Impact of aryloxy-linked quinazolines: A novel series of selective VEGFR-2 receptor tyrosine kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.01.137

Three series of 6,7-dimethoxyquinazoline derivatives substituted in the 4-position by aniline, N-methylaniline and aryloxy entities, targeting EGFR and VEGFR-2 tyrosine kinases, were designed and synthesized. Pharmacological activities of these compounds

Full text of DOI:10.1016/j.bmcl.2011.01.137

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2106–2112
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Impact of aryloxy-linked quinazolines: A novel series of selective VEGFR-2
receptor tyrosine kinase inhibitors
Antonio Garofalo a,b, Laurence Goossens a,b, , Perrine Six a,b, Amélie Lemoine a,b, Séverine Ravez a,b
,
⇑
Amaury Farce a,c, Patrick Depreux a,b
a Univ Lille Nord de France, F-59000 Lille, France
b UDSL, ICPAL, EA 4481, F-59006 Lille, France
c UDSL, UFR de Pharmacie, EA 4481, Laboratoire de Chimie Thérapeutique, F-59006 Lille, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Three series of 6,7-dimethoxyquinazoline derivatives substituted in the 4-position by aniline, N-methy-
laniline and aryloxy entities, targeting EGFR and VEGFR-2 tyrosine kinases, were designed and synthe-
sized. Pharmacological activities of these compounds have been evaluated for their enzymatic
inhibition of VEGFR-2 and EGFR and for their antiproliferative activities on various cancer cell lines.
We have studied the impact of the variation in the 4-position substitution of the quinazoline core. Sub-
stitution by aryloxy groups led to new compounds which are selective inhibitors of VEGFR-2 enzyme
with IC50 values in the nanomolar range in vitro.
Received 17 December 2010
Revised 28 January 2011
Accepted 29 January 2011
Available online 3 February 2011
Keywords:
Quinazoline
EGFR
Ó 2011 Elsevier Ltd. All rights reserved.
VEGFR2
RTK inhibitors
Anticancer agents
Receptor tyrosine kinases (RTKs) play crucial roles in signal
transduction pathways and cellular processes (proliferation, cell
cycle, cell metabolism, survival, apoptosis, DNA damage/repair,...).1,2
Most signal transduction pathways are mediated by protein
kinases, and aberrant kinase signaling leads to proliferation of can-
cer cells and also angiogenesis and growth of solid tumors such as
prostatic, colon, breast, and gastric cancers.3–5 Vascular endothelial
growth factor (VEGF) and receptor protein-tyrosine kinases are the
key regulators of angiogenesis, vasculogenesis, and developmental
hematopoiesis.6–8 VEGF is a mitogen and a survival factor for
vascular endothelial cells. The VEGF family of receptors consists
of three protein-tyrosine kinase receptors (VEGFR-1, VEGFR-2,
and VEGFR-3) and two non-protein kinase co-receptors (neuropi-
lin-1 and neuropilin-2). These components participate in new
blood vessel formation from angioblasts (vasculogenesis) and
new blood vessel formation from pre-existing vasculature (angio-
genesis). Interaction between VEGFR-1 and VEGFR-2 or VEGFR-2
and VEGFR-3 alters receptor tyrosine phosphorylation.9–11 The
VEGF receptor protein-tyrosine kinases consist of an extracellular
component containing seven immunoglobulin-like domains, a
single transmembrane segment, a juxtamembrane segment, and
an intracellular protein-tyrosine kinase domain. VEGFR-2 (Flk-1
mouse, KDR human) is the predominant mediator of VEGF-stimu-
lated endothelial cell migration, proliferation, survival, and
enhanced vascular permeability. Although VEGFR-2 has lower
affinity for VEGF than VEGFR-1, VEGFR-2 exhibits robust protein-
tyrosine kinase activity in response to its ligands. VEGFR-2 is
expressed at abnormally high levels in a large variety of human
solid tumors.12,13 Several researchers have been pursuing VEGFR-
2 kinase domain inhibitors to discover novel anti-angiogenic drugs.
The growing interest in this strategy for cancer treatment stems
from the envisioned favorable toxicity profile. Because VEGFR-2
is located essentially in endothelial cells and expressed by several
cancer cells, an angiogenesis inhibitor is not expected to affect
proliferation of normal cells, unlike conventional cytotoxic chemo-
therapy. Several successful strategies for the inhibition of angio-
genesis have been effectively demonstrated in preclinical and
clinical settings, as exemplified by the recent USFDA approval of
a humanized anti-VEGF monoclonal antibody (bevacizumab, Ava-
stinÒ) as a first-line treatment of metastatic colorectal cancer, in
combination with chemotherapy.14,15 Small molecule inhibitors
of VEGF receptor protein tyrosine kinases represent another
approach for inhibiting angiogenesis. The drug sorafenib
(NexavarÒ), from Bayer, has recently been approved by the USFDA
for the treatment of metastatic renal cell carcinomas.
⇑
Corresponding author. Address: Institut de Chimie Pharmaceutique Albert
Lespagnol, Univ Lille Nord de France, 3 rue du Professeur Laguesse, B.P.83 59006
Lille, France. Tel.: +33 (0) 3 20 96 47 02; fax: +33 (0) 3 20 96 49 06.
E-mail address: laurence.goossens@univ-lille2.fr (L. Goossens).
Quinazoline derivatives have attracted interest over the years
because of their multiple biological activities, notably as kinase
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.137

A. Garofalo et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2106–2112
2107
F
O
HN
N
Cl
HN
N
N
O
O
O
O
N
N
O
O
ZD1839 (Iressa, gefitinib)
Inhibition of EGFR
OSI-774 (Tarceva, erlotinib)
Inhibition of EGFR
Br
F
F
HN
O
HN
Cl
N
O
N
O
N
N
O
N
PD153035
Inhibition of EGFR
ZD6474 (vandetanib)
Inhibition of EGFR/VEGFR-2
Figure 1. Structure of ATP-mimic inhibitors for treatment in solid tumors.
inhibitors.16,17 The first generation of quinazoline as PD153035
exhibits a high affinity for EGFR but a poor in vivo activity.18,19
The clinical success of selective kinase inhibitors, such as gefitinib
(IressaÒ) and erlotinib (TarcevaÒ), as therapeutic agents for several
human cancers has prompted substantial interest in the further
development and clinical testing of such inhibitors for a wide vari-
ety of malignancies. Vandetanib (ZD6474), from AstraZeneca, a
once-daily oral anticancer drug in phase III, is considered to be a
dual tyrosine kinase inhibitors targeting EGFR and VEGFR-2
(Fig. 1).20–25 This quinazoline substituted with a halide at the
2- and 4-positions on the phenyl group shows a strong inhibitory
activity (IC50 = 500 nM for EGFR and 40 nM for VEGFR-2, in ELISAs
with recombinant enzymes).26
The 4-anilinoquinazoline scaffold has to be able to selectively
inhibit the tyrosine kinase activity of the epidermal growth factor
receptor (EGFR) and also to inhibit simultaneously the tyrosine
kinase activity of EGFR and VEGFR. But, to date, no selective
inhibition towards VEGFR tyrosine kinase activity was obtained
with the 4-anilinoquinazolines. However, an indole-ether quinazo-
line, AZD2171, is a highly potent ATP-competitive inhibitor of
recombinant KDR tyrosine kinase in vitro.27
the hydrophobic back pocket of the kinase active site, we envi-
sioned the replacement of the aniline moiety by an N-methyl ana-
log and aryloxy groups. According to the X-ray structural analysis,
Y
O
Y = F, Cl, Br, amid
O
O
c
N
N
5a-d
R
HN
Cl
N
O
O
O
a
N
N
O
N
3a-h
R = F, Cl, Br, urea, amid
2
X
X = F, Cl, Br
O
N
b
N
These compounds have been designed in view of the known
interactions between 4-anilinoquinazolines and the adenine-bind-
ing region of the ATP-binding site of these receptors and they have
shown a good cytotoxicity against several types of tumors in vitro.
We recently described the structure–activity relationships
(SAR) developed around anilinoquinazoline derivatives, exempli-
fied by the pharmacophore 1, as potent EGFR/VEGFR-2 dual kinase
inhibitors (Fig. 2).25,28
O
N
4a-c
Scheme 1. Reagents and conditions: (a) aniline derivatives, (CH3)2CHOH, reflux,
80–95%; (b) CH3I, NaH, DMF, room temperature, 25–40%; (c) phenol derivatives,
NaH, DMSO, 150 °C, 100 W, microwave, 59–65%.
With the aim of further exploring one of the key recognition
elements of the kinase inhibitors, which are presumed to bind into
O
R
Cl
N
N
N
N
O
O
O
O
N
N
H
O
N
N
alkyle
R1 : Cl, CH3
2
R2, R3 : alkyl, aminoalkyl
O
HN
N
R1
O
R :
N
R2
O
R3
O
1
6a (para)
6b (meta)
7a (para)
7b (meta)
Figure 2. Pharmacophore 1 of anilinoquinazoline derivatives, potent dual kinase
inhibitor.25,28
Scheme 2. Chemical structure of ureas 6–7.19

2108
A. Garofalo et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2106–2112
H
N
O
R
R2
O
Cl
N
O
O
N
O
O
O
O
O
O
a
b
N
N
N
N
10
11
2
R2 : 8, p-NH2
9, m-NH2
c
O
R'
O
N
N
H
N
H
O
O
N
R' : alkyl : 12
aromatic : 13-21
cyclohexyl : 22
Scheme 3. Reagents and conditions: (a) nBu4N+Brꢀ, 2-butanone, 20% NaOH, reflux, 80% for 8 and 93% for 9; (b) ROCOCl, NEt3, THF, room temperature, 50–60%; (c) isocyanate
derivatives, NEt3, CHCl3, room temperature, 25–60%.
the NH entity of anilinoquinazoline derivatives does not establish
interactions with EGFR or VEGFR-2 enzymatic active-site. Our
effort to develop small molecular, ATP-competitive dual EGFR/
VEGFR-2 inhibitors led to the development of three series of com-
pounds with different hydrogen bond donor and/or acceptor
groups at the 4-position on the quinazoline. We describe here
the chemistry, SAR, and biological testing for these series. The qui-
nazoline core was modified at position 4 of the aryl with different
bulky substituents such as amide, carbamate or urea, which are
commonly-used moieties in EGFR and VEGFR inhibitors with the
aim of strengthening the ligand–receptor interactions and there-
fore increasing affinity.
The kinase and cell data are summarized in Table 1. The first
SAR study focused on the quinazoline skeleton substituted at the
4-position by aniline, N-methylaniline or an aryloxy group
substituted by halides, amide, carbamate or urea. N-Methyl
analogs, in particular 4a, exhibited a good antiproliferative activity
despite a complete absence of EGF- and VEGF-R TK inhibitory
effect. Recently we reported that the methylation of the aniline
nitrogen of quinazolin derivatives is essential to obtain a drug
binds to DNA as a typical intercalating agent.19 Among the amide
analogs 3d and 5d, no kinase inhibition and no antiproliferative ef-
fect was observed at 10
gens showed some dual EGFR/VEGFR-2 kinase inhibition (3a–c
and 5a–c) with IC50 values <10 M. NH analogs 3a and 3b proved
to be moderately potent EGFR inhibitors (IC50 values of 0.3 and
0.4 M, respectively) and compared favourably with their aryloxy
lM concentration. Substitution by halo-
As shown in Scheme 1, 4-chloro-6,7-dimethoxyquinazoline (2)
was used as a starting material29,30 to synthesize according to
described procedures19,29 PD153035 (3a), anilines 3b–h, N-methyl
anilines 4a–c and ethers 5a–d analogs.
l
l
counterparts (5a and 5b). Introduction of donor–acceptor group
such as carbamate or urea is generally more tolerated. The car-
bamic acid methyl ester 3e–f showed no significant inhibition of
In Scheme 2, the corresponding ureas 6–7 were synthesized
according to described procedures.19
Selective reaction of the chloro derivative 2 with meta- or para-
aminophenol in the presence of tetra-n-butylammonium bromide
in a methyl ethyl ketone–sodium hydroxide mixture provided
intermediates 829 and 9.19 New carbamic acid esters 10–11 were
obtained by amidification with corresponding amines. Compounds
8 and 9 reacted with corresponding isocyanates in chloroform to
give the desired ureas 12, 13a–b,19 14a–b,19 15–22 in high yields
and after short reaction times (Scheme 3).
EGFR (>5
with oxygen at the 4-position (10 and 11) exhibited good levels
of inhibition against VEGFR-2 (0.7 and 0.6 M, respectively) but
showed a significant decrease in activity against EGFR (IC50
>10 M). All the urea-substituted aryloxy-quinazolines (13a–b,
14a–b) were selectively active against VEGFR-2 with IC50 values
<1 M. Compounds with para-substitution of urea on the aryl
13a, 14a (KDR IC50 = 0.07 M) were 10-fold more efficient than
lM) and VEGFR-2 (>5 lM). Replacing NH (3e and 3f)
l
l
l
l
Inhibitory activity of the synthesised compounds against EGFR
and VEGFR-2 tyrosine kinases was determined by measuring the
levels of phosphorylation of the tyrosine-specific peptides (poly
(Glu4-Tyr)substrate) in vitro. They were evaluated also for their
antiproliferative activity towards the human endothelial-like,
immortalized cell line EAhy926: this cell line is derived from the
fusion of human umbilical vein endothelial (HUVEC) cells with
the A549 human lung epithelial carcinoma cell line. These cells
were used because of their role in angiogenesis. The cytotoxicities
of the synthesised compounds were evaluated toward the hor-
mone-independent PC3 prostate cancer cells, MCF7 breast cancer
cells and HT29 colon cancer cells, by MTS test.
their meta-analogs (13b, 14b) and 100-fold more potent than the
compounds with NH linker (3g–h). The strong inhibitory activity
of these carbamates and ureas against VEGFR was not associated
with antiproliferative activity (cell-based assays IC50 >10 lM).
A SAR study for the urea-substituted aryloxy-quinazolines
13–22 was undertaken. As shown in Table 2, O-linkage resulted
in a selective inhibition of VEGFR-2. When urea was substituted
by an alkyl group 12, 22 (butyl and cyclohexyl), high potency for
both VEGFR-2 was achieved (IC50 <1 lM). Diaryl ureas derivatives
(15–19) resulted in excellent kinase inhibition with nanomolar IC50
value against KDR. Replacement of hydrogen with a halogen atom
on the phenyl ring (15–17) was reasonably well tolerated. Bi-sub-

A. Garofalo et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2106–2112
2109
Table 1
Enzymatic (EGFR/VEGFR-2) and cellular results for 6,7-dimethoxyquinazoline derivatives32
O
O
N
N
Compd
Enzymatic inhibitory (IC50
,
l
M)a
% Proliferative inhibitory (or IC50
, l
M)d
EGFRb
VEGFR-2c
Human prostate Human colorectal Human breast Human endothelial-like
PC3
7.40
33%
HT29
ND
4.20
MCF-7
30%
26%
immortalized EAhy926
ND
5.10
IressaÒ
Vandetanib
0.07
0.80
14.80
0.10
R
X
NH
PD153035 0.063
ND
>10
40%
42%
10.10
6.10
4.70
9.20
7.10
3.95
N-CH3 4a19
>10
O
5a
7.60
9.80
27%
6%
0%
0%
X
Br
F
NH
N-CH3 4b
3b31
0.38
>10
5.30
>10
6.50
22%
5.70
5.20
4.90
0%
4.50
46%
O
5b
>10
9.50
5%
4.07
0%
ND
X
F
Cl
Br
NH
N-CH3 4c
3c
5.70
>10
1.65
>10
1%
15%
0%
14%
31%
0%
0%
0%
O
5c
5.90
0.60
16%
0%
0%
0%
X
X
X
X
X
H
NH
O
3d
5d
>10
>10
>10
>10
2%
0%
0%
0%
12%
5%
0%
3%
N
O
O
O
O
H
N
NH
O
3e
10
6.90
>10
5.80
0.70
2%
0%
10%
1%
27%
4%
0%
6%
O
O
H
N
NH
O
3f
11
>10
>10
5.60
0.60
12%
28%
16%
0%
6%
0%
0%
0%
H
N
H
N
NH
N-CH3 6a
O
3g
>10
>10
>10
5.10
>10
0.06
35%
ND
23%
5.80
ND
12%
1.50
9.45
12%
8.01
6.80
15%
13a
H
N
H
N
NH
N-CH3 7a
3h
>10
>10
6.20
>10
43%
ND
45%
ND
7.15
15%
36%
27%
O
14a
>10
0.07
12%
9%
0%
24%
O
O
O
X
X
N-CH3 6b
O
>10
>10
>10
0.60
0%
19%
0%.
7%
0%
0%
0%
12%
13b
N
H
N
H
O
N-CH3 7b
>10
>10
>10
0.60
0%
19%
0%
2%
0%
0%
0%
0%
O
O
14b
X
N
H
N
H
ND: Not determined.
a
Compounds tested at a concentration of 10 lM. Values correspond to n = 3 (SD <10%).
Inhibition of EGFR (purified from human carcinoma A431 cells) tyrosine kinase activity.
Inhibition of VEGFR-2 (recombinant human protein) tyrosine kinase activity.
Cell proliferation was measured by MTS assay at 10 lM (at least three independent experiments).
b
c
d
stitution at the 3- and 4-position by fluorine and chorine on the
phenyl ring (17) led to an excellent inhibitory activity against VEG-
FR-2. ortho-position of halide such as in compound 16 resulted in a
weak decrease of activity against VEGFR-2. However, introduction
of a bulkier naphthalene group (20–21) was envisaged and toler-
ated only with 2-naphtyl substituent (20): an unfavorable steric
hindrance was observed with 1-naphtyl (21) resulting in IC50 value
ther evaluated in cell-based assays but were inactive towards dif-
ferent cancer cell lines.
Table 3 shows the effects of variations of the linker on the cen-
tral phenyl ring of compound 17. Replacement of oxygen with a
nitrogen atom (23 and 24)33 led to a dramatically reduced enzy-
matic inhibition. The O-linkage therefore appears essential in order
to obtain a VEGFR-2 selective inhibitor. To understand the ob-
served difference, we explored the binding mode of these aryloxy
quinazolines with KDR.
of 5.50
l
M. Its isomer position 20 was selectively active against
M. These quinazolines were fur-
VEGFR-2 with IC50 value of 0.03
l

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Table 2
Enzymatic (EGFR/VEGFR-2) and cellular results for aryloxy-quinazolines derivatives 12–22
O
O
N
N
Compd
Enzymatic inhibitory (IC50
,
l
M)a
% Proliferative inhibitory (or IC50
,
l
M)d
EGFRb
VEGFR-2c
Human prostate
Human colorectal
Human breast Human endothelial-like
PC3
7.40
33%
HT29
ND
4.20
MCF-7
30%
26%
immortalized EAhy926
ND
5.10
IressaÒ
Vandetanib
0.07
0.80
14.80
0.10
R
X
H
H
N
N
13a
>10
>10
>10
>10
>10
>10
>10
>10
>10
0.06
0.07
0.40
0.05
0.09
0.04
0.06
0.05
0.03
23%
12%
30%
2%
12%
9%
12%
0%
15%
24%
0%
O
O
O
O
O
O
O
O
O
O
O
H
N
H
N
14a
12
15
16
17
18
19
20
O
O
O
O
O
O
O
O
O
O
H
N
H
N
8%
10%
10%
6.40
30%
9.80
23%
6.80
H
N
H
N
13%
10%
26%
7.70
17%
0%
0%
Br
F
H
N
H
N
35%
36%
40%
23%
5%
5.10
9.00
5.00
42%
0%
F
H
N
H
N
Cl
F
H
N
H
N
CF3
H
N
H
N
H
N
H
N
H
N
H
N
21
>10
>10
5.50
1.00
8%
17%
8%
8%
0%
9%
H
N
H
N
2228
42%
31%
O
O
ND: Not determined.
a
Compounds tested at a concentration of 10 lM. Values correspond to n = 3 (SD <10%).
Inhibition of EGFR (purified from human carcinoma A431 cells) tyrosine kinase activity.
Inhibition of VEGFR-2 (recombinant human protein) tyrosine kinase activity.
Cell proliferation was measured by MTS assay at 10 lM (at least three independent experiments).
b
c
d

A. Garofalo et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2106–2112
2111
Table 3
Biological activities of N-(3-chloro-4-fluorophenyl)-N0-{4-[6,7-dimethoxyquinazolin-4-yloxy]phenyl}urea 17 and the anilino analogs 23–24
H
N
H
N
Cl
F
O
X
N
O
O
N
Compd
X
Enzymatic inhibitory
(IC50
M)a
% Proliferative inhibitory (or IC50, l
M)d
,
l
EGFRb
VEGFR-2c
Human prostate PC3 Human Colorectal HT29 Human breast MCF-7 Human endothelial-like immortalized EAhy926
23
24
17
NH
>10
4.30
>10
0.04
9.40
ND
36%
6.20
ND
26%
7.70
35%
30%
5.30
29%
9.00
N-CH3 >10
>10
O
ND: Not determined.
a
Compounds tested at a concentration of 10
Inhibition of EGFR (purified from human carcinoma A431 cells) tyrosine kinase activity.
Inhibition of VEGFR–2 (recombinant human protein) tyrosine kinase activity.
Cell proliferation was measured by MTS assay at 10 lM (at least three independent experiments).
l
M. Values correspond to n = 3 (SD <10%).
b
c
d
Figure 3 shows models of 17, 23 and 24 bound to VEGFR-2, gen-
erated by molecular modeling.34 As these models suggest, the ani-
lino-urea 23 bound to the ATP-site of KDR in the same manner as
its ether analog 17. Particularly, the quinazoline cores adopt essen-
tially volumes and conformations with identical orientations. We
observed interactions between the CO and NH carbamate group
with the backbone of Asp1046 and the Lys868–Glu885 salt bridge,
respectively. H–bond interaction with Cys919 (between a cystein
NH and the N1 nitrogen of quinazoline) was established. However,
substitution by aniline at the 4-position of quinazoline (23) led to a
displacement of the phenyl ring containing the urea group into the
active site of VEGFR-2. This position would have a negative impact
on the inhibition potency and limit donor-acceptor interaction of
the urea moiety. In contrast, the NMe analog 24 adopts volumes
and conformations in opposite directions compared to 17 and 23.
The phenyl ring of 24 is approximately perpendicular to the plane
of the quinazoline core and this structure is expected to limit inter-
action with the active-site of VEGFR-2. This prediction is in line
with the weaker activity observed for the quinazoline sub-series.
We also briefly investigated the replacement of the terminal aro-
matic urea residue with an aliphatic group (data no shown) but
this substitution further limited donor–acceptor interaction of
urea, resulting in lower enzymatic activity of these derivatives.
Consistent with the SARs in other series of urea class VEGFR-2
inhibitors, a diaryl urea moiety is favorable for optimal interaction
with the hydrophobic back pocket of KDR kinase.
In summary, we have described the discovery, SAR studies and
preliminary biological evaluation of a novel series of a VEGFR-2
selective tyrosine kinase inhibitor. We have investigated the
replacement of linkers at the 4-position of a quinazoline skeleton
by aryloxy, aniline and N-methylaniline entities. The diaryl urea
of aryloxy-quinazoline potently inhibited VEGFR-2 at nanomolar
concentrations. However their activity against VEGFR-2 is not
associated with their antiproliferative activity on cell-based assays.
Molecular modeling established the interactions of these com-
pounds with the active binding site. Further modifications may
be undertaken on the two phenyl groups of the urea moiety, and
varying the methoxy groups on the quinazoline core may result
in a better affinity and good antiproliferative activities.
Acknowledgments
The authors are grateful to the ‘Ligue Nationale Contre le
Cancer’ (Comity of Pas-de-Calais-France) for its financial support.
Figure 3. Docking mode of compound 17 and its anilino analogs 23-24 at the ATP–
site of VEGFR–2.

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A. Garofalo et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2106–2112
(Sigma) supplemented with 10% fetal bovine serum, glutamine (2 mM),
penicillin (100 IU/mL), and streptomycin (100 g/mL). HT29 colon cancer cell
and EAhy926, umbilical cell immortalized, were grown at 37 °C in a humidified
We are grateful to Michael Howsam for critical comments on the
manuscript.
l
atmosphere containing 5% CO2 in DMEM + Glutamax-I (Gibco) supplemented
with 10% fetal bovine serum, penicillin (100 IU/mL), and streptomycin (100
mL).
lg/
References and notes
In the cell proliferation assay, cells were plated in triplicate on 96-well plates
(3000 cells per well) and incubated for 24 h. Cells were then incubated in
culture medium that contained various concentrations of tested compounds,
each dissolved in less than 0.1% DMSO. After 72 h, cell growth was estimated
by the colorimetric MTS test.
1. Levitzki, A. Acc. Chem. Res. 2003, 36, 462.
2. Cohen, P. Nat. Rev. Drug Disc. 2002, 1, 309.
3. Cohen, P. Eur. J. Biochem. 2001, 268, 5001.
4. Zwick, E.; Bange, J.; Ullrich, A. Trends Mol. Med. 2002, 8, 17.
5. Traxler, P.; Bold, G.; Buchdunger, E.; Caravatti, G.; Furet, P.; Manley, P.; O’Reilly,
T.; Wood, J.; Zimmermann, J. Med. Res. Rev. 2001, 21, 499.
6. Lohela, M.; Bry, M.; Tammela, T.; Alitalo, K. Curr. Opin. Cell Biol. 2009, 21, 154–
165.
7. Rahimi, N. Exp. Eye Res. 2006, 83, 1005.
8. Roskoski, R., Jr. Biochem. Biophys. Res. Commun. 2004, 319, 1.
9. Carmeliet, P. Nat. Med. 2003, 9, 653.
10. Pandya, N. M.; Dhalla, N. S.; Santani, D. D. Vasc. Pharmacol. 2006, 44, 265.
11. Otrock, Z. K.; Mahfouz, R. A. R.; Makarem, J. A.; Shamseddine, A. I. Blood Cell
Mol. Dis. 2007, 39, 212.
12. Roskoski, R., Jr. Biochem. Biophys. Res. Commun. 2008, 375, 287.
13. Roskoski, R., Jr. Crit. Rev. Oncol. Hematol. 2007, 62, 179.
14. Fayette, J.; Soria, J. C.; Armand, J. P. Eur. Cancer Res. 2005, 41, 1109.
15. Sun, S.; Schiller, J. H. Crit. Rev. Oncol. Hematol. 2007, 62, 93.
16. Denny, W. A. Il Farmaco 2001, 56, 51.
In vitro kinase assays: Kinase assays were performed in 96-well plates
(Multiscreen Durapore, Millipore) using [c-
32P]ATP (Amersham Biosciences)
and the synthetic polymer poly(Glu4/Tyr) (Sigma Chemicals) as a phospho-
acceptor substrate. Tested compounds were dissolved in DMSO, final
concentration of DMSO in assay solutions was 0.1%, which was shown to
have no effect on kinase activity.
EGFR tyrosine kinase activity: 20 ng of EGFR (purified from human carcinoma
A431 cells, Sigma Chemicals) were incubated for 1 h at 28 °C using various
concentrations of tested compounds in kinase buffer (HEPES 50 mM pH 7.5,
BSA 0.1 mg/mL, MnCl2 10 mM, MgCl2 5 mM, Na3VO4 100
lM, DTT 0.5 mM,
poly(Glu4/Tyr) 250 g/mL, ATP 5 M, [ Ci).
l
l
c
-
32P]ATP 0.5
l
VEGFR-2 tyrosine kinase activity: 10 ng of VEGFR-2 (Recombinant Human
Protein, Invitrogen) were incubated for 1 h at 28 °C using various
concentrations of tested compounds in kinase buffer (Tris 50 mM pH 7.5,
BSA 25
glycerophosphate 5 mM, poly(Glu4/Tyr) 250
0.5 Ci).
The reaction was stopped by adding 20
l
g/mL, MnCl2 1.5 mM, MgCl2 10 mM, DTT 2.5 mM, Na3VO4 100
lM, b-
17. Fry, D. W. Exp. Cell Res. 2003, 284, 131.
l
g/mL, ATP M,
5
l
[c-
32P]ATP
18. Bridges, A. J.; Zhou, H.; Cody, D.; Rewcastle, G. W.; Mc-Michael, A.; Showalter,
H. D.; Fry, D. W.; Kraker, A. J.; Denny, W. A. J. Med. Chem. 1996, 39, 267.
19. Garofalo, A.; Goossens, L.; Baldeyrou, B.; Lemoine, A.; Ravez, S.; Six, P.; David-
Coordonnier, M. H.; Bonte, J. P.; Depreux, P.; Lansiaux, A.; Goossens, J. F. J. Med.
Chem. 2010, 53, 8089.
l
lL of trichloroacetic acid 100%. Wells
were screened out and washed 10 times with trichloroacetic acid 10%. Plates
were counted in a Top Count for 1 min per well.
33. Melting points were determined in open capillary tubes on a Büchi reference B-
530 digital melting point apparatus and are uncorrected. Kieselgel 60 F-254
commercial plates were used for analytical TLC as well as UV light with/
without iodine to follow the course of the reaction. Silica gel Kieselgel Si 60,
0.063–0.200 mm (Merck) was used for column chromatography. The
structures of all compounds were determined by IR (using a Bruker VECTOR
22 instrument) 1H NMR (300 MHz) spectra were recorded on a Bruker AC300P
NMR spectrometer in DMSO-d6 or in CDCl3 at room temperature. APCI+
(Atmospheric Pressure Chemical Ionization) mass spectra were obtained on an
LC–MS system Thermo Electon Surveyor MSQ.
20. Ranson, M.; Wardell, S. J. Clin. Pharm. Ther. 2004, 29, 95.
21. Shepherd, F. A.; Rodrigues Pereira, J.; Ciuleanu, T.; Tan, E. H.; Hirsh, V.;
Thongprasert, S.; Campos, D.; Maoleekoonpiroj, S.; Smylie, M.; Martins, R.; van
Kooten, M.; Dediu, M.; Findlay, B.; Tu, D.; Johnston, D.; Bezjak, A.; Clark, G.;
Santabárbara, P.; Seymour, L. N. Eng. J. Med. 2005, 353, 123.
22. Nelson, M. H.; Dolder, C. R. Ann. Pharmacother. 2006, 40, 261.
23. Ciardiello, F.; Bianco, R.; Caputo, R.; Caputo, R.; Damiano, V.; Troiani, T.; Melisi,
D.; De Vita, F.; De Placido, S.; Bianco, A. R.; Tortora, G. Clin. Cancer Res. 2004, 10,
784.
24. Escudier, B.; Eisen, T.; Stadler, W. M.; Szczylik, C.; Oudard, S.; Siebels, M.;
Negrier, S.; Chevreau, C.; Solska, E.; Desai, A. A.; Rolland, F.; Demkow, T.;
Hutson, T. E.; Gore, M.; Freeman, S.; Schwartz, B.; Shan, M.; Simantov, R.;
Bukowski, R. M. N. Eng. J. Med. 2009, 49, 332.
Compound 17: 1H NMR (DMSO) dH 3.95 (s, 3H), 3.97 (s, 3H), 7.20–7.40 (m, 3H),
7.50–7.70 (m, 6H), 8.52 (s, 1H), 8.90 (s, 1H), 8.92 (s, 1H). LC–MS (APCI+): m/z
469 (MH+) and 471 (M+2+H+)—tr (min): 4.02.
Compound 23: 1H NMR (DMSO) dH 3.95 (s, 3H), 3.97 (s, 3H), 7.02 (m, 1H), 7.19
(s, 1H), 7.31 (m, 1H), 7.48 (d, 2H, J = 9.00 Hz), 7.62 (d, 2H, J = 9,00 Hz), 7.95 (s,
1H), 8.08 (m, 1H), 8.54 (s, 1H), 8.58 (s, 1H), 9.21 (s, 1H), 10.11 (s, 1H). LC–MS
(APCI+): m/z 468 (MH+) and 470 (M+2+H+)—tr (min): 3.11.
25. Garofalo, A.; Goossens, L.; Lemoine, A.; Farce, A.; Arlot, Y.; Depreux, P. J. Enzyme
Inhib. Med. Chem. 2010, 25, 158.
26. Dent, P.; Curiel, D. T.; Fisher, P. B.; Grant, S. Drug Resist. Update 2009, 12, 65.
27. Wedge, S. R.; Kendrew, J.; Hennequin, L. F.; Valentine, P. J.; Barry, S. T.; Brave, S.
R.; Smith, N. R.; James, N. H.; Dukes, M.; Curwen, J. O.; Chester, R.; Jackson, J. A.;
Boffey, S. J.; Kilburn, L. L.; Barnett, S.; Richmond, G. H.; Wadsworth, P. F.;
Walker, M.; Bigley, A. L.; Taylor, S. T.; Cooper, L.; Beck, S.; Jürgensmeier, J. M.;
Ogilvie, D. J. Cancer Res. 2005, 15, 389.
Compound 24: 1H NMR (DMSO) dH 3.61 (s, 3H), 3.88 (s, 3H), 3.95 (s, 3H), 6,58 (d,
1H, H, Jo = 7.40 Hz), 6.98 (d, 2H, J = 8.40 Hz), 7.05–7.15 (m, 2H), 7.20 (d, 2H,
J = 8.40 Hz), 7.64 (s, 1H), 7.79 (d, 1H, J = 4,40 Hz), 7.91 (s, 1H), 8.61 (s, 1H), 8.87
(s, 1H). LC–MS (APCI+): m/z 482 (MH+) and 484 (M+2+H+)—tr (min): 3.43.
34. Molecular modeling: All the calculations have been carried out under the Sybyl
28. Garofalo, A.; Goossens, L.; Lemoine, A.; Ravez, S.; Six, P.; Howsam, M.; Farce, A.;
Depreux, P. MedChemComm 2011, 2, 65.
6.9.1 molecular modeling package running on Silicon Graphics Octane
2
workstations. The ligands were built from the internal fragments library of
Sybyl and their geometry was optimized by the Powell method available in the
Maximin2 procedure to a gradient of 0.001 Kcal/mol Å. The dielectric constant
was set to 4 to implicitly represent a biological medium the atomic charges
were attributed following the Gasteiger–Hückel method and the energy
minimization was run with the Tripos force field. The structure of the
protein co-crystallized with an inhibitor was obtained from the Protein Data
Bank (http://www.pdb.org) under the entry 1YWN.
29. Furuta, T.; Sakai, T.; Senga, T.; Osawa, T.; Kubo, K.; Shimizu, T.; Suzuki, R.;
Yoshino, T.; Endo, M.; Miwa, A. J. Med. Chem. 2006, 49, 2186.
30. Garofalo, A.; Goossens, L.; Lebegue, N.; Depreux, P. Synthetic Commun., in press.
31. Gibson, K. H.; Grundy, W.; Godfrey, A.; Woodburn, J. R.; Ashton, S. E.; Curry, B.
J.; Scarlett, L.; Barker, A. J.; Brown, D. S. Bioorg. Med. Chem. Lett. 1997, 7, 2723.
32. Cell culture and cell proliferation assays: Human prostate cancer cells PC3 and
breast cancer cell line MCF7 were grown at 37 °C in a humidified atmosphere
containing 5% CO2, respectively in RPMI-1640 medium (Sigma) and MEM