Synthesis and biological evaluation of di-aryl urea derivatives as c-Kit inhibitors

Article abstract of DOI:10.1016/j.bmc.2015.10.035

Inhibition of receptor tyrosine kinases (RTKs) continued to be a successful approach for the treatment of many types of human cancers and many potent small molecules kinase inhibitors have been discovered the last decade. In the present study, we describe

Full text of DOI:10.1016/j.bmc.2015.10.035

Bioorganic & Medicinal Chemistry 23 (2015) 7340–7347
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of di-aryl urea derivatives
as c-Kit inhibitors
Séverine Ravez a, Stéphane Arsenlis a,b, Amélie Barczyk a, Anthony Dupont a, Raphaël Frédérick c,
Stéphanie Hesse d, Gilbert Kirsch d, Patrick Depreux a,b, Laurence Goossens a,b,
⇑
a Univ. Lille, Institut de Chimie Pharmaceutique Albert Lespagnol—ICPAL, 3, rue du Professeur Laguesse, F-59006 Lille, France
b Univ. Lille, CHU Lille, EA 7365—GRITA—Groupe de Recherche sur les formes Injectables et les Technologies Associées, FED 4260, F-59000 Lille, France
c Université Catholique de Louvain, Medicinal Chemistry Research Group (CMFA), B1.73.10, Louvain Drug Research Institute (LDRI), B-1200 Bruxelles, Belgium
d Université de Lorraine, UMR CNRS 7565, Structure et Réactivité des Systèmes Moléculaires Complexes—Equipe 3 (HECRIN), F-57070 Metz Technopôle, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Inhibition of receptor tyrosine kinases (RTKs) continued to be a successful approach for the treatment of
many types of human cancers and many potent small molecules kinase inhibitors have been discovered
the last decade. In the present study, we describe the synthesis of thienopyrimidine derivatives and their
pharmacological evaluation against nine kinases (EGFR, PDGFR-ß, c-Kit, c-Met, Src, Raf, VEGFR-1, -2 and -
3). Most of the synthesized compounds showed from moderate to potent activities against c-Kit with IC50
values in the nanomolar range. Among them, 4-anilino(urea)thienopyrimidine analogs showed selectiv-
ity and potent c-Kit inhibition with IC50 values less than 6 nM. Docking simulation was performed for the
most promising compound 9 into the c-Kit active site to determine the potential binding mode. This
study reveal that the 4-anilino(urea)thienopyrimidine is an interesting scaffold to design novel potent
and selective c-Kit inhibitors which may make promising candidates for cancers where c-Kit receptors
are overexpressed.
Received 7 September 2015
Revised 22 October 2015
Accepted 23 October 2015
Available online 24 October 2015
Keywords:
c-Kit
Kinase inhibitors
Urea
Thienopyrimidine
Cancer
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
(PI3K) signaling cascades. Aberrant expression of c-Kit signaling
primarily results mostly in leukemia, tumors of gastrointestinal
Protein kinases are critical components of cellular signal trans-
duction cascades. They form one of the most important target
classes in drug development, as they are directly involved in many
diseases, including cancer. Therefore, small molecule kinase inhibi-
tors have been successfully introduced to the drug market as selec-
tive anticancer agents with few side effects.1
The c-Kit receptor (also known as CD117) was found to be a
subclass III tyrosine kinase receptor (RTK) and its constitutive acti-
vation via somatic mutations occurs in several cancers. The c-Kit
mutations allow ligand-independent activation of this receptor
and the constitutive downstream activation of the mitogen acti-
vated protein kinase (MAPK) and phosphatidylinositol 3-kinase
tract and germ cells.2,3
Imatinib mesylate (also known as Gleevec or Glivec) is a tyro-
sine kinase inhibitor targeting c-Kit, Abl and platelet-derived
growth factor receptors (PDGFR) (Fig. 1). It revolutionized the
treatment of chronic myeloid leukemia (CML) in 2001. Thus,
encouraged by its success in treating CML patients, scientists
explored its effect in other cancers and it was found to produce a
similar effect in several cancers where c-Kit was overexpressed.4
For example, the treatment of c-Kit mutant GIST with imatinib
mesylate produces responses in 80% of patients with over 90% of
these patients remaining progression free at one year.5 However,
the major drawback with imatinib and other c-Kit TKIs is the
development of resistance which is therapeutically challenging.6
Therefore, there is an effort to develop new drugs that inhibit this
receptor.
Abbreviations: ATP, adenosine 50-triphosphate; CML, chronic myeloid leukemia;
EGFR, epidermal growth factor receptor; FC, flash chromatography; GIST, gastroin-
testinal stromal tumor; MAPK, mitogen activated protein kinase; PDGFR, platelet-
derived growth factor receptor; PI3K, phosphatidylinositol 3-kinase; rt, room
temperature; TKI, tyrosine kinase inhibitor; VEGFR, vascular endothelial growth
factor receptor.
The 4-anilinoquinazoline scaffold has been broadly used as
protein kinase inhibitors because quinazoline generally forms
two hydrogen bonds with the kinase ATP binding site. The 4-
anilinoquinazoline class of inhibitors has led to several commercial
compounds.7 Among them, the EGFR-selective inhibitor gefitinib
⇑
Corresponding author. 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).
http://dx.doi.org/10.1016/j.bmc.2015.10.035
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

S. Ravez et al. / Bioorg. Med. Chem. 23 (2015) 7340–7347
7341
scaffold by thienopyrimidines and tetrahydrobenzothienopyrim-
idines and we synthesized also quinazoline derivatives without
methoxy groups in order to compare results between the different
series (Fig. 2). Based on previous results, we designed halogen
compounds, carbamic acid ester analogs and urea derivatives in
each series with the idea that these molecules could show EGFR,
dual EGFR/VEGFR-2, and VEGFR-2 inhibitions, respectively.
Contrary to what was expected, modulations of quinazoline
scaffold have led to discover new and potent c-Kit inhibitors.
Herein, we describe synthesis, in vitro enzymatic inhibition results
and molecular modeling of these compounds.
O
HN
N
N
N
N
N
H
N
Imatinib
(Gleevec, or Glivec)
IC50 C-Kit = 0.4 µM
IC50 Abl = 0.2 µM
IC50 PDGFR = 0.4 µM
Figure 1. Structure and IC50 values of imatinib.
2. Results and discussion
2.1. Chemistry
(Iressa) and the dual EGFR/VEGFR inhibitor vandetanib (Caprelsa)
exhibit potent inhibitory activities against receptors.
The synthesis of target compounds is illustrated in Scheme 1.
Displacement of the chloro substituent of heterocycle with com-
mercial or synthesized anilines in refluxing 2-propanol led to the
corresponding 4-anilino derivatives. Whereas, reaction of hetero-
cycle with appropriate phenol in presence of tetra-N-butylammo-
nium bromide in 2-butanone and 20% solution of sodium
hydroxide mixture allowed the access to 4-aryloxy compounds.
The target compounds were obtained with yields between 4%
and 99% due to their poor solubility in organic solvents which
made difficult the purification step.
Inspired by the potential inhibitory ability of these compounds,
quinazoline heterocycle has been chosen for further structural
modification in our group. We explored the substitution by several
anilino or aryloxy groups in C-4 position of the quinazoline. The
anilino compounds substituted by a carbamic acid ester in para
position show a dual EGFR/VEGFR-2 inhibition,8 while aryloxy
derivatives exhibit selectivity for VEGFR-2 versus EGFR. More
precisely, aryloxy compounds substituted by a bis-aryl urea group
in para position show potent inhibition of VEGFR, PDGFR and also
of c-Kit (IC50 in the nanomolar range).9–11
To determine the importance of quinazoline scaffold in these
structures, we envisaged the modulation of quinazoline by other
heterocycles containing two nitrogen atoms in position 1 and 3.
Indeed, we explored the modulation of 6,7-dimethoxyquinazoline
2.2. In vitro enzyme activity inhibition assay
Firstly, all compounds were evaluated on two tyrosine kinases:
EGFR, involved in cellular proliferation and VEGFR-2 which plays a
3-Cl,4-F
3-CH3,4-NHCOOEt
R =
X = NH or O
R
3-(X),4-NHCONH-(3-Br-Ph) with X = H, CH3 or Cl
X
6
5
5
8
5
4
4
4
4
5
O
O
6
7
7
6
7
3
3
3
3
N
N
N
N
6
8
2
2
2
2
S
S
N
N
N
N
7
8
9
1
1
1
1
series A
series B
series C
series D
Figure 2. Structures of designed compounds.
R
R
Cl
N
HN
O
N
N
1: 3-Cl,4-F
: 3-CH3,4-NHCOOEt
3: 4-NHCONH-(3-Br-Ph)
4: 3-Cl,4-F
N
N
2
5
: 3-CH3,4-NHCOOEt
6: 4-NHCONH-(3-Br-Ph)
N
N
R
R
10: 3-Cl,4-F
11
: 3-CH3,4-NHCOOEt
R
R
H2N
HO
Cl
N
HN
N
O
N
N
TBAB
7: 3-Cl,4-F
12: 4-NHCONH-(3-Br-Ph)
N
8
: 3-CH3,4-NHCOOEt
13
: 3-CH3,4-NHCONH-(3-Br-Ph)
i-PrOH
reflux
2h
2-butanone
20% NaOH (2: 1)
9: 4-NHCONH-(3-Br-Ph)
14: 4-NHCONH-(3-Br-Ph)
S
S
S
100°C
1h
18-99 %
R
R
18: 3-Cl,4-F
19
: 4-NHCONH-(3-Br-Ph)
Cl
N
HN
O
N
N
4-69 %
15: 3-Cl,4-F
16
: 3-CH3,4-NHCOOEt
17: 4-NHCONH-(3-Br-Ph)
N
N
20: 3-CH3,4-NHCONH-(3-Br-Ph)
21
: 4-NHCONH-(3-Br-Ph)
S
S
S
N
Scheme 1. Synthetic step for the quinazoline and thienopyrimidine derivatives (1–21).

7342
S. Ravez et al. / Bioorg. Med. Chem. 23 (2015) 7340–7347
crucial role in angiogenesis process. Inhibition assay was per-
formed by our team by measuring the levels of phosphorylation
of the tyrosine-specific peptides (poly(Glu Tyr)substrate) in vitro
factor receptor, PDGFR and c-Kit) in their catalytic domains, VEGFR
inhibitors are rarely selective for one kinase.12 For example, sorafe-
nib and sunitinib inhibit VEGFR-2 and also other kinases such as
VEGFR-1 and -3, PDGFR and Raf. . . For this reason, we investigated
a selectivity profile of newly synthesized urea analogs. Thus, the
selected urea compounds (3, 6, 9, 12, 17 and 19) were tested on a
panel of seven additional isolated kinase receptors including:
VEGFR-1, VEGFR-3, PDGFR-ß, c-Kit, C-Met, Src and Raf. This evalu-
ation was performed at ProQinase GmbH (Freiburg, Germany) by
a radiometric protein kinase assay (33PanQinaseÒ activity assay).
The inhibition data are summarized in Table 2.
Most of the compounds show no or low inhibition of EGFR,
C-Met, Raf and Src. However, selected ureas and more particu-
larly aryloxy derivatives, (6, 12 and 19) exhibit higher affinities
for the three isoforms of VEGFR. All compounds also inhibit the
PDGFR kinase activity (IC50 between 320 and 2820 nM) and
contrary to what was expected, they show potent inhibitions of
c-Kit. Indeed, all compounds show IC50 values on this kinase in
the nanomolar range and anilino derivatives (3, 9, 17) exhibit
better results than their aryloxy analogs (6, 12 and 19). Addition-
ally, we can observe a selectivity profile for c-Kit versus the other
tested kinase for aniline derivatives, and especially for compound
using [c-
32P]ATP (Table 1).
The 4-anilino derivatives substituted by halogens (1, 7 and 15)
show interesting affinity for EGFR and selectivity for this receptor
versus VEGFR-2, like the reference compound P1. However, modu-
lation of quinazoline by thienopyrimidine heterocycles decreases
the enzyme inhibition potential. Unlike the dual EGFR/VEGFR-2
inhibitor P2, the carbamic acid esters (2, 8 and 16) exhibit no or
low inhibition for both receptors. Insertion of bis-aryl urea group
on thienopyrimidine scaffolds led to keep inhibition of VEGFR-2
and selectivity for this kinase. Indeed, urea analogs 9 and 17 exhibit
IC50 values of 1160 and 310 nM, respectively. These values show a
decrease in activity against VEGFR-2 compared to the quinazoline
compounds.
Concerning the 4-aryloxy compounds, we observe an excellent
inhibitory activity and a general selectivity for VEGFR-2 versus EGFR
for most of the compounds. More exactly, urea analogs selectively
inhibit the VEGF receptor with IC50 values in nanomolar range.
As a result of the substantial sequence homology shared by three
isoforms of VEGFR and other RTKs (such as platelet-derived growth
Table 1
Structures and enzymatic results (EGFR, VEGFR-2) of targeted compounds
O
N
N
N
N
S
S
N
N
N
O
N
series B
series C
series D
series A
Enzymatic inhibition (IC50, nM)
Series
Compounds
X
EGFRa
VEGFR-2b
P1
1
A
B
—
—
400
30
5300
>10,000
F
7
15
C
D
—
—
2980
1330
>10,000
>10,000
HN
HN
Cl
P2
2
8
A
B
C
—
—
—
—
900
500
3820
>10,000
3140
H
N
O
>10,000
>10,000
>10,000
O
O
16
D
P3
3
9
A
B
C
—
—
—
—
7710
>10,000
>10,000
3950
12
91
1160
310
H
N
H
N
Br
17
D
HN
O
P4
4
10
18
A
B
C
—
—
—
—
>10,000
590
>10,000
>10,000
9500
>10,000
>10,000
3500
F
Cl
D
P5
5
11
A
B
C
—
—
—
>10,000
>10,000
>10,000
600
710
1140
H
N
O
O
O
O
O
P6
P7
P8
6
12
13
14
19
20
21
H
CH3
Cl
H
H
CH3
Cl
H
CH3
Cl
>10,000
>10,000
6
4
8
8
18
16
9
11
34
15
H
N
H
N
Br
A
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
3290
B
C
X
D
1260
a
Inhibition of EGFR (purified from human carcinoma A431 cells) tyrosine kinase activity.
Inhibition of VEGFR-2 (recombinant protein) tyrosine kinase activity.
b

S. Ravez et al. / Bioorg. Med. Chem. 23 (2015) 7340–7347
7343
Table 2
Enzymatic inhibitions (EGFR, VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-ß, wt c-Kit, c-Met, Src and raf) of urea analogs
#
Series
EGFRa
C-Metc
Rafc
Srcc
VEGFR-1c
VEGFR-2b
VEGFR-3c
PDGFR-bc
wt c-Kitc
P3
3
9
A
B
C
7720
>10,000
>10,000
3950
N.D.
>10,000
>10,000
>10,000
N.D.
>10,000
4020
>10,000
N.D.
>10,000
>10,000
7640
N.D.
5750
>10,000
7150
12
91
1160
310
N.D.
47
1980
420
N.D.
850
2120
2820
3
1
1
6
H
N
H
N
Br
Br
O
O
17
D
HN
O
P6
6
12
19
A
B
C
>10,000
>10,000
>10,000
>10,000
2310
>10,000
>10,000
>10,000
930
6220
>10,000
5840
5470
>10,000
>10,000
8460
46
650
1080
1700
6
8
18
11
9
7
6
5
320
570
2300
16
14
11
17
H
N
H
N
D
1020
N.D. Not determined.
a
Inhibition of EGFR (purified from human carcinoma A431 cells) tyrosine kinase activity.
Inhibition of VEGFR-2 (recombinant protein) tyrosine kinase activity.
Inhibition of VEGFR-1, VEGFR-3, PDGFR-ß, wt c-Kit, c-Met, Src and Raf was performed at ProQinase GmbH (Freiburg, Germany).
b
c
9. The results of this study allow us to conclude that the
thienopyrimidine heterocycle is a promising scaffold to design
novel kinase inhibitors and especially potent and selective c-Kit
inhibitors.
2.3. Docking study
In order to predict the possible binding mode of compounds
inside the binding site of c-Kit kinase, compounds were docked
into the ATP binding site of c-Kit kinase (PDB ID:4U0I) using
GOLDÒ software (Fig. 3).
As depicted in Fig. 3, the 4-anilinothienopyrimidine scaffold is
inserted in the adenine pocket of c-Kit ATP-binding site. The dock-
ing demonstrates that the nitrogen (N1) of the thienopyrimidine
forms a hydrogen bond with the NH of the peptide bond between
the Y672 and the C673. About the urea group, NH interacts with
the side chain of E640. The oxygen atom forms a hydrogen bond
with NH of the peptide bond between the C809 and D810. The
docking structure reveals that the bis-aryl urea group is essential
for the inhibition of c-Kit.
Recently, it was suggested that targeting multiple RTKs on GIST
is more effective than single agent RTK therapy targeting predom-
inantly c-Kit. Nevertheless, it was demonstrated that targeting
c-Kit and EGFR (imatinib and erlotinib or afatinib) or c-Kit and
C-Met (imatinib and amuvatinib) with novel combinations of
RTK inhibitors abrogates imatinib resistance in GIST.13 Hence,
induction of degradation of c-Kit by chemo-agents may provide a
new insight for treatment of GISTs. That is why the design of
potent and selective c-Kit inhibitors, such as compound 9, is a
potential and promising therapeutic strategy for the treatment of
this disease.
Figure 3. Docking of compound 9 in the binding site of c-Kit kinase (PDB ID: 4U0I). Pictures made using MOE software.

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S. Ravez et al. / Bioorg. Med. Chem. 23 (2015) 7340–7347
m/z calcd for C14H9ClFN3 274 [(M+H)+ for 35Cl] and 276 [(M+H)+
3. Conclusions
for 37Cl]. HRMS (ESI (M+H)+ m/z) calcd for C14H9ClFN3 274.0541.
Found 274.0535.
In summary, we have described the discovery, SAR study and
preliminary biological evaluation of selective and potent c-Kit inhi-
bitors. A small-sized library of novel thieno and tetrahydroben-
4.1.1.2. N-[2-Methyl-4-(quinazolin-4-ylamino)phenyl]carbamic
acid ethyl ester (2).
zothieno-pyrimidines was synthesized and
a dozen of urea
Compound 2 was obtained by crystalliza-
compounds were identified as kinase inhibitors. More precisely,
the 4-aryloxy compounds show multi-kinase inhibitions of VEGFR
(1, 2 and 3), PDGFR-b and c-Kit while their anilino analogs exhibit
potent and selective inhibition of c-Kit with IC50 values in the
nanomolar range. Indeed, the most active and selective compound
9 inhibits c-Kit at nanomolar concentration (IC50 = 1 nM), while
exhibits no significant inhibition against other tested kinase
(IC50 >1 160 nM). This 4-anilino(urea)thienopyrimidine can be
used as lead candidate for future c-Kit inhibitor development. First
results on cancer cell lines (prostate PC3, breast MCF7 and colon
HT29) show no or low inhibition of cellular proliferation at
tion from ethanol as a white solid (82%). Mp: 210–212 °C. IR
(cmꢀ1): 3484 (NH), 1706 (C@O). 1H NMR (DMSO-d6): d (ppm)
1.24–1.27 (t, 3H, J = 7.1 Hz, CH3), 2.24 (s, 3H, CH3), 4.09–4.15 (q,
2H, J = 7.1 Hz, CH2), 7.32–7.34 (m, 1H, ArH), 7.60–7.64 (m, 3H, 3
ArH), 7.76–7.78 (m, 1H, ArH), 7.83–7.87 (m, 1H, ArH), 8.54–8.56
(m, 2H, 2 ArH), 8.81 (br s, 1H, NH), 9.77 (br s, 1H, NH). 13C NMR
(DMSO-d6): d (ppm) 14.60, 18.01, 60.08, 115.26, 120.54, 123.00,
124.37, 126.07, 127.68, 129.15, 129.38, 132.25, 132.85, 149.62,
154.60, 157.80 (C@O). LC–MS (APCI+): m/z calcd for
C
C
18H18N4O2 323 [(M+H)+]. HRMS (ESI (M+H)+ m/z) calcd for
18H18N4O2 323.1502. Found 323.1496.
10 lM. Other investigations on several cancer cell lines must be
led. Thus, some modifications will be undertaken to increase DMPK
properties and further in vivo pharmacological evaluations with
the best candidate will be performed.
4.1.1.3. N-(3-Bromophenyl)-N0-[4-(quinazolin-4-ylamino)phe-
nyl]urea (3).
Compound 3 was obtained by crystallization
from methanol as
a white solid (99%). Mp: 232–234 °C. IR
(cmꢀ1): 3171 (NH), 1682 (C@O). 1H NMR (DMSO-d6): d (ppm)
7.17–7.35 (m, 3H, 3 ArH), 7.58–7.98 (m, 8H, 8 ArH), 8.73 (m, 2H,
2 ArH), 9.38 (br s, 1H, NH), 9.52 (br s, 1H, NH), 10.64 (br s, 1H,
4. Experimental section
4.1. General chemistry
NH). 13C NMR (DMSO-d6):
d (ppm) 114.37, 116.64, 118.09,
118.85, 120.00, 121.72, 123.72, 124.09, 124.29, 127.15, 130.70,
131.98, 134.28, 136.81, 141.60, 152.54, 153.02, 158.48 (C@O).
LC–MS (APCI+): m/z calcd for C21H16BrN5O434 [(M+H)+ for 79Br]
and 436 [(M+H)+ for 81Br]. HRMS (ESI (M+H)+ m/z) calcd for
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, 0.015–0.040 mm (Merck). The structure
of each compound was confirmed by IR (Bruker VECTOR 22 instru-
ment) and 1H NMR (300 MHz, Bruker AC300P spectrometer).
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
separation followed by APCI+ (atmospheric pressure chemical
ionization) mass spectral detection on an LC–MS system,
ThermoElecton 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).
C21H16BrN5O 434.0611. Found 434.0598.
4.1.1.4.
(7).
4-(3-Chloro-4-fluoroanilino)thieno[2,3-d]pyrimidine
Compound 7 was obtained by crystallization from ace-
tonitrile as a white solid (55%). Mp: 228–229 °C. IR (cmꢀ1): 3348
(NH). 1H NMR (DMSO-d6): d (ppm) 7.34–7.36 (dd, 1H, J = 9.0 and
9.0 Hz, ArH), 7.69–7.72 (m, 2H,
2 ArH), 7.77–7.79 (d, 1H,
J = 6.0 Hz, ArH), 8.11–8.14 (dd, 1H, J = 6.7 and 2.4 Hz, ArH), 8.47
(s, 1H, ArH), 9.71 (br s, 1H, NH). 13C NMR (DMSO-d6): d (ppm)
116.61, 116.82, 116.93 (J = 5.0 Hz), 118.80 (J = 20.1 Hz), 119.28,
121.38 (J = 6.9 Hz), 122.54, 123.55 (J = 5.7 Hz), 124.36, 136.57,
152.99 (J = 143.0 Hz), 166.62. LC–MS (APCI+): m/z calcd for
C
12H7ClFN3S280 [(M+H)+ for 35Cl] and 282 [(M+H)+ for 37Cl]. HRMS
(ESI (M+H)+ m/z) calcd for C12H7ClFN3S 280.0106. Found 280.0100.
4.1.1. General procedure for 4-anilino-quinazolines/
thienopyrimidines
4.1.1.5.
N-[2-Methyl-4-(thieno[2,3-d]pyrimidin-4-ylamino)
Compound 8 was
phenyl]carbamic acid ethyl ester (8).
To a stirred solution of commercial chloro derivative (1 equiv)
in isopropanol was added aniline (1.2 equiv). After 2 h at reflux,
the precipitate was filtered, washed with isopropanol. The
obtained residue was dissolved in a 10% K2CO3 solution and the
mixture was stirred 30 min at room temperature. Then, the aque-
ous layer was extracted with EtOAc and the organic layers were
dried over MgSO4. The solvent was removed under reduced
pressure.
obtained by crystallization from ethanol as a white solid (74%).
Mp: 224–226 °C. IR (cmꢀ1): 3250 (NH), 1681 (C@O). 1H NMR
(DMSO-d6): d (ppm) 1.24–1.27 (t, 3H, J = 7.0 Hz, CH3), 2.24 (s, 3H,
CH3), 4.10–4.15 (q, 2H, J = 7.1 Hz, CH2), 7.38–7.41(m, 1H, ArH),
7.57–7.58 (m, 2H, 2 ArH), 7.83–7.84 (d, 1H, J = 5.7 Hz, ArH), 8.10
(d, 1H, J = 5.7 Hz, ArH), 8.64 (s, 1H, ArH), 8.89 (br s, 1H, NH),
10.67 (br s, 1H, NH). 13C NMR (DMSO-d6): d (ppm) 14.58, 17.99,
60.18, 117.47, 120.44, 121.14, 124.97, 125.50, 132.10, 133.81,
149.63, 154.45, 154.57. LC–MS (APCI+): m/z calcd for C16H16N4O2S
329 [(M+H)+]. HRMS (ESI (M+H)+ m/z) calcd for C16H16N4O2S
329.1066. Found 329.1057.
4.1.1.1. 4-(3-Chloro-4-fluoroanilino)quinazoline (1).
Com-
pound 1 was obtained by crystallization from acetonitrile as a
white solid (99%). Mp: 216–218 °C. IR (cmꢀ1): 3122 (NH).1H NMR
(DMSO-d6): d (ppm) 7.41–7.46 (ddd, 1H, J = 9.0 and 3.1 Hz, ArH),
7.61–7.65 (m, 1H, ArH), 7.77–7.85 (m, 3H, 3 ArH), 8.19–8.22 (m,
1H, ArH), 8.53–8.55 (dd, 1H, J = 8.5 and 1.3 Hz, ArH), 8.60 (d, 1H,
J = 3.4 Hz, ArH), 9.95 (br s, 1H, NH). 13C NMR (DMSO-d6): d
(ppm). 115.60, 116.34 (d, J = 21.7 Hz), 118.58 (d, J = 18.3 Hz),
122.54 (d, J = 6.7 Hz), 123.09, 123.63, 126.13, 127.64, 132.92,
137.57, 149.65 (D, J = 224.0 Hz), 154.38, 157.60. LC–MS (APCI+):
4.1.1.6.
ylamino)phenyl]urea (9).
crystallization from methanol as
240–242 °C. IR (cmꢀ1): 3251 (NH), 1671 (C@O). 1H NMR (DMSO-
d6): (ppm) 7.13–7.15 (m, 1H, ArH), 7.22–7.26 (ddd, 2H,
J = 7.9 Hz, 2 ArH), 7.33–7.35 (m, 1H, ArH), 7.48–7.50 (d, 2H,
N-(3-Bromophenyl)-N0-[4-(thieno[2,3-d]pyrimidin-4-
Compound 9 was obtained by
white solid (48%). Mp:
a
d

S. Ravez et al. / Bioorg. Med. Chem. 23 (2015) 7340–7347
7345
J = 8.9 Hz, 2 ArH), 7.70–7.72 (m, 3H, 3 ArH), 7.85–7.87 (d, 1H,
J = 6.0 Hz, ArH), 7.89–7.90 (dd, 1H, J = 1.9 Hz, ArH), 8.46 (s, 1H,
ArH), 9.01 (br s, 1H, NH), 9.14 (br s, 1H, NH), 9.62 (br s, 1H, NH).
13C NMR (DMSO-d6): d (ppm) 116.64, 116.94, 118.66, 119.52,
120.26, 121.73, 122.34, 123.57, 124.12, 130.69, 133.45, 153.27,
154.86. LC–MS (APCI+): m/z calcd for C19H14BrN5OS440 [(M+H)+
for 79Br] and 442 [(M+H)+ for 81Br]. HRMS (ESI (M+H)+ m/z) calcd
for C19H14BrN5OS 440.0175. Found 440.0163.
solid (4%). Mp: 122–124 °C.1H NMR (DMSO-d6): d (ppm) 7.41–7.48
(m, 1H, ArH), 7.57 (dd, 1H, J = 9.0 and 9.0 Hz, ArH), 7.75–7.84 (m,
2H, ArH), 7.98–8.10 (m, 2H, 2 ArH), 8.34–8.40 (m, 1H, ArH), 8.65
(s, 1H, ArH). LC–MS (APCI+): m/z calcd for C14H8ClFN2O275 [(M
+H)+ for 35Cl] and 277 [(M+H)+ for 37Cl]. HRMS (ESI (M+H)+ m/z)
calcd for C14H8ClFN2O275.0382. Found 275.0377.
4.1.2.2.
N-[2-Methyl-4-(quinazolin-4-yloxy)phenyl]carbamic
Compound 5 was purified in methanol
acid ethyl ester (5).
and filtered while hot as a white solid (31%). Mp: 198–200 °C. IR
(cmꢀ1): 3171 (NH), 1713 (C@O). 1H NMR (DMSO-d6): d (ppm)
1.25–1.28 (t, 3H, J = 6.0 Hz, CH3), 2.25 (s, 3H, CH3), 4.11–4.17 (q,
2H, J = 6.0 Hz, CH2), 7.12–7.15 (dd, 1H, J = 8.7 and 2.7 Hz, ArH),
7.19 (d, 1H, J = 2.7 Hz, ArH), 7.43–7.45 (d, 1H, J = 8.7 Hz, ArH),
7.75–7.84 (m, 1H, ArH), 7.97–8.07 (m, 2H, 2 ArH), 8.34–8.40 (m,
1H, ArH), 8,74 (s, 1H, ArH), 8.93 (br s, 1H, NH). 13C NMR (DMSO-
d6): d (ppm) 14.57, 17.79, 60.22, 115.55, 119.58, 123.39, 123.52,
125.86, 127.51, 128.08, 133.63, 134.17, 134.62, 148.81, 151.08,
153.91, 154.60, 166.64 (C@O). LC–MS (APCI+): m/z calcd for
4.1.1.7.
4-(3-Chloro-4-fluoroanilino)-5,6,7,8-tetrahydrobenzo
Compound 15 was
[4,5]thieno[2,3-d]pyrimidine (15).
obtained by crystallization from acetonitrile as a white solid
(33%). Mp: 128–130 °C. IR (cmꢀ1): 3457 (NH). 1H NMR (DMSO-
d6): d (ppm) 1.85 (m, 4H, 2 CH2), 2.83 (m, 2H, 2 CH2), 3.12 (m,
2H, CH2), 7.40 (dd, 1H, J = 9.1 and 9.1 Hz, ArH), 7.60–7.67 (m, 1H,
ArH), 7.92 (dd, 1H, J = 6.9 and 2.5 Hz, ArH), 8.22 (s, 1H, ArH), 8.41
(br s, 1H, NH). 13C NMR (DMSO-d6): d (ppm) 21.92, 22.12, 25.05,
25.28, 116.28 (J = 21.5 Hz), 116.76, 118.61 (J = 18.6 Hz), 122.63
(J = 6.8 Hz), 123.70, 126.49, 133.28, 136.42, 151.90 (J = 257 Hz),
152.07, 165.94. LC–MS (APCI+): m/z calcd for C16H13ClFN3S 334
[(M+H)+ for 35Cl] and 336 [(M+H)+ for 37Cl]. HRMS (ESI (M+H)+
m/z) calcd for C16H13ClFN3S 334.0575. Found 334.0566.
C
C
18H17N3O3 324 [(M+H)+]. HRMS (ESI (M+H)+ m/z) calcd for
18H17N3O3 324.1342. Found 324.1331.
4.1.2.3. N-(3-Bromophenyl)-N0-{4-[(quinazolin-4-yl)oxy]phenyl}
urea (6). Compound 6 was purified in acetonitrile and filtered
4.1.1.8.
[2,3-d]pyrimidin-4-ylamino)phenyl]carbamic acid ethyl ester
(16). Compound 16 was obtained by crystallization from
N-[2-Methyl-4-(5,6,7,8-tetrahydrobenzo[4,5]thieno
while hot as a white solid (12%). Mp: 248–250 °C. IR (cmꢀ1): 3266
(NH), 1645 (C@O). 1H NMR (DMSO-d6): d (ppm) 7.23–7.25 (m, 1H,
ArH), 7.33–7.39 (m, 4H, 4 ArH), 7.61–7.64 (d, 2H, J = 8.9 Hz, ArH),
7.85–7.87 (m, 1H, ArH), 7.94–7.95 (m, 1H, ArH), 7.98–8.08
(m, 2H, 2 ArH), 8.44–8.46 (m, 1H, ArH), 8.79 (s, 1H, ArH), 8.94
(br s, 1H, NH), 8.99 (br s, 1H, NH). 13C NMR (DMSO-d6): d (ppm)
115.60, 117.05, 119.54, 120.40, 121.72, 122.35, 123.42, 124.34,
127.50, 128.06, 130.71, 134.60, 137.11, 141.40, 146.72, 151.07,
152.43, 153.93, 166.60 (C@O). LC–MS (APCI+): m/z calcd for
ethanol as a white solid (18%). Mp: 184–185 °C. IR (cmꢀ1): 3237
(NH), 1681 (C@O). 1H NMR (DMSO-d6): d (ppm) 1.23–1.25 (t, 3H,
J = 7.7 Hz, CH3), 1.86 (m, 4H, 2 CH2), 2.22 (s, 3H, CH3), 2.85 (m,
2H, CH2), 3.14 (m, 2H, CH2), 4.09–4.13 (q, 2H, J = 7.7 Hz, CH2),
7.31–7.50 (m, 3H, 3 ArH), 8.52 (s, 1H, ArH), 8.72 (br s, 1H, NH),
8.88 (br s, 1H, NH). 13C NMR (DMSO-d6): d (ppm) 14.59, 17.90,
21.74, 21.98, 24.96, 25.23, 60.15, 116.68, 121.70, 124.89, 125.58,
127.12, 132.10, 133.91, 134.12, 134.36, 149.35, 154.48, 154.92.
LC–MS (APCI+): m/z calcd for C20H22N4O2S 383 [(M+H)+]. HRMS
(ESI (M+H)+ m/z) calcd for C20H22N4O2S 383.1536. Found 383.1515.
C
21H15BrN4O2 435 [(M+H)+ for 79Br] and 437 [(M+H)+ for 81Br].
HRMS (ESI (M+H)+ m/z) calcd for C21H15BrN4O2 435.0451. Found
435.0432.
4.1.2.4. 4-(3-Chloro-4-fluoroaryloxy)-thieno[2,3-d]pyrimidine
4.1.1.9. N-(3-Bromophenyl)-N0-[4-(5,6,7,8-tetrahydrobenzo[4,5]
(10).
Compound 10 was obtained by crystallization from ace-
thieno[2,3-d]pyrimidin-4-ylamino)phenyl]urea (17).
Com-
tonitrile as a white solid (40%). Mp: 128–130 °C. 1H NMR (DMSO-
d6): d (ppm) 7.40–7.44 (m, 1H, ArH), 7.54–7.59 (dd, 1H, J = 9.0 Hz,
ArH), 7.67–7.68 (d, 1H, J = 6.0 Hz, ArH), 7.74–7.76 (dd, 1H, J = 6.3
and 3.0 Hz), 7.99 (d, 1H, J = 6.0 Hz, ArH), 8.66 (s, 1H, ArH). 13C
NMR (DMSO-d6): d (ppm) 117.39 (d, J = 23 Hz), 118.40, 118.48,
119.86 (d, J = 19 Hz), 122.90 (d, J = 7 Hz), 124.58, 127.66, 148.14,
152.81, 153.89 (D, J = 244 Hz), 162.85, 169.11. HRMS (ESI (M+H)+
m/z) calcd for C12H7ON2ClFS 280.9946. Found 280.9939.
pound 17 was obtained by crystallization from methanol as a
white solid (65%). Mp: 241–243 °C. IR (cmꢀ1): 3282 (NH), 1633
(C@O). 1H NMR (DMSO-d6): d (ppm) 1.86 (m, 4H, 2 CH2), 2.82
(m, 4H, 2 CH2), 3.13 (m, 2H, CH2), 7.15 (m, 1H, ArH), 7.23 (dd,
1H, J = 7.9 and 7.9 Hz, ArH), 7.30 (m, 1H, ArH), 7.45 (d, 2H,
J = 8.9 Hz, ArH), 7.58 (d, 2H, J = 8.9 Hz, ArH), 7.88 (dd, 1H, J = 1.5
and 1.5 Hz, ArH), 8.05 (s, 1H, NH), 8.33 (s, 1H, ArH), 8.72 (s, 1H,
NH), 8.88(s, 1H, NH). 13C NMR (DMSO-d6): d (ppm) 21.98, 22.13,
25.03, 25.39, 116.37, 116.92, 118.54, 120.29, 121.71, 123.32,
124.20, 126.59, 130.66, 132.53, 133.48, 135.24, 141.48, 152.16,
152.37, 155.05, 165.51. LC–MS (APCI+): m/z calcd for C23H20BrN5-
OS494 [(M+H)+ for 79Br] and 496 [(M+H)+ for 81Br]. HRMS (ESI (M
+H)+ m/z) calcd for C23H20BrN5OS 494.0644. Found 494.0629.
4.1.2.5.
N-[2-Methyl-4-(thieno[2,3-d]pyrimidin-4-yloxy)
Compound 11 was
phenyl]carbamic acid ethyl ester (11).
purified in methanol and filtered while hot as a white solid
(29%). Mp: 207–209 °C. IR (cmꢀ1): 3219 (NH), 1711 (C@O). 1H
NMR (DMSO-d6): d (ppm) 1.25 (t, 3H, J = 6.0 Hz, CH3), 2.22 (s, 3H,
CH3), 4.12 (q, 2H, J = 6.0 Hz, CH2), 7.08 (dd, 1H, J = 8.6 and 2.5 Hz,
ArH), 7.14 (d, 1H, J = 2.5 Hz, ArH), 7.39 (d, 1H, J = 8.6 Hz, ArH),
7.65 (d, 1H, J = 5.9 Hz, ArH), 7.96 (d, 1H, J = 5.9 Hz, ArH), 8,61 (s,
1H, ArH), 8.88 (s, 1H, NH). LC–MS (APCI+): m/z calcd for
4.1.2. General procedure for 4-aryloxy-quinazolines/thienopy-
rimidines substituted by halogen or urea
To a stirred solution of commercial chloro derivative (1 equiv)
and tetrabutylammonium bromide in 10 mL of a mixture of 20%
NaOH and 2-butanone (1:2) were added phenol (1 equiv). After
1 h at room temperature, the reaction was quenched by water,
and then the aqueous solution was extracted with EtOAc
(3 ꢁ 10 mL), washed with a solution of NaOH 1 N, and dried over
MgSO4. The solvent was removed under reduced pressure.
C
C
16H15N3O3S 330 [(M+H)+]. HRMS (ESI (M+H)+ m/z) calcd for
16H15N3O3S 330.0906. Found 330.0897.
4.1.2.6. N-(3-Bromophenyl)-N0-{4-[(thieno[2,3-d]pyrimidin-4-
yl)oxy]phenyl}urea (12). Compound 12 was purified in
acetonitrile and filtered while hot as a white solid (36%). Mp:
210–212 °C. IR (cmꢀ1): 3280 (NH), 1629 (C@O). 1H NMR (DMSO-
d6): d (ppm) 7.11–7.13 (m, 1H, ArH), 7.19–7.23 (m, 3H, 3 ArH),
7.38–7.40 (m, 1H, ArH), 7.58–7.60 (m, 2H, 2 ArH), 7.65–7.66
4.1.2.1. 4-(3-Chloro-4-fluoroaryloxy)quinazoline (4).
Com-
pound 4 was purified in heptane and filtered while hot as a white

7346
S. Ravez et al. / Bioorg. Med. Chem. 23 (2015) 7340–7347
(d, 1H, J = 5.9 Hz, ArH), 7.90–7.91 (dd, 1H, J = 1.8 Hz, ArH), 7.96–
7.97 (d, 1H, J = 5.9 Hz, ArH), 8.62 (s, 1H, ArH), 9.80 (br s, 2H, 2
163.05, 167.51. LC–MS (APCI+): m/z calcd for C23H19BrN4O2S495
[(M+H)+ for 79Br] and 497 [(M+H)+ for 81Br]. HRMS (ESI (M+H)+
m/z) calcd for C23H19BrN4O2S 495.0484. Found 495.0483.
NH). 13C NMR (DMSO-d6):
d (ppm) 116.96, 118.51, 119.44,
120.33, 121.66, 122.13, 123.99, 127.23, 130.57, 137.53, 141.87,
146.32, 149.00, 152.75, 152.98, 163.39, 168.89 (C@O). LC–MS
(APCI+): m/z calcd for C19H13BrN4O2S441 [(M+H)+ for 79Br] and
443 [(M+H)+ for 81Br]. HRMS (ESI (M+H)+ m/z) calcd for
4.1.2.11. N-(3-Bromophenyl)-N0-{3-methyl-4-[(5,6,7,8-tetrahy-
drobenzo[4,5]-thieno[2,3-d]pyrimidin-4-yl)oxy]phenyl}urea
(20).
Compound 20 was purified in methanol and filtered
C19H13BrN4O2S 441.0015. Found 441.0008.
while hot as white solid (64%). Mp: 208–210 °C. IR (cmꢀ1): 3278
(NH), 1641 (C@O). 1H NMR (DMSO-d6): d (ppm) 1.85 (m, 4H, 2
CH2), 2.80 (m, 2H, CH2), 2.07 (s, 3H, CH3), 2.99 (m, 2H, CH2),7.11
(m, 1H, ArH), 7.14 (m, 1H, 1 ArH), 7.23 (m, 2H, 2 ArH), 7.35–7.46
(m, 1H, ArH), 7.62 (m, 1H, ArH), 7.91 (m, 1H, ArH), 8.54 (s, 1H,
ArH), 8.80 (s, 1H, NH), 9.12 (s, 1H, NH). LC–MS (APCI+): m/z calcd
for C24H21BrN4O2S509 [(M+H)+ for 79Br] and 511 [(M+H)+ for
81Br]. HRMS (ESI (M+H)+ m/z) calcd for C24H21BrN4O2S 509.0641.
Found 509.0639.
4.1.2.7.
pyrimidin-4-yl)oxy]phenyl}urea (13).
N-(3-Bromophenyl)-N0-{3-methyl-4-[(thieno[2,3-d]
Compound 13 was
purified in methanol and filtered while hot as a white solid
(57%). Mp: 211–213 °C. IR (cmꢀ1): 3280 (NH), 1630 (C@O). 1H
NMR (DMSO-d6): d (ppm) 2.29 (s, 3H, CH3), 7.09–7.18 (m, 3H, 3
ArH), 7.22 (m, 1H, 2 ArH), 7.32–7.38 (m, 1H, ArH), 7.68 (m, 1H,
ArH), 7.82 (d, 1H, J = 5.8 Hz, ArH), 7.90 (m, 1H, ArH), 7.98 (d, 1H,
J = 5.8 Hz, ArH), 8.53 (s, 1H, ArH), 8.63 (s, 1H, NH), 9.77 (s, 1H,
NH). 13C NMR (DMSO-d6): d (ppm) 17.98, 116.70, 118.49, 118.54,
119.40, 120.09, 121.73, 122.93, 123.37, 124.01, 127.26, 130.42,
130.68, 134.90, 141.82, 147.28, 152.82, 153.01, 163.40, 168.91.
LC–MS (APCI+): m/z calcd for C20H15BrN4O2S455 [(M+H)+ for 79Br]
and 457 [(M+H)+ for 81Br]. HRMS (ESI (M+H)+ m/z) calcd for
C20H15BrN4O2S 455.0171. Found 455.0166.
4.1.2.12. N-(3-Bromophenyl)-N0-{3-chloro-4-[(5,6,7,8-tetrahy-
drobenzo[4,5]-thieno[2,3-d]pyrimidin-4-yl)oxy]phenyl}urea
(21).
Compound 21 was purified in methanol and filtered
while hot as a white solid (13%). Mp: 239–241 °C. IR (cmꢀ1):
3289 (NH), 1617 (C@O). 1H NMR (DMSO-d6): d (ppm) 1.85 (m,
4H, 4 ArH), 2.86 (m, 2H, 2 ArH), 3.00 (m, 2H, 2 ArH), 7.10–7.38
(m, 4H, 4 ArH), 7.45–7.62 (m, 1H, ArH), 7.80–7.97 (m, 1H, ArH),
8.09–8.12 (m, 1H, ArH), 8.42 (s, 1H, NH), 8.52 (s, 1H, ArH), 9.56
(s, 1H, NH). LC–MS (APCI+): m/z calcd for C23H18BrClN4O2S 528
[(M+H)+ for 35Cl/79Br], 530 [(M+H)+ for 35Cl/81Br], 530 [(M+H)+ for
37Cl/79Br], 532 [(M+H)+ for 37Cl/81Br]. HRMS (ESI (M+H)+ m/z) calcd
for C23H18BrClN4O2S 529.0095. Found 529.0091.
4.1.2.8.
pyrimidin-4-yl)oxy]phenyl}urea (14).
N-(3-Bromophenyl)-N0-{3-chloro-4-[(thieno[2,3-d]
Compound 14 was
purified in acetonitrile and filtered while hot as a white solid
(11%). Mp: 227–229 °C. IR (cmꢀ1): 3297 (NH), 1648 (C@O). 1H
NMR (DMSO-d6): d (ppm) 7.15–7.22 (m, 1H, ArH), 7.22–7.27 (m,
2H, 2 ArH), 7.31 (dd, 1H, J = 9.0 and 2.5 Hz, ArH), 7.57–7.59 (d, 1H,
J = 2.5 Hz, ArH), 7.67–7.70 (d, 1H, J = 5.9 Hz, ArH), 7.86–7.93 (m,
1H, ArH), 7.98 (d, 1H, J = 5.9 Hz, ArH), 8.20 (d, 1H, J = 9.0 Hz, ArH),
8.66 (s, 1H, ArH), 9.10 (br s, 2H, 2 NH). 13C NMR (DMSO-d6): d
(ppm) 116.97, 118.45, 118.51, 120.36, 121.37, 121.80, 122.50,
122.96, 123.03, 124.57, 127.50, 130.79, 133.70, 141.20, 146.83,
152.19, 152.88, 163.07, 169.04. LC–MS (APCI+): m/z calcd for
4.2. In vitro kinase assays
Kinase assays were performed in 96-well plates (Multiscreen
Durapore. Millipore) using [c-
32P]ATP (Perkin Elmer) and the syn-
thetic polymer poly(Glu4/Tyr) (Sigma Chemicals) as a phosphoac-
ceptor 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.
C
19H12BrClN4O2S474 [(M+H)+ for 35Cl/79Br], 476 [(M+H)+ for
35Cl/81Br], 476 [(M+H)+ for 37Cl/79Br], 478 [(M+H)+ for 37Cl/81Br].
HRMS (ESI (M+H)+ m/z) calcd for C19H12BrClN4O2S 474.9625. Found
474.962.
4.2.1. 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, Na3-
4.1.2.9. 4-(3-Chloro-4-fluoroaryloxy)-5,6,7,8-tetrahydrobenzo[4,
5]-thieno[2,3-d]pyrimidine
(18).
Compound
18
was
obtained by crystallization from cyclohexane as a white solid
(29%). Mp: 145–147 °C. 1H NMR (DMSO-d6): d (ppm) 1.85 (m,
4H, 2 CH2), 2.85 (m, 2H, CH2), 3.00 (m, 2H, CH2), 7.35–7.40 (m,
1H, ArH), 7.53 (dd, 1H, J = 9.0 and 9.0 Hz, ArH), 7.69 (dd, 1H,
J = 6.1 and 2.9 Hz), 8.51 (s, 1H, ArH). 13C NMR (DMSO-d6): d
(ppm) 21.73, 22.34, 24.96, 25.31, 117.25, 117.48, 118.25, 119.76,
119.96, 122.84, 122.92, 124.46, 126.82, 135.94, 148.19, 148.22,
151.85, 153.75, 156.18, 162.41, 167.75. LC–MS (APCI+): m/z calcd
for C16H12ClFN2OS335 [(M+H)+ for 35Cl] and 337 [(M+H)+ for
37Cl]. HRMS (ESI (M+H)+ m/z) calcd for C16H12ClFN2OS 335.0415.
Found 335.0405.
VO4 100
l
M, DTT 0.5 mM, poly(Glu4/Tyr) 250
Ci).
lg/mL, ATP 5 lM.
[c-
32P]ATP 0.5
l
4.2.2. 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
100
ATP 5
The reaction was stopped by adding 20
l
l
g/mL, MnCl2 1.5 mM, MgCl2 10 mM, DTT 2.5 mM, Na3VO4
M, ß-glycerophosphate 5 mM, poly(Glu4/Tyr) 250 g/mL,
M. [ Ci).
32P]ATP 0.5
l
l
c-
l
lL of trichloroacetic
4.1.2.10. N-(3-Bromophenyl)-N0-{4-[(5,6,7,8-tetrahydrobenzo[4,
5]-thieno[2,3-d]-pyrimidin-4-yl)oxy]phenyl}urea
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.
(19).
Compound 19 was purified in acetonitrile and filtered
while hot as a white solid (69%). Mp: 231–233 °C. IR (cmꢀ1):
3269 (NH), 1649 (C@O). 1H NMR (DMSO-d6): d (ppm) 1.86 (m,
4H, 2 CH2), 2.87 (m, 2H, CH2), 3.02 (m, 2H, CH2), 7.10–7.28 (m,
4H, 4 ArH), 7.30–7.39 (m, 1H, ArH), 7.53 (d, 2H, J = 8.8 Hz, ArH),
7.83–7.90 (m, 1H, ArH), 8.47 (s, 1H, ArH), 9.67 (s, 1H, NH), 9.74
(s, 1H, NH). 13C NMR (DMSO-d6): d (ppm) 21.78, 22.35, 24.96,
25.46, 116.88, 118.32, 119.30, 120.26, 121.65, 122.14, 123.98,
126.93, 130.58, 135.53, 137.34, 141.87, 146.44, 152.05, 152.69,
4.3. Molecular modeling
The compound was built using the Chembiodraw 3D module
and minimized. Docking simulation was then performed into
c-Kit (RCSB Protein Data Bank 4U0I) with the automated GOLD
program.14 The active site was defined including all residues in a
volume of 10 Å around ponatinib taken as reference.

S. Ravez et al. / Bioorg. Med. Chem. 23 (2015) 7340–7347
7347
3. Beham, A. W.; Schaefer, I. M.; Schüler, P.; Cameron, S.; Ghadimi, B. M. Int. J.
Colorectal Dis. 2012, 27, 689.
Acknowledgements
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Kinase assays were realized on the Binding-Platform of ICPAL,
Univ Lille 2, supported by PRIM (Pôle de RechercheInterdisci-
´
plinaire pour le Medicament). We thank the LaRMN (RMN Labora-
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tory, Faculty of Pharmacy, Univ Lille 2) and the CUMA (Centre
Universitaire de Mesures et d’Analyses, Univ Lille 2), for spectral
analysis.
7. Ravez, S.; Castillo-Aguilera, O.; Depreux, P.; Goossens, L. Expert Opin. Ther. Pat.
2015, 24, 1.
8. Garofalo, A.; Goossens, L.; Lemoine, A.; Ravez, S.; Six, P.; Howsam, M.; Farce, A.;
Depreux, P. Med. Chem. Commun. 2011, 2, 65.
9. Garofalo, A.; Goossens, L.; Six, P.; Lemoine, A.; Ravez, S.; Farce, A.; Depreux, P.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.10.035.
11. Ravez, S.; Barczyk, A.; Six, P.; Cagnon, A.; Garofalo, A.; Goossens, L.; Depreux, P.
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References and notes
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