Design and discovery of 4-anilinoquinazoline ureas as multikinase inhibitors targeting BRAF, VEGFR-2 and EGFR

Article abstract of DOI:10.1039/c3md00096f

4-Anilinoquinazoline ureas were envisaged according to the hybrid-design approach based upon two privileged pharmacophores in kinase drug discovery, i.e. 4-anilinoquinazoline and unsymmetrical diaryl urea. In our structure-activity relationships (SAR) campaign, title compounds were synthesized and profiled in biochemical assay for their kinase inhibitory activity. Title compounds 18-20 were found to be multikinase inhibitors with profound activity against BRAF, BRAF V600E, VEGFR-2 and EGFR. Molecular docking into DFG-out conformations of BRAF and VEGFR-2 suggested that they might be type II inhibitors.

Full text of DOI:10.1039/c3md00096f

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Design and discovery of 4-anilinoquinazoline ureas as
multikinase inhibitors targeting BRAF, VEGFR-2 and
EGFR
Cite this: DOI: 10.1039/c3md00096f
a
a
b
a
a
c
*
*
Qingwen Zhang, Yuanyuan Diao, Fei Wang, Ying Fu, Fei Tang, Qidong You
and Houyuan Zhoua
4-Anilinoquinazoline ureas were envisaged according to the hybrid-design approach based upon two
privileged pharmacophores in kinase drug discovery, i.e. 4-anilinoquinazoline and unsymmetrical diaryl
urea. In our structure–activity relationships (SAR) campaign, title compounds were synthesized and
profiled in biochemical assay for their kinase inhibitory activity. Title compounds 18–20 were found to
be multikinase inhibitors with profound activity against BRAF, BRAF V600E, VEGFR-2 and EGFR.
Molecular docking into DFG-out conformations of BRAF and VEGFR-2 suggested that they might be
type II inhibitors.
Received 28th March 2013
Accepted 26th April 2013
DOI: 10.1039/c3md00096f
www.rsc.org/medchemcomm
the aberrant signalling network represents an important para-
digm in targeted cancer drug discovery. The EGFR/Ras/RAF/
Introduction
Protein kinases have become the most important class of drug
target in the eld of oncology. Around 50–70% of current cancer
drug discovery programs are focused on protein kinase inhibi-
tors.1 Since the launch of imatinib (Gleevecꢀ) in 2001 for the
treatment of patients with Philadelphia chromosome positive
chronic myeloid leukemia (Ph + CML), some 20 small molecule
protein kinase inhibitors have been approved for the targeted
therapy of human cancers. A retrospective analysis of the
chemical space explored by the medicinal chemistry efforts
leading to these achievements found that 4-anilinoquinazoline
and unsymmetrical diaryl urea are two privileged pharmaco-
phores among others.2,3 4-Anilinoquinazoline is present in ve
launched products consisting of getinib (Iressaꢀ), erlotinib
(Tarcevaꢀ), lapatinib (Tykerbꢀ), vandetanib (Caprelsaꢀ), and
icotinib (Conmanaꢀ). Unsymmetrical diaryl urea is present in
two launched products, namely sorafenib (Nexavarꢀ) and its
uoro congener regorafenib (Stivargaꢀ) (Fig. 1).
MEK/ERK mitogen-activated protein kinase (MAPK) cascade is a
key signalling pathway involved in the regulation of cell prolif-
eration, survival and differentiation.5 Aberrant activation of this
pathway contributes to a wide range of human cancers.5 In fact,
BRAF was reported to be the most frequently mutated protein
kinase in human cancers.6 Meanwhile, VEGFR-2 has been
proven to be the principal mediator in tumour angiogenesis,
which is crucial for solid tumour development.7 The successful
launch of antiangiogenic drugs attests to the therapeutic value
of VEGFR-2 inhibition.8 Collectively, these facts justify the
Signalling pathways are not strictly linear processes but
rather involve a complex network of interconnected circuits.
When only a single pathway is targeted, redundancy and cross-
talk between these pathways allow for compensatory effects by
alternative pathways.4 Thus, multikinase inhibition targeting
aDivision of Medicinal Chemistry, Shanghai Institute of Pharmaceutical Industry, 1111
Zhongshan North One Road, Hongkou District, Shanghai 200437, China. E-mail:
chembiomed@163.com; Fax: +86 (0)21 6516 9893; Tel: +86 (0)21 5551 4600
bState Key Laboratory of New Drug and Pharmaceutical Process, Shanghai Institute of
Pharmaceutical Industry, 1111 Zhongshan North One Road, Hongkou District,
Shanghai 200437, China
cDepartment of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical
University, 24 Tongjia Xiang, Nanjing, Jiangsu 210009, China. E-mail: youqd@163.
com; Fax: +86 (0)25 8327 1351; Tel: +86 (0)25 8327 1351
Fig. 1 Launched kinase inhibitors containing 4-anilinoquinazoline (shown in
red) and unsymmetrical diaryl urea (shown in green) pharmacophores.
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development of multikinase inhibitors targeting BRAF, EGFR amide of aspartic acid in the DFG motif. R1 is expected to
and VEGFR-2.
occupy the lipophilic pockets created by the displacement of the
Sorafenib (BAY 43-9006) is an oral multikinase inhibitor DFG loop (BPIII and BPIV).10,15
approved by the U.S. Food and Drug Administration for the
treatment of patients with advanced renal cell carcinoma (RCC)
and patients with unresectable hepatocellular carcinoma
(HCC). It was shown to be a dual action RAF kinase and VEGFR
Results and discussion
Chemistry
inhibitor targeting both the RAF/MEK/ERK pathway and
receptor tyrosine kinases that promote angiogenesis.9 Sorafenib
is a type II inhibitor that stabilizes wild type BRAF and onco-
genic mutant BRAF V600E as well as VEGFR-2 all in their
inactive DFG-out conformations (PBD code 1UWH, 1UWJ and
4ASD, respectively).10–13 Type II inhibitors may have some
advantages including increased biochemical efficiency and the
potential for achieving an increased degree of selectivity
compared to type I inhibitors.14 Thus, sorafenib was taken as a
lead compound in our drug discovery endeavor to identify
multikinase inhibitors targeting BRAF, EGFR and VEGFR-2.
According to the hybrid-design approach of rational design
of inhibitors that bind to inactive kinase conformations, sor-
afenib is a rst-generation type II inhibitor.11 The binding
affinity of sorafenib is mainly derived from a combination of
hydrophobic and hydrogen bonding interactions of the biaryl
urea portion (“tail”) with the binding pocket created by the
signicant movement of the DFG motif. However, the binding
affinity contributed by the 2-methylcarbamoylpyridinyl portion
(“head”) interacting with the hinge region is relatively small.
We reasoned that substituting 2-methylcarbamoylpyridinyl
with a more efficient “head” portion might increase the overall
binding affinity. 4-Anilinoquinazoline is a well-known type I
scaffold that could deliver profound interactions with the
kinase hinge residues and hydrophobic pockets in and around
the adenine region.11 Furthermore, 4-anilinoquinazoline is a
well-known pharmacophore to deliver EGFR activity.2 Hence, we
employed 4-anilinoquinazoline as the “head” portion onto
which the unsymmetrical diaryl urea “tail” was attached to give
the novel hybrid scaffold (Fig. 2). This hybrid scaffold incor-
porates the two privileged pharmacophores into one single
molecule. Furthermore, this hybrid scaffold ts the generalized
pharmacophore model of type II inhibitors: the quinazoline
hinge-binding moiety (HBM) is connected through a nitrogen
atom to the central phenyl that is expected to occupy the DFG-
out pocket (BPII). The central phenyl and the R1 substituted
terminal phenyl are linked by urea which functions as the
hydrogen bond donor/acceptor to interact with the side chain of
a conserved glutamic acid in the C helix and with the backbone
Title compounds were prepared via the synthetic routes out-
lined in Scheme 1. 1-(4-Aminophenyl)-3-(4-chloro-3-(tri-
uoromethyl)phenyl)urea (1) was synthesized in a 10% yield by
the reaction of isocyanate prepared in situ from 4-chloro-3-(tri-
uoromethyl)aniline with benzene-1,4-diamine. Compounds 5–
7 were synthesized by nitro group reduction of compounds 2–4,
which were prepared in
triphosgene.16,17
a
one-pot procedure using
Compound 8 was prepared from commercially available 3-
hydroxy-4-methoxybenzaldehyde according to the literature.18,19
Title compounds 9–12 were synthesized by condensation of 8
with 1 or 5–7 under acidic conditions in reuxing isopropanol.
Compounds 13 and 15 were prepared from 7-uo-
roquinazolin-4(3H)-one, which was readily synthesized from
commercially available 2-amino-4-uorobenzoic acid according
to the literature.20–22 Compound 13 was condensed with 6 under
acidic conditions in reuxing isopropanol to deliver title
compound 14. Compound 15 was condensed under acidic
conditions in reuxing isopropanol with 5 and 6 to deliver
intermediate compounds 16 and 17, respectively. 16 and 17
were in turn reacted with aliphatic amine 2-(ethylamino)-
ethanol or 2-(piperazin-1-yl)ethanol in the presence of potas-
sium iodide in 1-methyl-2-pyrrolidinone (NMP) to give title
compounds 18–20 (Scheme 1).
Fig. 2 Evolution of the novel hybrid scaffold based upon the two privileged
pharmacophores 4-anilinoquinazoline (coloured in red) and unsymmetrical diaryl
urea (coloured in green). Potential hydrogen bond interactions are depicted as
dashed lines.
Scheme 1 Reagents and conditions: (a) Fe, NH4Cl, EtOH–THF–H2O, reflux, 83–
94%; (b) 1 or 5–7, HCl in i-PrOH, reflux, 11–61%; (c) 6, HCl in i-PrOH, reflux, 11%;
(d) 5 or 6, HCl in i-PrOH, reflux, 60–75%; (e) 2-(ethylamino)ethanol or 2-(piper-
azin-1-yl)ethanol, KI, NMP, 50 ꢀC, 20–82%.
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groups were ether-linked to C-7 of the quinazoline core to deliver
target molecules 14 and 18–20 (Table 2).
Structure–activity relationships
The same quinazoline moiety seen in getinib was used in title
compounds 9–12 (Table 1) to explore the structure–activity
relationships (SAR) of the R1-substituted urea moiety. 9–12 were
tested for their inhibitory activity against BRAF, BRAF V600E,
VEGFR-2 and EGFR. BRAF and BRAF V600E were tested in
LanthaScreen format (http://www.invitrogen.com) according to
the official protocol. VEGFR-2 and EGFR were tested in Caliper
mobility shi assay format.23
Compound 14 exhibited superior activity against BRAF and
comparable activity against BRAF V600E when compared with
sorafenib. However, 14 exhibited only minimal activity for
VEGFR-2 and EGFR. Incorporation of the more polar hydroxyl-
bearing group 4-(hydroxyethyl)piperazin-1-yl or ethyl(2-hydrox-
yethyl)amino led to much improved potency toward VEGFR-2
and EGFR (18–20). Meanwhile, the activity toward BRAF and
BRAF V600E was maintained (18 and 19) or even greatly
improved (20). Compared to sorafenib, compound 20 was
shown to be 5-fold more potent toward BRAF, and 3-fold more
potent toward BRAF V600E. Thus, the hydroxyl-bearing title
compounds 18–20 proved to be multikinase inhibitors with
profound activity toward BRAF, BRAF V600E, VEGFR-2 and
EGFR.
Overall, our 4-anilinoquinazoline urea scaffold seems to be a
viable platform to deliver profound activity toward both threo-
nine/serine kinases (BRAF and its oncogenic mutant BRAF
V600E) and angiogenesis related receptor tyrosine kinases
(VEGFR-2 and EGFR). Furthermore, it was observed that the
potency for these kinases is governed by both the R1-substituted
urea moiety and the quinazoline C-7 side chain.
Compound 9 was found to be 3-fold more active than sor-
afenib against VEGFR-2 (IC50 17 nM), whilst displayed moderate
activity against other 3 kinases. Compound 10 exhibited
substantial activity against EGFR (IC50 82 nM), whilst displayed
moderate activity against VEGFR-2. The 4-anilinoquinazoline
scaffold in 10 most likely contributes to the EGFR activity, since
sorafenib is devoid of potency against EGFR. When compared
with 9 having the 40-urea linker, 10 having the 30-urea linker
exhibited 5- and 3-fold improvement in potency toward BRAF and
BRAF V600E, respectively. This is consistent with SAR reported in
the literature.24 Substitution of 4-chloro-3-(triuoromethyl)-
phenyl for 3-chloro-4-uorophenyl gave compound 11, which
proved to be nearly 4-fold more potent for BRAF and equipotent
for BRAF V600E compared to sorafenib. However, compound 11
proved to be essentially inactive for VEGFR-2 and EGFR. Incor-
poration of 3-methylphenyl as the urea moiety (12) led to a slight
improvement in potency toward BRAF and BRAF V600E but
substantial loss of activity toward VEGFR-2 and EGFR when
compared with 10. Overall, 4-chloro-3-(triuoromethyl)phenyl
and 3-chloro-4-uorophenyl attached at the 30-position of the
central phenyl seem to be the R1-substituted urea moiety of
choice.
Molecular modeling
Molecular modeling was performed to establish the binding
mode of selected title compounds. Docking of 20 in an X-ray
crystal structure of sorafenib bound to BRAF (PDB code 1UWH)
is depicted in Fig. 3(a).12 20 could be comfortably accommo-
dated to the DFG-out conformation of BRAF. Quinazoline N1
accepts the hydrogen bond from the backbone amide NH of
Cys531 in the hinge region. The quinazoline core is sandwiched
by hydrophobic residues consisting of Ile462, Val470, Ala480,
Trp530, Phe582 and Phe594, forming favorable p–p interaction
(face-to-face) with the side chain of Trp530, and p–p interaction
(face-to-edge) with the side chain of Phe594. The meta-disub-
stituted central phenyl linking to the quinazoline core through
In order to further explore SAR, elaboration of the C-7 substit-
uent on the quinazoline ring was then undertaken. 4-Chloro-3-
(triuoromethyl)phenyl and 3-chloro-4-uorophenyl attached at
the 30-position of the central phenyl were employed. The C-6 group
was removed to allow a reduction in molecular weight and avoid
any potential metabolic liability. Tethered aliphatic tertiary amino
Table 1 Kinase inhibitory activity (IC50, nM) of title compounds 9–12
Compound
Position
R1
R2
BRAFa
BRAF V600Ea
VEGFR-2b
EGFRb
9
10
11
12
40
30
30
30
3-CF3-4-Cl
3-CF3-4-Cl
3-Cl-4-F
3-CH3
H
H
H
H
539
195
31
119
114
371
75
55
54
42
17
213
>10 000
8568
52
285
82
>10 000
2350
>10 000
Sorafenib
a
b
Tested in LanthaScreen format. Tested in Caliper mobility shi assay format.
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Table 2 Kinase inhibitory activity (IC50, nM) of title compounds 14 and 18–20
Compound
R1
A
BRAFa
BRAF V600Ea
VEGFR-2b
EGFRb
14
18
19
20
3-Cl-4-F
A1
A2
A3
A3
72
101
112
22
42
58
53
13
42
2165
75
91
95
52
3315
99
188
165
>10 000
3-CF3-4-Cl
3-CF3-4-Cl
3-Cl-4-F
Sorafenib
114
a
b
Tested in LanthaScreen format. Tested in Caliper mobility shi assay format.
4-NH occupies the hydrophobic pocket neighboring the ATP
Docking of 9 in an X-ray cocrystal structure of VEGFR-2 (PDB
binding site. Carbonyl and two NHs of urea form hydrogen code 2OH4) is depicted in Fig. 3(b).25 Similar to the binding
bond interactions with the backbone of Asp593 and the side mode of 20 in BRAF, 9 could be comfortably accommodated to
chain of Glu500, respectively. Terminal 4-chloro-3-(tri- the DFG-out conformation of VEGFR-2. The quinazoline core is
uoromethyl)phenyl extends into a second hydrophobic pocket positioned in a hydrophobic cle lined with Leu838, Ala864,
produced by the movement of Phe594 and lined with Val503, Phe916 and Leu1033. Quinazoline N1 accepts the hydrogen
Leu504, Ile512, Ile571 and His573.
bond from the backbone amide NH of Cys917 in the hinge
region. The para-disubstituted central phenyl linking to the
quinazoline core through 4-NH occupies the hydrophobic
pocket neighboring the ATP binding site. Carbonyl and two NHs
of urea form hydrogen bond interactions with the backbone of
Asp1044 and the side chain of Glu883, respectively. Terminal
4-chloro-3-(triuoromethyl)phenyl extends into
a
second
hydrophobic pocket created by the ip of Phe1045 and lined
with Ile866, Leu887, Ile890, Leu1017 and His1024.
Conclusion
In summary, we have described a hybrid-design approach based
upon two privileged pharmacophores, namely 4-anilinoquina-
zoline and unsymmetrical diaryl urea, to successfully deliver a
novel series of multikinase inhibitors. Title compounds 18–20
exhibited profound activity toward BRAF, BRAF V600E, VEGFR-2
and EGFR in biochemical screen. Molecular docking estab-
lished the interactions of 20 and 9 with the DFG-out confor-
mation of BRAF and VEGFR-2, respectively, suggesting that they
might be type II kinase inhibitors. The SAR presented herein
complemented by those independently reported by other
scientists could give a more complete landscape in this
eld.24,26–30 More work will have to be done to characterise the
therapeutic relevance of this series of small molecules.
Experimental section
Chemistry
General methods. 1H NMR was recorded on a Varian INOVA-
400 400 MHz spectrometer. Chemical shis (d) are in ppm
relative to the residual DMSO-d6 signal (2.50 ppm), and
coupling constants ( J) are reported in Hz. The following
Fig. 3 (a) Molecular modeling of 20 (magenta) bound to BRAF. (b) Molecular
modeling of 9 (magenta) bound to VEGFR-2. Hydrogen bonds are depicted as
dashed grey lines. Key interacting residues are illustrated in brown sticks.
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abbreviations are used for multiplicities: s ¼ singlet; br s ¼ broad
singlet; d ¼ doublet; dd ¼ double-doublet; t ¼ triplet; m ¼
multiplet. Mass spectra with electrospray ionization (MS-ESI)
were recorded on a Waters Micromass Q-Tof micro instrument.
1-(4-Aminophenyl)-3-(4-chloro-3-(triuoromethyl)phenyl)-
urea (1). Triphosgene (1.19 g, 4 mmol) was dissolved in anhy-
drous methylene chloride (16 mL). Under nitrogen and ice-
cooling, a mixture of 4-chloro-3-(triuoromethyl)aniline (1.96 g,
10 mmol) and diisopropylethylamine (DIEA) (1.55 g, 12 mmol)
in anhydrous methylene chloride was added slowly while
maintaining the temperature of the reaction mixture at 25–
1-(4-Chloro-3-(triuoromethyl)phenyl)-3-(4-(7-methoxy-6-(3-
morpholinopropoxy)quinazolin-4-ylamino)phenyl)urea (9).
A
mixture of 4-(3-(4-chloro-7-methoxyquinazolin-6-yloxy)propyl)
morpholine hydrochloride (8) (ref. 18 and 19) (0.37 g,
1.03 mmol), 1 (0.34 g, 1.03 mmol), isopropanol (10 mL) and
saturated HCl solution in isopropanol (2.5 mL) was stirred
under reux for 3 h. Aer cooling to room temperature, the
reaction mixture was diluted with methylene chloride (250 mL),
methanol (50 mL) and water (50 mL). The resulting mixture was
basied to pH 7.5 with 1 mol Lꢁ1 aqueous sodium hydroxide,
and extracted with methylene chloride. The combined organic
extract was washed with brine, dried over anhydrous sodium
sulfate, and evaporated in vacuo. The resulting residue was
puried by silica gel column chromatography eluting with
EtOAc–EtOH–Et3N 300 : 100 : 1 (v/v) to afford title compound 9
as an off-white solid (0.12 g, 18%). 1H NMR (DMSO-d6) d 9.39 (s,
1H, exchangeable), 9.10 (s, 1H, exchangeable), 8.77 (s, 1H,
exchangeable), 8.39 (s, 1H), 8.11 (d, J ¼ 2 Hz, 1H), 7.82 (s, 1H),
7.61–7.68 (m, 4H), 7.47 (m, 2H), 7.16 (s, 1H), 4.19 (m, 2H), 3.92
(s, 3H), 3.57–3.59 (m, 4H), 2.46 (m, 2H), 2.40 (m, 4H), 1.99
(m, 2H). MS-ESI m/z 631 (M + H)+.
ꢀ
35 C. Aer a further 30 min of stirring, a mixture of benzene-
1,4-diamine (1.08 g, 10 mmol) and DIEA (1.55 g, 12 mmol) in
anhydrous methylene chloride was added in one portion. The
resulting reaction mixture was stirred at room temperature for
24 h, and evaporated to dryness in vacuo. The residue was taken
in ethyl acetate, washed consecutively with 10% aqueous
potassium bisulfate, 5% aqueous sodium bicarbonate, and half-
saturated brine, dried over anhydrous magnesium sulfate, and
evaporated in vacuo. The residue was puried by basic Al2O3
column chromatography eluting with petroleum ether in EtOAc
1
(60–0%) to give 1 as an off-white solid (0.34 g, 10%): H NMR
1-(4-Chloro-3-(triuoromethyl)phenyl)-3-(3-(7-methoxy-6-(3-
(DMSO-d6) d 8.91 (s, 1H, exchangeable), 8.23 (s, 1H, exchange-
able), 8.07 (d, J ¼ 2.4 Hz, 1H), 7.55–7.61 (m, 2H), 7.07 (d, J ¼ 8.8
Hz, 2H), 6.53 (d, J ¼ 8.8 Hz, 2H), 4.76 (s, 2H, exchangeable).
1-(3-Aminophenyl)-3-(4-chloro-3-(triuoromethyl)phenyl)-
urea (5). A suspension of 2 (ref. 17) (6.00 g, 16.7 mmol) in 95%
ethanol (230 mL), THF (75 mL), and water (30 mL) was treated
with iron powder (5.59 g, 100 mmol) (activated with 1 mol Lꢁ1
HCl before use) and ammonium chloride (0.89 g, 16.7 mmol).
Aer being stirred under reux for 1.5 h, the mixture was
ltered through a pad of diatomaceous earth while still hot. The
ltrate was concentrated. The concentrate was diluted with
water and ethyl acetate, and the pH was adjusted to 9 with
ammonium hydroxide. The aqueous phase was separated, and
extracted with more ethyl acetate. The combined extracts were
dried over anhydrous sodium sulfate, and concentrated to
provide 5 as a pale yellow crystalline solid (5.15 g, 94%). 1H NMR
(DMSO-d6) d 8.97 (s, 1H, exchangeable), 8.44 (s, 1H, exchange-
able), 8.11 (s, 1H), 7.58 (d, J ¼ 1.6 Hz, 2H), 6.90 (t, J ¼ 8.0 Hz,
1H), 6.80 (t, J ¼ 2.0 Hz, 1H), 6.54–6.56 (m, 1H), 6.22–6.24
(m, 1H), 4.97 (s, 2H, exchangeable).
morpholinopropoxy)quinazolin-4-ylamino)phenyl)urea
(10).
This compound was prepared from 8 (1.72 g, 4.6 mmol) and 5
(1.51 g, 4.6 mmol) according to the procedure for 9 to afford title
compound 10 as a pale yellow solid (0.97 g, 61%). 1H NMR
(DMSO-d6) d 9.48 (s, 1H, exchangeable), 9.19 (s, 1H, exchange-
able), 8.91 (s, 1H, exchangeable), 8.49 (s, 1H), 8.17 (d, J ¼ 2.0 Hz,
1H), 8.02 (s, 1H), 7.92 (s, 1H), 7.63–7.66 (m, 2H), 7.52 (d, J ¼ 8.4
Hz, 1H), 7.33 (t, J ¼ 7.6 Hz, 1H), 7.23–7.24 (m, 2H), 4.26 (t, J ¼ 6.4
Hz, 2H), 3.98 (s, 3H), 3.65 (m, 4H), 2.53 (m, 2H), 2.48 (m, 4H),
2.06 (m, 2H). MS-ESI m/z 631 (M + H)+, 1261 (2M + H)+.
1-(3-Chloro-4-uorophenyl)-3-(3-(7-methoxy-6-(3-morpholi-
nopropoxy)quinazolin-4-ylamino)phenyl)urea
(11).
This
compound was prepared from 8 (1.41 g, 3.76 mmol) and 6
(1.12 g, 3.76 mmol) according to the procedure for 9 to afford
title compound 11 as an off-white solid (0.15 g, 11%). H NMR
(DMSO-d6) d 9.44 (s, 1H, exchangeable), 8.82 (s, 1H, exchange-
able), 8.77 (s, 1H, exchangeable), 8.47 (s, 1H), 7.82–7.98 (m, 3H),
7.21–7.47 (m, 6H), 4.23 (m, 2H), 3.96 (s, 3H), 3.61 (m, 4H), 2.52
(m, 2H), 2.43 (m, 4H), 2.02 (m, 2H). MS-ESI m/z 581 (M + H)+, 603
(M + Na)+, 1183 (2M + Na)+.
1
1-(3-Aminophenyl)-3-(3-chloro-4-uorophenyl)urea (6). This
compound was prepared from 3 (ref. 17) (10.22 g, 33 mmol)
according to the procedure for 5 to afford 6 as an off-white
crystalline solid (7.65 g, 83%). 1H NMR (DMSO-d6) d 8.68 (s, 1H,
exchangeable), 8.36 (s, 1H, exchangeable), 7.78 (dd, J ¼ 2.0, 6.8
Hz, 1H), 7.28 (m, 2H), 6.90 (t, J ¼ 8.0 Hz, 1H), 6.76 (s, 1H), 6.56
(d, J ¼ 7.6 Hz, 1H), 6.22 (d, J ¼ 7.6 Hz, 1H), 4.95 (s, 2H,
exchangeable).
1-(3-Aminophenyl)-3-m-tolylurea (7). This compound was
prepared from 4 (ref. 17) (1.71 g, 6.3 mmol) according to the
procedure for 5 to afford 7 as an off-white solid (1.29 g, 85%). 1H
NMR (DMSO-d6) d 8.41 (s, 1H, exchangeable), 8.27 (s, 1H,
exchangeable), 7.28 (s, 1H), 7.12–7.22 (m, 2H), 6.89 (t, J ¼ 8.0 Hz,
1H), 6.77 (t, J ¼ 2.0 Hz, 2H), 6.55 (dd, J ¼ 1.2, 7.6 Hz, 1H), 6.20
(dd, J ¼ 1.2, 8.0 Hz, 1H), 4.94 (s, 2H, exchangeable), 2.28 (s, 3H).
1-(3-(7-Methoxy-6-(3-morpholinopropoxy)quinazolin-4-yla-
mino)phenyl)-3-m-tolylurea (12). This compound was prepared
from 8 (0.45 g, 1.2 mmol) and 7 (0.29 g, 1.2 mmol) according to
the procedure for 9 to afford title compound 12 as a pale yellow
solid (0.21 g, 38%). 1H NMR (DMSO-d6) d 9.42 (s, 1H,
exchangeable), 8.66 (s, 1H, exchangeable), 8.54 (s, 1H,
exchangeable), 8.45 (m, 1H), 7.96 (s, 1H), 7.88 (s, 1H), 7.43 (d,
J ¼ 7.6 Hz, 1H), 7.14–7.30 (m, 6H), 6.79 (d, J ¼ 7.2 Hz, 1H), 4.22
(t, J ¼ 6.4 Hz, 2H), 3.94 (s, 3H), 3.60 (m, 4H), 2.41 (m, 6H), 2.28
(s, 3H), 2.02 (m, 2H). MS-ESI m/z 543 (M + H)+, 1085 (2M + H)+.
1-(3-Chloro-4-uorophenyl)-3-(3-(7-(3-morpholinopropoxy)-
quinazolin-4-ylamino)phenyl)urea (14). A mixture of 4-(3-(4-
chloroquinazolin-7-yloxy)propyl)morpholine hydrochloride (13)
(ref. 21) (0.34 g, 1 mmol), 6 (0.35 g, 1.25 mmol), isopropanol
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(8 mL) and saturated HCl solution in isopropanol (2 mL) was 2H), 7.49 (d, J ¼ 9.2 Hz, 1H), 7.33–7.41 (m, 4H), 4.34 (m, 2H),
stirred under reux for 4 h. Aer cooling to room temperature, 3.66–3.81 (m, 10H), 3.40 (m, 2H), 3.33 (m, 2H), 2.30 (m, 2H). MS-
the reaction mixture was evaporated in vacuo. The residue was ESI m/z 644 (M + H)+.
diluted with water (10 mL), basied to pH 9 with 1 mol Lꢁ1
1-(4-Chloro-3-(triuoromethyl)phenyl)-3-(3-(7-(3-(ethyl(2-
aqueous sodium hydroxide, and extracted with methylene hydroxyethyl)amino)propoxy)quinazolin-4-ylamino)phenyl)
chloride–methanol 3 : 1 (v/v) (3 ꢂ 60 mL). The combined urea (19). This compound was prepared from 16 (0.15 g,
organic extract was washed with water, dried over anhydrous 0.28 mmol) and 2-(ethylamino)ethanol (0.10 g, 1.12 mmol)
sodium sulfate, and evaporated in vacuo. The resulting residue according to the procedure for 18 to afford title compound 19 as
was puried by silica gel column chromatography eluting with a pale yellow powder (0.14 g, 82%). 1H NMR (DMSO-d6) d 9.73 (s,
EtOAc–EtOH–Et3N 90 : 10 : 0.5 (v/v) to afford title compound 14 1H, exchangeable), 9.20 (s, 1H, exchangeable), 8.91 (s, 1H,
as a yellow solid (0.06 g, 11%). 1H NMR (DMSO-d6) d 9.60 (s, 1H, exchangeable), 8.55 (s, 1H), 8.53 (d, J ¼ 9.2 Hz, 1H), 8.14 (s, 1H),
exchangeable), 8.80 (s, 1H, exchangeable), 8.74 (s, 1H, 8.02 (s, 1H), 7.61 (m, 2H), 7.53 (d, J ¼ 8.4 Hz, 1H), 7.19–7.32 (m,
exchangeable), 8.52 (s, 1H), 8.49 (d, J ¼ 9.2 Hz, 1H), 7.99 (s, 1H), 4H), 5.32 (br s, 1H, exchangeable), 4.27 (t, J ¼ 6.4 Hz, 2H), 3.77
7.81–7.82 (m, 1H), 7.49 (d, J ¼ 8.0 Hz, 1H), 7.22–7.33 (m, 5H), (m, 2H), 3.30 (m, 2H), 3.21 (m, 4H), 2.21 (t, J ¼ 7.2 Hz, 2H), 1.26
7.17 (d, J ¼ 2.4 Hz, 1H), 4.20 (t, J ¼ 6.4 Hz, 2H), 3.59 (m, 4H), (t, J ¼ 7.2 Hz, 3H). MS-ESI m/z 603 (M + H)+.
2.46–2.47 (m, 2H), 2.40 (t, J ¼ 4.4 Hz, 4H), 1.95 (quintet, J ¼
1-(3-Chloro-4-uorophenyl)-3-(3-(7-(3-(ethyl(2-hydroxyethyl)-
amino)propoxy)quinazolin-4-ylamino)phenyl)urea (20). This
6.8 Hz, 2H). MS-ESI m/z 601 (M + H)+, 1201 (2M + H)+.
1-(4-Chloro-3-(triuoromethyl)phenyl)-3-(3-(7-(3-chloropro- compound was prepared from 17 (0.14 g, 0.28 mmol) and 2-
poxy)quinazolin-4-ylamino)phenyl)urea (16). A mixture of 4- (ethylamino)ethanol (0.10 g, 1.12 mmol) according to the
chloro-7-(3-chloropropoxy)quinazoline (15) (ref. 22) (0.55 g, 2 procedure for 18 to afford title compound 20 as a white solid
mmol), 5 (0.66 g, 2 mmol), isopropanol (9 mL) and saturated HCl (0.03 g, 20%). 1H NMR (DMSO-d6) d 9.65 (s, 1H, exchangeable),
solution in isopropanol (2 mL) was stirred under reux for 3 h. 8.91 (s, 1H, exchangeable), 8.82 (s, 1H, exchangeable), 8.50–8.53
Aer cooling to room temperature, the reaction mixture was (m, 2H), 8.00 (s, 1H), 7.80–7.82 (m, 1H), 7.50 (d, J ¼ 8.4 Hz, 1H),
diluted with water, basied to pH 6–7 with 1 mol Lꢁ1 aqueous 7.19–7.35 (m, 6H), 5.30 (br s, 1H, exchangeable), 4.27 (t, J ¼
sodium hydroxide, and extracted consecutively with methylene 5.6 Hz, 2H), 3.76 (m, 2H), 3.20–3.29 (m, 6H), 2.20 (m, 2H), 1.25
chloride–methanol 1 : 3 (v/v) and methylene chloride. The (t, J ¼ 6.8 Hz, 3H). MS-ESI m/z 553 (M + H)+, 1105 (2M + H)+.
combined organic extract was washed with brine, dried over
anhydrous sodium sulfate, and evaporated in vacuo. The residue
was crystallized from methanol to afford 16 as an off-white ake
(0.83 g, 75%): 1H NMR (DMSO-d6)d 10.87 (br s, 1H, exchangeable),
9.49 (s, 1H, exchangeable), 9.20 (s, 1H, exchangeable), 8.76 (s, 1H),
8.65 (d, J ¼ 9.2 Hz, 1H), 8.14 (d, J ¼ 1.6 Hz, 1H), 7.96 (s, 1H), 7.62
(m, 2H), 7.44 (dd, J ¼ 2.4, 9.2 Hz, 2H), 7.36 (t, J ¼ 8.0 Hz, 1H), 7.25–
7.30 (m, 2H), 4.33 (t, J ¼ 6.4 Hz, 2H), 3.85 (t, J ¼ 6.4 Hz, 2H), 2.29
(dd, J ¼ 6.4, 12.4 Hz, 2H). MS-ESI m/z 550 (M + H)+, 1099 (2M + H)+.
1-(3-Chloro-4-uorophenyl)-3-(3-(7-(3-chloropropoxy)quina-
zolin-4-ylamino)phenyl)urea (17). This compound was prepared
Biology
Materials. BRAF, BRAF V600E, LanthaScreen Tb-anti-pMAP2K1
[pS217/221] Ab, Fluorescein-MAP2K1 (inactive) and Antibody
Dilution Buffer were purchased from Invitrogen. EGFR and
VEGFR-2 were purchased from Carna. ATP, DMSO, EDTA,
GW5074 and staurosporine were purchased from Sigma. 96-well
plate and 384-well plate were purchased from Corning.
LanthaScreen kinase assay
from 15 (0.26 g, 1 mmol) and 6 (0.28 g, 1 mmol) according to the A LanthaScreen kinase assay (http://www.invitrogen.com) was
procedure for 16 to afford 17 as an off-white solid (0.3 g, 60%). used to measure the potency of title compounds against BRAF and
1H NMR (DMSO-d6) d 9.70 (br s, 1H, exchangeable), 8.84 (s, 1H, BRAF V600E. GW-5074 was used as the reference compound.
exchangeable), 8.76 (s, 1H, exchangeable), 8.55 (s, 1H), 8.52 (d, Compounds were prepared in DMSO at 10 mM and serially
J ¼ 9.2 Hz, 1H), 8.00 (s, 1H), 7.81–7.83 (m, 1H), 7.49 (d, J ¼ 8.0 diluted in a 96-well plate as the source plate. 4 mL was transferred
Hz, 1H), 7.20–7.36 (m, 6H), 4.30 (m, 2H), 3.87 (t, J ¼ 6.8 Hz, 2H), to a new 96-well plate as the intermediate plate, and mixed with
2.27 (m, 2H). MS-ESI m/z 500 (M + H)+.
96 mL of 1ꢂ kinase buffer (50 mM HEPES, pH 7.5; 10 mM MgCl2;
1-(4-Chloro-3-(triuoromethyl)phenyl)-3-(3-(7-(3-(4-(2-hydrox- 1 mM EGTA; 0.01% Brij-35). 2.5 mL was transferred to a 384-well
yethyl)piperazin-1-yl)propoxy)quinazolin-4-ylamino)phenyl)urea assay plate in duplicate. 5 mL of BRAF 7 nM or BRAF V600E
(18). A mixture of 16 (0.15 g, 0.28 mmol), potassium iodide 0.7 nM, 2.5 mL of substrate solution containing Fluorescein-
(0.09 g, 0.56 mmol), 2-(piperazin-1-yl)ethanol (0.15 g, 1.12 mmol) MAP2K1 0.8 mM and ATP (for BRAF 2 mM, for BRAF V600E 6 mM)
and NMP (2 mL) was heated to 50 ꢀC for 32 hours with protection were added to the assay plate and incubated with the compound
from light. Aer cooling to room temperature, the reaction for 1 hour at room temperature. The reaction was stopped with
mixture was puried directly on silica gel eluting with EtOAc– the addition of 10 mL of detection solution (antibody 4 nM and
EtOH–Et3N from 300 : 100 : 1.5 to 100 : 100 : 1.5 (v/v), and nally EDTA 20 mM in Antibody Dilution Buffer) to each well of the assay
recrystallized from MeOH–EtOAc 6 : 1 (v/v) to give title plate. Aer mixing briey with a centrifuge and incubating for at
compound 18 as a pale yellow solid (0.07 g, 39%). 1H NMR least 30 minutes, the plate was read on a Victor instrument
(DMSO-d6) d 11.40 (br s, 1H, exchangeable), 9.81 (s, 1H, (Ex. 340 nm, Em. 520 nm). RFU values from the Victor program
exchangeable), 9.54 (s, 1H, exchangeable), 8.86 (s, 1H), 8.74 (d, were converted to percent inhibition values according to percent
J ¼ 8.8 Hz, 1H), 8.15 (s, 1H), 7.93 (s, 1H), 7.60 (dd, J ¼ 8.8,22.4 Hz, inhibition ¼ (max-sample RFU)/(max ꢁ min) ꢂ 100%, in which
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“max” means the RFU of no enzyme control and “min” means the
RFU of DMSO control. Plots of percent inhibition versus
compound concentration were tted by Graphpad 5.0 to calculate
IC50 of test compounds.
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A Caliper motility shi assay was used to measure the potency of
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Kinase reaction: Add 10 mL of kinase (VEGFR-2 4.5 nM or
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plate. Incubate at 28 ꢀC for 1 h. Add 25 mL of stop buffer
(100 mM HEPES, pH 7.5; 0.015% Brij-35; 0.2% Coating Reagent
#3; 50 mM EDTA) to stop the reaction.
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Acknowledgements
We are grateful to the Basic Research Key Program of
Shanghai Municipal (no. 09JC1413200) and the National
Science and Technology Major Project “Key New Drug
Research and Development” (no. 2009ZX09301-007) for
nancial support. We thank Shanghai ChemPartner Co., Ltd.
(Pudong New Area, Shanghai 201203, China) for generating
kinase inhibitory data.
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