Identification of type II inhibitors targeting BRAF using privileged pharmacophores

Article abstract of DOI:10.1111/cbdd.12198

V-RAF murine sarcoma viral oncogene homologue B1 (BRAF) is the most frequently mutated protein kinase in human cancers. The most common mutant BRAF V600E constitutively activates the RAS/RAF/MEK/ERK signaling pathway. BRAF has been validated as an important therapeutic target in human cancers. Phenylaminopyrimidine and unsymmetrical diaryl urea are two privileged pharmacophores in kinase inhibitor drug discovery. Herein, we describe the design of a novel hybrid pharmacophore, 4-phenylaminopyrimidine urea, using the above two pharmacophores. A new series of compounds were in turn synthesized and evaluated to successfully identify selective inhibitors of BRAF and oncogenic BRAF V600E. Once daily oral dosing of lead compound 3 demonstrated sustained antitumor efficacy in A549 human non-small-cell lung cancer xenograft model. Molecular docking suggested that compound 3 might be a type II kinase inhibitor binding to the DFG-out conformation of BRAF. A novel pharmacophore, 4-phenylaminopyrimidine urea (4-PAPU) was hybrid-designed to successfully identify selective inhibitors of BRAF. Docking suggested that they might be type II kinase inhibitors binding to the DFG-out conformation of BRAF.

Full text of DOI:10.1111/cbdd.12198

Chem Biol Drug Des 2014; 83: 27–36
Research Article
Identification of Type II Inhibitors Targeting BRAF
Using Privileged Pharmacophores
Qingwen Zhang1,*, Juan Wang2, Fei Wang3,
Administration; FGFR-1, fibroblast growth factor receptor 1;
FLT-3, Fms-like tyrosine kinase 3; IGF-1R, insulin-like growth
factor receptor 1; JAK-3, Janus kinase 3; MEK, MAPK/ERK
kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazo-
Xiuhua Chen2, Yunsong He1, Qidong You4 and
Houyuan Zhou1,*
lium bromide; PDB, Protein Data Bank; PDGFR, platelet
1Division of Medicinal Chemistry, Shanghai Institute of
Pharmaceutical Industry, 1111 Zhongshan North One
derived growth factor receptor; PI3Ka, phosphoinositol-3-
kinase a; PKCbII, protein kinase C bII; RET, rearranged during
transfection; ROCK-1, Rho-associated coiled-coiled contain-
ing protein kinase; TIE-2, tyrosine kinase with immunoglobulin
and epidermal growth factor homology domains-2; VEGFR,
Road, Hongkou District, Shanghai 200437, China
2Center for Pharmacology Evaluation and Research,
Shanghai Institute of Pharmaceutical Industry, 1111
Zhongshan North One Road, Hongkou District, Shanghai
200437, China
vascular endothelial growth factor receptor.
3State Key Laboratory of New Drug and Pharmaceutical
Received 3 February 2013, revised 1 July 2013 and accepted
for publication 9 July 2013
Process, Shanghai Institute of Pharmaceutical Industry,
1111 Zhongshan North One Road, Hongkou District,
Shanghai 200437, China
4Department of Medicinal Chemistry, School of Pharmacy,
China Pharmaceutical University, 24 Tongjia Xiang,
Nanjing, Jiangsu 210009, China
The RAS/RAF/MEK/ERK cascade is a key signaling path-
way involved in the regulation of cell proliferation, sur-
vival, and differentiation (1). Constitutive activation of the
RAS/RAF/MEK/ERK pathway has been implicated in a
number of human cancers affecting pancreas, colon,
lung, ovary and kidney, among others (2). The serine/
threonine kinase V-RAF murine sarcoma viral oncogene
homologue B1 (BRAF) is the most frequently mutated
protein kinase in human cancers (3). Point mutation of
valine to glutamic acid at position 600 (V600E) accounts
for most identified BRAF mutations (4) and results in
constitutive activation of the RAS/RAF/MEK/ERK pathway
(5). The mutational activation of BRAF in human cancers
supports its important role in human oncogenesis and
validates BRAF as an attractive target for pharmacologi-
cal intervention.
*Corresponding authors: Qingwen Zhang,
chembiomed@163.com and Houyuan Zhou,
zhou.houyuan@sipi.com.cn
V-RAF murine sarcoma viral oncogene homologue B1
(BRAF) is the most frequently mutated protein kinase
in human cancers. The most common mutant BRAF
V600E constitutively activates the RAS/RAF/MEK/ERK
signaling pathway. BRAF has been validated as an
important therapeutic target in human cancers. Phe-
nylaminopyrimidine and unsymmetrical diaryl urea are
two privileged pharmacophores in kinase inhibitor drug
discovery. Herein, we describe the design of a novel
hybrid pharmacophore, 4-phenylaminopyrimidine urea,
using the above two pharmacophores. A new series of
compounds were in turn synthesized and evaluated to
successfully identify selective inhibitors of BRAF and
oncogenic BRAF V600E. Once daily oral dosing of
lead compound 3 demonstrated sustained antitumor
efficacy in A549 human non-small-cell lung cancer
xenograft model. Molecular docking suggested that
compound 3 might be a type II kinase inhibitor binding
to the DFG-out conformation of BRAF.
Intensive drug discovery efforts have led to the successful
development of small molecule inhibitors of BRAF and
related family members for the treatment of human can-
cers. Sorafenib, an oral multikinase inhibitor approved by
U.S. Food and Drug Administration (FDA) for the treat-
ment of renal cell carcinoma (RCC) and hepatocellular
carcinoma (HCC), potently inhibits CRAF (IC50, 6 nmol/L),
wild-type BRAF (IC50, 22 nmol/L), and oncogenic mutant
BRAF V600E (IC50, 38 nmol/L) (6). In addition, sorafenib
demonstrates significant activity against several receptor
tyrosine kinases involved in neovascularization and tumor
progression, including vascular endothelial growth factor
receptor (VEGFR)-2, VEGFR-3, platelet derived growth
factor receptor (PDGFR), Fms-like tyrosine kinase 3 (FLT-3),
and stem cell factor receptor (c-KIT) (6). Vemurafenib,
Key words: drug design, kinase, pharmacophore, type II
inhibitor, urea
Abbreviations: ABL, Abelson kinase; ALK, anaplastic lym-
phoma kinase; BRAF, V-RAF murine sarcoma viral oncogene
homologue B1; CDK-2, cyclin-dependent kinase 2; c-KIT,
stem cell factor receptor; c-MET, mesenchymal epithelial tran-
sition factor; EGFR, epidermal growth factor receptor; ERK,
extracellular regulated kinase; FDA, U.S. Food and Drug
ª 2013 John Wiley & Sons A/S. doi: 10.1111/cbdd.12198
27

Zhang et al.
approved by FDA for the treatment of BRAF V600E muta-
tion-positive melanoma as determined by a companion
diagnostic, displays potent inhibition for BRAF V600E
(IC50, 31 nmol/L) and CRAF (IC50, 48 nmol/L) and high
selectivity in a panel of over 200 kinases, including wild-
type BRAF (IC50, 100 nmol/L) (7). Regorafenib, approved
by FDA for the treatment of colorectal cancer (CRC) and
gastrointestinal stromal tumor (GIST), potently inhibits
CRAF (IC50, 2.5 nmol/L), BRAF V600E (IC50, 19 nmol/L),
and BRAF (IC50, 28 nmol/L) in biochemical assay (8).
Regorafenib also inhibits a panel of angiogenic kinases
and the mutant oncogenic kinases c-KIT and rearranged
during transfection (RET) (8). Dabrafenib, which has
recently been approved by FDA for the treatment of mela-
noma with BRAF V600E mutation as detected by an FDA-
approved test, is a BRAF inhibitor with IC50 values of 0.7,
5.2 and 6.3 nmol/L for BRAF V600E, BRAF, and CRAF,
respectively (9). RAF265 is one of BRAF inhibitors currently
being evaluated in clinical trials (10) (Figure 1).
We have worked on the design, synthesis, and evaluation of
small molecule kinase inhibitors for years (11). Herein, we
report on
a privileged pharmacophores-based hybrid-
design approach to identify novel type II BRAF inhibitors.
Methods and Materials
Rational design using privileged pharmacophores
Unsymmetrical diaryl urea is a privileged pharmacophore
presented in many important protein kinase inhibitors such
as sorafenib, regorafenib, tivozanib, ABT-869, and
BIRB796 (Figure 2) (12). Phenylaminopyrimidine (PAP) is
another privileged pharmacophore in protein kinase inhibi-
tor drug discovery, which could be stratified into 2-phe-
nylaminopyrimidine (2-PAP) and 4-phenylaminopyrimidine
(4-PAP) (13). The former is presented in three launched
products consisting of imatinib, nilotinib, and pazopanib,
while the latter is contained in five launched products
Figure 1: Small molecule inhibitors
of V-RAF murine sarcoma viral
oncogene homologue B1 (BRAF)
launched or in clinical trials.
Figure 2: Representative kinase inhibitors containing unsymmetrical diaryl urea. The unsymmetrical diaryl urea pharmacophore is shown
in green.
28
Chem Biol Drug Des 2014; 83: 27–36

Identification of Type II Inhibitors Targeting BRAF
comprising of gefitinib, erlotinib, lapatinib, vandetanib, and
icotinib (Figure 3).
the aC helix and with the backbone amide of aspartic acid in
the DFG motif. The R1 substituted terminal phenyl is
expected to occupy the lipophilic BP-III and BP-IV pockets.
A hybrid-design approach was utilized to identify type II
ligands (14). Sorafenib was taken as a lead in our pursuit
of novel protein kinase inhibitors. We envisaged that
changing the O linkage to NH and replacing the 2-carbox-
amidopyridine group with 4-pyrimidinyl could combine the
two privileged kinase inhibitor pharmacophores, 4-PAP
and unsymmetrical diaryl urea, into one single pharmaco-
phore which we coined 4-phenylaminopyrimidine urea
(4-PAPU) (Figure 4).
According to the 4-PAPU pharmacophore, a series of title
compounds were in turn designed, synthesized, and eval-
uated to explore the structure–activity relationships (SAR)
(Table 1).
Synthetic chemistry
Title compounds 1–7 were prepared via the synthetic route
outlined in Scheme 1. Hydrochloride 10 was constructed by
condensation of commercially available 8 and 9 (17). Com-
pound 10 was neutralized (17) and then subjected to
Fe/NH4Cl reduction to deliver 11. Compound 10 was also
condensed with excessive morpholine and 1-methylpiper-
azine to give 12 and 13, respectively. Alternatively, 10 was
N-methylated with methyl iodide in the presence of potas-
sium carbonate to give 14, which was condensed with
excessive morpholine and 1-methylpiperazine in DMSO at
The novel 4-PAPU pharmacophore illustrated in Figure 4 fits
the pharmacophore model of type II inhibitors (15,16): The
pyrimidine group is the hinge-binding moiety (HBM) con-
nected through a nitrogen atom to the central phenyl that is
expected to occupy the DFG-out pocket (BP-II). The central
phenyl and the R1 substituted terminal phenyl are linked by
urea group which functions as the H-donor/acceptor to
interact with the side chain of a conserved glutamic acid in
Figure 3: Launched kinase inhibitors containing phenylaminopyrimidine (PAP). The PAP pharmacophore is shown in red.
Figure 4: Evolution of 4-phenylaminopyrimidine urea (4-PAPU) pharmacophore. The 4-phenylaminopyrimidine (4-PAP) pharmacophore is
colored in red. The unsymmetrical diaryl urea one is colored in green. Potential H-bond interactions are depicted as dashed lines.
Chem Biol Drug Des 2014; 83: 27–36
29

Zhang et al.
140 °C to give 15 and 16, respectively. Compound 12 was
subjected to catalytic hydrogenation in alcoholic solvent to
give 17, while 13, 15, and 16 were reduced by Fe/NH4Cl to
give 18, 19, and 20, respectively. Compounds 11, 17–20
were activated by forming urethane with 4-nitrophenyl
chloroformate (18) and then reacted with various substituted
aromatic amines in DMF in the presence of triethylamine at
40 °C to give title compounds 1–7. The synthetic proce-
dures of title compounds 3 and 7 are disclosed in detail
below as typical examples. Full experimental details and
characterization data could be found in our published patent
(19). Sorafenib was prepared according to literatures (20,21).
Table 1: Title compounds and their V-RAF murine sarcoma viral oncogene homologue B1 (BRAF) inhibitory activity (IC50, nmol/L)
Compound R1
R2
R3
BRAF
722
1068
271
BRAF V600E
1
4-Cl-3-CF3
H
H
H
H
Cl
298
626
142
2
3-Cl-4-F
morpholino
morpholino
4-methylpiperazin-1-yl >10000 7166
3
4-Cl-3-CF3
4-Cl-3-CF3
3-Cl-4-F
4
5
CH3 morpholino
366
232
6
4-Cl-3-CF3
4-Cl-3-CF3
CH3 morpholino
CH3 4-methylpiperazin-1-yl
1370 1369
5280 4993
7
Sorafenib
114
42
Scheme 1: Reagents and conditions: (a) concentrated HCl, H2O-acetone, reflux 2 h, 84%; (b) NaOAc,DMF, 85%; (c) Fe, NH4Cl, EtOH-
THF-H2O (6:2:1), reflux 3.5 h, 108%; (d) for 12, morpholine, KI, reflux 8 h, 96%; for 13, 1-methylpiperazine, DMSO, 140–150 °C 0.5 h,
100%; (e) CH3I,K2CO3, DMF, r.t. overnight, 100%; (f) morpholine or 1-methylpiperazine, DMSO, 140 °C 20 min, 15 95%, 16 98%; (g) for
17, 4 bar H2, Raney Ni, 60 °C 4 h, 68%; for 18–20, Fe, NH4Cl, EtOH-THF-H2O (6:2:1), reflux 2 h, 18 96%, 19 95%, 20 86%; (h)
4-nitrophenyl chloroformate, CH2Cl2, r.t. 2.5–5 h, 78–100%; (i) various substituted aromatic amines, Et3N, DMF, 40 °C 4.25–9 h, 10–71%.
30
Chem Biol Drug Des 2014; 83: 27–36

Identification of Type II Inhibitors Targeting BRAF
1H NMR spectra were recorded on a Varian INOVA-400
spectrometer (Varian, Inc., Palo Alto, CA) at 400 MHz. 13C
NMR spectra were measured on a Bruker AVIII 400
spectrometer (Bruker Corporation, Billerica, MA, USA) at
100 MHz. Chemical shifts (d) are in ppm relative to the
DMSO-d6 signal (1H 2.50 ppm, 13C 40 ppm). Mass spectra
were taken on a Waters Micromass Q-Tof microinstrument,
and ionization was positive ion electrospray (MS-ESI).
A
mixture of 4-chloro-3-(trifluoromethyl)aniline (0.43 g,
2.2 mmol), above obtained urethane hydrochloride (0.98 g,
2 mmol), and triethylamine (0.57 g, 5.6 mmol) in anhydrous
DMF (8 mL) was stirred at 40 °C for 4 h. The obtained reac-
tion was diluted with methylene chloride (90 mL), washed
consecutively with 1 mol/L aqueous sodium hydroxide
(3 9 30 mL) and water (2 9 30 mL), dried over anhydrous
sodium sulfate, and evaporated in vacuo. The resulting resi-
due was purified by silica gel column chromatography [elu-
ent, ethyl acetate:petroleum ether 3:1 (v/v)] to afford 3 as an
off-white solid (0.25 g, 25%). 1H NMR (DMSO-d6) d 9.07 (s,
1H, exchangeable), 8.95 (s, 1H, exchangeable), 8.72 (s, 1H,
exchangeable), 8.13 (s, 1H), 7.77 (s, 1H), 7.61 (s, 1H), 7.24
(d, J = 8.8 Hz, 1H), 7.18 (t, J = 8.4 Hz, 1H), 6.98 (d,
J = 7.6 Hz, 1H), 5.92 (s, 1H), 3.67 (m, 4H), 3.46 (m, 4H),
2.32 (s, 3H). 13C NMR (DMSO-d6) d 166.15, 163.47, 161.73,
152.88, 141.68, 139.89, 132.40, 129.44, 127.39, (127.69,
127.39, 127.08, 126.78) (q, J = 30 Hz), 123.43, (127.39,
124.67, 121.95, 119.26) (q, J = 272 Hz, CF3), 122.74,
117.20, 114.31, 112.42, 110.35, 81.79, 66.32, 44.52, 26.37.
MS-ESI m/z 507 (M+H)+, 529 (M+Na)+. Anal. Calcd for
C23H22ClF3N6O2·0.25H2O: C, 54.02%; H, 4.43%; N,
16.43%. Found: C, 53.95%; H, 4.36%; N, 16.62%.
Elemental analyses were performed on
a
Thermo
SCIENTIFIC FLASH 2000 Organic Elemental Analyzer.
2-Methyl-6-morpholino-N-(3-nitrophenyl)pyrimidin-
4-amine (12)
A mixture of 6-chloro-2-methyl-N-(3-nitrophenyl)pyrimidin-
4-amine hydrochloride (10) (9 g, 30 mmol), morpholine
(15 mL, 170 mmol) and several crystals of potassium
iodide was stirred under reflux for 8 h and then concen-
trated in vacuo. The obtained residue was filtered, washed
with water, and dried to give the crude product which was
recrystallized from toluene to afford 12 as orange crystals
1
(9.1 g, 96%). H NMR (DMSO-d6) d 9.47 (s, 1H, exchange-
able), 8.75 (t, J = 2.4 Hz, 1H), 7.95–7.97 (m, 1H), 7.71–
7.74 (m, 1H), 7.52 (t, J = 8 Hz, 1H), 5.83 (s, 1H), 3.67
(t, J = 4.8 Hz, 4H), 3.47 (t, J = 4.8 Hz, 4H), 2.35 (s, 3H).
1-(3-(6-Chloro-2-methylpyrimidin-4-ylamino)phenyl)-
3-(4-chloro-3-(trifluoromethyl)phenyl)urea (1)
N1-(2-Methyl-6-morpholinopyrimidin-4-yl)benzene-
1,3-diamine (17)
Title compound 1 was prepared in the manner similar to that
described for the preparation of title compound 3. A pale
yellow solid. 1H NMR (DMSO-d6) d 9.70 (s, 1H, exchange-
able), 9.09 (s, 1H, exchangeable), 8.81 (s, 1H, exchangeable),
8.13 (s, 1H), 7.83 (s, 1H), 7.60 (m, 2H), 7.34 (d, J = 8 Hz,
1H), 7.22–7.26 (m, 1H), 7.10 (d, J = 8 Hz, 1H), 6.64 (s, 1H),
2.41 (s, 3H). MS-ESI m/z 456 (M+H)+, 478 (M+Na)+.
A mixture of 12 (6.3 g, 20 mmol) and Raney Ni (ca. 1.5 g)
in ethanol (180 mL) was hydrogenated at 60 °C under
4 bar hydrogen for 4 h. The reaction was filtered, and the
filter cake was extracted with hot solvent mixture of DMF
(50 mL) and ethanol (200 mL). The filtrate and extraction
were combined and cooled to room temperature to crys-
tallize the product, which was filtered and dried in vacuo
to obtain 17 as pale yellow-green crystals (3.87 g, 68%).
1H NMR (DMSO-d6) d 8.59 (s, 1H, exchangeable), 6.91
(t, J = 8.0 Hz, 1H), 6.75 (t, J = 2.0 Hz, 1H), 6.67
(d, J = 8.0 Hz, 1H), 6.20 (d, J = 8.0 Hz, 1H), 5.81 (s, 1H),
4.93 (s, 2H, exchangeable), 3.66 (m, 4H), 3.42 (t, J= 4.8 Hz,
4H), 2.29 (s, 3H).
1-(3-Chloro-4-fluorophenyl)-3-(3-(2-methyl-
6-morpholinopyrimidin-4-ylamino)phenyl)urea (2)
Title compound 2 was prepared in the manner similar to
that described for the preparation of title compound 3. A
white solid. 1H NMR (DMSO-d6) d 8.91 (s, 1H, exchange-
able), 8.78 (s, 1H, exchangeable), 8.63 (s, 1H, exchange-
able), 7.80 (dd, J = 2.4, 6.8 Hz, 1H), 7.72 (s, 1H),
7.26–7.33 (m, 2H), 7.15 (m, 2H), 6.97 (m, 1H), 5.93 (s,
1H), 3.66 (m, 4H), 3.46 (m, 4H), 2.30 (s, 3H). MS-ESI m/z
457 (M+H)+, 479 (M+Na)+, 913 (2M+H)+. Anal. Calcd for
C22H22ClFN6O2·0.5H2O: C, 56.71%; H, 4.98%; N, 18.04%.
Found: C, 56.90%; H, 4.85%; N, 17.98%.
1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(3-(2-
methyl-6-morpholinopyrimidin-4-ylamino)phenyl)
urea (3)
The amine 17 (1.40 g, 5 mmol) was added in three por-
tions to a stirred solution of 4-nitrophenyl chloroformate
(1.22 g, 6 mmol) in anhydrous methylene chloride (26 mL)
at 0 °C. After stirring for 3.5 h at 20 °C, the precipitate
was filtered off, washed with methylene chloride, and dried
in vacuo to yield the urethane hydrochloride as an off-
white solid (2.15 g, 88%). 1H NMR (DMSO-d6) d 10.60 (s,
1H, exchangeable), 10.05 (s, 1H, exchangeable), 8.31–
8.33 (m, 2H), 7.68 (s, 1H), 7.53–7.56 (m, 2H), 7.32–7.41
(m, 2H), 7.11 (d, J = 7.2 Hz, 1H), 6.03 (s, 1H), 3.65
(m, 8H), 2.47(s, 3H).
1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(3-(2-
methyl-6-(4-methylpiperazin-1-yl)pyrimidin-
4-ylamino)phenyl)urea (4)
Title compound 4 was prepared in the manner similar to that
described for the preparation of title compound 3. An
off-white solid. 1H NMR (DMSO-d6)
d 9.07 (s, 1H,
exchangeable), 8.89 (s, 1H, exchangeable), 8.73 (s, 1H,
exchangeable), 8.13 (s, 1H), 7.76 (s, 1H), 7.60 (s, 2H),
Chem Biol Drug Des 2014; 83: 27–36
31

Zhang et al.
7.14–7.23 (m, 2H), 6.96 (d, J = 7.6 Hz, 1H), 5.92 (s, 1H),
3.47–3.50 (m, 4H), 3.34–3.37 (m, 4H), 2.30 (s, 3H), 2.21
(s, 3H);MS-ESI m/z 520 (M+H)+. Anal. Calcd for
1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(3-(methyl
(2-methyl-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)
amino)phenyl)urea (7)
C
24H25ClF3N7O: C, 55.44%; H, 4.85%; N, 18.86%. Found:
The amine 20 (4.73 g, 15.2 mmol) was dissolved in dry
methylene chloride (70 mL), and cooled to 0 °C. A solu-
tion of 4-nitrophenyl chloroformate (3.67 g, 18.2 mmol) in
dry methylene chloride (50 mL) was added dropwise.
After stirring the reaction at 20 °C for 2.5 h, the precipi-
tate formed was filtered off, washed with methylene
chloride and dried in vacuo to yield the urethane hydro-
chloride as a pale yellow solid (7.89 g, 100%). 1H NMR
C, 55.25%; H, 5.09%; N, 18.39%.
6-Chloro-N,2-dimethyl-N-(3-nitrophenyl)pyrimidin-
4-amine (14)
A mixture of 6-chloro-2-methyl-N-(3-nitrophenyl)pyrimidin-
4-amine hydrochloride (10) (10 g, 33.2 mmol), potassium
carbonate (13.8 g, 99.6 mmol), and methane iodide
(5.67 g, 40 mmol) in DMF (200 mL) was stirred at room
temperature overnight. The obtained reaction was poured
into ice-cold water (3 L) and stirred for 2 h. The resulting
precipitate was filtered off, washed, and dried to deliver 14
(9.26 g, 100%). 1H NMR (DMSO-d6) d 8.22–8.23 (m, 1H),
8.16–8.18 (m, 1H), 7.83–7.85 (m, H), 7.75 (t, J = 8 Hz,
1H), 6.47 (s, 1H), 3.46 (s, 3H), 2.39 (s, 3H).
(DMSO-d6)
d 9.62 (s, 1H, exchangeable), 8.11 (d,
J = 9.2 Hz, 2H), 7.48 (t, J = 2 Hz, 1H), 7.31–7.38
(m, 2H), 6.95 (d, J = 9.2 Hz, 2H), 6.88–6.91 (m, 1H),
5.64 (s, 1H), 3.46 (m, 4H), 3.39 (s, 3H), 3.03 (m, 4 H),
2.75 (s, 3H), 2.32 (s, 3H).
A
mixture of 4-chloro-3-(trifluoromethyl)aniline (0.59 g,
3 mmol), above obtained urethane hydrochloride (0.77 g,
1.5 mmol), and triethylamine (0.46 g, 4.5 mmol) in
anhydrous DMF (6 mL) was stirred at 40 °C for 9 h.
The obtained reaction was diluted with methylene
chloride (90 mL), washed consecutively with 0.5 mol/L
aqueous sodium hydroxide (20 mL) and water
(2 9 30 mL), dried over anhydrous sodium sulfate, and
evaporated in vacuo. The resulting residue was purified
by silica gel column chromatography [eluent, ethyl ace-
tate:ethanol 1:1 (v/v)] to afford 7 as an off-white solid
N,2-Dimethyl-6-(4-methylpiperazin-1-yl)-N-(3-
nitrophenyl)pyrimidin-4-amine (16)
A mixture of 14 (4.99 g, 17.9 mmol) and 1-methylpiper-
azine (10.77 g, 107.5 mmol) in DMSO (20 mL) was stirred
under nitrogen at 140 °C for 20 min. After cooled to
80 °C, the obtained reaction was poured into ice-cold
water (200 mL) under stirring and extracted with methy-
lene chloride (2 9 150 mL). The combined extracts was
washed with brine, dried over anhydrous sodium sulfate,
and evaporated in vacuo. The residue was dried to deliver
(0.20 g, 25%). 1H NMR (DMSO-d6)
d 9.13 (s, 1H,
exchangeable), 8.87 (s, 1H, exchangeable), 8.09 (d,
J = 2.4 Hz, 1H), 7.58–7.64 (m, 2H), 7.48 (s, 1H), 7.34
(t, J = 8.0 Hz, 1H), 7.24 (d, J = 8.0 Hz, 1H), 6.92 (d,
J = 8.0 Hz, 1H), 5.51 (s, 1H), 3.36 (m, 4H), 3.35 (s,
3H), 2.28–2.29 (m, 4H), 2.26 (s, 3H), 2.16 (s, 3H). 13C
1
16 as a solid (6.02 g, 98%). H NMR (DMSO-d6) d 8.12 (t,
J = 2.0 Hz, 1H), 8.00–8.02 (m, 1H), 7.75–7.77 (m, 1H),
7.65 (t, J = 8.0 Hz, 1H), 5.71 (s, 1H), 3.47 (t, J = 5.2 Hz,
4H), 3.41 (s, 3H), 2.32 (t, J = 5.2 Hz, 4H), 2.23 (s, 3H),
2.19 (s, 3H).
NMR (DMSO-d6)
d 165.76, 163.46, 163.16, 152.93,
146.10, 140.54, 139.76, 132.41, 130.24, (127.72,
127.37, 127.07, 126.77) (q, J = 30 Hz), 123.64, (127.37,
124.64, 121.93, 119.21) (q, J = 272 Hz, CF3),122.90,
120.54, 117.38, 116.72, 80.79, 54.68, 46.14, 43.94,
38.03, 26.60. MS-ESI m/z 534 (M+H)+, 1067 (2M+H)+.
Anal. Calcd for C25H27ClF3N7O·0.5H2O: C, 55.30%; H,
5.20%; N, 18.06%. Found: C, 55.06%; H, 5.34%; N,
17.68%.
N1-Methyl-N1-(2-methyl-6-(4-methylpiperazin-1-yl)
pyrimidin-4-yl)benzene-1,3-diamine (20)
A
suspension of 16 (6.02 g, 17.6 mmol) in ethanol
(360 mL), THF (120 mL), and water (60 mL) was treated
with ammonium chloride (0.94 g, 17.6 mmol) and iron
powder (5.91 g, 105.6 mmol) (activated with 1 mol/L HCl
before use). After being stirred under reflux for 2 h, the
mixture was filtered through a pad of diatomaceous earth
while still hot. The pad was washed with ethanol, and the
filtrate was concentrated. The concentrate was diluted
with water (100 mL) and ethyl acetate (200 mL), and the
pH was adjusted to 8 with ammonium hydroxide. The
aqueous phase was separated and extracted with more
ethyl acetate (2 9 50 mL). The combined extracts were
washed with brine, dried over sodium sulfate, filtered, and
concentrated to provide 20 as a tan solid (4.73 g, 86%).
1H NMR (DMSO-d6) d 7.07 (t, J = 8.0 Hz, 1H), 6.45 (m,
2H), 6.37–6.39 (m, 1H), 5.34 (s, 1H), 5.13 (s, 2H,
exchangeable), 3.29 (m, 7H), 2.29 (m, 4H), 2.26 (s, 3H),
2.16 (s, 3H).
1-(3-Chloro-4-fluorophenyl)-3-(3-(methyl
(2-methyl-6-morpholinopyrimidin-4-yl)amino)
phenyl)urea (5)
Title compound 5 was prepared in the manner similar to
that described for the preparation of title compound 7. A
white solid. 1H NMR (DMSO-d6) d 8.83 (s, 1H, exchange-
able), 8.78 (s, 1H, exchangeable), 7.79 (dd, J = 2.4,
6.8 Hz, 1H), 7.46–7.47 (m, 1H), 7.28–7.36 (m, 3H), 7.23
(d, J = 7.6 Hz, 1H), 6.91 (d, J = 7.2 Hz, 1H), 5.51 (s, 1H),
3.59 (t, J = 4.8 Hz, 4H), 3.36 (s, 3H), 3.33 (m, 4H), 2.28
(s, 3H). MS-ESI m/z 471 (M+H)+. Anal. Calcd for
C23H24ClFN6O2: C, 58.66%; H, 5.14%; N, 17.85%.
Found: C, 59.19%; H, 5.35%; N, 17.46%.
32
Chem Biol Drug Des 2014; 83: 27–36

Identification of Type II Inhibitors Targeting BRAF
1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(3-(methyl
(2-methyl-6-morpholinopyrimidin-4-yl)amino)
phenyl)urea (6)
measured with the MTT assay. Percentage of cell
growth inhibition was calculated as (ODvehicleÀODdrug)/
ODvehicle 9 100%. The IC50
s
were determined by
Title compound 6 was prepared in the manner similar to
that described for the preparation of title compound 7. A
white solid. 1H NMR (DMSO-d6) d 9.13 (s, 1H, exchange-
able), 8.87 (s, 1H, exchangeable), 8.09 (s, 1H), 7.61–7.62
(m, 2H), 7.48 (s, 1H), 7.35 (t, J = 8 Hz, 1H), 7.25 (d,
J = 8 Hz, 1H), 6.93 (d, J = 7.6 Hz, 1H), 5.51 (s, 1H), 3.59
(m, 4H), 3.37 (s, 3H), 3.26–3.37 (m, 4H), 2.28 (s, 3H). MS-
ESI m/z 521 (M+H)+.
interpolation from the dose–response curves.
In vivo antitumor efficacy of title compound 3 was evalu-
ated in A549 human NSCLC xenograft mouse model with
sorafenib as the positive control. A549 tumor cells were
implanted s.c. in the flank of male BALB/c nude mice
(SPF grade). Compounds were prepared in distilled water
with the aid of Tween-80 and were administered orally
once daily to mice bearing established A549 human
NSCLC at 25 mg/kg for 12 days. Relative tumor volume
(RTV) was used as the gauge for in vivo activity, while
body weight of test mouse was used as an indication of
toxicity. TV (tumor volume) was calculated using the equa-
tion length 9 (width)2/2. RTV was in turn calculated as
TVt/TV0 9 100%, wherein TV0 and TVt mean TV at d0 and
dt, respectively. Percent inhibition was calculated as
((mean RTV of vehicle group À mean RTV of compound
group)/mean RTV of vehicle group) 9 100%.
Biochemical assays
A LanthaScreen kinase assay (Invitrogena) was used to
measure the potency of title compounds against BRAF
and BRAF V600E. Compounds were prepared in DMSO
at 10 mmol/L and serially diluted in a 96-well plate as the
source plate. Four microlitre was transferred to a new 96-
well plate as the intermediate plate, and mixed with 96 lL
of 19 kinase buffer (50 mmol/L HEPES, pH 7.5; 10 mmol/
L MgCl2; 1 mmol/L EGTA; 0.01% Brij-35). Of 2.5 lL was
transferred to a 384-well assay plate in duplicates. Five mi-
crolitre of BRAF 7 nmol/L or BRAF V600E 0.7 nmol/L,
2.5 lL of substrate solution containing Fluorescein-
MAP2K1 0.8 lmol/L and ATP (for BRAF 2 lmol/L, for
BRAF V600E 6 lmol/L) were added to the assay plate
and incubated with the compounds for 1 h at room tem-
perature. The reaction was stopped with the addition of
10 lL of detection solution (antibody 4 nmol/L and EDTA
20 mmol/L in antibody dilution buffer) to each well of the
assay plate. After mixing briefly with centrifuge and incuba-
tion at least 30 min, the plate was read on a Victor instru-
ment (Ex. 340 nm, Em. 520 nm). RFU values from Victor
program were converted to percent inhibition values
according to percent inhibition = (max-sample RFU)/(max-
min) 9 100%, in which ‘max’ means the RFU of no
enzyme control and ‘min’ means the RFU of DMSO con-
trol. Plots of percent inhibition versus compound concen-
tration were fitted by Graphpad 5.0 to calculate IC50s of
the compounds.
Molecular modeling
Docking was carried out using AutoDock 4 (The Scripps
Research Institute, La Jolla, CA, USA). X-ray cocrystal
structure of BRAF [Protein Data Bank (PDB) code 1UWH)
was downloaded from RCSB Protein Data Bank and pre-
pared using AutoDockTools 1.4. Water molecules and
inorganic ions were removed. Default parameters in Auto-
Dock 4 were used in this study.
Results and Discussion
Preliminary SAR exploration study has been performed for
the title compounds designed based upon the 4-PAPU
pharmacophore (Table 1). Biochemical assay showed that
title compound 3, using 4-chloro-3-(trifluoromethyl)phenyl
seen in sorafenib as the R1 substituted urea moiety, is the
most potent inhibitor for both BRAF and BRAF V600E in
this series, although it is ca. threefold less active com-
pared to sorafenib. Substitution of 4-chloro-3-(trifluorom-
ethyl)phenyl for 3-chloro-4-fluorophenyl yields title
compound 2, which is ca. fourfold less potent relative to 3
for both BRAF and BRAF V600E. In contrast, 3-chloro-4-
fluorophenyl proved to be the better urea moiety than 4-
In vitro and in vivo growth inhibition studies
Title compound 3 was assayed in standard 3-(4,5-dim-
ethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
format for in vitro growth inhibitory activity against A549
human non-small-cell lung cancer (NSCLC), HCT116
colon cancer, CEM leukemia, and MCA-MB-435 mela-
noma cell lines with sorafenib as the positive control. All
tumor lines were maintained in DMEM supplemented with
10% fetal bovine serum in a humidified atmosphere with
5% CO2 at 37 °C. Sorafenib and 3 were dissolved in 5%
DMSO in PBS(À) (v/v) at a concentration of 1 mg/mL and
serially diluted with the same. Cancer cells (4000–5000
cells per well) were seeded in 96-well plates and 24 h after
plating were treated with the compounds, incubated at
37 °C with 5% CO2 for 72 h. Cell proliferation was
chloro-3-(trifluoromethyl)phenyl when comparing the IC50
s
of title compounds 5 and 6 in the N-methylated subseries.
Overall, both 4-chloro-3-(trifluoromethyl)phenyl and 3-
chloro-4-fluorophenyl seem to be the urea moiety of
choice.
It was found that assumed PK group R3 at the 2-methyl-
pyrimidine ring 6-position also plays important role in regu-
lating the inhibitory activity for BRAF and BRAF V600E.
Title compound 3, the most potent inhibitor in this series,
employs morpholino as R3. Substitution of morpholino for
Chem Biol Drug Des 2014; 83: 27–36
33

Zhang et al.
Figure 5: Kinase inhibitors containing 2-phenylaminopyrimidine urea (2-PAPU) pharmacophore. The 2-phenylaminopyrimidine (2-PAP)
pharmacophore is colored in red. The unsymmetrical diaryl urea one is colored in green.
chloro gives title compound 1, which is ca. 2.5-fold less
potent for both BRAF and BRAF V600E relative to 3.
Replacing morpholino with 4-methylpiperazin-1-yl delivers
title compound 4 which is inactive. When comparing the
IC50s of title compounds 6 and 7 in the N-methylated sub-
series, morpholino once again proved to be the better R3
group than 4-methylpiperazin-1-yl. Hence, morpholino
seems to be the R3 group of choice.
DFG-out conformation of BRAF (PBD code 1UWH) as well
as BRAF V600E (PBD code 1UWJ) (23). In the cocrystal
structure of sorafenib with BRAF, the diaryl urea motif
interacts with the DFG-out pocket (BP-II), with oxygen of
the urea group accepting an H-bond from the backbone
of Asp593 in the DFG motif and with NH of urea donating
an H-bond to the side chain of catalytic Glu500 in aC
helix. The trifluoromethyl substituent extends into the
hydrophobic pocket BP-III, while the chlorophenyl group
occupies a partial hydrophobic pocket BP-IV. 2-Carbox-
amidopyridine group at the opposite end binds into the
adenine pocket and forms two H-bonds with Cys531 in
the hinge region.
Methylation of the NH linking the central phenyl and the
2-methylpyrimidine ring gave ambiguous SAR results.
N-methylation appears to be detrimental when comparing
IC50 of compound 3 with that of compound 6. However,
when comparing IC50 of compound 4 with that of com-
pound 7, N-methylation appears to be beneficial.
Docking of title compound 3 in the crystal structure of
sorafenib bound to BRAF (PDB code 1UWH) is depicted
in Figure 6. Compound 3 is shown bound to the DFG-out
conformation of BRAF. Morpholino group binds in the
hydrophobic pocket surrounded by Trp530, Cys531 and
Phe582 and forms an H-bond with the backbone amide
NH of Cys531 in the hinge region. 2-Methylpyrimidine ring
is sandwiched by hydrophobic residues consisting of
Ile462, Ala480 and Phe594, forming p-p interaction (face-
to-edge) with side chain of Phe594. The meta-disubsti-
tuted phenyl linking 2-methylpyrimidine ring through NH
Title compound 3 was selected for further profiling. It was
found that 3 displays selectivity against a panel of kinases
comprising of 17 protein kinases [Abelson kinase (ABL),
anaplastic lymphoma kinase (ALK), Aurora A, cyclin-
dependent kinase 2 (CDK-2), epidermal growth factor
receptor (EGFR), fibroblast growth factor receptor
1
(FGFR-1), FLT-3, insulin-like growth factor receptor 1 (IGF-
1R), Janus kinase 3 (JAK-3), c-KIT, mesenchymal epithelial
transition factor (c-MET), p38a, PDGFRb, protein kinase C
bII (PKCbII), rho-associated coiled-coiled containing protein
kinase (ROCK)-1, tyrosine kinase with immunoglobulin and
epidermal growth factor homology domains-2 (TIE-2), and
VEGFR-2] as well as lipid kinase phosphoinositol-3-kinase
a (PI3Ka) (data not shown). It is noteworthy that kinase
inhibitors containing the 2-PAPU pharmacophore were
well documented. Notably, compound 21 was reported to
inhibit ABL transphosphorylation with an IC50 of 56 nmol/L
(18), while compound 22 was disclosed to be a potent
ABL inhibitor (IC50 <1 nmol/L) with BRAF IC50 of
180 nmol/L and p38a IC50 of 18 nmol/L (22) (Figure 5).
Collectively, our 4-PAPU pharmacophore appears to be a
scaffold expected to deliver profound activity for serine/
threonine kinase BRAF, while the 2-PAPU counterpart
seems to be a scaffold expected to deliver profound activ-
ity for tyrosine kinase ABL.
BRAF can be targeted by both type I and type II inhibitors
(10). For example, vemurafenib is a type I inhibitor bound
to the active DFG-in conformation of oncogenic mutant
BRAF V600E (PBD code 3OG7) (7). On the other hand,
sorafenib is a type II kinase inhibitor bound to the inactive
Figure 6: Molecular docking modeling of 3 (magenta) bound to
the DFG-out conformation of V-RAF murine sarcoma viral
oncogene homologue B1 (BRAF). Sorafenib in crystal structure is
colored in green. H-bonds are depicted as yellow dashed lines.
Key interacting residues are illustrated in brown sticks.
34
Chem Biol Drug Des 2014; 83: 27–36

Identification of Type II Inhibitors Targeting BRAF
BRAF and oncogenic BRAF V600E. Lead compound 3
demonstrated substantial oral antitumor efficacy in A549
human NSCLC xenograft model. Molecular docking
suggested that 3 might be a type II kinase inhibitor binding
to the DFG-out conformation of BRAF. These findings
warrant the further characterization and optimization
of this series to deliver compounds with improved drug-
likeness.
Acknowledgments
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
financial support. We thank Shanghai ChemPartner Co.,
Ltd. (Pudong New Area, Shanghai 201203, China) for
generating kinase inhibitory data.
Figure 7: Title compound 3 demonstrated oral antitumor efficacy
in A549 human non-small-cell lung cancer xenograft mouse
model. Daily oral administration of 3 at 25 mg/kg retarded tumor
growth during 12 days of treatment (n = 6 for compound groups,
n = 10 for vehicle group, error bars indicate standard deviation).
The authors declare no conflicts of interest.
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Chem Biol Drug Des 2014; 83: 27–36