Synthesis and biological evaluation of 2-amino-5-aryl-3-benzylthiopyridine scaffold based potent c-Met inhibitors

Article abstract of DOI:10.1016/j.bmc.2013.07.032

A series of 2-amino-N-benzylpyridine-3-carboxnamides, 2-amino-N- benzylpyridine-3-sulfonamides and 2-amino-3-benzylthiopyridines against c-Met were designed by means of bioisosteric replacement and docking analysis. Optimization of the 2-amino-3-benzylthiopyridine scaffold led to the identification of compound (R)-10b displaying c-Met inhibition with an IC ₅₀ up to 7.7 nM. In the cytotoxic evaluation, compound (R)-10b effectively inhibited the proliferation of c-Met addictive human cancer cell lines (IC₅₀ from 0.19 to 0.71 μM) and c-Met activation-mediated cell metastasis. At a dose of 100 mg/Kg, (R)-10b evidently inhibited tumor growth (45%) in NIH-3T3/TPR-Met xenograft model. Of note, (R)-10b could overcome c-Met-activation mediated gefitinib-resistance, which indicated its potential use for drug combination. Taken together, 2-amino-3-benzylthiopyridine scaffold was first disclosed and exhibited promising pharmacological profiles against c-Met, which left room for further exploration.

Full text of DOI:10.1016/j.bmc.2013.07.032

Accepted Manuscript
Synthesis and biological evaluation of 2-amino-5-aryl-3-benzylthiopyridine
scaffold based potent c-Met inhibitors
Dengyou Zhang, Xiaowei Zhang, Jing Ai, Yun Zhai, Zhongjie Liang, Ying
Wang, Yi Chen, Chunpu Li, Fei Zhao, Hualiang Jiang, Meiyu Geng, Cheng Luo,
Hong Liu
PII:
S0968-0896(13)00653-6
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.07.032
BMC 10996
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
1 March 2013
14 July 2013
15 July 2013
Please cite this article as: Zhang, D., Zhang, X., Ai, J., Zhai, Y., Liang, Z., Wang, Y., Chen, Y., Li, C., Zhao, F.,
Jiang, H., Geng, M., Luo, C., Liu, H., Synthesis and biological evaluation of 2-amino-5-aryl-3-benzylthiopyridine
scaffold based potent c-Met inhibitors, Bioorganic & Medicinal Chemistry (2013), doi: http://dx.doi.org/10.1016/
j.bmc.2013.07.032
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Synthesis and biological evaluation of 2-amino-5-aryl-3-
benzylthiopyridine scaffold based potent c-Met inhibitors
Dengyou Zhanga,†, Xiaowei Zhangb,† Jing Aib,†, Yun Zhaia, Zhongjie Lianga, Ying Wangb,
Yi Chenb, Chunpu Lia, Fei Zhaoa, Hualiang Jianga, c, Meiyu Geng b,*, Cheng Luo a, *and
Hong Liua,*
a State Key Laboratory of Drug Research, CAS Key Laboratory of Receptor Research,Shanghai Institute of Materia Medica, Shanghai Institutes for
Biological Sciences, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai 201203, PR China
b Division of Anti-Tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences,
Shanghai 201203, PR China
c School of Pharmacy, East China University of Science and Technology, Shanghai 200237, PR China
† These authors contributed equally to this study.
*
Correspondence: Hong Liu, Meiyu Geng and Cheng Luo
E-mail: hliu@mail.shcnc.ac.cn or mygeng@mail.shcnc.ac.cn or cluo@mail.shcnc.ac.cn
Keywords:
receptor tyrosine kinase
c-Met
2-amino-5-aryl-3-benzylthiopyridine
A B S T R A C T
A series of 2-amino-N-benzylpyridine-3-carboxnamides, 2-amino-N-benzylpyridine-3-sulfonamides and 2-amino-3-
benzylthiopyridines against c-Met were designed by means of bioisosteric replacement and docking analysis. Optimization
of the 2-amino-3-benzylthiopyridine scaffold led to the identification of compound (R)-10b displaying c-Met inhibition
with an IC50 up to 7.7 nM. In the cytotoxic evaluation, compound (R)-10b effectively inhibited the proliferation of c-Met
addictive human cancer cell lines (IC50 from 0.19 - 0.71 μM) and c-Met activation-mediated cell metastasis. At a dose of
100 mg/Kg, (R)-10b evidently inhibited tumor growth (45%) in NIH-3T3/TPR-Met xenograft model. Of note, (R)-10b
could overcome c-Met-activation mediated gefitinib-resistance, which indicated its potential use for drug combination.
Taken together, 2-amino-3-benzylthiopyridine scaffold was first disclosed and exhibited promising pharmacological
profiles against c-Met, which left room for further exploration.
© 2009 Elsevier Ltd. All rights reserved
1. Introduction
c-Met, also known as hepatocyte growth factor receptor, is a unique member of receptor tyrosine kinase. MET
amplification, MET mutation, HGF binding or interaction with other membrane receptors could lead to c-Met activation.1
Aberrant HGF/c-Met signaling has been identified in a wide range of most solid tumors and hematological malignancies.2,3
Furthermore, c-Met signaling might be responsible for the resistance mechanism in chemotherapy, radiotherapy, other RTK
based target therapy and so forth.4 To date, the main strategies employed to interrupt c-Met signaling pathway involve in
blocking the interaction between c-Met and HGF via antibodies5-16 or antagonists17-21 and interfering with the active site of
the kinase domain with small-molecule kinase inhibitors.22-47 Among the various strategies, targeting c-Met with small
molecules has captured extensive attention, resulting in a respectable number of compounds reaching the clinical stages.42
Recently, 2-aminopyridine based c-Met inhibitors have been widely investigated (Figure 1).26,28,43-47 Since researchers at
Pfizer reported the 2-amino-3-benzyloxypyridine (Figure 1, 2) was an excellent scaffold against c-Met , which culminated
in the discovery of crizotinib,26 pharmaceutical research community was keen to synthesize aminopyridine analogs in
pursuit of effective c-Met inhibitors. OSI presented a series of 4,5- or 2,3-fused aminopyridine derivatives44 (3, 4) with IC50
< 0.1 μM. Another interesting modification was the addition of one atom to the amino pyridine core (5) reported by
Xcovery.47 Lee et al. also reported a series of c-Met inhibitors with c-Met IC50 up to 8 nM in which C-3 position of amino
pyridine was substituted with benzoxazole (6).28
Figure 1
1

Generally, from the viewpoint of the interaction between small molecules and c-Met protein, these structures feature an
aminopyridine core responsible for H-bonding with the hinge region, an aryl fragment extending into the inside pocket and
a substituent R facing to the solvent accessible region (Figure 1). Furthermore, a flexible sp3 (2, 3, 5) or rigid sp2 (4, 6)
functionality as a linker was compatible at the C-3 position of the aminopyridine core. Despite various modifications of the
central aminopyridine cores, the aryl fragments and substituents R pursued by medicinal chemists, much less attention was
paid to the modification of the linker X between the aminopyridine core and aryl fragment. Therefore, there still remains
room for exploration of the new linkers, which might give rise to new generation of aminopyridine based c-Met inhibitors.
Undoubtedly, application of bioisosteres still plays an important role in contemporary drug design, which has often been
deployed to improve potency, alter selectivity and address pharmacokinetic issues or to obtain intellectual property. Here,
we questioned whether the known O-linker (2, 3, 5)could be simply replaced by other flexible atoms and the rigid linker (4,
6) could be changed into other bioisosteric functionalities. In combination with full consideration of the bio-isosteres and
synthetic feasibility, we propose three novel amino pyridine scaffolds with amide (8), sulfonamide (9) and S-linkers (10),
respectively (Figure 1). Besides, it is reasonable that 2,6-dichloro-3-fluorobenzyl fragment was kept due to its potential π-π
interaction with the residue Tyr1230 of c-Met kinase.
Initially, molecular modeling research was performed to ascertain the designed linker. Binding modes of representative
compounds (Table 1, 8b, 9b and 10b) were compared with a known c-Met inhibitor (crizotinib),26 as shown in Figure 2.
The amide linker compound 8b (Figure 2A) keeps the key H-bonding with Met 1160 and Pro1158. The longer bond length
of amide than that of oxygen orients the 2,6-dichloro-3-fluorobenzyl fragment forming parallel-displaced rather than face to
face π-π interaction (in crizotinib) with Tyr 1230. In addition, the 5-pyrazol-4-yl group goes through a lipophilic tunnel
surrounded by Ile 1084 and Tyr 1159. With regard to the sulfur linker, compound 10b (Figure 2C) displayed almost an
identical conformation as crizotinib. That the C-S bond is approximately 0.4 Å longer than C-O bond has a little impact on
π-π interaction between 2,6-dichloro-3-fluorobenzyl group and Tyr 1230, which may be ascribed to the smaller C-S-C angle
fitting the 2,6-dichloro-3-fluorobenzyl group into a right position for π-π stacking. Meanwhile, the aminopyridine core in
compound 10b H-bonds well with the hinge region. While for the sulfonamide linker compound 9b (Figure 2B and 2D), it
could adopt two completely different conformations in the active site. One conformation was reasonable and consistent with
crizotinib as respected. Yet the other one showed the almost inverted conformation, suggesting that the sulfonamide linker
might be incompatible with the inside pocket. Taken together, these observations theoretically indicated that the amide,
sulfur and sulfonamide linkers designed here were reasonable and might furnish novel promising c-Met inhibitors.
Figure 2
To test the design idea, a project for the synthesis of these novel aminopyridine scaffolds was launched. The scaffolds of 8
and 9 were readily constructed from corresponding starting materials whereas the preparation of the scaffold of 10 was
relatively complicated due to limited synthetic approaches. One indirect method for the construction of 10 has been
developed from the known N,N'-dimethyl-1-[(2-nitropyridin-3-yl)sulfanyl]formamide,48 which was used as a masked
synthon of 2-nitropyridine-3-thiol. Herein, we disclosed our efforts to the synthesis and biological evaluation of these new
amino pyridine derivatives.
2. Chemistry
The construction of 2-amino-N-benzyl-5-bromopyridine-3-carboxamide core 8a began with 1-(2,6-dichloro-3-
fluorophenyl)ethanone 11, as presented in Scheme 1. Reduction of 2,6-dichloro-3-fluoroacetophenone with NaBH4
afforded benzyl alcohol, which was converted to its mesylate 12. Substitution of 12 with sodium azide, followed by
reduction of the resulting benzyl azide with Zn/NH4Cl,49 delivered intermediate 13. Condensation of 13 with 2-amino-5-
bromopyridine-3-carboxylic acid smoothly furnished 8a.
Scheme 1
The chlorosulfonation50 of 2-amino-5-bromo pyridine afforded 2-amino-5-bromopyridine-3-sulfonyl chloride 15, which was
immediately used for coupling with benzyl amine 13 to give 2-amino-N-benzyl-5-bromopyridine-3-sulfonamide core 9a
(Scheme 2).
Scheme 2
As shown in Scheme 3, compared with 8a and 9a, the synthesis of 3-(benzylsulfanyl)-5-bromopyridin-2-amine core 10a
was not straightfoward. Initially, we aimed to obtain 2-nitropyridine-3-thiol by hydrolysis of the known intermediate 18.48
Unfortunately, attempt to purify the 2-nitropyridine-3-thiol on silica gel column proved unsuccessful due to its oxidative
2

dimerization. Therefore, we attempted to perform the reaction in one-pot sequential fashion in order to take advantage of
the thiol anion in situ. After extensive attempts, the intermediate 20 was readily prepared from 18 through sequential
hydrolysis of the dimethylcarbamothioate, trapping of the thiol anion by benzyl methanesulfonate 12 and reduction of the
nitro group. Next, bromination of 20 smoothly furnished 10a.
Scheme 3
With the key intermediate 8a, 9a and 10a in hand, we set out to derivatize the bromide on the C-5 position by employing
the pyrazole fragments, since they were frequently used to modulate the chemical properties and biological activity of
amino pyridine based c-Met inhibitors.42 First, a series of pyrazolyl-4-boronic esters were synthesized as depicted in
Scheme 4. Alkylation of 4-bromo-1H-pyrazole, followed by Miyaura boration, delivered boronic esters 25b-e. Acylation of
commercial available 23b proceeded well to afford 25a.
Scheme 4
Next, introduction of the pyrazole fragments at the C-5 position of 8a, 9a and 10a was readily achieved by Suzuki coupling
(Scheme 5). Further derivatization of the N-1 position of the pyrazole was also conducted. The intermediate 31 was
converted to its corresponding amide 10g through hydrolysis of the ester, followed by condensation of the resulting acid
with dimethylamine hydrochloride. The deprotection of the Boc groups of 28-30 smoothly gave compounds 8b, 9b and
10b. 10b was subsequently transformed to compounds 10h-j.
Scheme 5
To gain insight into SAR at C-5 position of 10a, other heteroaryl and aryl fragments were introduced through Suzuki
coupling of the intermediate 10a with corresponding boronic esters (10k-10o). Compounds 36a-l were prepared in a similar
manner as described for compound 10g in Scheme 6.
Scheme 6
The chiral synthesis of (R)-10b was depicted in Scheme 7. Reduction of the ketone 11 furnished the racemic alcohol
intermediate 37. Chemical resolution of 37 afforded the (S)-alcohol 38 by use of the Boc protected proline. Then the (R)-
10a intermediate was smoothly obtained in a similar manner as described for 10a in Scheme 3. Suzuki coupling of (R)-10a
with the boronic ester 25d, followed by the removal of the Boc protective group, gave the desired compound (R)-10b.
Scheme 7
3. Biology
3.1. ELISA kinase assay.
kinase activity was evaluated according to the enzyme-linked-immunosorbent assay. Briefly, 20 μg/mL poly (Glu,Tyr)4:1
(Sigma) was pre-coated as a substrate in 96-well plates. 50 μL 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, 1mmol/L DTT) was added to
each well. 1μL of various concentrations of indicated compounds diluted in 1% DMSO (v/v) (Sigma) were added to each
reaction well. 1% DMSO (v/v) was used as negative control. The kinase reaction initiated after the addition of purified
tyrosine kinase proteins diluted in 49 μL of kinase reaction buffer solution. After incubation for 60 min at 37°C, the plate
was washed three times with Phosphate Buffered Saline (PBS) containing 0.1% Tween 20 (T-PBS). 100 μL anti-
phosphotyrosine (PY99) antibody (1:1000 diluted in 5 mg/mL BSA T-PBS) was then added. After 30 min incubation at
37°C, the plate was washed three times. 100 μL horseradish peroxidase-conjugated goat anti-mouse IgG (1:2000 diluted in
5 mg/mL BSA T-PBS) was added. The plate was then incubated at 37°C for 30 min, and washed 3 times. 100 μL of a
solution containing 0.03% H2O2 and 2 mg/ml o-phenylenediamine 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 color changed, and the plate was read using a multi-
well spectrophotometer (SpectraMAX 190, Molecular Devices) at 490 nm. The inhibition rate (%) was calculated using the
following equation: [1-(A490/A490 control)] x 100%. IC50 values were calculated from the inhibition curves. For ATP
competition assay, various concentrations of ATP were diluted for the kinase reaction. TheLineweaver-Burke plot was
drawn by Prism.
3.2. Cell proliferation assay.
3

Cells were seeded in 96-well tissue culture plates. On the next day, cells were exposed to various concentrations of
compounds and further cultured for 72h. Finally, cell proliferation was determined by using sulforhodamineB (SRB;
Sigma) or CCK-8kit (Dojindo).
3.3. Western blot analysis.
Cells were cultured under regular growth conditions to exponential growth phase. Then the cells were treatment with
indicated concentration of compound for 2h at 37oC and lysed in 1×SDS sample buffer. Those cell lysates were
subsequently resolved on 10% SDS-PAGE, and transferred to nitrocellulose membranes. Membranes were probed with,
phospho c-Met and c-Met, phospho-EGFR and EGFR, phospho-ERK1/2 and ERK1/2, phospho-AKT and AKT (All from
Cell Signaling Technology) antibodies then subsequently with HRP-conjugated anti-rabbitIgG. Immunoreactive proteins
were detected using ECL Plus (GE Healthcare) and images were pictured by ImageQuant LAS 4010 (GE Healthcare).
3.4. Cell cycle analysis.
Cells were treated with compounds at indicated concentration for 24hrs, typsinized and fixed1h at -20oC by 70% ethanol.
Then, the fixed cells were stained with propidium iodide (10 μg/mL)for 30 minutes at 37 and cell cycle variables analyzed
by fluorescence-activated cell sorting analysis (BD).
3.5. Wounding Heal Assay.
MDCK cells were planted into the wells of 12-well plates and cultured 24-48 h until a uniform monolayer formed. All
wounding assays were performed in a serum-free medium by first pre-incubating the cells in the serum-free medium for 30
min before the start of the experiments. A micropipette was used to create a wound in the monolayer by scraping. After
washing to remove the detached cells, compounds at indicated concentration were added. Subsequently, the cells were
incubated with and without 50 ng/mL recombinant human HGF and placed in a cell incubator (37°C). They were then
image captured at 0 and 10h after incubation using phase-contrast microscope.
3.6. MDCK Scattering Assay.
MDCK cells were plated into 96-well plate at a density of 1,500 cells per well. After 24hrs incubation, MDCK cells formed
clusters; then, they were cultured with compounds and 50 ng/mL recombinant human HGF in FBS-free medium for another
24hrs.Cells were fixed by 4% paraformaldehyde for 15min at room temperature, and then stained by 0.2% violet purple,
washed with water, and allowed to dry before taking photos. Pictures were taken under microscope.
3.7. In vivo Antitumor Activity Assay.
Animal experiments were performed according to institutional ethical guidelines of animal care. Well-developed tumors
were cut into 1-mm3 fragments and transplanted s.c. into the right flank of nude mice using a trocar. When the tumor
volume reached 100 to 150 mm3, the mice were randomly assigned into control and treatment groups. Control groups were
given vehicle alone, and treatment groups received (R)-10b or crizotinib as indicated doses via oral administration 7 days
per week for 2 weeks. The sizes of the tumors were measured twice per week using microcaliper. The tumor volume (TV)
was calculated as: TV = (length × width2)/2, and the relative tumor volume (RTV) was calculated as: RTV = Vt/V0, where
Vt is the tumor volume on the day measured and V0 is the tumor volume on the first day of treatment. Relative tumor
volume was shown on indicated days as the median relative tumor volume SE indicated for groups of mice. T/C values
were calculated on the final day of study for drug-treated compared with vehicle-treated mice as (RTVTreated/RTVControl
)
×100%. Significant differences between the treated versus the control groups (P ≤ 0.05) were determined using Student’s t-
test.
4. Results and discussion
4.1 Identification and optimization of the 2-amino-3-benzylthiopyridine scaffold
As these novel amino pyridine derivatives against c-Met have not been previously synthesized and evaluated, the initial
goal was to identify the optimal linker. The c-Met enzymatic activities of 8b-d, 9b and 10b-10j were summarized in Table
1. To our delight, compound 10b exhibited c-Met inhibition with an IC50 of 16.4 nM, whereas compound 8b was nearly 20-
fold less active and compound 9b displayed no activity at 10 μM. Obviously, these data suggested that sulfur linker (one
atom) was more robust than amide and sulfonamide linkers (two atoms). The encouraging results prompted us to investigate
SAR about substituents on the pyrazole whilst keeping the sulfur as linker. It was observed that the incorporation of
sulfonyl (10h) and carbonyl (10i-j) on piperidine of 10b resulted in a remarkable decrease in potency which indicated the
basicity of piperidine might be an important factor for c-Met inhibition. Appending non-basic substituents at the N-1
position of the pyrazole (10c-e and 10g) was well tolerated, albeit less active than analog 10b. However, introduction of an
4

electron-withdraw group (10f) was not beneficial for the potency presumably due to the significant change of electron
density of the pyrazole.
Table 1
Subsequently, we extended our attention to explore SAR about other aryl/heteroaryl derivatives at the C-5 position of the 3-
(benzylthio)pyridin-2-amine scaffold. The result was shown in Table 2. Introduction of the “naked” pyrimidine (10k)
displayed moderate c-Met inhibition at 0.15 μM. The pyridinyl analogs 10l and 10m with the incorporation of polar
fragments were more effective and the IC50 reached to 26.5 nM and 66.2 nM respectively. Among the phenyl derivatives
(10n-o and 36a), benzamide analog 36a demonstrated potent c-Met inhibition with an IC50 of 25.8 nM. At this juncture, it
seemed that various aryl/heteroaryl substituents such as phenyl, pyrazoyl and pyridinyl were compatible at the C-5 position.
Table 2
Given the promising enzymatic activity and easy derivation, benzamide analogs at the C-5 postion were prepared for more
in-depth SAR exploration (Table 3). The simple change of the 1-methylpiperazine to morpholine (36b) or dimethyl amine
(36c) caused nearly 2-fold loss of activity. Introduction of the isopropyl, methylsulfonyl or hydroxyethyl group on the
piperazine also proved to be inefficient (36d-f). Replacement of the 1-methylpiperazine with the 4-(pyrrolidin-1-
yl)piperidine (36g) retained the potency at 22.1 nM. The N-mono substituted amide analog 36h showed an IC50 of 101 nM,
suggesting N,N•-disubstituents were preferred here. Bearing a fluoride atom adjacent to amide (36i-36l) eroded the c-Met
inhibition.
Table 3
Subsequently, the preliminary effects of 2-amino-3-benzylthiopyridine derivatives were evaluated on c-Met
phosphorylation and c-Met dependent proliferation in MKN and NIH3T3-TPR-Met cell lines (data not shown). The
inhibitory effect of compound 10b was clearly preferred over than other analogs. Therefore, compound 10b was chosen for
the further evaluation. In addition to consideration of configuration affecting the c-Met activity, the two enantiomers of
compound 10b were prepared. Apparently, the R enantiomer of 10b (for R, IC50 = 7.7 nM, for S, IC50 = 171.4 nM) was more
potent. Therefore, the (R)-10b was chosen for further evaluation.
4.2 Kinase Selectivity of (R)-10b
The selectivity of (R)-10b against tyrosine kinase activity was assessed by screening against c-Met family member, Ron,
along with other 15 tyrosine kinases. In contrast to its high potency against c-Met (IC50 = 7.7 nM), (R)-10b showed more
than 40-fold less potency against RON, Axl, ALK, and barely inhibited kinase activity against the other 13 tested tyrosine
kinases (IC50 > 1 μM)(Table 4). All these suggested that (R)-10b is a relatively selective inhibitor of c-Met.
Table 4
4.3 (R)-10b is an ATP Competitive Inhibitor of Met
To examine whether (R)-10b acts as an ATP competitive inhibitor, we evaluated the inhibitory potency of (R)-10b on
kinase enzymatic activities using competitive inhibitory assay by introduction of different concentrations of ATP. As shown
in Figure 3, Lineweaver-Burk plot for inhibition of Met tyrosine kinase by (R)-10b with respect to ATP concentration
showed all curves intersecting the y-intercept at zero, which indicates a competitive mechanism of inhibition.
Figure 3
4.4 (R)-10b Inhibits c-Met Phosphorylation and its Downstream Signaling Pathways
Furthermore, we investigated c-Met-targeting activity of (R)-10b in vitro and in vivo. Both natural (MKN-45, SNU-5
human gastric carcinoma cells and EBC-1 NSCLC cells that express elevated levels of constitutively active c-Met) and
genetical-modified (NIH-3T3/TPR-Met cells and BaF3/TPR-Met cells that stably express a constitutively active, ligand-
independent, oncogenic form of c-Met) c-Met expressing cell lines were selected. We found that (R)-10b inhibited c-Met
phosphorylation in a dose-dependent manner in all these cell lines (Figure 4). Furthermore, p44/42 and AKT, the key
downstream molecules of c-Met signaling were also inhibited upon treatment with (R)-10b, which support that (R)-10b
inhibits not only the c-Met activity, but subsequent c-Met downstream signaling as well.
Figure 4
4.5 (R)-10b Inhibits the Proliferation of Met-Addicted Human Cancer Cells
5

Further an extended panel of human cancer cell lines with different settings of c-Met expression/ activation was chosen to
test the anti-proliferation effect of (R)-10b. As shown in Table 5, (R)-10b effectively inhibited the proliferation of human
cancer cell lines, however, the IC50 values varied widely among these cancer cells. Notably, (R)-10b showed IC50 values of
0.29 0.05, 0.19 0.02, and 0.71 0.16 μM, respectively in c-Met addictive cancer cell lines MKN45, SNU-5 and EBC-1.
In contrast, (R)-10b exerted much less anti-proliferative effects with IC50 values around 10-20 μM in low c-Met-expressed
and activated cell lines such as MDA-MB-231, MCF-7 and NCI-H661. Moreover, (R)-10b significantly inhibited
proliferation of NIH-3T3/TPR-Met cells and BaF3/TPR-Met cells, with IC50 values of 0.61 0.16 μM and 0.58 0.15 μM,
respectively.
Table 5
4.6 (R)-10b Induces G1-phase Cell Cycle Arrest in MKN-45 and Ba/F3-TPR-Met Cells
To investigate the mechanisms associated with (R)-10b’s anti-proliferative activity, we explored the effects of (R)-10b on
the cell cycle distribution in MKN-45 cell and Ba/F3-TPR-Met cell. After cells were treated with vehicle or various
concentrations of (R)-10b for 24 h, DNA content in control group displayed a typical histogram observed in exponentially
growing cells, while increasing concentration of (R)-10b arrested cells in G1 phase, with 60.1% of cell population in G1
phase in the presence of 1 μM (R)-10b in MKN-45 cells (39.1% in control group) (Figure 5A). Consistently, (R)-10b
induced G1-phase cell cycle arrest, with 69.6% of cell population in G1 phase in the presence of 1 μM (R)-10b in
BaF3/TPR-Met cells (26.6% in control group) (Figure 5B). All these indicated that cell cycle arrest contributed to the anti-
proliferative effects of (R)-10b.
Figure 5
4.7 (R)-10b Inhibits HGF-induced MDCK Cell Scattering and Cell Migration
It’s well known that HGF/c-Met axis promotes cell scattering, a hallmark of cancer invasiveness and metastasis. It is well
documented that MDCK cells, which normally grow in clusters, exhibit disruption and scattering of cell colonies on HGF
treatment. To determine the effect of (R)-10b on cell scattering behavior, MDCK cells were treated with HGF in the
presence or absence of the agent. As shown in Figure 6A, MDCK cells treated with (R)-10b exhibited inhibitory effect on
HGF-induced cell scattering in a dose-dependent manner. At the dose of 3μM, this compound almost completely prevented
cell spreading, as evidence by the fact that MDCK cells remain in tight clusters. The enhanced cell migratory ability is
involved in the cell scattering phenotype. To examine the effect of (R)-10b on cell motility, MDCK cells were treated with
HGF in the presence of (R)-10b, and the migration was determined in vitro by a wound-healing assay. As shown in Figure
6B, (R)-10b strongly suppressed HGF-induced cell motility. These results indicated that (R)-10b strongly inhibited c-Met
activation-mediated cell metastasis.
Figure 6
4.8 (R)-10b Overcomes Drug Resistance in Gefitinib-Resistant Cell Line HCC827/GR5
Since MET amplification was previously shown to cause EGFR TKI acquired resistance,51 we further investigated whether
(R)-10b could overcome EGFR TKI resistance mediated by increased Met signaling. An EGFR TKI gefitinib resistant cell
line HCC827/GR5, which derives from a gefitinib sensitive cell line HCC827 and gets acquired resistance due to MET
amplification was chosen. As expected, the combination of (R)-10b and gefitinib substantial inhibited the growth of
HCC827/GR5 cells, whereas neither agent alone led to growth inhibition (Figure 7A).Moreover, in the resistant cells,
gefitinib or (R)-10b effectively reduced EGFR phosphorylation or c-Met phosphorylation, and they had no effects on AKT
and p44/42 phosphorylation. (R)-10b in combination with gefitnib fully suppressed the phosphorylation of AKT and p44/42
(Figure 7B). These results indicated that (R)-10b could overcome c-Met-activation mediated gefitinib-resistance, which
implies its potential use for drug combination.
Figure 7
4.9 (R)-10b Inhibits Tumor Growth in vivo
To evaluate the Met-inhibitory efficacy of (R)-10b in animal model, anti-tumor activity of the compound was investigated
in the NIH-3T3/TPR-Met xenograft model. In this model, tumor growth is specifically driven by constitutive c-Met
activation. We found that oral administration of (R)-10b at indicated doses for 14 days evidently inhibited tumor growth
(tumor growth inhibition of 100 mg/kg was 45.5%) (Figure 8). In addition, (R)-10b was well tolerated, as evidenced by no
significant loss of mice body weight after treatment (data not shown).
Figure 8
6

4.10 Pharmacokinnetic (PK) Profile of (R)-10b
The PK profile of compound (R)-10b was evaluated in rat after single iv (2 mg/kg) and po (10 mg/kg) administration. This
compound displayed a half-life time of 5.7 h and a poor oral bioavailability (F = 12.8%), which was attributed to its high
clearance (53 mL/min/Kg) and volume of distribution (20 L/Kg). This unfavorable PK profile of (R)-10b might account for
its moderate inhibition of tumor growth.
5. Conclusions
On the basis of reported aminopyridine c-Met inhibitors, a series of 2-amino-N-benzylpyridine-3-carboxnamides, 2-amino-
N-benzylpyridine-3-sulfonamides and 2-amino-3-benzylthiopyridines were designed by means of bioisosteric replacement
and docking analysis. We successfully prepared these novel aminopyridine scaffolds. Of particular note, deployment of
N,N'-dimethyl-1-[(2-nitropyridin-3-yl)sulfanyl]formamide as a masked synthon of 2-nitropyridine-3-thiol is the key to
construction of 2-amino-3-benzylthiopyridine derivatives.
Initial c-Met enzymatic assay found 2-amino-3-benzylthiopyridines were more robust than 2-amino-N-benzylpyridine-3-
carboxnamides and 2-amino-N-benzylpyridine-3-sulfonamides. Subsequent optimization gave rise to a racemic compound
10b with potent c-Met enzymatic IC50 at 16.4 nM. Further evaluation revealed its R configuration compound (R)-10b
displayed c-Met inhibition with an IC50 up to 7.7 nM. (R)-10b was demonstrated to act as an ATP competitive inhibitor.
(R)-10b exhibited dose-dependent inhibition on c-Met phosphorylation and corresponding downstream signaling pathways
as well. In the cytotoxic evaluation, compound (R)-10b effectively inhibited the proliferation a panel of human cancer cell
lines such as MKN45, SNU-5 and EBC-1cell lines. (R)-10b also demonstrated inhibition of c-Met activation mediated cell
metastasis. It was worth mentioning that (R)-10b could overcome c-Met-activation mediated gefitinib-resistance, which
implies its potential use for drug combination. At dose of 100 mg/Kg, (R)-10b evidently inhibited tumor growth (45%) in
NIH-3T3/TPR-Met xenograft model.
Although aminopyridine based c-Met inhibitors have been widely investigated, the scaffolds of 2-amino-N-benzylpyridine-
3-carboxnamide, 2-amino-N-benzylpyridine-3-sulfonamide and 2-amino-3-benzylthiopyridine against c-Met were first
prepared and biologically evaluated. Among these, 2-amino-3-benzylthiopyridine scaffold exhibited promising
pharmacological profiles, which warrant further exploration.
6. Experimental
Unless otherwise noted, the reagents (chemicals) were purchased from commercial sources, and used without further
purification. Analytical thin layer chromatography (TLC) was performed on HSGF 254 (0.15-0.2 mm thickness).
Compound spots were visualized by UV light (254 nm). Column chromatography was performed on silica gel FCP 200-300
or preparative HPLC (Agilent 1200 series HPLC system, ZORBAX Eclipse XDB-C18, 9.4 × 250 mm, 5 μm). Mircrowave
reactions were carried on CEM reactor. NMR spectra were run on 300 or 400 MHz instrument. Chemical shifts were
reported in parts per million (ppm, δ) downfield from tetramethylsilane. Proton coupling patterns are 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 measured on spectrometer. HPLC analysis of all compounds listed in Tables 1-3 was carried out on an
Agilent 1100 Series HPLC with an Agilent Zorbax Eclipse XDB-C18 (4.6 mm × 150 mm, 5 μm) reversed phase column
with two solvent systems (MeOH/H2O and MeOH/H2O/NH4OAc). All the assayed compounds displayed a purity of 95-99
% in both solvent systems.
6.1. 1-(2,6-Dichloro-3-fluorophenyl)ethyl methanesulfonate (12).
To a solution of 1-(2,6-dichloro-3-fluorophenyl)ethan-1-one 11 (20.00 g, 96.60 mmol) in 100 mL of MeOH was added
portionwise NaBH4 (7.31 g, 193.21 mmol). The resulting mixture was stirred at room temperature for 2h and then quenched
with 20 ml of water. After the removal of MeOH under vacuum, the residue was extracted with EA and washed with brine.
The organic layer was dried over anhydrous Na2SO4 and concentrated under vacuum to afford 1-(2,6-dichloro-3-
1
fluorophenyl)ethan-1-ol, which was pure enough for use in the next step. H NMR (400 MHz, CDCl3) δ 7.26 (dd, J = 4.8,
8.8 Hz, 1H), 7.02 (dd, J = 8.0, 8.8 Hz, 1H), 5.57 (q, J = 6.8 Hz, 1H), 2.86 (br, 1H), 1.64 (d, J = 6.8 Hz, 3H).
To a solution of 1-(2,6-dichloro-3-fluorophenyl)ethan-1-ol (19.00 g, 90.89 mmol) in 150 ml of CH2Cl2 was added Et3N
(13.27 mL, 95.43 mmol) and catalytic amount of DMAP. The resulting solution was cooled in an ice bath and added
dropwise MsCl (7.39 mL, 95.43 mmol). After complete addition of MsCl, the reaction mixture was maintained in the ice
bath for 1 h and then 30 ml of water was added to the reaction mixture. The organic phase was washed with brine, dried
over anhydrous Na2SO4 and concentrated under vacuum to afford 1-(2,6-dichloro-3-fluorophenyl)ethyl methanesulfonate
(12), which was pure enough for use in the next step. Yield: 84%; 1H NMR (300 MHz, CDCl3) δ 7.33 (dd, J = 4.8, 9.0 Hz,
1H), 7.12 (dd, J = 8.1, 9.0 Hz, 1H), 6.45 (q, J = 6.9 Hz, 1H), 2.91 (s, 3H), 1.84 (d, J = 6.9 Hz, 3H).
6.2. 1-(2,6-Dichloro-3-fluorophenyl)ethan-1-amine (13).
7

To a solution of 1-(2,6-Dichloro-3-fluorophenyl)ethyl methanesulfonate 19 (500 mg, 1.74 mmol) in 4 mL of DMF was
added NaN3 (226.4 mg, 3.48 mmol). The resulting mixture was stirred at 50 ºC for 1 h and diluted with 20 ml of water. The
mixture was extracted with EA (2 × 10 mL). The organic phase was washed with brine, dried over anhydrous Na2SO4 and
concentrated under vacuum to afford 2-(1-azidoethyl)-1,3-dichloro-4-fluorobenzene, which was pure enough for use in the
next step. 1H NMR (400 MHz, CDCl3) δ 7.31 (dd, J = 4.8, 8.8 Hz, 1H), 7.08 (dd, J = 8.0, 8.8 Hz, 1H), 5.55 (q, J = 6.8 Hz,
1H), 1.65 (d, J = 6.8 Hz, 3H).
2-(1-Azidoethyl)-1,3-dichloro-4-fluorobenzene (300 mg, 1.28 mmol) was suspended in 4 mL of EtOH/H2O (v/v, 3/1). To
this suspension was added Zn powder (109 mg, 1.67 mmol) and NH4Cl (171.4 mg, 3.20 mmol). The reaction mixture was
heated at 80 ºC for 1 h. Then the reaction mixture was filtered off and concentrated under vacuum. The residue was
partitioned between ethyl acetate and water. The organic phase was washed with brine, dried over anhydrous Na2SO4 and
concentrated under vacuum to afford 1-(2,6-Dichloro-3-fluorophenyl)ethan-1-amine (19), which was pure enough for use in
the next step. Yield: 71%; 1H NMR (400 MHz, CDCl3) δ 7.25 (dd, J = 4.8, 8.8 Hz, 1H), 6.99 (dd, J = 8.0, 8.8 Hz, 1H), 4.90
(q, J = 6.8 Hz, 1H), 2.32 (s, 2H), 1.56 (d, J = 6.8 Hz, 3H).
6.3. 2-Amino-5-bromo-N-[1-(2,6-dichloro-3-fluorophenyl)ethyl]pyridine-3-carboxamide (8a).
2-Amino-5-bromonicotinic acid (130 mg, 0.60 mmol) was dissolved in 5 mL of DMF. The solution was cooled in an ice
bath. Then to the solution were successively added HATU (228 mg, 0.66 mmol), DIPEA (126 μL, 0.72 mmol), 1-(2,6-
dichloro-3-fluorophenyl)ethan-1-amine (150 mg, 0.72 mmol). The resulting solution was stirred at room temperature for 2 h
and then the saturated sodium bicarbonate solution was added. The mixture was extracted with ethyl acetate (EA). The
combined organic layers were dried over anhydrous Na2SO4 and concentrated under vacuum. The crude product was
purified by flash column chromatography (0-20% EA/PE gradient) to afford 170 mg of 8a as a white solid (69.5% yield).
1H NMR (300 MHz, DMSO-d6) δ 9.12 (d, J = 5.4 Hz, 1H), 8.27 (d, J = 2.4 Hz, 1H), 8.15 (d, J = 2.4 Hz, 1H), 7.46 (dd, J =
5.1, 8.7 Hz, 1H), 7.36 (t, J = 8.7 Hz, 1H), 7.12 (s, 2H), 5.61-5.54 (m, 1H), 1.56 (d, J = 7.2 Hz, 3H).
6.4. 2-Amino-5-bromo-N-[1-(2,6-dichloro-3-fluorophenyl)ethyl]pyridine-3-sulfonamide (9a).
To the cooled 5 mL of chlorosulfonic acid at -15 ºC was added 700 mg of 2-amino-5-bromopyridine portionwise under
argon. The resulting mixture was then gradually heated to 160 ºC and maintained at this temperature for 3h. After cooling,
the reaction medium was slowly poured on ice and the resulting white precipitate was collected by filtration and washed
with cold water to give the crude 400 mg of 2-amino-5-bromopyridine-3-sulfonyl chloride 15, which was used immediately
in the next step.
To an ice cooled solution of 1-(2,6-dichloro-3-fluorophenyl)ethan-1-amine (306 mg, 1.47 mmol) and Et3N (205 μL, 1.47
mmol) was added 15 (400 mg, 1.47 mmol) portionwise. The resulting mixture was gradually warmed to room temperature
and stirred for overnight. The reaction mixture was diluted with 10 mL of water and extracted with 10 mL of CH2Cl2. The
organic phase was washed with brine, dried over anhydrous Na2SO4 and concentrated under vacuum. The crude product
was purified by flash column chromatography (0-40% EA/PE gradient) to afford 374 mg of 9a as a white solid (21% yield).
1H NMR (300 MHz, DMSO-d6) δ 8.78 (br, 1H), δ8.11 (d, J = 2.4 Hz, 1H), 7.48-7.24 (m, 3H), 6.77 (s, 2H), 5.19-5.12 (m,
1H), 1.50 (d, J = 7.2 Hz, 3H).
6.5. N,N'-dimethyl-1-[(2-nitropyridin-3-yl)oxy]methanethioamide (18).
To a solution of 2-nitropyridin-3-ol (10.00 g, 71.38 mmol) and dimethylcarbamothioic chloride (10.59 g, 85.66 mmol) in 50
mL of DMF was added DABCO (9.61 g, 85.66 mmol). The resulting mixture was stirred at room temperature for 24 h and
then diluted with 150 mL of water. The resulting mixture was extracted with 600 mL of acetyl acetate. The organic layer
was washed with water (3 × 100 mL), brine, dried over anhydrous Na2SO4 and concentrated under vacuum. The crude
product as a yellow solid was pure enough for use in the next step. 1H NMR (400 MHz, CDCl3) δ 8.47 (dd, J = 2.4, 4.4 Hz,
1H), 7.77 (dd, J = 2.4, 8.0 Hz, 1H), 7.70 (dd, J = 4.4, 8.0 Hz, 1H), 3.45 (s, 3 ), 3.40 (s, 3H).
6.6. N,N'-dimethyl-1-[(2-nitropyridin-3-yl)sulfanyl]formamide (19).
The obtained crude product 24 was suspended in 50 mL of Ph2O. The resulting mixture was heated at 160 ºC for 3h. After
cooling, The residue was purified on silica gel column (PE/EA, 4/1-2/1) and then the crude product 19 was recrystallized
from ether to afford 9.70 g of yellow solid (two steps: 60% yield). 1H NMR (300 MHz, CDCl3) δ 8.53 (dd, J = 1.8, 4.8 Hz,
1H), 8.18 (dd, J = 1.8, 8.1 Hz, 1H), 7.60 (dd, J = 4.8, 8.1 Hz, 1H), 3.12 (s, 3H), 3.04 (s, 3H).
6.7 3-{[1-(2,6-dichloro-3-fluorophenyl)ethyl]sulfanyl}pyridin-2-amine (20).
An eggplant-type flask was charged with N,N'-methyl-1-[(2-nitropyridin-3-yl)sulfanyl]formamide 25 (10.00 g, 44.01
mmol), KOH (5.43 g, 96.81 mmol). The flask was capped and then evacuated and refilled with argon three times. To the
flask was added 100 mL of MeOH/THF/H2O (2/2/1, v/v/v) via a syringe. The resulting mixture was stirred at room
temperature and monitored by TLC. After the complete consumption of 25, the organic solvents were removed under
vaccum. Under argon, to the residue was add a solution of 1-(2,6-dchloro-3-fluorophenyl)ethyl methanesulfonate 12 (12.64
g, 44.61 mmol) in 120 mL of THF. The resulting medium was stirred at room temperature for another 24 h. After the
8

removal of the solvent by evaporation under vacuum, the residue was extracted with 300 mL of EA. The organic layer was
washed with water (3 × 40 mL), brine, dried over anhydrous Na2SO4 and concentrated under vacuum to afford the crude
1
product 3-{[1-(2,6-dichloro-3-fluorophenyl)ethyl]sulfanyl}-2-nitropyridine. H NMR (300 MHz, CDCl3) δ 8.36 (d, J = 3.9
Hz, 1H), 7.88 (d, J = 7.8 Hz, 1H), 7.46 (m, 1H), 7.33-7.26 (m, 1H), 7.08-7.03 (m, 1H), 5.33-5.24 (m, 1H), 1.88-1.85 (m,
3H).
The crude product 3-{[1-(2,6-dichloro-3-fluorophenyl)ethyl]sulfanyl}-2-nitropyridine was dissolved in 120 mL of EtOH.
To the solution was added iron powder (12.06 g, 216.02 mmol), followed by 10 mL of HCl (1M). The resulting mixture
was stirred at 80 ºC for 8 h and filtered through a pad of celite. The filtrate was concentrated under vaccum. The resulting
residue was recrystallized from PE/EA (4/1). The white precipitate was collected by filtration. The corresponding filtrate
was concentrated under vaccum and the residue was further purified by flash column chromatography (0-15% EA/PE
1
gradient). The compound 20 was obtained as a white solid from 19 in 47% overall yield. H NMR (400 MHz, CDCl3) δ
8.01 (d, J = 4.8 Hz, 1H), 7.44-7.39 (m, 1H), 7.29-7.26 (m, 0.5H), 7.17 (dd, J = 4.8, 8.8 Hz, 0.5H), 7.01-6.97 (m, 1H), 6.53-
6.49 (m, 1H), 5.17 (s, 2H), 5.04-4.97 (m, 1H), 1.82 (dd, J = 2.4, 7.2 Hz, 3H).
6.8 5-Bromo-3-{[1-(2,6-dichloro-3-fluorophenyl)ethyl]sulfanyl}pyridin-2-amine (10a).
K2CO3 (1.67 g, 12.05 mmol) was added to an ice-cooled solution of 20 (3.48 g, 10.96 mmol) in 25 mL of CH2Cl2 , followed
by dropwise addition of a solution of bromine (617 μL, 12.05 mmol) in 10 mL of CH2Cl2. After the complete addition of
bromine, the resulting mixture was stirred for an additional 5 min and quenched with saturated sodium thiosulfate solution.
The mixture was extracted with CH2Cl2. The organic layer was washed with brine, dried over anhydrous Na2SO4 and
concentrated under vacuum. The crude product was purified flash column chromatography (0-7% EA/PE gradient) to afford
1
10a in 69% yield. H NMR (300 MHz, CDCl3) δ 8.03 (t, J = 2.1 Hz, 1H), 7.52 (dd, J = 2.4, 5.4 Hz, 1H), 7.32-7.26 (m,
0.5H), 7.21 (dd, J = 5.1, 9.0 Hz, 0.5H), 7.05-6.99 (m, 1H), 5.18 (s, 2H), 5.07-4.98 (m, 1H), 1.84 (dd, J = 2.7, 7.5 Hz, 3H).
6.9 Ethyl 2-(4-bromo-1H-pyrazol-1-yl)acetate (24).
To an ice-cooled solution of 4-bromo-1H-pyrazole (1.00 g, 6.80 mmol) in 10 mL of DMF was added portionwise sodium
hydride (327 mg, 8.16 mmol, 60% percent in oil). The resulting mixture was stirred for additional 1 h. Then to the mixture
was added ethyl 2-bromoacetate (0.83 mL, 7.48 mmol) and KI (226 mg, 1.36 mmol). The resulting mixture was gradually
heated to 80 ºC and stirred for 8 h. After cooled to room temperature, the reaction was quenched with 20 mL of water. The
mixture was extracted with EA (2 × 75 mL). The combined organic layer was washed with water (3 × 15 mL), brine (40
mL), dried over anhydrous Na2SO4 and concentrated under vacuum. The crude product was purified by flash column
chromatography (0-10% EA/PE gradient) to afford 24 as clear colorless oil in 83% yield. 1H NMR (300 MHz, CDCl3) δ
7.51 (s, 2H), 4.87 (s, 2H), 4.24 (q, J = 6.9 Hz, 2H), 1.29 (t, J = 6.9 Hz, 3H).
6.10 4-Bromo-1-(oxan-4-yl)-1H-pyrazole (26a). Representative experimental procedure for 24 and 26b.
To an ice-cooled solution of 4-bromo-1H-pyrazole (1.19 g, 8.07 mmol) in 10 mL of DMF was added portionwise sodium
hydride (355 mg, 8.88 mmol, 60% in oil). The resulting mixture was stirred for additional 1 h. Then to the mixture was
added oxan-4-yl methanesulfonate (1.60 g, 8.88 mmol), which was prepared in a similar manner as described for 12. The
resulting mixture was gradually heated to 100 ºC and stirred for 10 h. After cooled to room temperature, the reaction was
quenched with water. The mixture was extracted with EA. The organic layer was washed with brine, dried over anhydrous
Na2SO4 and concentrated under vacuum. The crude product was purified by silica gel column chromatography (EA/PE, 1/6)
to afford 33a as white solid in 61% yield. 1H NMR (300 MHz, DMSO-d6) δ 8.06 (s, 1H), 7.55 (s, 1H), 4.41-4.35 (m, 1H),
3.95-3.92 (m, 2H), 3.47-3.40 (m, 2H), 1.95-1.87 (m, 4H).
6.11 N,N'-dimethyl-4-(tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole-1-carboxamide (25a).
To an ice-cooled solution of 4-(tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (150 mg, 0.77 mmol), Et3N (269 μL, 1.93
mmol) and catalytic amount of DMAP in 5 mL of CH2Cl2 was added dimethylcarbamic chloride (178 μL, 1.93 mmol). The
resulting medium was stirred at room temperature for 34 h and diluted with water. The mixture was extracted with CH2Cl2.
The organic phase was washed with brine, dried over anhydrous Na2SO4 and concentrated under vacuum. The crude
product was purified by flash column chromatography (0-20% EA/PE gradient) o afford 25a as a white solid in 71% yield.
1H NMR (300 MHz, CDCl3) δ 8.40 (s, 1H), 7.87 (s, 1H), 3.21 (s, 6H), 1.33 (s, 12H).
6.12 2-(4-Bromo-1H-pyrazol-1-yl)ethan-1-ol (27).
To a solution of 4-bromo-1H-pyrazole (1.00 g, 6.80 mmol) in 20 mL of DMF was successively added 2-bromoethanol (0.53
mL, 7.48 mmol), Cs2CO3 (2.66 g, 8.16 mmol) and TBAI (503 mg, 1.36 mmol). The resulting mixture was heated at 90 ºC
for 2 h and diluted with 60 mL of water. The mixture was extracted with EA (3 × 100 mL). The combined organic layer was
washed with water (3 × 50 mL), brine (60 mL), dried over anhydrous Na2SO4 and concentrated under vacuum. The crude
product was purified by flash column chromatography (0-30% EA/PE gradient) o afford 27 as light yellow oil in 65% yield.
1H NMR (300 MHz, CDCl3) δ 7.49 (s, 1H), 7.48 (s, 1H), 4.23 (t, J = 4.8 Hz, 2H), 3.99 (t, J = 4.8 Hz, 2H), 2.73 (br, 1H).
9

6.13. 1-(Oxan-4-yl)-4-(tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (25c). Representative experimental
procedure for 25b, 25d and 25e.
To a solution of 4-bromo-1-(oxan-4-yl)-1H-pyrazole 33a (1.00 g, 4.33 mmol) and 4,4,5,5-tetramethyl-2-(tetramethyl-1,3,2-
dioxaborolan-2-yl)-1,3,2-dioxaborolane (1.32 g, 5.19 mmol) in 10 mL of DMF was added potassium acetate (1.27 g, 12.98
mmol), followed by 1,1'-Bis(diphenylphosphino)ferrocene-palladium(II)dichloride dichloromethane complex (177 mg, 0.22
mmol) under argon. The resulting mixture was stirred at 80 ºC for 10 h and then diluted with 40 mL of water. The mixture
was extracted with EA (3 × 30 mL). The combined organic phase was washed with water (3 × 30 mL), brine, dried over
anhydrous Na2SO4 and concentrated under vacuum. The crude product was purified by silica gel column chromatography
(EA/PE, 1/4) to afford 31c as white solid in 68% yield. 1H NMR (300 MHz, CDCl3) δ 7.80 (s, 1H), 7.75 (s, 1H), 4.42-4.31
(m, 1H), 4.12-4.07 (m, 2H), 3.57-3.49 (m, 2H), 2.13-1.99 (m, 4H), 1.32 (s, 12H).
6.14. 3-{[1-(2,6-Dichloro-3-fluorophenyl)ethyl]sulfanyl}-5-[1-(oxan-4-yl)-1H-pyrazol-4-yl]pyridine-2-amine (10c). Ge
neral experimental procedure for 8c-d and 10d-f.
A vial was charged with 10a (200 mg, 0.50 mmol), 25c (140 mg, 0.50 mmol), KF.2H2O (143 mg, 1.51 mmol), Pd(PPh3)4
(29 mg, 0.25 mmol) and 5 mL of DME/H2O/EtOH (v/v/v, 7/3/2). Then the vial was capped and heated at 110 ºC for 50 min
under microwave irradiation. The reaction mixture was extracted with EA. The organic layer was washed with brine, dried
over anhydrous Na2SO4 and concentrated under vacuum. The crude product was purified by flash column chromatography
(0-3% MeOH/CH2Cl2 gradient) and further separated by reverse HPLC (75-80% MeOH/H2O gradient) to afford compound
1
10c in 30% yield. HPLC: 95.07 %, tR = 3.15 min; H NMR (300 MHz, CDCl3) δ 8.12-8.11 (dd, J = 0.6, 2.1 Hz, 1H), 7.56 (s,
1H), 7.51-7.489 (m, 2H), 7.30-7.26 (m, 0.5H), 7.16 (dd, J = 5.1, 9.0 Hz, 0.5H), 7.01-6.95 (m, 1H), 5.25 (s, 2H), 5.08-5.00
(m, 1H), 4.37-4.28 (m, 1H), 4.13-4.08 (m, 2H), 3.58-3.46 (m, 2H), 2.12-2.00 (m, 4H), 1.85 (dd, J = 2.1, 7.5 Hz, 3H); MS
(ESI, m/z): 467.0 [M+H]+; HRMS (ESI) cacld for C21H22Cl2FN4OS ([M+H]+): 467.0875; found: 467.0891.
6.15. tert-Butyl 4-[4-(6-amino-5-{[1-(2,6-dichloro-3-fluorophenyl)ethyl]sulfamoyl}pyridin-3-yl)-1H-pyrazol-1-yl]pipe
ridine-1-carboxylate (29).
To a solution of 9a (200 mg, 0.45 mmol) and 25d (187 mg, 0.50 mmol) in 7 mL of dioxane was added 0.68 mL of sodium
carbonate solution (2 N), followed by Pd(PPh3)4 (26 mg, 0.23 mmol). The resulting mixture was evacuated and refilled with
argon three times. Then the reaction mixture was heated at 80 ºC for 24 h and was extracted with 80 mL of EA. After
washing with brine, the solution was dried over anhydrous Na2SO4 and concentrated under vacuum. The crude product was
purified by flash column chromatography (0-2% MeOH/CH2Cl2 gradient) and further recrystallized from MeOH to afford
29 as a white solid in 40% yield. 1H NMR (300 MHz, CDCl3) δ: 8.11 (d, J = 1.5 Hz, 1H), 7.79-7.76 (m, 1H), 7.63 (s, 1H),
7.57-7.54 (m, 1H), 7.02-6.91 (m, 1H), 6.77-6.72 (m, 1H), 6.28-6.17 (m, 1H), 5.51 (s, 2H), 5.47-5.37 (m, 1H), 4.35-4.25 (m,
3H), 2.96-2.85 (m, 2H), 2.19-2.16 (m, 2H), 2.01-1.89 (m, 2H), 1.60 (d, J =7.2 Hz, 3H), 1.49 (s, 9H).
6.16. 2-[4-(6-Amino-5-{[1-(2,6-dichloro-3-fluorophenyl)ethyl]sulfanyl}pyridin-3-yl)-1H-pyrazol-1-yl]-N,N'-dimethyl a
cetamide (10g).
0.20 mL of 2N NaOH was added to a solution of 31 (46 mg, 0.10 mmol) , prepared in a similar manner as described for
10c, in 2 mL of THF. The resulting medium was refluxed for 5 h. Additional 0.20 mL of 2N NaOH was added to the
reaction solution. Then the reaction was stirred for another 3 h under reflux. After concentration, the remaining residue was
acidified to PH 2~3 by adding 1N HCl and extracted with EA. The organic layer was dried over anhydrous Na2SO4 and
concentrated under vacuum to give a crude acid (43 mg). The crude acid was dissolved in 2 mL of DMF and the solution
was cooled in an ice bath. To the solution was successively added HATU (0.10 mmol), DIPEA (36 μL, 0.20 mmol) and
dimethylamine hydrochloride (10 mg, 0.12 mmol). The resulting medium was stirred at room temperature for 1h and
quenched with 2 mL of saturated NaHCO3. The mixture was extracted with 50 mL of EA. The organic layer was washed
with water (2 × 10 mL), brine, dried over anhydrous Na2SO4 and concentrated under vacuum. The crude product was
purified by flash column chromatography (0-3% MeOH/EA gradient) to afford compound 10g in 48% yield. HPLC: 96.90
1
%, tR = 2.32 min; H NMR (300 MHz, CDCl3) δ 8.14 (s, 1H), 7.63 (d, J = 2.7 Hz, 1H), 7.60 (d, J = 2.7 Hz, 1H), 7.54 (dd, J
= 2.1, 8.1 Hz, 1H), 7.32-7.26 (m, 0.5H), 7.18 (dd, J = 4.8, 8.7 Hz, 0.5H), 7.03-6.97 (m, 1H), 5.21 (s, 2H), 5.08-4.99 (m,
3H), 3.11 (s, 3H), 3.01 (s, 3H), 1.83 (dd, J = 3.3, 7.2 Hz, 3H); MS (ESI, m/z): 467.7 [M+H]+; HRMS (ESI) cacld for
C20H20Cl2FN5OSNa ([M+Na]+): 490.0647; found: 490.0630.
6.17. 3-{[1-(2,6-dichloro-3-fluorophenyl)ethyl]sulfanyl}-5-[1-(piperidin-4-yl)-1H-pyrazol-4-yl]pyridin-2-amine (10b).
Representative experimental procedure for 8b and 9b.
To the solution of 36 (20 mg, 0.035 mmol) in 2 mL of CH2Cl2 was added 0.5 mL of TFA. The resulting medium was stirred
at room temperature for 1h. After concentration, the residue was dissolved in CH2Cl2/MeOH (10/1). The solution was
neutralized with saturated NaHCO3 and then extracted with CH2Cl2. The organic phase was dried over anhydrous Na2SO4
1
and concentrated under vacuum to afford compound 10b in 80% yield. HPLC: 97.26 %, tR = 3.95 min; H NMR (400 MHz,
CDCl3) δ 8.15-8.14 (m, 1H), 7.57 (s, 1H), 7.52 (s, 1H), 7.50-7.49 (m, 1H), 7.30 (dd, J = 4.8, 8.8 Hz, 0.5H), 7.18 (dd, J =
4.8, 8.8 Hz, 0.5H), 7.03-6.98 (m, 1H), 5.13 (s, 2H), 5.08-5.02 (m, 1H), 4.31-4.24 (m, 1H), 3.36-3.32 (m, 2H), 2.90-2.83 (m,
10

2H), 2.25-2.22 (m, 2H), 2.06-1.97 (m, 2H), 1.84 (dd, J = 3.2, 7.2 Hz, 3H); MS (ESI, m/z): 466.0 [M+H]+; HRMS (ESI)
cacld for C21H23Cl2FN5OS ([M+H]+): 466.1035; found: 466.1031.
6.18. 2-{4-[4-(Amino-5-{[1-(2,6-dichloro-3-fluorophenyl)ethyl]sulfanyl}pyridin-3-yl)-1H-pyrazol-yl]piperidin-1-yl}-2-
oxol-ethyl acetate (32). Representative experimental procedure for 10h and 10i.
To an ice-cooled solution of 10b (120 mg, 0.26 mmol) and Et3N (39 μL, 0.28 mmol) in 5 mL of anhydrous CH2Cl2 was
added dropwise 2-chloro-2-oxoethyl acetate (29 μL, 0.27 mmol). The resulting medium was stirred for additional 15 min
and then diluted with 10 mL of water. The mixture was extracted with 50 mL of CH2Cl2. The organic phase was dried over
anhydrous Na2SO4 and concentrated under vacuum. The crude product was purified by flash column chromatography (0-
1
2% MeOH/CH2Cl2 gradient) to afford 32 in 48% yield. H NMR (300 MHz, CDCl3) δ 8.07 (s, 1H), 7.56-7.52 (m, 3H),
7.34-7.26 (m, 0.5H), 7.17 (dd, J = 5.1, 8.7 Hz, 0.5H), 7.04-6.99 (m, 1H), 5.78 (s, 2H), 5.07-5.03 (m, 1H), 4.78 (s, 2H), 4.70-
4.66 (m, 1H), 4.41-4.34 (m, 1H), 3.86-3.82 (m, 1H), 3.26 (t, J = 2.4 Hz, 1H), 2.87 (t, J = 2.4 Hz, 1H), 2.29-2.20 (m, 5H),
2.04-1.97 (m, 2H), 1.82 (dd, J = 1.8, 7.5 Hz, 3H).
6.19. 1-{4-[4-(6-Amino-5-{[1-(2,6-dichloro-3-fluorophenyl)ethyl]sulfanyl}pyridin-3-yl)-1H-pyrazol-1-yl]piperidin-1-
yl}-2-hydroxyethan-1-one (10j)
To a solution of 38 (70 mg, 0.12 mmol) in 5 mL of MeOH/H2O (3/2) was added monohydrate lithium hydroxide (25 mg,
0.60 mmol). The resulting mixture was stirred at room temperature for 2h. After concentration, the crude product was
purified by flash column chromatography (0-5% MeOH/CH2Cl2 gradient) to afford 10j in 58 yield. HPLC: 95.78 %, tR =
2.60 min; 1H NMR (300 MHz, CDCl3) δ 8.12 (d, J = 1.8Hz, 1H), 7.57 (s, 1H), 7.53 (d, J = 1.8 Hz, 1H), 7.51 (s, 1H), 7.31
(dd, J = 5.1, 8.7 Hz, 0.5H), 7.18 (dd, J = 5.1, 8.7 Hz, 0.5H), 7.03-6.96 (m, 1H), 5.32 (s, 2H), 5.09-5.02 (m, 1H), 4.73-4.69
(m, 1H), 4.43-4.33 (m, 1H), 4.22 (s, 2H), 3.76-3.66 (m, 1H), 3.23-3.13 (m, 1H), 3.00-2.91 (m, 1H), 2.27-2.23 (m, 2H), 2.07-
1.95 (m, 2H), 1.84 (dd, J = 1.8, 7.5 Hz 3H); MS (ESI, m/z): 523.9 [M+H]+; HRMS (ESI) cacld for C23H25Cl2FN5O2S
([M+H]+): 524.1090; found: 524.1075.
6.20. Methyl 4-(6-amino-5-{[1-(2,6-dichloro-3-fluorophenyl)ethyl]sulfanyl}pyridin-3-yl)-2-fluorobenzoate (34b).
To a solution of 10a (2.2 g, 5.55 mmol) and [3-fluoro-4-(methoxycarbonyl)phenyl]boronic acid (1.10 g, 5.55 mmol) in 44
mL of DME was added 11 mL of 1.5 M Na2CO3, followed by Pd(PPh3)2Cl2 (195 mg, 0.28 mmol) under argon. The
resulting mixture was stirred at 80 ºC for 16 h and extracted with EA. The organic phase was washed with brine, dried over
anhydrous Na2SO4 and concentrated under vacuum. The crude product was purified by silica gel column chromatography
1
(CH2Cl2/EA, 10/1) and further recrystallized from CH2Cl2 to afford 34 b as a white solid (1.80 g, 50.6% yield). H NMR
(400 MHz, DMSO-d6) δ 8.41 (t, J = 2.8 Hz, 1H), 7.86 (dt, J = 1.2, 8.0 Hz, 1H), 7.62-7.53 (m, 1.5H), 7.45 (dd, J = 1.6, 12.8
Hz, 1H), 7.39-7.32 (m, 2.5H), 6.62 (d, J = 3.2 Hz, 2H), 5.11-5.02 (m, 1H), 3.85 (s, 3H), 1.81 (dd, J = 1.6, 7.6 Hz, 3H).
6.21. 4-(6-Amino-5-{[1-(2,6-dichloro-3-fluorophenyl)ethyl]sulfanyl}pyridin-3-yl)-2-fluorobenzoic acid (35b).
40b (1.00 g, 2.13 mmol) was suspended in 20 mL of MeOH/THF/H2O (2/1/1). To this suspension was added LiOH.H2O
(447 mg, 10.65 mmol). The resulting mixture was stirred at room temperature for 10 h. After concentration, the residue was
acidified to PH 2~3 by adding 1N HCl. The white precipitate was filtered and dried to afford 35b as a white solid (900 mg,
93% yield). 1H NMR (400 MHz, DMSO-d6) δ 13.16 (br, 1H), 8.41 (t, J = 2.4 Hz, 1H), 7.85 (t, J = 8.0 Hz, 1H), 7.63 (d, J =
16.0 Hz, 1H), 7.56 (dd, J = 5.2, 8.8 Hz, 0.5H), 7.44-7.32 (m, 3.5H), 6.78 (s, 2H), 5.10-5.04 (m, 1H), 1.82 (dd, J = 2.4, 7.2
Hz, 3H).
6.22. 4-(6-Amino-5-{[1-(2,6-dichloro-3-fluorophenyl)ethyl]sulfanyl}pyridin-3-yl)-2-fluoro-N,N'-dimethylbenzamide
(36k). Representative experimental procedure for 36i, 36j and 36l.
To an ice-cooled suspension of 35b (120 mg, 0.26 mmol) in 4 mL of CH2Cl2 was successively added EDCI (56 mg, 0.29
mmol), HOBT (39 mg, 0.29 mmol), Et3N (40 μL, 0.29 mmol) and 316 μL of dimethyl amine (2M solution in THF). The
resulting mixture was stirred at room temperature for 6 h and quenched with saturated sodium carbonate solution. The
mixture was extracted with CH2Cl2. The organic phase was washed with brine, dried over anhydrous Na2SO4 and
concentrated under vacuum. The crude product was purified by flash column chromatography (60-70% EA/PE gradient) to
1
afford 36k in 69% yield. HPLC: 98.08 %, tR = 3.46 min; H NMR (400 MHz, CDCl3) δ 8.22 (s, 1H), 7.60 (s, 1H), 7.40 (t, J
= 7.6 Hz, 1H), 7.31-7.27 (m, 0.5H), 7.23-7.13 (m, 1.5H), 7.07 (d, J = 10.8 Hz, 1H), 7.02-6.96 (m, 1H), 5.44 (s, 2H), 5.10-
4.99 (m, 1H), 3.13 (s, 3H), 2.96 (s, 3H), 1.85-1.82 (m, 3H); EI-MS m/z (M+) 481; EI-HRMS calcd for (M+) 481.0594, found:
481.0588.
6.23. (1S)-1-(2,6-Dichloro-3-fluorophenyl)ethan-1-ol (38).
1-(2,6-Dichloro-3-fluorophenyl)ethan-1-ol 37 (24.00 g, 114.81 mmol) was dissolved in 300 mL of CH2ClCH2Cl. The
solution was cooled to -5 .To the solution was successively added Boc-L-proline (16.06 g, 74.63 mmol), EDCI (17.61 g,
91.85 mmol) and DMAP (1.68 g, 13.78 mmol). The resulting mixture was continued to stir overnight and diluted with 100
mL of water. The organic layer was collected, washed with brine, dried over anhydrous Na2SO4 and concentrated under
11

vacuum. The crude product was purified by silica gel column chromatography (PE/EA 100/1-50/1, v/v) to afford (1S)-1-
(2,6-dichloro-3-fluorophenyl)ethan-1-ol. (7.00 g, 58% yield). 1H NMR (400 MHz, CDCl3) δ 7.26 (dd, J = 4.8, 8.8 Hz, 1H),
7.02 (dd, J = 8.0, 8.8 Hz, 1H), 5.57 (q, J = 6.8 Hz, 1H), 2.86 (s, 1H), 1.64 (d, J = 6.8 Hz, 3H).
6.24. (1S)-1-(2,6-Dichloro-3-fluorophenyl)ethyl methanesulfonate ((S)-12).
(S)-12 was prepared in a similar manner as described for compound 12. 1H NMR (400 MHz, CDCl3) δ 7.33 (dd, J = 4.8, 8.8
Hz, 1H), 7.12 (dd, J = 8.0, 8.8 Hz, 1H), 6.45 (q, J = 6.8 Hz, 1H), 2.91 (s, 3H), 1.84 (d, J = 6.8 Hz, 3H).
6.25. 5-Bromo-3-{[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethyl]sulfanyl}pyridin-2-amine ((R)-10a).
1
(R)-10a was prepared in a similar manner as described for compound 10a. H NMR (400 MHz, CDCl3) δ 8.04-8.02 (m,
1H), 7.51 (dd, J = 2.4, 7.6 Hz, 1H), 7.29 (dd, J = 4.8, 8.8 Hz, 0.5H), 7.20 (dd, J = 4.8, 8.8 Hz, 0.5H), 7.04-6.99 (m, 1H),
5.16 (s, 2H), 5.06-4.98 (m, 1H), 1.83 (dd, J = 3.6, 7.2 Hz, 3H).
6.26. tert-Butyl-[4-(6-amino-5-{[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethyl]sulfanyl}pyridine-3-yl)-1H-pyrazol-1-yl]p-
iperidine-1-carboxylate ((R)-30).
To a solution of 42 (2.40 g, 6.06 mmol) and 26d (2.51 g, 6.66 mmol) in 48 mL of DMF was added 12 mL of 1.5 M Na2CO3,
followed by Pd(dppf)2Cl2 (247 mg, 0.30 mmol) under argon. The resulting mixture was stirred at 80
for 10 h and
extracted with EA. The organic phase was washed with brine, dried over anhydrous Na2SO4 and concentrated under
vacuum. The crude product was purified by reverse HPLC (MeOH/H2O, 10/1) to afford 43 as a white solid (1.70 g, 48.4%
yield). 1H NMR (400 MHz, CDCl3) δ 8.13-8.10 (m, 1H), 7.56 (s, 1H), 7.53-7.48 (m, 2H), 7.29 (dd, J = 5.2, 8.8 Hz, 0.5H),
7.17 (dd, J = 5.0, 8.8 Hz, 0.5H), 5.24 (s, 2H), 5.09-5.02 (m, 1H), 4.41-4.16 (m, 3H), 2.99-2.78 (m, 2H), 2.18-2.10 (m, 2H),
1.98-1.87 (m, 2H), 1.84 (dd, J = 3.2, 7.2 Hz, 3H), 1.48 (s, 12H).
6.27. 3-{[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethyl]sulfanyl}-5-[1-(piperidin-4-yl)-1H-pyrazol-4-yl]pyridin-2-amine
((R)-10b).
(R)-10b was prepared in a similar manner as described for compound 10b. [α]D18 +177 (C = 0.047, MeOH); 1H NMR (400
MHz, CDCl3) δ 8.12 (t, J = 2.0 Hz, 1H), 7.57 (s, 1H), 7.52 (s, 1H), 7.19 (t, J = 2.4 Hz, 1H), 7.30 (dd, J = 4.8, 8.8 Hz, 0.5H),
7.18 (dd, J = 4.8, 8.8 Hz, 0.5H), 7.02-6.98 (m, 1H), 5.16 (s, 2H), 5.09-5.02 (m, 1H), 4.26-4.19 (m, 1H), 3.29-3.26 (m, 2H),
2.83-2.76 (m, 2H), 2.19-2.14 (m, 2H), 1.98-1.88 (m, 2H), 1.83 (dd, J = 3.2, 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ
159.03, 158.47, 157.95, 156.56, 155.47, 146.25, 146.14, 142.21, 139.75, 139.69, 135.70, 130.38, 130.30, 130.09, 130.06,
129.47, 129.44, 128.64, 128.57, 123.06, 122.86, 122.70, 122.67, 122.47, 122.29, 120.02, 119.97, 119.03, 115.80, 115.75,
115.56, 115.52, 112.54, 59.77, 45.63, 45.38, 44.68, 33.85, 29.76, 19.30; MS (ESI, m/z): 466.1 [M+H]+.
Acknowledgments
HCC827and HCC827/GR6 were kindly gifted by Dr. Pasi A. Jänne (Dana-Farber Cancer Institute, Boston, MA). We
gratefully acknowledge financial support from National Basic Research Program of China (Grants 2009CB940903 and
2012CB518000), the National Natural Science Foundation of China (Grants 20721003 and 81025017), National S&T
Major Projects (2012ZX09103-101-072) and Silver Project (260644).National Science & Technology Major Project “Key
New Drug Creation and Manufacturing Program” (No.2012ZX09301001-007), National Program on Key Basic Research
Project of China (No.2012CB910704), the Natural Science Foundation of China for Innovation Research Group
(No.81021062) and the National Natural Science Foundation (No. 81102461).
References and notes
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14

F
F
Cl
Cl
O
Cl
Cl
F
R
O
Cl
HN
O
Cl
R
R
O
N
NH2
N
NH2
N
NH2
2
3
8
F
Cl
Cl
Cl
F
HN
S
F
Aryl
Cl
R
R
X
O
O
R
X
Cl
N
NH2
R
N
NH2
9
Cl
F
N
NH2
1
N
H
Cl
N
7
4
F
Cl
R
S
O
Cl
R
N
N
NH2
10
N
NH2
R
O
Cl
6
N
N
NH2
5
Figure 1. Representive modifications of the aminopyridine based c-Met inhibitors.
Figure 2. The binding model comparison of designed compounds with crizotinib. A. Binding pose of
compound 8b with c-Met; B and D. Binding pose of compound 9b with c-Met; C. Binding pose of
compound 10b with c-Met.
15

Scheme 1. Synthesis of 2-amino-N-benzyl-5-bromopyridine-3-carboxamide core 8aa
a Reagents and conditions: (i) NaBH4, MeOH, rt; MsCl, Et3N, 0 ºC -rt, 84% (2 steps) ; (ii) NaN3, DMF,
50 ºC; Zn, NH4Cl, EtOH/H2O (3:1), rt, 71%; (2 steps); (iii) 2-amino-5-bromopyridine-3-carboxylic acid,
HATU, DIPEA, DMF, 0 ºC-rt, 70%.
Scheme 2. Synthesis of 2-amino-N-benzyl-5-bromopyridine-3-sulfonamide core 9aa
a Reagents and conditions: (i) ClSO3H, -15 ºC-160 ºC; (ii) 13, CH2Cl2, Et3N, 0 ºC-rt, 21% (2 steps).
Scheme 3. Synthesis of 3-(benzylsulfanyl)-5-bromopyridin-2-amine core 10aa
a
Reagents and conditions: (i) DABCO, DMF, rt, 24h; (ii) Ph2O, 160 ºC, 3h; (iii) 12, KOH,
MeOH/THF/H2O (2:1:1), 0 ºC -rt; Fe/HCl, EtOH, reflux, 47% (3 steps); (iv) Br2, K2CO3, CH2Cl2, 0 ºC,
69%.
Scheme 4. Synthesis of pyrazolyl-4-boronic estera
16

a Reagents and conditions: (i) MsCl, Et3N, DMAP, CH2Cl2,0 ºC-rt, 94%; (ii) NaH, DMF, 0 ºC -100 ºC,
61-83%; (iii) dimethylcarbamic chloride, Et3N, DMAP, CH2Cl2, rt, 71%; (iv) 2-bromoethanol, Cs2CO3,
TBAI, DMF, 90 ºC, 65%; (v) Bis(pinacolato)diboron, Pd(dppf)Cl2, AcOK, DMF, 80 ºC, 8-73%.
Scheme 5. Synthesis of compounds 8b-d, 9b, 10b-ja
17

a
Reagents and conditions: (i) For 8a and 10a, Pd(PPh3)4, KF.2H2O, DME/H2O/EtOH (7:3:2), 110 ºC,
Mw, 19%-62%; For 9a, Pd(PPh3)4, Na2CO3, dioxane, 40%; (ii) For 31, aq.NaOH, THF; dimethylamine
hydrochloride, HATU, DIPEA, DMF, 0 ºC-rt, 48 (2 steps); (iii) For 28-30, CH2Cl2/TFA (4:1), rt, 80-
90%; (iv) For 10h, methanesulfonyl chloride, Et3N, CH2Cl2, 0 ºC-rt, 69%; For 10i, dimethylcarbamic
chloride, Et3N, CH2Cl2, 0 ºC-rt, 48%; For 10j, 2-chloro-2-oxoethyl acetate, Et3N, CH2Cl2, 0 ºC-rt, 48%;
LiOH.H2O, MeOH/H2O (3:2), rt, 58%.
Scheme 6. Synthesis of compounds 36a-la
18

a Reagents and conditions: (i)Pd(PPh3)Cl2, Na2CO3, DME/H2O(4:1), 80 ºC, 51% (33b); (ii)For 33a,
aq.NaOH, THF, 54% (2 steps); For 33b, LiOH.H2O, MeOH/THF/H2O (2:1:1), rt, 93%; (iii) For 36a-h,
HATU, DIPEA, DMF, 0 ºC-rt, 42-90%; For 36i-l, EDCI, HOBT, Et3N, CH2Cl2, 0 ºC-rt, 62-72%.
Scheme 7. Chiral synthesis of compounds (R)-10ba
N
NO2
Cl
O
Cl OMs
Cl OH
Cl
OH
i
ii
iii
S
+
Cl
Cl
Cl
Cl
N
O
F
F
F
F
11
37
38
(S)-12
19
iv
F
F
F
Cl
Cl
Cl
N
N
N
v
vi
N
HN
S
Cl
Br
S
Cl
BocN
S
Cl
N
NH2
N
NH2
N
NH2
a
(R)-10b
(R)-10a
(R)-30
Reagents and
conditions: (i) NaBH4, MeOH, rt; (ii) Boc-L-Pro, EDC, DMAP, ClCH2CH2Cl, -5 ºC, 58%; (iii) MsCl,
Et3N, 0 ºC-rt; (iv) KOH, MeOH/THF/H2O (2:1:1), 0 ºC-rt; Fe/HCl, EtOH, reflux; Br2, K2CO3, CH2Cl2,
0 ºC; (v) 25d, Pd(dppf)Cl2, Na2CO3, DMF/H2O,80 ºC, 48%; (vi) TFA/CH2Cl2, rt.
Table 1. SAR about the linker X and pyrazole derivatives at the C-5 position
19

2
No.
8b
R1
X
IC50 (nM)
No.
10e
R1
X
IC50 (nM)
HN
320.0 80.0
S
61.8 4.1
O
O
HN
HN
8c
99.0 20.0
10f
S
S
228.7 43.2
44.9 4.7
8d
280.0 40.0
10g
O
9b
19.4@10 μM
10h
10i
10j
S
S
58.7 1.1
O
N
N
10b
S
16.4 1.5
228.3 32.2
10c
10d
S
S
21.4 5.7
S
-
38.1 4.5
1.1 0.1
101.7 19.9
Crizotinib
-
Table 2. SAR about the aryl/heroaryl derivatives at the C-5 position
2
No.
10k
R
IC50 (nM)
No.
R
IC50 (nM)
130.2
0.1
151.8 22.6 10n
20

N
10l
N
26.5 0.0
10o
36a
50.5 2.5
25.8 5.2
N
10m
66.2 15.7
1.1 0.1
Crizotinib
-
Table 3. SAR about the benzamides derivatives at the C-5 position
IC50 (nM）
IC50 (nM）
No.
36a
R
Y
No.
36g
R
Y
H
25.8 5.2
H
22.1 0.3
36b
36c
H
H
45.3 3.8
44.9 8.7
36h
36i
H
101.0 12.4
64.9 9.3
F
240.3
66.9
36d
36e
H
H
36j
36k
36l
F
F
F
97.9 6.7
196.4 12.5
173.2 14.1
76.8 7.9
36f
H
-
81.7 21.9
1.1 0.1
Crizotinib
-
Table 4. Kinase Selectivity Profile of (R)-10b.
21

Kinase
Met
Enzyme IC50 (nM)
7.7 3.0
Kinase
EGFR
c-Kit
Enzyme IC50 (nM)
＞ 1000
＞ 1000
RON
336.4 111.9
446.5 261.8
348.9 153.1
＞ 1000
＞ 1000
Axl
c-Src
＞ 1000
ALK
Abl
＞ 1000
Tyro3
PDGFRα
PDGFRβ
FGFR1
RET
EPH-A2
EPH-B2
KDR
＞ 1000
＞ 1000
＞ 1000
＞ 1000
＞ 1000
＞ 1000
Flt-1
＞ 1000
Figure 3. Lineweaver-Burk plot of ATP competition assay of (R)-10b.
22

Figure 4. Dose-dependent inhibition of (R)-10b on c-Met phosphorylation and signal transduction
pathways in MKN-45 (A), SNU-5 (B), EBC-1 (C), BaF3/TPR-Met (D) and NIH3T3-TPR-Met (E).
Table 5. Antiproliferative activity of (R)-10b against human tumor cell lines.
(R)-10b
Crizotinib
IC50 (µM)
Crizotinib
IC50 (µM)
9.58 3.17
Cell line
Cell line
MCF-7
IC50 (µM)
IC50 (µM)
0.013 0.002
MKN-45
EBC-1
0.29 0.05
0.71 0.16
11.58 5.51
0.023 0.003
10.80 2.30
MDA-MB-231 12.10 3.45
23

0.016 0.003
17.25 2.66
11.47 2.68
10.97 1.58
12.85 0.29
14.82 0.85
SNU-5
0.19 0.02
SK-MEL-28
SK-OV-3
9.31 2.16
12.73 0.36
14.34 3.41
NCI-H441 15.51 2.22
NCI-H661 16.20 1.82
HCT-116
IC50s were calculated as means SD (μM) from three separate experiments.
Figure 5. Inhibitory effects of (R)-10b on cell growth. Cell cycle arrest induced by (R)-10b in MKN-
45 cell (A) and Ba/F3-TPR-Met cell (B).
24

Figure 6. (R)-10b inhibits motility of MDCK cell on scattering assay (A) and wounding heal assay (B).
Figure 7. (R)-10b Overcomes gefitinib-Resistance. (R)-10b plus gefitinib inhibit gefitinib-resistant
HCC827/GR5 cell proliferation (A), and downstream signal transduction pathways (B) in
HCC827/GR5 cell line.
25

Figure 8. In vivo effect of (R)-10b on tumor growth inhibition in the NIH-3T3/TPR-Met xenograft
model. (R)-10b and Crizotinib were administered by oral once daily for 2 weeks after the tumor
volume reached 100 to 150 mm3. Tumors were measured twice per week. Crizotinib was used as a
reference compound. Results are expressed as mean standard error (n = 6 per group). Asterisk
denotes p <0.05 compared to treatment with control, determined with Student’s t-test. Percent tumor
growth inhibition values (Inh.) were measured on the final day of study for drug-treated compared with
control mice and were calculated as 100%×{1-[(TreatedDay 14 - TreatedDay 0)/ (ControlDay 14 - ControlDay0)]}.
26

Graphical Abstract
A series of 2-amino-N-benzylpyridine-3-carboxnamides, 2-amino-N-benzylpyridine-3-sulfonamides and 2-amino-3-
benzylthiopyridines against c-Met were designed by means of bioisosteric replacement and docking analysis. Optimization
of the 2-amino-3-benzylthiopyridine scaffold identified compound (R)-10b displaying c-Met inhibition with an IC50 up to
7.7 nM. In the cytotoxic evaluation, compound (R)-10b effectively inhibited the proliferation of c-Met addictive human
cancer cell lines (IC50 from 0.19 - 0.71 µM) and c-Met activation-mediated cell metastasis. Of note, (R)-10b could overcome
c-Met-activation mediated gefitinib-resistance. (R)-10b could inhibit moderate inhibition of tumor growth (45%) in NIH-
3T3/TPR-Met xenograft model probably due to its high clearance and low bioavailability.
F
F
Cl
Cl
N
N
HN
S
Cl
R
X
Cl
N
NH2
N
NH2
(R)-10b
27