Discovery of Substituted 1H-Pyrazolo[3,4-b]pyridine Derivatives as Potent and Selective FGFR Kinase Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.6b00066

Fibroblast growth factor receptors (FGFRs) are important targets for cancer therapy. Herein, we describe the design, synthesis, and biological evaluation of a novel series of 1H-pyrazolo[3,4-b]pyridine derivatives as potent and selective FGFR kinase inhibitors. On the basis of its excellent in vitro potency and favorable pharmacokinetic properties, compound 7n was selected for in vivo evaluation and showed significant antitumor activity in a FGFR1-driven H1581 xenograft model. These results indicated that 7n would be a promising candidate for further drug development.

Full text of DOI:10.1021/acsmedchemlett.6b00066

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Letter
Discovery of Substituted 1H-Pyrazolo[3,4-b]pyridine
Derivatives as Potent and Selective FGFR Kinase Inhibitors
Bin Zhao, Yixuan Li, Pan Xu, Yang Dai, Cheng Luo, Yiming Sun, Jing Ai, Mei-Yu Geng, and Wenhu Duan
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00066 • Publication Date (Web): 20 Apr 2016
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ACS Medicinal Chemistry Letters
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Discovery of Substituted 1H-Pyrazolo[3,4-b]pyridine Derivatives as
Potent and Selective FGFR Kinase Inhibitors
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Bin Zhao†,ǁ, Yixuan Li‡,ǁ, Pan Xu§, Yang Dai‡, Cheng Luo§, Yiming Sun‡, Jing Ai*,‡, Meiyu Geng*,‡,
Wenhu Duan*,†
†Department of Medicinal Chemistry, ‡Division of Antitumor Pharmacology, and §Drug Discovery and Design Center, State
Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi
Road, Shanghai 201203, China
KEYWORDS: cancer, FGFR, inhibitor, pyrazolo[3,4-b]pyridine
ABSTRACT: Fibroblast growth factor receptors (FGFRs) are important targets for cancer therapy. Herein, we describe the design,
synthesis, and biological evaluation of a novel series of 1Hꢀpyrazolo[3,4ꢀb]pyridine derivatives as potent and selective FGFR kiꢀ
nase inhibitors. On the basis of its excellent in vitro potency and favorable pharmacokinetic properties, compound 7n was selected
for in vivo evaluation and showed significant antitumor activity in a FGFR1ꢀdriven H1581 xenograft model. These results indicated
that 7n would be a promising candidate for further drug development.
Fibroblast growth factors (FGFs) and their receptors reguꢀ
late a wide range of biological functions, including embryoꢀ
genesis, tissue repair, wound healing, and angiogenesis.1ꢀ3 The
fibroblast growth factor receptor (FGFR) family comprises
four highly conserved transmembrane tyrosine kinase recepꢀ
tors (FGFR1–4), which are differentially activated by binding
to a subset of 18 FGF ligands.3ꢀ4 Upon FGF binding, FGFR
undergoes dimerization and auto phosphorylation, resulting in
activation of downstream signaling pathways, such as the
MAPK and PLCγ pathways.4ꢀ5 These FGFR cascades play
crucial roles in key cell behaviors, such as proliferation, surꢀ
vival, differentiation, and migration, which makes FGFR sigꢀ
naling susceptible to subversion by cancer cells.6 Dysregulaꢀ
tion of FGFR signaling has been documented in clinical samꢀ
ples of bladder, lung, breast cancers, etc,7 and aberrant FGFR
Figure 1. Structures of selective FGFR inhibitors.
activation is closely correlated with metastatic progression and
poor prognosis.8ꢀ9 Knockdown studies and pharmaceutical
inhibition of FGFRs in preclinical models have further demonꢀ
strated that FGFRs are attractive targets for cancer theraꢀ
py.4,7,10
greater IC50 values.17 Nevertheless, PD173074 exhibits submiꢀ
cromolar inhibitory activity at the cellular level against
VEGFR2 (IC50 = 100–200 nM).17 The crystal structure of
PD173074 in complex with the FGFR1 kinase domain eluciꢀ
dates that its high affinity and selectivity for FGFR1 stem
from the presence of 3,5ꢀdimethoxy phenyl ring that adopts an
almost perpendicular orientation to the plane of the pyriꢀ
do[2,3ꢀd]pyrimidine rings and fills optimally a complementary
hydrophobic pocket.17 Guagnano et al. took advantage of this
finding and then developed a selective FGFR inhibitor, NVPꢀ
In recent years, many small molecules have been in clinical
development, such as nintedanib, dovitinib, and cediranib,
which are reported to target FGFR.11 However, because of the
high degree of homology of FGFRs with VEGFRs, most of
these compounds have multitarget specificity, which leads to
undesired side effects in their anticancer therapies.12ꢀ13 Thus,
discovery of highly selective FGFR inhibitors is an unmet
medical need. Currently, several selective FGFR inhibitors
have progressed robustly into clinical trials, such as NVPꢀ
BGJ398,14 AZD4547,15 and CH518328416 (Figure 1).
PD173074 (Figure 1), the first reported selective FGFR inꢀ
hibitor, inhibits FGFR1 with an IC50 value of 21.5 nM at the
molecular level, while inhibiting PDGFR, cꢀSrc and EGFR, as
well as several serine/threonine kinases with 1000ꢀfold or
Figure 2. Design of novel FGFR inhibitors.
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Scheme 1. Synthesis of Compounds 4a–k and 5a
an IC50 value of 24 nM.15 Based on these findings, we wished
1
2
3
4
5
6
7
8
to explore new scaffolds based on the structure of AZD4547,
which had higher potency and higher selectivity for FGFR
over VEGFR2 than AZD4547. By utilizing the scaffold hopꢀ
ping strategy and incorporation of chlorines at the 2ꢀ and 6ꢀ
positions of the phenyl ring, we designed novel 1Hꢀ
pyrazolo[3,4ꢀb]pyridine scaffold derivatives (Figure 2), which
demonstrated excellent in vitro and in vivo antitumor activities
and high selectivity for FGFR over VEGFR2. Herein, we reꢀ
port the synthesis and biological evaluation of these comꢀ
pounds.
aReagents and conditions: (a) AlMe3, toluene, rt to 60 °C, 4%–
28%.
9
Scheme 2. Synthesis of Compounds 7a–o, 9, 10, and 11a–ca
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As outlined in Schemes 1 and 2 (see Supporting Information
Schemes S4 and S5 for details), two synthetic routes were
employed for preparation of target compounds. Scheme 1 ilꢀ
lustrates the synthesis of compounds 4a–k and 5, which were
obtained by condensation of the appropriate amines (1a and 2)
and various substituted benzoic acid esters (3a–k) in the presꢀ
ence of trimethylaluminium at 60 °C. However, the yields of
compounds 4a–k and 5 synthesized via this route were unsatꢀ
isfactory (4%–28%). Therefore, an alternative procedure was
used to prepare 7a–o, 9, and 10 (Scheme 2). Various acyl
chlorides, prepared from the corresponding carboxylic acids
(6a–h, 6j, 6l–o and 8) or obtained commercially (6i and 6k),
were reacted with the appropriate amines (1a–c) in the presꢀ
ence of N,Nꢀdiisopropylethylamine at room temperature to
afford compounds 7a–o, 9, and 10 in the yields ranged from
18% to 69%. Deprotection of compounds 7j, 7m, and 7o proꢀ
vided target compounds 11a–c. The final products were evaluꢀ
ated for their inhibitory activity against FGFR1 and the prolifꢀ
eration of FGFR1ꢀamplified H1581 cells.
OMe
Cl
OMe
Cl
Cl
Cl
R1
a
H
Y
H
N
N
N
+
N
MeO
MeO
MeO
MeO
N
N
O
R1
NH2
HN
O
1a
6a-o
7a-o
OMe
OMe
X
X
X
N
N
N
R2
N
R2
b
N
N
N
+
N
MeO
N
N
X
HOOC
NH2
HN
9, 10
O
1b, 1c
OMe
8
OMe
Cl
Cl
Cl
H
H
N
R3
N
R3
N
N
MeO
c
N
N
Cl
HN
HN
O
O
7j, 7m, 7o
11a-c
aReagents and conditions: (a) for 7i and 7k, DIPEA, THF, 0 °C
to rt, 38%–41%; for 7a–h, 7j, and 7l–o, (i) oxalyl chloride, DMF,
CH2Cl2, 0 °C to rt; (ii) DIPEA, THF, 0 °C to rt, 18%–60%; (b) (i)
oxalyl chloride, DMF, CH2Cl2, 0 °C to rt; (ii) DIPEA, THF, 0 °C
to rt, 59%–69%; (c) for 11a and 11b, CF3COOH, CH2Cl2, rt,
83%–89%; for 11c, TBAF, THF, 0 °C to rt, 56%.
Table 1. SAR of Core Structure
BGJ398. NVPꢀBGJ398, currently in phase II clinical trials,
inhibits FGFR1–4 (IC50 = 0.9, 1.4, 1.0, and 60 nM, respectiveꢀ
ly) with low nanomolar potency at the molecular level and has
a minimum of 200ꢀfold selectivity for FGFR1 over all other
kinases evaluated (VEGFR2, IC50 = 180 nM).14 The cocrystal
structure of NVPꢀBGJ398 with FGFR1 elucidates that its
higher potency and selectivity compared to PD173074 result
from incorporation of chlorines at the 2ꢀ and 6ꢀpositions of the
phenyl ring. The two chlorine atoms of NVPꢀBGJ398 enforce
the plane of the phenyl ring adopting an almost perpendicular
orientation to the plane of the pseudobicyclic system consistꢀ
ing of an Nꢀpyrimidinꢀ4ꢀyl moiety; this conformation is closer
to the kinase bound conformation; therefore, incorporation of
chlorines decreases the deconjugation energy required to
achieve the optimal binding conformation and thus enhances
the potency of this scaffold.14 Besides, the two chlorine atoms
have favorable contacts with the gate keeper Val561 and
Ala640 in FGFR1, respectively, while the Ala640 residue is
replaced by a cysteine residue in VEGFR2.14 The increased
steric hindrance of cysteine compared to alanine might be the
determinant of selectivity of the compound against
VEGFR2.14 AZD4547, an investigational drug in phase II
clinical trials, has excellent in vivo efficacy and favorable
pharmacokinetic properties. AZD4547 inhibits FGFR1–4 (IC50
= 0.2, 2.5, 1.8, and 165 nM, respectively) with low nanomolar
potency at the molecular level, while inhibiting VEGFR2 with
IC50 (nM)a
Compd
X
Y
R1
VEGF
R2
H1581
Cell
FGFR1
3709.0 ±
165.3
548.4 ±
94.1
H
N
N
H
H
> 1000
9
365.9 ±
80.7
Cl
0.3 ± 0.1
1.7 ± 0.2
4a
324.3 ±
4.6
Cl
Cl
ꢀ
CH
N
H
Me
ꢀ
3.3 ± 0.0
> 5000
NDb
5
ND
ND
10
AZD45
47
67.2 ±
3.3
37.9 ±
2.8
ꢀ
1.2 ± 0.1
b
aValues are the mean ± SD of two independent assays. ND:
not determined.
Based on the structure of AZD4547, we utilized the scaffold
hopping strategy to design and synthesize compound 9. Much
to our disappointment, 9 showed weak activity against FGFR1
albeit displayed moderate potency against VEGFR2 (IC50
=
3709.0 and 548.4 nM, respectively) (Table 1). To enhance its
potency and selectivity, we introduced two chlorine atoms to
the ortho positions of the dimethoxyphenyl ring (4a). To our
delight, 4a exhibited high enzymatic and cellular activities
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against FGFR1 (IC50 = 0.3 and 1.7 nM, respectively), and had
a 1200ꢀfold selectivity for FGFR1 over VEGFR2 (IC50
To elucidate the interaction of 6ꢀ(2,6ꢀdichloroꢀ3,5ꢀ
dimethoxyphenyl)ꢀ1Hꢀpyrazolo[3,4ꢀb]pyridine derivatives
1
2
3
4
5
6
7
8
=
365.9 nM) (Table 1). Next, we assessed the importance of the
1Hꢀpyrazolo[3,4ꢀb]pyridine moiety. Replacement of the 1Hꢀ
pyrazolo[3,4ꢀb]pyridine with 1Hꢀindazole led to a significant
loss in enzymatic potency by 11ꢀfold (Table 1, 5 vs 4a). In
addition, Nꢀmethylation of 1Hꢀpyrazolo[3,4ꢀb]pyridine nucleꢀ
us completely eroded the activity in the enzymatic assay (10,
IC50 > 5 ꢁM), which implied that the N(1)ꢀH of pyraꢀ
zolopyridine moiety participated in Hꢀbonding interactions
within the FGFR1 kinase domain.
with FGFR, we proposed a binding mode of compound 4a in
the ATP site of FGFR1 using a reported crystal structure of
the FGFR1 kinase domain (PDB ID: 4WUN). As shown in
Figure 3A, 4a maintained all the Hꢀbonding interactions with
FGFR1 observed in AZD4547: the amide NH and the adjacent
nitrogen of pyrazolopyridine moiety formed critical Hꢀbonds
with the carbonyl and the amino group of Ala564, respectively;
the N(1)ꢀH of pyrazolopyridine nucleus was involved in a Hꢀ
bond with the backbone carbonyl group of Glu562; an addiꢀ
tional Hꢀbond occurred between the methoxy oxygen and the
amino group of Asp641. Furthermore, the two chlorine atoms,
which formed favorable hydrophobic contacts with Val561
and Ala640, respectively, enforced the tetraꢀsubstituted phenyl
ring to adopt the almost perpendicular orientation with respect
to the plane of the pyrazolopyridine rings. Overall, 4a fitted
well into the ATP site of FGFR1, and therefore we chose 4a as
the lead compound for further SAR exploration.
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Table 3. SAR of 4-Substituents on Phenyl Ring A
Figure 3. Proposed binding mode of compound 4a to the FGFR1
kinase domain. The atoms of 4a are colored as follows: carbon
silver, oxygen red, nitrogen blue, and chlorine green. (A) The
protein is shown in cartoon and key residues are colored as folꢀ
lows: carbon green, oxygen red, and nitrogen blue. Hydrogen
bonds are indicated by red dashes. (B) The protein is shown in
surface.
IC50 (nM)a
Compd
R3
FGFR1
H1581 Cell
Table 2. SAR of 3-Substituents on 1H-Pyrazolo[3,4-
b]pyridine
0.3 ± 0.1
1.7 ± 0.2
4a
H
NH2
NMe2
42.4 ± 2.9
8.8 ± 1.2
16.5 ± 0.5
> 1000
218.4 ± 1.1
294.2 ± 64.7
7i
11a
7k
3.9 ± 0.8
11.7 ± 0.8
8.8 ± 0.8
7.7 ± 0.6
6.9 ± 1.1
11.0 ± 0.5
7l
11b
4b
4c
1.4 ± 0.1
IC50 (nM)a
Compd
R2
558.0 ± 12.8
864.8 ± 30.7
8.2 ± 1.1
FGFR1
H1581 Cell
0.3 ± 0.1
1.7 ± 0.2
4a
4d
7.2 ± 0.9
0.5 ± 0.1
1.4 ± 0.1
0.2 ± 0.0
1.3 ± 0.2
0.7 ± 0.2
1.4 ± 0.2
2.3 ± 0.2
14.0 ± 1.3
14.5 ± 0.3
74.4 ± 12.6
27.6 ± 11.0
191.4 ± 16.7
525.2 ± 167.0
4e
4f
7a
7b
7c
4g
4h
14.2 ± 2.1
6.2 ± 0.5
7.8 ± 0.2
10.5 ± 1.8
15.6 ± 2.6
1.5 ± 0.4
7d
7e
7f
0.3 ± 0.01
0.6 ± 0.1
0.3 ± 0.1
0.7 ± 0.1
3.3 ± 1.1
< 0.5
7n
4i
11c
4.4 ± 0.2
0.3 ± 0.1
43.3 ± 1.8
4j
60.6 ± 20.7
7g
0.2 ± 0.0
1.2 ± 0.1
244.7 ± 21.6
37.9 ± 2.8
4k
4.1 ± 0.7
1.2 ± 0.1
3.7 ± 0.3
7h
AZD4547
ꢀ
AZD4547
ꢀ
37.9 ± 2.8
aValues are the mean ± SD of two independent assays.
aValues are the mean ± SD of two independent assays.
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Table 4. Pharmacokinetic Properties of Compounds 4e, 7n and 11c in ICR Micea
1
2
3
4
5
6
7
8
9
Dose
T1/2
(h)
Cmax
AUC0ꢀ∞
CL
Vd
F
Compd
4e
(mg/kg)
(ng/mL)
(ng·h/mL)
(mL/h/kg)
(mL/kg)
(%)
2 (iv)
12.5 (po)
2 (iv)
3.47
2.90
3.13
2.76
3.74
3.38
2.29
2.55
869.77
499.65
825.69
1648.10
796.28
801.97
1882.86
1350.03
1843.43
4618.74
2004.95
9009.61
1075.58
3509.55
2076.72
9127.04
1084.93
5427.18
ꢀ
40.21
ꢀ
ꢀ
ꢀ
997.53
4510.04
7n
12.5 (po)
2 (iv)
ꢀ
ꢀ
71.95
ꢀ
1859.47
10025.36
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12
13
14
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42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
11c
12.5 (po)
2 (iv)
ꢀ
963.06
ꢀ
ꢀ
52.30
ꢀ
3175.24
ꢀ
AZD4547
12.5 (po)
74.66
an = 3 animals/group. Data are the mean values.
We first examined the impact of various substituents at the
C3ꢀposition of the pyrazolopyridine scaffold on their biologiꢀ
cal activity (Table 2). Incorporation of a chlorine atom at the
2ꢀ or 3ꢀposition of phenyl ring of compound 4a reduced both
enzymatic and cellular potencies (7a and 7b), and the attachꢀ
ment of a methoxy group to the phenyl moiety also resulted in
a dramatic drop in potencies (7c and 7d). These results indiꢀ
cated that introduction of either the electronꢀwithdrawing
group or the electronꢀdonating group was detrimental to the
activity, which could be explained by the fact that the hydroꢀ
phobic pocket where the phenyl ring was accommodated was
too narrow to tolerate any bulky substituent (Figure 3B). In
addition, replacement of the phenyl ring with heterocyclic
bioisosteres afforded compounds 7e–h with weaker potencies
compared to 4a in both enzymatic and cellular assays. Accordꢀ
ingly, the 2, 3ꢀunsubstituted phenyl ring was optimal for good
activity against FGFR1.
extended out of the ATP binding pocket toward solvent (Figꢀ
ure 3).
To assess the pharmacokinetic properties of these 1Hꢀ
pyrazolo[3,4ꢀb]pyridine derivatives in vivo, we chose comꢀ
pounds 4e, 7n, and 11c for evaluation in male ICR mice
through intravenous and oral administration. The key pharmaꢀ
cokinetic parameters are summarized in Table 4. The eliminaꢀ
tion halfꢀlives (T1/2) of compounds 4e, 7n, and 11c were all
about three hours after both intravenous and oral administraꢀ
tion. The three compounds had similar maximum concentraꢀ
tions (Cmax) after intravenous administration, while 7n disꢀ
played the highest Cmax after oral administration. Moreover,
AUC values for 7n were much higher than that for 4e and 11c
after both intravenous and oral administration, and 7n had the
best oral bioavailability among them. In addition, the in vitro
plasma protein binding of compounds 4e, 7n, 11c and
AZD4547 were determined in mouse plasma, and they all
possessed high plasma protein binding rates (99.6%, 99.6%,
99.5%, and 99.3%, respectively). On the basis of its excellent
in vitro potency and favorable pharmacokinetic properties, 7n
was selected for further evaluation.
Next, we tested the influence of substituents at the 4ꢀ
position of phenyl ring A on their biological activity (Table 3).
Compound 7i with no substituents on phenyl ring A, showed
potent enzymatic potency against FGFR1 with an IC50 value of
42.4 nM but poor cellular potency (IC50 > 1 ꢁM). Incorporaꢀ
tion of an amino group at 4ꢀposition of the phenyl ring A imꢀ
proved both enzymatic and cellular potencies (11a vs 7i), and
the activity of 4ꢀdimethylamino analogue 7k was equipotent to
that of 11a. Though 4ꢀ(2ꢀ(dimethylamino)ethyl)(methyl)amino
analogue 7l displayed a substantial increase in potencies comꢀ
pared to 7k, it was one order of magnitude less potent than 4a
in both enzymatic and cellular assays. 4ꢀPiperazinyl analogue
11b was less active in enzymatic assay, but had similar celluꢀ
lar potency compared to 4a. 4ꢀpiperidinyl analogue 4b and 4ꢀ
morpholinyl analogue 4c exhibited comparable enzymatic
potency to 11b, but suffered a large enzymeꢀtoꢀcell shift. Reꢀ
placement of the methylpiperazine moiety of 4a by either
methylpiperidine (4d) or methylhomopiperazine (4e) reduced
enzymatic potency, but had little effect on cellular potency. In
order to further improve the cellular potency of this series of
compounds against FGFR1, various substituents were incorpoꢀ
rated at the 4’ꢀposition of piperazine moiety of 11b. Most of
the resulting analogues (4f–i, 7n, and 11c) demonstrated exꢀ
cellent inhibitory activity in both enzymatic and cellular asꢀ
says, except that analogues 4j and 4k suffered a sharp enꢀ
zymeꢀtoꢀcell shift, perhaps owing to the poor cellular penetraꢀ
tion. Thus, a range of substituents were tolerated at the 4’ꢀ
position of piperazine moiety, presumably because this region
Table 5. Kinase Selectivity Profile of Compound 7n
IC50 (nM)a
Kinase
AZD4547
7n
FGFR1
FGFR2
FGFR3
FGFR4
VEGFR2
0.3 ± 0.01
0.7 ± 0.1
2.0 ± 0.3
52.7 ± 0.6
422.7 ± 37.6
1.2 ± 0.1
0.4 ± 0.0
5.6 ± 1.8
45.7 ± 11.2
67.2 ± 3.3
aValues are the mean ± SD of two independent assays.
The selectivity of 7n was evaluated against a panel of 71
protein kinases, including FGFR family (FGFR2–4) and hoꢀ
mologous VEGFR2. As depicted in Table 5 and Supporting
Information Table S1, 7n exhibited similar strong potencies
against FGFR2 and 3 (IC50 = 0.7 and 2.0 nM, respectively)
and a moderate potency against FGFR4 (IC50 = 52.7 nM). IC50
values for all other kinases were more than 1 ꢁM or 400 nM
(VEGFR2, IC50 = 422.7 nM), indicating 7n is a selective
FGFR1–3 inhibitor.
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We finally investigated the in vivo antitumor efficacy of 7n
1
2
3
4
5
6
7
8
9
10
in H1581 xenograft model, which is specifically driven by
constitutive activation of FGFR1. Compound 7n was adminisꢀ
tered orally at doses of 12.5 mg/kg or 50 mg/kg once daily for
21 consecutive days. The results showed that, compared with
the vehicle group, 7n could suppress tumor growth in a doseꢀ
dependent manner, with the tumor growth inhibition rate
(TGI) of 54.0% and 100.4% at the doses of 12.5 mg/kg and 50
mg/kg, respectively (Figure 5), without significant body
weight loss (see Supporting Information Figure S1).
In summary, we reported the design, synthesis, and biologiꢀ
cal evaluation of a novel series of 1Hꢀpyrazolo[3,4ꢀb]pyridine
derivatives as potent and selective FGFR kinase inhibitors.
Systematic SAR explorations and preliminary assessment of
the pharmacokinetic properties resulted in the identification of
7n as a potential FGFR inhibitor for further evaluation. In
addition to excellent potencies against FGFR1–3, 7n exhibited
good selectivity for FGFR over other protein kinases. Besides,
the immunoblot analysis revealed that 7n suppressed FGFR
signaling in cancer cells. Moreover, 7n displayed potent antiꢀ
proliferative activities against a number of FGFRꢀdependent
cancer cell lines. Finally, significant antitumor efficacy was
observed for 7n in the FGFR1ꢀdriven H1581 xenograft model.
All these data indicated that 7n would be a promising lead
compound for further biological evaluation.
Figure 4. Inhibition of FGFR phosphorylation and downstream
signaling by Compound 7n in KG1 and KATOIII cells.
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In Western blot analysis, representative cancer cell lines
(KG1, KATOIII) were treated with 7n and positive drug
AZD4547 respectively. As shown in Figure 4, 7n inhibited the
phosphorylation of FGFR1/2 and the phosphorylation of PLCγ
and Erk, main downstream effectors of FGFR signaling,4 in a
doseꢀdependent manner in all the tested cell lines. These reꢀ
sults suggested that 7n exhibited an effective inhibition of
FGFR signaling.
Table 6. Effects of Compound 7n on Cell Proliferation
IC50 (nM)a
Cell lines
AZD4547
7n
0.7 ± 0.1
< 0.2
ASSOCIATED CONTENT
Supporting Information
H1581
KG1
37.9 ± 2.8
< 0.2
The Supporting Information is available free of charge on the
ACS Publications website at DOI:
Experimental details for the synthesis of all compounds, biologiꢀ
cal evaluation, and molecular modeling (Word)
SNU16
KATOIII
RT112
< 0.2
1.2 ± 0.1
20.2 ± 0.5
0.6 ± 0.3
< 0.4
< 0.2
AUTHOR INFORMATION
Corresponding Authors
aValues are the mean ± SD of two independent assays.
To elucidate the impact of 7n on FGFR mediated cancer cell
proliferation, five cell lines with known aberrant FGFR activaꢀ
tion were chosen, including FGFR1ꢀamplified H1581 cells,
FGFR1ꢀtranslocated KG1 cells, FGFR2ꢀamplified SNUꢀ16
cells and KATOIII cells, and FGFR3ꢀamplified RT112 cells.
Compound 7n showed significantly antiꢀproliferative effects
in all tested cell lines with IC50 values less than 1 nM, which
was more potent than AZD4547 (Table 6).
*(J.A.) Eꢀmail: jai@simm.ac.cn, phone: +86ꢀ21ꢀ50806600 ext.
2413;
*(M.G.) Eꢀmail: mygeng@simm.ac.cn, phone: +86ꢀ21ꢀ50806072;
*(W.D.) Eꢀmail: whduan@simm.ac.cn, phone: +86ꢀ21ꢀ50806032.
Author Contributions
ǁAuthors contributed equally to this work.
Funding Sources
We thank the National Natural Science Foundation of China (Nos.
81273365, 81573271, 81473243, and 81321092), National Sciꢀ
ence & Technology Major Project ‘‘Key New Drug Creation and
Manufacturing Program’’ (No. 2014ZX09304002ꢀ008ꢀ001), the
Shanghai Science and Technology Commission (No.
1315431901300) for their financial support. SAꢀSIBS Scholarship
Program is also gratefully acknowledged.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
MAPK, mitogenꢀactivated protein kinase; VEGFR, vascular enꢀ
dothelial growth factor receptor; PDGFR, plateletꢀderived growth
factor receptor; EGFR, epidermal growth factor receptor; DIPEA,
N,Nꢀdiisopropylethylamine; THF, tetrahydrofuran; DMF, N,Nꢀ
dimethylformamide; TBAF, tetrabutylammonium fluoride; SAR,
structure–activity relationship; ATP, adenosine triphosphate;
AUC, area under the curve
Figure 5. Antitumor efficacy of compound 7n in H1581 xenoꢀ
graft model. Results are expressed as the mean ± SEM (n = 6 for
inhibitorꢀtreated group, n = 12 for vehicle control group).
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