Novel 6-[(hetero)arylamino]thieno[3,2-b]pyridines: Synthesis and antitumoral activities

Article abstract of DOI:10.1016/j.ejmech.2010.09.030

Several novel 6-[(hetero)arylamino]thieno[3,2-b]pyridines were prepared by palladium-catalyzed C-N Buchwald-Hartwig coupling of the methyl 3-amino-6-bromothieno[3,2-b]pyridine-2-carboxylate with aryl and heteroarylamines, using different reaction conditio

Full text of DOI:10.1016/j.ejmech.2010.09.030

European Journal of Medicinal Chemistry 45 (2010) 5732e5738
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel 6-[(hetero)arylamino]thieno[3,2-b]pyridines: Synthesis and antitumoral
activities
,
a
b
b
Maria-João R.P. Queiroz a , Ricardo C. Calhelha , Luís A. Vale-Silva , Eugénia Pinto ,
*
M. São-José Nascimento b
a Centro de Química, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal
b Laboratório de Microbiologia, Centro de Estudos de Química Orgânica, Fitoquímica e Farmacologia/Centro de Química Medicinal - Faculdade de Farmácia da Universidade do Porto
(CEQOFFUP/CEQUIMED), Rua Aníbal Cunha 164, 4050-047 Porto, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
Several novel 6-[(hetero)arylamino]thieno[3,2-b]pyridines were prepared by palladium-catalyzed CeN
BuchwaldeHartwig coupling of the methyl 3-amino-6-bromothieno[3,2-b]pyridine-2-carboxylate with
aryl and heteroarylamines, using different reaction conditions. The antitumoral activity of the di(hetero)
arylamines obtained was evaluated against three representative human tumor cell lines, namely breast
adenocarcinoma (MCF-7), melanoma (A375-C5), and non-small cell lung cancer (NCI-H460) and some
structureeactivity relationships were established within each series. The most promising compounds
Received 9 March 2010
Received in revised form
31 August 2010
Accepted 1 September 2010
Available online 17 September 2010
were shown to be a benzothiazole derivative with GI50 3.5e6.9
GI50 13e21 M.
mM followed by an indole derivative with
Keywords:
m
Di(hetero)arylamines
Thieno[3,2-b]pyridines
Palladium
Ó 2010 Elsevier Masson SAS. All rights reserved.
BuchwaldeHartwig coupling
Antitumor activity
SARs
N
1. Introduction
N
R
S
N
NC
Thienopyridine derivatives have attracted much attention
because of their biological activities. 7-(Aryl)aminothieno[3,2-b]
pyridines I were shown to be potent inhibitors of the VEGFR-2
(Vascular Endothelial Growth Factor Receptor-2) which has been
S
HN
Cl
OMe
Cl
X
HN
R
X= O or NMe
N
identified as
a key component of the signaling pathway
H
responsible for the sprouting and maturation of new blood
vessels from the tumor leading to tumor growth and metastasis
[1]. Overexpression or overactivation of Src (the non-receptor
tyrosine kinase) is implicated in various diseases such as
cancer, osteoporosis and ischemic diseases. 7-[(2,4-Dichloro-
5-methoxyphenyl)amino]thieno[3,2-b]pyridine-6-carbonitriles II
with various groups at C-2 have shown to be inhibitors of Src
kinase activity [2].
I
II
Here we describe the synthesis of novel methyl 6-(heteroaryl)
aminothieno[3,2-b]pyridine-2-carboxylates by palladium-cata-
lyzed CeN BuchwaldeHartwig coupling [3] of the methyl 6-bro-
mothieno[3,2-b]pyridine-2-carboxylate (1), recently described by
us [4], with arylamines and heteroarylamines using different
conditions. All the methyl 6-[(hetero)arylamino]thieno[3,2-b]
pyridine-2-carboxylates obtained were studied as potential anti-
tumor compounds using three human tumor cell lines, represen-
tative of different types of tumors, and it was possible to establish
some structureeactivity relationships.
* 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.2010.09.030

M.-J.R.P. Queiroz et al. / European Journal of Medicinal Chemistry 45 (2010) 5732e5738
5733
Table 1
Reaction scheme and conditions, aminated components and product structures and yields.
NH2
NH2
i or ii
N
Ar
or
HetAr
N
Ar
+
or
CO2Me
CO2Me
HetAr
S
N
H
S
1
NH2
Br
2a-g and 4a-f
i) Pd(OAc)2 (16 mol%), rac-BINAP (18 mol %), Cs2CO3 (1.8 equiv.), dry toluene, 110 ºC, under Ar.
ii) Pd(OAc)2 (16 mol%), xantphos (18 mol%), Cs2CO3 (1.8 equiv.), dry dioxane, 120 oC, under Ar.
.
Aminated Component
Reaction Conditions
i, 21 h
Di(hetero)arylamine
NH2
NH2
MeO
MeO
N
CO2Me
MeO
MeO
S
N
H
2a, 60%
i, 4 h
i, 6 h
i, 21 h
NH2
N
CO2Me
NH2
S
N
H
OMe
2b, 70%
OMe
MeO
NH2
MeO
MeO
N
CO2Me
CO2Me
MeO
NH2
S
N
H
2c, 70%
OMe
OMe
NH2
N
S
MeO
MeO
N
H
MeO
MeO
NH2
2d, 50%
i, 22 h
Cl
NH3 Cl
NH2
Cl
N
CO2Me
NH2
S
N
H
2e, 48%
+
N
CO2Me
HN
S
Cl
N
CO2Me
3a, 5%
S
MeO
N
H
i, 25 h
Br
NH2
NH2
Br
N
CO2Me
NH2
S
N
H
2f, 50%
+
N
CO2Me
HN
S
Br
N
CO2Me
3b, 5%
S
N
H
(continued on next page)

5734
M.-J.R.P. Queiroz et al. / European Journal of Medicinal Chemistry 45 (2010) 5732e5738
Table 1 (continued )
Aminated Component
Reaction Conditions
i, 22 h
Di(hetero)arylamine
F
Br
NH2
F
Br
N
CO2Me
CO2Me
NH2
S
N
H
2g, 45%
ii, 4 h
N
S
NH2
N
NH2
NH2
N
S
N
H
S
4a, 54%
ii, 5 h
NH2
N
HN
N
CO2Me
H
S
N
H
4b, 53%
ii, 3 h
NH2
N
N
N
CO2Me
S
NH2
N
H
4c, 56%
NH2
ii, 4 h
N
N
NH2
N
N
CO2Me
S
N
H
4d, 45%
ii, 4 h
NH2
N
N
CO2Me
CO2Me
NH2
NH2
S
N
H
4e, 45%
ii, 2 h
NH2
N
N
N
N
S
N
N
H
4f, 55%
2. Results and discussion
dioxane as solvent, on the coupling of compound 1 with different
heteroarylamines. The corresponding 6-(heteroarylamino)thieno
[3,2-b]pyridines 4aef were obtained in good yields (Table 1, ii), thus
extended the scope of the reactivity of compound 1 in this type of
coupling. The use of BINAP as the ligand did not afford the dihe-
teroarylamines 4, so the use of Xantphos is crucial in the coupling
with the heteroarylamines used.
2.1. Chemistry
Several methyl 6-(arylamino)thieno[3,2-b]pyridine-2-carboxyl-
ates 2aeg were prepared by CeN BuchwaldeHartwig coupling of
compound
1 with different methoxylated and/or halogenated
anilines, in good yields, using the most general conditions, already
used by us in this type of coupling [5] for halogenated electron-defi-
cient rings and anilines (Table 1, i). When 2-chloro-5-methoxyaniline
hydrochloride and 2-bromoaniline were used as coupling compo-
nents the minor products 3a and 3b (Table 1), were also formed
resulting from the reaction of compound 1 with the 3-amino group of
the di(hetero)arylamines 2e and 2f previously formed, respectively.
Based in earlier experiences on CeN BuchwaldeHartwig
couplings using deactivated amines [5,6] as coupling components
we have used Xantphos as the ligand, Cs2CO3 as the base and
2.2. Biological activity
The in vitro growth inhibitory activity of all the di(hetero)aryl-
amines 2 and 4 prepared, was evaluated using three human tumor
cell lines, representing different tumor types, namely breast
adenocarcinoma (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 2. Doxorubicin was used as
a positive control.

M.-J.R.P. Queiroz et al. / European Journal of Medicinal Chemistry 45 (2010) 5732e5738
5735
Table 2
a Varian Unity Plus at 300 MHz on a Bruker Avance III at 400 MHz.
The 13C NMR spectra were measured in the same instruments at
75.4 and 100.6 MHz respectively. Mass spectra (EI) and HRMS were
performed by the mass spectrometry service of University of Vigo-
Spain.
Growth inhibitory activity of compounds 2 and 4 on the three human tumor cell
lines in study.
Compound
GI50
(
m
M)a
MCF-7
A375-C5
NCI-H460
Column chromatography was performed on MachereyeNagel
silica gel 230e400 mesh. Petroleum ether refers to the boiling
range 40e60 ꢀC. Ether refers to diethyl ether.
2a
2b
2c
2d
2e
2f
2g
4a
4b
4c
4d
4e
4f
123.6 ꢂ 6.1
26.3 ꢂ 0.8
30.0 ꢂ 3.7
>150.0
22.1 ꢂ 0.8
28.9 ꢂ 1.0
28.9 ꢂ 1.2
6.0 ꢂ 0.1
18.1 ꢂ 3.4
>150.0
>150.0
95.0 ꢂ 7.8
26.3 ꢂ 1.8
26.2 ꢂ 2.1
>150.0
21.7 ꢂ 1.6
30.9 ꢂ 1.5
31.3 ꢂ 1.8
6.4 ꢂ 0.5
15.8 ꢂ 2.5
>150.0
27.3 ꢂ 0.1
26.3 ꢂ 0.7
>150.0
25.8 ꢂ 2.2
34.1 ꢂ 2.4
34.0 ꢂ 3.3
3.5 ꢂ 0.0
17.3 ꢂ 2.8
>150.0
The ligand rac-BINAP [2,20-bis(diphenylphosphane)-1,10-binaphtyl,
97%], Xantphos [9,9-dimethyl-4,5-bis(diphenylphosphane)xanthene]
and the base Cs2CO3 were purchased from Sigma Aldrich.
4.1.1. General procedure for the synthesis of the di(hetero)
arylamines 2aeg
>150.0
>150.0
>150.0
>150.0
>150.0
A dry Schlenk tube was charged under Argon with dry toluene
(3e5 mL), the thienopyridine 1, Pd(OAc)2 (16 mol%), rac-BINAP
(18 mol%), Cs2CO3 (1.8 equiv.), the aniline (1.2 equiv) and the
mixture was heated at 110 ꢀC for several hours (Table 1). The
reactions were followed by TLC. After cooling, water (5 mL) and
ethyl acetate (5 mL) were added. The phases were separated, the
aqueous phase was extracted with more ethyl acetate (3 ꢁ 5 mL),
the organic phases were collected, dried (MgSO4) and filtered.
Removal of the solvent gave an oil which was submitted to column
chromatography using solvent gradient to isolate the products as
yellow solids after some washes with petroleum ether.
82.4 ꢂ 13.2
49.4 ꢂ 6.7
>150.0
>150.0
Doxorubicin was used as positive control (GI50: MCF-7 ¼ 43.3 ꢂ 2.6 nM; A375-
C5 ¼ 130.2 ꢂ 10.1 nM; NCI-H460 ¼ 35.6 ꢂ 1.6 nM).
a
The lowest concentrations causing 50% of cell growth inhibition (GI50) after
a continuous exposure of 48 h, expressed as means ꢂ SEM of three independent
experiments performed in duplicate.
From the analysis of Table 2 it is possible to infer that for the di
(hetero)arylamines2thepresenceof twomethoxygroupsinthe ortho
and para positions (2b) and in the meta and para positions (2c)
decreases significantly the GI50 values (25e34 mM) relatively to the
4.1.1.1. Methyl 3-amino-6-(4-methoxyphenylamino)thieno[3,2-b]pyri-
dine-2-carboxylate (2a). From compound 1 (150 mg, 0.540 mmol)
and 4-methoxyaniline (74.0 mg, 0.648 mmol), using solvent gradient
in the column chromatography from 50% ether/petroleum ether
till neat ether, compound 2a was isolated (100 mg, 60%),
monomethoxylated 2a and to the dimethoxylated with two methoxy
groups in the meta positions (2d). For the ortho-halogenated
compounds (2eeg) the GI50 values are comparable to those obtained
for compounds 2b and 2c, but are slightly lower for the chlorinated
compound bearing also a methoxy group (2e) (21e28
mM). The
m.p.199e201 ꢀC 1H NMR (CDCl3, 400 MHz):
d 3.84 (3H, s, OMe), 3.88
presenceofa fluorineatom in compound 2ggave identical GI50 values
to the corresponding unsubstituted ortho-brominated compound 2f
(28e37 mM).
The most active compounds are two of the diheteroarylamines
4. The benzothiazole derivative 4a presents the lowest GI50 values
(3H, s, OMe), 5.82 (1H, br s, NH), 6.13 (2H, br s, NH2), 6.94 (2H, d,
J ¼ 8.8 Hz, 30 and 50-H), 7.15 (2H, d, J ¼ 8.8 Hz, 20 and 60-H), 7.39 (1H,
d, J ¼ 2.8 Hz, HetAr-H), 8.23 (1H, br s, HetAr-H) ppm. 13C NMR (CDCl3,
100.6 MHz):
d
50.93 (OMe), 55.11 (OMe), 111.51 (CH), 114.57 (30and
50-CH), 123.51 (20 and 60-CH), 132.64 (C), 135.65 (C), 137.21 (CH),
138.59 (C), 141.40 (C), 147.51 (C), 156.27 (C), 165.05 (C]O), ppm. MS
(EI): m/z (%) 329 (Mþ, 36), 257 (100). HRMS Mþ calct. for
C16H15N3O3S 329.0834; found 329.0835.
(3.5e6.9 mM) and the indole derivative 4b is the second most active
with GI50 values 13e21 mM.
3. Conclusions
4.1.1.2. Methyl 3-amino-6-(2,4-dimethoxyphenylamino)thieno[3,2-b]
pyridine-2-carboxylate(2b). From compound 1 (150 mg, 0.540 mmol)
and 2,4-dimethoxyaniline (100 mg, 0.648 mmol), using solvent
gradient in the column chromatography from 50% ether/petroleum
ether till neat ether, compound 2b was isolated (135 mg, 70%), m.p.
Several new 6-[(hetero)aryl]aminothieno[3,2-b]pyridines were
prepared from the methyl 3-amino-6-bromothieno[3,2-b]pyridine-
2-carboxylate byCeN BuchwaldeHartwig using different ligands for
the coupling of arylamines and heteroarylamines. The di(hetero)
arylamines prepared were evaluated as antitumorals in three
representative human tumor cell lines and it was possible to estab-
lish some structureeactivity relationships. For the aniline deriva-
tives the most active compounds are a methoxylated ortho-chloro
156e158 ꢀC 1H NMR (CDCl3, 400 MHz):
d 3.84 (3H, s, OMe), 3.85 (3H,
s, OMe), 3.88 (3H, s, OMe), 5.95 (1H, br s, NH), 6.13 (2H, br s, NH2),
6.52 (1H, dd, J ¼ 8.9 and 2.8 Hz, 50-H), 6.58 (1H, d, J ¼ 2.8 Hz, 30-H),
7.26 (1H, d, J ¼ 8.9 Hz, 60-H), 7.47 (1H, d, J ¼ 2.8 Hz, HetAr-H), 8.29 (1H,
d, J ¼ 2.8 Hz, HetAr-H) ppm. 13C NMR (CDCl3, 100.6 MHz):
d 51.38
derivative (GI50 21e28
methoxy groups in the ortho and para positions and in the para and
meta positions (GI50 25e34 M) relative to the NeH bond. For the
mM) and the dimethoxylated derivatives with
(OMe), 55.65 (OMe), 55.68 (OMe), 96.19 (C), 99.69 (30-CH),104.02 (50-
CH), 112.23 (CH), 121.49 (60-CH), 122.87 (C), 136.04 (C), 138.38 (CH),
139.14 (C), 141.42 (C), 148.01 (C), 152.34 (C), 156.87 (C), 165.53 (C]O)
ppm. MS (EI): m/z (%) 359 (Mþ, 100), 312 (48). HRMS Mþ calct. for
C17H17N3O4S 359.0940, found 359.0940.
m
heterocyclic amine derivatives the most active is the benzothiazole
derivative, presenting very low GI50 values, followed by the indole
derivative. These compounds are also the most promising within all
the compounds studied. Further studies with the latter compounds
willbeenvisaged inorder to determinetheirmechanism(s)ofaction.
4.1.1.3. Methyl 3-amino-6-(3,4-dimethoxyphenylamino)thieno[3,2-b]
pyridine-2-carboxylate (2c). From compound 1 (150 mg, 0.540 mmol)
and 3,4-dimethoxyaniline (100 mg, 0.648 mmol), and using solvent
gradient in the column chromatography from 50% ether/petroleum
ether till neat ether, compound 2c was isolated (125 mg, 70%), m.p.
4. Experimental
4.1. Synthesis
187e189 ꢀC 1H NMR (CDCl3, 300 MHz):
d 3.87 (3H, s, OMe), 3.88 (3H,
Melting points were determined on a Stuart SMP3 apparatus
and are uncorrected. The 1H NMR spectra were measured on
s, OMe), 3.91 (3H, s, OMe), 5.85 (1H, br s, NH), 6.13 (2H, br s, NH2),
6.73e6.80 (2H, m, Ar-H), 6.89 (1H, d, J ¼ 8.7 Hz, 50-H), 7.43 (1H, d,

5736
M.-J.R.P. Queiroz et al. / European Journal of Medicinal Chemistry 45 (2010) 5732e5738
J ¼ 2.4 Hz, HetAr-H), 8.25 (1H, d, J ¼ 2.4 Hz, HetAr-H) ppm. 13C NMR
0.540 mmol) and 2-bromoaniline (120 mg, 0.648 mmol) and using
neat ether in the column chromatography, compound 2f was iso-
lated (102 mg, 50%), m.p.163e165 ꢀC 1H NMR (CDCl3, 400 MHz):
(CDCl3, 75.4 MHz):
d 51.41 (OMe), 56.00 (OMe), 56.20 (OMe), 96.23
(C), 106.80 (CH), 112.06 (50-CH), 112.51 (CH), 114.24 (CH), 133.59 (C),
136.05 (C), 137.78 (CH), 139.22 (C), 141.63 (C), 146.24 (C), 147.91 (C),
149.89 (C), 165.49 (C]O), ppm. MS (EI): m/z (%) 359 (Mþ, 53), 257
(53), 236 (69), 155 (100). HRMS Mþ calct. for C17H17N3O4S 359.0940,
found 359.0939.
d
3.90 (3H, s, OMe), 6.17 (2H, broad s, NH2), 6.24 (1H, broad s, NeH),
6.89e6.93 (1H, m, Ar-H), 7.25e7.34 (2H, m, Ar-H), 7.60 (1H, dd, J ¼ 8
and 1.6 Hz, Ar-H), 7.73 (1H, d, J ¼ 2.4 Hz, Ar-H), 8.43 (1H, broad s, Ar-
H) ppm. 13C NMR (CDCl3, 100.6 MHz):
d 51.52 (OMe), 97.71 (C),
114.11 (C), 117.32 (CH), 117.70 (CH), 123.24 (CH), 128.44 (CH), 133.42
(CH), 135.48 (C), 138.23 (C), 139.48 (C), 140.14 (CH), 141.20 (C),
147.64 (C), 165.37 (C]O) ppm. MS (EI): m/z (%) 379 (Mþ 81Br, 100),
377 (Mþ 79Br, 95), 347 (51), 345 (45). HRMS Mþ calct. for
C15H12BrN3O2S: Mþ 81Br 378.9813; found 378.9805. Mþ 79Br calct.:
376.9834; found 376.9839.
Compound 3b was also isolated as a minor polar product using
neat ether in the column chromatography as a yellow solid (10 mg,
5%), m.p. 201e203 ꢀC, after some washes with petroleum ether. 1H
4.1.1.4. Methyl 3-amino-6-(3,5-dimethoxyphenylamino)thieno[3,2-b]
pyridine-2-carboxylate (2d). From compound 1 (150 mg, 0.540 mmol)
and 3,5-dimethoxyaniline (100 mg, 0.648 mmol) and using solvent
gradient in the column chromatography from 50% ether/petroleum
ether till neat ether compound 2d was isolated (97 mg, 50%), m.p.
218e220 ꢀC 1H NMR (CDCl3 þ DMSO-d6, 400 MHz):
d
3.56 (6H, s,
2 ꢁ OMe), 3.64 (3H, s, OMe), 5.91 (1H, t, J ¼ 2.4 Hz, 40-H), 6.03 (2H, br
s, NH2), 6.13 (2H, d, J ¼ 2.0 Hz, 20 and 60-H), 7.50 (1H, d, J ¼ 2.4 Hz,
HetAr-H), 7.71 (1H, br s, NH), 8.19 (1H, d, J ¼ 2.4 Hz, HetAr-H) ppm.13C
NMR (CDCl3, 400 MHz): d 3.90 (3H, s, OMe), 3.94 (3H, s, OMe), 6.17
NMR (CDCl3
þ
DMSO-d6, 100.6 MHz):
d
50.76 (OMe), 54.73
(2H, broad s, NH2), 6.28 (1H, broad s, NeH), 6.92e6.97 (1H, m, Ar-
H), 7.27e7.32 (2H, m, Ar-H), 7.38 (1H, dd, J ¼ 8.0 and 1.6 Hz, Ar-H),
7.62 (1H, dd, J ¼ 8 and 1.2 Hz, Ar-H), 7.71 (1H, d, J ¼ 2.4 Hz, Ar-H),
7.74 (1H, d, J ¼ 2.4 Hz, Ar-H), 8.33 (1H, d, J ¼ 2.4 Hz, Ar-H), 8.47 (1H,
d, J ¼ 2.4 Hz, Ar-H), 8.94 (1H, broad s, NeH) ppm. 13C NMR (CDCl3,
(2 ꢁ OMe), 93.47 (40-CH), 95.53 (C), 96.75 (20and 60-CH), 113.74 (CH),
135.18 (C), 138.43 (C), 138.68 (CH), 139.66 (C), 142.89 (C), 147.34 (C),
161.03 (2 ꢁ C),164.75 (C]O) ppm. MS (EI): m/z (%) 359 (Mþ,100), 327
(40), 255 (41), 236 (60). HRMS Mþ calct. for C17H17N3O4S 359.0940;
found 359.0947.
100.6 MHz):
d 51.49 (OMe), 52.15 (OMe), 97.94 (C), 106.93 (C),
114.91 (C), 115.45 (CH), 118.74 (CH), 120.61 (CH), 123.95 (CH), 128.47
(CH), 133.54 (CH), 134.61 (C), 135.92 (C), 137.45 (C), 138.00 (C),
138.76 (C), 139.89 (CH), 140.94 (C), 141.47 (CH), 141.60 (C), 143.34
(C), 147.77 (C), 165.21 (C]O), 165.44 (C]O) ppm. MS (EI): m/z (%)
585 (Mþ 81Br, 100), 583 (Mþ 79Br 95), 521 (61), 519 (54). HRMS Mþ
calct. for C24H18BrN5O4S2: Mþ 81Br 584.9963; found 584.9947. Mþ
79Br 582.9984; found 582.9969.
4.1.1.5. Methyl 3-amino-6-(2-chloro-5-methoxyphenylamino)thieno
[3,2-b]pyridine-2-carboxylate (2e) and methyl 3-amino-6-[6-(2-chloro-
5-methoxyphenylamino)-2-(methoxycarbonyl)thieno[3,2-b]pyridin-3-
ylamino]thieno[3,2-b]pyridine-2-carboxylate (3a). From compound 1
(150 mg, 0.540 mmol) and 2-cloro-5-metoxianilina (126 mg,
0.648 mmol) and using solvent gradient in the column chroma-
tography from 10% AcOEt/petroleum ether till 30% AcOEt/petro-
leum ether, compound 2e was isolated (94.0 mg, 48%), m. p.
4.1.1.7. Methyl 6-(2-bromo-4-fluorophenylamino)-3-aminothieno[3,2-
138e140 ꢀC 1H NMR (CDCl3, 400 MHz):
d
3.77 (3H, s, OMe), 3.90
b]pyridine-2-carboxylate (2g). From compound
1
(150 mg,
(3H, s, OMe), 6.18 (2H, broad s, NH2), 6.25 (1H, broad s, NH), 6.52
(1H, dd, J ¼ 8.8 and 2.8 Hz, 40-H), 6.86 (1H, d, J ¼ 2.8 Hz, 60-H), 7.32
(1H, d, J ¼ 8.8 Hz, 30-H), 7.78 (1H, d, J ¼ 2.4 Hz, Ar-H), 8.45 (1H, d,
0.540 mmol) and 2-bromo-4-fluoroaniline (0.07 mL, 0.648 mmol)
and using solvent gradient in the column chromatography from
10% AcOEt/petroleum ether till 30% AcOEt/petroleum ether,
compound 2g was isolated (97.0 mg, 45%), m.p. 175e177 ꢀC 1H NMR
J ¼ 2.4 Hz, Ar-H) ppm. 13C NMR (CDCl3, 100.6 MHz):
d 51.54 (OMe),
55.59 (OMe), 97.88 (C), 103.23 (60-CH), 107.69 (40-CH), 114.82 (C),
118.04 (CH), 130.45 (30-CH), 135.46 (C), 137.81 (C), 139.12 (C), 140.41
(CH), 141.34 (C), 147.59 (C), 159.22 (C), 165.37 (C]O) ppm. MS (EI):
m/z (%)365 (Mþ 37Cl, 35), 363 (Mþ 35Cl, 100). HRMS Mþ calct. for
C16H14ClN3O3S: Mþ 37Cl 365.0415; found 365.0412. Mþ 35Cl calct.
363.0444; found 363.0436.
(CDCl3, 300 MHz): d 3.90 (3H, s, OMe), 6.03 (1H, broad s, NeH), 6.17
(2H, broad s, NH2), 7.02e7.09 (1H, m, Ar-H), 7.29e7.34 (1H, m, Ar-
H), 7.37e7.41 (1H, m, Ar-H), 7.58 (1H, d, J ¼ 2.4 Hz, Ar-H), 8.36 (1H,
d, J ¼ 2.4 Hz, Ar-H) ppm. 13C NMR (CDCl3, 75.4 MHz):
d 51.53 (OMe),
96.46 (C), 115.50 (C), 115.53 (d, J ¼ 22 Hz, CH), 115.91 (CH), 120.31 (d,
J ¼ 5 Hz, CH),120.50 (d, J ¼ 22 Hz, CH),135.58 (C),135.72 (d, J ¼ 3 Hz,
C), 138.97 (C), 139.29 (CH), 140.87 (C), 147.65 (C), 157.88 (d,
J ¼ 247 Hz, CF), 165.37 (C]O) ppm. MS (EI): m/z (%) 397 (Mþ 81Br,
99), 395 (Mþ 79Br, 98), 365 (51) 363 (49). HRMS Mþ calct. for
C15H11BrFN3O2S: Mþ 81Br 396.9719; found 396.9709. Mþ 79Br
394.9728; found 394.9739.
Compound 3a was also isolated as a minor product using 60%
AcOEt/petroleum ether in the column chromatography as a yellow
solid (10 mg, 5%), m.p. 205e207 ꢀC, after some washes with
petroleum ether. 1H NMR (CDCl3, 400 MHz):
d 3.78 (3H, s, OMe),
3.90 (3H, s, OMe), 3.94 (3H, s, OMe), 6.18 (2H, broad s, NH2), 6.27
(1H, broad s, NH), 6.55 (1H, dd, J ¼ 8.8 and 2.8 Hz, 40-H), 6.91 (1H, d,
J ¼ 2.8 Hz, 60-H), 7.33 (1H, d, J ¼ 8.8 Hz, 30-H), 7.71 (1H, d, J ¼ 2.4 Hz,
HetAr-H), 7.81 (1H, d, J ¼ 2.4 Hz, HetAr-H), 8.35 (1H, d, J ¼ 2.4 Hz,
HetAr-H), 8.48 (1H, d, J ¼ 2.4 Hz, HetAr-H), 8.94 (1H, broad s, NeH)
4.1.2. General procedure for the synthesis of the diheteroarylamines
4aef
A dry Schlenk tube was charged, under Argon, with dry dioxane
(3e5 mL), the thienopyridine 1, Pd(OAc)2 (16 mol%), Xantphos
(18 mol%), Cs2CO3 (1.8 equiv), the heteroarylamine (1.1 equiv) and
the mixture was heated at 110 ꢀC for several hours (Table 1). The
reaction work-up was done following the general procedure for the
synthesis of compounds 2.
ppm. 13C NMR (CDCl3, 100.6 MHz):
d 51.50 (OMe), 52.17 (OMe),
55.65 (OMe), 97.95 (C), 104.32 (60-CH), 107.18 (C), 108.28 (40-CH),
115.56 (C), 116.10 (CH), 120.61 (CH), 130.57 (30-CH), 134.62 (C),
135.86 (C), 137.46 (C), 137.58 (C), 138.35 (C), 140.22 (CH), 141.13 (C),
141.43 (CH), 141.60 (C), 143.30 (C), 147.78 (C), 159.19 (C), 165.20 (C]
O), 165.45 (C]O) ppm. MS (EI): m/z (%) 571 (Mþ 37Cl, 42), 569 (Mþ
35Cl, 100) HRMS Mþ calct. for C25H20ClN5O5S2: Mþ 37Cl 571.0565;
found 571.0573. Mþ 35Cl calct. 569.0594; found 569.0574.
4.1.2.1. Methyl 3-amino-6-(benzo[d]thiazol-2-ylamino)thieno[3,2-b]
pyridine-2-carboxylate (4a). From compound 1 (150 mg, 0.540 mmol)
and 2-aminobenzo[d]thiazole (80.0 mg, 0.648 mmol) and using
solvent gradient in the column chromatography from 50% AcOEt/
petroleum ether till AcOEt, compound 4a was isolated (103 mg, 54%),
4.1.1.6. Methyl 6-(2-bromophenylamino)-3-aminothieno[3,2-b]pyri-
dine-2-carboxylate (2f) and methyl 3-amino-6-[6-(2-bromopheny-
lamino)-2-(methoxycarbonyl)thieno[3,2-b]pyridin-3-ylamino]thieno
[3,2-b]pyridine-2-carboxylate (3b). From compound 1 (150 mg,
m.p. 167e169 ꢀC 1H NMR (CDCl3, 400 MHz):
d
3.91 (3H, s, OMe), 6.14
(2H, broad s, NH2), 7.01 (1H, broad s, NeH), 7.19 (1H, app dt, J ¼ 8.0

M.-J.R.P. Queiroz et al. / European Journal of Medicinal Chemistry 45 (2010) 5732e5738
5737
and 1.6 Hz, Ar-H), 7.42 (1H, dd, J ¼ 8.0 and 1.2 Hz, Ar-H), 7.55e7.63
(3H, m, Ar-H), 8.35 (1H, d, J ¼ 2.0 Hz, Ar-H) ppm. 13C NMR (CDCl3,
NeH) ppm. 13C NMR (DMSO-d6, 100.6 MHz):
d
51.20 (OMe), 94.10
(C), 113.43 (CH), 124.02 (CH), 124.27 (CH), 135.23 (C), 138.23 (C),
138.89 (CH), 139.01 (C), 139.42 (C), 140.45 (CH), 142.33 (CH), 148.27
(C), 164.60 (C]O) ppm. MS (EI): m/z (%) 300 (Mþ, 100), 268 (60).
HRMS Mþ calct. for C14H12N4O2S: 300.0681; found 300.0682.
100.6 MHz):
d 51.76 (OMe), 99.80 (C), 109.56 (C), 115.48 (C), 115.75
(CH), 124.68 (CH), 128.50 (CH), 131.80 (C), 132.64 (CH), 134.94 (C),
137.66 (CH), 139.86 (C), 144.80 (CH), 144.94 (C), 147.10 (C), 165.19 (C]
O) ppm. MS (EI): m/z (%) 356 (Mþ, 30), 273 (100), 230 (44). HRMS Mþ
calct. for C16H12N4O2S2: 356.0402; found 356.0414.
4.1.2.6. Methyl
3-amino-6-(pyrimidin-2-ylamino)thieno[3,2-b]pyri-
dine-2-carboxylate(4f). From compound 1 (150 mg, 0.540 mmol) and
2-aminopyrimidine (57.0 mg, 0.648 mmol) and using solvent
gradient in the column chromatography from 50% AcOEt/petroleum
ether till AcOEt, compound 4f was isolated (90.0 mg, 55%), m.p.
4.1.2.2. Methyl 3-amino-6-(1H-indol-5-ylamino)thieno[3,2-b]pyridine-
2-carboxylate (4b). From compound 1 (150 mg, 0.540 mmol) and 5-
aminoindole (80.0 mg, 0.648 mmol) and using solvent gradient in the
column chromatography from 50% AcOEt/petroleum ether till AcOEt,
compound 4b was isolated (96.0 mg, 53%), m.p. 227e229 ꢀC 1H NMR
271e273 ꢀC 1H NMR (DMSO-d6, 400 MHz):
d 3.79 (3H, s, OMe), 6.82
(2H, broad s, NH2), 6.97 (1H, t, J ¼ 4.8 Hz, 40-H), 8.58 (2H, d, J ¼ 4.8 Hz,
30 and 50-H), 7.82 (1H, d, J ¼ 2.4 Hz, Ar-H), 8.91 (1H, d, J ¼ 2.4 Hz, Ar-
H), 10.24 (1H, broad s, NeH) ppm. 13C NMR (DMSO-d6, 100.6 MHz):
(DMSO-d6, 400 MHz): d 3.74 (3H, s, OMe), 6.38 (1H, m, Ar-H), 6.72 (2H,
broad s, NH2), 6.98 (1H, dd, J ¼ 8.8 and 2.0 Hz, 60-H), 7.32e7.34 (1H, m,
Ar-H), 7.38e7.41 (2H, m, Ar-H), 7.50 (1H, d, J ¼ 2.4 Hz, Ar-H), 8.31 (1H,
d, J ¼ 2.4 Hz, Ar-H), 8.55 (1H, broad s, NeH), 11.06 (1H, broad s, NeH)
d
51.27 (OMe), 94.62 (C), 113.60 (40-CH),117.33 (CH),134.29 (C),136.88
(C), 139.80 (CH), 148.25 (C), 158.23 (30 and 50-CH), 159.60 (C), 164.55
(C]O) ppm. MS (EI): m/z (%) 301 (Mþ, 100), 269 (42). HRMS Mþ calct.
for C13H11N5O2S: 301.0633; found 301.0633.
ppm.13C NMR (DMSO-d6,100.6 MHz):
d 51.03 (OMe), 92.45 (C),100.98
(CH), 109.26 (CH), 112.21 (CH), 112.60 (CH), 116.98 (CH), 126.09 (CH),
128.25 (C), 132.23 (C), 133.03 (C), 135.69 (C), 136.95 (C), 137.58 (CH),
143.29 (C), 148.66 (C), 164.64 (C]O) ppm. MS (EI): m/z (%) 338 (Mþ,
100). HRMS Mþ calct. for C17H14N4O2S: 338.0837; found 338.0840.
4.2. In vitro antitumor evaluation
4.2.1. Material and methods
4.1.2.3. Methyl 3-amino-6-(isoquinolin-1-ylamino)thieno[3,2-b]pyri-
dine-2-carboxylate (4c). From compound 1 (150 mg, 0.540 mmol)
and 1-aminoisoquinoline (87.0 mg, 0.648 mmol) and using solvent
gradient in the column chromatography from 50% AcOEt/petroleum
ether till AcOEt, compound 4c was isolated (106 mg, 56%), m.p.
4.2.1.1. Reagents. Fetal bovine serum (FBS), L-glutamine, phosphate
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). Trichloroacetic acid (TCA) and Tris were sourced
from Merck (Darmstadt, Germany).
222e224 ꢀC 1H NMR (DMSO-d6, 400 MHz):
d 3.80 (3H, s, OMe), 6.85
(2H, broad s, NH2), 7.31 (1H, d, J ¼ 5.6 Hz, Ar-H), 7.68 (1H, broad t, Ar-
H), 7.74 (1H, broad t, Ar-H), 7.88 (1H, broad d, J ¼ 8.0 Hz, Ar-H), 8.10
(1H, d, J ¼ 5.6 Hz, Ar-H), 8.58 (1H, broad d, J ¼ 8.0 Hz, Ar-H), 9.02 (1H,
d, J ¼ 2.0 Hz, Ar-H), 9.09 (1H, d, J ¼ 2.0 Hz, Ar-H), 9.67 (1H, broad s,
4.2.1.2. Solutions of the compounds. Stocks 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).
NeH) ppm. 13C NMR (DMSO-d6,100.6 MHz):
d 51.27 (OMe), 94.70 (C),
113.93 (CH), 118.70 (C), 118.79 (CH), 123.17 (CH), 126.65 (CH), 126.99
(CH), 130.40 (CH), 134.15 (C), 136.95 (C), 137.64 (C), 139.89 (C), 140.24
(CH), 141.00 (CH), 148.31 (C), 151.95 (C), 164.60 (C]O) ppm. MS (EI):
m/z (%) 350 (Mþ, 93), 349 (Mþ ꢃ 1, 100), 317 (62). HRMS Mþ calct. for
C18H14N4O2S: 350.0837; found 350.0838.
4.1.2.4. Methyl 3-amino-6-(pyridin-2-ylamino)thieno[3,2-b]pyridine-
2-carboxylate (4d). From compound 1 (150 mg, 0.540 mmol) and 2-
aminopyridine (57.0 mg, 0.648 mmol) and using solvent gradient in
the column chromatography from 50% AcOEt/petroleum ether till
AcOEt, compound 4d was isolated (75.0 mg, 45%), m.p. 198e200 ꢀC,
after some washes with petroleum ether. 1H NMR (DMSO-d6,
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). They were routinely maintained as adherent
cell cultures in RPMI-1640 medium supplemented with 5% heat-
inactivated FBS, 2 mM glutamine and antibiotics (penicillin 100 U/
400 MHz):
d 3.78 (3H, s, OMe), 6.80 (2H, broad s, NH2), 6.85e6.89
(1H, m, Ar-H), 6.92e6.96 (1H, m, Ar-H), 7.63e7.68 (1H, m, Ar-H),
8.25e8.27 (1H, m, Ar-H), 8.65 (1H, d, J ¼ 2.4 Hz, Ar-H), 9.01 (1H, d,
J ¼ 2.4 Hz, Ar-H), 9.70 (1H, broad s, NH) ppm. 13C NMR (DMSO-d6,
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 ꢁ 105 cells/mL for MCF-7 and 0.75 ꢁ 105 cells/mL for
A375-C5 and NCI-H460, followed by a 24 h incubation.
100.6 MHz): d 51.21 (OMe), 94.02 (C), 111.74 (CH), 115.63 (CH), 115.65
(CH), 134.69 (C), 137.71 (C), 138.09 (CH), 138.85 (C), 139.17 (CH), 147.24
(CH), 148.39 (C), 155.02 (C), 164.59 (C]O) ppm. MS (EI): m/z (%) 300
(Mþ,100) 299 (Mþ ꢃ 1, 51) 267(54). HRMS Mþ calct. for C14H12N4O2S:
300.0681; found 300.0681.
4.2.1.4. Growth inhibition assay. The effects on the in vitro growth of
human tumor cell lines were evaluated according to the procedure
adopted by the NCI (USA) in their “In vitro Anticancer Drug
Discovery Screen”, using the protein-binding dye sulforhodamine B
to assess cell growth [7,8]. 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
4.1.2.5. Methyl 3-amino-6-(pyridin-3-ylamino)thieno[3,2-b]pyridine-
2-carboxylate (4e). From compound 1 (150 mg, 0.540 mmol) and
3-aminopyridine (57.0 mg, 0.648 mmol) and using solvent gradient
in the column chromatography from 50% AcOEt/petroleum ether
till AcOEt, compound 4e was isolated (75.0 mg, 45%), m.p.
150 mM (if possible). Following the exposure period adherent cells
210e212 ꢀC 1H NMR (DMSO-d6, 400 MHz):
d
3.77 (3H, s, OMe),
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
6.78 (2H, broad s, NH2), 7.30e7.34 (1H, m, Ar-H), 7.63e7.67 (1H, m,
Ar-H), 7.96 (1H, d, J ¼ 2.4 Hz, Ar-H), 8.16 (1H, m, Ar-H), 8.41 (1H, d,
J ¼ 2.4 Hz, Ar-H), 8.47 (1H, d, J ¼ 2.8 Hz, Ar-H), 8.99 (1H, broad s,

5738
M.-J.R.P. Queiroz et al. / European Journal of Medicinal Chemistry 45 (2010) 5732e5738
[2] (a) D.H. Boschelli, B. Wu, A.C.B. Sosa, J.J. Chen, J.M. Golas, F. Boschelli, Bioorg.
Med. Chem. Lett. 15 (2005) 4681e4684;
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 [7]. Doxorubicin
was tested in the same manner to be used as a positive control.
(b) D.H. Boschelli, B. Wu, A.C.B. Sosa, H. Durutlic, J.J. Chen, Y. Wang, J.M. Golas,
J. Lucas, F. Boschelli, J. Med. Chem. 48 (2005) 3891e3902.
[3] For reviews on palladium-catalyzed CeN BuchwaldeHartwig coupling see: (a)
J.F. Hartwig, Angew. Chem. Int. Ed. 37 (1998) 2046e2067;
(b) J.P. Wolfe, S. Wagaw, J.-F. Marcoux, S.L. Buchwald, Acc. Chem. Res. 31 (1998)
805e818;
Acknowledgments
(c) B.H. Yang, S.L. Buchwald, J. Organomet. Chem. 576 (1999) 125e146;
(d) A.R. Muci, S.L. Buchwald, Top. Curr. Chem. 219 (2002) 131e209;
(e) B. Schlummer, U. Scholz, Adv. Synth. Catal. 346 (2004) 1599e1626.
[4] R.C. Calhelha, M.-J.R.P. Queiroz, Tetrahedron Lett. 51 (2010) 281e283.
[5] (a) M.-J.R.P. Queiroz, A. Begouin, I.C.F.R. Ferreira, G. Kirsch, R.C. Calhelha,
S. Barbosa, L.M. Estevinho, Eur. J. Org. Chem. (2004) 3679e3685;
(b) M.-J.R.P. Queiroz, I.C.F.R. Ferreira, R.C. Calhelha, L. Estevinho, Bioorg. Med.
Chem. 15 (2007) 1788e1794;
Thanks are due to the Foundation for Science and Technology
(FCT, Portugal) and FEDER (European Communitarian Fund), for
financial support through the research centres, the research project
PTDC/QUI/68382/2006, the national NMR (Bruker 400) network
(REDE/1517/RMN/2005). R. C. Calhelha and L. Vale-Silva acknowl-
edge FCT for their PhD (SFRH/BD/29274/2006) and post-doctoral
(SFRH/BPD/29112/2006) grants, respectively.
(c) M.-J.R.P. Queiroz, R.C. Calhelha, G. Kirsch, Tetrahedron 63 (2007)
13000e13005.
[6] J. Yin, M.M. Zhao, M.A. Huffman, J.M. McNamara, Org. Lett.
4 (2002)
3481e3484.
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