Synthesis and study of antiproliferative activity of novel thienopyrimidines on glioblastoma cells

Article abstract of DOI:10.1016/j.ejmech.2010.02.032

The receptor tyrosine kinases (for example EGFR, PDGFR, VEGFR) are a transmembrane protein family which plays a crucial role in tumor growth, survival, metastasis dissemination and angiogenesis. During the past 10 years, many tyrosine kinase inhibitors (T

Full text of DOI:10.1016/j.ejmech.2010.02.032

European Journal of Medicinal Chemistry 45 (2010) 2473e2479
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and study of antiproliferative activity of novel thienopyrimidines
on glioblastoma cells
,
c
,
b
b
b
b
Stéphane Pédeboscq a , Denis Gravier , Françoise Casadebaig , Geneviève Hou , Arnaud Gissot ,
*
Francesca De Giorgi c, François Ichas c, Jean Cambar d, Jean-Paul Pometan a
,
b
a Service Pharmacie, Hôpital Saint-André, CHU de Bordeaux, 1 rue Jean Burguet, 33 075 Bordeaux, France
b Laboratoire de Chimie Organique, UFR de Pharmacie, Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat, 33 076 Bordeaux, France
c INSERM U916, Institut Bergonié, Université Victor Segalen Bordeaux 2, 229 cours de l'Argonne, 33 076 Bordeaux, France
d Laboratoire de Biologie Cellulaire, Laboratoire Santé, Travail, Environnement EA 3672, Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat, 33 076 Bordeaux, France
a r t i c l e i n f o
a b s t r a c t
Article history:
The receptor tyrosine kinases (for example EGFR, PDGFR, VEGFR) are a transmembrane protein family
which plays a crucial role in tumor growth, survival, metastasis dissemination and angiogenesis. During
the past 10 years, many tyrosine kinase inhibitors (TKIs) have been approved for cancer treatment
(imatinib, gefitinib, erlotinib, sunitinib, sorafenib). These compounds generally possess a pyrrolo- or
pyrimido- pyrimidine scaffold or approaching molecular structure. We synthesized 10 thienopyrimidine
compounds (including 5 newly synthesized) whose scaffold is very similar to the agents cited above. The
cytotoxicity of these agents was evaluated using a MTT assay and a flow cytometry technique on glio-
blastoma cell lines. Two compounds showed a similar cytotoxicity to the standard anti-EGFR gefitinib
Received 1 April 2009
Received in revised form
10 February 2010
Accepted 12 February 2010
Available online 18 February 2010
Keywords:
Thienopyrimidine
Epidermal growth factor receptor
Tyrosine kinase
Cytotoxicity
(IC50: gefitinib ¼ 51.9
m
M, 6b ¼ 61.8
m
M, 6c ¼ 41.2
m
M), suggesting a blockade of the EGFR pathway by
binding to the TK receptor.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Apoptosis
1. Introduction
The ATP binding site of tyrosine kinase (TK) receptors could
constitute a viable target for drug design [3,4] in these pathologies.
Novel targeted therapies against these receptors have been
synthesized and are now commonly used in clinical practice in
various cancerous pathologies. Among these new agents, the TKIs
(TK inhibitors) are small molecules that bind to the intracellular
domain of growth factor receptors, inhibit autophosphorylation
and consequently block the proliferation signaling pathway. These
agents then prevent the proliferation of cells which have amplifi-
cation or dysregulation of growth factor pathways. Compounds
based on quinazoline, indolin or pyridopyrimidine scaffolds [5e11]
have been synthesized and allow ATP-competitive binding on the
TK domain of the growth factor receptors.
During the past 10 years, many TKIs have been approved for
cancer treatment. Imatinib (anti Bcr/Abl kinase, c-kit, PDGFR) was
the first TKI commercialized and became the standard therapy for
chronic myelogenous leukemia (CML) with very impressive results,
and for gastrointestinal stromal tumors (GIST). Erlotinib and gefi-
tinib (anti-EGFR) are currently used in non-small cell lung cancer
The receptor tyrosine kinases (RTK) are a transmembrane
protein family which plays a crucial role in tumor growth, survival,
metastasis dissemination and angiogenesis. They are overex-
pressed in several types of tumors (in particular EGFR (epidermal
growth factor receptor), PDGFR (platelet-derived growth factor
receptor), FGFR (fibroblast growth factor receptor) and VEGFR
(vascular endothelial growth factor receptor)) and they mediate the
cellular transduction pathway consecutively to the binding of
growth factors to their extracellular domain. Proliferation signaling
is mediated after this binding by phosphorylation of the TK domain,
which leads to a cascade of downstream signaling pathways.
Abnormal signaling via these TK is linked to cancerous pathologies
and to pathologies other than cancer, for example, cardiovascular
and immunoinflammatory pathologies [1,2].
* Corresponding author. Service Pharmacie, Hôpital Saint-André, CHU de
Bordeaux, 1 rue Jean Burguet, 33 075 Bordeaux, France. Tel.: þ33 557 82 06 12; fax:
þ33 556 79 58 98.
(NSCLC), sunitinib (anti VEGFR-2, PDGFR-
(FMS-like tyrosine kinase 3)) in renal cancer and GIST, sorafenib
(anti Raf-kinase, VEGFR-2, PDGFR- and c-kit) in renal cancer and
b, c-kit and FLT-3
b
E-mail address: stephane.pedeboscq@chu-bordeaux.fr (S. Pédeboscq).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.02.032

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S. Pédeboscq et al. / European Journal of Medicinal Chemistry 45 (2010) 2473e2479
Table 1
hepatocellular carcinoma, and lapatinib (anti-EGFR and ErbB-2
(erythroblastic leukemia viral oncogene homolog 2)) in breast
cancer. These compounds generally possess a pyrrolo- or pyr-
imidopyrimidine scaffold or approaching molecular structure.
In a previous work we synthesized a series of 3-substituted
1,2,3,4-tetrahydroquinazoline and 3-substituted thieno[2,3-d]pyr-
imidin-4-one compounds which presented a noticeable platelet
antiaggregating power [12,13]. The most potent activity was
exhibited by the thienopyrimidinone derivatives. These thieno-
pyrimidine compounds present a similar scaffold to the agents
cited above. Several studies have shown that the presence of
Synthesized thieno[2,3-d]pyrimidine derivatives.
Compound
X
R1
R2
Reference
3a
3b
3c
3d
Anilino
Anilino
Anilino
Anilino
Dimethylamino
Anilino
Anilino
Anilino
Anilino
H
H
H
H
[25]
[22]
[26]
[25]
[27]
e
Methyl
Methoxy
H
e
Methoxy
e
3e
6aa
6ba
6ca
6da
6ea
H
H
H
H
Methyl
Methoxy
H
e
e
e
Methoxy
e
e
Dimethylamino
e
a
quinazoline skeleton substituted in position 4 by various
a
The newly synthesized compounds were characterized by spectroscopic data.
substituted anilino groups potentially increased the EGFR inhibi-
tory effect [14,15]. According to the results observed with the
former thienopyrimidinone derivatives as platelet antiaggregating
agents, the substitution of these compounds at the 4 position could
lead to new PDGFR or EGFR pathway inhibitors. Moreover, authors
recently show that compounds based on this thienopyrimidine
scaffold could present an interest in the inhibition of VEGFR and
PDGFR kinase domains [16]. This study describes the chemical
synthesis of 10 thienopyrimidine derivatives and evaluates their
activity on the epidermal growth factor receptor pathway.
displays high constitutive EGFR expression. This band was not
present for the U87-MG cell line, representing a negative control of
EGFR expression.
3.2. Cytotoxicity of the synthesized compounds
WeassessedthecytotoxicityofDMSO,whichwasusedasadilution
vehicle for the agents tested. DMSO showed very weak cytotoxicity
(<10%) for concentrations ꢀ1% of DMSO. These DMSO concentrations
2. Chemistry
corresponded to tested agent concentrations ꢀ200
mM. Consequently,
concentrations ꢀ200
mM can beconsidered specific of the cytotoxicity
The derivatives of thieno[2,3-d]pyrimidin-4-one were prepared
according to a method described by Gewald [17,18], which involves
the cyclization of the appropriate derivative of ethyl-2-amino-
thiophene-3-carboxylate with formamide. The use of phosphorus
oxychloride affords the corresponding chlorosubstituted
compounds which, by refluxing in dimethylformamide with the
appropriate aniline, lead to the final derivatives of 4-anilinothieno
[2,3-d]pyrimidine according to Scheme 1. Compounds synthesized
are summarized in Table 1 with the different substitution groups.
of the tested agent itself and can be taken into account.
Gefitinib, which was used as a reference, was very cytotoxic on
DBTRG.05-MG cells in
51.9 ꢁ 3.7 M. Fig. 2A shows that two synthesized compounds (6b
and 6c) exerted a comparable cytotoxicity to gefitinib with IC50 of
about 50 M, IC50 6c ¼ 41.2 ꢁ 1.2 M).
M (IC50 6b ¼ 61.8 ꢁ 0.9
Another group of synthesized agents (6a, 6d, 6e, 3d and 3e) was less
cytotoxic (Fig. 2A and B) with IC50 around 100e150 M (IC50
6a ¼ 109.1 ꢁ9.3 M, IC50 6d ¼ 117.2 ꢁ 8.3 M, IC50 6e ¼ 138.5 ꢁ
1.9 M, IC50 3e ¼ 149.4 ꢁ13.9 M). Finally,
M, IC50 3d ¼ 151.7 ꢁ 11.7
three compounds (3a, 3b and 3c) had very low cytotoxicity (IC50
3a ¼ 584.7 ꢁ 179.9 M, IC50 3b ¼ 474.1 ꢁ8.7 M, IC50 3c ¼ 571.0 ꢁ
61.1 M).
a dose-dependent manner. IC50 was
m
m
m
m
m
m
m
m
m
m
3. Results
m
m
m
3.1. Cell characterization
Expression of EGF receptor was assessed on DBTRG.05-MG and
U87-MG glioblastoma cell lines by immunocytochemistry and
immunoblotting. Both techniques gave very similar results. The
immunoblots in Fig. 1 show a 170 kDa band corresponding to EGFR
for the DBTRG.05-MG cell line, demonstrating that DBTRG.05-MG
3.3. Apoptosis induced on two glioblastoma cell lines
Cell lines of glioblastoma (U87-MG and DBTRG.05-MG) were
treated with increasing concentrations of 6b (which shows
O
Cl
X
NH
N
N
POCl3
S
N
H2N
R1
S
N
R2
N
S
1
2
3
O
Cl
X
NH
N
POCl3
N
R2
S
N
S
N
H2N
N
S
R1
4
5
6
R1
3, 6 : X =
R2
HN
Scheme 1. Synthesis of the 4-anilinothieno[2,3-d]pyrimidine derivatives.

S. Pédeboscq et al. / European Journal of Medicinal Chemistry 45 (2010) 2473e2479
2475
a
b
EGFR 170 kDa
Tubulin
55 kDa
DBTRG.05-MG
U87-MG
1,2
1
0,8
0,6
0,4
0,2
0
DBTRG.05-MG
U87-MG
Fig. 1. EGFR expression on DBTRG.05-MG and U87-MG cell lines measured by immunocytochemistry (A) and western blotting analysis (B) as described in Experimental protocols.
A: EGFR expression on DBTRG.05-MG left panel, on U87-MG right panel. B: Tubulin constitutes a control of the loaded quantity of protein. Histograms represent the ratio of EGFR
band normalized with the tubulin band quantified using Image J software.
important cytotoxicity on DBTRG.05-MG) and 3c (which induces no
cytotoxicity on DBTRG.05-MG). The drug-induced depolarization of
the mitochondrial membrane was used as a marker of apoptotic
cells and was analyzed by flow cytometry, as described in Experi-
mental protocols.
Two compounds (6b and 6c) showed a comparable cytotoxicity
to gefitinib. In their structure they contain an aromatic cycle. On the
other hand, the corresponding molecules of the 3 series (3b and 3c)
with the same C-4 substitution but without the aromaticity were
weakly cytotoxic (4 times less cytotoxic). It seems therefore that the
presence of this aromatic cycle near the thienopyrimidine scaffold
is essential for anti-EGFR activity. Given this result, we replaced the
aromatic substituent with an aliphatic group such as a dimethyla-
mino moiety. 6e was the least cytotoxic. In fact, replacement of the
anilino group by a dimethylamino group led to a smaller conjuga-
tion. Since conjugation seems to participate in RTK binding, our
finding confirmed the hypothesis about the importance of having
an aromatic group linked to the C-4.
Fig. 3 shows the percentage of apoptotic cells achieved with 6b
(Fig. 3A) and 3c (Fig. 3B). 6c induces apoptosis on DBTRG cell line
expressing EGFR (47% of apoptotic cells at 100
induction of apoptosis is seen on U87 cell line lacking this receptor
(4% of apoptotic cells at 100 M). On the other hand 3c induces no
mM) meanwhile no
m
apoptosis on DGTRG neither than on U87 (Fig. 3B).
Substitution on this phenyl ring and the position of substitution
could be of great importance. In fact, 6a compound with no
substitution and 6d compound with a substitution in the para
position were less cytotoxic than the substituted molecules in the
ortho position (6b substituted by a methyl group and 6c by
a methoxy group). 6b and 6c showed comparable cytotoxicity, so
the nature of the substitution seems to carry less importance than
the position itself. This ortho substitution might therefore repre-
sent a privileged conformational position for RTK binding and for
greater activity.
To assess whether the cytotoxic potency of these compounds is
linked to EGFR binding, induction of apoptosis has been evaluated
on glioblastoma cell lines expressing EGFR or not. We found that
one of the most potent synthesized compound (6b) exerted
a strong induction of apoptosis on the EGFR expressing DBTRG.05-
MG cell line while it did not induce any apoptosis on U87-MG cell
line which do not possess the EGF receptor. These data indicate
that the effect observed seems to be linked to EGFR pathway
blockade.
4. Discussion
In this work we evaluated the cytotoxicity of synthesized
substituted thienopyrimidines on glioblastoma cell lines in order to
assess the EGFR pathway blockade power of these agents. Cyto-
toxicity assays were performed using an MTT colorimetric assay
evaluating the number of surviving cells after incubation for 48 h
with the tested compounds.
In a previous study [19], gefitinib, which is used in NSCLC, was
cytotoxic on the DBTRG.05-MG cell line expressing EGF receptor, so
it can be used as a standard for assessing anti-EGFR agents. In our
hands, gefitinib was very cytotoxic.
Two series of thienopyrimidine compounds were synthesized:
the 3aee series with a cyclohexyl cycle and the 6aee series pos-
sessing a benzenic cycle. C-4 substitution permits to differentiate
the synthesized agents: by an anilino group (6a and 3a), by an ortho
methylanilino group (6b and 3b), by an ortho methoxyanilino
group (6c and 3c), by a para methoxyanilino group (6d and 3d) and
by a dimethylamino substituent (6e and 3e).

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S. Pédeboscq et al. / European Journal of Medicinal Chemistry 45 (2010) 2473e2479
120
scaffold of inhibitors such as quinazoline, pyridopyrimidine or
A
eventually thienopyrimidine [4]. Three binding modes have been
described differing in their inhibitor skeleton position. Most
compounds with the pyrimidine scaffold have two H-bonding
regions but a few have an additional 2-NH2 group that could
increase their binding potency. Our synthesized compounds do
not possess this 2-NH2 moiety but it can be hypothesized that
they can achieve the two other hydrogen bonds, on one hand,
with the 3-nitrogen atom and on the other, with the 4-anilino
group. Moreover, one of the most potent compound synthesized
(6c) could produce a third H-bonding with the oxygen atom of
the methoxy group.
100
80
60
40
20
0
6a
6b
6c
6d
6e
This work demonstrates that on glioblastoma cell lines har-
bouring EGF receptor, two of the new synthesized thienopyr-
imidine compounds exert strong cytotoxic activity comparable to
that of the standard anti-EGFR gefitinib. The effect seems to be
linked to the blockade of the EGFR pathway by binding to the TK
domain. These agents could therefore have potential in treating this
pathology and could be tested on other cell lines from other
cancerous pathologies.
GF
DMSO
0
1
10
100
1000
-20
120
B
100
80
60
40
20
0
5. Experimental protocols
5.1. Chemistry
Melting points were determined on a Kofler apparatus. IR
spectra were taken on a Shimadzu IR 470 Spectrometer. NMR
spectra were recorded in DMSO-d6 solution on a Bruker AMX 500
instrument (1H: 300 MHz, 13C: 75 MHz).
Thin layer chromatography (TLC) was performed using
aluminium precoated plates (silica gel SDS 60F 254 Whatman,
0.2 mm thickness). Column chromatography was carried out on
3a
3b
3c
3d
3e
GF
DMSO
silica gel 60e200 mm (Merck).
0
1
10
100
1000
5.1.1. General procedure for the preparation of thieno[2,3-d]
pyrimidin-4-one derivatives (Scheme 1: compounds 1 and 4)
The compounds were prepared according to previously
described methods [17,18].
-20
Fig. 2. Cytotoxicity of different thienopyrimidine compounds on DBTRG.05-MG cells.
Cytotoxicity of anti-EGFR gefitinib and vehicle DMSO were also plotted. X-axis in
M. A: compounds with a benzenic cycle. B: compounds with a cyclohexyl cycle.
Cell viability was determined using MTT assay as described in Experimental proto-
cols. Each drug concentration was tested in triplicate. Data are presented as
mean ꢁ SD.
5,6,7,8-Tetrahydrobenzo[b]thieno[2,3-d]pyrimidin-4(3H)-one [18]
Benzo[b]thieno[2,3-d]pyrimidin-4(3H)-one[17]
m
5.1.2. General procedure for the preparation of 4-chlorothieno[2,3-
d]pyrimidine derivatives (Scheme 1: compounds 2 and 5)
The compounds were prepared according to previously
described methods [22,23].
A general pharmacophore model for ATP and ATP-competi-
tive inhibitors in the active site of RTK, notably EGFR TK, has
been proposed [4,20,21]. It consists of a hydrophobic binding
site for the adenine ring of ATP as well as for the heterocyclic
4-Chloro-5,6,7,8-tetrahydrobenzo[b]thieno[2,3-d]pyrimidine [22]
4-Chlorobenzo[b]thieno[2,3-d]pyrimidine [23]
50
40
30
20
10
0
50
DBTRG
U87
40
30
20
10
0
0.0
0.1
1.0
10.0
50.0
100.0
0.0
0.1
1.0
10.0
50.0
100.0
6b concentration (µM)
3c concentration (µM)
Fig. 3. Induction of apoptosis by 6b (left panel) and 3c (right panel) on DBTRG.05-MG and U87-MG cell lines measured by assessment of mitochondrial potential by flow cytometry
as described in Experimental protocols. Each concentration was tested in triplicate. Data are presented as mean ꢁ SD.

S. Pédeboscq et al. / European Journal of Medicinal Chemistry 45 (2010) 2473e2479
2477
5.1.3. General procedure for the preparation of 4-anilinothieno[2,3-
d]pyrimidine and substituted derivatives [24] (Scheme 1:
compounds 3 and 6)
J ¼ 7.5 Hz, 1H, H8), 8.42 (s, 1H, H2), 8.67 (d, J ¼ 7.5 Hz, 1H, H5), 8.91
(s, NH), 13C NMR (75 MHz, DMSO-d6):
ppm 17.9 (CH3), 110.5 (C4a),
123.0 à 134.2 (C4b, C5 à C8 et C12 à C16), 134.9 (C8a), 137.3 (C11),
154.6 (C2), 156.2 (C4), 167.8 (C8b).
d
A
mixture of 0.01 mol of 4-chlorothieno[2,3-d]pyrimidine
(Scheme 1: compounds 2 and 5) and 0.02 mol of the appropriate
amine in 50 mL of dimethylformamide was heated at reflux for
20 h. The DMF was evaporated under reduced pressure. The reac-
tion mixture was treated with diethylether and the separated solid
was filtered and recrystallized from ethanol.
5.1.5.1.3. 4-(2-Methoxyanilino)benzo[b]thieno[2,3-d]pyrimidine
(6c). Compound 6c (R1]OCH3, R2]H): melting point: 172 ꢂC; yield
45%; IR KBr, cmꢃ1: 3442 (nNH);1H NMR (300 MHz, DMSO-d6):
d ppm
3.88 (s, 3H, OCH3), 7.04 (t, J ¼ 7.0 Hz, 1H), 7.21 (m, 2H), 7.64 (m, 2H),
7.99 (d, J ¼ 8 Hz, 1H), 8.18 (d, J ¼ 7 Hz, 1H, H8), 8.47 (d, J ¼ 7 Hz, 1H,
H5), 8.56 (s, 1H, H2), 8.63 (s, NH), 13C NMR (75 MHz, DMSO-d6):
1. 4-Anilino-5,6,7,8-tetrahydrobenzo[b]thieno[2,3-d]pyrimidine
[25] (3a)
d ppm 55.8 (OCH3), 110.9 (C4a), 111.2 à 126.3 (C5 à C8 et C13 à C16),
127.5 (C4b), 130.5 (C8a), 134.4 (C11), 151.0 (C12), 154.4 (C2), 155.3
(C4), 167.5 (C8b).
2. 4-(2-Methylanilino)-5,6,7,8-tetrahydrobenzo[b]thieno[2,3-d]
pyrimidine [22] (3b)
5.1.5.1.4. 4-(4-Methoxyanilino)benzo[b]thieno[2,3-d]pyrimidine
(6d). Compound 6d (R1]H, R2]OCH3): melting point: 150 ꢂC;
yield 65%; IR KBr, cmꢃ1: 3434 (nNH);1H NMR (300 MHz, DMSO-d6):
3. 4-(2-Methoxyanilino)-5,6,7,8-tetrahydrobenzo[b]thieno[2,3-d]
pyrimidine [26] (3c)
d
ppm 3.78 (s, OCH3), 6.98 (d, J ¼ 8.0 Hz, 2H), 7.51 (d, J ¼ 8.0 Hz, 2H),
7.59 (m, 2H, H6 and H7), 8.12 (d, J ¼ 7.0 Hz, 1H, H8), 8.49 (s, 1H, H2),
8.61 (d, J ¼ 7.0 Hz, 1H, H5), 8.97 (s, NH), 13C NMR (75 MHz, DMSO-
4. 4-(4-Methoxyanilino)-5,6,7,8-tetrahydrobenzo[b]thieno[2,3-d]
pyrimidine [25] (3d)
d6): d ppm 55.1 (OCH3), 110.8 (C4a), 113.5 (C13, C15), 125.8 (C12,
C16), 123.0 à 126.4 (C5 à C8), 130.9 (C4b), 131.5 (C8a), 134.3 (C11),
154.5 (C14), 156.0 (C2), 156.2 (C4), 167.8 (C8b).
5. Procedure for the preparation of 4-dimethylaminobenzo[b]thieno
[2,3-d]pyrimidine derivatives
5.1.5.2. 4-Dimethylaminobenzo[b]thieno[2,3-d]pyrimidine (Fig. 5)
(6e). Compound 6e: melting point: 96 ꢂC; yield 55%; 1H NMR
6. 4-Dimethylamino-5,6,7,8-tetrahydrobenzo[b]thieno[2,3-d]
pyrimidine [27] (3e)
(300 MHz, DMSO-d6):
d
ppm 3.11 (s, 6H, CH3), 8.03 (d, J ¼ 3.0 Hz,
1H, H8), 8.11 (d, J ¼ 3.0 Hz, 1H, H5), 8.62 (s, 1H, H2), 13C NMR
(75 MHz, DMSO-d6):
d ppm 40.5 (CH3), 112.6 (C4a), 123.0 à 126.2
(C5 à C8), 131.1 (C4b), 134.2 (C8a), 153.6 (C2), 161.4 (C4), 168.9 (C8b).
5.1.4. 4-Dimethylaminobenzo[b]thieno[2,3-d]pyrimidine (6e)
0.01 mol of 4-chlorothieno[2,3-d]pyrimidine (Scheme 1:
compounds 2 and 5) in 50 mL of dimethylformamide was heated at
reflux for 20 h. The DMF was evaporated under reduced pressure.
The reaction mixture was treated with diethylether and the sepa-
rated solid was filtered and recrystallized from ethanol.
5.2. Biological assays
5.2.1. Cell culture
Human glioblastoma cell line U87-MG (ECACC No: 89081402) and
DBTRG.05-MG (ECACC No: 93061119) was purchased from the Euro-
pean Collection of Cell Cultures (ECACC, Salisbury, United Kingdom).
DBTRG.05-MG cells were cultured in RPMI 1640 containing 10% heat-
inactivated FCS supplemented with L-glutamine (2 mM), pen-
5.1.5. Physical and spectroscopic data
5.1.5.1. 4-Anilinobenzo[b]thieno[2,3-d]pyrimidine and substituted
derivatives. The general structure of the synthesized compounds is
described in Fig. 4.
icillinestreptomycin (100 units/mLe100 mg/mL), sodium pyruvate
(1 mM) and hypoxanthineethymidine (1%). U87-MG cells were
cultured in EMEM containing 10% decomplemented FCS and sup-
plemented with L-glutamine (2 mM), penicillinestreptomycin
5.1.5.1.1. 4-Anilinobenzo[b]thieno[2,3-d]pyrimidine (6a). Com-
pound 6a (R1]R2]H): melting point: 166 ꢂC; yield 60%; IR KBr,
cmꢃ1: 3416 (nNH); 1H NMR (300 MHz, DMSO-d6):
d ppm 7.16 (t,
(100 units/mLe100 mg/mL), non-essential amino acids (1%), sodium
pyruvate (1 mM). Cells were cultured in 75 cm2 flasks (Greiner Bio-
One GmbH, Frickenhausen, Germany) and maintained at 37 ꢂC in
a fully humidified 5% CO2 atmosphere. Subcultures were assessed by
using trypsin/EDTA solution (1:250) for 5 min at 37 ꢂC.
J ¼ 7.0 Hz, 1H), 7.39 (t, J ¼ 7.0 Hz, 1H), 7.62 (m,4H), 8.14 (d, J ¼ 7.0 Hz,
1H, H8), 8.56 (s, 1H, H2), 8.58 (d, J ¼ 7.0 Hz, 1H, H5), 9.16 (s, NH), 13C
NMR (75 MHz, DMSO-d6): d ppm 111.6 (C4a), 123.1 à 128.3 (C5 à C8
et C12 à C16), 130.8 (C4b), 134.4 (C8a), 139.0 (C11), 154.5 (C2), 155.7
(C4), 168.2 (C8b).
5.1.5.1.2. 4-(2-Methylanilino)benzo[b]thieno[2,3-d]pyrimidine
5.2.2. Immunocytochemistry
(6b). Compound 6b (R1]CH3, R2]H): melting point: 142 ꢂC; yield
Detection of EGFR expression was carried out by immunocyto-
chemistry [28,29] on DBTRG.05-MG cell line and on U87-MG cell
line. Cells were seeded onto a glass coverslip. At confluence, the
cells were washed twice with PBS. For EGFR determination, cells
were fixed for 20 min at ꢃ20 ꢂC with a methyl alcohol/acetone
mixture (50/50, v/v). The cells were then incubated in the dark for 2
or 6 h with a primary mouse monoclonal anti-EGFR antibody
diluted (1:100) with 1% BSAePBS in order to determine EGFR. The
40%; IR KBr, cmꢃ1: 3375 (nNH);1H NMR (300 MHz, DMSO-d6):
d ppm
2.18 (s, CH3), 7.30 (m, 4H), 7.59 (m, 2H, H6 and H7), 8.14 (d,
H5 '
R1
R2
H
H5
H8
H3 '
N
N
CH3
CH3
H2 '
N
N
N
S
H2
N
H
S
Fig. 4. General structure of the 4-substituted benzo[b]thieno[2,3-d]pyrimidine
derivatives.
Fig. 5. Structure of 4-dimethylaminobenzo[b]thieno[2,3-d]pyrimidine.

2478
S. Pédeboscq et al. / European Journal of Medicinal Chemistry 45 (2010) 2473e2479
coverslip was rinsed twice with 1% BSAePBS for 15 min followed by
an additional washing step with PBS. The glass coverslip was dried
and cells were incubated for 1 h with the secondary FITC-conju-
gated goat anti-mouse antibody diluted (1:50) with 1% BSAePBS.
The coverslip was rinsed, dried and mounted in mineral oil on
a microscope slide. Fluorescent images of immunolabeled cells
were observed using an Olympus BH-2 fluorescent microscope and
captured with an Olympus C-5060 digital camera.
appropriate dilutions of tested compounds. Cells were incubated in
Krebs Ringer buffered saline (NaCl 130 mM, KCl 3.6 mM, HEPES
10 mM, NaHCO3 2 mM, NaH2PO4 0.5 mM, MgCl2 0.5 mM, CaCl2
1.5 mM, glucose 4.5 g/l, pH 7.42) supplemented with 200 nM
TMRM and maintained at 37 ꢂC in 5% CO2, 95% air atmosphere for
30 min. Verapamil (20 mM) was added during TMRM incubation to
block multidrug-resistant (MDR) pumps. Cells were then incubated
with trypsin at 37 ꢂC, resuspended in HBSS supplemented with 4%
fetal calf serum, and maintained at 4 ꢂC. 96-well microtiter plates
were then analyzed on a FACSCalibur flow cytometer (Becton
Dickinson) equipped with a 488-nm argon ion laser and fluores-
cence emission was measured in FL3 log mode. Forward scatter
(FSC) and side scatter (SSC) were used to gate out cellular frag-
ments. The number of apoptotic cells was calculated using the Cell
Quest software.
5.2.3. Immunoblot analysis of EGFR
Cells were cultured in 60 mm Petri dishes and incubated with
various concentrations of temozolomide, carboplatin, gefitinib or
bortezomib for 48 h. Cells were lysed at þ4 ꢂC with lysis buffer
(protease inhibitor cocktail (SigmaeAldrich) supplemented with
0.3% SDS and 0.75% b-mercaptoethanol), sonicated and cleared by
centrifugation. Supernatant was collected and protein concentra-
tion determined using the Bio-Rad Protein Assay kit (Bio-Rad
Laboratories GmbH, München, Deutschland). An equal volume of
Acknowledgements
Laemmli solution supplemented with
added to a supernatant aliquot and the mixture was heated for
10 min at 80 ꢂC. Samples (15
g of protein) were loaded on each
b-mercaptoethanol (5%) was
We would like to thank Mrs Chantal Medina for technical
assistance and competence on the FACSCalibur flow cytometer.
m
lane and proteins were separated by 10% SDS polyacrylamide gel
electrophoresis. Proteins were then transferred onto a PVDF (pol-
yvinylidene fluoride) membrane (NEN Life Science Product, Boston,
MA). Membrane was blocked for 2 h in TBST (Tris buffered saline
Tween 20) containing 10% low fat dry milk and probed with
primary antibody diluted in TBST with 5% low fat dry milk over-
night at 4 ꢂC. Primary antibody dilutions were as follows: 1/4000
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