Inhibition of tumor cell growth and angiogenesis by 7-Aminoalkoxy-4- aryloxy-quinazoline ureas, a novel series of multi-tyrosine kinase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2014.04.007

Several regulatory and signaling molecules governing angiogenesis are targets of interest for the development of drugs in the cancer, including growth factors such as Vascular Endothelial Growth Factor (VEGF) and Platelet-Derived Growth Factor (PDGF). A series of 4-aryloxy-6,7-dimethoxyquinazolines, previously synthesized in our laboratory, has shown a nanomolar inhibition of kinase enzymatic activity of VEGFR, PDGFR and c-Kit. We have therefore studied the impact of the variation in the 7-position substitution of the quinazoline core. Substitution by aminoalkoxy chains led to new highly potent ATP-competitive inhibitors of VEGFR, PDGFR and c-Kit enzyme with IC₅₀ values in the nanomolar range and this substitution has increased greatly antiproliferative activity on cancer cell lines (PC3, MCF7, HT29) and HUVEC (human umbilical vein endothelial cells). One of the most promising compounds (36) was assessed for its ability to limit the induction of web like network of capillary tubes by the human umbilical vascular endothelial cells (HUVEC) and for its ability to inhibit invasion.

Full text of DOI:10.1016/j.ejmech.2014.04.007

European Journal of Medicinal Chemistry 79 (2014) 369e381
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Inhibition of tumor cell growth and angiogenesis by 7-Aminoalkoxy-
4-aryloxy-quinazoline ureas, a novel series of multi-tyrosine kinase
inhibitors
,
,
,
,b
Séverine Ravez a b, Amélie Barczyk a b, Perrine Six a b, Aurélie Cagnon a
,
Antonio Garofalo a b, Laurence Goossens a , Patrick Depreux
,
,
b,
a,b
*
a Univ Lille Nord de France, F-59000 Lille, France
b Institut de Chimie Pharmaceutique Albert Lespagnol (ICPAL), UDSL, EA4481, 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:
Several regulatory and signaling molecules governing angiogenesis are targets of interest for the
development of drugs in the cancer, including growth factors such as Vascular Endothelial Growth Factor
(VEGF) and Platelet-Derived Growth Factor (PDGF). A series of 4-aryloxy-6,7-dimethoxyquinazolines,
previously synthesized in our laboratory, has shown a nanomolar inhibition of kinase enzymatic activity
of VEGFR, PDGFR and c-Kit. We have therefore studied the impact of the variation in the 7-position
substitution of the quinazoline core. Substitution by aminoalkoxy chains led to new highly potent
ATP-competitive inhibitors of VEGFR, PDGFR and c-Kit enzyme with IC50 values in the nanomolar range
and this substitution has increased greatly antiproliferative activity on cancer cell lines (PC3, MCF7,
HT29) and HUVEC (human umbilical vein endothelial cells). One of the most promising compounds (36)
was assessed for its ability to limit the induction of web like network of capillary tubes by the human
umbilical vascular endothelial cells (HUVEC) and for its ability to inhibit invasion.
Received 9 September 2013
Received in revised form
1 April 2014
Accepted 4 April 2014
Available online 5 April 2014
Keywords:
VEGFR
PDGFR-ß
c-Kit
Multi-kinase inhibitors
Anti-angiogenic agents
Cancer
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
Vascular endothelial growth factor (VEGF) has been identified as
the most common regulator of tumor angiogenesis, vascular
Hypoxia is a salient feature of many types of solid cancers and
arises as the result of spatial disorganization and flow based
disruption of abnormal microvasculature initiated by the growing
tumor. The impact of tumor hypoxia is multifaceted, with effects on
several aspects of tumor biology, including genetic instability,
angiogenesis, invasiveness, survival and metabolism [1]. Angio-
genesis, the formation of new blood vessels from the pre-existing
vasculature, is critical for development and subsequent growth of
tumors and is a fundamental process for the formation of metas-
tases [2]. Therefore, the inhibition of angiogenesis is an attractive
therapeutic approach to treat many human tumors [3].
permeability, endothelial cell activation, proliferation, and migra-
tion [4]. VEGF is a member of a family including of five homodi-
meric glycoprotein members (VEGF-A; VEGF-B; VEGF-C; VEGF-D
and placenta growth factor, PlGF) and exerts its activity through
binding to three high-affinity transmembrane receptors: VEGFR-1,
VEGFR-2 (also known as KDR) and VEGFR-3 [5]. These tyrosine
kinase receptors (TKRs) consist of an extracellular ligand-binding
domain connected to an intracellular tyrosine kinase domain
through a segment in the plasma membrane. Binding of VEGF to
VEGFR results in receptor dimerization and stimulation of the re-
ceptor associated tyrosine kinase activity, which leads to phos-
phorylation of tyrosine residues and initiates a signaling cascade
(such as ERK (extracellular-signal regulated kinase)/MAPK
(mitogen-activated protein kinase) or PI3K/Akt pathways) [6].
Preventing activation of VEGF receptors has been shown to
inhibit angiogenesis, tumor progression, and dissemination [7,8].
Bevacizumab, a recombinant humanized monoclonal antibody
against VEGF, has been approved as first-line therapy for metastatic
colorectal cancer [9]. In addition, small synthetic molecules that act
with an ATP-competitive mechanism by binding the ATP pocket of
Abbreviations: ATP, adenosine 50-triphosphate; DMSO, dimethyl sulfoxide;
EGFR, epidermal growth factor receptor; EtOAc, ethyl acetate; FC, flash chroma-
tography; HUVEC, human umbilical vein endothelial cell; MeOH, methanol; MKI,
multi-kinase inhibitor; PDGFR, platelet-derived growth factor receptor; RCC, renal
cell carcinoma; rt, room temperature; TKR, tyrosine kinase receptor; VEGFR,
vascular endothelial growth factor receptor.
* Corresponding author. Institut de Chimie Pharmaceutique Albert Lespagnol
(ICPAL), UDSL, EA4481, F-59006 Lille, France.
E-mail address: laurence.goossens@univ-lille2.fr (L. Goossens).
http://dx.doi.org/10.1016/j.ejmech.2014.04.007
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

370
S. Ravez et al. / European Journal of Medicinal Chemistry 79 (2014) 369e381
Table 1
the protein kinase domain have been approved for treatment of
several cancers [10].
IC50 values (nM) of kinase inhibition.
As a result of the substantial sequence homology shared by
three isoforms of VEGFR and other RTKs (such as platelet-derived
growth factor receptor, PDGFR and c-Kit) in their catalytic do-
mains, VEGFR inhibitors are not selective for one kinase [11].
However, their poor selectivity for VEGFRs may be an advantage
because PDGFR and c-Kit must be also inhibited in order to prevent
cancer evolution. Additionally, during VEGF inhibition, a hypoxic
environment can drive a shift from VEGF dependent signaling to
other proangiogenic pathways. For this reason, the use of multi-
targeted inhibitors should be of great therapeutic benefit. Multi-
kinase inhibitors (MKIs) are a new class of antiangiogenic thera-
peutics that inhibit kinase activities of several receptors, and recent
trial data have demonstrated the efficacy of MKIs in the treatment
of metastatic renal cell carcinoma (RCC), brain cancer or hepato-
cellular carcinoma [12,13,14]. Based on these positive clinical trial
results, various oral anti-angiogenic MKIs, like sunitinib, sorafenib
or pazopanib, have gained regulatory approval for use in RCC
(Fig. 1).
Previously, our group has described structure-activity relation-
ships (SAR) studies and preliminary biological evaluation of N-
(aromatic)-N0-{4-[(6,7-dimethoxyquinazolin-4-yl)oxy]-phenyl}
urea derivatives which inhibited VEGFR-2 at nanomolar concen-
trations [15,16,17].
More recently, to determine the kinase selectivity profile of
three selected compounds of this series (1e3), several kinases were
chosen for screening. The kinase panel consists of members of
VEGFR family (VEGFR-1, -2 and -3), PDGFR-ß, c-Kit, EGFR, c-Met, Src
and Raf. The data of kinase inhibition are summarized in Table 1.
The compounds (1e3) show low (or no) inhibitory efficacy
against four tested kinases of the panel (EGFR, c-Met, Src and Raf)
but inhibit VEGFR (-1, -2 and -3), PDGFR-ß and c-Kit at nanomolar
values. However, their excellent kinase inhibitory effect against
VEGFR (-1, -2 and -3), PDGFR-ß and c-Kit is not associated with an
antiproliferative activity on cell-based assays on PC3, HT29 and
Enzymatic inhibitory (IC50, nM)
Compound
1
2
3
X
H
CH3
Cl
EGFRa
>10,000
>10,000
35
>10,000
18
VEGFR-1b
VEGFR-2c
VEGFR-3b
PDGFR-ßb
c-Kitb
46
6
5
15
89
102
8
15
20
38
2000
3130
112
9
5
16
c-Metb
Srcb
2310
5470
934
>10,000
>10,000
2810
Rafb
a
Inhibition of EGFR (purified from human carcinoma A431 cells) tyrosine kinase
activity.
b
Inhibition of VEGFR-1, VEGFR-3, PDGFR-ß, c-Kit, c-Met, Src and Raf was per-
formed at ProQinase GmbH (Freiburg. Germany).
c
Inhibition of VEGFR-2 (recombinant human protein) tyrosine kinase activity.
evaluated for their antiproliferative activity towards five cell lines
(PC3, HT29, MCF7, HUVEC and MRC5) by MTS test.
2. Chemistry
We have synthesized new quinazoline compounds which were
divided in five groups distinguished by the nature of the ether
linker at the C-6 and C-7 positions of the quinazoline core: 6-
MCF7 (IC50 > 10 mM). In this study, we aimed to improve both the
butoxy-7-diethylaminoethoxy-
(series
A);
6-methoxy-7-
enzymatic and proliferative inhibitions of these N-(aromatic)-N0-
{4-[(6,7-dimethoxyquinazolin-4-yl)oxy]-phenyl}urea derivatives.
The effect of varying the O-substituent at the 7-position on the
quinazoline core was studied in the laboratory on 4-anilino de-
rivatives [18]. Enzymatic and antiproliferative activities were
influenced by the side chain at the 7-position of quinazoline.
Indeed, introduction of aminoalkyl such as piperidinoethoxy or
diethylaminoethoxy side chains, increased inhibitory activity
against VEGFR-2 and antiproliferative activity on cancer cell lines.
With regard to these observations, we designed a novel series of 7-
aminoalkoxy-4-aryloxy-quinazoline ureas and we report here
synthesis and biological evaluation. Inhibitory activity against nine
tyrosine kinases was determined and these compounds were
diethylaminoethoxy- (series B); 6-methoxy-7-piperidino-ethoxy-
(series C); 6-methoxy-7-pyrrolidinoethoxy- (series D) and 6-
methoxy-7-piperidinopropoxyquinazoline (series E) (Table 2).
The key intermediates 4-chloro-7-aminoalkoxyquinazolines
(16e18) were obtained in five steps for series C, D and E (Scheme 1).
Compounds (4e6) were synthesized from methyl vanillate by
alkylation reaction in the presence of appropriate commercial
chloroalkane and potassium carbonate in acetone. Nitration was
realized in the presence of a mixture of nitric acid and tin tetra-
chloride in dichloromethane [19]. Nitration occurs selectively at the
2-position due to electronic effects of different substituents on the
benzene ring. The position of nitro group was confirmed by 1H
NMR. The nitro derivatives (7e9) were obtained with yields
Fig. 1. Examples of multi-tyrosine kinase inhibitors marketed.

S. Ravez et al. / European Journal of Medicinal Chemistry 79 (2014) 369e381
371
Table 2
Target compounds
between 24 and 78%. Catalytic hydrogenation of the nitro group in
the presence of RaneyÒ nickel led to amino derivatives (10e12)
(41e79% of yields) which were converted by cyclization with
formamide in the presence of ammonium formate to obtain the
cyclised compounds (13e15) with yields between 59 and 75% [20].
Finally, cyclised compounds were reacted with phosphorus oxy-
chloride to obtain the key intermediates 4-chloroquinazolines (16e
18). [21]
As depicted in Scheme 2, the amino groups of commercial 4-
aminophenols were converted by condensation with commercial
3-bromophenylisocyanate in pyridine at room temperature to
obtain the corresponding ureas (21e23) in high yields and short
time reaction. [22]
The synthesis of the final products is illustrated in Scheme 3. The
4-chloroquinazolines 19 and 20, belonging respectively at series A
and B, were prepared according to described procedure using
respectively methyl vanillate and methyl 3,4-dihydroxybenzoate as
starting materials [23]. Reaction of chloride derivatives (16e20)
with appropriate phenolurea (21e23) in presence of tetra-N-
butylammonium bromide in 2-butanone and 20% solution of so-
dium hydroxide mixture allowed the obtaining of desired products
of various series with low yields (24e37).
Series
A
R1
R2
B
C
3. Results and discussion
D
In this section, we describe the biological evaluation of the
synthesized 7-aminoalkoxyquinazoline derivatives concerning
their inhibitory efficacy on several protein kinases and their anti-
proliferative activity. Vandetanib (EGFR/VEGFR inhibitor) and/or
antiangiogenic inhibitor cediranib (VEGFR, PDGFR and c-Kit
E
Scheme 1. Reagents and conditions: (i) aminoalkyl chloride, K2CO3, acetone, reflux; (ii) HNO3, SnCl4, CH2Cl2, ꢀ70 ꢁC; (iii) RaneyÒ Ni, H2, MeOH/CH2Cl2, rt; (iv) HCONH2, HCOONH4,
reflux; (v) POCl3, reflux.

372
S. Ravez et al. / European Journal of Medicinal Chemistry 79 (2014) 369e381
Scheme 2. Reagents and conditions: (i) pyridine, rt, nitrogen atmosphere.
inhibitor) were used as the reference substances in the following
evaluations.
inhibitory activity against VEGFR, PDGFR-ß and c-Kit with IC50
values < 12 nM. In addition, the length of the linker shows a sig-
nificant difference in activity against EGFR. Indeed, compounds
(35e37) of series E show an EGFR inhibition with micromolar IC50
while the compounds of other series, with aminoethoxy group,
3.1. Kinase selectivity
cause no EGFR inhibition at 10 mM concentration.
The synthesized compounds were tested on a panel of nine
isolated kinase receptors including: EGFR, VEGFR-1, VEGFR-2,
VEGFR-3, PDGFR-ß, c-Kit, c-Met, Src and Raf. Inhibitory activity
against EGFR and VEGFR-2 tyrosine kinases was determined by our
team by measuring the levels of phosphorylation of the tyrosine-
For kinases c-Met, Src and Raf, the inhibition values are low for
all compounds. However, no-substitution of the aromatic group by
a chlorine or methyl group (X ¼ H: 26, 29, 32) is tolerated and
causes a low increase in activity against c-Met and Src. Unlike, in-
hibition of Raf is improved when the aromatic group is substituted
by a chlorine (X ¼ Cl: 28, 31, 34, 37).
specific peptides (poly(Glu Tyr)substrate) in vitro using [g-
32P]
ATP. Inhibition of VEGFR-1, VEGFR-3, PDGFR-ß, c-Kit, c-Met, Src and
Raf was performed at ProQinase GmbH (Freiburg, Germany) by a
radiometric protein kinase assay (33PanQinaseÒ activity assay). The
inhibition of kinase data are summarized in Table 3.
All synthesized compounds show higher affinity for VEGFR (1, 2
and 3), PDGFR-ß and c-Kit (IC50 values at nanomolar range) than for
c-Met, Src, Raf and EGFR, like reference cediranib. The molecules
interfering simultaneously with multiple RTKs might be more
effective than single target agents. With the approval by FDA of
sorafenib and sunitinib e targeting VEGFR, PDGFR, FLT-3 and c-Kit
e a new generation of anti-cancer drugs, able to inhibit more than
one pathway, would probably play a major role.
Compounds of series A (24, 25), with a butoxy chain in C-6
position, show a decrease in activity against VEGFR, PDGFR-ß and c-
Kit compared to the reference compounds (1e3). Series B (26e28),
with a methoxy chain in C-6 position and a diethylaminoethoxy
group in C-7 position, were more efficient than their 6-butoxy-
analogs (24, 25) with IC50 values at nanomolar range against
VEGFR, PDGFR-ß and c-Kit. Cyclisation of diethylaminoethoxy
group in piperidino- (series C, compounds 29e31) or pyrrolidi-
noethoxy (series D, compounds 32e34) chain is reasonably well
tolerated. Replacement of piperidinoethoxy chain by piper-
idinopropoxy (series E, compounds 35e37) led to an excellent
3.2. Cellular inhibitory
Antiproliferative activities of compounds were evaluated to-
ward the hormone-independent PC3 prostate cancer cells, MCF7
breast cancer cells and HT29 colon cancer cells by MTS test. The
Scheme 3. Reagents and conditions: (i) nBu4NþBrꢀ, 2-butanone/20% NaOH, 100 ꢁC.

S. Ravez et al. / European Journal of Medicinal Chemistry 79 (2014) 369e381
373
Table 3
IC50 values (nM) of enzymatic inhibition (EGFR, VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-ß, c-Kit, c-Met, Src and raf) .
Enzymatic inhibitory (IC50, nM)
Series
R1
R2
X
Compd
EGFRa
VEGFR-1b
VEGFR-2c
VEGFR-3b
PDGFR-ßb
c-Kitb
c-Metb
Srcb
Rafb
Vandetanib
Cediranib
A
800
150
39
122
59
69
14
12
44
260
11
199
112
5300
38
61
N.D.
1530
55
N.D.
967
1770
3320
N.D.
156
1090
2130
N.D
>10,000
308
2100
>10,000
>10,000
H
CH3
24
25
395
168
273
B
C
D
E
H
CH3
Cl
26
27
28
>10,000
>10,000
>10,000
7
19
4
1
8
4
22
12
13
7
98
15
7
91
9
963
1100
1420
232
1900
283
245
400
60
H
CH3
Cl
29.HCl
30
31
>10,000
>10,000
>10,000
11
6
5
4
2
3
26
19
17
8
51
64
12
25
22
977
3440
4710
262
604
654
356
114
119
H
CH3
Cl
32
33
34
>10,000
>10,000
>10,000
5
5
5
3
2
1
23
14
8
4
36
17
6
13
13
706
2870
1850
239
548
254
205
103
63
H
CH3
Cl
35
36.HCl
37
9710
2300
1930
4
5
3
2
4
1
12
8
6
4
9
5
6
9
5
350
1390
619
268
120
73
155
126
66
N.D. Not determined.
a
Inhibition of EGFR (purified from human carcinoma A431 cells) tyrosine kinase activity.
Inhibition of VEGFR-1, VEGFR-3, PDGFR-ß, c-Kit, c-Met, Src and Raf was performed at ProQinase GmbH (Freiburg, Germany).
Inhibition of VEGFR-2 (recombinant human protein) tyrosine kinase activity.
b
c
synthesized compounds were evaluated also for their anti-
proliferative activity towards the human umbilical vein endothelial
cells (HUVEC) because of their role in angiogenesis (Table 4).
Introduction of aminoalkyl in C-7 position of quinazoline
allowed to a significant increase in inhibition effects on the growth
of three cancer cell lines PC3, HT29 and MCF7. The cellular results of
all compounds are equivalent or better than reference compounds
(vandetanib and cediranib). The best results are obtained for the
For one of most promising compounds (36), displaying potent
enzymatic inhibitory (IC50 on VEGFR-(1, 2 and 3), PDGFR-ß and c-
Kit < 9 nM), antiproliferative activities on several cancer cell lines
(zmM on PC3, HT29 and MCF7) and low proliferation inhibition on
MRC5, follow-up studies were realized.
For the first time, kinetic study of proliferation inhibition was
performed on HT29 with compound 36. The antiproliferative ac-
tivity of compound 36 was evaluated by MTS assay. The viability
was determined at 24, 48 and 72 h (Fig. 3).
inhibition of colon cancer cells HT29 (IC50 z 1 mM). The difference
between cellular and enzymatic results can be explained because
cancer cell lines PC3, HT29 and MCF7 are not only dependent on the
VEGFR signal.
Replacing O-methoxy by aminoalkoxy groups at the 7-position
allowed to keep good levels of inhibition against HUVEC
As outlined in Fig. 3, IC50 of compound 36 at 72 h is 1.00
HT29. At 48 h, the IC50 for compound 36 is close to that IC50 ob-
M).
Compound 36 was also assessed for its ability to limit the in-
duction of web like network of capillary tubes by the human um-
bilical vascular endothelial cells (HUVEC) and for its ability to
inhibit cell invasion.
mM on
tained at 72 h (IC50 48 h ¼ 1.75
m
(IC50 ꢂ 1
mM). Values are similar whatever the nature of the amine
(diethylamino-, piperidino-, pyrrolidino- moiety) or the length of
the linker (ethoxy or propoxy). IC50 values of proliferation inhibi-
tory in HUVEC are lower than cancer cell lines because normal
primary HUVEC are only dependent on the VEGFR signal.
3.3. Endothelial tube formation
Inhibition of proliferation of compounds was evaluated on non-
Endothelial cell tube formation assays are useful indicators of
angiogenesis potential. The antiangiogenic effect was evaluated on
HUVEC by treating with a reference compound (cediranib) and
compound 36. HUVEC were placed on MatrigelÒ in order to imitate
in vivo HUVEC and treated with several concentrations of cediranib
cancer cell lines (MRC5, human fetal lung fibroblast cells) at 1
mM.
The value of 1 M has been selected in accordance with the ob-
m
tained IC50 on HT29. In order to assess the selectivity of our com-
pounds for cancer cells versus non-cancer cells, percentages of
inhibition at 1 mM on HT29 are also shown in Fig. 2. Vandetanib and
or of compound 36 (0.1, 1, 5 and 10 mM) (Fig. 4).
cediranib were used as the reference substances.
The compound 36 inhibited significantly the formation of
endothelial tubes in a dose-dependent manner with lower con-
centrations than those of the reference compound, cediranib.
The results showed that the inhibition of proliferation of our
multi-kinase inhibitors against the cancer cell line HT29 was
significantly higher than that against the nonmalignant cell line
MRC5 at 1 mM, with selectivity similar to those of tyrosine kinase
3.4. Cell invasion
inhibitors marketed like vandetanib and cediranib. The inhibition
values on MRC5 are always below 15%, except for the compound 37
which present a very strong proliferation inhibition on MRC5 (40%
We investigated also the effects of compound 36 at several
concentrations (0.01, 0.1 and 1
mM) on the invasiveness of HUVEC
using a Boyden chamber assay. MatrigelÒ was applied to the filter
at 1 mM).

374
S. Ravez et al. / European Journal of Medicinal Chemistry 79 (2014) 369e381
Table 4
Cellular inhibition (PC3, prostate cancer cell; HT29, colon cancer cell; MCF7, breast cancer cell; HUVEC, human umbilical vein endothelial cell) results of compounds (24e37) .
Proliferation inhibitory (IC50, mM)a
Series
R1
R2
X
Compd
PC3
HT29
MCF7
HUVEC
Vandetanib
Cediranib
A
7.30 ꢃ 3.00
>10
1.76 ꢃ 0.78
1.61 ꢃ 0.49
1.10 ꢃ 0.09
1.29 ꢃ 0.54
9.57 ꢃ 0.41
7.94 ꢃ 1.91
0.91 ꢃ 0.05
1.45 ꢃ 1.11
4.39 ꢃ 1.69
0.27 ꢃ 0.14
1.53 ꢃ 0.73
0.78 ꢃ 0.22
H
CH3
24
25
3.77 ꢃ 0
3.94 ꢃ 0.07
B
C
D
E
H
CH3
Cl
26
27
28
29
30
31
32
33
34
35
36
37
2.76 ꢃ 0.44
>10
1.22 ꢃ 0.66
4.84 ꢃ 0.25
1.19 ꢃ 0.34
1.60 ꢃ 0.08
1.54 ꢃ 0.61
2.26 ꢃ 0.53
1.61 ꢃ 0.08
1.97 ꢃ 1.11
1.46 ꢃ 0.26
1.08 ꢃ 0.15
1.00 ꢃ 0.11
0.67 ꢃ 0.01
3.83 ꢃ 0.43
>10
4.97 ꢃ 1.75
7.16 ꢃ 1.06
5.19 ꢃ 1.09
5.48 ꢃ 0
3.90 ꢃ 0.26
6.79 ꢃ 1.68
5.07 ꢃ 1.35
2.79 ꢃ 1.54
5.02 ꢃ 1.19
2.78 ꢃ 0.74
0.79 ꢃ 0.17
1.39 ꢃ 0.60
0.34 ꢃ 0.11
0.33 ꢃ 0.23
0.49 ꢃ 0.36
0.48 ꢃ 0.06
0.38 ꢃ 0.03
0.51 ꢃ 0.26
0.48 ꢃ 0.06
0.12 ꢃ 0.01
0.33 ꢃ 0.17
0.17 ꢃ 0.02
4.74 ꢃ 1.00
6.58 ꢃ 1.17
5.39 ꢃ 1.03
7.03 ꢃ 0.60
5.32 ꢃ 0.10
6.59 ꢃ 1.19
3.38 ꢃ 0.16
1.33 ꢃ 0.20
4.09 ꢃ 0.13
1.94 ꢃ 0.03
H
CH3
Cl
H
CH3
Cl
H
CH3
Cl
Data are represented as the mean ꢃ SEM of at two experiments performed in triplicate. Higher concentrations were not used to avoid precipitation of the compound in the
culture medium.
a
Cell proliferation was realized by MTS assay.
membrane, and the number of HUVEC that penetrated the
MatrigelÒ and membrane was quantified (Fig. 5).
4. Conclusion
As shown in Fig. 5, compound 36 inhibited the invasiveness of
HUVEC in a dose-dependent manner. Treatment with compound
36 at 0.01, 0.1 and 1 mM inhibited invasion by 46%, 67%, and 77%,
In summary, a series of 7-aminoalkoxy-4-aryloxy-quinazoline
ureas have been synthesized. These derivatives are highly potent
inhibitors of VEGFR (1, 2 and 3), PDGFR-ß and c-Kit with IC50 in the
nanomolar range. Varying the methoxy groups on the 7-position of
the quinazoline scaffold by addition of a basic side chain (dieth-
ylamino-alkoxy, piperidino-alkoxy or pyrrolidino-alkoxy) led to
revealing a range of potent VEGFR, PDGFR-ß and c-Kit inhibitors
which exhibited a significant antiproliferative activities on various
cancer cell lines (PC3, HT29 and MCF7) and on HUVEC, but have
respectively.
Considering that tube formation and invasion are highly rele-
vant properties in the process of angiogenesis, our results showed
that potent multi-kinase inhibitor 36 inhibited angiogenesis by
preventing tube formation and suppressing endothelial cells
invasion.
100
90
***
80
70
60
**
**
**
**
50
40
30
20
10
0
**
**
*
*
*
**
*
MRC5
HT29
Compounds
Fig. 2. Effect of compounds (24e37) on the cell proliferation of human fetal lung fibroblast cells MRC5 and colon cancer cells HT29 at 1
mM. Cell proliferation was realized by MTS
assay. Data are represented as the mean ꢃ SEM of at two experiments performed in triplicate. Higher concentrations were not used to avoid precipitation of the compound in the
culture medium. *p < 0.05; **p < 0.01; ***p < 0.001 (Statistical analyzes were performed using Statistica software version 6 (StatSoftÒ). In order to examine the effect of our
compounds, we applied Wilcoxon non-parametric matched pairs signed ranks test. Results were taken as significant if p < 0.05).

S. Ravez et al. / European Journal of Medicinal Chemistry 79 (2014) 369e381
375
of each compound was confirmed by IR (Bruker VECTOR 22 in-
100
80
60
40
20
0
strument) and 1H NMR (300 MHz, Bruker AC300P spectrometer).
24H
48H
72H
Chemicals shifts (d) are reported in parts per million downfield
from TMS. J values are in hertz, and the splitting patterns are
abbreviated as follows: s, singlet; d, doublet; t, triplet; q, quartet; m,
multiplet. The purity of the compounds was tested by HPLC sepa-
ration followed by APCIþ (atmospheric pressure chemical ioniza-
tion) mass spectral detection on an LC-MS system, Thermo Electon
Surveyor MSQ, and was >95%. Purity of all tested compounds is
superior of 98%. HRMS experiments were performed on Q Exactive
Benchtop LC-MS/MS (Thermo Scientific).
-9
-8
-7
-6
-5
-4
log[36], M
5.1.1. General procedure for aminoalkoxy derivatives (4e6)
To a solution of methyl vanillate (6 g, 32.93 mmol) in acetone
(100 mL), were added the appropriate chloroalkane (39.52 mmol)
and K2CO3 (131.72 mmol). After 5 h at reflux, the mixture was
cooled to room temperature and filtered. The solvent was removed
under reduced pressure and the residue dissolved in a 10% solution
of K2CO3. The aqueous solution was extracted with EtOAc, dried
over MgSO4 and the solvent was removed under reduced pressure.
IC50 24H
4.16 µM
IC50 48H
1.75 µM
IC50 72H
1.00 µM
Fig. 3. Proliferation inhibition of compound 36 on HT29 at 24 h, 48 h and 72 h. Cell
proliferation was realized by MTS assay. Data are represented as the mean ꢃ SEM of at
two experiments performed in triplicate. Higher concentrations were not used to avoid
precipitation of the compound in the culture medium.
5.1.1.1. Methyl
Brown oil (95%). TLC: Rf 0.8 (CH2Cl2:MeOH(NH3) 9:1 v/v). IR (cmꢀ1):
2933 (piperidine), 1713 (C]O). 1H NMR (DMSO-d6):
(ppm) 1.40e
1.47 (m, 2H, CH2), 1.57e1.65 (m, 4H, 2 CH2), 2.51e2.54 (m, 4H, 2
CH2), 2.84 (t, 2H, J ¼ 6.2 Hz, CH2), 3.90 (s, 3H, OCH3), 3.91 (s, 3H,
OCH3), 4.21 (t, 2H, J ¼ 6.2 Hz, CH2), 6.90 (d, 1H, J ¼ 8.1 Hz, ArH), 7.54
(d, 1H, J ¼ 1.8 Hz, ArH), 7.66 (dd, 1H, J ¼ 8.1, 1.8 Hz, ArH). LC-MS
(APCIþ): m/z calcd for C16H23NO4 294 [(MþH)þ].
3-methoxy-4-piperidinoethoxybenzoate
(4).
lower proliferation inhibition against the normal cells MRC5.
Further investigations suggested that multi-kinase inhibitor 36
strongly represses the angiogenic process by inhibiting endothelial
cell invasion and preventing tube formation with lower concen-
trations than those of the reference compound (cediranib). The new
quinazoline molecules with a highly potent inhibition of VEGFR,
PDGFR-ß and c-Kit and superior antiproliferative activities on
cancer cell lines compared to the reference compounds (1e3) have
been described, and the objective is thus fully reached.
d
5.1.1.2. Methyl
Brown oil (89%). TLC: Rf 0.9 (CH2Cl2:MeOH(NH3) 9:1 v/v). IR (cmꢀ1):
2950 (pyrrolidine), 1711 (C]O). 1H NMR (DMSO-d6):
(ppm) 1.81
3-methoxy-4-pyrrolidinoethoxybenzoate
(5).
Further pharmacological evaluations in vivo with several 7-
aminoalkoxy-4-aryloxy-quinazoline ureas are currently under
investigation.
d
(m, 4H, 2 CH2), 2.64 (m, 4H, 2CH2), 2.99 (t, 2H, J ¼ 6.0 Hz, CH2), 3.89
(s, 3H, OCH3), 3.91 (s, 3H, OCH3), 4.21 (t, 2H, J ¼ 6.0 Hz, CH2), 6.90 (d,
1H, J ¼ 8.4 Hz, ArH), 7.54 (d,1H, J ¼ 1.8 Hz, ArH), 7.65 (dd,1H, J ¼ 8.4,
1.8 Hz, ArH). LCeMS (APCIþ): m/z calcd for C15H21NO4 280
[(MþH)þ].
5. Experimental methods
5.1. General chemistry
Melting points were determined with a Büchi 535 capillary
melting point apparatus and are uncorrected. Macherey Nagel
PolygramÒ sil G/UV254 commercial plates were used for analytical
TLC as well as UV light and/or with iodine to follow the course of
the reaction. Flash chromatography (FC) was performed with silica
gel Macherey Nagel Si 60, 0.015e0.040 mm (Merck). The structure
5.1.1.3. Methyl
Brown oil (94%). TLC: Rf 0.9 (CH2Cl2:MeOH(NH3) 9:1 v/v). IR (cmꢀ1):
2935 (piperidine), 1709 (C]O). 1H NMR (DMSO-d6):
(ppm) 1.37e
1.48 (m, 2H, CH2), 1.51e1.60 (m, 4H, 2CH2), 2.03 (m, 2H, CH2), 2.37e
2.41 (m, 4H, 2 CH2), 2.46 (t, 2H, J ¼ 7.4 Hz, CH2), 3.88 (s, 3H, OCH3),
3.90 (s, 3H, OCH3), 4.12 (t, 2H, J ¼ 7.0 Hz, CH2), 6.90 (d,1H, J ¼ 8.4 Hz,
3-methoxy-4-piperidinopropoxybenzoate
(6).
d
Fig. 4. Effect of cediranib and compound 36 on tube formation Images depicting the formation of HUVEC capillary-like tubular network by treatment with cediranib or compound
36 (0.1, 1, 5 and 10 M).
m

376
S. Ravez et al. / European Journal of Medicinal Chemistry 79 (2014) 369e381
Fig. 5. Invasion inhibition on HUVEC in a Boyden chamber assay of cediranib and compound 36 Images depicting the penetration of HUVEC through the filter membrane. The
number of HUVEC that penetrated the membrane was quantified.
ArH), 7.53 (d, 1H, J ¼ 1.9 Hz, ArH), 7.63 (dd, 1H, J ¼ 8.5, 2.0 Hz, ArH).
LCeMS (APCIþ): m/z calcd for C17H25NO4 308 [(MþH)þ].
4H, 2 CH2), 3.84 (s, 3H, OCH3), 3.96 (s, 3H, OCH3), 4.53 (t, 2H,
J ¼ 5.7 Hz, CH2), 7.39 (s, 1H, ArH), 7.78 (s, 1H, ArH). LC-MS (APCIþ):
m/z calcd for C15H20N2O6 325 [(MþH)þ].
5.1.2. General procedure for nitro derivatives (7e9)
To
a
stirred mixture of aminoalkoxy derivatives (4e6)
5.1.2.3. Methyl 2-nitro-4-piperidinopropoxy-5-methoxybenzoate (9).
Yellow oil (78%). TLC: Rf 0.7 (CH2Cl2:MeOH(NH3) 9:1 v/v). IR (cmꢀ1):
2935 (piperidine), 1731 (C]O), 1520 (NO2). 1H NMR (DMSO-d6):
(15.30 mmol) in dichloromethane (100 mL), a mixture of nitric acid
(45.90 mmol) and tin tetrachloride (45.90 mmol) diluted in 20 mL
of dichloromethane was slowly added, while maintaining the
temperature at ꢀ70 ꢁC. After the addition, the mixture was stirred
for an additional 4 h at room temperature. The mixture was
neutralized by 10% K2CO3 solution and then the aqueous solution
was extracted with dichloromethane, dried over CaCl2 and the
solvent was removed under reduced pressure.
d
(ppm) 1.38e1.49 (m, 2H, CH2), 1.54e1.65 (m, 4H, 2CH2), 2.05 (m,
2H, CH2), 2.37e2.45 (m, 4H, 2CH2), 2.49 (t, 2H, J ¼ 7.3 Hz, CH2), 3.90
(s, 3H, OCH3), 3.90 (s, 3H, OCH3), 4.16 (t, 2H, J ¼ 7.3 Hz, CH2), 7.07 (s,
1H, ArH), 7.48 (s, 1H, ArH). LC-MS (APCIþ): m/z calcd for C17H24N2O6
353 [(MþH)þ].
5.1.3. General procedure for methyl-2-aminobenzoate derivatives
(10e12)
5.1.2.1. Methyl 2-nitro-4-piperidinoethoxy-5-methoxybenzoate (7).
Yellow oil (58%). TLC: Rf 0.6 (CH2Cl2:MeOH(NH3) 9:1 v/v). IR (cmꢀ1):
2485 (piperidine), 1730 (C]O), 1522 (NO2). 1H NMR (DMSO-d6):
To a solution of nitro compounds (7e9) (15.30 mmol) in 100 mL
of a mixture of methanol and dichloromethane (5:5), was added
RaneyÒ nickel. After 18 h at room temperature in hydrogen atmo-
sphere, the mixture was filtered. The solvent was removed under
reduced pressure and the residue was purified by FC (CH2Cl2/
MeOH, 9:1).
d
(ppm) 1.45 (m, 2H, CH2), 1.62e1.69 (m, 4H, 2 CH2), 2.55e2.59 (m,
4H, 2CH2), 2.85 (t, 2H, J ¼ 9.0 Hz, CH2), 3.90 (s, 3H, OCH3), 3.94 (s,
3H, OCH3), 4.24 (t, 2H, J ¼ 9.0 Hz, CH2), 7.07 (s, 1H, ArH), 7.52 (s, 1H,
ArH). LCeMS (APCIþ): m/z calcd for C16H22N2O6 339 [(MþH)þ].
5.1.2.2. Methyl 2-nitro-4-pyrrolidinoethoxy-5-methoxybenzoate (8).
Yellow oil (24%). TLC: Rf 0.6 (CH2Cl2:MeOH(NH3) 9:1 v/v). IR (cmꢀ1):
2470 (pyrrolidine), 1709 (C]O), 1531 (NO2). 1H NMR (DMSO-d6):
5.1.3.1. Methyl 2-amino-4-piperidinoethoxy-5-methoxybenzoate
(10). Brown oil (79%). TLC: Rf 0.6 (CH2Cl2:MeOH(NH3) 9:1 v/v). IR
(cmꢀ1): 3442 (NH2), 2935 (piperidine), 1663 (C]O). 1H NMR
d
(ppm) 3.34 (m, 4H, 2CH2), 3.45e3.47 (m, 2H, CH2), 3.58e3.77 (m,
(DMSO-d6): d (ppm) 1.33e1.40 (m, 2H, CH2), 1.42e1.52 (m, 4H, 2

S. Ravez et al. / European Journal of Medicinal Chemistry 79 (2014) 369e381
377
CH2), 2.37e2.45 (m, 4H, 2 CH2), 2.65 (t, 2H, J ¼ 6.0 Hz, CH2), 3.63 (s,
3H, OCH3), 3.74 (s, 3H, OCH3), 4.01 (t, 2H, J ¼ 6.0 Hz, CH2), 6.37 (s,
2H, NH2), 6.40 (s, 1H, ArH), 7.12 (s, 1H, ArH). LCeMS (APCIþ): m/z
calcd for C16H24N2O4 309 [(MþH)þ].
(100 mL). The organic layer was washed with a 1 M solution of
K2CO3, brine and dried over CaCl2, and the solvent was removed
under reduced pressure.
5.1.5.1. 4-Chloro-6-methoxy-7-piperidinoethoxyquinazoline
(16).
5.1.3.2. Methyl 2-amino-4-pyrrolidinoethoxy-5-methoxybenzoate
(11). Brown oil (51%). TLC: Rf 0.5 (CH2Cl2:MeOH(NH3) 9:1 v/v). IR
(cmꢀ1): 3534 (NH2), 2950 (pyrrolidine), 1684 (C]O). 1H NMR
Beige solid (71%). TLC: Rf 0.5 (CH2Cl2:MeOH(NH3) 9:1 v/v). Mp:
132e134 ꢁC. IR (cmꢀ1): 2935 (piperidine), 1087 (CeCl). 1H NMR
(DMSO-d6):
d (ppm) 1.44e1.50 (m, 2H, CH2), 1.59e1.67 (m, 4H,
(DMSO-d6):
d
(ppm) 1.79e2.05 (m, 4H, 2 CH2), 2.99e3.24 (m, 4H, 2
2CH2), 2.55e2.58 (m, 4H, 2CH2), 2.93 (t, 2H, J ¼ 6.0 Hz, CH2), 4.06 (s,
3H, OCH3), 4.35 (t, 2H, J ¼ 6.0 Hz, CH2), 7.35 (s, 1H, ArH), 7.39 (s, 1H,
ArH), 8.87 (s, 1H, ArH). LCeMS (APCIþ): m/z calcd for C16H20ClN3O2
321 [(MþH)þ for 35Cl], 323 [(Mþ2þH)þ for 37Cl].
CH2), 3.57 (t, 2H, J ¼ 5.5 Hz, CH2), 3.66 (s, 3H, OCH3), 3.74 (s, 3H,
OCH3), 4.32 (t, 2H, J ¼ 5.1 Hz, CH2), 6.41 (s, 1H, ArH), 6.46 (s, 2H,
NH2), 7.17 (s,1H, ArH). LC-MS (APCIþ): m/z calcd for C15H22N2O4 295
[(MþH)þ].
5.1.5.2. 4-Chloro-6-methoxy-7-pyrrolidinoethoxyquinazoline
(17).
5.1.3.3. Methyl 2-amino-4-piperidinopropoxy-5-methoxybenzoate
(12). Yellow oil (41%). TLC: Rf 0.6 (CH2Cl2:MeOH(NH3) 9:1 v/v). IR
(cmꢀ1): 3424 (NH2), 2935 (piperidine), 1678 (C]O). 1H NMR
Beige solid (49%). TLC: Rf 0.6 (CH2Cl2:MeOH(NH3) 9:1 v/v). Mp:
155e157 ꢁC. IR (cmꢀ1): 2950 (pyrrolidine), 1090 (CeCl). 1H NMR
(DMSO-d6):
d (ppm) 1.70 (m, 4H, 2CH2), 2.51e2.57 (m, 4H, 2CH2),
(DMSO-d6):
d
(ppm) 1.35e1.83 (m, 6H, 3 CH2), 2.15e2.24 (m, 2H,
2.90 (t, 2H, J ¼ 6.0 Hz, CH2), 4.00 (s, 3H, OCH3), 4.32 (t, 2H, J ¼ 6.0 Hz,
CH2), 7.38 (s, 1H, ArH), 7.47 (s, 1H, ArH), 8.87 (s, 1H, ArH). LCeMS
(APCIþ): m/z calcd for C15H18ClN3O2 307 [(M þ H)þ for 35Cl], 309
[(Mþ2þH)þ for 37Cl].
CH2), 2.71e3.19 (m, 4H, 2 CH2), 3.35 (m, 2H, CH2), 3.67 (s, 3H, OCH3),
3.75 (s, 3H, OCH3), 4.01 (t, 2H, J ¼ 5.8 Hz, CH2), 6.39 (s,1H, ArH), 6.46
(s, 2H, NH2), 7.14 (s, 1H, ArH). LCeMS (APCIþ): m/z calcd for
C
17H26N2O4 323 [(MþH)þ].
5.1.5.3. 4-Chloro-6-methoxy-7-piperidinopropoxyquinazoline (18).
Beige solid (70%). TLC: Rf 0.6 (CH2Cl2:MeOH(NH3) 9:1 v/v). Mp:
151e153 ꢁC. IR (cmꢀ1): 2935 (piperidine), 1086 (CeCl). 1H NMR
5.1.4. General procedure for quinazolin-4-one derivatives (13e15)
To a solution of methyl-2-amino-benzoate derivatives (10e12)
(3.39 mmol) in formamide (5 mL) was added ammonium formate
(10.19 mmol). The reaction mixture was stirred for 24 h under
reflux conditions. The reaction was quenched by water and the
mixture was alkalinized by 10% K2CO3 solution. The precipitated
was collected by filtration, washed with H2O and dried in vacuo.
(DMSO-d6):
d (ppm) 1.35e1.42 (m, 2H, CH2), 1.44e1.55 (m, 4H,
2CH2), 1.92 (m, 2H, CH2), 2.30e2.34 (m, 4H, 2 CH2), 2.41 (t, 2H,
J ¼ 6.3 Hz, CH2), 4.00 (s, 3H, OCH3), 4.26 (t, 2H, J ¼ 6.2 Hz, CH2), 7.40
(s, 1H, ArH), 7.46 (s, 1H, ArH), 8.88 (s, 1H, ArH). LC-MS (APCIþ): m/z
calcd for C17H22ClN3O2 335 [(MþH)þ for 35Cl], 337 [(Mþ2þH)þ for
37Cl].
5.1.4.1. 6-Methoxy-7-piperidinoethoxyquinazolin-4-one
(13).
White solid (59%). TLC: Rf 0.4 (CH2Cl2:MeOH(NH3) 9:1 v/v). Mp:
5.1.6. General procedure for urea derivatives (21e23)
155e157 ꢁC. IR (cmꢀ1): 2935 (piperidine), 1650 (C]O). 1H NMR
To a stirred solution of aminophenol derivatives (0.10 g,
(DMSO-d6):
d
(ppm) 1.33e1.45 (m, 2H, CH2), 1.46e1.55 (m, 4H, 2
0.69 mmol) in
5
mL of pyridine was added 3-
CH2), 2.42e2.51 (m, 4H, 2 CH2), 2.72 (t, 2H, J ¼ 5.4 Hz, CH2), 3.86 (s,
3H, OCH3), 4.20 (t, 2H, J ¼ 5.3 Hz, CH2), 7.15 (s, 1H, ArH), 7.44 (s, 1H,
ArH), 7.94 (s, 1H, ArH), 7.98 (s, 1H, NH). LCeMS (APCIþ): m/z calcd
for C16H21N3O3 304 [(MþH)þ].
bromophenylisocyanate (0.69 mmol). After 1 h at room tempera-
ture under nitrogen atmosphere, the solvent was removed under
reduced pressure. The residue was washed by CH2Cl2 and
recrystallized.
5.1.4.2. 6-Methoxy-7-pyrrolidinoethoxyquinazolin-4-one
(14).
5.1.6.1. N-(3-Bromo-phenyl)-N0-(4-hydroxyphenyl)urea
(21).
Beige solid (75%). TLC: Rf 0.4 (CH2Cl2:MeOH(NH3) 9:1 v/v). Mp:
Crystallization from acetonitrile gave pure 21 as a white solid (71%).
182e184 ꢁC. IR (cmꢀ1): 2950 (pyrrolidine), 1658 (C]O). 1H NMR
TLC: Rf 0.7 (CH2Cl2:MeOH 9:1 v/v). Mp: 227e229 ꢁC. IR (cmꢀ1):
(DMSO-d6):
d
(ppm) 1.82e1.87 (m, 4H, 2 CH2), 2.70e2.79 (m, 4H,
3301 (OH), 1635 (C]O). 1H NMR (DMSO-d6):
d (ppm) 6.68 (m, 2H,
2CH2), 3.07 (t, 2H, J ¼ 6.2 Hz, CH2), 3.97 (s, 3H, OCH3), 4.31 (t, 2H,
J ¼ 6.0 Hz, CH2), 7.15 (s, 1H, ArH), 7.55 (s, 1H, ArH), 8.00 (s, 1H, ArH),
8.01 (s, 1H, NH). LCeMS (APCIþ): m/z calcd for C15H19N3O3 290
[(MþH)þ].
ArH), 7.08e7.30 (m, 5H, ArH), 7.82 (m, 1H, ArH), 8.38 (s, 1H, NH),
8.71 (s, 1H, NH), 9.10 (s, 1H, OH). LCꢀMS (APCIþ): m/z calcd for
C
13H11BrN2O2 307 [(MþH)þ for 79Br], 309 [(MþH)þ for 81Br].
5.1.6.2. N-(3-Bromo-phenyl)-N0-(3-methyl-4-hydroxyphenyl)urea
(22). Crystallization from acetonitrile gave pure 22 as a beige solid
(89%). Mp: 204e206 ꢁC. TLC: Rf 0.6 (CH2Cl2:MeOH 9:1 v/v). IR
5.1.4.3. 6-Methoxy-7-piperidinopropoxyquinazolin-4-one
(15).
Beige solid (75%). TLC: Rf 0.4 (CH2Cl2:MeOH(NH3) 9:1 v/v). Mp:
172e174 ꢁC. IR (cmꢀ1): 2935 (piperidine), 1662 (C]O). 1H NMR
(cmꢀ1): 3288 (OH), 1635 (C]O). 1H NMR (DMSO-d6):
d (ppm) 2.15
(DMSO-d6):
d
(ppm) 1.31e1.42 (m, 2H, CH2), 1.43e1.53 (m, 4H,
(s, 3H, CH3), 6.55 (dd, 1H, J ¼ 2.7 Hz and J ¼ 9.0 Hz, ArH), 6.61 (d, 1H,
J ¼ 2.7 Hz, ArH), 7.08e7.33 (m, 4H, ArH), 7.75 (s, 1H, NH), 7,89 (m,
1H, ArH), 8.95 (s, 1H, NH), 9.12 (s, 1H, OH). LCꢀMS (APCIþ): m/z
calcd for C14H13BrN2O2 321 [(MþH)þ for 79Br], 323 [(MþH)þ for
81Br].
2CH2), 1.91 (m, 2H, CH2), 2.33e2.36 (m, 4H, 2CH2), 2.37 (t, 2H,
J ¼ 7.1 Hz, CH2), 3.85 (s, 3H, OCH3), 4.11 (t, 2H, J ¼ 7.1 Hz, CH2), 7.04
(s, 1H, ArH), 7.42 (s, 1H, ArH), 7.96 (s, 1H, ArH), 8.00 (s, 1H, NH). LCe
MS (APCIþ): m/z calcd for C17H23N3O3 318 [(MþH)þ].
5.1.5. General procedure for 4-chloroquinazoline derivatives (16e
18)
5.1.6.3. N-(3-Bromo-phenyl)-N0-(3-chloro-4-hydroxyphenyl)urea
(23). Crystallization from acetonitrile gave pure 23 as a beige solid
(61%). Mp: 178e180 ꢁC. TLC: Rf 0.6 (CH2Cl2:MeOH 9:1 v/v). IR
A solution of quinazolin-4-one derivatives (13e15) (15.0 mmol)
in phosphorus oxychloride (30 mL) was refluxed for 6 h. After
removal of the solvent, the residue was dissolved in ice-water
(50 mL) and the mixture was neutralized by 10% K2CO3 solution.
The precipitate was collected by filtration and dissolved in CH2Cl2
(cmꢀ1): 3283 (OH), 1643 (C]O). 1H NMR (DMSO-d6):
d (ppm) 6.72
(dd, 1H, J ¼ 2.4 Hz and J ¼ 9.1 Hz, ArH), 6.84 (d, 1H, J ¼ 2.4 Hz, ArH),
7.09e7.32 (m, 3H, ArH), 7.77 (m, 2H, ArH), 8.07 (s, 1H, NH), 9.34 (s,
1H, NH), 9.64 (s, 1H, OH). LCꢀMS (APCIþ): m/z calcd for

378
S. Ravez et al. / European Journal of Medicinal Chemistry 79 (2014) 369e381
C13H10BrClN2O2 340 [(MþH)þ for 35Cl/79Br], 342 [(MþH)þ for
35Cl/81Br], 342 [(MþH)þ for 37Cl/79Br], 344 [(MþH)þ for 37Cl/81Br].
5.1.7.4. N-(3-Bromo-phenyl)-N0-{4-3-methyl-[(6-methoxy-7-
diethylaminoethoxyquinazolin-4-yl)oxy]-phenyl}urea (27).
Compound 27 was synthesized from 20 and 22 using the general
procedure described above to afford as a white solid collected by
filtration and washed with CH2Cl2 (28%). TLC: Rf 0.6
(CH2Cl2:MeOH(NH3) 9:1 v/v). Mp: 128e130 ꢁC. IR (cmꢀ1): 1640
5.1.7. General procedure for final products (24e37)
To a stirred solution of chloro derivatives (16e20) (0.10 g,
0.32 mmol) and tetrabutylammonium bromide in 10 mL of a
mixture of a 20% solution of NaOH and 2-butanone (1:2) were
added urea derivatives (0.32 mmol) (21e23). After 1 h at room
temperature, the reaction was quenched by water, and then the
aqueous solution was extracted with EtOAc (3 ꢄ 10 mL), washed
with a 1 M solution of NaOH, and dried over MgSO4. The solvent
was removed under reduced pressure.
(C]O). 1H NMR (DMSO-d6):
d (ppm) 1.00 (m, 6H, 2 CH3), 2.31 (s, 3H,
CH3), 2.62 (m, 4H, CH2), 2.92 (m, 2H, CH2), 3.98 (s, 3H, OCH3), 4.26
(m, 2H, CH2), 7.05e7.42 (m, 6H, ArH), 7.52 (s, 1H, ArH), 7.80e7.90
(m, 2H, ArH), 8.53 (s, 1H, ArH), 8.59 (s, 1H, NH), 10.05 (s, 1H, NH). 13
C
NMR (DMSO-d6):
d (ppm) 10.19, 18.81, 47.61, 50.11, 56.61, 66.08,
101.43, 108.13, 110.45, 116.88, 119.92, 120.25, 122.14, 122.70, 123.92,
124.31, 130.32, 131,12, 135.30, 142.41, 147.79, 149.01, 150.44, 152.85,
153.41, 154.68, 165.50. LCꢀMS (APCIþ): m/z calcd for C29H34BrN5O4
594 [(MþH)þ for 79Br], 596 [(MþH)þ for 81Br]. HRMS (ESI (MþH)þ
m/z) calcd for C29H34BrN5O4 594.1710 found 594.1686.
5 . 1. 7 . 1. N - ( 3 - B r o m o - p h e n y l ) - N 0- { 4 - [ ( 6 - b u t o x y - 7 -
diethylaminoethoxyquinazolin-4-yl)oxy]phenyl-}urea
(24).
Compound 24 was synthesized from 19 and 21 using the general
procedure described above to afford as a white solid collected by
filtration and washed with methanol (22%). TLC: Rf 0.5
(CH2Cl2:MeOH(NH3) 9:1 v/v). Mp: 187e189 ꢁC. IR (cmꢀ1): 1650 (C]
5.1.7.5. N-(3-Bromo-phenyl)-N0-{4-3-chloro-[(6-methoxy-7-
diethylaminoethoxyquinazolin-4-yl)oxy]-phenyl}urea
(28).
O). 1H NMR (DMSO-d6):
d
(ppm) 0.98e1.05 (m, 9H, 3CH3),1.48e1.53
Compound 28 was synthesized from 20 and 23 using the general
procedure described above to afford as a white solid collected by
filtration and washed with MeOH (13%). TLC: Rf 0.6
(CH2Cl2:MeOH(NH3) 9:1 v/v). Mp: 157e159 ꢁC. IR (cmꢀ1): 1648
(m, 2H, CH2), 1.82e1.85 (m, 2H, CH2), 2.62e2.65 (m, 4H, 2CH2),
2.90e2.95 (m, 2H, CH2), 4.10e4.30 (m, 4H, 2 CH2), 7.12 (m, 1H, ArH),
7.25 (m, 3H, 3 ArH), 7.40 (m, 2H, 2 ArH), 7.52 (m, 3H, 3 ArH), 7.91 (s,
1H, ArH), 8.52 (s, 1H, ArH), 8.88 (s, 1H, NH), 8.99 (s, 1H, NH). 13C
(C]O). 1H NMR (DMSO-d6):
d
(ppm) 1.00 (t, 6H, J ¼ 6.8 Hz, 2 CH3),
NMR (DMSO-d6):
d
(ppm) 14.24, 19.01, 19.25, 31.22, 47.84, 51.09,
2.59 (m, 4H, CH2), 2.88 (t, 2H, J ¼ 5.9 Hz, CH2), 3.95 (s, 3H, OCH3),
4.13 (t, 2H, J ¼ 5.9 Hz, CH2), 7.28e7.58 (m, 7H, ArH), 7.90 (m, 1H,
ArH), 8.19 (m,1H, ArH), 8.47 (s,1H, NH), 8.59 (s,1H, ArH), 9.61 (s,1H,
68.99, 101.92, 108.02, 110.26, 117.32, 120.12, 120.35, 122.58, 123.47,
124.38, 124.62, 131.05, 131.27, 135.57, 143.59, 148.45, 149.85, 150.04,
152.70, 153.22, 155.26, 165.47. LCꢀMS (APCIþ): m/z calcd for
NH). 13C NMR (DMSO-d6):
d (ppm) 12.32, 47.45, 51.36, 56.51, 68.08,
C
31H36BrN5O4 622 [(MþH)þ for 79Br], 624 [(MþH)þ for 81Br]. HRMS
101.17, 107.80, 109.97, 117.43, 120.84, 122.00, 122.26, 122.80, 123.24,
123.58, 125.10, 131.26, 133.80, 141.58, 147.75, 149.33, 150.65, 152.55,
152.57, 155.53, 165.10. LCꢀMS (APCIþ): m/z calcd for
(ESI (MþH)þ m/z) calcd for C31H36BrN5O4 622.2023 found 622.1999.
5.1.7.2. N-(3-Bromo-phenyl)-N0-{3-methyl-4-[(6-butoxy-7-
C
28H29BrClN5O4 614 [(M þ H)þ for 35Cl/79Br], 616 [(MþH)þ for
diethylaminoethoxyquinazolin-4-yl)oxy]-phenyl}urea
(25).
35Cl/81Br], 616 [(MþH)þ for 37Cl/79Br], 618 [(MþH)þ for 37Cl/81Br].
HRMS (ESI (MH)þ m/z) calcd for C28H29BrClN5O4 616.1134 found
616.1090.
Compound 25 was synthesized from 19 and 22 using the general
procedure described above to afford as a beige solid collected by
crystallization
(CH2Cl2:MeOH(NH3) 9:1 v/v). Mp: 169e171 ꢁC. IR (cmꢀ1): 1699 (C]
O). 1H NMR (DMSO-d6):
(ppm) 0.98e1.05 (m, 9H, 3 CH3), 1.5 (m,
from
acetonitrile
(52%).
TLC:
Rf
0.5
5 .1. 7 . 6 . N - ( 3 - B r o m o - p h e n y l ) - N 0- { 4 - [ ( 6 - m e t h o x y - 7 -
d
piperidinoethoxyquinazolin-4-yl)oxy]-phenyl}urea
hydrochloride
2H, CH2), 1.75 (m, 2H, CH2), 2.36 (s, 3H, CH3), 2.63 (m, 4H, 2CH2),
2.91 (m, 2H, CH2), 4.10e4.15 (m, 4H, 2 CH2), 6.99e7.15 (m, 3H, 3
ArH), 7.15e7.38 (m, 3H, 3 ArH), 7.51 (s, 1H, ArH), 7.82 (m, 1H, ArH),
7.91 (s, 1H, ArH), 8.20 (s, 1H, NH), 8.52 (s, 1H, ArH), 9.48 (s, 1H, NH).
(29). Compound 29 was synthesized from 16 and 21 using the
general procedure described above to afford as a pink solid
collected by precipitation in a mixture of diethyl ether/hydrochloric
acid 6 N in isopropanol (15%). TLC: Rf 0.6 (CH2Cl2:MeOH(NH3) 9:1 v/
v). Mp: 222e224 ꢁC. IR (cmꢀ1): 2380 (NHþ); 1707 (C]O). 1H NMR
13C NMR (DMSO-d6):
d (ppm) 12.01, 14.19, 18.44, 19.22, 31.14, 47.75,
50.89, 68.67, 101.81, 107.78, 110.21, 117.16, 120.08, 120.56, 122.23,
123.04, 124.01, 124.59, 130.49, 131.20, 135.07, 142.14, 148.09, 149.08,
149.98, 152.69, 153.17, 155.42, 165.42. LCꢀMS (APCIþ): m/z calcd for
(DMSO-d6): d (ppm) 0.92e1.06 (m, 2H, CH2), 1.28e1.41 (m, 4H, 2
CH2),1.69e1.75 (m, 4H, 2CH2), 3.59e3.62 (m, 4H, 2 CH2), 4.02 (s, 3H,
OCH3), 7.13e7.37 (m, 5H, 5 ArH), 7.50e758 (m, 3H, 3ArH), 7.66 (s,
1H, ArH), 7.86 (s, 1H, ArH), 8.67 (s, 1H, ArH), 9.44 (s, 1H, NH), 9.53 (s,
C
32H38BrN5O4 636 [(MþH)þ for 79Br], 638 [(MþH)þ for 81Br]. HRMS
(ESI (MþH)þ m/z) calcd for C32H38BrN5O4 636.2179 found 636.2157.
1H, NH), 10.51 (s, 1H, NHþ). 13C NMR (DMSO-d6):
d (ppm) 24.32,
26.17, 54.62, 56.47, 57.58, 68.12, 102.17, 107.92, 111.10, 116.18, 120.12,
120.72, 122.34, 123.22, 124.12, 124.67, 130.72, 131.20, 135.10, 142.10,
148.18, 149.22, 151.62, 152.73, 153.32, 155.08, 165.50. LCꢀMS
(APCIþ): m/z calcd for C29H30BrN5O4 592 [(MþH)þ for 79Br], 594
[(Mþ2þH)þ for 81Br]. HRMS (ESI (MþH)þ m/z) calcd for
5 .1. 7 . 3 . N - ( 3 - B r o m o - p h e n y l ) - N 0- { 4 - [ ( 6 - m e t h o x y - 7 -
diethylaminoethoxyquinazolin-4-yl)oxy]-phenyl}urea
(26).
Compound 26 was synthesized from 20 and 21 using the general
procedure described above to afford as a white solid collected by
filtration and washed with MeOH (15%). TLC: Rf 0.5
(CH2Cl2:MeOH(NH3) 9:1 v/v). Mp: 144e146 ꢁC. IR (cmꢀ1): 1699
C29H30BrN5O4 592.1553 found 592.1532.
(C]O). 1H NMR (DMSO-d6):
d
(ppm) 1.02 (m, 6H, CH3), 2.69 (m, 4H,
5.1.7.7. N-(3-Bromo-phenyl)-N0-{3-methyl-4-[(6-methoxy-7-
piperidinoethoxyquinazolin-4-yl)oxy]-phenyl}urea (30).
CH2), 2.98 (m, 2H, CH2), 3.98 (s, 3H, OCH3), 4.30 (m, 2H, CH2), 7.11e
7.62 (m, 9H, ArH), 7.88 (s, 1H, ArH), 8.51 (s, 1H, ArH), 8.92 (s, 1H,
Compound 30 was synthesized from 16 and 22 using the general
procedure described above to afford as a white solid collected by
filtration and washed with MeOH (12%). TLC: Rf 0.5
(CH2Cl2:MeOH(NH3) 9:1 v/v). Mp: 206e208 ꢁC. IR (cmꢀ1): 2935
NH), 9.00 (s, 1H, NH). 13C NMR (DMSO-d6):
d (ppm) 12.31, 47.48,
51.42, 56.48, 97.74, 101.18, 107.82, 110.07, 118.22, 121.85, 122.03,
122.28, 122.74, 123.27, 123.52, 125.18, 131.27, 133.92, 142.02, 148.13,
149.32, 151.33, 152.41, 152.49, 156.01, 165.32. LCꢀMS (APCIþ): m/z
calcd for C28H30BrN5O4 580 [(M þ H)þ for 79Br], 582 [(MþH)þ for
81Br]. HRMS (ESI (MþH)þ m/z) calcd for C28H30BrN5O4 580.1553
found 580.1514.
(piperidine), 1643 (C]O). 1H NMR (DMSO-d6):
d (ppm) 1.34e1.42
(m, 2H, CH2), 1.45e1.56 (m, 4H, 2 CH2), 2.28 (s, 3H, CH3), 2.46e2.50
(m, 4H, 2 CH2), 2.75 (t, 2H, J ¼ 6.6 Hz, CH2), 3.97 (s, 3H, OCH3), 4.29
(t, 2H, J ¼ 6.6 Hz, CH2), 7.01e7.16 (m, 3H, 3ArH), 7.21e7.32 (m, 2H,

S. Ravez et al. / European Journal of Medicinal Chemistry 79 (2014) 369e381
379
2ArH), 7.41 (s, 1H, ArH), 7.54 (s, 1H, ArH), 7.81e7.91 (m, 2H, 2 ArH),
C
29H30BrN5O4 592 [(MþH)þ for 79Br], 594 [(MþH)þ for 81Br]. HRMS
8.10 (s, 1H, NH), 8.53 (s, 1H, ArH), 9.24 (s, 1H, NH). 13C NMR (DMSO-
(ESI (MþH)þ m/z) calcd for C29H30BrN5O4 592.1553 found 592.1534.
d6):
d (ppm) 18.29, 24.37, 26.02, 54.88, 56.46, 57.50, 67.36, 101.23,
107.97, 110.12, 117,26, 120.08, 120.67, 122.22, 123.24, 124.01, 124.65,
130.66, 131.18, 135.00, 142.06, 148.20, 149.21, 150.55, 152.72, 153.10,
155.39, 165.42. LCꢀMS (APCIþ): m/z calcd for C30H32BrN5O4 606
[(MþH)þ for 79Br], 608 [(Mþ2þH)þ for 81Br]. HRMS (ESI (MþH)þ m/
z) calcd for C30H32BrN5O4 606.1710 found 606.1686.
5.1.7.11. N-(3-Bromo-phenyl)-N0-{3-chloro-4-[(6-methoxy-7-
pyrrolidinoethoxyquinazolin-4-yl)oxy]-phenyl}urea
(34).
Compound 34 was synthesized from 17 and 23 using the general
procedure described above to afford as a beige solid collected by
filtration and washed with MeOH (9%). TLC: Rf 0.5
(CH2Cl2:MeOH(NH3) 9:1 v/v). Mp: 187e189 ꢁC. IR (cmꢀ1): 2950
(pyrrolidine), 1645 (C]O). 1H NMR (DMSO-d6):
d (ppm) 1.70 (m,
5.1.7.8. N-(3-Bromo-phenyl)-N0-{3-chloro-4-[(6-methoxy-7-
4H, 2 CH2), 2.60e2.65 (m, 4H, 2CH2), 2.90 (t, 2H, J ¼ 6.7 Hz, CH2),
4.00 (s, 3H, OCH3), 4.32 (t, 2H, J ¼ 6.7 Hz, CH2), 7.12e7.48 (m, 5H,
5ArH), 7.55e7.62 (m, 2H, 2 ArH), 7.91 (s, 1H, ArH), 8.12e8.20 (m, 1H,
ArH), 8.48 (s, 1H, NH), 8.60 (s, 1H, ArH), 9.61 (s, 1H, NH). 13C NMR
piperidinoethoxyquinazolin-4-yl)oxy]-phenyl}urea
(31).
Compound 31 was synthesized from 16 and 23 using the general
procedure described above to afford as a white solid collected by
crystallization
(CH2Cl2:MeOH(NH3) 9:1 v/v). Mp: 204e206 ꢁC. IR (cmꢀ1): 2935
(piperidine), 1649 (C]O). 1H NMR (DMSO-d6):
(ppm) 1.32e1.42
from
acetonitrile
(13%).
TLC:
Rf
0.6
(DMSO-d6): d (ppm) 23.58, 54.37, 54.42, 56.36, 68.37, 101.12, 107.84,
111.02, 117.52, 120.05, 120.08, 120.92, 122.33, 122.80, 122.86, 124.72,
131.13, 137.54, 141.99, 147.33, 148.98, 150.37, 152.71, 152.91, 155.31,
165.37. LCꢀMS (APCIþ): m/z calcd for C28H27BrClN5O4 612 [(MþH)þ
for 35Cl/79Br], 614 [(MþH)þ for 35Cl/81Br], 614 [(MþH)þ for
37Cl/79Br], 616 [(MþH)þ for 37Cl/81Br]. HRMS (ESI (MþH)þ m/z)
calcd for C28H27BrClN5O4 614.0978 found 614.0955.
d
(m, 2H, CH2), 1.44e1.58 (m, 4H, 2CH2), 2.49 (m, 4H, 2CH2), 2.74 (t,
2H, J ¼ 6.0 Hz, CH2), 3.97 (s, 3H, OCH3), 4.29 (t, 2H, J ¼ 6.0 Hz, CH2),
7.29e7.37 (m, 3H, 3 ArH), 7.41 (m, 1H, ArH), 7.53e7.57 (m, 2H,
2ArH), 7.84 (dd, 1H, J ¼ 9.6, 2.4 Hz, ArH), 8.16 (d,1H, J ¼ 9.6 Hz, ArH),
8.43 (s, 1H, ArH), 8.51 (s, 1H, ArH), 8.56 (s, 1H, NH), 9.58 (s, 1H, NH).
13C NMR (DMSO-d6):
d (ppm) 24.30, 26.11, 54.58, 56.52, 57.65, 68.07,
5 .1. 7 .12 . N - ( 3 - B r o m o - p h e n yl ) - N 0- { 4 - [ ( 6 - m e t h o x y - 7 -
piperidinopropoxyquinazolin-4-yl)oxy]-phenyl}urea
Compound 35 was synthesized from 18 and 21 using the general
procedure described above to afford as a white solid collected by
crystallization
(CH2Cl2:MeOH(NH3) 9:1 v/v). Mp: 179e181 ꢁC. IR (cmꢀ1): 2935
(piperidine), 1712 (C]O). 1H NMR (DMSO-d6):
(ppm) 1.68e1.73
101.92, 106.99, 11.12, 116.20, 120.32, 120.41, 122.40, 123.18, 124.10,
124.70, 131.81, 132.21, 135.12, 142.15, 148.27, 149.21, 151.70, 152.71,
153.08, 155.68, 165.13. LCꢀMS (APCIþ): m/z calcd for
(35).
C
29H29BrClN5O4 626 [(MþH)þ for 35Cl/79Br], 628 [(MþH)þ for
35Cl/81Br], 628 [(MþH)þ for 37Cl/79Br], 630 [(MþH)þ for 37Cl/81Br].
HRMS (ESI (MþH)þ m/z) calcd for C29H29BrClN5O4 628.2714 found
628.2783.
from
acetonitrile
(26%).
TLC:
Rf
0.5
d
(m, 4H, 2CH2), 2.15e2.25 (m, 4H, 2CH2), 2.75e2.78 (m, 2H, CH2),
2.93e2.99 (m, 2H, CH2), 3.05e3.17 (m, 2H, CH2), 4.00 (s, 3H, OCH3),
4.15e4.21 (m, 2H, CH2), 7.13e7.32 (m, 6H, 6ArH), 7.62 (m, 2H, 2ArH),
7.68 (s, 1H, ArH), 7.75 (s, 1H, ArH), 8.27 (s, 1H, NH), 8.37 (s, 1H, ArH),
5 .1. 7 . 9 . N - ( 3 - B r o m o - p h e n y l ) - N 0- { 4 - [ ( 6 - m e t h o x y - 7 -
pyrrolidinoethoxyquinazolin-4-yl)oxy]-phenyl}urea
(32).
9.30 (s, 1H, NH). 13C NMR (DMSO-d6):
d (ppm) 24.62, 26.07, 26.42,
Compound 32 was synthesized from 17 and 21 using the general
procedure described above to afford as a gray solid collected by
filtration and washed with MeOH (24%). TLC: Rf 0.6
(CH2Cl2:MeOH(NH3) 9:1 v/v). Mp: 179e181 ꢁC. IR (cmꢀ1): 2950
54.77, 56.02, 56.50, 67.48, 100.89, 107.52, 110.81, 116.38, 120.87,
121.03, 122.91, 122.99, 123.20, 123.66, 125.13, 125.18, 132.03, 133.95,
142.64, 147.71, 149.30, 151.69, 153.03, 157.60, 165.37. LCꢀMS
(APCIþ): m/z calcd for C30H32BrN5O4 606 [(MþH)þ for 79Br], 608
[(MþH)þ for 81Br]. HRMS (ESI (MþH)þ m/z) calcd for C30H32BrN5O4
606.1701 found 606.17017.
(pyrrolidine), 1503 (C]O). 1H NMR (DMSO-d6):
d (ppm) 1.64e1.82
(m, 4H, 2CH2), 2.54e2.62 (m, 4H, 2CH2), 2.93 (m, 2H, CH2), 3.98 (s,
3H, OCH3), 4.30 (m, 2H, CH2), 7.12e7.15 (m, 2H, 2ArH), 7.20e7.23
(m, 2H, 2ArH), 7.35 (m, 1H, ArH), 7.40 (m, 1H, ArH), 7.56 (m, 3H,
3ArH), 7.87 (s, 1H, ArH), 8.54 (s, 1H, ArH), 8.94 (s, 1H, NH), 8.99 (s,
5.1.7.13. N-(3-Bromo-phenyl)-N0-{3-methyl-4-[(6-methoxy-7-
piperidinopropoxyquinazolin-4-yl)oxy]-phenyl}urea
(36). Compound 36 was synthesized from 18 and 22 using the
general procedure described above to afford as a beige solid
collected by precipitation in a mixture of diethyl ether/hydrochloric
acid 6 N in isopropanol (18%). TLC: Rf 0.5 (CH2Cl2:MeOH(NH3) 9:1 v/
v). Mp > 250 ꢁC. IR (cmꢀ1): 2510 (NHþ); 1705 (C]O). 1H NMR
1H, NH). 13C NMR (DMSO-d6):
d (ppm) 23.58, 54.35, 54.49, 56.46,
hydrochloride
68.18, 101.24, 107.81, 110.14, 117.48, 119.91, 119.92, 120.85, 122.15,
122.81, 122.82, 124.70, 131.09, 137.44, 141.98, 147.35, 149.16, 150.51,
152.68, 152.94, 155.27, 165.40. LCꢀMS (APCIþ): m/z calcd for
C
28H28BrN5O4 578 [(MþH)þ for 79Br], 580 [(Mþ2þH)þ for 81Br].
HRMS (ESI (MþH)þ m/z) calcd for C28H28BrN5O4 578.1397 found
578.1372.
(DMSO-d6):
d (ppm) 1.70e1.80 (m, 4H, 2CH2), 2.22e2.31 (m, 4H, 2
CH2), 2.94e2.98 (m, 2H, CH2), 3.10e3.16 (m, 2H, CH2), 3.21e3.29 (m,
2H, CH2), 4.01 (s, 3H, OCH3), 4.27e4.30 (m, 2H, CH2), 4,33 (s, 3H,
CH3), 7.03e7.51 (m, 5H, 5ArH), 7.47 (m, 2H, 2ArH), 7.61 (s, 1H, ArH),
7.88 (s, 1H, ArH), 8.54 (s, 1H, NH), 8.69 (s, 1H, ArH), 9.99 (s, 1H, NH),
5.1.7.10. N-(3-Bromo-phenyl)-N0-{3-methyl-4-[(6-methoxy-7-
pyrrolidinoethoxyquinazolin-4-yl)oxy]-phenyl}urea
(33).
Compound 33 was synthesized from 17 and 22 using the general
procedure described above to afford as a white solid collected by
filtration and washed with MeOH (25%). TLC: Rf 0.6
(CH2Cl2:MeOH(NH3) 9:1 v/v). Mp: 193e197 ꢁC. IR (cmꢀ1): 2950
10.17 (s, 1H, NHþ). 13C NMR (DMSO-d6):
d (ppm) 24.54, 27.01, 27.05,
53.98, 56.32, 56.59, 68.50, 101.32, 107.57, 110.83, 114.03, 120.02,
121.38, 122.47, 122.91, 123.05, 123.47, 125.32, 125.87, 132.04, 133.48,
143.05, 149.15, 149.73, 152.99, 153.05, 158.02, 165.48. LCꢀMS
(APCIþ): m/z calcd for C31H34BrN5O4 620 [(M þ H)þ for 79Br], 622
[(MþH)þ for 81Br]. HRMS (ESI (MþH)þ m/z) calcd for C31H34BrN5O4
620.1866 found 620.1842.
(pyrrolidine), 1651 (C]O). 1H NMR (DMSO-d6):
d (ppm) 1.70e1.72
(m, 4H, 2CH2), 2.28 (s, 3H, CH3), 2.51e2.55 (m, 4H, 2 CH2), 2.90 (t,
2H, J ¼ 6.1 Hz, CH2), 3.99 (s, 3H, OCH3), 4.30 (t, 2H, J ¼ 6.1 Hz, CH2),
7.08e7.35 (m, 6H, 6ArH), 7.41 (s, 1H, ArH), 7.56 (s, 1H, ArH), 7.81e
7.84 (m, 1H, ArH), 8.11 (s, 1H, NH), 8.58 (s, 1H, ArH), 9.30 (s, 1H, NH).
13C NMR (DMSO-d6):
d
(ppm) 18.30, 23.62, 54.43, 54.49, 56.44,
5.1.7.14. N-(3-Bromo-phenyl)-N0-{3-chloro-4-[(6-methoxy-7-
68.37, 101.19, 107.77, 110.11, 117.26, 120.08, 120.68, 122.22, 123.25,
124.00, 124.63, 130.65, 131.15, 135.00, 142.07, 148.20, 149.19, 150.52,
152.70, 153.11, 155.34, 165.41. LCꢀMS (APCIþ): m/z calcd for
piperidinopropoxyquinazolin-4-yl)oxy]-phenyl}urea
Compound 37 was synthesized from 18 and 23 using the general
procedure described above to afford as a white solid collected by
(37).

380
S. Ravez et al. / European Journal of Medicinal Chemistry 79 (2014) 369e381
filtration and washed with MeOH (21%). TLC: Rf 0.7
(CH2Cl2:MeOH(NH3) 9:1 v/v). Mp: 198e200 ꢁC. IR (cmꢀ1): 2935
5.2.7. In vitro endothelial tube formation assay
MatrigelÒ was thawed overnight at 4 ꢁC in an ice bath, and then
(piperidine), 1708 (C]O). 1H NMR (DMSO-d6):
d
(ppm) 1.69e1.78
50 mL of solution was used to coat 96-well plates. The plates were
(m, 4H, 2CH2), 2.15e2.28 (m, 4H, 2CH2), 2.68e2.72 (m, 2H, CH2),
3.01e3.12 (m, 2H, CH2), 3.15e3.23 (m, 2H, CH2), 3.99 (s, 3H, OCH3),
4.20e4.25 (m, 2H, CH2), 7.10e7.15 (m, 5H, 5 ArH), 7.36e7.42 (m, 2H,
2 ArH), 7.56 (s, 1H, ArH), 7.78 (s, 1H, ArH), 8.34 (s, 1H, NH), 8.43 (s,
then incubated at 37 ꢁC for 60 min to ensure complete gelation of
the matrix. HUVEC cells were then seeded into 96-well plates at a
cell density of 20 000 cells/well and allowed to incubate for 10 h at
37 ꢁC. Cells were treated with different concentrations of com-
1H, ArH), 9.27 (s, 1H, NH). 13C NMR (DMSO-d6):
d
(ppm) 24.59,
pound (0.1, 1, 5 and 10 mM). The morphological changes of the cells
26.06, 26.46, 54.56, 55.44, 56.47, 67.69, 101.15,107.67, 109.93, 117.43,
120.85, 121.98, 122.27, 122.77, 123.21, 123.56, 125.05, 125.10, 131.25,
133.80, 141.57, 147.73, 149.33, 150.62, 152.55, 155.59, 165.09. LCꢀMS
(APCIþ): m/z calcd for C30H31BrClN5O4 640 [(MþH)þ for 35Cl/79Br],
642 [(MþH)þ for 35Cl/81Br], 642 [(MþH)þ for 37Cl/79Br], 644
[(MþH)þ for 37Cl/81Br]. HRMS (ESI (MþH)þ m/z) calcd for
and tubes formed were observed under inverted microscope and
photographed with MoticamÒ.
5.2.8. Cell invasion assay
In vitro invasion assay was carried out using invasion chamber,
coated with MatrigelÒ matrix, with 6.4 mm diameter PET mem-
brane (8 micron pore size, BD Biocat). The bottom chambers were
filled with ECGM medium with 20 ng/mL VEGF and the top
chambers were seeded with ECGM medium (without growth fac-
tors) and HUVEC (1.105 cells per well). The top chamber contained
C
30H31BrClN5O4 640.1299 found 640.1304.
5.2. Biological assays and methods
5.2.1. In vitro kinase assays
vehicle or compound at various concentrations (0.01, 0.1 or 1 mM).
Kinase assays were performed in 96-well plates (Multiscreen
Cells were allowed to migrate for 24 h. Non migrated cells were
scraped with a cotton swab, and migrated cells were fixed with
100% methanol and stained with 0.05% crystal violet. The number
of HUVEC that penetrated the membrane was quantified by manual
counting and photographed with MoticamÒ. The percentage of
migrated cells was expressed on the basis of vehicle control wells.
Durapore. Millipore) using [g-
32P]ATP (Perkin Elmer) and the syn-
thetic polymer poly(Glu4/Tyr) (Sigma Chemicals) as a phos-
phoacceptor substrate. Tested compounds were dissolved in DMSO.
The final concentration of DMSO in assay solutions was 0.1%, which
was shown to have no effect on kinase activity.
5.2.2. EGFR tyrosine kinase activity
Acknowledgment
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
Kinase assays were realized on the Binding-Platform of ICPAL,
UDSL, supported by PRIM (Pôle de Recherche Interdisciplinaire
pour le Medicament). We thank the LaRMNS, RMN Laboratory,
Faculty of Pharmacy, UDSL. The authors thank Céline Lenglart and
Nathalie Duhal from the CUMA (Centre Universitaire de Mesures et
d’Analyses), UDSL, for mass spectrometry analysis.
100
mM, DTT 0.5 mM, poly(Glu4/Tyr) 250
mg/mL, ATP 5 mM [g-
32P]
ATP 0.5
mCi).
5.2.3. VEGFR-2 tyrosine kinase activity
10 ng of VEGFR-2 (Recombinant Human Protein, Invitrogen)
Appendix A. Supplementary data
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
mL, MnCl2 1.5 mM, MgCl2 10 mM, DTT 2.5 mM, Na3VO4 100 M, ß-
glycerophosphate 5 mM, poly(Glu4/Tyr) 250 g/mL, ATP 5
32P]ATP 0.5
Ci).
The reaction was stopped by adding 20
mg/
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.04.007.
m
m
mM
[
g-
m
References
m
L of trichloroacetic acid,
100%. Wells were washed 10 times with trichloroacetic acid, 10%.
Plates were counted in a Top Count (Perkin Elmer) for 1 min per
well.
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