Synthesis and biological evaluation of new [1,2,4]triazolo[4,3-a]pyridine derivatives as potential c-Met inhibitors

Article abstract of DOI:10.1016/j.bmc.2016.05.057

A series of [1,2,4]triazolo[4,3-a]pyrazine derivatives (4a–4i) were designed, synthesized and evaluated for their c-Met kinase inhibition and antitumor activity against SNU5 gastric cell line in vitro. Among these compounds, 4d was found to show the highest activity against c-Met and high selectivity against the tumor cells which are believed to be dependent on the c-Met oncogene amplification, because 4d selectively inhibited c-Met while had no effect on other 59 kinases. In vivo efficacy study on human gastric (MKN-45) and human non-small cell lung (NCI-H1993) tumor xenograft in nude mouse demonstrated that 4d·CH₃SO₃H had a better inhibiting activity than SGX-523 in a dose-dependent manner. When tested in mice, compound 4d·CH₃SO₃H was found to have biological half-lives and plasma exposure values higher than those of JNJ-38877605, and its long-term toxicity and acute toxicity turned out to be acceptable, all of which indicates that 4d·CH₃SO₃H is a desirable drug candidate.

Full text of DOI:10.1016/j.bmc.2016.05.057

Accepted Manuscript
Synthesis and biological evaluation of new [1,2,4] triazol [4,3-a] pyridine de-
rivatives as potential c-Met inhibitors
Junjun Zhao, Lei Fang, Xiaobing Zhang, Yan Liang, Shaohua Gou
PII:
S0968-0896(16)30387-X
http://dx.doi.org/10.1016/j.bmc.2016.05.057
BMC 13041
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Accepted Date:
18 April 2016
25 May 2016
27 May 2016
Please cite this article as: Zhao, J., Fang, L., Zhang, X., Liang, Y., Gou, S., Synthesis and biological evaluation of
new [1,2,4] triazol [4,3-a] pyridine derivatives as potential c-Met inhibitors, Bioorganic & Medicinal Chemistry
(2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.05.057
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Synthesis and biological evaluation of new [1,2,4] triazol [4,3-a] pyridine derivatives as potential c-Met
inhibitors
Junjun Zhao,a,b,# Lei Fang,a, # Xiaobing Zhang,b Yan Liang,b Shaohua Gou a,*
aJiangsu Province Hi-Tech Key Laboratory for Bio-medical Research, School of Chemistry and Chemical Engineering, Southeast University,
Nanjing 211189, China
b Lianyungang Hongchuang Pharmaceutical Co., Ltd, Lianyungang 222000, China
Corresponding author: Tel./fax: +86-25-83272381; e-mail: sgou@seu.edu.cn.
# Author Contributions: J. Zhao and L. Fang contributed equally to this work.
Key words: antitumor; c-Met inhibitors; selectivity; pharmacokinetic profile
Abstract A series of [1,2,4]triazolo[4,3-a]pyrazine derivatives (4a-4i) were designed, synthesized and evaluated for
their c-Met kinase inhibition and antitumor activity against SNU5 gastric cell line in vitro. Among these compounds,
4d was found to show the highest activity against c-Met and high selectivity against the tumor cells which are
believed to be dependent on the c-Met oncogene amplification, because 4d selectively inhibited c-Met while had no
effect on other 59 kinases. In vivo efficacy study on human gastric (MKN-45) and human non-small cell lung (NCI-
H1993) tumor xenograft in nude mouse demonstrated that 4d.CH3SO3H had a better inhibiting activity than SGX-
523 in a dose-dependent manner. When tested in mice, compound 4d.CH3SO3H was found to have biological half-
lives and plasma exposure values higher than those of JNJ-38877605, and its long-term toxicity and acute toxicity
turned out to be acceptable, all of which indicates that 4d.CH3SO3H is a desirable drug candidate.
Introduction
c-Met is a prototypic member of the receptor tyrosine kinase (RTK) family. The ligand for c-Met is a growth
factor known as hepatocyte growth factor (HGF).1 c-Met over expression or enhanced activation relative to normal
tissues has been noted in many human cancers such as gastric, colorectal, pancreatic, lung, head and neck, ovarian,
renal, glioma, metastatic melanoma, prostatic, and breast carcinomas.2,3 Thus, targeting the ATP binding site of c-
1

Met is a popular strategy for inhibition of the kinase, and many small molecules selectively targeting the ATP
binding site of c-Met kinase have been identified and exerted significant therapeutic effects in treating human
cancers clinically4,5.
The reported ATP-competitive c-Met inhibitors have been categorized into two classes based on their chemo type
and predicted binding mode (Figure 1).6 Class I inhibitors bind in a U-shaped conformation to the ATP-binding site
at the entrance of the kinase pocket and wrap around Met1211. In contrast, class II inhibitors, which are delineated
by the kinase linker strand (or hinge) to the deep hydrophobic Ile1145 pocket near the C-helix region, bind to c-Met
with an extended conformation that stretches from the ATP-binding site. In general, class I inhibitors showed high
selectivity to c-Met kinase,7-14 while class II inhibitors may also inhibit other kinases, depending on their substitution
patterns.15-19
Figure 1. General chemical structures of class I and class II c-Met inhibitors.
In an ongoing effort to design novel and selective inhibitors of the c-Met enzyme, there are several approaches in
class I currently being tested in the clinic. Supergen’s SGX-523 (1)20, Pfizer’s PF-04217903 (2)21, and Janssen’s
JNJ-3887705 (3)22 in Figure 2 are currently in Phase I or II clinical trials. These compounds displayed high potency
and good selectivity to c-Met, however, they also displayed poor overall solubility and exhibited short duration.
Moreover, SGX-523 was a potent c-Met inhibitor which was withdrawn from Phase I clinical trial due to a
reversible acute renal failure in one dose group caused by the insoluble metabolite compound of oxidation of
quinoline in the kidney. In this context, searching for more efficient and higher selective c-Met inhibitors is still in
great need. Recently, Boezio et al reported AMG 337 as a potent and selective c-Met inhibitor with high unbound
target coverage.23 Several reviews have also summarized current advances in the development of c-Met inhibitors.24-
26 In order to discover novel c-Met inhibitors and build diversification of chemical library, we continued to develop
structurally relevant novel compounds 4 by introducing substitution at 3-position to prevent oxidation of quinolone
(Figure 2). Furthermore, we also replaced [1,2,3]triazolo[4,5-b]pyridine with [1,2,4]triazolo[4,3-a]pyrazine and
changed the side chain into tricyclic scaffold, hoping such modifications might improve the solubility and the
2

pharmacokinetic properties. Herein, we report the synthesis and the biological characterization of a new class of
[1,2,4]triazolo[4,3-a]pyrazine derivatives.
Figure 2. Core modifications lead to the triazolopyridinone chemotype.
Results and discussion
Chemistry For the synthesis of the derivatives, a similar synthetic procedure previously used for SGX-523 was
employed.27 Generally, the aimed products 4a~4h could be divided into two fragments of compound 8 and
compound 11. Compound 8 was synthesized first from compound 5 (2-fluoro-4-chloro-pyridine), which was
coupled with (1-methyl-1H-pyrazol-4-yl)boronic acid to afford compound 6. Then 6 was treated with hydrazine
hydrate and subsequent CS2 to afford [1,2,4]triazolo[4,3-a]pyrazine (8). Compound 11 was prepared from
compound 9 (3-amino-6-bromoquinolin-4-ol) with ClCH2COCl or BrCH2CH2Br yielded compound 10 which
underwent a reaction with R2X to give the compound 11. The final products 4a~4h were provided by the coupling
reaction of compounds 8 and 11 catalyzed by Pd2(dba)3 (Scheme 1). To improve the aqueous solubility and stability
of the typical compound, 4d was converted into its methanesulfonate salt for further researches. For the synthesis of
the optically active compounds (R)- and (S)-4i, the starting material 12 was used instead of 9. Compound 12
underwent a substitution reaction with (R)- or (S)-pyrrolidin-2-ylmethanol to offer the (R)- and (S)-13. After a
coupling reaction, compounds (R)- and (S)-13 were converted to (R)- and (S)-14, which further reacted with 8 to
afford the chiral product (R)- and (S)-4i, respectively (Scheme 2).
3

Scheme 1. Synthetic Route of Compounds 4a~4h. Reagents and conditions: a) compound 5, (1-methyl-1H-pyrazol-
4-yl)boronic acid, Pd(OAc)2, 2-dicyclohexylphosphino-2',4',6'- triisopropylbiphenyl, K3PO4, dioxane/water (v:v =
10:1), reflux, 12 h; b) compound 6, hydrazine hydrate, ethanol, reflux, 12h; c) compound 7, CS2, KOH,
o
ethanol/water (v:v=4:1), reflux, 46h; d) compound 9, K2CO3, DMF, ClCH2COCl or BrCH2CH2Br, 80 C, 14h; e)
compound 10a, t-BuONa, DMF, R2X, rt 6h; f) compound 8 and 10 or 11, Pd2(dba)3, 4,5-Bis(diphenylphosphino)-
9,9-dimethylxanthene, DMF, DIEA, 100 oC, 12h.
4

Scheme 2. Synthetic Route of Optically Active Compounds 4i. Reagents and conditions: a) compound 12, THF,
o
o
NaH, (R)- or (S)-pyrrolidin-2-ylmethanol, 40 C, 3 h; b) compound 13, Cs2CO3, CuI, DMF, 100 C, 16 h; c)
compound 8b and 14, Pd2(dba)3, 4,5-Bis(diphenylphosphino)-9,9-dimethylxanthene, DMF, DIEA, 100 oC, 12 h.
The derivatives were characterized by 1H NMR and ESI-MS. The analytical data for the target compounds, which
can be seen from experiments section, showed that the characteristic signals of 1HNMR for indazole were 7.20~8.10
ppm, 7.60~8.90 ppm for quinoline, and pyrazol-substituents data of 6-position in indazole were consistent with the
reference.27 ESI-MS gave information about all the [M+H]+ peaks corresponding to molecular weight of confirmed
novel compounds. Further 13CNMR and elemental analysis confirmed that the structures were fixed with the
structures proposed.
Evaluation of biological activity The compounds prepared in this study were examined for their ability to inhibit
c-Met activity using an enzyme assay with a recombinant c-Met kinase domain and in a cellular assay that
determined the inhibition of gastric cells SNU5. The biological activity of the [1,2,4]triazolo[4,3-a]pyrazine with a
general structure 4 are shown in Table 1. A simple compound, 4a, showed inhibition of c-met kinase activity with
IC50 of 9.3 nM and inhibition of SNU5 with IC50 of 85.8 nM. Replacing the R1 of H with F in compound 4b resulted
in increases of potency (IC50 of inhibition of kinase activity 7.9 nM, inhibition of SNU5 17.8 nM), suggesting that
the F atom was better filled with the pyrazol ring, which was further confirmed by comparing the corresponding data
5

between 4c and 4d, 4f and 4g. However, transposing the H in 4c to OCF2H in 4e led to a significant loss of potency.
The key modification led to improved potency against c-Met involved replacing the H group with a methyl or a 2-
methoxyethyl group at nitrogen atom of R2 group. This change resulted in an increase in activity, and the methyl
group showed the best fills with the R2 group (4a vs 4c or 4f, 4b vs 4d or 4g). To probe whether a carbonyl group
might affect the potency of 4 to c-Met, compound 4h was synthesized and tested, which resulted in a loss of potency.
Moreover, (R)-4i demonstrated an improvement over (S)-4i. Noticeably, compound 4d was found to show the
highest activity against c-Met and SNU5 cell line.
Table 1. Activity of [1,2,4]Triazolo[4,3-a]pyrazine derivatives against c-Met
c-Met IC50 (nM)
R2
Entry
Compound
R1
Y
enzymea
9.3
cellb
85.8
17.8
3.0
H
H
carbonyl
carbonyl
carbonyl
carbonyl
carbonyl
carbonyl
carbonyl
CH2
1
2
4a
4b
F
H
Me
7.9
H
3
12.0
2.9
4c
F
Me
4
2.0
4d
OCF2H
Me
5
>1000
20.1
11.4
42.5
37.0
122.7
3.1
>1000
19.1
4.5
4e
H
F
F
F
F
2-methoxyethyl
2-methoxyethyl
c-propanecarbonyl
6
4f
7
4g
8
12.3
29.2
327.1
3.9
4h
(R)-pyrrolidine
9
(R)-4i
(S)-4i
SGX-523
(S)-pyrrolidine
10
-
11
aInhibition of kinase activity. b Inhibition of gastric cells SNU5.
In order to investigate the interaction mode of the target compounds with c-Met, we further performed a
molecular modeling study of 4d and SGX-523 with the MOE (Molecular Operating Environment) software. Initial
minimization was carried out via the homology modeling function of MOE. The generated model was firstly
minimized with a few thousand cycles of minimization using the ABNR (adopted-basis Newton-Raphson) method,
and then the ligands were docked by positioning them in the active site in accordance with the published crystal
structure of c-Met (PDB code: 3dkf). It was revealed that both 4d and SGX-523 could bind to the ATP-binding
region of c-MET kinase, and the superposition of 4d and SGX-523 (Figure 3, A) showed that the backbones of the
two ligands, which were located at the hinge region of kinase domain, overlapped quite well. This suggested that 4d
may interact with the corresponding residues in a similar manner to SGX-523. Indeed, the key interactions of the
6

aromatic ring system with Tyr1248 and the H-bond formed between quinoline ring and Met1160 (Figure 3, B) were
observed for both 4d and SGX-523. This may explain the potent c-Met inhibition activity of 4d and SGX-523.
A
B
Figure 3. A) The superposition of 4d (shown in red) and SGX-523 (shown in green) in the 3D docking study. B)
The illustration of the interaction mode of 4d with the corresponding active sites of c-Met kinase (PDB code: 3dkf).
Based on the data in Table 1, compound 4d with the highest activity against both c-Met and SNU5 cell line was
selected to perform comprehensive screening for the inhibition effect on 60 different kinases, and staurosporine, a
potent but nonspecific inhibitors of protein kinases,28 was used as positive control. The data in Table 2 revealed that
staurosporine had potent activity towards almost all of 60 kinases without selectivity, while 4d significantly
7

inhibited c-Met with IC50 value of 0.7 nM, but had low or even no effect on the other kinases. The result
demonstrated that 4d is a selective c-Met inhibitor.
Table 2. Inhibitory activity of 4d against 60 different kinases
IC50 (nM)
Staurosporine
IC50 (nM)
Staurosporine
Kinase
Kinase
4d
4d
ABL
AKT1
>30000
>30000
>30000
>30000
>30000
8690
196.2
9.0
HER2
HER4
IGF1R
INSR
JAK1
JAK2
JAK3
KDR
>30000
>30000
>30000
>30000
>30000
21052
115.0
28.3
200.7
73.6
12.5
0.4
ALK
6.7
ARG
75.9
2.4
AurA
AXL
3.0
BRK
>30000
>30000
>30000
>30000
>30000
>30000
>30000
>30000
>30000
>30000
>30000
>30000
>30000
>30000
>30000
>30000
>30000
>30000
>30000
>30000
>30000
>30000
11161
247.0
65.0
2.6
>30000
>30000
>30000
>30000
>30000
0.7
0.5
BTK
4.1
CDK2
LCK
1.8
CKIT
4.1
LTK
3.6
ECK
262.6
74.2
10.6
0.6
LYNa
c-Met
MUSK
p38a
2.1
EGFR
69.2
3.8
EGFR L858R
EGFR T790M
EGFR T790ML858R
EphA1
EphB1
EphB2
ERK2
>30000
>30000
>30000
>30000
>30000
>30000
>30000
>30000
>30000
>30000
>30000
2829
10.5
0.6
1.1
PDGFRa
PDGFRb
PKACa
PKCa
PKACz
PKN1
PKN2
RET
49.8
127.9
198.8
659.1
1.8
0.6
2.3
1.2
216.8
1.2
FER
FES
8.1
2.2
FLT1
5.4
4.6
FLT3
0.2
RON
102.7
0.2
FLT4
0.9
ROS
FGFR1
FGFR2
FGFR3
FGFR4
FYN
6.3
SRC
15453
4.7
5.8
SYK
>30000
>30000
>30000
>30000
>30000
1.3
15.6
148.3
4.4
TIE2
213.2
0.5
TRK-A
TYK2
YES
0.6
GSK3b
16184
18.6
4.4
8

With its excellent potencies against both enzyme and SNU5 cell, [1,2,4]triazolo[4,3-a]pyrazine derivative 4d was
further evaluated in other cancer cell lines in vitro with SGX-523 as positive control (Table 3). Compound 4d
showed significant inhibition of cell lines with the c-Met oncogene amplification including hepatoma cell lines
HCCLM3 (33nM), MHCC97-H (6nM), MHCC97-L (7nM), gastric cancer cell line MKN-45 (6nM), and lung
cancer cell line NCI- H1993 (14nM). In contrast, compound 4d showed little activity against no c-Met gene
amplification cell lines such as hepatoma cell line Huh-7 (>1000nM), gastric cancer cell line NCI-N87 (>1000nM),
and lung cancer cell lines NCI-1975 (>1000nM) and A549 (>1000nM). It is clear that 4d inhibits c-Met kinase
activity with high selectivity.
Table 3. Activity of 4d and SGX-523 against different cell lines
Cellular IC50 (nM)
HCCL MHCC97- MHCC97- MKN-
NCI-
NCI-
N87
NCI-
1975
Huh-7
A549
M3
33
H
6
L
7
45
6
H1993
14
25
>10000
>10000
>10000 >10000 >10000
>10000 >10000 >10000
4d
SGX-523
24
11
18
15
Western Blot Assay Typical compound 4d in its methanesulfonate was next selected to perform a western blot
assay to investigate its effect on the phosphorylation of tyrosine kinases as well as the activation of the downstream
signal transduction pathways. In the c-MET highly expressing HCCLM3 cells, 4d.CH3SO3H significantly induced
the phosphorylation of the tyrosine kinases at the concentration of 100nM, 300nM, or 1000nM, and the downstream
phosphorylation of ERK and AKT was partially or completely inhibited. In the Huh-7 cells which were not c-Met
gene amplification, 4d.CH3SO3H showed no effect on the inhibition of phosphorylated of ERK and AKT. This result
suggested that 4d.CH3SO3H inhibited the HCCLM3 cell by inhibiting the downstream signal transduction pathways
of c-Met. In addition, compared with SGX-523, 4d.CH3SO3H showed a similar or a better activity (Figure 4).
Figure 4. The effect of 4d.CH3SO3H and SXG-523 on HCCLM3 cells as well as 4d.CH3SO3H on Huh-7 cells.
9

In vivo efficacy study Two different human tumor cell lines comprising human gastric (MKN-45) and human
non-small cell lung (NCI-H1993) were used to study the efficacy of compound 4d.CH3SO3H and SGX-523 on in
vivo tumor xenograft in nude mouse (Figures 5 and 6). At all of the test concentrations (10, 30, 60 mg/kg),
4d.CH3SO3H significantly inhibited the tumor (MKN-45) growth in nude mice and the inhibitory rates were 29.5%,
34.2%, and 61.4%, respectively. While the inhibition rate of SGX-523 (10 mg/kg) was 11.2%, which is obviously
lower than 4d.CH3SO3H at the same dosage. For the inhibition of NCI-H1993 tumor growth, the rates of
4d.CH3SO3H (10, 30, 60 mg/kg) were 24.4%, 19.5%, and 24.9%, but SGX-523 (10 mg/kg) showed no inhibitory
effect. The results clearly indicated that 4d.CH3SO3H had a better inhibiting activity than SGX-523 in a dose-
dependent manner.
Figure 5. Efficacy of 4d.CH3SO3H and SGX-523 on resistant human gastric MKN-45 tumor xenograft in nude mice.
10

Figure 6. Efficacy of 4d.CH3SO3H and SGX-523 on resistant human non-small cell lung NCI-H1993 tumor
xenograft in nude mice.
Toxicity studies in mice Long-term toxicity studies were performed in male and female SD mice to determine
potential adverse effects and maximum tolerated dose (MTD) for 4d.CH3SO3H. Both male and female SD mice were
treated for 28 days with compound 4d.CH3SO3H at 15, 75, or 225 (mg/kg)/day and then rehabilitate for another 28
days. The body weights of all treated mice remained normal for the duration of the study when compared to the
vehicle-treated controls. No biologically significant changes in clinical pathology were observed on day 56. The No
Observed Adverse Effect Level (NOAEL) in mice long-term toxicity studies were estimated to be 225 (mg/kg)/day.
Acute toxicity studies were performed in male and female SD mice for 28 days with compound 4d.CH3SO3H at 300,
or 600 (mg/kg). No biologically significant changes in clinical pathology were observed. The NOAEL in mice acute
toxicity studies were estimated to be 600 (mg/kg)/day.
Pharmacokinetic study With its excellent in vitro enzyme and cell potencies, the pharmacokinetic properties of
4d.CH3SO3H were also evaluated in vivo, and the results were summarized in Table 4. Compared with JNJ-
38877605, 4d.CH3SO3H, at the same dose in rats, demonstrated a significantly higher plasma concentration (8628
ng/mL vs 2373 ng/mL), longer half-life (5.55 h vs 2.97 h) and mean residence time (9.10 h vs 4.90 h), lower
clearance (0.686 mL/min/kg vs 5.81 mL/min/kg), suggesting that the designed [1,2,4]triazolo[4,3-a]pyrazine
derivative, 4d.CH3SO3H, showed a better performance than Janssen’s JNJ-38877605 in pharmacokinetic. So this
designed compound should be well worth studying based on preliminary biological tests. Further research is
currently under investigation and will be reported in due course.
11

Table 4. Pharmacokinetic profile of [1,2,4]triazolo[4,3-a]pyrazine derivative 4d.CH3SO3H a
doseb
AUC
CL/F
Vz/F
Cmax
Compound
t1/2 (h)
MRT (h)
(ng/mL)
(mg/kg)
(ng/mL*h)
(mL/min/kg)
0.68±0.07
5.81±2.5
(mL/kg)
327±37
4d.CH3SO3H
5
5
8628±592 122487±13875 5.55±0.78
2373±1444 17325±9505 2.97±0.25
9.10±0.85
4.90±0.62
JNJ-38877605
1480±613
aPharmacokinetic parameters following administration of 4d.CH3SO3H in each half of male and female Sprague-
b
Dawley rats: 8 animals were divided into two groups, 4 animals per study. Dosed as a solution of stroke-
physiological saline solution with 5% DMSO.
Conclusion
A series of [1,2,4]triazolo[4,3-a]pyrazine derivatives 4 were designed and synthesized. The synthesized
compounds were evaluated for their biological activity against both c-Met and gastric cancer cell line SNU5, and
most of them showed good cytotoxicity. In particular, compound 4d showed an excellent potency against activity
both kinase and the tumor cells. Comprehensive screening for the inhibition of 60 different kinases revealed that 4d
could selectively inhibit c-Met while had no effect on other kinases, indicating the cellular activity of 4d is probably
dependent on the c-Met oncogene amplification. In vivo efficacy study on human gastric (MKN-45) and human non-
small cell lung (NCI-H1993) tumor xenograft in nude mouse demonstrated that 4d.CH3SO3H had a better inhibiting
activity than SGX-523 in a dose-dependent manner. Moreover, Compound 4d.CH3SO3H also showed a better
performance than JNJ-38877605 in pharmacokinetics, and a higher level of NOAEL in mice long-term toxicity (225
mg/kg.day) and acute toxicity (600 mg/kg.day). Further studies on structural optimization and biological activities
about these derivatives are still underway in our laboratory.
EXPERIMENTAL SECTION
Chemistry All chemicals were obtained from commercial purchase and solvents were purified and dried by
standard procedures. Flash chromatography (FC): silica gel (SiO2; 40 mm, 200~300 mesh). Melting points were
1
uncorrected and were determined using a capillary apparatus (RDCSY-I). H NMR and 13C NMR Spectra: Bruker
AVANCE-400 Digital NMR Spectrometer, ESI-MS: Thermo Finnigan LCQ advantage MAX. Elemental analysis
was performed on a Vario EL III apparatus.
2-Fluoro-5-(1-methyl-1H-pyrazol-4-yl)pyridine (6a). Typical procedure 1: a mixture of 5-chloro-2-
fluoropyridine 5a (6.60 g, 50.2 mmol), (1-methyl-1H-pyrazol-4-yl)boronic acid (6.30 g, 50.1 mmol), Pd(OAc)2
12

(0.46 g, 2.1 mmol), 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (1.99 g, 4.1 mmol), and K3PO4 (26.30 g,
123.7 mmol) in dioxane/water (80 mL/8 mL) was stirred at 100oC under N2 atmosphere until the reaction was
completed by TLC. After the solution was concentrated under reduced pressure, the product was purified via column
1
chromatography to afford compound 6a (6.80 g, 77%). HNMR (400MHz, CDCl3, ppm) δ 8.22-8.13 (m, 3 H), 7.88
(d, J = 0.4 Hz, 1 H), 7.30 (d, J = 6.8 Hz, 1 H) 3.90 (s, 3 H). MS m/z (ESI): [M+H]+ 178.1.
2,3-Difluoro-5-(1-methyl-1H-pyrazol-4-yl)pyridine (6b). Following the typical procedure 1, the reaction of 5-
chloro-2,3-difluoropyridine 5b (7.41 g, 49.4 mmol), (1-methyl-1H-pyrazol-4-yl)boronic acid (6.31 g, 50.1 mmol),
Pd(OAc)2 (0.46 g, 2.1 mmol), 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (1.96 g, 4.1 mmol), and K3PO4
1
(26.24 g, 123.6 mmol) in dioxane/water (80 mL/8 mL) afforded compound 6b (6.10 g, 76%). HNMR (400MHz,
CDCl3, ppm) δ 8.32-8.25 (m, 3 H), 8.01 (d, J = 0.4 Hz, 1 H), 3.88 (s, 3 H). MS m/z (ESI): [M+H]+ 196.1
6-(1-Methyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-a] pyridine-3-thiol (8a). Typical procedure 2: a mixture of 2-
fluoro-5-(1-methyl-1H-pyrazol-4-yl)pyridine 6a (1.60 g, 9.0 mmol), and 80% hydrazine hydrate (1.81 g, 46.1 mmol)
in ethanol (25 mL) was stirred under reflux for 12h. After the reaction solution was cooled down to room
temperature, white solid crystallized. The solid was filtered out and washed with ethanol to afford intermediate 7a,
which was used in the next step directly.
To a solution of 7a and CS2 (1.03 g, 13.4 mmol) in EtOH/H2O (20 mL/5 mL), KOH (0.40 mg, 7.0 mmol) was
added, the resulting mixture was stirred under reflux for 46 h. After concentrated under reduced pressure, NaOH
solution (1 M, 30 mL) was added. The solution was extracted by CH2Cl2 twice, and the aqueous layer was treated
with HCl solution (1 M). The residue was filtered and washed with water to afford product 8a (1.30 g, 55% for 2
steps). 1HNMR (400MHz, CDCl3, ppm) δ 14.89 (s, 1 H), 8.39 (s, 1 H), 8.28~8.19 (m, 2 H), 8.04 (s, 1 H), 7.88 (dd, J
= 1.2 and 12.4 Hz, 1 H), 3.88 (s, 3 H). MS m/z (ESI): [M+H]+ 249.9.
8-Fluoro-6-(1-methyl-1H-pyrazol-4-yl)-[1,2,4] triazolo [4,3 -a]pyridine-3-thiol (8b). Following the typical
procedure 2, the reaction of 2,3-difluoro-5-(1-methyl-1H-pyrazol-4-yl)pyridine 6b (1.81 g, 9.2 mmol), and 80%
hydrazine hydrate (1.80 g, 46.1 mmol) in ethanol (25 mL) afforded intermediate 7b used in the next step directly.
The reaction of 7b, CS2 (1.01 g, 13.4 mmol) with KOH (0.40 g, 7.0 mmol) in EtOH/H2O (20 mL/5 mL) afforded
product 8b (1.12 g, 46.9% for 2 steps). 1HNMR (400MHz, CDCl3, ppm) δ 14.89 (s, 1 H), 8.39 (s, 1H), 8.26 (s, 1H),
8.04 (s, 1 H), 7.88 (dd, J = 1.2 and 12.4 Hz, 1 H), 3.88 (s, 3 H). MS m/z (ESI): [M+H]+ 249.9.
13

8-(Difluoromethoxy)-6-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-a]pyridine-3-thiol (8c). The reaction of
8-fluoro-6-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]triazolo [4,3-a]pyridine-3-thiol 8b (0.38 mg, 1.5 mmol) with NaOH
(0.09 g, 2.3 mmol) in DMF (10 mL) was stirred and heated to 100 oC for 16h. The mixture was added into water and
filtered, then the filtered cake, K2CO3 (0.41 g, 3 mmol), ethyl chlorodifluoroacetate (0.28 g, 1.8 mmol), and DMF (5
mL) were mixed and stirred at 70oC for 10 h. The reaction mixture was added into water and extracted with CH2Cl2.
After the solution was concentrated under reduced pressure, the crude product was collected and purified via column
chromatography (CH2Cl2:MeOH = 25:1) to afford compound 8c (0.20 g, 45%). MS m/z (ESI): [M+H]+ 298.1.
9-Bromo-2H-[1,4]oxazino[3,2-c]quinolin-3(4H)-one (10a). Typical procedure 3: a mixture of 3-amino-6-
bromoquinolin-4-ol (0.30 g, 1.3 mmol), K2CO3 (0.52 g, 3.8 mmol), DMF (7 mL) was stirred at room temperature,
o
then 2-chloroacetyl chloride (0.16 g, 1.3 mmol) was dropwise added and the reaction mixture was stirred at 80 C
for 14 h. The mixture was added into water and filtered, then the filtered cake was washed with water to afford
product 10a (0.20 mg, 57%). MS m/z (ESI): [M+H]+ 278.9.
9-Bromo-3,4-dihydro-2H-[1,4]oxazino[3,2-c]quinoline (10b). Following the typical procedure 3, the reaction
of 3-amino-6-bromoquinolin-4-ol (0.26 g, 1.1 mmol), K2CO3 (0.90 g, 6.5 mmol) and 1,2-dibromoethane (0.62 g, 3.3
mmol) in DMF (10 mL) afforded product 10b (0.10 g, 35%). MS m/z (ESI): [M+H]+ 264.9.
9-Bromo-4-methyl-2H-[1,4]oxazino[3,2-c]quinolin-3(4H)-one (11a). Typical procedure 4: a mixture of 10a
(1.01 g, 3.6 mmol) and t-BuONa (0.51 g, 5.4 mmol) in DMF (20 mL) was stirred at room temperature for 15 min,
then MeI (0.56 g, 3.9 mmol) was added. The reaction was stirred at room temperature for 6 h, then the mixture was
added into a blending solution of water/ethanol=1/1 and filtered. The resulting filtered cake was washed with
water/ethanol=1/1 to afford product 11a (0.69 g, 66%). MS m/z (ESI): [M+H]+ 292.9.
9-Bromo-4-(2-methoxyethyl)-2H-[1,4]oxazino[3,2-c]quinolin-3(4H)-one (11b). Following the typical
procedure 4, the reaction of 10a (0.10 g, 0.4 mmol), t-BuONa (0.05 g, 0.5 mmol), and 1-bromo-2-methoxyethane
(0.07 g, 0.5 mmol) in DMF (5 mL) afforded product 11b (0.06 g, 46%). MS m/z (ESI): [M+H]+ 337.1.
(9-Bromo-2H-[1,4]oxazino[3,2-c]quinolin-4(3H)-yl) (cyclopropyl)methanone (11c). Following the typical
procedure 4, the reaction of 10b (0.08 g, 0.3 mmol), N-ethyl-N-isopropylpropan-2-amine (0.11 ml, 0.6 mmol), and
cyclopropanecarbonyl chloride (30 ul, 0.3 mmol) in CH2Cl2 (10 mL) afforded product 11c (0.06 g, 60%). MS m/z
(ESI): [M+H]+ 333.0.
14

9-((6-(1-Methyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-a]pyridin-3-yl)thio)-2H-[1,4]oxazino[3,2-c]quinolin-
3(4H)-one (4a). Typical procedure 5: a mixture of 8a (51 mg, 0.22 mmol), 10a (55 mg, 0.20 mmol), Pd2(dba)3 (12
mg, 0.02 mmol), and 4,5-Bis(diphenylphosphino)-9,9-dimethylxanthene (24 mg, 0.04 mmol) in DMF (5 mL) was
stirred at room temperature until the solid was dissolved. Then sodium t-butoxide (39 mg, 0.40 mmol) was added
o
and the reaction was stirred at 100 C for 12 h. After the solution was concentrated under reduced pressure, the
crude product was collected, and purified via column chromatography (CH2Cl2:MeOH = 20/1) to afford compound
4a (34 mg, 37%). m.p. 128-129 oC. 1HNMR (400MHz, DMSO-d6, ppm) δ 11.09 (d, J =10.4 Hz, 1 H), 8.64 (s, 1H),
8.46 (s, 1 H), 8.35 (s, 1 H), 8.11-7.95 (m, 2 H), 7.97-7.76 (m, 3 H), 7.47 (dd, J = 9.2 and 2.0 Hz, 1 H), 4.87 (s, 2 H),
3.86 (s, 3 H); 13CNMR (100MHz, DMSO-d6, ppm) δ 160.5, 144.0, 143.3, 142.9, 142.6, 136.4, 136.0, 135.8, 134.6,
131.7, 129.0, 124.0, 122.1, 121.0, 119.8, 118.5, 116.3, 114.0, 112.0, 68.0, 38.7; MS m/z (ESI): [M+H]+ 430.1;
Element Analysis for C21H15N7O2S (%) C: 58.73%, H: 3.52%, N: 22.83% Found C: 58.76%, H: 3.51%, N: 22.85%.
9-((8-Fluoro-6-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-a]pyridin-3-yl)thio)-2H-[1,4]oxazino
[3,2-
c]quinolin-3(4H)-one (4b). Following the typical procedure 5, the reaction of 8b (118 mg, 0.52 mmol), 10a (120
mg, 0.43 mmol), Pd2(dba)3 (25 mg, 0.04 mmol), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (50 mg, 0.09
mmol), and sodium t-butoxide (50 mg, 0.52 mmol) in DMF (5 mL) afforded compound 4b (16 mg, 8%). m.p. 115-
116 oC. 1HNMR (400MHz, DMSO-d6, ppm) δ 11.09 (d, J =10.4 Hz, 1 H), 8.56 (s, 1H), 8.47 (s, 1 H), 8.38 (s, 1 H),
8.08 (s, 1 H), 7.92 (d, J = 2.0 Hz, 1 H), 7.86 (dd, J = 10.4 and 8.0 Hz, 2 H), 7.49 (dd, J = 9.2 and 2.0 Hz, 1 H), 4.89
(s, 2 H), 3.86 (s, 3 H); 13CNMR (100MHz, DMSO-d6, ppm) δ 161.2, 150.1, 148.0, 147.7, 143.6, 138.4, 137.3, 136.8,
134.2 (d, J = 172 Hz, 1 C), 131.7, 129.3, 124.0, 122.5, 121.2, 119.8, 118.6, 116.3, 114.0, 111.2, 68.1, 38.7; MS m/z
(ESI): [M+H]+ 448.1; Element Analysis for C21H14FN7O2S (%) C: 56.37%, H: 3.15%, N: 21.91% Found C: 56.36%,
H: 3.12%, N: 21.95%.
4-Methyl-9-((6-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]
triazolo[4,3-a]pyridin-3-yl)thio)-2H-[1,4]oxazino[3,2-
c]quinolin-3(4H)-one (4c). Following the typical procedure 5, the reaction of 8a (44 mg, 0.19 mmol), 11a (46 mg,
0.16 mmol), Pd2(dba)3 (10 mg, 0.02 mmol), 4,5-Bis(diphenylphosphino)-9,9-dimethylxanthene (50 mg, 0.03 mmol),
and sodium t-butoxide (20 mg, 0.19 mmol) in DMF (5 mL) afforded compound 4c (12 mg, 16%). m.p. 167-169 oC.
1HNMR (400MHz, DMSO-d6, ppm) δ 8.56 (s, 1H), 8.16-8.12 (m, 1 H), 7.94-7.90 (m, 1 H), 7.87-7.79 (m, 2 H), 7.61
(d, J = 0.8 Hz, 1 H), 7.56 (d, J = 0.8 Hz, 1 H), 7.41 (dd, J = 9.2 and 2.8 Hz, 1 H), 7.20 (s, 1 H), 4.80 (s, 2 H), 3.88 (s,
3 H), 3.43 (s, 3 H); 13CNMR (100MHz, DMSO-d6, ppm) δ 160.6, 144.0, 143.3, 142.9, 142.6, 136.4, 136.0, 135.8,
15

134.6, 131.7, 129.0, 124.0, 122.1, 121.0, 119.8, 118.5, 116.3, 114.0, 112.0, 68.1, 38.7, 27.8; MS m/z (ESI): [M+H]+
444.1; Element Analysis for C22H17N7O2S (%) C: 59.58%, H: 3.86%, N: 22.11% Found C: 59.55%, H: 3.88%, N:
22.10%.
9-((8-Fluoro-6-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-a]pyridin-3-yl)thio)-4-methyl-2H-[1,4]oxazino
[3,2-c]quinolin-3(4H)-one (4d.CH3SO3H and its methanesulfonate). Following the typical procedure 5, the reaction
of 8b (93 mg, 0.38 mmol), 11a (100 mg, 0.34 mmol), Pd2(dba)3 (20 mg, 0.03 mmol), 4,5-bis(diphenylphosphino)-
9,9-dimethylxanthene (40 mg, 0.07 mmol), and sodium t-butoxide (40 mg, 0.41 mmol) in DMF (5 mL) afforded
compound 4d (36 mg, 23%). m.p. 253-255 oC. MS m/z (ESI): [M+H]+ 462.2.
A mixture of 4d (12.0 g, 26.0 mmol) and CH3SO3H (10.0 g, 104.0 mmol) in DMF (400 mL), was stirred at rt for
2h. Then the reaction solution was filtered, and the solid was washed with ethyl acetate to afford compound
1
4d.CH3SO3H (10.2 g, 70.4%). m.p. over 300 oC. HNMR (400MHz, DMSO-d6, ppm) δ 10.07 (bs, 1 H), 9.05 (s, 1
H), 8.55 (s, 1 H), 8.37 (s, 1 H), 8.10-7.96 (m, 3 H), 7.82(d, J = 12 Hz, 1 H), 7.74 (d, J = 9.6 Hz, 1 H), 5.20 (s, 2 H),
3.85 (s, 3 H), 3.42 (s, 3 H), 2.45 (s, 3 H); 13CNMR (100MHz, DMSO-d6, ppm) δ 161.2, 150.0 (d, J = 17.6 Hz, 1 C),
148.0, 147.7, 143.6 (d, J = 24.4 Hz, 1 C), 138.4, 137.3, 136.8, 134.2 (d, J = 172 Hz, 1 C), 131.7, 129.2, 124.0, 122.5,
121.2 (d, J = 5.2 Hz, 1 C), 119.8, 118.6, 116.3, 114.0, 111.2 (d, J = 12 Hz, 1 C), 68.0, 39.7, 38.7, 27.9; IR (KBr, cm-
1) 3421, 3060, 2939, 2617, 1699, 1647, 1577, 1476, 1431, 1393, 1346, 1160, 1231, 1035, 880, 776; MS m/z (ESI):
[M+H]+ 462.1; Element Analysis for C22H16FN7O2S.CH3SO3H (%) C: 49.54%, H: 3.62%, N: 17.58% Found C:
49.56%, H: 3.61%, N: 17.53%.
9-((8-(Difluoromethoxy)-6-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-a]pyridin-3-yl)thio)-4-methyl-2H-
[1,4]oxazino[3,2-c]quinolin-3(4H)-one (4e). Following the typical procedure 5, the reaction of 8c (105 mg, 0.35
mmol), 11a (108 mg, 0.37 mmol), Pd2(dba)3 (21 mg, 0.04 mmol), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene
(38 mg, 0.07 mmol), and sodium t-butoxide (42 mg, 0.43 mmol) in DMF (5 mL) afforded compound 4e (28 mg,
17%). m.p. 221-222 oC. 1HNMR (400MHz, DMSO-d6, ppm) δ 8.58 (s, 1 H), 8.02 (s, 1 H), 8.00 (d, J = 2.0 Hz, 1 H),
7.87 (d, J = 8.8 Hz, 1 H), 7.50 (t, J = 73.2 Hz, 1 H), 7.60 (s, 1 H), 7.56 (s, 1 H), 7.44 (dd, J = 8.8 and 2.0 Hz, 1 H),
7.15 (s, 1 H), 4.82 (s, 2 H), 3.88 (s, 3 H), 3.44 (s, 3 H); 13CNMR (100MHz, DMSO-d6, ppm) δ 160.6, 146.1, 145.3,
144.0, 142.5, 136.9, 136.0, 135.9, 134.0 (d, J = 172 Hz, 1 C), 131.9, 129.8, 124.9, 122.6, 122.0, 119.9, 118.5, 117.2
(t, J = 175 Hz, 1 C), 116.3, 114.8, 113.1, 68.1, 38.7, 27.9; MS m/z (ESI): [M+H]+ 509.8; Element Analysis for
C23H17F2N7O3S (%) C: 54.22%, H: 3.36%, N: 19.24% Found C: 53.26%, H: 3.32%, N: 19.22%.
16

4-(2-Methoxyethyl)-9-((6-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-a]pyridin-3-yl)thio)-2H-
[1,4]oxazino[3,2-c]quinolin-3(4H)-one (4f). Following the typical procedure 5, the reaction of 8a (50 mg, 0.22
mmol), 11b (56 mg, 0.17 mmol), Pd2(dba)3 (10 mg, 0.02 mmol), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene
(20 mg, 0.03 mmol), and sodium t-butoxide (25 mg, 0.25 mmol) in DMF (5 mL) afforded compound 4f (20 mg,
24%). m.p. 172-173 oC. 1HNMR (400MHz, DMSO-d6, ppm) δ 8.90 (s, 1 H), 8.70-8.62 (m, 1 H), 8.35 (s, 1 H), 8.10-
7.98 (m, 2 H), 7.95-7.79 (m, 3 H), 7.50 (dd, J = 9.2 and 2.1 Hz, 1 H), 4.96 (s, 2 H), 4.25 (t, J =5.2 Hz, 2 H), 3.86 (s,
3 H), 3.57 (t, J = 5.2 Hz, 2 H), 3.21 (s, 3 H); 13CNMR (100MHz, DMSO-d6, ppm) δ 160.9, 144.0, 143.3, 142.9,
142.6, 136.4, 136.0, 135.8, 134.6, 131.7, 129.0, 124.0, 122.1, 121.0, 119.8, 118.5, 116.3, 114.0, 112.0, 70.4, 69.5,
58.7, 46.3, 38.7; MS m/z (ESI): [M+H]+ 488.1; Element Analysis for C24H21N7O3S (%) C: 59.13%, H: 4.34%, N:
20.11% Found C: 59.11%, H: 4.32%, N: 20.15%.
9-((8-Fluoro-6-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]tri-azolo[4,3-a]pyridin-3-yl)thio)-4-(2-methoxyethyl)-2H-
[1,4]oxazino[3,2-c]quinolin-3(4H)-one (4g). Following the typical procedure 5, the reaction of 8b (75 mg, 0.30
mmol), 11b (84 mg, 0.25 mmol), Pd2(dba)3 (15 mg, 0.03 mmol), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene
(30 mg, 0.05 mmol), and sodium t-butoxide (30 mg, 0.30 mmol) in DMF (5 mL) afforded compound 4g (20 mg,
15%). m.p. 148-149 oC. 1HNMR (400MHz, DMSO-d6, ppm) δ 8.91 (s, 1 H), 8.08-7.98 (m, 2 H), 7.93 (d, J = 8.8Hz,
1 H), 7.67 (s, 1 H), 7.61 (s, 1 H), 7.50 (dd, J = 8.8 and 2.0 Hz, 1 H), 7.17 (d, J = 10.4 Hz, 1 H), 4.87 (s, 2 H), 4.23 (t,
J =5.2 Hz, 2 H), 3.95 (s, 3 H), 3.70 (t, J = 5.2 Hz, 2 H), 3.33 (s, 3 H); 13CNMR (100MHz, DMSO-d6, ppm) δ 161.5,
150.6, 148.5, 147.9, 143.4, 138.4, 137.3, 136.6, 134.0 (d, J = 175 Hz, 1 C), 131.2, 129.3, 124.0, 122.5, 121.0, 119.5,
118.6, 116.3, 114.0, 111.0, 70.2, 69.9, 58.5, 46.3, 38.9; MS m/z (ESI): [M+H]+ 506.1; Element Analysis for
C24H20FN7O3S (%) C: 57.02%, H: 3.99%, N: 19.40% Found C: 57.06%, H: 4.01%, N: 19.45%.
(R)-9-((8-Fluoro-6-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-a]pyridin-3-yl)thio)-2,3,12,12a-
tetrahydro-1H-pyrrolo[1',2':4,5][1,4]oxazino[3,2-c]quinolone ((R)-4i). Following the typical procedure 5, the
reaction of 8b (65 mg, 0.26 mmol), (R)-14 (80 mg, 0.26 mmol), Pd2(dba)3 (24 mg, 0.03 mmol), 4,5-
Bis(diphenylphosphino)-9,9-dimethylxanthene (30 mg, 0.05 mmol), and sodium t-butoxide (30 mg, 0.31 mmol) in
DMF (5 mL) afforded compound (R)-4i (15 mg, 12%). m.p. 133-134 oC. 1HNMR (400MHz, DMSO-d6, ppm) δ 8.41
(s, 1 H), 8.05 (s, 1 H), 8.00 (s, 1 H), 7.90 (d, J = 8.8 Hz, 1 H), 7.67 (s, 1 H), 7.58 (s, 1 H), 7.45 (d, J = 8.8 Hz, 1 H),
7.15 (d, J = 10.8 Hz, 1 H), 4.30 (dd, J = 10.0 and 2.4 Hz, 1 H), 3.95 (m, 4 H), 3.56 (t, J =8.8 Hz, 1 H), 3.40 (m, 2 H),
2.31 (m, 1 H), 2.02 (m, 1 H), 1.84 (m, 2 H); 13CNMR (100MHz, DMSO-d6, ppm) δ 150.8, 148.6, 147.5, 143.4,
17

138.0, 137.9, 136.0, 134.2 (d, J = 172 Hz, 1 C), 133.0, 129.6, 124.0, 122.5, 121.0, 119.9, 118.5, 116.0, 114.2, 112.6,
68.1, 62.2, 52.0, 38.7, 24.1, 22.2; MS m/z (ESI): [M+H]+ 473.9; Element Analysis for C24H20FN7OS (%) C: 60.88%,
H: 4.26%, N: 20.71% Found C: 60.86%, H: 4.22%, N: 20.75%.
(S)-9-((8-Fluoro-6-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-a]pyridin-3-yl)thio)-2,3,12,12a-tetrahydro-
1H-pyrrolo[1',2':4,5][1,4]oxazino[3,2-c]quinolone ((S)-4i). Following the typical procedure 5, the reaction of 8b
(65 mg, 0.26 mmol), (S)-14 (96 mg, 0.31 mmol), Pd2(dba)3 (29 mg, 0.03 mmol), 4,5-Bis(diphenylphosphino)-9,9-
dimethylxanthene (36 mg, 0.06 mmol), and sodium t-butoxide (36 mg, 0.37 mmol) in DMF (5 mL) afforded
compound (R)-4i (22 mg, 15%). m.p. 130-131 oC. 1HNMR (400MHz, DMSO-d6, ppm) δ 8.34 (s, 1 H), 8.04 (d, J =
1.2 Hz, 1 H), 7.94 (d, J = 2.0 Hz, 1 H), 7.81 (d, J = 8.8 Hz, 1 H), 7.64 (m, 2 H), 7.36 (dd, J = 8.8 and 2.4 Hz, 1 H),
7.12 (dd, J = 9.6 and 0.8 Hz, 1 H), 4.26 (dd, J = 10.0 and 3.2 Hz, 1 H), 3.92-3.86 (m, 4 H), 3.51 (m, 1 H), 3.37 (m, 2
H), 2.26 (m, 1 H), 1.98 (m, 1 H), 1.77 (m, 2 H); 13CNMR (100MHz, DMSO-d6, ppm) δ 151.6, 148.0, 147.5, 143.6,
138.4, 137.3, 136.8, 134.2 (d, J = 172 Hz, 1 C), 131.7, 129.3, 124.0, 122.5, 121.2, 119.8, 118.6, 116.3, 114.0, 111.9,
66.3, 60.5, 52.1, 38.7, 26.0, 21.7; MS m/z (ESI): [M+H]+ 473.9; Element Analysis for C24H20FN7OS (%) C: 60.88%,
H: 4.26%, N: 20.71% Found C: 60.85%, H: 4.23%, N: 20.75%.
Tyrosine Kinase Assays Tyrosine kinase assay was conducted with the help of the Beckman Coulter (Fullerton,
CA) Biomek FX robotic instrument or automated microtiter plate washer for most manipulations. In the assay, 20
mM test compounds or reference standard in DMSO was added to plate wells. The final DMSO concentration was
4%. Then, 5 mM ATP solution diluted in kinase reaction buffer was added to each well. Subsequently, the kinase
reaction was initiated by the addition of purified tyrosine kinase proteins diluted in 10 mL of kinase reaction buffer
o
solution. After incubation for 1 h at 37 C, 100 mL of antiphosphotyrosine (PY99) antibody (1:500 dilution) diluted
o
in TPBS containing 5 mg/ml BSA was added. After 1 h incubation at 37 C, finally, 100 mL of a solution containing
0.03% H2O2 and 2 mg/ml o-phenylenediamine in 0.1 M citrate buffer, pH 5.5, was added and samples were
incubated at room temperature until color emerged. The plate was read using a multiwell spectrophotometer
(SPECTRAMAX 190) at 400, 445, and 520 nm. The inhibition rate (%) was calculated using the following equation:
coumarin emission 445 nm/fluorescein emission 520 nm. IC50 values were obtained by Logit method and were
determined from the results of at least three independent tests. The results were analyzed by Graphpad Prism 5.0
software.
18

Cell Proliferation Assay SNU-5 HCCLM3, MHCC97-H, MHCC97-L, MKN-45, NCI-H1993 Huh-7, NCI-N87,
NCI-1975, or A549 cell lines were used for the cell proliferation assays. Cells were maintained in RPMI-1640
medium supplemented with 10% fetal bovine serum. Cells were plated in 96-well plates. On the next day, the tested
compounds were dosed at 0.6% DMSO and the cells were cultured for 3 days. At the end of incubation, cell survival
was determined by the CellTiter-Glo. The IC50 values were obtained from the growth curves using GraphPad Prism
5.0.
Molecular modeling. Generally, homology modeling was carried out using the MOE software (Chemical
Computing Group Inc.). Initial minimization was performed within the homology modeling function of MOE. The
model from MOE was minimized with a few thousand cycles of minimization using the ABNR (adopted-basis
Newton-Raphson) method. Ligands were modeled by positioning them in the active site in accordance with the
published crystal structure of the c-Met kinase complexed with SGX-523 (PDB code: 3dkf). The entire complex was
then subjected to alternate cycles of minimization and dynamics. Each dynamics run was short, about 3ps. The
intent was to get a satisfactory structure for the complex that was consistent with the published crystal structure.
Western Blot Assay Synergy H1 is a flexible monochromator-based multi-mode microplate reader that was
purchased from American Bio-Tek Company. Dissolve the relative compounds in DMSO to obtain the solutions
having a known concentration of 10 mM; store the solutions in -20 oC. Dilute the solutions using nutrient solution
without serous, to obtain the solutions having a known concentration of 0.1 um, 0.3 um and 1 um. Add drugs of
different concentrations to inoculated cells which are in the logarithmic phase on a six pore plates; collect the cells
based on the corresponding figure at certain time after that. Centrifuge the cell lysis buffer to obtain protein lysate;
transfer the protein lysate to cell lysate and put it in the boiling water until denaturation; store it under -80C or
conduct electrophoresis on SDC-PAGE; transfer the protein to PVDF membrane by semi-dry blotter; put the PVDF
membrane in blocking buffer for 1 hour at room temperature under sealed condition. Detect the cleaned PVDF
membrane by Odyssey fluorescence imaging.
In Vivo Efficacy Study BALB/c small nude mice, female for 6~7 weeks, are purchased from Shanghai SLAC
Laboratory Animal CO. LTD; feed the mice under grade SPF. MKN-45 or NCI-H1993 cells (5x106) were
inoculated subcutaneously into the BALB/c nude mice; when the tumor grows up to 100-150 mm3, allocate the mice
in groups randomly (D0). Respectively dose each group of mice by intragastric administration with dosage of 10
19

mg/kg, 30 mg/kg and 60 mg/kg; weigh the weight of mice twice every week and record the data. Gross tumor
volume (V): V=1/2ab2. In which, a means length; b means width.
Pharmacokinetic Assay BALB/c small nude mice, half male and half female for 6~7 weeks, are purchased from
Shanghai SLAC Laboratory Animal CO. LTD; feed the mice under grade SPF. HCCLM3 cells were inoculated
subcutaneously into the BALB/c nude mice; when the tumor grows up to 100-200 mm3, allocate the mice randomly.
Dose the mice by intragastric administration with single dose at 10 mg/kg, and sample the blood and tumor from the
mice at 0.25, 1, 2.0, 4.0, 6.0, 8.0, 12.0, 24.0, and 36.0 h after dosage. Centrifuge the blood to separate plasma. Detect
the content of drugs in plasma and tumor by a quantitative LC-MS assay in Suzhou Kang Run medical testing
company.
Long-term Toxicity Studies in Mice BALB/c small nude mice, half male and half female for 6~7 weeks, are
purchased from Shanghai SLAC Laboratory Animal CO. LTD; feed the mice under grade SPF. Successively dose
the mice by intragastric administration with dosage of 15 mg/kg, 75 mg/kg and 225 mg/kg once a day for 4 weeks;
feed the mice for 4 weeks to recovery after dosage; weigh the mice of the same group at the start of the test, after
administration and after the recovery time. Dissect the mice after bloodletting to die, weigh the corresponding
visceral organs, observe and check the tissue.
Acute Toxicity Studies in Mice BALB/c small nude mice, half male and half female for 6~7 weeks, are
purchased from Shanghai SLAC Laboratory Animal CO. LTD; feed the mice under grade SPF. Dose the mice by
intragastric administration with dosage of 300 mg/kg or 600 mg/kg by single-dose; observe the mice for 14 days
after administration; weigh the mice of the same group at the start of the test and after the observing time. Dissect
the mice after bloodletting to die, weigh the corresponding visceral organs, observe and check the tissue.
ACKNOWLEDGMENT
This work is supported by Jiangsu Province Hi-Tech Key Laboratory for Biomedical Research and Jiangsu Hansoh
Pharmaceutical Corporation together with a project funded by the Priority Academic Program Development of
Jiangsu Higher Education Institutions. Dr. Fang is thankful to the support of Natural Science Foundation of Jiangsu
Province (No. BK20151402) and the Priority Academic Program Development of Jiangsu Higher Education
Institutions.
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Graphical abstract
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