Discovering potent inhibitors against c-Met kinase: Molecular design, organic synthesis and bioassay

Article abstract of DOI:10.1039/c1ob06186k

The receptor tyrosine kinase c-Met is an attractive target for therapeutic treatment of cancers nowadays. The discovery of small molecule inhibitors is of special interest in the blockade of the c-Met kinase pathway. Here, we initiated our study from comp

Full text of DOI:10.1039/c1ob06186k

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PAPER
Discovering potent inhibitors against c-Met kinase: molecular design, organic
synthesis and bioassay†
Zhongjie Liang,‡a Xiao Ding,‡a Jing Ai,‡b Xiangqian Kong,a Limin Chen,a Liang Chen,a Cheng Luo,*a,c
Meiyu Geng,*b Hong Liu,*a Kaixian Chena and Hualiang Jianga
Received 17th July 2011, Accepted 29th September 2011
DOI: 10.1039/c1ob06186k
The receptor tyrosine kinase c-Met is an attractive target for therapeutic treatment of cancers
nowadays. The discovery of small molecule inhibitors is of special interest in the blockade of the c-Met
kinase pathway. Here, we initiated our study from compound 1a, a novel inhibitor against c-Met kinase.
A substructure similarity search against the SPECS database and chemical synthesis methods were
performed to obtain a series of pyrazolidine-3,5-dione derivatives. Through the enzyme-based assay
against c-Met kinase, 4 compounds (1c, 1e, 1m and 1o) showed potential inhibitory activity, with IC50
values mostly less than 10 mM. Based on the structure–activity relationship (SAR) and binding mode
analysis, a focused combinatorial library was designed by the LD1.0 program. Taking into account
ADMET properties and synthesis accessibility, seven candidate compounds (5a–g) were successfully
synthesized. The activity of the most potent compounds 5b (IC50 = 0.46 mM) was 20 fold higher than
that of the lead 1a. Taken together, our findings identified the pyrazolidine-3,5-dione derivatives as
potent inhibitors against c-Met kinase and demonstrated the efficiency of the strategy in the
development of small molecules against c-Met kinase.
PI3K-AKT-mTOR cascade and so on.2 When deregulated, the
Introduction
over activation of the kinase has been implicated in tumor cell
c-Met kinase, which is encoded by proto-oncogene MET
proliferation, angiogenesis, invasion/metastasis and resistance to
(mesenchymal-epithelial transition factor), is the cell-surface re-
apoptosis and cytotoxic therapies, which implied the kinase as
an attractive therapeutic target.4 Consequently, inhibiting the
HGF/c-Met kinase pathway would have potent anti-tumor effects
via multiple complementary mechanisms. To date, numerous
examples of successful therapeutic interventions with c-Met kinase
inhibitors have been reported, which in turn has proved the kinase
as a target of priority.5–9
ceptor for hepatocyte growth factor (HGF). The kinase, together
with its natural ligand HGF, is an important paracrine mediator of
epithelial-mesenchymal cell interactions involved in the regulation
of multiple cellular activities, including cell migration, mitogenesis,
morphogenesis, and angiogenesis.1 The kinase activation is conse-
quently essential for embryonic development and wound healing.2
Moreover, c-Met kinase can be over-activated via a variety of
mechanisms, including ligand-dependent activation (autocrine
or paracrine), gene amplification, gene mutation and cross-talk
with other receptors (heterodimerization).3 The activation gives
rise to receptor dimerization and recruitment of several SH2
domain-containing signal transducers that activate a number of
pathways including the GRB2-SOS-RAS-RAF-MEK-ERK axis,
Nowadays, several different strategies targeting the HGF/c-
Met kinase pathway have been explored,1,5 each focusing on one
of the serial steps that regulate c-Met kinase activation. The
strategies include: (a) blocking the interaction between c-Met
kinase and HGF by antagonists or neutralizing antibodies, such
as NK1 and NK2 isoforms,10,11 (b) blocking the phosphorylation
of tyrosine residues in the kinase domain by small molecule
inhibitors which compete for ATP binding at the active site, and
(c) blocking the c-Met kinase dependent signaling by interfering
with c-Met associated signal transducers or downstream signaling
components.12 Amongst these strategies, inhibition of the kinase
activity by small molecules is a pharmacologically attractive
method that has been demonstrated for other tyrosine kinases.13
Recently, we identified compound (1a) from screening of our in-
house SPECS database as an inhibitor against c-Met kinase with
an IC50 value of 9.5 mM. By virtue of the cost-inefficient and time-
consuming process of conventional drug discovery over decades,
focused combinatorial library design has been proved to be a
aDrug Discovery and Design Center, State Key Laboratory of Drug Research,
Shanghai Institute of Materia Medica, Chinese Academy of Sciences,
Shanghai, 201203, China. E-mail: hliu@mail.shcnc.ac.cn; Fax: +86-21-
50807188; Tel: +86-21-50806600
bDivision of Anti-Tumor Pharmacology, State Key Laboratory of Drug
Research, Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, Shanghai, 201203, China. E-mail: mygeng@mail.shcnc.ac.cn
cCenter for Systems Biology, Soochow University, Jiangsu, 215006, China.
E-mail: cluo@mail.shcnc.ac.cn
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c1ob06186k
‡ These authors contributed equally to this study.
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To further develop better inhibitors based on this scaffold, a
substructure similarity search against the SPECS database and
chemical synthesis were performed on the pyrazolidine-3,5-dione
scaffold. By in vitro assay against c-Met kinase, 18 compounds
(1a–r) stand out as c-Met kinase inhibitors, whose structures are
shown in Table 1. The compounds labeled with asterisks were
synthesized through the routes outlined in Scheme 1, and the
details of the synthetic procedures and structural characterizations
are described in the Experimental section. The primary inhibitory
activities of these compounds at 10 mM against c-Met kinase were
determined, and IC50 values were measured for the compounds
with better inhibitory activities (>50%), which are also listed
in Table 1. The detail bioassay protocol is described in the
Experimental section.
SAR analysis indicated that substituent R2 of the A ring was
essential for the inhibitory activity against c-Met kinase. Among
these derivatives, esters proved to be the favored substituent for
the R2 group (1a–1c). Replacing the ester group with a methyl
group (1a and 1d–1e), a methoxy group (1a and 1f) or a nitro
group (1a and 1g) actually reduced the enzyme potency to 48.1%,
23.6% and 43.2%, respectively. The removal of the ester groups
made the compounds basically lose the inhibitory activity (1a
and 1j). Furthermore, H-bond donating groups on R5 or R6
of the D ring were also essential for the inhibitory activities.
Consequently, a carboxyl group and an ester group (1a–1e, 1h–1i)
on R6 afforded good enzyme potency. Replacing R6 with a chloro
group decreased the inhibitory activity (1b and 1k) and the change
of the chloro group to R4 also decreased the activity (1k and 1l).
The introduction of the H-bond donating group sulfamide to R5
afforded compounds 1m, 1n and 1o with inhibition of 56.2%,
43.4% 57.6%, respectively. In addition, replacing the furan ring
(C ring) with a benzene ring weakened the inhibitory activity to
42.2% (1c and 1p). It is also worth noting that the inhibitory
activities were abolished when the pyrazolidine-3,5-dione ring was
replaced by a 3-methyl-1H-pyrazol-5(4H)-one ring (1p and 1q) or
a 3-ethoxy-1H-pyrazol-5(4H)-one ring (1b and 1r).
Fig. 1 Flow chart of the discovery strategy for c-Met kinase inhibitors in
this study.
complementary method in rational drug design.14 Here we present
a structure based molecular design to identify the pyrazolidine-
3,5-dione derivatives as potent c-Met kinase inhibitors. Firstly,
we performed a substructure similarity search against the SPECS
database on the pyrazolidine-3,5-dione scaffold. Meanwhile, the
chemical synthesis method was also employed. After evaluating for
their inhibitory activity by in vitro enzymatic assay, 18 compounds
(including 1a) were identified as inhibitors against c-Met kinase.
In addition, among them, 4 compounds (1c, 1e, 1m and 1o)
showed moderate potency, with IC50 values less than 10 mM.
Secondly, based on the structure–activity relationship (SAR) and
analysis of their binding modes compared with X-ray crystal
structures, fragments of advantage were extracted for the design
of a focused combinatorial library containing 720 molecules by
the LD1.0 program. Taking into account the ADMET properties
and feasibility of chemical synthesis, seven compounds (5a–g) were
successfully synthesized ultimately. The activity of the most potent
compounds 5b (IC50 = 0.46 mM) was ~20 fold higher than that of
the lead compound 1a. The detailed design procedure is shown in
Fig. 1. Overall, our study demonstrated that the pyrazolidine-3,5-
dione could be a better lead scaffold for c-Met kinase inhibitors,
which will accelerate the discovery of new inhibitors against c-Met
kinase.
Binding modes of pyrazolidine-3,5-dione derivatives to c-Met
kinase
To explore the binding modes of the derivatives to c-Met kinase, a
molecular docking method was performed on the 18 compounds
(1a–r). The results revealed that the compounds displayed a bent
“U-shaped” binding mode as inhibitors of type I, as shown
in Fig. 2(a). It indicates that the pyrazolidine-3,5-dione core is
sandwiched into a narrow cleft formed by the P-loop of the N-
lobe and the activation loop of the C-lobe.
Taking compound 1a for example, in the hinge region of c-
Met kinase, the atom N2H and atom O of the 3-carbonyl of
the pyrazolidine-3,5-dione ring H-bond with the backbone CO
and NH of Met1160 respectively. The A ring partially forms a p-
stacking interaction with the phenyl ring of Tyr1159, with the ester
substituent for the R2 group pointed to the solvent. Similarly, the
D ring also partially forms p-stacking interaction with Tyr1230
in the activation loop. Meanwhile, the carboxyl substituent for
the R6 group H-bonds with the backbone CO of Tyr1230 and the
backbone NH of Arg1086 respectively in the P-loop. In addition,
residues Ile1084, Val1092, Ala1108 and Leu1157 of the N-lobe
form a stable hydrophobic environment for the compound, as
Results and discussion
Identification of pyrazolidine-3,5-dione derivatives as c-Met kinase
inhibitors
Compound 1a from our in-house database was identified as an
inhibitor against c-Met kinase, with an IC50 value of 9.5 mM.
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Table 1 Chemical structures and c-Met kinase inhibitory activities of compounds (1a–1r)
Compound
No
R1
R2
R3
R4
R5
R6
Inhibition (%)
IC50 (mM)
9.5
1a
1b*
1c*
1d
1e
H
H
H
H
H
H
H
H
H
CH3
H
H
H
H
COOCH3
COOCH3
COOEt
CH3
CH3
OCH3
H
H
H
H
H
H
H
CH3
OCH3
H
H
CH3
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
Cl
H
H
Cl
Cl
F
F
H
H
Cl
H
H
COOH
COOH
COOH
COOEt
COOCH3
COOH
COOH
COOH
COOH
COOH
Cl
49.3
42.2
63.1
48.1
56.8
23.6
43.2
38.2
67.8
9.2
21.0
35.6
56.2
43.4
6.5
7.8
1f*
1g*
1h*
1i*
1j
NO2
COOCH3
COOEt
H
COOCH3
COOCH3
COOCH3
CH3
10.3
10.4
1k
1l
1m
1n
H
H
H
SO2NH2
H
1o
Cl
CH3
H
H
H
57.6
42.2
9.1
1p*
1q*
1r*
6.8
8.0
Scheme 1 Reagents and conditions: a) ethyl malonyl chloride, Et3N, CH2Cl2; b) NaOH, EtOH; c) AcOH, reflux.
shown in Fig. 2(b). The binding mode analysis was quite in
agreement with the SAR results aforementioned. By virtue of
the key H-bond between the 3-carbonyl of the pyrazolidine-3,5-
dione ring and the hinge region, the inhibitory activities were
totally abolished when the 3-carbonyl was substituted. Since the
substituent R2 is pointed to the solvent and may form a van der
Waal interaction with the backbone of the hinge region, the more
hydrophilic groups such as esters and hydrophobic methyl are
favored for the activities. The carboxyl group and ester group on
R5 or R6 are likely to H-bond with the activation loop and P-loop,
which in turn explains why the H-bond donors or acceptors are
favored here.
As triazolopyridazines were reported to be the potent, selective,
ATP-competitive inhibitors of c-Met kinase,15 the binding modes
were compared with compound 1a. (Fig. 2(c)). The alignment
shows that instead of the quinoline in the hinge region, compound
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a pyrazolidine-3,5-dione ring and a triazolopyridazine would be a
good design to be investigated.
Focused combinatorial library design
Based on the SAR and binding mode analysis for the pyrazolidine-
3,5-dione derivatives, a focused combinatorial library was de-
signed, with structural fragments extracted from the potent
derivatives and reported inhibitors. The LD1.0 program16 was
used during the focused library design. The binding mode
analysis aforementioned indicates that the pyrazolidine-3,5-dione
analogues can be divided into four parts. As shown in Fig. 3(a):
part A is pointed into the solvent (site A), part B H-bonds with
the hinge region (site B), part C is a linker between part A
and B, probably forming p-stacking interaction with Tyr1230 if
the length and orientation are reasonable, and the last part D
is probably an aromatic ring with substituents H-bonding with
the activation loop and P-loop. The LD1.0 program optimized
9 fragments for part A, 2 fragments for part B, 5 fragments
for linker part C and 8 fragments for part D. The chemical
structures of the fragments are shown in Fig. 3(b). Amongst them,
fragments A1–A4 were designed with ester and methyl substituents
on the benzyl ring aforementioned. As the hydrophilic substituents
pointed into the solvent this probably make the compounds in the
correct orientation. Fragments A5–A9 were designed based on
the reported inhibitors, which proved to be favored for potent
activity. Fragments B1–B2 were designed with the atom N2H
and 3-carbonyl in the ring retained, which form H-bonds with
Fig. 2 Binding mode analysis of compound 1a with c-Met kinase. (a)
c-Met kinase is shown in cartoon, with the N-lobe shown in pink, while the
C-lobe shown in purple. Compound 1a occupies the ATP pocket between
the two lobes and is shown in green sticks. (b) Detailed interactions of
compound 1a with surrounding key residues were analyzed. Compound 1a
is shown in green sticks, while surrounding key residues are shown in gray
sticks. (c) Compound 1a and triazolopyridazine molecule (in yellow sticks)
were compared in the ATP pocket. The kinase is shown in electrostatic
surface (the color blue indicates the positive-charge, while red indicates
negative-charged).
1a possesses the pyrazolidine-3,5-dione ring forming the key H-
bonds. Intrigued by the triazolopyridazine forming a stable p-
stacking interaction with Tyr1230, a hybrid structure containing
Fig. 3 (a) The pyrazolidine-3,5-dione scaffold was divided into four parts (A-B-C-D). (b) Building blocks for focused library generation. Fragments for
parts A, B, C and D. The symbol * represents the site where fragments connect to each other to form a complete structure.
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Table 2 Chemical structures and c-Met kinase inhibitory activities of 7 compounds selected from the focused library
Compound
No
R1
R2
R3
R4
R5
R6
Inhibition (%)
IC50 (mM)
5a*
5b
H
CH3
H
CH3
H
H
COOCH3
CH3
COOCH3
CH3
COOCH3
COOEt
H
OH
H
H
OCH3
H
H
H
H
H
H
H
F
H
COOEt
H
H
F
COOH
NO2
H
COOCH3
COOH
COOH
49.2
92.1
74.2
48.2
75.2
81.8
48.6
0.46
0.91
5c
5d
5e*
5f*
5g*
6.9
7.5
the hinge region of the kinase. Different aromatic rings were
designed for the linker part C, especially for the fragments C5
which was extracted from the X-ray structure. As for part D,
different H-bonds donors and acceptors were tried on the benzyl
ring, which were expected to H-bond with the activation loop and
P-loop.
Then LD1.0 connected these fragments together according
to sort A-B-C-D, producing a library containing 720 (9 ¥ 2 ¥
5 ¥ 8) molecules. All molecules in the designed library were
first filtered by the drug-likeness of LD1.0, and then ranked
by the scoring function of DOCK4.0,17 AutoDock3.0.518,19 As
AutoDock3.0.5 has a better ability to optimize binding poses
as well as predicting binding energy. Besides the binding energy
prediction by the score functions aforementioned, the ADMET
properties and the favorable shape complementarity and poten-
tial in forming H-bonds with c-Met kinase were also analy-
zed.
The binding mode analysis aforementioned indicated that part
A was pointed to the solvent flanked by the side-chain of residue
Tyr1159 and residue Lys1161 (site A). Among these fragments,
the morpholine group, which appears to serve as a solubilizing
group and forms no ligand–protein interactions, was extracted
from the reported inhibitor AM7 (PDB ID code: 2RFN).20 Part
B resides in the adenine site surrounded by residues Ile1084,
Val1092, Ala1108 and Met1160 (site B), and the backbone of
Met1160 anticipates the key H-bonds with compounds. The linker
part C resides in the site C lined by residues Leu1140, Leu1157,
Met1211, Ala1221, Asp1222, Ala1226 and Tyr1230. Considering
the hydrophobic characteristic and potential for forming H-bond
and p-stacking interactions, fragment C5 has an advantage. While,
part D probably H-bonds with the residues Arg1086 in the P-
loop, Asp1164, Asn1167, Arg1208, Asn1209, and Tyr1230 and
Asp1231 in the activation loop. Consequently, the carboxyl, ester,
hydroxyl and nitro substituents were favored on the benzyl ring.
Unfortunately, related target compounds with morpholine and
pyridine in site A and triazolopyridazine in site C have not been
afforded due difficulties synthesising the intermediate. Finally, 7
compounds (5a–g) were successfully synthesized for biological
testing as shown in Table 2.
Structure–activity relationship (SAR) analysis
In vitro assay indicates that, seven compounds (5a–5g) in Table 2
showed good c-Met kinase inhibitory activity and the activity
of the most potent one 5b (0.46 mM) increased 20 fold. The
results illustrated that the 3,4-dimethylbenzene group (A3) is the
most optimized fragment for part A; a furan group (C1) is the
optimized fragment compared with a benzene group (C2) for
part C; Fragments for part D are structurally more diverse than
fragments for part C and a 2-hydroxy-4-nitrophenyl group (D3)
was indicated to be the most favorable. Nevertheless, the fragment
triazolopyridazine predicted to be favored was not added to the
pyrazolidine-3,5-dione ring by virtue of the unstable intermediates
in synthesis. Accordingly, the building blocks for parts B and C
are fixed at fragments B1 and C1 and the fragments for parts A
an D are a little bit flexible. The reason underlying the structure–
activity relationship can be addressed from the binding modes of
c-Met kinase with the compounds, which displayed a bent “U-
shaped” binding mode in the ATP site. The R2 group of phenyl
ring A was directed toward a solvent-exposed area, which provides
an opportunity for the introduction of polar, water-solubilizing
substituents. Meanwhile, hydrophobic substituents may also be
favored through van der Waals interactions with the backbone
of Lys1161 His1162 and Gly1163. The most potent compound
5b incorporating methyl groups in ring A supported this view.
Part B resides in the hinge region and the key H-bond interaction
is formed between 3-carbonyl of pyrazolidine-3,5-dione ring and
Met1160. The inhibitory activities were abolished when the 3-
carbonyl was substituted. Compounds with H-bond donor or
acceptor groups (carboxyl-group, ester-group, hydroxyl group) on
R5 and R6 possess potent inhibitory activity due to forming an H-
bond interaction with Arg1086 in the P-loop, Asp1164, Arg1208,
and Tyr1230 and Asp1231 in the activation loop. Introduction of
other groups here actually decreased the inhibitory activity.
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removed at first and the polar hydrogen atoms were added. The
potential of the enzyme was assigned with Kollman united charge
encoded in SYBYL software package. The molecular geometry
of compounds was also modeled using the SYBYL assigned with
Gasteiger–Hu¨ckel charge. Some of the torsion angles were defined,
allowing the conformation search for compounds during the
docking process. The Lamarckian genetic algorithm (LGA) was
applied to deal with the protein–inhibitor interactions. A Solis and
Wets local search was performed for the energy minimization on a
user-specified proportion of the population. The docked structures
of compounds were generated after a reasonable number of
evaluations. The docking simulation protocol is listed as follows:
First, a PDBQ file of the kinase was created. Atomic salvation
parameters and fragmental volumes were assigned to the enzyme
using the ADDSOL module. Second, the grid map with 80 ¥
Conclusion
The discovery of c-Met kinase inhibitors is now of special interest
in the treatment of cancer. In the present study, a series of
pyrazolidine-3,5-dione derivatives were designed and synthesized
based on the hit compound 1a. All the compounds were evaluated
for their ability to inhibit enzymatic activity of c-Met kinase by
in vitro assay. Through substructure similarity search against the
SPECS database and chemical synthesis methods, 4 compounds
(1c, 1e, 1m and 1o) tested by the enzyme-based assay against c-
Met kinase showed potential inhibitory activity, with IC50 values
mostly less than 10 mM. Based on the rich information of
structure–activity relationship (SAR) and binding mode analysis,
a focused combinatorial library was designed employing the
LD1.0 program. Finally, seven compounds (5a–g) were proved
to be potent inhibitors through in vitro assay. The activity of
the most potent compounds 5b (IC50 = 0.46 mM) was more
than 20 times higher than that of the lead 1a. Although, the
theoretically favored fragments, such as morpholine, pyridine
and triazolopyridazine, were not incorporated in our compounds,
the pyrazolidine-3,5-dione is no doubt an optimized scaffold as
inhibitor against c-Met kinase. Taking into account the rigid
characteristic, further optimization of introducing alkyl with
heteroatoms between the four parts and searching for other
favored rings for the A and C parts, are still under way. Overall,
the potent compounds discovered in this study demonstrated the
feasibility for pyrazolidine-3,5-dione derivatives as c-Met kinase
inhibitors and the efficiency of the strategy for the inhibitor design.
˚
80 ¥ 80 points and a spacing of 0.375 A were calculated using
the AutoGrid program to evaluate the binding energies between
the inhibitors and the enzyme. The grid center was set at the
active site position (43.566 36.747 37.807) and the affinity and
electrostatic potential grids were calculated for each type of atom
in the inhibitors. The energetic configuration of a particular
inhibitor was found by tri-linear interpolation of affinity values
and electrostatic interaction of the eight grid points around each
atom of the inhibitor. Third, some important parameters for LGA
calculations were reasonably setup according to requirements of
the Amber force field and our problem. Thirty docking simulations
were performed for each compound and conformations of multiple
runs were then clustered with the root-mean-square deviation
˚
(RMSD) tolerance of 1.0 A. Finally, the proper conformation
and position compared with the complex structure was extracted
from the optimized complex.
Experimental
Computational details
Molecular docking by DOCK4.0. To explore the detailed
interactions of pyrazolidine-3,5-dione derivatives with c-Met
kinase, the docking programs DOCK4.017 and AutoDock3.0.518,19
were employed. We used DOCK4.0 as the primary screening tool
targeting at the active site of the X-ray crystal structure of c-Met
kinase (PDB entry: 3CD8)15 complexed with triazolopyridazine.
Chemistry
The reagents (chemicals) were purchased from Lancaster, Acros,
and Shanghai Chemical Reagent Co. and used without further
purification. Analytical thin-layer chromatography (TLC) was
HSGF 254 (150–200 mm thickness; Yantai Huiyou Co., China).
Nuclear magnetic resonance (NMR) spectroscopy was performed
on a Bruker AMX-400 and AMX-300 NMR (IS as TMS).
Chemical shifts were reported in parts per million (ppm, d)
downfield from tetramethylsilane. Proton coupling patterns were
described as singlet (s), doublet (d), triplet (t), quartet (q), multiplet
(m), and broad (br). Low- and high-resolution mass spectra
(LRMS and HRMS) were given with electric, electrospray, and
matrix-assisted laser desorption ionization (EI, ESI, and MALDI)
produced by a Finnigan MAT-95, LCQ-DECA spectrometer and
IonSpec 4.7 T. The purity of final compounds was assessed by the
analytical HPLC method and found to be >95%. An Agilent 1100
series HPLC with an Agilent Zorbax Exlipse SB-C18 (25–4.6 mm,
5 mm particle sizes) reversed-phase column was used for analytical
HPLC analyses. The elution buffer was an A/B gradient, where
A = H2O and B = CH3OH.
˚
Residues around the inhibitor at a radius of 6 A were isolated for
the construction of the grid for docking screening. This radius was
large enough to include all of the residues involved in compound
binding. During the docking procedure, Kollman all-atom charges
were assigned to the protein, while Geisterger–Hu¨ckel charges
were assigned to the compounds. In the docking search, the
conformational flexibilities of the compounds were considered. We
used 50 configurations per ligand-binding cycle and 60 maximum
anchor orientations in the anchor-first docking algorithm. All
docked configurations were energy minimized by the use of 100
maximum iterations and 1 minimization cycle. Prior to screening
the compounds in the designed library, we tested this docking
protocol by screening a small molecule database that contained
1000 compounds that consisted of 24 known inhibitors15,20–24 and
976 decoy molecules. The result that inhibitors mostly reside at the
top-scoring rank proved the feasibility of the protocol. The same
test method was performed on the protocol of AutoDock3.03.
Methyl 4-(3,5-dioxopyrazolidin-1-yl)benzoate 4a. 4-Hydr-
azino-methylbenzoate hydrochloride (9.2 mmol) was stirred for
15 min at room temperature in a mixture of 10% aqueous Na2CO3
solution (20 mL) and CH2Cl2(40 mL). The separated aqueous
Molecular docking by AutoDock3.0.5. The X-ray crystal struc-
ture of c-Met kinase (PDB entry: 3CD815) was also used as
the receptor model by the AutoDock program. The ligand was
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solution was extracted with CH2Cl2. The combined organic
phases were dried over Na2SO4, filtered and concentrated. The
residue was dissolved in anhydrous THF (50 mL) and Et3N (10.1
mmol) was added. The reaction mixture was cooled to -10 ◦C
and a solution of ethyl malonyl chloride (9.4 mmol) in anhydrous
THF (25 mL) was added dropwise. The reaction mixture was
allowed to warm to room temperature and stirred overnight. The
reaction mixture was diluted with H2O (100 mL) and extracted
with EtOAc. The combined organic phases were washed with
H2O, brine, dried over Na2SO4, filtered and concentrated to yield
crude compound as a brown residue. The crude compound was
dissolved in EtOH (100 mL) and ethanolic 1 N NaOH solution
(18.3 mmol) was added. The reaction mixture was stirred for
30 min at room temperature. The reaction mixture was then
acidified by addition of aqueous 1 N HCl. The precipitate formed
was collected by filtration, washed with water, and dried on the
sintered glass to yield 4a as a white solid. Yield: 70.2%. 1H NMR
(300 Hz, DMSO-d6): d 8.01–7.89 (m, 2H), 7.83–7.79 (m, 2H),
3.83 (s, 3H), 3.67 (s, 2H).
7.62 (t, J = 3.0 Hz, 1H), 3.86 (s, 3H). EI-MS m/z 466 [M+]. HRMS
(EI) m/z calcd for C23H15ClN2O7 [M+] 466.0568, found 466.0589.
3-(5-((1-(4-(Methoxycarbonyl)phenyl)-3,5-dioxopyrazolidin-4-
ylidene)methyl)furan-2-yl)benzoic acid 1b. General procedures for
the synthesis of target compounds. The intermediate 4a (1 mmol)
and 3-(5-formylfuran-2-yl)benzoic acid (1 mmol) were dissolved
in glacial acetic acid (5 mL). The reaction mixture was refluxed
for 3 h. The precipitate formed was collected by filtration, washed
with water, and dried on the sintered glass to yield 1b as a yellow
solid. Yield: 78.6%. Mp 232–234 ◦C. HPLC: 100.00%, tR = 1.562
1
min. H NMR (DMSO-d6) d: 8.42–8.63 (m, 1H), 8.18–8.26 (m,
1H), 7.91–8.08 (m, 6H), 7.64–7.74 (m, 2H), 7.54–7.61 (m, 1H),
3.86 (s, 3H). EI-MS m/z 432 [M+]. HRMS (EI) m/z calcd for
16C23H16N2O7 [M+] 432.0958, found 432.0962.
3-(5-((1-(4-(Ethoxycarbonyl)phenyl)-3,5-dioxopyrazolidin-4-yl-
idene)methyl)furan-2-yl)benzoic acid 1c. In the same manner as
described in the preparation of 1b, 1c was prepared from 4b and 3-
◦
(5-formylfuran-2-yl)benzoic acid. Yield: 72.1%. Mp 241–246 C.
Ethyl 4-(3,5-dioxopyrazolidin-1-yl)benzoate 4b. As described
in the preparation of 4a, 4b was prepared from 4-hydrazino-
ethylbenzoate hydrochloride in the same manner. Yield: 69.3%.
1H NMR (300 Hz, DMSO-d6): d 8.26 (m, 1H), 7.49 (m, 1H), 7.74
(m, 1H), 7.56 (m, 1H), 4.36–4.31 (m, 2H), 3.87 (s, 2H), 1.36–1.23
(m, 3H).
HPLC: 98.22%, tR = 1.125 min. 1H NMR (DMSO-d6) d: 8.42–8.62
(m, 1H), 8.19–8.28 (m, 1H), 7.90–8.08 (m, 6H), 7.56–7.74 (m, 3H),
4.33 (q, J = 7.1 Hz, 2H), 1.34 (t, J = 7.1 Hz, 3H). EI-MS m/z 446
[M+]. HRMS (EI) m/z calcd for C24H18N2O7 [M+] 446.1114, found
446.1120.
Ethyl 2-chloro-5-(5-((3,5-dioxo-1-p-tolylpyrazolidin-4-ylidene)-
methyl)furan-2-yl)benzoate 1d. Mp 212–216 ◦C. HPLC: 96.12%,
tR = 6.235 min. 1H NMR (DMSO-d6) d: 8.38–8.53 (m, 1H), 8.31–
8.37 (m, 1H), 8.14 (d, J = 8.5 Hz, 1H), 7.76 (d, J = 8.5 Hz, 1H),
7.54–7.72 (m, 4H), 7.26 (d, J = 8.1 Hz, 2H), 4.40 (q, J = 7.1 Hz,
2H), 2.26–2.37 (m, 3H), 1.38 (t, 3H). EI-MS m/z 450 [M+]. HRMS
(EI) m/z calcd for C24H19ClN2O5 [M+] 450.0982, found 450.0986.
1-(4-Methoxyphenyl)pyrazolidine-3,5-dione 4c. In the same
manner as described in the preparation of 4a, 4c was prepared
from (4-methoxyphenyl)hydrazine hydrochloride. Yield: 73.5%.1H
NMR (300 Hz, DMSO-d6): d 8.02–7.98 (m, 2H), 7.83–7.79 (m,
2H), 3.83 (s, 3H), 3.67 (s, 2H).
1-(4-Nitrophenyl)pyrazolidine-3,5-dione 4d. In the same man-
ner as described in the preparation of 4a, 4d was prepared from (4-
nitrophenyl)hydrazine hydrochloride. Yield: 60.6%.1H NMR (300
Hz, DMSO-d6): d 8.86–8.74 (m, 2H), 8.25–8.13 (m, 2H), 3.72(s,
2H).
Methyl
2-chloro-5-(5-((3,5-dioxo-1-p-tolylpyrazolidin-4-yli-
dene)methyl)furan-2-yl)benzoate 1e. Mp 228–231 ◦C. HPLC:
97.55%, tR = 6.336 min. 1H NMR (DMSO-d6) d: 8.49 (d, J = 3.5
Hz, 1H), 8.36 (br s, 1H), 8.06–8.19 (m, 1H), 7.76 (d, J = 8.5 Hz,
1H), 7.54–7.70 (m, 4H), 7.26 (d, J = 8.5 Hz, 2H), 3.93 (s, 3H),
2.32 (s, 3H). EI-MS m/z 436 [M+]. HRMS (EI) m/z calcd for
C23H17ClN2O5 [M+] 436.0826, found 436.0829.
Ethyl 4-(3-ethoxy-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzoate
4e. To
a
solution of methyl 4-(3,5-dioxopyrazolidin-1-
yl)benzoate (4a, 1 mmol) in ethanol was added concentrated
H2SO4 drop wise. The resulting mixture was refluxed overnight.
Solvent was evaporated. NaHCO3 solution was added to the
residue and the solution was extracted with EtOAc. The organic
layer was dried over Na2SO4, filtrated and concentrated to give the
2-Fluoro-5-(5-((1-(4-methoxyphenyl)-3,5-dioxopyrazolidin-4-yli-
dene)methyl)furan-2-yl)benzoic acid 1f. In the same manner as
described in the preparation of 1b, 1f was prepared from 4c and 2-
fluoro-5-(5-formylfuran-2-yl)benzoic acid. Yield: 79.6%. Mp 221–
1
desired compound 4e as a yellow solid. Yield: 92.3%. H NMR
225 ◦C. HPLC: 100.00%, tR = 1.987 min. H NMR (300 MHz,
1
(300 Hz, DMSO-d6): d 8.02–7.99 (m, 2H), 7.94–7.92 (m, 2H),
4.37–4.30 (m, 2H), 4.26–4.21(m, 2H), 3.74 (s, 2H), 1.36–1.23 (m,
6H).
DMSO-d6) d: 7.65–7.74 (m, 3H), 7.44–7.59 (m, 3H), 7.34–7.43
(m, 2H), 6.82–7.01 (m, 2H), 3.98 (s, 3H). EI-MS m/z 422 [M+].
HRMS (EI) m/z calcd for C22H15FN2O6 [M+] 422.0914, found
422.0926.
Methyl 4-(3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzoa-
te 4f. In the same manner as described in the preparation of 4a,
4f was prepared from 3-oxobutanoic acid. Yield: 90.1%.1H NMR
(300 Hz, DMSO-d6): d 8.02–7.99 (m, 2H), 7.94–7.92 (m, 2H),
3.84(s, 3H), 3.72 (s, 2H), 2.12 (s, 3H).
2 - Fluoro - 5 - (5 - ((1 - (4 - nitrophenyl) - 3,5 - dioxopyrazolidin - 4-
ylidene)methyl)furan-2-yl)benzoic acid 1g. In the same manner as
described in the preparation of 1b, 1g was prepared from 4d and 2-
fluoro-5-(5-formylfuran-2-yl)benzoic acid. Yield: 76.3%. Mp 198–
◦
1
2-Chloro-5-(5-((1-(4-(methoxycarbonyl)phenyl)-3,5-dioxopyra-
201 C. HPLC: 100.00%, tR = 1.235 min. H NMR (DMSO-d6)
d: 8.24–8.51 (m, 4H), 8.01–8.21 (m, 3H), 7.65 (s, 1H), 7.38–
7.53 (m, 2H). EI-MS m/z 437 [M+]. HRMS (EI) m/z calcd for
C21H12FN3O7 [M+] 437.0659, found 437.0664.
zolidin-4-ylidene)methyl)furan-2-yl)benzoic acid 1a. Mp 237–
◦
1
239 C. HPLC: 100%, tR = 1.428 min. H NMR (DMSO-d6) d:
8.28–8.61 (m, 2H), 7.85–8.17 (m, 5H), 7.73 (d, J = 8.5 Hz, 2H),
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3-(5-((1-(4-(Methoxycarbonyl)phenyl)-3,5-dioxopyrazolidin-4-
ylidene)methyl)furan-2-yl)-4-methylbenzoic acid 1h. In the same
manner as described in the preparation of 1b, 1h was prepared
from 4a and 3-(5-fo◦rmylfuran-2-yl)-4-methylbenzoic acid. Yield:
74.4%. Mp 221–224 C. HPLC: 100.00%, tR = 1.637 min. 1H NMR
(DMSO-d6) d: 8.57 (d, J = 3.2 Hz, 1H), 7.89–8.10 (m, 4H), 7.85
(t, J = 6.6 Hz, 1H), 7.73 (d, J = 7.4 Hz, 1H), 7.55–7.64 (m, 1H),
7.36–7.46 (m, 1H), 7.24–7.33 (m, 1H), 3.87 (s, 3H), 2.61 (s, 3H).
EI-MS m/z 446 [M+]. HRMS (EI) m/z calcd for C24H18N2O7 [M+]
446.1114, found 446.1121.
4-(5-((1-(3-Chloro-4-methylphenyl)-3,5-dioxopyrazolidin-4-yli-
dene)methyl)furan-2-yl)-N-(thiazol-2-yl)benzenesulfonamide 1o.
Mp:240–241 ◦C. HPLC: 98.22%, tR = 1.412 min. 1H NMR
(DMSO-d6): d 8.55–8.54 (m, 1H), 8.14–8.10 (m, 2H), 7.94–7.91
(m, 2H), 7.69–7.56 (m, 3H), 7.31–7.25 (m, 3H), 6.89–6.88 (m,
1H), 2.32 (s, 3H). EI-MS m/z 540 [M+]. HRMS (EI) m/z calcd
for C24H17S2N4O5Cl [M+] 540.0329 found 540.0335.
3¢-((1-(4-(Ethoxycarbonyl)phenyl)-3,5-dioxopyrazolidin-4-yli-
dene)methyl)biphenyl-3-carboxylic acid 1p. In the same manner
as described in the preparation of 1b, 1p was prepared from 4b
and 3¢-formyl-[1,1¢-biphenyl]-3-carboxylic acid. Yield: 77.3%. Mp
203–206 ◦C. HPLC: 100.00%, tR = 1.725 min. 1H NMR (DMSO-
d6) d: 8.33 (d, J = 1.8 Hz, 1H), 8.20–8.42 (m, 1H), 7.85–8.17 (m,
7H), 7.55–7.86 (m, 4H), 7.28–7.51 (m, 1H), 4.29–4.33 (m, 2H),
1.30–1.34 (m, 3H). EI-MS m/z 456[M+]. HRMS (EI) m/z calcd
for C26H20N2O6 [M+] 456.1321, found 456.1327.
3-(5-((1-(4-(Ethoxycarbonyl)phenyl)-3,5-dioxopyrazolidin-4-
ylidene)methyl)furan-2-yl)-4-methoxybenzoic acid 1i. In the same
manner as described in the preparation of 1b, 1i was prepared
from 4b and 3-(5-formylfuran-2-yl)-4-methoxybenzoic acid. Yield:
78.2%. Mp 205–209 ◦C. HPLC: 97.46%, tR = 1.542 min. 1H NMR
(DMSO-d6) d: 8.62–8.74 (m, 1H), 8.39–8.58 (m, 1H), 7.96–8.08
(m, 4H), 7.92 (d, J = 8.9 Hz, 1H), 7.75 (d, J = 3.9 Hz, 1H), 7.38–
7.47 (m, 1H), 7.33 (d, J = 8.9 Hz, 1H), 4.32 (d, J = 7.1 Hz, 2H),
4.07 (s, 3H), 1.34 (t, 3H). EI-MS m/z 476 [M+]. HRMS (EI) m/z
calcd for C25H20N2O8 [M+] 476.1220, found 476.1215.
3¢-((3-Ethoxy-1-(4-(ethoxycarbonyl)phenyl)-5-oxo-1H-pyrazol-
4(5H)-ylidene)methyl)biphenyl-3-carboxylic acid 1q. In the same
manner as described in the preparation of 1b, 1q was prepared
from 4e and 3¢-formyl-[1,1¢-biphenyl]-3-carboxylic acid. Yield:
62.5%. Mp 211–214 ◦C. HPLC: 97.36%, tR = 1.685 min. 1H NMR
(DMSO-d6) d: 8.58–8.68 (m, 1H), 8.32 (d, J = 12.0 Hz, 1H), 7.89–
8.17 (m, 9H), 7.59–7.71 (m, 2H), 4.45–4.59 (m, 2H), 4.29–4.37 (m,
2H), 1.35 (d, J = 7.4 Hz, 6H). EI-MS m/z 484 [M+]. HRMS (EI)
m/z calcd for C28H24N2O6 [M+] 484.1634 found 484.1639.
2-Chloro-5-(5-((3,5-dioxo-1-(m-tolyl)pyrazolidin-4-ylidene)-
methyl)furan-2-yl)benzoic acid 1j. Mp 194-197 ◦C. HPLC: 100%,
tR = 1.723 min.1H NMR (DMSO-d6) d: 8.53 (d, J = 2.7 Hz, 1H),
8.29–8.36 (m, 1H), 8.05–8.12 (m, 1H), 7.67–7.79 (m, 2H), 7.46–
7.64 (m, 3H), 7.33 (t, J = 7.8 Hz, 1H), 7.02 (d, J = 7.1 Hz, 1H),
2.35 (s, 3H). EI-MS m/z 422 [M+]. HRMS (EI) m/z calcd for
C22H15ClN2O5 [M+] 422.0669, found 422.0652.
3-(5-((1-(4-(Methoxycarbonyl)phenyl)-3-methyl-5-oxo-1H-pyr-
azol-4(5H)-ylidene)methyl)furan-2-yl)benzoic acid 1r. In the
same manner as described in the preparation of 1b, 1r was prepared
from 4f a◦nd 3-(5-formylfuran-2-yl)benzoic acid. Yield: 68.6%. Mp
221–225 C. HPLC: 100.00%, tR = 1.632 min. 1H NMR (DMSO-
d6): d 8.66–8.65 (m, 1H), 8.27 (s, 1H), 8.16–8.03 (m, 6H), 7.83 (s,
1H), 7.70–7.58 (m, 3H), 2.37 (s, 3H). EI-MS m/z 430 [M+]. HRMS
(EI) m/z calcd for C24H18N2O6 [M+] 430.1165 found 430.1171.
Methyl 4-(4-((5-(3-chlorophenyl)furan-2-yl)methylene)-3,5-di-
oxopyrazolidin-1-yl)benzoate 1k. Mp 196–199 ◦C. HPLC: 100%,
1
tR = 5.359 min. H NMR (DMSO-d6) d: 8.28 (m, 1H), 8.13 (d,
J = 4.6 Hz, 1H), 7.62–7.92 (m, 5H), 7.50 (d, J = 8.5 Hz, 2H),
7.35–7.43 (m, 1H). EI-MS m/z 422 HRMS (EI) m/z calcd for
C22H15ClN2O5 [M+] 422.0669, found 422.0675.
2 - Fluoro - 5 - (5 - ((1 - (4 - (methoxycarbonyl)phenyl) - 3,5 - dioxo-
pyrazolidin-4-ylidene)methyl)furan-2-yl)benzoic acid 5a. In the
same manner as described in the preparation of 1b, 5a was pre-
pared from 4a and 2-fluoro-5-(5-formylfuran-2-yl)benzoic acid.
Methyl
4-(4-((5-(3-chloro-2-methylphenyl)furan-2-yl)methy-
lene)-3,5-dioxopyrazolidin-1-yl)benzoate 1l. Mp 183–187 ◦C.
HPLC: 95.26%, tR = 5.369 min. 1H NMR (DMSO-d6) d: 8.53 (d,
J = 2.8 Hz, 1H), 8.01–8.13 (m, 3H), 7.84–8.01 (m, 3H), 7.67–7.75
(m, 1H), 7.54 (d, J = 7.4 Hz, 2H), 3.86 (s, 3H), 2.41 (s, 3H).
EI-MS m/z 436 [M+]. HRMS (EI) m/z calcd for C23H17ClN2O5
[M+] 436.0826, found 436.0833.
◦
Yield: 73.9%. Mp 209–212 C. HPLC: 100.00%, tR = 1.874 min.
1H NMR (DMSO-d6) d: 8.50–8.63 (m, 1H), 8.42 (d, J = 6.7 Hz,
1H), 8.16–8.26 (m, 1H), 7.99–8.07 (m, 3H), 7.88–7.99 (m, 1H), 7.71
(d, J = 2.8 Hz, 1H), 7.41–7.58 (m, 2H), 3.86 (s, 3H). EI-MS m/z
450[M+]. HRMS (EI) m/z calcd for C23H15FN2O7 [M+] 450.0863,
found 450.0871.
Methyl 4-(3,5-dioxo-4-((5-(4-sulfamoylphenyl)furan-2-yl)methy-
lene)pyrazolidin-1-yl)benzoate 1m. Mp 189–192 ◦C. HPLC:
100.00%, tR = 1.023 min.1H NMR (DMSO-d6) d: 8.55 (d, J =
3.5 Hz, 1H), 8.18 (d, J = 8.1 Hz, 2H), 8.01–8.09 (m, 2H), 7.96 (dd,
J = 8.5, 1.8 Hz, 4H), 7.72 (d, J = 11.0 Hz, 1H), 7.62 (t, J = 4.1 Hz,
1H), 7.52 (s, 2H), 3.86 (s, 3H). EI-MS m/z 467 [M+]. HRMS (EI)
m/z calcd for C22H17SN2O7 [M+] 467.0787 found 467.0780.
1-(3,4-Dimethylphenyl)-4-((5-(5-hydroxy-2-nitrophenyl)furan-2-
yl)methylene)pyrazolidine-3,5-dione 5b. Mp:274-278 ◦C. HPLC:
1
95.13%, tR = 1.123 min. H NMR (DMSO-d6): d 8.91–8.87 (m,
1H), 8.264 (s, 1H), 8.22–8.19 (m, 1H), 7.77–7.45 (m, 4H), 7.22–7.19
(m, 2H), 2.28 (s, 3H), 2.24 (s, 3H).; EI-MS m/z 419 [M+]. HRMS
(EI) m/z calcd for C22H17N3O6 [M+] 419.1117 found 419.1125.
4-(5-((3,5-Dioxo-1-p-tolylpyrazolidin-4-ylidene)methyl)furan-2-
yl)-N-(thiazol-2-yl)benzenesulfonamide 1n. Mp 212–216 ◦C.
HPLC: 95.29%, tR = 1.365 min. H NMR (DMSO-d6) d: 12.88
(br s, 3H), 8.54 (d, J = 3.2 Hz, 1H), 8.12 (dd, J = 8.3, 3.4 Hz, 3H),
7.93 (d, J = 8.1 Hz, 3H), 7.52–7.73 (m, 4H), 7.21–7.34 (m, 3H),
6.88 (d, 1H), 2.32 (s, 3H). EI-MS m/z 506 [M+]. HRMS (EI) m/z
calcd for C24H18S2N4O5 [M+] 506.0719 found 506.0725.
Ethyl
4-(5-((1-(4-(methoxycarbonyl)phenyl)-3,5-dioxopyra-
zolidin-4-ylidene)methyl)furan-2-yl)benzoate 5c. Mp 195–197
◦C. HPLC:95.96%, tR = 5.892.1H NMR (DMSO-d6) d: 8.53 (d,
J = 3.5 Hz, 1H), 7.99–8.17 (m, 5H), 7.79–8.00 (m, 3H), 7.69 (d,
J = 9.9 Hz, 1H), 7.62 (t, J = 3.7 Hz, 1H), 4.35 (q, J = 7.1 Hz, 2H),
3.86 (s, 3H), 1.35 (t, J = 7.1 Hz, 3H). EI-MS m/z 460 [M+]. HRMS
(EI) m/z calcd for C25H20N2O7 [M+] 460.1271 found 460.1278.
1
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Methyl
3-(5-((1-(3,4-dimethylphenyl)-3,5-dioxopyrazolidin-4-
0.03% H2O2 and 2 mg ml-1 o-phenylenediamine in 0.1 M citrate
buffer, pH 5.5, was added and samples were incubated at room
temperature until color emerged. The reaction was terminated
by the addition of 50 ml of 2 M H2SO4, and the plate was read
using a multiwell spectrophotometer (VERSAmaxTM, Molecular
Devices, Sunnyvale, CA,USA) at 490 nm. The inhibition rate (%)
was calculated using the following equation: [1 - (A490/A490
control)] ¥ 100%. IC50 values were calculated from the inhibition
curves.
◦
ylidene)methyl)furan-2-yl)benzoate 5d. Mp 204–207 C. HPLC:
96.23%, tR = 5.356 min. H NMR (DMSO-d6) d: 8.42–8.59 (m,
2H), 8.25 (br. s., 1H), 8.03 (d, J = 7.8 Hz, 1H), 7.66–7.76 (m, 2H),
7.41–7.64 (m, 3H), 7.21 (d, J = 8.1 Hz, 1H), 3.93 (s, 3H), 2.27 (s,
3H), 2.23 (s, 3H). EI-MS m/z 416 [M+]. HRMS (EI) m/z calcd
for C24H20N2O5 [M+] 416.1372 found 416.1378.
1
4-Methoxy-3-(5-((1-(4-(methoxycarbonyl)phenyl)-3,5-dioxo-
pyrazolidin-4-ylidene)methyl)furan-2-yl)benzoic acid 5e. In the
same manner as described in the preparation of 1b, 5e was
prepared from 4a and 3-(5-form◦ylfuran-2-yl)-4-methoxybenzoic
acid. Yield: 78.8%. Mp 216–219 C. HPLC: 100.00%, tR = 1.215
Acknowledgements
1
min. H NMR (DMSO-d6) d: 8.66 (d, J = 8.5 Hz, 1H), 8.43–
We gratefully acknowledge the financial support from the National
Natural Science Foundation of China (20972174, 81025017,
30725046, 91029704 and 21021063), the State Key Program
of Basic Research of China grant (2009CB918502), Shanghai
Committee of Science and Technology (10410703900), the Chinese
Academy of Sciences (XDA01040305), Guangdong S&T Dept.
(2010A030100006) and Program of Shanghai Subject Chief Sci-
entist (10XD1405100).
8.57 (m, 1H), 8.03 (s, 4H), 7.90–7.99 (m, 1H), 7.72 (s, 1H),
7.29–7.42 (m, 2H), 4.07 (s, 3H), 3.87 (s, 3H). EI-MS m/z 462
[M+]. HRMS (EI) m/z calcd for C24H18N2O8 [M+] 462.1063 found
462.1071.
5-(5-((1-(4-(Ethoxycarbonyl)phenyl)-3,5-dioxopyrazolidin-4-yl-
idene)methyl)furan-2-yl)-2-fluorobenzoic acid 5f. In the same
manner as described in the preparation of 1b, 5f was prepared
from 4b and 2-fluoro-5-(5-formylfuran-2-yl)benzoic acid. Yield:
62.8%. Mp 212–214 ◦C. HPLC: 100.00%, tR = 1.921 min.1H NMR
(DMSO-d6) d: 8.25–8.55 (m, 2H), 7.93–8.11 (m, 4H), 7.85–7.92
(m, 1H), 7.69 (d, J = 7.3 Hz, 2H), 7.52–7.61 (m, 1H), 4.30 (q,
J = 6.8 Hz, 2H), 1.32 (t, J = 7.1 Hz, 3H). EI-MS m/z 464 [M+].
HRMS (EI) m/z calcd for C24H17FN2O7 [M+] 464.1020 found
464.1027.
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3¢-((1-(4-(Methoxycarbonyl)phenyl)-3,5-dioxopyrazolidin-4-yl-
idene)methyl)biphenyl-3-carboxylic acid 5g. In the same manner
as described in the preparation of 1b, 5g was prepared from 4a
and 3¢-formyl-[1,1¢-biphenyl]-3-carboxylic acid. Yield: 65.3%. Mp:
◦
1
214–215 C. HPLC: 99.15%, tR = 1.326 min. H NMR (DMSO-
d6) d: 8.87–9.03 (m, 1H), 8.43–8.62 (m, 1H), 8.32 (s, 1H), 7.86–
8.12 (m, 8H), 7.60–7.73 (m, 2H), 3.85 (m, 3H); EI-MS m/z 442
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ELISA kinase assay
The tyrosine kinase activities were evaluated according to the re-
ported protocol.25 Briefly, in enzyme-linked-immunosorbent assay
(ELISA), 20 mg ml-1 Poly(Glu,Tyr)4 : 1 (Sigma) was pre-coated as
a substrate in 96-well plates. 50 ml of 10 mM ATP solution diluted
in kinase reaction buffer (50 mM HEPES pH 7.4, 50 mM MgCl2,
0.5 mM MnCl2, 0.2 mM Na3VO4, 1mM DTT) was added to each
well. Various concentrations of compounds diluted in 10 ml of 1%
DMSO (v/v) were added to each reaction well, with 1% DMSO
(v/v) used as the negative control. The kinase reaction was initi-
ated by the addition of purified c-Met kinase proteins diluted in
40 mlof kinasereaction buffer solution. After incubation for 60min
at 37 ◦C, the plate was washed three times with Phosphate Buffered
Saline (PBS) containing 0.1% Tween 20 (T-PBS). Next, 100 ml
anti-phosphotyrosine (PY99) antibody (1 : 500 diluted in 5 mg
◦
ml-1 BSA T-PBS) was added. After 30 min incubation at 37 C,
the plate was washed three times. 100 ml horseradish peroxidase-
conjugated goat anti-mouse IgG (1 : 2000 diluted in 5 mg ml-1 BSA
T-PBS) was added. The plate was reincubated at 37 ◦C for 30 min,
and washed as before. Finally, 100 ml of a solution containing
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