Design, synthesis, and antiproliferative activity assessment of non-ATP-competitive fibroblast growth factor receptor 1 inhibitors

Article abstract of DOI:10.1134/S1070363216120355

Fibroblast growth factor receptor 1 (FGFR1) is considered a therapeutic target for multiple cancers, including gastric cancer. FGFR1 inhibitors, being ATP competitors, can prevent the kinase domain and the downstream signaling cascade from phosphorylation

Full text of DOI:10.1134/S1070363216120355

ISSN 1070-3632, Russian Journal of General Chemistry, 2016, Vol. 86, No. 12, pp. 2744–2751. © Pleiades Publishing, Ltd., 2016.
Design, Synthesis, and Antiproliferative Activity Assessment
of Non-ATP-Competitive
Fibroblast Growth Factor Receptor 1 Inhibitors1
S. Yinga,2, Jia Wanga,2, C. Xua,2, Y. Kanga, X. Zhanga, L. Shia, L. Fana,
Z. Wanga, J. Zhoua, X. Wua, J. Wua*, W. Lia,b**, and G. Lianga
a Chemical Biology Research Center, College of Pharmaceutical Sciences, Wenzhou Medical University,
Wenzhou, Zhejiang, 325035 China
*e-mail: wjzwzmu@163.com
b College of Information Science and Computer Engineering, Wenzhou Medical University,
Wenzhou, Zhejiang, 325035 China
**e-mail: lwlwzmu@163.com
Received January 9, 2016
Abstract—Fibroblast growth factor receptor 1 (FGFR1) is considered a therapeutic target for multiple cancers,
including gastric cancer. FGFR1 inhibitors, being ATP competitors, can prevent the kinase domain and the
downstream signaling cascade from phosphorylation and thus have the potential to treat cancers associated with
aberrant FGFR1 activation. However, untargeted inhibition may cause numerous side effects. Thus, a non-ATP
competitive FGFR1 inhibitor should be urgently identified and explored. In this study, we designed and
synthesized 17 derivatives of nordihydroguaiaretic acid (NDGA), a known ATP-independent FGFR3 inhibitor.
In the kinase activity assay, 3,5-bis(2-fluorobenzylidene)piperidin-4-one (1B) showed the highest kinase
inhibitory activity among all derivatives and was thus identified as a non-ATP-competitive FGFR1 inhibitor. In
the biological effect evaluation, 1B restrained the FGFR−FRS2−ERK signaling pathway in a dose-dependent
manner and inhibited the growth of two gastric cancer cell lines. Overall, 1B can be considered as a potential
candidate for treating gastric cancer and as an outstanding lead compound for the discovery of novel non-ATP-
competitive FGFR1 inhibitors.
Keywords: antitumor activity, design and synthesis, FGFR1 inhibitors, gastric cancer, NDGA analogs, non-
ATP-competitive protein kinase inhibitor
DOI: 10.1134/S1070363216120355
INTRODUCTION
including cell proliferation, growth, differentiation,
migration, and survival [3]. As a result of over-
expression or mutation, the deregulation of FGFR
activity could be closely associated with the develop-
ment of different types of tumors, such as breast cancer
[4], lung cancer [5], oral squamous cell carcinoma [6],
and gastric cancer (GC) [7], by increasing cell prolifera-
tion and angiogenesis while inhibiting cell apoptosis.
Fibroblast growth factor receptors (FGFRs), a
well-known subfamily of receptor tyrosine kinases
(RTKs), include four members, namely, FGFR1 to
FGFR4, which are composed of an extracellular receptor
domain, a single-pass trans-membrane domain, and a
carboxy-terminal cytoplasmic domain [1]. Ligand
binding induces FGFR dimerization, which causes the
phosphorylation of intracellular receptor kinase
domains and activates the intracellular FGFR substrate
2α (FRS2α) and phospholipase Cγ1 (PLCγ1) [2].
Activated FRS2α initiates downstream signaling
through the RAS-MAPK-ERK pathway or the PI3K-
AKT pathway to induce a series of biological effects,
Based on the important effect of FGFR on
tumorigenesis, FGFR is considered to have great
potential for targeted tumor therapy [8]. The blockage
action of the kinase domain gives rise to the inhibition
of the phosphorylation of FGFR and its substrate, such
that the pathological state caused by aberrant FGFR
activation is mitigated [9, 10]. Small molecule
inhibitors of FGFR are universally becoming
increasingly accepted to possess a high therapeutic
1 The text was submitted by the authors in English.
2 S. Ying, Jia Wang, and C. Xu contributed equally to this work.
2744

DESIGN, SYNTHESIS, AND ANTIPROLIFERATIVE ACTIVITY ASSESSMENT
2745
significance [11]. Since the phosphorylation of the
HO
HO
CH3
receptor kinase domain is triggered by ATP binding,
the major FGFR inhibitors generally imitate the
structure of ATP, and then compete with ATP for the
binding site, resulting in the inactivation of kinase
conformation. However, almost all RTKs encoded by
the human genome possess a similar 3D structure of
the intercellular kinase domain. As a consequence of
the untargeted inhibition among different RTKs, ATP
competitive inhibitors have an inevitable defect in that
they induce a series of adverse effects. Given the poor
selectivity between the subtype FGFRs of ATP
competitive inhibitors, the discovery of novel non-
ATP competitive inhibitors may overcome the problem
via binding to the atypical allosteric site or generating
a hydrophobic interaction with the specific residues
[12]. The development of this class of inhibitor
presents good prospects.
OH
OH
CH3
NDGA
Delete the chiral
carbon atom
Introduce a Micheal
addition receptor
O
R1
R1
n
Series A: n = 0, 2, 3
Nordihydroguaiaretic Acid (NDGA), obtained from
the creosote bush Larrea divaricatta, has been reported
to exhibit strong activity as a non-ATP competitive
inhibitor of FGFR3, inhibiting the autophosphorylation
of FGFR3 both in vivo and in vitro [13]. Increasing
concentrations of ATP did not affect Km at various
concentrations of NDGA, indicating that NDGA
restricted the activation of FGFR in an ATP-inde-
pendent manner. Notably, among all the potent non-
ATP competitive inhibitors, NDGA is the only one
obtained from a natural source and has a low toxicity and
could thus be used as a leading compound for further
structural modification and medicinal chemistry research.
Add in nitrogen atom with
or without substitute group
O
R3
R3
N
R2
Series B
Fig. 1. Design of series A and B compounds on the basis of
NDGA structure.
On the basis of high homology between FGFR3
and FGFR1, our research group determined the
FGFR1 inhibitory potential of NDGA and found it to
be more preferably active than its FGFR3 inhibitory
activity in our early study. In this research, we
designed and synthesized two classes of NDGA
derivatives. Through the kinase activity screening of
17 NDGA derivatives, we found that 1B not only had a
lower IC50 relative to FGFR1 kinase in an ATP-
dependent manner compared with NDGA, but also
showed excellent anti-tumor activity against GC.
biological activity of small molecular inhibitor of
kinase may greatly enhance via a Micheal addition
reaction with the particular cysteine residue within the
ATP-binding site after adding electrophilic functional
groups into its structure such as α,β-unsaturated
carbonyls [14]. One famous example was Afatinib
(BIBW2992), an irreversible covalent ErbB Family
Blocker. Thus, we introduced a Micheal addition
receptor into the middle linker [14]. Furthermore, we
also added one nitrogen atom into the previous
aliphatic ring, which might provide site that forms
added hydrogen bond with the protein residues and
improve their water solubility, as well as be convenient
for the next Structure-Activity Relationship (SAR)
Analysis. The synthesis procedure was a simple, one-
step reaction, and the yield ranged from 44 to 82.7%.
The structural information of all compounds was
shown in (Table 1).
RESULTS AND DISCUSSION
Design and synthesis. In this research, we designed
and synthesized two classes NDGA derivatives (A and
B class). Firstly, in consideration of the isolation and
purification of chemicals containing chiral atom were
always difficult, we deleted the chiral carbon atoms
(Fig. 1) In the light of literature reported that the
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 86 No. 12 2016

2746
S. YING et al.
Table 1. Synthetic process and chemical structures of series
A and B compounds
pounds (Table 2). Compounds 11A and 1B exhibited
considerable inhibitory activity, with the cor-
responding half maximal inhibitory concentration
values (IC50) of 11 and 14 μM, respectively. In contrast
to NDGA, which has an IC50 of 24.5 μM, our active
compound possessed approximately twice as much
activity of its lead compound at the kinase level. By
analyzing the result of the inhibitory effect, our
preliminary SAR analysis was performed. Among the
A class compounds, 4A and 11A both had relative
better potent FGFR1 kinase inhibitory activity,
whereas the compound with 2-bromine substituent
may be superior to the other series A compounds. As
shown in Table 2, only 1B exhibited a particular
CHO
O
R1
O
+
n
CH3CH2OH
40% NaOH
R1
R1
n
A
CHO
O
FGFR1 inhibitory effect among the
B
class
compounds, whereas others all had a negligible effect.
The detailed SAR has to be performed after we
synthesize more B class compounds and determine
their inhibitory action in the future.
R3
+
N
R2
O
1B inhibits the activity of FGFR1 kinase in a non-
ATP competitive manner. Given that NDGA is
noncompetitive with ATP, we suppose that 1B might
be similar to its lead compound. To confirm the action
mechanism of 1B with FGFR1, an ATP-competitive
assay was performed by using a kinase inhibitory
activity screening assay. In the absence of a com-
pound, the ATP concentration was gradually increased.
The percentage conversion of the FGFR1 substrate
steadily rode and became constant when the ATP
concentration reached 2000 μM. When the compound
of 1B was added, FGFR1 phosphorylation was inhi-
bited. The percentage conversion recovered because of
the increased ATP concentration, but the initial no-
compound status can no longer be achieved. Although
the ATP concentration reached a level of 4000 μM, no
influence of the inhibitory activity caused by the
compound could be observed (Fig. 2a).
CH3CH2OH
40% NaOH
R3
R3
N
R2
B
Comp.
Comp.
R1
2,4-Cl
na
R2
R3
2-F
no.
1A
2A
3A
4A
5A
6A
7A
8A
9A
10A
11A
no.
1B
2B
3B
4B
5B
6B
2
2
2
2
2
2
3
3
0
0
0
H
H
H
2-Cl
2,6-F
2-CF3
2-Cl
4-F
2-F
2-Br
CH3
CH3
2,3-Cl
2,5-F
2,4-Cl
2,3-Cl
2,4-Cl
2-Cl
CH2CH3 2-F
As a standard index to describe the biological
activity of an inhibitor, the curve of graph B (Fig. 2b)
shows that the IC50 value of 1B was irrelevant to the
increase in ATP concentration. All results indicated
that the inhibitory effect of 1B of FGFR1
phosphorylation occurred in a non-ATP competitive
manner, which was similar to the case of NDGA.
Moreover, the enzyme kinetic parameters (α value and
Ki value, as shown in Fig. 2) more intuitively identified
with our prediction.
2-Br
a
When n is equal to 2, 3, or 0, the middle connecting ring
respectively corresponds to cyclopentanone, cyclohexanone, or
acetone.
Screening for the inhibitory activity for FGFR1
kinase. By screening for the inhibition of FGFR1
kinase, we assessed the inhibitory effects of 17 com-
Inhibitory activity of 1B relative to the
intercellular FGFR1 kinase and its downstream
signaling protein in GC cell lines. The intercellular
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DESIGN, SYNTHESIS, AND ANTIPROLIFERATIVE ACTIVITY ASSESSMENT
2747
(a)
(b)
40
ATP, μM
1
2
3
1
2
3
4
5
6
7
8
100
60
30
20
20
10
0
4
–20
0
1000
3000
5000
0.0 0.5 1.0 1.5 2.0
log 1B, μM
ATP, μM
Fig. 2. Inhibitory mode of 1B relative to FGFR1 kinase was identified as independent of ATP concentration by kinase inhibitory
activity screening assay. The conversion data were fitted with GraphPad for global fitting using “mixed model
inhibition.” (a) Influence of increasing ATP concentration on the conversion of substrate peptide inhibited by 1B, μM: (1) 0.00,
(2) 2.75, (3) 11.00, (4) 44.00. (b) 1B Maintained a stable capability to inhibit the phosphorylation of FGFR1 at all ATP
concentrations, as indicated by similar IC50 values: (1) 4192, (2) 2096, (3) 1048, (4) 524, (5) 262, (6) 131, (7) 66, (8) 33. Inhibitory
kinetics parameters of 1B: νmax = 30.32 μM/s, α = 2.411, Ki = 20.91 μM, Km = 117.43 μM.
FGFR1 kinase terminal was the core section of the
whole receptor for exerting its biological effects and
activating its downstream signaling pathways. Studies
found that FGFR1 expression was amplified in SGC-
7901 cells, which was thus selected for further
research [15]. Among the 17 NDGA derivatives, 1B
showed the most potent inhibitory activity relative to
FGFR1 according to kinase activity screening. Thus,
1B was selected as the active compound for the
following anti-proliferative evaluation and determina-
tion of the effect of downstream signaling. By Western
blot analysis, Our data (Fig. 3a) reveal that compound
1B exhibited an apparent inhibitory effect on the bFGF-
induced phosphorylation of FGFR1 at a concentration
of 5 μM. In previous studies on FGFR signaling, FRS2
has been universally recognized as being phos-
phorylated at multiple sites by activated FGFR to
initiate downstream signaling, and the activation of the
downstream MAPK/ERK pathway could be crucial to
cell proliferation. Thus, we tested the effects of 1B on
the activation of FRS2 and ERK1/2 in SGC-7901 cells.
Figure 3b intuitively shows that 1B also remarkably
inhibits the phosphorylation of the downstream
signaling protein, such as FRS2 and ERK, in a dose-
dependent manner.
the determination of the anti-proliferative capability of
compound 1B. With the use of an MTT assay, all GC
cell lines (4000 cells/well) were treated with various
concentrations of 1B for 72 h. The viability of tumor
cells significantly declined (data not shown). Further-
more, we selected these GC cell lines to test the anti-
proliferative activity of NDGA for a comparison with
1B. The result (Table 3) shows that 1B displays
excellent anti-proliferative effect on the GC cell lines
with an IC50 value of 1.5 μM for SGC-7901, and
1.6 μM for MGC-803. Meanwhile, the value of NDGA
was over 30-fold higher than that of 1B (Table 3).
Table 2. Inhibition rate (IR) and the IC50 value of series A
and B compounds
IR, %a
IR, %
Comp.
IC50, Comp.
IC50,
59.1±3.5
95.3±2.1
74
86
32.7±8.2
91.2±0.7
80.9±1.6
24.2±0.8
–6.0±3.0
–3.4±6.6
–1.1±5.6
13.4±7.7
–
11
14
–
1A
2A
3A
4A
5A
6A
7A
8A
9A
10A
11A
1B
41.5±3.3 >100
99.8±0.2
25.7±4.2
39.6±2.8
20
2B
b
–
3B
–
–
–
4B
1B Inhibited the growth of GC cell lines. The
FGF-FGFR signaling pathway has been demonstrated
to be significant to the survival, angiogenesis,
proliferation, and migration of tumors. Wen reported
that microRNA-133b could serve as a tumor-
suppressive gene to down-regulate FGFR1 expression
and suppress GC cell growth [15]. Therefore, two GC
cells lines (MGC-803 and SGC-7901) were chosen for
54.2±4.8 >100
5B
–
41.9±7.5
58.1±6.4
–
–
6B
29
a
IR indicates the inhibition rate of compounds relative to the
FGFR1 kinase at a concentration of 20 μM. (–) indicates that
b
the IC50 value was undetermined.
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2748
S. YING et al.
1B
NDGA
(a)
Concentration, μM
bFGF (20 ng/mL)
P-FGFR1
–
–
–
+
1
+
5
+
10
+
20
+
FGFR1
P-FRS2
FRS2
P-ERK
ERK
GAPDH
(b)
1.0
2.5
3
2
ns
0.8
ns
2.0
**
ns
**
**
**
0.6
0.4
0.2
0.0
1.5
***
1.0
***
***
***
1
0
**
***
***
***
0.5
0.0
***
– + + + + +
– – 1 5 10 20
– + + + + +
– – 1 5 10 20
– + + + + +
– – 1 5 10 20
bFGF (20 ng/mL)
Concentration, μM
1B NDGA
P-FGFR1/FGFR1
1B NDGA
P-ERK/ERK
1B NDGA
P-FRS2/FRS2
Fig. 3. 1B Inhibited the phosphorylation of FGFR1, FRS2, and ERK dependent on ATP concentration. (a) SGC-7901 cells were
treated by 1B for 1 h after being starved for 24 h and then stimulated with 20 ng/mL of bFGF for 10 min. Phosphorylation levels of
FGFR1, FRS2, and ERK in cell lysates were measured by Western blot analysis. (b) Column figures show the normalized optical
density as a percentage of total control.
Over the past two decades, the exploitation of
FGFR inhibitors has attracted considerable attention
from various research institutions and pharmaceutical
companies. Numerous small-molecule FGFR
inhibitors that bind to the intercellular kinase domain
have been designed and synthesized [8, 9]. Among
these inhibitors, some excellent ones are being
evaluated in clinical trials to determine their anti-tumor
activity. By analyzing the protein–ligand crystal
structure, a selective FGFR1-3 inhibitor AZD4547 was
designed and investigated for potential treatment
capability against cancer under a phase 2 clinical trial
(NCT01795768) [16]. Another highly active and
selective FGFR inhibitor, FIIN-1, displays high
inhibitory activity in various FGFR-dependent tumor
cells by forming a covalent bond with Cys486 in the
FGFR1 ATP-binding domain [17]. Despite these
highly active inhibitors, we note that they both work
through an ATP-dependent mechanism. However, the
ATP-binding pocket of FGFRs is highly similar to that
of other RTKs, such as VEGFR and PDGFR. The poor
selectivity between distinct RTKs may cause numerous
Table 3. 1B exhibited excellent inhibition of the growth of
the GC cells. Two GC cells were treated with 1B at
concentrations (0.48, 2.4, 5, 12, and 60 μM) for 72 h. The
MTT assay provides the respective IC50 values of the
compounds. In contrast to NDGA, 1B showed over 30-fold
activity upgrade
IC50, μM
Compound
MGC-803
1.6±0.2
SGC-7901
1.5±0.6
1B
NDGA
46.9±7.1
65.1±5.8
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DESIGN, SYNTHESIS, AND ANTIPROLIFERATIVE ACTIVITY ASSESSMENT
2749
side effects, such as nausea, diarrhea, as well as a
decrease in thrombocytes and erythrocytes [9]. In
addition, the effect of these ATP structural imitators
may be offset by the high physiological or intracellular
concentrations of ATP. These inevitable weaknesses
have caused researchers to look toward non-ATP
competitive inhibitors. Given that such inhibitors will
be less likely to compete with ATP for binding; their
selectivity may be significantly improved.
solved in 10 mL of ethanol (piperidin-4-one hydro-
chloride was dissolved in a 10 : 1 mixture of ethanol
and distilled water). Corresponding benzaldehyde
(4 mmol) was added to the solution at 5 to 8°C, and
0.5 mL of 40% aqueous NaOH was added dropwise as
a catalyst with stirring. The reaction was monitored by
TLC on silica gel. When the reaction was complete,
distilled water was added to precipitate the product
completely, after which it was filtered off in a vacuum
and purified by column chromatography used eluent
petroleum ether–ethyl acetate as eluent (6 : 1–2 : 1).
The structures were characterized by 1H-NMR
spectroscopy and confirmed by mass spectrometry.
The spectral data of known products (1A–11A, 1B,
4B, and 5B) are consistent with those reported in the
published literature by our research team [20–22].
Unreported compounds (2B, 3B, 6B) are shown below.
GC is the second most common cause of cancer
death worldwide [18]. Several recent studies proved
that over-expression of FGFR1 was closely associated
with the formation and development of GC. Some
studies discovered that over-expression of the mRNA
and protein of FGFR1 in GC tissue was observed in 12
(50%) of 24 and 37 (61%) of 61 separately [19].
Conversely, knockdown of FGFR1 or the use of
microRNA for the negative regulation of FGFR1
activity could successfully inhibit the growth of GC
cells. Developing an anti-GC drug by targeting FGFR1
is becoming increasingly urgent and much promising.
In this research, we successfully discovered an
excellent leading compound of FGFR1 inhibitor for
treating GC. At the cellular level, 1B was found to
show excellent inhibitory activity in FGFR1 over-
expressing GC cells via a potent mechanism that
suppresses the FGFR1/FRS2α/ERK1/2 pathway. This
compound also exhibits better anti-tumor effects than
NDGA, not only in the level of FGFR1 kinase, but
also in the cellular level. Thus, 1B may be a promising
candidate for treating GC.
(3E,5E)-3,5-Bis(2,6-difluorobenzylidene)pipe-
ridin-4-one (2B). Light yellow powder, yield 33%, mp
164.1–166.9°C. 1H NMR spectrum (CDCl3), δ, ppm (J,
Hz): 7.602 s (2H, 2Ar-CH=CH), 7.322–7.372 m (2H,
2H4), 6.949 d (4H, 2H3, 2H5; J = 7.8), 3.994 s (4H,
2N–CH2). ESI MS spectrum, m/z: 348.0 [M + 1]+.
Found, %: C 65.51; H 3.83; F 21.68; N 4.13; O 4.81.
C19H15F2NO. Calculated, %: C 65.71; H 3.77; F 21.88;
N 4.03; O 4.61. M 347.31.
(3E,5E)-3,5-Bis[2-(trifluoromethyl)benzylidene]-
piperidin-4-one (3B). Yellow powder, yield 82%, mp
153.5–155.7°C. 1H NMR spectrum (CDCl3), δ, ppm (J,
Hz): 7.996 s (2H, 2Ar-CH=C), 7.735 d (2H, 2Ar-H3,
J = 7.8), 7.563 t (2H, 2Ar-H5, J = 7.8), 7.462 t (2Ar-H4,
J = 7.8), 7.254 d (2H, 2Ar-H6, J = 7.8), 3.872 s (4H,
CH2–N–CH2). ESI-MS m/z: 412.1 [M + 1]+. Found, %:
C 61.34; H 3.65; F 27.51; N 3.61; O 3.87.
C21H15F6NO. Calculated, %: C 61.32; H 3.68; F 27.71;
N 3.41; O 3.89. M 411.34.
EXPERIMENTAL
Synthesis of series A and B compounds. Reagents
and solvents for the synthesis were commercially
available and obtained from Sigma-Aldrich (St Louis,
Missouri, USA) and Aladdin (Shanghai, China), which
were used without further purification. Silica gel
(GF254) for column chromatography (200–300 mesh)
was obtained from Aladdin. Melting points were
measured on a Fisher-Johns melting apparatus and
were uncorrected. Electron-spray ionization mass
spectra (ESI-MS) data were collected in positive mode
on a Bruker Esquire 3000t spectrometer. The 1H-NMR
spectra data was recorded on a 600 MHz spectrometer
(Bruker Corporation, Switzerland) with TMS as an
internal standard.
(3E,5E)-3,5-Bis(2-fluorobenzylidene)-1-ethyl-
piperidin-4-one (6B). Light yellow powder, yield
76%, mp 130.0–133.17°C. 1H NMR spectrum (CDCl3),
δ, ppm (J, Hz): 7.907 s (2H, 2Ar-CH=C), 7.367–7.382
m (2H, 2Ar-H6), 7.344–7.357 m (2H, 2Ar-H4), 7.175–
7.199 m (2H, 2Ar-H5), 7.114–7.144 m (2H, Ar-H3) ,
3.717 d (4H, 2N–CH2–C, J = 7.8), 2.567 q (2H,
N–CH2, J = 7.2), 1.010 t (3H, CH3, J = 7.2). ESI-MS
m/z: 340.7 [M + 1]+. Found, %: C74.13; H 5.89; F
11.08; N 4.31; O 4.64. C21H19F2NO Calculated, %: C
74.32; H 5.64; F 11.20; N 4.13; O 4.71. M 339.38.
Kinase activity screening. The kinase activity of
Brief general procedure of synthesis (for details,
FGFR1 was determined by Mobility Shift Assay on a
see [20–22]). Corresponding ketone (2 mmol) was dis-
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2750
S. YING et al.
Caliper Life Sciences Labchip EZ Reader. The enzyme
solution was prepared in a 1.25×kinase base buffer
(62.5 mM HEPES, 0.001875% Brij-35, 12.5 mM
MgCl2, 2.5 mM DTT). Stop buffer (100 mM HEPES,
0.015% Brij-35, 0.2% coating reagent no. 3, 50 mM
EDTA) was got ready. A test compound was dissolved
in DMSO and then diluted to a specific concentration
with water in a 384-well plate. 5 μL of compound
solution were diluted with 10 μL of 2.5× enzyme
solution, in wells. 10 mM EDTA was used as the low
control. The mixture was incubated at room tem-
perature for 10 min, after which 10 μL of peptide
solution (2.5×, FAM-P22, add FAM-labeled peptide
and ATP in the 1.25× kinase base buffer) was added to
each well of the 384-well assay plate. After incubation
at 28oC for 1 h, 25 μL of stop buffer was added into
the mixture to stop the reaction. Conversion data were
then collected on the EZ reader, the percent inhibition
of kinase activity was calculated by the following formula:
glycine, sodium dodecyl sulfate (SDS), pre-stained
protein marker, and nonfat dry milk were from Bio-
Rad (Germany). pFRS2α (Tyr 196) was purchased
from Cell Signaling Technology (Beverly, MA, USA).
pFGFR1, p-ERK1/2, ERK1/2, FRS2, and GAPDH
were obtained from Santa Cruz Biotechnology, Inc.
(CA, USA).
MTT cell proliferation assay. MGC803 and
SGC7901 Cells (4000 cells/well) were seeded on a 96-
well plate. The next day, cells were treated with
different concentrations (0, 0.480, 2.400, 5.000,
12.000, and 60.000 μM) of the test compound. After
72-h incubation, 25 µL of MTT (5 mg/mL) were added
to each well for 4 hours at 37°C in the dark. The MTT
solution was removed, and 150 µL of DMSO was
added to each well to solubilize the MTT metabolic
product; then the absorbance was read at 490 nm.
Western blot analysis. SGC-7901 cells (treated
with the test compound after 24-h starvation, and then
stimulated with 20 ng/mL bFGF for 10 min) were
lysed in a lysis buffer for 15 min on ice. The cell
lysates were centrifuged at 4°C to remove insoluble
components. Protein (90 μg) from each sample was
subjected to SDS-PAGE and transferred onto PVDF
membranes. After incubation with 5% nonfat dry milk
in TBST, the membranes were incubated with primary
antibodies (anti-pFGFR1, anti-FGFR1, anti- pFRS2,
anti-FRS2, anti-pERK, anti-ERK, anti-GAPDH, Santa
Cruz) at 4ºC overnight and then with the horseradish
peroxidase-labeled secondary antibody at room
temperature for 1 h. The results were analyzed by
Quantity One software to determine the relative ratio.
Inhibition % = (max–conversion)/(max–min) × 100%,
where (max) stands for the DMSO control, (min)
stands for low control, (conversion) means the average
of two experimental values given by EZ reader.
To determine the half maximal inhibitory concentra-
tion of the test compound relative to kinase activity,
10 gradient concentrations of the compound (100,
33.330, 11.110, 3.700, 1.230, 0.410, 0.140, 0.046,
0.015, and 0.005 μM) were set up. The inhibition ratios
for different concentrations relative to kinase were
determined and calculated, and the concentration–inhibi-
tion rate curve was fit using the GraphPad Prism software.
ATP competitive test. The same kinase inhibition
assay with four concentrations of the test compounds
(0 to 100 μM) and eight concentrations of ATP
(12.500, 6.250, 3.125, 1.563, 0.781, 0.391, 0.195, and
0.098 mM) was used to determine the conversion of
the peptide substrate, catalyzed by FGFR1 kinase,
within 1 h. The inhibition curve was fit using the
GraphPad Prism software.
CONCLUSIONS
In conclusion, we have designed and synthesized
two types of compounds employing NDGA as the lead
compound. Among these compounds, 1B exhibits a
satisfactory effect in terms of inhibiting the kinase
activity of FGFR1 and suppressing the proliferation of
two GC cell lines in vitro. This study discovered a
potent non-ATP competitive FGFR1 inhibitor, as well
as a new FGFR1 kinase inhibitor scaffold–3,5-
dibenzylidenepiperidin-4-one. This scaffold was first
recommended as an FGFR inhibitor and potential
candidate for GC treatment applications.
Cells and reagents. Human GC cells (SGC-7901,
and MGC-803) were purchased from the Cell Bank of
the Chinese Academy of Sciences (Shanghai, China).
All cells were maintained in RPMI 1640 medium
(Invitrogen, CA, USA) with 10% FBS (HyClone,
Logan, UT, USA); then incubated at 37ºC in an atmo-
sphere of 5% CO2. Methylthiazoletetrazolium (MTT)
and dimethylsulfoxide (DMSO) were from Sigma-
Aldrich (St. Louis, MO,USA). Forty percent acryl-
amide, Coomassie Brilliant Blue, TEMED, Tris,
ACKNOWLEDGMENTS
The kinase activity screening and ATP competitive
assay both were authorized to ChemPartner (Shanghai,
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 86 No. 12 2016

DESIGN, SYNTHESIS, AND ANTIPROLIFERATIVE ACTIVITY ASSESSMENT
vol. 10, no. 2, p. 116. doi 10.1038/nrc2780
2751
China). The work was supported by National Natural
Science Foundation of China (81402839, 81272462),
ZheJiang Province Natural Science Funding of China
(LY17H160059, LY13H300005), Technology Foundation
for Medical Science of Zhejiang Province (2012KYA129),
Key Laboratory of Functional Protein Research of
Guangdong Higher Education Institutes, Jinan
University, the Fundamental Research Funds for the
Central Universities (X. Wu), Guangdong Provincial
‘‘Thousand-Hundred-Ten Talent Project’’ (X. Wu),
University Students in Zhejiang Science and Tech-
nology Innovation Projects (2013R413023,
2015R413051).
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