Design, synthesis, and biological evaluation of thieno[2,3-d]pyrimidine derivatives as novel dual c-Met and VEGFR-2 kinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2017.11.010

Both c-Met and VEGFR-2 are important targets for cancer therapies. Here we report a series of potent dual c-Met and VEGFR-2 inhibitors bearing thieno[2,3-d]pyrimidine scaffold. The cell proliferation assay in vitro demonstrated that most target compounds had inhibition potency both on c-Met and VEGFR-2 with IC₅₀ values in nanomolar range, especially compound 12j and 12m. Based on the further enzyme assay in vitro, compound 12j was considered as the most potent one, the IC₅₀ values of which were 25 nM and 48 nM for c-Met and VEGFR-2, respectively. Following that, we docked the compound 12j with the proteins c-Met and VEGFR-2, and interpreted the SAR of these analogues. All the results indicate that 12j is a dual inhibitors of c-Met and VEGFR-2 that holds promising potential.

Full text of DOI:10.1016/j.bmc.2017.11.010

Accepted Manuscript
Design, synthesis, and biological evaluation of thieno[2,3-d]pyrimidine deriv-
atives as novel dual c-Met and VEGFR-2 kinase inhibitors
Jieming Li, Weijie Gu, Xinzhou Bi, Huilan Li, Chen Liao, Chunxia Liu,
Wenlong Huang, Hai Qian
PII:
S0968-0896(17)31790-X
https://doi.org/10.1016/j.bmc.2017.11.010
BMC 14062
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
8 September 2017
2 November 2017
3 November 2017
Please cite this article as: Li, J., Gu, W., Bi, X., Li, H., Liao, C., Liu, C., Huang, W., Qian, H., Design, synthesis,
and biological evaluation of thieno[2,3-d]pyrimidine derivatives as novel dual c-Met and VEGFR-2 kinase
inhibitors, Bioorganic & Medicinal Chemistry (2017), doi: https://doi.org/10.1016/j.bmc.2017.11.010
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Design, synthesis, and biological evaluation of thieno[2,3-d]pyrimidine
derivatives as novel dual c-Met and VEGFR-2 kinase inhibitors
Jieming Lia, Weijie Gua, Xinzhou Bia, Huilan Lia, Chen Liaoa, Chunxia Liua, Wenlong
Huanga,b,*, Hai Qiana,b,*
a Center of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical
University, 24 Tongjiaxiang, Nanjing 210009, PR China
b
Jiangsu Key Laboratory of Drug Discovery for Metabolic Disease, China Pharmaceutical
University, 24 Tongjiaxiang, Nanjing 210009, PR China
* Corresponding authors. Tel./fax: +86 25 83271051.
E-mail addresses: ydhuangwenlong@126.com (W. Huang), qianhai24@163.com (H. Qian)
Abstract
Both c-Met and VEGFR-2 are important targets for cancer therapies. Here we
report a series of potent dual c-Met and VEGFR-2 inhibitors bearing
thieno[2,3-d]pyrimidine scaffold. The cell proliferation assay in vitro demonstrated
that most target compounds had inhibition potency both on c-Met and VEGFR-2 with
IC50 values in nanomolar range, especially compound 12j and 12m. Based on the
further enzyme assay in vitro, compound 12j was considered as the most potent one,
the IC50 values of which were 25 nM and 48 nM for c-Met and VEGFR-2,
respectively. Following that, we docked the compound 12j with the proteins c-Met
and VEGFR-2, and interpreted the SAR of these analogues. All the results indicate
that 12j is a dual inhibitors of c-Met and VEGFR-2 that holds promising potential.
Keywords: c-Met; VEGFR-2; Kinase; Thieno[2,3-d]pyrimidine; Cancer.
1. Introduction
Cancer, already the leading cause of death in many high-income countries, is set
to become a major cause of morbidity and mortality in the next few decades in every
region of the world, irrespective of level of resource.1 It was also predicted that global
cancer cases could increase to 22 million per year by 2030.2 Therefore, the
identification of novel biological targets and the discovery of more specific
chemotherapeutics are significant objectives of current research, especially for the
most invasive tumors.
The c-Met receptor tyrosine kinase (RTK) is the receptor for hepatocyte growth
factor/scatter factor (HGF).3 Binding of HGF to c-Met induces dimerization of c-Met
and phosphorylation of multiple tyrosine residues in the intracellular region, leading
to activation of c-Met signaling pathways. c-Met signaling plays important roles in
cell growth, survival, motility and morphogenesis during embryonic development as
well as in cancer cell proliferation, invasion, metastasis, tumor angiogenesis and drug

resistance.3, 4 What is more, it was reported that dysregulation of c-Met correlated
with a poor prognosis in clinical studies.5 Thus, c-Met is an attractive therapeutic
target for cancer therapy.5
Vascular endothelial growth factor receptor 2 (VEGFR-2, also known as KDR) is
a tyrosine kinase receptor expressed in endothelial cells. Binding of VEGF to VEGFR
induces a conformational change in VEGFR, followed by receptor dimerization and
phosphorylation of tyrosine residues. The VEGF/VEGFR2 signaling pathway plays
an important role in tumor angiogenesis, which stimulates tumor growth by supplying
oxygen and nutrients.6, 7 Its overexpression has been reported to correlate with the
degree of vascularity, aggressive disease and poor prognosis in the majority of human
solid tumors, making the VEGF/VEGFR axis an attractive molecular target for cancer
therapy.8-11
c-Met has been shown to collaborate synergistically with VEGFR-2, resulting in
promoting angiogenesis of development and progression of various human
cancers.12-14 Thus, molecules that simultaneously inhibit both c-Met and VEGFR-2
may have broader advantages over either c-Met-selective or VEGFR-2-selective
inhibitor as they can disrupt multiple signaling pathways involved in tumor
proliferation, metastasis and angiogenesis.15 As previously reported, several kinase
inhibitors, including cabozantinib and foretinib, can suppress c-Met and VEGFR-2
kinases simultaneously.16, 17 Therefore, we started the synthetic studies of dual
inhibitors of c-Met and VEGFR-2 with potent antitumor efficacy.
Figure 1. Kinase inhibitors using thieno[2,3-d]pyrimidine skeleton
As shown in Fig. 1, the thieno[2,3-d]pyrimidine skeleton had been widely used
in FGFR1 inhibitors18, B-Raf inhibitors19, FLT3 inhibitors20, and EGFR kinase
inhibitors.21 Cabozantinib16 (Fig. 2A) and Foretinib17 (Fig. 2B), compounds for
treatment of medullary thyroid cancer, were highly potent c-Met and VEGFR-2
inhibitor.22 The different biological profiles of these compounds indicated that the
cyclopropane-1,1-dicarboxamide moiety might be critical for the potent dual
inhibition of c-Met and VEGFR-2.23 Thus thieno[2,3-d]pyrimidine skeleton was
introduced to the side chain moiety, which conformed to ‘5 atoms regulation’ and
might form hydrogen bonds with amino acid residues of c-Met and VEGFR-2.24
Molecular docking study of the pyrimidine nucleus revealed that it could penetrate
into the ATP binding pocket of the c-Met and VEGFR-2 receptors (PDB code: 3LQ8,
3U6J), the nitrogen atom of pyrimidine ring could interact with the amino acid

residues (Met1160 of the c-Met protein and Cys919 of the VEGFR-2) in the hinge
region. In an attempt to pursue new antitumor agents potently inhibited both c-Met
and VEGFR-2, a series of new thieno[2,3-d]pyrimidine derivatives were synthesized
and their inhibitory activities were evaluated. In addition, the preliminary
structure-activity relationships and possible enzyme binding modes were also
illustrated.
Figure 2. Design of novel inhibitor for both c-Met and VEGFR-2 kinases.
2. Chemistry
The compound 1 was prepared by condensation of diethyl malonate and
1,2-dibromoethane under 50 % sodium hydroxide solution. The compound 1 was
coupled with equal equivalence 4-fluoroaniline by utilizing sulfoxide chloride and
triethylamine to obtain 2. On the other way, 3 was synthesized by benzyl alcohol and
3,4-difluoronitrobenzene under basic condition at reflux temperature. The amine of 4
was prepared by reduction of the nitro of 3. The compound 5 was prepared by
condensation of 2 and 4 under basic condition at room temperature. Under the
hydrogen atmosphere, the benzyl of 5 had been removed by hydrogenolysis to
generate 6 (see Scheme 1).
The synthesis of thieno[2,3-d]pyrimidine derivatives 10, 11 and 12a-12r were
shown in Scheme 2. The compound 7 was synthesized starting from benzy
acetoacetate, ethyl cyanoacetate and S8 by Gewald reaction, then it cyclized with
formamidine acetate to obtain bicyclic compound 8. The chloro-substituted
intermediate 9 was afforded by the chlorination of the compound 8 at the presence of
POCl3, and then the target compound 10 were obtained by a condensation reaction of
9 and 6. A convenient hydrolyzation of 10 utilizing anhydrous AlCl3 resulted in 11,
which then reacted with different substituted amino or alcohol to generate 12a-12r.

Scheme 1. Reagents and conditions: (a) Dibromoethane, TEBA, NaOH/H2O, r.t., 3 h; (b)
4-Fluoroaniline, SOCl2, Et3N, THF, 0 °C→r.t., 3h; (c) Benzyl alcohol, KOH, CH3CN, r.t.,
overnight; (d) Iron powder, ammonium formate, toluene/H2O, reflux, 8 h; (e) Compd 2 and 4,
SOCl2, Et3N, THF, 0 °C→r.t., overnight; (f) Pd/C, H2, THF/MeOH, r.t., 7 h. Total yield: 18.43 %.
Scheme 2. Reagents and conditions: (a) S8, Et3N, EtOH, r.t., 16 h; (b) Formamidine acetate, DMF,
100 °C, 16 h; (c) POCl3, Et3N, CH3CN, 0 °C→ 60 °C, 3.5 h; (d) Compd 6, DABCO, CH3CN,
reflux, 2 h; (e) PhOMe, AlCl3, MeNO2, DCM, r.t., 5 h; (f) Amine or alcohol , EDC·HCl, DMAP,
r.t., overnight. Total yield: 10.03-14.97 %. For full details, see Supporting Information.
3. Results and discussion
3.1. Cell proliferation inhibition and SAR study of target compounds
The effects of the compounds on cell proliferation were evaluated using
BaF3-TPR-Met cells which were the c-Met dependent cell line, and in
VEGF-stimulated human umbilical vein endothelial cells (HUVEC).
All compounds depicted in Table 1 were firstly screened for inhibition against
BaF3-TPR-Met. Also included were the activities of reference compound
Cabozantinib. Among the tested compounds, 12j, 12k, 12m, 12n and 12q showed the
most potent inhibitory activities, which were then selected to further test their
inhibitory activities against HUVEC and enzymatic activities in vitro. The selected
compounds were evaluated for their anticancer activity against HUVEC by CCK-8
assay. The results are summarized in Table 2.
As shown in Table 1 and Table 2, most of the tested compounds displayed
moderate to high inhibitory activities against BaF3-TPR-Met. 12j and 12m exhibited
nearly equivalent inhibition percent compared with the positive control Cabozantinib.
The cellular assays revealed that the introduction of different types of groups had a
large influence on activity, which suggested that the R group might be directed toward
a solvent exposed area and contributed to cellular inhibitory efficacy. At first,
different esters (10, 12a-12b) and amides (12c-12r) were introduced to the solvent
region. The results showed that amide substitutes had more potent inhibitory activities
than ester substitutes. In addition, when amino was cyclized (12c-12e, 12h-12i), the
activities were reduced. The possible reason was that the tumor cells were in an acidic
environment, and tertiary amine can be protonated in this microenvironment, resulting
in a positive electrical center, which enhanced the polarity of the end of the solvent
area. 12k and 12m demonstrated much higher inhibitory activities than that of 12l and
12n, respectively, which implied that two carbon atoms in the linker of the solvent
area had more potent inhibitory activities than three carbon atoms. It is possible that

the connecting arm of the two carbon atoms was more favorable for the binding of the
polar group to the receptor in the solvent region. Additionally, 12j exhibited the most
potent inhibitory activity, suggesting that polar long chain substituents were better
tolerated. However, when the amide was linked to the aryl group (12o, Log P 4.41),
the activity reduced significantly as shown in Table 1. It can be inferred that the
inhibitory activity has a certain correlation with the Log P value of the compound. It
seemed that compounds with higher inhibition percent had lower Log P values.
According to the above analysis, we can draw the conclusion that introduction of
polar flexible chain in the solvent region was beneficial to the improvement of
inhibitory rate.
Table 1
Structures, Log Ps and inhibition percent of target compounds against BaF3-TRP-Met and
HUVEC in vitro.
Inhibition %a
Compd
R
Log Pb
BaF3-TRP-Met
10.52
HUVEC
6.17
5.17
3.78
3.93
4.09
3.28
3.80
4.12
3.58
4.06
3.96
10
8.72
4.56
11
2.13
12a
12b
12c
12d
12e
12f
12g
12h
18.13
16.37
61.54
31.12
20.29
60.05
26.64
23.01
12.74
10.83
15.85
10.53
24.42
13.72
15.48
12i
61.02
23.84
3.07

12j
95.97
79.52
3.32
12k
12l
90.86
88.64
68.92
33.29
3.56
3.70
12m
12n
12o
95.37
92.83
26.67
73.62
61.34
32.85
3.37
3.75
4.41
12p
12q
86.58
92.63
27.48
66.67
4.17
3.41
12r
88.16
97.72
29.74
72.83
4.04
4.28
Cabozantinibc
a The tested concentration was 1 μM.
b Log P values were predicted at http://www.vcclab.org/.
c Used as positive control.
3.2. Enzymatic activities of optimized compounds
The inhibitory activities of optimized compounds against c-Met and VEGFR-2
kinases were determined by the ELISA assay using anti-phosphotyrosine antibodies.
After the determination of the compounds on cell proliferation against
BaF3-TPR-Met and HUVEC, compounds 12j, 12k, 12m, 12n and 12q were further
assayed with the enzymatic activities against c-Met and VEGFR-2 by ELISA, using
cabozantinib as comparison (Table 2). The enzymatic activities of these compounds
showed similar structure–activity relationships (SARs) summarized in the cellular
level. All the optimized compounds showed more potent activities against c-Met than
against VEGFR-2 indicating that c-Met might have a wider range of molecular
recognition in the back pocket than VEGFR-2. Particularly, compound 12j displayed a
better enzymatic potency than other compounds on c-Met and VEGFR-2 kinase with
IC50 values of 0.025 μM and 0.048 μM, respectively.
Table 2
Enzymatic and cellular activities in vitro of selected compounds
BaF3-TRP-Met
IC50a (μM)
HUVEC
IC50a (μM)
c-Met
IC50a (μM)
VEGFR-2
IC50a (μM)
Compd

0.051±0.004
0.081±0.003
0.055±0.008
0.086±0.006
0.086±0.007
0.139±0.024
0.089±0.005
0.153±0.023
0.025±0.003
0.047±0.002
0.036±0.007
0.059±0.007
0.048±0.006
0.083±0.004
0.054±0.005
0.081±0.005
12j
12k
12m
12n
12q
0.078±0.007
0.024±0.0032
0.123±0.015
ND
0.052±0.003
0.074±0.009
Cabozantinibb
0.0072±0.0021
0.0040±0.0013
ND, not determined.
a Data presented is the mean ± SD value of three independent determinations.
b Used as positive control.
3.3. Molecular docking studies
A
B
Figure 3. (A) The model of 12j bound to c-Met (PDB code: 3LQ8). (B) The model of 12j bound
to VEGFR-2 (PDB code: 3U6J). Red dashed lines represent H-bonds.
In order to better understand the interaction between compounds and kinases,
molecular docking studies on the potent compound 12j were performed using the
Glide docking protocol.
Fig. 3A demonstrates that compound 12j could dock into the binding site of
c-Met kinase (PDB: 3UQ8). The backbone NH of Met 1160 donated a hydrogen bond
to the nitrogen of pyrimidine, and the carbonyl oxygen of Phe 1223 could form
hydrogen bond with the NH of cyclopropane-1,1-dicarboxamide in the side region.
Moreover, 4- fluoro-phenyl ring occupied a deep hydrophobic pocket defined by Asp
1222 and Phe 1134.
The binding model of compound 12j and the ATP-binding cavity of VEGFR-2
kinase (PDB: 3U6J) is depicted in Fig. 3B. In this binding model, hydrogen bonds
between the hinge-region Cys 919 and the nitrogen of the pyrimidine ring were
observed. Moreover, NH and carbonyl oxygen of cyclopropane-1,1-dicarboxamide in
the side region could form two significant hydrogen bonds with the backbone N-H of
Asp 1046 and Lys 868, respectively. In addition, oxygen of sulfuryl can form new
hydrogen bond with Asn 923 residue. Finally, 4- fluoro-phenyl ring occupied a deep
hydrophobic pocket defined by Val 898 and Ile 892.
Therefore, we concluded that both the thieno[2,3-d]pyrimidine and
4-fluoro-phenyl-cyclopropane-1,1-dicarboxamide moieties were crucial to the

analogues for good potencies on both c-Met and VEGFR-2 as shown by the data in
Table 2.
4. Conclusion
In summary, to develop new dual c-Met and VEGFR-2 inhibitors as potential
anticancer agents, a series of novel thieno[2,3-d]pyrimidine derivatives were designed
and synthesized. Compound 12j showed the greatest inhibitory activities against
BaF3-TPR-Met and HUVEC with IC50 values of 0.051 μM and 0.086 μM,
respectively and also exhibited the most potent inhibitory activity against c-Met and
VEGFR-2 with IC50 values of 0.025 μM and 0.048 μM, respectively. Molecular
docking of the most potent inhibitor 12j into ATP binding site of c-Met and VEGFR-2
was performed and the result suggested that compound 12j could bind well with the
active site of c-Met and VEGFR-2. The above results demonstrated that compound
12j had emerged as a promising candidate for further development. Although none of
our designed derivatives are better than cabozantinib at both enzymatic and cellular
levels, this study has provided us with the clear SARs that are responsible for
inhibition of c-Met/VEGFR-2 and will aid in the design of more potent dual
c-Met/VEGFR-2 inhibitors.
5. Experimental section
5.1. Chemistry
All reagents and solvents were obtained from commercial sources and used
without further purification. Melting points were determined on RY-1 melting point
apparatus, and were not corrected. Mass spectra (MS) were acquired using a Waters
UPLC/MS (ACQUITY UPLC/TQD (Waters UPLC with the ACQUITY TQD [Waters
Corporation, Milford, MA, USA])) operating in electron spray ionization mode (ESI+)
and was used to confirm the purity of each compound. TLCs and preparative TLCs
were performed on silica gel GF/UV 254. Purifications by column chromatography
were carried out over silica gel (200–300 mesh) and visualized under UV light at 254
nm and 365 nm. Proton nuclear magnetic resonance (1H NMR) spectra were recorded
on Bruker ACF-300/500 MHz instruments. Chemical shifts were reported as δ values
(ppm) downfield from internal tetramethylsilane of the indicated organic solution.
Peak multiplicities were expressed as follows: s, singlet; d, doublet; t, triplet; q,
quartet; dd, doublet of doublet; br, broad singlet; m, multiplet. Coupling constants (J
values) were given in hertz (Hz).
The physical characteristics, 1H NMR, 13C NMR and MS data for all
intermediates and target compounds, were reported in Supporting Information.
5.2. Cell proliferation assay
BaF3-TPR-Met and HUVECs were seeded in 96-well plates in RPMI 1640
medium with 10% fetal bovine serum (FBS) and human endothelial serum free
medium (Invitrogen), respectively. The following day, serial dilutions of compounds
or 0.1% DMSO were added to each well. BaF3-TPR-Met cells were then incubated
for further 72 h. Proliferation of HUVECs was stimulated with 60 ng/mL recombinant
human VEGF (R&D Systems, Minneapolis, MN) for 120 h. After incubation, cell
proliferation was determined using Cell Counting Kit-8 (DOJINDO Laboratories,

Kumamoto, Japan). IC50 values were calculated by nonlinear regression analysis using
GraphPad Prism (GraphPad Software, Inc.).
5.3. In vitro c-Met and VEGFR-2 enzyme assay
The effects of the compounds on the activities of two tyrosine kinases were
determined using enzyme-linked immunosorbent assays (ELISAs) with purified
recombinant proteins. Briefly, 20 μg/mL poly (Glu, Tyr) 4:1 (Sigma) was pre-coated
in 96-well plates as a substrate. A 50 μL aliquot of 10 μmol/L ATP solution diluted in
kinase reaction buffer (50 mmol/L HEPES [pH 7.4], 50 mmol/L MgCl2, 0.5 mmol/L
MnCl2, 0.2 mmol/L Na3VO4, and 1 mmol/L DTT) was added to each well; 1 μL of
various concentrations of indicated compounds diluted in 1% DMSO (v/v) (Sigma)
was then added to each reaction well. DMSO (1%, v/v) was used as the negative
control. The kinase reaction was initiated by the addition of purified tyrosine kinase
proteins diluted in 49 μL of kinase reaction buffer. After incubation for 60 min at
37 ℃, the plate was washed three times with phosphate-buffered saline (PBS)
containing 0.1% Tween 20 (T-PBS). Anti-phosphotyrosine (PY99) antibody was then
added. After a 30-min incubation at 37 ℃, the plate was washed three times, and
horseradish peroxidase-conjugated goat anti-mouse IgG was added. The plate was
then incubated at 37 ℃for 30 min and washed 3 times. A 100 μL aliquot of a solution
containing 0.03% H2O2 and 2 mg/ml ophenylenediamine in 0.1 mol/L citrate buffer
(pH 5.5) was added. The reaction was terminated by the addition of 50 μL of 2 mol/L
H2SO4 as the color changed, and the plate was analyzed using a multi-well
spectrophotometer (SpectraMAX 190, Molecular Devices) at 490 nm. The inhibition
percent (%) was calculated using the following equation: [1-(A490/A490 control)]×100%.
The IC50 values were calculated from the inhibition curves in two separate
experiments.
5.4. Molecular modeling
All ligand molecules were drawn in ChemDraw 2014, and saved as sdf style.
Then ligands were processed at a simulated pH of 7.4 ± 1.0 to generate all possible
tautomers, stereoisomers, and protonation states and were finally minimized at the
OPLS 2005 force field with Ligand preparation protocol of Maestro 10.2. The crystal
structure of c-Met crystal structure (PDB code: 3LQ8)17 and VEGFR-2 crystal
structure (PDB code: 3U6J)25 were downloaded from the Protein Data Bank (PDB).
The protein was opened with Glide generation protocol of Maestro 10.2. Prior to
ligand docking, hydrogens were added and the crystallized ligand was removed. Then
a 10-Å box centered on the geometrical center of the ligand binding site was
generated for grid calculation. The docking studies were processed with the Glide
docking protocol. When docking finished, only one docking conformation was saved.
Acknowledgments
This study was supported by National Natural Science Foundation of China (No.
81673299).
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Design, synthesis, and biological evaluation of thieno[2,3-d]pyrimidine
derivatives as novel dual c-Met and VEGFR-2 kinase inhibitors
Jieming Lia, Weijie Gua, Xinzhou Bia, Huilan Lia, Chen Liaoa, Chunxia Liua, Wenlong
Huanga,b,*, Hai Qiana,b,*
a Center of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjiaxiang,
Nanjing 210009, PR China
b Jiangsu Key Laboratory of Drug Discovery for Metabolic Disease, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing
210009, PR China
* Corresponding authors. Tel./fax: +86 25 83271051.
E-mail addresses: ydhuangwenlong@126.com (W. Huang), qianhai24@163.com (H. Qian)
The cyclopropane-1, 1-dicarboxamide moiety might be critical for the potent dual inhibition of
c-Met and VEGFR-2. Thieno[2,3-d]pyrimidine as the scaffold of potent kinase inhibitor was
combined with the cyclopropane-1,1-dicarboxamide moiety to obtain potent c-Met and VEGFR-2
inhibitor.