell lines. Indeed, compound III showed selectivity against
thien-2-yl-thieno[2,3-b]pyridin-2-yl)arylmethanones
selectivity against a tumorigenic cell line, in very low EC50 values
1]. The methyl (3-amino-6-benzo[d]thiazol-2-ylamino)thieno[3,2-
I
showed
MCF-7 and NCI-H460, presenting very low GI50 values (1
mM) and
[
compound IV presented GI50 values in the 3e4 M range for the
m
b]pyridine-2-carboxylate II was previously prepared by us and
showed to be the most active compound in a series of methyl
three cell lines. The effects of compounds III and IV on the cell cycle
profile of NCI-H460 cells pointed to different mechanisms of action
for each compound [3]. Several methyl 3-amino-6-[(hetero)aryle-
thynyl]thieno[3,2-b]pyridine-2-carboxylates have also been pre-
pared by us and studied against the same human tumor cell lines,
compounds Vaec being the most potent ones. Effects of these three
*
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 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.09.023

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M.-J.R.P. Queiroz et al. / European Journal of Medicinal Chemistry 69 (2013) 855e862
4N
3
5
Rn = H unless stated
2
C-O Coupling
6
S
2a R1 = F, 70%
7
1
6
'
2
OH
O1
2b R = F, 63%
5
4'
'
'
3
2
c R = F, 65%
i)
1
R1
R3
R1 2'
R3
2d R = OMe, 50%
3
'
2
2e R = OMe, 55%
N
POBr3
N
R2
2
R
3
2
f R = OMe, 45%
S
5 o
6h
S
4
3
6
C
H N
2
N
1
5
3a R = F, 65%
OH
Br
ii)
2
2
3
b R = F, 83%
R1
R3
6
S1
6'
3
1
, 85%
7
3c R = F, 80%
C-N coupling
R2
HN
1
5'
3d R = OMe, 80%
1
'
2
4
'
3e R = OMe, 83%
R1
3' R3 3f R = OMe, 85%
3
2
'
R2
i) CuI (20 mol%), N, N-dimethylglycine (30mol%), Cs
2 3
CO (4 equiv.),dry dioxane, 110 ºC, 18h.
ii) Pd(OAc) (6mol%) BINAP (8mol%), Cs CO (2 equiv.), dry toluene, 100 ºC, 2h.
2
,
2
3
Scheme 1. Synthesis of di(hetero)arylethers 2aef and di(hetero)arylamines 3aef by copper (CeO) and palladium (CeN) catalyzed couplings, respectively.
compounds on the cell cycle profile of the NCI-H460 cells were
observed and compound Vb was found to significantly induce
apoptosis at its GI50 in this cell line [4]. Recently we have prepared
methoxylated di(hetero)arylethers functionalizing the 6-position
of the same thieno[3,2-b]pyridine moiety. The compounds with a
methoxy group in ortho (VIa) or meta (VIb) relative to the ether
Several N-(1H-indol-5-yl)-2-arylthieno[3,2-b]pyridin-7-amines
[8], aminothieno[3,2-c]pyridine 1,3-diarylureas [9], thieno[3,2-b]
pyridine arylethers phenylacetylthioureas [10], thieno[3,2-b]pyri-
dine arylethers N -arylmalonamides [11], and N-(3-fluoro-4-(2-
arylthieno[3,2-b]pyridin-7-yloxy)phenyl)-2-oxo-3-
phenylimidazolidine-1-carboxamides [12] have been previously
described as inhibitors of the vascular endothelial growth factor
receptor (VEGFR-2), a mediator of the biological function of the
vascular endothelial growth factor (VEGF), related to angiogenesis
and metastasis.
3
function showed very low GI50 values (1e2.5
mM). These com-
pounds are fluorescent and have been encapsulated into nano-
liposomes for drug delivery purposes [5]. In another study,
compound Vd showed a very low GI50 value (1.2 mM) in the HepG2
hepatocellular carcinoma cells with no hepatotoxicity to porcine
liver primary cells. Effects of this compound on the cell cycle of
HepG2 were verified [6]. By functionalizing the amino group of the
In the present study, new fluorinated and methoxylated di(he-
tero)arylethers and di(hetero)arylamines were prepared by func-
tionalizing the 7-position of the thieno[3,2-b]pyridine, using
copper (CeO) or palladium (CeN) catalyzed couplings, respectively.
The compounds were evaluated for their growth inhibitory activity
towards human tumor cell lines, and the most active ones were
further studied regarding their effects on NCI-H460 cell cycle pro-
file and programmed cell death. The hepatotoxicity of the com-
pounds was assessed using non-tumor porcine liver primary cells.
3
-aminothieno[3,2-b]pyridine-2-carboxylate, several amino dia-
rylamines were obtained. Among them, compound VII was the
most active presenting very low GI50 values (1 M) for all the cell
m
lines referred above including HepG2, without hepatotoxicity to
porcine liver primary cells. Effects on the cell cycle profile of the
NCI-H460 cells treated with compound VII were observed [7].
2
. Results and discussion
NH2
N
NH2
N
2.1. Synthesis
R1
CO Me
2
S
N
H
S
The reaction of the 7-bromothieno[3,2-b]pyridine (1), also
prepared by us from the commercial thieno[3,2-b]pyridin-7-ol and
POBr , with methoxy or fluorophenols and anilines gave rise in
3
moderate to good yields to the new di(hetero)arylethers 2aef, by a
copper-catalyzed (CeO) Ullmann coupling with N,N-dimethyl
glycine as the ligand [13], previously used by us in the synthesis of
other di(hetero)arylethers [5], and in good to high yields to the new
di(hetero)arylamines 3aef by a palladium-catalyzed (CeN) Buch-
waldeHartwig coupling [14] with BINAP as the ligand, usually
performed in our group [2] (Scheme 1).
S
O
N
II
I
S
NH2
R1 = H, o-OMe, m-OMe, m-F
or di-o and m-OMe
N
NH2
CO Me
2
S
NH2
N
IV
NH2
CO Me
CO Me
2
N
S
2
S
R2
S
O
III
R1 VI a) R
1
2
NH2
= OMe, R = OMe
S
N
b) R = H, R2 = OMe
1
2.2. Growth inhibitory activity on human tumor cell lines and on
porcine liver primary cells
CO Me
R1
2
S
H N
R2
R3
2
OMe
The tumor cell growth inhibitory activity of the di(hetero)ary-
lethers 2aef and di(hetero)arylamines 3aef was evaluated in five
human tumor cell lines: MCF-7 (breast adenocarcinoma), NCI-H460
(non-small cell lung carcinoma), HCT15 (colon adenocarcinoma),
HepG2 (hepatocellularcarcinoma) and HeLa (cervical carcinoma)
using the sulforhodamine B assay as previously described [2e7].
V a) R , R = H, R3 =OMe
1
2
HN
1
2
3
N
b) R = NH , R = R = H
VII
2
1
2
3
c) R , R = H, R = NH
CO Me
2
2
1
3
2
S
d) R , R = H, R = NH2
Structures of compounds I-VII
This allowed the determination of the GI50 values (mM),

M.-J.R.P. Queiroz et al. / European Journal of Medicinal Chemistry 69 (2013) 855e862
857
corresponding to the concentration of the compounds which
2.3. Effects on cell cycle profile and programmed cell death of NCI-
H460 cells
inhibited 50% of cell growth (Table 1). Furthermore, the toxicity of
the compounds towards non-tumor cells was studied using porcine
liver primary cells (PLP2).
From the results obtained (Table 1) it is possible to establish
some structureeactivity relationships. Among the di(hetero)ary-
lethers 2aef, compound 2e, m-methoxylated, showed the lowest
Treatment of NCI-H460 cells with the GI50 concentration of
compounds 2e, 3d and 3e showed that all the compounds caused
pronounced alterations in the cell cycle profile of these cells (Fig. 1).
In addition, a shoulder in the left of the G1 peak was evident
following treatment with all the compounds, which was indicative
of a sub-G1 peak and suggestive of cell death.
GI50 values in the different tumor cell lines (0.39e3.14
mM), in
particular in the HCT15 cell line (0.39 M). The other methoxylated
m
compounds, 2d and 2f, were not very efficient inhibitors of cell
growth. The fluorinated compounds 2aec presented no significant
growth inhibitory activity against the five cell lines studied, pre-
senting high GI50 values.
The di(hetero)arylethers 2a and 2f presented GI50 values in the
PLP2 cells similar to the GI50 values obtained in most of the human
tumor cell lines (except in the HepG2 and in the HeLa cells, in which
the GI50 values were lower than in the primary cells). Nonetheless,
the di(hetero)arylethers 2bee presented lower GI50 values in the
tumor cell lines than in the PLP2.
This was further confirmed with the TUNEL assay. Indeed, a
strong and significant increase in programmed cell death was
observed following 24 h and 48 h of treatment with the compounds
(Fig. 2). When cells were treated with the compounds for 24 h,
compounds 3d and 3e caused a stronger increase than compound
2e in the levels of cell death. However, when cells were treated for
48 h all compounds caused similar effects in the levels of cell death,
which was higher than 30%.
3. Conclusions
Regarding the di(hetero)arylamines 3aef, the most active
compounds are 3d and 3e. These compounds, with o-methoxy or
m-methoxy groups relative to the NH, presented the lowest GI50
values in the tumor cell lines studied but also presented low GI50
values in the non-tumor PLP2 cells. Nevertheless for compound 3d,
The fluorinated and methoxylated di(hetero)arylethers and
di(hetero)arylamines were prepared by copper (CeO) or palla-
dium (CeN) catalyzed couplings of 7-bromothieno[3,2-b]pyri-
dine with ortho, meta and para fluoro or methoxy phenols or
anilines in moderate to high yields, after chromatographic pu-
rification and were fully characterized. The compounds obtained
were evaluated for their growth inhibitory activity on several
human tumor cell lines. The most active compounds, were
shown to be a di(hetero)arylether with a methoxy group in the
meta position relative to the ether function and two di(hetero)
arylamines with a methoxy group either in the ortho or in the
meta position relative to the NH. These were further tested at
their GI50 concentrations on NCI-H460 cells causing pronounced
alterations in the cell cycle profile and a strong and significant
increase in the programmed death of these cells. The fluorinated
and the other methoxylated compounds presented high
GI50 values in all the cell lines tested. Furthermore, the hepa-
totoxicity of the compounds was assessed using porcine
liver primary cells (PLP2). The results showed that one of the
most active compounds, the di(hetero)arylether 2e, was
nontoxic to non-tumor cells at their GI50 concentrations, com-
pound 2e being the most promising antitumoral. The hepato-
toxicity of the two di(hetero)arylamines 3d and 3e needs to be
further studied.
the GI50 for the HeLa cell line (1.40
the GI50 (6.56 M) for the PLP2 cells. Despite this, the GI50 values for
this compound in the other cell lines, except for HepG2, were also
lower (5.40e5.91 M) than the GI50 value for PLP2, but they are
mM) was very much lower than
m
m
very much closer. Also, for compound 3e, which was the most
active compound, the GI50 determined for most of the tumor cell
lines (0.09e0.31
mM), with the exception of the HepG2 cells
(
5.02 M), were slightly lower than the GI50 in PLP2 cells (1.94 m
m
M).
The fluorinated compounds 3aec presented much higher GI50
values than the methoxylated ones, for the inhibitory growth ac-
tivity in the tumor cell lines studied and presented higher GI50
values in the PLP2 cells than in most of the tumor cell lines studied.
On the other hand, the p-methoxylated compound 3f presented
similar GI50 values in the tumor cells and primary cells.
The most active compounds in the tumor cell lines, 2e, 3d and
3
e, were chosen to be further studied regarding their effect on cell
cycle profile and programmed cell death on one of the most sen-
sitive cell lines, NCI-H460. This cell line was also chosen since it
represents non-small cell lung cancer, one of the cancers which
currently cause most deaths worldwide.
Table 1
Growth inhibitory activity of the synthesized di(hetero)arylethers 2aef and di(hetero)arylamines 3aef on various human tumor cell lines and in non-tumor porcine liver
primary cells (PLP2).
GI50 (uM)a
HepG2
HeLa
MCF-7
NCI-H460
HCT15
PLP2
2
2
2
2
2
2
3
3
3
3
3
3
a
b
c
d
e
f
a
b
c
d
e
f
16.43 ꢀ 0.74
42.65 ꢀ 3.12
45.10 ꢀ 1.97
62.68 ꢀ 4.22
2.70 ꢀ 0.41
53.24 ꢀ 4.65
25.8 ꢀ 2.81
32.02 ꢀ 2.33
26.26 ꢀ 2.56
18.00 ꢀ 1.29
5.02 ꢀ 0.06
22.08 ꢀ 2.11
5.38 ꢀ 1.11
50.26 ꢀ 2.47
34.51 ꢀ 4.09
33.83 ꢀ 2.30
28.94 ꢀ 7.03
3.14 ꢀ 0.12
49.4 ꢀ 1.52
26.37 ꢀ 2.93
69.24 ꢀ 5.18
77.49 ꢀ 3.83
1.40 ꢀ 0.06
0.09 ꢀ 0.00
20.42 ꢀ 2.24
3.28 ꢀ 0.67
74.29 ꢀ 1.98
101.83 ꢀ 8.40
98.48 ꢀ 7.52
90.89 ꢀ 5.01
1.56 ꢀ 0.23
93.31 ꢀ 4.86
94.37 ꢀ 4.26
63.71 ꢀ 3.78
80.74 ꢀ 6.01
5.88 ꢀ 0.86
0.25 ꢀ 0.03
20.20 ꢀ 1.31
4.32 ꢀ 1.03
77.96 ꢀ 3.98
102.65 ꢀ 8.54
100.60 ꢀ 5.90
94.17 ꢀ 4.47
1.04 ꢀ 0.04
97.22 ꢀ 4.48
103.47 ꢀ 1.61
88.08 ꢀ 4.91
91.15 ꢀ 5.21
0.39 ꢀ 0.03
86.05 ꢀ 4.24
86.65 ꢀ 6.04
70.65 ꢀ 5.67
91.16 ꢀ 5.34
5.91 ꢀ 0.21
0.31 ꢀ 0.04
25.92 ꢀ 1.83
1.91 ꢀ 0.06
87.24 ꢀ 4.29
>125
>125
>125
26.65 ꢀ 1.98
94.45 ꢀ 6.09
114.91 ꢀ 6.92
119.46 ꢀ 5.97
100.41 ꢀ 4.41
6.56 ꢀ 0.2
1.94 ꢀ 0.16
22.73 ꢀ 2.02
2.06 ꢀ 0.03
86.77 ꢀ 5.39
79.51 ꢀ 1.94
77.47 ꢀ 2.50
78.46 ꢀ 6.56
5.40 ꢀ 0.89
0.28 ꢀ 0.03
19.44 ꢀ 1.44
5.77 ꢀ 0.95
Ellipticine
a
GI50 values correspond to the compound concentration which inhibited 50% of cell growth. Results are from three independent experiments (performed in triplicate), and
are expressed as mean ꢀ standard deviation (SD).

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M.-J.R.P. Queiroz et al. / European Journal of Medicinal Chemistry 69 (2013) 855e862
859
Gel 60, 230e400 mesh and the dry flash on silica was performed on
Panreac, Silica Gel 60, 70e230 mesh. Ether refers to diethylether.
ꢁ
Petroleum ether refers to the boiling range 40e60 C.
4.1.1. 7-Bromothieno[3,2-b]pyridine (1)
From thieno[3,2-b]pyridin-7-ol (300 mg, 2.00 mmol) and
ꢁ
POBr
3
(2.80 g, 10.0 mmol) and the mixture was heated at 65 C for
6
h. After cooling, NaOH (aq) (5 mL), water (5 mL) and chloroform
5 mL) were added. The phases were separated and the aqueous
phase was extracted with more chloroform (2 ꢂ 5 mL). The
organic phase was dried (MgSO ) and filtered. Removal of the
solvent gave compound 1as a yellow solid (363 mg, 85%), m.p.
(
4
ꢁ
1
6
7e68 C. H NMR (400 MHz, CDCl
3
):
d
7.46 (1H, d, J ¼ 5.2 Hz, 6-
H), 7.67 (1H, d, J ¼ 5.6 Hz, HetAreH), 7.83 (1H, d, J ¼ 5.6 Hz,
13
HetAreH), 8.51 (1H, d, J ¼ 5.2 Hz, 5-H) ppm. C NMR (100.6 MHz,
CDCl ): 121.7 (6-CH), 125.9 (CH), 126.9 (C), 131.4 (CH), 135.7 (C),
47.5 (5-CH), 156.4 (C) ppm. HRMS (EI-TOF): calcd for C
3
d
7
9
1
[
7
H
4
BrNS
þ
81
þ
M] 212.9248. Found 212.9248. Calcd for C H BrNS [M]
7 4
214.9227. Found 214.9227.
4.1.2. General procedure for the synthesis of di(hetero)arylethers
2aef
A dry Schlenk tube was charged under Ar with dry dioxane
(3 mL), the fluoro or methoxyphenol (1.1 equiv.), N,N-dimethyl
glycine (30 mol%), CuI (20 mol%), Cs
2
CO
3
(2 equiv.) and compound
ꢁ
1. The mixture was heated with stirring under Ar at 100 C for 18 h.
After cooling water (5 mL) and ethyl acetate (5 mL) were added. The
phases were separated and the aqueous phase was extracted with
more ethyl acetate (2 ꢂ 5 mL). The organic phase was dried
4
(MgSO ) and filtered. Removal of the solvent gave an oil which was
submitted to column chromatography.
4.1.2.1. 7-(2-Flurophenoxy)thieno[3,2-b]pyridine (2a).
Compound 1 (100 mg, 0.470 mmol), 2-fluorophenol, and after pu-
rification by column chromatography using a solvent gradient from
5
0% to 65% ether/petroleum ether, compound 2a was obtained as a
1
3
white oil (81.0 mg, 70%). H NMR (400 MHz, CDCl ): d 6.54 (1H, d,
Fig. 2. Analysis of programmed cell death of NCI-H460 cells treated with the com-
pounds. Cells were treated for 24 h (A) or 48 h (B) with medium, DMSO, or the GI50
concentrations of compounds 2e, 3d and 3e. Levels of programmed cell death were
determined with the TUNEL assay. Results are the mean ꢀ SEM of three independent
experiments. Statistical significance was tested by two tailed paired Student’s t-Test
using DMSO as a negative control. * Indicates p < 0.05.
J ¼ 4.8 Hz, 6-H), 7.22e7.33 (4H, m, AreH), 7.64 (1H, d, J ¼ 5.6 Hz,
HetAreH), 7.80 (1H, d, J ¼ 5.6 Hz, HetAreH), 8.54 (1H, br s, 5-H) ppm.
1
3
0
C NMR (100.6 MHz, CDCl
3
):
d
103.2 (6-CH), 117.5 (d, J ¼ 18.1 Hz, 3 -
0
CH), 122.4 (C), 123.6 (CH), 124.9 (CH), 125.2 (d, J ¼ 4.0 Hz, 4 -CH),
0
0
1
1
27.4 (d, J ¼ 7.0 Hz, 6 -CH), 131.3 (CH), 140.6 (d, J ¼ 12.1 Hz, 1 -C),
48.6 (5-CH), 153.3 (C), 157.0 (d, J ¼ 251.5 Hz, CF), 160.1 (C) ppm.
þ
HRMS (EI-TOF): calcd for
245.0305.
13 8
C H FNOS [M ] 245.0311. Found
4
. Materials and methods
4
.1. Chemistry
4.1.2.2. 7-(3-Flurophenoxy)thieno[3,2-b]pyridine (2b). From com-
pound 1 (100 mg, 0.470 mmol), 3-fluorophenol, and after purifi-
cation by column chromatography using a solvent gradient from
ꢁ
Melting points ( C) were determined in a Stuart SMP3 and are
1
13
uncorrected. H and C NMR spectra were recorded on a Varian
Unity Plus at 300 and 75.4 MHz, respectively or on a Bruker Avance
III at 400 and 100.6 MHz, respectively. Two dimensional He
correlations were performed to attribute some signals. Mass
spectra (MS) EI-TOF or ESI-TOF and HRMS on the M or on the
30% to 50% ether/petroleum ether, compound 2b was obtained as a
1
white oil (72.0 mg, 63%). H NMR (400 MHz, CDCl
3
):
d
6.64 (1H, d,
1
13
C
J ¼ 5.2 Hz, 6-H), 6.92e7.04 (3H, m, AreH), 7.39e7.44 (1H, m, AreH),
7.60 (1H, d, J ¼ 5.6 Hz, HetAreH), 7.77 (1H, d, J ¼ 5.6 Hz, HetAreH),
þ
13
8.56 (1H, br s, 5-H) ppm. C NMR (100.6 MHz, CDCl
3
):
d
104.7 (6-
þ
[
M þ H] were performed by the mass spectrometry service of the
CH), 108.7 (d, J ¼ 24.1 Hz, CH), 112.8 (d, J ¼ 21.1 Hz, CH), 116.4 (d,
0
University of Vigo, C.A.C.T.I., Spain. The reactions were monitored
by thin layer chromatography (TLC) using MachereyeNagel pre-
coated aluminum silica gel 60 sheets (0.20 mm) with UV254 indi-
cator. Column chromatography was performed on Panreac, Silica
J ¼ 3.0 Hz, 6 -CH), 123.0 (C), 125.2 (CH), 130.9 (CH), 131.0 (d,
0
0
J ¼ 10.0 Hz, 5 -CH),149.0 (5-CH),154.9 (d, J ¼ 11.1 Hz,1 -C),158.9 (C),
159.6 (C), 163.4 (d, J ¼ 248.4 Hz, CF) ppm. HRMS (EI-TOF): calcd for
þ
C
13
H
8
FNOS [M ] 245.0311. Found 245.0313.
Fig. 1. Analysis of cell cycle profile by flow cytometry of NCI-H460 cells treated with the compounds. Cells were treated for 24 h with medium (A), DMSO (B), or the GI50 con-
centrations of compounds 2e (C), 3d (D) or 3e (E). The left panel corresponds to dot plots of forward versus side scatter (FSC versus SSC) and shows the gated population. In the right
panel, the histograms indicate the cell cycle profile of the gated population following the exclusion of cellular aggregates and debris. All images are representative of three in-
dependent experiments.

860
M.-J.R.P. Queiroz et al. / European Journal of Medicinal Chemistry 69 (2013) 855e862
4
.1.2.3. 7-(4-Flurophenoxy)thieno[3,2-b]pyridine (2c). From com-
filtered. Removal of the solvent gave a solid which was submitted to
a dry flash in silica using ether or AcOEt and a solid was obtained.
pound 1 (100 mg, 0.470 mmol) and 4-fluorophenol, and after pu-
rification by column chromatography using a solvent gradient from
4
0% to 60% ether/petroleum ether, compound 2c was obtained as a
4.1.3.1. N-(2-Fluorophenyl)thieno[3,2-b]pyridin-7-amine
From compound 1 (200 mg, 0.940 mmol) and 2-fluoroaniline,
compound 3a was obtained as a yellow solid (148 mg, 65%), m.p.
(3a).
1
white oil (75.0 mg, 65%). H NMR (400 MHz, CDCl
3
):
d
6.54 (1H, d,
J ¼ 5.2 Hz, 6-H), 7.13e7.20 (4H, m, AreH), 7.60 (1H, d, J ¼ 5.6 Hz,
ꢁ
1
HetAreH), 7.76 (1H, d, J ¼ 5.6 Hz, HetAreH), 8.52 (1H, br s, 5-H)
134e135 C. H NMR (400 MHz, DMSO-d
6
):
d
6.43 (1H, d, J ¼ 5.2 Hz,
13
ppm. C NMR (100.6 MHz, CDCl
3
):
d
103.9 (6-CH), 116.9 (d,
6-H), 7.24e7.37 (4H, m, AreH), 7.42 (1H, d, J ¼ 5.6 Hz, HetAreH),
0
0
0
0
J ¼ 23.1 Hz, 3 and 5 -CH), 122.6 (d, J ¼ 9.0 Hz, 2 and 6 -CH), 124.9
7.95 (1H, d, J ¼ 5.6 Hz, HetAreH), 8.26 (1H, d, J ¼ 5.2 Hz, 5-H), 8.76
0
13
(
1
C), 125.2 (CH), 130.9 (CH), 148.9 (5-CH), 149.5 (d, J ¼ 3.0 Hz, 1 -C),
(1H, br s, NH) ppm. C NMR (100.6 MHz, DMSO-d
6
):
d
102.4 (6-CH),
0
58.8 (C), 160.2 (d, J ¼ 244.5 Hz, CF), 160.5 (C) ppm. HRMS (EI-TOF):
116.4 (d, J ¼ 20.0 Hz, 3 -CH), 119.1 (C), 124.9 (d, J ¼ 6.0 Hz, CH), 125.0
þ
0
calcd for C13
H
8
FNOS [M ] 245.0311. Found 245.0314.
(CH), 126.9 (d, J ¼ 8 Hz, CH), 127.2 (d, J ¼ 12.0 Hz, 1 -C), 127.50 (CH),
1
30.1 (CH), 146.8 (C), 148.01 (5-CH), 157.4 (C), 156.7 (d, J ¼ 246.5 Hz,
þ
9 2
H FN S [M ] 244.0470. Found
4.1.2.4. 7-(2-Methoxyphenoxy)thieno[3,2-b]pyridine
(2d). From
CF) ppm. HRMS (EI-TOF): calcd for C13
244.0473.
compound 1 (140 mg, 0.488 mmol), 2-methoxyphenol, and after
purification by column chromatography using a solvent gradient
from 25% ether/petroleum ether to 30% ether/petroleum ether,
4.1.3.2. N-(3-Fluorophenyl)thieno[3,2-b]pyridin-7-amine
(3b).
1
compound 2d was obtained as a yellow oil (55.0 mg, 50%). H NMR
From compound 1 (200 mg, 0.940 mmol) and 3-fluoroaniline,
compound 3b was obtained as a yellow solid (180 mg, 83%), m.p.
(
300 MHz, CDCl
.02e7.07 (2H, m, AreH), 7.19e7.22 (1H, m, AreH), 7.27e7.29 (1H,
m, AreH), 7.57 (1H, d, J ¼ 5.4 Hz, HetAreH), 7.73 (1H, d, J ¼ 5.4 Hz,
3
):
d
3.76 (3H, s, OMe), 6.43 (1H, d, J ¼ 5.7 Hz, 6-H),
ꢁ
1
7
186e187 C. H NMR (400 MHz, DMSO-d ): d 6.86e6.89 (1H, m, Are
6
H), 7.00 (1H, d, J ¼ 5.2 Hz, 6-H), 7.06e7.13 (2H, m, AreH), 7.37e7.39
(1H, m, AreH), 7.46 (1H, d, J ¼ 5.6 Hz, HetAreH), 8.00 (1H, d,
J ¼ 5.6 Hz, HetAreH), 8.36 (1H, d, J ¼ 5.2 Hz, 5-H), 9.00 (1H, br s, NH)
13
HetAreH), 8.46 (1H, d, J ¼ 5.7 Hz, 5-H) ppm. C NMR (75.4 MHz,
CDCl ): 55.8 (OMe), 103.3 (6-CH), 113.0 (CH), 121.2 (CH), 121.9 (C),
23.00 (CH), 125.1 (CH), 127.1 (CH), 130.5 (CH), 141.9 (C), 148.9 (5-
3
d
13
1
ppm. C NMR (100.6 MHz, DMSO-d
6
):
d
103.2 (6-CH), 107.6 (d,
0
CH), 151.8 (C), 158.6 (C), 160.4 (C) ppm. HRMS (EI-TOF): calcd for
C
J ¼ 24.0 Hz, CH), 109.4 (d, J ¼ 22.0 Hz, CH), 116.7 (d, J ¼ 2.0 Hz, 6 -
þ
0
14
H
11NO
2
S: [M ] 257.0511. Found 257.0513.
CH), 120.8 (C), 125.1 (CH), 130.3 (CH), 130.7 (d, J ¼ 10.0 Hz, 5 -CH),
0
1
42.4 (d, J ¼ 10 Hz, 1 -C), 145.2 (C), 148.3 (5-CH), 157.7 (C), 162.6 (d,
þ
4.1.2.5. 7-(3-Methoxyphenoxy)thieno[3,2-b]pyridine
(2e). From
J ¼ 244.0 Hz, CF) ppm. HRMS (EI-TOF): calcd for C13
H
9
FN
2
S [M ]
compound 1 (140 mg, 0.488 mmol) and 3-methoxyphenol, and
after purification by column chromatography using a solvent
gradient from 30% ether/petroleum ether to 70% ether/petroleum
ether, compound 2ewas obtained as a yellow oil (61.0 mg, 55%). H
NMR (400 MHz, CDCl
244.0470. Found 244.0470.
4.1.3.3. N-(4-Fluorophenyl)thieno[3,2-b]pyridin-7-amine
From compound 1 (200 mg, 0.940 mmol) and 4-fluoroaniline,
compound 3c was obtained a yellow solid (180 mg, 80%), m.p. 184e
(3c).
1
3
):
d
3.82 (3H, s, OMe), 6.62 (1H, d, J ¼ 5.6 Hz,
0
ꢁ
1
6
-H), 6.75 (1H, apparent t, J ¼ 2.4 Hz, 2 -H), 6.77e6.79 (1H, m, Are
185 C. H NMR (400 MHz, DMSO-d
6
):
d
6.75 (1H, d, J ¼ 5.2 Hz, 6-H),
0
0
0
0
0
H), 6.83e6.86 (1H, m, AreH), 7.34 (1H, apparent t, J ¼ 8.4 Hz, 5 -H),
7.20e7.24 (2H, m, 3 and 5 -H), 7.29e7.32 (2H, m, 2 and 6 -H), 7.42
(1H, d, J ¼ 5.6 Hz, HetAreH), 7.96 (1H, d, J ¼ 5.6 Hz, HetAreH), 8.27
7.58 (1H, d, J ¼ 5.2 Hz, HetAreH), 7.74 (1H, d, J ¼ 5.2 Hz, HetAreH),
8
d
1
3
13
.51 (1H, d, J ¼ 5.6 Hz, 5-H) ppm. C NMR (100.6 MHz, CDCl
3
):
(1H, d, J ¼ 5.2 Hz, 5-H), 8.78 (1H, br s, NH) ppm. C NMR
0
0
55.5 (OMe), 104.4 (6-CH), 106.8 (2 -CH), 111.6 (CH), 112.9 (CH),
(100.6 MHz, DMSO-d
6
):
d
101.6 (6-CH), 115.9 (d, J ¼ 22.0 Hz, 3 and
0
0
0
0
122.8 (C), 125.2 (CH), 130.5 (5 -CH), 130.6 (CH), 149.1 (5-CH), 154.8
5 -CH), 119.5 (C), 124.7 (d, J ¼ 8.0 Hz, 2 and 6 -CH), 125.1 (CH), 130.0
0
(
C), 158.9 (C), 160.1 (C), 161.1 (C) ppm. HRMS (EI-TOF): calcd for
(CH), 136.2 (d, J ¼ 3 Hz, 1 -C), 146.5 (C), 148.1 (5-CH), 157.5 (C), 158.6
þ
þ
C
14
H
11NO
2
S: [M ] 257.0511. Found 257.0509.
(d, J ¼ 240.4 Hz, CF) ppm. HRMS (EI-TOF): calcd for C13
H
9
FN
2
S [M ]
2
44.0470. Found 244.0474.
4.1.2.6. 7-(4-Methoxyphenoxy)thieno[3,2-b]pyridine
(2f). From
compound 1 (140 mg, 0.488 mmol) and 4-methoxyphenol, and
after purification by column chromatography using a solvent
gradient from 30% ether/petroleum ether to 70% ether/petroleum
ether, compound 2f was obtained as a yellow solid (50.0 mg, 45%),
4.1.3.4. N-(2-Methoxyphenyl)thieno[3,2-b]pyridin-7-amine
(3d).
From compound 1 (100 mg, 0.470 mmol) and 2-methoxyaniline,
compound 3d was obtained a yellow solid (95.0 mg, 80%), m.p.
ꢁ
1
6
130e131 C. H NMR (400 MHz, DMSO-d ): d 3.73 (s, 3H, OMe), 6.37
ꢁ
1
m.p. 70.5e72.1 C. H NMR (400 MHz, CDCl
3
):
d
3.86 (3H, s, OMe),
(1H, d, J ¼ 5.2 Hz, 6-H), 6.97e7.01 (1H, m, AreH), 7.12e7.14 (1H, m,
0
0
6
.53 (1H, d, J ¼ 5.2 Hz, 6-H), 6.98 (2H, d, J ¼ 9.2 Hz, 3 and 5 -H), 7.13
AreH), 7.22e7.29 (2H, m, 2ꢂ AreH), 7.38 (1H, d, J ¼ 5.6 Hz, HetAre
0
0
(
(
2H, d, J ¼ 9.2 Hz, 2 and 6 -H), 7.59 (1H, d, J ¼ 5.6 Hz, HetAreH), 7.75
H), 7.91 (1H, d, J ¼ 5.6 Hz, HetAreH), 8.20 (1H, br s, 5-H), 8.43 (1H,
13
13
1H, d, J ¼ 5.6 Hz, HetAreH), 8.49 (1H, d, J ¼ 5.2 Hz, 5-H) ppm.
C
br s, NH) ppm. C NMR (100.6 MHz, DMSO-d
6
):
d
55.4 (OMe), 102.2
0
NMR (100.6 MHz, CDCl
3
):
d
55.6 (OMe), 103.6 (6-CH), 115.1 (3 and
(6-CH),112.2 (CH),118.6 (C),120.6 (CH),124.6 (CH),126.9 (CH),127.0
(CH), 127.53 (C), 130.17 (CH), 147.29 (5-CH), 147.85 (C), 154.27 (C),
0
0
0
5
-CH), 122.2 (2 and 6 -CH), 122.5 (C), 125.1 (CH),130.7 (CH), 147.0
þ
(
C), 148.9 (5-CH), 157.4 (C), 158.5 (C), 161.1 (C) ppm. HRMS (EI-TOF):
156.69 (C) ppm. HRMS (EI-TOF): calcd for C14
256.0670. Found 256.0675.
H
12
N
2
OS [M ]
þ
calcd for C14
H11NO
2
S: [M ] 257.0511. Found 257.0512.
4
3
.1.3. General procedure for the synthesis of di(hetero)arylamines
aef
A dry Schlenk tube was charged under Ar with dry toluene
3 mL), the fluoro or methoxyanilines (1.1 equiv.), Pd(OAc) (6 mol
), BINAP (8 mol%), Cs CO (2 equiv.) and compound 1. The mixture
4.1.3.5. N-(3-Methoxyphenyl)thieno[3,2-b]pyridin-7-amine
From compound 1 (100 mg, 0.470 mmol) and 3-methoxyaniline,
compound 3e was obtained a yellow solid (99.0 mg, 83%), m.p.
(3e).
ꢁ
1
(
%
2
146e147 C. H NMR (400 MHz, CDCl
3
):
d
3.82 (3H, s, OMe), 6.67
0
0
2
3
(1H, br s, NH), 6.74e6.77 (1H, m, 4 -H), 6.84e6.85 (1H, m, 2 -H),
ꢁ
0
was heated with stirring under Ar at 100 C for 2 h. After cooling
water (5 mL) and ethyl acetate (5 mL) were added. The phases were
separated and the aqueous phase was extracted with more ethyl
acetate (2 ꢂ 5 mL). The organic phase was dried (MgSO
6.87e6.89 (1H, m, 6 -H), 6.98 (1H, d, J ¼ 5.2 Hz, 6-H), 7.31 (1H,
0
apparent, J ¼ 8.4 Hz, 5 -H), 7.55 (1H, d, J ¼ 5.6 Hz, HetAreH), 7.65
13
(1H, d, J ¼ 5.6 Hz, HetAreH), 8.39 (1H, d, J ¼ 5.2 Hz, 5-H), ppm.
C
0
4
) and
NMR (100.6 MHz, CDCl
3
):
d
55.4 (OMe), 102.4 (6-CH), 108.5 (2 -CH),

M.-J.R.P. Queiroz et al. / European Journal of Medicinal Chemistry 69 (2013) 855e862
861
0
0
1
(
10.3 (4 -CH), 114.9 (6 -CH), 120.7 (C), 125.5 (CH), 128.8 (CH), 130.3
30 min on ice [15]. Cellular DNA content was analyzed using a FACS
Calibur (BD) flow cytometer. Cell cycle profile was subsequently
analyzed using the Flow Jo 7.6.5 software (Tree Star, Inc., Ashland,
OR, USA) after cell debris and aggregates exclusion [4,7].
0
5 -CH), 140.20 (C), 146.7 (C), 147.52 (5-CH), 156.5 (C), 160.63 (C)
ppm. HRMS (EI-TOF): calcd for C14
56.0678.
þ
H
12
N
2
OS [M ] 256.0670. Found
2
For programmed cell death analysis, following 24 h and 48 h
treatment cells were fixed in 4% paraformaldehyde for 30 min and
further kept in PBS at 4 C until analysis. Cytospins were then
4
.1.3.6. N-(4-Methoxyphenyl)thieno[3,2-b]pyridin-7-amine(3f).
ꢁ
From compound 1 (100 mg, 0.470 mmol) and 4-methoxyaniline,
compound 3f was obtained a yellow solid (102 mg, 85%). m.p. 184e
prepared and cells permeabilized in ice cold 0.1% Triton X-100 in
0.1% sodium citrate for 2 min. Programmed cell death was assessed
using the “in situ cell death detection kit-fluorescein” (Roche, Basel
Switzerland). Briefly, cells were incubated with the TUNEL reaction
mix (enzyme diluted 1:20) for 1 h, as previously described [16].
Slides were mounted with Vectashield Mounting Media with DAPI
(Vector Laboratories). Cells were observed in a DM2000 microscope
(LEICA) and a semi-quantitative evaluation of the levels of pro-
grammed cell death was performed by counting a minimum of 500
cells per slide, except in the cases of 2e and 3d treatment at 48 h, in
which only w300 cells were counted.
ꢁ
1
185 C. H NMR (400 MHz, DMSO-d
6
): d 3.76 (3H, s, OMe), 6.61 (1H,
0
0
d, J ¼ 5.2 Hz, 6-H), 6.97 (2H, d, J ¼ 8.8 Hz, 3 and 5 -H), 7.21 (2H, d,
0
0
J ¼ 8.8 Hz, 2 and 6 -H), 7.39 (1H, d, J ¼ 5.6 Hz, HetAreH), 8.21 (1H,
13
br s, 5-H), 8.60 (1H, br s, NH), ppm. C NMR (100.6 MHz, DMSO-d
6
):
0
0
d
55.3 (OMe),101.1 (6-CH), 114.4 (3 and 5 -CH),118.9 (C), 125.1 (CH),
0 0
125.6 (2 and 6 -CH), 129.8 (CH), 132.4 (C), 147.5 (C), 148.1 (5-CH),
þ
12 2
156.4 (C), 157.4 (C) ppm. HRMS (EI-TOF): calcd for C14H N OS [M ]
2
56.0670. Found 256.0677.
4.2. Growth inhibitory activity on human tumor cell lines and on
porcine liver primary cells
4.4. Statistical analysis
Five human tumor cell lines were used: MCF-7 (breast adeno-
carcinoma) from DSMZ (Leibniz-Institut DSMZ
e
Deutsche
Results were expressed as mean ꢀ standard deviation (SD) or
Sammlung von Mikroorganismen und Zellkulturen GmbH), NCI-
H460 (non-small cell lung carcinoma), HCT15 (colon carcinoma),
HeLa (cervical carcinoma) and HepG2 (hepatocellular carcinoma)
from ECACC (European Collection of Cell Cultures). Cells were
standard error (SEM) of at least three independent experiments,
performed in duplicate. Statistical significance was tested with a
two tailed paired Student’s t-Test in relation to control (DMSO
treatment). *p < 0.05.
ꢁ
routinely maintained as adherent cell cultures at 37 C, in a hu-
2
midified air incubator containing 5% CO . Cells were cultured in: i)
Acknowledgments
RPMI-1640 medium supplemented with 10% heat-inactivated fetal
bovine serum (FBS) and 2 mM glutamine, for the MCF-7, NCI-H460
and HCT15 cell lines and ii)DMEM supplemented with 10% FBS,
The authors would like to thank to the Foundation for the Sci-
ence and Technology (FCTePortugal) for financial support through
the NMR Portuguese network (Bruker 400 Avance III-Univ Minho);
to FCT and FEDER-COMPETE/QREN/EU for financial support
through the research unities PEst-C/QUI/UI686/2011 and PEst-OE/
AGR/UI0690/2011, the research project PTDC/QUI-QUI/111060/
2
mM glutamine,100 U/mL penicillin and 100 mg/mL streptomycin,
for HeLa and HepG2 cells). To assess the growth inhibitory activity
of the compounds, the Sulforhodamine B assay was performed
according to a procedure previously described by the authors [2e
7
]. For this, each cell line was plated at an appropriate density
2
6
009 and the post-Doctoral grants attributed to R.C.C. (SFRH/BPD/
8344/2010) and R.T.L. (SRH/BPD/68787/2010). IPATIMUP is an
3
(
7.5 ꢂ 10 cells/well for MCF-7, NCI-H460 and HCT15 cells or
4
1.0 ꢂ 10 cells/well for HeLa and HepG2 cells) in 96-well plates.
Associate Laboratory of the Portuguese Ministry of Science, Tech-
nology and Higher Education and is partially supported by FCT.
For the evaluation of toxicity towards non-tumor cells, a pri-
mary cell culture was prepared from a freshly harvested porcine
liver obtained from a local slaughter house, according to a proce-
dure previously established by some of us [6,7]; these cells were
designated as PLP2 cells. Cell culture was continued with direct
monitoring every two to three days using a phase contrast micro-
scope. Before confluence was reached, cells were sub-cultured and
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.09.023.
4
plated in 96-well plates at a density of 1.0 ꢂ 10 cells/well, and
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1
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[
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NCI-H460 cells (1 ꢂ 10 cells/well) were plated in 6-well plates
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