2-Aminopyrimidine Derivatives as New Selective Fibroblast Growth Factor Receptor 4 (FGFR4) Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.7b00091

A series of 2-aminopyrimidine derivatives were designed and synthesized as highly selective FGFR4 inhibitors. One of the most promising compounds 2n tightly bound FGFR4 with a K_d value of 3.3 nM and potently inhibited its enzymatic activity with an IC₅₀ value of 2.6 nM, but completely spared FGFR1/2/3. The compound selectively suppressed proliferation of breast cancer cells harboring dysregulated FGFR4 signaling with an IC₅₀ value of 0.38 μM. Furthermore, 2n exhibited extraordinary target specificity in a Kinome-wide screen against 468 kinases, with S(35) and S(10) selectivity scores of 0.01 and 0.007 at 1.0 μM, respectively.

Full text of DOI:10.1021/acsmedchemlett.7b00091

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Letter
2-Aminopyrimidine Derivatives as New Selective
Fibroblast Growth Factor Receptor 4 (FGFR4) Inhibitors
Cheng Mo, Zhang Zhang, Christopher P. Guise, Xueqiang Li, Jinfeng Luo, Zheng-Chao Tu,
Yong Xu, Adam V. Patterson, Jeff Bruce Smaill, Xiaomei Ren, Xiaoyun Lu, and Ke Ding
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.7b00091 • Publication Date (Web): 31 Mar 2017
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2-Aminopyrimidine Derivatives as New Selective Fibroblast Growth
Factor Receptor 4 (FGFR4) Inhibitors
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Cheng Mo,a,b Zhang Zhang,c Christopher P. Guise,e,d Xueqiang Li,a,b Jinfeng Luo,a Zhengchao
Tu,a Yong Xu,a Adam V. Patterson,e,d Jeff B. Smaill,e,d* Xiaomei Ren,c* Xiaoyun Lu,c* Ke Dingc*
a State Key Laboratory of Respiratory Diseases, Guangzhou Institutes of Biomedicine and Health, Chinese Academy
of Sciences, # 190 Kaiyuan Avenue, Guangzhou 510530, China
b University of Chinese Academy of Sciences, # 19 Yuquan Road, Beijing 100049, China
c School of Pharmacy, Jinan University, # 601 Huangpu Avenue West, Guangzhou 510632, China.
d Auckland Cancer Society Research Centre, University of Auckland, #92019 Private Bag, Auckland 1142, New Zea-
land
e Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, #92019 Private Bag, Auckland 1142,
New Zealand
KEYWORDS: selective FGFR4 inhibitor, targeted therapy, breast cancer, hepatocellular carcinoma
ABSTRACT: A series of 2-aminopyrimidine derivatives were designed and synthesized as highly selective FGFR4 inhibi-
tors. One of the most promising compounds 2n tightly bound FGFR4 with a Kd value of 3.3 nM and potently inhibited its
enzymatic activity with an IC50 value of 2.6 nM, but completely spared FGFR1/2/3. The compound selectively suppressed
proliferation of breast cancer cells harboring dysregulated FGFR4 signaling with an IC50 value of 0.38 μM. Furthermore,
2n exhibited extraordinary target specificity in a Kinome-wide screen against 468 kinases, with S(35) and S(10) selectivity
scores of 0.01 and 0.007 at 1.0 µM, respectively.
The fibroblast growth factor receptor (FGFR) family
comprises four functional members (i.e., FGFR1, FGFR2,
FGFR3, FGFR4), which consist of a conserved extracellular
ligand-binding domain, a single transmembrane region
and a cytosolic region with a split tyrosine kinase domain.1-
such as amplification,10 point mutation11 and single nucleo-
tide polymorphism,12 were also detected in human breast
tumor tissues. It has been demonstrated that silencing of
FGF19/FGFR4 signaling by siRNA or introduction of an
anti-FGF19 antibody potently suppresses proliferation and
induces apoptosis of triple-negative breast cancer cells,11, 13
offering promise in a disease setting that scarcely benefits
from currently available therapies.14 A synergistic interac-
2
FGFRs are high affinity receptors for the fibroblast
growth factors (FGFs) which includes 18 members.1
FGF/FGFR signaling is tightly regulated by ligand specifici-
ty, and temporal and spatial expression of the signaling
components, and is involved in many fundamental biolog-
ical processes, such as metabolism, embryonic develop-
ment and adult tissue homeostasis.2-3
tion was also observed between FGFR4 signaling blockade
15
and doxorubicin chemotherapy.13,
Collective evidence
also suggests that FGF19/FGFR4 signaling is an oncogenic
driver in a subset of hepatocellular carcinoma (HCC) pa-
tients. In an analysis of 281 HCC patients, approximately
48% of the tumors expressed FGF19, which was associated
with larger tumor size, more advanced stage and early re-
currence.16 In addition, about 30% of HCC samples tested
display increased expression of FGFR417 and 25% over-
express β-Klotho protein.18 These data collectively indicate
that targeting the FGF19/FGFR4 signaling pathway may
provide a new promising strategy for the management of a
defined subgroup of cancer patients.19,20
FGFR4 is mainly expressed in liver, lung, lymphoid and
breast tissues and specifically utilizes endocrine FGF19 as
the intracellular ligand.4-5 Upon binding with a complex
formed by FGFR4 and its co-receptor, transmembrane β-
Klotho protein,4 FGFR19 promotes receptor dimerization,
autophosphorylation and activation of the signaling path-
way, which plays significant roles in regulation of bile acid
homeostasis,6 and maintenance of glucose and protein
metabolism.7 FGFR4 and/or FGF19 aberrations (i.e., ampli-
fication, gene fusion or mutation) have been recently de-
tected in a variety of human cancers. For instance, FGF19
is amplified in approximate 15% of breast cancer,8 and high
expression of FGF19 was closely correlated to poor progno-
sis of the disease.9 Various genetic alterations of FGFR4,
A number of small molecule FGFR inhibitors with differ-
ent selectivity profiles have been developed into clinical
investigation for the management of FGFR-driven human
cancers.21 Unfortunately, hyperphosphatemia and tissue
calcification are common “on-target” clinical adverse ef-
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fects for most of the drugs investigated,22-23 due to their
graft and patient-derived xenograft (PDX) models of
HCC.25 Most recently, BLU554, a structurally related deriv-
ative of inhibitor 1, was advanced into early stage clinical
investigation (clinical trial ID NCT02508467). The clinical
report of the compound is eagerly awaited. Nevertheless, it
is still highly valuable to identify new selective FGFR4 in-
hibitors for anticancer drug discovery. Herein, we describe
the design, synthesis and biological evaluation of a series
of 2-aminopyrimidine derivatives as new selective FGFR4
inhibitors.26
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strong inhibitory activity against FGFR1 and FGFR3, block-
ing FGF23-mediated signaling.24 Given the fact that the
FGFR family share significant sequence homology in their
kinase domains (Figure 1A), it is a considerable challenge
to develop selective FGFR4 inhibitors over the other family
members. Most recently, BLU9931 (1, Figure 1B) was dis-
covered as the first selective FGFR4 inhibitor. The struc-
ture-based design of this molecule involved the covalent
targeting of a unique Cysteine 552 (Cys552) residue in the
hinge region of the receptor (Figure 1C).25 The compound
exhibited extraordinary target specificity and demonstrat-
ed significant anti-proliferative activity against a panel of
human hepatocellular carcinoma (HCC) cells with activat-
ed FGFR4 signaling. Moreover, the compound also
demonstrated promising in vivo efficacy in several xeno-
A
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The X-Ray co-crystal structure of compound 1 with
FGFR4 revealed that the compound binds with the ATP-
binding pocket of the receptor, forming a critical covalent
bond with Cys552 (Figure 1C). It was also shown that Ca,
Cb,
B
O
O
O
Cl
Cl
b
Cl
O
N
O
c
Ring-opening strategy
Z
Optimization
a
d
N
O
N
O
Cl
A
HN
F
N
Cl
Cl
H
HN
N
HN
N
N
Cl
H
H
N
N
O
O
O
1
BLU9931
2a, Z=O; 2b, Z=NH
2n
0°
CH3
CH3
CH3
H
H
H
H
n-butane conformation
180
°
H
H
H
H
CH3
Anti-conformation
Lowest Energy Conformation
Syn-conformation
Highest Energy Conformation
C
D
Figure 1. Design of new selective FGFR4 inhibitors. (A) Sequence alignment of the FGFR1/2/3/4 kinase domains using the
multiple sequence viewer in Maestro. (B) Design of new selective FGFR4 inhibitors. (C) Superimposition of FGFR4-
BLU9931 (PDB: 4xcu; multicolor; residues, green) with FGFR1 (PDB: 3tt0; constant color, brown; residues, purple). (D)
Predicted binding mode of 2a (orange, carbon atoms; blue, nitrogen atoms; red, oxygen atoms; green, chlorine atoms) in a
crystal structure of FGFR4 (PDB:4xcu) with BLU9931 (cyan) aligned to it.
Cc and Cd atoms in this inhibitor were in an almost copla-
nar configuration (Figure 1B/C), and there were no obvious
direct interactions between the phenyl ring A (Figure 1B)
and the protein. It is well-known that the lowest energy
conformation of n-butane is an anti-form configuration
where the dihedral angle of the four carbon atoms is ap-
proximately ±180° (Figure 1B).27 Thus, an n-butane-like
moiety might be utilized as a bioisostere of the phenyl ring
A moiety to mimic the conformation of compound 1.
Based on this hypothesis, a series of 2-aminopyrimidine
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pound 1 was significantly less potent against FGFR1/2/3,
derivatives were designed and synthesized (Figure 1B).
Encouragingly, a preliminary computational study sug-
gested that compound 2a, which harbors a methyleneoxyl
moiety to potentially mimic the conformation of the par-
ent compound 1, nicely bound into the ATP-binding pock-
et of FGFR4 with a similar mode to that of compound 1
(Figure 1D). The warhead acrylamide is predicted to cova-
lently react with the sulfhydryl side chain of Cys552, which
derives the paralog selectivity.25 The 2-aminopyrimidine
core forms two pairs of hydrogen bonds with the backbone
amino and carbonyl group of Ala553, respectively. The
tetra-substituted phenyl group, which adopts an almost
perpendicular orientation to the 2-aminopyrimidine core,
makes favorable van der Waals contacts with the hydro-
phobic back pocket of the ATP binding site, with one
methoxyl forming a hydrogen bond with the backbone
carboxamide NH group of Asp630. It is also noteworthy
that compound 2a possesses less aromatic ring count and
higher Fsp3 value than that of compound 1, which might
benefit its drug-like physico-chemical properties.28-29
with our data comparing favorably to the reported data.25
Whereas, the pan FGFRs inhibitor FIIN-1 exhibited IC50
values of 3.6, 2.3, 6.0 and 261.3 nM against FGFR1, FGFR2,
FGFR3 and FGFR4, respectively. We were delighted to find
that our first designed compound 2a potently suppressed
the enzymatic activity of FGFR4 with an IC50 value of 11.1
nM, which was only 2-fold less potent than the positive
control 1, yet entirely spared FGFR1/2/3 with IC50 values >
10,000 nM (Table 1). Investigation also suggested that the
methyleneoxyl linker in compound 2a could be replaced
by a methyleneamino moiety (2b) to demonstrate slightly
decreased FGFR4 suppression with an IC50 value of 43.4
nM, while maintaining the paralog selectivity. Compound
2a harbored a methyl substitution at the 3’- position,
which was adopted from the original parental molecule 1
and was considered to be important for the selective inhi-
bition against FGFR4. Thus, we first investigated the po-
tential impact of the methyl substituent position on the
paralog sparing FGFR4 inhibition. It was shown that this
substitutent position indeed had a great influence on the
target inhibitory potency and paralog selectivity. When
the methyl group was shifted to the 6’- position, the result-
ing molecule 2c demonstrated a total loss of all FGFR in-
hibitory activity. The 5’-methyl substituted compound 2d
exhibited slightly increased FGFR4 inhibitory potency with
an IC50 value of 7.5 nM, but its selectivity was decreased
due to increased inhibition of FGFR1. Whereas, a 4’- me-
thyl substitutent (2e) resulted in a slight decrease in
FGFR4 inhibitory potency and similar FGFR1 inhibition as
that observed in the 5’-position (2d). Although removal of
the methyl group barely affected the FGFR4 inhibition
potency, the resulting molecule 2f displayed obviously
improved suppression against both FGFR1 and FGFR2 with
IC50 values of 431.0 and 311.9 nM, respectively, suggesting
significant loss of its target specificity. Thus, the results
collectively suggested that 3’- substitution is an optimal
strategy for designing new selective FGFR4 inhibitors. Fur-
ther investigation also revealed that the 3’- position is well
tolerated to a variety of substituted groups such as ethyl
(2g), methoxyl (2h), chloro- (2l) and trifluoromethyl (2m)
moieties which demonstrated potent and paralog-selective
inhibition of FGFR4. Encouragingly, the 3’-chloro substi-
tuted analogue 2l exhibited an obviously improved FGFR4
inhibition potency with an IC50 value of 3.8 nM, but re-
tained excellent selectivity against FGFR1/2/3 at a high
concentration of 10,000 nM. Not surprisingly, the 3’-fluro
substituted compound 2k displayed a similar potency and
selectivity profile to that of the 3’-unsubstituted molecule
2f. Notably, increases in size of a 3’-alkoxy group resulted
in stepwise loss of FGFR4 inhibitory potency (compare 2h,
2i and 2j). Our investigation suggested that substitution at
the 5’- position of the molecules could retain strong FGFR4
inhibition. Thus, several 3’, 5’- disubstituted compounds,
i.e., 2n, 2o, 2p, were also designed and synthesized. These
displayed potent and selective suppression of FGFR4. Par-
ticularly, the 3’-chloro-5’-fluoro- analogue 2n which exhib-
ited the best FGFR4 inhibitory activity with an IC50 value
of 2.6 nM, 2-fold more potent than compound 1. Com-
pound 2n also displayed an obviously improved paralog
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Scheme 1. Synthesis of Compounds 2a-2z*
*Reagents and conditions: (A) K2CO3, Bu4N+·I-, DMF, 60℃
, 2.0 h; (B) Pd(OAc)2, XantPhos, Cs2CO3, 100℃, Overnight;
(C) TFA, DCM, rt, Overnight; (D) Acryloyl chloride, DIEA,
dry DCM, 0℃, 2.0 h. R: substituted phenyl.
The designed inhibitors were readily prepared by using
a Buchwald-Hartwig cross-coupling reaction as the key
step (Scheme 1).30 Briefly, commercially available 2-
chloropyrimidin-5-ol was treated with different substitut-
ed benzyl bromides giving the corresponding intermedi-
ates 5 through a nucleophilic substitution reaction. Com-
pounds 5 were coupled with various Boc-protected o-
phenylenediamines under palladium catalysis to afford the
Buchwald-Hartwig coupling products, which were depro-
tected to produce the arylamines 7. Intermediates 7 were
reacted with acryloyl chloride to produce the designed
molecules 2a, 2c-2g and 2k-2l with good to moderate
yields. The other designed inhibitors were synthesized by
utilizing a similar protocol (Supporting Information).
Kinase inhibitory activities of the designed compounds
against FGFR4 and the family members FGFR1/2/3 were
evaluated by using a well-established FRET-based Z’-Lyte
assay.25 The first reported selective FGFR4 inhibitor 1 and a
FGFR1/2/3 inhibitor FIIN-131 were used as a positive control
to validate the screening conditions. Under our assay con-
ditions, it exhibited strong inhibitory potency against
FGFR4 with an IC50 value of 6.0 nM, further to this com-
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selectivity over the reported inhibitor 1. Further investiga- ducted on the tetra-substituted phenyl moiety which was
1
2
3
4
5
6
7
8
tion also revealed that warhead acrylamide was crucial for
potent FGFR4 inhibition. Replacement of the acrylamide
predicted to approach the hydrophobic back pocket of the
ATP binding site. It was shown that the tetra-substitution
was critical for the molecules to demonstrate strong
FGFR4 inhibition. Removal of one or more substituents
caused significant potency loss against FGFR4. For in-
stance, removal of a methoxyl group (2s) resulted in a 6.3-
fold potency loss, whereas deletion of a chloro- group (2t)
reduces potency 3-fold. The other chloro- or methoxyl
deleted compounds (2u-2z) displayed a 6.0 to 122-fold loss
of potency. Thus, compounds 2a, 2h, 2l, 2o, and 2n stood
out as representatives for further investigation due to their
strong FGFR4 inhibitory potency and extraordinary pa-
ralog specificity.
with an α, β-saturated propionamide moiety (2q) caused a
totally abolishment of FGFR inhibition. It is generally ac-
cepted that a benzyloxy group possesses a potential
Table 1. Kinase inhibitory activities of compounds 2a-2z
Z
R
N
9
HN
N
Ra
H
N
10
11
12
13
14
15
16
17
18
19
20
21
22
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24
25
26
27
28
29
30
31
32
33
34
35
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59
60
3
O
Cl
O
6
4
5
O
Y:
Rb
Cl
Kinase inhibitory activity* (IC50, nM)
FGFR1 FGFR2 FGFR3 FGFR4
Compd
Z
Ra/Rb
R
A
B
O
Me/H
Y
>10,000
>10,000
>10,000
11.1
2a
N
O
Me/H
6’-Me
Y
Y
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
43.4
2b
2c
>10,000
O
O
5’-Me
4’-Me
Y
Y
825.3
975.1
>10,000
>10,000
>10,000
>10,000
7.5
2d
2e
28.1
O
H/H
Y
431.0
311.9
1,200
8.2
2f
O
O
O
O
O
Et/H
MeO/H
EtO/H
iPrO/H
F/H
Y
Y
Y
Y
Y
>10,000
>10,000
>10,000
>10,000
617.7
>10,000
>10,000
>10,000
>10,000
396.8
>10,000
>10,000
>10,000
>10,000
4,300
25.6
4.8
2g
2h
2i
Figure 2. Compound 2n selectively targets FGFR4. (A)
Compound 2n dose-dependently inhibits the activation of
FGFR4 and downstream proteins in MDA-MB-453 cells
with FGFR4Y367C. The cells were treated with or without
compound 2n for 4h at the indicated concentrations, re-
spectively. Cells were then harvested for Western blot
analysis. (B) KinomeScan kinase selectivity profile for
compound 2n. Compound 2n was profiled at a concentra-
tion of 1.0 μΜ against a diverse panel of 468 kinases by
DiscoveRx.
72.9
202.3
5.3
2j
2k
O
Cl/H
Y
>10,000
>10,000
>10,000
3.8
2l
O
O
O
O
CF3/H
Cl/F
Y
Y
Y
Y
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
24.9
2.6
2m
2n
2o
2p
Cl/ Cl
Cl/ CF3
8.5
17.1
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
669.2
2q
2r
Taking compound 2n as an example, the strong FGFR4
inhibition of the compounds was further validated by in-
vestigating its inhibition of the activation of FGFR4 and
downstream signaling partners in MDA-MB-453 cells har-
boring an activating FGFR4Y367C mutation (Figure 2A).11 It
was shown that the compound diminished the phosphory-
lation of FGFR4 and inhibited the activation of down-
stream FRS2, ERK1/2 and AKT in a dose-dependent man-
ner, while the total amount of corresponding proteins re-
mained unchanged as determined by western blot analy-
sis. The phosphorylation of FGFR4, ERK1/2 and FRS2 was
almost totally abolished after a treatment of 0.03 μM of
compound 2n, although its effect on p-Akt was less obvi-
ous.
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
24.0
12.3
38.7
152.4
98.0
2s
2t
O
O
O
O
O
Cl/H
Cl/H
Cl/H
Cl/H
Cl/H
2u
2v
2w
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
22.7
273.5
465.7
2x
2y
2z
O
O
O
Cl/H
Cl/H
Cl/H
239.9
3.6
246.9
2.3
243.5
6.0
6.0
1
261.3
FIIN-1
*Kinase activity assays were performed using a FRET-
based Z’-Lyte biochemical assay.25 Data are means of three
independent experiments, and the variations are <20%.
The binding affinity of compound 2n for FGFR4 was also
determined by using an active-site-dependent competition
binding assay (DiscoveRx Corporation, San Diego, USA). It
was shown that compound 2n tightly bound to the ATP-
binding site of the kinase with a binding constant (Kd)
value of 3.3 nM, validating its strong kinase inhibition
against FGFR4. We further profiled the target specificity of
this compound against a panel of 468 kinases (including
403 nonmutated kinases) using the DiscoveRx screening
platform at a concentration of 1.0 μM, which was about
metabolic liability and introduction of steric hindrance is a
feasible strategy to minimize oxidative metabolism and
improve the metabolic stability of the molecule. Based on
this rationale, compound 2r in which a methyl group was
introduced at the benzyloxyl linker, was designed and syn-
thesized. Disappointingly, this modification caused a dra-
matic 220-fold loss in potency. Modification was also con-
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compound 2n dose-dependently induced apoptosis in
300 times higher than its Kd value with FGFR4. The results
1
2
3
4
5
6
7
8
revealed that 2n demonstrated impressive target specifici-
ty with S(10) and S(35) selectivity scores of 0.007 and 0.01
at 1.0 µM, respectively (Table S2). For instance, 2n showed
almost 100% competition rate (99.5% inhibition, ctrl% =
0.5) with FGFR4 at 1.0 μM, and only showed obvious bind-
ing to a very small portion of the kinases investigated. The
main “off-target” hits included calcium/calmodulin de-
pendent protein kinase ID (CAMK1D, 94.7% inhibition),
G-protein coupled receptor kinase 4 (GRK4, 92.8% inhibi-
tion), and SRSF Protein Kinase 3 (SPRK3, 88% inhibition).
MDA-MB-453 cells (Figure S2).
In summary, a series of 2-aminopyrimidine derivatives
were designed and synthesized as highly selective FGFR4
inhibitors. These compounds potently suppressed FGFR4
activity with low nanomolar IC50 values and excellent iso-
type selectivity. One of the most promising compounds 2n
tightly bound with FGFR4 with a Kd value of 3.3 nM and
potently inhibited its enzymatic activity with an IC50 value
of 2.6 nM, but completely spared FGFR1/2/3. The com-
pound selectively suppressed proliferation of breast cancer
cells harboring dysregulated FGFR4 signaling with an IC50
value of 0.38 μM. Furthermore, it exhibited extraordinary
target specificity in a Kinome-wide screen against 468 ki-
nases, with S(10) and S(35) selectivity scores of 0.007 and
0.01 at 1.0 µM, respectively. Compound 2n may serve as
new lead compound for future anticancer drug discovery.
Further structural optimization of the compound and in
vivo antitumor efficacy investigation are ongoing.
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Table 2. Antiproliferative activities of the designed com-
pounds against a panel of breast cancer cells
Cell growth inhibition (IC50, µM)
Compd
MDA-MB-453a
MDA-MB-231b
MCF-7b
2a
2h
2l
0.22±0.13
0.21±0.16
0.65±0.42
0.87±0.75
0.38±0.17
0.32±0.14
>10
2.14±1.08
>10
>10
1.18±0.89
>10
2o
2n
1
>10
>10
>10
>10
ASSOCIATED CONTENT
3.76±2.35
2.34±0.88
a Cancer cells harboring an FGFR4Y367C mutation that also
Supporting Information
b
Experimental procedures for the synthesis, 1H NMR and 13C
NMR for final compounds, kinase selectivity, and details of in
vitro assays.
This material is available free of charge via the Internet at
http://pubs.acs.org.
demonstrate overexpression of FGFR4. Cancer cells that
have low FGFR4 expression under IP-Western blotting.
The IC50 values were determined using CCK8. Data are
mean values ± standard deviation (SD) of three independ-
ent experiments.
AUTHOR INFORMATION
Corresponding Author
The growth inhibitory effects of the compounds 2a, 2h,
2l, 2o, and 2n were evaluated against a panel of breast
cancer cell lines (Table 2). MDA-MB-453 breast cancer
cells predominantly express a mutated form of FGFR4,
FGFR4Y367C, which causes spontaneous receptor dimeriza-
tion and constitutive activation in a ligand-independent
manner.11 Moreover, FGFR4Y367C had been identified as an
important driver gene for the proliferation of MDA-MB-
453 cells. On the another hand, MCF-7 and MDA-MB-231
breast cancer cells harbor low level of FGFR4 and serve as
negative control cell models to investigate the target speci-
ficity of the inhibitors.13 It was shown that almost all of the
selected FGFR4 inhibitors exhibited promising anti-
proliferative activity against MDA-MB-453 breast cancer
cells with similar potencies to that of inhibitor 1. However,
none of them displayed obvious cell growth inhibition
against MCF-7 and MDA-MB-231 breast cancer cells ex-
pressing low levels of FGFR4. For instance, compound 2n
potently inhibited the proliferation of MDA-MB-453 breast
cancer cells with an IC50 value of 0.38 μM, which is compa-
rable to that of compound 1, but was unable to suppress
the growth of MCF-7 and MDA-MB-231 cancer cells at a 10
µM concentration. These data further supported the
strong and selective antagonism of the new inhibitors
against FGFR4. It is also noteworthy that all of the new
inhibitors appear to display better target specificity than
the original compound 1 in the cell based assays, which is
consistent with the data from the biochemical kinase inhi-
bition assays. Further investigation also demonstrated that
*Tel: +86-20-85228025; Fax: +86-20-85224766; Email:
luxy2016@jnu.edu.cn (X. L.); ren_xiaomei@gibh.ac.cn (X.R.);
j.smaill@auckland.ac.nz(J. S.); dingke@jnu.edu.cn (K. D.)
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
The authors appreciate the financial support from National
Natural Science Foundation of China (81425021 and 81673285),
Guangdong Province (2013A022100038, 2015A030312014 and
2015A030306042,
2016A050502041),
Guangzhou
City
(201508030036), Jinan University and the Health Research
Coun-cil of New Zealand (13/1020).
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